European Pharmacopoeia 5th Ed., 2005

Preface EUROPEAN PHARMACOPOEIA 5.0 I. PREFACE The European Pharmacopoeia was inaugurated in 1964 through the Conventio...

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Preface

EUROPEAN PHARMACOPOEIA 5.0

I. PREFACE The European Pharmacopoeia was inaugurated in 1964 through the Convention on the Elaboration of a European Pharmacopoeia. The present Fifth Edition of the European Pharmacopoeia is therefore published at the time where the 40th Anniversary of the Pharmacopoeia can be celebrated. The work on the Pharmacopoeia has gone through a remarkable development since the first difficult years. Elaboration and approval of monographs and other texts proceed by an effective and smoothly running process producing public quality standards that keep pace with scientific progresses. The work is remarkable because of its volume - the Fifth Edition presents close to 2000 monographs and other texts - and because all technical requirements have to be adopted by the European Pharmacopoeia Commission by unanimous decision. The monographs of the Pharmacopoeia are legally enforced in the countries being signatories to the Convention on the Elaboration of a European Pharmacopoeia. In addition to the 31 European countries and the European Union now being parties to the Convention, the work on the Pharmacopoeia is followed by 16 European and non-European countries and the WHO as observers. The quality standards of the European Pharmacopoeia have, therefore, an impact on the quality of medicines, which goes far beyond the European region. The Fifth Edition of the European Pharmacopoeia will become effective on 1st January 2005. Like the Fourth Edition, the present main volumes will be added to by three annual supplements implementing the decisions of each of the three annual Sessions of the European Pharmacopoeia Commission. The presentation of the Pharmacopoeia in a main volume and three annual supplements was initiated by the publication of the Fourth Edition. The intention was to increase the flexibility of the publication scheme and, in particular, to shorten the time span between adoption and enforcement. The shortening of the time span, which has indeed been successful, is possible only thanks to a very flexible attitude by those countries that make national translations of the European Pharmacopoeia monographs. A very low number of rapid revisions implemented in the past three years is another result of the new publication scheme. The Fourth Edition is completed with the publication of Supplement 4.8 since it is impracticable to work with more than the eight supplements. The Commission decided therefore to proceed to the Fifth Edition by consolidation of the Fourth Edition after three years, only. The change from First to Second Edition was caused by major changes in the general methods, while the change from Second to Third Edition was due to the wish to consolidate the work achieved and to change the form of presentation from a loose-leaf format into a main volume followed by annual supplements. The change from Fourth Edition to Fifth Edition continues the work of making the publication of the Pharmacopoeia as user-friendly as possible. It is assumed that the publication of this Fifth Edition will proceed by publication of supplements over the next three years. The eight founder countries of the Convention realised in 1964 that manufacturing and quality control standards for medicinal products on the European market had to be harmonised for reasons of public health and to facilitate the free movement of medicines. Since 1964 the world has changed and the market for medicinal products has become global. Accordingly, international harmonisation among the three major pharmacopoeias of the world, the European Pharmacopoeia, the Japanese Pharmacopoeia and the United States Pharmacopeia, has been in progress since

1990 when the Pharmacopoeial Discussion Group was set up to co-ordinate the harmonisation work. In the first years, the work was focused on the harmonisation of monographs on widely used excipients. In the absence of harmonised general methods this was a difficult work, which has now been speeded up by ‘harmonisation by attribute’ meaning that there may be tests that cannot be fully harmonised before the concerned general method is harmonised. At the stage where the monographs are harmonised, detailed information will be provided in the monograph and in a chapter of the Pharmacopoeia devoted to information on international harmonisation. In recent years, harmonisation of a wide range of general methods has been in progress, partly because of an impact from the International Conference on Harmonisation (ICH). Implementation in the Pharmacopoeia of harmonised general methods, for example for a dosage form specification, needs careful consideration because the specification must be met by products already on the market as well as new products submitted to the regulatory process. The European Pharmacopoeia Commission supports strongly the international harmonisation. It is not the harmonisation work itself that gives rise to the greatest problems, rather the implementation, which has to be decided in mutual agreement with the registration authorities. The links between the European Pharmacopoeia Commission and European regulators have been steadily strengthened during the years, as have the links with the pharmaceutical manufacturers and their associations. The new European Directives 2001/82/EC and 2001/83/EC on medicines for human use and veterinary use maintain the mandatory character of the European Pharmacopoeia monographs in the preparation of dossiers for marketing authorisation of medicines, which was instituted in the first directive 75/318/EEC in 1975. It means that the monographs of the European Pharmacopoeia must therefore be updated to keep pace with products on the market, with scientific progress, and with regulatory developments. In the field of active pharmaceutical substances, the European Pharmacopoeia Commission decided at its March 2002 Session that the principles and terminology of the revised ICH Q3A impurity testing guideline Impurities in new drug substances should as far as possible be implemented in the monographs on active substances, both new and already published. A change in terminology has been introduced in the Impurities section of monographs published in Supplement 4.6 and later where the term ‘specified impurities’ is used for impurities that have a defined individual acceptance criterion. A revision of the general monograph Substances for pharmaceutical use (2034) was also presented in Supplement 4.6 to implement the threshold values for reporting, identification and qualification of organic impurities in active substances of the revised ICH guideline. For the Fifth Edition a new chapter, 5.10. Control of impurities in substances for pharmaceutical use has been developed with great assistance by the chairs of the chemical Groups of Experts and other experts from the Commission, and by consultations of the Groups of Experts. The next step will be revision of monographs to ensure that they contain related substances tests and lists on specified and other detectable impurities. Monographs containing a related substances test based on TLC will be considered for revision. Major revision work will thus proceed during the coming years. Hopefully, these revisions can be completed with the publication of the Sixth Edition. In the meantime, users of the Pharmacopoeia must consult the new Chapter 5.10 i

Preface


these characteristics. The section does not necessarily give acceptance criteria for the concerned properties ; this is usually left as an option for labelling by the manufacturers and where specified, the values are indicative only. This is a new development which is in agreement with the policy of the European Pharmacopoeia Commission to make monographs and other texts appropriate to the needs The aim of the revision is to ensure that the related of regulatory authorities and manufacturers of starting substances test and impurity lists reflect the purity of materials and medicinal products. The intention is to provide pharmaceutical substances being authorised for the manufacturers of excipient materials and manufacturers European market. The goal cannot be met without of medicinal products a ’common language’, to facilitate close collaboration with the registration authorities and the establishment of product-specific specifications, and to consultations regarding the specifications for impurities. provide regulators with data generated by methods that have A procedure for co-operation with the CPMP/CVMP been independently assessed. Quality Working Party has been established. It will It is the intention of the European Pharmacopoeia certainly contribute to ensure the validity of the European Pharmacopoeia monographs. The Certification of Suitability Commission to continue the work by drafting sections on functionality-related characteristics in monographs of Monographs of the European Pharmacopoeia might on excipients available in more than one physical grade. be a valuable source of information on the purity of Introduction of the concept of functionality-related pharmaceutical substances. The procedure is, however, characteristics presupposes that the relevant general confidential and will be kept so. In cases where a new impurity is present and calls for revision of the monograph, methods are available in the Pharmacopoeia. The European Pharmacopoeia Commission has therefore established a this can be done only when the manufacturer provides the Working Party on synthetic polymers to investigate the concerned Group of Experts with the information required need for general methods for polymers and a Working for updating. Party on powder characterisation methods. The provision The growing number of monographs on pharmaceutical of the needed general methods, for example in the field of substances and the need to keep them updated means powder characterisation, is also included in international a great workload on the Groups of Experts. In 2001, harmonisation among the pharmacopoeias. the number of chemical groups was increased and some The achievements of the European Pharmacopoeia reallocations of experts between the groups took place. Commission during the past three years would not have There is, however, still a need for more experts with access been possible without the participation of the great number to experimental facilities as permanent members of the of experts from industry, academia and national authorities, Groups of Experts or as members on an ad hoc basis. In who have given their time and expertise to the work of addition to the reorganisation of the system of Groups of Groups of Experts and Working Parties. The Commission Experts and Working Parties the working procedures for is indebted to all these experts whose work is given on a the elaboration of monographs have been expanded. In voluntary basis. The Commission is equally indebted to the addition to Procedure 1, the traditional elaboration by a Chairs of the Groups and Working Parties who have the Group of Experts, and Procedure 2, adaptation of national responsibility of guiding the work through and bringing it to monographs, which is now considered almost complete, term according to tight time limits. The Chairs are thanked Procedures 3 and 4 have been established in recent years. for their contributions within the Groups and also for their Procedure 3 applies to substances produced by only one advice and counsel to the Commission itself. manufacturer and which are close to patent expiry. The manufacturer and the national pharmacopoeia authority The work of the European Pharmacopoeia Commission is of the country where the substance is produced carry out strongly dependent on an effective Secretariat. The role of the preliminary drafting stages and check the requirements the Secretariat is to obtain and process all the information experimentally. The draft is reviewed by the working party and reports needed for the Groups of Experts, Working also responsible for the adaptation procedure and then Parties and for the Commission, to undertake laboratory processed in the usual way by public inquiry. Procedure 3 work to support the experts and to ensure the availability of has proved successful. The Commission decided in 2002 to all the reference standards needed to allow the requirements establish a modified version, Procedure 4. This procedure in the monographs to be tested. The prompt publication of implies collaboration between the manufacturer of the the Pharmacopoeia main volumes and Supplements and substance and the EDQM on the draft monograph and the on-line electronic version is possible, only, because of experimental checking by the EDQM laboratory and dedicated and hard work by the staff at the Secretariat. laboratories of national pharmacopoeia authorities before Along with the growing volume of the European publication for public inquiry. At present, Procedure 4 is run Pharmacopoeia and its adjustment to the regulatory process, as a pilot project supervised by members of the European the use of the Pharmacopoeia and its interpretation has Pharmacopoeia Commission. It is the aim of the Commission become rather complex. The journal of the European to have a full coverage of monographs on substances no Pharmacopoeia, Pharmeuropa, is a valuable source of longer subject to a patent and being present on more than information. General chapters for information will appear one European market. It requires the collaboration with the in the Pharmacopoeia during the publication of the Fifth innovators and manufacturers of active substances, which Edition as a result of the international harmonisation, and has been established during recent years. because the European Pharmacopoeia Commission has agreed on the elaboration of other chapters for information. The Fifth Edition of the European Pharmacopoeia During the past two years, the staff at the EDQM have has a number of excipient monographs containing offered training courses to users of the Pharmacopoeia. a non-mandatory section on functionality-related The Commission is grateful to the EDQM for having characteristics. The aim is to provide users with a list taken this initiative, which also strengthens the role of of physical and physicochemical characteristics that are the Pharmacopoeia and the links to its users. The links critical to the typical uses of the concerned excipient, to users of the Pharmacopoeia are also strengthened by and to provide the general methods required to assess on impurity control for the interpretation of monographs published in the past and therefore adapted to a style that has now been changed as described above. Users can in addition find information on representative chromatograms, reagents and columns used in drafting the monographs on the EDQM web site.

ii

Preface


the frequent workshops and conferences organised by the EDQM. This activity is highly valued by the Commission as it gives the opportunity to Commission members to exchange viewpoints and to discuss new developments with experts from authorities, industry and academia. The EDQM web site is another valuable source for information on the work programme and other activities of the Commission, its Groups and the EDQM. During the past three years I have had the honour to serve the European Pharmacopoeia Commission as its elected chair. The task has been challenging but, certainly, rewarding because of the insight it has given me into the many quality aspects of the development, manufacture and marketing of medicinal products. I wish to thank members of the European Pharmacopoeia Commission for their support and collaborative spirit within and in between the Sessions of

the Commission. The two vice-chairs of the Commission are thanked for good collaboration and support during the years we have joined the Presidium. I will also thank the staff at the EDQM, in particular the secretaries to the Groups, for their kindness, enthusiasm and hard work for the benefit of the Pharmacopoeia. Finally, I wish to express warm thanks to the Director of EDQM, Dr. Agnes Artiges, and her deputy as secretary to the European Pharmacopoeia Commission, Mr. Peter Castle. I have appreciated our collaboration during the three years and wish to express heartfelt thanks to both for their support to the chair and for the tremendous work they are doing to develop the European Pharmacopoeia and its role in the European regulatory system. Professor, Dr. Henning G. Kristensen Chair of the European Pharmacopoeia Commission

iii

Introduction


II. INTRODUCTION The European Pharmacopoeia is prepared under the auspices of the Council of Europe in accordance with the terms of the Convention on the elaboration of a European Pharmacopoeia (European Treaty Series No. 50) as amended by the Protocol to the Convention (European Treaty Series No. 134), signed by the Governments of Austria, Belgium, Bosnia and Herzegovina, Croatia, Cyprus, the Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Luxembourg, the Netherlands, Norway, Portugal, Romania, Serbia and Montenegro, Slovak Republic, Slovenia, Spain, Sweden, Switzerland, “the Former Yugoslav Republic of Macedonia”, Turkey, the United Kingdom, and by the European Community.

European Pharmacopoeia monographs and other texts are designed to be appropriate to the needs of: — regulatory authorities ; — those engaged in the control of quality ; — manufacturers of starting materials and medicinal products. The European Pharmacopoeia is widely used internationally. It is the intention of the Commission to work closely with users of the Pharmacopoeia in order to satisfy better their needs and facilitate their co-operation. To this end improved procedures are being developed for obtaining advice on priorities for elaborating new monographs and enhancing the quality of the Pharmacopoeia.

The preparation of the Pharmacopoeia is the responsibility of the European Pharmacopoeia Commission (“the Commission”), appointed in accordance with Article 5 of the above-mentioned Convention. It is composed of delegations appointed by the Contracting Parties. Each delegation consists of not more than 3 members chosen for their competence in matters within the functions of the Commission.

TECHNICAL SECRETARIAT AND LABORATORY The European Pharmacopoeia Commission has a Technical Secretariat with scientific and administrative staff, situated in Strasbourg. The European Pharmacopoeia Laboratory is situated within the Secretariat and, amongst other duties, is in charge of the establishment and monitoring of all reference substances, preparations and spectra needed for the monographs of the Pharmacopoeia. The Technical Secretariat is an administrative division of the European Directorate for the Quality of Medicines (EDQM) of the Council of Europe.

Observers from non-Member States and international organisations are admitted to Sessions of the Commission in accordance with the Rules of Procedures. Observers are at present admitted from : Albania, Algeria, Australia, Bulgaria, Canada, China, Georgia, Lithuania, Malaysia, Malta, Morocco, Poland, Senegal, Syria, Tunisia, Ukraine, and the World Health Organisation.

GENERAL PRINCIPLES General rules for interpretation of the texts of the Pharmacopoeia are given in the General Notices. The The functions of the Commission established by Article 6 of following information should also be noted. the Convention as amended by the Protocol are : The general principles applied in the elaboration of monographs of the European Pharmacopoeia are laid Article 6 down in technical guides. The Technical Guide for the “Subject to the provision of Article 4 of the present Elaboration of Monographs, which deals mainly with Convention, the functions of the Commission shall be : monographs on chemical substances, is available as a special issue of Pharmeuropa (see below under Publications). (a) to determine the general principles applicable to the Other technical guides are being prepared to deal with elaboration of the European Pharmacopoeia ; aspects specific to monographs on other groups of (b) to decide upon methods of analysis for that purpose ; products. The principles applied are revised from time to (c) to arrange for the preparation of and to adopt monographs time without complete retrospective application so that to be included in the European Pharmacopoeia and ; monographs published already may not always follow the (d) to recommend the fixing of the time limits within which latest recommendations, but wherever an issue with impact on public health is identified, monographs are revised. its decisions of a technical character relating to the The procedures for the tests and assays published in the European Pharmacopoeia shall be implemented within individual monographs have been validated, according to the territories of the Contracting Parties.” current practice at the time of their elaboration, for the In accordance with the terms of the Convention, the purpose for which they are intended. Contracting Parties undertake to take the necessary It is recognised that general chapters are used elsewhere measures to ensure that the monographs of the European than in the monographs of the Pharmacopoeia ; in these Pharmacopoeia shall become the official standards circumstances users are recommended to consult the applicable within their respective territories. Technical Guide which gives extensive information on the PURPOSE OF THE EUROPEAN PHARMACOPOEIA application of many of the methods. The purpose of the European Pharmacopoeia is to promote General monographs. The standards of the European Pharmacopoeia are represented by general and specific public health by the provision of recognised common monographs. The use of general monographs has developed standards for use by health-care professionals and others in recent years to provide standards that best fulfil the concerned with the quality of medicines. Such standards are to be of appropriate quality as a basis for the safe use of aims stated above and meet the needs of users. It is now usually necessary to apply one or more general medicines by patients and consumers. Their existence : monographs along with any specific monograph. Since — facilitates the free movement of medicinal products in it is not practically possible to include in each specific Europe ; monograph a cross-reference to applicable or potientially — ensures the quality of medicinal products exported from applicable general monographs, cross-referencing has been Europe. discontinued except where it is necessary to avoid ambiguity. v

Introduction

A list of general monographs is included in each new edition and supplement to aid users in identifying those that are needed for use with a specific monograph. Use of animals. In accordance with the European Convention on the protection of animals used for experimental and other scientific purposes (1986), the Commission is committed to the reduction of animal usage, wherever possible, in pharmacopoeia testing and encourages those associated with its work to seek alternative procedures. An alternative or modified method is adopted by the Commission once it has been clearly demonstrated that it offers satisfactory control for pharmacopoeial purposes. Considerable progress was made in this area while the 4th Edition was in force and while the 5th Edition was being prepared. Hydrates. With the publication of the 4th Edition, the policy on monograph titles for hydrated forms was changed. For all monographs published for the first time in the 4th Edition or subsequent editions, the degree of hydration, where applicable, is indicated in the monograph title. In previous editions, the policy was to indicate the degree of hydration only where several forms exist. If a monograph on both an anhydrous and a hydrated form of a given substance are published, then “anhydrous” will be included in the title of the relevant form. In order to avoid placing an unnecessary burden on manufacturers for relabelling, this policy will not be applied retrospectively to monographs published already, unless there is reason to believe that this is justified as a public health measure, notably for safety reasons where the substance contains a large proportion of water. Chiral substances. Monographs on chiral substances that describe a particular enantiomer have a test to confirm enantiomeric purity, usually by measurement of optical rotation. Monographs that describe racemates are, in this respect, heterogeneous because of changes of policy during the 3rd Edition. Older monographs do not always have a test to show racemic character. During the course of the 3rd Edition, a test for racemic character was included in all new and revised monographs on racemates, using measurement of optical rotation. When it was shown that in many cases a test for optical rotation, even with narrow limits around zero rotation, was not necessarily sufficiently discriminating because of the low specific optical rotation of the enantiomers, the Commission modified the policy applied. A test for racemic character using optical rotation is now included only if there is information on the specific optical rotation of the enantiomers that indicates that such a test would be discriminating in terms of enantiomeric purity. If other techniques, such as circular dichroism, can serve the intended purpose, they will be prescribed instead of optical rotation. Polymorphism. Where a substance shows polymorphism, this is usually stated under Characters. In general, no particular crystalline form is required in monographs ; exceptionally, in a few monographs, the crystalline form required is specified, for example, via an infrared absorption spectrophotometric identification test where the spectrum is required to be recorded using the substance in the solid state without recrystallisation, the chemical reference substance provided being of the required crystalline form. However, for substances other than these exceptional cases, depending on the use of a given substance in a dosage form, it may be necessary for a manufacturer to ensure that a particular crystalline form is used. The information given under Characters is intended to alert users to the need to evaluate this aspect during the development of a dosage form. The monograph on Substances for pharmaceutical use (2034) and 5.9. Polymorphism should also be consulted. vi


Specificity of assays. For the elaboration of monographs on chemical substances, the approach generally preferred by the Commission is to provide control of impurities via a well designed Tests section rather than by the inclusion of an assay that is specific for the active moiety. It is therefore the full set of requirements of a monograph that is designed to ensure that the product is of suitable quality. Impurities. Following a review of policy on control of impurities, a new general chapter 5.10. Control of impurities in substances for pharmaceutical use has been included in the 5th Edition. Together with the general monograph Substances for pharmaceutical use (2034), it describes the policy of controlling impurities in specific monographs and provides explanations on how the limits in the related substances test should be understood. Currently the test is a limit test (comparison of peaks areas). In the future (next Edition) and in order to be in line with licensing practice and international collaboration, this test will progressively be changed to utilise a quantitative acceptance criterion. At present, some of the current monographs already satisfy this approach. Except where required for the application of the monograph, in which case the name is followed by “CRS”, impurities are not provided as reference substances nor can they be provided for experimental purposes. Chromatographic columns. As an aid to users, information is made available via the web site www.pheur.org on chromatographic columns that have been found satisfactory during development of monographs and general methods. Information is also given on other equipment and reagents where this is considered useful. This information is given without warranty and does not imply that other columns, equipment or reagents than those specified are not suitable. Residual solvents. The requirements for residual solvents are given in the monograph Substances for pharmaceutical use (2034) together with the general chapters 2.4.24. Identification and control of residual solvents and 5.4. Residual solvents. Thus all active substances and excipients are subject to relevant control of residual solvents, even where no test is specified in the individual monograph. The requirements have been aligned with the ICH guideline on this topic. Reference substances, reference preparations and reference spectra. Where necessary for application of a monograph, reference substances, reference preparations and reference spectra are established and provided to users. They are chosen for their suitability for the purposes stated in the monograph and are not necessarily suitable for other uses. Any necessary information for proper use is given, for example a declared content, but no complete certificate of analysis is provided since this is not relevant for the intended use. No expiry date is attributed to reference substances and preparations, which are subjected to regular periodic monitoring to ensure their continued suitability. Where an assigned value for a given attribute, for example chemical content, is provided, no uncertainty for the assigned value is indicated. The reference substances, preparations and spectra are provided to enable the analyst to determine compliance or otherwise with a monograph. The uncertainty of an assigned value is not to be taken into account when judging compliance, since the uncertainty is already allowed for in the prescribed limits. Medical devices. All editions of the Pharmacopoeia have contained monographs on articles that are regarded as medical devices. For Member States of the European Union, a unified framework for standardisation of medical devices is now provided by a Directive (93/42/EEC). Following


an agreement between the various parties involved, the Commission has decided that the monographs on medical devices will be deleted once standards have been developed as foreseen by the Directive. Specifications included in the section on containers will be adapted to take account of future standards developed within the framework of the Directive. The monographs on surgical sutures remain in the Pharmacopoeia but they have been modified to conform to the requirements of the Directive and are now to be seen as standards of the type foreseen there. This adaptation of the monographs has involved deletion of some monographs on specific types of sutures in favour of a more general approach. Homoeopathic preparations. A general monograph on homoeopathic preparations was added to the Pharmacopoeia during the 2nd Edition. A number of monographs on substances used in homoeopathic preparations are now also included and further monographs are in preparation. All of these texts have been grouped in a separate section. It is understood that when the same substance is used in both homoeopathic and other preparations then the monograph in the main body of the Pharmacopoeia applies. Patents. The description in the Pharmacopoeia of articles subject to protection by patent does not confer or imply any right to the use of such patents by any person or persons other than the proprietors of the patents concerned. Protected species. Monographs, notably those on herbal drugs, may cover material obtained from protected species. Inclusion of these monographs is without prejudice to the provisions for protection of these species by national and international law. CERTIFICATION PROCEDURE A procedure for the certification of suitability of monographs of the Pharmacopoeia with respect to control of the purity of a product from a given source has been established [see Public Health Committee (Partial Agreement) Resolution AP-CSP (99) 4 or any subsequent revision available from EDQM and on the web site (www.pheur.org)] as an aid to the use of monographs in applications for marketing authorisation. The certification procedure also applies to herbal drugs, herbal drug preparations and transmissible spongiform encephalopathy (TSE) risk. Certificates may be granted with respect to published monographs. Details of the operation of this scheme are

Introduction

available from the Secretariat and on the EDQM web site. A daily updated list of certificates granted is available on-line on the EDQM web site. A list of voided or suspended certificates is also published in Pharmeuropa. PUBLICATIONS The European Pharmacopoeia is available in English and French versions in the form of a book with 3 supplements per year, and in electronic form (internet and CD-ROM). Pharmeuropa, the European Pharmacopoeia Forum, is published 4 times per year as an aid in the elaboration of monographs and as a vehicle for information on pharmacopoeial and related matters. It is available on subscription from EDQM. Web site. Information on activities and many other aspects of the European Pharmacopoeia is to be found on the EDQM web site (www.pheur.org). Implementation. The date on which monographs are to be implemented is fixed by a resolution of the Public Health Committee (Partial Agreement) of the Council of Europe, following a recommendation by the Commission. This date is usually about 6 months after publication. Where a monograph is to be implemented at a date earlier than the next publication date of the Pharmacopoeia or a supplement, a Resolution of the Public Health Committee gives the full text to be implemented. The text is also published in Pharmeuropa for information and posted on the web site as part of the Resolution. Revision programme. Monographs and other texts of the Pharmacopoeia are revised as necessary following a decision of the Commission. Revision proposals are published in Pharmeuropa. INTERNATIONAL HARMONISATION The European Pharmacopoeia is engaged in a process of harmonisation with the Japanese Pharmacopoeia and the United States Pharmacopeia, within an informal structure referred to as the Pharmacopoeial Discussion Group (PDG). The activities are developed in co-ordination with those of the International Conference on Harmonisation (ICH). Information on the status of harmonised texts is given in chapter 5.8. Pharmacopoeial harmonisation. Harmonised general chapters have a preliminary statement indicating interchangeability with the other two pharmacopoeias.

vii

Members of the Commission


III. EUROPEAN PHARMACOPOEIA COMMISSION COMPOSITION OF THE COMMISSION, LIST OF EXPERTS AND OF THE SECRETARIAT AS OF 30 NOVEMBER 2003 CHAIR AND VICE-CHAIRS OF THE COMMISSION Chair

Henning G.

KRISTENSEN

Vice-chairs

Dries Liisa

DE KASTE TURAKKA

Hungary

Hilda Jozsef J.

KÖSZEGI-SZALAI LIPTAK

Iceland

Gudrun Ingolf J.

BALDURSDOTTIR PETERSEN

Ireland

T.A. Michael Joan

McGUINN MORRIS O’RIORDAN

MEMBERS OF THE COMMISSION Austria

Kristof Andreas Christian

LISZKA MAYRHOFER NOE

Italy

CIGNITTI FARINA OREFICI

Belgium

Luc Jos Paule

ANGENOT HOOGMARTENS JACQMAIN

Maurizio Anna Graziella

Latvia

Janis

OZOLINS

Aida

MEHMEDAGIC

Luxembourg

Bosnia and Herzegovina

Jacqueline Jean-Louis

GENOUX-HAMES ROBERT

Croatia

Dragica Ivana Laila

BEGIC STARESINIC-SERNHORST STEFANINI ORESIC

Netherlands

Dries Jan Willem Pieter H.

DE KASTE DORPEMA VREE

Norway

Cyprus

Louis

PANAYI

Gunhild Valborg Randi

BRUGAARD HOLTEN WINSNES

Portugal

José Manuel CORREIA NEVES SOUSA LOBO Rui MORGADO

Romania

Daniele

ENACHE

Marija Stana

MASKOVIC MICIC

Czech Republic Jiri M. Denmark

PORTYCH TRAVNICKOVA

Kirsten BRØNNUM-HANSEN Steen Honoré HANSEN Eva SANDBERG

Estonia

Signe

LEITO

Serbia and Montenegro

Finland

Jussi Kaarina Liisa

HOLMALAHTI SINIVUO TURAKKA

Slovak Republic N. Ruzena J.

France

Jean-Paul An Alain

FOURNIER LÊ NICOLAS

Slovenia

Martina Evgen Uros

CVELBAR TOMAZIN URLEB

Germany

Ulrike Dietrich D.

HOLZGRABE KRÜGER SCHNÄDELBACH

Spain

Franco Jordi Alexandra

FERNANDEZ GONZALEZ RUIZ COMBALIA VARDULAKI

Greece

Michael A. Alexandra

KOUPPARIS TSOKA

Sweden

Lennart Marianne Christina

AKERBLOM EK GRAFFNER

CHALABALA MARTINCOVA SLANY

ix

Alternate Members


Switzerland

Werner Silvia Helena

ERNI WEBER BRUNNER WINDEMANN

“The Former Yugoslav Republic of Macedonia”

Aneta Tatjana

DIMITROVSKA PERUSEVSKA

Turkey

Orhan Yilmaz Hayriye

Slovak Republic Daniel Ladislav

GRANCAI SOVIK

Slovenia

Maja Barbara Ales

LUSIN RAZINGER-MIHOVEC ROTAR

Sweden

Torbjörn

ARVIDSSON

CANBOLAT CAPAN MIHCAK

Switzerland

Andreas Uwe Eugen

BRUTSCHE VÖLKER WACHBERGER

United Kingdom Derek H. Gerard A.David

CALAM LEE WOOLFSON

United Kingdom Aileen M.T. John M.

European Commission

Nicolas

ROSSIGNOL

EMEA

Emer

COOKE

ALTERNATE MEMBERS Austria

J. Josef

KURZ TRENKER

Belgium

Jacques Luc Arnold J.

DE BEER DELATTRE VLIETINCK

Denmark

Sven Lars J Lene

FRØKJAER HUSAGER THOMSEN

Estonia

Juhan

RUUT

Finland

Hannele

SALOMIES

France

BOURQUIN Thierry Hendrick Jan DE JONG VILAIN Caroline

Germany

Gerhard Rainer Rainer

FRANZ MOHR SEITZ

Greece

Evangelos A.

PETRODASKALAKIS TSANTILI-KAKOULIDOU

Hungary

Tamas L.

PAÀL

Ireland

Edward

BOURKE

Italy

Massimo Agostino Loredana

DI MUZIO MACRI NICOLETTI

Luxembourg

Mariette

BACKES-LIES

Netherlands

DE ROOIJ-LAMME Ellen Peter M.J.M. JONGEN Jan-Anton NORDER

x

LEE MIDGLEY

EXPERTS Susan Maqbool Jean-Marc Lennart Ferhan Concepcion Hansruedy Julio Hanspeter Cyrille Luc Gunnar Astrid Jean-Claude Torbjörn Wilfried Nataliya Nikolaevna Jérôme Sylvie Paolo Teresa Kenneth Elizabeth Ann K. Hüsnü Can Michel Alain Françoise Denis Leandro David N. Brita Kathrin Serge Pietro Jean-Pierre Hanno Mikael Johannes Giovanni Colin Nicole Thierry Bernard

AGERHOLM AHMED AIACHE AKERBLOM AKTAN ALONSO VERDURAS ALTORFER ALVAREZ-BUILLA AMSTUTZ ANDRES ANGENOT ANTONI ARBIN ARGOUD ARVIDSSON ARZ ASMOLOVA AUCOUTURIER AUDOLY AURELI AZCONA LLANEZA BÄCKSTRÖM BAKER BASER BAUER BAYOL BEAUJEAN BELLENOT BELLENTANI BENTLEY BERGH BERNARD-SUMMERMATTER BESSET BIANCHINI BINDER BINDER BISRAT BLÜMEL BOCCARDI BOOTH BORNSTEIN BOURQUIN BOUYSSIERE

Experts


Brigitte Harald Per O. Einar M. Adrian F. Kirsten Lukas Peter Volker Marian Rosario Jörg Roger Peter R. Derek H. Kandemir Salvador François Gunnar Sergio A.J. Richard Pierre Xavier Vivienne Maurizio Klaus Juan Giovanni Laurence Pierre-Albert Stéphane Giordano Bruno Klaus Gérard Jacques-Christian Alastair Jacqueline Jacques Josep M. Hendrick Jan Dries Carmen Anna Ellen Paul Clemens Louis H.T. Luc Reto Joseph Jan S. Jan J. Roland Johannes Eric Erik Milada Thomas Jan Willem

BRAKE BREIVIK BREMER BREVIK BRISTOW BRØNNUM-HANSEN BRUCKNER BRUEGGER BUEHLER BUKOVSKY BULLIDO BUND BURGENER BYRON CALAM CANEFE CANIGUERAL CANO CARLIN CAROLI CAWS CAWTHORNE CHAMINADE CHENIVESSE CHRIST CIANFRIGLIA CICHUTEK CLARAMUNT CAMPANA COLLI COLLIERE COMPAGNON CORNEN CORSI CUSSLER DAMIEN DARBORD DAVIDSON DAYAN-KENIGSBERG DE BEER DE CIURANA i GAY DE JONG DE KASTE DE LA MORENA CRIADO DE PASQUALE DE ROOIJ-LAMME DECLERCK DECRISTOFORO DEDEREN DELATTRE DELLA CASA DEMEESTER DEN HARTIGH DENYER DIJKSTERHUIS DOBBELAER DODT DOELKER DOEVENDANS DOLEZALOVA DOLL DORPEMA

Maria Helena Robert Anil Siegfried Erling Marianne Torben Ulrich Magnus Bengt Jean-Pierre Øystein Bernard M. Charles J. Gemma L.M. Peter Rainer V’Iain Øystein Ton Ulf Lucien Isabelle Jean-Paul Bruno Gerhard Florence Nicola Rose E. Maria Cristina Andreas Jesus Didier Andrea Olga Nicole Michel Chris T. Marcel Christina Tatjana Daniel Marta Norbert Gerhard Peter Kjell-Olov Jean Louis Emanuel Giuseppe Michèle Robert Sylvie Klaus Lilian Franz-Josef Steen Honoré Paul Goetz Vassiliki Kaare

DOS ANJOS RODRIGUES AMARAL DRILLIEN DUDANI EBEL EHRIN EK ELHAUGE ENGEL ERICKSON ERLANDSSON ETCHEGARAY EVENSEN EVERETT FALLAIS FEENSTRA-BIELDERS FEIGENWINTER FENDT FENTON-MAY FODSTAD FÖRCH FORSMAN FOSSE FOURASTÉ FOURNIER FRANK FRANZ FUCHS FUZZATI GAINES DAS GALLI GARDI GARICANO AISA GARONNAT GAZZANIGA GELDOF GIBELIN GIRARD GODDARD GOVERDE GRAFFNER GRAFNETTEROVA GRANCAI GRANSTRÖM GREIDZIAK GROHMANN GRONSKI GRÖNVIK GROSSIORD GUADAGNINO GUALANDI GUITTET GURNY GUYOMARD-DEVANLAY HABERER HAMILTON HAMMERSCHMIDT HANSEN HARGREAVES HARNISCHFEGER HARTOFYLAX HASLOV

xi

Experts

Heribert Mary Alice Ingrid Irmtraud Keith Peter Jaakko-Juhani B. François Rikke Valborg Ulrike Ronald Ernö Rolf Anthony R. Ronny Lars J Herwig Marianne Miia M.B. Jana Christa Mats E. Edgar Robert Peter M.J.M. Karl-Henrik Juan Ignacio Mats Jan Hans Ernst Lawrence Isabelle Damien Marylène Claus Brigitte Frans Hilda Katjusa Henning G. Nikolaus G. Burt H. M. Michael Ursula Harry V. Francesco Fritz Reinhard Mervi Christophe Daniel Annie Maria Grazia John Joyce An

xii


HÄUSLER HEFFORD HEGGER HELD HELLIWELL HENRYS HIMBERG HINSCH HIRSCH HOFF-JØRGENSEN HOLTEN HOLZGRABE HOOGERBRUGGE HORVATH HOVIK HUBBARD HÜBINETTE HUSAGER IGEL INZELT-KOVACS JAKAVA-VILJANEN JAMES JERABKOVA JERSCH JOHANSSON JOHN JOHNSON JONGEN JÖNSSON JORQUERA NIETO JOSEFSON KARLSEN KEEGSTRA KELLER KELLY KEMPF KERLOC’H KOBISCH KOCH KOPP KORSE KÖSZEGI-SZALAI KREFT KRISTENSEN KRIZ KROES KROON KRÜGER KUKRAL KUPPERS LA TORRE LACKNER LANGE LANKINEN LAROCHE LARZUL LASSERRE LAVAGGI LAVDIOTIS LAWRENCE LÊ

Marie-Frédérique Aileen M.T. Eva Ulla Franck Silvano Céline Patrick Heiner Bruce Antonio Warren C. Georges Carla Ana Maria Andreas Alessandro D.L. Jos H.A. Andreas Bernard Geerd J. Jacques John M. Giovanni Marianne Michael Miquel Tony Birgitte Thomas Patrick Manfred Jacques Laurence Carl Zdenka B.W. Hartwig Michael Vera Philip S. Ria Robin A.J. Steven C. Alain Loredana Vittorio Jochen Dagmar Ningur Werner Hok Liang Edgar Rose-Marie Bo Graziella Carsten Inge R.A. A.

LE POTIER LEE LEMBERKOVICS LENNMARKER LEVEILLER LONARDI LORTEAU-SOURGEN LOUIS LUDWIG MADSEN MANES ARMENGOL MANN MANSVELD MARCHIORO MARQUES GONCALVES MARTI MARTINI MASSART MATHÔT MAYRHOFER MAZIERE MEYER MICHAUD MIDGLEY MIGLIACCIO MIKAELSSON MILCHARD MIR MOFFAT MØLLGAARD MONTAG-LESSING MONTENOISE MOOS MOREL MOUILLOT MROZ MRVOVA MÜLLER MÜLLER MUTZ MYSLIVCOVA NEWLANDS NIBBELING NICHOLAS NICHOLS NICOLAS NICOLETTI NISTRIO NORWIG NOVA NOYANALPAN OBEXER OEI OHST ÖLANDER OLSSON OREFICI OVERBALLE-PETERSEN OVERBY JENSEN PACKER PADILLA

Experts


Béatrice Roya Berit Smestad Maurice Teresa Caroline J.M. Skevos Geoffrey F. Jean-Paul Roger D. Aude Wolfgang Bertrand Gilles Stephen Poly Agustín Juhani Roberto Brian Bernard Miluse Ivan Martin Ain Jean Alain H.H.T. Alessandro Eike Franz Ascensao Maria Markus Valérie Therese Hans Peter Jean-Louis Yves Eugène Joachim Véronique Michael Ales D. Maria-Sol Jordi Christoph Alain Michael Corinne Piero A. Eva Magali Gabriele Marjolijn J.J.C. Jeannot Martin Ernst Heinz D.

PANTERNE PARKER PAULSEN PENSAERT PEPPER PERSON PHILIANOS PHILLIPS PICAULT PICKETT PLANTEFEVE POHLER POIRIER PONCHEL POOLE POPOVA PORTELA MOREIRA POSTI POZZI PRIESTLY PRILLEUX PSEIDLOVA PSIKAL PUNZENGRUBER RAAL RABIANT RAGON RAYMAKERS REGOLA REICH REIGEL RIBEIRO FARINHA RICHTER RIDOUX RINGBOM RINIKER ROBERT ROCHÉ ROETS RÖHMEL ROSILIO RÖSSLER ROTAR RUDD RUIZ RUIZ COMBALIA SAAL SABOURAUD SAENGER SAINT-REQUIER SALVADORI SANDBERG SAUTEL SCHÄFFNER SCHALK SCHEFFER SCHELCHER SCHIESTL SCHLÄFLI SCHMITTER SCHNÄDELBACH

Theres Henrik Dieter Harald Volker Michael Roland Gerhard Rainer Carlo Dorothea Carlo Hanfried Ekrem François Kaarina Carl Einar Wenche Mikael Jan W.H. William Henry Glenn Robert Axel Juerg Roland Otto Borut Rainer Adriana Maryse Karl Gustav Lennart Pierre-Cyril Robin C. Maria Keith G. Liisa Michael Peter Michel Uros Miguel Angel Lars Luisa Anja F.J. Willem G. Heim Cees Hans Hans P. Bernard M.M. Daniel Jos Philippe Alexandra Paul Michel Alfons Geert

SCHNEIDER SCHULTZ SCHULZ SCHULZ SCHULZE SCHWANIG SEGONDS SEIPKE SEITZ SERAFINI SESARDIC SESSA SEYFARTH SEZIK SIMONDET SINIVUO SJØGREN SKARE SKOOG SMEETS SMITH SMITH SOUSSAIN STAHLBOM STALDER STERN STICHER STRUKELJ SUCHI SUPPA SURGOT SVENSSON SVENSSON TCHORELOFF THORPE TOLLIS TRUMAN TURAKKA TÜRCK TURECEK ULMSCHNEIDER URLEB USERA VAELDS FREDERIKSEN VALVO VAN ARKENS VAN DE VAART VAN DER SLUIS VAN DER VELDE VAN DER VLIES VAN DOORNE VAN EGMOND VAN GENUGTEN VAN GYSEGEM VAN ROMPAY VANNIER VARDULAKI VARLEY VEILLARD VERBRUGGEN VERDONK

xiii

Secretariat of the European Pharmacopoeia Commission

Christopher H. Michel Peep Philippe Eva Arnold J. Pieter H. Claude Stephen Silvia Marjolein Volker A.J. Elisabeth M. Randi Maria Bengt Bernhard David A.David Kaskashan Giorgio Max Tatjana Stéphan Ange

VERMAAT VERT VESKI VILLATTE VITKOVA VLIETINCK VREE WASTIEL WATERS WEBER BRUNNER WEDA WESSELY WEST WILLIAMSON WINSNES WIRZ WITTGREN WOLF WOOD WOOLFSON ZAIDI ZANNI ZELLER ZEUGIN MISEV ZIENTARA ZOLA

SECRETARIAT OF THE EUROPEAN PHARMACOPOEIA COMMISSION Director (European Directorate for the Quality of Medicines) Agnès

ARTIGES

Scientific Officers (Technical Secretariat, Laboratory and Biological Standardisation): Peter John Jean-Marc Stefan Anne-Sophie Karl-Heinz Catherine Emmanuelle Arnold Marie-Emmanuelle

xiv

CASTLE (Secretary to the Commission) MILLER SPIESER ALMELING BOUIN BUCHHEIT CHAMBERLIN CHARTON DAAS BEHR-GROSS


Brigitte Andrea Isabelle Catherine Ellen Pascale Guy Ulrich Monica Laure Michael

JACQUEL LODI MERCIER MILNE PEL POUKENS-RENWART RAUTMANN ROSE SORINAS-JIMENO TACONET WIERER

Publication Claude Hans-Joachim Stephan Lynne Valérie Catherine Alice Rachel

COUNE BIGALKE BREHIN HENDERSON MÉAU-BOUDES NICOLAS ROBERTS TURNER

Quality Assurance Vincent Pierre

EGLOFF LEVEAU

Translation Michelle Benoît Rex

BACQUE BERNARD HUISH

Public relations Caroline

LARSEN-LE TARNEC

Expert consultants Syed Laik Raymond

ALI BOUDET-DALBIN

The European Pharmacopoeia Commission and the European Directorate for the Quality of Medicines also wishes to thank the Secretariat for their contribution towards the publication: BYLINSKI Isabelle Anne ESPIN FROMWEILER Sandra KNAUP Carole

Contents of the 5th Edition


IV. CONTENTS OF THE 5th EDITION The 5th Edition consists of all texts published in the 4th Edition, which may subsequently have been revised or corrected, and new texts. For the information of the reader, lists are given below of monographs and general chapters that are new, or that have been revised, corrected or deleted, and texts whose title has been changed for the 5th Edition. The version date (01/2005 for volume 5.0) and the reference number (four digits for monographs and five digits for general chapters) are specified above the title of each text (monographs and general chapters). The version date makes it possible to identify the successive versions of revised texts in different volumes of the 5th Edition. Corrections that are indicated by the word “corrected” under the version date are to be taken into account from the publication date of the volume. For the 5th Edition, the following systematic modifications have been made to the texts of the Pharmacopoeia. -

The term “specified impurities” has replaced “qualified impurities” in the Impurities section of monographs

-

-

-

-

in accordance with the texts on Substances for pharmaceutical use (2034) and 5.10. Control of impurities in substances for pharmaceutical use. This term, which is compliant with the ICH guidelines, applies to impurities for which a specific acceptance criterion has been defined. In cases covered by the general monograph on Substances for pharmaceutical use (2034), the test for sterility and the corresponding information in the Labelling section are no longer included in specific monographs. Chromatograms published for information no longer appear in the Pharmacopoeia; they are now available on the Internet site: www.pheur.org. A reference to available biological reference preparations has been added to the monographs concerned. The solubility in ether has been deleted from the Characters section and from the reagent descriptions. The reference to storage in a cool place has been deleted from the monographs and reagent descriptions.

A vertical line in the margin indicates where part of a text has been revised or corrected. A horizontal line in the margin indicates where part of a text has been deleted. It is to be emphasized that these indications, which are not necessarily exhaustive, are given for information and do not form an official part of the texts. Editorial changes are not indicated. Individual copies of texts will not be supplied.

NEW TEXTS INCLUDED IN THE 5th EDITION Clofazimine (2054) Closantel sodium dihydrate for veterinary use (1716) 2.4.30. Ethylene glycol and diethylene glycol in ethoxylated substances Colestyramine (1775) 2.6.24. Avian viral vaccines: tests for extraneous agents in Coriander oil (1820) seed lots (previously texts 2.6.3, 2.6.4, 2.6.5 and Dipivefrine hydrochloride (1719) 2.6.6) Dodecyl gallate (2078) 2.6.25. Avian live virus vaccines: tests for extraneous agents in batches of finished product (previously Edrophonium chloride (2106) texts 2.6.3, 2.6.4, 2.6.5 and 2.6.6) Formoterol fumarate dihydrate (1724) 5.9. Polymorphism Human coagulation factor VIII (rDNA) (1643) 5.10. Control of impurities in substances for Hydromorphone hydrochloride (2099) pharmaceutical use Insulin aspart (2084) 5.11. Characters section in monographs Insulin lispro (2085) Isradipine (2110) MONOGRAPHS The monographs below appear for the first time in the Lactobionic acid (1647) European Pharmacopoeia. They will be implemented on Lysine acetate (2114) 1 January 2005 at the latest. Mitomycin (1655) Monographs Octyl gallate (2057) Botulinum toxin type A for injection (2113) Oleyl alcohol (2073) Ciprofibrate (2013) Oxeladin hydrogen citrate (1761) Propylene glycol dicaprylocaprate (2122) Clioquinol (2111) GENERAL CHAPTERS

xv



Pyrantel embonate (1680) Ribavirin (2109) Salmon oil, farmed (1910) Tiamulin for veterinary use (1660) Tiamulin hydrogen fumarate for veterinary use (1659) Vinorelbine tartrate (2107)

Vaccines for veterinary use

Vaccines for human use

Sodium fluoride (18F) injection (2100)

Meningococcal group C conjugate vaccine (2112)

Sodium iodide (131I) solution for radiolabelling (2121)

Avian viral tenosynovitis vaccine (live) (1956) Bovine leptospirosis vaccine (inactivated) (1939) Infectious chicken anaemia vaccine (live) (2038) Radiopharmaceutical preparations

REVISED TEXTS IN THE 5th EDITION GENERAL CHAPTERS

Etilefrine hydrochloride (1205)

1.

General notices

Eucalyptus oil (0390)

2.2.1.

Clarity and degree of opalescence of liquids

Famotidine (1012)

2.2.3.

Potentiometric determination of pH

Goserelin (1636)

2.2.5.

Relative density

Human anti-D immunoglobulin (0557)

2.2.24. Absorption spectrophotometry, infrared

Hyoscine butylbromide (0737)

2.2.34. Thermal analysis

Hyoscine hydrobromide (0106)

2.2.40. Near-infrared spectrophotometry

Hyoscyamine sulphate (0501)

2.2.46. Chromatographic separation techniques

Insulin, human (0838)

2.4.8.

Heavy metals

Iohexol (1114)

2.6.9.

Abnormal toxicity

Iopamidol (1115)

2.6.14. Bacterial endotoxins

Josamycin propionate (1982)

2.7.16. Assay of pertussis vaccine (acellular)

Ketoprofen (0922)

2.9.11. Test for methanol and 2-propanol

Lactose, anhydrous (1061)

3.2.1.

Glass containers for pharmaceutical use

Lactose monohydrate (0187)

4.

Reagents

Lavender oil (1338)

5.2.8.

Minimising the risk of transmitting animal spongiform encephalopathy agents via human and veterinary medicinal products

Paraffin, light liquid (0240) Paraffin, liquid (0239) Penbutolol sulphate (1461)

MONOGRAPHS

Povidone, iodinated (1142) The monographs below have been technically revised Primidone (0584) since their last publication. They will be implemented on Propyl gallate (1039) 1 January 2005. Propylene glycol (0430) General monographs Protirelin (1144) Substances for pharmaceutical use (2034) Risperidone (1559) Monographs Sulfamethoxazole (0108) Adrenaline tartrate (0254) Ammonium glycyrrhizate (1772) Bitter-fennel fruit oil (1826) Buserelin (1077) Cefapirin sodium (1650) Codergocrine mesilate (2060) Dexpanthenol (0761) Doxycycline hyclate (0272) Epirubicin hydrochloride (1590) Estradiol benzoate (0139)

xvi

Thiamazole (1706) Vaccines for human use BCG for immunotherapy (1929) BCG vaccine, freeze-dried (0163) Diphtheria and tetanus vaccine (adsorbed) (0444) Diphtheria and tetanus vaccine (adsorbed) for adults and adolescents (0647) Diphtheria, tetanus and hepatitis B (rDNA) vaccine (adsorbed) (2062) Diphtheria, tetanus and pertussis vaccine (adsorbed) (0445)



Diphtheria, tetanus, pertussis and poliomyelitis (inactivated) vaccine (adsorbed) (2061) Diphtheria, tetanus, pertussis, poliomyelitis (inactivated) and haemophilus type b conjugate vaccine (adsorbed) (2066)


Diphtheria vaccine (adsorbed) (0443)

Avian infectious laryngotracheitis vaccine (live) (1068)

Diphtheria vaccine (adsorbed) for adults and adolescents (0646) Haemophilus type b conjugate vaccine (1219)

Avian infectious bronchitis vaccine (live) (0442) Avian infectious bursal disease vaccine (live) (0587) Avian infectious encephalomyelitis vaccine (live) (0588) Canine leptospirosis vaccine (inactivated) (0447) Duck viral hepatitis type I vaccine (live) (1315)

Meningococcal polysaccharide vaccine (0250)

Fowl-pox vaccine (live) (0649)

Tetanus vaccine (adsorbed) (0452)

Marek’s disease vaccine (live) (0589)

Typhoid polysaccharide vaccine (1160)

Newcastle disease vaccine (live) (0450)

CORRECTED TEXTS IN THE 5th EDITION The texts below from the 4th Edition have been modified and specify “01/2005:XXXX-corrected” above the title. These modifications are to be taken into account from the publication date of the 5th Edition. GENERAL CHAPTERS

Azithromycin (1649)

2.4.18. Free formaldehyde

Benfluorex hydrochloride (1601)

2.4.24. Identification and control of residual solvents

Benzyl alcohol (0256)

2.5.34. Acetic acid in synthetic peptides

Calcium hydrogen phosphate, anhydrous (0981)

2.6.15. Prekallikrein activator

Calcium hydroxide (1078)

2.6.21. Nucleic acid amplification techniques

Carbidopa (0755)

2.7.6.

Castor oil, hydrogenated (1497)

Assay of diphtheria vaccine (adsorbed)

2.7.11. Assay of human coagulation factor IX

Castor oil, virgin (0051)

2.7.14. Assay of hepatitis A vaccine

Cefaclor (0986)

2.7.15. Assay of hepatitis B vaccine (rDNA)

Cefadroxil monohydrate (0813)

5.3.

Cefamandole nafate (1402)

Statistical analysis of results of biological assays and tests

MONOGRAPHS General monographs Vaccines for veterinary use (0062)

Cefixime (1188) Cefotaxime sodium (0989) Cefradine (0814) Chlorhexidine digluconate solution (0658) Ciclosporin (0994)

Monographs

Cilazapril (1499)

Acetylcholine chloride (1485)

Ciprofloxacin (1089)

Acetylcystein (0967)

Clarithromycin (1651)

Acitretin (1385)

Clobazam (1974)

Air, medicinal (1238)

Clobetasone butyrate (1090)

Almagate (2010)

Detomidine hydrochloride for veterinary use (1414)

Alteplase for injection (1170)

Dihydralazine sulphate, hydrated (1310)

Amantadine hydrochloride (0463)

Diphenhydramine hydrochloride (0023)

Amikacin sulphate (1290)

Disodium phosphate, anhydrous (1509)

Amoxicillin sodium (0577)

Domperidone maleate (1008)

Ampicillin sodium (0578)

Doxepin hydrochloride (1096)

Anise oil (0804)

Ebastine (2015)

Aspartame (0973)

Econazole (2049)

Azelastine hydrochloride (1633)

Econazole nitrate (0665)

xvii



Eleutherococcus (1419)

Papaverine hydrochloride (0102)

Erythropoietin concentrated solution (1316)

Pefloxacin mesilate dihydrate (1460)

Fibrin sealant kit (0903)

Pepsin powder (0682)

Fish oil, rich in omega-3-acids (1912)

Phenoxymethylpenicillin (0148)

Foscarnet sodium hexahydrate (1520)

Phenoxymethylpenicillin potassium (0149)

Framycetin sulphate (0180)

Piroxicam (0944)

Gentamicin sulphate (0331)

Polymyxin B sulphate (0203)

Glycerol (0496)

Polysorbate 20 (0426)

Glycerol (85 per cent) (0497)

Potassium acetate (1139)

Glycine (0614)

Potassium clavulanate, diluted (1653)

Goldenrod (1892)

Povidone (0685)

Goldenrod, European (1893)

Pravastatin sodium (2059)

Gonadorelin acetate (0827)

Primaquine diphosphate (0635)

Guaifenesin (0615)

Propanol (2036)

Halofantrine hydrochloride (1979)

Propranolol hydrochloride (0568)

Heparins, low-molecular-mass (0828)

Propylene glycol dilaurate (2087)

Heptaminol hydrochloride (1980)

Propylene glycol monolaurate (1915)

Human anti-D immunoglobulin for intravenous administration (1527)

Pseudoephedrine hydrochloride (1367)

Human plasma for fractionation (0853) Human plasma (pooled and treated for virus inactivation) (1646)

Rifabutin (1657) Sodium alendronate (1564) Sodium hyaluronate (1472)

Hyaluronidase (0912)

Sodium polystyrene sulphonate (1909)

Hydroxycarbamide (1616)

Somatropin (0951)

Ifosfamide (1529)

Somatropin bulk solution (0950)

Imipramine hydrochloride (0029)

Somatropin for injection (0952)

Insulin, bovine (1637)

Sorbitol, liquid (crystallising) (0436)

Insulin, porcine (1638)

Sorbitol, liquid (non-crystallising) (0437)

Interferon alfa-2 concentrated solution (1110)

Spiramycin (0293)

Interferon gamma-1b concentrated solution (1440)

Star anise oil (2108)

Ioxaglic acid (2009)

Sucrose (0204)

Isoflurane (1673)

Tamoxifen citrate (1046)

Ivermectin (1336)

Thiamine hydrochloride (0303)

Lemon oil (0620)

Ticlopidine hydrochloride (1050)

Lisinopril dihydrate (1120)

DL-α-Tocopheryl

Lobeline hydrochloride (1988)

RRR-α-Tocopheryl hydrogen succinate (1259)

Lomustine (0928)

Tramazoline hydrochloride monohydrate (1597)

Magaldrate (1539)

Triacetin (1106)

Maltitol, liquid (1236)

Vancomycin hydrochloride (1058)

Methylprednisolone hydrogen succinate (1131)

Vinblastine sulphate (0748)

Methylthioninium chloride (1132)

Vincristine sulphate (0749)

Minoxidil (0937)

Water for injections (0169)

Mometasone furoate (1449)

Water, highly purified (1927)

Netilmicin sulphate (1351)

Water, purified (0008)

Nifedipine (0627)

Willow bark (1583)

Nifuroxazide (1999)

Xylose (1278)

Omega-3-acid ethyl esters 60 (2063)

Zopiclone (1060)

xviii

hydrogen succinate (1258)




Equine influenza vaccine (inactivated) (0249)

Measles, mumps and rubella vaccine (live) (1057)

Foot-and-mouth disease (ruminants) vaccine (inactivated) (0063)

Measles vaccine (live) (0213) Mumps vaccine (live) (0538)

Swine erysipelas vaccine (inactivated) (0064)

Pertussis vaccine (acellular, co-purified, adsorbed) (1595)

Tetanus vaccine for veterinary use (0697)

Rubella vaccine (live) (0162)

Radiopharmaceutical preparations


Iobenguane (123I) injection (1113)

Clostridium novyi (type B) vaccine for veterinary use (0362)

L-Methionine

Clostridium perfringens vaccine for veterinary use (0363)

Homoeopathic preparations

Clostridium septicum vaccine for veterinary use (0364)

Iron for homoeopathic preparations (2026)

([11C]methyl) injection (1617)

TEXTS WHOSE TITLE HAS CHANGED FOR THE 5th EDITION The titles of the following texts have been changed in the 5th Edition. MONOGRAPHS

GENERAL CHAPTERS

2.2.34. Thermal analysis (previously Thermogravimetry) Monographs Amfetamine sulphate (0368) (previously Amphetamine 2.7.4. Assay of human coagulation factor VIII (previously sulphate) Assay of blood coagulation factor VIII) Riboflavin (0292) (previously Riboflavine) Riboflavin sodium phosphate (0786) (previously 3.1.4. Polyethylene without additives for containers Riboflavine sodium phosphate) for parenteral preparations and for ophthalmic Tosylchloramide sodium (0381) (previously Chloramine) preparations (previously Polyethylene without Triacetin (1106) (previously Glycerol triacetate) additives for containers for preparations for parenteral use and for ophthalmic preparations) Vaccines for veterinary use 3.1.5. Polyethylene with additives for containers for Avian infectious bronchitis vaccine (live) (0442) parenteral preparations and for ophthalmic (previously Avian infectious bronchitis vaccine (live), freeze-dried) preparations (previously Polyethylene with additives for containers for preparations for Avian infectious bursal disease vaccine (live) (0587) parenteral use and for ophthalmic preparations) (previously Avian infectious bursal disease (Gumboro disease) vaccine (live), freeze-dried) 3.1.6. Polypropylene for containers and closures Avian infectious laryngotracheitis vaccine (live) (1068) for parenteral preparations and ophthalmic (previously Avian infectious laryngotracheitis vaccine preparations (previously Polypropylene for (live) for chickens) containers and closures for preparations for Canine leptospirosis vaccine (inactivated) (0447) parenteral and ophthalmic use) (previously Leptospira vaccine for veterinary use) Duck viral hepatitis type I vaccine (live) (1315) 5.2.8. Minimising the risk of transmitting animal (previously Duck viral hepatitis vaccine (live)) spongiform encephalopathy agents via human and veterinary medicinal products Fowl-pox vaccine (live) (0649) (previously Fowl-pox vaccine (live), freeze-dried) (previously Minimising the risk of transmitting animal spongiform encephalopathy agents via Newcastle disease vaccine (live) (0450) (previously medicinal products) Newcastle disease vaccine (live), freeze-dried)

TEXTS DELETED FOR THE 5th EDITION The following texts were deleted on 1 January 2005. GENERAL CHAPTERS 2.6.3. Tests for extraneous viruses using fertilised eggs 2.6.4. Test for leucosis viruses 2.6.5. Test for extraneous viruses using cell cultures 2.6.6. Test for extraneous agents using chicks

MONOGRAPHS Carbenicillin sodium (0812)

xix

CONTENTS VOLUME 1 I.

PREFACE

i

II. INTRODUCTION

v

III. EUROPEAN PHARMACOPOEIA COMMISSION

ix

IV. CONTENTS OF THE FIFTH EDITION

xv

GENERAL CHAPTERS 1.

General Notices

1

2.

Methods of Analysis

13

2.1. Apparatus

15

2.2. Physical and physicochemical methods

21

2.3. Identification

93

2.4. Limit tests

101

2.5. Assays

125

2.6. Biological tests

143

2.7. Biological assays

185

2.8. Methods in Pharmacognosy

213

2.9. Pharmaceutical technical procedures

223

Materials for Containers, and Containers

265

3.1. Materials used for the manufacture of containers

267

3.2. Containers

301

4.

Reagents

319

5.

General Texts

441

3.

GENERAL MONOGRAPHS

567

MONOGRAPHS ON DOSAGE FORMS

597

MONOGRAPHS ON VACCINES FOR HUMAN USE

633

MONOGRAPHS ON VACCINES FOR VETERINARY USE

713

MONOGRAPHS ON IMMUNOSERA FOR HUMAN USE

799

MONOGRAPHS ON IMMUNOSERA FOR VETERINARY USE

807

MONOGRAPHS ON RADIOPHARMACEUTICAL PREPARATIONS

815

MONOGRAPHS ON SUTURES FOR HUMAN USE

871

MONOGRAPHS ON SUTURES FOR VETERINARY USE

883

MONOGRAPHS ON HOMOEOPATHIC PREPARATIONS

891

VOLUME 2 MONOGRAPHS INDEX Note : on the first page of each chapter/section there is a list of contents.

903 2739


1. General notices

01/2005:10100 functionality-related characteristics that are important for the use of the substance may be appended to the monograph for information. Test methods for determination of one or more 1.1. GENERAL STATEMENTS of these characteristics may be given, also for information. The General Notices apply to all monographs and other texts General monographs. Substances and preparations that are of the European Pharmacopoeia. the subject of an individual monograph are also required The official texts of the European Pharmacopoeia are to comply with relevant, applicable general monographs. published in English and French. Translations in other Cross-references to applicable general monographs are not languages may be prepared by the signatory States of the normally given in individual monographs. European Pharmacopoeia Convention. In case of doubt General monographs apply to all substances and preparations or dispute, the English and French versions are alone within the scope of the Definition section of the general authoritative. monograph, except where a preamble limits the application, In the texts of the European Pharmacopoeia, the word for example to substances and preparations that are the “Pharmacopoeia” without qualification means the European subject of a monograph of the Pharmacopoeia. Pharmacopoeia. The official abbreviation Ph. Eur. may be General monographs on dosage forms apply to all used to indicate the European Pharmacopoeia. preparations of the type defined. The requirements are not The use of the title or the subtitle of a monograph implies necessarily comprehensive for a given specific preparation that the article complies with the requirements of the and requirements additional to those prescribed in the relevant monograph. Such references to monographs in the general monograph may be imposed by the competent texts of the Pharmacopoeia are shown using the monograph authority. title and reference number in italics. Conventional terms. The term “competent authority” A preparation must comply throughout its period of validity ; means the national, supranational or international body or a distinct period of validity and/or specifications for opened organisation vested with the authority for making decisions or broached containers may be decided by the competent concerning the issue in question. It may, for example, be a authority. The subject of any other monograph must comply national pharmacopoeia authority, a licensing authority or throughout its period of use. The period of validity that is an official control laboratory. assigned to any given article and the time from which that The expression “unless otherwise justified and authorised” period is to be calculated are decided by the competent means that the requirements have to be met, unless the authority in the light of experimental results of stability competent authority authorises a modification or an studies. exemption where justified in a particular case. Unless otherwise indicated in the General Notices or in Statements containing the word “should” are informative or the monographs, statements in monographs constitute advisory. mandatory requirements. General chapters become In certain monographs or other texts, the terms “suitable” mandatory when referred to in a monograph, unless such and “appropriate” are used to describe a reagent, reference is made in a way that indicates that it is not the intention to make the text referred to mandatory but rather micro-organism, test method etc. ; if criteria for suitability are not described in the monograph, suitability is demonstrated to cite it for information. to the satisfaction of the competent authority. The active ingredients (medicinal substances), excipients (auxiliary substances), pharmaceutical preparations and Interchangeable methods. Certain general chapters contain other articles described in the monographs are intended a statement that the text in question is harmonised with for human and veterinary use (unless explicitly restricted the corresponding text of the Japanese Pharmacopoeia to one of these uses). An article is not of Pharmacopoeia and/or the United States Pharmacopeia and that these texts quality unless it complies with all the requirements are interchangeable. This implies that if a substance or stated in the monograph. This does not imply that preparation is found to comply with a requirement using an performance of all the tests in a monograph is necessarily interchangeable method from one of these pharmacopoeias a prerequisite for a manufacturer in assessing compliance it complies with the requirements of the European with the Pharmacopoeia before release of a product. The Pharmacopoeia. In the event of doubt or dispute, the text of manufacturer may obtain assurance that a product is of the European Pharmacopoeia is alone authoritative. Pharmacopoeia quality from data derived, for example, from validation studies of the manufacturing process and from 01/2005:10200 in-process controls. Parametric release in circumstances deemed appropriate by the competent authority is thus not 1.2. OTHER PROVISIONS APPLYING precluded by the need to comply with the Pharmacopoeia. TO GENERAL CHAPTERS AND The tests and assays described are the official methods upon which the standards of the Pharmacopoeia are based. MONOGRAPHS With the agreement of the competent authority, alternative Quantities. In tests with numerical limits and assays, the methods of analysis may be used for control purposes, quantity stated to be taken for examination is approximate. provided that the methods used enable an unequivocal The amount actually used, which may deviate by not more decision to be made as to whether compliance with the than 10 per cent from that stated, is accurately weighed standards of the monographs would be achieved if the official methods were used. In the event of doubt or dispute, or measured and the result is calculated from this exact quantity. In tests where the limit is not numerical, but usually the methods of analysis of the Pharmacopoeia are alone depends upon comparison with the behaviour of a reference authoritative. in the same conditions, the stated quantity is taken for Certain materials that are the subject of a pharmacopoeial monograph may exist in different grades suitable for different examination. Reagents are used in the prescribed amounts. purposes. Unless otherwise indicated in the monograph, Quantities are weighed or measured with an accuracy the requirements apply to all grades of the material. In commensurate with the indicated degree of precision. For some monographs, particularly those on excipients, a list of weighings, the precision corresponds to plus or minus 5 units General Notices (1) apply to all monographs and other texts

5

1. General notices


after the last figure stated (for example, 0.25 g is to be interpreted as 0.245 g to 0.255 g). For the measurement of volumes, if the figure after the decimal point is a zero or ends in a zero (for example, 10.0 ml or 0.50 ml), the volume is measured using a pipette, a volumetric flask or a burette, as appropriate ; otherwise, a graduated measuring cylinder or a graduated pipette may be used. Volumes stated in microlitres are measured using a micropipette or microsyringe. It is recognised, however, that in certain cases the precision with which quantities are stated does not correspond to the number of significant figures stated in a specified numerical limit. The weighings and measurements are then carried out with a sufficiently improved accuracy. Apparatus and procedures. Volumetric glassware complies with Class A requirements of the appropriate International Standard issued by the International Organisation for Standardisation. Unless otherwise prescribed, analytical procedures are carried out at a temperature between 15 °C and 25 °C. Unless otherwise prescribed, comparative tests are carried out using identical tubes of colourless, transparent, neutral glass with a flat base ; the volumes of liquid prescribed are for use with tubes having an internal diameter of 16 mm but tubes with a larger internal diameter may be used provided the volume of liquid used is adjusted (2.1.5). Equal volumes of the liquids to be compared are examined down the vertical axis of the tubes against a white background, or if necessary against a black background. The examination is carried out in diffuse light.

contamination (Purified water in containers) are not relevant. The term “distilled water” indicates purified water prepared by distillation. The term “ethanol” without qualification means anhydrous ethanol. The term “alcohol” without qualification means ethanol (96 per cent). Other dilutions of ethanol are indicated by the term “ethanol” or “alcohol” followed by a statement of the percentage by volume of ethanol (C2H6O) required. EXPRESSION OF CONTENT In defining content, the expression “per cent” is used according to circumstances with one of two meanings : — per cent m/m (percentage, mass in mass) expresses the number of grams of substance in 100 grams of final product, — per cent V/V (percentage, volume in volume) expresses the number of millilitres of substance in 100 millilitres of final product. The expression “parts per million (ppm)” refers to mass in mass, unless otherwise specified.

TEMPERATURE Where an analytical procedure describes temperature without a figure, the general terms used have the following meaning : — in a deep-freeze : below − 15 °C, — in a refrigerator : 2 °C to 8 °C, Any solvent required in a test or assay in which an indicator — cold or cool : 8 °C to 15 °C, is to be used is previously neutralised to the indicator, unless — room temperature : 15 °C to 25 °C. a blank test is prescribed. Water-bath. The term “water-bath” means a bath of boiling water unless water at another temperature is indicated. Other methods of heating may be substituted provided the 01/2005:10300 temperature is near to but not higher than 100 °C or the indicated temperature. 1.3. GENERAL CHAPTERS Drying and ignition to constant mass. The terms “dried to constant mass” and “ignited to constant mass” mean that CONTAINERS 2 consecutive weighings do not differ by more than 0.5 mg, the second weighing following an additional period of drying Materials used for containers are described in general chapter 3.1. General names used for materials, particularly or of ignition respectively appropriate to the nature and plastic materials, each cover a range of products varying not quantity of the residue. only in the properties of the principal constituent but also in Where drying is prescribed using one of the expressions the additives used. The test methods and limits for materials “in a desiccator” or “in vacuo”, it is carried out using the depend on the formulation and are therefore applicable only conditions described under 2.2.32. Loss on drying. for materials whose formulation is covered by the preamble to the specification. The use of materials with different formulations, and the test methods and limits applied to REAGENTS them, are subject to agreement by the competent authority. The proper conduct of the analytical procedures described in the Pharmacopoeia and the reliability of the results depend, The specifications for containers in general chapter 3.2 in part, upon the quality of the reagents used. The reagents have been developed for general application to containers of the stated category but in view of the wide variety of are described in general chapter 4. It is assumed that reagents of analytical grade are used ; for some reagents, tests containers available and possible new developments, the publication of a specification does not exclude the use, in to determine suitability are included in the specifications. justified circumstances, of containers that comply with other specifications, subject to agreement by the competent SOLVENTS authority. Where the name of the solvent is not stated, the term Reference may be made within the monographs of the “solution” implies a solution in water. Pharmacopoeia to the definitions and specifications for containers provided in chapter 3.2. Containers. The general Where the use of water is specified or implied in the monographs for pharmaceutical dosage forms may, under analytical procedures described in the Pharmacopoeia or the heading Definition/Production, require the use of for the preparation of reagents, water complying with the requirements of the monograph on Purified water (0008) is certain types of container ; certain other monographs may, under the heading Storage, indicate the type of container used, except that for many purposes the requirements for bacterial endotoxins (Purified water in bulk) and microbial that is recommended for use.

6

See the information section on general monographs (cover pages)

1. General notices


1.4. MONOGRAPHS

01/2005:10400 Solubility. In statements of solubility in the section headed Characters, the terms used have the following significance referred to a temperature between 15 °C and 25 °C.

TITLES Monograph titles are in English and French in the respective versions and there is a Latin subtitle. RELATIVE ATOMIC AND MOLECULAR MASSES The relative atomic mass (Ar) or the relative molecular mass (Mr) is shown, as and where appropriate, at the beginning of each monograph. The relative atomic and molecular masses and the molecular and graphic formulae do not constitute analytical standards for the substances described. DEFINITION Statements under the heading Definition constitute an official definition of the substance, preparation or other article that is the subject of the monograph. Limits of content. Where limits of content are prescribed, they are those determined by the method described under Assay. Vegetable drugs. In monographs on vegetable drugs, the definition indicates whether the subject of the monograph is, for example, the whole drug or the drug in powdered form. Where a monograph applies to the drug in several states, for example both to the whole drug and the drug in powdered form, the definition states this. PRODUCTION Statements under the heading Production draw attention to particular aspects of the manufacturing process but are not necessarily comprehensive. They constitute instructions to manufacturers. They may relate, for example, to source materials, to the manufacturing process itself and its validation and control, to in-process testing or to testing that is to be carried out by the manufacturer on the final article either on selected batches or on each batch prior to release. These statements cannot necessarily be verified on a sample of the final article by an independent analyst. The competent authority may establish that the instructions have been followed, for example, by examination of data received from the manufacturer, by inspection of manufacture or by testing appropriate samples.

Descriptive term

Approximate volume of solvent in millilitres per gram of solute

Very soluble

less than

1

Freely soluble

from

1

to

10

Soluble

from

10

to

30

Sparingly soluble

from

30

to

100 1000 10 000

Slightly soluble

from

100

to

Very slightly soluble

from

1000

to

Practically insoluble

more than

10 000

The term “partly soluble” is used to describe a mixture where only some of the components dissolve. The term “miscible” is used to describe a liquid that is miscible in all proportions with the stated solvent. IDENTIFICATION The tests given in the identification section are not designed to give a full confirmation of the chemical structure or composition of the product ; they are intended to give confirmation, with an acceptable degree of assurance, that the article conforms to the description on the label. Certain monographs have subdivisions entitled “First identification” and “Second identification”. The test or tests that constitute the “First identification” may be used for identification in all circumstances. The test or tests that constitute the “Second identification” may be used for identification provided it can be demonstrated that the substance or preparation is fully traceable to a batch certified to comply with all the other requirements of the monograph.

TESTS AND ASSAYS Scope. The requirements are not framed to take account of all possible impurities. It is not to be presumed, for example, that an impurity that is not detectable by means of the prescribed tests is tolerated if common sense and good pharmaceutical practice require that it be absent. See also below under Impurities. Calculation. Where the result of a test or assay is required to be calculated with reference to the dried or anhydrous substance or on some other specified basis, the determination of loss on drying, water content or other property is carried out by the method prescribed in the relevant test in the monograph. The words “dried substance” The absence of a section on Production does not imply or “anhydrous substance” etc. appear in parenthesis after that attention to features such as those referred to above the result. is not required. A product described in a monograph of Limits. The limits prescribed are based on data obtained the Pharmacopoeia is manufactured in accordance with in normal analytical practice ; they take account of normal a suitable quality system in accordance with relevant analytical errors, of acceptable variations in manufacture and international agreements and supranational and national compounding and of deterioration to an extent considered regulations governing medicinal products for human or acceptable. No further tolerances are to be applied to the veterinary use. limits prescribed to determine whether the article being Where in the section under the heading Production a examined complies with the requirements of the monograph. monograph on a vaccine defines the characteristics of In determining compliance with a numerical limit, the the vaccine strain to be used, any test methods given for calculated result of a test or assay is first rounded to the confirming these characteristics are provided for information number of significant figures stated, unless otherwise as examples of suitable methods. Similarly, test methods for prescribed. The last figure is increased by one when the part choice of vaccine composition are provided for information rejected is equal to or exceeds one half-unit, whereas it is not as examples of suitable methods. modified when the part rejected is less than a half-unit. Indication of permitted limit of impurities. For comparative CHARACTERS tests, the approximate content of impurity tolerated, or The statements under the heading Characters are not to be the sum of impurities, may be indicated for information interpreted in a strict sense and are not requirements. only. Acceptance or rejection is determined on the basis General Notices (1) apply to all monographs and other texts

7

1. General notices


of compliance or non-compliance with the stated test. If the use of a reference substance for the named impurity is not prescribed, this content may be expressed as a nominal concentration of the substance used to prepare the reference solution specified in the monograph, unless otherwise described. Vegetable drugs. For vegetable drugs, the sulphated ash, total ash, water-soluble matter, alcohol-soluble matter, water content, content of essential oil and content of active principle are calculated with reference to the drug that has not been specially dried, unless otherwise prescribed in the monograph. Equivalents. Where an equivalent is given, for the purposes of the Pharmacopoeia only the figures shown are to be used in applying the requirements of the monograph.

IMPURITIES A list of all known and potential impurities that have been shown to be detected by the tests in a monograph may be given for information. See also 5.10. Control of impurities in substances for pharmaceutical use. FUNCTIONALITY-RELATED CHARACTERISTICS A list of functionality-related characteristics that are not the subject of official requirements but which are nevertheless important for the use of a substance may be appended to a monograph, for information (see also above 1.1. General statements).

REFERENCE SUBSTANCES, REFERENCE PREPARATIONS AND REFERENCE SPECTRA Certain monographs require the use of a reference substance, a reference preparation or a reference spectrum. These STORAGE are chosen with regard to their intended use as prescribed The information and recommendations given under the in the monographs of the Pharmacopoeia and are not heading Storage do not constitute a pharmacopoeial necessarily suitable in other circumstances. The European requirement but the competent authority may specify Pharmacopoeia Commission does not accept responsibility particular storage conditions that must be met. for any errors arising from use other than as prescribed. The articles described in the Pharmacopoeia are stored The reference substances, the reference preparations and in such a way as to prevent contamination and, as far as the reference spectra are established by the European possible, deterioration. Where special conditions of storage Pharmacopoeia Commission and may be obtained from the are recommended, including the type of container (see 1.3. Technical Secretariat. They are the official standards to be General chapters) and limits of temperature, they are stated used in cases of arbitration. A list of reference substances, in the monograph. reference preparations and reference spectra may be obtained from the Technical Secretariat. The following expressions are used in monographs under Storage with the meaning shown. Local standards may be used for routine analysis, provided they are calibrated against the standards established by the In an airtight container means that the product is stored European Pharmacopoeia Commission. in an airtight container (3.2). Care is to be taken when the container is opened in a damp atmosphere. A low moisture Any information necessary for proper use of the reference content may be maintained, if necessary, by the use of a substance or reference preparation is given on the label desiccant in the container provided that direct contact with or in the accompanying leaflet or brochure. Where no the product is avoided. drying conditions are stated in the leaflet or on the label, Protected from light means that the product is stored either the substance is to be used as received. No certificate of analysis or other data not relevant to the prescribed use in a container made of a material that absorbs actinic light sufficiently to protect the contents from change induced by of the product are provided. No expiry date is indicated : such light or in a container enclosed in an outer cover that the products are guaranteed to be suitable for use when provides such protection or stored in a place from which all dispatched. The stability of the contents of opened containers cannot be guaranteed. such light is excluded. Chemical Reference Substances. The abbreviation CRS LABELLING indicates a Chemical Reference Substance established by In general, labelling of medicines is subject to supranational the European Pharmacopoeia Commission. Some Chemical Reference Substances are used for the microbiological assay and national regulation and to international agreements. of antibiotics and their activity is stated, in International The statements under the heading Labelling therefore are Units, on the label or on the accompanying leaflet and not comprehensive and, moreover, for the purposes of the defined in the same manner as for Biological Reference Pharmacopoeia only those statements that are necessary Preparations. to demonstrate compliance or non-compliance with the monograph are mandatory. Any other labelling statements Biological Reference Preparations. The majority of the are included as recommendations. When the term “label” is primary biological reference preparations referred to in the used in the Pharmacopoeia, the labelling statements may European Pharmacopoeia are the appropriate International appear on the container, the package, a leaflet accompanying Standards and Reference Preparations established by the package or a certificate of analysis accompanying the the World Health Organisation. Because these reference article, as decided by the competent authority. materials are usually available only in limited quantities, the Commission has established Biological Reference Preparations (indicated by the abbreviation BRP) where WARNINGS appropriate. Where applicable, the potency of the Biological Materials described in monographs and reagents specified Reference Preparations is expressed in International Units. for use in the Pharmacopoeia may be injurious to health For some Biological Reference Preparations, where an unless adequate precautions are taken. The principles of international standard or reference preparation does not good quality control laboratory practice and the provisions exist, the potency is expressed in European Pharmacopoeia of any appropriate regulations are to be observed at all Units. times. Attention is drawn to particular hazards in certain monographs by means of a warning statement ; absence of Reference spectra. The reference spectrum is accompanied such a statement is not to be taken to mean that no hazard by information concerning the conditions used for sample exists. preparation and recording the spectrum. 8


1.5. Abbreviations and symbols


01/2005:10500

Lp/10 dose

PFU

The smallest quantity of toxin that, in the conditions of the test, when mixed with 0.1 IU of antitoxin and administered by the specified route, causes paralysis in the test animals within a given period The largest quantity of a toxin that, in the conditions of the test, when mixed with 0.1 IU of antitoxin and administered by the specified route, does not cause symptoms of toxicity in the test animals within a given period The quantity of toxin or toxoid that flocculates in the shortest time with 1 IU of antitoxin The statistically determined quantity of virus that may be expected to infect 50 per cent of the cell cultures to which it is added The statistically determined quantity of virus that may be expected to infect 50 per cent of fertilised eggs into which it is inoculated The statistically determined quantity of a virus that may be expected to infect 50 per cent of the animals into which it is inoculated The statistically determined dose of a vaccine that, in the conditions of the tests, may be expected to protect 50 per cent of the animals against a challenge dose of the micro-organisms or toxins against which it is active The statistically determined dose of a vaccine that, in the conditions of the tests, may be expected to induce specific antibodies in 50 per cent of the animals for the relevant vaccine antigens Pock-forming units or plaque-forming units

SPF

Specified-pathogen-free.

1.5. ABBREVIATIONS AND SYMBOLS A

Absorbance

Lo/10 dose

Specific absorbance Ar

Relative atomic mass Specific optical rotation

bp BRP

Boiling point

CRS

Chemical Reference Substance

Lf dose

Biological Reference Preparation Relative density

IU

International Unit

λ M

Wavelength

Mr mp

Relative molecular mass

CCID50 EID50

Molarity Melting point

ID50

Refractive index Ph. Eur. U. ppm

European Pharmacopoeia Unit

R

Substance or solution defined under 4. Reagents Used in chromatography to indicate the ratio of the distance travelled by a substance to the distance travelled by the solvent front Used in chromatography to indicate the ratio of the distance travelled by a substance to the distance travelled by a reference substance Substance used as a primary standard in volumetric analysis (chapter 4.2.1)

Rf

Rst

RV

Parts per million

Abbreviations used in the monographs on immunoglobulins, immunosera and vaccines LD50 The statistically determined quantity of a substance that, when administered by the specified route, may be expected to cause the death of 50 per cent of the test animals within a given period MLD Minimum lethal dose L+/10 dose

L+ dose

lr/100 dose

The smallest quantity of a toxin that, in the conditions of the test, when mixed with 0.1 IU of antitoxin and administered by the specified route, causes the death of the test animals within a given period The smallest quantity of a toxin that, in the conditions of the test, when mixed with 1 IU of antitoxin and administered by the specified route, causes the death of the test animals within a given period The smallest quantity of a toxin that, in the conditions of the test, when mixed with 0.01 IU of antitoxin and injected intracutaneously causes a characteristic reaction at the site of injection within a given period

General Notices (1) apply to all monographs and other texts

PD50

ED50

Collections of micro-organisms ATCC American Type Culture Collection 10801 University Boulevard Manassas, Virginia 20110-2209, USA C.I.P. Collection de Bactéries de l’Institut Pasteur B.P. 52, 25 rue du Docteur Roux 75724 Paris Cedex 15, France IMI International Mycological Institute Bakeham Lane Surrey TW20 9TY, Great Britain I.P. Collection Nationale de Culture de Microorganismes (C.N.C.M.) Institut Pasteur 25, rue du Docteur Roux 75724 Paris Cedex 15, France NCIMB National Collection of Industrial and Marine Bacteria Ltd 23 St Machar Drive Aberdeen AB2 1RY, Great Britain

9

1.6. Units (SI) used in the Pharmacopoeia

NCPF


The derived units may be formed by combining the base units according to the algebraic relationships linking the corresponding quantities. Some of these derived units have special names and symbols. The SI units used in the European Pharmacopoeia are shown in Table 1.6-2.

National Collection of Pathogenic Fungi London School of Hygiene and Tropical Medicine Keppel Street London WC1E 7HT, Great Britain National Collection of Type Cultures Central Public Health Laboratory Colindale Avenue London NW9 5HT, Great Britain National Collection of Yeast Cultures AFRC Food Research Institute Colney Lane Norwich NR4 7UA, Great Britain Statens Serum Institut 80 Amager Boulevard, Copenhagen, Denmark

NCTC

NCYC

S.S.I.

Some important and widely used units outside the International System are shown in Table 1.6-3. The prefixes shown in Table 1.6-4 are used to form the names and symbols of the decimal multiples and submultiples of SI units. NOTES 1. In the Pharmacopoeia, the Celsius temperature is used (symbol t). This is defined by the equation :

where T0 = 273.15 K by definition. The Celsius or centigrade temperature is expressed in degree Celsius (symbol °C). The unit “degree Celsius” is equal to the unit “kelvin”. 2. The practical expressions of concentrations used in the Pharmacopoeia are defined in the General Notices.

01/2005:10600

1.6. UNITS OF THE INTERNATIONAL SYSTEM (SI) USED IN THE PHARMACOPOEIA AND EQUIVALENCE WITH OTHER UNITS INTERNATIONAL SYSTEM OF UNITS (SI) The International System of Units comprises 3 classes of units, namely base units, derived units and supplementary units(1). The base units and their definitions are set out in Table 1.6-1.

3. The radian is the plane angle between two radii of a circle which cut off on the circumference an arc equal in length to the radius. 4. In the Pharmacopoeia, conditions of centrifugation are defined by reference to the acceleration due to gravity (g) :

5. Certain quantities without dimensions are used in the Pharmacopoeia : relative density (2.2.5), absorbance (2.2.25), specific absorbance (2.2.25) and refractive index (2.2.6). 6. The microkatal is defined as the enzymic activity which, under defined conditions, produces the transformation (e.g. hydrolysis) of 1 micromole of the substrate per second.

Table 1.6.-1. – SI base units Unit

Quantity

Definition

Name

Symbol

Name

Symbol

Length

l

metre

m

The metre is the length of the path travelled by light in a vacuum during a time interval of 1/299 792 458 of a second.

Mass

m

kilogram

kg

The kilogram is equal to the mass of the international prototype of the kilogram.

Time

t

second

s

Electric current

I

ampere

A

The ampere is that constant current which, maintained in two straight parallel conductors of infinite length, of negligible circular cross-section and placed 1 metre apart in vacuum would produce between these conductors a force equal to 2 × 10− 7 newton per metre of length.

Thermodynamic temperature

T

kelvin

K

The kelvin is the fraction 1/273.16 of the thermodynamic temperature of the triple point of water.

Amount of substance

n

mole

mol

The mole is the amount of substance of a system containing as many elementary entities as there are atoms in 0.012 kilogram of carbon-12*.

Luminous intensity

Iv

candela

cd

The candela is the luminous intensity in a given direction of a source emitting monochromatic radiation with a frequency of 540 × 1012 hertz and whose energy intensity in that direction is 1/683 watt per steradian.

The second is the duration of 9 192 631 770 periods of the radiation corresponding to the transition between the two hyperfine levels of the ground state of the caesium-133 atom.

* When the mole is used, the elementary entities must be specified and may be atoms, molecules, ions, electrons, other particles or specified groups of such particles. (1) The definitions of the units used in the International System are given in the booklet “ Le Système International d’Unités (SI) ” published by the Bureau International des Poids et Mesures, Pavillon de Breteuil, F-92310 Sèvres.

10


1.6. Units (SI) used in the Pharmacopoeia


Table 1.6.-2. – SI units used in the European Pharmacopoeia and equivalence with other units Unit

Quantity

Expression in SI Expression in other Conversion of other units into SI units base units SI units m− 1

Name

Symbol

Name

Symbol

Wave number

ν

one per metre

1/m

Wavelength

λ

µm nm

Area

A, S

micrometre nanometre square metre

m2

10− 6m 10− 9m m2

Volume

V

cubic metre

m3

m3

Frequency

ν

hertz

Hz

s− 1

Density

ρ

kilogram per cubic metre

kg/m3

kg·m− 3

Velocity

v

metre per second

m/s

m·s− 1

Force

F

newton

N

m·kg·s− 2

Pressure

p

pascal

Pa

m− 1·kg·s− 2

N·m− 2

1 dyne/cm2 = 10− 1 Pa = 10− 1 N·m− 2 1 atm = 101 325 Pa = 101.325 kPa 1 bar = 105 Pa = 0.1 MPa 1 mm Hg = 133.322 387 Pa 1 Torr = 133.322 368 Pa 1 psi = 6.894 757 kPa

Dynamic viscosity

η

pascal second

Pa·s

m− 1·kg·s− 1

N·s·m− 2

1 P = 10− 1 Pa·s = 10− 1 N·s·m− 2 1 cP = 1 mPa·s

Kinematic viscosity

ν

square metre per second

m2/s

m2·s− 1

Pa·s·m3·kg− 1 N·m·s·kg− 1

1 St = 1 cm2·s− 1 = 10− 4 m2·s− 1

Energy

W

joule

J

m2·kg·s− 2

N·m

Power Radiant flux

P

watt

W

m2·kg·s− 3

N·m·s− 1 J·s− 1

1 erg/s = 1 dyne·cm·s− 1 = 10− 7 W = 10− 7 N·m·s− 1 = 10− 7 J·s− 1

Absorbed dose (of radiant energy)

D

gray

Gy

m2·s− 2

J·kg− 1

1 rad = 10− 2 Gy

Electric potential, electromotive force

U

volt

V

m2· kg·s− 3·A− 1

W·A− 1

Electric resistance

R

ohm

Ω

m2· kg·s− 3·A− 2

V·A− 1

Quantity of electricity

Q

coulomb

C

A·s

Activity of a radionuclide

A

becquerel

Bq

s− 1

Concentration (of amount of substance), molar concentration

c

mole per cubic metre

mol/m3

mol·m− 3

1 mol/l = 1M = 1 mol/dm3 = 103 mol·m− 3

Mass concentration

ρ

kilogram per cubic metre

kg/m3

kg·m− 3

1 g/l = 1 g/dm3 = 1 kg·m− 3

Table 1.6.-3. – Units used with the International System Unit

Quantity

Value in SI units

Time

Symbol

minute

min

1 min = 60 s

hour

h

1 h = 60 min = 3600 s

day

d

1 d = 24 h = 86 400 s

Plan angle

degree

°

1° = (π/180) rad

Volume

litre

l

1 l = 1 dm3 = 10− 3 m3

Mass

tonne

t

1 t = 103 kg

Rotational frequency

revolution per minute

r/min

1 r/min = (1/60) s− 1

Time


1 ml = 1 cm3 = 10− 6 m3

1 g/ml = 1 g/cm3 = 103 kg·m− 3

1 dyne = 1 g·cm·s− 2 = 10− 5 N 1 kp = 9.806 65 N

1 erg = 1 cm2·g·s− 2 = 1 dyne·cm = 10− 7 J 1 cal = 4.1868 J

1 Ci = 37·109 Bq = 37·109 s− 1

Table 1.6.-4. – Decimal multiples and sub-multiples of units Factor

Prefix

Prefix

Symbol

10

deci

d

P

10

−2

centi

c

T

10− 3

milli

m

10

−6

micro

µ

−9

nano

n

exa

E

15

peta

1012

tera

10 10

Factor −1

Symbol

18

10

9

giga

G

10

6

mega

M

10

10

3

kilo

k

10− 12

pico

p

hecto

h

10− 15

femto

f

atto

a

102 10

1

deca

da

10

− 18

11

2.1.3. Ultraviolet ray lamps for analytical purposes


2.1. APPARATUS

01/2005:20102 01/2005:20101

2.1.1. DROPPERS The term “drops” means standard drops delivered from a standard dropper as described below. Standard droppers (Figure 2.1.1-1) are constructed of practically colourless glass. The lower extremity has a circular orifice in a flat surface at right angles to the axis.

2.1.2. COMPARATIVE TABLE OF POROSITY OF SINTERED-GLASS FILTERS(1) Table 2.1.2.-1 Porosity number (Ph. Eur.) (2)

Maximum diameter of pores in micrometres

Germany

France

United Kingdom

1.6

less than 1.6

5f

–

–

1 - 2.5

5

–

5

4

1.6 - 4

–

–

–

–

4-6

–

5

–

10

4 - 10

4f

–

4

16

10 - 16

4

4

–

40

16 - 40

3

3

3

–

40 - 50

–

–

2

100

–

40 - 100

2

2

–

–

100 - 120

–

–

1

160

100 - 160

1

1

–

–

150 - 200

0

0

–

250

160 - 250

–

–

–

200 - 500

–

00

–

–

Special Uses Diameters in micrometres < 2.5

Bacteriological filtration

4 - 10

Ultra-fine filtration, separation of micro-organisms of large diameter

10 - 40

Analytical filtration, very fine filtration of mercury, very fine dispersion of gases

40 - 100

Fine filtration, filtration of mercury, fine dispersion of gases

100 - 160

Filtration of coarse materials, dispersion and washing of gases, support for other filter materials

160 - 500

Filtration of very coarse materials, dispersion and washing of gases.

01/2005:20103

Figure 2.1.1.-1. – Standard dropper Dimensions in millimetres Other droppers may be used provided they comply with the following test. Twenty drops of water R at 20 ± 1 °C flowing freely from the dropper held in the vertical position at a constant rate of one drop per second weighs 1000 ± 50 mg.

2.1.3. ULTRAVIOLET RAY LAMPS FOR ANALYTICAL PURPOSES

Mercury vapour in quartz lamps is used as the source of ultraviolet light. A suitable filter may be fitted to eliminate the visible part of the spectrum emitted by the lamp. When the Pharmacopoeia prescribes in a test the use of ultraviolet light of wavelength 254 nm or 365 nm, an instrument consisting of a mercury vapour lamp and a filter which gives an emission band with maximum intensity at about 254 nm or 365 nm is used. The lamp used should be capable of The dropper must be carefully cleaned before use. Carry out revealing without doubt a standard spot of sodium salicylate with a diameter of about 5 mm on a support of silica gel G R, three determinations on any given dropper. No result may deviate by more than 5 per cent from the mean of the three the spot being examined while in a position normal to the radiation. determinations.

(1) The given limits are only approximate. (2) The European Pharmacopoeia has adopted the system proposed by the International Organisation for Standardisation (ISO).


17

2.1.4. Sieves


Maximum tolerance(4) for an aperture (+ X) : no aperture size For this purpose apply 5 µl of a 0.4 g/l solution of sodium salicylate R in alcohol R(3) for lamps of maximum output at shall exceed the nominal size by more than X, where : 254 nm and 5 µl of a 2 g/l solution in alcohol R(1) for lamps of maximum output at 365 nm. The distance between the lamp and the chromatographic plate under examination used in a pharmacopoeial test should never exceed the distance used to carry out the above test. w = width of aperture. Tolerance for mean aperture (± Y) : the average aperture size shall not depart from the nominal size by more than ± Y, where : 01/2005:20104

2.1.4. SIEVES

Intermediary tolerance (+ Z) : not more than 6 per cent of the total number of apertures shall have sizes between “nominal + X” and “nominal + Z”, where :

Sieves are constructed of suitable materials with square meshes. For purposes other than analytical procedures, sieves with circular meshes may be used, the internal diameters of which are 1.25 times the aperture of the square mesh of the corresponding sieve size. There must be no reaction between the material of the sieve and the substance being sifted. Degree of comminution is prescribed in the monograph using the sieve number, which is the size of the mesh in micrometres, given in parenthesis after the name of the substance (Table 2.1.4.-1).

Wire diameter d : the wire diameters given in Table 2.1.4.-1 apply to woven metal wire cloth mounted in a frame. The nominal sizes of the wire diameters may depart from these values within the limits dmax and dmin. The limits define a permissible range of choice ± 15 per cent of the recommended nominal dimensions. The wires in a test sieve shall be of a similar diameter in warp and weft directions.

Table 2.1.4.-1 (values in micrometers) Sieve numbers (Nominal dimensions of apertures)

Wire diameters

Tolerances for apertures Maximum tolerance for an aperture

Tolerance for mean aperture

Intermediary tolerance

Recommended nominal dimensions

+X

±Y

+Z

d

dmax

dmin

11 200

770

350

560

2500

2900

2100

8000

600

250

430

2000

2300

1700

5 600

470

180

320

1600

1900

1300

4000

370

130

250

1400

1700

1200

2 800

290

90

190

1120

1300

950

2000

230

70

150

900

1040

770

1 400

180

50

110

710

820

600

1000

140

30

90

560

640

480

710

112

25

69

450

520

380

500

89

18

54

315

360

270

355

72

13

43

224

260

190

250

58

9.9

34

160

190

130

180

47

7.6

27

125

150

106

125

38

5.8

22

90

104

77

90

32

4.6

18

63

72

54

63

26

3.7

15

45

52

38

45

22

3.1

13

32

37

27

38

–

–

–

30

35

24

Admissible limits

(3) The alcohol R used must be free from fluorescence. (4) See the International Standard ISO 3310/1 (1975).

18


2.1.6. Gas detector tubes


2.1.5. TUBES FOR TESTS

01/2005:20105 given in the leaflet supplied with the tube. If the oil used is not cited in the leaflet, the tube manufacturer must verify the reactivity and if necessary provide a tube specific for this oil. COMPARATIVE

Tubes used for comparative tests are matched tubes of colourless glass with a uniform internal diameter. The base is transparent and flat. A column of the liquid is examined down the vertical axis of the tube against a white background, or if necessary, against a black background. The examination is carried out in diffused light. It is assumed that tubes with an internal diameter of 16 mm will be used. Tubes with a larger internal diameter may be used instead but the volume of liquid examined must then be increased so that the depth of liquid in the tubes is not less than where the prescribed volume of liquid and tubes 16 mm in internal diameter are used. 01/2005:20106

2.1.6. GAS DETECTOR TUBES

1. Gas supply

5. Indicator tube

2. Pressure regulator

6. Indicator tube pump

3. Needle valve

7. End open to atmosphere

4. “Y”-piece

Gas detector tubes are cylindrical, sealed tubes consisting of an inert transparent material and are constructed to allow the passage of gas. They contain reagents adsorbed onto inert substrates that are suitable for the visualisation of the substance to be detected and, if necessary, they also contain preliminary layers and/or adsorbent filters to eliminate substances that interfere with the substance to be detected. The layer of indicator contains either a single reagent for the detection of a given impurity or several reagents for the detection of several substances (monolayer tube or multilayer tube). The test is carried out by passing the required volume of the gas to be examined through the indicator tube. The length of the coloured layer or the intensity of a colour change on a graduated scale gives an indication of the impurities present. The calibration of the detector tubes is verified according to the manufacturer’s instructions. Operating conditions. Examine according to the manufacturer’s instructions or proceed as follows : The gas supply is connected to a suitable pressure regulator and needle valve. Connect the flexible tubing fitted with a Y-piece to the valve and adjust the flow of gas to be examined to purge the tubing in order to obtain an appropriate flow (Figure 2.1.6.-1). Prepare the indicator tube and fit to the metering pump, following the manufacturer’s instructions. Connect the open end of the indicator tube to the short leg of the tubing and operate the pump by the appropriate number of strokes to pass a suitable volume of gas to be examined through the tube. Read the value corresponding to the length of the coloured layer or the intensity of the colour on the graduated scale. If a negative result is achieved, indicator tubes can be verified with a calibration gas containing the appropriate impurity. In view of the wide variety of available compressor oils, it is necessary to verify the reactivity of the oil detector tubes for the oil used. Information on the reactivity for various oils is

Figure 2.1.6.-1. – Apparatus for gas detector tubes Carbon dioxide detector tube. Sealed glass tube containing adsorbent filters and suitable supports for hydrazine and crystal violet indicators. The minimum value indicated is 100 ppm with a relative standard deviation of at most ± 15 per cent. Sulphur dioxide detector tube. Sealed glass tube containing adsorbent filters and suitable supports for the iodine and starch indicator. The minimum value indicated is 0.5 ppm with a relative standard deviation of at most ± 15 per cent. Oil detector tube. Sealed glass tube containing adsorbent filters and suitable supports for the sulphuric acid indicator. The minimum value indicated is 0.1 mg/m3 with a relative standard deviation of at most ± 30 per cent. Nitrogen monoxide and nitrogen dioxide detector tube. Sealed glass tube containing adsorbent filters and suitable supports for an oxidising layer (Cr(VI) salt) and the diphenylbenzidine indicator. The minimum value indicated is 0.5 ppm with a relative standard deviation of at most ± 15 per cent. Carbon monoxide detector tube. Sealed glass tube containing adsorbent filters and suitable supports for di-iodine pentoxide, selenium dioxide and fuming sulphuric acid indicators. The minimum value indicated is 5 ppm or less, with a relative standard deviation of at most ± 15 per cent. Hydrogen sulphide detector tube. Sealed glass tube containing adsorbent filters and suitable supports for an appropriate lead salt indicator. The minimum value indicated is 1 ppm or less, with a relative standard deviation of at most ± 10 per cent. Water vapour detector tube. Sealed glass tube containing adsorbent filters and suitable supports for the magnesium perchlorate indicator. The minimum value indicated is 67 ppm or less, with a relative standard deviation of at most ± 20 per cent.


19

2.2.1. Clarity and degree of opalescence of liquids


2.2. PHYSICAL AND PHYSICOCHEMICAL METHODS

formazin polymer consists of chains of different lengths, which fold into random configurations. This results in a wide assay of particle shapes and sizes, which analytically fits the possibility of different particle sizes and shapes that are found in the real samples. Due to formazin’s reproducibility, scattering characteristics and traceability, instrument 01/2005:20201 calibration algorithms and performance criteria are mostly based on this standard.

2.2.1. CLARITY AND DEGREE OF OPALESCENCE OF LIQUIDS

Turbidity standard. The formazin suspension prepared by mixing equal volumes of the hydrazine sulphate solution and the hexamethylenetetramine solution is defined as a 4000 NTU (nephelometric turbidity units) primary reference standard. Reference suspensions I, II, III and IV have values of 3 NTU, 6 NTU, 18 NTU and 30 NTU respectively. Stabilised formazin suspensions that can be used to prepare stable, diluted turbidity standards are available commercially and may be used after comparison with the standards prepared as described. Formazin has several desirable characteristics that make it an excellent turbidity standard. It can be reproducibly prepared from assayed raw materials. The physical characteristics make it a desirable light-scatter calibration standard. The

INSTRUMENTAL METHODS INTRODUCTION The degree of opalescence may also be determined by instrumental measurement of the light absorbed or scattered on account of submicroscopic optical density inhomogeneities of opalescent solutions and suspensions. 2 such techniques are nephelometry and turbidimetry. For turbidity measurement of coloured samples, ratio turbidimetry and nephelometry with ratio selection are used. The light scattering effect of suspended particles can be measured by observation of either the transmitted light (turbidimetry) or the scattered light (nephelometry). Ratio turbidimetry combines the principles of both nephelometry and turbidimetry. Turbidimetry and nephelometry are useful for the measurement of slightly opalescent suspensions. Reference suspensions produced under well-defined conditions must be used. For quantitative measurements the construction of calibration curves is essential, since the relationship between the optical properties of the suspension and the concentration of the dispersed phase is at best semi-empirical. The determination of opalescence of coloured liquids is done with ratio turbidimeters or nephelometers with ratio selection since colour provides a negative interference, attenuating both incident and scattered light and lowering the turbidity value. The effect is so great for even moderately coloured samples that conventional nephelometers cannot be used. The instrumental assessment of clarity and opalescence provides a more discriminatory test that does not depend on the visual acuity of the analyst. Numerical results are more useful for quality monitoring and process control, especially in stability studies. For example, previous numerical data on stability can be projected to determine whether a given batch of dosage formulation or active pharmaceutical ingredient will exceed shelf-life limits prior to the expiry date. NEPHELOMETRY When a suspension is viewed at right angles to the direction of the incident light, the system appears opalescent due to the reflection of light from the particles of the suspension (Tyndall effect). A certain portion of the light beam entering a turbid liquid is transmitted, another portion is absorbed and the remaining portion is scattered by the suspended particles. If measurement is made at 90° to the light beam, the light scattered by the suspended particles can be used for the determination of their concentration, provided the number and size of particles influencing the scattering remain constant. The reference suspension must maintain a constant degree of turbidity and the sample and reference suspensions must be prepared under identical conditions. The Tyndall effect depends both upon the number of particles and their size. Nephelometric measurements are more reliable in low turbidity ranges, where there is a linear relationship between nephelometric turbidity unit (NTU) values and relative detector signals. As the degree of turbidity increases, not all the particles are exposed to the incident light and the scattered radiation of other particles is hindered on its way to the detector. The maximum nephelometric values at which reliable measurements can


23

VISUAL METHOD Using identical test tubes of colourless, transparent, neutral glass with a flat base and an internal diameter of 15-25 mm, compare the liquid to be examined with a reference suspension freshly prepared as described below, the depth of the layer being 40 mm. Compare the solutions in diffused daylight 5 min after preparation of the reference suspension, viewing vertically against a black background. The diffusion of light must be such that reference suspension I can readily be distinguished from water R, and that reference suspension II can readily be distinguished from reference suspension I. A liquid is considered clear if its clarity is the same as that of water R or of the solvent used when examined under the conditions described above, or if its opalescence is not more pronounced than that of reference suspension I. Hydrazine sulphate solution. Dissolve 1.0 g of hydrazine sulphate R in water R and dilute to 100.0 ml with the same solvent. Allow to stand for 4-6 h. Hexamethylenetetramine solution. In a 100 ml glassstoppered flask, dissolve 2.5 g of hexamethylenetetramine R in 25.0 ml of water R. Primary opalescent suspension (formazin suspension). To the solution of hexamethylenetetramine in the flask add 25.0 ml of hydrazine sulphate solution. Mix and allow to stand for 24 h. This suspension is stable for 2 months, provided it is stored in a glass container free from surface defects. The suspension must not adhere to the glass and must be well mixed before use. Standard of opalescence. Dilute 15.0 ml of the primary opalescent suspension to 1000.0 ml with water R. This suspension is freshly prepared and may be stored for at most 24 h. Reference suspensions. Prepare the reference suspensions according to Table 2.2.1.-1. Mix and shake before use. Table 2.2.1.-1 I

II

III

IV

Standard of opalescence

5.0 ml

10.0 ml

30.0 ml

50.0 ml

Water R

95.0 ml

90.0 ml

70.0 ml

50.0 ml

2.2.2. Degree of coloration of liquids


be made lie between 1750-2000 NTU. Linearity must be demonstrated by constructing a calibration curve using at least 4 concentrations. TURBIDIMETRY The optical property expressed as turbidity is the interaction between light and suspended particles in liquid. This is an expression of the optical property that causes light to be scattered and absorbed rather than transmitted in a straight line through the sample. The quantity of a solid material in suspension can be determined by the measurement of the transmitted light. A linear relationship between turbidity and concentration is obtained when the particle sizes are uniform and homogeneous in the suspension. This is true only in very dilute suspensions containing small particles. Linearity between turbidity and concentration must be established by constructing a calibration curve using at least 4 concentrations. RATIO TURBIDIMETRY In ratio turbidimetry the relationship of the transmission measurement to the 90° scattered light measurement is determined. This procedure compensates for the light that is diminished by the colour of the sample. The influence of colour of the sample may also be eliminated by using an infrared light-emitting diode (IR LED) at 860 nm as light source of the instrument. The instrument’s photodiode detectors receive and measure scattered light at a 90° angle from the sample as well as measuring the forward scatter (light reflected) in front of the sample along with the measurement of light transmitted directly through the sample. The measuring results are given in NTU(ratio) and are obtained by calculating the ratio of the 90° angle scattered light measured to the sum of the components of forward scattered and transmitted light values. In ratio turbidimetry the influence of stray light becomes negligible. Nephelometers are used for measurements of the degree of opalescence of colourless liquids. Measurements of reference suspensions I-IV with a ratio turbidimeter show a linear relationship between the concentrations and measured NTU values. Reference suspensions I-IV (Ph. Eur.) may be used as calibrators for the instrument. Table 2.2.1.-2 Formazin suspensions

Opalescent values (NTU)

Reference suspension I

3

Reference suspensions II

6

Reference suspension III

18

Reference suspension IV

30

Standard of opalescence

60

Primary opalescent suspension

01/2005:20202

4000

INSTRUMENTAL DETERMINATION OF OPALESCENCE Requirements in monographs are expressed in terms of the visual examination method with the defined reference suspensions. Instrumental methods may also be used for determining compliance with monograph requirements once the suitability of the instrument as described below has been established and calibration with reference suspensions I-IV and with water R or the solvent used has been performed. Apparatus. Ratio turbidimeters or nephelometers with selectable ratio application use as light source a tungsten lamp with spectral sensitivity at about 550 nm operating at a filament colour temperature of 2700 K or IR LED having an emission maximum at 860 nm with a 60 nm spectral bandwidth. Other suitable light sources may also be used. Silicon photodiodes and photomultipliers are commonly 24

used as detectors and record changes in light scattered or transmitted by the sample. The light scattered at 90 ± 2.5° is detected by the primary detector. Other detectors are those to detect back and forward scatter as well as transmitted light. The instruments used are calibrated against standards of known turbidity and are capable of automatic determination of turbidity. The test results expressed in NTU units are obtained directly from the instrument and compared to the specifications in the individual monographs. Instruments complying with the following specifications are suitable. — Measuring units : NTU. NTU is based on the turbidity of a primary reference standard of formazin. FTU (Formazin Turbidity Units) or FNU (Formazin Nephelometry Units) are also used which are equivalent to NTU in low regions (up to 40 NTU). These units are used in all 3 instrumental methods, nephelometry, turbidimetry and ratio turbidimetry. — Measuring range : 0.01-1100 NTU. — Resolution : 0.01 NTU within the range of 0-10 NTU, 0.1 NTU within the range of 10-100 NTU and 1 NTU for the range > 100 NTU. The instrument is calibrated and controlled with reference standards of formazin. — Accuracy: 0-10 NTU : ± 0.01 NTU. 10-1000 NTU : ± 5 per cent. — Repeatability: 0-10 NTU : ± 0.01 NTU. 10-1000 NTU : ± 2 per cent of the measured value. — Calibration : with 4 reference suspensions of formazin in the range of interest. Reference suspensions described in this chapter or suitable reference standards calibrated against the primary reference suspensions may be used. — Stray light : this is a significant source of error in low level turbidimetric measurement ; stray light reaches the detector of an optical system, but does not come from the sample < 0.15 NTU for the range 0-10 NTU, < 0.5 NTU for the range 10-1000 NTU. Instruments complying with the above characteristics and verified using the reference suspensions described under Visual method may be used instead of visual examination for determination of compliance with monograph requirements. Instruments with range or resolution, accuracy and repeatability capabilities other than those mentioned above may be used provided they are sufficiently validated and are capable for the intended use. The test methodology for the specific substance/product to be analysed must also be validated to demonstrate its analytical capability. The instrument and methodology should be consistent with the attributes of the product to be tested.

2.2.2. DEGREE OF COLORATION OF LIQUIDS The examination of the degree of coloration of liquids in the range brown-yellow-red is carried out by one of the 2 methods below, as prescribed in the monograph. A solution is colourless if it has the appearance of water R or the solvent or is not more intensely coloured than reference solution B9. METHOD I Using identical tubes of colourless, transparent, neutral glass of 12 mm external diameter, compare 2.0 ml of the liquid to be examined with 2.0 ml of water R or of the solvent or of the reference solution (see Tables of reference


2.2.2. Degree of coloration of liquids


solutions) prescribed in the monograph. Compare the colours in diffused daylight, viewing horizontally against a white background.

iodide R. Titrate the liberated iodine with 0.1 M sodium thiosulphate, using 0.5 ml of starch solution R, added towards the end of the titration, as indicator. The end-point is reached when the solution shows a slight pale brown colour.

METHOD II

1 ml of 0.1 M sodium thiosulphate is equivalent to 24.97 mg of CuSO4,5H2O.

Using identical tubes of colourless, transparent, neutral Standard solutions glass with a flat base and an internal diameter of 15 mm to 25 mm, compare the liquid to be examined with water R or the solvent or the reference solution (see Tables of reference Using the 3 primary solutions, prepare the 5 standard solutions as follows : solutions) prescribed in the monograph, the depth of the layer being 40 mm. Compare the colours in diffused daylight, viewing vertically against a white background. Table 2.2.2.-1 REAGENTS Primary solutions Yellow solution. Dissolve 46 g of ferric chloride R in about 900 ml of a mixture of 25 ml of hydrochloric acid R and 975 ml of water R and dilute to 1000.0 ml with the same mixture. Titrate and adjust the solution to contain 45.0 mg of FeCl3,6H2O per millilitre by adding the same acidic mixture. Protect the solution from light. Titration. Place in a 250 ml conical flask fitted with a ground-glass stopper, 10.0 ml of the solution, 15 ml of water R, 5 ml of hydrochloric acid R and 4 g of potassium iodide R, close the flask, allow to stand in the dark for 15 min and add 100 ml of water R. Titrate the liberated iodine with 0.1 M sodium thiosulphate, using 0.5 ml of starch solution R, added towards the end of the titration, as indicator. 1 ml of 0.1 M sodium thiosulphate is equivalent to 27.03 mg of FeCl3,6H2O. Red solution. Dissolve 60 g of cobalt chloride R in about 900 ml of a mixture of 25 ml of hydrochloric acid R and 975 ml of water R and dilute to 1000.0 ml with the same mixture. Titrate and adjust the solution to contain 59.5 mg of CoCl2,6H2O per millilitre by adding the same acidic mixture.

Volume in millilitres Standard solution

Red Yellow Blue Hydrochloric acid solution solution solution (10 g/l HCl)

B (brown)

3.0

3.0

2.4

1.6

BY (brownish-yellow)

2.4

1.0

0.4

6.2

Y (yellow)

2.4

0.6

0.0

7.0

GY (greenish-yellow)

9.6

0.2

0.2

0.0

R (red)

1.0

2.0

0.0

7.0

Reference solutions for Methods I and II Using the 5 standard solutions, prepare the following reference solutions. Table 2.2.2.-2. - Reference solutions B Volumes in millilitres Reference solution

Standard solution B

Hydrochloric acid (10 g/l HCl)

B1

75.0

25.0

B2

50.0

50.0

B3

37.5

62.5

B4

25.0

75.0

B5

12.5

87.5

B6

5.0

95.0

B7

2.5

97.5

Titration. Place in a 250 ml conical flask fitted with a ground-glass stopper, 5.0 ml of the solution, 5 ml of dilute hydrogen peroxide solution R and 10 ml of a 300 g/l solution of sodium hydroxide R. Boil gently for 10 min, allow to cool and add 60 ml of dilute sulphuric acid R and 2 g of potassium iodide R. Close the flask and dissolve the precipitate by shaking gently. Titrate the liberated iodine with 0.1 M sodium thiosulphate, using 0.5 ml of starch solution R, added towards the end of the titration, as indicator. The end-point is reached when the solution turns pink.

B8

1.5

98.5

B9

1.0

99.0

1 ml of 0.1 M sodium thiosulphate is equivalent to 23.79 mg of CoCl2,6H2O.

Reference solution

Standard solution BY

BY1

100.0

0.0

BY2

75.0

25.0

BY3

50.0

50.0

BY4

25.0

75.0

BY5

12.5

87.5

BY6

5.0

95.0

BY7

2.5

97.5

Blue primary solution. Dissolve 63 g of copper sulphate R in about 900 ml of a mixture of 25 ml of hydrochloric acid R and 975 ml of water R and dilute to 1000.0 ml with the same mixture. Titrate and adjust the solution to contain 62.4 mg of CuSO4,5H2O per millilitre by adding the same acidic mixture. Titration. Place in a 250 ml conical flask fitted with a ground-glass stopper, 10.0 ml of the solution, 50 ml of water R, 12 ml of dilute acetic acid R and 3 g of potassium General Notices (1) apply to all monographs and other texts

Table 2.2.2.-3. - Reference solutions BY Volumes in millilitres Hydrochloric acid (10 g/l HCl)

25

2.2.3. Potentiometric determination of pH


Table 2.2.2.-4. - Reference solutions Y Volumes in millilitres Reference solution

Standard solution Y


Y1

100.0

0.0

Y2

75.0

25.0

Y3

50.0

50.0

Y4

25.0

75.0

Y5

12.5

87.5

Y6

5.0

95.0

Y7

2.5

97.5

in which E is the potential, expressed in volts, of the cell containing the solution to be examined and Es is the potential, expressed in volts, of the cell containing the solution of known pH (pHs), k is the change in potential per unit change in pH expressed in volts, and calculated from the Nernst equation. Table 2.2.3.-1. – Values of k at different temperatures

Table 2.2.2.-5. - Reference solutions GY

Temperature (°C)

k (V)

15

0.0572

20

0.0582

25

0.0592

30

0.0601

35

0.0611

Volumes in millilitres

The potentiometric determination of pH is made by measuring the potential difference between 2 appropriate electrodes immersed in the solution to be examined : 1 of these electrodes is sensitive to hydrogen ions (usually a GY1 25.0 75.0 glass electrode) and the other is the reference electrode (for GY2 15.0 85.0 example, a saturated calomel electrode). GY3 8.5 91.5 Apparatus. The measuring apparatus is a voltmeter with an input resistance at least 100 times that of the electrodes used. GY4 5.0 95.0 It is normally graduated in pH units and has a sensitivity 3.0 97.0 GY5 such that discrimination of at least 0.05 pH unit or at least GY6 1.5 98.5 0.003 V may be achieved. GY7 0.75 99.25 Method. Unless otherwise prescribed in the monograph, all measurements are made at the same temperature (20-25 °C). Table 2.2.3.-2 shows the variation of pH with respect to Table 2.2.2.-6. - Reference solutions R temperature of a number of reference buffer solutions Volumes in millilitres used for calibration. For the temperature correction, when necessary, follow the manufacturer’s instructions. Reference Standard solution R Hydrochloric acid solution (10 g/l HCl) The apparatus is calibrated with the buffer solution of R1 potassium hydrogen phthalate (primary standard) and 0.0 100.0 1 other buffer solution of different pH (preferably one R2 75.0 25.0 shown in Table 2.2.3.-2). The pH of a third buffer solution R3 50.0 50.0 of intermediate pH read off on the scale must not differ by more than 0.05 pH unit from the value corresponding to R4 37.5 62.5 this solution. Immerse the electrodes in the solution to be R5 25.0 75.0 examined and take the reading in the same conditions as for the buffer solutions. R6 12.5 87.5 When the apparatus is in frequent use, checks must be R7 5.0 95.0 carried out regularly. If not, such checks should be carried out before each measurement. Storage For Method I, the reference solutions may be stored in sealed All solutions to be examined and the reference buffer tubes of colourless, transparent, neutral glass of 12 mm solutions must be prepared using carbon dioxide-free external diameter, protected from light. water R. For Method II, prepare the reference solutions immediately PREPARATION OF REFERENCE BUFFER SOLUTIONS before use from the standard solutions. Potassium tetraoxalate 0.05 M. Dissolve 12.61 g of C4H3KO8,2H2O in carbon dioxide-free water R and dilute to 1000.0 ml with the same solvent. 01/2005:20203 Potassium hydrogen tartrate, saturated at 25 °C. Shake an excess of C4H5KO6 vigorously with carbon dioxide-free water R at 25 °C. Filter or decant. Prepare immediately 2.2.3. POTENTIOMETRIC before use. DETERMINATION OF pH Potassium dihydrogen citrate 0.05 M. Dissolve 11.41 g of C6H7KO7 in carbon dioxide-free water R and dilute to The pH is a number which represents conventionally the 1000.0 ml with the same solvent. Prepare immediately before hydrogen ion concentration of an aqueous solution. For use. practical purposes, its definition is an experimental one. The pH of a solution to be examined is related to that of a Potassium hydrogen phthalate 0.05 M. Dissolve 10.13 g of reference solution (pHs) by the following equation : C8H5KO4, previously dried for 1 h at 110 ± 2 °C, in carbon dioxide-free water R and dilute to 1000.0 ml with the same solvent. Reference solution

26

Standard solution GY



2.2.5. Relative density


Table 2.2.3.-2. – pH of reference buffer solutions at various temperatures Temperature (°C)

Potassium tetraoxalate 0.05 M

Potassium hydrogen tartrate saturated at 25 °C

Potassium dihydrogen citrate 0.05 M

Potassium hydrogen phthalate 0.05 M

Potassium dihydrogen phosphate 0.025 M + disodium hydrogen phosphate 0.025 M

Potassium dihydrogen phosphate 0.0087 M + disodium hydrogen phosphate 0.0303 M

Disodium tetraborate 0.01 M

Sodium carbonate 0.025 M + sodium bicarbonate 0.025 M

Calcium hydroxide, saturated at 25°C

C4H3KO8,2H2O

C4H5KO6

C6H7KO7

C8H5KO4

KH2PO4+ Na2HPO4

KH2PO4+ Na2HPO4

Na2B4O7, 10H2O

Na2CO3+ NaHCO3

Ca(OH)2

15

1.67

3.80

4.00

6.90

7.45

9.28

10.12

12.81

20

1.68

3.79

4.00

6.88

7.43

9.23

10.06

12.63

25

1.68

3.56

3.78

4.01

6.87

7.41

9.18

10.01

12.45

30

1.68

3.55

3.77

4.02

6.85

7.40

9.14

9.97

12.29

35

1.69

3.55

3.76

4.02

6.84

7.39

9.10

9.93

12.13

+ 0.001

− 0.0014

− 0.0022

+ 0.0012

− 0.0028

−0.0028

−0.0082

− 0.0096

−0.034

(1) pH variation per degree Celsius.

Potassium dihydrogen phosphate 0.025 M + disodium hydrogen phosphate 0.025 M. Dissolve 3.39 g of KH2PO4 and 3.53 g of Na2HPO4, both previously dried for 2 h at 120 ± 2 °C, in carbon dioxide-free water R and dilute to 1000.0 ml with the same solvent. Potassium dihydrogen phosphate 0.0087 M + disodium hydrogen phosphate 0.0303 M. Dissolve 1.18 g of KH2PO4 and 4.30 g of Na2HPO4, both previously dried for 2 h at 120 ± 2 °C, in carbon dioxide-free water R and dilute to 1000.0 ml with the same solvent. Disodium tetraborate 0.01 M. Dissolve 3.80 g of Na2B4O7,10H2O in carbon dioxide-free water R and dilute to 1000.0 ml with the same solvent. Store protected from atmospheric carbon dioxide. Sodium carbonate 0.025 M + sodium hydrogen carbonate 0.025 M. Dissolve 2.64 g of Na2CO3 and 2.09 g of NaHCO3 in carbon dioxide-free water R and dilute to 1000.0 ml with the same solvent. Store protected from atmospheric carbon dioxide. Calcium hydroxide, saturated at 25 °C. Shake an excess of calcium hydroxide R with carbon dioxide-free water R and decant at 25 °C. Store protected from atmospheric carbon dioxide. STORAGE Store buffer solutions in suitable chemically resistant, tight containers, such as type I glass bottles or plastic containers suitable for aqueous solutions.

Table 2.2.4.-1 Reaction

pH

Indicator

Colour

Alkaline

>8

Litmus paper red R

Blue

Thymol blue solution R (0.05 ml)

Grey or violet-blue

Phenolphthalein solution R (0.05 ml)

Colourless or pink


Grey

Phenolphthalein paper R

Red


Violet-blue

Slightly alkaline 8.0 – 10.0

Strongly alkaline

Neutral

> 10

6.0 – 8.0 Methyl red solution R

Neutral to methyl 4.5 – 6.0 Methyl red solution R red Neutral to phenolphtalein

< 8.0

Acid

0.15 per cent or a daily intake of > 1.0 mg (whichever is the lower)

Human use or human and veterinary use

> 2 g/day

> 0.03 per cent

> 0.05 per cent

> 0.05 per cent

Veterinary use only

Not applicable

> 0.1 per cent

> 0.2 per cent

> 0.5 per cent


587



01/2005:0153 preparation are designed to maintain adequate immunogenic properties, to render the preparation harmless and to prevent contamination with extraneous agents.

VACCINES FOR HUMAN USE Vaccina ad usum humanum

For a combined vaccine, where there is no monograph to cover a particular combination, the vaccine complies with the monograph for each individual component, with any necessary modifications approved by the competent authority. DEFINITION Vaccines for human use are preparations containing antigenic substances capable of inducing a specific and active immunity in man against an infecting agent or the toxin or the antigen elaborated by it. They shall have been shown to have acceptable immunogenic activity in man with the intended vaccination schedule. Vaccines for human use may contain : organisms inactivated by chemical or physical means that maintain adequate immunogenic properties ; living organisms that are naturally avirulent or that have been treated to attenuate their virulence whilst retaining adequate immunogenic properties ; antigens extracted from the organisms or secreted by them or produced by genetic engineering ; the antigens may be used in their native state or may be detoxified by chemical or physical means and may be aggregated, polymerised or conjugated to a carrier to increase their immunogenicity. Terminology used in monographs on vaccines for human use is defined in chapter 5.2.1. Bacterial vaccines are suspensions of various degrees of opacity in colourless or almost colourless liquids, or may be freeze-dried. The concentration of living or inactivated bacteria is expressed in terms of International Units of opacity or, where appropriate, is determined by direct cell count or, for living bacteria, by viable count. Bacterial toxoids are prepared from toxins by diminishing their toxicity to a non-detectable level or by completely eliminating it by physical or chemical procedures whilst retaining adequate immunogenic properties. The toxins are obtained from selected strains of micro-organisms. The method of production is such that the toxoid does not revert to toxin. Toxoids may be liquid or freeze-dried. They may be purified and adsorbed. Adsorbed toxoids are suspensions of white or grey particles dispersed in colourless or pale yellow liquids and may form a sediment at the bottom of the container. Viral vaccines are prepared from viruses grown in animals, in fertilised eggs, in suitable cell cultures or in suitable tissues or by culture of genetically engineered cells. They are liquids that vary in opacity according to the type of preparation or may be freeze-dried. Liquid preparations and freeze-dried preparations after reconstitution may be coloured if a pH indicator such as phenol red has been used in the culture medium. PRODUCTION General provisions. Requirements for production including in-process testing are included in individual monographs. Where justified and authorised, certain tests may be omitted where it can be demonstrated, for example by validation studies, that the production process consistently ensures compliance with the test. Unless otherwise justified and authorised, vaccines are produced using a seed-lot system. The methods of 588

Unless otherwise justified and authorised, in the production of a final lot of vaccine, the number of passages of a virus, or the number of subcultures of a bacterium, from the master seed lot shall not exceed that used for production of the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy. Vaccines are as far as possible free from ingredients known to cause toxic, allergic or other undesirable reactions in man. Suitable additives, including stabilisers and adjuvants may be incorporated. Penicillin and streptomycin are not used at any stage of production nor added to the final product ; however, master seed lots prepared with media containing penicillin or streptomycin may, where justified and authorised, be used for production. Substrates for propagation. Substrates for propagation comply with the relevant requirements of the Pharmacopoeia (5.2.2, 5.2.3) or in the absence of such requirements with those of the competent authority. Processing of cell banks and subsequent cell cultures is done under aseptic conditions in an area where no other cells are being handled. Serum and trypsin used in the preparation of cell suspensions shall be shown to be free from extraneous agents. Seed lots. The strain of bacterium or virus used in a master seed lot is identified by historical records that include information on the origin of the strain and its subsequent manipulation. Suitable measures are taken to ensure that no micro-organism other than the seed strain is present in a seed lot. Culture media. Culture media are as far as possible free from ingredients known to cause toxic, allergic or other undesirable reactions in man ; if inclusion of such ingredients is necessary, it shall be demonstrated that the amount present in the final lot is reduced to such a level as to render the product safe. Approved animal (but not human) serum may be used in the growth medium for cell cultures but the medium used for maintaining cell growth during virus multiplication shall not contain serum, unless otherwise stated. Cell culture media may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration although it is preferable to have a medium free from antibiotics during production. Propagation and harvest. The seed cultures are propagated and harvested under defined conditions. The purity of the harvest is verified by suitable tests as defined in the monograph. Control cells. For vaccines produced in cell cultures, control cells are maintained and tested as prescribed. In order to provide a valid control, these cells must be maintained in conditions that are rigorously identical with those used for the production cell cultures, including use of the same batches of media and media changes. Control eggs. For live vaccines produced in eggs, control eggs are incubated and tested as prescribed in the monograph. Purification. Where applicable, validated purification procedures may be applied. Inactivation. Inactivated vaccines are produced using a validated inactivation process whose effectiveness and consistency have been demonstrated. Where there are recognised potential contaminants of a harvest, for example in vaccines produced in eggs from healthy, non-SPF flocks, the inactivation process is also validated with respect to the



potential contaminants. A test for inactivation is carried out as soon as possible after the inactivation process, unless otherwise justified and authorised. Stability of intermediates. During production of vaccines, intermediates are obtained at various stages and are stored, sometimes for long periods. Such intermediates include : — seed lots, — live or inactivated harvests from bacterial or viral cultures, — purified harvests that may consist of toxins or toxoids, polysaccharides, bacterial or viral suspensions, — purified antigens, — adsorbed antigens, — conjugated polysaccharides, — final bulk vaccine, — vaccine in the final closed container stored at a temperature lower than that used for stability studies and intended for release without re-assay. Except where they are used within a short period of time, stability studies are carried out on the intermediates in the intended storage conditions to establish the expected extent of degradation. For final bulk vaccine, stability studies may be carried out on representative samples in conditions equivalent to those intended to be used for storage. For each intermediate (except for seed lots), a period of validity applicable for the intended storage conditions is established, where appropriate in the light of stability studies. Final bulk. The final bulk is prepared by aseptically blending the ingredients of the vaccine. Adsorbents. Vaccines may be adsorbed on aluminium hydroxide, aluminium phosphate, calcium phosphate or other suitable adsorbent ; the adsorbents are prepared in special conditions which confer the appropriate physical form and adsorptive properties.


For vaccines for administration by a non-parenteral route, the final lot is prepared by distributing the final bulk under suitable conditions into sterile, tamper-proof containers. Degree of adsorption. During development of an adsorbed vaccine, the degree of adsorption is evaluated as part of the consistency testing. A release specification for the degree of adsorption is established in the light of results found for batches used in clinical testing. From the stability data generated for the vaccine it must be shown that at the end of the period of validity the degree of adsorption will not be less than for batches used in clinical testing. Stability. During development studies, maintenance of potency of the final lot throughout the period of validity shall be demonstrated ; the loss of potency in the recommended storage conditions is assessed and excessive loss even within the limits of acceptable potency may indicate that the vaccine is unacceptable. Expiry date. Unless otherwise stated, the expiry date is calculated from the beginning of the assay or from the beginning of the first assay for a combined vaccine. For vaccines stored at a temperature lower than that used for stability studies and intended for release without re-assay, the expiry date is calculated from the date of removal from cold storage. If, for a given vaccine, an assay is not carried out, the expiry date is calculated from the date of an approved stability-indicating test or failing this from the date of freeze-drying or the date of filling into the final containers. For a combined vaccine where components are presented in separate containers, the expiry date is that of the component which expires first. The expiry date applies to vaccines stored in the prescribed conditions. Animal tests. In accordance with the provisions of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes, tests must be carried out in such a way as to use the minimum number of animals and to cause the least pain, suffering, distress or lasting harm. The criteria for judging tests in monographs must be applied in the light of this. For example, if it is indicated that an animal is considered to show positive, infected etc. when typical clinical signs or death occur then as soon as sufficient indication of a positive result is obtained the animal in question shall be either humanely destroyed or given suitable treatment to prevent unnecessary suffering. In accordance with the General Notices, alternative test methods may be used to demonstrate compliance with the monograph and the use of such tests is particularly encouraged when this leads to replacement or reduction of animal use or reduction of suffering.

Antimicrobial preservatives. Antimicrobial preservatives are used to prevent spoilage or adverse effects caused by microbial contamination occurring during the use of a vaccine. Antimicrobial preservatives are not included in freeze-dried products. For single-dose liquid preparations, inclusion of antimicrobial preservatives is not normally acceptable. For multidose liquid preparations, the need for effective antimicrobial preservation is evaluated taking into account likely contamination during use and the maximum recommended period of use after broaching of the container. If an antimicrobial preservative is used, it shall be shown that it does not impair the safety or efficacy of the vaccine. TESTS Addition of antibiotics as antimicrobial preservatives is not Vaccines comply with the tests prescribed in individual normally acceptable. monographs including, where applicable, the following : During development studies, the effectiveness of the antimicrobial preservative throughout the period of validity Aluminium (2.5.13) : maximum 1.25 mg of aluminium (Al) per single human dose where an aluminium adsorbent has shall be demonstrated to the satisfaction of the competent been used in the vaccine, unless otherwise stated, authority. The efficacy of the antimicrobial preservative is evaluated as Calcium (2.5.14) : maximum 1.3 mg of calcium (Ca) per single human dose where a calcium adsorbent has been used described in chapter 5.1.3. If neither the A criteria nor the in the vaccine, unless otherwise stated, B criteria can be met, then in justified cases the following criteria are applied to vaccines for human use : bacteria, no Formaldehyde (2.4.18) : maximum 0.2 g/l of free increase at 24 h and 7 days, 3 log reduction at 14 days, no formaldehyde is present in the final product where increase at 28 days ; fungi, no increase at 14 days and 28 days. formaldehyde has been used in the preparation of the Final lot. For vaccines for parenteral administration, the final vaccine, unless otherwise stated. lot is prepared by aseptically distributing the final bulk into Phenol (2.5.15) : maximum 2.5 g/l is present in the final product where phenol has been used in the preparation of sterile tamper-proof containers which, after freeze-drying where applicable, are closed so as to exclude contamination. the vaccine, unless otherwise stated. General Notices (1) apply to all monographs and other texts

589



Water (2.5.12) : maximum 3.0 per cent m/m for freeze-dried vaccines, unless otherwise stated.

live animals. The strain of bacterium used may have been modified by genetic engineering. The identity, antigenic potency and purity of each bacterial culture used is carefully STORAGE controlled. Store protected from light. Unless otherwise stated, the Bacterial vaccines contain inactivated or live bacteria or storage temperature is 5 ± 3 °C ; liquid adsorbed vaccines their antigenic components ; they are liquid preparations of must not be allowed to freeze. various degrees of opacity or they may be freeze-dried. Bacterial toxoids are prepared from toxins by diminishing LABELLING their toxicity to a very low level or by completely eliminating The label states : it by physical or chemical means whilst retaining adequate — the name of the preparation, immunising potency. The toxins are obtained from selected — a reference identifying the final lot, strains of specified micro-organisms grown in suitable media or are obtained by other suitable means, for example, — the recommended human dose and route of chemical synthesis. administration, The toxoids may be : — the storage conditions, — liquid, — the expiry date, — precipitated with alum or other suitable agent, — the name and amount of any antimicrobial preservative, — the name of any antibiotic, adjuvant, flavour or stabiliser — purified and/or adsorbed on aluminium phosphate, aluminium hydroxide, calcium phosphate or other present in the vaccine, adsorbent prescribed in the monograph. — the name of any constituent that may cause adverse Bacterial toxoids are clear or slightly opalescent liquids. reactions and any contra-indications to the use of the Adsorbed toxoids are suspensions or emulsions. Certain vaccine, toxoids may be freeze-dried. — for freeze-dried vaccines : Unless otherwise indicated, statements and requirements — the name or composition and the volume of the given below for bacterial vaccines apply equally to bacterial reconstituting liquid to be added, vaccines, bacterial toxoids and products containing a — the time within which the vaccine is to be used after combination of bacterial cells and toxoid. reconstitution. VIRAL VACCINES Viral vaccines are prepared by growth in suitable cell cultures 01/2005:0062 (5.2.4), in tissues, in micro-organisms, in fertilised eggs or, corrected where no other possibility is available, in live animals, or by other suitable means. The strain of virus used may have VACCINES FOR VETERINARY USE been modified by genetic engineering. They are liquid or freeze-dried preparations of one or more viruses or viral subunits or peptides. Vaccina ad usum veterinarium Live viral vaccines are prepared from viruses of attenuated In the case of combined vaccines, for each component that virulence or of natural low virulence for the target species. is the subject of a monograph in the Pharmacopoeia, the Inactivated viral vaccines are treated by a validated provisions of that monograph apply to that component, procedure for inactivation of the virus and may be purified modified where necessary as indicated (see Tests (Safety) and concentrated. below, Evaluation of safety of veterinary vaccines (5.2.6) VECTOR VACCINES and Evaluation of efficacy of veterinary vaccines (5.2.7)). Vector vaccines are liquid or freeze-dried preparations of one DEFINITION or more types of live micro-organisms (bacteria or viruses) that are non-pathogenic or have low pathogenicity for the Vaccines for veterinary use are preparations containing target species and in which have been inserted one or more antigenic substances and are administered for the purpose genes encoding antigens that stimulate an immune response of inducing a specific and active immunity against disease provoked by bacteria, toxins, viruses, fungi or parasites. The protective against other microorganisms. vaccines, live or inactivated, confer active immunity that may be transferred passively via maternal antibodies against PRODUCTION The methods of preparation, which vary according to the the immunogens they contain and sometimes also against type of vaccine, are such as to maintain the identity and antigenically related organisms. Vaccines may contain immunogenicity of the antigen and to ensure freedom from bacteria, toxins, viruses or fungi, living or inactivated, contamination with extraneous agents. parasites, or antigenic fractions or substances produced by these organisms and rendered harmless whilst retaining Substances of animal origin used in the production of all or part of their antigenic properties ; vaccines may also vaccines for veterinary use comply with the requirements of contain combinations of these constituents. The antigens chapter 5.2.5. Other substances used in the preparation of may be produced by recombinant DNA technology. Suitable vaccines for veterinary use comply with requirements of the adjuvants may be included to enhance the immunising Pharmacopoeia (where a relevant monograph exists) and properties of the vaccines. are prepared in a manner that avoids contamination of the Terminology used in monographs on vaccines for veterinary vaccine. use is defined in chapter 5.2.1. SUBSTRATES FOR PRODUCTION Cell cultures used in the production of vaccines for veterinary BACTERIAL VACCINES AND BACTERIAL TOXOIDS use comply with the requirements of chapter 5.2.4. Bacterial vaccines and bacterial toxoids are prepared from cultures grown on suitable solid or liquid media, or by other Where a monograph refers to chicken flocks free from suitable means ; the requirements of this section do not specified pathogens (SPF), these flocks comply with the apply to bacterial vaccines prepared in cell cultures or in requirements prescribed in chapter 5.2.2. 590



For production of inactivated vaccines, where vaccine organisms are grown in poultry embryos, such embryos are derived either from SPF flocks (5.2.2) or from healthy non-SPF flocks free from the presence of certain agents and their antibodies, as specified in the monograph. It may be necessary to demonstrate that the inactivation process is effective against specified potential contaminants. For the production of a master seed lot and for all passages of a micro-organism up to and including the working seed lot, eggs from SPF flocks (5.2.2) are used. Where it is unavoidable to use animals or animal tissues in the production of veterinary vaccines, such animals shall be free from specified pathogens, as appropriate to the source species and the target animal for the vaccine. MEDIA At least the qualitative composition must be recorded of media used for seed culture preparation and for production. The grade of each named ingredient is specified. Where media or ingredients are claimed as proprietary, this is indicated and an appropriate description recorded. Ingredients that are derived from animals are specified as to the source species and country of origin, and must comply with the criteria described in chapter 5.2.5. Preparation processes for media used, including sterilisation procedures, are documented.


seed lot. Each master seed lot is assigned a specific code for identification purposes. Production of vaccine is not normally undertaken using virus more than 5 passages from the master seed lot. In the tests on the master seed lot described below, the organisms used are not normally more than 5 passages from the master seed lot at the start of the tests, unless otherwise indicated. Where the master seed lot is contained within a permanently infected master cell seed, the following tests are carried out on an appropriate volume of virus from disrupted master cell seed. Where relevant tests have been carried out on disrupted cells to validate the suitability of the master cell seed, these tests need not be repeated. Propagation. The master seed lot and all subsequent passages are propagated on cells, on embryonated eggs or in animals that have been shown to be suitable for vaccine production (see above), and, where applicable, using substances of animal origin that meet the requirements prescribed in chapter 5.2.5. Identification. A suitable method to identify the vaccine strain and to distinguish it as far as possible from related strains must be used. Bacterial and fungal contamination. The master seed lot complies with the test for sterility (2.6.1).

Mycoplasmas (2.6.7). The master seed lot complies with The addition of antibiotics during the manufacturing process the test for mycoplasmas (culture method and indicator cell is normally restricted to cell culture fluids and other media, culture method). egg inocula and material harvested from skin or other Absence of extraneous viruses. Monographs may contain tissues. requirements for freedom from extraneous agents, otherwise BACTERIAL SEED LOTS the requirements stated below apply. General requirements. The genus and species (and varieties Preparations of monoclonal or polyclonal antibodies where appropriate) of the bacteria used in the vaccine are containing high levels of neutralising antibody to the virus stated. Bacteria used in manufacture are handled in a of the seed lot are made on a batch basis, using antigen that seed-lot system wherever possible. Each master seed lot is not derived from any passage level of the virus isolate is tested as described below. A record of the origin, date giving rise to the master seed virus. Each batch of serum is of isolation, passage history (including purification and maintained at 56 °C for 30 min to inactivate complement. characterisation procedures) and storage conditions is Each batch is shown to be free of antibodies to potential maintained for each master seed lot. Each master seed lot is contaminants of the seed virus and is shown to be free of assigned a specific code for identification purposes. any non-specific inhibiting effects on the ability of viruses to infect and propagate within cells (or eggs, where applicable). Propagation. The minimum and maximum number of subcultures of each master seed lot prior to the production If such a serum cannot be obtained, other methods are used to remove or neutralise the seed virus specifically. stage are specified. The methods used for the preparation of seed cultures, preparation of suspensions for seeding, techniques for inoculation of seeds, titre and concentration If the seed lot virus would interfere with the conduct and sensitivity of a test for extraneous viruses, a sample of the of inocula and the media used, are documented. It shall be master seed lot is treated with a minimum amount of the demonstrated that the characteristics of the seed material monoclonal or polyclonal antibody so that the vaccine (for example, dissociation or antigenicity) are not changed by these subcultures. The conditions under which each seed virus is neutralised as far as possible or removed. The final virus-serum mixture shall, if possible, contain at least the lot is stored are documented. virus content of 10 doses of vaccine per 0.1 ml for avian Identity and purity. Each master seed lot is shown to vaccines and per millilitre for other vaccines. For avian contain only the species and strain of bacterium stated. A vaccines, the testing to be carried out on seed lots is given brief description of the method of identifying each strain by in chapters 2.6.3, 2.6.4, 2.6.5 and 2.6.6. For mammalian biochemical, serological and morphological characteristics vaccines, the seed lot or the mixture of seed lot and antiserum and distinguishing it as far as possible from related strains is is tested for freedom from extraneous agents as follows. recorded, as is also the method of determining the purity of The mixture is inoculated onto cultures of at least 70 cm2 of the strain. If the master seed lot is shown to contain living the required cell types. The cultures may be inoculated at organisms of any kind other than the species and strain any suitable stage of growth up to 70 per cent confluency. stated, then it is unsuitable for vaccine production. At least 1 monolayer of each type must be retained as a VIRUS SEED LOTS control. The cultures must be monitored daily for a week. At General requirements. Viruses used in manufacture are the end of this period the cultures are freeze thawed 3 times, handled in a seed-lot system. Each master seed lot is tested centrifuged to remove cell debris and re-inoculated onto the as described below. A record of the origin, date of isolation, same cell type as above. This is repeated twice. The final passage history (including purification and characterisation passage must produce sufficient cells in appropriate vessels procedures) and storage conditions is maintained for each to carry out the tests below.


591



Cytopathic and haemadsorbing agents are tested for using the methods described in the relevant sections on testing cell cultures (5.2.4) and techniques such as immuno-fluorescence are used for detection of specific contaminants for the tests in cell cultures. The master seed lot is inoculated onto : — primary cells of the species of origin of the virus, — cells sensitive to viruses pathogenic for the species for which the vaccine is intended, — cells sensitive to pestiviruses. If the master seed lot is shown to contain living organisms of any kind, other than the virus of the species and strain stated, or foreign viral antigens, then it is unsuitable for vaccine production. INACTIVATION Inactivated vaccines are subjected to a validated inactivation procedure. The testing of the inactivation kinetics described below is carried out once for a given production process. The rest of this section applies to each production run. When conducting tests for inactivation, it is essential to take account of the possibility that under the conditions of manufacture, organisms may be physically protected from inactivant. Inactivation kinetics. The inactivating agent and the inactivation procedure shall be shown, under conditions of manufacture, to inactivate the vaccine micro-organism. Adequate data on inactivation kinetics shall be obtained. Normally, the time required for inactivation shall be not more than 67 per cent of the duration of the inactivation process. Aziridine. If an aziridine compound is used as the inactivating agent then it shall be shown that no inactivating agent remains at the end of the inactivation procedure. This may be accomplished by neutralising the inactivating agent with thiosulphate and demonstrating residual thiosulphate in the inactivated harvest at the completion of the inactivation procedure. Formaldehyde. If formaldehyde is used as the inactivating agent, then a test for free formaldehyde is carried out as prescribed under Tests. Other inactivating agents. When other inactivation methods are used, appropriate tests are carried out to demonstrate that the inactivating agent has been removed or reduced to an acceptable residual level. Inactivation and/or detoxification testing. A test for complete inactivation and/or detoxification is performed immediately after the inactivation and/or detoxification procedure and, if applicable, the neutralisation or removal of the inactivating or detoxifying agent. Bacterial vaccines. The test selected shall be appropriate to the vaccine bacteria being used and shall consist of at least 2 passages in production medium or, if solid medium has been used for production, in a suitable liquid medium or in the medium prescribed in the monograph. The product complies with the test if no evidence of any live micro-organism is observed. Bacterial toxoids. The test selected shall be appropriate to the toxin or toxins present and shall be the most sensitive available. Viral vaccines. The test selected shall be appropriate to the vaccine virus being used and must consist of at least 2 passages in cells, embryonated eggs or, where no other suitably sensitive method is available, in animals. The quantity of cell samples, eggs or animals shall be sufficient to ensure appropriate sensitivity of the test. For tests in cell cultures, not less than 150 cm2 of cell culture monolayer is 592

inoculated with 1.0 ml of inactivated harvest. The product complies with the test if no evidence of the presence of any live virus or other micro-organism is observed. CHOICE OF VACCINE COMPOSITION AND CHOICE OF VACCINE STRAIN For the choice of vaccine composition and choice of vaccine strain, important aspects to be evaluated include safety, efficacy and stability. General requirements for evaluation of safety and efficacy are given in chapter 5.2.6 and chapter 5.2.7. These requirements may be made more explicit or supplemented by the requirements of specific monographs. For live vaccines, a maximum virus titre or bacterial count acceptable from the point of view of safety is established during development studies. This is then used as the maximum acceptable titre for each batch of vaccine at release. Potency and immunogenicity. The tests given under the headings Potency and Immunogenicity in monographs serve 2 purposes : — the Potency section establishes by a well-controlled test in experimental conditions, the minimum acceptable vaccinating capacity for all vaccines within the scope of the definition, which must be guaranteed throughout the period of validity ; — well-controlled experimental studies are normally a part of the overall demonstration of efficacy of a vaccine (see chapter 5.2.7) ; the test referred to in the section ‘Immunogenicity’ (which is usually a cross-reference to the Potency section) is suitable as a part of this testing. For most vaccines, the tests cited under Potency or Immunogenicity are not suitable for the routine testing of batches. For live vaccines, the minimum acceptable virus titre or bacterial count that gives satisfactory results in the Potency test and other efficacy studies is established during development. For routine testing it must be demonstrated for each batch that the titre or count at release is such that at the end of the period of validity, in the light of stability studies, the vaccine, stored in the recommended conditions, will contain not less than the minimum acceptable virus titre or bacterial count determined during development studies. For inactivated vaccines, if the test described under Potency is not used for routine testing, a batch potency test is established during development. The aim of the batch potency test is to ensure that each batch of vaccine would, if tested, comply with the test described under Potency or Immunogenicity. The acceptance criteria for the batch potency test are therefore established by correlation with the test described under Potency. Where a batch potency test is described in a monograph, this is given as an example of a test that is considered suitable, after establishment of correlation with the potency test ; other test models can also be used. Route of administration. During development of a vaccine, safety and immunogenicity are demonstrated for each route of administration to be recommended. The following is a non-exhaustive list of such routes of administration : — intramuscular, — subcutaneous, — intravenous, — ocular, — oral, — nasal, — foot-stab, — wing web,




— intradermal, — intraperitoneal, — in ovo. Methods of administration. During development of a vaccine, safety and immunogenicity are demonstrated for each method of administration to be recommended. The following is a non-exhaustive list of such methods of administration : — injection, — drinking water, — spray, — eye-drop, — scarification, — implantation, — immersion. Categories of animal. Monographs may indicate that a given test is to be carried out for each category of animal of the target species for which the product is recommended or is to be recommended. The following is a non-exhaustive list of categories that are to be taken into account. — Mammals: — pregnant animals/non-pregnant animals, — animals raised primarily for breeding/animals raised primarily for food production, — animals of the minimum age or size recommended for vaccination. — Avian species: — birds raised primarily for egg production/birds raised primarily for production of meat, — birds before point of lay/birds after onset of lay. — Fish: — broodstock fish/fish raised primarily for food production. Stability. Evidence of stability is obtained to justify the proposed period of validity. This evidence takes the form of the results of virus titrations, bacterial counts or potency tests carried out at regular intervals until 3 months beyond the end of the shelf life on not fewer than 3 representative consecutive batches of vaccine kept under recommended storage conditions together with results from studies of moisture content (for freeze-dried products), physical tests on the adjuvant, chemical tests on substances such as the adjuvant constituents and preservatives and pH, as appropriate. Where applicable, studies on the stability of the reconstituted vaccine are carried out, using the product reconstituted in accordance with the proposed recommendations. FINAL BULK VACCINE The final bulk vaccine is prepared by combining one or more batches of antigen that comply with all the relevant requirements with any auxiliary substances, such as adjuvants, stabilisers, antimicrobial preservatives and diluents. Antimicrobial preservatives. Antimicrobial preservatives are used to prevent spoilage or adverse effects caused by microbial contamination occurring during use of a vaccine which is expected to be no longer than 10 h after first broaching. Antimicrobial preservatives are not included in freeze-dried products but, if justified, taking into account the maximum recommended period of use after reconstitution, they may be included in the diluent for multi-dose freeze-dried products. For single-dose liquid preparations, inclusion of antimicrobial preservatives is not acceptable unless justified and authorised, but may be acceptable, for

example where the same vaccine is filled in single-dose and multidose containers and is used in non-food-producing species. For multidose liquid preparations, the need for effective antimicrobial preservation is evaluated taking into account likely contamination during use and the maximum recommended period of use after broaching of the container. During development studies the effectiveness of the antimicrobial preservative throughout the period of validity shall be demonstrated to the satisfaction of the competent authority. The efficacy of the antimicrobial preservative is evaluated as described in chapter 5.1.3 and in addition samples are tested at suitable intervals over the proposed in use shelf-life. If neither the A criteria nor the B criteria can be met, then in justified cases the following criteria are applied to vaccines for veterinary use : bacteria, no increase from 24 h to 7 days, 3 log reduction at 14 days, no increase at 28 days ; fungi, no increase at 14 days and 28 days. Addition of antibiotics as antimicrobial preservative is generally not acceptable. Test for inactivation and/or detoxification. For inactivated vaccines, where the auxiliary substances would interfere with a test for inactivation and/or detoxification, a test for inactivation or detoxification is carried out during preparation of the final bulk, after the different batches of antigen have been combined but before addition of auxiliary substances ; the test for inactivation or detoxification may then be omitted on the final bulk and the batch. Where there is a risk of reversion to toxicity, the test for detoxification performed at the latest stage of the production process at which the sensitivity of the test is not compromised (e.g. after the different batches of antigen have been combined but before the addition of auxiliary substances) is important to demonstrate a lack of reversion to toxicity. In-process tests. Certain tests may be carried out on the final bulk vaccine rather than on the batch or batches prepared from it ; such tests include those for antimicrobial preservatives, free formaldehyde and the potency determination for inactivated vaccines. BATCH Unless otherwise prescribed in the monograph, the final bulk vaccine is distributed aseptically into sterile, tamper-proof containers which are then closed so as to exclude contamination. Only a batch that complies with each of the requirements given below under Identification, Tests and Potency or in the relevant individual monograph may be released for use. With the agreement of the competent authority, certain of the batch tests may be omitted where in-process tests give an equal or better guarantee that the batch would comply or where alternative tests validated with respect to the Pharmacopoeia method have been carried out. The identification test can often be conveniently combined with the batch potency test to avoid unnecessary use of animals. For a given vaccine, a validated in vitro test can be used to avoid the unnecessary use of animals. It is recognised that, in accordance with General Notices (1.1. General statements), for an established vaccine the routine application of the safety test will be waived by the competent authority in the interests of animal welfare when a sufficient number of consecutive production batches have been produced and found to comply with the test, thus demonstrating consistency of the manufacturing process. Significant changes to the manufacturing process may require resumption of routine testing to re-establish consistency. The number of consecutive batches to be tested


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of direct inoculation may be used. Where the volume of liquid in each container is at least 20 ml, the minimum volume to be used for each culture medium is 10 per cent of the contents or 5 ml, whichever is less. The appropriate number of items to be tested (2.6.1) is 1 per cent of the batch with a minimum of 4 and a maximum of 10. For avian live viral vaccines, for non-parenteral use only, the requirement for sterility is usually replaced by requirements for absence of pathogenic micro-organisms and for a maximum of 1 non-pathogenic micro-organism per dose. Extraneous agents. Monographs prescribe a set of measures that taken together give an acceptable degree of assurance that the final product does not contain infectious extraneous agents. These measures include : 1) Production within a seed-lot system and a cell-seed system, wherever possible ; 2) Extensive testing of seed lots and cell seed for extraneous agents ; 3) Requirements for SPF flocks used for providing substrates for vaccine production ; 4) Testing of substances of animal origin, which must, wherever possible, undergo an inactivation procedure ; 5) For live vaccines, testing of the final product for infectious extraneous agents ; such tests are less extensive than those carried out at earlier stages because of the guarantees given by in-process testing. In cases of doubt, the tests intended for the seed lot of a live vaccine may also be applied to the final product. If an extraneous agent is found in such a test, the vaccine does not comply the monograph. Avian live viral vaccines comply with the tests for extraneous agents in batches of finished products (2.6.4). Mycoplasmas (2.6.7). Where prescribed in a monograph, the vaccine complies with the test for mycoplasmas (culture method). Safety. In general, 2 doses of an inactivated vaccine and/or 10 doses of a live vaccine are injected by a recommended route. It may be necessary to reduce the prescribed number of doses under certain circumstances or amend the method of re-constitution and injection, for example for a combined IDENTIFICATION vaccine, where it is difficult to reconstitute 10 doses of the For inactivated vaccines, the identification prescribed in live component in 2 doses of the inactivated component. monographs is usually an antibody induction test since this The animals are observed for the longest period stated in is applicable to all vaccines. the monographs. No abnormal local or systemic reaction occurs. Where several batches are prepared from the same TESTS final bulk, the safety test is carried out on the first batch and then omitted for further batches prepared from the same The monographs also indicate tests to be carried out on final bulk. each particular vaccine. During development studies, the type and degree of reactions All hen eggs, chickens and chicken cell cultures for use expected with the vaccine are defined in the light of safety in quality control tests shall be derived from an SPF flock testing. This definition is then used as part of the operating (5.2.2). procedure for the batch safety test to evaluate acceptable Formaldehyde (2.4.18 ; use Method B if sodium and unacceptable reactions. metabisulphite has been used to neutralise excess The immune status of animals to be used for the safety formaldehyde). Where formaldehyde has been used in the test is specified in the individual monograph. For most preparation, the concentration of free formaldehyde is not greater than 0.5 g/l, unless a higher amount has been shown monographs, one of the 3 following categories is specified : to be safe. 1) the animals must be free from antibodies against the virus/bacterium/toxin etc. contained in the vaccine, Phenol (2.5.15). When the vaccine contains phenol, the concentration is not greater than 5 g/l. 2) the animals are preferably free from antibodies but animals with a low level of antibody may be used as long as Sterility (2.6.1). Where prescribed in the monograph, vaccines comply with the test for sterility. Where the volume the animals have not been vaccinated and the administration of liquid in a container is greater than 100 ml, the method of of the vaccine does not cause an anamnestic response, 3) the animals must not have been vaccinated against the membrane filtration is used wherever possible. Where the method of membrane filtration cannot be used, the method disease the vaccine is intended to prevent. depends on a number of factors such as the type of vaccine, the frequency of production of batches and experience with the vaccine during development safety testing and during application of the batch safety test. Without prejudice to the decision of the competent authority in the light of information available for a given vaccine, testing of 10 consecutive batches is likely to be sufficient for most products. For products with an inherent safety risk, it may be necessary to continue to conduct the safety test on each batch. Animal tests. In accordance with the provisions of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes, tests must be carried out in such a way as to use the minimum number of animals and to cause the least pain, suffering, distress or lasting harm. The criteria for judging tests in monographs must be applied in the light of this. For example, if it is indicated that an animal is considered to be positive, infected etc. when typical clinical signs occur then as soon as it is clear that the result will not be affected the animal in question shall be either humanely killed or given suitable treatment to prevent unnecessary suffering. In accordance with the General Notices, alternative test methods may be used to demonstrate compliance with the monograph and the use of such tests is particularly encouraged when this leads to replacement or reduction of animal use or reduction of suffering. Physical tests. A vaccine with an oily adjuvant is tested for viscosity by a suitable method and shown to be within the limits set for the product. The stability of the emulsion shall be demonstrated. Chemical tests. Tests for the concentrations of appropriate substances such as aluminium and preservatives are carried out to show that these are within the limits set for the product. pH. The pH of liquid products and diluents is measured and shown to be within the limits set for the product. Water. Where applicable, the freeze-drying process is checked by a determination of water and shown to be within the limits set for the product.

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As a general rule, category 1 is specified for live vaccines. For other vaccines, category 2 is usually specified but where most animals available for use in tests would comply with category 1, this may be specified for inactivated vaccines also. Category 3 is specified for some inactivated vaccines where determination of antibodies prior to testing is unnecessary or impractical. For poultry vaccines, as a general rule the use of SPF birds is specified. For avian vaccines, the safety test is generally carried out using 10 SPF chickens (5.2.2), except that for vaccines not recommended for use in chickens it is carried out using 10 birds of one of the species for which the vaccine is recommended, the birds being free from antibodies against the disease agent for which the vaccine is intended to provide protection. POTENCY See Choice of vaccine composition and choice of vaccine strain under Production.

Vegetable fatty oils

unsaponifiable matters. Vegetable fatty oils are obtained from the seeds, the fruit or the pit/stone/kernel of various plants by expression and/or solvent extraction, then possibly refined and hydrogenated. A suitable antioxidant may be added if necessary. Virgin oil : an oil obtained from raw materials of special quality by mechanical procedures (e.g. by cold expression or centrifugation). Refined oil : an oil obtained by expression and/or solvent extraction, and subsequently, either alkali refining (followed by bleaching and deodorisation) or physical refining. Hydrogenated oil : an oil obtained by expression and/or solvent extraction, and subsequently, either alkali refining or physical refining, then possible bleaching, followed by drying, hydrogenation and subsequent bleaching and deodorisation. Only phosphoric acid and alkali refined oils are used in the preparation of parenteral dosage forms.

STORAGE Store protected from light at a temperature of 5 ± 3 °C, unless otherwise indicated. Liquid preparations are not to be allowed to freeze, unless otherwise indicated.

PRODUCTION OBTAINING OF A CRUDE OIL When the plant has a high oil content, the oil is generally obtained by expression under heating followed by an LABELLING extraction ; when the plant has a low oil content, the oil is The label states : generally obtained by direct extraction. — that the preparation is for veterinary use, Mechanical procedures — the volume of the preparation and the number of doses A. Expression in the container, High-pressure screw-pressing. It consists of some or all — the route of administration, of the following steps : cleaning, drying, dehulling or — the type or types of bacteria or viruses used and for live decorticating, grinding, cooking and flaking. vaccines the minimum and the maximum number of live During cleaning the foreign matter is eliminated. Drying bacteria or the minimum and the maximum virus titre, may be necessary if the seed moisture content is higher — where applicable, for inactivated vaccines, the minimum than desirable for downstream processing. Decorticating is potency in International Units, useful to obtain a high-protein meal by reduction of fibre — where applicable, the name and amount of antimicrobial and to reduce impurities in the oil. Cooking serves various preservative or other substance added to the vaccine, purposes : completion of the breakdown of oil cells, lowering — the name of any substance that may cause an adverse of the viscosity of the oil, coagulation of the protein in the reaction, meal, adjustment of the moisture level, sterilisation of the — for freeze-dried vaccines : seed, detoxifying undesirable seed constituents (gossypol for cottonseed) and fixing certain phosphatides in the cake — the name or composition and the volume of the thus lowering subsequent refining losses. The efficacy of the reconstituting liquid to be added, — the period within which the vaccine is to be used after expression process is such that only 3 per cent to 6 per cent of the oil is left in the cake. reconstitution, Wet screw-pressing. The bunches are loaded into cages — for vaccines with an oily adjuvant, that if the vaccine is (for palm fruit) and moved into a horizontal steriliser with accidentally injected into man, urgent medical attention application of live steam and heating. The purposes of this is necessary, steriliser are inactivation of enzymes, loosening of the fruit — the animal species for which the vaccine is intended, on the bunch, coagulation of proteins, etc. After heating — the indications for the vaccine, in a digester, the pulp is fed to a screw-press. The oil is — the instructions for use, centrifugally clarified and vacuum-dried. — any contra-indications to the use of the product including Pre-pressing followed by solvent extraction. The same any required warning on the dangers of administration sequence of steps is performed as above. The main function of an overdose, of pre-pressing is to obtain a cake of excellent permeability — the doses recommended for different species. for the following solvent extraction stage. The extraction is performed either in a percolation type or in an immersion 01/2005:1579 type apparatus. The efficacy of the solvent extraction process is such that residual oil levels in meal are generally below 1 per cent. VEGETABLE FATTY OILS B. Centrifugation Olea herbaria Centrifugation separates the oily phase from the aqueous phase which contains water, water-soluble components and DEFINITION residual solid particles. This operation can be carried out Vegetable fatty oils are mainly solid or liquid triglycerides using : of fatty acids. They may contain small amounts of other — self-cleaning bowl or disc centrifuges, lipids such as waxes, free fatty acids, partial glycerides or General Notices (1) apply to all monographs and other texts

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Vegetable fatty oils


— super-decanters, which are horizontal turbines equipped with a cylindrical bowl that tapers slightly at one end and which contains a continuously turning screw that scrapes the sides of the bowl. The screw and the bowl rotate at different speeds. The solid particles are discarded from the tapered end of the bowl and the oil flows out from the other end. Solvent extraction. Prior to extraction, the following steps are carried out : the seeds are tempered for about a week at a temperature below 24 °C in order to loosen the hull from the seed and allow the seed moisture to attain equilibrium. Then the seeds are cleaned, ground, dehulled and flaked. The most widely used solvent is a mixture of mainly n-hexane and methylpentanes (bp : 65-70 °C) commonly referred to as “hexane”. Due to the major fire and explosive risks of this mixture, liquified gases and supercritical gases may also be used. REFINING The objective of refining is to remove impurities and contaminants of the oil with the least possible damage to the triglycerides and with minimal loss of oil. The contents of the following substances are reduced : — free fatty acids which may cause deterioration of the oil by oxidation, smoked taste when heated and sharp flavour (by alkali refining), — water which favours the enzymatic hydrolysis reactions (by alkali refining, drying), — partial glycerides which may cause foaming and bitter taste (by neutralisation, washing), — phosphatides and phosphorous compounds which have emulsifying properties, may cause deposits, a darkening of the oil when heated, a cloudy appearance and bad organoleptic stability (by alkali refining), — colouring matters such as chlorophyll (by alkali refining), carotenoids (by bleaching), — glycolipids which may form colloidal solutions with water, — free hydrocarbons, paraffin, waxes and resinous materials, — metals (Fe, Cu, Pb, Sn, Pt, Pd, etc.) which are strong oxidation catalysts, — pigments such as gossypol (in cottonseed oil) or mycotoxins such as aflatoxin (mainly in arachis oil), — pesticides, — oxidation products (aldehydes, peroxides), — proteins having possible allergic reactions, — unsaponifiable matters (e.g. lignins, sterols, tocopherols and other vitamins), — polycyclic aromatic hydrocarbons. Alkali refining. It involves the following steps : degumming, neutralisation using alkali, washing and drying. Degumming. During this step of the refining, i.e. treatment with water and/or phosphoric acid, and/or sodium chloride, the phosphatides, phosphorous compounds and metals are eliminated. The use of this step depends on the nature of the oil. Neutralisation with alkali. This step reduces the free fatty acid content below 0.1 per cent ; the fatty acids are converted into oil-insoluble soaps, also called “soapstocks”. Other substances may be removed by adsorption on these soaps : mucilaginous substances, phosphatides, oxidation products, colouring matters, etc. All substances that become insoluble in the oil on hydration are removed. Neutralisation with alkali has the disadvantage of saponifying a portion of neutral oil if the neutralisation is not well conducted.

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Washing. This operation consists in removing the excess of soaps and alkali as well as the remaining traces of metals, phosphatides and other impurities, using hot water. Drying. The remaining water is eliminated under vacuum before any further steps, such as bleaching. Physical refining. It involves a steam treatment of the oil under high vacuum at a temperature greater than 235 °C. This technique must be applied to oils naturally low in phosphatides and metals (palm, coconut, olive) or from which phosphatides and metals have been removed by an acid treatment using concentrated phosphoric acid followed by an adsorptive treatment with activated bleaching earth (for sunflower, rapeseed, soya-bean). Moreover it cannot be used for heat sensitive oils (cottonseed oil) which darken. Bleaching. The common method of bleaching is by adsorption treatment of the oil, which is generally heated at 90 °C for 30 min under vacuum, with bleaching earth (natural or activated) or carbon (activated or not) ; synthetic silica adsorbents may also be added. Substances which have not been totally removed during refining are eliminated, for example carotenoids and chlorophyll. Deodorisation. Deodorisation eliminates odours, volatile substances and residual extraction solvents ; it involves injecting dry vapour into the oil which is kept under vacuum at a high temperature. Different temperatures are used according to the oil : 200 °C to 235 °C for 1 h 30 min to 3 h or greater than 240 °C for 30 min. One of the main side reactions is thermic decolourisation due to the destruction of carotenoids when the temperature is greater than 150 °C. This technique provokes a loss of substances which may be distilled (free fatty acids, sterols, tocopherols, part of the refined oil) and may cause cis-trans isomerisation of the unsaturated fatty-acid double bonds. WINTERISATION Elimination of solids and waxes by filtration at low temperature (also called dewaxing). These solids and waxes could affect the appearance of the oil and cause deposits. HYDROGENATION The hydrogenation of the dried and/or bleached oil is performed using a catalyst (e.g. Ni, Pt, Pd), at a temperature of about 100 °C to 200 °C under hydrogen pressure. The catalyst is then removed by filtration at 90 °C. The hydrogen must be pure : free of poisons for the catalyst, water-free, low in carbon dioxide, methane and nitrogen contents. Small amounts of trans-fatty acids or polymers may be obtained. CHROMATOGRAPHIC PURIFICATION In high purity applications, mainly for parenteral uses, the oil may be further purified by passing the oil through a column containing an activated earth. A solvent may sometimes be used to improve the efficiency. High polarity molecules such as oxidised materials, acids, alcohols, partial glycerides and free sterols are preferentially removed. When the oil is used in the preparation of parenteral dosage forms, the limits set in the monograph for the acid value, the peroxide value and the water content may be different. LABELLING The label states : — where applicable, that the oil was obtained by expression or extraction, — where applicable, that the oil is suitable for use in the manufacture of parenteral dosage forms, — the name and concentration of any added antioxidant.


Capsules


01/2005:1502 Large-volume parenterals Infusions and injections supplied in containers with a nominal content of more than 100 ml. GLOSSARY The following introductory text provides definitions and/or Small-volume parenterals Infusions and injections supplied in containers with a explanations of terms that may be found in, or used in association with, the general monographs on dosage forms, nominal content of 100 ml or less. but that are not defined within them. Where relevant, reference is made to other equivalent terms that may be found in other publications or contexts. 01/2005:0016 This glossary is published for information.

CAPSULES

Standard Term Standard Terms for describing the pharmaceutical form of a medicinal product, the routes of administration and the containers used have been established by the European Pharmacopoeia Commission and are provided in a separate publication on Standard Terms. Active substance Equivalent terms : active ingredient, drug substance, medicinal substance, active pharmaceutical ingredient. Vehicle A vehicle is the carrier, composed of one or more excipients, for the active substance(s) in a liquid preparation. Basis A basis is the carrier, composed of one or more excipients, for the active substance(s) in semi-solid and solid preparations. Conventional-release dosage forms Conventional-release dosage forms are preparations showing a release of the active substance(s) which is not deliberately modified by a special formulation design and/or manufacturing method. In the case of a solid dosage form, the dissolution profile of the active substance depends essentially on its intrinsic properties. Equivalent term : immediate-release dosage form. Modified-release dosage forms Modified-release dosage forms are preparations where the rate and/or place of release of the active substance(s) is different from that of a conventional-release dosage form administered by the same route. This deliberate modification is achieved by a special formulation design and/or manufacturing method. Modified-release dosage forms include prolonged-release, delayed-release and pulsatile-release dosage forms. Prolonged-release dosage forms Prolonged-release dosage forms are modified-release dosage forms showing a slower release of the active substance(s) than that of a conventional-release dosage form administered by the same route. Prolonged-release is achieved by a special formulation design and/or manufacturing method. Equivalent term : extended-release dosage form. Delayed-release dosage forms Delayed-release dosage forms are modified-release dosage forms showing a release of the active substance(s) which is delayed. Delayed release is achieved by a special formulation design and/or manufacturing method. Delayed-release dosage forms include gastro-resistant preparations as defined in the general monographs on solid oral dosage forms. Pulsatile-release dosage forms Pulsatile-release dosage forms are modified-release dosage forms showing a sequential release of the active substance(s). Sequential release is achieved by a special formulation design and/or manufacturing method.

TESTS Uniformity of content (2.9.6). Unless otherwise prescribed or justified and authorised, capsules with a content of active substance less than 2 mg or less than 2 per cent of the total mass comply with test B for uniformity of content of single-dose preparations. If the preparation has more than one active substance, the requirement applies only to those ingredients which correspond to the above conditions.


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Capsulae The requirements of this monograph do not necessarily apply to preparations that are presented as capsules intended for use other than by oral administration. Requirements for such preparations may be found, where appropriate, in other general monographs, for example Rectal preparations (1145) and Vaginal preparations (1164). DEFINITION Capsules are solid preparations with hard or soft shells of various shapes and capacities, usually containing a single dose of active substance. They are intended for oral administration. The capsule shells are made of gelatin or other substances, the consistency of which may be adjusted by the addition of substances such as glycerol or sorbitol. Excipients such as surface-active agents, opaque fillers, antimicrobial preservatives, sweeteners, colouring matter authorised by the competent authority and flavouring substances may be added. The capsules may bear surface markings. The contents of capsules may be solid, liquid or of a paste-like consistency. They consist of one or more active substances with or without excipients such as solvents, diluents, lubricants and disintegrating agents. The contents do not cause deterioration of the shell. The shell, however, is attacked by the digestive fluids and the contents are released. Where applicable, containers for capsules comply with the requirements of Materials used for the manufacture of containers (3.1 and subsections) and Containers (3.2 and subsections). Several categories of capsules may be distinguished : — hard capsules, — soft capsules, — gastro-resistant capsules, — modified-release capsules, — cachets. PRODUCTION In the manufacture, packaging, storage and distribution of capsules, suitable means are taken to ensure their microbial quality ; recommendations on this aspect are provided in the text on Microbiological quality of pharmaceutical preparations (5.1.4).

Capsules


Uniformity of mass (2.9.5). Capsules comply with the test for uniformity of mass of single-dose preparations. If the test for uniformity of content is prescribed for all the active substances, the test for uniformity of mass is not required. Dissolution. A suitable test may be carried out to demonstrate the appropriate release of the active substance(s), for example one of the tests described in Dissolution test for solid dosage forms (2.9.3). Where a dissolution test is prescribed, a disintegration test may not be required.

substances dispensed in soft capsules may attack the disc ; in such circumstances and where authorised, the disc may be omitted. Operate the apparatus for 30 min, unless otherwise justified and authorised and examine the state of the capsules. If the capsules fail to comply because of adherence to the discs, repeat the test on a further 6 capsules omitting the discs. The capsules comply with the test if all 6 have disintegrated.

STORAGE Store at a temperature not exceeding 30 °C.

DEFINITION Modified-release capsules are hard or soft capsules in which the contents or the shell or both contain special excipients or are prepared by a special process designed to modify the rate, the place or the time at which the active substance(s) are released. Modified release capsules include prolonged-release capsules and delayed-release capsules.

LABELLING The label states the name of any added antimicrobial preservative.

Hard capsules DEFINITION Hard capsules have shells consisting of two prefabricated cylindrical sections one end of which is rounded and closed, the other being open. PRODUCTION The active substance(s) usually in solid form (powder or granules) are filled into one of the sections which is then closed by slipping the other section over it. The security of the closure may be strengthened by suitable means. TESTS Disintegration. Hard capsules comply with the test for disintegration of tablets and capsules (2.9.1). Use water R as the liquid medium. When justified and authorised, 0.1 M hydrochloric acid or artificial gastric juice R may be used as the liquid medium. If the capsules float on the surface of the water, a disc may be added. Operate the apparatus for 30 min, unless otherwise justified and authorised and examine the state of the capsules. The capsules comply with the test if all 6 have disintegrated.

Modified-release capsules

PRODUCTION A suitable test is carried out to demonstrate the appropriate release of the active substance(s).

Gastro-resistant capsules DEFINITION Gastro-resistant capsules are delayed-release capsules that are intended to resist the gastric fluid and to release their active substance or substances in the intestinal fluid. Usually they are prepared by filling capsules with granules or with particles covered with a gastro-resistant coating or in certain cases, by providing hard or soft capsules with a gastro-resistant shell (enteric capsules). PRODUCTION For capsules filled with granules or filled with particles covered with a gastro-resistant coating, a suitable test is carried out to demonstrate the appropriate release of the active substance(s).

TESTS Disintegration. For capsules with a gastro-resistant shell carry out the test for disintegration (2.9.1) with the following DEFINITION Soft capsules have thicker shells than those of hard capsules. modifications. Use 0.1 M hydrochloric acid as the liquid medium and operate the apparatus for 2 h, or other such time The shells consist of one part and are of various shapes. as may be authorised, without the discs. Examine the state of the capsules. The time of resistance to the acid medium PRODUCTION varies according to the formulation of the capsules to be Soft capsules are usually formed, filled and sealed in one examined. It is typically 2 h to 3 h but even with authorised operation but for extemporaneous use, the shell may be deviations it must not be less than 1 h. No capsule shows prefabricated. The shell material may contain an active signs of disintegration or rupture permitting the escape of substance. the contents. Replace the acid by phosphate buffer solution Liquids may be enclosed directly ; solids are usually dissolved pH 6.8 R. When justified and authorised, a buffer solution or dispersed in a suitable vehicle to give a solution or of pH 6.8 with added pancreas powder (for example, 0.35 g dispersion of a paste-like consistency. of pancreas powder R per 100 ml of buffer solution) may be There may be partial migration of the constituents from the used. Add a disc to each tube. Operate the apparatus for capsule contents into the shell and vice versa because of the 60 min and examine the state of the capsules. If the capsules fail to comply because of adherence to the discs, repeat the nature of the materials and the surfaces in contact. test on a further 6 capsules omitting the discs. The capsules comply with the test if all 6 have disintegrated. TESTS Dissolution. For capsules prepared from granules or Disintegration. Soft capsules comply with the test for particles already covered with a gastro-resistant coating, a disintegration of tablets and capsules (2.9.1). Use water R as the liquid medium. When justified and authorised, 0.1 M suitable test is carried out to demonstrate the appropriate release of the active substance(s), for example the test hydrochloric acid or artificial gastric juice R may be used as the liquid medium. Add a disc to each tube. Liquid active described in Dissolution test for solid dosage forms (2.9.3).

Soft capsules

600



Ear preparations

Cachets

of mass of single-dose preparations. If the test for uniformity of content is prescribed for all the active substances, the test for uniformity of mass is not required.

DEFINITION Cachets are solid preparations consisting of a hard shell STORAGE containing a single dose of one or more active substances. Store uncoated medicated chewing gums protected from The cachet shell is made of unleavened bread usually from humidity and light. rice flour and consists of 2 prefabricated flat cylindrical sections. Before administration, the cachets are immersed in 01/2005:0652 water for a few seconds, placed on the tongue and swallowed with a draught of water. LABELLING The label states the method of administration of the cachets.

EAR PREPARATIONS Auricularia

DEFINITION 01/2005:1239 Ear preparations are liquid, semi-solid or solid preparations intended for instillation, for spraying, for insufflation, for application to the auditory meatus or as an ear wash. CHEWING GUMS, MEDICATED Ear preparations usually contain one or more active substances in a suitable vehicle. They may contain Masticabilia gummis medicata excipients, for example, to adjust tonicity or viscosity, to adjust or stabilise the pH, to increase the solubility of DEFINITION Medicated chewing gums are solid, single-dose preparations the active substances, to stabilise the preparation or to with a base consisting mainly of gum that are intended to be provide adequate antimicrobial properties. The excipients do not adversely affect the intended medicinal action of the chewed but not swallowed. preparation or, at the concentrations used, cause toxicity or They contain one or more active substances which are undue local irritation. released by chewing. After dissolution or dispersion of the Preparations for application to the injured ear, particularly active substances in saliva, chewing gums are intended to where the ear-drum is perforated, or prior to surgery are be used for : sterile, free from antimicrobial preservatives and supplied in — local treatment of mouth diseases, single-dose containers. — systemic delivery after absorption through the buccal Ear preparations are supplied in multi-dose or single-dose mucosa or from the gastrointestinal tract. containers, provided, if necessary, with a suitable administration device which may be designed to avoid the PRODUCTION introduction of contaminants. Medicated chewing gums are made with a tasteless Unless otherwise justified and authorised, aqueous ear masticatory gum base that consists of natural or synthetic preparations supplied in multidose containers contain elastomers. They may contain other excipients such as fillers, softeners, sweetening agents, flavouring substances, a suitable antimicrobial preservative at a suitable stabilisers and plasticisers and authorised colouring matter. concentration, except where the preparation itself has adequate antimicrobial properties. Medicated chewing gums are manufactured by compression Where applicable, containers for ear preparations comply or by softening or melting the gum bases and adding successively the other substances. In the latter case, chewing with the requirements of Materials used for the manufacture gums are then further processed to obtain the desired gum of containers (3.1 and subsections) and Containers (3.2 and presentation. The medicated chewing gums may be coated, subsections). for example, if necessary to protect from humidity and light. Several categories of ear preparations may be distinguished : — ear-drops and sprays, Unless otherwise justified and authorised, a suitable test is — semi-solid ear preparations, carried out to demonstrate the appropriate release of the active ingredient(s). — ear powders, In the manufacture, packaging, storage and distribution of — ear washes, medicated chewing gums, suitable means must be taken to — ear tampons. ensure their microbial quality ; recommendations related PRODUCTION to this aspect are provided in the general chapter on Microbiological quality of pharmaceutical preparations During the development of an ear preparation, the (5.1.4). formulation for which contains an antimicrobial preservative, the effectiveness of the chosen preservative shall be TESTS demonstrated to the satisfaction of the competent authority. Uniformity of content (2.9.6). Unless otherwise prescribed A suitable test method together with criteria for judging the preservative properties of the formulation are provided in or justified and authorised, medicated chewing gums with the text on Efficacy of antimicrobial preservation (5.1.3). a content of active ingredient less than 2 mg or less than 2 per cent of the total mass comply with test A for uniformity In the manufacture, packaging, storage and distribution of content of single-dose preparations. If the preparation of ear preparations, suitable means are taken to ensure contains more than one active substance, the requirement their microbial quality ; recommendations on this aspect applies only to those active substances which correspond to are provided in the text on Microbiological quality of the above conditions. pharmaceutical preparations (5.1.4). Uniformity of mass (2.9.5). Uncoated medicated chewing Sterile ear preparations are prepared using materials gums and, unless otherwise justified and authorised, coated and methods designed to ensure sterility and to avoid medicated chewing gums comply with the test for uniformity the introduction of contaminants and the growth of General Notices (1) apply to all monographs and other texts

601

Eye preparations


micro-organisms ; recommendations on this aspect are provided in the text on Methods of preparation of sterile products (5.1.1). In the manufacture of ear preparations containing dispersed particles, measures are taken to ensure a suitable and controlled particle size with regard to the intended use. TESTS Uniformity of content (2.9.6). Unless otherwise prescribed or justified and authorised, single-dose ear preparations with a content of active substance less than 2 mg or less than 2 per cent of the total mass comply with test B for uniformity of content of single-dose preparations. If the preparation has more than one active substance, the requirement applies only to those ingredients that correspond to the above conditions. Uniformity of mass (2.9.5). Single-dose ear preparations comply with the test for uniformity of mass of single-dose preparations. If the test for uniformity of content is prescribed for all the active substances, the test for uniformity of mass is not required. Sterility (2.6.1). Where the label indicates that the ear preparation is sterile, it complies with the test for sterility. STORAGE If the preparation is sterile, store in a sterile, airtight, tamper-proof container.

Semi-solid ear preparations comply with the requirements of the monograph on Semi-solid preparations for cutaneous application (0132). They are supplied in containers fitted with a suitable applicator.

Ear powders DEFINITION Ear powders comply with the requirements of the monograph on Powders for cutaneous application (1166). They are supplied in containers fitted with a suitable device for application or insufflation.

Ear washes DEFINITION Ear washes are preparations intended to cleanse the external auditory meatus. They are usually aqueous solutions with apH within physiological limits. Ear washes intended for application to injured parts or prior to a surgical operation are sterile. TESTS Deliverable mass or volume (2.9.28). Ear washes supplied in single-dose containers comply with the test.

LABELLING The label states : — the name of any added antimicrobial preservative, — where applicable, that the preparation is sterile, — for multidose containers, the period after opening the container after which the contents must not be used. This period does not exceed 4 weeks, unless otherwise justified and authorised.

Ear tampons DEFINITION Ear tampons are intended to be inserted into the external auditory meatus. They comply with the requirements of the monograph on Medicated tampons (1155). 01/2005:1163

Ear-drops and sprays DEFINITION Ear-drops and sprays are solutions, emulsions or suspensions of one or more active substances in liquids suitable for application to the auditory meatus without exerting harmful pressure on the ear-drum (for example, water, glycols or fatty oils). They may also be placed in the auditory meatus by means of a tampon impregnated with the liquid. Emulsions may show evidence of phase separation but are readily redispersed on shaking. Suspensions may show a sediment which is readily dispersed on shaking to give a suspension which remains sufficiently stable to enable the correct dose to be delivered. Ear drops are usually supplied in multidose containers of glass or suitable plastic material that are fitted with an integral dropper or with a screw cap of suitable materials incorporating a dropper and rubber or plastic teat. Alternatively, such a cap assembly is supplied separately. Sprays are usually supplied in multi-dose containers fitted with an appropriate applicator. When sprays are supplied in pressurised containers, these comply with the requirements of the monograph on Pressurised pharmaceutical preparations (0523).

Semi-solid ear preparations DEFINITION Semi-solid ear preparations are intended for application to the external auditory meatus, if necessary by means of a tampon impregnated with the preparation. 602

EYE PREPARATIONS Ophthalmica DEFINITION Eye preparations are sterile liquid, semi-solid or solid preparations intended for administration upon the eyeball and/or to the conjunctiva or for insertion in the conjunctival sac. Where applicable, containers for eye preparations comply with the requirements of Materials used for the manufacture of containers (3.1 and subsections) and Containers (3.2 and subsections). Several categories of eye preparations may be distinguished : — eye drops, — eye lotions, — powders for eye drops and eye lotions, — semi-solid eye preparations, — ophthalmic inserts. PRODUCTION During the development of an eye preparation, the formulation for which contains an antimicrobial preservative, the effectiveness of the chosen preservative shall be demonstrated to the satisfaction of the competent authority. A suitable test method together with criteria for judging the preservative properties of the formulation are provided in the text on Efficacy of antimicrobial preservation (5.1.3).


Eye preparations


Eye preparations are prepared using materials and methods designed to ensure sterility and to avoid the introduction of contaminants and the growth of micro-organisms ; recommendations on this aspect are provided in the text on Methods of preparation of sterile products (5.1.1). In the manufacture of eye preparations containing dispersed particles, measures are taken to ensure a suitable and controlled particle size with regard to the intended use. TESTS Sterility (2.6.1). Eye preparations comply with the test for sterility. Applicators supplied separately also comply with the test for sterility. Remove the applicator with aseptic precautions from its package and transfer it to a tube of culture medium so that it is completely immersed. Incubate and interpret the results as described in the test for sterility. Deliverable mass or volume (2.9.28). Liquid and semi-solid eye preparations supplied in single-dose containers comply with the test. STORAGE Unless otherwise prescribed, store in a sterile, airtight, tamper-proof container.

TESTS Particle size. Unless otherwise justified and authorised, eye-drops in the form of a suspension comply with the following test : introduce a suitable quantity of the suspension into a counting cell or with a micropipette onto a slide, as appropriate, and scan under a microscope an area corresponding to 10 µg of the solid phase. For practical reasons, it is recommended that the whole sample is first scanned at low magnification (e.g. × 50) and particles greater than 25 µm are identified. These larger particles can then be measured at a larger magnification (e.g. × 200 to × 500). For each 10 µg of solid active substance, not more than 20 particles have a maximum dimension greater than 25 µm, and not more than 2 of these particles have a maximum dimension greater than 50 µm. None of the particles has a maximum dimension greater than 90 µm. LABELLING The label states for multidose containers, the period after opening the container after which the contents must not be used. This period does not exceed 4 weeks, unless otherwise justified and authorised.

Eye lotions

DEFINITION Eye lotions are sterile aqueous solutions intended for use in washing or bathing the eye or for impregnating eye dressings. Eye lotions may contain excipients, for example to adjust the tonicity or the viscosity of the preparation or to adjust or stabilise the pH. These substances do not adversely affect Eye-drops the intended action or, at the concentrations used, cause undue local irritation. DEFINITION Eye-drops are sterile aqueous or oily solutions or suspensions Eye lotions supplied in multidose containers contain a suitable antimicrobial preservative in appropriate of one or more active substances intended for instillation concentration except when the preparation itself has into the eye. adequate antimicrobial properties. The antimicrobial Eye-drops may contain excipients, for example, to adjust preservative chosen is compatible with the other ingredients the tonicity or the viscosity of the preparation, to adjust of the preparation and remains effective throughout the or stabilise the pH, to increase the solubility of the active period of time during which the eye lotions are in use. substance, or to stabilise the preparation. These substances If eye lotions are prescribed without an antimicrobial do not adversely affect the intended medicinal action or, at preservative, they are supplied in single-dose containers. Eye the concentrations used, cause undue local irritation. lotions intended for use in surgical procedures or in first-aid Aqueous preparations supplied in multidose containers treatment do not contain an antimicrobial preservative and contain a suitable antimicrobial preservative in appropriate are supplied in single-dose containers. concentration except when the preparation itself has Eye lotions examined under suitable conditions of visibility, adequate antimicrobial properties. The antimicrobial are practically clear and practically free from particles. preservative chosen must be compatible with the other The containers for multidose preparations do not contain ingredients of the preparation and must remain effective more than 200 ml of eye lotion, unless otherwise justified throughout the period of time during which eye-drops are and authorised. in use. LABELLING If eye-drops are prescribed without antimicrobial preservatives they are supplied wherever possible in The label states : single-dose containers. Eye-drops intended for use in surgical — where applicable, that the contents are to be used on one procedures do not contain antimicrobial preservatives and occasion only, are supplied in single-dose containers. — for multidose preparations, the period after opening the Eye-drops that are solutions, examined under suitable container after which the contents must not be used. conditions of visibility, are practically clear and practically This period does not exceed 4 weeks, unless otherwise free from particles. justified and authorised. Eye-drops that are suspensions may show a sediment that is readily redispersed on shaking to give a suspension which Powders for eye-drops and powders for eye remains sufficiently stable to enable the correct dose to be lotions delivered. Multidose preparations are supplied in containers that allow DEFINITION successive drops of the preparation to be administered. The Powders for the preparation of eye-drops and eye lotions are containers contain at most 10 ml of the preparation, unless supplied in a dry, sterile form to be dissolved or suspended otherwise justified and authorised. in an appropriate liquid vehicle at the time of administration. LABELLING The label states the name of any added antimicrobial preservative.


603

Foams, medicated


They may contain excipients to facilitate dissolution or dispersion, to prevent caking, to adjust the tonicity, to adjust or stabilise the pH or to stabilise the preparation. After dissolution or suspension in the prescribed liquid, they comply with the requirements for eye-drops or eye lotions, as appropriate. TESTS Uniformity of content (2.9.6). Unless otherwise prescribed or justified and authorised, single-dose powders for eye-drops and eye lotions with a content of active substance less then 2 mg or less than 2 per cent of the total mass comply with test B for uniformity of content of single-dose preparations. If the preparation has more than one active substance, the requirement applies only to those substances which correspond to the above condition. Uniformity of mass (2.9.5). Single-dose powders for eye-drops and eye lotions comply with the test for uniformity of mass of single-dose preparations. If the test for uniformity of content is prescribed for all the active substances, the test for uniformity of mass is not required.

Semi-solid eye preparations

PRODUCTION In the manufacture of ophthalmic inserts, means must be taken to ensure a suitable dissolution behaviour. TESTS Uniformity of content (2.9.6). Ophthalmic inserts comply, where applicable, with test A for uniformity of content. LABELLING The label states : — where applicable, the total quantity of active substance per insert, — where applicable, the dose released per unit time. 01/2005:1105

FOAMS, MEDICATED

DEFINITION Semi-solid eye preparations are sterile ointments, creams or gels intended for application to the conjunctiva. They contain one or more active substances dissolved or dispersed in a suitable basis. They have a homogeneous appearance. Semi-solid eye preparations comply with the requirements of the monograph on Semi-solid preparations for cutaneous application (0132). The basis is non-irritant to the conjunctiva. Semi-solid eye preparations are packed in small, sterilised collapsible tubes fitted or provided with a cannula and having a content of not more than 5 g of the preparation. The tubes must be well-closed to prevent microbial contamination. Semi-solid eye preparations may also be packed in suitably designed single-dose containers. The containers, or the nozzles of tubes, are of such a shape as to facilitate administration without contamination. Tubes are tamper-proof. TESTS Particle size. Semi-solid eye preparations containing dispersed solid particles comply with the following test : spread gently a quantity of the preparation corresponding to at least 10 µg of solid active substance as a thin layer. Scan under a microscope the whole area of the sample. For practical reasons, it is recommended that the whole sample is first scanned at a small magnification (e.g. × 50) and particles greater than 25 µm are identified. These larger particles can then be measured at a larger magnification (e.g. × 200 to × 500). For each 10 µg of solid active substance, not more than 20 particles have a maximum dimension greater than 25 µm, and not more than 2 of these particles have a maximum dimension greater than 50 µm. None of the particles has a maximum dimension greater than 90 µm.

Ophthalmic inserts DEFINITION Ophthalmic inserts are sterile, solid or semi-solid preparations of suitable size and shape, designed to be inserted in the conjunctival sac, to produce an ocular effect. They generally consist of a reservoir of active substance embedded in a matrix or bounded by a rate-controlling 604

membrane. The active substance, which is more or less soluble in physiological fluids, is released over a determined period of time. Ophthalmic inserts are individually distributed into sterile containers.

Musci medicati Additional requirements for medicated foams may be found, where appropriate, in other general monographs, for example on Rectal preparations (1145), Vaginal preparations (1164) and Liquid preparations for cutaneous application (0927). DEFINITION Medicated foams are preparations consisting of large volumes of gas dispersed in a liquid generally containing one or more active substances, a surfactant ensuring their formation and various other excipients. Medicated foams are usually intended for application to the skin or mucous membranes. Medicated foams are usually formed at the time of administration from a liquid preparation in a pressurised container. The container is equipped with a device consisting of a valve and a push button suitable for the delivery of the foam. Medicated foams intended for use on severely injured skin and on large open wounds are sterile. Medicated foams supplied in pressurised containers comply with the requirements of the monograph on Pressurised pharmaceutical preparations (0523). PRODUCTION Sterile medicated foams are prepared using materials and methods designed to ensure sterility and to avoid the introduction of contaminants and the growth of micro-organisms ; recommendations on this aspect are provided in the text on Methods of preparation of sterile products (5.1.1). TESTS Relative foam density. Maintain the container at about 25 °C for at least 24 h. Taking care not to warm the container, fit a rigid tube 70 mm to 100 mm long and about 1 mm in internal diameter onto the push button. Shake the container to homogenise the liquid phase of the contents and dispense 5 ml to 10 ml of foam to waste. Tare a flat-bottomed dish with a volume of about 60 ml and about 35 mm high. Place the end of the rigid tube attached to the push button in the corner of the dish, press the push button and fill the dish uniformly, using a circular motion. After the foam has


Granules


completely expanded, level off by removing the excess foam Sterility (2.6.1). When the label indicates that the with a slide. Weigh. Determine the mass of the same volume preparation is sterile, it complies with the test for sterility. of water R by filling the same dish with water R. LABELLING The relative foam density is equivalent to the ratio : The label states, where applicable, that the preparation is sterile. m

=

mass of the test sample of foam, in grams,

e

=

mass of same volume of water R, in grams.

Carry out three measurements. None of the individual values deviate by more than 20 per cent from the mean value. Duration of expansion. The apparatus (Figure 1105.-1) consists of a 50 ml burette, 15 mm in internal diameter, with 0.1 ml graduations and fitted with a 4 mm single bore stopcock. The graduation corresponding to 30 ml is at least 210 mm from the axis of the stopcock. The lower part of the burette is connected by means of a plastic tube not longer than 50 mm and 4 mm in internal diameter to the foam-generating container equipped with a push button fitted to this connection. Maintain the container at about 25 °C for at least 24 h. Shake the container, taking care not to warm it, to homogenise the liquid phase of the contents and dispense 5 ml to 10 ml of the foam to waste. Connect the push button to the outlet of the burette. Press the button and introduce about 30 ml of foam in a single delivery. Close the stopcock and at the same time start the chronometer and read the volume of foam in the burette. Every 10 s read the growing volume until the maximum volume is reached. Carry out three measurements. None of the times needed to obtain the maximum volume is more than 5 min.

50

40

30

20

10

0

Figure 1105.-1. – Apparatus for the determination of the duration of expansion General Notices (1) apply to all monographs and other texts

01/2005:0499

GRANULES Granulata Requirements for granules to be used for the preparation of oral solutions or suspensions are given in the monograph on Liquid preparations for oral use (0672). Where justified and authorised, the requirements of this monograph do not apply to granules for veterinary use. DEFINITION Granules are preparations consisting of solid, dry aggregates of powder particles sufficiently resistant to withstand handling. They are intended for oral administration. Some are swallowed as such, some are chewed and some are dissolved or dispersed in water or another suitable liquid before being administered. Granules contain one or more active substances with or without excipients and, if necessary, colouring matter authorised by the competent authority and flavouring substances. Granules are presented as single-dose or multidose preparations. Each dose of a multidose preparation is administered by means of a device suitable for measuring the quantity prescribed. For single-dose granules, each dose is enclosed in an individual container, for example a sachet or a vial. Where applicable, containers for granules comply with the requirements of Materials used for the manufacture of containers (3.1 and subsections) and Containers (3.2 and subsections). Several categories of granules may be distinguished : — effervescent granules, — coated granules, — gastro-resistant granules, — modified-release granules. PRODUCTION In the manufacture, packaging, storage and distribution of granules, suitable means are taken to ensure their microbial quality ; recommendations on this aspect are provided in the text on Microbiological quality of pharmaceutical preparations (5.1.4). TESTS Uniformity of content (2.9.6). Unless otherwise prescribed or justified and authorised, single-dose granules with a content of active substance less than 2 mg or less than 2 per cent of the total mass comply with test B for uniformity of content of single-dose preparations. If the preparation has more than one active substance, the requirement applies only to those substances which correspond to the above conditions. Uniformity of mass (2.9.5). Single-dose granules except for coated granules comply with the test for uniformity of mass of single-dose preparations. If the test for uniformity of content is prescribed for all the active substances, the test for uniformity of mass is not required. 605

Intramammary preparations for veterinary use


Gastro-resistant granules

Uniformity of mass of delivered doses from multidose containers (2.9.27). Granules supplied in multidose containers comply with the test. STORAGE If the preparation contains volatile ingredients or the contents have to be protected, store in an airtight container.

Effervescent granules DEFINITION Effervescent granules are uncoated granules generally containing acid substances and carbonates or hydrogen carbonates which react rapidly in the presence of water to release carbon dioxide. They are intended to be dissolved or dispersed in water before administration. TESTS Disintegration. Place one dose of the effervescent granules in a beaker containing 200 ml of water R at 15-25 °C ; numerous bubbles of gas are evolved. When the evolution of gas around the individual grains ceases, the granules have disintegrated, being either dissolved or dispersed in the water. Repeat the operation on 5 other doses. The preparation complies with the test if each of the 6 doses used disintegrates within 5 min. STORAGE In an airtight container.

DEFINITION Gastro-resistant granules are delayed-release granules that are intended to resist the gastric fluid and to release the active substance(s) in the intestinal fluid. These properties are achieved by covering the granules with a gastro-resistant material (enteric-coated granules) or by other suitable means. PRODUCTION A suitable test is carried out to demonstrate the appropriate release of the active substance(s). TESTS Dissolution. Carry out a suitable test to demonstrate the appropriate release of the active substance(s), for example the test described in Dissolution test for solid dosage forms (2.9.3). 01/2005:0945

INTRAMAMMARY PREPARATIONS FOR VETERINARY USE Praeparationes intramammariae ad usum veterinarium

DEFINITION Intramammary preparations for veterinary use are sterile preparations intended for introduction into the mammary Coated granules gland via the teat canal. There are two main categories : DEFINITION those intended for administration to lactating animals, and those intended for administration to animals at the end of Coated granules are usually multidose preparations and lactation or to non-lactating animals for the treatment or consist of granules coated with one or more layers of prevention of infection. mixtures of various excipients. Intramammary preparations for veterinary use are solutions, PRODUCTION emulsions or suspensions or semi-solid preparations The substances used as coatings are usually applied as a containing one or more active substances in a suitable solution or suspension in conditions in which evaporation vehicle. They may contain excipients such as stabilising, of the vehicle occurs. emulsifying, suspending and thickening agents. Suspensions may show a sediment which is readily dispersed on shaking. TESTS Emulsions may show evidence of phase separation but are Dissolution. A suitable test may be carried out to readily redispersed on shaking. demonstrate the appropriate release of the active Unless otherwise justified and authorised, intramammary substance(s), for example one of the tests described in preparations for veterinary use are supplied in containers for Dissolution test for solid dosage forms (2.9.3). use on one occasion only for introduction in a single teat canal of an animal. Modified-release granules If supplied in multidose containers, aqueous preparations contain a suitable antimicrobial preservative at a suitable DEFINITION concentration, except where the preparation itself has Modified-release granules are coated or uncoated granules adequate antimicrobial properties. Precautions for which contain special excipients or which are prepared by administration and for storage between administrations special procedures, or both, designed to modify the rate, the must be taken. place or the time at which the active substance or substances Where applicable, containers for intramammary preparations are released. for veterinary use comply with the requirements of Materials Modified-release granules include prolonged-release granules used for the manufacture of containers (3.1 and subsections) and delayed-release granules. and Containers (3.2 and subsections). PRODUCTION A suitable test is carried out to demonstrate the appropriate release of the active substance(s). TESTS Dissolution. Carry out a suitable test to demonstrate the appropriate release of the active substance(s), for example the test described in Dissolution test for solid dosage forms (2.9.3). 606

PRODUCTION During the development of a intramammary preparation for veterinary use, the formulation for which contains an antimicrobial preservative, the effectiveness of the chosen preservative shall be demonstrated to the satisfaction of the competent authority. A suitable test method together with criteria for judging the preservative properties of the formulation are provided in the text on Efficacy of antimicrobial preservation (5.1.3).



Intramammary preparations for veterinary use are prepared using materials and methods designed to ensure sterility and to avoid the introduction of contaminants and the growth of micro-organisms ; recommendations on this aspect are provided in the text on Methods of preparation of sterile products (5.1.1). In the manufacture of intramammary preparations for veterinary use containing dispersed particles, measures are taken to ensure a suitable and controlled particle size with regard to the intended use. TESTS Deliverable mass or volume. Squeeze out as much as possible of the contents of ten containers according to the instructions on the label. The mean mass or volume does not differ by more than 10 per cent from the nominal mass or volume. Sterility (2.6.1). Intramammary preparations for veterinary use comply with the test for sterility ; use the technique of membrane filtration or, in justified cases, direct inoculation of the culture media. Squeeze out the contents of ten containers and mix thoroughly. For each medium, use 0.5 g to 1 g (or 0.5 ml to 1 ml as appropriate) taken from the mixed sample. STORAGE Store in a sterile, airtight, tamper-proof container.

Liquid preparations for cutaneous application

PRODUCTION For continuous release, the intraruminal device is designed to release the active substance(s) at a defined rate over a defined period of time. This may be achieved by erosion, corrosion, diffusion, osmotic pressure or any other suitable chemical, physical or physico-chemical means. For pulsatile-release, the intraruminal device is designed to release a specific quantity of active substance(s) at one or several defined intermediate times. This may be achieved by corrosion by ruminal fluids of the metallic elements of the intraruminal device which leads to sequential release of the constituent units which are usually in the form of tablets. In the manufacture of intraruminal devices, means are taken to ensure an appropriate release of the active substance(s). In the manufacture, packaging, storage and distribution of intraruminal devices, suitable means are taken to ensure their microbial quality ; recommendations on this aspect are provided in the text on Microbiological quality of pharmaceutical preparations (5.1.4). TESTS Uniformity of content (2.9.6). Unless otherwise justified and authorised, constituent tablet units of intraruminal devices in which the active substances are present at levels less than 2 mg or less than 2 per cent of the total mass comply with test A for uniformity of content of single-dose preparations. If the preparation contains more than one active substance, the requirement applies only to those substances which correspond to the above conditions. Uniformity of mass (2.9.5). Unless otherwise justified and authorised, the constituent tablet units of intraruminal devices comply with the test for uniformity of mass. If the test for uniformity of content is prescribed for all active substances, the test for uniformity of mass is not required.

LABELLING The label states : — the name of the active substance(s) and the mass or number of International Units of the active substance(s) that may be delivered from the container using normal technique, — whether the preparation is intended for use in a lactating LABELLING animal or a non-lactating animal, The label states : — in the case of multidose containers, the name of any — for continuous-release devices, the dose released per unit added antimicrobial preservative. time, — for pulsatile-release devices, the dose released at specified times. 01/2005:1228

INTRARUMINAL DEVICES Praeparationes intraruminales The requirements of this monograph do not apply to preparations (sometimes known as boluses), such as large conventional tablets, capsules or moulded dosage forms which give immediate or prolonged release of the active substance(s). Such preparations comply with the relevant parts of the monographs on Capsules (0016) or Tablets (0478). DEFINITION Intraruminal devices are solid preparations each containing one or more active substances. They are intended for oral administration to ruminant animals and are designed to be retained in the rumen to deliver the active substance(s) in a continuous or pulsatile manner. The period of release of the active substance(s) may vary from days to weeks according to the nature of the formulation and/or the delivery device. Intraruminal devices may be administered using a balling gun. Some intraruminal devices are intended to float on the surface of the ruminal fluid while others are intended to remain on the floor of the rumen or reticulum. Each device has a density appropriate for its intended purpose. General Notices (1) apply to all monographs and other texts

01/2005:0927

LIQUID PREPARATIONS FOR CUTANEOUS APPLICATION Praeparationes liquidae ad usum dermicum Where justified and authorised, the requirements of this monograph do not apply to preparations intended for systemic and veterinary use. DEFINITION Liquid preparations for cutaneous application are preparations of a variety of viscosities intended for local or transdermal delivery of active ingredients. They are solutions, emulsions or suspensions which may contain one or more active substances in a suitable vehicle. They may contain suitable antimicrobial preservatives, antioxidants and other excipients such as stabilisers, emulsifiers and thickeners. Emulsions may show evidence of phase separation but are readily redispersed on shaking. Suspensions may show a sediment which is readily dispersed on shaking to give a suspension which is sufficiently stable to enable a homogeneous preparation to be delivered. 607

Liquid preparations for oral use


Where applicable, containers for liquid preparations for cutaneous application comply with the requirements of Materials used for the manufacture of containers (3.1 and subsections) and Containers (3.2 and subsections). When liquid preparations for cutaneous application are dispensed in pressurised containers, the containers comply with the requirements of the monograph on Pressurised pharmaceutical preparations (0523). Preparations specifically intended for use on severely injured skin are sterile. Several categories of liquid preparations for cutaneous application may be distinguished, for example : — shampoos, — cutaneous foams. PRODUCTION During the development of a liquid preparation for cutaneous application, the formulation for which contains an antimicrobial preservative, the effectiveness of the chosen preservative shall be demonstrated to the satisfaction of the competent authority. A suitable test method together with criteria for judging the preservative properties of the formulation are provided in the text on Efficacy of antimicrobial preservation (5.1.3). In the manufacture, packaging, storage and distribution of liquid preparations for cutaneous application, suitable means are taken to ensure their microbial quality ; recommendations on this aspect are provided in the text on Microbiological quality of pharmaceutical preparations (5.1.4). Sterile liquid preparations for cutaneous application are prepared using materials and methods designed to ensure sterility and to avoid the introduction of contaminants and the growth of micro-organisms ; recommendations on this aspect are provided in the text on Methods of preparation of sterile products (5.1.1). In the manufacture of liquid preparations for cutaneous application containing dispersed particles, measures are taken to ensure a suitable and controlled particle size with regard to the intended use. TESTS Deliverable mass or volume (2.9.28). Liquid preparations for cutaneous application supplied in single-dose containers comply with the test. Sterility (2.6.1). Where the label indicates that the preparation is sterile, it complies with the test for sterility. STORAGE If the preparation is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states : — the name of any added antimicrobial preservative, — where applicable, that the preparation is sterile.

Cutaneous foams DEFINITION Cutaneous foams comply with the requirements of the monograph on Medicated foams (1105). 01/2005:0672

LIQUID PREPARATIONS FOR ORAL USE Praeparationes liquidae peroraliae Where justified and authorised, the requirements of this monograph do not apply to liquid preparations for oral use intended for veterinary use. DEFINITION Liquid preparations for oral use are usually solutions, emulsions or suspensions containing one or more active substances in a suitable vehicle ; they may, however, consist of liquid active substances used as such (oral liquids). Some preparations for oral use are prepared by dilution of concentrated liquid preparations, or from powders or granules for the preparation of oral solutions or suspensions, for oral drops or for syrups, using a suitable vehicle. The vehicle for any preparations for oral use is chosen having regard to the nature of the active substance(s) and to provide organoleptic characteristics appropriate to the intended use of the preparation. Liquid preparations for oral use may contain suitable antimicrobial preservatives, antioxidants and other excipients such as dispersing, suspending, thickening, emulsifying, buffering, wetting, solubilising, stabilising, flavouring and sweetening agents and colouring matter, authorised by the competent authority. Emulsions may show evidence of phase separation but are readily redispersed on shaking. Suspensions may show a sediment which is readily dispersed on shaking to give a suspension which remains sufficiently stable to enable the correct dose to be delivered. Where applicable, containers for liquid preparations for oral use comply with the requirements of Materials used for the manufacture of containers (3.1 and subsections) and Containers (3.2 and subsections). Several categories of preparations may be distinguished : — oral solutions, emulsions and suspensions, — powders and granules for oral solutions and suspensions, — oral drops, — powders for oral drops, — syrups, — powders and granules for syrups.

PRODUCTION During the development of a preparation for oral use, the formulation for which contains an antimicrobial preservative, the effectiveness of the chosen preservative shall be Shampoos demonstrated to the satisfaction of the competent authority. A suitable test method together with criteria for judging the DEFINITION preservative properties of the formulation are provided in Shampoos are liquid or, occasionally semi-solid preparations the text on Efficacy of antimicrobial preservation (5.1.3). intended for application to the scalp and subsequent washing In the manufacturing, packaging, storage and distribution away with water. Upon rubbing with water they usually form of liquid preparations for oral use, suitable means are taken a foam. to ensure their microbial quality ; recommendations on this aspect are provided in the text on Microbiological quality of They are emulsions, suspensions or solutions. Shampoos pharmaceutical preparations (5.1.4). normally contain surface active agents. 608



Liquid preparations for oral use

TESTS Uniformity of content (2.9.6). Unless otherwise prescribed or justified and authorised, single-dose powders and single-dose granules with a content of active substance less than 2 mg or less than 2 per cent of the total mass TESTS comply with test B for uniformity of content of single-dose Uniformity of content (2.9.6). Unless otherwise prescribed preparations. If the preparation has more than one active or justified and authorised, single-dose preparations that are substance, the requirement applies only to those substances suspensions comply with the following test. After shaking, that correspond to the above conditions. empty each container as completely as possible and carry Uniformity of mass (2.9.5). Single-dose powders and out the test on the individual contents. They comply with single-dose granules comply with the test for uniformity of test B for uniformity of content of single-dose preparations. mass of single-dose preparations. If the test for uniformity of Uniformity of mass. Single-dose preparations that are content is prescribed for all the active substances, the test solutions or emulsions comply with the following test : for uniformity of mass is not required. weigh individually the contents of 20 containers, emptied LABELLING as completely as possible, and determine the average mass. Not more than 2 of the individual masses deviate by more The label states : than 10 per cent from the average mass and none deviates — the method of preparation of the solution or suspension, by more than 20 per cent. — the conditions and the duration of storage after Dose and uniformity of dose of oral drops. Into a suitable, constitution. graduated cylinder, introduce by means of the dropping device the number of drops usually prescribed for one dose Oral drops or introduce by means of the measuring device, the usually prescribed quantity. The dropping speed does not exceed DEFINITION 2 drops per second. Weigh the liquid, repeat the addition, Oral drops are solutions, emulsions or suspensions which weigh again and carry on repeating the addition and are administered in small volumes such as drops by the weighing until a total of 10 masses are obtained. No single means of a suitable device. mass deviates by more than 10 per cent from the average mass. The total of 10 masses does not differ by more than LABELLING 15 per cent from the nominal mass of 10 doses. If necessary, The label states the number of drops per millilitre of measure the total volume of 10 doses. The volume does not preparation or per gram of preparation if the dose is differ by more than 15 per cent from the nominal volume of measured in drops. 10 doses. Deliverable mass or volume (2.9.28). Liquid preparations Powders for oral drops for oral use supplied in single-dose containers comply with the test. DEFINITION Powders for the preparation of oral drops generally conform Uniformity of mass of delivered doses from multidose to the definition of Oral powders (1165). They may contain containers (2.9.27). Liquid preparations for oral use excipients to facilitate dissolution or suspension in the supplied in multidose containers comply with the test. prescribed liquid or to prevent caking. LABELLING After dissolution or suspension, they comply with the The label states the name of any added antimicrobial requirements for oral drops. preservative. TESTS Uniformity of content (2.9.6). Unless otherwise prescribed Oral solutions, emulsions and suspensions or justified and authorised, single-dose powders for oral drops with a content of active substance less than 2 mg DEFINITION or less than 2 per cent of the total mass comply with Oral solutions, emulsions and suspensions are supplied test B for uniformity of content of single-dose preparations. in single-dose or multi-dose containers. Each dose from a If the preparation has more than one active substance, multi-dose container is administered by means of a device suitable for measuring the prescribed volume. The device is the requirement applies only to those substances that correspond to the above conditions. usually a spoon or a cup for volumes of 5 ml or multiples Uniformity of mass (2.9.5). Single-dose powders for thereof or an oral syringe for other volumes. oral drops comply with the test for uniformity of mass single-dose preparations. If the test for uniformity of Powders and granules for oral solutions of content is prescribed for all the active substances, the test and suspensions for uniformity of mass is not required. In the manufacture of liquid preparations for oral use containing dispersed particles, measures are taken to ensure a suitable and controlled particle size with regard to the intended use.

DEFINITION Powders and granules for the preparation of oral solutions or suspensions generally conform to the definitions in the monographs on Oral powders (1165) or Granules (0499) as appropriate. They may contain excipients in particular to facilitate dispersion or dissolution and to prevent caking. After dissolution or suspension, they comply with the requirements for oral solutions or oral suspensions, as appropriate. General Notices (1) apply to all monographs and other texts

Syrups DEFINITION Syrups are aqueous preparations characterised by sweet taste and a viscous consistency. They may contain sucrose at a concentration of at least 45 per cent m/m. The sweet taste can also be obtained by using other polyols or sweetening agents. Syrups usually contain aromatic or other flavouring agents. Each dose from a multi-dose container is 609

Nasal preparations


administered by means of a device suitable for measuring the prescribed volume. The device is usually a spoon or a cup for volumes of 5 ml or multiples thereof. LABELLING The label states the name and concentration of the polyol or sweetening agent.

Powders and granules for syrups DEFINITION Powders and granules for syrups generally conform to the definitions in the monograph on Oral powders (1165) or Granules (0499). They may contain excipients to facilitate dissolution. After dissolution, they comply with the requirements for syrups.

— — — —

nasal powders, semi-solid nasal preparations, nasal washes, nasal sticks.

PRODUCTION During the development of a nasal preparation, the formulation for which contains an antimicrobial preservative, the effectiveness of the chosen preservative shall be demonstrated to the satisfaction of the competent authority. A suitable test method together with criteria for judging the preservative properties of the formulation are provided in the text on Efficacy of antimicrobial preservation (5.1.3). In the manufacture, packaging, storage and distribution of nasal preparations, suitable means are taken to ensure their microbial quality ; recommendations on this aspect are provided in the text on Microbiological quality of pharmaceutical preparations (5.1.4). Sterile nasal preparations are prepared using materials and methods designed to ensure sterility and to avoid the introduction of contaminants and the growth of micro-organisms ; recommendations on this aspect are provided in the text on Methods of preparation of sterile products (5.1.1). In the manufacture of nasal preparations containing dispersed particles, measures are taken to ensure a suitable and controlled particle size with regard to the intended use.

TESTS Uniformity of content (2.9.6). Unless otherwise prescribed or justified and authorised, single-dose powders and granules for syrups with a content of active substance less than 2 mg or less than 2 per cent of the total mass comply with test B for uniformity of content of single-dose preparations. If the preparation has more than one active substance, the requirement applies only to those substances that correspond to the above conditions. Uniformity of mass (2.9.5). Single-dose powders and granules for syrups comply with the test for uniformity of TESTS mass of single-dose preparations. If the test for uniformity of Sterility (2.6.1). Where the label states that the preparation content is prescribed for all the active substances, the test is sterile, it complies with the test for sterility. for uniformity of mass is not required. STORAGE If the preparation is sterile, store in a sterile, airtight, 01/2005:0676 tamper-proof container.

NASAL PREPARATIONS Nasalia DEFINITION Nasal preparations are liquid, semi-solid or solid preparations intended for administration to the nasal cavities to obtain a systemic or local effect. They contain one or more active substances. Nasal preparations are as far as possible non-irritating and do not adversely affect the functions of the nasal mucosa and its cilia. Aqueous nasal preparations are usually isotonic and may contain excipients, for example, to adjust the viscosity of the preparation, to adjust or stabilise the pH, to increase the solubility of the active substance, or to stabilise the preparation. Nasal preparations are supplied in multidose or single-dose containers, provided, if necessary, with a suitable administration device which may be designed to avoid the introduction of contaminants. Unless otherwise justified and authorised, aqueous nasal preparations supplied in multidose containers contain a suitable antimicrobial preservative in appropriate concentration, except where the preparation itself has adequate antimicrobial properties. Where applicable, the containers comply with the requirements of Materials used for the manufacture of containers (3.1 and subsections) and Containers (3.2 and subsections). Several categories of nasal preparations may be distinguished : — nasal drops and liquid nasal sprays, 610

LABELLING The label states : — the name of any added antimicrobial preservative, — where applicable, that the preparation is sterile.

Nasal drops and liquid nasal sprays DEFINITION Nasal drops and liquid nasal sprays are solutions, emulsions or suspensions intended for instillation or spraying into the nasal cavities. Emulsions may show evidence of phase separation but are easily redispersed on shaking. Suspensions may show a sediment which is readily dispersed on shaking to give a suspension which remains sufficiently stable to enable the correct dose to be delivered. Nasal drops are usually supplied in multidose containers provided with a suitable applicator. Liquid nasal sprays are supplied in containers with atomising devices or in pressurised containers fitted with a suitable adapter and with or without a metering dose valve, which comply with the requirements of the monograph on Pressurised pharmaceutical preparations (0523). The size of droplets of the spray is such as to localise their deposition in the nasal cavity. TESTS Unless otherwise prescribed or justified and authorised, nasal drops supplied in single-dose containers and single doses of metered nasal sprays intended for systemic action, comply with the following tests.



Oromucosal preparations

Uniformity of mass. Nasal drops that are solutions comply with the following test : weigh individually the contents of ten containers emptied as completely as possible, and determine the average mass. Not more than two of the individual masses deviate by more than 10 per cent from the average mass and none deviates by more than 20 per cent. Metered-dose nasal sprays that are solutions comply with the following test : discharge once to waste. Wait for not less than 5 s and discharge again to waste. Repeat this procedure for a further three actuations. Weigh the mass of the container, discharge once to waste and weigh the remaining mass of the container. Calculate the difference between the two masses. Repeat the procedure for a further nine containers. They comply with the test if not more than two of the individual values deviate by more than 25 per cent from the average value and none deviates by more than 35 per cent. Uniformity of content (2.9.6). Nasal drops that are suspensions or emulsions comply with the following test : empty each container as completely as possible and carry out the test on the individual content. They comply with test B of uniformity of content. Uniformity of delivered dose. Metered-dose nasal sprays that are suspensions or emulsions comply with the following test. Use an apparatus capable of quantitatively retaining the dose leaving the actuator of the atomising device. Shake a container for 5 s and discharge once to waste. Wait for not less than 5 s, shake for 5 s and discharge again to waste. Repeat this procedure for a further three actuations. After 2 s, fire one dose of the metered-dose nasal spray into the collecting vessel by actuating the atomising device. Collect the contents of the collecting vessel by successive rinses. Determine the content of active substance in the combined rinses. Repeat the procedure for a further nine containers. Unless otherwise justified and authorised, the preparation complies with the test if not more than one of the individual contents is outside the limits of 75 per cent to 125 per cent and none is outside the limits of 65 per cent and 135 per cent of the average content. If two or three individual contents are outside the limits of 75 per cent to 125 per cent but within the limits of 65 per cent to 135 per cent, repeat the test for twenty more containers. The preparation complies with the test if not more than three individual contents of the thirty individual contents are outside the limits of 75 per cent to 125 per cent and none is outside the limits of 65 per cent to 135 per cent of the average content.

The containers are adapted to deliver the product to the site of application.


611

Nasal washes DEFINITION Nasal washes are generally aqueous isotonic solutions intended to cleanse the nasal cavities. Nasal washes intended for application to injured parts or prior to a surgical operation are sterile. TESTS Deliverable mass or volume (2.9.28). Nasal washes supplied in single-dose containers comply with the test.

Nasal sticks DEFINITION Nasal sticks comply with the monograph on Sticks (1154). 01/2005:1807

OROMUCOSAL PREPARATIONS Praeparationes buccales This monograph does not apply to dental preparations or to preparations such as chewable tablets (0478) , medicated chewing gums (1239), oral lyophilisates and other solid or semi-solid preparations that are intended to be chewed or dispersed in the saliva before being swallowed. Where justified and authorised, this monograph does not apply to preparations for veterinary use.

DEFINITION Oromucosal preparations are solid, semi-solid or liquid preparations, containing one or more active substances intended for administration to the oral cavity and/or the throat to obtain a local or systemic effect. Preparations intended for a local effect may be designed for application to a specific site within the oral cavity such as the gums (gingival preparations) or the throat (oropharyngeal preparations). Preparations intended for a systemic effect are designed to be absorbed primarily at one or more sites on the oral mucosa (e.g. sublingual preparations). Mucoadhesive preparations are intended to be retained in the oral cavity by adhesion to the mucosal epithelium and may modify systemic drug absorption at the site of application. For many oromucosal preparations, it is likely that some proportion of the active substance(s) will be swallowed and may be absorbed via the gastrointestinal tract. Oromucosal preparations may contain suitable antimicrobial Nasal powders preservatives and other excipients such as dispersing, DEFINITION suspending, thickening, emulsifying, buffering, wetting, Nasal powders are powders intended for insufflation into the solubilising, stabilising, flavouring and sweetening agents. Solid preparations may in addition contain glidants, nasal cavity by means of a suitable device. lubricants and excipients capable of modifying the release of They comply with the requirements of the monograph on the active substance(s). Powders for cutaneous application (1166). Where applicable, containers for oromucosal preparations The size of the particles is such as to localise their deposition comply with the requirements for Materials used for the in the nasal cavity and verified by adequate methods of manufacture of containers (3.1 and subsections) and particle-size determination. Containers (3.2 and subsections). Several categories of preparations for oromucosal use may Semi-solid nasal preparations be distinguished : — gargles, DEFINITION Semi-solid nasal preparations comply with the requirements — mouthwashes, of the monograph on Semi-solid preparations for cutaneous — gingival solutions, — oromucosal solutions and oromucosal suspensions, application (0132).



— semi-solid oromucosal preparations (including for example gingival gel, gingival paste, oromucosal gel, oromucosal paste), — oromucosal drops, oromucosal sprays and sublingual sprays (including oropharyngeal sprays), — lozenges and pastilles, — compressed lozenges, — sublingual tablets and buccal tablets, — oromucosal capsules, — mucoadhesive preparations. PRODUCTION During the development of an oromucosal preparation containing an antimicrobial preservative, the effectiveness of the chosen preservative shall be demonstrated to the satisfaction of the competent authority. A suitable test method together with the criteria for judging the preservative properties of the formulation are provided in 5.1.3 Efficacy of antimicrobial preservation. In the manufacture, packaging, storage and distribution of oromucosal preparations, suitable means are taken to ensure their microbiological quality ; recommendations on this aspect are provided in the text on Microbiological quality of pharmaceutical preparations (5.1.4). In the manufacture of semi-solid and liquid oromucosal preparations containing dispersed particles, measures are taken to ensure a suitable and controlled particle size with regard to the intended use.

They are supplied as ready-to-use solutions or concentrated solutions to be diluted. They may also be prepared from powders or tablets to be dissolved in water before use. Mouthwashes may contain excipients to adjust the pH which, as far as possible, is neutral.

Gingival solutions DEFINITION Gingival solutions are intended for administration to the gingivae by means of a suitable applicator.

Oromucosal solutions and oromucosal suspensions DEFINITION Oromucosal solutions and oromucosal suspensions are liquid preparations intended for administration to the oral cavity by means of a suitable applicator. Oromucosal suspensions may show a sediment which is readily dispersible on shaking to give a suspension which remains sufficiently stable to enable the correct dose to be delivered.

Semi-solid oromucosal preparations

DEFINITION Semi-solid oromucosal preparations are hydrophilic gels or pastes intended for administration to the oral cavity or to a TESTS specific part of the oral cavity such as the gingivae (gingival Uniformity of content (2.9.6). Unless otherwise prescribed gel, gingival paste). They may be provided as single-dose or justified and authorised, single-dose preparations with a preparations. content of active substance less than 2 mg or less than 2 per Semi-solid oromucosal preparations comply with the cent of the total mass comply with test A (compressed and moulded dosage forms) or test B (capsules) for the uniformity requirements of the monograph on Semi-solid preparations for cutaneous use (0132). of content of single-dose preparations. If the preparation contains more than one active substance, this requirement applies only to those substances that correspond to the Oromucosal drops, oromucosal sprays and above conditions. sublingual sprays Uniformity of mass (2.9.5). Solid single-dose preparations DEFINITION comply with the test for uniformity of mass. If the test for the uniformity of content is prescribed, or justified and Oromucosal drops, oromucosal sprays and sublingual sprays authorised for all active substances, the test for uniformity are solutions, emulsions or suspensions intended for local or of mass is not required. systemic effect. They are applied by instillation or spraying into the oral cavity or onto a specific part of the oral cavity LABELLING such as spraying under the tongue (sublingual spray) or into The label states the name of any added antimicrobial the throat (oropharyngeal spray). preservative. Emulsions may show evidence of phase separation but are readily redispersed on shaking. Suspensions may show a Gargles sediment which is readily dispersed on shaking to give a suspension which remains sufficiently stable to enable the DEFINITION correct dose to be delivered. Gargles are aqueous solutions intended for gargling to Liquid oromucosal sprays are supplied in containers with obtain a local effect. They are not to be swallowed. They are atomising devices or in pressurised containers having a supplied as ready-to-use solutions or concentrated solutions suitable adaptor, with or without a metering dose valve, to be diluted. They may also be prepared from powders or which comply with the requirements of the monograph on tablets to be dissolved in water before use. Pressurised pharmaceutical preparations (0523). Gargles may contain excipients to adjust the pH which, as far The size of the droplets of the spray is such as to localise as possible, is neutral. their deposition in the oral cavity or the throat as intended.

Mouthwashes

TESTS Unless otherwise prescribed or justified and authorised, DEFINITION oromucosal drops supplied in single-dose containers, single-doses of metered oromucosal sprays and sublingual Mouthwashes are aqueous solutions intended for use in contact with the mucous membrane of the oral cavity, usually sprays intended for systemic action comply with the following tests. after dilution with water. They are not to be swallowed. 612



Uniformity of mass. Oromucosal drops that are solutions comply with the following test : determine the individual masses of the contents of 10 containers emptied as completely as possible, and calculate the average mass. Not more than 2 of the individual masses deviate by more than 10 per cent from the average mass and none deviates by more than 20 per cent. Metered-dose oromucosal sprays and sublingual sprays that are solutions comply with the following test : discharge once to waste. Wait for not less than 5 s and discharge again to waste. Repeat this procedure for a further 3 actuations. Weigh the mass of the container, discharge once to waste and weigh the remaining mass of the container. Calculate the difference between the 2 masses. Repeat the procedure for a further 9 containers. The preparation complies with the test if not more than 2 of the individual masses deviate by more than 25 per cent from the average value and none deviates by more than 35 per cent. Uniformity of content (2.9.6). Oromucosal drops that are suspensions or emulsions comply with the following test : empty each container as completely as possible and carry out the test on the individual contents. They comply with test B of uniformity of content. Uniformity of delivered dose. Metered-dose oromuscal sprays and sublingual sprays that are suspensions or emulsions comply with the following test : use an apparatus capable of quantitatively retaining the dose leaving the actuator of the atomising device. Shake a container for 5 s and discharge once to waste. Wait for not less than 5 s, shake for 5 s and discharge again to waste. Repeat this procedure for a further 3 actuations. After 2 s, fire one dose of the metered-dose oromucosal spray into the collecting vessel by actuating the atomising device. Collect the contents of the collecting vessel by successive rinses. Determine the content of active substance in the combined rinses. Repeat the procedure for a further 9 containers. Unless otherwise justified and authorised, the preparation complies with the test if not more than one of the individual contents is outside the limits of 75 per cent to 125 per cent and none is outside the limits of 65 per cent to 135 per cent of the average content. If 2 or 3 individual contents are outside the limits of 75 per cent to 125 per cent but within the limits of 65 per cent to 135 per cent, repeat the test for 20 more containers. The preparation complies with the test if not more than 3 individual contents of the 30 contents are outside the limits of 75 per cent to 125 per cent and none is outside the limits of 65 per cent to 135 per cent of the average content.

Lozenges and pastilles DEFINITION


Compressed lozenges DEFINITION Compressed lozenges are solid, single-dose preparations intended to be sucked to obtain a local or systemic effect. They are prepared by compression and are often rhomboid in shape. Compressed lozenges conform with the general definition of tablets. PRODUCTION In the manufacture of compressed lozenges, means are taken to ensure that they possess a suitable mechanical strength to resist handling without crumbling or breaking. This may be demonstrated by examining the Friability of uncoated tablets (2.9.7) and the Resistance to crushing of tablets (2.9.8). TESTS Dissolution. For compressed lozenges intended for a systemic effect, a suitable test is carried out to demonstrate the appropriate release of the active substance(s).

Sublingual tablets and buccal tablets DEFINITION Sublingual tablets and buccal tablets are solid, single-dose preparations to be applied under the tongue or to the buccal cavity, respectively, to obtain a systemic effect. They are prepared by compression of mixtures of powders or granulations into tablets with a shape suited for the intended use. Sublingual tablets and buccal tablets conform to the general definition of tablets. PRODUCTION In the manufacture of sublingual tablets and buccal tablets, means are taken to ensure that they possess suitable mechanical strength to resist handling without crumbling or breaking. This may be demonstrated by examining the Friability of uncoated tablets (2.9.7) and the Resistance to crushing of tablets (2.9.8). TESTS Dissolution. Unless otherwise justified and authorised, a suitable test is carried out to demonstrate the appropriate release of the active substance(s).

Oromucosal capsules DEFINITION Oromucosal capsules are soft capsules to be chewed or sucked.

Mucoadhesive preparations

DEFINITION Mucoadhesive preparations contain one or more active substances intended for systemic absorption through the buccal mucosa over a prolonged period of time. They may be supplied as mucoadhesive buccal tablets or as other mucoadhesive solid or semi-solid preparations. Mucoadhesive buccal tablets are prepared by compression of mono- or multi-layered tablets. They usually contain Lozenges are hard preparations prepared by moulding. Pastilles are soft, flexible preparations prepared by moulding hydrophilic polymers, which on wetting with the saliva produce a flexible hydrogel that adheres to the buccal of mixtures containing natural or synthetic polymers or mucosa. gums and sweeteners. Lozenges and pastilles are solid, single-dose preparations intended to be sucked to obtain, usually, a local effect in the oral cavity and the throat. They contain one or more active substances, usually in a flavoured and sweetened base, and are intended to dissolve or disintegrate slowly in the mouth when sucked.


613

Parenteral preparations


PRODUCTION During the development of a parenteral preparation, the formulation for which contains an antimicrobial preservative, the effectiveness of the chosen preservative shall be demonstrated to the satisfaction of the competent authority. A suitable test method together with criteria for judging the preservative properties of the formulation are provided under Efficacy of antimicrobial preservation (5.1.3). TESTS Parenteral preparations are prepared using materials Dissolution. Unless otherwise justified and authorised, a and methods designed to ensure sterility and to avoid suitable test is carried out to demonstrate the appropriate the introduction of contaminants and the growth of release of the active substance(s). micro-organisms ; recommendations on this aspect are provided in the text on Methods of preparation of sterile products (5.1.1). 01/2005:0520 Water used in the manufacture of parenteral preparations complies with the requirements of water for injections in bulk stated in the monograph on Water for injections (0169). PARENTERAL PREPARATIONS PRODUCTION In the manufacture of mucoadhesive buccal tablets, means are taken to ensure that they possess suitable mechanical strength to resist handling without crumbling or breaking. This may be demonstrated by examining the Friability of uncoated tablets (2.9.7) and the Resistance to crushing of tablets (2.9.8).

TESTS Particulate contamination : sub-visible particles (2.9.19). For preparations for human use, solutions for infusion or The requirements of this monograph do not necessarily solutions for injection supplied in containers with a nominal apply to products derived from human blood, to content of more than 100 ml comply with the test. immunological preparations, or radiopharmaceutical preparations. Special requirements may apply to For preparations for veterinary use, when supplied in preparations for veterinary use depending on the species containers with a nominal content of more than 100 ml of animal for which the preparation is intended. and when the content is equivalent to a dose of more than 1.4 ml per kilogram of body mass, solutions for infusion or DEFINITION solutions for injection comply with the test for particulate Parenteral preparations are sterile preparations intended for contamination : sub-visible particles. administration by injection, infusion or implantation into Products for which the label states that the product is to be the human or animal body. used with a final filter are exempt from these requirements. Parenteral preparations may require the use of excipients, Sterility (2.6.1). Parenteral preparations comply with the for example to make the preparation isotonic with blood, to test for sterility. adjust the pH, to increase solubility, to prevent deterioration of the active substances or to provide adequate antimicrobial STORAGE properties but not to adversely affect the intended medicinal Store in a sterile, airtight, tamper-proof container. action of the preparation or, at the concentrations used, to cause toxicity or undue local irritation. LABELLING Containers for parenteral preparations are made as far as The label states : possible from materials that are sufficiently transparent — the name and concentration of any added antimicrobial to permit the visual inspection of the contents, except for preservative, implants and in other justified and authorised cases. — where applicable, that the solution is to be used in Where applicable, the containers for parenteral preparations conjunction with a final filter, comply with the requirements for Materials used for the — where applicable, that the preparation is free from manufacture of containers (3.1 and subsections) and bacterial endotoxins or that it is apyrogenic. Containers (3.2 and subsections). Parenteral preparations are supplied in glass containers Injections (3.2.1) or in other containers such as plastic containers (3.2.2, 3.2.2.1 and 3.2.9) and prefilled syringes. The tightness DEFINITION of the container is ensured by suitable means. Closures Injections are sterile solutions, emulsions or suspensions. ensure a good seal, prevent the access of micro-organisms They are prepared by dissolving, emulsifying or suspending and other contaminants and usually permit the withdrawal of a part or the whole of the contents without removal of the the active substance(s) and any added excipients in Water for closure. The plastic materials or elastomers (3.2.9) of which injections (0169), in a suitable, sterile non-aqueous liquid or in a mixture of these vehicles. the closure is composed are sufficiently firm and elastic Solutions for injection, examined under suitable conditions to allow the passage of a needle with the least possible shedding of particles. Closures for multidose containers are of visibility, are clear and practically free from particles. sufficiently elastic to ensure that the puncture is resealed Emulsions for injection do not show any evidence of phase when the needle is withdrawn. separation. Suspensions for injection may show a sediment which is readily dispersed on shaking to give a suspension Several categories of parenteral preparations may be which remains sufficiently stable to enable the correct dose distinguished : to be withdrawn. — injections, Multidose preparations. Multidose aqueous injections — infusions, contain a suitable antimicrobial preservative at an — concentrates for injections or infusions, appropriate concentration except when the preparation itself — powders for injections or infusions, has adequate antimicrobial properties. When it is necessary — implants. to present a preparation for parenteral use in a multidose

Parenteralia

614



Parenteral preparations

container, the precautions to be taken for its administration and more particularly for its storage between successive withdrawals are given. Antimicrobial preservatives. Aqueous preparations which are prepared using aseptic precautions and which cannot be terminally sterilised may contain a suitable antimicrobial preservative in an appropriate concentration. No antimicrobial preservative is added when : — the volume to be injected in a single dose exceeds 15 ml, unless otherwise justified, — the preparation is intended for administration by routes where, for medical reasons, an antimicrobial preservative is not acceptable, such as intracisternally, epidurally, intrathecally or by any route giving access to the cerebrospinal fluid, or intra- or retro-ocularly. Such preparations are presented in single-dose containers.

PRODUCTION In the manufacture of infusions containing dispersed particles, measures are taken to ensure a suitable and controlled particle size with regard to the intended use. The volume of the infusion in the container is sufficient to permit the withdrawal and administration of the nominal dose using a normal technique (2.9.17).

PRODUCTION In the manufacture of injections containing dispersed particles, measures are taken to ensure a suitable and controlled particle size with regard to the intended use. Single-dose preparations. The volume of the injection in a single-dose container is sufficient to permit the withdrawal and administration of the nominal dose using a normal technique.

DEFINITION Concentrates for injections or infusions are sterile solutions intended for injection or infusion after dilution. They are diluted to a prescribed volume with a prescribed liquid before administration. After dilution, they comply with the requirements for injections or for infusions.

TESTS Uniformity of content (2.9.6). Unless otherwise prescribed or justified and authorised, single-dose suspensions for injection with a content of active substance less than 2 mg or less than 2 per cent of the total mass comply with test A for uniformity of content of single-dose preparations. If the preparation contains more than one active substance, the requirement applies only to those substances that correspond to the above conditions. Bacterial endotoxins - pyrogens. A test for bacterial endotoxins (2.6.14) is carried out or, where justified and authorised, the test for pyrogens (2.6.8). Recommendations on the limits for bacterial endotoxins are given in chapter 2.6.14. Preparations for human use. The preparation complies with a test for bacterial endotoxins (2.6.14) or with the test for pyrogens (2.6.8). Preparations for veterinary use. When the volume to be injected in a single dose is 15 ml or more and is equivalent to a dose of 0.2 ml or more per kilogram of body mass, the preparation complies with a test for bacterial endotoxins (2.6.14) or with the test for pyrogens (2.6.8). Any preparation. Where the label states that the preparation is free from bacterial endotoxins or apyrogenic, respectively, the preparation complies with a test for bacterial endotoxins (2.6.14) or with the test for pyrogens (2.6.8), respectively.

TESTS Bacterial endotoxins - pyrogens. They comply with a test for bacterial endotoxins (2.6.14) or, where justified and authorised, with the test for pyrogens (2.6.8). For the latter test, inject 10 ml per kilogram of body mass into each rabbit, unless otherwise justified and authorised.

Concentrates for injections or infusions

TESTS Bacterial endotoxins - pyrogens. They comply with the requirements prescribed for injections or for infusions, after dilution to a suitable volume.

Powders for injections or infusions DEFINITION Powders for injections or infusions are solid, sterile substances distributed in their final containers and which, when shaken with the prescribed volume of a prescribed sterile liquid, rapidly form either clear and practically particle-free solutions or uniform suspensions. After dissolution or suspension, they comply with the requirements for injections or for infusions. Freeze-dried products for parenteral use are considered as powders for injections or infusions. PRODUCTION The uniformity of content and uniformity of mass of freeze-dried products for parenteral use are ensured by the in-process control of the amount of the solution prior to freeze-drying.

TESTS Uniformity of content (2.9.6). Unless otherwise prescribed or justified and authorised, powders for injections or infusions with a content of active substance less than 2 mg or less than 2 per cent of the total mass or with a unit mass equal to or less than 40 mg comply with test A for uniformity of content of single-dose preparations. If the preparation Infusions contains more than one active substance, the requirement DEFINITION applies only to those substances that correspond to the above conditions. Infusions are sterile, aqueous solutions or emulsions with water as the continuous phase ; they are usually made Uniformity of mass (2.9.5). Powders for injections or isotonic with blood. They are principally intended for infusions comply with the test for uniformity of mass of administration in large volume. Infusions do not contain any single-dose preparations. If the test for uniformity of content added antimicrobial preservative. is prescribed for all the active substances, the test for uniformity of mass is not required. Solutions for infusion, examined under suitable conditions of visibility, are clear and practically free from particles. Bacterial endotoxins - pyrogens. They comply with the requirements prescribed for injections or for infusions, after Emulsions for infusion do not show any evidence of phase dissolution or suspension in a suitable volume of liquid. separation. General Notices (1) apply to all monographs and other texts

615

Patches, transdermal


PRODUCTION In the manufacture, packaging, storage and distribution of transdermal patches suitable means are taken to ensure their microbial quality ; recommendations on this aspect are provided in the text on Microbiological quality of pharmaceutical preparations (5.1.4).

LABELLING The label states the instructions for the preparation of injections and infusions.

Implants DEFINITION Implants are sterile, solid preparations of a size and shape suitable for parenteral implantation and release the active substance(s) over an extended period of time. Each dose is provided in a sterile container.

TESTS Uniformity of content (2.9.6). Unless otherwise prescribed or justified and authorised, transdermal patches comply with test C for uniformity of content of single-dose preparations. Dissolution. A suitable test may be required to demonstrate the appropriate release of the active substance(s), for 01/2005:1011 example one of the tests described in Dissolution test for transdermal patches (2.9.4). The disc assembly method, the cell method or the rotating cylinder method may be used, PATCHES, TRANSDERMAL as suitable, according to the composition, dimensions and shape of the patch. Emplastra transcutanea A membrane may be used. It can be of various materials, such as inert porous cellulose or silicones, and must not DEFINITION affect the release kinetics of the active substance(s) from Transdermal patches are flexible pharmaceutical the patch. Furthermore, it must be free of substances that preparations of varying sizes, containing one or more active may interfere with its performance (for example grease). substances. They are intended to be applied to the unbroken The membrane may be suitably treated before the tests, skin in order to deliver the active substance(s) to the systemic for example, by maintaining it in the medium to be used in circulation after passing through the skin barrier. the test for 24 h. Apply the membrane above the releasing surface of the patch, avoiding the formation of air bubbles. Transdermal patches normally consist of an outer covering which supports a preparation which contains the active The test conditions and the requirements are to be authorised substance(s). The transdermal patches are covered on the by the competent authority. site of the release surface of the preparation by a protective liner, which is removed before applying the patch to the skin. STORAGE Store at room temperature, unless otherwise indicated. The outer covering is a backing sheet impermeable to the active substance(s) and normally impermeable to water, designed to support and protect the preparation. The outer LABELLING covering may have the same dimensions as the preparation The label states, where applicable, the total quantity of active substance(s) per patch, the dose released per unit time and or it may be larger. In the latter case the overlapping border of the outer covering is covered by pressure-sensitive the area of the releasing surface. adhesive substances which assure the adhesion of the patch to the skin. The preparation contains the active substance(s) together 01/2005:1166 with excipients such as stabilisers, solubilisers or substances intended to modify the release rate or to enhance transdermal POWDERS FOR CUTANEOUS absorption. It may be a single layer or multi-layer solid or semi-solid matrix, and in this case it is the composition APPLICATION and structure of the matrix which determines the diffusion pattern of the active substance(s) to the skin. The matrix Pulveres ad usum dermicum may contain pressure-sensitive adhesives which assure the Where justified and authorised, the requirements of adhesion of the preparation to the skin. The preparation this monograph do not apply to powders for cutaneous may exist as a semi-solid reservoir one side of which is a application intended for veterinary use. membrane which may control the release and the diffusion of the active substance(s) from the preparation. The pressure-sensitive adhesive substances may, in this case, be DEFINITION applied to some or all parts of the membrane, or only around Powders for cutaneous application are preparations consisting of solid, loose, dry particles of varying degrees of the border of the membrane of the outer covering. fineness. They contain one or more active substances, with When applied to the dried, clean and unbroken skin, the or without excipients and, if necessary, colouring matter transdermal patch adheres firmly to the skin by gentle authorised by the competent authority. pressure of the hand or the fingers and can be peeled off without causing appreciable injury to the skin or detachment Powders for cutaneous application are presented as of the preparation from the outer covering. The patch must single-dose powders or multidose powders. They are free not be irritant or sensitising to the skin, even after repeated from grittiness. Powders specifically intended for use on large open wounds or on severely injured skin are sterile. applications. The protective liner generally consists of a sheet of plastic or Multidose powders for cutaneous application may be metal material. When removed, the protective liner does not dispensed in sifter-top containers, containers equipped with detach the preparation (matrix or reservoir) or the adhesive a mechanical spraying device or in pressurised containers. from the patch. Powders dispensed in pressurised containers comply Transdermal patches are normally individually enclosed in with the requirements of Pressurised pharmaceutical sealed sachets. preparations (0523). 616


Premixes for medicated feeding stuffs for veterinary use


Where applicable, containers for powders comply with the requirements of Materials used for the manufacture of containers (3.1 and subsections) and Containers (3.2 and subsections). PRODUCTION In the manufacture of powders for cutaneous application, measures are taken to ensure a suitable particle size with regard to the intended use. In the manufacture, packaging, storage and distribution of powders for cutaneous application, suitable means are taken to ensure their microbial quality ; recommendations on this aspect are provided in the text on Microbiological quality of pharmaceutical preparations (5.1.4). Sterile powders for cutaneous application are prepared using materials and methods designed to ensure sterility and to avoid the introduction of contaminants and the growth of micro-organisms ; recommendations on this aspect are provided in the text on Methods of preparation of sterile products (5.1.1).

Where applicable, containers for oral powders comply with the requirements of Materials used for the manufacture of containers (3.1 and subsections) and Containers (3.2 and subsections). Multidose oral powders require the provision of a measuring device capable of delivering the quantity prescribed. Each dose of a single-dose powder is enclosed in an individual container, for example a sachet or a vial. PRODUCTION In the manufacture of oral powders, means are taken to ensure a suitable particle size with regard to the intended use. In the manufacture, packaging, storage and distribution of oral powders, suitable means are taken to ensure their microbial quality ; recommendations on this aspect are provided in the text on Microbiological quality of pharmaceutical preparations (5.1.4).

TESTS Uniformity of content (2.9.6). Unless otherwise prescribed TESTS or justified and authorised, single-dose oral powders with a Fineness. If prescribed, the fineness of a powder is content of active substance less than 2 mg or less than 2 per determined by the sieve test (2.9.12) or another appropriate cent of the total mass comply with test B for uniformity of method. content of single-dose preparations. If the preparation has Uniformity of content (2.9.6). Unless otherwise prescribed more than one active substance, the requirement applies only to those substances which correspond to the above or justified and authorised, single-dose powders for conditions. cutaneous application with a content of active substance less than 2 mg or less than 2 per cent of the total mass Uniformity of mass (2.9.5). Single-dose oral powders comply with test B for uniformity of content of single-dose comply with the test for uniformity of mass of single-dose preparations. If the preparation has more than one active preparations. If the test for uniformity of content is substance, the requirement applies only to those substances prescribed for all the active substances, the test for which correspond to the above conditions. uniformity of mass is not required. Uniformity of mass (2.9.5). Single-dose powders for Uniformity of mass of delivered doses from multidose cutaneous application comply with the test for uniformity of containers (2.9.27). Oral powders supplied in multidose mass of single-dose preparations. If the test for uniformity of containers comply with the test. content is prescribed for all the active substances, the test STORAGE for uniformity of mass is not required. If the preparation contains volatile ingredients, or the Sterility (2.6.1). Where the label indicates that the contents have to be protected, store in an airtight container. preparation is sterile, it complies with the test for sterility. LABELLING The label states : — that the preparation is for external use, — where applicable, that the preparation is sterile.

Effervescent powders

Effervescent powders are presented as single-dose or multidose preparations and generally contain acid substances and carbonates or hydrogen carbonates which react rapidly in the presence of water to release carbon dioxide. They are intended to be dissolved or dispersed in 01/2005:1165 water before administration.

POWDERS, ORAL Pulveres perorales Requirements for powders to be used for the preparation of oral solutions or suspensions are given in the monograph for Liquid preparations for oral use (0672). Where justified and authorised, the requirements of this monograph do not apply to oral powders intended for veterinary use. DEFINITION Oral powders are preparations consisting of solid, loose, dry particles of varying degrees of fineness. They contain one or more active substances, with or without excipients and, if necessary, colouring matter authorised by the competent authority and flavouring substances. They are generally administered in or with water or another suitable liquid. They may also be swallowed directly. They are presented as single-dose or multidose preparations. General Notices (1) apply to all monographs and other texts

STORAGE In an airtight container. 01/2005:1037

PREMIXES FOR MEDICATED FEEDING STUFFS FOR VETERINARY USE Praeadmixta ad alimenta medicata ad usum veterinarium DEFINITION Mixtures of one or more active substances, usually in suitable bases, that are prepared to facilitate feeding the active substances to animals. They are used exclusively in the preparation of medicated feeding stuffs. 617

Preparations for inhalation


Premixes occur in granulated, powdered, semi-solid or liquid form. Used as powders or granules, they are free-flowing and homogeneous ; any aggregates break apart during normal handling. Used in liquid form, they are homogeneous suspensions or solutions which may be obtained from thixotropic gels or structured liquids. The particle size and other properties are such as to ensure uniform distribution of the active substance(s) in the final feed. Unless otherwise justified and authorised, the instructions for use state that the concentration of a premix in granulated or powdered form is at least 0.5 per cent in the medicated feeding stuff. PRODUCTION Active substance. An active substance intended for incorporation into a medicated premix complies with the requirements of the relevant monograph of the European Pharmacopoeia, unless already otherwise justified and authorised for existing premixes. TESTS Loss on drying (2.2.32). Unless otherwise justified and authorised, for premixes occurring in granulated or powdered form, maximum 15.0 per cent, determined on 3.000 g by drying in an oven at 100-105 °C for 2 h. LABELLING The label states : — the category of animal for which the premix is intended, — the instructions for the preparation of the medicated feeding stuffs from the premix and the basic feed, — where applicable, the time that must elapse between the cessation of feeding of the medicated feeding stuff and collection of the material intended for human consumption.

PRODUCTION During the development of a preparation for inhalation which contains an antimicrobial preservative, the effectiveness of the chosen preservative shall be demonstrated to the satisfaction of the competent authority. A suitable test method together with the criteria for judging the preservative properties of the formulation are described in the text on Efficacy of antimicrobial preservation (5.1.3). The size of aerosol particles to be inhaled is controlled so that a significant fraction is deposited in the lung. The fine-particle characteristics of preparations for inhalation are determined by the method for Aerodynamic assessment of fine particles (2.9.18). In assessing the uniformity of delivered dose of a multidose inhaler, it is not sufficient to test a single inhaler. Manufacturers must substitute procedures which take both inter- and intra-inhaler dose uniformity into account. A suitable procedure based on the intra-inhaler test would be to collect each of the specified doses at the beginning, middle and end of the number of doses stated on the label from separate inhalers. Pressurised metered-dose inhalers are tested for leakage. All inhalers are tested for extraneous particulate contamination. LABELLING For metered-dose preparations the label states : — the delivered dose, except for preparations for which the dose has been established as a metered-dose or as a predispensed-dose, — where applicable, the number of deliveries from the inhaler to provide the minimum recommended dose, — the number of deliveries per inhaler. The label states, where applicable, the name of any added antimicrobial preservative.

Liquid preparations for inhalation 01/2005:0671 Three categories of liquid preparations for inhalation may be distinguished : PREPARATIONS FOR INHALATION A. preparations intended to be converted into vapour, B. liquid preparations for nebulisation, C. pressurised metered-dose preparations for inhalation. Inhalanda Liquid preparations for inhalation are solutions or DEFINITION dispersions. Preparations for inhalation are liquid or solid preparations Dispersions are readily dispersible on shaking and they intended for administration as vapours or aerosols to the remain sufficiently stable to enable the correct dose to be lung in order to obtain a local or systemic effect. They delivered. Suitable excipients may be used. contain one or more active substances which may be A. PREPARATIONS INTENDED TO BE CONVERTED INTO dissolved or dispersed in a suitable vehicle. VAPOUR Preparations for inhalation may, depending on the type of preparation, contain propellants, co-solvents, diluents, DEFINITION antimicrobial preservatives, solubilising and stabilising Preparations intended to be converted into vapour are agents, etc. These excipients do not adversely affect the solutions, dispersions or solid preparations. They are usually functions of the mucosa of the respiratory tract or its cilia. added to hot water and the vapour generated is inhaled. Preparations for inhalation are supplied in multidose or B. LIQUID PREPARATIONS FOR NEBULISATION single-dose containers. When supplied in pressurised DEFINITION containers, they comply with the requirements of the monograph on Pressurised pharmaceutical Liquid preparations for inhalation intended to be converted preparations (0523). into aerosols by continuously operating nebulisers or metered-dose nebulisers are solutions, suspensions or Preparations intended to be administered as aerosols emulsions. Suitable co-solvents or solubilisers may be used (dispersions of solid or liquid particles in a gas) are to increase the solubility of the active substances. administered by one of the following devices : Liquid preparations for nebulisation in concentrated form — nebuliser, for use in continuously operating nebulisers are diluted to — pressurised metered-dose inhaler, the prescribed volume with the prescribed liquid before use. Liquids for nebulisation may also be prepared from powders. — dry-powder inhaler. 618




components of the apparatus are airtight so that when a vacuum is applied to the base of the filter, all of the air drawn through the collection tube passes through the inhaler. Unless otherwise prescribed in the instructions to the patient, shake the inhaler for 5 s and discharge one delivery to waste. Fire the inverted inhaler into the apparatus, depressing the valve for a sufficient time to ensure complete discharge. Repeat the procedure until the number of deliveries that constitute the minimum recommended dose have been sampled. Quantitatively collect the contents of the apparatus and determine the amount of active substance. Repeat the procedure for a further 2 doses. Discharge the device to waste, waiting not less than 5 s between actuations until (n/2)+1 deliveries remain, where n is the number of deliveries stated on the label. Collect 4 doses using the procedure described above. Discharge the device to waste, waiting not less than 5 s between actuations until 3 doses remain. Collect these 3 doses using the procedure described above. C. PRESSURISED METERED-DOSE PREPARATIONS FOR For preparations containing more than one active substance, carry out the test for uniformity of delivered dose for each INHALATION active substance. DEFINITION Unless otherwise justified and authorised, the preparation Pressurised metered-dose preparations for inhalation are complies with the test if 9 out of 10 results lie between solutions, suspensions or emulsions supplied in special 75 per cent and 125 per cent of the average value and all lie containers equipped with a metering valve and which are between 65 per cent and 135 per cent. If 2 or 3 values lie held under pressure with suitable propellants or suitable outside the range of 75 per cent to 125 per cent, repeat the mixtures of liquefied propellants, which can act also as test for 2 more inhalers. Not more than 3 of the 30 values lie solvents. Suitable co-solvents, solubilisers and stabilisers outside the range 75 per cent to 125 per cent and no value may be added. lies outside the range 65 per cent to 135 per cent. The delivered dose is the dose delivered from the inhaler Fine particle dose. Using an apparatus and procedure to the patient. For some preparations, the dose has described in Aerodynamic assessment of fine particles been established as a metered-dose. The metered-dose is (2.9.18 - apparatus C or D), calculate the fine particle dose. determined by adding the amount deposited within the device to the delivered dose. It may also be determined Number of deliveries per inhaler. Take one inhaler and directly. discharge the contents to waste, actuating the valve at intervals of not less than 5 s. The total number of deliveries TESTS so discharged from the inhaler is not less than the number Uniformity of delivered dose. Containers usually operate stated on the label (this test may be combined with the test in an inverted position. For containers that operate in an upright position, an equivalent test is applied using methods for uniformity of delivered dose). that ensure the complete collection of the delivered dose. In all cases, prepare the inhaler as directed in the instructions Powders for inhalation to the patient. DEFINITION The dose collection apparatus must be capable of Powders for inhalation are presented as single-dose powders quantitatively capturing the delivered dose. or multidose powders. To facilitate their use, active The following apparatus and procedure may be used. substances may be combined with a suitable carrier. They The apparatus (Figure 0671.-1) consists of a filter-support are generally administered by dry-powder inhalers. In base with an open-mesh filter-support, such as a stainless pre-metered systems, the inhaler is loaded with powders steel screen, a collection tube that is clamped or screwed pre-dispensed in capsules or other suitable pharmaceutical to the filter-support base, and a mouthpiece adapter to forms. For devices using a powder reservoir, the dose is ensure an airtight seal between the collection tube and created by a metering mechanism within the inhaler. the mouthpiece. Use a mouthpiece adapter which ensures The delivered dose is the dose delivered from the inhaler. that the front face of the inhaler mouthpiece is flush with For some preparations, the dose has been established as a the front face of the sample collection tube. The vacuum metered dose or as a predispensed dose. The metered dose connector is connected to a system comprising a vacuum source and a flow regulator. The source should be adjusted is determined by adding the amount deposited within the device to the delivered dose. It may also be determined to draw air through the complete assembly, including the directly. filter and the inhaler to be tested, at 28.3 ± 1.5 litres/min. Air should be drawn continuously through the apparatus TESTS to avoid loss of the active substance into the atmosphere. The filter support base is designed to accommodate 25 mm Uniformity of delivered dose. In all cases, prepare the diameter filter disks. The filter disk and other materials used inhaler as directed in the instructions to the patient. The dose in the construction of the apparatus must be compatible with collection apparatus must be capable of quantitatively the active substance and solvents that are used to extract the capturing the delivered dose. A dose collection apparatus active substance from the filter. One end of the collection similar to that described for the evaluation of pressurised tube is designed to hold the filter disk tightly against the metered-dose inhalers may be used provided that the filter-support base. When assembled, the joints between the dimensions of the tube and the filter can accommodate The pH of the liquid preparations for use in continuously operating nebulisers is not lower than 3 and not higher than 8.5. Suspensions and emulsions are readily dispersible on shaking and they remain sufficiently stable to enable the correct dose to be delivered. Aqueous preparations for nebulisation supplied in multidose containers may contain a suitable antimicrobial preservative at a suitable concentration except where the preparation itself has adequate antimicrobial properties. Continuously operating nebulisers are devices that convert liquids into aerosols by high-pressure gases, ultrasonic vibration or other methods. They allow the dose to be inhaled at an appropriate rate and particle size which ensures deposition of the preparation in the lungs. Metered-dose nebulisers are devices that convert liquids into aerosols by high-pressure gases, ultrasonic vibration or other methods. The volume of liquid to be nebulised is metered so that the aerosol dose can be inhaled with one breath.


619



Figure 0671.-1. – Dose collection apparatus for pressurised metered-dose preparations Dimensions in millimetres atmosphere. Switch on the pump, open the two way valve the measured flow rate. A suitable tube is defined in Figure 0671.-1. Connect the tube to a flow system according and adjust the flow control valve until the pressure drop to the scheme specified in Figure 0671.-2 and Table 0671.-1. across the inhaler is 4.0 kPa (40.8 cm H2O) as indicated by the differential pressure meter. Remove the inhaler from the Unless otherwise stated, determine the test flow rate and mouthpiece adapter and without touching the flow control duration using the dose collection tube, the associated flow valve, connect a flow meter to the inlet of the sampling system, a suitable differential pressure meter and a suitable apparatus. If the flow rate is above 100 litres/min adjust the volumetric flow meter, calibrated for the flow leaving the flow control valve to obtain a flow rate of 100 ± 5 litres/min. meter, according to the following procedure. Note the volumetric airflow rate and define this as the test flow rate, Q, in litres per minute. Define the test flow Prepare the inhaler for use and connect it to the inlet of duration, T, in seconds so that a volume of 4 litres of air is the apparatus using a mouthpiece adapter to ensure an airtight seal. Use a mouthpiece adapter which ensures that drawn through the inhaler. Ensure that critical flow occurs in the flow control valve by the front face of the inhaler mouthpiece is flush with the the following procedure. With the inhaler in place and the front face of the sample collection tube. Connect one port test flow rate Q, measure the absolute pressure on both sides of a differential pressure meter to the pressure reading point, P1, in Figure 0671.-2 and let the other be open to the of the control valve (pressure reading points P2 and P3 in 620




Figure 0671.-2). A ratio P3/P2 ≤ 0.5 indicates critical flow. Switch to a more powerful pump and re-measure the test flow rate if critical flow is not indicated.

Table 0671.-1. – Specifications of the apparatus described in figure 0671.-2 Code

Item

Description

Predispensed systems. Prepare the inhaler as directed in the instructions to the patient and connect it to the apparatus using an adapter which ensures a good seal. Draw air through the inhaler using the predetermined conditions. Repeat the procedure until the number of deliveries which constitute the minimum recommended dose have been sampled. Quantitatively collect the contents of the apparatus and determine the amount of active substance.

A

Sample collection tube

Capable of quantitatively capturing the delivered dose, e.g. Dose collection tube similar to that described in Fig. 0671.-1 with dimensions of 34.85 mm ID × 12 cm length (e.g. product number XX40 047 00, Millipore Corporation, Bedford, MA 01732 with modified exit tube, ID ≥ 8 mm, fitted with Gelman product number 61631), or equivalent.

B

Filter

47 mm filter, e.g. A/E glass fibre filter (Gelman Sciences, Ann Arbor, MI 48106), or equivalent.

C

Connector

ID ≥ 8 mm, e.g., short metal coupling, with low-diameter branch to P3.

D

Vacuum tubing

8 ± 0.5 mm ID × 50 ± 10 cm length, e.g., silicone tubing with an OD of 14 mm and an ID of 8 mm.

E

Two-way solenoid valve

Minimum airflow resistance orifice having an ID of ≥ 8 mm and a maximum response time of 100 ms (e.g. type 256-A08, Bürkert GmbH, D-74653 Ingelfingen), or equivalent.

F

Vacuum pump

Pump must be capable of drawing the required flow rate through the assembled apparatus with the dry powder inhaler in the mouthpiece adapter (e.g. product type 1023, 1423 or 2565, Gast Manufacturing Inc., Benton Harbor, MI 49022), or equivalent. Connect the pump to the solenoid valve using short and/or wide (≥ 10 mm ID) vacuum tubing and connectors to minimise pump capacity requirements.

G

Timer

Timer capable of driving the solenoid valve for the required time period (e.g. type G814, RS Components International, Corby, NN17 9RS, UK), or equivalent.

P1

Pressure tap

2.2 mm ID, 3.1 mm OD, flush with internal surface of the sample collection tube, centred and burr-free, 59 mm from its inlet.

P1 P2 P3

Pressure measurements

Differential pressure to atmosphere (P1) or absolute pressure (P2 and P3).

Repeat the procedure for a further 9 doses. Reservoir systems. Prepare the inhaler as directed in the instructions to the patient and connect it to the apparatus using an adapter which ensures a good seal. Draw air through the inhaler under the predetermined conditions. Repeat the procedure until the number of deliveries which constitute the minimum recommended dose have been sampled. Quantitatively collect the contents of the apparatus and determine the amount of active substance. Repeat the procedure for a further 2 doses. Discharge the device to waste until (n/2)+1 deliveries remain, where n is the number of deliveries stated on the label. If necessary, store the inhaler to discharge electrostatic charges. Collect 4 doses using the procedure described above. Discharge the device to waste until 3 doses remain. If necessary, store the inhaler to discharge electrostatic charges. Collect 3 doses using the procedure described above. For preparations containing more than 1 active substance, carry out the test for uniformity of delivered dose for each active substance.

The preparation complies with the test if 9 out of 10 results lie between 75 per cent and 125 per cent of the average value H Flow control Adjustable regulating valve with maximum and all lie between 65 per cent and 135 per cent. If 2 or valve Cv ≥ 1, (e.g. type 8FV12LNSS, Parker Han3 values lie outside the range of 75 per cent to 125 per cent, nifin plc., Barnstaple, EX31 1NP, UK), or equivalent. repeat the test for 2 more inhalers. Not more than 3 of the 30 values lie outside the range 75 per cent to 125 per cent Fine particle dose. Using the apparatus and procedure and no value lies outside the range 65 per cent to 135 per described in Aerodynamic assessment of fine particles cent. (2.9.18 - apparatus C or D), calculate the fine particle dose. In justified and authorised cases, these ranges may be Number of deliveries per inhaler for multidose inhalers. extended but no value should be greater than 150 per cent Discharge doses from the inhaler until empty, at the or less than 50 per cent of the average value. predetermined flow rate. Record the deliveries discharged.

Figure 0671.-2. – Apparatus suitable for measuring the uniformity of delivered dose for powder inhalers General Notices (1) apply to all monographs and other texts

621

Preparations for irrigation


The total number of doses delivered is not less than the number stated on the label (this test may be combined with the test for uniformity of delivered dose).

01/2005:1116

PREPARATIONS FOR IRRIGATION Praeparationes ad irrigationem DEFINITION

01/2005:0523

PRESSURISED PHARMACEUTICAL PREPARATIONS Praeparationes pharmaceuticae in vasis cum pressu Additional requirements for preparations presented in pressurised containers may be found, where appropriate, in other general monographs, for example Preparations for inhalation (0671), Liquid preparations for cutaneous application (0927), Powders for cutaneous application (1166), Nasal preparations (0676) and Ear preparations (0652).

Preparations for irrigation are sterile, aqueous large volume preparations intended to be used for irrigation of body cavities, wounds and surfaces, for example during surgical procedures.

DEFINITION Pressurised pharmaceutical preparations are presented in special containers under pressure of a gas and contain one or more active substances. The preparations are released from Preparations for irrigation are either solutions prepared by the container, upon actuation of an appropriate valve, in the dissolving one or more active substances, electrolytes or form of an aerosol (dispersion of solid or liquid particles in a osmotically active substances in water complying with the gas, the size of the particles being adapted to the intended requirements for Water for injections (0169) or they consist use) or of a liquid or semisolid jet such as a foam. The of such water alone. In the latter case, the preparation may pressure for the release is generated by suitable propellants. be labelled as water for irrigation. Irrigation solutions are The preparations consist of a solution, an emulsion or a usually adjusted to be isotonic with blood. suspension and are intended for local application to the skin Examined in suitable conditions of visibility, preparations for or to mucous membranes of various body orifices, or for irrigation are clear and practically free from particles. inhalation. Suitable excipients may also be used, for example solvents, solubilisers, emulsifying agents, suspending agents Preparations for irrigation are supplied in single-dose and lubricants for the valve to prevent clogging. containers. The containers and closures comply with the Propellants. The propellants are either gases liquefied requirements for containers for preparations for parenteral under pressure or compressed gases or low-boiling liquids. use (3.2.1 and 3.2.2) but the administration port of the Liquefied gases are, for example, fluorinated hydrocarbons container is incompatible with intravenous administration equipment and does not allow the preparation for irrigation and low-molecular-mass hydrocarbons (such as propane and butane). Compressed gases are, for example, carbon dioxide, to be administered with such equipment. nitrogen and nitrous oxide. Mixtures of these propellants may be used to obtain optimal PRODUCTION solution properties and desirable pressure, delivery and Preparations for irrigation are prepared using materials spray characteristics. and methods designed to ensure sterility and to avoid Containers. The containers are tight and resistant to the the introduction of contaminants and the growth of internal pressure and may be made of metal, glass, plastic or micro-organisms ; recommendations on this aspect are combinations of these materials. They are compatible with provided in the text on Methods of preparation of sterile their contents. Glass containers are protected with a plastic products (5.1.1). coating. Spraying device. The valve keeps the container tightly TESTS closed when not in use and regulates the delivery of Deliverable mass or volume (2.9.28). Preparations for the contents during use. The spray characteristics are irrigation supplied in single-dose containers comply with influenced by the type of spraying device, in particular by the test. the dimensions, number and location of orifices. Some Sterility (2.6.1). Preparations for irrigation comply with the valves provide a continuous release, others (“metering dose valves”) deliver a defined quantity of product upon each test for sterility. valve actuation. Bacterial endotoxins (2.6.14) : less than 0.5 IU/ml. The various valve materials in contact with the contents are Pyrogens (2.6.8). Preparations for which a validated test compatible with them. for bacterial endotoxins cannot be carried out comply with Requirements for pressurised pharmaceutical preparations. the test for pyrogens. Inject per kilogram of the rabbits Pressurised preparations are provided with a delivery device mass, 10 ml of the preparation, unless otherwise justified appropriate for the intended application. and authorised. Special requirements may be necessary for the selection of propellants, for particle size and the single-dose delivered LABELLING by the metering valves. The label states : LABELLING — that the preparation is not to be used for injection, The label states : — that the preparation is to be used for one occasion only — the method of use, and that any unused portion of preparation is to be — any precautions to be taken, discarded. 622


Rectal preparations


— for a container with a metering dose valve, the amount of Dissolution. A suitable test may be required to demonstrate the appropriate release of the active substance(s) from solid, active substance in a unit-spray. single-dose preparations, for example the dissolution test for suppositories and soft capsules (2.9.3). Where a dissolution test is prescribed, a disintegration test 01/2005:1145 may not be required.

RECTAL PREPARATIONS Rectalia DEFINITION Rectal preparations are intended for rectal use in order to obtain a systemic or local effect, or they may be intended for diagnostic purposes. Where applicable, containers for rectal preparations comply with the requirements for Materials used for the manufacture of containers (3.1 and subsections) and Containers (3.2 and subsections). Several categories of rectal preparations may be distinguished : — suppositories, — rectal capsules, — rectal solutions, emulsions and suspensions, — powders and tablets for rectal solutions and suspensions, — semi-solid rectal preparations, — rectal foams, — rectal tampons.

LABELLING The label states the name of any added antimicrobial preservative.

Suppositories DEFINITION Suppositories are solid, single-dose preparations. The shape, volume and consistency of suppositories are suitable for rectal administration. They contain one or more active substances dispersed or dissolved in a suitable basis which may be soluble or dispersible in water or may melt at body temperature. Excipients such as diluents, adsorbents, surface-active agents, lubricants, antimicrobial preservatives and colouring matter, authorised by the competent authority, may be added if necessary.

PRODUCTION Suppositories are prepared by compression or moulding. If necessary, the active substance(s) are previously ground and sieved through a suitable sieve. When prepared by moulding, the medicated mass, sufficiently liquified by heating, is poured into suitable moulds. The suppository solidifies on PRODUCTION cooling. Various excipients are available for this process, such as hard fat, macrogols, cocoa butter, and various During the development of a rectal preparation, the formulation for which contains an antimicrobial preservative, gelatinous mixtures consisting of, for example, gelatin, water and glycerol. Where applicable, the determination of the the effectiveness of the chosen preservative shall be demonstrated to the satisfaction of the competent authority. softening time of lipophilic suppositories (2.9.22) and/or the A suitable test method together with criteria for judging the determination of the resistance to rupture of suppositories (2.9.24) are carried out. preservative properties of the formulation are provided in the text on Efficacy of antimicrobial preservation (5.1.3). A suitable test is carried out to demonstrate the appropriate release of the active substance(s) from suppositories In the manufacture, packaging, storage and distribution intended for modified release or for prolonged local action. of rectal preparations, suitable means are taken to ensure their microbial quality ; recommendations on this aspect In the manufacture of suppositories containing dispersed are provided in the text on Microbiological quality of active substances, measures are taken to ensure a suitable pharmaceutical preparations (5.1.4). and controlled particle size. In the manufacture of semi-solid and liquid rectal TESTS preparations containing dispersed particles measures are taken to ensure a suitable and controlled particle size with Disintegration. Unless intended for modified release or regard to the intended use. for prolonged local action, they comply with the test for disintegration of suppositories and pessaries (2.9.2). For TESTS suppositories with a fatty base, examine after 30 min and for Uniformity of content (2.9.6). Unless otherwise prescribed suppositories with a water-soluble base after 60 min, unless otherwise justified and authorised. or justified and authorised, solid single-dose preparations with a content of active substance less than 2 mg or less than 2 per cent of the total mass comply with test A (tablets) Rectal capsules or test B (suppositories, rectal capsules) for uniformity of content of single-dose preparations. If the preparation DEFINITION contains more than one active substance, this requirement Rectal capsules (shell suppositories) are solid, single-dose applies only to those substances that correspond to the preparations generally similar to soft capsules as defined in above conditions. the monograph on Capsules (0016) except that they may Uniformity of mass (2.9.5). Solid, single-dose preparations have lubricating coatings. They are of elongated shape, are comply with the test for uniformity of mass. If the test for smooth and have a uniform external appearance. uniformity of content is prescribed for all active substances, the test for uniformity of mass is not required. PRODUCTION Deliverable mass or volume (2.9.28). Liquid and semi-solid A suitable test is carried out to demonstrate the appropriate rectal preparations supplied in single-dose containers comply release of the active substance(s) from rectal capsules with the test. intended for modified release or for prolonged local action.


623

Semi-solid preparations for cutaneous application


TESTS Disintegration. Unless intended for modified release or for prolonged local action, they comply with the test for disintegration of suppositories and pessaries (2.9.2). Examine the state of the capsules after 30 min, unless otherwise justified and authorised.

Rectal solutions, emulsions and suspensions DEFINITION Rectal solutions, emulsions and suspensions are liquid preparations intended for rectal use in order to obtain a systemic or local effect, or they may be intended for diagnostic purposes. Rectal solutions, emulsions and suspensions are supplied in single-dose containers and they contain one or more active substances dissolved or dispersed in water, glycerol or macrogols or other suitable solvents. Emulsions may show evidence of phase separation but are readily redispersed on shaking. Suspensions may show a sediment which is readily dispersible on shaking to give a suspension which remains sufficiently stable to enable the correct dose to be delivered. Rectal solutions, emulsions and suspensions may contain excipients, for example to adjust the viscosity of the preparation, to adjust or stabilise the pH, to increase the solubility of the active substance(s) or to stabilise the preparation. These substances do not adversely affect the intended medical action or, at the concentrations used, cause undue local irritation. Rectal solutions, emulsions and suspensions are supplied in containers containing a volume in the range of 2.5 ml to 2000 ml. The container is adapted to deliver the preparation to the rectum or it is accompanied by a suitable applicator.

Powders and tablets for rectal solutions and suspensions DEFINITION Powders and tablets intended for the preparation of rectal solutions or suspensions are single-dose preparations which are dissolved or dispersed in water at the time of administration. They may contain excipients to facilitate dissolution or dispersion or to prevent aggregation of the particles. After dissolution or suspension, they comply with the requirements for rectal solutions or rectal suspensions, as appropriate. TESTS Disintegration. Tablets for rectal solutions or suspensions disintegrate within 3 min when tested according to the test for disintegration of tablets and capsules (2.9.1) but using water R at 15 °C to 25 °C. LABELLING The label states : — the method of preparation of the rectal solution or suspension, — the conditions and duration of storage of the solution or suspension after constitution.

Semi-solid rectal preparations DEFINITION Semi-solid rectal preparations are ointments, creams or gels. 624

They are often supplied as single-dose preparations in containers provided with a suitable applicator. Semi-solid rectal preparations comply with the requirements of the monograph on Semi-solid preparations for cutaneous application (0132).

Rectal foams DEFINITION Rectal foams comply with the requirements of the monograph on Medicated foams (1105).

Rectal tampons DEFINITION Rectal tampons are solid, single-dose preparations intended to be inserted into the lower part of the rectum for a limited time. They comply with the requirements of the monograph on Medicated tampons (1155). 01/2005:0132

SEMI-SOLID PREPARATIONS FOR CUTANEOUS APPLICATION Praeparationes molles ad usum dermicum The requirements of this monograph apply to all semi-solid preparations for cutaneous application. Where appropriate, additional requirements specific to semi-solid preparations intended to be applied to particular surfaces or mucous membranes may be found in other general monographs, for example Ear preparations (0652), Nasal preparations (0676), Rectal preparations (1145), Eye preparations (1163) and Vaginal preparations (1164). DEFINITION Semi-solid preparations for cutaneous application are intended for local or transdermal delivery of active substances, or for their emollient or protective action. They are of homogeneous appearance. Semi-solid preparations for cutaneous application consist of a simple or compound basis in which, usually, one or more active substances are dissolved or dispersed. According to its composition, the basis may influence the activity of the preparation. The basis may consist of natural or synthetic substances and may be single phase or multiphase. According to the nature of the basis, the preparation may have hydrophilic or hydrophobic properties ; it may contain suitable excipients such as antimicrobial preservatives, antioxidants, stabilisers, emulsifiers, thickeners and penetration enhancers. Semi-solid preparations for cutaneous application intended for use on severely injured skin are sterile. Where applicable, containers for semi-solid preparations for cutaneous application comply with the requirements for Materials used for the manufacture of containers (3.1 and subsections) and Containers (3.2 and subsections). Several categories of semi-solid preparations for cutaneous application may be distinguished : — ointments, — creams, — gels, — pastes,



— poultices, — medicated plasters. According to their structure, ointments, creams and gels generally show viscoelastic behaviour and are non-newtonian in character e.g. plastic, pseudoplastic or thixotropic type flow at high shear rates. Pastes frequently exhibit dilatancy. PRODUCTION During the development of semi-solid preparations for cutaneous application whose formulation contains an antimicrobial preservative, the necessity for and the effectiveness of the chosen preservative shall be demonstrated to the satisfaction of the competent authority. A suitable test method together with criteria for judging the preservative properties of the formulation are provided in Efficacy of antimicrobial preservation (5.1.3). In the manufacture, packaging, storage and distribution of semi-solid preparations for cutaneous application, suitable steps are taken to ensure their microbiological quality ; recommendations on this are provided in Microbiological Quality of Pharmaceutical Preparations (5.1.4). Sterile semi-solid preparations for cutaneous application are prepared using materials and methods designed to ensure sterility and to avoid the introduction of contaminants and the growth of micro-organisms ; recommendations on this are provided in Methods of Preparation of Sterile Products (5.1.1). In the manufacture of semi-solid preparations for cutaneous application, suitable measures are taken to ensure that the defined rheological properties are fulfilled. Where appropriate, the following non-mandatory tests may be carried out : measurement of consistency by penetrometry (2.9.9), viscosity (apparent viscosity) (2.2.10) and a suitable test to demonstrate the appropriate release of the active substance(s). In the manufacture of semi-solid preparations for cutaneous application containing (an) active substance(s) which is/are not dissolved in the basis (e.g. emulsions or suspensions), measures are taken to ensure appropriate homogeneity of the preparation to be delivered. In the manufacture of semi-solid preparations for cutaneous application containing dispersed particles, measures are taken to ensure a suitable and controlled particle size with regard to the intended use. TESTS Deliverable mass or volume (2.9.28). Semi-solid preparations for cutaneous application supplied in single-dose containers comply with the test. Sterility (2.6.1). Where the label indicates that the preparation is sterile, it complies with the test for sterility. STORAGE If the preparation contains water or other volatile ingredients, store in an airtight container. If the preparation is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states : — the name of any added antimicrobial preservative, — where applicable, that the preparation is sterile.

Ointments DEFINITION An ointment consists of a single-phase basis in which solids or liquids may be dispersed. General Notices (1) apply to all monographs and other texts

Semi-solid preparations for cutaneous application

Hydrophobic Ointments Hydrophobic ointments can absorb only small amounts of water. Typical bases used for their formulation are hard, liquid and light liquid paraffins, vegetable oils, animal fats, synthetic glycerides, waxes and liquid polyalkylsiloxanes. Water-emulsifying Ointments Water-emulsifying ointments can absorb larger amounts of water and thereby produce water-in-oil or oil-in-water emulsions depending on the nature of the emulsifiers : water-in-oil emulsifying agents such as wool alcohols, sorbitan esters, monoglycerides and fatty alcohols, or oil-in-water emulsifying agents such as sulphated fatty alcohols, polysorbates, macrogol cetostearyl ether or esters of fatty acids with macrogols may be used for this purpose. Their bases are those of the hydrophobic ointments. Hydrophilic Ointments Hydrophilic ointments are preparations having bases that are miscible with water. The bases usually consist of mixtures of liquid and solid macrogols (polyethylene glycols). They may contain appropriate amounts of water.

Creams DEFINITION Creams are multiphase preparations consisting of a lipophilic phase and an aqueous phase. Lipophilic Creams Lipophilic creams have as the continuous phase the lipophilic phase. They contain water-in-oil emulsifying agents such as wool alcohols, sorbitan esters and monoglycerides. Hydrophilic Creams Hydrophilic creams have as the continuous phase the aqueous phase. They contain oil-in-water emulsifying agents such as sodium or trolamine soaps, sulphated fatty alcohols, polysorbates and polyoxyl fatty acid and fatty alcohol esters combined, if necessary, with water-in-oil emulsifying agents.

Gels DEFINITION Gels consist of liquids gelled by means of suitable gelling agents. Lipophilic Gels Lipophilic gels (oleogels) are preparations whose bases usually consist of liquid paraffin with polyethylene or fatty oils gelled with colloidal silica or aluminium or zinc soaps. Hydrophilic Gels Hydrophilic gels (hydrogels) are preparations whose bases usually consist of water, glycerol or propylene glycol gelled with suitable gelling agents such as starch, cellulose derivatives, carbomers and magnesium-aluminium silicates.

Pastes DEFINITION Pastes are semi-solid preparations for cutaneous application containing large proportions of solids finely dispersed in the basis.

Poultices DEFINITION Poultices consist of a hydrophilic heat-retentive basis in which solid or liquid active substances are dispersed. They are usually spread thickly on a suitable dressing and heated before application to the skin. 625

Sticks


Medicated plasters

LABELLING The label states : DEFINITION — the quantity of active substance(s) per stick, Medicated plasters are flexible preparations containing — for urethral sticks and sticks to be inserted into wounds one or more active substances. They are intended to be that they are sterile. applied to the skin. They are designed to maintain the active substance(s) in close contact with the skin such that these 01/2005:0478 may be absorbed slowly, or act as protective or keratolytic agents. TABLETS Medicated plasters consist of an adhesive basis, which may be coloured, containing one or more active substances, Compressi spread as a uniform layer on an appropriate support made of natural or synthetic material. It is not irritant or sensitising The requirements of this monograph do not necessarily to the skin. The adhesive layer is covered by a suitable apply to preparations that are presented as tablets intended protective liner, which is removed before applying the plaster for use other than by oral administration. Requirements to the skin. When removed, the protective liner does not for such preparations may be found, where appropriate, detach the preparation from the outer, supporting layer. in other general monographs ; for example Rectal Medicated plasters are presented in a range of sizes directly preparations (1145), Vaginal preparations (1164) and adapted to their intended use or as larger sheets to be cut Oromucosal preparations (1807). This monograph does not before use. Medicated plasters adhere firmly to the skin apply to lozenges, oral lyophilisates, oral pastes and oral when gentle pressure is applied and can be peeled off without gums. Where justified and authorised, the requirements of causing appreciable injury to the skin or detachment of the this monograph do not apply to tablets for veterinary use. preparation from the outer, supporting layer. DEFINITION TESTS Tablets are solid preparations each containing a single Dissolution. A suitable test may be required to demonstrate dose of one or more active substances and usually obtained by compressing uniform volumes of particles. Tablets are the appropriate release of the active substance(s), for intended for oral administration. Some are swallowed whole, example one of the tests described in Dissolution test for some after being chewed, some are dissolved or dispersed in transdermal patches (2.9.4). water before being administered and some are retained in the mouth where the active substance is liberated. 01/2005:1154 The particles consist of one or more active substances with or without excipients such as diluents, binders, disintegrating STICKS agents, glidants, lubricants, substances capable of modifying the behaviour of the preparation in the digestive tract, colouring matter authorised by the competent authority and Styli flavouring substances. Additional requirements for sticks may be found, where Tablets are usually right, circular solid cylinders, the end appropriate, in other general monographs, for example surfaces of which are flat or convex and the edges of which Nasal preparations (0676). may be bevelled. They may have lines or break-marks and DEFINITION may bear a symbol or other markings. Tablets may be coated. Sticks are solid preparations intended for local application. Where applicable, containers for tablets comply with the They are rod-shaped or conical preparations consisting of requirements for Materials used for the manufacture of one or more active substances alone or which are dissolved containers (3.1 and subsections) and Containers (3.2 and or dispersed in a suitable basis which may dissolve or melt subsections). at body temperature. Several categories of tablets for oral use may be Urethral sticks and sticks for insertion into wounds are distinguished : sterile. — uncoated tablets, — coated tablets, PRODUCTION — effervescent tablets, In the manufacture, packaging, storage and distribution of — soluble tablets, sticks, suitable means are taken to ensure their microbial quality ; recommendations on this aspect are provided in — dispersible tablets, the text on Microbiological quality of pharmaceutical — orodispersible tablets, preparations (5.1.4). — gastro-resistant tablets, Urethral sticks and other sterile sticks are prepared using — modified-release tablets. materials and methods designed to ensure sterility and to avoid the introduction of contaminants and the growth PRODUCTION of micro-organisms ; recommendations on this aspect are Tablets are usually prepared by compressing uniform provided in the text on Methods of preparation of sterile volumes of particles or particle aggregates produced by products (5.1.1). granulation methods. In the manufacture of tablets, means In the manufacture of sticks means are taken to ensure that are taken to ensure that they possess a suitable mechanical the preparation complies with a test for mass uniformity or, strength to avoid crumbling or breaking on handling or where appropriate, a test for uniformity of content. subsequent processing. This may be demonstrated by examining the Friability of uncoated tablets (2.9.7) and TESTS the Resistance to crushing of tablets (2.9.8). Chewable Sterility (2.6.1). Urethral sticks and sticks for insertion into tablets are prepared to ensure that they are easily crushed by chewing. wounds comply with the test for sterility. 626


Tablets


For tablets for which subdivision is authorised, it is demonstrated to the satisfaction of the competent authority that the subdivided parts comply either with test A for Uniformity of content of single-dose preparations (2.9.6) or with the test for Uniformity of mass of single-dose preparations (2.9.5), as appropriate. In the manufacture, packaging, storage and distribution of tablets, suitable means are taken to ensure their microbiological quality ; recommendations on this aspect are provided in the text on Microbiological quality of pharmaceutical preparations (5.1.4). TESTS

Coated tablets DEFINITION Coated tablets are tablets covered with one or more layers of mixtures of various substances such as natural or synthetic resins, gums, gelatin, inactive and insoluble fillers, sugars, plasticisers, polyols, waxes, colouring matter authorised by the competent authority and sometimes flavouring substances and active substances. The substances used as coatings are usually applied as a solution or suspension in conditions in which evaporation of the vehicle occurs. When the coating is a very thin polymeric coating, the tablets are known as film-coated tablets.

Uniformity of content (2.9.6). Unless otherwise prescribed or justified and authorised, tablets with a content of active substance less than 2 mg or less than 2 per cent of the total mass comply with test A for uniformity of content of single-dose preparations. If the preparation has more than one active substance, the requirement applies only to those substances which correspond to the above conditions.

Coated tablets have a smooth surface which is often coloured and may be polished ; a broken section, when examined under a lens, shows a core surrounded by one or more continuous layers with a different texture.

Dissolution. A suitable test may be carried out to demonstrate the appropriate release of the active substance(s), for example one of the tests described in Dissolution test for solid dosage forms (2.9.3).

the test on a further 6 tablets, replacing water R with 0.1 M hydrochloric acid. The tablets comply with the test if all 6 have disintegrated in the acid medium.

PRODUCTION

Where justified, uniformity of mass or uniformity of content Unless otherwise justified and authorised, coated tablets of coated tablets other than film-coated tablets may be other than film-coated tablets comply with test A for ensured by control of the cores. uniformity of content of single-dose preparations irrespective of their content of active substance(s). TESTS Uniformity of mass (2.9.5). Uncoated tablets and, unless Disintegration. Coated tablets other than film-coated otherwise justified and authorised, film-coated tablets tablets comply with the test for disintegration of tablets comply with the test for uniformity of mass of single-dose and capsules (2.9.1). Use water R as the liquid. Add a disc preparations. If the test for uniformity of content is to each tube. Operate the apparatus for 60 min, unless prescribed or justified and authorised for all the active otherwise justified and authorised, and examine the state of substances, the test for uniformity of mass is not required. the tablets. If any of the tablets has not disintegrated, repeat

Where a dissolution test is prescribed, a disintegration test may not be required.

Uncoated tablets DEFINITION Uncoated tablets include single-layer tablets resulting from a single compression of particles and multi-layer tablets consisting of concentric or parallel layers obtained by successive compression of particles of different composition. The excipients used are not specifically intended to modify the release of the active substance in the digestive fluids. Uncoated tablets conform to the general definition of tablets. A broken section, when examined under a lens, shows either a relatively uniform texture (single-layer tablets) or a stratified texture (multi-layer tablets) but no signs of coating. TESTS Disintegration. Uncoated tablets comply with the test for disintegration of tablets and capsules (2.9.1). Use water R as the liquid. Add a disc to each tube. Operate the apparatus for 15 min, unless otherwise justified and authorised, and examine the state of the tablets. If the tablets fail to comply because of adherence to the discs, repeat the test on a further 6 tablets omitting the discs. The tablets comply with the test if all 6 have disintegrated. Chewable tablets are not required to comply with the test. General Notices (1) apply to all monographs and other texts

Film-coated tablets comply with the disintegration test prescribed above except that the apparatus is operated for 30 min, unless otherwise justified and authorised. If coated tablets or film-coated tablets fail to comply because of adherence to the discs, repeat the test on a further 6 tablets omitting the discs. The tablets comply with the test if all 6 have disintegrated. Chewable coated tablets are not required to comply with the test.

Effervescent tablets DEFINITION Effervescent tablets are uncoated tablets generally containing acid substances and carbonates or hydrogen carbonates which react rapidly in the presence of water to release carbon dioxide. They are intended to be dissolved or dispersed in water before administration. TESTS Disintegration. Place 1 tablet in a beaker containing 200 ml of water R at 15-25 °C ; numerous bubbles of gas are evolved. When the evolution of gas around the tablet or its fragments ceases the tablet has disintegrated, being either dissolved or dispersed in the water so that no agglomerates of particles remain. Repeat the operation on 5 other tablets. The tablets comply with the test if each of the 6 tablets used disintegrates in the manner prescribed within 5 min, unless otherwise justified and authorised. 627

Tampons, medicated


Soluble tablets

PRODUCTION For tablets prepared from granules or particles already covered with a gastro-resistant coating, a suitable test is carried out to demonstrate the appropriate release of the active substance(s).

DEFINITION Soluble tablets are uncoated or film-coated tablets. They are intended to be dissolved in water before administration. The solution produced may be slightly opalescent due to the TESTS added excipients used in the manufacture of the tablets. Disintegration. For tablets covered with a gastro-resistant TESTS coating carry out the test for disintegration (2.9.1) with the Disintegration. Soluble tablets disintegrate within 3 min following modifications. Use 0.1 M hydrochloric acid as the when examined by the test for disintegration of tablets and liquid. Operate the apparatus for 2 h, or other such time capsules (2.9.1), but using water R at 15-25 °C. as may be justified and authorised, without the discs and examine the state of the tablets. The time of resistance to the acid medium varies according to the formulation of the Dispersible tablets tablets to be examined. It is typically 2 h to 3 h but even with authorised deviations is not less than 1 h. No tablet DEFINITION shows signs of either disintegration (apart from fragments Dispersible tablets are uncoated or film-coated tablets of coating) or cracks that would allow the escape of the intended to be dispersed in water before administration contents. Replace the acid by phosphate buffer solution giving a homogeneous dispersion. pH 6.8 R and add a disc to each tube. Operate the apparatus for 60 min and examine the state of the tablets. If the tablets TESTS fail to comply because of adherence to the discs, repeat the Disintegration. Dispersible tablets disintegrate within 3 min test on a further 6 tablets omitting the discs. The tablets when examined by the test for disintegration of tablets and comply with the test if all 6 have disintegrated. capsules (2.9.1), but using water R at 15-25 °C. Dissolution. For tablets prepared from granules or particles Fineness of dispersion. Place 2 tablets in 100 ml of water R already covered with a gastro-resistant coating, a suitable and stir until completely dispersed. A smooth dispersion test is carried out to demonstrate the appropriate release is produced, which passes through a sieve screen with a of the active substance(s), for example the test described in nominal mesh aperture of 710 µm. Dissolution test for solid dosage forms (2.9.3).

Orodispersible tablets

Tablets for use in the mouth

DEFINITION Orodispersible tablets are uncoated tablets intended to be placed in the mouth where they disperse rapidly before being swallowed. TESTS Disintegration. Orodispersible tablets disintegrate within 3 min when examined by the test for disintegration of tablets and capsules (2.9.1).

Modified-release tablets DEFINITION Modified-release tablets are coated or uncoated tablets which contain special excipients or which are prepared by special procedures, or both, designed to modify the rate, the place or the time at which the active substance(s) are released. Modified-release tablets include prolonged-release tablets, delayed-release tablets and pulsatile-release tablets. PRODUCTION A suitable test is carried out to demonstrate the appropriate release of the active substance(s).

Gastro-resistant tablets DEFINITION Gastro-resistant tablets are delayed-release tablets that are intended to resist the gastric fluid and to release their active substance(s) in the intestinal fluid. Usually they are prepared from granules or particles already covered with a gastro-resistant coating or in certain cases by covering tablets with a gastro-resistant coating (enteric-coated tablets). Tablets covered with a gastro-resistant coating conform to the definition of coated tablets. 628

DEFINITION Tablets for use in the mouth are usually uncoated tablets. They are formulated to effect a slow release and local action of the active substance(s) or the release and absorption of the active substance or substances at a defined part of the mouth. They comply with the requirements of the monograph on Oromucosal preparations (1807). 01/2005:1155

TAMPONS, MEDICATED Tamponae medicatae Additional requirements for medicated tampons may be found, where appropriate, in other general monographs, for example Rectal preparations (1145), Vaginal preparations (1164) and Ear preparations (0652). DEFINITION Medicated tampons are solid, single-dose preparations intended to be inserted into the body cavities for a limited period of time. They consist of a suitable material such as cellulose, collagen or silicone impregnated with one or more active substances. PRODUCTION In the manufacture, packaging, storage and distribution of medicated tampons, suitable means are taken to ensure their microbial quality ; recommendations on this aspect are provided in the text on Microbiological quality of pharmaceutical preparations (5.1.4). LABELLING The label states the quantity of active substance(s) per tampon.



Vaginal preparations

01/2005:1164 more active substances dispersed or dissolved in a suitable basis that may be soluble or dispersible in water or may melt at body temperature. Excipients such as diluents, adsorbents, VAGINAL PREPARATIONS surface-active agents, lubricants, antimicrobial preservatives and colouring matter authorised by the competent authority Vaginalia may be added, if necessary. DEFINITION PRODUCTION Vaginal preparations are liquid, semi-solid or solid Pessaries are usually prepared by moulding. Where preparations intended for administration to the vagina appropriate in the manufacture of pessaries, measures are usually in order to obtain a local effect. They contain one or taken to ensure a suitable and controlled particle size of the more active substances in a suitable basis. active substance(s). If necessary, the active substance(s) are Where appropriate, containers for vaginal preparations previously ground and sieved through a suitable sieve. comply with the requirements for Materials used for the When prepared by moulding, the medicated mass, sufficiently manufacture of containers (3.1 and subsections) and liquified by heating, is poured into suitable moulds. The Containers (3.2 and subsections). pessary solidifies on cooling. Various excipients are available Several categories of vaginal preparations may be for this process, such as hard fat, macrogols, cocoa butter, distinguished : and various gelatinous mixtures consisting, for example, of gelatin, water and glycerol. — pessaries, A suitable test is carried out to demonstrate the appropriate — vaginal tablets, release of the active substance(s) from pessaries intended for — vaginal capsules, prolonged local action. — vaginal solutions, emulsions and suspensions, Where appropriate, the determination of the resistance to — tablets for vaginal solutions and suspensions, rupture of pessaries (2.9.24) is carried out. — semi-solid vaginal preparations, TESTS — vaginal foams, Disintegration. Unless intended for prolonged local action, — medicated vaginal tampons. they comply with the test for disintegration of suppositories and pessaries (2.9.2). Examine the state of the pessaries PRODUCTION after 60 min, unless otherwise justified and authorised. In the manufacturing, packaging, storage and distribution of vaginal preparations, suitable means are taken to ensure Vaginal tablets their microbial quality ; recommendations on this aspect are provided in the text on Microbiological quality of DEFINITION pharmaceutical preparations (5.1.4). Vaginal tablets are solid, single-dose preparations. TESTS They generally conform to the definitions of uncoated or Uniformity of content (2.9.6). Unless otherwise prescribed film-coated tablets given in the monograph on Tablets (0478). or justified and authorised, solid single-dose preparations PRODUCTION with a content of active substance less than 2 mg or less than 2 per cent of the total mass comply with test A (vaginal A suitable test is carried out to demonstrate the appropriate tablets) or test B (pessaries, vaginal capsules) for uniformity release of the active substance(s) from vaginal tablets intended for prolonged local action. of content of single-dose preparations. If the preparation has more than one active substance, the requirement applies only to those substances which correspond to the above conditions. Uniformity of mass (2.9.5). Solid single-dose vaginal preparations comply with the test for uniformity of mass of single-dose preparations. If the test for uniformity of content is prescribed for all the active substances, the test for uniformity of mass is not required. Deliverable mass or volume (2.9.28). Liquid and semi-solid vaginal preparations supplied in single-dose containers comply with the test. Dissolution. A suitable test may be carried out to demonstrate the appropriate release of the active substance(s) from solid single-dose preparations, for example one of the tests described in Dissolution test for solid dosage forms (2.9.3). When a dissolution test is prescribed, a disintegration test may not be required.

Pessaries

TESTS Disintegration. Unless intended for prolonged local action, they comply with the test for disintegration of suppositories and pessaries (special method for vaginal tablets, 2.9.2). Examine the state of the tablets after 30 min, unless otherwise justified and authorised.

Vaginal capsules DEFINITION Vaginal capsules (shell pessaries) are solid, single-dose preparations. They are generally similar to soft capsules, differing only in their shape and size. Vaginal capsules have various shapes, usually ovoid. They are smooth and have a uniform external appearance. PRODUCTION A suitable test is carried out to demonstrate the appropriate release of the active substance(s) from vaginal capsules intended for prolonged local action.

DEFINITION Pessaries are solid, single-dose preparations. They have various shapes, usually ovoid, with a volume and consistency suitable for insertion into the vagina. They contain one or

TESTS Disintegration. Unless intended for prolonged local action, they comply with the test for disintegration of suppositories and pessaries (2.9.2). Examine the state of the capsules after 30 min, unless otherwise justified and authorised.


629

Veterinary liquid preparations for cutaneous application

Vaginal solutions, emulsions and suspensions

Vaginal foams

DEFINITION Vaginal solutions, emulsions and suspensions are liquid preparations intended for a local effect, for irrigation or for diagnostic purposes. They may contain excipients, for example to adjust the viscosity of the preparation, to adjust or stabilise the pH, to increase the solubility of the active substance(s) or to stabilise the preparation. The excipients do not adversely affect the intended medical action or, at the concentrations used, cause undue local irritation. Vaginal emulsions may show evidence of phase separation but are readily redispersed on shaking. Vaginal suspensions may show a sediment that is readily dispersed on shaking to give a suspension which remains sufficiently stable to enable a homogeneous preparation to be delivered. They are supplied in single-dose containers. The container is adapted to deliver the preparation to the vagina or it is accompanied by a suitable applicator. PRODUCTION In the manufacture of vaginal suspensions measures are taken to ensure a suitable and controlled particle size with regard to the intended use.

Tablets for vaginal solutions and suspensions DEFINITION Tablets intended for the preparation of vaginal solutions and suspensions are single-dose preparations which are dissolved or dispersed in water at the time of administration. They may contain excipients to facilitate dissolution or dispersion or to prevent caking. Apart from the test for disintegration, tablets for vaginal solutions or suspensions conform with the definition for Tablets (0478). After dissolution or dispersion, they comply with the requirements for vaginal solutions or vaginal suspensions, as appropriate. TESTS Disintegration. Tablets for vaginal solutions or suspensions disintegrate within 3 min when tested according to the test for disintegration of tablets and capsules (2.9.1), but using water R at 15 °C to 25 °C. LABELLING The label states : — the method of preparation of the vaginal solution or suspension, — the conditions and duration of storage of the solution or suspension after constitution.

Semi-solid vaginal preparations DEFINITION Semi-solid vaginal preparations are ointments, creams or gels. They are often supplied in single-dose containers. The container is provided with a suitable applicator. Semi-solid vaginal preparations comply with the requirements of the monograph on Semi-solid preparations for cutaneous application (0132). 630


DEFINITION Vaginal foams comply with the requirements of the monograph on Medicated foams (1105).

Medicated vaginal tampons DEFINITION Medicated vaginal tampons are solid, single-dose preparations intended to be inserted in the vagina for a limited time. They comply with the requirements of the monograph on Medicated tampons (1155). 01/2005:1808

VETERINARY LIQUID PREPARATIONS FOR CUTANEOUS APPLICATION Praeparationes liquidae veterinariae ad usum dermicum Unless otherwise justified and authorised, veterinary liquid preparations for cutaneous application comply with the requirements of the monograph on Liquid preparations for cutaneous application (0927). In addition to these requirements, the following statements apply to veterinary liquid preparations for cutaneous application. DEFINITION Veterinary liquid preparations for cutaneous application are liquid preparations intended to be applied to the skin to obtain a local and/or systemic effect. They are solutions, suspensions or emulsions which may contain one or more active substances in a suitable vehicle. They may be presented as concentrates in the form of wettable powders, pastes, solutions or suspensions, which are used to prepare diluted suspensions or emulsions of active substances. They may contain suitable antimicrobial preservatives, antioxidants and other excipients such as stabilisers, emulsifiers and thickeners. Several categories of veterinary liquid preparations for cutaneous application may be distinguished : — cutaneous foams (see Liquid preparations for cutaneous application (0927)), — dip concentrates, — pour-on preparations, — shampoos (see Liquid preparations for cutaneous application (0927)), — spot-on preparations, — sprays, — teat dips, — teat sprays, — udder-washes.

Dip concentrates DEFINITION Dip concentrates are preparations containing one or more active substances, usually in the form of wettable powders, pastes, solutions or suspensions, which are used to prepare diluted solutions, suspensions or emulsions of active substances. The diluted preparations are applied by complete immersion of the animal.



Veterinary liquid preparations for cutaneous application

Pour-on preparations

Teat dips

DEFINITION Pour-on preparations contain one or more active substances for the prevention and treatment of ectoparasitic and/or endoparasitic infestations of animals. They are applied in volumes which are usually greater than 5 ml by pouring along the animal’s dorsal midline.

DEFINITION Teat dips contain one or more disinfectant active substances, usually in the form of solutions into which the teats of an animal are dipped pre- and, where necessary, post-milking to reduce the population of pathogenic micro-organisms on the surfaces. Teat dips may be supplied/presented as ready-to-use preparations or they may be prepared by dilution of teat dip concentrates. Pre- and post-milking teat dips often differ in formulation. Teat dips usually contain emollients to promote skin hydration, to soften the skin and allow healing of lesions that would otherwise harbour bacteria.

Spot-on preparations

DEFINITION Spot-on preparations contain one or more active substances for the prevention and treatment of ectoparasitic and/or endoparasitic infestations of animals. They are applied Teat sprays in volumes which are usually less than 10 ml, to a small area DEFINITION on the head or back, as appropriate, of the animal. Teat sprays contain one or more disinfectant active substances, usually in the form of solutions which are Sprays sprayed onto the teats of an animal pre- and, where DEFINITION necessary, post-milking to reduce the population of pathogenic micro-organisms on the surfaces. Teat sprays Sprays contain one or more active substances that are may be supplied/presented as ready-to-use preparations or intended to be applied externally for therapeutic or prophylactic purposes. They are delivered in the form of an they may be prepared by dilution of teat spray concentrates. aerosol by the actuation of an appropriate valve or by means Pre- and post-milking sprays often differ in formulation. Teat of a suitable atomising device that is either an integral part sprays usually contain emollients to promote skin hydration, to soften the skin and allow healing of lesions that would of the container or is supplied separately. otherwise harbour bacteria. Sprays may be presented in pressurised containers (see Pressurised pharmaceutical preparations (0523)). When Udder-washes so presented, sprays usually consist of one or more active substances in a suitable vehicle held under pressure with DEFINITION suitable propellants or suitable mixtures of propellants. When otherwise presented, sprays are supplied in well-closed Udder-washes contain one or more disinfectant active containers. substances, usually in the form of solutions which are sprayed onto the udder and teats of an animal to remove mud PRODUCTION and faecal contamination before the application of teat dips During the development and manufacture of a spray, or sprays. Udder-washes are usually prepared by the dilution measures are taken to ensure that the assembled product either of concentrated preparations or of ready-to-use teat conforms to a defined spray rate and spray pattern. dips or teat sprays.


631


BCG for immunotherapy

01/2005:1929 Virulent mycobacteria. Examine the working seed lot as prescribed under Tests, using 10 guinea-pigs. PROPAGATION AND HARVEST BCG FOR IMMUNOTHERAPY The bacteria are grown in a suitable medium for not more than 21 days by surface or submerged culture. The culture BCG ad immunocurationem medium does not contain substances known to cause toxic or allergic reactions in human beings or to cause the bacteria DEFINITION to become virulent for guinea-pigs. The culture is harvested BCG for immunotherapy is a freeze-dried preparation of live and suspended in a sterile liquid medium that protects the bacteria derived from a culture of the bacillus of Calmette viability of the culture as determined by a suitable method of and Guérin (Mycobacterium bovis BCG) whose capacity for viable count. treatment has been established. FINAL BULK It complies with the monograph Vaccines for human The final bulk is prepared from a single harvest or by pooling use (0153). a number of single harvests. A stabiliser may be added ; if the stabiliser interferes with the determination of bacterial PRODUCTION concentration on the final bulk, the determination is carried GENERAL PROVISIONS out before addition of the stabiliser. Only final bulk that complies with the following requirements BCG for immunotherapy shall be produced by a staff may be used in the preparation of the final lot. consisting of healthy persons who do not work with other infectious agents ; in particular they shall not work with Bacterial and fungal contamination. Carry out the test for virulent strains of Mycobacterium tuberculosis, nor shall sterility (2.6.1), using 10 ml of final bulk for each medium. they be exposed to a known risk of tuberculosis infection. The final bulk complies with the test for sterility, except for Staff are examined periodically for tuberculosis. BCG for the presence of mycobacteria. immunotherapy is susceptible to sunlight : the procedures Count of viable units. Determine the number of viable units for production shall be so designed that all products are per millilitre by viable count on solid medium using a protected from direct sunlight and from ultraviolet light at method suitable for the product to be examined or by a all stages of manufacture, testing and storage. suitable biochemical method. Carry out the test in parallel Production is based on a seed-lot system. The production on a reference preparation of the same strain. method shall have been shown to yield consistently BCG Bacterial concentration. Determine the total bacterial products that can be used for treatment of superficial concentration by a suitable method, either directly by bladder cancer and are safe. The product is prepared from cultures which are separated from the master seed lot by as determining the mass of the micro-organisms, or indirectly by an opacity method that has been calibrated in relation to the few subcultures as possible and in any case not more than mass of the micro-organisms ; if the bacterial concentration is 8 subcultures. During the course of these subcultures the determined before addition of a stabiliser, the concentration preparation is not freeze-dried more than once. in the final bulk is established by calculation. The total If a bioluminescence test or other biochemical method is bacterial concentration is within the limits approved for the used instead of viable count, the method is validated against particular product. the viable count for each stage of the process at which it The ratio of the count of viable units to the total bacterial is used. concentration is not less than that approved for the SEED LOTS particular product. The strain used to establish the master seed lot is chosen for and maintained to preserve its characteristics, its capacity to FINAL LOT treat and prevent superficial bladder cancer, and its relative The final bulk is distributed into sterile containers and freeze-dried to a moisture content favourable to the stability absence of pathogenicity for man and laboratory animals. of the product ; the containers are closed either under The strain used shall be identified by historical records vacuum or under an inert gas. that include information on its origin and subsequent Except where the filled and closed containers are stored at a manipulation. temperature of − 20 °C or lower, the expiry date is not later Before establishment of a working seed lot a batch is than 4 years from the date of harvest. prepared and reserved for use as the comparison product. When a new working seed lot is established, a suitable test Only a final lot that complies with the following requirement for delayed hypersensitivity in guinea-pigs is carried out on for count of viable units and with each of the requirements a batch of product prepared from the new working seed given below under Identification, Tests and Assay may be lot ; the product is shown to be not significantly different in released for use. Provided the test for virulent mycobacteria activity from the comparison product. Antimicrobial agent has been carried out with satisfactory results on the final sensitivity testing is also carried out. bulk, it may be omitted on the final lot. Only a working seed lot that complies with the following Count of viable units. Determine the number of viable units requirements may be used for propagation. per millilitre of the reconstituted product by viable count on solid medium using a method suitable for the product to be Identification. The bacteria in the working seed lot examined, or by a suitable biochemical method. The ratio of are identified as Mycobacterium bovis BCG using the count of viable units after freeze-drying to that before is microbiological techniques, which may be supplemented not less than that approved for the particular product. by molecular biology techniques (for example, nucleic acid amplification and restriction-fragment-length polymorphism). Bacterial and fungal contamination. Carry out the test for IDENTIFICATION sterility (2.6.1), using 10 ml for each medium. The working BCG for immunotherapy is identified by microscopic examination of the bacilli in stained smears demonstrating seed lot complies with the test for sterility, except for the their acid-fast property and by the characteristic appearance presence of mycobacteria. General Notices (1) apply to all monographs and other texts

635

BCG vaccine, freeze-dried


of colonies grown on solid medium. Alternatively, molecular biology techniques (for example, nucleic acid amplification) may be used.

PRODUCTION

Temperature stability. Maintain samples of the freeze-dried product at 37 °C for 4 weeks. Determine the number of viable units in the heated product and in unheated product as described below. The number of viable units in the heated product is within the limits approved for the particular product but in any case not less than 20 per cent of that in unheated product. Water. Not more than the limit approved for the particular product, determined by a suitable method.

BACTERIAL SEED LOTS The strain used to establish the master seed lot is chosen for and maintained to preserve its characteristics, its capacity to sensitise man to tuberculin and to protect animals against tuberculosis, and its relative absence of pathogenicity for man and laboratory animals. The strain used shall be identified by historical records that include information on its origin and subsequent manipulation.

GENERAL PROVISIONS BCG vaccine shall be produced by a staff consisting of healthy persons who do not work with other infectious TESTS agents ; in particular they shall not work with virulent strains Virulent mycobacteria. Inject subcutaneously or of Mycobacterium tuberculosis, nor shall they be exposed to intramuscularly into each of 6 guinea-pigs, each weighing a known risk of tuberculosis infection. Staff are examined 250-400 g and having received no treatment likely to interfere periodically for tuberculosis. BCG vaccine is susceptible to with the test, a quantity of the product to be examined sunlight : the procedures for the preparation of the vaccine equivalent to at least 1/25 of 1 human dose. Observe the shall be designed so that all cultures and vaccines are animals for at least 42 days. At the end of this period, kill the protected from direct sunlight and from ultraviolet light at guinea-pigs and examine by autopsy for signs of infection all stages of manufacture, testing and storage. with tuberculosis, ignoring any minor reactions at the site of injection. Animals that die during the observation period Production of the vaccine is based on a seed-lot system. The production method shall have been shown to yield are also examined for signs of tuberculosis. The product complies with the test if none of the guinea-pigs shows signs consistently BCG vaccines that induce adequate sensitivity to tuberculin in man, that have acceptable protective potency of tuberculosis and if not more than 1 animal dies during in animals and are safe. The vaccine is prepared from the observation period. If 2 animals die during this period cultures which are derived from the master seed lot by as and autopsy does not reveal signs of tuberculosis, repeat few subcultures as possible and in any case not more than the test on 6 other guinea-pigs. The product complies with 8 subcultures. During the course of these subcultures the the test if not more than 1 animal dies during the 42 days following the injection and autopsy does not reveal any sign preparation is not freeze-dried more than once. of tuberculosis. If a bioluminescence test or other biochemical method is used instead of viable count, the method is validated against Bacterial and fungal contamination. The reconstituted product complies with the test for sterility (2.6.1) except for the viable count for each stage of the process at which it is used. the presence of mycobacteria.

ASSAY Determine the number of viable units in the reconstituted product by viable count on solid medium using a method suitable for the product to be examined or by a suitable validated biochemical method. The number is within the range stated on the label. Determine the number of viable units in the comparison control in parallel. LABELLING The label states : — the minimum and the maximum number of viable units per dose in the reconstituted product, — that the product must be protected from direct sunlight.

A suitable batch of vaccine is prepared from the first working seed lot and is reserved for use as the comparison vaccine. When a new working seed lot is established, a suitable test for delayed hypersensitivity in guinea-pigs is carried out on a batch of vaccine prepared from the new working seed lot ; the vaccine is shown to be not significantly different in activity from the comparison vaccine. Antimicrobial agent sensitivity testing is also carried out. Only a working seed lot that complies with the following requirements may be used for propagation. Identification. The bacteria in the working seed lot are identified as Mycobacterium bovis BCG using microbiological techniques, which may be supplemented by molecular biology techniques (for example, nucleic acid amplification and restriction-fragment-length polymorphism).

Bacterial and fungal contamination. Carry out the test for sterility (2.6.1), using 10 ml for each medium. The working seed lot complies with the test for sterility except for the 01/2005:0163 presence of mycobacteria. Virulent mycobacteria. Examine the working seed lot as prescribed under Tests, using 10 guinea-pigs.

BCG VACCINE, FREEZE-DRIED

PROPAGATION AND HARVEST The bacteria are grown in a suitable medium for not more than 21 days by surface or submerged culture. The culture medium does not contain substances known to cause toxic DEFINITION or allergic reactions in humans or to cause the bacteria to Freeze-dried BCG vaccine is a preparation of live bacteria become virulent for guinea-pigs. The culture is harvested derived from a culture of the bacillus of Calmette and Guérin and suspended in a sterile liquid medium that protects the (Mycobacterium bovis BCG) whose capacity to protect viability of the vaccine as determined by a suitable method against tuberculosis has been established. of viable count.

Vaccinum tuberculosis (BCG) cryodesiccatum

636


Cholera vaccine


FINAL BULK VACCINE The final bulk vaccine is prepared from a single harvest or by pooling a number of single harvests. A stabiliser may be added ; if the stabiliser interferes with the determination of bacterial concentration in the final bulk vaccine, the determination is carried out before addition of the stabiliser. Only final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Bacterial and fungal contamination. Carry out the test for sterility (2.6.1), using 10 ml for each medium. The final bulk vaccine complies with the test for sterility except for the presence of mycobacteria. Count of viable units. Determine the number of viable units per millilitre by viable count on solid medium using a method suitable for the vaccine to be examined or by a suitable biochemical method. Carry out the test in parallel on a reference preparation of the same strain. Bacterial concentration. Determine the total bacterial concentration by a suitable method, either directly by determining the mass of the micro-organisms, or indirectly by an opacity method that has been calibrated in relation to the mass of the organisms ; if the bacterial concentration is determined before addition of a stabiliser, the concentration in the final bulk vaccine is established by calculation. The total bacterial concentration is within the limits approved for the particular product. The ratio of the count of viable units to the total bacterial concentration is not less than that approved for the particular product. FINAL LOT The final bulk vaccine is distributed into sterile containers and freeze-dried to a moisture content favourable to the stability of the vaccine ; the containers are closed either under vacuum or under an inert gas.

TESTS Virulent mycobacteria. Inject subcutaneously or intramuscularly into each of 6 guinea-pigs, each weighing 250-400 g and having received no treatment likely to interfere with the test, a quantity of vaccine equivalent to at least 50 human doses. Observe the animals for at least 42 days. At the end of this period, kill the guinea-pigs and examine by autopsy for signs of infection with tuberculosis, ignoring any minor reactions at the site of injection. Animals that die during the observation period are also examined for signs of tuberculosis. The vaccine complies with the test if none of the guinea-pigs shows signs of tuberculosis and if not more than 1 animal dies during the observation period. If 2 animals die during this period and autopsy does not reveal signs of tuberculosis repeat the test on 6 other guinea-pigs. The vaccine complies with the test if not more than 1 animal dies during the 42 days following the injection and autopsy does not reveal any sign of tuberculosis. Bacterial and fungal contamination. The reconstituted vaccine complies with the test for sterility (2.6.1) except for the presence of mycobacteria. Excessive dermal reactivity. Use 6 healthy, white or pale-coloured guinea-pigs, each weighing not less than 250 g and having received no treatment likely to interfere with the test. Inject intradermally into each guinea-pig, according to a randomised plan, 0.1 ml of the reconstituted vaccine and of 2 tenfold serial dilutions of the vaccine and identical doses of the comparison vaccine. Observe the lesions formed at the site of the injection for 4 weeks. The vaccine complies with the test if the reaction it produces is not markedly different from that produced by the comparison vaccine. Temperature stability. Maintain samples of the freeze-dried vaccine at 37 °C for 4 weeks. Determine the number of viable units in the heated vaccine and in unheated vaccine as described below. The number of viable units in the heated vaccine is not less than 20 per cent that in unheated vaccine. Water. Not more than the limit approved for the particular product, determined by a suitable method.

Except where the filled and closed containers are stored at a temperature of − 20 °C or lower, the expiry date is not later ASSAY than 4 years from the date of harvest. Determine the number of viable units in the reconstituted Only a final lot that complies with the following requirement vaccine by viable count on solid medium using a method suitable for the vaccine to be examined or by a suitable for count of viable units and with each of the requirements validated biochemical method. The number is within given below under Identification, Tests and Assay may be released for use. Provided the test for virulent mycobacteria the range stated on the label. Determine the number of viable units in the comparison vaccine in parallel. has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Provided the test for excessive dermal reactivity has been carried out with satisfactory results on the working seed lot and on 5 consecutive final lots produced from it, the test may be omitted on the final lot.

LABELLING The label states : — the minimum and maximum number of viable units per millilitre in the reconstituted vaccine, — that the vaccine must be protected from direct sunlight. Count of viable units. Determine the number of viable units per millilitre of the reconstituted vaccine by viable count on solid medium using a method suitable for the vaccine to be examined or by a suitable biochemical method. The ratio of 01/2005:0154 the count of viable units after freeze-drying to that before is not less than that approved for the particular product.

CHOLERA VACCINE

IDENTIFICATION BCG vaccine is identified by microscopic examination of the bacilli in stained smears demonstrating their acid-fast property and by the characteristic appearance of colonies grown on solid medium. Alternatively, molecular biology techniques (for example nucleic acid amplification) may be used. General Notices (1) apply to all monographs and other texts

Vaccinum cholerae DEFINITION Cholera vaccine is a homogeneous suspension of a suitable strain or strains of Vibrio cholerae containing not less than 8 × 109 bacteria in each human dose. The human dose does not exceed 1.0 ml. 637

Cholera vaccine, freeze-dried


PRODUCTION The vaccine is prepared using a seed-lot system. The vaccine consists of a mixture of equal parts of vaccines prepared from smooth strains of the 2 main serological types, Inaba and Ogawa. These may be of the classical biotype with or without the El-Tor biotype. A single strain or several strains of each type may be included. All strains must contain, in addition to their type O antigens, the heat-stable O antigen common to Inaba and Ogawa. If more than one strain each of Inaba and Ogawa are used, these may be selected so as to contain other O antigens in addition. The World Health Organisation recommends new strains which may be used if necessary, in accordance with the regulations in force in the signatory States of the Convention on the Elaboration of a European Pharmacopoeia. In order to comply with the requirements for vaccination certificates required for international travel, the vaccine must contain not less than 8 × 109 organisms of the classical biotype. Each strain is grown separately. The bacteria are inactivated either by heating the suspensions (for example, at 56 °C for 1 h) or by treatment with formaldehyde or phenol or by a combination of the physical and chemical methods. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for immunosera and vaccines for human use (2.6.9) modified as follows : inject 0.5 ml of the vaccine into each mouse and 1.0 ml into each guinea pig. IDENTIFICATION It is identified by specific agglutination tests. TESTS Phenol (2.5.15). If phenol has been used in the preparation, the concentration is not more than 5 g/l. Antibody production. Test the ability of the vaccine to induce antibodies (such as agglutinating, vibriocidal or haemagglutinating antibodies) in the guinea-pig, the rabbit or the mouse. Administer the vaccine to a group of at least 6 animals. At the end of the interval of time necessary for maximum antibody formation, determined in preliminary tests, collect sera from the animals and titrate them individually for the appropriate antibody using a suitable method. The vaccine to be examined passes the test if each serotype has elicited a significant antibody response. Sterility (2.6.1). It complies with the test for sterility.

suspension containing not less than 8 × 109 bacteria in each human dose. The human dose does not exceed 1.0 ml of the reconstituted vaccine. PRODUCTION The vaccine is prepared using a seed-lot system. The vaccine consists of a mixture of equal parts of vaccines prepared from smooth strains of the 2 main serological types, Inaba and Ogawa. These may be of the classical biotype with or without the El-Tor biotype. A single strain or several strains of each type may be included. All strains must contain, in addition to their type O antigens, the heat-stable O antigen common to Inaba and Ogawa. If more than one strain each of Inaba and Ogawa are used, these may be selected so as to contain other O antigens in addition. The World Health Organisation recommends new strains which may be used if necessary in accordance with the regulations in force in the signatory States of the Convention on the Elaboration of a European Pharmacopoeia. In order to comply with the requirements for vaccination certificates required for international travel, the vaccine must contain not less than 8 × 109 organisms of the classical biotype. Each strain is grown separately. The bacteria are inactivated either by heating the suspensions (for example, at 56 °C for 1 h) or by treatment with formaldehyde or by a combination of the physical and chemical methods. Phenol is not used in the preparation. The vaccine is distributed into sterile containers and freeze-dried to a moisture content favourable to the stability of the vaccine. The containers are then closed so as to exclude contamination. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for immunosera and vaccines for human use (2.6.9) modified as follows : inject 0.5 ml of the vaccine into each mouse and 1.0 ml into each guinea pig. IDENTIFICATION The vaccine reconstituted as stated on the label is identified by specific agglutination tests. TESTS Phenol (2.5.15). If phenol has been used in the preparation, the concentration is not more than 5 g/l.

LABELLING The label states : — the method used to inactivate the bacteria, — the number of bacteria in each human dose.

Antibody production. Test the ability of the vaccine to induce antibodies (such as agglutinating, vibriocidal or haemagglutinating antibodies) in the guinea-pig, the rabbit or the mouse. Administer the reconstituted vaccine to a group of at least 6 animals. At the end of the interval of time necessary for maximum antibody formation, determined in preliminary tests, collect sera from the animals and titrate them individually for the appropriate antibody using a suitable method. The vaccine to be examined passes the test 01/2005:0155 if each serotype has elicited a significant antibody response.

CHOLERA VACCINE, FREEZE-DRIED Vaccinum cholerae cryodesiccatum

LABELLING

DEFINITION Freeze-dried cholera vaccine is a preparation of a suitable strain or strains of Vibrio cholerae. The vaccine is reconstituted as stated on the label to give a uniform 638

Sterility (2.6.1). The reconstituted vaccine complies with the test for sterility.

The label states : — the method used to inactivate the bacteria, — the number of bacteria in each human dose.



Diphtheria and tetanus vaccine for adults and adolescents

01/2005:0444 IDENTIFICATION A. Diphtheria toxoid is identified by a suitable immunochemical method (2.7.1). The following method, DIPHTHERIA AND TETANUS VACCINE applicable to certain vaccines, is given as an example. (ADSORBED) Dissolve in the vaccine to be examined sufficient sodium citrate R to give a 100 g/l solution. Maintain at 37 °C for Vaccinum diphtheriae et tetani adsorbatum about 16 h and centrifuge until a clear supernatant liquid is obtained. The clear supernatant liquid reacts with a DEFINITION suitable diphtheria antitoxin, giving a precipitate. Diphtheria and tetanus vaccine (adsorbed) is a preparation B. Tetanus toxoid is identified by a suitable immunochemical of diphtheria formol toxoid and tetanus formol toxoid with method (2.7.1). The following method, applicable to a mineral adsorbent. The formol toxoids are prepared from certain vaccines, is given as an example. The clear the toxins produced by the growth of Corynebacterium supernatant liquid obtained as described in identification diphtheriae and Clostridium tetani, respectively. test A reacts with a suitable tetanus antitoxin, giving a precipitate. PRODUCTION TESTS GENERAL PROVISIONS Specific toxicity of the diphtheria and tetanus components. Aluminium (2.5.13) : maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium The production method is validated to demonstrate that the product, if tested, would comply with the following test: phosphate is used as the adsorbent. inject subcutaneously 5 times the single human dose stated Free formaldehyde (2.4.18) : maximum 0.2 g/l. on the label into each of 5 healthy guinea-pigs, each weighing Antimicrobial preservative. Where applicable, determine 250-350 g, that have not previously been treated with any the amount of antimicrobial preservative by a suitable material that will interfere with the test. If within 42 days of chemical method. The content is not less than the minimum the injection any of the animals shows signs of or dies from amount shown to be effective and is not greater than 115 per diphtheria toxaemia or tetanus, the vaccine does not comply cent of the quantity stated on the label. with the test. If more than 1 animal dies from non-specific Sterility (2.6.1). The vaccine complies with the test for causes, repeat the test once ; if more than 1 animal dies in sterility. the second test, the vaccine does not comply with the test. BULK PURIFIED DIPHTHERIA AND TETANUS TOXOIDS The bulk purified diphtheria and tetanus toxoids are prepared as described in the monographs on Diphtheria vaccine (adsorbed) (0443) and Tetanus vaccine (adsorbed) (0452) and comply with the requirements prescribed therein. FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption of suitable quantities of bulk purified diphtheria toxoid and tetanus toxoid onto a mineral carrier such as hydrated aluminium phosphate or aluminium hydroxide ; the resulting mixture is approximately isotonic with blood. Suitable antimicrobial preservatives may be added. Certain antimicrobial preservatives, particularly those of the phenolic type, adversely affect the antigenic activity and must not be used. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85 per cent and not greater than 115 per cent of the intended amount. Sterility (2.6.1). Carry out the test for sterility using 10 ml for each medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containers are closed so as to prevent contamination. Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the test for antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided the free formaldehyde content has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. General Notices (1) apply to all monographs and other texts

ASSAY Diphtheria component. Carry out one of the prescribed methods for the assay of diphtheria vaccine (adsorbed) (2.7.6). The lower confidence limit (P = 0.95) of the estimated potency is not less than 30 IU per single human dose. Tetanus component. Carry out one of the prescribed methods for the assay of tetanus vaccine (adsorbed) (2.7.8). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. LABELLING The label states : — the minimum number of International Units of each component per single human dose, — where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults, — the name and the amount of the adsorbent, — that the vaccine must be shaken before use, — that the vaccine is not to be frozen. 01/2005:0647

DIPHTHERIA AND TETANUS VACCINE (ADSORBED) FOR ADULTS AND ADOLESCENTS Vaccinum diphtheriae et tetani adulti et adulescentis adsorbatum DEFINITION Diphtheria and tetanus vaccine (adsorbed) for adults and adolescents is a preparation of diphtheria formol toxoid and tetanus formol toxoid with a mineral adsorbent. The 639

Diphtheria and tetanus vaccine for adults and adolescents


formol toxoids are prepared from the toxins produced by the IDENTIFICATION growth of Corynebacterium diphtheriae and Clostridium A. Diphtheria toxoid is identified by a suitable tetani, respectively. It shall have been demonstrated to the immunochemical method (2.7.1). The following method, competent authority that the quantity of diphtheria toxoid applicable to certain vaccines, is given as an example. used does not produce adverse reactions in subjects from Dissolve in the vaccine to be examined sufficient sodium the age groups for which the vaccine is intended. citrate R to give a 100 g/l solution. Maintain at 37 °C for about 16 h and centrifuge until a clear supernatant liquid is obtained. The clear supernatant liquid reacts with a suitable diphtheria antitoxin, giving a precipitate. If a PRODUCTION satisfactory result is not obtained with a vaccine adsorbed on aluminium hydroxide, carry out the test as follows. GENERAL PROVISIONS Centrifuge 15 ml of the vaccine to be examined and Specific toxicity of the diphtheria and tetanus components. suspend the residue in 5 ml of a freshly prepared mixture The production method is validated to demonstrate that of 1 volume of a 56 g/l solution of sodium edetate R and the product, if tested, would comply with the following test : 49 volumes of a 90 g/l solution of disodium hydrogen inject subcutaneously 5 times the single human dose stated phosphate R. Maintain at 37 °C for not less than 6 h and on the label into each of 5 healthy guinea-pigs, each weighing centrifuge. The clear supernatant liquid reacts with a 250-350 g, that have not previously been treated with any suitable diphtheria antitoxin, giving a precipitate. material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from B. Tetanus toxoid is identified by a suitable immunochemical diphtheria toxaemia or tetanus, the vaccine does not comply method (2.7.1). The following method, applicable to with the test. If more than 1 animal dies from non-specific certain vaccines, is given as an example. The clear causes, repeat the test once ; if more than 1 animal dies in supernatant liquid obtained during identification the second test, the vaccine does not comply with the test. test A reacts with a suitable tetanus antitoxin, giving a precipitate. BULK PURIFIED DIPHTHERIA TOXOID AND TETANUS TOXOIDS The bulk purified diphtheria and tetanus toxoids are prepared TESTS as described in the monographs on Diphtheria vaccine (adsorbed) (0443) and Tetanus vaccine (adsorbed) (0452) Aluminium (2.5.13) : maximum 1.25 mg per single human and comply with the requirements prescribed therein. dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. FINAL BULK VACCINE The vaccine is prepared by adsorption of suitable quantities Free formaldehyde (2.4.18) : maximum 0.2 g/l. of bulk purified diphtheria toxoid and tetanus toxoid onto Antimicrobial preservative. Where applicable, determine a mineral carrier such as hydrated aluminium phosphate or the amount of antimicrobial preservative by a suitable aluminium hydroxide ; the resulting mixture is approximately chemical method. The content is not less than the minimum isotonic with blood. Suitable antimicrobial preservatives may amount shown to be effective and is not greater than 115 per be added. Certain antimicrobial preservatives, particularly cent of the quantity stated on the label. those of the phenolic type, adversely affect the antigenic Sterility (2.6.1). The vaccine complies with the test for activity and must not be used. sterility. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85 per cent and not greater than 115 per cent of the intended amount.

ASSAY Diphtheria component. Carry out one of the prescribed methods for the assay of diphtheria vaccine (adsorbed) (2.7.6).

Sterility (2.6.1). Carry out the test for sterility using 10 ml for each medium.

The lower confidence limit (P = 0.95) of the estimated potency is not less than 2 IU per single human dose.

FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containers are closed so as to prevent contamination.

Tetanus component. Carry out one of the prescribed methods for the assay of tetanus vaccine (adsorbed) (2.7.8).

Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the test for antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot.

The lower confidence limit (P = 0.95) of the estimated potency is not less than 20 IU per single human dose. LABELLING The label states : — the minimum number of International Units of each component per single human dose,

Provided the free formaldehyde content has been determined — the name and the amount of the adsorbent, on the bulk purified toxoids or on the final bulk and it has been shown that the content in the final lot will not exceed — that the vaccine must be shaken before use, 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. — that the vaccine is not to be frozen. 640



DIP-TET-HBV

01/2005:2062 FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria DIPHTHERIA, TETANUS AND toxoid, tetanus toxoid and HBsAg onto a mineral carrier such HEPATITIS B (rDNA) VACCINE as aluminium hydroxide or hydrated aluminium phosphate. Suitable antimicrobial preservatives may be added. (ADSORBED) Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Vaccinum diphtheriae, tetani et Antimicrobial preservative. Where applicable, determine hepatitidis B (ADNr) adsorbatum the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85 per cent DEFINITION and not greater than 115 per cent of the intended content. Diphtheria, tetanus and hepatitis B (rDNA) vaccine Sterility (2.6.1). Carry out the test for sterility using 10 ml (adsorbed) is a combined vaccine composed of : diphtheria for each medium. formol toxoid ; tetanus formol toxoid ; hepatitis B surface FINAL LOT antigen (HBsAg) ; a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate. Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements The formol toxoids are prepared from the toxins produced given below under Identification, Tests and Assay may be by the growth of Corynebacterium diphtheriae and released for use. Clostridium tetani, respectively. Provided the test for antimicrobial preservative and the HBsAg is a component protein of hepatitis B virus ; the assays for the diphtheria and tetanus components have been antigen is obtained by recombinant DNA technology. carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. PRODUCTION Provided the content of free formaldehyde has been GENERAL PROVISIONS determined on the bulk purified antigens or on the final The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may clinical efficacy and safety in man. be omitted on the final lot. The content of bacterial endotoxins (2.6.14) in the bulk If an in vivo assay is used for the hepatitis B component, purified diphtheria toxoid and tetanus toxoid is determined provided it has been carried out with satisfactory results on to monitor the purification procedure and to limit the the final bulk vaccine, it may be omitted on the final lot. amount in the final vaccine. For each component, the content of bacterial endotoxins is less than the limit approved Osmolality (2.2.35). The osmolality of the vaccine is within the limits approved for the particular preparation. for the particular vaccine and in any case the contents are such that the final vaccine contains less than 100 IU per IDENTIFICATION single human dose. A. Diphtheria toxoid is identified by a suitable Reference vaccine(s). Provided valid assays can be immunochemical method (2.7.1). The following method, performed, monocomponent reference vaccines may be used applicable to certain vaccines, is given as an example. for the assays on the combined vaccine. If this is not possible Dissolve in the vaccine to be examined sufficient sodium because of interaction between the components of the citrate R to give a 100 g/l solution. Maintain at 37 °C for combined vaccine or because of the difference in composition about 16 h and centrifuge until a clear supernatant liquid between monocomponent reference vaccine and the test is obtained. The clear supernatant liquid reacts with a vaccine, a batch of combined vaccine shown to be effective suitable diphtheria antitoxin, giving a precipitate. in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative B. Tetanus toxoid is identified by a suitable immunochemical batch, strict adherence to the production process used for method (2.7.1). The following method, applicable to the batch tested in clinical trials is necessary. The reference certain vaccines, is given as an example. The clear vaccine may be stabilised by a method that has been shown supernatant liquid obtained during identification to have no effect on the assay procedure. test A reacts with a suitable tetanus antitoxin, giving a precipitate. Specific toxicity of the diphtheria and tetanus components. The production method is validated to demonstrate that C. The assay or, where applicable, the electrophoretic the product, if tested, would comply with the following test : profile, serves also to identify the hepatitis B component inject subcutaneously 5 times the single human dose stated of the vaccine. on the label into each of 5 healthy guinea-pigs, each weighing TESTS 250-350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of Aluminium (2.5.13) : maximum 1.25 mg per single human the injection any of the animals shows signs of or dies from dose, if aluminium hydroxide or hydrated aluminium diphtheria toxaemia or tetanus, the vaccine does not comply phosphate is used as the adsorbent. with the test. If more than 1 animal dies from non-specific Free formaldehyde (2.4.18) : maximum 0.2 g/l. causes, repeat the test once ; if more than 1 animal dies in the second test, the vaccine does not comply with the test. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable PRODUCTION OF THE COMPONENTS chemical method. The content is not less than the minimum The production of the components complies with the amount shown to be effective and is not greater than 115 per requirements of the monographs on Diphtheria vaccine cent of the quantity stated on the label. (adsorbed) (0443), Tetanus vaccine (adsorbed) (0452) and Sterility (2.6.1). It complies with the test for sterility. Hepatitis B vaccine (rDNA) (1056). General Notices (1) apply to all monographs and other texts

641

Diphtheria, tetanus and pertussis (acellular, component) vaccine

Pyrogens (2.6.8). It complies with the test for pyrogens. Inject the equivalent of 1 human dose into each rabbit. ASSAY Diphtheria component. Carry out one of the prescribed methods for the assay of diphtheria vaccine (adsorbed) (2.7.6). The lower confidence limit (P = 0.95) of the estimated potency is not less than 30 IU per single human dose. Tetanus component. Carry out one of the prescribed methods for the assay of tetanus vaccine (adsorbed) (2.7.8). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. Hepatitis B component. It complies with the assay of hepatitis B vaccine (2.7.15). LABELLING The label states : — the minimum number of International Units of diphtheria and tetanus toxoid per single human dose, — the amount of HBsAg per single human dose, — the type of cells used for production of the HBsAg component, — where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults, — the name and the amount of the adsorbent, — that the vaccine must be shaken before use, — that the vaccine is not to be frozen. 01/2005:1931

DIPHTHERIA, TETANUS AND PERTUSSIS (ACELLULAR, COMPONENT) VACCINE (ADSORBED) Vaccinum diphtheriae, tetani et pertussis sine cellulis ex elementis praeparatum adsorbatum


PRODUCTION GENERAL PROVISIONS The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. The content of bacterial endotoxins (2.6.14) in the bulk purified diphtheria toxoid, tetanus toxoid and pertussis components is determined to monitor the purification procedure and to limit the amount in the final vaccine. For each component, the content of bacterial endotoxins is less than the limit approved for the particular vaccine and, in any case, the contents are such that the final vaccine contains less than 100 IU per single human dose. Reference vaccine(s). Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. PRODUCTION OF THE COMPONENTS The production of the components complies with the requirements of the monographs on Diphtheria vaccine (adsorbed) (0443), Tetanus vaccine (adsorbed) (0452) and Pertussis vaccine (acellular, component, adsorbed) (1356). FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption of suitable quantities of bulk purified diphtheria toxoid, tetanus toxoid and pertussis components separately or together onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. Suitable antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85 per cent and not greater than 115 per cent of the intended content. Sterility (2.6.1). Carry out the test for sterility using 10 ml for each medium. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for absence of residual pertussis toxin, irreversibility of pertussis toxoid, free formaldehyde and antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Osmolality (2.2.35). The osmolality of the vaccine is within the limits approved for the particular preparation.

DEFINITION Diphtheria, tetanus and pertussis (acellular, component) vaccine (adsorbed) is a combined vaccine composed of: diphtheria formol toxoid ; tetanus formol toxoid ; individually purified antigenic components of Bordetella pertussis ; a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani, respectively. The vaccine contains either pertussis toxoid or a pertussis-toxin-like protein free from toxic properties, produced by expression of a genetically modified form of the corresponding gene. Pertussis toxoid is prepared from pertussis toxin by a method that renders the latter harmless while maintaining adequate immunogenic properties and avoiding reversion to toxin. The vaccine may also contain filamentous haemagglutinin, pertactin (a 69 kDa IDENTIFICATION outer-membrane protein) and other defined components A. Diphtheria toxoid is identified by a suitable of B. pertussis such as fimbrial-2 and fimbrial-3 antigens. immunochemical method (2.7.1). The following method, The latter 2 antigens may be copurified. The antigenic applicable to certain vaccines, is given as an example. composition and characteristics are based on evidence of Dissolve in the vaccine to be examined sufficient sodium protection and freedom from unexpected reactions in the citrate R to give a 100 g/l solution. Maintain at 37 °C for target group for which the vaccine is intended. 642



about 16 h and centrifuge until a clear supernatant liquid is obtained. The clear supernatant liquid reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.7.1). The following method, applicable to certain vaccines, is given as an example. The clear supernatant liquid obtained as described in identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The pertussis components are identified by a suitable immunochemical method (2.7.1). The following method, applicable to certain vaccines, is given as an example. The clear supernatant liquid obtained as described in identification test A reacts with specific antisera to the pertussis components of the vaccine. TESTS Absence of residual pertussis toxin and irreversibility of pertussis toxoid. This test is not necessary for the product obtained by genetic modification. Use 3 groups each of not fewer than 5 histamine-sensitive mice. Inject intraperitoneally into the first group twice the single human dose of the vaccine stored at 2-8 °C. Inject intraperitoneally into the second group twice the single human dose of the vaccine incubated at 37 °C for 4 weeks. Inject diluent into the third group of mice. After 5 days, inject into each mouse 2 mg of histamine base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 h. The test is invalid if 1 or more control mice die following histamine challenge. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. If 1 mouse dies in either or both of the first and second groups, the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined ; the vaccine complies with the test if, in both of the groups given the vaccine, not more than 5 per cent of the total number of mice die following histamine challenge. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows : inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 2 g/l of gelatin and challenge with histamine as above ; the strain is suitable if more than 50 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine show symptoms of sensitisation. Aluminium (2.5.13) : maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.4.18) : maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than the minimum amount shown to be effective and is not greater than 115 per cent of the quantity stated on the label. Sterility (2.6.1). The vaccine complies with the test for sterility. ASSAY Diphtheria component. Carry out one of the prescribed methods for the assay of diphtheria vaccine (adsorbed) (2.7.6). The lower confidence limit (P = 0.95) of the estimated potency is not less than the minimum potency stated on the label. Unless otherwise justified and authorised, the minimum potency stated on the label is 30 IU per single human dose. General Notices (1) apply to all monographs and other texts

Diphtheria, tetanus and pertussis vaccine (adsorbed)

Tetanus component. Carry out one of the prescribed methods for the assay of tetanus vaccine (adsorbed) (2.7.8). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. Pertussis component. The vaccine complies with the assay of pertussis vaccine (acellular) (2.7.16). LABELLING The label states : — the minimum number of International Units of diphtheria and tetanus toxoid per single human dose, — the names and amounts of the pertussis components per single human dose, — where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults, — the name and the amount of the adsorbent, — that the vaccine must be shaken before use, — that the vaccine is not to be frozen, — where applicable, that the vaccine contains a pertussis toxin-like protein produced by genetic modification. 01/2005:0445

DIPHTHERIA, TETANUS AND PERTUSSIS VACCINE (ADSORBED) Vaccinum diphtheriae, tetani et pertussis adsorbatum DEFINITION Diphtheria, tetanus and pertussis vaccine (adsorbed) is a preparation of diphtheria formol toxoid and tetanus formol toxoid with a mineral adsorbent to which a suspension of inactivated Bordetella pertussis has been added. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani, respectively. PRODUCTION GENERAL PROVISIONS Specific toxicity of the diphtheria and tetanus components. The production method is validated to demonstrate that the product, if tested, would comply with the following test : inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs, each weighing 250-350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria toxaemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from non-specific causes, repeat the test once ; if more than 1 animal dies in the second test, the vaccine does not comply with the test. BULK PURIFIED DIPHTHERIA AND TETANUS TOXOIDS, BULK INACTIVATED B. PERTUSSIS SUSPENSION The bulk purified diphtheria and tetanus toxoids and the inactivated B. pertussis suspension are prepared as described in the monographs on Diphtheria vaccine (adsorbed) (0443), Tetanus vaccine (adsorbed) (0452) and Pertussis vaccine (adsorbed) (0161), respectively, and comply with the requirements prescribed therein. FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption of suitable quantities of bulk purified diphtheria toxoid and tetanus toxoid onto a mineral carrier such as hydrated aluminium 643

Diphtheria, tetanus and pertussis vaccine (adsorbed)


phosphate or aluminium hydroxide and admixture of an appropriate quantity of a suspension of inactivated B. pertussis ; the resulting mixture is approximately isotonic with blood. The B. pertussis concentration of the final bulk vaccine does not exceed that corresponding to an opacity of 20 IU per single human dose. If 2 or more strains of B. pertussis are used, the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units. Suitable antimicrobial preservatives may be added to the bulk vaccine. Certain antimicrobial preservatives, particularly those of the phenolic type, adversely affect the antigenic activity and must not be used. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85 per cent and not greater than 115 per cent of the intended amount. Sterility (2.6.1). Carry out the test for sterility using 10 ml for each medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containers are closed so as to prevent contamination. Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for specific toxicity of the pertussis component, antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided the free formaldehyde content has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. IDENTIFICATION A. Diphtheria toxoid is identified by a suitable immunochemical method (2.7.1). The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine to be examined sufficient sodium citrate R to give a 100 g/l solution. Maintain at 37 °C for about 16 h and centrifuge until a clear supernatant liquid is obtained ; reserve the precipitate for identification test C. The clear supernatant liquid reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.7.1). The following method, applicable to certain vaccines, is given as an example. The clear supernatant liquid obtained during identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. Dissolve in the vaccine to be examined sufficient sodium citrate R to give a 100 g/l solution. Maintain at 37 °C for about 16 h and centrifuge to obtain a bacterial precipitate. Other suitable methods for separating the bacteria from the adsorbent may also be used. Identify pertussis vaccine by agglutination of the bacteria from the resuspended precipitate by antisera specific to B. pertussis or by the assay.

or distribute males and females equally between the groups. Allow the animals access to food and water for at least 2 h before injection and during the test. Inject each mouse of the vaccine group intraperitoneally with 0.5 ml, containing a quantity of the vaccine equivalent to not less than half the single human dose. Inject each mouse of the control group with 0.5 ml of a 9 g/l sterile solution of sodium chloride R, preferably containing the same amount of antimicrobial preservative as that injected with the vaccine. Weigh the groups of mice immediately before the injection and 72 h and 7 days after the injection. The vaccine complies with the test if : (a) at the end of 72 h the total mass of the group of vaccinated mice is not less than that preceding the injection ; (b) at the end of 7 days the average increase in mass per vaccinated mouse is not less than 60 per cent of that per control mouse ; and (c) not more than 5 per cent of the vaccinated mice die during the test. The test may be repeated and the results of the tests combined. Aluminium (2.5.13) : maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.4.18) : maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than the minimum amount shown to be effective and is not greater than 115 per cent of the quantity stated on the label. Sterility (2.6.1). The vaccine complies with the test for sterility. ASSAY Diphtheria component. Carry out one of the prescribed methods for the assay of diphtheria vaccine (adsorbed) (2.7.6). The lower confidence limit (P = 0.95) of the estimated potency is not less than 30 IU per single human dose. Tetanus component. Carry out one of the prescribed methods for the assay of tetanus vaccine (adsorbed) (2.7.8). If the test is carried out in guinea-pigs, the lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose ; if the test is carried out in mice, the lower confidence limit (P = 0.95) of the estimated potency is not less than 60 IU per single human dose. Pertussis component. Carry out the assay of pertussis vaccine (2.7.7). The estimated potency is not less than 4 IU per single human dose and the lower confidence limit (P = 0.95) of the estimated potency is not less than 2 IU per single human dose. LABELLING The label states : — the minimum number of International Units of each component per single human dose, — where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults,

TESTS — the name and the amount of the adsorbent, Specific toxicity of the pertussis component. Use not fewer — that the vaccine must be shaken before use, than 5 mice each weighing 14 g to 16 g for the vaccine group and for the saline control. Use mice of the same sex — that the vaccine is not to be frozen. 644



Diphtheria, tetanus, pertussis and haemophilus type b vaccine

01/2005:1932 The content of bacterial endotoxins (2.6.14) in bulk purified diphtheria toxoid, tetanus toxoid, pertussis components and PRP conjugate is determined to monitor the purification DIPHTHERIA, TETANUS, PERTUSSIS procedure and to limit the amount in the final vaccine. For each component, the content of bacterial endotoxins is less (ACELLULAR, COMPONENT) AND the limit approved for the particular vaccine ; if the HAEMOPHILUS TYPE b CONJUGATE than vaccine is presented with the haemophilus component in a separate container, the contents of the diphtheria, tetanus VACCINE (ADSORBED) and pertussis antigens are in any case such that the final vial for these components contains less than 100 IU per single Vaccinum diphtheriae, tetani, pertussis human dose. sine cellulis ex elementis praeparatum The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal et haemophili stirpe b coniugatum toxicity for immunosera and vaccines for human use (2.6.9). adsorbatum During development studies and wherever revalidation is necessary, it shall be demonstrated by tests in animals DEFINITION that the vaccine induces a T-cell dependent B-cell immune response to PRP. Diphtheria, tetanus, pertussis (acellular, component) and haemophilus type b conjugate vaccine (adsorbed) is a Reference vaccine(s). Provided valid assays can be combined vaccine composed of: diphtheria formol toxoid ; performed, monocomponent reference vaccines may be used tetanus formol toxoid ; individually purified antigenic for the assays on the combined vaccine. If this is not possible components of Bordetella pertussis ; polyribosylribitol because of interaction between the components of the phosphate (PRP) covalently bound to a carrier protein ; a combined vaccine or because of the difference in composition mineral absorbent such as aluminium hydroxide or hydrated between monocomponent reference vaccine and the test aluminium phosphate. The product may be presented with vaccine, a batch of combined vaccine shown to be effective the haemophilus type b component in a separate container, in clinical trials or a batch representative thereof is used as the contents of which are mixed with the other components a reference vaccine. For the preparation of a representative immediately before use. batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference The formol toxoids are prepared from the toxins produced vaccine may be stabilised by a method that has been shown by the growth of Corynebacterium diphtheriae and to have no effect on the assay procedure. Clostridium tetani respectively. PRODUCTION OF THE COMPONENTS The vaccine contains either pertussis toxoid or a The production of the components complies with the pertussis-toxin-like protein free from toxic properties requirements of the monographs on Diphtheria vaccine produced by expression of a genetically modified form of (adsorbed) (0443), Tetanus vaccine (adsorbed) (0452), the corresponding gene. Pertussis toxoid is prepared from pertussis toxin by a method that renders the toxin harmless Pertussis vaccine (acellular, component, adsorbed) (1356) and Haemophilus type b conjugate vaccine (1219). while maintaining adequate immunogenic properties FINAL BULK VACCINE and avoiding reversion to toxin. The acellular pertussis component may also contain filamentous haemagglutinin, Different methods of preparation may be used : a final pertactin (a 69 kDa outer-membrane protein) and other bulk vaccine may be prepared by adsorption, separately or defined components of B. pertussis such as fimbrial-2 and together, of suitable quantities of bulk purified diphtheria fimbrial-3 antigens. The latter 2 antigens may be copurified. toxoid, tetanus toxoid, acellular pertussis components and The antigenic composition and characteristics are based PRP conjugate onto a mineral carrier such as aluminium on evidence of protection and freedom from unexpected hydroxide or hydrated aluminium phosphate ; or 2 final reactions in the target group for which the vaccine is bulks may be prepared and filled separately, one containing intended. the diphtheria, tetanus and pertussis components, the other the haemophilus component, which may be freeze-dried. PRP is a linear copolymer composed of repeated units of 3-β-D-ribofuranosyl-(1→1)-ribitol-5-phosphate [(C10H19O12P)n], Suitable antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following with a defined molecular size and derived from a suitable requirements may be used in the preparation of the final lot. strain of Haemophilus influenzae type b. The carrier protein, when conjugated to PRP, is capable of inducing Antimicrobial preservative. Where applicable, determine a T-cell-dependent B-cell immune response to the the amount of antimicrobial preservative by a suitable polysaccharide. chemical method. The amount is not less than 85 per cent and not greater than 115 per cent of the intended content. PRODUCTION Sterility (2.6.1). Carry out the test for sterility using 10 ml for each medium. GENERAL PROVISIONS The production method shall have been shown to yield FINAL LOT consistently vaccines comparable with the vaccine of proven Only a final lot that is satisfactory with respect to the test clinical efficacy and safety in man. for osmolality shown below and with respect to each of the If the vaccine is presented with the haemophilus component requirements given below under Identification, Tests and in a separate vial, as part of consistency studies the assays of Assay may be released for use. the diphtheria, tetanus and pertussis components are carried Provided the tests for absence of residual pertussis out on a suitable number of batches of vaccine reconstituted toxin, irreversibility of pertussis toxoid and antimicrobial preservative and the assay have been carried out with as for use. For subsequent routine control, the assays of satisfactory results on the final bulk vaccine, they may be these components may be carried out without mixing with omitted on the final lot. the haemophilus component. General Notices (1) apply to all monographs and other texts

645

Diphtheria, tetanus, pertussis and haemophilus type b vaccine

Provided the free formaldehyde content has been determined on the bulk purified antigens or the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. Osmolality. (2.2.35). The osmolality of the vaccine, reconstituted where applicable, is within the limits approved for the particular preparation. pH (2.2.3). The pH of the vaccine, reconstituted if necessary, is within the range approved for the particular product. Free PRP. Unbound PRP is determined after removal of the conjugate, for example by anion-exchange, size-exclusion or hydrophobic chromatography, ultrafiltration or other validated methods. The amount of free PRP is not greater than that approved for the particular product. IDENTIFICATION If the vaccine is presented with the haemophilus component in a separate vial : identification tests A, B and C are carried out using the vial containing the diphtheria, tetanus and pertussis components ; identification test D is carried out on the vial containing the haemophilus components. A. Diphtheria toxoid is identified by a suitable immunochemical method (2.7.1). The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine to be examined sufficient sodium citrate R to give a 100 g/l solution. Maintain at 37 °C for about 16 h and centrifuge until a clear supernatant liquid is obtained. The clear supernatant liquid reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.7.1). The following method, applicable to certain vaccines, is given as an example. The clear supernatant liquid obtained as described in identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The pertussis components are identified by a suitable immunochemical method (2.7.1). The following method, applicable to certain vaccines, is given as an example. The clear supernatant liquid obtained as described in identification test A reacts with a specific antisera to the pertussis components of the vaccine. D. The haemophilus component is identified by a suitable immunochemical method (2.7.1) for PRP. TESTS If the product is presented with the haemophilus component in a separate container : the tests for absence of residual pertussis toxin, irreversibility of pertussis toxoid, aluminium, free formaldehyde, antimicrobial preservative and sterility are carried out on the container with the diphtheria, tetanus and pertussis components ; the tests for PRP content, water (where applicable), sterility and pyrogens are carried out on the container with the haemophilus component. If the haemophilus component is freeze-dried, some tests may be carried out on the freeze-dried product rather than on the bulk conjugate where the freeze-drying process may affect the component to be tested. Absence of residual pertussis toxin and irreversibility of pertussis toxoid. This test is not necessary for the product obtained by genetic modification. Use 3 groups each of not fewer than 5 histamine-sensitive mice. Inject intraperitoneally into the first group twice the single human dose of the vaccine stored at 2-8 °C. Inject intraperitoneally into the second group twice the single human dose of the vaccine incubated at 37 °C for 4 weeks. Inject diluent into 646


the third group of mice. After 5 days, inject into each mouse 2 mg of histamine base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 h. The test is invalid if 1 or more control mice die following histamine challenge. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. If 1 mouse dies in either or both of the first and second groups, the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined ; the vaccine complies with the test if, in both of the groups given the vaccine, not more than 5 per cent of the total number of mice die following histamine challenge. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows : inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 2 g/l of gelatin and challenge with histamine as above ; the strain is suitable if more than 50 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine show symptoms of sensitisation. PRP : minimum 80 per cent of the amount of PRP stated on the label. PRP is determined either by assay of ribose (2.5.31) or phosphorus (2.5.18), by an immunochemical method (2.7.1) or by anion-exchange liquid chromatography (2.2.29) with pulsed-amperometric detection. Aluminium (2.5.13) : maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.4.18) : maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than the minimum amount shown to be effective and is not greater than 115 per cent of the quantity stated on the label. Water (2.5.12) : maximum 3.0 per cent for the freeze-dried haemophilus component. Sterility (2.6.1). It complies with the test for sterility. Pyrogens (2.6.8). It complies with the test for pyrogens. Inject per kilogram of the rabbit’s mass a quantity of the vaccine equivalent to : 1 µg of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria protein as carrier ; 0.1 µg of PRP for a vaccine with tetanus toxoid as carrier ; 0.025 µg of PRP for a vaccine with OMP as carrier. ASSAY Diphtheria component. Carry out one of the prescribed methods for the assay of diphtheria vaccine (adsorbed) (2.7.6). The lower confidence limit (P = 0.95) of the estimated potency is not less than the minimum potency stated on the label. Unless otherwise justified and authorised, the minimum potency stated on the label is 30 IU per single human dose. Tetanus component. Carry out one of the prescribed methods for the assay of tetanus vaccine (adsorbed) (2.7.8). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. Pertussis component. The vaccine complies with the assay of pertussis vaccine (acellular) (2.7.16). LABELLING The label states : — the minimum number of International Units of diphtheria and tetanus toxoid per single human dose,



Diphtheria, tetanus, pertussis and hepatitis B vaccine

Reference vaccine(s). Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. PRODUCTION OF THE COMPONENTS The production of the components complies with the 01/2005:1933 requirements of the monographs on Diphtheria vaccine (adsorbed) (0443), Tetanus vaccine (adsorbed) (0452), DIPHTHERIA, TETANUS, PERTUSSIS Pertussis vaccine (acellular, component, adsorbed) (1356) and Hepatitis B vaccine (rDNA) (1056). (ACELLULAR, COMPONENT) AND FINAL BULK VACCINE HEPATITIS B (rDNA) VACCINE The final bulk vaccine is prepared by adsorption, separately (ADSORBED) or together, of suitable quantities of bulk purified diphtheria toxoid, tetanus toxoid, acellular pertussis components and Vaccinum diphtheriae, tetani, pertussis hepatitis B surface antigen onto a mineral carrier such as sine cellulis ex elementis praeparatum et aluminium hydroxide or hydrated aluminium phosphate. Suitable antimicrobial preservatives may be added. hepatitidis B (ADNr) adsorbatum Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. DEFINITION Diphtheria, tetanus, pertussis (acellular, component) and Antimicrobial preservative. Where applicable, determine hepatitis B (rDNA) vaccine (adsorbed) is a combined vaccine the amount of antimicrobial preservative by a suitable composed of: diphtheria formol toxoid ; tetanus formol chemical method. The amount is not less than 85 per cent toxoid ; individually purified antigenic components of and not greater than 115 per cent of the intended content. Bordella pertussis ; hepatitis B surface antigen ; a mineral Sterility (2.6.1). Carry out the test for sterility using 10 ml adsorbent such as aluminium hydroxide or hydrated for each medium. aluminium phosphate. FINAL LOT The formol toxoids are prepared from the toxins produced Only a final lot that is satisfactory with respect to the test by the growth of Corynebacterium diphtheriae and for osmolality and with respect to each of the requirements Clostridium tetani, respectively. given below under Identification, Tests and Assay may be The vaccine contains either pertussis toxoid or a released for use. pertussis-toxin-like protein free from toxic properties, produced by expression of a genetically modified form of Provided the tests for absence of residual pertussis the corresponding gene. Pertussis toxoid is prepared from toxin, irreversibility of pertussis toxoid and antimicrobial pertussis toxin by a method that renders the latter harmless preservative and the assays for the diphtheria, tetanus and while maintaining adequate immunogenic properties pertussis components have been carried out with satisfactory and avoiding reversion to toxin. The vaccine may also results on the final bulk vaccine, they may be omitted on contain filamentous haemagglutinin, pertactin (a 69 kDa the final lot. outer-membrane protein) and other defined components Provided the content of free formaldehyde has been of B. pertussis such as fimbrial-2 and fimbrial-3 antigens. determined on the bulk purified antigens or on the final The latter 2 antigens may be copurified. The antigenic bulk and it has been shown that the content in the final lot composition and characteristics are based on evidence of will not exceed 0.2 g/l, the test for free formaldehyde may protection and freedom from unexpected reactions in the be omitted on the final lot. target group for which the vaccine is intended. If an in vivo assay is used for the hepatitis B component, Hepatitis B surface antigen is a component protein of provided it has been carried out with satisfactory results on hepatitis B virus ; the antigen is obtained by recombinant the final bulk vaccine, it may be omitted on the final lot. DNA technology. Osmolality (2.2.35). The osmolality of the vaccine is within PRODUCTION the limits approved for the particular preparation. GENERAL PROVISIONS IDENTIFICATION The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven A. Diphtheria toxoid is identified by a suitable clinical efficacy and safety in man. immunochemical method (2.7.1). The following method, applicable to certain vaccines, is given as an example. The content of bacterial endotoxins (2.6.14) in the bulk Dissolve in the vaccine to be examined sufficient sodium purified diphtheria toxoid, tetanus toxoid and pertussis citrate R to give a 100 g/l solution. Maintain at 37 °C for components is determined to monitor the purification about 16 h and centrifuge until a clear supernatant liquid procedure and to limit the amount in the final vaccine. For is obtained. The clear supernatant liquid reacts with a each component, the content of bacterial endotoxins is less suitable diphtheria antitoxin, giving a precipitate. than the limit approved for the particular vaccine. — the names and amounts of the pertussis components per single human dose, — the number of micrograms of PRP per single human dose, — the type and nominal amount of carrier protein per single human dose, — where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults, — the name and the amount of the adsorbent, — that the vaccine must be shaken before use, — that the vaccine is not to be frozen, — where applicable, that the vaccine contains a pertussis toxin-like protein produced by genetic modification.


647

DIP-TET-PERa-IPV


B. Tetanus toxoid is identified by a suitable immunochemical method (2.7.1). The following method, applicable to certain vaccines, is given as an example. The clear supernatant liquid obtained as described in identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The pertussis components are identified by a suitable immunochemical method (2.7.1). The following method, applicable to certain vaccines, is given as an example. The clear supernatant liquid obtained as described in identification test A reacts with a specific antisera to the pertussis components of the vaccine. D. The assay or, where applicable, the electrophoretic profile, serves also to identify the hepatitis B component of the vaccine.

TESTS Absence of residual pertussis toxin and irreversibility of pertussis toxoid. This test is not necessary for the product obtained by genetic modification. Use 3 groups each of not fewer than 5 histamine-sensitive mice. Inject intraperitoneally into the first group twice the single human dose of the vaccine stored at 2-8 °C. Inject intraperitoneally into the second group twice the single human dose of the vaccine incubated at 37 °C for 4 weeks. Inject diluent into the third group of mice. After 5 days, inject into each mouse 2 mg of histamine base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 h. The test is invalid if 1 or more control mice die following histamine challenge. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. If 1 mouse dies in either or both of the first and second groups, the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined ; the vaccine complies with the test if, in both of the groups given the vaccine, not more than 5 per cent of the total number of mice die following histamine challenge. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows : inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 2 g/l of gelatin and challenge with histamine as above ; the strain is suitable if more than 50 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine show symptoms of sensitisation.

ASSAY Diphtheria component. Carry out one of the prescribed methods for the assay of diphtheria vaccine (adsorbed) (2.7.6). The lower confidence limit (P = 0.95) of the estimated potency is not less than the minimum potency stated on the label. Unless otherwise justified and authorised, the minimum potency stated on the label is 30 IU per single human dose. Tetanus component. Carry out one of the prescribed methods for the assay of tetanus vaccine (adsorbed) (2.7.8). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. Pertussis component. The vaccine complies with the assay of pertussis vaccine (acellular) (2.7.16). Hepatitis B component. The vaccine complies with the assay of hepatitis B vaccine (2.7.15). LABELLING The label states : — the minimum number of International Units of diphtheria and tetanus toxoid per single human dose, — the names and amounts of the pertussis components per single human dose, — the amount of HBsAg per single human dose, — the type of cells used for production of the hepatitis B component, — where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults, — the name and the amount of the adsorbent, — that the vaccine must be shaken before use, — that the vaccine is not to be frozen, — where applicable, that the vaccine contains a pertussis toxin-like protein produced by genetic modification. 01/2005:1934

DIPHTHERIA, TETANUS, PERTUSSIS (ACELLULAR, COMPONENT) AND POLIOMYELITIS (INACTIVATED) VACCINE (ADSORBED) Vaccinum diphtheriae, tetani, pertussis sine cellulis ex elementis praeparatum et poliomyelitidis inactivatum adsorbatum

DEFINITION Diphtheria, tetanus, pertussis (acellular, component) and poliomyelitis (inactivated) vaccine (adsorbed) is a combined vaccine containing : diphtheria formol toxoid ; tetanus Free formaldehyde (2.4.18) : maximum 0.2 g/l. formol toxoid ; individually purified antigenic components of Bordetella pertussis ; suitable strains of human polioviruses Antimicrobial preservative. Where applicable, determine 1, 2 and 3 grown in suitable cell cultures and inactivated by the amount of antimicrobial preservative by a suitable chemical method. The content is not less than the minimum a validated method ; a mineral adsorbent such as aluminium amount shown to be effective and is not greater than 115 per hydroxide or hydrated aluminium phosphate. The formol toxoids are prepared from the toxins produced cent of the quantity stated on the label. by the growth of Corynebacterium diphtheriae and Sterility (2.6.1). The vaccine complies with the test for Clostridium tetani respectively. sterility. The vaccine contains either pertussis toxoid or a Pyrogens (2.6.8). The vaccine complies with the test for pertussis-toxin-like protein free from toxic properties pyrogens. Inject the equivalent of 1 human dose into each produced by expression of a genetically modified form of rabbit. the corresponding gene. Pertussis toxoid is prepared from Aluminium (2.5.13) : maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent.

648



DIP-TET-PERa-IPV

Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85 per cent and not greater than 115 per cent of the intended content. Sterility (2.6.1). Carry out the test for sterility using 10 ml for each medium. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. PRODUCTION Provided the tests for absence of residual pertussis GENERAL PROVISIONS toxin, irreversibility of pertussis toxoid and antimicrobial The production method shall have been shown to yield preservative and the assays for the diphtheria, tetanus and consistently vaccines comparable with the vaccine of proven pertussis components have been carried out with satisfactory clinical efficacy and safety in man. results on the final bulk vaccine, they may be omitted on The production method is validated to demonstrate that the the final lot. Provided the free formaldehyde content has been determined product, if tested, would comply with the test for abnormal toxicity for immunosera and vaccines for human use (2.6.9). on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed The content of bacterial endotoxins (2.6.14) in bulk purified 0.2 g/l, the test for free formaldehyde may be omitted on diphtheria toxoid, tetanus toxoid, pertussis components the final lot. and purified, inactivated monovalent poliovirus harvests is Provided that the determination of D-antigen content has determined to monitor the purification procedure and to been carried out with satisfactory results during preparation limit the amount in the final vaccine. For each component, of the final bulk before addition of the adsorbent, it may be the content of bacterial endotoxins is less than the limit omitted on the final lot. approved for the particular vaccine and, in any case, the Provided that the in vivo assay for the poliomyelitis contents are such that the final vaccine contains less than component has been carried out with satisfactory results on 100 IU per single human dose. the final bulk vaccine, it may be omitted on the final lot. Reference vaccine(s). Provided valid assays can be Osmolality (2.2.35). The osmolality of the vaccine is within performed, monocomponent reference vaccines may be used the limits approved for the particular preparation. for the assays on the combined vaccine. If this is not possible because of interaction between the components of the IDENTIFICATION combined vaccine or because of the difference in composition A. Diphtheria toxoid is identified by a suitable between monocomponent reference vaccine and the test immunochemical method (2.7.1). The following method, vaccine, a batch of combined vaccine shown to be effective applicable to certain vaccines, is given as an example. in clinical trials or a batch representative thereof is used as Dissolve in the vaccine to be examined sufficient sodium a reference vaccine. For the preparation of a representative citrate R to give a 100 g/l solution. Maintain at 37 °C for batch, strict adherence to the production process used for about 16 h and centrifuge until a clear supernatant liquid the batch tested in clinical trials is necessary. The reference is obtained. The clear supernatant liquid reacts with a vaccine may be stabilised by a method that has been shown suitable diphtheria antitoxin, giving a precipitate. to have no effect on the assay procedure. B. Tetanus toxoid is identified by a suitable immunochemical PRODUCTION OF THE COMPONENTS method (2.7.1). The following method, applicable to The production of the components complies with the certain vaccines, is given as an example. The clear requirements of the monographs on Diphtheria vaccine supernatant liquid obtained as described in identification (adsorbed) (0443), Tetanus vaccine (adsorbed) (0452), test A reacts with a suitable tetanus antitoxin, giving a Pertussis vaccine (acellular, component, adsorbed) (1356) precipitate. and Poliomyelitis vaccine (inactivated) (0214). C. The pertussis components are identified by a suitable immunochemical method (2.7.1). The following method, FINAL BULK VACCINE applicable to certain vaccines, is given as an example. The final bulk vaccine is prepared by adsorption onto a The clear supernatant liquid obtained as described in mineral carrier such as aluminium hydroxide or hydrated identification test A reacts with a specific antisera to the aluminium phosphate, separately or together, of suitable pertussis components of the vaccine. quantities of bulk purified diphtheria toxoid, tetanus D. The vaccine is shown to contain human polioviruses 1, toxoid, acellular pertussis components and admixture of 2 and 3 by a suitable immunochemical method (2.7.1) suitable quantities of purified monovalent harvests of such as the determination of D-antigen by enzyme-linked human polioviruses 1, 2 and 3 or a suitable quantity of a immunosorbent assay (ELISA). trivalent pool of such purified monovalent harvests. Suitable antimicrobial preservatives may be added. TESTS Only a final bulk vaccine that complies with the following Absence of residual pertussis toxin and irreversibility requirements may be used in the preparation of the final lot. of pertussis toxoid. This test is not necessary for the product obtained by genetic modification. Use 3 groups Bovine serum albumin. Determined on the poliomyelitis each of not fewer than 5 histamine-sensitive mice. Inject components by a suitable immunochemical method (2.7.1) intraperitoneally into the first group twice the single human after virus harvest and before addition of the adsorbent in dose of the vaccine stored at 2-8 °C. Inject intraperitoneally the preparation of the final bulk vaccine, the amount of into the second group twice the single human dose of the bovine serum albumin is such that the content in the final vaccine will be not more than 50 ng per single human dose. vaccine incubated at 37 °C for 4 weeks. Inject diluent into pertussis toxin by a method that renders the toxin harmless while maintaining adequate immunogenic properties and avoiding reversion to toxin. The vaccine may also contain filamentous haemagglutinin, pertactin (a 69 kDa outer-membrane protein) and other defined components of B. pertussis such as fimbrial-2 and fimbrial-3 antigens. The latter 2 antigens may be copurified. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended.


649

DIP-TET-PERa-HBV-IPV-HIB


LABELLING The label states : — the minimum number of International Units of diphtheria and tetanus toxoid per single human dose, — the names and amounts of the pertussis components per single human dose, — the types of poliovirus contained in the vaccine, — the nominal amount of poliovirus of each type (1, 2 and 3), expressed in European Pharmacopoeia units of D-antigen, per single human dose, — the type of cells used for production of the poliomyelitis component, The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows : inject intravenously — where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for threefold dilutions of a reference pertussis toxin preparation reinforcing doses or for administration to adults, in phosphate-buffered saline solution containing 2 g/l of gelatin and challenge with histamine as above ; the strain is — the name and the amount of the adsorbent, suitable if more than 50 per cent of the animals are sensitised — that the vaccine must be shaken before use, by 50 ng of pertussis toxin and none of the control animals — that the vaccine is not to be frozen, injected with only diluent and challenged similarly with — where applicable, that the vaccine contains a pertussis histamine show symptoms of sensitisation. toxin-like protein produced by genetic modification. Aluminium (2.5.13) : maximum 1.25 mg per single human dose if aluminium hydroxide or hydrated aluminium 01/2005:2067 phosphate is used as the adsorbent. the third group of mice. After 5 days, inject into each mouse 2 mg of histamine base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 h. The test is invalid if 1 or more control mice die following histamine challenge. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. If 1 mouse dies in either or both of the first and second groups, the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined ; the vaccine complies with the test if, in both of the groups given the vaccine, not more than 5 per cent of the total number of mice die following histamine challenge.

Free formaldehyde (2.4.18) : maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than the minimum amount shown to be effective and is not greater than 115 per cent of the quantity stated on the label. Sterility (2.6.1). It complies with the test for sterility. ASSAY Diphtheria component. Carry out one of the prescribed methods for the assay of diphtheria vaccine (adsorbed) (2.7.6). The lower confidence limit (P = 0.95) of the estimated potency is not less than the minimum potency stated on the label.

DIPHTHERIA, TETANUS, PERTUSSIS (ACELLULAR, COMPONENT), HEPATITIS B (rDNA), POLIOMYELITIS (INACTIVATED) AND HAEMOPHILUS TYPE b CONJUGATE VACCINE (ADSORBED) Vaccinum diphtheriae, tetani, pertussis sine cellulis ex elementis praeparatum, hepatitidis B (ADNr), poliomyelitidis inactivatum et haemophili stirpe b coniugatum adsorbatum

DEFINITION Diphtheria, tetanus, pertussis (acellular, component), hepatitis B (rDNA), poliomyelitis (inactivated) and haemophilus type b conjugate vaccine (adsorbed) is a Tetanus component. Carry out one of the prescribed methods for the assay of tetanus vaccine (adsorbed) (2.7.8). combined vaccine composed of : diphtheria formol toxoid ; tetanus formol toxoid ; individually purified antigenic The lower confidence limit (P = 0.95) of the estimated components of Bordetella pertussis ; hepatitis B surface potency is not less than 40 IU per single human dose. antigen (HBsAg) ; human polioviruses 1, 2 and 3 grown in Pertussis component. The vaccine complies with the assay suitable cell cultures and inactivated by a suitable method ; polyribosylribitol phosphate (PRP) covalently bound to of pertussis vaccine (acellular) (2.7.16). a carrier protein. The antigens in the vaccine may be Poliomyelitis component adsorbed on a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. The product may be D-antigen content. As a measure of consistency of presented with the haemophilus component in a separate production, determine the D-antigen content for human polioviruses 1, 2 and 3 by a suitable immunochemical method container, the contents of which are mixed with the other components immediately before or during use. (2.7.1) following desorption using a reference preparation The formol toxoids are prepared from the toxins produced calibrated in European Pharmacopoeia D-antigen units. by the growth of Corynebacterium diphtheriae and For each type, the content, expressed with reference to the amount of D-antigen stated on the label, is within the limits Clostridium tetani respectively. approved for the particular product. The vaccine contains either pertussis toxoid or a pertussis-toxin-like protein free from toxic properties Poliomyelitis vaccine (inactivated) BRP is calibrated in produced by expression of a genetically modified form of European Pharmacopoeia units and intended for use in the the corresponding gene. Pertussis toxoid is prepared from assay of D-antigen. The European Pharmacopoeia unit and pertussis toxin by a method that renders the toxin harmless the International Unit are equivalent. while maintaining adequate immunogenic properties In vivo test. The vaccine complies with the in vivo assay of and avoiding reversion to toxin. The acellular pertussis poliomyelitis vaccine (inactivated) (2.7.20). component may also contain filamentous haemagglutinin, Unless otherwise justified and authorised, the minimum potency stated on the label is 30 IU per single human dose.

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pertactin (a 69 kDa outer-membrane protein) and other defined components of B. pertussis such as fimbrial-2 and fimbrial-3 antigens. The latter 2 antigens may be copurified. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended. Hepatitis B surface antigen is a component protein of hepatitis B virus ; the antigen is obtained by recombinant DNA technology. PRP is a linear copolymer composed of repeated units of 3-βD-ribofuranosyl-(1 → 1)-ribitol-5-phosphate [(C10H19O12P)n], with a defined molecular size and derived from a suitable strain of Haemophilus influenzae type b. The carrier protein, when conjugated to PRP, is capable of inducing a T-cell-dependent B-cell immune response to the polysaccharide. PRODUCTION GENERAL PROVISIONS The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. If the vaccine is presented with the haemophilus component in a separate vial, as part of consistency studies the assays of the diphtheria, tetanus, pertussis, hepatitis B and poliomyelitis components are carried out on a suitable number of batches of vaccine reconstituted as for use. For subsequent routine control, the assays of these components may be carried out without mixing with the haemophilus component. The content of bacterial endotoxins (2.6.14) in bulk purified diphtheria toxoid, bulk purified tetanus toxoid, bulk purified pertussis components, the hepatitis B surface antigen, the purified, inactivated monovalent poliovirus harvests and bulk PRP conjugate is determined to monitor the purification procedure and to limit the amount in the final vaccine. For each component, the content of bacterial endotoxins is not greater than the limit approved. During development studies and wherever revalidation is necessary, a test for pyrogens in rabbits (2.6.8) is carried out by injection of a suitable dose of the final lot. The vaccine is shown to be acceptable with respect to absence of pyrogenic activity. The production method is validated to demonstrate that the product, if tested, would comply with the following test for specific toxicity of the diphtheria and tetanus component : inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs, each weighing 250-350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria, toxaemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from non-specific causes, repeat the test once ; if more than 1 animal dies in the second test, the vaccine does not comply with the test. During development studies and wherever revalidation is necessary, it shall be demonstrated by tests in animals that the vaccine induces a T-cell-dependent B-cell immune response to PRP. The stability of the final lot and relevant intermediates is evaluated using one or more indicator tests. For the haemophilus component, such tests may include determination of molecular size, determination of free PRP in the conjugate and kinetics of depolymerisation. Taking account of the results of the stability testing, release General Notices (1) apply to all monographs and other texts


requirements are set for these indicator tests to ensure that the vaccine will be satisfactory at the end of the period of validity. Reference vaccine(s). Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. PRODUCTION OF THE COMPONENTS The production of the components complies with the requirements of the monographs on Diphtheria vaccine (adsorbed) (0443), Tetanus vaccine (adsorbed) (0452), Pertussis vaccine (acellular, component, adsorbed) (1356), Hepatitis B vaccine (rDNA) (1056), Poliomyelitis vaccine (inactivated) (0214) and Haemophilus type b conjugate vaccine (1219). FINAL BULKS Vaccine with all components in the same container. The final bulk is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, bulk purified tetanus toxoid, bulk purified acellular pertussis components and bulk purified hepatitis B surface antigen onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate and admixture of a suitable quantity of PRP conjugate and suitable quantities of purified and inactivated, monovalent harvests of human polioviruses 1, 2 and 3 or a suitable quantity of a trivalent pool of such monovalent harvests. Suitable antimicrobial preservatives may be added. Vaccine with the haemophilus component in a separate container. The final bulk of diphtheria, tetanus, pertussis, hepatitis B and poliovirus component is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, bulk purified tetanus toxoid, bulk purified acellular pertussis components and bulk purified hepatitis B surface antigen onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate and admixture of suitable quantities of purified and inactivated, monovalent harvests of human polioviruses 1, 2 and 3 or a suitable pool of such monovalent harvests. This final bulk is filled separately. Suitable antimicrobial preservatives may be added. The final bulk of the haemophilus component is prepared by dilution of the bulk conjugate to the final concentration with a suitable diluent. A stabiliser may be added. Only final bulks that comply with the following requirements may be used in the preparation of the final lot. Bovine serum albumin. Determined on the poliomyelitis components by a suitable immunochemical method (2.7.1) after purification of the harvests and before preparation of the final bulk vaccine, before addition of the adsorbent, the amount of bovine serum albumin is such that the content in the final vaccine will be not more than 50 ng per single human dose. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85 per cent and not greater than 115 per cent of the intended content. 651



Sterility (2.6.1). Carry out the test for sterility using 10 ml for each medium. FINAL LOT Where the haemophilus component is in a separate container, the final bulk of the haemophilus component is freeze-dried. Only a final lot that is satisfactory with respect to the test for osmolality shown below and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for osmolality, for absence of residual pertussis toxin and irreversibility of pertussis toxoid and for antimicrobial preservative and the diphtheria, tetanus and pertussis component assays have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided the free formaldehyde content has been determined on the bulk purified antigens and the purified monovalent harvests or the trivalent pool of polioviruses or the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the trivalent pool of inactivated monovalent harvests of polioviruses or on the final bulk vaccine, it may be omitted on the final lot. If an in vivo assay is used for the hepatitis B component, provided it has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Provided the in vivo assay for the poliomyelitis component has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Free PRP. For vaccines with all components in the same container, the free PRP content is determined on the non-absorbed fraction. Unbound PRP is determined on the haemophilus component after removal of the conjugate, for example by anion-exchange, size-exclusion or hydrophobic chromatography, ultrafiltration or other validated methods. The amount of free PRP is not greater than that approved for the particular product. Bacterial endotoxins (2.6.14) : less than the limit approved for the particular product. Osmolality (2.2.35). The osmolality of the vaccine, reconstituted where applicable, is within the limits approved for the particular preparation. IDENTIFICATION If the vaccine is presented with the haemophilus component in a separate vial : identification tests A, B, C, D and E are carried out using the vial containing the diphtheria, tetanus, pertussis, hepatitis B and poliomyelitis components ; identification test F is carried out on the vial containing the haemophilus components. A. Diphtheria toxoid is identified by a suitable immunochemical method (2.7.1). The following method is given as an example. Dissolve in the vaccine to be examined sufficient sodium citrate R to give a 100 g/l solution. Maintain at 37 °C for about 16 h and centrifuge until a clear supernatant liquid is obtained. The clear supernatant liquid reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.7.1). The following method is given as an example. The clear supernatant liquid obtained during identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. 652

C. The clear supernatant liquid obtained during identification test A reacts with a specific antisera to the pertussis components of the vaccine when examined by suitable immunochemical methods (2.7.1). D. The hepatitis B component is identified by a suitable immunochemical method (2.7.1), for example the in vitro assay or by a suitable electrophoretic method (2.2.31). E. The vaccine is shown to contain human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.7.1), such as determination of D-antigen by enzyme-linked immunosorbent assay (ELISA). F. The PRP and the haemophilus carrier protein are identified by a suitable immunochemical method (2.7.1). TESTS If the product is presented with the haemophilus component in a separate container : the tests for absence of residual pertussis toxin and irreversibility of pertussis toxoid, free formaldehyde, aluminium, antimicrobial preservative and sterility are carried out on the container with the diphtheria, tetanus, pertussis, poliomyelitis and hepatitis B components ; the tests for PRP content, water, antimicrobial preservative, aluminium (where applicable) and sterility are carried out on the container with the haemophilus component. Some tests for the haemophilus component are carried out on the freeze-dried product rather than on the bulk conjugate where the freeze-drying process may affect the component to be tested. Absence of residual pertussis toxin and irreversibility of pertussis toxoid. This test is not necessary for the product obtained by genetic modification. Use 3 groups each of not fewer than 5 histamine-sensitive mice. Inject intraperitoneally into the first group twice the single human dose of the vaccine stored at 2-8 °C. Inject intraperitoneally into the second group twice the single human dose of the vaccine incubated at 37 °C for 4 weeks. Inject diluent into the third group of mice. After 5 days, inject into each mouse 2 mg of histamine base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 h. The test is invalid if 1 or more control mice die following histamine challenge. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. If 1 mouse dies in either or both of the first and second groups, the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined ; the vaccine complies with the test if, in both of the groups given the vaccine, not more than 5 per cent of the total number of mice die following histamine challenge. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows : inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 2 g/l of gelatin and challenge with histamine as above ; the strain is suitable if more than 50 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine show symptoms of sensitisation. PRP : minimum 80 per cent of the amount of PRP stated on the label, for a vaccine with the haemophilus component in a separate container. For a vaccine with all components in the same container : the PRP content determined on the non-absorbed fraction is not less than that approved for the product.



PRP is determined either by assay of ribose (2.5.31) or phosphorus (2.5.18), by an immunochemical method (2.7.1) or by anion-exchange liquid chromatography (2.2.29) with pulsed-amperometric detection. Aluminium (2.5.13) : maximum 1.25 mg of aluminium (Al) per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.4.18) : maximum 0.2 g/l of free formaldehyde per single human dose. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than the minimum amount shown to be effective and is not greater than 115 per cent of the quantity stated on the label. Water (2.5.12) : maximum 3.0 per cent for the freeze-dried haemophilus component. Sterility (2.6.1). It complies with the test for sterility. ASSAY Diphtheria component. Carry out one of the prescribed methods for the assay of diphtheria vaccine (adsorbed) (2.7.6). The lower confidence limit (P = 0.95) of the estimated potency is not less than the minimum potency stated on the label. Unless otherwise justified and authorised, the minimum potency stated on the label is 30 IU per single human dose. Tetanus component. Carry out one of the prescribed methods for the assay of tetanus vaccine (adsorbed) (2.7.8). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. Pertussis component. The vaccine complies with the assay of pertussis vaccine (acellular) (2.7.16). Hepatitis B component. The vaccine complies with the assay of hepatitis B vaccine (2.7.15). Poliomyelitis component D-antigen content. As a measure of consistency of production, determine the D-antigen content for human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.7.1) using a reference preparation calibrated in European Pharmacopoeia D-antigen units. For each type, the content, expressed with reference to the amount of D-antigen stated on the label, is within the limits approved for the particular product. Poliomyelitis vaccine (inactivated) BRP is calibrated in European Pharmacopoeia units and intended for use in the assay of D antigen. The European Pharmacopoeia unit and the International Unit are equivalent. In vivo test. The vaccine complies with the in vivo assay of poliomyelitis vaccine (inactivated) (2.7.20).

DIP-TET-PERa-IPV-HIB

— the type and nominal amount of carrier protein per single human dose, — where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults, — the name and the amount of the adsorbent, — that the vaccine must be shaken before use, — that the vaccine is not to be frozen, — where applicable, that the vaccine contains a pertussis toxin-like protein produced by genetic modification. 01/2005:2065

DIPHTHERIA, TETANUS, PERTUSSIS (ACELLULAR, COMPONENT), POLIOMYELITIS (INACTIVATED) AND HAEMOPHILUS TYPE b CONJUGATE VACCINE (ADSORBED) Vaccinum diphtheriae, tetani, pertussis sine cellulis ex elementis praeparatum poliomyelitidis inactivatum et haemophili stirpe b coniugatum adsorbatum

DEFINITION Diphtheria, tetanus, pertussis (acellular, component), poliomyelitis (inactivated) and haemophilus type b conjugate vaccine (adsorbed) is a combined vaccine composed of: diphtheria formol toxoid ; tetanus formol toxoid ; individually purified antigenic components of Bordetella pertussis ; suitable strains of human polioviruses 1, 2 and 3 grown in suitable cell cultures and inactivated by a suitable method ; polyribosylribitol phosphate (PRP) covalently bound to a carrier protein ; a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate. The product is presented with the haemophilus component in a separate container the contents of which are mixed with the other components immediately before use. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani respectively. The vaccine contains either pertussis toxoid or a pertussis-toxin-like protein free from toxic properties produced by expression of a genetically modified form of the corresponding gene. Pertussis toxoid is prepared from pertussis toxin by a method that renders the toxin harmless while maintaining adequate immunogenic properties and avoiding reversion to toxin. The acellular pertussis component may also contain filamentous haemagglutinin, LABELLING pertactin (a 69 kDa outer-membrane protein) and other The label states : defined components of B. pertussis such as fimbrial-2 and — the minimum number of International Units of diphtheria fimbrial-3 antigens. The latter 2 antigens may be co-purified. The antigenic composition and characteristics are based and tetanus toxoid per single human dose, — the names and amounts of the pertussis components per on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is single human dose, intended. — the amount of HBsAg per single human dose, PRP is a linear copolymer composed of repeated units of — the nominal amount of poliovirus of each type (1, 2 3-β-D-ribofuranosyl-(1→1)-ribitol-5-phosphate [(C10H19O12P)n], and 3), expressed in European Pharmacopoeia Units of with a defined molecular size and derived from a suitable D-antigen, per single human dose, strain of Haemophilus influenzae type b. The carrier — the types of cells used for production of the poliomyelitis protein, when conjugated to PRP, is capable of inducing and the hepatitis B components, a T-cell-dependent B-cell immune response to the — the number of micrograms of PRP per single human dose, polysaccharide. General Notices (1) apply to all monographs and other texts

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PRODUCTION GENERAL PROVISIONS The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. The content of bacterial endotoxins (2.6.14) in bulk purified diphtheria toxoid, tetanus toxoid, pertussis components, purified, inactivated monovalent poliovirus harvests and bulk PRP conjugate is determined to monitor the purification procedure and to limit the amount in the final vaccine. For each component, the content of bacterial endotoxins is less than the limit approved for the particular vaccine and, in any case, the contents are such that the final vaccine contains less than 100 IU per single human dose. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for immunosera and vaccines for human use (2.6.9), and with the following test for specific toxicity of the diphtheria and tetanus components : inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs, each weighing 250-350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria, toxaemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from non-specific causes, repeat the test once ; if more than 1 animal dies in the second test, the vaccine does not comply with the test. During development studies and wherever revalidation is necessary, it shall be demonstrated by tests in animals that the vaccine induces a T-cell dependent B-cell immune response to PRP. As part of consistency studies the assays of the diphtheria, tetanus, pertussis and poliomyelitis components are carried out on a suitable number of batches of vaccine reconstituted as for use. For subsequent routine control, the assays of these components may be carried out without mixing with the haemophilus component. Reference vaccine(s). Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. PRODUCTION OF THE COMPONENTS The production of the components complies with the requirements of the monographs on Diphtheria vaccine (adsorbed) (0443), Tetanus vaccine (adsorbed) (0452), Pertussis vaccine (acellular, component, adsorbed) (1356), Poliomyelitis vaccine (inactivated) (0214) and Haemophilus type b conjugate vaccine (1219). FINAL BULKS The final bulk of the diphtheria, tetanus, pertussis and poliomyelitis components is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, bulk purified tetanus toxoid and bulk purified acellular pertussis components onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate and admixture of suitable quantities of purified, 654

monovalent harvests of human polioviruses 1, 2 and 3 or a suitable quantity of a trivalent pool of such monovalent harvests. Suitable antimicrobial preservatives may be added. The final bulk of the haemophilus component is prepared by dilution of the bulk conjugate to the final concentration with a suitable diluent. A stabiliser may be added. Only final bulks that comply with the following requirements may be used in the preparation of the final lot. Bovine serum albumin. Determined on the poliomyelitis components by a suitable immunochemical method (2.7.1) during preparation of the final bulk vaccine, before addition of the adsorbent, the amount of bovine serum albumin is such that the content in the final vaccine will be not more than 50 ng per single human dose. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85 per cent and not greater than 115 per cent of the intended content. Sterility (2.6.1). Carry out the test for sterility using 10 ml for each medium. FINAL LOT The final bulk of the haemophilus component is freeze-dried. Only a final lot that is satisfactory with respect to the test for osmolality shown below and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the test for absence of residual pertussis toxin and irreversibility of pertussis toxoid, the test for antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided that the free formaldehyde content has been determined on the bulk purified antigens and the purified monovalent harvests or the trivalent pool of polioviruses or the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. Provided that the in vivo assay for the poliomyelitis component has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Osmolality (2.2.35). The osmolality of the vaccine, reconstituted where applicable, is within the limits approved for the particular preparation. Free PRP. Unbound PRP is determined on the haemophilus component after removal of the conjugate, for example by anion-exchange, size-exclusion or hydrophobic chromatography, ultrafiltration or other validated methods. The amount of free PRP is not greater than that approved for the particular product. IDENTIFICATION Identification tests A, B, C and D are carried out using the vial containing the diphtheria, tetanus, pertussis and poliomyelitis components ; identification test E is carried out on the vial containing the haemophilus component. A. Diphtheria toxoid is identified by a suitable immunochemical method (2.7.1). The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine to be examined sufficient sodium citrate R to give a 100 g/l solution. Maintain at 37 °C for about 16 h and centrifuge until a clear supernatant liquid is obtained. The clear supernatant liquid reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.7.1). The following method, applicable to certain vaccines, is given as an example. The clear



supernatant liquid obtained during identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The pertussis components are identified by suitable immunochemical methods (2.7.1). The following method, applicable to certain vaccines, is given as an example. The clear supernatant liquid obtained during identification test A reacts with specific antisera to the pertussis components of the vaccine. D. The vaccine is shown to contain human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.7.1), such as determination of D-antigen by enzyme-linked immunosorbent assay (ELISA). E. The haemophilus component is identified by a suitable immunochemical method (2.7.1) for PRP. TESTS The tests for absence of residual pertussis toxin, irreversibility of pertussis toxoid, aluminium, free formaldehyde, antimicrobial preservative and sterility are carried out on the container with the diphtheria, tetanus, pertussis and poliomyelitis components ; the tests for PRP content, water, sterility and pyrogens are carried out on the container with the haemophilus component. Some tests for the haemophilus component may be carried out on the freeze-dried product rather than on the bulk conjugate where the freeze-drying process may affect the component to be tested. Absence of residual pertussis toxin and irreversibility of pertussis toxoid. This test is not necessary for the product obtained by genetic modification. Use 3 groups each of not fewer than 5 histamine-sensitive mice. Inject intraperitoneally into the first group twice the single human dose of the vaccine stored at 2-8 °C. Inject intraperitoneally into the second group twice the single human dose of the vaccine incubated at 37 °C for 4 weeks. Inject diluent into the third group of mice. After 5 days, inject into each mouse 2 mg of histamine base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 h. The test is invalid if 1 or more control mice die following histamine challenge. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. If 1 mouse dies in either or both of the first and second groups, the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined ; the vaccine complies with the test if, in both of the groups given the vaccine, not more than 5 per cent of the total number of mice die following histamine challenge. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows : inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 2 g/l of gelatin and challenge with histamine as above ; the strain is suitable if more than 50 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine show symptoms of sensitisation. PRP : minimum 80 per cent of the amount of PRP stated on the label. PRP is determined either by assay of ribose (2.5.31) or phosphorus (2.5.18), by an immunochemical method (2.7.1) or by anion-exchange liquid chromatography (2.2.29) with pulsed-amperometric detection. Aluminium (2.5.13) : maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.4.18) : maximum 0.2 g/l. General Notices (1) apply to all monographs and other texts


Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than the minimum amount shown to be effective and is not greater than 115 per cent of the quantity stated on the label. Water (2.5.12) : maximum 3.0 per cent for the haemophilus component. Sterility (2.6.1). It complies with the test for sterility. Pyrogens (2.6.8). It complies with the test for pyrogens. Inject per kilogram of the rabbit’s mass a quantity of the vaccine equivalent to : 1 µg of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria protein as carrier ; 0.1 µg of PRP for a vaccine with tetanus toxoid as carrier ; 0.025 µg of PRP for a vaccine with OMP as a carrier. ASSAY Diphtheria component. Carry out one of the prescribed methods for the assay of diphtheria vaccine (adsorbed) (2.7.6). Unless otherwise justified and authorised, the lower confidence limit (P = 0.95) of the estimated potency is not less than 30 IU per single human dose. Tetanus component. Carry out one of the prescribed methods for the assay of tetanus vaccine (adsorbed) (2.7.8). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. Pertussis component. It complies with the assay of pertussis vaccine (acellular) (2.7.16). Poliomyelitis component D-antigen content. As a measure of consistency of production, determine the D-antigen content for human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.7.1) using a reference preparation calibrated in Ph. Eur. Units of D-antigen. For each type, the content, expressed with reference to the amount of D-antigen stated on the label, is within the limits approved for the particular product. Poliomyelitis vaccine (inactivated) BRP is calibrated in Ph. Eur. Units and intended for use in the assay of D-antigen. The Ph. Eur. Unit and the IU are equivalent. In vivo test. The vaccine complies with the in vivo assay of poliomyelitis vaccine (inactivated) (2.7.20). LABELLING The label states : — the minimum number of International Units of diphtheria and tetanus toxoid per single human dose, — the names and amounts of the pertussis components per single human dose, — the nominal amount of poliovirus of each type (1, 2 and 3), expressed in European Pharmacopoeia Units of D-antigen, per single human dose, — the type of cells used for production of the poliomyelitis component, — the number of micrograms of PRP per single human dose, — the type and nominal amount of carrier protein per single human dose, — where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults, — the name and the amount of the adsorbent, — that the vaccine must be shaken before use, — that the vaccine is not to be frozen, — where applicable, that the vaccine contains a pertussis toxin-like protein produced by genetic modification. 655

DIP-TET-PERw-IPV


01/2005:2061 or a suitable quantity of a trivalent pool of such purified monovalent harvests. Suitable antimicrobial preservatives DIPHTHERIA, TETANUS, PERTUSSIS may be added. a final bulk vaccine that complies with the following AND POLIOMYELITIS (INACTIVATED) Only requirements may be used in the preparation of the final lot. VACCINE (ADSORBED) Bovine serum albumin. Determined on the poliomyelitis components by a suitable immunochemical method (2.7.1) Vaccinum diphtheriae, tetani, pertussis et during preparation of the final bulk vaccine, before addition poliomyelitidis inactivatum adsorbatum of the adsorbent, the amount of bovine serum albumin is such that the content in the final vaccine will be not more DEFINITION than 50 ng per single human dose. Diphtheria, tetanus, pertussis and poliomyelitis (inactivated) Antimicrobial preservative. Where applicable, determine vaccine (adsorbed) is a combined vaccine containing : the amount of antimicrobial preservative by a suitable diphtheria formol toxoid ; tetanus formol toxoid ; an chemical method. The amount is not less than 85 per cent inactivated suspension of Bordetella pertussis ; suitable and not greater than 115 per cent of the intended content. strains of human polioviruses 1, 2 and 3 grown in suitable Sterility (2.6.1). Carry out the test for sterility using 10 ml cell cultures and inactivated by a validated method ; a mineral adsorbent such as aluminium hydroxide or hydrated for each medium. aluminium phosphate. FINAL LOT Only a final lot that is satisfactory with respect to the test The formol toxoids are prepared from the toxins produced for osmolality and with respect to each of the requirements by the growth of Corynebacterium diphtheriae and given below under Identification, Tests and Assay may be Clostridium tetani respectively. released for use. PRODUCTION Provided that the tests for specific toxicity of the pertussis GENERAL PROVISIONS component and antimicrobial preservative, and the assays for the diphtheria, tetanus and pertussis components have The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. clinical efficacy and safety in man. Provided that the free formaldehyde content has been Reference vaccine(s). Provided valid assays can be performed, monocomponent reference vaccines may be used determined on the bulk purified antigens, the inactivated for the assays on the combined vaccine. If this is not possible B. pertussis suspension and the purified monovalent harvests or the trivalent pool of polioviruses or on the final because of interaction between the components of the combined vaccine or because of the difference in composition bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may between monocomponent reference vaccine and the test be omitted on the final lot. vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as Provided that the in vivo assay for the poliomyelitis a reference vaccine. For the preparation of a representative component has been carried out with satisfactory results on batch, strict adherence to the production process used for the final bulk vaccine, it may be omitted on the final lot. the batch tested in clinical trials is necessary. The reference vaccine may be stabilised by a method that has been shown Osmolality (2.2.35). The osmolality of the vaccine is within the limits approved for the particular preparation. to have no effect on the assay procedure. Specific toxicity of the diphtheria and tetanus components. IDENTIFICATION The production method is validated to demonstrate that A. Diphtheria toxoid is identified by a suitable the product, if tested, would comply with the following test : immunochemical method (2.7.1). The following method, inject subcutaneously 5 times the single human dose stated applicable to certain vaccines, is given as an example. on the label into each of 5 healthy guinea-pigs, each weighing Dissolve in the vaccine to be examined sufficient sodium 250-350 g, that have not previously been treated with any citrate R to give a 100 g/l solution. Maintain at 37 °C for material that will interfere with the test. If within 42 days of about 16 h and centrifuge until a clear supernatant liquid the injection any of the animals shows signs of or dies from is obtained. The clear supernatant liquid reacts with a diphtheria toxaemia or tetanus, the vaccine does not comply suitable diphtheria antitoxin, giving a precipitate. with the test. If more than 1 animal dies from non-specific B. Tetanus toxoid is identified by a suitable immunochemical causes, repeat the test once ; if more than 1 animal dies in method (2.7.1). The following method, applicable to the second test, the vaccine does not comply with the test. certain vaccines, is given as an example. The clear PRODUCTION OF THE COMPONENTS supernatant liquid obtained during identification test A reacts with a suitable tetanus antitoxin, giving a The production of the components complies with the precipitate. requirements of the monographs on Diphtheria vaccine (adsorbed) (0443), Tetanus vaccine (adsorbed) (0452), C. The centrifugation residue obtained in identification A Pertussis vaccine (adsorbed) (0161) and Poliomyelitis may be used. Other suitable methods for separating the vaccine (inactivated) (0214). bacteria from the adsorbent may also be used. Identify pertussis vaccine by agglutination of the bacteria from FINAL BULK VACCINE the resuspended precipitate by antisera specific to The final bulk vaccine is prepared by adsorption onto a B. pertussis or by the assay of the pertussis component mineral carrier such as aluminium hydroxide or hydrated prescribed under Assay. aluminium phosphate, separately or together, of suitable quantities of bulk purified diphtheria toxoid and bulk D. The vaccine is shown to contain human polioviruses 1, purified tetanus toxoid and admixture of suitable quantities 2 and 3 by a suitable immunochemical method (2.7.1) of an inactivated suspension of B. pertussis and purified such as the determination of D-antigen by enzyme-linked monovalent harvests of human polioviruses 1, 2 and 3 immunosorbent assay (ELISA). 656



TESTS Specific toxicity of the pertussis component. Use not fewer than 5 healthy mice each weighing 14-16 g for the vaccine group and for the saline control. Use mice of the same sex or distribute males and females equally between the groups. Allow the animals access to food and water for at least 2 h before injection and during the test. Inject each mouse of the vaccine group intraperitoneally with 0.5 ml, containing a quantity of the vaccine equivalent to not less than half the single human dose. Inject each mouse of the control group with 0.5 ml of a 9 g/l sterile solution of sodium chloride R, preferably containing the same amount of antimicrobial preservative as that injected with the vaccine. Weigh the groups of mice immediately before the injection and 72 h and 7 days after the injection. The vaccine complies with the test if : (a) at the end of 72 h the total mass of the group of vaccinated mice is not less than that preceding the injection ; (b) at the end of 7 days the average increase in mass per vaccinated mouse is not less than 60 per cent of that per control mouse ; and (c) not more than 5 per cent of the vaccinated mice die during the test. The test may be repeated and the results of the tests combined. Aluminium (2.5.13) : maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.4.18) : maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than the minimum amount shown to be effective and is not greater than 115 per cent of the quantity stated on the label. Sterility (2.6.1). It complies with the test for sterility. ASSAY Diphtheria component. Carry out one of the prescribed methods for the assay of diphtheria vaccine (adsorbed) (2.7.6). The lower confidence limit (P = 0.95) of the estimated potency is not less than 30 IU per single human dose. Tetanus component. Carry out one of the prescribed methods for the assay of tetanus vaccine (adsorbed) (2.7.8). If the test is carried out in guinea pigs, the lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose ; if the test is carried out in mice, the lower confidence limit (P = 0.95) of the estimated potency is not less than 60 IU per single human dose. Pertussis component. Carry out the assay of pertussis vaccine (2.7.7). The estimated potency is not less than 4 IU per single human dose and the lower confidence limit (P = 0.95) of the estimated potency is not less than 2 IU per single human dose. Poliomyelitis component D-antigen content. As a measure of consistency of production, determine the D-antigen content for human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.7.1) using a reference preparation calibrated in European Pharmacopoeia Units of D-antigen. For each type, the content, expressed with reference to the amount of D-antigen stated on the label, is within the limits approved for the particular product. Poliomyelitis vaccine (inactivated) BRP is calibrated in European Pharmacopoeia Units and intended for use in the assay of D-antigen. The European Pharmacopoeia Unit and the International Unit are equivalent. General Notices (1) apply to all monographs and other texts

DIP-TET-PERw-IPV-HIB

In vivo test. The vaccine complies with the in vivo assay of poliomyelitis vaccine (inactivated) (2.7.20). LABELLING The label states : — the minimum number of International Units of diphtheria and tetanus toxoid per single human dose, — the minimum number of International Units of pertussis vaccine per single human dose, — the nominal amount of poliovirus of each type (1, 2 and 3), expressed in European Pharmacopoeia Units of D-antigen, per single human dose, — the type of cells used for production of the poliomyelitis component, — where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults, — the name and the amount of the adsorbent, — that the vaccine must be shaken before use, — that the vaccine is not to be frozen.

01/2005:2066

DIPHTHERIA, TETANUS, PERTUSSIS, POLIOMYELITIS (INACTIVATED) AND HAEMOPHILUS TYPE b CONJUGATE VACCINE (ADSORBED) Vaccinum diphtheriae, tetani, pertussis, poliomyelitidis inactivatum et haemophili stirpe b coniugatum adsorbatum DEFINITION Diphtheria, tetanus, pertussis, poliomyelitis (inactivated) and haemophilus type b conjugate vaccine (adsorbed) is a combined vaccine composed of: diphtheria formol toxoid ; tetanus formol toxoid ; an inactivated suspension of Bordetella pertussis ; suitable strains of human polioviruses 1, 2 and 3 grown in suitable cell cultures and inactivated by a suitable method ; polyribosylribitol phosphate (PRP) covalently bound to a carrier protein ; a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate. The product is presented with the haemophilus component in a separate container, the contents of which are mixed with the other components immediately before use. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani respectively. PRP is a linear copolymer composed of repeated units of 3-β-D-ribofuranosyl-(1→1)-ribitol-5-phosphate [(C10H19O12P)n], with a defined molecular size and derived from a suitable strain of Haemophilus influenzae type b. The carrier protein, when conjugated to PRP, is capable of inducing a T-cell-dependent B-cell immune response to the polysaccharide. PRODUCTION GENERAL PROVISIONS The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. 657



During development studies and wherever revalidation is necessary, it shall be demonstrated by tests in animals that the vaccine induces a T-cell dependent B-cell immune response to PRP.

Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85 per cent and not greater than 115 per cent of the intended content.

As part of consistency studies the assays of the diphtheria, tetanus, pertussis and poliomyelitis components are carried out on a suitable number of batches of vaccine reconstituted as for use. For subsequent routine control, the assays of these components may be carried out without mixing with the haemophilus component.

Sterility (2.6.1). Carry out the test for sterility using 10 ml for each medium.

Bovine serum albumin. Determined on the poliomyelitis components by a suitable immunochemical method (2.7.1) during preparation of the final bulk vaccine, before addition of the adsorbent, the amount of bovine serum albumin is such that the content in the final vaccine will be not more than 50 ng per single human dose.

may be used. Other suitable methods for separating the bacteria from the adsorbent may also be used. Identify pertussis vaccine by agglutination of the bacteria from the resuspended precipitate by antisera specific to B. pertussis or by the assay of the pertussis component prescribed under Assay.

FINAL LOT The final bulk of the haemophilus component is freeze-dried.

Only a final lot that is satisfactory with respect to the test for osmolality shown below and with respect to each of the requirements given below under Identification, Tests and Reference vaccine(s). Provided valid assays can be performed, monocomponent reference vaccines may be used Assay may be released for use. for the assays on the combined vaccine. If this is not possible Provided that the tests for specific toxicity of the pertussis because of interaction between the components of the component and antimicrobial preservative, and the assays combined vaccine or because of the difference in composition for the diphtheria, tetanus and pertussis components have between monocomponent reference vaccine and the test been carried out with satisfactory results on the final bulk vaccine, a batch of combined vaccine shown to be effective vaccine, they may be omitted on the final lot. in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative Provided that the free formaldehyde content has been determined on the bulk purified antigens, the inactivated batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference B. pertussis suspension and the purified monovalent vaccine may be stabilised by a method that has been shown harvests or the trivalent pool of polioviruses or on the final bulk and it has been shown that the content in the final lot to have no effect on the assay procedure. will not exceed 0.2 g/l, the test for free formaldehyde may Specific toxicity of the diphtheria and tetanus components. be omitted on the final lot. The production method is validated to demonstrate that the product, if tested, would comply with the following test : Provided that the in vivo assay for the poliomyelitis inject subcutaneously 5 times the single human dose stated component has been carried out with satisfactory results on on the label into each of 5 healthy guinea-pigs, each weighing the final bulk vaccine, it may be omitted on the final lot. 250-350 g, that have not previously been treated with any Osmolality (2.2.35). The osmolality of the vaccine, material that will interfere with the test. If within 42 days of reconstituted where applicable, is within the limits approved the injection any of the animals shows signs of or dies from for the particular preparation. diphtheria toxaemia or tetanus, the vaccine does not comply Free PRP. Unbound PRP is determined on the haemophilus with the test. If more than 1 animal dies from non-specific component after removal of the conjugate, for example causes, repeat the test once ; if more than 1 animal dies in by anion-exchange, size-exclusion or hydrophobic the second test, the vaccine does not comply with the test. chromatography, ultrafiltration or other validated methods. PRODUCTION OF THE COMPONENTS The amount of free PRP is not greater than that approved The production of the components complies with the for the particular product. requirements of the monographs on Diphtheria vaccine (adsorbed) (0443), Tetanus vaccine (adsorbed) (0452), Pertussis vaccine (adsorbed) (0161), Poliomyelitis vaccine IDENTIFICATION (inactivated) (0214) and Haemophilus type b conjugate Identification tests A, B, C and D are carried out using vaccine (1219). the vial containing the diphtheria, tetanus, pertussis and poliomyelitis components ; identification test E is carried FINAL BULKS out on the vial containing the haemophilus component. The final bulk of the diphtheria, tetanus, pertussis and poliomyelitis components is prepared by adsorption, A. Diphtheria toxoid is identified by a suitable separately or together, of suitable quantities of bulk purified immunochemical method (2.7.1). The following method, diphtheria toxoid, and bulk purified tetanus toxoid onto a applicable to certain vaccines, is given as an example. mineral carrier such as aluminium hydroxide or hydrated Dissolve in the vaccine to be examined sufficient sodium aluminium phosphate and admixture of suitable quantities citrate R to give a 100 g/l solution. Maintain at 37 °C for of an inactivated suspension of B. pertussis and of purified, about 16 h and centrifuge until a clear supernatant liquid monovalent harvests of human polioviruses 1, 2 and 3 or is obtained. The clear supernatant liquid reacts with a a suitable quantity of a trivalent pool of such monovalent suitable diphtheria antitoxin, giving a precipitate. harvests. Suitable antimicrobial preservatives may be added. B. Tetanus toxoid is identified by a suitable immunochemical method (2.7.1). The following method, applicable to The final bulk of the haemophilus component is prepared by certain vaccines, is given as an example. The clear dilution of the bulk conjugate to the final concentration with supernatant liquid obtained during identification a suitable diluent. A stabiliser may be added. test A reacts with a suitable tetanus antitoxin, giving a precipitate. Only final bulks that comply with the following requirements may be used in the preparation of the final lot. C. The centrifugation residue obtained in identification A

658




D. The vaccine is shown to contain human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.7.1), such as determination of D-antigen by enzyme-linked immunosorbent assay (ELISA). E. The haemophilus component is identified by a suitable immunochemical method (2.7.1) for PRP.

TESTS The tests for specific toxicity of the pertussis component, aluminium, free formaldehyde, antimicrobial preservative and sterility are carried out on the container with diphtheria, tetanus, pertussis and poliomyelitis components ; the tests for PRP, water, sterility and pyrogens are carried out on the container with the haemophilus component.

diphtheria toxoid or CRM 197 diphtheria protein as carrier ; 0.1 µg of PRP for a vaccine with tetanus toxoid as carrier ; 0.025 µg of PRP for a vaccine with OMP as carrier. ASSAY Diphtheria component. Carry out one of the prescribed methods for the assay of diphtheria vaccine (adsorbed) (2.7.6). The lower confidence limit (P = 0.95) of the estimated potency is not less than 30 IU per single human dose. Tetanus component. Carry out one of the prescribed methods for the assay of tetanus vaccine (adsorbed) (2.7.8). If the test is carried out in guinea-pigs, the lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose ; if the test is carried out in mice, the lower confidence limit (P = 0.95) of the estimated potency is not less than 60 IU per single human dose.

Pertussis component. Carry out the assay of pertussis Some tests for the haemophilus component may be carried vaccine (2.7.7). out on the freeze-dried product rather than on the bulk The estimated potency is not less than 4 IU per single conjugate where the freeze-drying process may affect the human dose and the lower confidence limit (P = 0.95) of the component to be tested. estimated potency is not less than 2 IU per single human Specific toxicity of the pertussis component. Use not fewer dose. than 5 healthy mice each weighing 14-16 g, for the vaccine Poliomyelitis component group and for the saline control. Use mice of the same sex or distribute males and females equally between the groups. D-antigen content. As a measure of consistency of Allow the animals access to food and water for at least 2 h production, determine the D-antigen content for human before injection and during the test. Inject each mouse of polioviruses 1, 2 and 3 by a suitable immunochemical the vaccine group intraperitoneally with 0.5 ml, containing a method (2.7.1) using a reference preparation calibrated quantity of the vaccine equivalent to not less than half the in European Pharmacopoeia Units of D-antigen. For single human dose. Inject each mouse of the control group each type, the content, expressed with reference to the with 0.5 ml of a 9 g/l sterile solution of sodium chloride R, amount of D-antigen stated on the label, is within the limits preferably containing the same amount of antimicrobial approved for the particular product. Poliomyelitis vaccine preservative as that injected with the vaccine. Weigh the (inactivated) BRP is calibrated in European Pharmacopoeia groups of mice immediately before the injection and 72 h Units and intended for use in the assay of D-antigen. The and 7 days after the injection. The vaccine complies with the European Pharmacopoeia Unit and the International Unit test if : (a) at the end of 72 h the total mass of the group of are equivalent. vaccinated mice is not less than that preceding the injection ; In vivo test. The vaccine complies with the in vivo assay of (b) at the end of 7 days the average increase in mass per poliomyelitis vaccine (inactivated) (2.7.20). vaccinated mouse is not less than 60 per cent of that per control mouse ; and (c) not more than 5 per cent of the vaccinated mice die during the test. The test may be repeated LABELLING and the results of the tests combined. The label states : PRP : minimum 80 per cent of the amount of PRP stated — the minimum number of International Units of diphtheria on the label. PRP is determined either by assay of ribose and tetanus toxoid per single human dose, (2.5.31) or phosphorus (2.5.18), by an immunochemical method (2.7.1) or by anion-exchange liquid chromatography — the minimum number of International Units of pertussis (2.2.29) with pulsed-amperometric detection. vaccine per single human dose, Aluminium (2.5.13) : maximum 1.25 mg per single human — the nominal amount of poliovirus of each type (1, 2 dose, if aluminium hydroxide or hydrated aluminium and 3), expressed in European Pharmacopoeia Units of phosphate is used as the adsorbent. D-antigen, per single human dose, Free formaldehyde (2.4.18) : maximum 0.2 g/l. — the type of cells used for production of the poliomyelitis component, Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable — the number of micrograms of PRP per single human dose, chemical method. The content is not less than the minimum amount shown to be effective and is not greater than 115 per — the type and nominal amount of carrier protein per single human dose, cent of the quantity stated on the label. Water (2.5.12) : maximum 3.0 per cent for the haemophilus — where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for component. reinforcing doses or for administration to adults, Sterility (2.6.1). It complies with the test for sterility. — the name and the amount of the adsorbent, Pyrogens (2.6.8). It complies with the test for pyrogens. — that the vaccine must be shaken before use, Inject per kilogram of the rabbit’s mass a quantity of the — that the vaccine is not to be frozen. vaccine equivalent to : 1 µg of PRP for a vaccine with General Notices (1) apply to all monographs and other texts

659

Diphtheria vaccine (adsorbed)


01/2005:0443 50 000 minimal reacting doses for guinea-pig skin in 1 Lf (diphtheria toxin BRP is suitable for use as the reference toxin). Dilute the toxin in 100 Lf/ml diphtheria toxoid to a DIPHTHERIA VACCINE (ADSORBED) suitable concentration, for example 2 × 10− 4 Lf/ml. Prepare serial twofold dilutions of the diluted diphtheria toxin and Vaccinum diphtheriae adsorbatum use undiluted test samples (50 µl/well). Distribute them in the wells of a sterile tissue culture plate containing DEFINITION a medium suitable for Vero cells. To ascertain that any Diphtheria vaccine (adsorbed) is a preparation of diphtheria cytotoxic effect noted is specific to diphtheria toxin, prepare in parallel dilutions where the toxin is neutralised by a formol toxoid with a mineral adsorbent. The formol toxoid suitable concentration of diphtheria antitoxin, for example is prepared from the toxin produced by the growth of 100 IU/ml. Include control wells without toxoid or toxin and Corynebacterium diphtheriae. with non-toxic toxoid at 100 Lf/ml on each plate to verify PRODUCTION normal cell growth. Add cell suspension to each well, seal the plates and incubate at 37 °C for 5-6 days. Cytotoxic effect GENERAL PROVISIONS is judged to be present where there is complete metabolic Specific toxicity. The production method is validated to inhibition of the Vero cells, indicated by the pH indicator demonstrate that the product, if tested, would comply of the medium. Confirm cytopathic effect by microscopic with the following test : inject subcutaneously 5 times examination or suitable staining such as MTT dye. The the single human dose stated on the label into each of 5 test is invalid if 5 × 10− 5 Lf/ml of reference diphtheria healthy guinea-pigs, each weighing 250-350 g, that have not toxin in 100 Lf/ml toxoid has no cytotoxic effect on Vero previously been treated with any material that will interfere cells or if the cytotoxic effect of this amount of toxin is not with the test. If within 42 days of the injection any of the neutralised in the wells containing diphtheria antitoxin. The animals shows signs of or dies from diphtheria toxaemia, the bulk purified toxoid complies with the test if no toxicity vaccine does not comply with the test. If more than 1 animal neutralisable by antitoxin is found in either sample. dies from non-specific causes, repeat the test once ; if more Antigenic purity. Not less than 1500 Lf per milligram of than 1 animal dies in the second test, the vaccine does not protein nitrogen. comply with the test. FINAL BULK VACCINE BULK PURIFIED TOXOID For the production of diphtheria toxin, from which toxoid is The final bulk vaccine is prepared by adsorption of a suitable quantity of bulk purified toxoid onto a mineral carrier such prepared, seed cultures are managed in a defined seed-lot as hydrated aluminium phosphate or aluminium hydroxide ; system in which toxinogenicity is conserved and, where the resulting mixture is approximately isotonic with blood. necessary, restored by deliberate reselection. A highly Suitable antimicrobial preservatives may be added. Certain toxinogenic strain of Corynebacterium diphtheriae with antimicrobial preservatives, particularly those of the phenolic known origin and history is grown in a suitable liquid type, adversely affect the antigenic activity and must not be medium. At the end of cultivation, the purity of each used. culture is tested and contaminated cultures are discarded. Toxin-containing culture medium is separated aseptically Only a final bulk vaccine that complies with the following from the bacterial mass as soon as possible. The toxin requirements may be used in the preparation of the final lot. content (Lf per millilitre) is checked to monitor consistency Antimicrobial preservative. Where applicable, determine of production. Single harvests may be pooled to prepare the amount of antimicrobial preservative by a suitable the bulk purified toxoid. The toxin is purified to remove chemical method. The amount is not less than 85 per cent components likely to cause adverse reactions in humans. The and not greater than 115 per cent of the intended amount. purified toxin is detoxified with formaldehyde by a method Sterility (2.6.1). Carry out the test for sterility using 10 ml that avoids destruction of the immunogenic potency of the for each medium. toxoid and reversion of the toxoid to toxin, particularly on exposure to heat. Alternatively, purification may be carried FINAL LOT out after detoxification. The final bulk vaccine is distributed aseptically into sterile, Only bulk purified toxoid that complies with the following tamper-proof containers. The containers are closed so as to requirements may be used in the preparation of the final prevent contamination. bulk vaccine. Only a final lot that is satisfactory with respect to each of Sterility (2.6.1). Carry out the test for sterility using 10 ml the requirements given below under Identification, Tests for each medium. and Assay may be released for use. Provided the test for antimicrobial preservative and the assay have been carried Absence of toxin and irreversibility of toxoid. Using out with satisfactory results on the final bulk vaccine, they the same buffer solution as for the final vaccine, without may be omitted on the final lot. adsorbent, prepare a solution of bulk purified toxoid at 100 Lf/ml. Divide the solution into 2 equal parts. Maintain Provided the free formaldehyde content has been determined 1 part at 5 ± 3 °C and the other at 37 °C for 6 weeks. Carry on the bulk purified antigens or on the final bulk and it has out a test in Vero cells for active diphtheria toxin using been shown that the content in the final lot will not exceed 50 µl/well of both samples. The sample should not contain 0.2 g/l, the test for free formaldehyde may be omitted on antimicrobial preservatives and detoxifying agents should be the final lot. determined to be below the concentration toxic to Vero cells. IDENTIFICATION Non-specific toxicity may be eliminated by dialysis. Use freshly trypsinised Vero cells at a suitable concentration, Diphtheria toxoid is identified by a suitable immunochemical method (2.7.1). The following method, applicable to certain for example 2.5 × 105 ml− 1 and a reference diphtheria vaccines, is given as an example. Dissolve in the vaccine to toxin diluted in 100 Lf/ml diphtheria toxoid. A suitable be examined sufficient sodium citrate R to give a 100 g/l reference diphtheria toxin will contain either not less than solution. Maintain at 37 °C for about 16 h and centrifuge 100 LD50/ml or 67 to 133 lr/100 in 1 Lf and 25 000 to 660



Diphtheria vaccine (adsorbed) for adults and adolescents

BULK PURIFIED TOXOID until a clear supernatant liquid is obtained. The clear supernatant liquid reacts with a suitable diphtheria antitoxin, The bulk purified toxoid is prepared as described in the giving a precipitate. monograph on Diphtheria vaccine (adsorbed) (0443) and complies with the requirements prescribed therein. TESTS FINAL BULK VACCINE Aluminium (2.5.13) : maximum 1.25 mg per single human The final bulk vaccine is prepared by adsorption of a suitable dose, if aluminium hydroxide or hydrated aluminium quantity of bulk purified toxoid onto a mineral carrier such phosphate is used as the absorbent. as hydrated aluminium phosphate or aluminium hydroxide ; Free formaldehyde (2.4.18) : maximum 0.2 g/l. the resulting mixture is approximately isotonic with blood. Suitable antimicrobial preservatives may be added. Certain Antimicrobial preservative. Where applicable, determine antimicrobial preservatives, particularly those of the phenolic the amount of antimicrobial preservative by a suitable chemical method. The content is not less than the minimum type, adversely affect the antigenic activity and must not be amount shown to be effective and is not greater than 115 per used. cent of the quantity stated on the label. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Sterility (2.6.1). The vaccine complies with the test for sterility. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable ASSAY chemical method. The amount is not less than 85 per cent Carry out one of the prescribed methods for the assay of and not greater than 115 per cent of the intended amount. diphtheria vaccine (adsorbed) (2.7.6). Sterility (2.6.1). Carry out the test for sterility using 10 ml The lower confidence limit (P = 0.95) of the estimated for each medium. potency is not less than 30 IU per single human dose. FINAL LOT LABELLING The final bulk vaccine is distributed aseptically into sterile, The label states : tamper-proof containers. The containers are closed so as to — the minimum number of International Units per single prevent contamination. human dose, Only a final lot that is satisfactory with respect to each of — where applicable, that the vaccine is intended for primary the requirements given below under Identification, Tests vaccination of children and is not necessarily suitable for and Assay may be released for use. Provided the test for reinforcing doses or for administration to adults, antimicrobial preservative and the assay have been carried — the name and the amount of the adsorbent, out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. — that the vaccine must be shaken before use, — that the vaccine is not to be frozen. Provided the free formaldehyde content has been determined on the bulk purified toxoid or on the final bulk and it has 01/2005:0646 been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on DIPHTHERIA VACCINE (ADSORBED) the final lot.

FOR ADULTS AND ADOLESCENTS

IDENTIFICATION toxoid is identified by a suitable immunochemical Vaccinum diphtheriae adulti et adulescentis Diphtheria method (2.7.1). The following method, applicable to adsorbatum certain vaccines, is given as an example. Dissolve in the vaccine to be examined sufficient sodium citrate R to give DEFINITION a 100 g/l solution. Maintain at 37 °C for about 16 h and Diphtheria vaccine (adsorbed) for adults and adolescents centrifuge until a clear supernatant liquid is obtained. The is a preparation of diphtheria formol toxoid with a mineral clear supernatant liquid reacts with a suitable diphtheria adsorbent. The formol toxoid is prepared from the toxin antitoxin, giving a precipitate. If a satisfactory result is not produced by the growth of Corynebacterium diphtheriae. obtained with a vaccine adsorbed on aluminium hydroxide, It shall have been demonstrated to the competent authority carry out the test as follows. Centrifuge 15 ml of the vaccine that the quantity of diphtheria toxoid used does not produce to be examined and suspend the residue in 5 ml of a freshly adverse reactions in subjects from the age groups for which prepared mixture of 1 volume of a 56 g/l solution of sodium the vaccine is intended. edetate R and 49 volumes of a 90 g/l solution of disodium hydrogen phosphate R. Maintain at 37 °C for not less than PRODUCTION 6 h and centrifuge. The clear supernatant liquid reacts with GENERAL PROVISIONS a suitable diphtheria antitoxin, giving a precipitate. Specific toxicity. The production method is validated to TESTS demonstrate that the product, if tested, would comply with the following test : inject subcutaneously 5 times the single Aluminium (2.5.13) : maximum 1.25 mg per single human human dose stated on the label into each of 5 healthy dose, if aluminium hydroxide or hydrated aluminium guinea-pigs, each weighing 250-350 g, that have not phosphate is used as the adsorbent. previously been treated with any material that will interfere Free formaldehyde (2.4.18) : maximum 0.2 g/l. with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria toxaemia, the Antimicrobial preservative. Where applicable, determine vaccine does not comply with the test. If more than 1 animal the amount of antimicrobial preservative by a suitable chemical method. The content is not less than the minimum dies from non-specific causes, repeat the test once ; if more amount shown to be effective and is not greater than 115 per than 1 animal dies in the second test, the vaccine does not cent of the quantity stated on the label. comply with the test. General Notices (1) apply to all monographs and other texts

661

Haemophilus type b conjugate vaccine


Sterility (2.6.1). The vaccine complies with the test for sterility. ASSAY Carry out one of the prescribed methods for the assay of diphtheria vaccine (adsorbed) (2.7.6). The lower confidence limit (P = 0.95) of the estimated potency is not less than 2 IU per single human dose.

No complex products of animal origin are included in the menstruum used for preservation of strain viability, either for freeze-drying or for frozen storage.

It is recommended that PRP produced by the seed lot be characterised using nuclear magnetic resonance spectrometry (2.2.33). H. INFLUENZAE TYPE b POLYSACCHARIDE (PRP) H. influenzae type b is grown in a liquid medium that does not contain high-molecular-mass polysaccharides ; LABELLING if any ingredient of the medium contains blood-group The label states : substances, the process shall be validated to demonstrate — the minimum number of International Units per single that after the purification step they are no longer detectable. human dose, The bacterial purity of the culture is verified by methods — the name and the amount of the adsorbent, of suitable sensitivity. These may include inoculation into suitable media, examination of colony morphology, — that the vaccine must be shaken before use, microscopic examination of Gram-stained smears and culture — that the vaccine is not to be frozen. agglutination with suitable specific antisera. The culture may be inactivated. PRP is separated from the culture medium and purified by a suitable method. Volatile matter, 01/2005:1219 including water, in the purified polysaccharide is determined by a suitable method such as thermogravimetry (2.2.34) ; the HAEMOPHILUS TYPE b CONJUGATE result is used to calculate the results of certain tests with reference to the dried substance, as prescribed below.

VACCINE

Only PRP that complies with the following requirements Vaccinum haemophili stirpe b coniugatum may be used in the preparation of the conjugate. Identification. PRP is identified by an immunochemical DEFINITION method (2.7.1) or other suitable method, for example 1H nuclear magnetic resonance spectrometry (2.2.33). Haemophilus type b conjugate vaccine is a liquid or freeze-dried preparation of a polysaccharide, derived Molecular-size distribution. The percentage of PRP eluted from a suitable strain of Haemophilus influenzae type b, before a given K0 value or within a range of K0 values is covalently bound to a carrier protein. The polysaccharide, determined by size-exclusion chromatography (2.2.30) ; an polyribosylribitol phosphate, referred to as PRP, is acceptable value is established for the particular product a linear copolymer composed of repeated units of and each batch of PRP must be shown to comply with this 3-β-D-ribofuranosyl-(1→1)-ribitol-5-phosphate [(C10H19O12P)n], limit. Limits for currently approved products, using the with a defined molecular size. The carrier protein, when indicated stationary phases, are shown for information conjugated to PRP, is capable of inducing a T-cell-dependent in Table 1219.-1. Where applicable, the molecular-size B-cell immune response to the polysaccharide. distribution is also determined after chemical modification of the polysaccharide. PRODUCTION Liquid chromatography (2.2.29) with multiple-angle laser GENERAL PROVISIONS light-scattering detection may also be used for determination The production method shall have been shown to yield of molecular-size distribution. consistently haemophilus type b conjugate vaccines of adequate safety and immunogenicity in man. The production A validated determination of the degree of polymerisation or of the weight-average molecular weight and the dispersion of of PRP and of the carrier are based on seed-lot systems. The production method is validated to demonstrate that the molecular masses may be used instead of the determination of molecular size distribution. product, if tested, would comply with the test for abnormal toxicity for immunosera and vaccines for human use (2.6.9). Ribose (2.5.31). Not less than 32 per cent, calculated with During development studies and wherever revalidation of the reference to the dried substance. manufacturing process is necessary, it shall be demonstrated Phosphorus (2.5.18) : 6.8 per cent to 9.0 per cent, calculated by tests in animals that the vaccine consistently induces a with reference to the dried substance. T-cell-dependent B-cell immune response. Protein (2.5.16). Not more than 1.0 per cent, calculated The stability of the final lot and relevant intermediates is with reference to the dried substance. Use sufficient PRP to evaluated using one or more indicator tests. Such tests may allow detection of proteins at concentrations of 1 per cent include determination of molecular size, determination of or greater. free PRP in the conjugate and the immunogenicity test in Nucleic acid (2.5.17). Not more than 1.0 per cent, calculated mice. Taking account of the results of the stability testing, with reference to the dried substance. release requirements are set for these indicator tests to ensure that the vaccine will be satisfactory at the end of the Bacterial endotoxins (2.6.14) : less than 25 IU per microgram period of validity. of PRP. BACTERIAL SEED LOTS Residual reagents. Where applicable, tests are carried out to determine residues of reagents used during inactivation The seed lots of H. influenzae type b are shown to be free from contamination by methods of suitable sensitivity. These and purification. An acceptable value for each reagent is established for the particular product and each batch of PRP may include inoculation into suitable media, examination must be shown to comply with this limit. Where validation of colony morphology, microscopic examination of Gram-stained smears and culture agglutination with suitable studies have demonstrated removal of a residual reagent, the test on PRP may be omitted. specific antisera. 662


Haemophilus type b conjugate vaccine


Table 1219.-1. – Product characteristics and specifications for PRP and carrier protein in currently approved products Carrier Type

Conjugation

Haemophilus polysaccharide

Purity

Nominal amount per dose

Type of PRP

Nominal amount per dose

Coupling method

Procedure

Diphtheria toxoid

> 1500 Lf per milligram of nitrogen

18 µg

Size-reduced PRP K0 : 0.6-0.7, using cross-linked agarose for chromatography R

25 µg

cyanogen bromide activation of PRP

activated diphtheria toxoid (D-AH+), cyanogen bromide-activated PRP

Tetanus toxoid

> 1500 Lf per milligram of nitrogen

20 µg

PRP ≥ 50 % ≤ K0 : 0.30, using cross-linked agarose for chromatography R

10 µg

carbodi-imide mediated

ADH-activated PRP (PRP-cov.AH) + tetanus toxoid + EDAC

CRM 197 diphtheria protein

> 90 % of diphtheria protein

25 µg

Size-reduced PRP Dp = 15-35 or 10-35

10 µg

reductive amination (1-step method) or N-hydroxysuccinimide activation

direct coupling of PRP to CRM 197 (cyanoborohydride activated)

Meningococcal group B outer membrane protein (OMP)

outer membrane protein vesicles : ≤ 8 % of lipopolysaccharide

125 µg or 250 µg

Size-reduced PRP K0 < 0.6, using cross-linked agarose for chromatography R or Mw > 50 × 103

7.5 µg or 15 µg

thioether bond

PRP activation by CDI PRPIM + BuA2 + BrAc = PRPBuA2-BrAc + thioactivated OMP

ADH = adipic acid dihydrazide BrAc = bromoacetyl chloride BuA2 = butane-1,4-diamide CDI = carbonyldiimidazole

Dp = degree of polymerisation EDAC = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide IM = imidazolium Mw = weight-average molecular weight

CARRIER PROTEIN The carrier protein is chosen so that when the PRP is conjugated it is able to induce a T-cell-dependent B-cell immune response. Currently approved carrier proteins and coupling methods are listed for information in Table 1219.-1. The carrier proteins are produced by culture of suitable micro-organisms ; the bacterial purity of the culture is verified ; the culture may be inactivated ; the carrier protein is purified by a suitable method. Only a carrier protein that complies with the following requirements may be used in the preparation of the conjugate. Identification. The carrier protein is identified by a suitable immunochemical method (2.7.1). Sterility (2.6.1). Carry out the test using for each medium 10 ml or the equivalent of one-hundred doses, whichever is less. Diphtheria toxoid. Diphtheria toxoid is produced as described in Diphtheria vaccine (adsorbed) (0443) and complies with the requirements prescribed therein for bulk purified toxoid. Tetanus toxoid. Tetanus toxoid is produced as described in Tetanus vaccine (adsorbed) (0452) and complies with the requirements prescribed therein for bulk purified toxoid, except that the antigenic purity is not less than 1500 Lf per milligram of protein nitrogen. Diphtheria protein CRM 197. It contains not less than 90 per cent of diphtheria CRM 197 protein, determined by a suitable method. Suitable tests are carried out, for validation or routinely, to demonstrate that the product is nontoxic. OMP (meningococcal group B Outer Membrane Protein complex). OMP complies with the following requirements for lipopolysaccharide and pyrogens. Lipopolysaccharide. Not more than 8 per cent of lipopolysaccharide, determined by a suitable method. Pyrogens (2.6.8). Inject into each rabbit 0.25 µg of OMP per kilogram of body mass.

BULK CONJUGATE PRP is chemically modified to enable conjugation ; it is usually partly depolymerised either before or during this procedure. Reactive functional groups or spacers may be introduced into the carrier protein or PRP prior to conjugation. As a measure of consistency, the extent of derivatisation is monitored. The conjugate is obtained by the covalent binding of PRP and carrier protein. Where applicable, unreacted but potentially reactogenic functional groups are made unreactive by means of capping agents ; the conjugate is purified to remove reagents. Only a bulk conjugate that complies with the following requirements may be used in the preparation of the final bulk vaccine. For each test and for each particular product, limits of acceptance are established and each batch of conjugate must be shown to comply with these limits. Limits applied to currently approved products for some of these tests are listed for information in Table 1219.-2. For a freeze-dried vaccine, some of the tests may be carried out on the final lot rather than on the bulk conjugate where the freeze-drying process may affect the component being tested. PRP. The PRP content is determined by assay of phosphorus (2.5.18) or by assay of ribose (2.5.31) or by an immunochemical method (2.7.1). Protein. The protein content is determined by a suitable chemical method (for example, 2.5.16). PRP to protein ratio. Determine the ratio by calculation. Molecular-size distribution. Molecular-size distribution is determined by size-exclusion chromatography (2.2.30). Free PRP. Unbound PRP is determined after removal of the conjugate, for example by anion-exchange, size-exclusion or hydrophobic chromatography, ultrafiltration or other validated methods. Free carrier protein. Determine the content by a suitable method, either directly or by deriving the content by calculation from the results of other tests. The amount is within the limits approved for the particular product.


663

Hepatitis A (inactivated) and hepatitis B (rDNA) vaccine


Table 1219.-2. – Bulk conjugate requirements for currently approved products Test

Protein carrier Diphtheria toxoid

Tetanus toxoid

CRM 197

OMP

Free PRP

< 37 %

< 20 %

< 25 %

< 15 %

Free protein

7 days

3 01/2005:1102

FELINE CALICIVIROSIS VACCINE (LIVE), FREEZE-DRIED Vaccinum calicivirosis felinae vivum cryodesiccatum DEFINITION Freeze-dried feline calicivirosis vaccine (live) is a preparation of one or more suitable strains of feline calicivirus.

PRODUCTION The virus is grown in suitable cell lines (5.2.4). The viral suspension is harvested and mixed with a suitable stabilising solution. The mixture is subsequently freeze-dried. CHOICE OF VACCINE STRAIN The vaccine strain shall have been shown to be satisfactory with respect to safety (including safety for pregnant queens if such use is not contra-indicated), absence of reversion Sterility. The reconstituted vaccine complies with the test for sterility prescribed in Vaccines for veterinary use (0062). to virulence, and immunogenicity. The following tests may be used during demonstration of safety (5.2.6) and efficacy (5.2.7). POTENCY Safety. To 10 cats of the minimum age stated for vaccination Carry out a potency test for each strain of feline calicivirus and which do not have antibodies against feline calicivirus, in the vaccine. administer by a recommended route, a quantity of virus equivalent to 10 times the maximum titre that may be Use cats 8 to 12 weeks old and that do not have expected in a batch of vaccine. Observe the cats for 21 days. antibodies against feline calicivirus. Vaccinate 10 cats by a No abnormal local or systemic reaction occurs. recommended route and according to the recommended Reversion to virulence. To 2 cats which do not have schedule. Keep 10 cats as controls. 4 weeks after the last antibodies against feline calicivirus administer by a injection, administer intranasally to each vaccinated and recommended route a quantity of virus (for example, control cat a quantity of a virulent strain of calicivirus of approximately 10 doses) that will allow maximum recovery the same type as the vaccine strain sufficient to produce of virus for the passages described below. 5 days later, kill typical signs of the disease (hyperthermia, buccal ulcers, the cats, remove the nasal mucus, tonsils and the trachea, respiratory signs) in at least 8 of the control cats. Observe mix, homogenise in 10 ml of buffered saline and decant ; the cats for 14 days after challenge ; collect nasal washings inoculate the supernatant intranasally into 2 other cats ; daily on days 2 to 14 to test for virus excretion. Note daily the body temperature and signs of disease using the scoring carry out this operation five times. Verify the presence of virus at each passage. If the virus is not recovered, carry out system shown below. The vaccine complies with the test if a second series of passages. Observe the cats given the last the score for the vaccinated cats is significantly lower than passage for 21 days and compare the reactions observed with that for the controls. 758


Feline infectious enteritis vaccine (inactivated)


those seen in the cats in the safety test described above. The Observed signs strain complies with the test if no evidence of an increase in Death virulence is seen compared to the original virus. Depressed state Immunogenicity. The test described under Potency is Temperature ≥ 39.5 °C suitable to demonstrate immunogenicity. Temperature ≤ 37 °C BATCH TESTING If the test for potency has been carried out with satisfactory Ulcer (nasal or oral) results on a representative batch of vaccine, this test may — small and few in number be omitted as a routine control on other batches of vaccine — large and numerous prepared from the same seed lot, subject to agreement by the Nasal discharge competent authority.

IDENTIFICATION When neutralised by one or more monospecific antisera, the reconstituted vaccine no longer infects susceptible cell cultures into which it is inoculated.

TESTS Safety. Administer 10 doses of vaccine , by a recommended route to 2 cats, 8 to 12 weeks old and having no antibodies against feline calicivirus. Observe the cats for 14 days. No abnormal local or systemic reaction occurs. Bacterial and fungal contamination. The reconstituted vaccine complies with the test for sterility prescribed in Vaccines for veterinary use (0062). Mycoplasmas (2.6.7). The reconstituted vaccine complies wtith the test for mycoplasmas. Extraneous viruses. Neutralise the vaccine using one or more monospecific antisera and inoculate into suitable cell cultures ; make at least one passage and maintain the cultures for 14 days. Examine the cultures for cytopathic effects and carry out tests for haemadsorbing agents. No signs of viral contamination occur in the cell cultures.

Score 10 2 1 2 1 3

— slight — copious

1

Ocular discharge

1

Weight loss

2

2

Virus excretion (total number of days) : ≤ 4 days

1

5-7 days

2

>7 days

3 01/2005:0794

FELINE INFECTIOUS ENTERITIS (FELINE PANLEUCOPENIA) VACCINE (INACTIVATED) Vaccinum panleucopeniae felinae infectivae inactivatum DEFINITION Feline infectious enteritis (feline panleucopenia) vaccine (inactivated) is a liquid or freeze-dried preparation of feline panleucopenia virus or canine parvovirus inactivated by a suitable method.

Virus titre. Titrate the reconstituted vaccine in susceptible cell cultures at a temperature favourable to replication of the virus. One dose of the vaccine contains not less than the quantity of virus equivalent to the minimum titre stated on the label.

PRODUCTION The virus is propagated in suitable cell cultures (5.2.4). The virus is harvested and may be purified and concentrated. The test for inactivation is carried out using a quantity of inactivated virus equivalent to not less than 100 doses of the vaccine by a validated method such as the following : inoculate into suitable non-confluent cells POTENCY and after incubation for 8 days, make a subculture using trypsinised cells. After incubation for a further 8 days, Carry out a potency test for each strain of feline calicivirus examine the cultures for residual live parvovirus by an in the vaccine. immunofluorescence test. The immunofluorescence test may be supplemented by a haemagglutination test or other Use cats 8 to 12 weeks old that do not have antibodies suitable tests on the supernatant of the cell cultures. No against feline calicivirus. Vaccinate 10 cats by one of the live virus is detected. recommended routes and according to the recommended The vaccine may contain an adjuvant and may be freeze-dried. schedule. Keep 10 cats as controls. 4 weeks after the last CHOICE OF VACCINE COMPOSITION injection, administer intranasally to each vaccinated and control cat a quantity of a virulent strain of calicivirus of the The vaccine is shown to be satisfactory with respect to safety same type as the vaccine strain sufficient to produce typical (5.2.6) and efficacy (5.2.7) in cats. The following test may be signs of the disease (hyperthermia, buccal ulcers, respiratory used during demonstration of immunogenicity. signs) in not fewer than 8 of the control cats. Observe the Immunogenicity. Use 10 susceptible cats, 8 to 12 weeks cats for 14 days after challenge ; collect nasal washings daily old. Draw a blood sample from each cat and test individually on days 2 to 14 to test for virus excretion. Note daily the for antibodies against feline panleucopenia virus and canine body temperature and signs of disease using the scoring parvovirus to determine susceptibility. Vaccinate 5 cats by system shown below. The vaccine complies with the test if the recommended schedule. Carry out leucocyte counts the score for the vaccinated cats is significantly lower than 8 days and 4 days before challenge and calculate the mean of that for the controls. the 2 counts to serve as the initial value. 20 to 22 days after General Notices (1) apply to all monographs and other texts

759

Feline infectious enteritis vaccine (live)


at least 1:20. The test is not valid if either control cat the last vaccination, challenge each cat by the intraperitoneal develops antibodies against canine parvovirus or feline injection of a suspension of pathogenic feline panleucopenia panleucopenia virus. virus. Observe the cats for 14 days. Carry out leucocyte counts on the fourth, sixth, eighth and tenth days after B. Vaccinate according to the recommended schedule, challenge. The test is not valid unless the 5 control cats all 2 cats, 8 to 12 weeks old and having antibody titres less show on not fewer than one occasion a diminution in the than 4 ND50 (neutralising dose 50 per cent) per 0.1 ml number of leucocytes of at least 75 per cent of the initial of serum measured by the method described below. value ; these animals may die from panleucopenia. The 14 days after vaccination, examine the serum of each vaccine complies with the test if the 5 vaccinated cats remain animal as follows. Heat the serum at 56 °C for 30 min in excellent health and show no sign of leucopenia ; that is and prepare serial dilutions using a medium suitable for to say, the diminution in the number of leucocytes does not feline cells. Add to each dilution an equal volume of a exceed, in any of the four counts, 50 per cent of the initial virus suspension containing an amount of virus such that value. when the volume of serum-virus mixture appropriate for the assay system is inoculated into cell cultures, each BATCH TESTING culture receives approximately 104 CCID50. Incubate the Batch potency test. For routine testing of batches of vaccine mixtures at 37 °C for 1 h and inoculate four feline cell a test based on production of haemagglutination-inhibiting cultures with a suitable volume of each mixture. Incubate antibodies in guinea-pigs may be used instead of test A or B the cell cultures at 37 °C for 7 days, passage and incubate described under Potency if a satisfactory correlation with for a further 7 days. Examine the cultures for evidence of the test for immunogenicity has been established. specific cytopathic effects and calculate the antibody titre. The vaccine complies with the test if the mean titre is not IDENTIFICATION less than 32 ND50 per 0.1 ml of serum. If one cat fails to respond, repeat the test using 2 more cats and calculate When injected into animals, the vaccine stimulates the the result as the mean of the titres obtained from all of production of antibodies against the parvovirus present in the 3 cats that have responded. the vaccine. TESTS Safety. Use cats of the minimum age recommended for vaccination and preferably having no antibodies against feline panleucopenia virus or against canine parvovirus or, where justified, use cats with a low level of such antibodies as long as they have not been vaccinated against feline panleucopenia virus or against canine parvovirus, and administration of the vaccine does not cause an anamnestic response. Administer by a recommended route a double dose of vaccine to each of 2 cats. Observe the animals for 14 days. No abnormal local or systemic reaction occurs. Sterility. The vaccine complies with the test for sterility prescribed in the monograph on Vaccines for veterinary use (0062).

01/2005:0251

FELINE INFECTIOUS ENTERITIS (FELINE PANLEUCOPENIA) VACCINE (LIVE) Vaccinum panleucopeniae felinae infectivae vivum DEFINITION Feline infectious enteritis (feline panleucopenia) vaccine (live) is a preparation of a suitable strain of feline panleucopenia virus.

PRODUCTION The virus is propagated in suitable cell cultures (5.2.4). The virus suspension is harvested and may be purified and Carry out test A or test B. concentrated. It is mixed with a suitable stabilising solution. A. Use 4 cats, 8 to 12 weeks old. Draw a blood sample from The vaccine may be freeze-dried. each cat and test individually for antibodies against feline CHOICE OF VACCINE STRAIN panleucopenia virus and canine parvovirus to determine The vaccine strain shall have been shown to be satisfactory susceptibility. Inject by a recommended route one dose with respect to safety (including safety for pregnant of vaccine into each of 2 cats. After 21 days, draw a queens if such use is not contra-indicated or if the virus is blood sample from each cat and separate the serum excreted in the faeces), absence of increase in virulence and from each sample. Inactivate each serum by heating immunogenicity. at 56 °C for 30 min. To 1 volume of each serum add The following tests may be used during demonstration of 9 volumes of a 200 g/l suspension of light kaolin R in phosphate buffered saline pH 7.4 R. Shake each mixture safety (5.2.6) and efficacy (5.2.7). for 20 min. Centrifuge, collect the supernatant liquid Safety. Each test is carried out for each recommended and mix with 1 volume of a concentrated suspension of route of administration. Use 5 cats, of the minimum pig erythrocytes. Allow to stand at 4 °C for 60 min and age recommended for vaccination and free from specific centrifuge. The dilution of the serum obtained is 1:10. haemagglutination-inhibiting antibodies against feline Using each serum, prepare a series of twofold dilutions. panleucopenia virus and canine parvovirus. Make counts To 0.025 ml of each of the latter dilutions add 0.025 ml of of leucocytes in circulating blood on days 8 and 4 before a suspension of canine parvovirus or feline panleucopenia injection of the vaccine strain and calculate the mean virus antigen containing 4 haemagglutinating units. of the 2 counts to serve as the initial value. Administer Allow to stand at 37 °C for 30 min and add 0.05 ml of a to each cat by a recommended route a quantity of virus suspension of pig erythrocytes containing 30 × 106 cells corresponding to at least 10 times the maximum virus titre that may be expected in a batch of vaccine and at the lowest per millilitre. Allow to stand at 4 °C for 90 min and level of attenuation. Observe the animals for 21 days. Make note the last dilution of serum that still completely leucocyte counts on the fourth, sixth, eighth and tenth days inhibits haemagglutination. The vaccine complies with after inoculation. The strain complies with the test if: the the test if both vaccinated cats have developed titres of POTENCY

760



Feline leukaemia vaccine (inactivated)

cats remain in good health and there is no abnormal local or systemic reaction ; for each animal and for each blood count, the number of leucocytes is not less than 50 per cent of the initial value.

Virus titre. Reconstitute the vaccine, if necessary, and titrate in suitable cell cultures. One dose of vaccine contains not less than the quantity of virus equivalent to the minimum titre stated on the label.

Increase in virulence. Use 2 cats of the minimum age recommended for vaccination and which do not have haemagglutination-inhibiting antibodies against feline panleucopenia virus and canine parvovirus. Administer to each cat, by a recommended route, a quantity of virus suitable to allow maximum recovery of the virus for subsequent passages. From the second to the tenth day after administration of the virus, collect the faeces from each cat and check for the presence of the virus ; pool faeces containing virus. Administer 1 ml of the suspension of pooled faeces by the oronasal route to each of 2 other cats of the same age and susceptibility ; repeat this operation a further 4 times. Verify the presence of virus at each passage. If the virus is not found, carry out a second series of passages ; if the virus is not found in one of the second series of passages, the vaccine strain complies with the test. The vaccine strain complies with the test if : no cat dies or shows signs attributable to the vaccine ; no indication of increase of virulence compared to the original vaccinal virus is observed ; account is taken, notably, of the count of white blood cells, of results of histological examination of the thymus and of the titre of excreted virus.

POTENCY Use 10 susceptible cats, 8 to 12 weeks old. Draw a blood sample from each cat and test individually for antibodies against feline panleucopenia virus and canine parvovirus to determine susceptibility. Vaccinate 5 cats by the recommended schedule. Carry out leucocyte counts 8 days and 4 days before challenge and calculate the mean of the 2 counts to serve as the initial value. 20 to 22 days after the last vaccination, challenge each cat by the intraperitoneal injection of a suspension of pathogenic feline panleucopenia virus. Observe the cats for 14 days. Carry out leucocyte counts on the fourth, sixth, eighth and tenth days after challenge. The test is not valid unless the 5 control cats all show on not fewer than one occasion a diminution in the number of leucocytes of at least 75 per cent of the initial value ; these animals may die from panleucopenia. The vaccine complies with the test if the 5 vaccinated cats remain in excellent health and show no sign of leucopenia ; that is to say, the diminution in the number of leucocytes does not exceed, in any of the four counts, 50 per cent of the initial value.

Immunogenicity. The test described under Potency is suitable to demonstrate immunogenicity of the strain.

LABELLING The label states that the vaccine should not be used in pregnant queens (unless it has been shown to be safe in such conditions).

BATCH TESTING If the test for potency has been carried out with satisfactory results on a representative batch of vaccine, this test may be omitted as a routine control on other batches of vaccine prepared from the same seed lot, subject to agreement by the competent authority. IDENTIFICATION Carry out replication of the vaccine virus in a susceptible cell line in a substrate suitable for a fluorescent antibody test or peroxidase test. Prepare suitable controls. Test a proportion of the cells with monoclonal antibodies specific for feline panleucopenia virus and a proportion with monoclonal antibodies specific for canine parvovirus. Feline panleucopenia virus is detected but no canine parvovirus is detected in the cells inoculated with the vaccine.

01/2005:1321

FELINE LEUKAEMIA VACCINE (INACTIVATED) Vaccinum leucosis felinae inactivatum DEFINITION Feline leukaemia vaccine (inactivated) is a preparation of immunogens from a suitable strain of feline leukaemia virus.

PRODUCTION The immunogens consist either of a suitable strain of feline leukaemia virus inactivated in such a manner that adequate immunogenicity is maintained or of a fraction of the virus TESTS with adequate immunogenic properties ; the immunogenic Safety. Use 2 cats of the minimum age recommended fraction may be produced by recombinant DNA technology. for vaccination and having no antibodies against feline Where applicable, the test for inactivation is carried out panleucopenia virus or against canine parvovirus. Administer using a quantity of inactivated virus equivalent to not less 10 doses of the vaccine to each cat, by a recommended than 25 doses of vaccine and two passages in the same type route. Observe the animals for 14 days. No abnormal local of cell cultures as used for the production of the vaccine or systemic reaction occurs. or in cell cultures shown to be at least as sensitive ; no live Extraneous viruses. Neutralise the vaccine using a suitable virus is detected. monospecific antiserum against feline panleucopenia virus The vaccine may contain an adjuvant. and inoculate into suitable cell cultures ; make at least one CHOICE OF VACCINE COMPOSITION passage and maintain the cultures for 14 days. Examine The choice of the feline leukaemia virus strains and/or of the cultures for cytopathic effects and carry out tests for the antigens included in the vaccine composition is made haemadsorbing agents. No signs of viral contamination in such a manner as to ensure the safety (5.2.6) (including occur in the cell cultures. safety for pregnant queens if the vaccine may be used in Bacterial and fungal contamination. The vaccine, such animals) and immunogenicity (5.2.7) of the vaccine. reconstituted if necessary, complies with the test for sterility The following tests may be used during demonstration of prescribed in the monograph on Vaccines for veterinary safety and immunogenicity. use (0062). Safety. Carry out the test for each intended route of Mycoplasmas (2.6.7). The vaccine complies with the test administration. Use animals that do not have antibodies for mycoplasmas. against gp 70 antigen of feline leukaemia virus nor display General Notices (1) apply to all monographs and other texts

761

Feline viral rhinotracheitis vaccine (inactivated)


TESTS Safety. Use two cats of the minimum age recommended for vaccination and that do not have antibodies against feline leukaemia virus. Inject by a recommended route A. Vaccinate, according to the intended schedule, not a double dose of vaccine into each animal. Observe the fewer than ten cats of the minimum age recommended animals for 14 days. The vaccine complies with the test if no for vaccination. Keep five cats as controls. Record the abnormal local or systemic reaction is produced. rectal temperature of each cat on the day before each vaccination, at the time of vaccination, 4 h and 8 h later, Inactivation. If the vaccine contains inactivated virus, and once per day during the four following days. Observe carry out a test for residual live feline leukaemia virus by the animals for at least 4 weeks after the last vaccination. making two passages on susceptible cell cultures. No virus No abnormal local or systemic reaction occurs during is detected. If the vaccine contains an adjuvant, if possible the test. 1, 2 and 4 weeks after the last vaccination, separate the adjuvant from the liquid phase by a method submit the animals to suitable tests for evidence of an that does not inactivate the virus nor interfere in any other immunosuppressive effect. The vaccine complies with the way with the detection of virus. test if no significant difference is observed in vaccinated Sterility. The vaccine complies with the test for sterility animals compared with controls. prescribed in Vaccines for veterinary use (0062). B. Inject two doses of vaccine by one of the intended POTENCY routes to not fewer than ten cats of the minimum age Use not fewer than twenty-five susceptible cats of the recommended for vaccination. At the end of the period of time stated in the instructions for use, inject one dose minimum age recommended for vaccination, free from antibodies against the antigens of feline leukaemia virus and of vaccine to each animal. Where the instructions for against the feline oncogene membrane antigen (anti-FOCMA use recommend it, administer a third injection after the antibodies), and showing no viraemia or antigenaemia at the period indicated. Observe the animals for 14 days after time of the test. Vaccinate not fewer than fifteen cats by a the last administration. The vaccine complies with the recommended route in accordance with the instructions for test if no abnormal local or systemic reaction occurs use. Keep not fewer than ten cats as controls. Observe the during the test. animals for at least 14 days after the last administration of C. If the vaccine is not contra-indicated for use in pregnant vaccine. Inject by the peritoneal or oronasal route, on one queens, inject two doses of vaccine into each of not fewer or several occasions, a quantity of a virulent strain of feline than ten cats at different stages of pregnancy. Observe leukaemia virus sufficient to induce persistent viraemia or the cats until parturition and note any effects on gestation antigenaemia in not fewer than 80 per cent of susceptible and the offspring. The vaccine complies with the test if animals ; use for the challenge an epidemiologically relevant no abnormal local or systemic reaction occurs during strain consisting predominantly of type A virus. Observe the the test. animals for 15 weeks and, from the third week onwards, test each week for viraemia or antigenaemia (p27 protein) by Immunogenicity. The test described under Potency is suitable methods such as immunofluorescence on circulating suitable to demonstrate immunogenicity of the vaccine. leucocytes or enzyme-linked immunosorbent assay. A cat is considered persistently infected if it shows positive viraemia IN-PROCESS CONTROL TESTS or antigenaemia for three consecutive weeks or on five During production, suitable immunochemical tests are occasions, consecutively or not, between the third and the carried out for the evaluation of the quality and purity of the viral antigens included in the vaccine composition. The fifteenth week. The test is not valid if fewer than 80 per cent values found are within the limits approved for the particular of the control cats are persistently infected. The vaccine complies with the test if not fewer than 80 per cent of the vaccine. vaccinated cats show no persistent infection. BATCH TESTING LABELLING Potency. The test described under Potency is not carried out for routine testing of batches of vaccine. It is carried out, The label states the antigen or antigens contained in the for a given vaccine, on one or more occasions, as decided by vaccine. or with the agreement of the competent authority ; where the test is not carried out, a suitable validated alternative method is used, the criteria for acceptance being set with reference to a batch of vaccine that has given satisfactory 01/2005:1207 results in the test described under Potency. viraemia or antigenaemia at the time of the test ; absence of antibodies and antigen is demonstrated by enzyme-linked immunosorbent assay (2.7.1).

Bacterial endotoxins. For vaccines produced by recombinant DNA technology with a bacterial host cell such as Escherichia coli, a test for bacterial endotoxins (2.6.14) is carried out on each final lot or, where the nature of the adjuvant prevents performance of a satisfactory test, on the antigen immediately before addition of the adjuvant. The value found is within the limit approved for the particular vaccine and which has been shown to be safe for cats. IDENTIFICATION When injected into healthy, seronegative cats, the vaccine stimulates the production of specific antibodies against the antigen or antigens stated on the label. 762

FELINE VIRAL RHINOTRACHEITIS VACCINE (INACTIVATED) Vaccinum rhinotracheitidis viralis felinae inactivatum DEFINITION Feline viral rhinotracheitis vaccine (inactivated) is a preparation of a suitable strain of feline rhinotracheitis virus (feline herpesvirus 1) that has been inactivated while maintaining adequate immunogenic properties or of an inactivated fraction of the virus having adequate immunogenic properties.


Feline viral rhinotracheitis vaccine (live), freeze-dried


PRODUCTION The vaccine strain is grown in suitable cell cultures (5.2.4). The viral suspension is harvested and inactivated. The test for inactivation is carried out using 2 passages in cell cultures of the same type as those used for preparation of the vaccine or in cell cultures shown to be at least as sensitive ; the quantity of virus used in the test is equivalent to not less than 25 doses of vaccine. No live virus is detected. The virus may be fragmented and the fragments may be purified and concentrated. The vaccine may be adjuvanted ; it may be freeze-dried. CHOICE OF VACCINE COMPOSITION The vaccine is shown to be satisfactory with respect to safety and immunogenicity in cats. The following test may be used during demonstration of efficacy (5.2.7). Immunogenicity. The test described under Potency, carried out using vaccine prepared from the most attenuated virus that will be attained during production, is suitable to demonstrate immunogenicity of the strain. BATCH TESTING The test described under Potency is not necessarily carried out for routine testing of batches of vaccine. It is carried out, for a given vaccine, on one or more occasions, as decided by or with the agreement of the competent authority. Where the test is not carried out, a suitable alternative validated test is carried out, the criteria for acceptance being set with reference to a batch of vaccine that has given satisfactory results in the test described under Potency. The following test may be used after a satisfactory correlation with the test described under Potency has been established. Batch potency test. Use a group of 15 seronegative mice. Administer half a dose of the vaccine to each mouse and 7 days later repeat the administration. 21 days after the first injection, take blood samples and determine the level of antibodies against feline rhinotracheitis virus by a suitable immunochemical method (2.7.1), such as an immunofluorescence technique using pools of serum from groups of 3 mice. The antibody levels are not significantly lower than those obtained with a batch of vaccine that has given satisfactory results in the test described under Potency.

Sterility. The reconstituted vaccine complies with the test for sterility prescribed under Vaccines for veterinary use (0062).

IDENTIFICATION When administered to susceptible animals, the vaccine stimulates the production of specific serum antibodies against feline rhinotracheitis virus or against the fraction of the virus used to produce the vaccine. TESTS Safety. Use cats 8 to 12 weeks old and preferably having no feline rhinotracheitis virus antibodies or antibodies to a fraction of the virus, or, where justified, use cats with a low level of such antibodies as long as they have not been vaccinated against viral rhinotracheitis and administration of the vaccine does not cause an anamnestic response. Administer a double dose of vaccine by a recommended route to each of 2 cats. Observe the cats for 14 days. No abnormal local or systemic reaction occurs. Inactivation. Carry out a test for residual infectious feline rhinotracheitis virus using 10 doses of vaccine and 2 passages in cell cultures of the same type as those used for preparation of the vaccine or in other suitably sensitive cell cultures. No live virus is detected. If the vaccine contains an adjuvant that interferes with the test, where possible separate the adjuvant from the liquid phase by a method that does not inactivate or otherwise interfere with detection of live virus. General Notices (1) apply to all monographs and other texts

POTENCY Use cats 8 to 12 weeks old and which do not have antibodies against feline rhinotracheitis virus or against a fraction of the virus. Vaccinate 10 cats according to the instructions for use and keep 10 cats as controls. 4 weeks after the last administration of vaccine, administer intranasally to each of the 20 cats, a quantity of virulent feline rhinotracheitis virus sufficient to produce in susceptible cats typical signs of disease such as fever, nasal discharge and cough. Observe the cats for 14 days ; collect nasal washings daily on days 2 to 14 after challenge to test for virus excretion. Note daily the body temperature and signs of disease using the scoring system shown below. If any sign of disease is observed on more than one day, record the score once only. The vaccine complies with the test if the score for the vaccinated cats is significantly lower than that for the controls. Sign

Score

Death

10

Depressed state Temperature :

2

39.5 °C - 40.0 °C

1

≥ 40.0 °C

2

≤ 37.0 °C

3

Glossitis

3

Nasal discharge, slight

1

Nasal discharge, copious

2

Cough Sneezing

2

Sneezing, paroxysmal

2

Ocular discharge, slight

1

Ocular discharge, serious Conjunctivitis

2

Weight loss ≥ 5.0 per cent

5

1

2

Virus excretion (total number of days) : ≤ 4 days

1

5-7 days

2

> 7 days

3 01/2005:1206

FELINE VIRAL RHINOTRACHEITIS VACCINE (LIVE), FREEZE-DRIED Vaccinum rhinotracheitidis viralis felinae vivum cryodesiccatum DEFINITION Freeze-dried feline viral rhinotracheitis vaccine (live) is a preparation of a suitable strain of feline rhinotracheitis virus (feline herpesvirus 1). PRODUCTION The vaccine strain is grown in suitable cell cultures (5.2.4). The viral suspension is harvested and mixed with a suitable stabilising solution. The mixture is subsequently freeze-dried. 763

Foot-and-mouth disease (ruminants) vaccine (inactivated)


CHOICE OF VACCINE STRAIN Only a virus strain shown to be satisfactory with respect to safety, reversion to virulence and immunogenicity may be used in the preparation of the vaccine ; if the vaccine is not contra-indicated in pregnant queens, the safety for such use is demonstrated. The following tests may be used during demonstration of safety (5.2.6) and efficacy (5.2.7). Safety. Administer by a recommended route to each of ten cats, of the minimum age stated for vaccination and which do not have antibodies against feline rhinotracheitis virus, a quantity of virus equivalent to ten times the maximum titre that may be expected in a batch of vaccine. Observe the cats for 21 days. They remain in good health and show no abnormal local or systemic reaction. Reversion to virulence. Administer by a recommended route to each of two cats that do not have antibodies against feline rhinotracheitis virus, a quantity of virus that will allow optimal re-isolation of the virus for subsequent passages (for example ten times the minimum titre to be stated on the label). Kill the cats 2 to 4 days after the inoculation and remove the nasal mucus, tonsils and local lymphatic ganglia and the trachea ; mix the samples, homogenise in 10 ml of buffered saline and allow to settle ; inoculate 1 ml of supernatant intranasally into two cats ; repeat this operation not fewer than five times ; verify the presence of virus at each passage ; if the virus is not recovered, carry out a second series of passages. Observe the cats given the last passage for 21 days and compare any reactions with those seen in the safety test described above. No evidence of an increase in virulence is seen compared to the original virus. Immunogenicity. The test described under Potency is suitable to demonstrate immunogenicity of the strain. BATCH TESTING If the test for potency has been carried out with satisfactory results on a representative batch of vaccine, this test may be omitted as a routine control on other batches of vaccine prepared from the same seed lot, subject to agreement by the competent authority.

POTENCY Use cats 8 to 12 weeks old and which do not have antibodies against feline viral rhinotracheitis virus. Vaccinate ten cats according to the instructions for use and keep ten cats as controls. Four weeks after the last administration of vaccine, administer intranasally to each cat a quantity of virulent feline rhinotracheitis virus sufficient to produce in susceptible cats typical signs of disease such as fever, nasal discharge and cough. Observe the cats for 14 days ; collect nasal washings daily on days 2 to 14 after challenge to test for virus excretion. Note daily the body temperature and signs of disease using the scoring system shown below. If any sign of disease is observed on more than one day, record the score once only. The vaccine complies with the test if the score for the vaccinated cats is significantly lower than that for the controls.

IDENTIFICATION When mixed with a monospecific antiserum, the reconstituted vaccine no longer infects susceptible cell cultures into which it is inoculated.

Score

Death

10

Depressed state Temperature :

2

39.5 °C - 40.0 °C

1

≥ 40.0 °C

2

≤ 37.0 °C

3

Glossitis

3

Nasal discharge, slight

1

Nasal discharge, copious

2

Cough Sneezing

2

Sneezing, paroxysmal

2

Ocular discharge, slight

1

Ocular discharge, serious Conjunctivitis

2 2

Weight loss ≥ 5.0 per cent

5

1

Virus excretion (total number of days) :

TESTS Safety. Administer ten doses of the vaccine in a suitable volume by a recommended route to two cats, 8 to 12 weeks old, that do not have antibodies against feline rhinotracheitis virus. Observe the cats for 14 days. They remain in good health and show no abnormal local or systemic reaction. Bacterial and fungal contamination. The reconstituted vaccine complies with the test for sterility prescribed in the monograph on Vaccines for veterinary use (0062). Mycoplasmas (2.6.7). The reconstituted vaccine complies with the test for mycoplasmas. Extraneous viruses. Neutralise the vaccinal virus using a monospecfic antiserum and inoculate into suitable cell cultures ; make at least one passage and maintain the cultures for 14 days. No cytopathic effect develops ; the cells show no evidence of the presence of haemadsorbing agents or other signs of viral contamination. Virus titre. Titrate the reconstituted vaccine in susceptible cell cultures at a temperature favourable to replication of the virus. One dose of vaccine contains not less than the quantity of virus corresponding to the minimum virus titre stated on the label. 764

Sign

≤ 4 days

1

5-7 days

2

> 7 days

3 01/2005:0063 corrected

FOOT-AND-MOUTH DISEASE (RUMINANTS) VACCINE (INACTIVATED) Vaccinum aphtharum epizooticarum inactivatum ad ruminantes DEFINITION Foot-and-mouth disease (ruminants) vaccine (inactivated) is a liquid preparation containing one or more types or sub-types of foot-and-mouth disease virus inactivated without impairing their immunogenic activity. PRODUCTION The virus is grown either in suspensions of cattle-tongue epithelium obtained immediately after slaughter of healthy animals, or, preferably, in suitable cell cultures (5.2.4). The



Foot-and-mouth disease (ruminants) vaccine (inactivated)

virus is separated from cellular material by filtration or other TESTS suitable procedures and the virus is inactivated in suitable Safety. Use three non-vaccinated cattle not less than conditions. 6 months old having serum free from foot-and-mouth disease antibodies and coming from regions free from The vaccine is prepared from inactivated antigen by foot-and-mouth disease. Inoculate each animal intradermally blending with one or more adjuvants. The antigen may into the tongue at not fewer than twenty points, using 0.1 ml be concentrated and purified by a number of procedures. of the vaccine at each point. Observe the animals for not less The concentrated antigen is used for the preparation of than 4 days. No lesions or signs of foot-and-mouth disease vaccine immediately or after storage at a temperature not occur. At the end of the observation period, inject into the exceeding − 90 °C, unless stability of the antigen at a higher same animals by the prescribed route three times the dose temperature has been demonstrated. stated on the label. Observe the animals for 6 days after this inoculation. No lesions or clinical signs of foot-and-mouth Validation of the inactivation procedure. During disease occur on the feet or tongue and any reaction at the inactivation, the virus titre is monitored by a sensitive and site of injection remains insignificant. reproducible technique. The inactivation procedure is not satisfactory unless the decrease in virus titre, plotted Sterility. It complies with the test for sterility prescribed in logarithmically, is linear and extrapolation indicates that the monograph on Vaccines for veterinary use (0062). there is less than 1 infectious virus unit per 104 litres of liquid preparation at the end of inactivation. POTENCY BULK INACTIVATED ANTIGEN The potency of the vaccine is expressed as the number Inactivation. A proportion of each batch of bulk inactivated of 50 per cent cattle protective doses (PD50) contained in antigen representing at least 200 doses is tested for freedom the dose stated on the label. The PD50 is determined in from infectious virus by inoculation into sensitive cell animals given primary vaccination and challenged by the cultures. For this purpose, the sample of the inactivated inoculation of 10 000 ID50 of virulent bovine virus of the antigen is concentrated to allow testing of such large same type or sub-type as that used in the preparation of the samples in cell cultures. It must be shown that the selected vaccine in the conditions described below. concentration and assay systems are not detrimental to detection of infectious virus within the test sample and that The vaccine contains at least 3 PD50 per dose for cattle. the concentrated inactivated antigen does not interfere with Carry out a potency test for each type and sub-type of virus replication or cause toxic changes. foot-and-mouth disease virus present in the vaccine. Antigenicity. The antigen content of each batch of bulk inactivated antigen is determined by an in vitro method (for example, 146S-particle measurement by sucrose density gradient centrifugation and ultraviolet spectophotometry at 259 nm).

Use cattle not less than 6 months old, obtained from areas free from foot-and-mouth disease, which have never been vaccinated against foot-and-mouth disease and are free from antibodies neutralising the different types of foot-and-mouth disease virus. Vaccinate not fewer than three groups of not fewer than five cattle per group by the route stated on the Safety. A representative sample of the bulk inactivated label. Use a different dose of the vaccine for each group. antigen may be diluted and blended with the adjuvants for Administer the different doses by injecting different volumes safety testing in cattle, as described below. of the vaccine rather than by dilution of the vaccine. Sterility. The bulk inactivated antigen, the adjuvants and all For example, if the label states that the injection of 2 ml dilution buffers comply with the test for sterility prescribed corresponds to the administration of 1 dose of vaccine, a in the monograph on Vaccines for veterinary use (0062). 1/4 dose of vaccine would be obtained by injecting 0.5 ml, and a 1/10 dose would be would be obtained by injecting Potency. A representative sample of the bulk inactivated 0.2 ml. Three weeks after the vaccination, challenge the antigen may be diluted and blended with the adjuvants for vaccinated animals and a control group of two animals with potency testing in cattle, as described below. a suspension of virus that has been obtained from cattle and that is fully virulent and of the same type or sub-type as In situations of extreme urgency and subject to agreement that used in the preparation of the vaccine, by inoculating a by the competent authority, a batch of vaccine may total of 10 000 ID50 intradermally into two sites on the upper be released before completion of the tests and the surface of the tongue (0.1 ml per site). Observe the animals determination of potency if a test for sterility has been for 8 days and then slaughter. Unprotected animals show carried out on the bulk inactivated antigen and all other lesions at sites other than the tongue. Protected animals components of the vaccine and if the test for safety and may display lingual lesions. The test is not valid unless each the determination of potency have been carried out on a control animal shows lesions on at least three feet. From the representative batch of vaccine prepared from the same number of protected animals in each group, calculate the bulk inactivated antigen. In this context, a batch is not PD50 content of the vaccine. considered to be representative unless it has been prepared using the same amount of antigen and with the same STORAGE formulation as the batch to be released. The vaccine must not be frozen. IDENTIFICATION

LABELLING The label states :

The serum of a susceptible animal that has been immunised with the vaccine neutralises the types or sub-types of the virus used to prepare the vaccine, when tested by a suitably sensitive method. General Notices (1) apply to all monographs and other texts

— the method of preparation, — the types and sub-types of virus used to prepare the vaccine. 765

Fowl-pox vaccine (live)


01/2005:0649 to be recommended for vaccination and from an SPF flock (5.2.2). Carry out this passage operation not fewer than 5 times ; verify the presence of the virus at each passage. FOWL-POX VACCINE (LIVE) Care must be taken to avoid contamination by virus from previous passages. If the virus is not found at a passage level, carry out a second series of passages. Carry out the Vaccinum variolae gallinaceae vivum test for safety (section 2-4-1) using the unpassaged vaccine virus and the maximally passaged virus that has been 1. DEFINITION recovered. Administer the virus by the route recommended Fowl-pox vaccine (live) is a preparation of a suitable strain of for vaccination likely to be the least safe. The vaccine avian pox virus. This monograph applies to vaccines intended virus complies with the test if no indication of increase in for administration to chickens for active immunisation. virulence of the maximally passaged virus compared with the unpassaged virus is observed. If virus is not recovered at 2. PRODUCTION any passage level in the first and second series of passages, the vaccine virus also complies with the test. 2-1. PREPARATION OF THE VACCINE The vaccine virus is grown in embryonated hens’ eggs or in 2-4-3. Immunogenicity. A test is carried out for each route cell cultures. and method of administration to be recommended using in each case chickens not older than the youngest age 2-2. SUBSTRATE FOR VIRUS PROPAGATION to be recommended for vaccination. The quantity of the 2-2-1. Embryonated hens’ eggs. If the vaccine virus is grown vaccine virus administered to each chicken is not greater in embryonated hens’ eggs, they are obtained from flocks than the minimum virus titre to be stated on the label and free from specified pathogens (SPF) (5.2.2). the virus is at the most attenuated passage level that will 2-2-2. Cell cultures. If the vaccine virus is grown in cell be present in a batch of the vaccine. Use for the test not cultures, they comply with the requirements for cell cultures fewer than 30 chickens of the same origin and from an SPF for production of veterinary vaccines (5.2.4). flock (5.2.2). Vaccinate by a recommended route not fewer than 20 chickens. Maintain not fewer than 10 chickens 2-3. SEED LOTS as controls. Challenge each chicken after 21 days by the 2-3-1. Extraneous agents. The master seed lot complies feather-follicle route with a sufficient quantity of virulent with the tests for extraneous agents in seed lots (2.6.24). In fowl-pox virus. Observe the chickens at least daily for these tests on the master seed lot, the organisms used are 21 days after challenge. Record the deaths and the number not more than 5 passages from the master seed lot at the of surviving chickens that show clinical signs of disease. start of the tests. Examine each surviving chicken for macroscopic lesions : cutaneous lesions of the comb, wattle and other unfeathered 2-4. CHOICE OF VACCINE VIRUS areas of the skin and diphtherical lesions of the mucous The vaccine virus shall be shown to be satisfactory with respect to safety (5.2.6) and efficacy (5.2.7) for the chickens membranes of the oro-pharyngeal area. for which it is intended. The test is not valid if: The following tests for safety (section 2-4-1), increase in — during the observation period after challenge fewer virulence (section 2-4-2) and immunogenicity (section than 90 per cent of the control chickens die or show 2-4-3) may be used during demonstration of safety and severe clinical signs of fowl pox, including notable immunogenicity. macroscopical lesions of the skin or mucous membranes of the oro-pharyngeal area, 2-4-1. Safety. Carry out the test for each route and method of administration to be recommended for vaccination using — and/or during the period between vaccination and in each case chickens not older than the youngest age to challenge, more than 10 per cent of the control or be recommended for vaccination. Use vaccine virus at the vaccinated chickens show abnormal clinical signs or die least attenuated passage level that will be present between from causes not attributable to the vaccine. the master seed lot and a batch of the vaccine. For each test The vaccine virus complies with the test if during the use not fewer than 20 chickens, from an SPF flock (5.2.2). observation period after challenge not less than 90 per cent Administer to each chicken a quantity of the vaccine virus of the vaccinated chickens survive and show no notable equivalent to not less than 10 times the maximum virus titre clinical signs of disease, including macroscopical lesions of likely to be contained in a dose of the vaccine. Observe the the skin and mucous membranes of the oro-pharyngeal area. chickens at least daily for 21 days. The test is not valid if more than 10 per cent of the chickens die from causes not 3. BATCH TESTS attributable to the vaccine virus. The vaccine virus complies 3-1. Identification. Carry out an immunostaining test in cell with the test if no chicken shows notable clinical signs of cultures to demonstrate the presence of the vaccine virus. fowl pox or dies from causes attributable to the vaccine virus. For egg adapted strains, inoculate the vaccine into eggs and 2-4-2. Increase in virulence. Administer by a suitable route notice the characteristic lesions. a quantity of the vaccine virus that will allow recovery of 3-2. Bacteria and fungi virus for the passages described below to each of 5 chickens Vaccines intended for administration by injection, not older than the minimum age to be recommended scarification or piercing of the wing web comply with the for vaccination and from an SPF flock (5.2.2). Use the test for sterility prescribed in the monograph Vaccines for vaccine virus at the least attenuated passage level that veterinary use (0062). will be present between the master seed lot and a batch Vaccines not intended for administration by injection, of the vaccine. Prepare 4 to 7 days after administration a scarification or piercing of the wing web either comply suspension from the induced skin lesions of each chicken with the test for sterility prescribed in the monograph and pool these samples. Administer 0.2 ml of the pooled Vaccines for veterinary use (0062) or with the following samples by cutaneous scarification of the comb or other unfeathered part of the body, or by another suitable method test : carry out a quantitative test for bacterial and to each of 5 other chickens not older than the minimum age fungal contamination ; carry out identification tests for 766


Furunculosis vaccine (inactivated) for salmonids


microorganisms detected in the vaccine ; the vaccine does not contain pathogenic microorganisms and contains not more than 1 non-pathogenic microorganism per dose. Any liquid supplied with the vaccine complies with the test for sterility prescribed in the monograph Vaccines for veterinary use (0062). 3-3. Mycoplasmas. The vaccine complies with the test for mycoplasmas (2.6.7). 3-4. Extraneous agents. The vaccine complies with the tests for extraneous agents in batches of finished product (2.6.25). 3-5. Safety. Use not fewer than 10 chickens from an SPF flock (5.2.2) of the youngest age recommended for vaccination. For vaccines recommended for use in chickens older than 6 weeks, chickens 6 weeks old may be used. Administer 10 doses of the vaccine to each chicken by a recommended route. Observe the chickens at least daily for 21 days. The test is not valid if more than 20 per cent of the chickens show abnormal clinical signs or die from causes not attributable to the vaccine. The vaccine complies with the test if no chicken shows notable clinical signs of disease or dies from causes attributable to the vaccine. 3-6. Virus titre. Titrate the vaccine virus by inoculation into embryonated hens’ eggs from an SPF flock (5.2.2) or into suitable cell cultures (5.2.4). The vaccine complies with the test if 1 dose contains not less than the minimum virus titre stated on the label. 3-7. Potency. The vaccine complies with the requirements of the test prescribed under Immunogenicity (section 2-4-3) when administered according to the recommended schedule by a recommended route and method. It is not necessary to carry out the potency test for each batch of the vaccine if it has been carried out on a representative batch using a vaccinating dose containing not more than the minimum virus titre stated on the label.

satisfactory with respect to safety (5.2.6) and efficacy (5.2.7) in the species of fish for which it is intended. The following tests may be used during demonstration of safety and immunogenicity. Safety A. During development of the vaccine, safety is tested on 3 different batches. A test is carried out in each species of fish for which the vaccine is intended. The fish used are from a population that does not have specific antibodies against A. salmonicida subsp. salmonicida and has not been vaccinated against or exposed to furunculosis. The test is carried out in the conditions recommended for the use of the vaccine with a water temperature not less than 10 °C. An amount of vaccine corresponding to twice the recommended dose per mass unit is administered intraperitoneally into each of not fewer than 50 fish of the minimum body mass recommended for vaccination. The fish are observed for 21 days. No abnormal local or systemic reaction occurs. The test is invalid if more than 6 per cent of the fish die from causes not attributable to the vaccine. B. Safety is also demonstrated in field trials by administering the intended dose to a sufficient number of fish in not fewer than 2 sets of premises. Samples of 30 fish are taken on 3 occasions (after vaccination, at the middle of the rearing period and at slaughter) and examined for local reactions in the body cavity. Moderate lesions involving localised adhesions between viscera or between viscera and the abdominal wall and slight opaqueness and/or sparse pigmentation of the peritoneum are acceptable. Extensive lesions including adhesions between greater parts of the abdominal organs, massive pigmentation and/or obvious thickening and opaqueness of greater areas of the peritoneum are unacceptable if they occur in more than 10 per cent of the fish in any sample. Such lesions include adhesions that give the viscera a “one-unit” appearance and/or lead to manifest laceration of the peritoneum following evisceration.

01/2005:1521 Immunogenicity. The test described under Potency is suitable to demonstrate the immunogenicity of the vaccine.

FURUNCULOSIS VACCINE (INACTIVATED, OIL-ADJUVANTED, INJECTABLE) FOR SALMONIDS

Vaccinum furunculosidis ad salmonidas inactivatum cum adiuvatione oleosa ad iniectionem DEFINITION Furunculosis vaccine (inactivated, oil-adjuvanted, injectable) for salmonids is prepared from cultures of one or more suitable strains of Aeromonas salmonicida subsp. salmonicida.

BATCH TESTING Batch potency test. For routine testing of batches of vaccine, the test described under Potency may be carried out using not fewer than 30 fish per group ; alternatively, a suitable validated test based on antibody response may be carried out, the criteria being set with reference to a batch of vaccine that has given satisfactory results in the test described under Potency. The following test may be used after a satisfactory correlation with the test described under Potency has been established.

Use fish from a population that does not have specific antibodies against A. salmonicida subsp. salmonicida and that are within defined limits for body mass. Carry out the test at a defined temperature. Inject intraperitoneally into each of not fewer than 25 fish one dose of vaccine, PRODUCTION according to the instructions for use. Perform mock The strains of A. salmonicida are cultured and harvested vaccination on a control group of not fewer than 10 fish. separately. The harvests are inactivated by a suitable method. Collect blood samples at a defined time after vaccination. They may be purified and concentrated. Whole or disrupted Determine for each sample the level of specific antibodies cells may be used and the vaccine may contain extracellular against A. salmonicida subsp. salmonicida by a suitable products of the bacterium released into the growth medium. immunochemical method (2.7.1). The vaccine complies with The vaccine contains an oily adjuvant. the test if the mean level of antibodies is not significantly CHOICE OF VACCINE COMPOSITION lower than that found for a batch that gave satisfactory results in the test described under Potency. The test is The strains included in the vaccine are shown to be not valid if the control group shows antibodies against suitable with respect to production of antigens of assumed A. salmonicida subsp. salmonicida. immunological importance. The vaccine is shown to be General Notices (1) apply to all monographs and other texts

767

Infectious bovine rhinotracheitis vaccine (live), freeze-dried


01/2005:0696

IDENTIFICATION When injected into fish that do not have specific antibodies against A. salmonicida, the vaccine stimulates the production of such antibodies. TESTS Safety. Use not fewer than 10 fish of one of the species for which the vaccine is intended, having, where possible, the minimum body mass recommended for vaccination ; if fish of the minimum body mass are not available, use fish not greater than twice this mass. Use fish from a population that preferably does not have specific antibodies against A. salmonicida subsp. salmonicida or, where justified, use fish from a population with a low level of such antibodies as long as they have not been vaccinated against or exposed to furunculosis and administration of the vaccine does not cause an anamnestic response. Carry out the test in the conditions recommended for use of the vaccine with a water temperature not less than 10 °C. Administer intraperitoneally to each fish an amount of vaccine corresponding to twice the recommended dose per mass unit. Observe the animals for 21 days. No abnormal local or systemic reaction attributable to the vaccine occurs. The test is invalid if more than 10 per cent of the fish die from causes not attributable to the vaccine. Sterility. The vaccine complies with the test for sterility prescribed in the monograph on Vaccines for veterinary use (0062). POTENCY Carry out the test according to a protocol defining limits of body mass for the fish, water source, water flow and temperature limits, and preparation of a standardised challenge. Vaccinate not fewer than 100 fish by a recommended route, according to the instructions for use. Perform mock vaccination on a control group of not fewer than 100 fish ; mark vaccinated and control fish for identification. Keep all the fish in the same tank or mix equal numbers of controls and vaccinates in each tank if more than one tank is used. Carry out challenge by injection at a fixed time interval after vaccination, defined according to the statement regarding development of immunity. Use for challenge a culture of A. salmonicida subsp. salmonicida whose virulence has been verified. Observe the fish daily until at least 60 per cent specific mortality is reached in the control group. Plot for both vaccinates and controls a curve of specific mortality against time from challenge and determine by interpolation the time corresponding to 60 per cent specific mortality in controls. The test is invalid if the specific mortality is less than 60 per cent in the control group 21 days after the first death in the fish. Read from the curve for vaccinates the mortality (M) at the time corresponding to 60 per cent mortality in controls. Calculate the relative percentage survival (RPS) from the expression :

INFECTIOUS BOVINE RHINOTRACHEITIS VACCINE (LIVE), FREEZE-DRIED Vaccinum rhinotracheitidis infectivae bovinae vivum cryodesiccatum DEFINITION Freeze-dried infectious bovine rhinotracheitis vaccine (live) is a preparation of one or more attenuated strains of infectious bovine rhinotracheitis virus (bovine herpesvirus 1). PRODUCTION The virus is grown in suitable cell cultures (5.2.4). The viral suspension is collected and mixed with a suitable stabilising solution. The mixture is subsequently freeze-dried. CHOICE OF VACCINE STRAIN Only a virus strain shown to be satisfactory with respect to the following characteristics may be used in the preparation of the vaccine : safety (including absence of abortigenicity and passage through the placenta) ; reversion to virulence and immunogenicity. The strain may have markers. The following tests may be used during demonstration of safety (5.2.6) and efficacy (5.2.7). Safety. Use 5 calves 3 months old, or of the minimum age to be recommended for vaccination if this is less than 3 months, and that do not have antibodies against infectious bovine rhinotracheitis virus. Administer to each calf, by the intended route, a quantity of virus corresponding to 10 doses of vaccine. The calves are observed for 21 days. No abnormal local or systemic reaction occurs. Abortigenicity and passage through the placenta. 24 pregnant cows that do not have antibodies against infectious bovine rhinotracheitis virus are used for the test : 8 of the cows are in the fourth month of pregnancy, 8 in the fifth month and 8 in the sixth or seventh month. A quantity of virus equivalent to ten doses of vaccine is administered by the intended route to each cow. The cows are observed until the end of pregnancy. If abortion occurs, tests for infectious bovine rhinotracheitis virus are carried out ; neither the virus nor viral antigens are present in the foetus or placenta. A test for antibodies against infectious bovine rhinotracheitis virus is carried out on calves born at term before ingestion of colostrum ; no such antibodies are found. Reversion to virulence. Suitable samples are taken from the 5 calves used for the test for safety at a time when the vaccinal virus can be easily detected. The presence and titre of virus in the samples are verified. The samples are then mixed and administered intranasally to 2 other calves of the same age having no antibodies against bovine rhinotracheitis virus. 5 further serial passages are carried out. The presence of the virus is verified at each passage. No abnormal local or systemic reaction occurs.

The vaccine complies with the test if the RPS is not less than Immunogenicity. The test described under Potency is suitable to demonstrate immunogenicity. 80 per cent. BATCH TESTING If the test for potency has been carried out with satisfactory LABELLING results on a representative batch of vaccine, this test may be omitted as a routine control on other batches of vaccine The label states information on the time needed for development of immunity after vaccination under the range prepared from the same seed lot, subject to agreement by the competent authority. of conditions corresponding to the recommended use. 768


Infectious chicken anaemia vaccine (live)


1. DEFINITION Infectious chicken anaemia vaccine (live) is a preparation of a suitable strain of chicken anaemia virus. This monograph applies to vaccines intended for administration to breeder chickens for active immunisation, to prevent excretion of the virus, to prevent or reduce egg transmission and to protect passively their future progeny.

2. PRODUCTION 2-1. PREPARATION OF THE VACCINE The vaccine virus is grown in embryonated hens’ eggs or in cell cultures. 2-2. SUBSTRATE FOR VIRUS PROPAGATION 2-2-1. Embryonated hens’ eggs. If the vaccine virus is grown in embryonated hens’ eggs, they are obtained from flocks free from specified pathogens (SPF) (5.2.2). 2-2-2. Cell cultures. If the vaccine virus is grown in cell cultures, they comply with the requirements for cell cultures for production of veterinary vaccines (5.2.4). 2-3. SEED LOTS 2-3-1. Extraneous agents. The master seed lot complies with the tests for extraneous agents in seed lots (2.6.24). In these tests on the master seed lot, the organisms used are not more than 5 passages from the master seed lot at the start of the tests. 2-4. CHOICE OF VACCINE VIRUS The vaccine virus shall be shown to be satisfactory with respect to safety (5.2.6) and efficacy (5.2.7) for the chickens for which it is intended. The following tests for safety (section 2-4-1), increase in virulence (section 2-4-2) and immunogenicity (section 2-4-3) may be used during the demonstration of safety and immunogenicity. 2-4-1. Safety 2-4-1-1. General test. Carry out the test for each route and method of administration to be recommended for vaccination in chickens not older than the youngest age to be recommended for vaccination and from an SPF flock (5.2.2). Use vaccine virus at the least attenuated passage level that will be present between the master seed lot and a batch of the vaccine. For each test use not fewer than 20 chickens. Administer to each chicken a quantity of the vaccine virus not less than 10 times the maximum virus titre likely to be contained in 1 dose of the vaccine. 14 days after vaccination, collect blood samples from half of the chickens and determine the haematocrit value. Kill these chickens and carry out post-mortem examination. Note any pathological changes attributable to chicken anaemia virus, such as thymic atrophy and specific bone-marrow lesions. Observe the remaining chickens at least daily for 21 days. The vaccine virus complies with the test if during the observation period no chicken shows notable clinical signs of chicken anaemia or dies from causes attributable to the vaccine virus. 2-4-1-2. Safety for young chicks. Use not fewer than twenty 1-day-old chicks from an SPF flock (5.2.2). Administer to each chick by the oculonasal route a quantity of the vaccine virus equivalent to not less than the maximum titre likely to be contained in 1 dose of the vaccine. Observe the chicks at least daily. Record the incidence of any clinical signs attributable to the vaccine virus, such as depression, and any deaths. 14 days after vaccination, collect blood samples from half of the chicks and determine the haematocrit value. Kill these chicks and carry out post-mortem examination. Note any pathological changes attributable to chicken anaemia virus, such as thymic atrophy and specific bone marrow lesions. Observe the remaining chicks at least daily for 21 days. Assess the extent to which the vaccine strain is pathogenic for 1-day-old susceptible chicks from the results of the clinical observations and mortality rates and the proportion of chicks examined at 14 days that show anaemia (haematocrit value less than 27 per cent) and signs of infectious chicken anaemia on post-mortem examination. The results are used to formulate the label statement on safety for young chicks.


769

IDENTIFICATION A. Reconstitute the vaccine as stated on the label. When mixed with a suitable quantity of a monospecific antiserum, the reconstituted vaccine is no longer able to infect susceptible cell cultures into which it is inoculated. B. Any markers of the strain are verified. TESTS Safety. Use 2 calves 3 months old or of the minimum age recommended for vaccination if this is less than 3 months, having no antibodies against bovine rhinotracheitis virus. Administer 10 doses of the reconstituted vaccine to each calf by a recommended route. Observe the calves for 21 days. No abnormal local or systemic reaction occurs. Bacterial and fungal contamination. The reconstituted vaccine complies with the test for sterility prescribed in the monograph on Vaccines for veterinary use (0062). Mycoplasmas (2.6.7). The reconstituted vaccine complies with the test for mycoplasmas. Extraneous viruses. Neutralise the vaccine using a monospecific antiserum and inoculate into suitable cell cultures. Maintain the cultures for 14 days and make a passage at 7 days. The cell cultures show no signs of viral contamination. Virus titre. Titrate the reconstituted vaccine in susceptible cell cultures at a temperature favourable to replication of the virus. One dose of the vaccine contains not less than the quantity of virus equivalent to the minimum virus titre stated on the label. POTENCY Use susceptible calves, 2 to 3 months old and free from antibodies neutralising infectious bovine rhinotracheitis virus. Administer to five calves, by the route stated on the label a volume of the reconstituted vaccine containing a quantity of virus equivalent to the minimum virus titre stated on the label. Keep 2 calves as controls. After 21 days, administer intranasally to the seven calves a quantity of infectious bovine rhinotracheitis virus sufficient to produce typical signs of disease such as fever, ocular and nasal discharge and ulceration of the nasal mucosa in a susceptible calf. Observe the animals for 21 days. The vaccinated calves show no more than mild signs ; the controls show typical signs. In not fewer than 4 of the 5 vaccinated calves, the maximum virus titre found in the nasal mucus is at least 100 times lower than the average of the maximum titres found in the control calves ; the average number of days on which virus is excreted is at least 3 days less in vaccinated calves than in the control calves.

01/2005:2038

INFECTIOUS CHICKEN ANAEMIA VACCINE (LIVE) Vaccinum anaemiae infectivae pulli vivum

Infectious chicken anaemia vaccine (live)


2-4-2. Increase in virulence. The test for increase in virulence consists of the administration of the vaccine virus at the least attenuated passage level that will be present between the master seed lot and a batch of the vaccine to a group of five 1-day-old chicks from an SPF flock (5.2.2), sequential passages, 5 times where possible, to further similar groups of 1-day-old chicks and testing of the final recovered virus for increase in virulence. If the properties of the vaccine virus allow sequential passage to 5 groups via natural spreading, this method may be used, otherwise passage as described below is carried out and the maximally passaged virus that has been recovered is tested for increase in virulence. Care must be taken to avoid contamination by virus from previous passages. Administer by the intramuscular route a quantity of the vaccine virus that will allow recovery of virus for the passages described below. Prepare 7 to 9 days after administration a suspension from the liver of each chick and pool these samples. Depending on the tropism of the virus, other tissues such as spleen or bone marrow may be used. Administer 0.1 ml of the pooled samples by the intramuscular route to each of 5 other chicks of the same age and origin. Carry out this passage operation at least 5 times ; verify the presence of the virus at each passage. If the virus is not found at a passage level, carry out a second series of passages. Carry out the tests for safety (section 2-4-1) using the unpassaged vaccine virus and the maximally passaged vaccine virus that has been recovered. The vaccine virus complies with the test if no indication of increase in virulence of the maximally passaged virus compared with the unpassaged virus is observed. If virus is not recovered at any passage level in the first and second series of passages, the vaccine virus also complies with the test. 2-4-3. Immunogenicity. A test is carried out for each route and method of administration to be recommended using chickens from an SPF flock (5.2.2) not older than the youngest age to be recommended for vaccination. The test for prevention of virus excretion is intended to demonstrate absence of egg transmission. The quantity of the vaccine virus administered to each chicken is not greater than the minimum titre to be stated on the label and the virus is at the most attenuated passage level that will be present in a batch of the vaccine. 2-4-3-1 Passive immunisation of chickens. Vaccinate according to the recommended schedule not fewer than 10 breeder chickens not older than the minimum age recommended for vaccination and from an SPF flock (5.2.2) ; keep not fewer than 10 unvaccinated breeder chickens of the same origin and from an SPF flock (5.2.2). At a suitable time after excretion of vaccine virus has ceased, collect fertilised eggs from each vaccinated and control breeder chicken and incubate them. Challenge at least 3 randomly chosen 1-day-old chickens from each vaccinated and control breeder chicken by intramuscular administration of a sufficient quantity of virulent chicken anaemia virus. Observe the chickens at least daily for 14 days after challenge. Record the deaths and the surviving chickens that show clinical signs of disease. At the end of the observation period determine the haematocrit value of each surviving chicken. Kill these chickens and carry out post-mortem examination. Note any pathological signs attributable to chicken anaemia virus, such as thymic atrophy and specific bone-marrow lesions. The test is not valid if : — during the observation period after challenge fewer than 90 per cent of the chickens of the control breeder chickens die or show severe clinical signs of infectious

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chicken anaemia, including haematocrit value under 27 per cent, and/or notable macroscopic lesions of the bone marrow and thymus, — and/or during the period between vaccination and egg collection more than 10 per cent of vaccinated or control breeder chickens show notable clinical signs of disease or die from causes not attributable to the vaccine. The vaccine complies with the test if during the observation period after challenge not fewer than 90 per cent of the chickens of the vaccinated breeder chickens survive and show no notable clinical signs of disease and/or macroscopic lesions of the bone marrow and thymus. 2-4-3-2. Prevention of virus excretion. Vaccinate according to the recommended schedule not fewer than 10 chickens not older than the minimum age recommended for vaccination and from an SPF flock (5.2.2). Maintain separately not fewer than 10 chickens of the same age and origin as controls. At a suitable time after excretion of vaccine virus has ceased, challenge all the chickens by intramuscular administration of a sufficient quantity of virulent chicken anaemia virus. Collect blood and faecal samples from the chickens on days 3, 5 and 7 after challenge and carry out a test for virus isolation to determine whether or not the chickens are viraemic and are excreting the virus. The test is not valid if : — fewer than 70 per cent of the control chickens are viraemic and excrete the virus at one or more times of sampling, — and/or during the period between vaccination and challenge more than 10 per cent of control or vaccinated chickens show abnormal clinical signs or die from causes not attributable to the vaccine. The vaccine complies with the test if not fewer than 90 per cent of the vaccinated chickens do not develop viraemia or excrete the virus. 3 BATCH TESTING 3-1. Identification. The vaccine, diluted if necessary and mixed with a monospecific chicken anaemia virus antiserum, no longer infects susceptible cell cultures or eggs from an SPF flock (5.2.2) into which it is inoculated. 3-2. Bacteria and fungi. Vaccines intended for administration by injection comply with the test for sterility prescribed in the monograph Vaccines for veterinary use (0062). Vaccines not intended for administration by injection either comply with the test for sterility prescribed in the monograph Vaccines for veterinary use (0062) or with the following test : carry out a quantitative test for bacterial and fungal contamination ; carry out identification tests for microorganisms detected in the vaccine ; the vaccine does not contain pathogenic microorganisms and contains not more than 1 non-pathogenic microorganism per dose. Any liquid supplied with the vaccine complies with the test for sterility prescribed in the monograph Vaccines for veterinary use (0062). 3-3. Mycoplasmas. The vaccine complies with the test for mycoplasmas (2.6.7). 3-4. Extraneous agents. The vaccine complies with the tests for extraneous agents in batches of finished product (2.6.25). 3-5. Safety. Use not fewer than 10 chickens not older than the minimum age recommended for vaccination and from an SPF flock (5.2.2). Administer by a recommended route to each chicken 10 doses of the vaccine. Observe the chickens at least daily for 21 days. The test is not valid if more than 20 per cent of the chickens show abnormal clinical signs or die from causes not attributable to the vaccine. The


Mannheimia vaccine (inactivated) for cattle


PRODUCTION Production of the vaccine is based on a seed-lot system. The seed material is cultured in a suitable medium ; each strain is cultivated separately and identity is verified using a suitable method. During production, various parameters such as growth rate are monitored by suitable methods ; the values are within the limits approved for the particular product. Purity and identity of the harvest are verified using suitable methods. After cultivation, the bacterial suspensions are collected separately and inactivated by a suitable method. The vaccine may contain an adjuvant and may be freeze-dried. CHOICE OF VACCINE COMPOSITION The choice of composition and the strains to be included in the vaccine is based on epidemiological data on the prevalence of the different serovars of M. haemolytica and on the claims being made. The vaccine is shown to be satisfactory with respect to safety (5.2.6) and efficacy (5.2.7) in cattle. As part of the studies to demonstrate the suitability of the vaccine with respect to these characteristics the following tests may be carried out. Safety A. The test is carried out for each route of administration to be stated on the label and in animals of each category for which the vaccine is intended. For each test, use not fewer than 10 animals that preferably do not have antibodies against the serovars of M. haemolytica or against the leucotoxin present in the vaccine. Where justified, animals with a known history

of no previous mannheimia vaccination and with low antibody titres (measured in a sensitive test system such as an ELISA) may be used. Administer to each animal a double dose of vaccine containing not less than the maximum potency that may be expected in a batch of vaccine. Administer a single dose of vaccine to each animal after the recommended interval. Observe the animals for at least 14 days after the last administration. Record body temperature the day before vaccination, at vaccination, 2 h, 4 h and 6 h later and then daily for 4 days ; note the maximum temperature increase for each animal. No abnormal local or systemic reaction occurs ; the average body temperature increase for all animals does not exceed 1.5 °C and no animal shows a rise greater than 2 °C. If the vaccine is intended for use or may be used in pregnant cows, vaccinate the cows at the relevant stages of pregnancy and prolong the observation period until 1 day after parturition. The vaccine complies with the test if no animal shows abnormal local or systemic reactions or clinical signs of disease or dies from causes attributable to the vaccine. In addition, if the vaccine is intended for use in pregnant cows, no significant effects on the pregnancy and offspring are demonstrated. B. The animals used for the field trials are also used to evaluate safety. Carry out a test in each category of animals for which the vaccine is intended. Use not fewer than 3 groups of 20 animals with corresponding groups of not fewer than 10 controls in 3 different locations. Examine the injection sites for local reactions after vaccination. Record body temperatures the day before vaccination, at vaccination and on the 2 days following vaccination. The vaccine complies with the test if no animal shows abnormal local or systemic reactions or clinical signs of disease or dies from causes attributable to the vaccine. The average body temperature increase for all animals does not exceed 1.5 °C and no animal shows a rise greater than 2 °C. In addition, if the vaccine is intended for use in pregnant cows, no significant effects on the pregnancy and offspring are demonstrated. Immunogenicity. As part of the studies to demonstrate the suitability of the vaccine with respect to immunogenicity, the test described under Potency may be carried out for each proposed route of administration and using vaccine of minimum potency. BATCH TESTING Batch potency test. The test described under Potency is not carried out for routine testing of batches of vaccine. It is carried out, for a given vaccine, on one or more occasions, as decided by or with the agreement of the competent authority. Where the test is not carried out, a suitable validated test is carried out, the criteria for acceptance being set with reference to the results obtained with a batch of vaccine that has given satisfactory results in the test described under Potency. Bacterial endotoxins. A test for bacterial endotoxins (2.6.14) is carried out on the final lot or, where the nature of the adjuvant prevents performance of a satisfactory test, on the bulk antigen or the mixture of bulk antigens immediately before addition of the adjuvant. The maximum acceptable amount of bacterial endotoxins is that found for a batch of vaccine that has been shown satisfactory in safety test A given under Choice of vaccine composition or in the safety test described under Tests, carried out using 10 animals. Where the latter test is used, note the maximum temperature increase for each animal ; the average body temperature increase for all animals does not exceed 1.5 °C. The method


771

vaccine complies with the test if no chicken shows notable clinical signs of disease or dies from causes attributable to the vaccine. 3-6. Virus titre. Titrate the vaccine virus by inoculation into suitable cell cultures (5.2.4) or eggs from an SPF flock (5.2.2). The vaccine complies with the test if 1 dose contains not less than the minimum virus titre stated on the label. 3-7. Potency. The vaccine complies with the requirements of the tests prescribed under Immunogenicity (sections 2-4-3-1 and 2-4-3-2) when administered by a recommended route and method. It is not necessary to carry out the potency test for each batch of the vaccine if it has been carried out on a representative batch using a vaccinating dose containing not more than the minimum virus titre stated on the label. 4. LABELLING The label states to which extent the vaccine virus causes disease if it spreads to susceptible young chicks.

01/2005:1944

MANNHEIMIA VACCINE (INACTIVATED) FOR CATTLE Vaccinum mannheimiae inactivatum ad bovidas DEFINITION Mannheimia vaccine (inactivated) for cattle is a preparation from cultures of one or more suitable strains of Mannheimia haemolytica (formerly Pasteurella haemolytica). This monograph applies to vaccines intended for administration to cattle of different ages for protection against respiratory diseases caused by M. haemolytica.

Mannheimia vaccine (inactivated) for sheep


chosen for determining the amount of bacterial endotoxin present in the vaccine batch used in the safety test for determining the maximum acceptable level of endotoxin is used subsequently for testing of each batch. IDENTIFICATION When injected into healthy seronegative animals, the vaccine stimulates the production of specific antibodies against the serovars of M. haemolytica and/or against the leucotoxin present in the vaccine. TESTS Safety. Use 2 cattle of the minimum age recommended for vaccination that have not been vaccinated against mannheimiosis. Administer a double dose of vaccine to each animal by a recommended route. Observe the animals for 14 days. Record body temperature the day before vaccination, at vaccination, 2 h, 4 h and 6 h later and then daily for 2 days. The animals remain in good health and no abnormal local or systemic reaction occurs ; a transient temperature increase not exceeding 2 °C may occur. Sterility. It complies with the test for sterility prescribed in the monograph on Vaccines for veterinary use (0062). POTENCY Carry out a test for each serovar for which protection is claimed on the label. Use not fewer than 16 animals of the minimum age recommended for vaccination, free from antibodies against M. haemolytica and against the leucotoxin of M. haemolytica. Vaccinate not fewer than 8 of the animals by a recommended route and according to the recommended schedule. Keep 8 animals as controls. 21 days after the last vaccination, challenge all the animals by the intratracheal route or by another appropriate route, with a suitable quantity of a low-passage, virulent strain of a serovar of M. haemolytica. Observe the animals for a further 7 days ; to avoid unnecessary suffering, severely ill animals are killed and are then considered to have died from the disease. During the observation period, the animals are examined for signs of disease for example, increased body temperature, dullness, abnormal breathing and the mortality is recorded. Kill surviving animals at the end of the observation period. Post-mortem examination is carried out on any animal that dies and those killed at the end of the observation period. The lungs are examined and the extent of lung lesions due to mannheimiosis is evaluated. Samples of lung tissue are collected for re-isolation of the challenge organisms. The clinical observations and lung lesions are scored and the results obtained for these parameters and the bacterial re-isolation results compared for the 2 groups. The test is invalid if signs of M. haemolytica infection occur in less than 70 per cent of the control animals. The vaccine complies with the requirements of the test if there is a significant difference between the scores obtained for the clinical and post-mortem observations in the vaccinates compared to the controls. For vaccines with a claim for a beneficial effect on the extent of infection against the serovar, the results for the infection rates are also significantly better for the vaccinates compared to the controls. LABELLING The label states : — the serovar(s) of M. haemolytica against which protection is claimed, — the serovar(s) of M. haemolytica and/or the leucotoxin present in the vaccine. 772

01/2005:1946

MANNHEIMIA VACCINE (INACTIVATED) FOR SHEEP Vaccinum mannheimiae inactivatum ad ovem DEFINITION Mannheimia vaccine (inactivated) for sheep is a preparation of one or more suitable strains of Mannheimia haemolytica (formerly Pasteurella haemolytica). This monograph applies to vaccines intended for administration to sheep for active immunisation and to protect passively their future progeny against disease caused by M. haemolytica. PRODUCTION Production of the vaccine is based on a seed lot system. The seed material is cultured in a suitable medium ; each strain is cultivated separately and identity is verified using a suitable method. During production, various parameters such as growth rate are monitored by suitable methods ; the values are within the limits approved for the particular product. Purity and identity of the harvest are verified using suitable methods. After cultivation, the bacterial suspensions are collected separately and inactivated by a suitable method. The vaccine may contain an adjuvant and may be freeze-dried. CHOICE OF VACCINE COMPOSITION The choice of composition and the strains to be included in the vaccine is based on epidemiological data on the prevalence of the different serovars of M. haemolytica and on the claims being made for the product, for example active and/or passive protection. The vaccine is shown to be satisfactory with respect to safety (5.2.6) and efficacy (5.2.7) in sheep. As part of the studies to demonstrate the suitability of the vaccine with respect to these characteristics the following tests may be carried out. Safety A. The test is carried out for each of the routes of administration to be stated on the label and in animals of each category (for example, young sheep, pregnant ewes) for which the vaccine is intended. For each test, use not fewer than 10 animals that preferably do not have antibodies against the serovars of M. haemolytica or against the leucotoxin present in the vaccine. Where justified, animals with a known history of no previous mannheimia vaccination and with low antibody titres (measured in a sensitive test system such as an ELISA) may be used. Administer to each animal a double dose of vaccine containing not less than the maximum potency that may be expected in a batch of vaccine. Administer a single dose of vaccine to each animal after the recommended interval. Observe the animals for at least 14 days after the last administration. Record body temperature the day before vaccination, at vaccination, 2 h, 4 h and 6 h later and then daily for 4 days ; note the maximum temperature increase for each animal. No abnormal local or systemic reaction occurs ; the average body temperature increase for all animals does not exceed 1.5 °C and no animal shows a rise greater than 2 °C. If the vaccine is intended for use or may be used in pregnant ewes, vaccinate the ewes at the relevant stages of pregnancy and prolong the observation period until 1 day after lambing.



Mannheimia vaccine (inactivated) for sheep

The vaccine complies with the test if no animal shows abnormal local reactions or clinical signs of disease or dies from causes attributable to the vaccine. In addition, if the vaccine is intended for use in pregnant ewes, no significant effects on the pregnancy and offspring are demonstrated. B. The animals used for the field trials are also used to evaluate safety. Carry out a test in each category of animals for which the vaccine is intended. Use not fewer than 3 groups of 20 animals with corresponding groups of not fewer than 10 controls in 3 different locations. Examine the injection sites for local reactions after vaccination. Record body temperatures the day before vaccination, at vaccination and on the 2 days following vaccination. The vaccine complies with the test if no animal shows abnormal local or systemic reactions or clinical signs of disease or dies from causes attributable to the vaccine. The average body temperature increase for all animals does not exceed 1.5 °C and no animal shows a rise greater than 2 °C. In addition, if the vaccine is intended for use in pregnant ewes, no significant effects on the pregnancy and offspring are demonstrated. Immunogenicity. As part of the studies to demonstrate the suitability of the vaccine with respect to immunogenicity, the tests described under Potency may be carried out for each proposed route of administration and using vaccine of minimum potency. BATCH TESTING Batch potency test. The relevant test or tests described under Potency are not carried out for routine testing of batches of vaccine. They are carried out, for a given vaccine, on one or more occasions, as decided by or with the agreement of the competent authority. Where the relevant test or tests are not carried out, a suitable validated batch potency test is carried out, the criteria for acceptance being set with reference to the results obtained with a batch of vaccine that has given satisfactory results in the test(s) described under Potency. Bacterial endotoxins. A test for bacterial endotoxins (2.6.14) is carried out on the final lot or, where the nature of the adjuvant prevents performance of a satisfactory test, on the bulk antigen or the mixture of bulk antigens immediately before addition of the adjuvant. The maximum acceptable amount of bacterial endotoxins is that found for a batch of vaccine that has been shown satisfactory in safety test A given under Choice of vaccine composition or in the safety test described under Tests, carried out using 10 animals. Where the latter test is used, note the maximum temperature increase for each animal ; the average body temperature increase for all animals does not exceed 1.5 °C. The method chosen for determining the amount of bacterial endotoxin present in the vaccine batch used in the safety test for determining the maximum acceptable level of endotoxin is used subsequently for testing of each batch.

before vaccination, at vaccination, 2 h, 4 h and 6 h later and then daily for 2 days. The animals remain in good health and no abnormal local or systemic reaction occurs ; a transient temperature increase not exceeding 2 °C may occur. Sterility. It complies with the test for sterility prescribed in the monograph on Vaccines for veterinary use (0062).


773

POTENCY Active immunisation. For vaccines with claims for active immunisation due to M. haemolytica, carry out a test for each serovar of M. haemolytica for which protection is claimed on the label. Use not fewer than 20 lambs of the minimum age recommended for vaccination, free from antibodies against M. haemolytica and against the leucotoxin of M. haemolytica. Vaccinate not fewer than 10 of the animals by a recommended route and according to the recommended schedule. Keep 10 animals as controls. 21 days after the last vaccination, challenge all the lambs by the intratracheal route or by another appropriate route, with a suitable quantity of a low-passage, virulent strain of a serovar of M. haemolytica. Where necessary for a given serovar, prechallenge with parainfluenza type 3 (PI3) virus or another appropriate respiratory pathogen may be used. Observe the animals for a further 7 days ; to avoid unnecessary suffering, severely ill animals are killed and are then considered to have died from the disease. During the observation period, the animals are examined for signs of disease (for example, increased body temperature, dullness, abnormal respiration) and the mortality is recorded. Kill surviving animals at the end of the observation period. Post-mortem examination is carried out on any animal that dies and those killed at the end of the observation period. The lungs are examined and the extent of lung lesions due to mannheimiosis is evaluated. Samples of lung tissue are collected for re-isolation of the challenge organisms. The clinical observations and lung lesions are scored and the results obtained for these parameters and the bacterial re-isolation results compared for the 2 groups. The test is invalid if signs of M. haemolytica infection occur in less than 70 per cent of the control lambs. The vaccine complies with the requirements of the test if there is a significant difference between the scores obtained for the clinical and post-mortem observations in the vaccinates compared to the controls. For vaccines with a claim for a beneficial effect on the extent of infection against the serovar, the results for the infection rates are also significantly better for the vaccinates compared to the controls. Passive protection. For vaccines with claims for passive protection against mannheimiosis carry out a test for each serovar of M. haemolytica for which protection is claimed on the label. Use at least 6 ewes that preferably do not have antibodies against the serovars of M. haemolytica or against the leucotoxin present in the vaccine. Where justified, animals IDENTIFICATION with a known history of no previous mannheimia vaccination, When injected into healthy seronegative animals, the vaccine from a source with a low incidence of respiratory disease stimulates the production of specific antibodies against the and with low antibody titres (measured in a sensitive serovars of M. haemolytica and/or against the leucotoxin test system such as an ELISA) may be used. Vaccinate present in the vaccine. the animals by 1 of the recommended routes, at the recommended stages of pregnancy and according to the TESTS recommended schedule. A challenge study is conducted with 20 newborn, colostrum-deprived lambs. 10 of these Safety. Use 2 sheep of the minimum age recommended for vaccination or, if not available, of an age as close as possible lambs are given colostrum from the vaccinated ewes and to the minimum recommended age, and that have not been 10 control lambs are given colostrum or colostrum substitute vaccinated against mannheimiosis. Administer a double dose without detectable antibodies to M. haemolytica. When of vaccine to each animal by a recommended route. Observe the lambs are at the age claimed for the duration of the passive protection, challenge by the intratracheal route with the animals for 14 days. Record body temperature the day

Marek’s disease vaccine (live)


a suitable quantity of a low-passage, virulent strain of a serovar of M. haemolytica. Observe the animals for a further 7 days ; to avoid unnecessary suffering, severely ill animals are killed and are then considered to have died from the disease. Observe the animals and assess the effect of the challenge on the offspring of the vaccinates and the controls as described in the test for active immunisation. The test is invalid if clinical signs or lesions of M. haemolytica infection occur in less than 70 per cent of the control lambs. The vaccine complies with the requirements of the test if there is a significant difference between the scores obtained for the clinical and post-mortem observations in the lambs from the vaccinates compared to those from the controls. For vaccines with a claim for a beneficial effect on the extent of infection against the serovar, the results for the infection rates are also significantly better for the lambs from the vaccinates compared to those from the controls. LABELLING The label states : — the serovar(s) of M. haemolytica against which protection is claimed, — the serovar(s) of M. haemolytica and/or the leucotoxin present in the vaccine, — for vaccines for passive protection, the length of time for which the passive protection is claimed. 01/2005:0589

MAREK’S DISEASE VACCINE (LIVE) Vaccinum morbi Marek vivum 1. DEFINITION Marek’s disease vaccine (live) is a preparation of a suitable strain or strains of Marek’s disease virus (gallid herpesvirus 2 or 3) and/or turkey herpesvirus (meleagrid herpesvirus 1). This monograph applies to vaccines intended for administration to chickens for active immunisation. 2. PRODUCTION 2-1. PREPARATION OF THE VACCINE The vaccine virus is grown in cell cultures. If the vaccine contains more than 1 type of virus, the different types are grown separately. The vaccine may be freeze-dried or stored in liquid nitrogen. 2-2. SUBSTRATE FOR VIRUS PROPAGATION 2-2-1. Cell cultures. The cell cultures comply with the requirements for cell cultures for production of veterinary vaccines (5.2.4). 2-3. SEED LOTS 2-3-1. Extraneous agents. The master seed lot complies with the tests for extraneous agents in seed lots (2.6.24). In these tests on the master seed lot, the organisms used are not more than 5 passages from the master seed lot at the start of the tests. 2-4. CHOICE OF VACCINE VIRUS The vaccine virus shall be shown to be satisfactory with respect to safety (5.2.6) and efficacy (5.2.7) for the chickens for which it is intended. The tests shown below for safety of the strain (section 2-4-1), increase in virulence (section 2-4-2) and immunogenicity (section 2-4-3) may be used during the demonstration of safety and immunogenicity. Additional testing may be 774

needed to demonstrate safety in breeds of chickens known to be particularly susceptible to Marek’s disease virus, unless the vaccine is to be contra-indicated. 2-4-1. Safety. Carry out the test for the route to be recommended for vaccination likely to be the least safe and in the category of chickens for which the vaccine is intended likely to be the most susceptible for Marek’s disease. Use vaccine virus at the least attenuated passage level that will be present between the master seed lot and a batch of the vaccine. Use not fewer than eighty 1-day-old chickens from a flock free from specified pathogens (SPF) (5.2.2). Divide them randomly into 2 groups of not fewer than 40 chickens and maintain the groups separately. Carry out examination for macroscopic lesions of each chicken that dies and of the surviving chickens at the end of the observation periods. Administer to each chicken of one group (I) a quantity of the vaccine virus equivalent to not less than 10 times the maximum virus titre likely to be contained in 1 dose of the vaccine. Administer by a suitable route to each chicken of the other group (II) a quantity of virulent Marek’s disease virus that will cause mortality and/or severe macroscopic lesions of Marek’s disease in not fewer than 70 per cent of the effective number of chickens within 70 days (initial number reduced by the number that die within the first 7 days of the test). Observe the chickens of group II at least daily for 70 days and those of group I at least daily for 120 days. The test is not valid if 1 or more of the following apply : more than 10 per cent of the chickens in any group die within the first 7 days of the test ; fewer than 70 per cent of the effective number of chickens in group II show macroscopic lesions of Marek’s disease. The vaccine virus complies with the test if : — no chicken of group I shows notable clinical signs or macroscopic lesions of Marek’s disease or dies from causes attributable to the vaccine virus, — at 120 days the number of surviving chickens of group I is not fewer than 80 per cent of the effective number. 2-4-2. Increase in virulence. The test for increase in virulence is required for Marek’s disease virus vaccine strains but not for turkey herpesvirus vaccine strains, which are naturally apathogenic. Administer by the intramuscular route a quantity of the vaccine virus that will allow recovery of virus for the passages described below to each of five 1-day-old chickens from an SPF flock (5.2.2). Use vaccine virus at the least attenuated passage level that will be present between the master seed lot and a batch of the vaccine. Prepare 5 to 7 days later a suspension of white blood cells of each chicken and pool these samples. Administer a suitable volume of the pooled samples by the intraperitoneal route to each of 5 other chickens that are 1-day-old and from an SPF flock (5.2.2). Carry out this passage operation not fewer than 5 times ; verify the presence of the virus at each passage. Care must be taken to avoid contamination by virus from previous passages. If the virus is not found at a passage level, carry out a second series of passages. Carry out the safety test (section 2-4-1) using the unpassaged vaccine virus and the maximally passaged virus that has been recovered. Administer the virus by the route to be recommended for vaccination likely to be the least safe for use in these birds. The vaccine virus complies with the test if no indication of increase in virulence of the maximally passaged virus compared with the unpassaged virus is observed. If virus is not recovered at any passage level in the first and second series of passages, the vaccine virus also complies with the test. 2-4-3. Immunogenicity. A test is carried out for each route and method of administration to be recommended using in each case chickens of the youngest age to be recommended for vaccination. The quantity of the vaccine



virus administered to each chicken is not greater than the minimum virus titre to be stated on the label and the virus is at the most attenuated passage level that will be present in a batch of the vaccine. Use for the test not fewer than 60 chickens of the same origin and from an SPF flock (5.2.2). Vaccinate by a recommended route not fewer than 30 chickens. Maintain not fewer than 30 chickens as controls. Challenge each chicken after 9 days by a suitable route with a sufficient quantity of virulent Marek’s disease virus. Observe the chickens at least daily for 70 days after challenge. Record the deaths and the number of surviving chickens that show clinical signs of disease. At the end of the observation period kill all the surviving chickens and carry out examination for macroscopic lesions of Marek’s disease. The test is not valid if : — during the observation period after challenge fewer than 70 per cent of the control chickens die or show severe clinical signs or macroscopic lesions of Marek’s disease, — and/or during the period between the vaccination and challenge more than 10 per cent of the control or vaccinated chickens show abnormal clinical signs or die from causes not attributable to the vaccine. The vaccine virus complies with the test if the percentage relative protection, calculated using the following expression, is not less than 80 per cent :

V

=

percentage of challenged vaccinated chickens that survive to the end of the observation period without notable clinical signs or macroscopic lesions of Marek’s disease,

C

=

percentage of challenged control chickens that survive to the end of the observation period without notable clinical signs or macroscopic lesions of Marek’s disease.

Myxomatosis vaccine (live) for rabbits

vaccine complies with the test if 1 dose contains not less than the minimum virus titre stated on the label. 3-6-2. Vaccines containing more than one type of virus. For vaccines containing more than 1 type of virus, titrate each virus by inoculation into suitable cell cultures (5.2.4), reading the results by immunostaining using antibodies. The vaccine complies with the test if 1 dose contains for each vaccine virus not less than the minimum virus titre stated on the label. 3-7. Potency. The vaccine complies with the requirements of the test prescribed under Immunogenicity (section 2-4-3) when administered according to the recommended schedule by a recommended route and method. It is not necessary to carry out the potency test for each batch of the vaccine if it has been carried out on a representative batch using a vaccinating dose containing not more than the minimum virus titre stated on the label. 01/2005:1943

MYXOMATOSIS VACCINE (LIVE) FOR RABBITS Vaccinum myxomatosidis vivum ad cuniculum DEFINITION Myxomatosis vaccine (live) for rabbits is a preparation of a suitable strain of either myxoma virus that is attenuated for rabbits or Shope fibroma virus. The vaccine is intended for the active immunisation of rabbits against myxomatosis.

3. BATCH TESTS 3-1. Identification. Carry out an immunostaining test in cell cultures using monoclonal antibodies to demonstrate the presence of each type of virus stated on the label. 3-2. Bacteria and fungi. The vaccine and, where applicable, the liquid supplied with it comply with the requirement for sterility prescribed in the monograph Vaccines for veterinary use (0062). 3-3. Mycoplasmas. The vaccine complies with the test for mycoplasmas (2.6.7). 3-4. Extraneous agents. The vaccine complies with the tests for extraneous agents in batches of finished product (2.6.25). 3-5. Safety. Use not fewer than 10 chickens from an SPF flock (5.2.2) and not older than the youngest age recommended for vaccination. Administer by a recommended route and method to each chicken 10 doses of the vaccine. Observe the chickens at least daily for 21 days. The test is not valid if more than 20 per cent of the chickens show abnormal clinical signs or die from causes not attributable to the vaccine. The vaccine complies with the test if no chicken shows notable clinical signs of disease or dies from causes attributable to the vaccine. 3-6. Virus titre 3-6-1 Vaccines containing one type of virus. Titrate the vaccine virus by inoculation into suitable cell cultures (5.2.4). If the virus titre is determined in plaque-forming units (PFU), only primary plaques are taken into consideration. The

PRODUCTION The virus is propagated in suitable cell cultures (5.2.4). The viral suspension is harvested, titrated and may be mixed with a suitable stabilising solution. The vaccine may be freeze-dried. CHOICE OF VACCINE STRAIN The vaccine is shown to be satisfactory with respect to safety, absence of increase in virulence and immunogenicity. The following tests may be used during demonstration of safety, absence of increase in virulence (5.2.6) and efficacy (5.2.7). Safety. The test is carried out for each route of administration to be stated on the label. Use at least 10 rabbits of the minimum age to be recommended for vaccination and that do not have antibodies against myxoma virus. Administer to each rabbit by a recommended route a quantity of virus corresponding to not less than 10 times the maximum titre that may be expected in a dose of vaccine. Observe the rabbits for 28 days. Record the body temperature the day before vaccination, at vaccination, 4 h after vaccination and then daily for 4 days ; note the maximum temperature increase for each animal. No abnormal local or systemic reaction occurs ; the average temperature increase does not exceed 1 °C and no animal shows a rise greater than 2 °C. A local reaction lasting less than 28 days may occur. If the vaccine is intended for use in pregnant rabbits, administer the virus to not less than 10 pregnant rabbits according to the schedule to be recommended on the label. Prolong the observation period until 1 day after parturition. The rabbits remain in good health and there is no abnormal local or systemic reaction. No adverse effects on the pregnancy or the offspring are noted. Increase in virulence. (This test is performed only for vaccines based on attenuated strains of myxoma virus). Administer by a recommended route to each of 2 rabbits, 5 to 7 weeks old and which do not have antibodies against


775

Neonatal piglet colibacillosis vaccine (inactivated)


myxoma virus, a quantity of virus that will allow recovery of virus for the passages described below. Use vaccine virus at the least attenuated passage level that will be present between the master seed lot and a batch of the vaccine. Kill the rabbits 5 to 10 days after inoculation and remove from each rabbit organs, or tissues with sufficient virus to allow passage ; homogenise the organs and tissues in a suitable buffer solution, centrifuge the suspension and use the supernatant for further passages. Inoculate the supernatant into suitable cell cultures to verify the presence of virus. Administer by an appropriate route, at a suitable rate, a suitable volume of the supernatant to each of 2 other rabbits of the same age and the same susceptibility. This operation is then repeated at least 5 times. If the virus has disappeared, a second series of passages is carried out. Inoculate virus from the highest recovered passage level to rabbits, observe for 28 days and compare any reactions that occur with those seen in the test for safety described above. There is no indication of an increase in virulence as compared with the non-passaged virus. If virus is not recovered in either of 2 series of passages, the vaccine virus also complies with the test. Immunogenicity. The test described under Potency may be used to demonstrate the immunogenicity of the strain. BATCH POTENCY TEST If the test for potency has been carried out with satisfactory results on a representative batch of vaccine, using a vaccinating dose containing not more than the minimum virus titre stated on the label, this test may be omitted as a routine control on other batches of vaccine prepared from the same seed lot. IDENTIFICATION Carry out an immunofluorescence test in suitable cell cultures, using a monospecific antiserum.

Administer 1 dose of vaccine to each of not fewer than 10 of the rabbits according to the instructions for use. Keep not less than 5 other rabbits as controls. Not less than 21 days after the last vaccination, administer by a suitable route to each rabbit a quantity of a virulent strain of myxoma virus sufficient to cause typical signs of myxomatosis in a susceptible rabbit. Observe the rabbits for a further 21 days. The test is not valid if fewer than 90 per cent of the control rabbits display typical signs of myxomatosis. A vaccine containing myxoma virus complies with the test if not fewer than 90 per cent of vaccinated rabbits show no signs of myxomatosis. A vaccine containing Shope fibroma virus complies with the test if not fewer than 75 per cent of vaccinated rabbits show no signs of myxomatosis. LABELLING The label states, where applicable, that a local reaction may occur. 01/2005:0962

NEONATAL PIGLET COLIBACILLOSIS VACCINE (INACTIVATED) Vaccinum colibacillosis fetus a partu recentis inactivatum ad suem DEFINITION Neonatal piglet colibacillosis vaccine (inactivated) is prepared from cultures of one or more suitable strains of Escherichia coli, carrying one or more adhesins or enterotoxins. This monograph applies to vaccines administered by injection to sows and gilts for protection of newborn piglets against enteric forms of colibacillosis.

TESTS Safety. Use not fewer than 2 rabbits, not older than the minimum age recommended for vaccination, that do not have antibodies against myxoma virus and rabbit haemorrhagic disease virus and that have been reared in suitable isolation conditions to avoid contact with myxoma virus. Administer by a recommended route to each rabbit 10 doses of vaccine. Observe the rabbits at least daily for 14 days. No abnormal local or systemic reaction occurs. Extraneous agents. At the end of the 14 day observation period of the safety test, administer by a recommended route to each rabbit, a further 10 doses of vaccine. After 14 days take a blood sample from each rabbit and carry out a test for antibodies against rabbit haemorrhagic disease virus. No antibodies are found. Bacterial and fungal contamination. The vaccine, reconstituted if necessary, complies with the test for sterility prescribed in the monograph on Vaccines for Veterinary Use (0062). Mycoplasmas (2.6.7). The vaccine, reconstituted if necessary, complies with the test for mycoplasmas. Virus titre. Reconstitute the vaccine, if necessary, and titrate in suitable cell cultures. 1 dose of the vaccine contains not less than the quantity of virus equivalent to the minimum virus titre stated on the label.

PRODUCTION The E. coli strains used for production are cultured separately in a suitable medium. The cells or toxins are processed to render them safe and are blended. The vaccine may contain one or more suitable adjuvants. CHOICE OF VACCINE COMPOSITION The E. coli strains used in the production of the vaccine are shown to be satisfactory with respect to expression of antigens and the vaccine is shown to be satisfactory with respect to safety and immunogenicity. The following tests may be used during demonstration of safety (5.2.6) and efficacy (5.2.7). Expression of antigens. The expression of antigens that stimulate a protective immune response is verified by a suitable immunochemical method (2.7.1) carried out on the antigen obtained from each of the vaccine strains under the conditions used for the production of the vaccine. Safety A. Administer a double dose of vaccine by a recommended route to each of not fewer than 10 pregnant sows that have not been vaccinated against colibacillosis. Administer 1 dose of vaccine to each of the animals after the recommended interval. Observe the animals until farrowing. Record body temperature the day before vaccination, at vaccination, 2 h, 4 h and 6 h later and then daily for 4 days ; note the maximum temperature increase POTENCY for each animal. Note any effects on gestation or the offspring. No abnormal local or systemic reaction occurs : Use not fewer than 15 susceptible rabbits of the minimum the average temperature increase for all animals does not age to be recommended for vaccination, free from antibodies exceed 1.5 °C and no animal shows a rise greater than against myxoma virus and reared in suitable isolation 2 °C. conditions to ensure absence of contact with myxoma virus. 776



B. The animals used for field trials are also used to evaluate safety. Use not fewer than 3 groups each of not fewer than 20 animals with corresponding groups of not fewer than 10 controls. Examine the injection site for local reactions after vaccination. Record body temperature the day before vaccination, at vaccination, at the time interval after which a rise in temperature, if any, was seen in test A, and daily during the 2 days following vaccination ; note the maximum temperature increase for each animal. No abnormal local or systemic reaction occurs : the average temperature increase for all animals does not exceed 1.5 °C and no animal shows a rise greater than 2 °C. Immunogenicity. The suitability of the vaccine with respect to immunogenicity may be demonstrated by the test described under Potency. BATCH TESTING Batch potency test. The test described under Potency is not carried out for routine testing of batches of vaccine. It is carried out, for a given vaccine, on one or more occasions, as decided by or with the agreement of the competent authority ; where the test is not carried out, a suitable validated test is carried out, the criteria for acceptance being set with reference to a batch of vaccine that has given satisfactory results in the test described under Potency. The following test may be used after a suitable correlation with the test described under Potency has been established by a statistical evaluation.

Neonatal piglet colibacillosis vaccine (inactivated)

amount of bacterial endotoxins is that found for a batch of vaccine that has been shown satisfactory in safety test A given under Choice of vaccine composition or in the safety test described under Tests, carried out using 10 piglets. Where the latter test is used, note the maximum temperature increase for each animal ; the average temperature increase for all animals does not exceed 1.5 °C. The method chosen for determining the amount of bacterial endotoxin present in the vaccine batch used in the safety test for determining the maximum acceptable level of endotoxin is used subsequently for testing of each batch. IDENTIFICATION In animals free from specific antibodies against the antigens stated on the label, the vaccine stimulates the production of antibodies against these antigens.

TESTS Safety. Use pigs preferably having no specific antibodies against the antigens stated on the label or, where justified, pigs with a low level of such antibodies as long as they have not been vaccinated against colibacillosis and administration of the vaccine does not cause an anamnestic response. Administer to each of 2 pigs a double dose of vaccine by a recommended route. Observe the animals for 14 days. Record body temperature before vaccination, at vaccination, 2 h, 4 h and 6 h later and then daily for 2 days. No abnormal local or systemic reaction occurs ; a transient temperature increase not exceeding 2 °C may occur. Use pigs not less than 3 weeks old and free from specific antibodies against the antigens stated on the label : vaccinate Sterility. The vaccine complies with the test for sterility each of 5 pigs by the route and according to the schedule prescribed in the monograph on Vaccines for veterinary stated on the label. Maintain 2 pigs as unvaccinated use (0062). controls. Alternatively, if the nature of the antigens allows reproducible results to be obtained, a test in laboratory POTENCY animals (for example, guinea-pigs, mice, rabbits or rats) may Carry out the test with a challenge strain representing each be carried out. To obtain a valid assay, it may be necessary type of antigen against which the vaccine is intended to to carry out a test using several groups of animals, each protect : if a single strain with all the necessary antigens is receiving a different dose. For each dose, carry out the not available, repeat the test using different challenge strains. test as follows. Vaccinate not fewer than 5 animals with a single injection of a suitable dose. Maintain not fewer than Use not fewer than 8 susceptible gilts free from specific 2 animals as unvaccinated controls. Where the schedule antibodies against the antigens stated on the label. Take not stated on the label requires a booster injection to be given, a fewer than 4 at random and vaccinate these at the stage of booster vaccination may also be given in this test provided it pregnancy and according to the recommended vaccination has been demonstrated that this will still provide a suitably scheme. Within 12 h of their giving birth, take not fewer sensitive test system. At a given interval within the range than 15 healthy piglets from the vaccinated animals and of 14 to 21 days after the last injection, collect blood from 15 healthy piglets from the unvaccinated controls, taking at each animal and prepare serum samples. Use a suitable least 3 from each litter. Challenge all the piglets orally with a validated test such as an enzyme-linked immunosorbent pathogenic strain of E. coli before or after colostrum feeding assay (2.7.1) to measure the antibody response to each of and using the same conditions for vaccinated animals and the antigens stated on the label. The antibody levels are not controls. The strain used must not be one used in the significantly less than those obtained with a batch that has manufacture of the vaccine. Return the piglets to their dam given satisfactory results in the test described under Potency and observe for 8 days. and there is no significant increase in antibody titre in the On each day, note clinical signs in each piglet and score controls. using the following scale : Where seronegative animals are not available, seropositive 0 no signs animals may be used in the above test. During the 1 slight diarrhoea development of a test with seropositive animals, particular care will be required during the validation of the test system 2 marked diarrhoea (watery faeces) to establish that the test is suitably sensitive and to specify 3 dead acceptable pass, fail and retest criteria. It will be necessary to take into account the range of possible prevaccination Total scores for each piglet over 8 days are calculated. The titres and establish the acceptable minimum titre rise after test is not valid unless at least 40 per cent of the piglets vaccination in relation to these. from the control animals die and not more than 15 per Bacterial endotoxins. A test for bacterial endotoxins (2.6.14) cent of the piglets from the control animals show no signs of illness. The vaccine complies with the test if there is a is carried out on the final lot or, where the nature of the adjuvant prevents performance of a satisfactory test, on the significant reduction in score in the group of piglets from the vaccinated gilts compared with the group from the bulk antigen or the mixture of bulk antigens immediately unvaccinated controls. before addition of the adjuvant. The maximum acceptable General Notices (1) apply to all monographs and other texts

777

Neonatal ruminant colibacillosis vaccine (inactivated)


stocks, by the route and according to the schedule stated For some adhesins (for example, F5 and F41), there is on the label. At least 30 animals from the same stocks published evidence that high mortality cannot be achieved are assigned to control groups. The animals are observed under experimental conditions. If challenge has to be for 14 days after the last dose. No abnormal local or carried out with a strain having such adhesins : the test is systemic reaction is noted and, in particular, no rise in not valid if fewer than 70 per cent of the control piglets show temperature of more than 1.5 °C occurs within 2 days of clinical signs expected with the challenge strain ; the vaccine administration of each dose of vaccine. complies with the test if there is a significant reduction in score in the group of piglets from the vaccinated gilts Immunogenicity. The suitability of the vaccine with respect compared with the group from the unvaccinated controls. to immunogenicity must be demonstrated for each species for which it is intended. This may be demonstrated by the LABELLING test described under Potency. The label states the antigen or antigens contained in the vaccine that stimulate a protective immune response. BATCH TESTING Batch potency test. The test described under Potency is not 01/2005:0961 carried out for routine testing of batches of vaccine. It is carried out, for a given vaccine, on one or more occasions, as decided by or with the agreement of the competent NEONATAL RUMINANT authority ; where the test is not carried out, a suitable validated test is carried out, the criteria for acceptance COLIBACILLOSIS VACCINE being set with reference to a batch of vaccine that has given (INACTIVATED) satisfactory results in the test described under Potency. The following test may be used after a suitable correlation Vaccinum colibacillosis fetus a partu with the test described under Potency has been established by a statistical evaluation. recentis inactivatum ad ruminantes DEFINITION Neonatal ruminant colibacillosis vaccine (inactivated) is prepared from cultures of one or more suitable strains of Escherichia coli, carrying one or more adhesin factors or enterotoxins. This monograph applies to vaccines administered by injection to dams for protection of newborn offspring against enteric forms of colibacillosis. PRODUCTION The E. coli strains used for production are cultured separately in a suitable medium. The cells or toxins are processed to render them safe and are blended. The vaccine may contain one or more suitable adjuvants. CHOICE OF VACCINE COMPOSITION The E. coli strains used in the production of the vaccine are shown to be satisfactory with respect to expression of antigens and the vaccine is shown to be satisfactory with respect to safety and immunogenicity. The following tests may be used during demonstration of safety (5.2.6) and efficacy (5.2.7). Expression of antigens. The expression of antigens that stimulate a protective immune response is verified by a suitable immunochemical method (2.7.1) carried out on the antigen obtained from each of the vaccine strains under the conditions used for the production of the vaccine. Safety A. A double dose of vaccine is administered to each of 10 pregnant animals of each of the species for which the vaccine is intended and that have not been vaccinated against colibacillosis. 1 dose is administered to each animal after the interval stated on the label. The animals are observed until parturition has occurred. Record body temperature the day before vaccination, at vaccination, 2 h, 4 h and 6 h later and then daily for 4 days ; note the maximum temperature increase for each animal. No abnormal local or systemic reaction occurs ; the average temperature increase for all animals does not exceed 1.5 °C and no animal shows a rise greater than 2 °C. Any effects on gestation or the offspring are noted. B. Safety is demonstrated in field trials for each species for which the vaccine is intended by administering the intended dose to at least 60 animals from 3 different 778

To obtain a valid assay, it may be necessary to carry out a test using several groups of animals, each receiving a different dose. For each dose required, carry out the test as follows. Vaccinate not fewer than 5 animals (for example rabbits, guinea-pigs, rats or mice), free from specific antibodies against the antigens stated on the label, using one injection of a suitable dose. Maintain 2 animals as unvaccinated controls. Where the schedule stated on the label requires a booster injection to be given, a booster vaccination may also be given in this test provided it has been demonstrated that this will still provide a suitably sensitive test system. At a given interval within the range of 14 to 21 days after the last injection, collect blood from each animal and prepare serum samples. Use a suitable validated test such as an enzyme-linked immunosorbent assay (2.7.1) to measure the antibody response to each of the protective antigens stated on the label. The antibody levels are not significantly less than those obtained with a batch that has given satisfactory results in the test described under Potency and there is no significant increase in antibody titre in the controls. Where seronegative animals are not available, seropositive animals may be used in the above test. During the development of a test with seropositive animals, particular care will be required during the validation of the test system to establish that the test is suitably sensitive and to specify acceptable pass, fail and retest criteria. It will be necessary to take into account the range of possible prevaccination titres and establish the acceptable minimum titre rise after vaccination in relation to these. Bacterial endotoxins. A test for bacterial endotoxins (2.6.14) is carried out on the final lot or, where the nature of the adjuvant prevents performance of a satisfactory test, on the bulk antigen or the mixture of bulk antigens immediately before addition of the adjuvant. The maximum acceptable amount of bacterial endotoxins is that found for a batch of vaccine that has been shown satisfactory in safety test A given under Choice of vaccine composition or in the safety test described under Tests, carried out using 10 animals. Where the latter test is used, note the maximum temperature increase for each animal ; the average temperature increase for all animals does not exceed 1.5 °C. The method chosen for determining the amount of bacterial endotoxin present



in the vaccine batch used in the safety test for determining the maximum acceptable level of endotoxins is used subsequently for testing of each batch. IDENTIFICATION In animals free from specific antibodies against the antigens stated on the label, the vaccine stimulates the production of antibodies against these antigens. TESTS Safety. Use animals of one of the species for which the vaccine is recommended and preferably having no specific antibodies against the antigens stated on the label or, where justified, use animals with a low level of such antibodies as long as they have not been vaccinated against colibacillosis and administration of the vaccine does not cause an anamnestic response. Administer by a recommended route a double dose of vaccine to each of 2 animals. Observe the animals for 14 days. Record body temperature before vaccination, at vaccination, 2 h, 4 h and 6 h later and then daily for 2 days. No abnormal local or systemic reaction occurs ; a transient temperature increase not exceeding 2 °C may occur. Sterility. The vaccine complies with the test for sterility prescribed in the monograph on Vaccines for veterinary use (0062). POTENCY Carry out the test with a challenge strain representing each type of antigen against which the vaccine is intended to protect : if a single strain with all the necessary antigens is not available, repeat the test using different challenge strains. Use not fewer than 15 susceptible animals of one of the species for which the vaccine is recommended and which are free from specific antibodies against the antigens stated on the label. Take not fewer than 10 at random and vaccinate these at the recommended stage of pregnancy and according to the recommended schedule. Collect colostrum from all animals after parturition and store the samples individually in conditions that maintain antibody levels. Take not fewer than 15 newborn unsuckled animals and house them in an environment ensuring absence of enteric pathogens. Allocate a colostrum sample from not fewer than 10 vaccinated dams and not fewer than 5 controls to the offspring. After birth, feed the animals with the colostrum sample allocated to it. After feeding the colostrum and within 12 h of birth, challenge the animals orally with a pathogenic strain of E. coli and observe for 10 days. The strain must not be one used in the manufacture of the vaccine. On each day, note clinical signs in each animal and score using the following scale : 0

no signs

1

slight diarrhoea

2

marked diarrhoea (watery faeces)

3

dead

Total scores for each animal over 10 days are calculated. The test is not valid unless 80 per cent of the offspring from the control animals die or show severe signs of disease. The vaccine complies with the test if there is a significant reduction in score in the group of animals from vaccinated dams compared with the group from the unvaccinated controls. LABELLING The label states the antigen or antigens contained in the vaccine that stimulate a protective immune response. General Notices (1) apply to all monographs and other texts

Newcastle disease vaccine (inactivated)

01/2005:0870

NEWCASTLE DISEASE VACCINE (INACTIVATED) Vaccinum pseudopestis aviariae inactivatum DEFINITION Newcastle disease vaccine (inactivated) [also known as avian paramyxovirus 1 vaccine (inactivated) for vaccines intended for some species] consists of an emulsion or a suspension of a suitable strain of Newcastle disease virus (avian paramyxovirus 1) that has been inactivated in such a manner that immunogenic activity is retained. PRODUCTION The virus is propagated in embryonated eggs from healthy flocks or in suitable cell cultures (5.2.4). The test for inactivation is carried out in embryonated eggs or suitable cell cultures and the quantity of inactivated virus used is equivalent to not less than ten doses of vaccine. No live virus is detected. The vaccine may contain an adjuvant. CHOICE OF VACCINE COMPOSITION The vaccine is shown to be satisfactory with respect to safety (5.2.6) and immunogenicity (5.2.7) for each species and category of birds for which it is intended. The following tests may be used during demonstration of immunogenicity. Immunogenicity. For domestic fowl, the test with virulent challenge (test B) described under Potency is suitable for demonstrating immunogenicity. For other species of birds (for example, pigeons or turkeys), test C described under Potency is suitable for demonstrating immunogenicity. BATCH TESTING Batch potency test For vaccines used in domestic fowl, carry out test A described under Potency but, if the nature of the product does not allow valid results to be obtained with test A or if the batch does not comply with test A, carry out test B. A test using fewer than twenty birds per group and a shorter observation period after challenge may be used if this has been shown to give a valid potency test. For vaccines used in species other than domestic fowl, carry out a suitable validated test for which a satisfactory correlation has been established with test C described under Potency, the criteria for acceptance being set with reference to a batch that has given satisfactory results in the latter test. A test in chickens from a flock free from specified pathogens (5.2.2) and consisting of a measure of the serological response to graded amounts of vaccine (for example, 1/25, 1/50 and 1/100 dose with serum sampling 17 to 21 days later) may be used. IDENTIFICATION When injected into animals free from antibodies against Newcastle disease virus, the vaccine stimulates the production of such antibodies. TESTS Safety. If the vaccine is intended for use in domestic fowl, vaccinate ten chickens from a flock free from specified pathogens (5.2.2). If the vaccine is not for use in domestic fowl, use ten birds of one of the species for which the vaccine is intended, free from antibodies against Newcastle disease virus. Inject twice the vaccinating dose by a recommended 779

Newcastle disease vaccine (inactivated)


haemagglutinating units and red blood cells. The test route into each of ten birds, 14 to 28 days old. Observe the system used must include negative and positive control birds for 21 days. No abnormal local or systemic reaction sera, the latter having an HI titre of 5.0 log2 to 6.0 log2. occurs. The vaccine complies with the test if the mean HI titre of Inactivation. Inject two-fifths of a dose into the allantoic the vaccinated group is equal to or greater than 4.0 log2 cavity of each of ten embryonated hen eggs, 9 to 11 days and that of the unvaccinated group is 2.0 log2 or less. If old, from flocks free from specified pathogens (5.2.2) (SPF the HI titres are not satisfactory, carry out test B. eggs) and incubate. Observe for 6 days and pool separately Haemagglutination-inhibition test. Inactivate the test the allantoic fluid from eggs containing live embryos, and sera by heating at 56 °C for 30 min. Add 0.025 ml to the that from eggs containing dead embryos, excluding those first row of wells in a microtitre plate. Add 0.025 ml of a dying within 24 h of the injection. Examine embryos that buffered 9 g/l solution of sodium chloride R at pH 7.2 to die within 24 h of injection for the presence of Newcastle pH 7.4 to the rest of the wells. Prepare twofold dilutions disease virus : the vaccine does not comply with the test if of the sera across the plate. To each well add 0.025 ml of Newcastle disease virus is found. a suspension containing 4 haemagglutinating units of Inject into the allantoic cavity of each of ten SPF eggs, 9 inactivated Newcastle disease virus. Incubate the plate at to 11 days old, 0.2 ml of the pooled allantoic fluid from the 4 °C for 1 h. Add 0.025 ml of a 1 per cent V/V suspension live embryos and, into each of ten similar eggs, 0.2 ml of of red blood cells collected from chickens, 3 to 4 weeks old the pooled fluid from the dead embryos and incubate for and free from antibodies against Newcastle disease virus. 5 to 6 days. Test the allantoic fluid from each egg for the Incubate the plate at 4 °C for 1 h. The HI titre is equal to presence of haemagglutinins using chicken erythrocytes. the highest dilution that produces complete inhibition. The vaccine complies with the test if there is no evidence of B. Use chickens 21 to 28 days old and from a flock free haemagglutinating activity and if not more than 20 per cent from specified pathogens (5.2.2). For vaccination, of the embryos die at either stage. If more than 20 per cent use not fewer than three groups, each of not fewer of the embryos die at one of the stages, repeat that stage ; than twenty chickens ; keep a group of ten chickens the vaccine complies with the test if there is no evidence of as controls. Choose a number of different volumes of haemagglutinating activity and not more than 20 per cent of the vaccine corresponding to the number of groups : the embryos die at that stage. for example, volumes equivalent to 1/25, 1/50 and Antibiotics may be used in the test to control extraneous 1/100 of a dose. Allocate a different volume to each bacterial infection. vaccination group. Inject intramuscularly into each chicken the volume of vaccine allocated to its group. 17 to Extraneous agents. Inject a double dose by a recommended 21 days later, challenge all the chickens by intramuscular route into each of ten chickens, 14 to 28 days old and from a injection of 6 log10 embryo LD50 of the Herts (Weybridge flock free from specified pathogens (5.2.2). After 3 weeks, 33/56) strain of Newcastle disease virus. Observe the inject one dose by the same route. Collect serum samples chickens for 21 days. Calculate the PD50 by standard from each chicken 2 weeks later and carry out tests for statistical methods from the number of chickens that antibodies to the following agents by the methods prescribed survive in each vaccinated group without showing any for chicken flocks free from specified pathogens (5.2.2) : clinical evidence of Newcastle disease during the 21 days. avian encephalomyelitis virus, avian infectious bronchitis The vaccine complies with the test if the smallest dose virus, avian leucosis viruses, egg-drop syndrome virus, avian stated on the label corresponds to not less than 50 PD50 bursal disease virus, avian infectious laryngotracheitis virus, and the lower confidence limit is not less than 35 PD50 influenza A virus, Marek’s disease virus. The vaccine does per dose. If the lower confidence limit is less than 35 not stimulate the formation of antibodies against these PD50 per dose, repeat the test ; the vaccine must be shown agents. to contain not less than 50 PD50 in the repeat test. The Sterility. The vaccine complies with the test for sterility test is not valid unless all the control birds die within prescribed in the monograph on Vaccines for veterinary 6 days of challenge. use (0062). C. Vaccinate not fewer than twenty birds of the target species, free from antibodies against avian paramyxovirus 1, in POTENCY accordance with the recommendations for use. Maintain For vaccines for use in domestic fowl carry out test A. as unvaccinated controls a group of not fewer than ten If an unsatisfactory result is obtained with test A but a birds of the same age, from the same source and free from satisfactory result is obtained with test B, the vaccine antibodies against avian paramyxovirus 1. The test is complies with the requirements. For vaccines for use only invalid if serum samples obtained at the time of the first in other species, such as pigeons, carry out test C. vaccination show the presence of antibodies against avian A. Inject intramuscularly into each of not fewer than ten paramyxovirus 1 in either vaccinates or controls or if tests chickens, 21 to 28 days old and from a flock free from carried out at the time of challenge show such antibodies specified pathogens (5.2.2), a volume of the vaccine in controls. 4 weeks after the last vaccination, challenge equivalent to 1/50 of a dose. 17 to 21 days later, collect each bird intramuscularly with a sufficient quantity of serum samples from each vaccinated chicken and from virulent avian paramyxovirus 1. The test is not valid if each of not fewer than five control chickens of the same fewer than 90 per cent of the control birds die or show age and from the same source. Measure the antibody serious signs of Newcastle disease virus infection. The levels in the sera by the haemagglutination-inhibition vaccine complies with the test if not fewer than 90 per (HI) test using the technique described below or cent of the vaccinated birds survive and show no serious an equivalent technique with the same numbers of signs of avian paramyxovirus 1 infection.

780


Newcastle disease vaccine (live)


NEWCASTLE DISEASE (LIVE)

01/2005:0450 2-4-2. Amino-acid sequence. Determine the sequence of a fragment of RNA from the vaccine virus containing the region encoding for the F0 cleavage site by a suitable VACCINE method. The encoded amino-acid sequence is shown to be one of the following :

Vaccinum pseudopestis aviariae vivum 1. DEFINITION

F2

Site

Cleavage site ∨ 111 112 113 114 115 116 117 Gly Gly

Lys Gln

Gly Arg

Leu

F1 118

119

Ile

Gly

Newcastle disease vaccine (live) is a preparation of a suitable or Gly Gly Arg Gln Gly Arg Leu Ile Gly strain of Newcastle disease virus (avian paramyxovirus 1). or Gly Glu Arg Gln Glu Arg Leu Val Gly This monograph applies to vaccines intended for administration to chickens and/or other avian species for or equivalent with leucine at 117 and no basic amino acids at active immunisation. sites 111, 112, 114 and 115. 2. PRODUCTION 2-4-3. Safety. Carry out the test for each route and method of administration to be recommended for vaccination and 2-1. PREPARATION OF THE VACCINE in each avian species for which the vaccine is intended, The vaccine virus is grown in embryonated hens’ eggs or in using in each case birds not older than the youngest age to cell cultures. be recommended for vaccination. Use vaccine virus at the 2-2. SUBSTRATE FOR VIRUS PROPAGATION least attenuated passage level that will be present between 2-2-1. Embryonated hens’ eggs. If the vaccine virus is grown the master seed lot and a batch of the vaccine. For tests in chickens, use not fewer than 20 chickens, from an SPF in embryonated hens’ eggs, they are obtained from flocks flock (5.2.2). For species other than the chicken, use not free from specified pathogens (SPF) (5.2.2). fewer than 20 birds that do not have antibodies against 2-2-2. Cell cultures. If the vaccine virus is grown in cell Newcastle disease virus. Administer to each bird a quantity cultures, they comply with the requirements for cell cultures of the vaccine virus equivalent to not less than 10 times the for production of veterinary vaccines (5.2.4). maximum virus titre likely to be contained in 1 dose of the vaccine. Observe the birds at least daily for 21 days. The 2-3. SEED LOTS test is not valid if more than 10 per cent of the birds show 2-3-1. Extraneous agents. The master seed lot complies abnormal clinical signs or die from causes not attributable to with the tests for extraneous agents in seed lots (2.6.24). In the vaccine virus. The vaccine virus complies with the test if these tests on the master seed lot, the organisms used are no bird shows notable clinical signs of Newcastle disease or not more than 5 passages from the master seed lot at the dies from causes attributable to the vaccine virus. start of the tests. 2-4-4. Increase in virulence. The test for increase in 2-4. CHOICE OF VACCINE VIRUS virulence consists of the administration of the vaccine virus The vaccine virus shall be shown to be satisfactory with at the least attenuated passage level that will be present respect to safety (5.2.6) and efficacy (5.2.7) for the birds for between the master seed lot and a batch of the vaccine to which it is intended. a group of 5 birds not more than 2 weeks old, sequential The following tests for intracerebral pathogenicity index passages, 5 times where possible, to further similar groups (section 2-4-1), amino-acid sequence (section 2-4-2), safety and testing of the final recovered virus for increase in (section 2-4-3), increase in virulence (section 2-4-4) and virulence. If the properties of the vaccine virus allow immunogenicity (section 2-4-5) may be used during the sequential passage to 5 groups via natural spreading, this demonstration of safety and immunogenicity. method may be used, otherwise passage as described below 2-4-1. Intracerebral pathogenicity index. Use vaccine virus is carried out and the maximally passaged virus that has been recovered is tested for increase in virulence. Care must at the least attenuated passage level that will be present in be taken to avoid contamination by virus from previous a batch of the vaccine. Inoculate the vaccine virus into the allantoic cavity of embryonated hens’ eggs, 9- to 11- days-old, passages. Carry out the test in a target species, using the from an SPF flock (5.2.2). Incubate the inoculated eggs for chicken if it is one of the target species. For the test in chickens, use chickens from an SPF flock (5.2.2). For other a suitable period and harvest and pool the allantoic fluids. species, carry out the test in birds that do not have antibodies Use not fewer than ten 1-day-old chickens (i.e. more than against Newcastle disease virus. Administer by eye-drop a 24 h but less than 40 h after hatching), from an SPF flock (5.2.2). Administer by the intracerebral route to each chick quantity of the vaccine virus that will allow recovery of virus for the passages described below. Observe the birds for the 0.05 ml of the pooled allantoic fluids containing not less than 108.0 EID50 or, if this virus quantity cannot be achieved, period shown to correspond to maximum replication of the not less than 107.0 EID50. Observe the chickens at least daily vaccine virus, kill them and prepare a suspension from the brain of each bird and from a suitable organ depending on for 8 days after administration and score them once every 24 h. A score of 0 is attributed to a chicken if it is clinically the tropism of the strain (for example, mucosa of the entire trachea, intestine, pancreas) ; pool the samples. Administer normal, 1 if it shows clinical signs of disease and 2 if it is 0.05 ml of the pooled samples by eye-drop to each of 5 other dead. The intracerebral pathogenicity index is the mean of the scores per chicken per observation over the 8 day period. birds of the same species, age and origin. Carry out this passage operation not fewer than 5 times ; verify the presence If an inoculum of not less than 108.0 EID50 is used, the vaccine of the virus at each passage. If the virus is not found at a virus complies with the test if its intracerebral pathogenicity passage level, carry out a second series of passages. index is not greater than 0.5 ; if an inoculum of not less than 107.0 EID50 but less than 108.0 EID50 is used, the vaccine virus A. Carry out the test for intracerebral pathogenicity index (section 2-4-1) using unpassaged vaccine virus and the complies with the test if its intracerebral pathogenicity index maximally passaged virus that has been recovered. is not greater than 0.4. General Notices (1) apply to all monographs and other texts

781

Newcastle disease vaccine (live)


B. Carry out the test for amino-acid sequence (section 2-4-2) using unpassaged vaccine virus and the maximally passaged virus that has been recovered. C. Carry out the test for safety (section 2-4-3) using unpassaged vaccine virus and the maximally passaged virus that has been recovered. Administer the virus by the route to be recommended for vaccination likely to be the least safe and to the avian species for which the vaccine is intended that is likely to be the most susceptible to Newcastle disease. The vaccine virus complies with the test if, in the tests 2-4-4A, 2-4-4B and 2-4-4C, no indication of increase in virulence of the maximally passaged virus compared with the unpassaged virus is observed. If virus is not recovered at any passage level in the first and second series of passages, the vaccine virus also complies with the test. 2-4-5. Immunogenicity. For each avian species for which the vaccine is intended, a test is carried out for each route and method of administration to be recommended using in each case birds not older than the youngest age to be recommended for vaccination. The quantity of the vaccine virus administered to each bird is not greater than the minimum titre to be stated on the label and the virus is at the most attenuated passage level that will be present in a batch of the vaccine. 2-4-5-1. Vaccines for use in chickens. Use not fewer than 30 chickens of the same origin and from an SPF flock (5.2.2). Vaccinate by a recommended route not fewer than 20 chickens. Maintain not fewer than 10 chickens as controls. Challenge each chicken after 21 days by the intramuscular route with not less than 105.0 embryo LD50 of the Herts (Weybridge 33/56) strain of Newcastle disease virus. Observe the chickens at least daily for 14 days after challenge. Record the deaths and the number of surviving chickens that show clinical signs of disease. The test is not valid if 6 days after challenge fewer than 100 per cent of the control chickens have died or if during the period between vaccination and challenge more than 10 per cent of the vaccinated or control chickens show abnormal clinical signs or die from causes not attributable to the vaccine. The vaccine virus complies with the test if during the observation period after challenge not fewer than 90 per cent of the vaccinated chickens survive and show no notable clinical signs of Newcastle disease. 2-4-5-2. Vaccines for use in avian species other than the chicken. Use not fewer than 30 birds of the species for which the vaccine is intended for Newcastle disease, of the same origin and that do not have antibodies against avian paramyxovirus 1. Vaccinate by a recommended route not fewer than 20 birds. Maintain not fewer than 10 birds as controls. Challenge each bird after 21 days by the intramuscular route with a sufficient quantity of virulent avian paramyxovirus 1. Observe the birds at least daily for 21 days after challenge. Record the deaths and the surviving birds that show clinical signs of disease. The test is not valid if : — during the observation period after challenge fewer than 90 per cent of the control birds die or show severe clinical signs of Newcastle disease, — or if during the period between the vaccination and challenge more than 10 per cent of the vaccinated or control birds show abnormal clinical signs or die from causes not attributable to the vaccine. The vaccine virus complies with the test if during the observation period after challenge not fewer than 90 per cent of the vaccinated birds survive and show no notable clinical signs of Newcastle disease. For species where there 782

is published evidence that it is not possible to achieve this level of protection, the vaccine complies with the test if there is a significant reduction in morbidity and mortality of the vaccinated birds compared with the control birds. 3. BATCH TESTS 3-1. Identification 3-1-1. Identification of the vaccine virus. The vaccine, diluted if necessary and mixed with a monospecific Newcastle disease virus antiserum, no longer provokes haemagglutination of chicken red blood cells or infects embryonated hens’ eggs from an SPF flock (5.2.2) or susceptible cell cultures (5.2.4) into which it is inoculated. 3-1-2. Identification of the virus strain. The strain of vaccine virus is identified by a suitable method, for example using monoclonal antibodies. 3-2. Bacteria and fungi Vaccines intended for administration by injection comply with the test for sterility prescribed in the monograph Vaccines for veterinary use (0062). Vaccines not intended for administration by injection either comply with the test for sterility prescribed in the monograph Vaccines for veterinary use (0062) or with the following test : carry out a quantitative test for bacterial and fungal contamination ; carry out identification tests for microorganisms detected in the vaccine ; the vaccine does not contain pathogenic microorganisms and contains not more than 1 non-pathogenic microorganism per dose. Any liquid supplied with the vaccine complies with test for sterility prescribed in the monograph Vaccines for veterinary use (0062). 3-3. Mycoplasmas. The vaccine complies with the test for mycoplasmas (2.6.7). 3-4. Extraneous agents. The vaccine complies with the tests for extraneous agents in batches of finished product (2.6.25). 3-5. Safety. For vaccines recommended for use in chickens, use not fewer than 10 chickens from an SPF flock (5.2.2) and of the youngest age recommended for vaccination. For vaccines recommended for use only in avian species other than the chicken, use not fewer than 10 birds of the species likely to be most sensitive to Newcastle disease, that do not have antibodies against Newcastle disease virus and of the minimum age recommended for vaccination. Administer to each bird by eye-drop, or parenterally if only parenteral administration is recommended, 10 doses of the vaccine in a volume suitable for the test. Observe the birds at least daily for 21 days. The test is not valid if more than 20 per cent of the birds show abnormal clinical signs or die from causes not attributable to the vaccine. The vaccine complies with the test if no bird shows notable clinical signs of disease or dies from causes attributable to the vaccine. 3-6. Virus titre. Titrate the vaccine virus by inoculation into embryonated hens’ eggs from an SPF flock (5.2.2) or into suitable cell cultures (5.2.4). The vaccine complies with the test if 1 dose contains not less than the minimum virus titre stated on the label. 3-7. Potency. Depending on the indications, the vaccine complies with 1 or both of the tests prescribed under Immunogenicity (section 2-4-5) when administered according to the recommended schedule by a recommended route and method. If the test in section 2-4-5-2. Vaccine for use in avian species other than the chicken is conducted and the vaccine is recommended for use in more than 1 avian species, the test is carried out with birds of that species for which the vaccine is recommended which is likely to be the most susceptible to avian paramyxovirus 1. It is not necessary



Pasteurella vaccine (inactivated) for sheep

to carry out the potency test for each batch of the vaccine if it has been carried out on a representative batch using a vaccinating dose containing not more than the minimum virus titre stated on the label.

The vaccine complies with the test if no animal shows abnormal local reactions or clinical signs of disease or dies from causes attributable to the vaccine. In addition, if the vaccine is intended for use in pregnant ewes, no significant effects on the pregnancy and offspring are demonstrated. B. The animals used for the field trials are also used to 01/2005:2072 evaluate safety. Carry out a test in each category of animals for which the vaccine is intended. Use not fewer PASTEURELLA VACCINE than 3 groups of 20 animals with corresponding groups of not fewer than 10 controls in 3 different locations. (INACTIVATED) FOR SHEEP Examine the injection sites for local reactions after Record body temperatures the day before Vaccinum pasteurellae inactivatum ad ovem vaccination. vaccination, at vaccination and on the 2 days following vaccination. The vaccine complies with the test if no DEFINITION animal shows abnormal local or systemic reactions or Pasteurella vaccine (inactivated) for sheep is a preparation of clinical signs of disease or dies from causes attributable one or more suitable strains of Pasteurella trehalosi. This to the vaccine. The average body temperature increase monograph applies to vaccines intended for administration for all animals does not exceed 1.5 °C and no animal to sheep to protect against disease caused by P. trehalosi. shows a rise greater than 2 °C. In addition, if the vaccine is intended for use in pregnant ewes, no significant effects PRODUCTION on the pregnancy and offspring are demonstrated. Production of the vaccine is based on a seed-lot system. Immunogenicity. As part of the studies to demonstrate the The seed material is cultured in a suitable medium ; each suitability of the vaccine with respect to immunogenicity, strain is cultivated separately and identity is verified using the test described under Potency may be carried out for a suitable method. During production, various parameters each proposed route of administration and using vaccine such as growth rate are monitored by suitable methods ; of minimum potency. the values are within the limits approved for the particular BATCH TESTING product. Purity and identity of the harvest are verified using suitable methods. After cultivation, the bacterial Batch potency test. The test described under Potency is not suspensions are collected separately and inactivated by a carried out for routine testing of batches of vaccine. It is suitable method. The vaccine may contain an adjuvant and carried out, for a given vaccine, on one or more occasions, may be freeze-dried. as decided by or with the agreement of the competent authority. Where the test is not carried out, a suitable CHOICE OF VACCINE COMPOSITION validated batch potency test is carried out, the criteria for The choice of composition and the strains to be included acceptance being set with reference to the results obtained in the vaccine is based on epidemiological data on the with a batch of vaccine that has given satisfactory results in prevalence of the different serovars of P. trehalosi. The the test described under Potency. vaccine is shown to be satisfactory with respect to safety (5.2.6) and efficacy (5.2.7) in sheep. As part of the studies Bacterial endotoxins. A test for bacterial endotoxins (2.6.14) to demonstrate the suitability of the vaccine with respect to is carried out on the final lot or, where the nature of the these characteristics the following tests may be carried out. adjuvant prevents performance of a satisfactory test, on the bulk antigen or the mixture of bulk antigens immediately Safety before addition of the adjuvant. The maximum acceptable A. The test is carried out for each of the routes of amount of bacterial endotoxins is that found for a batch of administration to be stated on the label and in animals of vaccine that has been shown satisfactory in safety test A each category (for example, young sheep, pregnant ewes) given under Choice of vaccine composition or in the safety for which the vaccine is intended. test described under Tests, carried out using 10 animals. For each test, use not fewer than 10 animals that Where the latter test is used, note the maximum temperature preferably do not have antibodies against the serovars of increase for each animal ; the average body temperature P. trehalosi or against leucotoxin present in the vaccine. increase for all animals does not exceed 1.5 °C. The method Where justified, animals with a known history of no chosen for determining the amount of bacterial endotoxin previous pasteurella vaccination and with low antibody present in the vaccine batch used in the safety test for titres (measured in a sensitive test system such as an determining the maximum acceptable level of endotoxin is ELISA) may be used. used subsequently for testing of each batch. Administer to each animal a double dose of vaccine containing not less than the maximum potency that may IDENTIFICATION When injected into healthy seronegative animals, the vaccine be expected in a batch of vaccine. Administer a single stimulates the production of specific antibodies against dose of vaccine to each animal after the recommended interval. Observe the animals for at least 14 days after the the serovars of P. trehalosi and/or against the leucotoxin present in the vaccine. last administration. Record body temperature the day before vaccination, at vaccination, 2 h, 4 h and 6 h later and then daily for 4 days ; note the maximum temperature TESTS increase for each animal. No abnormal local or systemic Safety. Use 2 sheep of the minimum age recommended for vaccination or, if not available, of an age as close as possible reaction occurs ; the average body temperature increase to the minimum recommended age, and that have not been for all animals does not exceed 1.5 °C and no animal shows a rise greater than 2 °C. If the vaccine is intended vaccinated against Pasteurella. Administer a double dose of vaccine to each animal by a recommended route. Observe for use or may be used in pregnant ewes, vaccinate the ewes at the relevant stages of pregnancy and prolong the the animals for 14 days. Record body temperature the day before vaccination, at vaccination, 2 h, 4 h and 6 h later and observation period until 1 day after lambing. General Notices (1) apply to all monographs and other texts

783

Porcine actinobacillosis vaccine (inactivated)


then daily for 2 days. The animals remain in good health and no abnormal local or systemic reaction occurs ; a transient temperature increase not exceeding 2 °C may occur. Sterility. It complies with the test for sterility prescribed in the monograph on Vaccines for veterinary use (0062).

of A. pleuropneumoniae, and treated to render them harmless ; fractions of toxins derived from suitable strains of A. pleuropneumoniae and treated if necessary to render them harmless. This monograph applies to vaccines intended for protection of pigs against actinobacillosis.

PRODUCTION The seed material is cultured in a suitable medium ; each strain is cultivated separately. During production, various parameters such as growth rate, protein content and quantity of relevant antigens are monitored by suitable methods ; the values are within the limits approved for the particular product. Purity and identity are verified on the harvest using suitable methods. After cultivation, the bacterial suspensions are collected separately and inactivated by a suitable method. They may be detoxified, purified and concentrated. The vaccine may contain an adjuvant. CHOICE OF VACCINE COMPOSITION The choice of strains is based on epidemiological data. The vaccine is shown to be satisfactory with respect to safety (5.2.6) and efficacy (5.2.7) in pigs. The following tests may be used during demonstration of safety and immunogenicity. Safety A. Carry out a test in each category of animals for which the vaccine is intended and by each of the recommended routes of administration. Use animals that do not have antibodies against the serotypes of A. pleuropneumoniae or its toxins present in the vaccine. Administer a double dose of vaccine by a recommended route to each of not fewer than 10 animals. Administer a single dose of vaccine to each of the animals after the interval recommended in the instructions for use. Observe the animals for 14 days after vaccination. Record body temperature the day before vaccination, at vaccination, 2 h, 4 h and 6 h later and then daily for 4 days ; note the maximum temperature increase for each animal. No abnormal local or systemic reaction occurs ; the average temperature increase for all animals does not exceed 1.5 °C and no animal shows a rise greater than 2 °C. If the vaccine is intended for use in pregnant sows, for the test in this category of animals, prolong the observation period up to farrowing and note any effects on gestation or the offspring. B. The animals used for field trials are also used to evaluate safety. Carry out a test in each category of animals LABELLING for which the vaccine is intended. Use not fewer than The label states : 3 groups each of not fewer than 20 animals with — the serovar(s) of P. trehalosi against which protection is corresponding groups of not fewer than 10 controls. claimed, Examine the injection site for local reactions after vaccination. Record body temperature the day before — the serovar(s) of P. trehalosi and/or the leucotoxin vaccination, at vaccination, at the time interval after present in the vaccine. which a rise in temperature, if any, was seen in test A, and daily during the 2 days following vaccination ; note 01/2005:1360 the maximum temperature increase for each animal. No abnormal local or systemic reaction occurs ; the average temperature increase for all animals does not exceed PORCINE ACTINOBACILLOSIS 1.5 °C and no animal shows a rise greater than 2 °C. VACCINE (INACTIVATED) Immunogenicity. The test described under Potency may be used to demonstrate the immunogenicity of the vaccine. Vaccinum actinobacillosis inactivatum BATCH TESTING ad suem Batch potency test. The test described under Potency is not DEFINITION carried out for routine testing of batches of vaccine. It is carried out, for a given vaccine, on one or more occasions, Porcine actinobacillosis vaccine (inactivated) is a as decided by or with the agreement of the competent liquid preparation which has one or more of the authority ; where the test is not carried out, a suitable following components : inactivated Actinobacillus validated test is carried out, the criteria for acceptance pleuropneumoniae of a suitable strain or strains ; toxins, being set with reference to a batch of vaccine that has given proteins or polysaccharides derived from suitable strains

POTENCY Carry out a test for each serovar of P. trehalosi for which protection is claimed on the label. Use not fewer than 20 lambs of the minimum age recommended for vaccination, free from antibodies against P. trehalosi and against the leucotoxin of P. trehalosi. Vaccinate not fewer than 10 of the animals by a recommended route and according to the recommended schedule. Keep 10 animals as controls. 21 days after the last vaccination, infect all the lambs by injection, using the subcutaneous or other suitable route, with a suitable quantity of a low-passage, virulent strain of a serovar of P. trehalosi. Observe the animals for a further 7 days ; to avoid unnecessary suffering, severely ill animals are killed and are then considered to have died from the disease. During the observation period, the animals are examined for any signs of disease (for example, severe dullness, excess salivation) and the mortality is recorded. Kill surviving animals at the end of the observation period. Post-mortem examination is carried out on any animal that dies and those killed at the end of the observation period. The lungs, pleura, liver and spleen are examined for haemorrhages and the extent of lung consolidation due to pasteurellosis is evaluated. Samples of lung, liver and spleen tissue are collected for re-isolation of the challenge organisms. The mortality, clinical observations and the post-mortem lesions are scored and the results obtained for these parameters and the bacterial re-isolation results compared for the 2 groups. The test is invalid if clinical signs or lesions of P. trehalosi infection occur in less than 70 per cent of the control lambs. The vaccine complies with the requirements of the test if there is a significant difference between the scores obtained for the clinical and post-mortem observations in the vaccinates compared to the controls. For vaccines with a claim for a beneficial effect on the extent of infection against the serovar, the results for the infection rates are also significantly better for the vaccinates compared to the controls.

784



Porcine influenza vaccine (inactivated)

satisfactory results in the test described under Potency. The following test may be used after a satisfactory correlation with the test described under Potency has been established. Inject a suitable dose subcutaneously into each of 5 seronegative mice, weighing 18-20 g. Where the schedule stated on the label requires a booster injection to be given, a booster vaccination may also be given in this test provided it has been demonstrated that this will still provide a suitably sensitive test system. Before the vaccination and at a given interval within the range of 14-21 days after the last injection, collect blood from each animal and prepare serum samples. Determine individually for each serum the titre of specific antibodies against each antigenic component stated on the label, using a suitable validated test such as enzyme-linked immunosorbent assay (2.7.1). The vaccine complies with the test if the antibody levels are not significantly lower than those obtained for a batch that has given satisfactory results in the test described under Potency. Bacterial endotoxins. A test for bacterial endotoxins (2.6.14) is carried out on the final bulk or, where the nature of the adjuvant prevents performance of a satisfactory test, on the bulk antigen or mixture of bulk antigens immediately before addition of the adjuvant. The maximum acceptable amount of bacterial endotoxins is that found for a batch of vaccine that has been shown satisfactory in safety test A described under Choice of vaccine composition or the safety test described under Tests, carried out using 10 pigs. Where the latter test is used, note the maximum temperature increase for each animal ; the average temperature increase for all animals does not exceed 1.5 °C. The method chosen for determining the amount of bacterial endotoxin present in the vaccine batch used in the safety test for determining the maximum acceptable level of endotoxin is used subsequently for batch testing.

or intratracheally or by aerosol with a suitable quantity of a serotype of A. pleuropneumoniae. Observe the animals for 7 days ; to avoid unnecessary suffering, severely ill control animals are killed and are then considered to have died from the disease. Kill all surviving animals at the end of the observation period. Carry out a post-mortem examination on all animals. Examine the lungs, the tracheobronchial lymph nodes and the tonsils for the presence of A. pleuropneumoniae. Evaluate the extent of lung lesions at post-mortem examination. Each of the 7 lobes of the lungs is allotted a maximum possible lesion score (2) of 5. The area showing pneumonia and/or pleuritis of each lobe is assessed and expressed on a scale of 0 to 5 to give the pneumonic score per lobe (the maximum total score possible for each complete lung is 35). Calculate separately for the vaccinated and the control animals the total score (the maximum score per group is 245, if 7 pigs are used per group). The vaccine complies with the test if the vaccinated animals, when compared with controls, show lower incidence of: mortality ; typical clinical signs (dyspnoea, coughing and vomiting) ; typical lung lesions ; re-isolation of A. pleuropneumoniae from the lungs, the tracheobronchial lymph nodes and the tonsils. Where possible, the incidence is analysed statistically and shown to be significantly lower for vaccinates.

IDENTIFICATION When injected into healthy seronegative animals, the vaccine stimulates the production of specific antibodies against the antigenic components of A. pleuropneumoniae stated on the label.

01/2005:0963

TESTS Safety. Use 2 pigs of the minimum age stated for vaccination and which do not have antibodies against the serotypes of A. pleuropneumoniae or its toxins present in the vaccine. Administer to each pig a double dose of vaccine by a recommended route. Observe the animals for 14 days. Record body temperature the day before vaccination, at vaccination, 2 h, 4 h and 6 h later and then daily for 2 days. No abnormal local or systemic reaction occurs ; a transient temperature increase not exceeding 2 °C may occur. Sterility. The vaccine complies with the test for sterility prescribed in the monograph on Vaccines for veterinary use (0062).

LABELLING The label states : — the antigens present in the vaccine, — the serotypes of A. pleuropneumoniae for which the vaccine affords protection.

PORCINE INFLUENZA VACCINE (INACTIVATED) Vaccinum influenzae inactivatum ad suem DEFINITION Porcine influenza vaccine (inactivated) is an aqueous suspension, an oily emulsion or a freeze-dried preparation of one or more inactivated strains of swine or human influenza virus. Suitable strains contain both haemagglutinin and neuraminidase.

PRODUCTION The virus is propagated in the allantoic cavity of fertilised hen eggs from a healthy flock or in suitable cell cultures (5.2.4). Each virus strain is cultivated separately. After cultivation, the viral suspensions are collected separately and inactivated by a method that avoids destruction of the POTENCY The challenge strain for the potency test is chosen to ensure immunogenicity. If necessary, they may be purified. challenge with each Ap toxin (1) produced by the serotypes An amplification test for residual infectious influenza virus stated on the label; it may be necessary to carry out more is carried out on each batch of antigen immediately after than one test using a different challenge strain for each test. inactivation by passage in the same type of substrate as that used for production (eggs or cell cultures) or a substrate Vaccinate according to the recommended schedule not shown to be at least as sensitive. The quantity of inactivated fewer than 7 pigs, of the minimum age recommended virus used in the test is equivalent to not less than 10 doses for vaccination, which do not have antibodies against of the vaccine. No live virus is detected. A. pleuropneumoniae and Ap toxins. Keep not fewer than 7 unvaccinated pigs of the same age as controls. 3 weeks The vaccine may contain one or more suitable adjuvants ; after the last vaccination, challenge all the pigs intranasally it may be freeze-dried. (1) The nomenclature of the toxins of A. pleuropneumoniae is described by J. Frey et al., Journal of General Microbiology, 1993, 139, 1723-1728. (2) The system of lung scores is described in detail by P.C.T. Hannan, B.S. Bhogal, J.P. Fish, Research in Veterinary Science, 1982, 33, 76-88.


785

Porcine influenza vaccine (inactivated)


CHOICE OF VACCINE COMPOSITION The choice of strains is based on the antigenic types and sub-types observed in Europe. The vaccine is shown to be satisfactory with respect to safety and immunogenicity for pigs. The following tests may be used during demonstration of safety (5.2.6) and efficacy (5.2.7). Safety A. A test is carried out in each category of animal for which the vaccine is intended (sows, fattening pigs). The animals used do not have antibodies against swine influenza virus. 2 doses of vaccine are injected by the intended route into each of not fewer than 10 animals. After 14 days, 1 dose of vaccine is injected into each of the animals. The animals are observed for a further 14 days. During the 28 days of the test, no abnormal local or systemic reaction is produced. B. The animals used in the test for immunogenicity are also used to evaluate safety. The rectal temperature of each vaccinated animal is measured at the time of vaccination and 24 h and 48 h later. No abnormal effect on rectal temperature is noted nor other systemic reactions (for example, anorexia). The injection site is examined for local reactions at slaughter. No abnormal local reaction occurs. C. The animals used for field trials are also used to evaluate safety. A test is carried out in each category of animals for which the vaccine is intended (sows, fattening pigs). Not fewer than 3 groups each of not fewer than 20 animals in at least 2 locations are used with corresponding groups of not fewer than 10 controls. The rectal temperature of each animal is measured at the time of vaccination and 24 h and 48 h later. No abnormal effect on rectal temperature is noted. The injection site is examined for local reactions at slaughter. No abnormal local reaction occurs.

IDENTIFICATION When injected into healthy, susceptible animals, the vaccine stimulates the production of specific antibodies against the influenza virus subtypes included in the vaccine. The antibodies may be detected by a suitable immunochemical method (2.7.1). TESTS Safety. Use 2 pigs, free from antibodies against swine influenza virus and not older than the minimum age stated for vaccination. Inject into each pig a double dose of vaccine by the route stated on the label. Observe the animals for 14 days and then inject into each animal 1 dose of vaccine. Observe the animals for 14 days. No abnormal local or systemic reaction occurs during the 28 days of the test. Inactivation. If the vaccine has been prepared in eggs, inoculate 0.2 ml into the allantoic cavity of each of 10 fertilised hen eggs, 9 to 11 days old. Incubate at a suitable temperature for 3 days. The death of any embryo within 24 h of inoculation is considered as non-specific mortality and the egg is discarded. The test is not valid unless at least 80 per cent of the eggs survive. Collect the allantoic fluid of each egg, pool equal quantities and carry out a second passage on fertilised eggs in the same manner. Incubate for 4 days ; the allantoic fluid of these eggs shows no haemagglutinating activity.

If the vaccine has been prepared in cell cultures, carry out a suitable test for residual infectious influenza virus using 2 passages in the same type of cell culture as used in the production of vaccine. No live virus is detected. If the vaccine contains an oily adjuvant that interferes with this test, where possible separate the aqueous phase from the vaccine by means that do not diminish the capacity to detect residual infectious influenza virus. Extraneous viruses. On the pigs used for the safety test, Immunogenicity. The test described under Potency carried carry out tests for antibodies. The vaccine does not stimulate the formation of antibodies other than those against out using an epidemiologically relevant challenge strain or strains is suitable to demonstrate the immunogenicity of the influenza virus. In particular, no antibodies against viruses pathogenic for pigs or against viruses which could interfere vaccine. with the diagnosis of infectious diseases of pigs (including IN-PROCESS TESTS viruses of the pestivirus group) are detected. For vaccines produced in eggs, the content of bacterial Sterility. The vaccine complies with the test for sterility endotoxins is determined on the virus harvest to monitor prescribed in the monograph on Vaccines for veterinary production. use (0062). BATCH POTENCY TEST The test described below under Potency is not carried out POTENCY for routine testing of batches of vaccine. It is carried out, for a given vaccine, on one or more occasions, as decided Carry out a potency test for each subtype used in the preparation of the vaccine. Use not fewer than 20 pigs of by or with the agreement of the competent authority ; the minimum age recommended for vaccination and that do where the test is not carried out, a suitable validated test not have antibodies against swine influenza virus. Vaccinate is carried out, the criteria for acceptance being set with not fewer than 10 pigs as recommended on the label and reference to a batch of vaccine that has given satisfactory results in the test described under Potency. The following keep not fewer than 10 pigs as unvaccinated controls. Take a blood sample from all control pigs immediately before test may be used after a satisfactory correlation with the challenge. 3 weeks after the last administration of vaccine, test described under Potency has been established by a challenge all the pigs with a suitable quantity of a virulent statistical evaluation. influenza field virus by the intratracheal route. Kill half of the vaccinated and control pigs 24 h after challenge and the Inject subcutaneously into each of 5 seronegative other half 72 h after challenge. For each pig, measure the guinea-pigs, 5 to 7 weeks old, a quarter of the dose stated quantity of influenza virus in 2 lung tissue homogenates, on the label. Collect blood samples before the vaccination one from the left apical, cardiac and diaphragmatic lobes, and 21 days after vaccination. Determine for each sample and the other from the corresponding right lung lobes. Take the level of specific antibodies against each virus subtype equivalent samples from each animal. The test is invalid if in the vaccine by haemagglutination-inhibition or another suitable test. The vaccine complies with the test if the level antibodies against influenza virus are found in any control pig immediately before challenge. The vaccine complies with of antibodies is not lower than that found for a batch of the test if, at both times of measurement, the mean virus vaccine that gave satisfactory results in the potency test in titre in the pooled lung tissue samples of vaccinated pigs is pigs (see Potency). 786


Porcine parvovirosis vaccine (inactivated)


significantly lower than that for control pigs, when analysed by a suitable statistical method such as the Wilcoxon Mann-Whitney test. 01/2005:0965

PORCINE PARVOVIROSIS VACCINE (INACTIVATED) Vaccinum parvovirosis inactivatum ad suem DEFINITION Inactivated porcine parvovirosis vaccine consists of a suspension of inactivated porcine parvovirus or of a noninfectious fraction of the virus. PRODUCTION The virus is grown in suitable cell cultures (5.2.4). The viral suspension is harvested ; the virus is inactivated by a method that avoids destruction of the immunogenicity and may be fragmented (inactivation may be by fragmentation) ; the virus or viral fragments may be purified and concentrated at a suitable stage of the process. A test for residual infectious porcine parvovirus is carried out on each batch of antigen immediately after inactivation and on the final bulk or, if the vaccine contains an adjuvant, on the bulk antigen or the mixture of bulk antigens immediately before the addition of adjuvant. The quantity used in the test is equivalent to not less than 100 doses of the vaccine. The bulk harvest is inoculated into suitable non-confluent cells ; after incubation for 7 days, a subculture is made using trypsinised cells. After incubation for a further 7 days, the cultures are examined for residual live parvovirus by an immunfluorescence test. No live virus is detected. The vaccine may contain one or more suitable adjuvants. CHOICE OF VACCINE COMPOSITION The vaccine is shown to be satisfactory with respect to safety (including absence of adverse effects on fertility, gestation, farrowing or offspring) and immunogenicity in pigs. The following tests may be used during demonstration of safety (5.2.6) and efficacy (5.2.7). Safety A. A test is carried out in each category of animals for which the vaccine is intended and by each of the recommended routes. The animals used do not have antibodies against porcine parvovirus or against a fraction of the virus. Two doses of vaccine are injected by the intended route into each of not fewer than ten animals. After 14 days, one dose of vaccine is injected into each of the animals. The animals are observed for a further 14 days. During the 28 days of the test, no abnormal local or systemic reaction occurs. If the vaccine is intended for use in pregnant sows, for the test in this category of animal, the observation period is prolonged up to farrowing and any effects on gestation or the offspring are noted. B. The animals used in the test for immunogenicity are also used to evaluate safety. The rectal temperature of each vaccinated animal is measured at the time of vaccination and 24 h and 48 h later. No abnormal effect on body temperature is noted nor other systemic reactions (for example, anorexia). The injection site is examined for local reactions after vaccination and at slaughter. No abnormal local reaction occurs. C. The animals used for field trials are also used to evaluate safety. A test is carried out in each category of animals for which the vaccine is intended (sows, gilts). Not fewer General Notices (1) apply to all monographs and other texts

than three groups each of not fewer than twenty animals are used with corresponding groups of not fewer than ten controls. The rectal temperature of each animal is measured at the time of vaccination and 24 h and 48 h later. No abnormal effect on body temperature is noted. The injection site is examined for local reactions after vaccination and at slaughter. No abnormal local reaction occurs. Immunogenicity. The test described under Potency may be used to demonstrate the immunogenicity of the vaccine. BATCH POTENCY TEST The test described below is not carried out for routine testing of batches of vaccine. It is carried out, for a given vaccine, on one or more occasions, as decided by or with the agreement of the competent authority ; where the test is not carried out,a suitable validated test is carried out, the criteria for acceptance being set with reference to a batch of vaccine that has given satisfactory results in the test described underPotency. The following test may be used after a satisfactory correlation with the test described under Potency has been established by a statistical evaluation. Vaccinate subcutaneously not fewer than five guinea-pigs, 5 to 7 weeks old, according to the vaccination scheme stated on the label using one-fourth of the prescribed dose volume. Take blood samples after the period corresponding to maximum antibody production and carry out tests on the serum for specific antibodies by a haemagglutination-inhibition test or other suitable test. The antibody titres are not less than those obtained with a batch of vaccine shown to be satisfactory in the test in pigs (see Potency). IDENTIFICATION The vaccine stimulates the formation of specific antibodies against porcine parvovirus or the fraction of the virus used in the production of the vaccine when injected into susceptible animals on one or, if necessary, more than one occasion. TESTS Safety. Use two pigs 6 weeks to 6 months old and having no antibodies against porcine parvovirus virus or against a fraction of the virus. Inject into each animal by one of the routes stated on the label a double dose of vaccine. Observe the animals for 14 days and then inject a single dose of vaccine into each pig. Observe the animals for a further 14 days. No abnormal local or systemic reaction occurs during the 28 days of the test. Inactivation. This test may be omitted by the manufacturer if a test for inactivation has been carried out on the bulk vaccine, immediately before the addition of the adjuvant, where applicable. Use a quantity of vaccine equivalent to ten doses. If the vaccine contains an oily adjuvant, break the emulsion and separate the phases. If the vaccine contains a mineral adjuvant, carry out an elution to liberate the virus. Concentrate the viral suspension 100 times by ultrafiltration or ultracentrifugation. None of the above procedures must be such as to inactivate or otherwise interfere with detection of live virus. Carry out a test for residual live virus in suitable non-confluent cells ; after incubation for 7 days, make a subculture using trypsinised cells. After incubation for a further 7 days, examine the cultures for residual live parvovirus by an immunfluorescence test. No live virus is detected. Extraneous viruses. On the pigs used for the safety test carry out tests for antibodies. The vaccine does not stimulate the formation of antibodies - other than those against porcine parvovirus - against viruses pathogenic for pigs or 787

Porcine progressive atrophic rhinitis vaccine (inactivated)

against viruses which could interfere with the diagnosis of infectious diseases in pigs (including the viruses of the pestivirus group). Sterility. The vaccine complies with the test for sterility prescribed in the monograph on Vaccines for veterinary use (0062). POTENCY Vaccinate according to the recommended schedule not less than seven gilts, 5 to 6 months old, which do not have antibodies against porcine parvovirus or against a fraction of the virus. The interval between vaccination and service is that stated on the label. Mate the gilts on two consecutive days immediately following signs of oestrus. Keep not less than five unvaccinated mated gilts of the same age as controls. At about the 40th day of gestation, challenge all gilts using a suitable strain of porcine parvovirus. Slaughter the gilts at about the 90th day of gestation and examine their foetuses for infection with porcine parvovirus as demonstrated by the presence of either virus or antibodies. The vaccine complies with the test if not fewer than 80 per cent of the total number of piglets from vaccinated gilts are protected from infection. The test is not valid unless : not fewer than seven vaccinated gilts and five control gilts are challenged ; not fewer than 90 per cent of piglets from the control gilts are infected ; and the average number of piglets per litter for the vaccinated gilts is not less than six. 01/2005:1361

PORCINE PROGRESSIVE ATROPHIC RHINITIS VACCINE (INACTIVATED) Vaccinum rhinitidis atrophicantis ingravescentis suillae inactivatum DEFINITION Porcine progressive atrophic rhinitis vaccine (inactivated) is a preparation containing either the dermonecrotic exotoxin of Pasteurella multocida, treated to render it harmless while maintaining adequate immunogenic activity, or a genetically modified form of the exotoxin which has adequate immunogenic activity and which is free from toxic properties ; the vaccine may also contain cells and/or antigenic components of one or more suitable strains of P. multocida and/or Bordetella bronchiseptica. This monograph applies to vaccines administered to sows and gilts for protection of their progeny. PRODUCTION The bacterial strains used for production are cultured separately in suitable media. The toxins and/or cells are treated to render them safe. Detoxification. A test for detoxification of the dermonecrotic exotoxin of P. multocida is carried out immediately after detoxification. The concentration of detoxified exotoxin used in the test is not less than that in the vaccine. The suspension complies with the test if no toxic dermonecrotic exotoxin is detected. The test for detoxification is not required where the vaccine is prepared using a toxin-like protein free from toxic properties, produced by expression of a modified form of the corresponding gene. Antigen content. The content of the dermonecrotic exotoxin of P. multocida in the detoxified suspension or the toxin-like protein in the harvest is determined by a suitable immunochemical method (2.7.1), such as an enzyme-linked 788


immunosorbent assay and the value found is used in the formulation of the vaccine. The content of other antigens stated on the label is also determined (2.7.1). The vaccine may contain a suitable adjuvant. CHOICE OF VACCINE COMPOSITION The strains used for the preparation of the vaccine are shown to be satisfactory with respect to the production of the dermonecrotic exotoxin and the other antigens claimed to be protective. The vaccine is shown to be satisfactory with respect to safety (5.2.6) and efficacy (5.2.7). Production of antigens. The production of antigens claimed to be protective is verified by a suitable bioassay or immunochemical method (2.7.1), carried out on the antigens obtained from each of the vaccine strains under the conditions used for the production of the vaccine. The following tests may be used during demonstration of safety and immunogenicity. Safety A. Carry out the test for each route of administration stated on the label. Use pigs that do not have antibodies against the components of the vaccine, that are from a herd or herds where there are no signs of atrophic rhinitis and that have not been vaccinated against atrophic rhinitis. If the vaccine is intended for use in pregnant animals, carry out the test in pregnant sows or gilts, vaccinating them at the recommended stage of pregnancy. Administer a double dose of vaccine by a recommended route to each of not fewer than 10 sows or gilts. Administer a single dose of vaccine to each of the animals after the recommended interval. Observe the pigs until farrowing. Record body temperature the day before vaccination, at vaccination, 2 h, 4 h and 6 h later and then daily for 4 days ; note the maximum temperature increase for each animal. Note any effects on gestation and the offspring. No abnormal local or systemic reaction occurs ; the average temperature increase for all animals does not exceed 1.5 °C and no animal shows a rise greater than 2 °C. B. The animals used for field trials are also used to evaluate safety. Use not fewer than 3 groups each of not fewer than 20 animals with corresponding groups of not fewer than 10 controls. Examine the injection site for local reactions after vaccination. Record body temperature the day before vaccination, at vaccination, at the time interval after which a rise in temperature, if any, was seen in test A, and daily during the 2 days following vaccination ; note the maximum temperature increase for each animal. No abnormal local or systemic reaction occurs ; the average temperature increase for all animals does not exceed 1.5 °C and no animal shows a rise greater than 2 °C. Immunogenicity. The test described under Potency is suitable for demonstration of immunogenicity. BATCH TESTING Batch potency test. The test described under Potency is not carried out for routine testing of batches of vaccine. It is carried out, for a given vaccine, on one or more occasions, as decided by or with the agreement of the competent authority ; where the test is not carried out, a suitable validated test is carried out, the criteria for acceptance being set with reference to a batch of vaccine that has given satisfactory results in the test described under Potency. The following test may be used after a satisfactory correlation with the test described under Potency has been established. Use not fewer than 5 pigs not less than 3 weeks old and that do not have antibodies against the components of the vaccine. Vaccinate each pig by a recommended route and according to the recommended schedule. Maintain not fewer



than 2 pigs of the same origin as unvaccinated controls under the same conditions. Alternatively, if the nature of the antigens allows reproducible results to be obtained, a test in susceptible laboratory animals may be carried out. To obtain a valid assay, it may be necessary to carry out a test using several groups of animals, each receiving a different quantity of vaccine. For each quantity of vaccine, carry out the test as follows : vaccinate not fewer than 5 animals with a suitable quantity of vaccine. Maintain not fewer than 2 animals of the same species and origin as unvaccinated controls. Where the schedule stated on the label requires a booster injection to be given, a booster vaccination may also be given in this test provided it has been demonstrated that this will still provide a suitably sensitive test system. At a given interval within the range of 14 to 21 days after the last administration, collect blood from each animal and prepare serum samples. Use a validated test such as an enzyme-linked immunosorbent assay to measure the antibody response to each of the antigens stated on the label. The test is not valid and must be repeated if there is a significant antibody titre in the controls. The vaccine complies with the test if the antibody responses of the vaccinated animals are not significantly less than those obtained with a batch of vaccine that has given satisfactory results in the test or tests (as applicable) described under Potency. Where animals seronegative for the antigens stated on the label are not available, seropositive animals may be used in the above test. During the development of a test with seropositive animals, particular care will be required during the validation of the test system to establish that the test is suitably sensitive and to specify acceptable pass, fail and retest criteria. It will be necessary to take into account the range of prevaccination antibody titres and to establish the acceptable minimum antibody titre rise after vaccination in relation to these. Bacterial endotoxins. A test for bacterial endotoxins (2.6.14) is carried out on the batch or, where the nature of the adjuvant prevents performance of a satisfactory test, on the bulk antigen or the mixture of bulk antigens immediately before addition of the adjuvant. The maximum acceptable amount of bacterial endotoxins is that found for a batch of vaccine shown satisfactory in safety test A given under Choice of vaccine composition or in the safety test described under Tests, carried out using 10 pigs. Where the latter test is used, note the maximum temperature increase for each animal ; the average temperature increase for all animals does not exceed 1.5 °C. The method chosen for determining the amount of bacterial endotoxin present in the vaccine batch used in the safety test for determining the maximum acceptable level of endotoxin is used subsequently for testing of each batch.

Porcine progressive atrophic rhinitis vaccine (inactivated)

Sterility (2.6.1). The vaccine complies with the test for sterility prescribed in the monograph on Vaccines for veterinary use (0062). POTENCY Use pigs that do not have antibodies against the components of the vaccine, that are from a herd or herds where there are no signs of atrophic rhinitis and that have not been vaccinated against atrophic rhinitis. A. Vaccines containing dermonecrotic exotoxin of P. multocida (with or without cells of P. multocida). Use not fewer than 12 breeder pigs. Vaccinate not fewer than 6 randomly chosen pigs at the stage of pregnancy or non-pregnancy and by the route and schedule stated on the label. Maintain not fewer than 6 of the remaining pigs as unvaccinated controls under the same conditions. From birth allow all the piglets from the vaccinated and unvaccinated breeder pigs to feed from their own dam. Constitute from the progeny 2 challenge groups each of not fewer than 30 piglets chosen randomly, taking not fewer than 3 piglets from each litter. On the 2 consecutive days preceding challenge, the mucosa of the nasal cavity of the piglets may be treated by instillation of 0.5 ml of a solution of acetic acid (10 g/l C2H4O2) in isotonic buffered saline pH 7.2. Challenge each piglet at 10 days of age by the intranasal route with a sufficient quantity of a toxigenic strain of P. multocida. At the age of 42 days, kill the piglets of the 2 groups and dissect the nose of each of them transversally at premolar-1. Examine the ventral and dorsal turbinates and the nasal septum for evidence of atrophy or distortion and grade the observations on the following scales : Turbinates 0

no atrophy

1

slight atrophy

2

moderate atrophy

3

severe atrophy

4

very severe atrophy with almost complete disappearance of the turbinate

The maximum score is 4 for each turbinate and 16 for the sum of the 2 dorsal and 2 ventral turbinates. Nasal septum 0

no deviation

1

very slight deviation

2

deviation of the septum

The maximum total score for the turbinates and the nasal septum is 18. The test is not valid and must be repeated if fewer than 80 per cent of the progeny of each litter of the unvaccinated breeder pigs have a total score of at least 10. The vaccine complies with the test if a significant reduction in the total score has been demonstrated in the TESTS group from the vaccinated breeder pigs compared to that Safety. Use not fewer than 2 pigs that do not have antibodies from the unvaccinated breeder pigs. against P. multocida and that preferably do not have B. Vaccines containing P. multocida dermonecrotic exotoxin antibodies against B. bronchiseptica. Administer to each pig (with or without cells of P. multocida) and cells and/or a double dose of vaccine by a recommended route. Observe antigenic components of B. bronchiseptica. the pigs for 14 days. Record body temperature the day before Use not fewer than 24 breeder pigs. Vaccinate not fewer vaccination, at vaccination, 2 h, 4 h and 6 h later and then than 12 randomly chosen pigs at the stage of pregnancy daily for 2 days. No abnormal local or systemic reaction or non-pregnancy and by the route and schedule stated occurs ; a transient temperature increase not exceeding 2 °C on the label. Maintain not fewer than 12 of the remaining may occur. IDENTIFICATION In animals free from specific antibodies against the antigens stated on the label, the vaccine stimulates the production of antibodies against these antigens.


789

Rabies vaccine (inactivated) for veterinary use


pigs as unvaccinated controls under the same conditions. From birth allow all the piglets from the vaccinated and unvaccinated breeder pigs to feed from their own dam. Using groups of not fewer than 6 pigs, constitute from their progeny 2 challenge groups from vaccinated pigs and 2 groups from unvaccinated pigs each group consisting of not fewer than 30 piglets chosen randomly, taking not fewer than 3 piglets from each litter. On the 2 consecutive days preceding challenge, the mucosa of the nasal cavity of the piglets may be treated by instillation of 0.5 ml of a solution of acetic acid (10 g/l C2H4O2) in isotonic buffered saline pH 7.2. For a group of piglets from not fewer than 6 vaccinated pigs and a group from not fewer than 6 controls, challenge each piglet by the intranasal route at 10 days of age with a sufficient quantity of a toxigenic strain of P. multocida. For the other group of piglets from not fewer than 6 vaccinated pigs and the other group from not fewer than 6 controls, challenge each piglet at 7 days of age by the intranasal route with a sufficient quantity of B. bronchiseptica. In addition, challenge each piglet at 10 days of age by the intranasal route with a sufficient quantity of a toxigenic strain of P. multocida. At the age of 42 days, kill the piglets of the 4 groups and dissect the nose of each of them transversally at premolar-1. Examine the ventral and dorsal turbinates and the nasal septum for evidence of atrophy or distortion and grade the observations on the scale described above. The test is not valid and must be repeated if fewer than 80 per cent of the progeny of each litter of the unvaccinated breeder pigs have a total score of at least 10. The vaccine complies with the test if a significant reduction in the total score has been demonstrated in the groups from the vaccinated breeder pigs compared to the corresponding group from the unvaccinated breeder pigs. LABELLING The label states the protective antigens present in the vaccine. 01/2005:0451

RABIES VACCINE (INACTIVATED) FOR VETERINARY USE Vaccinum rabiei inactivatum ad usum veterinarium DEFINITION Rabies vaccine (inactivated) for veterinary use is a liquid or freeze-dried preparation of fixed rabies virus inactivated by a suitable method. PRODUCTION The vaccine is prepared from virus grown either in suitable cell lines or in primary cell cultures from healthy animals (5.2.4). The virus suspension is harvested on one or more occasions within 28 days of inoculation. Multiple harvests from a single production cell culture may be pooled and considered as a single harvest. The rabies virus is inactivated by a suitable method. Inactivation. The test for residual live rabies virus is carried out by inoculation of the inactivated virus into the same type of cell culture as that used in the production of the vaccine or a cell culture shown to be at least as sensitive ; the quantity of inactivated virus used in the test is equivalent to not less than 25 doses of the vaccine. After incubation for 790

4 days, a subculture is made using trypsinised cells ; after incubation for a further 4 days, the cultures are examined for residual live rabies virus by an immunofluorescence test. No live virus is detected. Antigen content. The content of rabies virus glycoprotein is determined by a suitable immunochemical method (2.7.1). The content is within the limits approved for the particular preparation. The vaccine may contain one or more adjuvants. CHOICE OF VACCINE COMPOSITION The vaccine is shown to be satisfactory with respect to immunogenicity for each species for which it is recommended. The suitability of the vaccine with respect to immunogenicity for carnivores (cats and dogs) is demonstrated by direct challenge. For other species, if a challenge test has been carried out for the vaccine in cats or dogs, an indirect test is carried out by determining the antibody level following vaccination of not fewer than twenty animals according to the recommended schedule ; the vaccine is satisfactory if, after the period claimed for protection, the mean rabies virus antibody level in the serum of the animals is not less than 0.5 IU/ml and if not more than 10 per cent of the animals have an antibody level less than 0.1 IU/ml. The test described below may be used to demonstrate immunogenicity in cats and dogs. Immunogenicity. Use not fewer than 35 susceptible animals of the minimum age recommended for vaccination. Take a blood sample from each animal and test individually for antibodies against rabies virus to determine susceptibility. Administer by the recommended route to each of not fewer than 25 animals one dose of vaccine. Keep not fewer than 10 animals as controls. Observe all the animals for a period equal to the claimed duration of immunity. No animal shows signs of rabies. On the last day of the claimed period for duration of immunity or later, challenge all animals by intramuscular injection of virulent rabies virus of a strain approved by the competent authority. Observe the animals for 90 days. Animals that die from causes not attributable to rabies are eliminated. The test is not valid if the number of such deaths reduces the number of vaccinated animals in the test to fewer than 25. The test is not valid unless at least eight control animals (or a statistically equivalent number if more than 10 control animals are challenged) show signs of rabies and the presence of rabies virus in their brain is demonstrated by the fluorescent-antibody test or some other suitable method. The vaccine complies with the test if not more than 2 of the 25 vaccinated animals (or a statistically equivalent number if more than 25 vaccinated animals are challenged) show signs of rabies. BATCH TESTING The test described under Potency is not necessarily carried out for routine testing of batches of vaccine. It is carried out, for a given vaccine, on one or more occasions, as decided by or with the agreement of the competent authority ; where the test is not carried out, a suitable validated alternative method is used, the criteria for acceptance being set with reference to a batch of vaccine that has given satisfactory results in the test described above for immunogenicity or in the test described under Potency. The following test may be used after a suitable correlation with the test described above for immunogenicity or the test described under Potency has been established. Batch potency test. Use 5 mice each weighing 18 g to 20 g. Vaccinate each mouse subcutaneously or intramuscularly using one-fifth of the recommended dose volume. Take blood samples 14 days after the injection and test the sera individually for rabies antibody using the rapid



Rabies vaccine (inactivated) for veterinary use

out a simplified test using one dilution of the vaccine to be examined. Such a test enables the analyst to determine that the vaccine has a potency significantly higher than the required minimum but will not give full information on the validity of each individual potency determination. It allows a considerable reduction in the number of animals required for the test and should be considered by each laboratory in accordance with the provisions of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes. Selection and distribution of the test animals. Use in the IDENTIFICATION test healthy female mice about 4 weeks old and from the same stock. Distribute the mice into at least 10 groups of When injected into animals, the vaccine stimulates the not fewer than 10 mice. production of specific neutralising antibodies. Preparation of the challenge suspension. Inoculate a TESTS group of mice intracerebrally with the CVS strain of rabies Safety. If the vaccine is intended for more than one species virus and when the mice show signs of rabies, but before including one belonging to the order of Carnivora, carry out they die, kill the mice and remove the brains and prepare the test in dogs. Otherwise use one of the species for which a homogenate of the brain tissue in a suitable diluent. Separate gross particulate matter by centrifugation and use the vaccine is intended. Administer, by a recommended route, a double dose of vaccine to each of 2 animals having the supernatant liquid as challenge suspension. Distribute the suspension in small volumes in ampoules, seal and no antibodies against rabies virus. Observe the animals for store at a temperature below − 60 °C. Thaw one ampoule 14 days. No abnormal local or systemic reaction occurs. of the suspension and make serial dilutions in a suitable Inactivation. Carry out the test using a pool of the contents diluent. Allocate each dilution to a group of mice and inject of 5 containers. intracerebrally into each mouse 0.03 ml of the dilution For vaccines which do not contain an adjuvant, carry out allocated to its group. Observe the animals for 14 days and a suitable amplification test for residual infectious rabies record the number in each group that, between the fifth and virus using the same type of cell culture as that used in the the fourteenth days, develop signs of rabies. Calculate the production of the vaccine or a cell culture shown to be at ID50 of the undiluted suspension. least as sensitive. No live virus is detected. Determination of potency of the vaccine to be examined. For vaccines that contain an adjuvant, inject intracerebrally Prepare at least three serial dilutions of the vaccine to into each of not fewer than 10 mice each weighing 11 g be examined and three similar dilutions of the reference to 15 g 0.03 ml of a pool of at least 5 times the smallest preparation. Prepare the dilutions such that those stated dose. To avoid interference from any antimicrobial containing the largest quantity of vaccine may be expected preservative or the adjuvant, the vaccine may be diluted not to protect more than 50 per cent of the animals into more than 10 times before injection. In this case or if the which they are injected and those containing the smallest vaccine strain is pathogenic only for unweaned mice, carry quantities of vaccine may be expected to protect less than out the test on mice 1 to 4 days old. Observe the animals 50 per cent of the animals into which they are injected. for 21 days. If more than 2 animals die during the first Allocate each dilution to a different group of mice and inject 48 h, repeat the test. From the third to the twenty-first days intraperitoneally into each mouse 0.5 ml of the dilution following the injection, the animals show no signs of rabies allocated to its group. 14 days after the injection prepare a and immunofluorescence tests carried out on the brains of suspension of the challenge virus such that, on the basis the animals show no indication of the presence of rabies of the preliminary titration, it contains about 50 ID50 in virus. each 0.03 ml. Inject intracerebrally into each vaccinated mouse 0.03 ml of this suspension. Prepare 3 suitable serial Sterility. The vaccine complies with the test for sterility dilutions of the challenge suspension. Allocate the challenge prescribed in the monograph on Vaccines for veterinary suspension and the 3 dilutions one to each of 4 groups of use (0062). 10 unvaccinated mice and inject intracerebrally into each mouse 0.03 ml of the suspension or one of the dilutions POTENCY allocated to its group. Observe the animals in each group The potency of rabies vaccine is determined by comparing for 14 days. The test is not valid if more than 2 mice of any the dose necessary to protect mice against the clinical effects group die within the first 4 days after challenge. Record of the dose of rabies virus defined below, administered the numbers in each group that show signs of rabies in the intracerebrally, with the quantity of a reference preparation, period 5 days to 14 days after challenge. calibrated in International Units, necessary to provide the The test is not valid unless : same protection. The International Unit is the activity of a stated quantity of — for both the vaccine to be examined and the reference preparation the 50 per cent protective dose lies between the International Standard. The equivalence in International the smallest and the largest dose given to the mice, Units of the International Standard is stated by the World Health Organisation. — the titration of the challenge suspension shows that 0.03 ml of the suspension contained at least 10 ID50, Rabies vaccine (inactivated) for veterinary use BRP is calibrated in International Units against the International — the confidence limits (P = 0.95) are not less than 25 per Standard. cent and not more than 400 per cent of the estimated potency, The test described below uses a parallel-line model with at least 3 points for the vaccine to be examined and the — the statistical analysis shows a significant slope and no reference preparation. Once the analyst has experience significant deviations from linearity or parallelism of the with the method for a given vaccine, it is possible to carry dose-response lines. fluorescent focus inhibition test described for Human rabies immunoglobulin (0723). The amount of antibody is not less than that produced by a vaccine that has been found satisfactory with respect to immunogenicity as described above or in the test described under Potency. Antigen content. The quantity of rabies virus glycoprotein per dose, determined by a suitable immunochemical method (2.7.1), is not significantly lower than that of a batch that has been found satisfactory with respect to immunogenicity as described above or in the test described under Potency.


791

Rabies vaccine (live, oral) for foxes


The vaccine complies with the test if the estimated potency is not less than 1 IU in the smallest prescribed dose. LABELLING The label states : — the type of cell culture used to prepare the vaccine and the species of origin, — the minimum number of International Units per dose, — the minimum period for which the vaccine provides protection.

BATCH TESTING If the test for potency has been carried out with satisfactory results on a representative batch of vaccine, this test may be omitted as a routine control on other batches of vaccine prepared from the same seed lot. IDENTIFICATION A. When mixed with a monospecific rabies antiserum, the vaccine is no longer able to infect susceptible cell cultures into which it is inoculated. B. A test is carried out to demonstrate the presence of the genetic marker.

01/2005:0746

RABIES VACCINE (LIVE, ORAL) FOR FOXES

TESTS Extraneous viruses (a) Mix the vaccine with a specific neutralising rabies virus antiserum. It no longer provokes cytopathic effects in susceptible cell cultures. It shows no evidence of haemagglutinating or haemadsorbing agents.

(b) Inoculate 1 in 10 and 1 in 1000 dilutions of the vaccine into susceptible cell cultures. Incubate at 37 °C. After 2, Vaccinum rabiei perorale vivum ad vulpem 4 and 6 days, stain the cells with a panel of monoclonal antibodies that do not react with the vaccine strain but that react with other strains of rabies virus (for example, street DEFINITION virus, Pasteur strain). The vaccine shows no evidence of contaminating rabies virus. Rabies vaccine (live, oral) for foxes is a preparation of an immunogenic strain of an attenuated rabies virus. The Bacterial and fungal contamination. The vaccine complies vaccine is incorporated in a bait in such a manner as to with the test for sterility prescribed in the monograph on enable the tests prescribed below to be performed aseptically. Vaccines for veterinary use (0062). Mycoplasmas (2.6.7). The vaccine complies with the test PRODUCTION for mycoplasmas. The attenuated virus strain is grown in suitable cell cultures Virus titre. Titrate the vaccine in suitable cell cultures. (5.2.4) ; if the cell cultures are of mammalian origin, they are One dose of the vaccine contains not less than the quantity shown to be free from rabies virus. The virus suspension of virus equivalent to the minimum titre stated on the label. is harvested on one or more occasions within 14 days of inoculation. Multiple harvests from a single cell lot may POTENCY be pooled and considered as a single harvest. The viral Use not fewer than thirty-five foxes, at least three months old, suspension may be mixed with a suitable stabiliser. The vaccine may be freeze-dried or liquid. A freeze-dried vaccine free from rabies-neutralising antibodies. Administer orally and with the bait stated on the label to each of not fewer has to be reconstituted before use. than twenty-five animals a volume of the vaccine containing CHOICE OF VACCINE STRAIN a quantity of virus equivalent to the minimum titre stated Only a virus strain shown to be satisfactory with respect on the label. Keep not fewer than ten animals as controls. to immunogenicity (see Potency) and the following Observe all the animals for 180 days. No animal shows characteristics may be used in the preparation of the vaccine : signs of rabies. The test is not valid if fewer than twenty-five — when administered orally at the dose and by the method vaccinated animals survive after this observation period. On the 180th day after vaccination, challenge all foxes by the recommended for use to forty foxes, it causes no sign of intramuscular injection of virulent rabies virus of a strain rabies within 180 days of administration, approved by the competent authority. Observe the animals — when administered orally at ten times the for 90 days. Animals that die from causes not attributable recommended dose to each of ten foxes, it causes to rabies are eliminated. The test is not valid if the number no sign of rabies within 180 days of administration, of such deaths reduces the number of vaccinated animals in the test to fewer than twenty-five. The vaccine complies — when administered orally at ten times the with the test if not more than two of twenty-five vaccinated recommended dose to each of ten dogs, it causes animals (or a statistically equivalent number if more than no sign of rabies within 180 days of administration, twenty-five vaccinated animals are challenged) show signs — when administered orally at ten times the of rabies. The test is not valid unless at least nine control recommended dose to each of ten cats, it causes animals (or a statistically equivalent number if more than ten no sign of rabies within 180 days of administration, control animals are challenged) show signs of rabies and the presence of rabies virus in their brain is demonstrated by the — in natural and experimental conditions, the virus strain fluorescent-antibody test or some other reliable method. does not spread from one animal to another in wild rodents, LABELLING — the virus strain has one or more stable genetic markers that may be used to discriminate the vaccine strain from other rabies virus strains.

792

The label states the nature of the genetic marker of the virus strain.


Swine-fever vaccine (live), classical, freeze-dried


01/2005:0064 Sterility. It complies with the test for sterility prescribed in corrected the monograph on Vaccines for veterinary use (0062). POTENCY If the vaccine contains more than 1 serotype, a test for 2 serotypes may be carried out on a single group by injecting each challenge serotype on different flanks of the Vaccinum erysipelatis suillae inactivatum animals. Validation and acceptance criteria are applied separately to the respective injection sites. If the vaccine DEFINITION contains more than 1 serotype, the potency test may also Swine erysipelas vaccine (inactivated) is a preparation of one be carried out using a separate group for each serotype. or more suitable strains of Erysipelothrix rhusiopathiae Use not fewer than 15 pigs not less than 12 weeks old, (E. insidiosa) inactivated by a suitable method. This weighing not less than 20 kg and that do not have antibodies monograph applies to vaccines intended to protect pigs against swine erysipelas. Divide the animals into 2 groups. against swine erysipelas. Vaccinate a group of not fewer than 10 pigs according to the recommended schedule. Maintain a group of not fewer than PRODUCTION 5 pigs as unvaccinated controls. 3 weeks after vaccination, The vaccine may contain an adjuvant. challenge the vaccinated animals and the control group by separate intradermal injections of 0.1 ml of a virulent strain CHOICE OF VACCINE COMPOSITION The vaccine is shown to be satisfactory with respect to safety of each of serotype 1 and serotype 2 of E. rhusiopathiae. Observe the animals for 7 days. The vaccine complies with (5.2.6) and efficacy (5.2.7). the test if not fewer than 90 per cent of the vaccinated Immunogenicity. The test described under Potency is animals remain free from diamond skin lesions at the suitable to demonstrate immunogenicity of the vaccine with injection site. The test is invalid if fewer than 80 per cent of respect to E. rhusiopathiae serotypes 1 and 2. If claims control animals show typical signs of disease, i.e. diamond are made concerning another serotype, then a further test skin lesions at the injection sites. to demonstrate immunogenicity against this serotype is Swine erysipelas bacteria serotype 1 BRP and swine necessary. erysipelas bacteria serotype 2 BRP are suitable for use as BATCH TESTING challenge strains. Batch potency test. The test described under Potency is not LABELLING carried out for routine testing of batches of vaccine. It is carried out, for a given vaccine, on one or more occasions, as The label states the serotypes of E. rhusiopathiae included decided by or with the agreement of the competent authority. in the vaccine. Where the test is not carried out, a suitable validated alternative test is carried out, the criteria for acceptance 01/2005:0065 being set with reference to a batch of vaccine that has given satisfactory results in the test described under Potency. The SWINE-FEVER VACCINE (LIVE), following test may be used after a satisfactory correlation CLASSICAL, FREEZE-DRIED with the test described under Potency has been established. Use mice of a suitable strain (for example, NMRI) weighing Vaccinum pestis classicae suillae vivum 17-20 g, from a uniform stock and that do not have antibodies against swine erysipelas. Administer the vaccine cryodesiccatum to be examined to a group of 10 mice. Inject a suitable DEFINITION dose (usually 1/10 of the pig dose) subcutaneously into Freeze-dried classical swine-fever vaccine (live) is a each mouse. At a given interval (for example, 21-28 days), depending on the vaccine to be examined, bleed the animals preparation obtained from a strain of classical swine-fever virus which has lost its pathogenicity for the pig by under anaesthesia. Pool the sera, using an equal volume adaptation either to cell cultures or to the rabbit. from each mouse. Determine the level of antibodies by a suitable immunochemical method (2.7.1), for example, PRODUCTION enzyme-linked immunosorbent assay with erysipelas For vaccine prepared in rabbits, the seed-lot (or the vaccine) ELISA coating antigen BRP. The antibody level is not is made from the homog-enised organs and/or blood of significantly less than that obtained with a batch that has given satisfactory results in the test described under Potency. rabbits from healthy colonies, sacrificed at the peak of the temperature rise following intravenous inoculation of the IDENTIFICATION virus. The vaccine is freeze-dried. Injected into animals that do not have antibodies against CHOICE OF VACCINE STRAIN E. rhusiopathiae, it stimulates the production of such Only a virus strain shown to be satisfactory with respect to antibodies. the following characteristics may be used in the preparation of the vaccine : safety ; non-transmissibility ; irreversibility of TESTS attenuation ; and immunogenic properties. The following Safety. Use pigs of the minimum age recommended for tests may be used during demonstration of safety (5.2.6) and vaccination and preferably having no antibodies against efficacy (5.2.7). swine erysipelas or, where justified, use pigs with a low level The dose of vaccine used throughout the following tests of such antibodies as long as they have not been vaccinated is determined by the manufacturer on the basis of prior against Swine erysipelas and administration of the vaccine experiments. does not cause an anamnestic response. Administer a Tests in pigs double dose of vaccine by a recommended route to each of 2 pigs. Observe the animals for 14 days. No abnormal local Selection of animals. The piglets are 6 to 7 weeks old. or systemic reaction occurs. The sows are primiparae. All animals are healthy and must

SWINE ERYSIPELAS VACCINE (INACTIVATED)


793

Swine-fever vaccine (live), classical, freeze-dried


have had no contact with swine-fever virus and serologically must be free from swine-fever and bovine viral diarrhoea antibodies. They must have a week in which to adapt themselves to the new quarters where the tests are to be carried out.

B. For non-lapinised vaccines prepared in cell cultures, the serum of pigs immunised with the vaccine neutralises the virus used in the preparation of the vaccine.

Safety

Safety. Use three piglets complying with the requirements prescribed for the selection of animals under Choice of Vaccine Strain. Inject intramuscularly into each piglet ten doses of the reconstituted vaccine as a single injection. Observe the animals for 21 days. The temperature curve remains normal and the animals remain in apparent good health and display normal growth.

(a) Each of five piglets receives intramuscularly as a single injection ten doses of vaccine (group a). (b) Five piglets are immunodepressed by the daily injection of 2 mg of prednisolone per kilogram of body mass for five consecutive days and on the third day they receive one dose of vaccine (group b).

TESTS

Extraneous viruses. Mix the vaccine with a monospecific antiserum and inoculate into susceptible cell cultures. No cytopathic effect is produced.

The animals of groups (a) and (b) are observed for 21 days. They must remain in good health. The temperature curve and the weight curve must not differ significantly from those Carry out a haemagglutination test using chicken red blood cells and the supernatant liquid of the cell cultures. The of control animals. test is negative. Carry out a haemadsorption test on the cell cultures. The test is negative. (c) Ten non-immune pregnant sows each receive two doses of vaccine intramuscularly as a single injection between Use ten mice each weighing 11 g to 15 g. Inject intracerebrally the twenty-fifth and thirty-fifth days of gestation. Ten into each mouse 0.03 ml of the vaccine reconstituted so that non-immune pregnant sows of the same age and of the 1 ml contains 1 dose. Observe the animals for 21 days. If same origin receive instead of the two doses of vaccine an more than two mice die within the first 48 h, repeat the test. equal volume of a 9 g/l solution of sodium chloride R. The From the third to the twenty-first day after the injection, the vaccinal virus does not cause abnormalities in the gestation mice show no abnormalities attributable to the vaccine. or in the piglets. Bacterial and fungal contamination. The vaccine to be examined complies with the test for sterility prescribed in Non-transmissibility. Twelve piglets of the same origin are the monograph on Vaccines for veterinary use (0062). kept together. Six are vaccinated in the normal way and the six others are kept as contact controls. After 40 days, Mycoplasmas (2.6.7). The vaccine complies with the test all the pigs are challenged by intramuscular inoculation of for mycoplasmas. a sufficient quantity of the challenge virus (see Potency) to kill an unvaccinated piglet in 7 days. The vaccinated piglets resist challenge whereas the contact piglets must display the POTENCY typical signs of swine fever. The potency is expressed as the number of 50 per cent protective doses (PD50) for pigs contained in the dose Irreversibility of attenuation. Each of two piglets receives one dose of vaccine intramuscularly. Seven days later, 5 ml indicated on the label. The vaccine contains at least 100 PD50 of blood is taken from each of the piglets and the samples are per dose. pooled. 5 ml of the pooled blood is injected intramuscularly Use piglets complying with the requirements for selection of into each of two other piglets. This operation is repeated animals described under Choice of Vaccine Strain. To two six times. The animals must not display any sign of swine groups of five piglets inject intramuscularly : fever and must show normal growth. — 1/40 of a dose of the vaccine to be examined into each Immunogenic properties. The immunogenic properties piglet of the first group, may be demonstrated by the method described for the determination of potency. The quantity of the vaccinal virus — 1/160 of a dose of the vaccine to be examined into each piglet of the second group. corresponding to one dose of vaccine contains at least 100 PD50. Use two piglets as controls. IDENTIFICATION

Prepare the dilutions using buffered salt solution pH 7.2 R. On the fourteenth day after the injection, inoculate intramuscularly into each vaccinated and control animal a sufficient quantity of challenge virus to kill an unvaccinated piglet in 7 days. The challenge virus preparation consists of blood of pigs infected experimentally by virus that has not been submitted to passage in cell cultures. The control animals die within the seven days of inoculation. Observe the vaccinated animals for 14 days. From the number of animals which survive without showing any sign of swine fever, calculate the number of PD50 contained in the vaccine using the usual statistical methods.

A. For vaccines prepared in rabbits and lapinised vaccines prepared in cell cultures, inject intravenously 0.5 ml of the vaccine reconstituted as stated on the label into one or more non-immunised rabbits and one or more rabbits immunised either with an identical dose of a vaccine of the same type injected by the same route at least 10 days and at most 2 months beforehand or with a sufficient dose of antiserum administered a few hours before the injection of the vaccine. Measure the temperature of the rabbits in the morning and the evening starting 24 h after the injection and continuing until the fifth day after LABELLING the injection. The vaccine is identified by its specific pyrogenic character leading to a rise in temperature of at The label states that the vaccine has been prepared in cell least 1.5 °C in the non-immunised rabbits only. cultures or in rabbits as appropriate. 794



Tetanus vaccine for veterinary use

01/2005:0697 shortest practical time interval before adsorption. Prepare corrected a dilution of the detoxified harvest so that the guinea-pigs each receive 10 times the amount of toxoid (measured in Lf units) that will be present in a dose of vaccine. Divide TETANUS VACCINE the dilution into 2 equal parts. Keep one part at 5 ± 3 °C FOR VETERINARY USE and the other at 37 °C for 6 weeks. Attribute each dilution to a separate group of guinea-pigs and inject into each guinea-pig the dilution attributed to its group. Observe the Vaccinum tetani ad usum veterinarium animals for 21 days. The toxoid complies with the test if no guinea-pig shows clinical signs of disease or dies from causes DEFINITION attributable to the neurotoxin of C. tetani. Tetanus vaccine for veterinary use is a preparation of the BATCH POTENCY TEST neurotoxin of Clostridium tetani treated in a manner that eliminates toxicity while maintaining adequate immunogenic Where the test described under Potency is used as the batch potency test, the vaccine complies with the test if the properties. antibody titre in International Units is not less than that found for a batch of vaccine shown to be satisfactory with PRODUCTION respect to immunogenicity in the target species. The C. tetani strain used for production is cultured in a suitable medium. The toxin is purified and then detoxified or it may be detoxified before purification. The antigenic purity IDENTIFICATION If the nature of the adjuvant allows it, carry out test A. is determined in Lf units of tetanus toxoid per milligram of protein and shown to be not less than the value approved for Otherwise carry out test B. the particular product. A. Dissolve in the vaccine sufficient sodium citrate R to give a 100 g/l solution. Maintain the solution at 37 °C CHOICE OF VACCINE COMPOSITION for about 16 h and centrifuge until a clear supernatant The C. tetani strain used in the preparation of the vaccine liquid is obtained. The supernatant reacts with a suitable is shown to be satisfactory with respect to the production tetanus antitoxin, giving a precipitate. of the neurotoxin. The vaccine is shown to be satisfactory with respect to safety and immunogenicity for each species B. When injected into susceptible animals, the vaccine of animal for which it is intended. As part of the studies to provokes the formation of antibodies against the demonstrate these characteristics, the tests described below neurotoxin of C. tetani. may be used. Production of antigens. The production of the neurotoxin TESTS of C. tetani is verified by a suitable immunochemical method Safety. Inject 5 ml of the vaccine subcutaneously as 2 equal (2.7.1) carried out on the neurotoxin obtained from the divided doses, at separate sites into each of 5 healthy vaccine strain under the conditions used for the production guinea-pigs, each weighing 350 g to 450 g, that have not of the vaccine. previously been treated with any material that will interfere Safety. Carry out the test for each recommended route of with the test. No abnormal local or systemic reaction occurs. administration and species of animal for which the vaccine If within 21 days of the injection any of the animals shows is intended ; use animals of the minimum age recommended signs of or dies from tetanus, the vaccine does not comply for vaccination and of the most sensitive category for the with the test. If more than 1 animal dies from non-specific species. causes, repeat the test. If any animal dies in the second test, Use not fewer than 15 animals, free from antitoxic antibodies the vaccine does not comply with the test. for each test. Administer a double dose of vaccine to each Sterility. The vaccine complies with the test for sterility animal. Administer a single dose of vaccine to each animal prescribed in the monograph on Vaccines for veterinary after the interval stated on the label. Observe the animals use (0062). until 14 days after the last administration. If the vaccine is intended for use in pregnant animals, vaccinate the animals POTENCY at the stage of pregnancy and according to the scheme Administer 1 dose of vaccine subcutaneously to each of at stated on the label and prolong the observation period until 1 day after parturition. The vaccine complies with the least 5 susceptible guinea-pigs. After 28 days, administer again 1 dose subcutaneously to each guinea-pig. 14 days test if no animal shows abnormal local or systemic signs of disease or dies from causes attributable to the vaccine. If the after the second dose, collect blood from each guinea-pig vaccine is intended for use in pregnant animals, in addition and prepare serum samples. Determine for each serum no significant effects on the gestation and the offspring are the titre of antibodies against the neurotoxin of C. tetani using a suitable immunochemical method (2.7.1) such as a demonstrated. toxin-binding-inhibition test (ToBI test) and a homologous Immunogenicity. The test described under Potency may reference serum. Determine the average antibody titre of be used to demonstrate immunogenicity. It shall also be the serum samples. demonstrated for each target species that the vaccine, Clostridia (multicomponent) rabbit antiserum BRP, administered by the recommended route, stimulates an immune response consistent with the claims for the product Clostridium tetani guinea-pig antiserum for vaccines for veterinary use BRP and Clostridium tetani rabbit (for example, induction of antitoxic antibodies or induction antiserum BRP are suitable as reference sera. of protective levels of antitoxic antibodies). Tetanus vaccine intended for use in animals other than DETOXIFIED HARVEST horses complies with the test if the average antibody titre is Absence of toxin and irreversibility of toxoid : carry out not less than 7.5 IU/ml. a test for reversion to toxicity on the detoxified harvest Tetanus vaccine intended for use in horses complies with the using 2 groups of 5 guinea-pigs, each weighing 350 g to 450 g ; if the vaccine is adsorbed, carry out the test with the test if the average antibody titre is not less than 30 IU/ml. General Notices (1) apply to all monographs and other texts

795

Vibriosis (cold-water) vaccine (inactivated) for salmonids


For tetanus vaccine presented as a combined vaccine for use C. Safety is demonstrated in addition in field trials by administering the intended dose to a sufficient number in animals other than horses, the above test may be carried of fish distributed in not fewer than two sets of premises. out in susceptible rabbits instead of guinea-pigs. The vaccine No abnormal reaction occurs. complies with the test if the average antibody titre of the sera of the vaccinated rabbits is not less than 2.5 IU/ml. Immunogenicity.The test described under Potency, carried out for each recommended route of administration, is 01/2005:1580 suitable to demonstrate immunogenicity of the vaccine. BATCH TESTING VIBRIOSIS (COLD-WATER) VACCINE Batch potency test. For routine testing of batches of vaccine, the test described under Potency may be carried out using (INACTIVATED) FOR SALMONIDS groups of not fewer than thirty fish of one of the species for which the vaccine is intended ; alternatively, a suitable Vaccinum vibriosidis aquae frigidae validated test based on antibody response may be carried inactivatum ad salmonidas out, the criteria being set with reference to a batch of vaccine that has given satisfactory results in the test described under DEFINITION Potency. The following test may be used after a satisfactory Cold-water vibriosis vaccine (inactivated) for salmonids is correlation with the test described under Potency has been prepared from cultures of one or more suitable strains of established. Vibrio salmonicida. Use fish from a population that does not have specific antibodies against V. salmonicida and that are within PRODUCTION specified limits for body mass. Carry out the test at a defined The strains of V. salmonicida are cultured and harvested separately. The harvests are inactivated by a suitable method. temperature. Inject into each of not fewer than twenty-five They may be purified and concentrated. Whole or disrupted fish one dose of vaccine, according to the instructions for cells may be used and the vaccine may contain extracellular use. Perform mock vaccination on a control group of not products of the bacterium released into the growth medium. fewer than ten fish. Collect blood samples at a defined time after vaccination. Determine for each sample the level of CHOICE OF VACCINE COMPOSITION specific antibodies against V. salmonicida by a suitable The strain or strains of V. salmonicida used are shown to be immunochemical method (2.7.1). The vaccine complies with suitable with respect to production of antigens of assumed the test if the mean level of antibodies is not significantly immunological importance. The vaccine is shown to be lower than that found for a batch that gave satisfactory satisfactory with respect to safety (5.2.6) and immunogenicity results in the test described under Potency. The test is (5.2.7) in the species of fish for which it is intended. The not valid if the control group shows antibodies against following tests may be used during demonstration of safety V. salmonicida. and immunogenicity. Safety. Safety is tested in three different batches using test A, IDENTIFICATION test B or both, depending on the recommendations for use. When injected into fish that do not have specific antibodies against V. salmonicida, the vaccine stimulates the A. Vaccines intended for administration by injection. production of such antibodies. A test is carried out in each species of fish for which the vaccine is intended. The fish used are from a TESTS population that does not have specific antibodies against Safety. Use not fewer than ten fish of one of the species V. salmonicida and which has not been vaccinated for which the vaccine is intended, having, where possible, against nor exposed to cold-water vibriosis. The test is the minimum body mass recommended for vaccination ; carried out in the conditions recommended for the use if fish of the minimum body mass are not available, use of the vaccine with a water temperature not less than 10 °C. An amount of vaccine corresponding to twice the fish not greater than twice this mass. Use fish from a recommended dose per mass unit for fish of the minimum population that does not have specific antibodies against body mass recommended for vaccination is administered V. salmonicida and that has not been vaccinated against nor exposed to cold-water vibriosis. Carry out the test in the intraperitoneally to each of not fewer than fifty fish of conditions recommended for use of the vaccine with a water the minimum recommended body mass. The fish are temperature not less than 10 °C. For vaccines administered observed for 21 days. No abnormal local or systemic reaction occurs. The test is invalid if more than 6 per cent by injection or immersion, inject intraperitoneally into of the fish die from causes not attributable to the vaccine. each fish an amount of vaccine corresponding to twice the recommended dose per mass unit. For vaccines administered B. Vaccines intended for administration by immersion. by immersion only, use a bath with double the recommended A test is carried out in each species of fish for which concentration and bathe the fish for twice the recommended the vaccine is intended. The fish used are from a population that does not have specific antibodies against immersion time. Observe the animals for 21 days. No abnormal local or systemic reaction attributable to the V. salmonicida and which has not been vaccinated vaccine occurs. The test is invalid if more than 10 per cent of against nor exposed to cold-water vibriosis. The test is carried out in the conditions recommended for the use of the fish die from causes not attributable to the vaccine. the vaccine with a water temperature not less than 10 °C. Sterility. The vaccine complies with the test for sterility prescribed in the monograph on Vaccines for veterinary Prepare an immersion bath at twice the recommended use (0062). concentration. Not fewer than fifty fish, having not less than the minimum body mass recommended for POTENCY vaccination are used. The fish are bathed for twice the recommended time. The fish are observed for 21 days. No Carry out the test according to a protocol defining limits of body mass for the fish, water source, water flow and abnormal local or systemic reaction occurs. The test is invalid if more than 6 per cent of the fish die from causes temperature limits, and preparation of a standardised challenge. Vaccinate not fewer than one hundred fish by a not attributable to the vaccine. 796



Vibriosis vaccine (inactivated) for salmonids

not have specific antibodies against the relevant serovars recommended route, according to the instructions for use. of V. anguillarum or where applicable V. ordalii and which Perform mock vaccination on a control group of not fewer has not been vaccinated against nor exposed to vibriosis. than one hundred fish ; mark vaccinated and control fish The test is carried out in the conditions recommended for identification. Keep all the fish in the same tank or mix for the use of the vaccine with a water temperature not equal numbers of controls and vaccinates in each tank if less than 10 °C. An amount of vaccine corresponding to more than one tank is used. Carry out challenge by injection twice the recommended dose per mass unit for fish of at a fixed time interval after vaccination, defined according the minimum body mass recommended for vaccination to the statement regarding development of immunity. Use is administered intraperitoneally to each of not fewer for challenge a culture of V. salmonicida whose virulence than fifty fish of the minimum recommended body mass. has been verified. Observe the fish daily until at least 60 per The fish are observed for 21 days. No abnormal local or cent specific mortality is reached in the control group. Plot systemic reaction occurs. The test is invalid if more than for both vaccinates and controls a curve of specific mortality 6 per cent of the fish die from causes not attributable to against time from challenge and determine by interpolation the vaccine. the time corresponding to 60 per cent specific mortality in controls. The test is invalid if the specific mortality is less than 60 per cent in the control group 21 days after the B. Vaccines intended for administration by immersion. A test is carried out in each species of fish for which the first death in the fish. Read from the curve for vaccinates vaccine is intended. The fish used are from a population the mortality (M) at the time corresponding to 60 per cent that does not have specific antibodies against the relevant mortality in controls. Calculate the relative percentage serovars of V. anguillarum or where applicable V. ordalii survival (RPS) from the expression : and which has not been vaccinated against nor exposed to vibriosis. The test is carried out in the conditions recommended for the use of the vaccine with a water temperature not less than 10 °C. Prepare an immersion The vaccine complies with the test if the RPS is not less bath at twice the recommended concentration. Not than 60 per cent for vaccines administered by immersion and fewer than fifty fish having the minimum body mass 90 per cent for vaccines administered by injection. recommended for vaccination are used. The fish are bathed for twice the recommended time. The fish are LABELLING observed for 21 days. No abnormal local or systemic The label states information on the time needed for reaction occurs. The test is invalid if more than 6 per cent development of immunity after vaccination under the range of the fish die from causes not attributable to the vaccine. of conditions corresponding to the recommended use. C. Safety is also demonstrated in field trials by administering the intended dose to a sufficient number of fish distributed in not fewer than two sets of premises. No 01/2005:1581 abnormal reaction occurs.

VIBRIOSIS VACCINE (INACTIVATED) FOR SALMONIDS

Immunogenicity.The test described under Potency, carried out for each recommended route of administration is suitable to demonstrate immunogenicity of the vaccine.

BATCH TESTING Batch potency test. For routine testing of batches of vaccine, the test described under Potency may be carried out using groups of not fewer than thirty fish of one of the species DEFINITION for which the vaccine is intended ; alternatively, a suitable Vibriosis vaccine (inactivated) for salmonids is prepared from validated test based on antibody response may be carried cultures of one or more suitable strains or serovars of Vibrio out, the criteria being set with reference to a batch of vaccine anguillarum ; the vaccine may also include Vibrio ordalii. that has given satisfactory results in the test described under Potency. The following test may be used after a satisfactory PRODUCTION correlation with the test described under Potency has been The strains of V. anguillarum and V. ordalii are cultured established. and harvested separately. The harvests are inactivated by a Use fish from a population that does not have specific suitable method. They may be purified and concentrated. antibodies against the relevant serovars of V. anguillarum Whole or disrupted cells may be used and the vaccine may contain extracellular products of the bacterium released into and where applicable V. ordalii and that are within specified limits for body mass. Carry out the test at a defined the growth medium. temperature. Inject into each of not fewer than twenty-five CHOICE OF VACCINE COMPOSITION fish one dose of vaccine, according to the instructions for The strains of V. anguillarum and V. ordalii used are use. Perform mock vaccination on a control group of not shown to be suitable with respect to production of antigens fewer than ten fish. Collect blood samples at a defined of assumed immunological importance. The vaccine is time after vaccination. Determine for each sample the shown to be satisfactory with respect to safety (5.2.6) and level of specific antibodies against the different serovars of immunogenicity (5.2.7) in the species of fish for which V. anguillarum and against V. ordalii included in the vaccine it is intended. The following tests may be used during by a suitable immunochemical method (2.7.1). The vaccine demonstration of safety and immunogenicity. complies with the test if the mean levels of antibodies are not Safety. Safety is tested in three different batches using test A, significantly lower than those found for a batch that gave test B or both, depending on the recommendations for use. satisfactory results in the test described under Potency. The A. Vaccines intended for administration by injection. A test test is not valid if the control group shows antibodies against is carried out in each species of fish for which the vaccine the relevant serovars of V. anguillarum or, where applicable, is intended. The fish used are from a population that does against V. ordalii.

Vaccinum vibriosidis ad salmonidas inactivatum


797

Vibriosis vaccine (inactivated) for salmonids


one hundred fish by a recommended route, according to the instructions for use. Perform mock vaccination on a control group of not fewer than one hundred fish ; mark vaccinated and control fish for identification. Keep all the fish in the same tank or mix equal numbers of controls and vaccinates in each tank if more than one tank is used. TESTS Safety. Use not fewer than ten fish of one of the species for Carry out challenge by injection at a fixed time interval after vaccination, defined according to the statement regarding which the vaccine is intended, having, where possible, the development of immunity. Use for challenge cultures of minimum body mass recommended for vaccination ; if fish V. anguillarum or V. ordalii whose virulence has been of the minimum body mass are not available, use fish not verified. Observe the fish daily until at least 60 per cent greater than twice this mass. Use fish from a population specific mortality is reached in the control group. Plot for that does not have specific antibodies against the relevant serovars of V. anguillarum and, where applicable, V. ordalii both vaccinates and controls a curve of specific mortality against time from challenge and determine by interpolation and which has not been vaccinated against nor exposed to vibriosis. Carry out the test in the conditions recommended the time corresponding to 60 per cent specific mortality in controls. The test is invalid if the specific mortality is for the use of the vaccine with a water temperature not less than 60 per cent in the control group 21 days after the less than 10 °C. For vaccines administered by injection or immersion, inject intraperitoneally into each fish an amount first death in the fish. Read from the curve for vaccinates the mortality (M) at the time corresponding to 60 per cent of vaccine corresponding to twice the recommended dose per mass unit. For vaccines administered by immersion only, mortality in controls. Calculate the relative percentage survival (RPS) from the expression : use a bath with double the recommended concentration and bathe the fish for twice the recommended immersion time. Observe the animals for 21 days. No abnormal local or systemic reaction attributable to the vaccine occurs. The test is invalid if more than 10 per cent of the fish die from causes The vaccine complies with the test if the RPS is not less not attributable to the vaccine. than 60 per cent for vaccines administered by immersion and 75 per cent for vaccines administered by injection. Sterility. The vaccine complies with the test for sterility prescribed in the monograph on Vaccines for veterinary LABELLING use (0062). The label states : — the serovar or serovars of V. anguillarum included in POTENCY the vaccine, Carry out a separate test for each species and each serovar — where applicable, that the vaccine includes V. ordalii, included in the vaccine. Carry out the test according to a protocol defining limits of body mass for the fish, water — information on the time needed for development of source, water flow and temperature limits, and preparation immunity after vaccination under the range of conditions of a standardised challenge. Vaccinate not fewer than corresponding to the recommended use.

IDENTIFICATION When injected into fish that do not have specific antibodies against V. anguillarum and, where applicable, V. ordalii, the vaccine stimulates the production of such antibodies.

798



Diphtheria antitoxin

01/2005:0085 of volumes of the test toxin and sufficient of a suitable liquid to bring the total volume to 5.0 ml. Allow the mixtures to stand at room temperature, protected from light, for 60 min. BOTULINUM ANTITOXIN Using four mice for each mixture, inject a dose of 1.0 ml intraperitoneally into each mouse. Observe the mice for 96 h. Immunoserum botulinicum The test dose of toxin is the quantity in 1.0 ml of the mixture DEFINITION made with the smallest amount of toxin capable of causing, despite partial neutralisation by the reference preparation, Botulinum antitoxin is a preparation containing antitoxic globulins that have the power of specifically neutralising the the death of all four mice injected with the mixture within the observation period. toxins formed by Clostridium botulinum type A, type B or type E, or any mixture of these types. Determination of potency of the antitoxin. Prepare solutions of each reference preparation in a suitable liquid PRODUCTION such that each contains 0.25 IU of antitoxin per millilitre. It is obtained by fractionation from the serum of horses, Prepare solutions of each test toxin in a suitable liquid such or other mammals, that have been immunised against that each contains 2.5 test doses per millilitre. Cl. botulinum type A, type B and type E toxins. Using each toxin solution and the corresponding reference IDENTIFICATION preparation in turn, determine the potency of the antitoxin. It specifically neutralises the types of Cl. botulinum toxins Prepare mixtures of the solution of the test toxin and the stated on the label, rendering them harmless to susceptible antitoxin to be examined such that each contains 2.0 ml animals. of the solution of the test toxin, one of a graded series of volumes of the antitoxin to be examined, and sufficient of POTENCY a suitable liquid to bring the total volume to 5.0 ml. Also Not less than 500 IU of antitoxin per millilitre for each prepare mixtures of the solution of the test toxin and the of types A and B and not less than 50 IU of antitoxin per solution of the reference preparation such that each contains millilitre for type E. 2.0 ml of the solution of the test toxin, one of a graded series of volumes of the solution of the reference preparation The potency of botulinum antitoxin is determined by centred on that volume (2.0 ml) that contains 0.5 IU, and comparing the dose necessary to protect mice against the sufficient of a suitable liquid to bring the total volume to lethal effects of a fixed dose of botulinum toxin with the quantity of the standard preparation of botulinum antitoxin 5.0 ml. Allow the mixtures to stand at room temperature, necessary to give the same protection. For this comparison a protected from light, for 60 min. Using four mice for each mixture, inject a dose of 1.0 ml intraperitoneally into each reference preparation of each type of botulinum antitoxin, mouse. Observe the mice for 96 h. calibrated in International Units, and suitable preparations of botulinum toxins, for use as test toxins, are required. The The mixture that contains the largest volume of antitoxin potency of each test toxin is determined in relation to the that fails to protect the mice from death contains 0.5 IU. This specific reference preparation ; the potency of the botulinum quantity is used to calculate the potency of the antitoxin in antitoxin to be examined is determined in relation to the International Units per millilitre. potency of the test toxins by the same method. The test is not valid unless all the mice injected with International Units of the antitoxin are the specific mixtures containing 2.0 ml or less of the solution of the neutralising activity for botulinum toxin type A, type B and reference preparation die and all those injected with mixtures type E contained in stated amounts of the International containing more survive. Standards which consist of dried immune horse sera of types A, B and E. The equivalence in International Units of the LABELLING International Standard is stated from time to time by the The label states the types of Cl. botulinum toxin neutralised World Health Organisation. by the preparation. Selection of animals. Use mice having body masses such that the difference between the lightest and the heaviest does not exceed 5 g. Preparation of test toxins. CAUTION : Botulinum toxin is 01/2005:0086 extremely toxic : exceptional care must be taken in any procedure in which it is employed. Prepare type A, B and E DIPHTHERIA ANTITOXIN toxins from sterile filtrates of approximately 7-day cultures in liquid medium of Cl. botulinum types A, B and E. To the filtrates, add 2 volumes of glycerol, concentrate, if necessary, Immunoserum diphthericum by dialysis against glycerol and store at or slightly below 0 °C. DEFINITION Selection of test toxins. Select toxins of each type for use Diphtheria antitoxin is a preparation containing antitoxic as test toxins by determining for mice the L+/10 dose and globulins that have the power of specifically neutralising the the LD50, the observation period being 96 h. The test toxins toxin formed by Corynebacterium diphtheriae. contain at least 1000 LD50 in an L+/10 dose. PRODUCTION Determination of test doses of the toxins (L+/10 dose). Prepare solutions of the reference preparations in a suitable It is obtained by fractionation from the serum of horses, liquid such that each contains 0.25 IU of antitoxin per or other mammals, that have been immunised against millilitre. Using each solution in turn, determine the diphtheria toxin. test dose of the corresponding test toxin. Prepare mixtures of the solution of the reference preparation IDENTIFICATION It specifically neutralises the toxin formed by C. diphtheriae, and the test toxin such that each contains 2.0 ml of the solution of the reference preparation, one of a graded series rendering it harmless to susceptible animals. General Notices (1) apply to all monographs and other texts

801

Gas-gangrene antitoxin, mixed


ASSAY Not less than 1000 IU of antitoxin per millilitre for antitoxin obtained from horse serum. Not less than 500 IU of antitoxin per millilitre for antitoxin obtained from the serum of other mammals. The potency of diphtheria antitoxin is determined by comparing the dose necessary to protect guinea-pigs or rabbits against the erythrogenic effects of a fixed dose of diphtheria toxin with the quantity of the standard preparation of diphtheria antitoxin necessary to give the same protection. For this comparison a reference preparation of diphtheria antitoxin, calibrated in International Units, and a suitable preparation of diphtheria toxin, for use as a test toxin, are required. The potency of the test toxin is determined in relation to the reference preparation ; the potency of the diphtheria antitoxin to be examined is determined in relation to the potency of the test toxin by the same method. The International Unit of antitoxin is the specific neutralising activity for diphtheria toxin contained in a stated amount of the International Standard, which consists of a quantity of dried immune horse serum. The equivalence in International Units of the International Standard is stated by the World Health Organisation. Preparation of test toxin. Prepare diphtheria toxin from cultures of C. diphtheriae in a liquid medium. Filter the culture to obtain a sterile toxic filtrate and store at 4 °C. Selection of test toxin. Select a toxin for use as a test toxin by determining for guinea-pigs or rabbits the lr/100 dose and the minimal reacting dose, the observation period being 48 h. The test toxin has at least 200 minimal reacting doses in the lr/100 dose. Minimal reacting dose. This is the smallest quantity of toxin which, when injected intracutaneously into guinea-pigs or rabbits, causes a small, characteristic reaction at the site of injection within 48 h. The test toxin is allowed to stand for some months before being used for the assay of antitoxin. During this time its toxicity declines and the lr/100 dose may be increased. Determine the minimal reacting dose and the lr/100 dose at frequent intervals. When experiment shows that the lr/100 dose is constant, the test toxin is ready for use and may be used for a long period. Store the test toxin in the dark at 0 °C to 5 °C. Maintain its sterility by the addition of toluene or other antimicrobial preservative that does not cause a rapid decline in specific toxicity. Determination of test dose of toxin (lr/100 dose). Prepare a solution of the reference preparation in a suitable liquid such that it contains 0.1 IU of antitoxin per millilitre. Prepare mixtures of the solution of the reference preparation and of the test toxin such that each contains 1.0 ml of the solution of the reference preparation, one of a graded series of volumes of the test toxin and sufficient of a suitable liquid to bring the total volume to 2.0 ml. Allow the mixtures to stand at room temperature, protected from light, for 15 min to 60 min. Using two animals for each mixture, inject a dose of 0.2 ml intracutaneously into the shaven or depilated flanks of each animal. Observe the animals for 48 h. The test dose of toxin is the quantity in 0.2 ml of the mixture made with the smallest amount of toxin capable of causing, despite partial neutralisation by the reference preparation, a small but characteristic erythematous lesion at the site of injection. Determination of potency of the antitoxin. Prepare a solution of the reference preparation in a suitable liquid such that it contains 0.125 IU of antitoxin per millilitre. 802

Prepare a solution of the test toxin in a suitable liquid such that it contains 12.5 test doses per millilitre. Prepare mixtures of the solution of the test toxin and of the antitoxin to be examined such that each contains 0.8 ml of the solution of the test toxin, one of a graded series of volumes of the antitoxin to be examined and sufficient of a suitable liquid to bring the total volume to 2.0 ml. Also prepare mixtures of the solution of the test toxin and the solution of the reference preparation such that each contains 0.8 ml of the solution of the test toxin, one of a graded series of volumes of the solution of the reference preparation centred on that volume (0.8 ml) that contains 0.1 IU and sufficient of a suitable liquid to bring the total volume to 2.0 ml. Allow the mixtures to stand at room temperature, protected from light, for 15 min to 60 min. Using two animals for each mixture, inject a dose of 0.2 ml intracutaneously into the shaven or depilated flanks of each animal. Observe the animals for 48 h. The mixture that contains the largest volume of antitoxin that fails to protect the guinea-pigs from the erythematous effects of the toxin contains 0.1 IU. This quantity is used to calculate the potency of the antitoxin in International Units per millilitre. The test is not valid unless all the sites injected with mixtures containing 0.8 ml or less of the solution of the reference preparation show erythematous lesions and at all those injected with mixtures containing more there are no lesions. 01/2005:0090

GAS-GANGRENE ANTITOXIN, MIXED Immunoserum gangraenicum mixtum DEFINITION Mixed gas-gangrene antitoxin is prepared by mixing gas-gangrene antitoxin (novyi), gas-gangrene antitoxin (perfringens) and gas-gangrene antitoxin (septicum) in appropriate quantities. IDENTIFICATION It specifically neutralises the alpha toxins formed by Clostridium novyi (former nomenclature : Clostridium oedematiens), Clostridium perfringens and Clostridium septicum, rendering them harmless to susceptible animals. ASSAY Gas-gangrene antitoxin (novyi), not less than 1000 IU of antitoxin per millilitre ; gas-gangrene antitoxin (perfringens), not less than 1000 IU of antitoxin per millilitre ; gas-gangrene antitoxin (septicum) not less than 500 IU of antitoxin per millilitre. Carry out the assay for each component, as prescribed in the monographs on Gas-gangrene antitoxin (novyi) (0087), Gas-gangrene antitoxin (perfringens) (0088) and Gas-gangrene antitoxin (septicum) (0089). 01/2005:0087

GAS-GANGRENE ANTITOXIN (NOVYI) Immunoserum gangraenicum (Clostridium novyi) DEFINITION Gas-gangrene antitoxin (novyi) is a preparation containing antitoxic globulins that have the power of neutralising the alpha toxin formed by Clostridium novyi (Former



Gas-gangrene antitoxin (perfringens)

Prepare mixtures of the solution of the test toxin and the antitoxin to be examined such that each contains 0.8 ml of the solution of the test toxin, one of a graded series of volumes of the antitoxin to be examined and sufficient of IDENTIFICATION a suitable liquid to bring the total volume to 2.0 ml. Also It specifically neutralises the alpha toxin formed by Cl. novyi, prepare mixtures of the solution of the test toxin and the rendering it harmless to susceptible animals. solution of the reference preparation such that each contains 0.8 ml of the solution of the test toxin, one of a graded ASSAY series of volumes of the solution of the reference preparation Not less than 3750 IU of antitoxin per millilitre. centred on that volume (0.8 ml) that contains 10 IU and The potency of gas-gangrene antitoxin (novyi) is determined sufficient of a suitable liquid to bring the total volume to 2.0 ml. Allow the mixtures to stand at room temperature, by comparing the dose necessary to protect mice or other protected from light, for 60 min. Using six mice for each suitable animals against the lethal effects of a fixed dose of Cl. novyi toxin with the quantity of the standard preparation mixture, inject a dose of 0.2 ml intramuscularly into each of gas-gangrene antitoxin (novyi) necessary to give the same mouse. Observe the mice for 72 h. protection. For this comparison a reference preparation of The mixture that contains the largest volume of antitoxin gas-gangrene antitoxin (novyi), calibrated in International that fails to protect the mice from death contains 10 IU. This Units, and a suitable preparation of Cl. novyi toxin for use quantity is used to calculate the potency of the antitoxin in as a test toxin are required. The potency of the test toxin International Units per millilitre. is determined in relation to the reference preparation ; the The test is not valid unless all the mice injected with potency of the gas-gangrene antitoxin (novyi) to be examined mixtures containing 0.8 ml or less of the solution of the is determined in relation to the potency of the test toxin by reference preparation die and all those injected with mixtures the same method. containing a larger volume survive. The International Unit of antitoxin is the specific neutralising activity for Cl. novyi toxin contained in a stated amount of the International Standard, which consists of a quantity of 01/2005:0088 dried immune horse serum. The equivalence in International Units of the International Standard is stated by the World GAS-GANGRENE ANTITOXIN Health Organisation. (PERFRINGENS) Selection of animals. Use mice having body masses such that the difference between the lightest and the heaviest does not exceed 5 g. Immunoserum gangraenicum Preparation of test toxin. Prepare the test toxin from a (Clostridium perfringens) sterile filtrate of an approximately 5-day culture in liquid DEFINITION medium of Cl. novyi. Treat the filtrate with ammonium sulphate R, collect the precipitate, which contains the toxin, Gas-gangrene antitoxin (perfringens) is a preparation dry in vacuo over diphosphorus pentoxide R, powder and containing antitoxic globulins that have the power store dry. of specifically neutralising the alpha toxin formed by Selection of test toxin. Select a toxin for use as a test toxin Clostridium perfringens. It is obtained by fractionation from the serum of horses, or other mammals, that have been by determining for mice the L+ dose and the LD50, the observation period being 72 h. The test toxin has an L+ dose immunised against Cl. perfringens alpha toxin. of 0.5 mg or less and contains not less than 25 LD50 in each IDENTIFICATION L+ dose. It specifically neutralises the alpha toxin formed by Determination of test dose of toxin (L+ dose). Prepare a Cl. perfringens, rendering it harmless to susceptible animals. solution of the reference preparation in a suitable liquid such that it contains 12.5 IU of antitoxin per millilitre. ASSAY Prepare a solution of the test toxin in a suitable liquid such Not less than 1500 IU of antitoxin per millilitre. that 1 ml contains a precisely known amount such as 10 mg. The potency of gas-gangrene antitoxin (perfringens) is Prepare mixtures of the solution of the reference preparation determined by comparing the dose necessary to protect and the solution of the test toxin such that each contains mice or other suitable animals against the lethal effects 0.8 ml of the solution of the reference preparation, one of of a fixed dose of Cl. perfringens toxin with the quantity a graded series of volumes of the solution of the test toxin of the standard preparation of gas-gangrene antitoxin and sufficient of a suitable liquid to bring the total volume (perfringens) necessary to give the same protection. For to 2.0 ml. Allow the mixtures to stand at room temperature, this comparison a reference preparation of gas-gangrene protected from light, for 60 min. Using six mice for each antitoxin (perfringens), calibrated in International Units, mixture, inject a dose of 0.2 ml intramuscularly into each and a suitable preparation of Cl. perfringens toxin for use mouse. Observe the mice for 72 h. as a test toxin are required. The potency of the test toxin The test dose of toxin is the quantity in 0.2 ml of the mixture is determined in relation to the reference preparation ; the made with the smallest amount of toxin capable of causing, potency of the gas-gangrene antitoxin (perfringens) to be despite partial neutralisation by the reference preparation, examined is determined in relation to the potency of the test the death of all six mice injected with the mixture within toxin by the same method. the observation period. The International Unit of antitoxin is the specific neutralising Determination of potency of the antitoxin. Prepare a activity for Cl. perfringens toxin contained in a stated solution of the reference preparation in a suitable liquid amount of the International Standard, which consists of a such that it contains 12.5 IU of antitoxin per millilitre. quantity of dried immune horse serum. The equivalence in Prepare a solution of the test toxin in a suitable liquid such International Units of the International Standard is stated by the World Health Organisation. that it contains 12.5 test doses per millilitre. nomenclature : Clostridium oedematiens). It is obtained by fractionation from the serum of horses, or other mammals, that have been immunised against Cl. novyi alpha toxin.


803

Gas-gangrene antitoxin (septicum)


Selection of animals. Use mice having body masses such that the difference between the lightest and the heaviest does not exceed 5 g. Preparation of test toxin. Prepare the test toxin from a sterile filtrate of an approximately 5-day culture in liquid medium of Cl. perfringens. Treat the filtrate with ammonium sulphate R, collect the precipitate, which contains the toxin, dry in vacuo over diphosphorus pentoxide R, powder and store dry. Selection of test toxin. Select a toxin for use as a test toxin by determining for mice the L+ dose and the LD50, the observation period being 48 h. The test toxin has an L+ dose of 4 mg or less and contains not less than 20 LD50 in each L+ dose. Determination of test dose of toxin (L+ dose). Prepare a solution of the reference preparation in a suitable liquid such that it contains 5 IU of antitoxin per millilitre.

01/2005:0089

GAS-GANGRENE ANTITOXIN (SEPTICUM) Immunoserum gangraenicum (Clostridium septicum) DEFINITION Gas-gangrene antitoxin (septicum) is a preparation containing antitoxic globulins that have the power of specifically neutralising the alpha toxin formed by Clostridium septicum. It is obtained by fractionation from the serum of horses, or other mammals, that have been immunised against Cl. septicum alpha toxin. IDENTIFICATION It specifically neutralises the alpha toxin formed by Cl. septicum, rendering it harmless to susceptible animals.

Prepare a solution of the test toxin in a suitable liquid such that 1 ml contains a precisely known amount such as 10 mg. ASSAY Prepare mixtures of the solution of the reference preparation Not less than 1500 IU of antitoxin per millilitre. The potency of gas-gangrene antitoxin (septicum) is and the solution of the test toxin such that each contains determined by comparing the dose necessary to protect 2.0 ml of the solution of the reference preparation, one of mice or other suitable animals against the lethal effects of a graded series of volumes of the solution of the test toxin a fixed dose of Cl. septicum toxin with the quantity of the and sufficient of a suitable liquid to bring the total volume to 5.0 ml. Allow the mixtures to stand at room temperature, standard preparation of gas-gangrene antitoxin (septicum) necessary to give the same protection. For this comparison a protected from light, for 60 min. Using six mice for each reference preparation of gas-gangrene antitoxin (septicum), mixture, inject a dose of 0.5 ml intravenously into each calibrated in International Units, and a suitable preparation mouse. Observe the mice for 48 h. of Cl. septicum toxin for use as a test toxin are required. The test dose of toxin is the quantity in 0.5 ml of the mixture The potency of the test toxin is determined in relation to made with the smallest amount of toxin capable of causing, the reference preparation ; the potency of the gas-gangrene despite partial neutralisation by the reference preparation, antitoxin (septicum) to be examined is determined in relation the death of all six mice injected with the mixture within to the potency of the test toxin by the same method. the observation period. The International Unit of antitoxin is the specific neutralising activity for Cl. septicum toxin contained in a stated amount Determination of potency of the antitoxin. Prepare a of the International Standard, which consists of a quantity of solution of the reference preparation in a suitable liquid dried immune horse serum. The equivalence in International such that it contains 5 IU of antitoxin per millilitre. Units of the International Standard is stated by the World Prepare a solution of the test toxin in a suitable liquid such Health Organisation. that it contains five test doses per millilitre. Selection of animals. Use mice having body masses such that the difference between the lightest and the heaviest Prepare mixtures of the solution of the test toxin and the does not exceed 5 g. antitoxin to be examined such that each contains 2.0 ml Preparation of test toxin. Prepare the test toxin from a of the solution of the test toxin, one of a graded series of sterile filtrate of an approximately 5-day culture in liquid volumes of the antitoxin to be examined and sufficient of medium of Cl. septicum. Treat the filtrate with ammonium a suitable liquid to bring the total volume to 5.0 ml. Also sulphate R, collect the precipitate, which contains the toxin, prepare mixtures of the solution of the test toxin and the solution of the reference preparation such that each contains dry in vacuo over diphosphorus pentoxide R, powder and store dry. 2.0 ml of the solution of the test toxin, one of a graded series of volumes of the solution of the reference preparation Selection of test toxin. Select a toxin for use as a test toxin by determining for mice the L+ dose and the LD50, the centred on that volume (2.0 ml) that contains 10 IU and observation period being 72 h. The test toxin has an L+ dose sufficient of a suitable liquid to bring the total volume to of 0.5 mg or less and contains not less than 25 LD50 in each 5.0 ml. Allow the mixtures to stand at room temperature, L+ dose. protected from light, for 60 min. Using six mice for each mixture, inject a dose of 0.5 ml intravenously into each Determination of test dose of toxin (L+ dose). Prepare a mouse. Observe the mice for 48 h. solution of the reference preparation in a suitable liquid such that it contains 5 IU of antitoxin per millilitre. The mixture that contains the largest volume of antitoxin Prepare a solution of the test toxin in a suitable liquid such that fails to protect the mice from death contains 10 IU. This that 1 ml contains a precisely known amount such as 20 mg. quantity is used to calculate the potency of the antitoxin in Prepare mixtures of the solution of the reference preparation International Units per millilitre. and the solution of the test toxin such that each contains The test is not valid unless all the mice injected with 2.0 ml of the solution of the reference preparation, one of mixtures containing 2.0 ml or less of the solution of the a graded series of volumes of the solution of the test toxin reference preparation die and all those injected with mixtures and sufficient of a suitable liquid to bring the total volume containing a larger volume survive. to 5.0 ml. Allow the mixtures to stand at room temperature, 804



Tetanus antitoxin for human use

necessary to give the same protection. In countries where the paralysis method is not obligatory the lethal method may be used. For this method the number of animals and the procedure are identical with those described for the paralysis method but the end-point is the death of the animal rather than the onset of paralysis and the L+/10 dose is used instead of the Lp/10 dose. For this comparison a reference preparation of tetanus antitoxin, calibrated in International Units, and a suitable preparation of tetanus toxin, for use as a test toxin, are required. The potency of the test toxin is determined in relation to the reference preparation ; the potency of the tetanus antitoxin to be examined is determined in relation to the potency of the test toxin by the same method. The International Unit of antitoxin is the specific neutralising activity for tetanus toxin contained in a stated amount of the International Standard which consists of a quantity of dried immune horse serum. The equivalence in International Units of the International Standard is stated by the World Health Organisation. Selection of animals. If mice are used, the body masses should be such that the difference between the lightest and the heaviest does not exceed 5 g. Preparation of test toxin. Prepare the test toxin from a sterile filtrate of an approximately 9-day culture in liquid medium of Cl. tetani. To the filtrate add 1 to 2 volumes of glycerol and store slightly below 0 °C. Alternatively, treat the filtrate with ammonium sulphate R, collect the precipitate, which contains the toxin, dry in vacuo over diphosphorus pentoxide R, powder and store dry, either in sealed ampoules or in vacuo over diphosphorus pentoxide R. Determination of test dose of toxin (Lp/10 dose). Prepare a solution of the reference preparation in a suitable liquid such that it contains 0.5 IU of antitoxin per millilitre. If the test toxin is stored dry, reconstitute it using a suitable liquid. Prepare mixtures of the solution of the reference preparation 01/2005:0091 and the test toxin such that each contains 2.0 ml of the solution of the reference preparation, one of a graded series of volumes of the test toxin and sufficient of a suitable TETANUS ANTITOXIN liquid to bring the volume to 5.0 ml. Allow the mixtures to FOR HUMAN USE stand at room temperature, protected from light, for 60 min. Using six mice for each mixture, inject a dose of 0.5 ml Immunoserum tetanicum subcutaneously into each mouse. Observe the mice for 96 h. Mice that become paralysed may be killed. ad usum humanum The test dose of toxin is the quantity in 0.5 ml of the mixture DEFINITION made with the smallest amount of toxin capable of causing, Tetanus antitoxin for human use is a preparation containing despite partial neutralisation by the reference preparation, antitoxic globulins that have the power of specifically paralysis in all six mice injected with the mixture within the neutralising the toxin formed by Clostridium tetani. observation period. Determination of potency of the antitoxin. Prepare a PRODUCTION solution of the reference preparation in a suitable liquid It is obtained by fractionation from the serum of horses, or such that it contains 0.5 IU of antitoxin per millilitre. other mammals, that have been immunised against tetanus Prepare a solution of the test toxin in a suitable liquid such toxin. that it contains five test doses per millilitre. IDENTIFICATION Prepare mixtures of the solution of the test toxin and the It specifically neutralises the toxin formed by Cl. tetani, antitoxin to be examined such that each contains 2.0 ml rendering it harmless to susceptible animals. of the solution of the test toxin, one of a graded series of volumes of the antitoxin to be examined and sufficient of POTENCY a suitable liquid to bring the total volume to 5.0 ml. Also Not less than 1000 IU of antitoxin per millilitre when prepare mixtures of the solution of the test toxin and the intended for prophylactic use. Not less than 3000 IU of solution of the reference preparation such that each contains antitoxin per millilitre when intended for therapeutic use. 2.0 ml of the solution of the test toxin, one of a graded The potency of tetanus antitoxin is determined by comparing series of volumes of the solution of the reference preparation centred on that volume (2.0 ml) that contains 1 IU and the dose necessary to protect guinea-pigs or mice against the paralytic effects of a fixed dose of tetanus toxin with the sufficient of a suitable liquid to bring the total volume to 5.0 ml. Allow the mixtures to stand at room temperature, quantity of the standard preparation of tetanus antitoxin protected from light, for 60 min. Using six mice for each mixture, inject a dose of 0.5 ml intravenously into each mouse. Observe the mice for 72 h. The test dose of toxin is the quantity in 0.5 ml of the mixture made with the smallest amount of toxin capable of causing, despite partial neutralisation by the reference preparation, the death of all six mice injected with the mixture within the observation period. Determination of potency of the antitoxin. Prepare a solution of the reference preparation in a suitable liquid such that it contains 5 IU of antitoxin per millilitre. Prepare a solution of the test toxin in a suitable liquid such that it contains five test doses per millilitre. Prepare mixtures of the solution of the test toxin and the antitoxin to be examined such that each contains 2.0 ml of the solution of the test toxin, one of a graded series of volumes of the antitoxin to be examined and sufficient of a suitable liquid to bring the total volume to 5.0 ml. Also prepare mixtures of the solution of the test toxin and the solution of the reference preparation such that each contains 2.0 ml of the solution of the test toxin, one of a graded series of volumes of the solution of the reference preparation centred on that volume (2.0 ml) that contains 10 IU and sufficient of a suitable liquid to bring the total volume to 5.0 ml. Allow the mixtures to stand at room temperature, protected from light, for 60 min. Using six mice for each mixture, inject a dose of 0.5 ml intravenously into each mouse. Observe the mice for 72 h. The mixture that contains the largest volume of antitoxin that fails to protect the mice from death contains 10 IU. This quantity is used to calculate the potency of the antitoxin in International Units per millilitre. The test is not valid unless all the mice injected with mixtures containing 2.0 ml or less of the solution of the reference preparation die and all those injected with mixtures containing more survive.


805

Viper venom antiserum, European


protected from light, for 60 min. Using six mice for each mixture, inject into each mouse subcutaneously a dose of 0.5 ml. Observe the mice for 96 h. Mice that become paralysed may be killed. The mixture that contains the largest volume of antitoxin that fails to protect the mice from paralysis contains 1 IU. This quantity is used to calculate the potency of the antitoxin in International Units per millilitre. The test is not valid unless all the mice injected with mixtures containing 2.0 ml or less of the solution of the reference preparation show paralysis and all those injected with mixtures containing more do not.

Determination of the test dose of venom. Prepare graded dilutions of the reconstituted venom in a 9 g/l solution of sodium chloride R or other isotonic diluent in such a manner that the middle dilution contains in 0.25 ml the dose expected to be the LD50. Dilute with an equal volume of the same diluent. Using at least four mice, each weighing 18 g to 20 g, for each dilution, inject 0.5 ml intravenously into each mouse. Observe the mice for 48 h and record the number of deaths. Calculate the LD50 using the usual statistical methods. Determination of the potency of the antiserum to be examined. Dilute the reconstituted test venom so that 0.25 ml contains the test dose of 5 LD50 (test venom solution). Prepare serial dilutions of the antiserum to be examined in a 9 g/l solution of sodium chloride R or other isotonic diluent, 01/2005:0145 the dilution factor being 1.5 to 2.5. Use a sufficient number and range of dilutions to enable a mortality curve between 20 per cent and 80 per cent mortality to be established and VIPER VENOM ANTISERUM, to permit an estimation of the statistical variation. Prepare mixtures such that 5 ml of each mixture contains EUROPEAN 2.5 ml of one of the dilutions of the antiserum to be examined and 2.5 ml of the test venom solution. Allow the mixtures to Immunoserum contra venena viperarum stand in a water-bath at 37 °C for 30 min. Using not fewer than six mice, each weighing 18 g to 20 g, for each mixture, europaearum inject 0.5 ml intravenously into each mouse. Observe the DEFINITION mice for 48 h and record the number of deaths. Calculate European viper venom antiserum is a preparation containing the PD50, using the usual statistical methods. At the same time verify the number of LD50 in the test dose of venom, antitoxic globulins that have the power of neutralising the using the method described above. Calculate the potency of venom of one or more species of viper. The globulins are obtained by fractionation of the serum of animals that have the antiserum from the expression : been immunised against the venom or venoms. IDENTIFICATION It neutralises the venom of Vipera ammodytes, or Vipera aspis, or Vipera berus, or Vipera ursinii or the mixture of these venoms stated on the label, rendering them harmless to susceptible animals.

Tv

=

number of LD50 in the test dose of venom.

In each mouse dose of the venom-antiserum mixture at the end point there is one LD50 of venom remaining unneutralised by the antiserum and it is this unneutralised ASSAY venom that is responsible for the deaths of 50 per cent of the mice inoculated with the mixture. The amount of venom Each millilitre of the preparation to be examined contains neutralised by the antiserum is thus one LD50 less than the sufficient antitoxic globulins to neutralise not less than total amount contained in each mouse dose. Therefore, 100 mouse LD50 of Vipera ammodytes venom or Vipera aspis venom and not less than 50 mouse LD50 of the venoms as the potency of the antiserum is defined in terms of the number of LD50 of venom that are neutralised. rather than of other species of viper. the number of LD50 in each mouse dose, the expression The potency of European viper venom antiserum is required in the calculation of potency is Tv − 1 rather than Tv. determined by estimating the dose necessary to protect mice Alternatively, the quantity of test venom in milligrams that against the lethal effects of a fixed dose of venom of the is neutralised by 1 ml or some other defined volume of the relevant species of viper. antiserum to be examined may be calculated. Selection of test venoms. Use venoms which have the LABELLING normal physicochemical, toxicological and immunological characteristics of venoms from the particular species of The label states the venom or venoms against which the vipers. They are preferably freeze-dried and stored in the antiserum is effective. dark at 5 ± 3 °C. Warning : Because of the allergenic properties of viper venoms, inhalation of venom dust should be avoided by Select a venom for use as a test venom by determining the suitable precautions. LD50 for mice, the observation period being 48 h.

806


Clostridium perfringens beta antitoxin for veterinary use


01/2005:0340 intra-peritoneally into each mouse. Observe the mice for 72 h. If all the mice die, the amount of toxin present in CLOSTRIDIUM PERFRINGENS BETA 0.5 ml of the mixture is in excess of the test dose. If none of mice dies, the amount of toxin present in 0.5 ml of the ANTITOXIN FOR VETERINARY USE the mixture is less than the test dose. Prepare fresh mixtures such that 5.0 ml of each mixture contains 2.0 ml of the Immunoserum clostridii perfringentis beta solution of the reference preparation (10 IU) and one of a series of graded volumes of the solution of the test toxin ad usum veterinarium separated from each other by steps of not more than 20 per DEFINITION cent and covering the expected end-point. Allow the mixtures to stand at room temperature for 30 min. Clostridium perfringens beta antitoxin for veterinary use is a preparation containing principally the globulins that have Using not fewer than two mice for each mixture, inject a dose the power of specifically neutralising the beta toxin formed of 0.5 ml intravenously or intraperitoneally into each mouse. by Clostridium perfringens (types B and C). It consists of Observe the mice for 72 h. Repeat the determination at least the serum or a preparation obtained from the serum of once and combine the results of the separate tests that have animals immunised against Cl. perfringens beta toxin. been made with mixtures of the same composition so that a series of totals is obtained, each total representing the IDENTIFICATION mortality due to a mixture of a given composition. The test It specifically neutralises the beta toxin of Cl. perfringens dose of toxin is the amount present in 0.5 ml of that mixture rendering it harmless to susceptible animals. which causes the death of one half of the total number of mice injected with it. POTENCY The potency of the crude serum is not less than 1000 IU/ml Determination of the potency of the antitoxin to be examined when obtained from horses and not less than 250 IU/ml Preliminary test. Dissolve a quantity of the test toxin in a when obtained from cattle. suitable liquid so that 2.0 ml contains 10 times the test dose. The potency of the concentrated serum is not less than Prepare mixtures of the solution of the test toxin and the 3000 IU/ml when obtained from horses and not less than antitoxin to be examined such that each mixture contains 1000 IU/ml when obtained from cattle. The International Unit is the specific neutralising activity for 2.0 ml of the solution of the test toxin, one of a series of graded volumes of the antitoxin to be examined and sufficent Cl. perfringens beta toxin contained in a stated amount of of a suitable liquid to bring the final volume to 5.0 ml. Allow the International Standard, which consists of a quantity of dried immune horse serum. The equivalence in International the mixtures to stand at room temperature for 30 min. Using not fewer than two mice for each mixture, inject a dose of Units of the International Standard is stated by the World 0.5 ml intravenously or intraperitoneally into each mouse. Health Organisation. Observe the mice for 72 h. If none of the mice dies, 0.5 ml The potency of clostridium perfringens beta antitoxin is of the mixture contains more than 1 IU. If all the mice die, determined by comparing the dose necessary to protect 0.5 ml of the mixture contains less than 1 IU. mice or other suitable animals against the toxic effects of a Final test. Prepare mixtures of the solution of the test fixed dose of Cl. perfringens beta toxin with the quantity toxin and of the antitoxin to be examined such that 5.0 ml of a reference preparation of clostridium perfringens beta of each mixture contains 2.0 ml of the solution of the test antitoxin, calibrated in International Units, necessary to toxin and one of a series of graded volumes of the antitoxin give the same protection. For this comparison, a suitable to be examined, separated from each other by steps of not preparation of Cl. perfringens beta toxin for use as a test more than 20 per cent and covering the expected end-point toxin is required. The dose of the test toxin is determined as determined by the preliminary test. Prepare further in relation to the reference preparation ; the potency of the mixtures such that 5.0 ml of each mixture contains 2.0 ml of clostridium perfringens beta antitoxin to be examined is the solution of the test toxin and one of a series of graded determined in relation to the reference preparation using volumes of the solution of the reference preparation, in order the test toxin. to confirm the test dose of the toxin. Allow the mixtures Preparation of test toxin. Prepare the test toxin from to stand at room temperature for 30 min. Using not fewer a sterile filtrate of an early culture in liquid medium of than two mice for each mixture, proceed as described in the Cl. perfringens type B or type C and dry by a suitable preliminary test. The test mixture which contains, in 0.5 ml, method. 1 IU is that mixture which kills the same or almost the Select the test toxin by determining for mice the L+ dose same number of mice as the reference mixture containing, and the LD50, the observation period being 72 h. A suitable in 0.5 ml, 1 IU. Repeat the determination at least once and beta toxin contains not less than one L+ dose in 0.2 mg and calculate the average of all valid estimates. Estimates are not less than 25 LD50 in each L+ dose. valid only if the reference preparation gives a result within 20 per cent of the expected value. Determination of test dose of toxin. Prepare a solution of the reference preparation in a suitable liquid so that it The confidence limits (P = 0.95) have been estimated to be : contains 5 IU/ml. Prepare a solution of the test toxin in 85 per cent and 114 per cent when two animals per dose a suitable liquid so that 1 ml contains a precisely known are used, amount such as 10 mg. Prepare mixtures of the solution of 91.5 per cent and 109 per cent when four animals per dose the reference preparation and the solution of the test toxin are used, such that each mixture contains 2.0 ml of the solution of 93 per cent and 108 per cent when six animals per dose are the reference preparation (10 IU), one of a series of graded used. volumes of the solution of the test toxin and sufficient of a suitable liquid to bring the total volume to 5.0 ml. Allow LABELLING the mixtures to stand at room temperature for 30 min. Using not fewer than two mice, each weighing 17 g to 22 g, The label states whether the product consist of crude concentrated serum. for each mixture, inject a dose of 0.5 ml intravenously or 810



Clostridium perfringens epsilon antitoxin for veterinary use

01/2005:0341 for 30 min. Using not fewer than two mice, each weighing 17 g to 22 g, for each mixture, inject a dose of 0.5 ml intravenously or intraperitoneally into each mouse. Observe CLOSTRIDIUM PERFRINGENS the mice for 72 h. If all the mice die, the amount of toxin EPSILON ANTITOXIN FOR present in 0.5 ml of the mixture is in excess of the test dose. If none of the mice dies, the amount of toxin present in VETERINARY USE 0.5 ml of the mixture is less than the test dose. Prepare similar fresh mixtures such that 5.0 ml of each mixture contains 2.0 ml of the solution of the reference preparation (1 IU) and one of a series of graded volumes of the solution of the test toxin, separated from each other by DEFINITION steps of not more than 20 per cent and covering the expected end-point. Allow the mixtures to stand at room temperature Clostridium perfringens epsilon antitoxin for veterinary for 30 min. Using not fewer than two mice for each mixture, use is a preparation containing the globulins that have the power of specifically neutralising the epsilon toxin formed by inject a dose of 0.5 ml intravenously or intraperitoneally Clostridium perfringens type D. It consists of the serum or a into each mouse. Observe the mice for 72 h. Repeat the preparation obtained from the serum of animals immunised determination at least once and add together the results of the separ-ate tests that have been made with mixtures of against Cl. perfringens epsilon toxin. the same composition so that a series of totals is obtained, each total representing the mortality due to a mixture of a IDENTIFICATION given composition. The test dose of the toxin is the amount It specifically neutralises the epsilon toxin of Cl. perfringens present in 0.5 ml of that mixture which causes the death of type D, rendering it harmless to susceptible animals. one half of the total number of mice injected with it. Determination of the potency of the antitoxin to be POTENCY The potency of the crude serum is not less than 120 IU/ml examined when obtained from horses and not less than 100 IU/ml Preliminary test. Dissolve a quantity of the test toxin in a when obtained from cattle. suitable liquid so that 2.0 ml contains 10 times the test dose (solution of the test toxin). Prepare mixtures of the solution The potency of the concentrated serum is not less than of the test toxin and of the antitoxin to be examined such 300 IU/ml when obtained from horses and not less than that each mixture contains 2.0 ml of the solution of the test 150 IU/ml when obtained from cattle. toxin, one of a series of graded volumes of the antitoxin to The International Unit is the specific neutralising activity for be examined and sufficient of a suitable liquid to bring the Cl. perfringens epsilon toxin contained in a stated amount final volume to 5.0 ml. Allow the mixtures to stand at room of the International Standard, which consists of a quantity of temperature for 30 min. Using not fewer than two mice dried immune horse serum. The equivalence in International for each mixture, inject a dose of 0.5 ml intravenously or Units of the International Standard is stated by the World intra-peritoneally into each mouse. Observe the mice for Health Organisation. 72 h. If none of the mice dies, 0.5 ml of the mixture contains more than 0.1 IU. If all the mice die, 0.5 ml of the mixture The potency of clostridium perfringens epsilon antitoxin is determined by comparing the dose necessary to protect mice contains less than 0.1 IU. or other suitable animals against the toxic effects of a fixed Final test. Prepare mixtures of the solution of the test dose of Cl. perfringens epsilon toxin with the quantity of toxin and of the antitoxin to be examined such that 5.0 ml a reference preparation of clostridium perfringens epsilon of each mixture contains 2.0 ml of the solution of the test anti-toxin, calibrated in International Units, necessary to toxin and one of a series of graded volumes of the antitoxin give the same protection. For this comparison, a suitable to be examined, separated from each other by steps of not preparation of Cl. perfringens epsilon toxin for use as a test more than 20 per cent and covering the expected end-point toxin is required. The dose of the test toxin is determined as determined by the preliminary test. Prepare further in relation to the reference preparation, the potency of the mixtures such that 5.0 ml of each mixture contains 2.0 ml of antitoxin to be examined is determined in relation to the the solution of the test toxin and one of a series of graded reference preparation using the test toxin. volumes of the solution of the reference preparation to confirm the test dose of the toxin. Allow the mixtures to Preparation of test toxin. Prepare the test toxin from stand at room temperature for 30 min. Using not fewer a sterile filtrate of an early culture in liquid medium of than two mice for each mixture proceed as described in the Cl. perfringens type D and dry by a suitable method. Select the test toxin by determining for mice the L+/10 dose preliminary test. The test mixture which contains 0.1 IU in and the LD50, the observation period being 72 h. A suitable 0.5 ml is that mixture which kills the same or almost the same number of mice as the reference mixture containing epsilon toxin contains not less than one L+/10 dose in 0.1 IU in 0.5 ml. Repeat the determination at least once and 0.005 mg and not less than 20 LD50 in each L+/10 dose. calculate the average of all valid estimates. Estimates are Determination of test dose of toxin. Prepare a solution valid only if the reference preparation gives a result within of the reference preparation in a suitable liquid so that it 20 per cent of the expected value. contains 0.5 IU of antitoxin per millilitre. Prepare a solution The confidence limits (P = 0.95) have been estimated to be : of the test toxin in a suitable liquid so that 1 ml contains a precisely known amount such as 1 mg. Prepare mixtures of 85 per cent and 114 per cent when 2 animals per dose are the solution of the reference preparation and the solution used, of the test toxin such that each mixture contains 2.0 ml of 91.5 per cent and 109 per cent when 4 animals per dose the solution of the reference preparation (1 IU), one of a series of graded volumes of the solution of the test toxin and are used, 93 per cent and 108 per cent when 6 animals per dose are sufficient of a suitable liquid to bring the total volume to used. 5.0 ml. Allow the mixtures to stand at room temperature

Immunoserum clostridii perfringentis epsilon ad usum veterinarium


811

Tetanus antitoxin for veterinary use


Lp/10 dose (Limes paralyticum). This is the smallest quantity of toxin which when mixed with 0.1 IU of antitoxin and injected subcutaneously into mice (or guinea-pigs) causes tetanic paralysis in the animals on or before the fourth day after injection. 01/2005:0343 Paralytic dose 50 per cent. This is the quantity of toxin which when injected subcutaneously into mice (or guinea-pigs) causes tetanic paralysis in one half of the TETANUS ANTITOXIN FOR animals on or before the fourth day after injection. VETERINARY USE Determination of test dose of toxin. Reconstitute or dilute the reference preparation with a suitable liquid so that it Immunoserum tetanicum contains 0.5 IU per millilitre. Measure or weigh a quantity of the test toxin and dilute with or dissolve in a suitable liquid. ad usum veterinarium Prepare mixtures of the solution of the reference preparation and the solution of the test toxin so that each mixture will DEFINITION contain 0.1 IU of antitoxin in the volume chosen for injection Tetanus antitoxin for veterinary use is a preparation and one of a series of graded volumes of the solution of the containing principally the globulins that have the power test toxin, separated from each other by steps of not more of specifically neutralising the neurotoxin formed by Clostridium tetani. It consists of the serum or a preparation than 20 per cent and covering the expected end-point. Adjust each mixture with a suitable liquid to the same final volume obtained from the serum of animals immunised against (0.4 ml to 0.6 ml if mice are used for the test or 4.0 ml if tetanus toxin. guinea-pigs are used). Allow the mixtures to stand at room temperature for 60 min. Using not fewer than two animals IDENTIFICATION It specifically neutralises the neurotoxin formed by Cl. tetani, for each mixture, inject the chosen volume subcutaneously into each animal. Observe the animals for 96 h and make rendering it harmless to susceptible animals. daily records of the degree of tetanus developing in each group of animals. Repeat the test at least once and calculate POTENCY The potency of the crude serum is not less than 300 IU/ml the test dose as the mean of the different tests. The test dose of the toxin is the amount present in that mixture which when obtained from horses and not less than 150 IU/ml causes tetanic paralysis in one half of the total number of when obtained from cattle. animals injected with it. The potency of the concentrated serum is not less than When the test dose of the toxin has been determined a 1000 IU/ml when obtained from horses and not less than concentrated solution of the test toxin may be prepared 500 IU/ml when obtained from cattle. in a mixture consisting of 1 volume of a 9 g/l solution of The International Unit is the specific neutralising activity sodium chloride R and 1 or 2 volumes of glycerol R. This for tetanus toxin contained in a stated amount of the concentrated solution may be stored frozen and diluted as International Standard which consists of a quantity of dried immune horse serum. The equivalence in International Units required. The specific activity of such a solution should be of the International Standard is stated by the World Health redetermined at frequent intervals. Organisation. Determination of the potency of the antitoxin to be The potency of tetanus antitoxin is determined by comparing examined the dose necessary to protect mice (or guinea-pigs) against Preliminary test. Measure or weigh a quantity of the test the toxic effects of a fixed dose of tetanus toxin with the toxin and dilute with or dissolve in a suitable liquid so that quantity of a reference preparation of tetanus antitoxin, the solution contains five test doses per millilitre (solution calibrated in International Units, necessary to give the same of the test toxin). Prepare mixtures of the solution of the protection. In countries where the paralysis method is not test toxin and the antitoxin to be examined so that for each obligatory, the lethal method may be used. For this method mixture the volume chosen for injection contains the test the number of animals and the procedure are identical with dose of toxin and one of a series of graded volumes of the those described for the paralysis method but the end-point antitoxin to be examined. Adjust each mixture to the same is the death of the animal rather than the onset of paralysis final volume with a suitable liquid. Allow the mixtures to and the L+/10 dose and the LD50 are used instead of the stand at room temperature for 60 min. Using not fewer than Lp/10 dose and the paralytic dose 50 per cent. For this two animals for each mixture, inject the chosen volume comparison, a suitable preparation of tetanus toxin for subcutaneously into each animal. Observe the animals use as a test toxin is required. The dose of the test toxin for 96 h and make daily records of the degree of tetanus is determined in relation to the reference preparation ; the developing in each group of animals. Using the results, potency of the antitoxin to be examined is determined in select suitable mixtures for the final test. relation to the reference preparation using the test toxin. Final test. Prepare mixtures of the solution of the test toxin Preparation of test toxin. Prepare the test toxin from a and the antitoxin to be examined so that for each mixture sterile filtrate of an 8 to 10 day culture in liquid medium the volume chosen for the injection contains the test dose of of Cl. tetani. A test toxin may be prepared by adding this toxin and one of a series of graded volumes of the antitoxin to filtrate to glycerol R in the proportion of 1 volume of filtrate be examined, separated from each other by steps of not more to 1 to 2 volumes of glycerol R. The solution of test toxin than 20 per cent and covering the expected end-point as may be stored at or slightly below 0 °C. The toxin may also determined in the preliminary test. Prepare further mixtures be dried by a suitable method. with the same amount of test toxin and graded volumes of Select the test toxin by determining for mice the Lp/10 dose the reference preparation, centred on 0.1 IU in the volume and the paralytic dose 50 per cent. A suitable toxin contains chosen for injection, to confirm the test dose of the toxin. Adjust each mixture to the same final volume with a suitable not less than 1000 times the paralytic dose 50 per cent in liquid. Allow the mixtures to stand at room temperature for one Lp/10 dose. LABELLING The label states whether the product consists of crude or concentrated serum.

812



60 min. Using not fewer than two animals for each mixture, inject the chosen volume subcutaneously into each animal. Observe the animals for 96 h and make daily records of the degree of tetanus developing in each group of animals. The test mixture which contains 0.1 IU in the volume injected is that mixture which causes tetanic paralysis in the same, or almost the same, number of animals as the reference mixture containing 0.1 IU in the volume injected. Repeat the determination at least once and calculate the mean of all valid estimates. Estimates are valid only if the reference preparation gives a result within 20 per cent of the expected value.


Tetanus antitoxin for veterinary use

The confidence limits (P = 0.95) have been estimated to be : 85 per cent and 114 per cent when 2 animals per dose are used, 91.5 per cent and 109 per cent when 3 animals per dose are used, 93 per cent and 108 per cent when 6 animals per dose are used. LABELLING The label states whether the product consists of crude or concentrated serum.

813


Ammonia (13N) injection

01/2005:1492 CHEMICAL PURITY Aluminium. In a test-tube about 12 mm in internal diameter, mix 1 ml of acetate buffer solution pH 4.6 R and 2 ml of a 1 AMMONIA (13N) INJECTION in 20 dilution of the preparation to be examined in water R. Add 0.05 ml of a 10 g/l solution of chromazurol S R. After Ammoniae (13N) solutio iniectabilis 3 min, the colour of the solution is not more intense than that of a standard prepared at the same time and in the DEFINITION same manner using 2 ml of a 1 in 20 dilution of aluminium Ammonia (13N) injection is a sterile solution of [13N]ammonia standard solution (2 ppm Al) R (2 ppm). for diagnostic use. The injection contains not less than The injection may be released for use before completion of 90.0 per cent and not more than 110.0 per cent of the the test. declared nitrogen-13 radioactivity at the date and time RADIONUCLIDIC PURITY stated on the label. Not less than 99 per cent of the total (a) Half-life. The half-life is between 9 min and 11 min. radioactivity corresponds to nitrogen-13 in the form of [13N]ammonia. Not less than 99.0 per cent of the total (b) Gamma emitting impurities. Retain a sample of the radioactivity corresponds to nitrogen-13. preparation to be examined for 2 h. Examine the gamma-ray spectrum of the decayed material for the presence of PRODUCTION radionuclidic impurities, which should, where possible, be identified and quantified. The total gamma radioactivity due RADIONUCLIDE PRODUCTION to these impurities does not exceed 1.0 per cent of the total Nitrogen-13 is a radioactive isotope of nitrogen which may radioactivity. be produced by various nuclear reactions, such as proton irradiation of carbon-13 or oxygen-16, or deuteron irradiation The injection may be released for use before completion of of carbon-12. tests (a) and (b). RADIOCHEMICAL SYNTHESIS RADIOCHEMICAL PURITY [13N]Ammonia may be prepared by proton irradiation Examine by liquid chromatography (2.2.29). of water followed by the reduction of the resulting Test solution. The preparation to be examined. [13N]nitrates/nitrites mixture with a reducing agent. The 13 Reference solution. Dilute 1.0 ml of dilute ammonia R2 to [ N]ammonia formed is distilled from the reaction mixture 10.0 ml with water R. and trapped in a slightly acidic solution. 13 The chromatographic procedure may be carried out using : Other methods may produce [ N]ammonia “in-target” by proton irradiation of water containing a small amount of — a column 0.04 m long and 4.0 mm in internal diameter ethanol or acetic acid, or by proton irradiation of a slurry packed with cation exchange resin R (10 µm), of [13C]carbon powder in water. The resulting solution can — as mobile phase at a flow rate of 2 ml/min 0.002 M nitric be purified, to remove radionuclidic and radiochemical acid, impurities, using anion and cation exchange columns. — a suitable radioactivity detector, CHARACTERS — a conductivity detector, A clear, colourless solution. — a loop injector, Nitrogen-13 has a half-life of 9.96 min and emits positrons maintaining the column at a constant temperature between with a maximum energy of 1.198 MeV, followed by 20 °C and 30 °C. annihilation gamma radiation of 0.511 MeV. Inject separately the test solution and the reference solution. The chromatogram obtained with the radioactivity IDENTIFICATION detector and the test solution shows a principal peak with A. Record the gamma-ray spectrum using a suitable approximately the same retention time as the peak in the instrument. The only gamma photons have an energy of chromatogram obtained with the reference solution and 0.511 MeV and, depending on the measurement geometry, the conductivity detector. Not less than 99 per cent of the a sum peak of 1.022 MeV may be observed. total radioactivity corresponds to nitrogen-13 in the form B. It complies with test (a) for radionuclidic purity (see of ammonia. Tests). The injection may be released for use before completion of C. Examine the chromatograms obtained in the test the test. for radiochemical purity. The principal peak in the RADIOACTIVITY radiochromatogram obtained with the test solution has approximately the same retention time as the principal Measure the radioactivity using suitable equipment by peak in the radiochromatogram obtained with the comparison with a standardised fluorine-18 solution or reference solution. by using an instrument calibrated with the aid of such a solution. Standardised fluorine-18 solutions are available TESTS from laboratories recognised by the competent authority. pH (2.2.3). The pH of the injection is 5.5 to 8.5. IMPURITIES Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical A. [13N]O2−, preparations (0125). The injection may be released for use before completion of the test. B. [13N]O3−, Bacterial endotoxins (2.6.14) : less than 175/V IU/ml, V being the maximum recommended dose in millilitres. The C. [18F−], injection may be released for use before completion of the D. H2[15O]. test. General Notices (1) apply to all monographs and other texts

817

Carbon monoxide (15O)


01/2005:1607 Injection : test sample, reference gas (b). Examine the chromatogram obtained with the thermal conductivity detector and calculate the content of carbon CARBON MONOXIDE (15O) monoxide. 15 Carbonei monoxidum ( O) RADIONUCLIDIC PURITY Oxygen-15 : minimum 99 per cent of the total radioactivity. DEFINITION Mixture of carbon [15O]monoxide in the gaseous phase and a A. Gamma spectrometry. Comparison : standardised fluorine-18 solution, or by suitable vehicle such as Medicinal air (1238), for diagnostic using an instrument calibrated with the aid of such a use. solution. Standardised fluorine-18 solutions and/or Purity : standardisation services are available from the competent — minimum 99 per cent of the total radioactivity authority. corresponds to oxygen-15, Results : the spectrum obtained with the solution to be — minimum 97 per cent of the total radioactivity examined does not differ significantly from that obtained corresponds to oxygen-15 in the form of carbon monoxide with a standardised fluorine-18 solution. (CO). B. Half-life : 1.9 min to 2.2 min. PRODUCTION The preparation may be released for use before completion of the test. RADIONUCLIDE PRODUCTION Oxygen-15 is a radioactive isotope of oxygen which may RADIOCHEMICAL PURITY be produced by various nuclear reactions such as proton Carbon [15O]monoxide. Gas chromatography (2.2.28) : use irradiation of nitrogen-15 or deuteron irradiation of the normalisation procedure. nitrogen-14. Test sample. Carbon [15O]monoxide as described under RADIOCHEMICAL SYNTHESIS radiochemical synthesis. In order to recover oxygen-15 as molecular oxygen from the nitrogen target gas, carrier oxygen is added at concentrations Reference gas (a). Nitrogen gas mixture R. generally ranging from 0.2 per cent V/V to 1.0 per cent V/V. Reference gas (b). Nitrogen R, containing 2.0 per cent V/V of carbon monoxide R1. After irradiation, the target gas is usually reacted with activated charcoal at a temperature of about 950 °C. The Column : activated charcoal is preconditioned before use by flushing — size : l = 1.8 m, Ø1 = 6.3 mm and Ø2 = 3.2 mm, an inert gas at the production flow rate at a temperature of 15 about 950 °C for not less than 1 h. The carbon [ O]monoxide — stationary phase : GC concentrical column R, Carrier gas : helium for chromatography R. obtained is purified by passage through a carbon dioxide scavenger, such as soda lime, before mixing with the vehicle. Flow rate : 65 ml/min. Temperature : CHARACTERS — column : 40 °C, Appearance : colourless gas. Half-life and nature of radiation of oxygen-15 : see Table of — injection port : 40 °C, — thermal conductivity detector : 70 °C. physical characteristics of radionuclides (5.7). Detection : thermal conductivity detector and radioactivity IDENTIFICATION detector connected in series. A. Gamma spectrometry. Injection : loop injector. Results : the only gamma photons have an energy of 0.511 MeV and, depending on the measurement geometry, Run time : 10 min. Retention times : oxygen, nitrogen and carbon monoxide a sum peak of 1.022 MeV may be observed. eluting from the inner column = about 0.4 min ; carbon B. It complies with the test for radionuclidic purity (see dioxide eluting from the inner column = about 0.8 min ; Tests). oxygen eluting from the outer column = about 2.1 min ; C. Examine the chromatograms obtained in the test for nitrogen eluting from the outer column = about 3.1 min ; radiochemical purity. carbon monoxide eluting from the outer column = about Results : the principal peaks in the chromatogram 6.2 min. obtained with the test gas using the radioactivity detector System suitability : reference gas (a) : are similar in retention times to the principal peaks corresponding to carbon monoxide in the chromatogram — 5 clearly separated principal peaks are observed in the chromatogram obtained using the thermal conductivity obtained with reference gas (a) using the thermal detector, conductivity detector. — resolution : minimum of 1.5 between the peaks due to TESTS carbon dioxide eluting from the inner column and oxygen eluting from the outer column, in the chromatogram The following tests are performed on carbon [15O]monoxide obtained using the thermal conductivity detector. as described under radiochemical synthesis before mixing with the vehicle. Limits : examine the chromatogram obtained with the radioactivity detector and calculate the percentage content Carbon monoxide. Gas chromatography (2.2.28) as of oxygen-15 substances from the peak areas. described in the test for radiochemical purity. 15 The concentration of carbon monoxide in the test sample is — carbon [ O]monoxide : minimum 97 per cent of the total radioactivity. determined before administration and is used to calculate the amount of carbon monoxide to be administered to the — disregard the first peak corresponding to components patient. co-eluting from the inner column. 818



Cyanocobalamin (57Co) capsules

RADIOACTIVITY The radioactive concentration is determined before administration. Measure the radioactivity using suitable equipment by comparison with a standardised fluorine-18 solution or by measurement in an instrument calibrated with the aid of such a solution.

RADIONUCLIDIC PURITY Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised chromium-51 solution. RADIOCHEMICAL PURITY Examine by zone electrophoresis (2.2.31), using a paper strip as the support and a solution containing 0.2 g/l of barbital sodium R and 10 g/l of sodium nitrate R as the electrolyte solution. A paper with the following characteristics is 2 01/2005:0266 suitable : mass per unit area 120 g/m ; thickness 0.22 mm ; capillary rise 105 mm to 115 mm per 30 min. Apply to the paper 10 µl of the injection as a 3 mm band at CHROMIUM (51Cr) EDETATE a position 10 cm from the cathode. Apply an electric field of about 30 V per centimetre for 30 min using a stabilised INJECTION current. [51Cr]chromium edetate moves about 5 cm towards the anode. [51Cr]Chromate moves about 10 cm towards the Chromii (51Cr) edetatis solutio iniectabilis anode and [51Cr]chromic ion moves about 7 cm towards the cathode. Determine the distribution of the radioactivity DEFINITION using a suitable detector. Not less than 95 per cent of the 51 Chromium ( Cr) edetate injection is a sterile solution total radioactivity is found in the band corresponding to containing chromium-51 in the form of a complex of [51Cr]chromium edetate. chromium(III) with ethylenediaminetetraacetic acid, the latter being present in excess. It may be made isotonic RADIOACTIVITY by the addition of sodium chloride and may contain a Measure the radioactivity using suitable equipment by suitable antimicrobial preservative such as benzyl alcohol. Chromium-51 is a radioactive isotope of chromium and may comparison with a standardised chromium-51 solution or be prepared by the neutron irradiation of chromium, either by measurement in an instrument calibrated with the aid of of natural isotopic composition or enriched in chromium-50. such a solution. The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the declared chromium-51 radioactivity at the date and hour stated on the label. Not 01/2005:0710 less than 95 per cent of the radioactivity corresponds to chromium-51 in the form of chromium edetate. The injection contains a variable quantity of chromium (Cr) not exceeding CYANOCOBALAMIN (57Co) CAPSULES 1 mg per millilitre. CHARACTERS A clear, violet solution. Chromium-51 has a half-life of 27.7 days and emits gamma radiation. IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised chromium-51 solution. Standardised chromium-51 solutions are available from laboratories recognised by the competent authority. The gamma photon has an energy of 0.320 MeV. B. Examine the electropherogram obtained in the test for radiochemical purity. The distribution of radioactivity contributes to the identification of the preparation. TESTS pH (2.2.3). The pH of the solution is 3.5 to 6.5. Chromium. Prepare a reference solution (1 mg per millilitre of Cr) as follows : dissolve 0.96 g of chromic potassium sulphate R and 2.87 g of sodium edetate R in 50 ml of water R, boil for 10 min, cool, adjust to pH 3.5 to 6.5 using dilute sodium hydroxide solution R and dilute to 100.0 ml with water R. Measure the absorbance (2.2.25) of the injection to be examined and the reference solution at the absorption maximum at 560 nm. The absorbance of the injection to be examined is not greater than that of the reference solution. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. General Notices (1) apply to all monographs and other texts

Cyanocobalamini (57Co) capsulae DEFINITION Cyanocobalamin (57Co) capsules contain [57Co]-α-(5,6dimethylbenzimidazol-1-yl)cobamide cyanide and may contain suitable auxiliary substances. Cobalt-57 is a radioactive isotope of cobalt and may be produced by proton irradiation of nickel. Cyanocobalamin (57Co) may be prepared by the growth of suitable micro-organisms on a medium containing (57Co) cobaltous ion. Not less than 90 per cent of the cobalt-57 is in the form of cyanocobalamin. The capsules comply with the requirements for hard capsules in the monograph on Capsules (0016), unless otherwise justified and authorised. CHARACTERS Hard gelatin capsules. Cobalt-57 has a half-life of 271 days and emits gamma radiation. IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised cobalt-57 solution. Standardised cobalt-57 and cobalt-58 solutions are available from laboratories recognised by the competent authority. The most prominent gamma photon of cobalt-57 has an energy of 0.122 MeV. B. Examine the chromatograms obtained in the test for radiochemical purity. The principal peak in the radio-chromatogram obtained with the test solution has a retention time similar to that of the peak in the chromatogram obtained with the reference solution. 819

Cyanocobalamin (57Co) solution


01/2005:0269

TESTS Disintegration. The capsules comply with the test for disintegration of tablets and capsules (2.9.1) except that one capsule is used in the test instead of six. Uniformity of content. Determine by measurement in a suitable counting assembly and under identical geometrical conditions the radioactivity of each of not less than ten capsules. Calculate the average radioactivity per capsule. The radioactivity of no capsule differs by more than 10 per cent from the average. The relative standard deviation is less than 3.5 per cent.

CYANOCOBALAMIN (57Co) SOLUTION Cyanocobalamini (57Co) solutio DEFINITION

Cyanocobalamin (57Co) solution is a solution of [57Co]-α-(5,6-dimethylbenzimidazol-1-yl)cobamide cyanide and may contain a stabiliser and an antimicrobial preservative. Cobalt-57 is a radioactive isotope of cobalt and RADIONUCLIDIC PURITY may be produced by the irradiation of nickel with protons of Record the gamma-ray spectrum using a suitable 57 instrument calibrated with the aid of standardised cobalt-57 suitable energy. Cyanocobalamin ( Co) may be prepared by the growth of suitable micro-organisms on a medium and cobalt-58 solutions. The spectrum does not differ 57 containing ( Co) cobaltous ion. The solution contains not significantly from that of the stan-dardised cobalt-57 solution. less than 90.0 per cent and not more than 110.0 per cent Determine the relative amounts of cobalt-57, cobalt-56 and cobalt-58 present. Cobalt-56 has a half-life of 78 days and its of the declared cobalt-57 radioactivity at the date stated on presence is shown by gamma photons of energy 0.847 MeV. the label. Not less than 90 per cent of the cobalt-57 is in the form of cyanocobalamin. Cobalt-58 has a half-life of 70.8 days and its presence is shown by gamma photons of energy 0.811 MeV. Not more than 0.1 per cent of the total radioactivity is due to cobalt-56, CHARACTERS cobalt-58 and other radionuclidic impurities. A clear, colourless or slightly pink solution. Cobalt-57 has a half-life of 271 days and emits gamma radiation. RADIOCHEMICAL PURITY Examine by liquid chromatography (2.2.29). Test solution. Dissolve the contents of a capsule in 1.0 ml of IDENTIFICATION water R and allow to stand for 10 min. Centrifuge at 2000 A. Record the gamma-ray spectrum using a suitable r/min for 10 min. Use the supernatant. instrument. The spectrum does not differ significantly from that of a standardised cobalt-57 solution. Reference solution. Dissolve 10 mg of cyanocobalamin CRS Standardised cobalt-57 and cobalt-58 solutions are in the mobile phase and dilute to 100 ml with the mobile available from laboratories recognised by the competent phase. Dilute 2 ml of the solution to 100 ml with the mobile authority. The most prominent gamma photon of phase. Use within 1 h. cobalt-57 has an energy of 0.122 MeV. The chromatographic procedure may be carried out using : B. Examine the chromatograms obtained in the test for radiochemical purity. The principal peak in the — a stainless steel column 0.25 m long and 4 mm in radiochromatogram obtained with the solution to be internal diameter packed with octylsilyl silica gel for examined has a retention time similar to that of the chromatography R (5 µm), peak in the chromatogram obtained with the reference solution. — as mobile phase at a flow rate of 1.0 ml/min a mixture prepared as follows : mix 26.5 volumes of methanol R and 73.5 volumes of a 10 g/l solution of disodium hydrogen TESTS phosphate R adjusted to pH 3.5 using phosphoric acid R pH (2.2.3). The pH of the solution is 4.0 to 6.0. and use within 2 days, RADIONUCLIDIC PURITY — a radioactivity detector adjusted for cobalt-57, Record the gamma-ray spectrum using a suitable instrument calibrated with the aid of standardised cobalt-57 and — as detector a spectrophotometer set at 361 nm, cobalt-58 solutions. The spectrum does not differ — a loop injector. significantly from that of the standardised cobalt-57 solution. Determine the relative amounts of cobalt-57, cobalt-56 and Inject 100 µl of the test solution and record the chromatogram cobalt-58 present. Cobalt-56 has a half-life of 78 days and its for three times the retention time of cyanocobalamin. presence is shown by gamma photons of energy 0.847 MeV. Determine the peak areas and calculate the percentage of Cobalt-58 has a half-life of 70.8 days and its presence is cobalt-57 present as cyanocobalamin. Inject 100 µl of the shown by gamma photons of energy 0.811 MeV. Not more reference solution and record the chromatogram for 30 min. than 0.1 per cent of the total radioactivity is due to cobalt-56, cobalt-58 and other radionuclidic impurities. RADIOACTIVITY RADIOCHEMICAL PURITY Examine by liquid chromatography (2.2.29). The average radioactivity determined in the test for Reference solution. Dissolve 10 mg of cyanocobalamin CRS uniformity of content is not less than 90.0 per cent and in the mobile phase and dilute to 100 ml with the mobile not more than 110.0 per cent of the declared cobalt-57 phase. Dilute 2 ml of the solution to 100 ml with the mobile radioactivity, at the date stated on the label. phase. Use within 1 h. The chromatographic procedure may be carried out using :

STORAGE Store in an airtight container, protected from light, at a temperature of 2 °C to 8 °C. 820

— a stainless steel column 0.25 m long and 4 mm in internal diameter packed with octylsilyl silica gel for chromatography R (5 µm),


Cyanocobalamin (58Co) capsules


TESTS Disintegration. The capsules comply with the test for disintegration of tablets and capsules (2.9.1) except that one capsule is used in the test instead of six. Uniformity of content. Determine by measurement in a suitable counting assembly and under identical geometrical conditions the radioactivity of each of not less than ten capsules. Calculate the average radioactivity per capsule. The radioactivity of no capsule differs by more than 10 per cent from the average. The relative standard deviation is less than 3.5 per cent. RADIONUCLIDIC PURITY Record the gamma-ray spectrum using a suitable instrument calibrated with the aid of standardised cobalt-58, cobalt-57 and cobalt-60 solutions. The spectrum does not differ RADIOACTIVITY significantly from that of the standardised cobalt-58 solution. Measure the radioactivity using suitable counting equipment Standardised cobalt-58, cobalt-57 and cobalt-60 solutions are available from laboratories recognised by the competent by comparison with a standardised cobalt-57 solution. authority. Determine the relative amounts of cobalt-58, cobalt-57 and cobalt-60 present. Cobalt-57 has a half-life of STORAGE Store protected from light at a temperature of 2 °C to 8 °C. 272 days and its presence is shown by gamma photons of energy 0.122 MeV. Cobalt-60 has a half-life of 5.27 years and its presence is shown by gamma photons of energies 1.173 MeV and 1.333 MeV. Not more than 1 per cent of the total radioactivity is due to cobalt-60 and not more than 2 per 01/2005:1505 cent of the total radioactivity is due to cobalt-57, cobalt-60 and other radionuclidic impurities. 58 CYANOCOBALAMIN ( Co) CAPSULES RADIOCHEMICAL PURITY Examine by liquid chromatography (2.2.29). Test solution. Dissolve the contents of a capsule in 1.0 ml Cyanocobalamini (58Co) capsulae of water R and allow to stand for 10 min. Centrifuge at 2000 r/min for 10 min. Use the supernatant. DEFINITION 58 58 Cyanocobalamin ( Co) capsules contain [ Co]-α-(5,6Reference solution. Dissolve 10 mg of cyanocobalamin CRS dimethylbenzimidazol-1-yl)cobamide cyanide and may in the mobile phase and dilute to 100 ml with the mobile contain suitable auxiliary substances. Cobalt-58 is a phase. Dilute 2 ml of the solution to 100 ml with the mobile radioactive isotope of cobalt and may be produced by phase. Use within 1 h of preparation. neutron irradiation of nickel. Cyanocobalamin (58Co) may The chromatographic procedure may be carried out using : be prepared by the growth of suitable micro-organisms on — a stainless steel column 0.25 m long and 4 mm in a medium containing (58Co) cobaltous ion. Not less than internal diameter packed with octylsilyl silica gel for 84 per cent of the cobalt-58 is in the form of cyanocobalamin. chromatography R (5 µm), The capsules comply with the requirements for hard capsules in the monograph on Capsules (0016), unless otherwise — as mobile phase at a flow rate of 1.0 ml/min a mixture justified and authorised. The average radioactivity is not prepared as follows : mix 26.5 volumes of methanol R and less than 90.0 per cent and not more than 110.0 per cent 73.5 volumes of a 10 g/l solution of disodium hydrogen of the declared cobalt-58 radioactivity at the date stated on phosphate R, adjusted to pH 3.5 with phosphoric acid R the label. and use within 2 days, — as mobile phase at a flow rate of 1.0 ml/min a mixture prepared as follows : mix 26.5 volumes of methanol R and 73.5 volumes of a 10 g/l solution of disodium hydrogen phosphate R adjusted to pH 3.5 using phosphoric acid R and use within 2 days, — a radioactivity detector adjusted for cobalt-57, — as detector a spectrophotometer set at 361 nm, — a loop injector. Inject 100 µl of the solution to be examined and record the chromatogram for three times the retention time of cyanocobalamin. Determine the peak areas and calculate the percentage of cobalt-57 present as cyanocobalamin. Inject 100 µl of the reference solution and record the chromatogram for 30 min.

CHARACTERS Hard gelatin capsules. Cobalt-58 has a half-life of 70.9 days and emits beta (β+) radiation and gamma radiation. IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised cobalt-58 solution. Standardised cobalt-58 solutions are available from laboratories recognised by the competent authority. The most prominent gamma photons of cobalt-58 have energies of 0.511 MeV (annihilation radiation) and 0.811 MeV. B. Examine the chromatograms obtained in the test for radiochemical purity. The principal peak in the radiochromatogram obtained with the test solution has a retention time similar to that of the peak in the chromatogram obtained with the reference solution. General Notices (1) apply to all monographs and other texts

— a radioactivity detector adjusted for cobalt-58, — as detector a spectrophotometer set at 361 nm, — a loop injector. Inject 100 µl of the test solution and record the chromatogram for three times the retention time of cyanocobalamin. Determine the peak areas and calculate the percentage of cobalt-58 present as cyanocobalamin. Inject 100 µl of the reference solution and record the chromatogram for 30 min. RADIOACTIVITY The average radioactivity determined in the test for uniformity of content is not less than 90.0 per cent and not more than 110.0 per cent of the declared cobalt-58 radioactivity, at the date stated on the label. STORAGE Store in an airtight container, protected from light, at a temperature of 2 °C to 8 °C. 821

Cyanocobalamin (58Co) solution


01/2005:0270 — as mobile phase at a flow rate of 1.0 ml/min a mixture prepared as follows : mix 26.5 volumes of methanol R and 58 73.5 volumes of a 10 g/l solution of disodium hydrogen CYANOCOBALAMIN ( Co) SOLUTION phosphate R adjusted to pH 3.5 using phosphoric acid R and use within 2 days, Cyanocobalamini (58Co) solutio — a radioactivity detector adjusted for cobalt-58, DEFINITION — as detector a spectrophotometer set at 361 nm, Cyanocobalamin (58Co) solution is a solution of — a loop injector. [58Co]-α-(5,6-dimethylbenzimidazol-1-yl)cobamide cyanide Inject 100 µl of the solution to be examined and record and may contain a stabiliser and an antimicrobial the chromatogram for three times the retention time of preservative. Cobalt-58 is a radioactive isotope of cobalt cyanocobalamin. Determine the peak areas and calculate and may be produced by neutron irradiation of nickel. the percentage of cobalt-58 present as cyanocobalamin. 58 Cyanocobalamin ( Co) may be prepared by the growth of Inject 100 µl of the reference solution and record the 58 suitable micro-organisms on a medium containing ( Co) chromatogram for 30 min. cobaltous ion. The solution contains not less than 90.0 per cent and not more than 110.0 per cent of the declared RADIOACTIVITY cobalt-58 radioactivity at the date stated on the label. Not Measure the radioactivity using suitable counting equipment less than 90 per cent of the cobalt-58 is in the form of by comparison with a standardised cobalt-58 solution or by cyanocobalamin. measurement in an instrument calibrated with the aid of such a solution. CHARACTERS A clear, colourless or slightly pink solution. STORAGE Cobalt-58 has a half-life of 70.8 days and emits beta (β+) Store protected from light at a temperature of 2 °C to 8 °C. radiation and gamma radiation. IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised cobalt-58 solution. Standardised cobalt-58, cobalt-57 and cobalt-60 solutions are available from laboratories recognised by the competent authority. The most prominent gamma photons of cobalt-58 have energies of 0.511 MeV (annihilation radiation) and 0.811 MeV. B. Examine the chromatograms obtained in the test for radiochemical purity. The principal peak in the radiochromatogram obtained with the solution to be examined has a retention time similar to that of the peak in the chromatogram obtained with the reference solution. TESTS pH (2.2.3). The pH of the solution is 4.0 to 6.0. RADIONUCLIDIC PURITY Record the gamma-ray spectrum using a suitable instrument having adequate resolution and calibrated with the aid of standardised cobalt-58, cobalt-57 and cobalt-60 solutions. The spectrum does not differ significantly from that of the standardised cobalt-58 solution. Determine the relative amounts of cobalt-58, cobalt-57 and cobalt-60 present. Cobalt-57 has a half-life of 271 days and its presence is shown by gamma photons of energy 0.122 MeV. Cobalt-60 has a half-life of 5.27 years and its presence is shown by gamma photons of energies 1.173 MeV and 1.332 MeV. Not more than 1 per cent of the total radioactivity is due to cobalt-60 and not more than 2 per cent of the total radioactivity is due to cobalt-57, cobalt-60 and other radionuclidic impurities. RADIOCHEMICAL PURITY Examine by liquid chromatography (2.2.29). Reference solution. Dissolve 10 mg of cyanocobalamin CRS in the mobile phase and dilute to 100 ml with the mobile phase. Dilute 2 ml of the solution to 100 ml with the mobile phase. Use within 1 h. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4 mm in internal diameter packed with octylsilyl silica gel for chromatography R (5 µm), 822

01/2005:1325

FLUDEOXYGLUCOSE (18F) INJECTION Fludeoxyglucosi (18F) solutio iniectabilis

DEFINITION Fludeoxyglucose (18F) injection is a sterile solution of 2-[18F]fluoro-2-deoxy-D-glucopyranose (2-[18F]fluoro-2-deoxy-D-glucose) for diagnostic use. The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the declared fluorine-18 radioactivity at the date and time stated on the label. Not less than 95 per cent of the radioactivity corresponds to fluorine-18 in the form of 2-[18F]fluoro-2-deoxy-D-glucose and 2-[18F]fluoro-2-deoxy-D-mannose, with the 2-[18F]fluoro-2-deoxy-D-mannose fraction not exceeding 10 per cent of the total radioactivity. Not less than 99.0 per cent of the radioactivity corresponds to fluorine-18. The content of 2-fluoro-2-deoxy-D-glucose is not more than 10 mg per maximum recommended dose of injection. PRODUCTION RADIONUCLIDE PRODUCTION Fluorine-18 is a radioactive isotope of fluorine which may be produced by various nuclear reactions induced by proton irradiation of oxygen-18, deuteron irradiation of neon-20, helium-3 or helium-4 irradiation of oxygen-16. RADIOCHEMICAL SYNTHESIS 2-[18F]Fluoro-2-deoxy-D-glucose may be prepared by various chemical synthetic pathways, which lead to different products in terms of specific radioactivity, by-products and possible impurities.



Most widely used is the method of phase transfer catalysed nucleophilic substitution of 1,3,4,6-tetra-O-acetyl-2-Otrifluoromethanesulphonyl-β-D-mannopyranose with [18F]fluoride. Generally, [18F]fluoride is adsorbed on an anion-exchange resin and eluted with a solution of potassium carbonate which is then evaporated to dryness. Addition of a phase transfer catalyst such as an aminopolyether in dry acetonitrile may be used to enhance the nucleophilicity of the [18F]fluoride so that it reacts easily with the tetra-acetylated mannosyltriflate at elevated temperature. Hydrolysis under either alkaline or acidic conditions yields 2-[18F]fluoro-2-deoxy-D-glucose. Hydrolysis using hydrochloric acid may lead to the formation of 2-chloro-2-deoxy-D-glucose. Hydrolysis under alkaline conditions may lead to the formation of 2-[18F]fluoro-2-deoxy-D-mannose as a by-product. Variations of the method substitute the aminopolyether by a tetra-alkyl ammonium salt, or use solid phase catalysed nucleophilic substitution on derivatised anion-exchange resin, e.g. derivatised with 4-(4-methylpiperidino)pyridine. Electrophilic pathways for production of 2-[18F]fluoro-2-deoxyD-glucose proceed by the reaction of molecular [18F]fluorine or [18F]acetylhypofluorite with 3,4,6-tri-O-acetyl-D-glucal. [18F]Acetylhypofluorite is obtained by conversion of molecular [18F]fluorine on a solid complex of acetic acid and potassium acetate. The production of molecular [18F]fluorine requires the addition of small amounts of fluorine to the neon target gas, usually from 0.1 per cent to 1 per cent, resulting in the reduction of the specific radioactivity of the end-product. Hydrolysis of the O-acetyl protected [18F]fluorinated sugar yields 2-[18F]fluoro-2-deoxy-D-glucose and usually small amounts of 2-[18F]fluoro-2-deoxy-D-mannose. The preparation can be purified by serial chromatography on combinations of ion-retardation resin, ion-exchange resin, alumina and octadecyl derivatised silica gel. Removal of the phase transfer catalyst can be achieved by different methods, all using combinations of separation cartridges. Production systems and their performance comply with the requirements set by the competent authority. STARTING MATERIALS 1. Target materials Each batch of target material must be tested in special production runs before its use in routine fluorine-18 production and manufacture of the preparation, to ensure that under specified conditions, the target yields fluorine-18 in the desired quantity and quality. 2. Precursors for organic synthesis It is recommended to test the precursors in production runs before their use for the manufacture of the preparation, to ensure that under specified production conditions, the precursors yield the preparation in the desired quantity and quality. 1,3,4,6-Tetra-O-acetyl-2-O-trifluoromethanesulphonylβ-D-mannopyranose. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of 1,3,4,6-tetra-O-acetyl-2-Otrifluoromethanesulphonyl-β-D-mannopyranose. Melting point (2.2.14) : 119 °C to 122 °C. 3,4,6-Tri-O-acetyl-D-glucal. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of 3,4,6-tri-O-acetyl-D-glucal. Melting point (2.2.14) : 53 °C to 55 °C. CHARACTERS A clear, colourless or slightly yellow solution. General Notices (1) apply to all monographs and other texts

Fludeoxyglucose (18F) injection

Fluorine-18 has a half-life of 109.8 min and emits positrons with a maximum energy of 0.633 MeV, followed by annihilation gamma radiation of 0.511 MeV. IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The only gamma photons have an energy of 0.511 MeV ; and depending on the measurement geometry, a sum peak of 1.022 MeV may be observed. B. It complies with the test for radionuclidic purity (see Tests). C. Examine the chromatograms obtained in test (a) for radiochemical purity. The principal peak in the radiochromatogram obtained with the test solution has approximately the same retention time as the principal peak in the chromatogram obtained with reference solution (b). TESTS pH (2.2.3). The pH of the injection is 4.5 to 8.5. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. Bacterial endotoxins (2.6.14) : less than 175/V IU/ml, V being the maximum recommended dose in millilitres. The injection may be released for use before completion of the test. CHEMICAL PURITY Particular tests for chemical purity may be omitted if the substances mentioned are not used or cannot be formed in the production process (a) 2-Fluoro-2-deoxy-D-glucose and 2-chloro-2-deoxy-Dglucose. Examine by liquid chromatography (2.2.29). Test solution. The preparation to be examined. Reference solution (a). Dissolve 10 mg of glucose R in water R and dilute to 100 ml with the same solvent. Reference solution (b). Dissolve 10 mg of 2-fluoro-2-deoxy-D-glucose R in water R and dilute to V with the same solvent, V being the maximum recommended dose in millilitres. Reference solution (c). Dissolve 1.0 mg of 2-chloro-2-deoxy-D-glucose R in water R and dilute to 2.0 ml with the same solvent. Dilute 1 ml of this solution to V with the same solvent, V being the maximum recommended dose in millilitres. The chromatographic procedure may be carried out using : — a column 0.25 m long and 4.0 mm in internal diameter packed with strongly basic anion-exchange resin for chromatography R (10 µm), — as mobile phase at a flow rate of 1 ml/min 0.1 M sodium hydroxide protected against contamination by carbon dioxide, — a suitable radioactivity detector for radiochemical purity testing, — a detector suitable for carbohydrates in the required concentration range, — a loop injector, maintaining the column at a constant temperature between 20 °C and 30 °C. Equilibrate the column with the mobile phase until a stable baseline is achieved. Inject separately reference solutions (a), (b) and (c). If the validation studies exclude the formation of 2-chloro-2-deoxy-D-glucose inject separately reference 823

Fludeoxyglucose (18F) injection


solutions (a) and (b). Continue the chromatography for twice the retention time of D-glucose, 2-fluoro-2-deoxy-D-glucose and when required, 2-chloro-2-deoxy-D-glucose respectively. Inject the test solution. The chromatogram obtained with the detector for carbohydrates shows a principal peak corresponding to D-glucose (test solutions from nucleophilic pathways) or 2-fluoro-2-deoxy-D-glucose (test solutions from electrophilic pathways). When the chromatograms are recorded in the prescribed conditions, 2-chloro-2-deoxy-D-glucose elutes after 2-fluoro-2-deoxy-D-glucose, but their corresponding peaks may not be completely resolved. In the chromatogram obtained with the test solution, the areas of the peaks corresponding to 2-fluoro-2-deoxy-D-glucose and 2-chloro-2-deoxy-D-glucose are not greater than the areas of the peaks in the chromatograms obtained with reference solution (b) and/or reference solution (c) (10 mg of 2-fluoro-2-deoxy-D-glucose per V and 0.5 mg of 2-chloro-2-deoxy-D-glucose per V respectively). (b) Aminopolyether. This test is performed only on the bulk solution before addition of sodium chloride by the producer and it is not intended for the final preparation to be injected. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R. Test solution. The preparation to be examined. Reference solution. Dissolve 0.110 g of aminopolyether R in water R and dilute to 10.0 ml with the same solvent. Dilute 0.2 ml of this solution to V with the same solvent, V being the maximum recommended dose in millilitres. Apply separately to the plate 2 µl of the test solution and 2 µl of the reference solution. Develop over a path of about 8 cm using a mixture of 1 volume of ammonia R and 9 volumes of methanol R. Allow the plate to dry in air for 15 min. Expose the plate to iodine vapour for at least 10 min. In the chromatogram obtained with the test solution the spot corresponding to aminopolyether is not more intense than the spot in the chromatogram obtained with the reference solution (2.2 mg per V). (c) Tetra-alkyl ammonium salts. Examine by liquid chromatography (2.2.29). Test solution. The preparation to be examined. Reference solution. Dilute 2.1 ml of 0.1 M tetrabutylammonium hydroxide to 20 ml with water R. Dilute 1 ml of this solution to V with the same solvent, V being the maximum recommended dose in millilitres. The chromatographic procedure may be carried out using : — a column 0.125 m long and 4.0 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 0.6 ml/min a mixture of 25 volumes of a 0.95 g/l solution of toluenesulphonic acid R and 75 volumes of acetonitrile R, — as detector a spectrophotometer set at 254 nm, — a loop injector, maintaining the column at a constant temperature between 20 °C and 30 °C. Equilibrate the column with the mobile phase until a stable baseline is obtained. Inject the reference solution. Continue the chromatography for twice the retention time of tetrabutylammonium ions. Inject the test solution. In the chromatogram obtained with the test solution, the area of the peak corresponding to tetrabutylammonium ions is not greater than the area of the peak in the chromatogram obtained with the reference solution (2.75 mg per V). 824

(d) Solid phase derivatisation agent 4-(4-methylpiperidino)pyridine. Examine by ultraviolet spectrophotometry (2.2.25). Test solution. The preparation to be examined. Reference solution. Dissolve 20 mg of 4-(4methylpiperidino)pyridine R in water R and dilute to 100.0 ml with the same solvent. Dilute 0.1 ml of this solution to V with the same solvent, V being the maximum recommended dose in millilitres. Measure the absorbance of the test solution and the reference solution at the maximum of 263 nm. The absorbance of the test solution is not greater than that of the reference solution (0.02 mg per V). (e) Residual solvents (2.4.24). The concentration of acetonitrile does not exceed 4.1 mg per V, V being the maximum recommended dose in millilitres. The injection may be released for use before completion of the test. RADIONUCLIDIC PURITY Record the gamma-ray spectrum using a suitable instrument. The half-life is between 105 min and 115 min. The injection may be released for use before completion of the test. RADIOCHEMICAL PURITY A. Examine by liquid chromatography (2.2.29) as described in test (a) for chemical purity. When the chromatograms obtained with the radioactivity detector are recorded in the prescribed conditions, the principal peak in the chromatogram obtained with the test solution has the same retention time as the peak obtained with reference solution (b) using the carbohydrate detector. The retention times of 2-[18F]fluoro-2-deoxy-D-mannose and [18F]fluoride are approximately 90 per cent and approximately 50 per cent of that of 2-[18F]fluoro-2-deoxy-D-glucose respectively. Other peaks in the chromatogram may be due to partially acetylated 2-[18F]fluoro-2-deoxy-D-glucose derivatives. Calculate the percentage content of [18F]fluorinated substances from the areas of the peaks in the chromatogram obtained with the test solution. The sum of the percentages of radioactivity corresponding to 2-[18F]fluoro-2-deoxy-D-glucose and 2-[18F]fluoro-2-deoxy-D-mannose is not less than 95 per cent of the total radioactivity with the 2-[18F]fluoro-2-deoxy-D-mannose fraction not exceeding 10 per cent of the total radioactivity. The method may underestimate or miss unhydrolysed or partially hydrolysed 2-[18F]fluoro-2-deoxytetra-acetyl-Dglucose, since these intermediate reaction products may further hydrolyse to the desired end-product under the chromatographic conditions. B. Examine by thin-layer chromatography (2.2.27) using a TLC silica gel plate R. Test solution. The preparation to be examined. Apply 2 µl to 10 µl to the plate. Develop over a path of 8 cm using a mixture of 5 volumes of water R and 95 volumes of acetonitrile R. Allow the plate to dry in air for 15 min. Determine the distribution of radioactivity using a suitable detector. Not less than 95 per cent of the total radioactivity is found in the spot corresponding to 2-fluoro-2-deoxy-D-glucose (Rf about 0.45). Possible contaminants are [18F]fluoride (Rf 0.0) ; partially acetylated 2-[18F]fluoro-2-deoxy-D-glucose derivatives (Rf about 0.8-0.95).



Flumazenil (N-[11C]methyl) injection

Synthesis of [5-methyl-11C]flumazenil The most widely used method to obtain [5-methyl11 C]flumazenil is the N-alkylation of demethylflumazenil with iodo[11C]methane in alkaline conditions in a solvent such as dimethylformamide or acetone. The resulting [5-methyl-11C]flumazenil can be purified by semi-preparative liquid chromatography. For example, a column packed with LABELLING octadecylsilyl silica gel for chromatography eluted with a The accompanying information specifies the particular mixture of ethanol and water is suitable. synthetic pathway of production. The label on the actual PRECURSOR FOR SYNTHESIS container states the maximum recommended dose in Demethylflumazenil millilitres. Melting point (2.2.14) : 286 °C to 289 °C. 01/2005:1917 Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of demethylflumazenil. FLUMAZENIL (N-[11C]METHYL) RADIOACTIVITY Measure the radioactivity using suitable counting equipment by comparison with a standardised fluorine-18 solution or using an instrument calibrated with the aid of such a solution. Standardised fluorine-18 solutions are available from laboratories recognised by the competent authority.

INJECTION

Flumazenil (N-[11C]methyl) solutio iniectabilis

DEFINITION Sterile solution of ethyl 8-fluoro-5-[11C]methyl-6-oxo-5,6dihydro-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate which may contain a stabiliser such as ascorbic acid. Content : 90 per cent to 110 per cent of the declared carbon-11 radioactivity at the date and time stated on the label. Content of flumazenil : maximum 50 µg in the maximum recommended dose in millilitres.

CHARACTERS Appearance : clear, colourless solution. Half-life and nature of radiation of carbon-11 : see Table of physical characteristics of radionuclides (5.7). IDENTIFICATION A. Gamma-ray spectrometry. Results : the only gamma photons have an energy of 0.511 MeV and, depending on the measurement geometry, a sum peak of 1.022 MeV may be observed. B. It complies with test B for radionuclidic purity (see Tests). C. Examine the chromatograms obtained in the test for radiochemical purity. Results : the principal peak in the radiochromatogram obtained with the test solution is similar in retention time to the principal peak in the chromatogram obtained with reference solution (a).

TESTS pH (2.2.3) : 6.0 to 8.0. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use PRODUCTION before completion of the test. RADIONUCLIDE PRODUCTION Bacterial endotoxins (2.6.14) : less than 175/V IU/ml, V Carbon-11 is a radioactive isotope of carbon which is most being the maximum recommended dose in millilitres. The commonly produced by proton irradiation of nitrogen. injection may be released for use before completion of the Depending on the addition of either trace amounts of oxygen test. or small amounts of hydrogen, the radioactivity is obtained Flumazenil and impurity A. Liquid chromatography (2.2.29). as [11C]carbon dioxide or [11C]methane, respectively. Test solution. The preparation to be examined. RADIOCHEMICAL SYNTHESIS 11 Reference solution (a). Dissolve 2.5 mg of flumazenil R in [5-Methyl- C]flumazenil may be prepared by 5 ml of methanol R. N-alkylation of ethyl 8-fluoro-6-oxo-5,6-dihydro-4Himidazo[1,5-a][1,4]benzodiazepine-3-carboxylate Reference solution (b). Dissolve 2.5 mg of (demethylflumazenil) with iodo[11C]methane or [11C]methyl demethylflumazenil R in 50 ml of methanol R. trifluoromethanesulphonate. Reference solution (c). To 0.1 ml of reference solution (a) add 0.1 ml of reference solution (b) and dilute to V with a Synthesis of iodo[11C]methane Iodo[11C]methane may be produced from [11C]carbon dioxide 0.9 g/l solution of sodium chloride R, V being the maximum recommended dose in millilitres. or from [11C]methane. The most frequently used method is Reference solution (d). Dilute 0.1 ml of reference solution (a) reduction of [11C]carbon dioxide with lithium aluminium to 50 ml with methanol R. Dilute 1.0 ml of this solution to V hydride. The [11C]methanolate formed is reacted with with a 0.9 g/l solution of sodium chloride R, V being the hydriodic acid. Alternatively [11C]methane, either obtained directly in the target or by on-line processes from [11C]carbon maximum recommended dose in millilitres. dioxide, is reacted with iodine. Column : — size : l = 0.15 m, Ø = 3.9 mm, Synthesis of [11C]methyl trifluoromethanesulphonate 11 [ C]methyl trifluoromethanesulphonate may be — stationary phase : spherical octadecylsilyl silica gel for prepared from iodo[11C]methane using a solid support chromatography R (5 µm) with a specific surface area of such as graphitised carbon, impregnated with silver 440 m2/g, a pore size of 100 nm and a carbon loading of trifluoromethanesulphonate. 19 per cent, General Notices (1) apply to all monographs and other texts

825

Gallium (67Ga) citrate injection


— temperature : maintain at a constant temperature between IMPURITIES 20-30 °C. Mobile phase : methanol R, water R (45:55 V/V). Flow rate : 1 ml/min. Detection : spectrophotometer at 260 nm and radioactivity detector connected in series. Injection : 100 µl. A. R = H : ethyl 8-fluoro-6-oxo-5,6-dihydro-4Himidazo[1,5-a][1,4]benzodiazepine-3-carboxylate (demethylflumazenil),

Run time : 10 min. Relative retention with reference to flumazenil : impurity A = about 0.74.

B. R = CH2-CO-CH3 : ethyl 8-fluoro-6-oxo-9-(2-oxopropyl)5,6-dihydro-4H-imidazo[1,5-a][1,4]benzodiazepine3-carboxylate (acetone addition compound of demethylflumazenil).

System suitability : reference solution (c) : — resolution : minimum 2.5 between the peaks due to flumazenil and impurity A.

01/2005:0555

Limits : examine the chromatogram obtained with the spectrophotometer : — flumazenil : not more than the area of the corresponding peak in the chromatogram obtained with reference solution (c) (50 µg/V), — impurity A : not more than the area of the corresponding peak in the chromatogram obtained with reference solution (c) (5 µg/V), — any other impurity : not more than the area of the principal peak in the chromatogram obtained with reference solution (d) (1 µg/V). Residual solvents are limited according to the principles defined in the general chapter (5.4), using the general method (2.4.24). The preparation may be released for use before completion of the test. RADIONUCLIDIC PURITY Carbon-11 : minimum 99 per cent of the total radioactivity. The preparation may be released for use before completion of the test. A. Gamma-ray spectrometry. Results : the spectrum obtained with the solution to be examined does not differ significantly from that obtained with a standardised fluorine-18 solution. B. Half-life : 19.9 min to 20.9 min. RADIOCHEMICAL PURITY Liquid chromatography (2.2.29) as described in the test for flumazenil and impurity A, with the following modifications. Injection : test solution and reference solution (a) ; if necessary, dilute the test solution to a radioactivity concentration suitable for the detector. Limit : examine the chromatogram obtained with the radioactivity detector : — [5-methyl-11C]flumazenil : minimum 95 per cent of the total radioactivity. RADIOACTIVITY Determine the radioactivity using a calibrated instrument. LABELLING The label states the maximum recommended dose in millilitres. 826

GALLIUM (67Ga) CITRATE INJECTION Gallii (67Ga) citratis solutio iniectabilis DEFINITION Gallium (67Ga) citrate injection is a sterile solution of gallium-67 in the form of gallium citrate. It may be made isotonic by the addition of sodium chloride and sodium citrate and may contain a suitable antimicrobial preservative such as benzyl alcohol. Gallium-67 is a radioactive isotope of gallium and may be obtained by the irradiation, with protons of suitable energy, of zinc which may be enriched in zinc-68. Gallium-67 may be separated from zinc by solvent extraction or column chromatography. The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the declared gallium-67 radioactivity at the date and hour stated on the label. Not more than 0.2 per cent of the total radioactivity is due to gallium-66. CHARACTERS A clear, colourless solution. Gallium-67 has a half-life of 3.26 days and emits gamma radiation. IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised gallium-67 solution when measured either by direct comparison or by use of an instrument calibrated with the aid of such a solution. Standardised gallium-67 solutions are available from laboratories recognised by the competent authority. The most prominent gamma photons have energies of 0.093 MeV, 0.185 MeV and 0.300 MeV. B. To 0.2 ml of the injection to be examined add 0.2 ml of a solution containing 1 g/l of ferric chloride R and 0.1 per cent V/V of hydrochloric acid R and mix. Compare the colour with that of a solution containing 9 g/l of benzyl alcohol R and 7 g/l of sodium chloride R treated in the same manner. A yellow colour develops in the test solution only. TESTS pH (2.2.3). The pH of the injection is 5.0 to 8.0. Zinc. To 0.1 ml of the injection to be examined add 0.9 ml of water R, 5 ml of acetate buffer solution pH 4.7 R, 1 ml of a 250 g/l solution of sodium thiosulphate R and 5.0 ml of a dithizone solution prepared as follows : dissolve 10 mg



Human albumin injection, iodinated (125I)

of dithizone R in 100 ml of methyl ethyl ketone R allow to Results : the spectrum obtained with the preparation stand for 5 min, filter and immediately before use dilute the to be examined does not differ significantly from that solution to ten times its volume with methyl ethyl ketone R. obtained with a standardised iodine-125 solution, apart Shake vigorously for 2 min and separate the organic layer. from any differences attributable to the presence of Measure the absorbance (2.2.25) of the organic layer at iodine-126. The most prominent photon has an energy 530 nm, using the organic layer of a blank solution as the of 0.027 MeV, corresponding to the characteristic X-ray compensation liquid. The absorbance is not greater than that of tellurium, gamma photons of an energy of 0.035 MeV of the organic layer obtained with 0.1 ml of zinc standard are also present. Iodine-126 has a half-life of 13.11 days solution (5 ppm Zn) R treated in the same manner. and its most prominent gamma photons have energies of 0.388 MeV and 0.666 MeV. Sterility. It complies with the test for sterility B. Examine by a suitable immunoelectrophoresis technique prescribed in the monograph on Radiopharmaceutical (2.7.1). Using antiserum to normal human serum, preparations (0125). The injection may be released for use compare normal human serum and the preparation to be before completion of the test. examined, both diluted if necessary. The main component RADIONUCLIDIC PURITY of the preparation to be examined corresponds to the Record the gamma-ray spectrum using a suitable instrument. main component of the normal human serum. The diluted The spectrum does not differ significantly from that of a solution may show the presence of small quantities of standardised gallium-67 solution, apart from any differences other plasma proteins. attributable to the presence of gallium-66. Gallium-66 has a half-life of 9.4 h and its most prominent gamma photon has TESTS an energy of 1.039 MeV. Not more than 0.2 per cent of the pH (2.2.3) : 5.0 to 9.0. total radioactivity is due to gallium-66. Albumin RADIOACTIVITY Reference solution. Dilute human albumin solution R with Measure the radioactivity using suitable counting equipment a 9 g/l solution of sodium chloride R to a concentration of by comparison with a standardised gallium-67 solution or 5 mg of albumin per millilitre. by measurement in an instrument calibrated with the aid of To 1.0 ml of the preparation to be examined and to 1.0 ml of such a solution. the reference solution add 4.0 ml of biuret reagent R and mix. After exactly 30 min, measure the absorbance (2.2.25) 01/2005:1922 of each solution at 540 nm, using as the compensation liquid a 9 g/l solution of sodium chloride R treated in the same manner. From the absorbances measured, calculate HUMAN ALBUMIN INJECTION, the content of albumin in the injection to be examined in 125 IODINATED ( I) milligrams per millilitre. Sterility. It complies with the test for sterility Iodinati (125I) humani albumini prescribed in the monograph on Radiopharmaceutical preparations (0125). solutio iniectabilis Bacterial endotoxins (2.6.14) : less than 175/V IU/ml, DEFINITION V being the maximum recommended dose in millilitres. Sterile, endotoxin-free solution of human albumin labelled RADIONUCLIDIC PURITY with iodine-125. It may contain a suitable buffer and an antimicrobial preservative. The human albumin used Iodine-125 : minimum 99.0 per cent of the total radioactivity. complies with the requirements of the monograph on Gamma-ray and X-ray spectroscopy. Human albumin solution (0255). Comparison : standardised solution of iodine-125. Content : 90 per cent to 110 per cent of the declared Determine the relative amounts of iodine-125 and iodine-126 iodine-125 radioactivity at the date stated on the label. present. Purity : RADIOCHEMICAL PURITY — minimum of 99.0 per cent of the total radioactivity Iodine-125 in albumin fractions II to V, iodine-125 corresponds to iodine-125, corresponding to unbound iodide. Size-exclusion — minimum of 80 per cent of the total radioactivity is chromatography (2.2.30). associated with the albumin fractions II to V, Test solution. Mix 0.25 ml of the preparation to be examined — maximum of 5 per cent of the total radioactivity with 0.25 ml of the mobile phase. Use immediately after corresponds to unbound iodide. mixing. Content of albumin : 95 per cent to 105 per cent of the Reference solution. Human albumin solution R or another declared albumin content stated on the label. appropriate human albumin standard diluted with the mobile CHARACTERS phase to a suitable albumin concentration. Appearance : clear, colourless to yellowish solution. Column : Half-life and nature of radiation of iodine-125 : see Table of — size : l = 0.6 m, Ø = 7.5 mm, physical characteristics of radionuclides (5.7). — stationary phase : silica gel for size-exclusion chromatography R, IDENTIFICATION — temperature : 25 °C. A. Gamma-ray and X-ray spectrometry. Mobile phase : dissolve 11.24 g of potassium dihydrogen Comparison : standardised iodine-125 solution, or by phosphate R, 42.0 g of disodium hydrogen phosphate R, using a calibrated instrument. Standardised iodine-125 11.70 g of sodium chloride R in 2000 ml of water R. solutions and/or standardisation services are available from the competent authority. Flow rate : 0.6 ml/min. General Notices (1) apply to all monographs and other texts

827

Indium (111In) chloride solution


Detection : spectrophotometer at 280 nm and radioactivity detector set for iodine-125 connected in series. Injection : loop injector. Run time : 85 min. Retention times : Fraction

Description of the compound

Retention time (min)

1

I

High molecular mass compound

18 - 20

2

II

Poly III albumin

23 - 24

3

III

Poly II albumin

25 - 26

4

IV

Poly I albumin

28

5

V

Human serum albumin

29 - 31

6

VI

Iodide

43 - 45

Peak No.

IDENTIFICATION A. Carry out the test after allowing sufficient time for short-lived impurities such as indium-110m to decay. Record the gamma-ray and X-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised indium-111 solution apart from any differences due to the presence of indium-114m, when measured either by direct comparison or by using an instrument calibrated with the aid of such a solution. Standardised indium-111 and indium-114m solutions are available from laboratories recognised by the competent authority. The most prominent gamma photons of indium-111 have energies of 0.171 MeV and 0.245 MeV. B. To 100 µl of silver nitrate solution R2 add 50 µl of the solution. A white precipitate is formed. C. It complies with the test for pH (see Tests). D. Examine the chromatogram obtained in the test for radiochemical purity. The principal peak has an Rf value of 0.5 to 0.8.

The main peak in the chromatogram obtained with the reference solution corresponds to fraction V. Limits : — radioactivity in fractions II to V : minimum 80 per cent of the total radioactivity applied to the column, — iodine-125 in fraction VI : maximum 5 per cent of the total radioactivity.

TESTS pH (2.2.3). The pH of the solution is 1.0 to 2.0. Cadmium. Not more than 0.40 µg/ml, determined by electrothermal atomic absorption spectrometry (2.2.23, Method I). Test solution. Dilute 0.05 ml of the solution to be examined to a suitable volume with a suitable concentration of RADIOACTIVITY hydrochloric acid R. Measure the radioactivity using suitable equipment by Reference solutions. Prepare the reference solutions using comparison with a standardised iodine-125 solution or by cadmium standard solution (0.1 per cent Cd) R, diluted measurement with a calibrated instrument. as necessary with the same concentration of hydrochloric acid R as in the solution to be examined. LABELLING Measure the absorbance at 228.8 nm using a cadmium The label states : hollow-cathode lamp as source of radiation. — the amount of albumin, Copper. Not more than 0.15 µg/ml, determined by — the maximum volume to be injected. electrothermal atomic absorption spectrometry (2.2.23, Method I). Test solution. Dilute 0.1 ml the solution to be examined 01/2005:1227 to a suitable volume with a suitable concentration of hydrochloric acid R. INDIUM (111In) CHLORIDE SOLUTION Reference solutions. Prepare the reference solutions using copper standard solution (0.1 per cent) R diluted Indii (111In) chloridi solutio as necessary with the same concentration of hydrochloric acid R as the solution to be examined. DEFINITION Measure the absorbance at 324.8 nm using a copper Indium (111In) chloride solution is a sterile solution of hollow-cathode lamp as source of radiation. indium-111 as the chloride in aqueous hydrochloric acid containing no additives. Indium-111 is a radioactive isotope Iron. Not more than 0.60 µg/ml, determined by of indium and may be produced by the irradiation of cadmium electrothermal atomic absorption spectrometry (2.2.23, Method I). with protons of suitable energy. The solution contains not less than 90.0 per cent and not more than 110.0 per cent of Test solution. Dilute 0.1 ml of the solution to be examined the declared indium-111 radioactivity at the date and hour to a suitable volume with a suitable concentration of stated on the label. Not more than 0.25 per cent of the total hydrochloric acid R. radioactivity is due to radionuclides other than indium-111. Reference solutions. Prepare the reference solutions Not less than 95 per cent of the radioactivity corresponds to using iron standard solution (0.1 per cent Fe) R diluted indium-111 in the form of ionic indium(III). The method of as necessary with the same concentration of hydrochloric preparation is such that no carrier is added and the specific acid R as the solution to be examined. radioactivity is not less than 1.85 GBq of indium-111 per Measure the absorbance at 248.3 nm using an iron microgram of indium. hollow-cathode lamp as source of radiation. CHARACTERS Sterility. It complies with the test for sterility A clear, colourless solution. prescribed in the monograph on Radiopharmaceutical Indium-111 has a half-life of 2.8 days and emits gamma preparations (0125). The solution may be released for use radiation and X-rays. before completion of the test. 828



Indium (111In) oxine solution

RADIONUCLIDIC PURITY Record the gamma-ray and X-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised solution of indium-111 apart from any differences due to the presence of indium-114m. Indium-114m. Carry out the test after allowing sufficient time for short-lived impurities such as indium-110m to decay. Take a volume equivalent to 30 MBq and record the gamma-ray spectrum using a suitable detector with a shield of lead, 6 mm thick, placed between the sample and the detector. The response in the region corresponding to the 0.558 MeV photon and the 0.725 MeV photon of indium-114m does not exceed that obtained using 75 kBq of a standardised solution of indium-114m (0.25 per cent) measured under the same conditions, when all measurements are calculated with reference to the date and hour of administration. Standardised indium-111 and indium-114m solutions are available from laboratories recognised by the competent authority. RADIOCHEMICAL PURITY Examine by thin-layer chromatography (2.2.27) using silica gel as the coating substance on a glass-fibre sheet. Apply to the plate 5 µl of the solution to be examined. Develop immediately over a path of 15 cm using a 9.0 g/l solution of sodium chloride R adjusted to pH 2.3 ± 0.05 with dilute hydrochloric acid R. Allow the plate to dry in a current of cold air. Determine the distribution of radioactivity using a suitable detector. Indium-111 chloride migrates with an Rf value of 0.5 to 0.8. Not less than 95 per cent of the total radioactivity of the chromatogram corresponds to indium-111 chloride.

label. Not more than 0.25 per cent of the total radioactivity is due to radionuclides other than indium-111. Not less than 90 per cent of the radioactivity corresponds to indium-111 complexed with oxine. The method of preparation is such that no carrier is added and the specific radioactivity is not less than 1.85 GBq of indium-111 per microgram of indium.


829

CHARACTERS A clear, colourless solution. Indium-111 has a half-life of 2.8 days and emits gamma radiation and X-rays.

IDENTIFICATION A. Carry out the test after allowing sufficient time for short-lived impurities such as indium-110m to decay. Record the gamma-ray and X-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised indium-111 solution apart from any differences due to the presence of indium-114m, when measured either by direct comparison or by using an instrument calibrated with the aid of such a solution. Standardised indium-111 and indium-114m solutions are available from laboratories recognised by the competent authority. The most prominent gamma photons of indium-111 have energies of 0.171 MeV and 0.245 MeV. B. Place 5 mg to 10 mg of magnesium oxide R in a glass container of approximately 20 mm in internal diameter. Add 20 µl of the solution to be examined. Examine in ultraviolet light at 365 nm. Bright yellow fluorescence is produced. C. The distribution of radioactivity between the organic RADIOACTIVITY and aqueous phases in the test for radiochemical purity contributes to the identification of the preparation. Measure the radioactivity using suitable counting equipment by comparison with a standardised indium-111 solution or by measurement in an instrument calibrated with the aid of TESTS such a solution. pH (2.2.3). The pH of the solution is 6.0 to 7.5. Sterility. It complies with the test for sterility 01/2005:1109 prescribed in the monograph on Radiopharmaceutical preparations (0125). The solution may be released for use before completion of the test. INDIUM (111In) OXINE SOLUTION RADIONUCLIDIC PURITY Record the gamma-ray and X-ray spectrum using a suitable Indii (111In) oxini solutio instrument. The spectrum does not differ significantly from that of a standardised solution of indium-111, apart from any differences due to the presence of indium-114m. Indium-114m. Carry out the test after allowing sufficient time for short-lived impurities such as indium-110m to decay. Take a volume equivalent to 30 MBq and record the gamma-ray spectrum using a suitable detector with a shield of lead, 6 mm thick, placed between the sample and the detector. The response in the region corresponding to the 0.558 MeV photon and the 0.725 MeV photon of indium-114m does not exceed that obtained using 75 kBq of a standardised solution of indium-114m (0.25 per cent) measured under the same conditions, when all measurements are calculated with 111 C27H18[ In]N3O3 Mr 547.2 reference to the date and hour of administration. (It should be noted that indium (111In) oxine solution is a precursor DEFINITION used in the in vitro labelling of white blood cells or platelets 111 Indium ( In) oxine solution is a sterile solution of indium-111 prior to their re-injection into the patient. It is not intended in the form of a complex with 8-hydroxyquinoline. It may for direct administration). Standardised indium-111 and contain suitable surface active agents and may be made indium-114m solutions are available from laboratories iso-tonic by the addition of sodium chloride and a suitable recognised by the competent authority. buffer. Indium-111 is a radioactive isotope of indium and may RADIOCHEMICAL PURITY be produced by the irradiation of cadmium with protons of suitable energy. The solution contains not less than 90.0 per To a silanised separating funnel containing 3 ml of a 9 g/l cent and not more than 110.0 per cent of the declared solution of sodium chloride R add 100 µl of the solution indium-111 radioactivity at the date and hour stated on the to be examined and mix. Add 6 ml of octanol R and shake

Indium (111In) pentetate injection


vigorously. Allow the phases to separate and then run the lower layer into a suitable vial for counting. Allow the upper layer to drain completely into a similar vial. Add 1 ml of octanol R to the funnel, shake vigorously and drain into the vial containing the organic fraction. Add 5 ml of dilute hydrochloric acid R to the funnel, shake vigorously and drain these rinsings into a third vial. Seal each vial and, using a suitable instrument, measure the radioactivity in each. Calculate the radiochemical purity by expressing the radioactivity of the indium-111 oxine complex, found in the organic phase, as a percentage of the radioactivity measured in the three solutions. Not less than 90 per cent of the radioactivity corresponds to indium-111 complexed with oxine. RADIOACTIVITY Measure the radioactivity using suitable counting equipment by comparison with a standardised indium-111 solution or by measurement in an instrument calibrated with the aid of such a solution.

01/2005:0670

INDIUM (111In) PENTETATE INJECTION Indii (111In) pentetatis solutio iniectabilis DEFINITION Indium (111In) pentetate injection is a sterile and apyrogenic solution containing indium-111 in the form of indium diethylenetriaminepenta-acetate. It may contain calcium and may be made isotonic by the addition of sodium chloride and a suitable buffer. Indium-111 is a radioactive isotope of indium which may be obtained by proton irradiation, of appropriate energy, of cadmium which may be enriched with cadmium-111 or cadmium-112. The injection contains not less than 90 per cent and not more than 110 per cent of the declared indium-111 radioactivity at the date and hour stated on the label. The radioactivity due to indium-114m is not greater than 0.2 per cent of the total radioactivity at the date and hour of administration. Not less than 95 per cent of the radioactivity corresponds to indium-111 complexed with pentetate. CHARACTERS A clear, colourless solution. Indium-111 has a half-life of 2.8 days and emits gamma radiation and X-rays. IDENTIFICATION A. Record the gamma-ray and X-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised indium-111 solution apart from any differences due to the presence of indium-114m, when measured either by direct comparison or by using an instrument calibrated with the aid of such a solution. Standardised indium-111 and indium-114m solutions are available from laboratories recognised by the competent authority. The most prominent gamma photons of indium-111 have energies of 0.171 MeV and 0.245 MeV. B. Examine the chromatogram obtained in the test for radiochemical purity. The distribution of radioactivity contributes to the identification of the preparation. 830

TESTS pH (2.2.3). The pH of the injection is 7.0 to 8.0. Cadmium. Not more than 5 µg of Cd per millilitre, determined by atomic absorption spectrometry (2.2.23, Method II). Test solution. Mix 0.1 ml of the injection to be examined with 0.9 ml of a mixture of 1 volume of hydrochloric acid R and 99 volumes of water R. Reference solutions. Prepare the reference solutions using cadmium standard solution (0.1 per cent Cd) R and diluting with a mixture of 1 volume of hydrochloric acid R and 99 volumes of water R. Measure the absorbance at 228.8 nm using a cadmium hollow-cathode lamp as source of radiation and an air-acetylene flame. Uncomplexed diethylenetriaminepenta-acetic acid. In a micro test-tube, mix 100 µl of the injection to be examined with 100 µl of a freshly prepared 1 g/l solution of hydroxynaphthol blue, sodium salt R in 1 M sodium hydroxide. Add 50 µl of a 0.15 g/l solution of calcium chloride R. The solution remains pinkish-violet or changes from blue to pinkish-violet (0.4 mg/ml). Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. Bacterial endotoxins (2.6.14) : less than 14/V IU/ml, V being the maximum recommended dose in millilitres. RADIONUCLIDIC PURITY Record the gamma-ray and X-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised solution of indium-111 apart from any differences due to the presence of indium-114m. Indium-114m. Retain a sample of the injection to be examined for a sufficient time to allow the indium-111 radioactivity to decay to a sufficiently low level to permit the detection of radionuclidic impurities. Record the gamma-ray spectrum of the decayed material in a suitable instrument calibrated with the aid of a standardised indium-114m solution. Indium-114m has a half-life of 49.5 days and its most prominent gamma photon has an energy of 0.190 MeV. The radioactivity due to indium-114m is not greater than 0.2 per cent of the total radioactivity at the date and hour of administration. RADIOCHEMICAL PURITY Examine by thin-layer chromatography (2.2.27) using silica gel as the coating substance on a glass-fibre sheet. Heat the plate at 110 °C for 10 min. Use a plate such that during development the mobile phase migrates over a distance of 10 cm to 15 cm in about 10 min. Apply to the plate 5 µl to 10 µl of the injection to be examined and allow to dry. Develop over a path of 10 cm to 15 cm using a 9 g/l solution of sodium chloride R. Allow the plate to dry in air. Determine the distribution of radioactivity using a suitable detector. Indium pentetate complex migrates near to the solvent front. The radioactivity of the spot corresponding to indium pentetate complex represents not less than 95 per cent of the total radioactivity of the chromatogram. RADIOACTIVITY Measure the radioactivity using suitable counting equipment by comparison with a standardised indium-111 solution or by measurement in an instrument calibrated with the aid of such a solution.


Iobenguane (123I) injection


01/2005:1113 chromatograms obtained with the test solution and reference corrected solution (b). Calculate the concentration as iobenguane base by multiplying the result obtained in the assay by 0.85. 123 Sterility. It complies with the test for sterility IOBENGUANE ( I) INJECTION prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. Iobenguani (123I) solutio iniectabilis Bacterial endotoxins (2.6.14) : less than 175/V IU/ml, V being the maximum recommended dose in millilitres. RADIONUCLIDIC PURITY Record the gamma-ray spectrum using a suitable instrument. Determine the relative amounts of iodine-125, tellurium-121 and other radionuclidic impurities present. No radionuclides with longer half-lives than iodine-125 are detected. For the determination of iodine-125, tellurium-121 and other radionuclidic impurities, retain the solution to be examined C8H10[123I]N3 for a sufficient time to allow the radioactivity of iodine-123 to decrease to a level which permits the detection of DEFINITION radionuclidic impurities. Record the gamma-ray spectrum 123 Iobenguane ( I) injection is a sterile, bacterial-endotoxin and the X-ray spectrum of the decayed material using a free solution of 1-(3-[123I]iodobenzyl)guanidine or its salts. It suitable instrument. Not more than 0.35 per cent of the total may contain a suitable buffer, a suitable labelling catalyst radioactivity is due to radionuclides other than iodine-123. such as ionic copper, a suitable labelling stabiliser such as The injection may be released for use before completion of ascorbic acid and antimicrobial preservatives. Iodine-123 is a the test. radioactive isotope of iodine and may be obtained by proton RADIOCHEMICAL PURITY irradiation of xenon enriched in xenon-124 (not less than 98 per cent) followed by the decay of caesium-123 formed via Examine by liquid chromatography (2.2.29). xenon-123. The injection contains not less than 90.0 per cent Test solution. The injection to be examined. and not more than 110.0 per cent of the declared iodine-123 Reference solution (a). Dissolve 0.100 g of sodium iodide R radioactivity at the date and hour stated on the label. Not in the mobile phase and dilute to 100 ml with the mobile less than 95 per cent of the radioactivity corresponds phase. to iodine-123 in the form of iobenguane. The specific Reference solution (b). Dissolve 20.0 mg of iobenguane radioactivity is not less than 10 GBq of iodine-123 per gram sulphate CRS in 50 ml of the mobile phase and dilute to of iobenguane base. Not more than 0.35 per cent of the total 100.0 ml with the mobile phase. radioactivity is due to radionuclides other than iodine-123. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.0 mm in internal CHARACTERS diameter packed with silica gel for chromatography R A clear, colourless or slightly yellow solution. (5 µm), Iodine-123 has a half-life of 13.2 h and emits gamma radiation — as mobile phase at a flow rate of 1.0 ml/min a mixture of and X-rays. 1 volume of an 80 g/l solution of ammonium nitrate R, 2 volumes of dilute ammonia R2 and 27 volumes of IDENTIFICATION methanol R, — a suitable radioactivity detector, A. Record the gamma-ray and X-ray spectrum using a suitable instrument. The spectrum does not differ significantly — a spectrophotometer set at 254 nm and provided with a from that of a standardised iodine-123 solution apart from flow-cell, any differences attributable to the presence of iodine-125, — a 10 µl loop injector. tellurium-121 and other radionuclidic impurities. The Inject the test solution and the reference solutions. Not less most prominent gamma photon of iodine-123 has than 95 per cent of the radioactivity of the chromatogram is an energy of 0.159 MeV. Iodine-125 has a half-life of 59.4 days and emits an X-ray of 0.027 MeV and a photon of found in the peak corresponding to iobenguane. Not more 0.035 MeV. Tellurium-121 has a half-life of 19.2 days and than 4 per cent of the radioactivity is found in the peak the most prominent photons have energies of 0.507 MeV corresponding to iodide and not more than 1 per cent of the and 0.573 MeV. Standardised iodine-123, iodine-125 and radioactivity is found in other peaks. tellurium-121 solutions are available from laboratories RADIOACTIVITY recognised by the competent authority. Measure the radioactivity using a suitable counting B. Examine the chromatogram obtained in the test for radiochemical purity. The distribution of the radioactivity apparatus by comparison with a standardised iodine-123 solution or by measurement in an instrument calibrated with contributes to the identification of the preparation. the aid of such a solution. TESTS pH (2.2.3). The pH of the solution is 3.5 to 8.0. Specific radioactivity. The specific radioactivity is calculated from the results obtained in the test for radiochemical purity. Determine the content of iobenguane sulphate from the areas of the peaks corresponding to iobenguane in the General Notices (1) apply to all monographs and other texts

STORAGE Store protected from light. LABELLING The label states the specific radioactivity expressed in GBq of iodine-123 per gram of iobenguane base. 831

Iobenguane (131I) injection for diagnostic use


01/2005:1111 Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use INJECTION FOR before completion of the test.

IOBENGUANE (131I) DIAGNOSTIC USE

Bacterial endotoxins (2.6.14) : less than 175/V IU/ml, V being the maximum recommended dose in millilitres. RADIONUCLIDIC PURITY Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised iodine-131 solution. Determine the relative amounts of iodine-131, iod-ine-133, iodine-135 and other radionuclidic impurities present. Iodine-133 has a half-life of 20.8 h and its most prominent gamma photons have energies of 0.530 MeV and 0.875 MeV. Iodine-135 has a half-life of 6.55 h and its most prominent gamma photons have energies of 0.527 MeV, 1.132 MeV and 1.260 MeV. Not less than 99.9 per cent of the total radioactivity is due to iodine-131.

Iobenguani (131I) solutio iniectabilis ad usum diagnosticum

RADIOCHEMICAL PURITY Examine by liquid chromatography (2.2.29). Test solution. The injection to be examined.

C8H10[131I]N3 DEFINITION Iobenguane (131I) injection for diagnostic use is a sterile, bacterial endotoxin-free solution of 1-(3-[131I]iodobenzyl)guanidine or its salts. It may contain a suitable buffer. It may also contain a suitable labelling catalyst such as ionic copper and a suitable labelling stabiliser such as ascorbic acid. It may contain antimicrobial preservatives. Iodine-131 is a radioactive isotope of iodine and may be obtained by neutron irradiation of tellurium or by extraction of uranium fission products. The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the declared iodine-131 radioactivity at the date and hour stated on the label. Not less than 94 per cent of the radioactivity corresponds to iodine-131 in the form of iobenguane. The specific radioactivity is not less than 20 GBq of iodine-131 per gram of iobenguane base. CHARACTERS

Reference solution (a). Dissolve 0.100 g of sodium iodide R in the mobile phase and dilute to 100 ml with the mobile phase. Reference solution (b). Dissolve 20.0 mg of iobenguane sulphate CRS in 50 ml of the mobile phase and dilute to 100.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.0 mm in internal diameter packed with silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.0 ml/min a mixture of 1 volume of an 80 g/l solution of ammonium nitrate R, 2 volumes of dilute ammonia R2 and 27 volumes of methanol R, — a suitable radioactivity detector,

A clear, colourless or slightly yellow solution. Iodine-131 has a half-life of 8.04 days and emits beta and gamma radiation.

— a spectrophotometer set at 254 nm and provided with a flow-cell, — a 10 µl loop injector.

Inject the test solution and the reference solutions. Not less than 94 per cent of the radioactivity of the chromatogram is A. Record the gamma-ray spectrum using a suitable found in the peak corresponding to iobenguane. Not more instrument. The spectrum does not differ significantly than 5 per cent of the radioactivity is found in the peak from that of a standardised iodine-131 solution by direct corresponding to iodide and not more than 1 per cent of the comparison with such a solution. Standardised iodine-131 radioactivity is found in other peaks. solutions are available from laboratories recognised by the competent authority. The most prominent gamma photon of iodine-131 has an energy of 0.365 MeV. RADIOACTIVITY B. Examine the chromatogram obtained in the test for Measure the radioactivity using a suitable counting radiochemical purity. The distribution of the radioactivity apparatus by comparison with a standardised iodine-131 contributes to the identification of the preparation. solution or by measurement in an instrument calibrated with the aid of such a solution. TESTS IDENTIFICATION

pH (2.2.3). The pH of the solution is 3.5 to 8.0.

STORAGE

Specific radioactivity. The specific radioactivity is calculated from the results obtained in the test for radiochemical purity. Determine the content of iobenguane sulphate from the areas of the peaks corresponding to iobenguane in the chromatograms obtained with the test solution and reference solution (b). Calculate the concentration as iobenguane base by multiplying the result obtained in the assay by 0.85. 832

Store protected from light. LABELLING The label states the specific radioactivity expressed in gigabecquerels of iodine-131 per gram of iobenguane base.



Krypton (81mKr) inhalation gas

01/2005:1112 RADIONUCLIDIC PURITY Record the gamma-ray spectrum using a suitable instrument. 131 spectrum does not differ significantly from that of a IOBENGUANE ( I) INJECTION FOR The standardised iodine-131 solution. Determine the relative THERAPEUTIC USE amounts of iodine-131, iodine-133, iodine-135 and other radionuclidic impurities present. Iodine-133 has a half-life of 20.8 h and its most prominent gamma photons have energies Iobenguani (131I) solutio iniectabilis of 0.530 MeV and 0.875 MeV. Iodine-135 has a half-life of ad usum therapeuticum 6.55 h and its most prominent gamma photons have energies of 0.527 MeV, 1.132 MeV and 1.260 MeV. Not less than 99.9 per cent of the total radioactivity is due to iodine-131. RADIOCHEMICAL PURITY Examine by liquid chromatography (2.2.29). Test solution. The injection to be examined. Reference solution (a). Dissolve 0.100 g of sodium iodide R in the mobile phase and dilute to 100 ml with the mobile phase. C8H10 [131I] N3 Reference solution (b). Dissolve 20.0 mg of iobenguane sulphate CRS in 50 ml of the mobile phase and dilute to DEFINITION 100.0 ml with the mobile phase. Iobenguane (131I) injection for therapeutic use The chromatographic procedure may be carried out using : is a sterile, bacterial endotoxin-free solution of 131 — a stainless steel column 0.25 m long and 4.0 mm in internal 1-(3-[ I]iodobenzyl)guanidine or its salts. It may contain diameter packed with silica gel for chromatography R a suitable buffer, a suitable labelling catalyst such as ionic (5 µm), copper, a suitable labelling stabiliser such as ascorbic acid and antimicrobial preservatives. Iodine-131 is a radioactive — as mobile phase at a flow rate of 1.0 ml/min a mixture of isotope of iodine and may be obtained by neutron irradiation 1 volume of an 80 g/lsolution of ammonium nitrate R, of tellurium or by extraction of uranium fission products. The 2 volumes of dilute ammonia R2 and 27 volumes of injection contains not less than 90.0 per cent and not more methanol R, than 110.0 per cent of the declared iodine-131 radioactivity — a suitable radioactivity detector, at the date and hour stated on the label. Not less than 92 per — a spectrophotometer set at 254 nm and provided with a cent of the radioactivity corresponds to iodine-131 in the flow-cell, form of iobenguane. The specific radioactivity is not less — a 10 µl loop injector. than 400 GBq of iodine-131 per gram of iobenguane base. Inject the test solution and the reference solutions. Not less CHARACTERS than 92 per cent of the radioactivity of the chromatogram is found in the peak corresponding to iobenguane. Not more A clear, colourless or slightly yellow solution. than 7 per cent of the radioactivity is found in the peak Iodine-131 has a half-life of 8.04 days and emits beta and corresponding to iodide and not more than 1 per cent of the gamma radiation. radioactivity is found in other peaks. IDENTIFICATION RADIOACTIVITY A. Record the gamma-ray spectrum using a suitable Measure the radioactivity using a suitable counting instrument. The spectrum does not differ significantly apparatus by comparison with a standardised iodine-131 from that of a standardised iodine-131 solution by direct solution or by measurement in an instrument calibrated with comparison with such a solution. Standardised iodine-131 the aid of such a solution. solutions are available from laboratories recognised by the competent authority. The most prominent gamma STORAGE photon of iodine-131 has an energy of 0.365 MeV. Store protected from light. B. Examine the chromatogram obtained in the test for radio-chemical purity. The distribution of the radioactivity LABELLING contributes to the identification of the preparation. The label states the specific radioactivity expressed in gigabecquerels of iodine-131 per gram of iobenguane base. TESTS pH (2.2.3). The pH of the solution is 3.5 to 8.0. 01/2005:1533 Specific radioactivity. The specific radioactivity is calculated from the results obtained in the test for radiochemical KRYPTON (81mKr) INHALATION GAS purity. Determine the content of iobenguane sulphate from the areas of the peaks corresponding to iobenguane in the Kryptonum (81mKr) ad inhalationem chromatograms obtained with the test solution and reference solution (b). Calculate the concentration as iobenguane base DEFINITION by multiplying the result obtained in the assay by 0.85. Krypton (81mKr) inhalation gas is a mixture of krypton-81m and a suitable vehicle such as air. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical Krypton-81m is formed by decay of its parent radionuclide preparations (0125). The injection may be released for use rubidium-81. Rubidium-81 has a half-life of 4.58 h. before completion of the test. The krypton-81m formed is separated from the rubidium-81 Bacterial endotoxins (2.6.14) : less than 175/V IU/ml, with a flow of a suitable gas in a rubidium/krypton V being the maximum recommended dose in millilitres. generator. Rubidium-81 is produced by proton irradiation of General Notices (1) apply to all monographs and other texts

833

L-Methionine

([11C]methyl) injection


krypton isotopes or by helium-3 or helium-4 irradiation of bromine. After separation of rubidium-81 from the target, it is retained by a suitable support. Krypton-81m is eluted at a suitable flow rate with a vehicle such as air. The level of moisture required in the eluent depends on the type of generator used. The transport tube for administration has a defined length and inner diameter. The radioactivity concentration is determined before administration. The radioactivity due to radionuclides other than krypton-81m is not greater than 0.1 per cent, expressed as a percentage of the total radioactivity in the preparation and calculated with reference to the date and time of administration. CHARACTERS A clear, colourless gas. Krypton-81m has a half-life of 13.1 s and emits gamma radiation. IDENTIFICATION A. Record the gamma-ray and X-ray spectrum using a suitable instrument. The gamma photon of krypton-81m has an energy of 0.190 MeV. B. The half-life is 11.8 s to 14.4 s. TESTS RADIONUCLIDIC PURITY Elute the generator as prescribed. Pass a sufficient amount (2 litres to 10 litres) of eluate at a suitable flow rate through a suitable absorber such as water. Determine the amount of radioactivity eluted. Allow the krypton-81m to decay for 5 min and record the gamma and X-ray spectrum of the residual radioactivity on the absorber using a suitable instrument. Examine the gamma-ray and X-ray spectrum of the absorber for the presence of radioactive impurities, which must be identified and quantified. The absorbed radioactivity is not more than 0.1 per cent of the radioactivity passed through the absorber, calculated with reference to the date and time of administration. RADIOACTIVITY Determine the radioactive concentration of the preparation using suitable equipment such as an ionisation chamber or a gamma ray spectrometer. The measurement equipment may be calibrated by reference to a primary calibrated instrument at a laboratory recognised by the competent authority. The radioactivity is measured under defined operating conditions, such as gas flow rate and measurement geometry, that are identical to those used for the calibration of the instrument. STORAGE The storage conditions apply to the generator. LABELLING The labelling conditions apply to the generator. 834

01/2005:1617 corrected

L-METHIONINE

([11C]METHYL) INJECTION

([11C]methyl) solutio iniectabilis

L-Methionini

DEFINITION Sterile solution of (2S)-2-amino-4-([11C]methylsulphanyl)butanoic acid for diagnostic use. Content : 90 per cent to 110 per cent of the declared carbon-11 radioactivity at the date and time stated on the label. Purity : — minimum of 99 per cent of the total radioactivity corresponds to carbon-11, — minimum of 95 per cent of the total radioactivity corresponds to carbon-11 in the form of L-[methyl-11C]methionine and D-[methyl-11C]methionine, — maximum of 10 per cent of the total radioactivity corresponds to carbon-11 in the form of D-[methyl-11C]methionine. Content of methionine : maximum of 2 mg per maximum recommended dose in millilitres. PRODUCTION RADIONUCLIDE PRODUCTION Carbon-11 is a radioactive isotope of carbon which is most commonly produced by proton irradiation of nitrogen. Depending on the addition of either trace amounts of oxygen or small amounts of hydrogen, the radioactivity is obtained as [11C]carbon dioxide or [11C]methane. RADIOCHEMICAL SYNTHESIS L-[Methyl-11C]methionine can be prepared by various chemical synthetic pathways. All methods rely on the alkylation of the sulphide anion of L-homocysteine with [11C]methyl iodide or [11C]methyl triflate. Variations in the procedures used to generate the sulphide anion of L-homocysteine and methods to obtain [11C]methyl iodide lead to negligible differences with respect to quality in terms of specific radioactivity, enantiomeric purity and possible chemical and radiochemical impurities. Synthesis of [11C]methyl iodide [11C]Methyl iodide can be obtained either starting from [11C]carbon dioxide or from [11C]methane. The most frequently used method is the reduction of [11C]carbon dioxide with lithium aluminium hydride. The formed [11C]methanol is reacted with hydroiodic acid. Alternatively [11C]methane, either obtained directly in the target or by on-line processes from [11C]carbon dioxide, is reacted with iodine. Synthesis of [11C]methyl triflate [11C]methyl triflate can be prepared from [11C]methyl iodide using a silver triflate-impregnated solid support such as graphitised carbon. Synthesis of L-[methyl-11C]methionine The most widely used method to obtain L-[methyl11 C]methionine is the alkylation of the sulphide anion, generated from L-homocysteine thiolactone, with



L-Methionine

([11C]methyl) injection

[11C]methyl iodide or [11C]methyl triflate in alkaline conditions in a solvent such as acetone. The L-[methyl-11C]methionine obtained can be purified by semi-preparative liquid chromatography. For example, a column packed with octadecylsilyl silica gel for chromatography eluted with a 9 g/l solution of sodium chloride is suitable. L-Homocysteine thiolactone hydrochloride Specific optical rotation (2.2.7) : + 20.5 to + 21.5, determined on a 10 g/l solution at 25 °C. Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of L-homocysteine thiolactone hydrochloride.

Detection : spectrophotometer at 225 nm and radioactivity detector connected in series. Injection : loop injector. Run time : 10 min. Relative retention with reference to methionine (retention time = about 2.6 min) : impurity B = about 0.8, impurity A = about 2.7. System suitability : reference solution (a) : — resolution : minimum of 2.5 between the peaks due to methionine and impurity B. Limits : examine the chromatogram obtained with the spectrophotometer : — impurity A : not more than the area of the corresponding CHARACTERS peak in the chromatogram obtained with reference Appearance : clear, colourless solution. solution (a) (0.6 mg/V), Half-life and nature of radiation of carbon-11 : see Table of — impurity B : not more than the area of the corresponding physical characteristics of radionuclides (5.7). peak in the chromatogram obtained with reference solution (a) (2 mg/V), IDENTIFICATION — methionine : not more than the area of the corresponding A. Gamma-ray spectrometry. peak in the chromatogram obtained with reference Results : the only gamma photons have an energy of solution (a) (2 mg/V). 0.511 MeV and, depending on the measurement geometry, Residual solvents (2.4.24) : maximum 50 mg/V for a sum peak of 1.022 MeV may be observed. the concentration of acetone, V being the maximum B. It complies with the test for radionuclidic purity (see recommended dose in millilitres. The preparation may be Tests). released for use before completion of the test. C. Examine the chromatograms obtained in the test for RADIONUCLIDIC PURITY radiochemical purity. Carbon-11 : minimum 99 per cent of the total radioactivity. Results : the principal peak in the radiochromatogram A. Gamma-ray spectroscopy. obtained with the test solution is similar in retention time Comparison : standardised fluorine-18 solution, or by to the principal peak in the chromatogram obtained with using an instrument calibrated with the aid of such a reference solution (b). solution. Standardised fluorine-18 solutions and/or standardisation services are available from the competent TESTS authority. pH (2.2.3) : 4.5 to 8.5. Results : the spectrum obtained with the solution to be Sterility. It complies with the test for sterility examined does not differ significantly from that obtained prescribed in the monograph on Radiopharmaceutical with a standardised fluorine-18 solution. preparations (0125). The injection may be released for use B. Half-life : 19.9 min to 20.9 min. before completion of the test. The preparation may be released for use before completion Bacterial endotoxins (2.6.14) : less than 175/V IU/ml, V of the test. being the maximum recommended dose in millilitres. The RADIOCHEMICAL PURITY injection may be released for use before completion of the test. L-[Methyl-11C]methionine and impurity E. Liquid chromatography (2.2.29) as described in the test for CHEMICAL PURITY impurity A, impurity B and methionine. Impurity A, impurity B and methionine. Liquid Injection : test solution and reference solution (b). chromatography (2.2.29). Limits : examine the chromatogram obtained with the Test solution. The preparation to be examined. radioactivity detector : Reference solution (a). Dissolve 0.6 mg of L-homocysteine — total of L-[methyl-11C]methionine and impurity E : thiolactone hydrochloride R, 2 mg of DL-homocysteine R minimum of 95 per cent of the total radioactivity, and 2 mg of DL-methionine R in water R and dilute to V, — other peaks in the chromatogram may be due to V being the maximum recommended dose in millilitres. impurity C, impurity D and impurity F. Reference solution (b). Dissolve 2 mg of L-methionine R in ENANTIOMERIC PURITY the same solvent as used in the test solution and dilute to 10 ml with the same solvent. Impurity E. Thin-layer chromatography (2.2.27). Column : Test solution. The preparation to be examined. — size : l = 0.25 m, Ø = 4.6 mm, Reference solution (a). Dissolve 2 mg of L-methionine R in water R and dilute to 10 ml with the same solvent. — stationary phase : spherical octadecylsilyl silica gel Reference solution (b). Dissolve 4 mg of DL-methionine R in for chromatography R (5 µm) with a specific surface of water R and dilute to 10 ml with the same solvent. 220 m2/g, a pore size of 8 nm and a carbon loading of 6.2 per cent, Plate : TLC octadecysilyl silica gel plate for chiral separations R. — temperature : 25 °C. Mobile phase : methanol R, water R (50:50 V/V). Mobile phase : 1.4 g/l solution of potassium dihydrogen phosphate R. Application : 2-10 µl. Flow rate : 1 ml/min. Development : over a path of 8 cm. General Notices (1) apply to all monographs and other texts

835

Norcholesterol injection, iodinated (131I)


Drying : in air for 5 min. Detection : spray with a 2 g/l solution of ninhydrin R in ethanol R and heat at 60 °C for 10 min. Determine the distribution of radioactivity using a suitable detector. Retention factors : L-[methyl-11C]methionine = about 0.58 ; impurity E = about 0.51. System suitability : the chromatogram obtained with reference solution (b) shows 2 clearly separated spots. Limits : — total of L-[methyl-11C]methionine and impurity E : minimum 95 per cent of the total radioactivity, — impurity E : maximum 10 per cent of the total radioactivity. The preparation may be released for use before completion of the test. RADIOACTIVITY Measure the radioactivity using suitable equipment by comparison with a standardised fluorine-18 solution or by measurement in an instrument calibrated with the aid of such a solution. LABELLING The accompanying information specifies the maximum recommended dose in millilitres. IMPURITIES

A. (3S)-3-aminodihydrothiophen-2(3H)-one (L-homocysteine thiolactone),

B. (2S)-2-amino-4-sulphanylbutanoic acid (L-homocysteine),

C. (2RS)-2-amino-4-([11C]methylsulphonyl)butanoic acid (DL-[methyl-11C]methionine S,S-dioxide),

D. (2RS)-2-amino-4-([11C]methylsulphinyl)butanoic acid (DL-[methyl-11C]methionine S-oxide),

E. (2R)-2-amino-4-([11C]methylsulphanyl)butanoic acid (D-[methyl-11C]methionine), F. [11C]methanol 836

01/2005:0939

NORCHOLESTEROL INJECTION, IODINATED (131I) Norcholesteroli iodinati (131I) solutio iniectabilis

DEFINITION Iodinated (131I) norcholesterol injection is a sterile, bacterial endotoxin-free solution of 6β-[131I]iodomethyl-19-norcholest-5(10)-en-3β-ol. It may contain a suitable emulsifier such as polysorbate 80 and a suitable antimicrobial preservative such as benzyl alcohol. Iodine-131 is a radioactive isotope of iodine and may be obtained by neutron irradiation of tellurium or by extraction from uranium fission products. The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the declared iodine-131 radioactivity at the date and hour stated on the label. Not less than 85 per cent of the radioactivity corresponds to iodine-131 in the form of 6β-[131I]iodomethyl-19-norcholest-5(10)-en-3β-ol. Not more than 5 per cent of the radioactivity corresponds to iodine-131 in the form of iodide. The specific radioactivity is 3.7 GBq to 37 GBq per gram of 6β-iodomethylnorcholesterol. CHARACTERS A clear or slightly turbid, colourless or pale yellow solution. Iodine-131 has a half-life of 8.04 days and emits beta and gamma radiation. IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised iodine-131 solution by direct comparison with such a solution. The most prominent photon of iodine-131 has an energy of 0.365 MeV. Standardised iodine-131 solutions are available from laboratories recognised by the competent authority. B. Examine the chromatogram obtained in test (a) for radiochemical purity. The distribution of radioactivity contributes to the identification of the preparation. TESTS pH (2.2.3). The pH of the solution is between 3.5 and 8.5. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. Bacterial endotoxins (2.6.14) : less than 175/V IU/ml, V being the maximum recommended dose in millilitres. RADIONUCLIDIC PURITY Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised iodine-131 solution. Determine the relative amounts of iodine-131, iodine-133, iodine-135 and other



radionuclidic impurities present. Iodine-133 has a half-life of 20.8 h and its most prominent gamma photons have energies of 0.530 MeV and 0.875 MeV. Iodine-135 has a half-life of 6.55 h and its most prominent gamma photons have energies of 0.527 MeV, 1.132 MeV and 1.260 MeV. Not less than 99.9 per cent of the total radioactivity is due to iodine-131. RADIOCHEMICAL PURITY a) Examine by thin-layer chromatography (2.2.27) using silica gel GF254 R as the coating substance. Test solution. The injection to be examined. Carrier solution. Dissolve 10 mg of potassium iodide R, 20 mg of potassium iodate R and 0.1 g of sodium hydrogen carbonate R in distilled water R and dilute to 10 ml with the same solvent. Apply to the plate up to 5 µl of the test solution and 10 µl of the carrier solution on the same spot. Develop over a path of 15 cm (about 60 min) using chloroform R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Determine the distribution of radioactivity using a suitable detector. In the chromatogram obtained, not less than 85 per cent of the total radioactivity is found in the spot corresponding to 6β-iodomethyl-19-norcholest-5(10)-en-3β-ol at an Rf value of about 0.5. Iodide ion remains near the starting-line. b) Examine by thin-layer chromatography (2.2.27) using silica gel GF254 R as the coating substance. Test solution. The injection to be examined. Carrier solution. Dissolve 10 mg potassium iodide R, 20 mg of potassium iodate R and 0.1 g of sodium hydrogen carbonate R in distilled water R and dilute to 10 ml with the same solvent. Apply to the plate 10 µl of the carrier solution and then up to 5 µl of the test solution on the same spot. Develop over a path of 15 cm (about 90 min) using a mixture of equal volumes of chloroform R and ethanol R. Allow the plate to dry in air. Expose the plate to ultraviolet light at 254 nm for 5 min. A yellow spot corresponding to iodide develops at an Rf value of about 0.5. Determine the distribution of radioactivity using a suitable detector. The main peak of radio-activity is near to the solvent front. Other iodocholesterols migrate near the solvent front. In the chromatogram obtained, not more than 5 per cent of the total radioactivity is found in the spot corresponding to iodide.

Oxygen (15O)

— minimum 97 per cent of the total radioactivity corresponds to oxygen-15 in the form of oxygen (O2). PRODUCTION RADIONUCLIDIC PRODUCTION Oxygen-15 is a radioactive isotope of oxygen which may be produced by various nuclear reactions such as proton irradiation of nitrogen-15 or deuteron irradiation of nitrogen-14. RADIOCHEMICAL SYNTHESIS In order to recover oxygen-15 as molecular oxygen from the nitrogen target gas, carrier oxygen is added at concentrations generally ranging from 0.2 per cent V/V to 1.0 per cent V/V. After irradiation, the target gas is usually passed through activated charcoal and a carbon dioxide scavenger, such as soda lime, before mixing with the vehicle. CHARACTERS Appearance : colourless gas. Half-life and nature of radiation of oxygen-15 : see Table of physical characteristics of radionuclides (5.7). IDENTIFICATION A. Gamma spectrometry. Results : the only gamma photons have an energy of 0.511 MeV and, depending on the measurement geometry, a sum peak of 1.022 MeV may be observed. B. It complies with the test for radionuclidic purity (see Tests). C. Examine the chromatograms obtained in the test for radiochemical purity. Results : the retention times of the principal peaks in the chromatogram obtained with the test gas using the radioactivity detector are similar to those of the principal peaks corresponding to oxygen in the chromatogram obtained with the reference gas using the thermal conductivity detector.

TESTS The following tests are performed on [15O]oxygen as described under radiochemical synthesis before mixing with the vehicle. RADIONUCLIDIC PURITY Oxygen-15 : minimum 99 per cent of the total radioactivity. RADIOACTIVITY Measure the radioactivity using suitable counting equipment A. Gamma spectrometry. by comparison with a standardised iodine-131 solution or Comparison : standardised fluorine-18 solution, or by by measurement in an instrument calibrated with the aid of using an instrument calibrated with the aid of such a such a solution. solution. Standardised fluorine-18 solutions and/or standardisation services are available from the competent STORAGE authority. Store protected from light at a temperature not exceeding Results : the spectrum obtained with the solution to be − 18 °C. examined does not differ significantly from that obtained with a standardised fluorine-18 solution. 01/2005:1620 B. Half-life : 1.9 min to 2.2 min. The preparation may be released for use before completion OXYGEN (15O) of the test. RADIOCHEMICAL PURITY Oxygenium (15O) Oxygen-15 in the form of O2. Gas chromatography (2.2.28) : use the normalisation procedure. DEFINITION Test sample. [15O]oxygen as described under radiochemical Mixture of [15O]oxygen in the gaseous phase and a suitable synthesis. vehicle such as Medicinal air (1238), for diagnostic use. Reference gas. Nitrogen gas mixture R. Purity : Column : — minimum 99 per cent of the total radioactivity corresponds to oxygen-15, — size : l = 1.8 m, Ø1 = 6.3 mm and Ø2 = 3.2 mm, General Notices (1) apply to all monographs and other texts

837

Raclopride ([11C]methoxy) injection


— stationary phase: GC concentrical column R. Carrier gas : helium for chromatography R. Flow rate : 65 ml/min. Temperature : — column : 40 °C, — injection port : 40 °C, — thermal conductivity detector : 70 °C. Detection : thermal conductivity detector and radioactivity detector connected in series. Injection : loop injector. Run time : 10 min. Retention times : oxygen, nitrogen and carbon monoxide eluting from the inner column = about 0.4 min ; carbon dioxide eluting from the inner column = about 0.8 min ; oxygen eluting from the outer column = about 2.1 min ; nitrogen eluting from the outer column = about 3.1 min ; carbon monoxide eluting from the outer column = about 6.2 min. System suitability : reference gas : — 5 clearly separated principal peaks are observed in the chromatogram obtained using the thermal conductivity detector, — resolution : minimum of 1.5 between the peaks due to carbon dioxide eluting from the inner column and oxygen eluting from the outer column, in the chromatogram obtained using the thermal conductivity detector. Limits : examine the chromatogram obtained with the radioactivity detector and calculate the percentage content of oxygen-15 substances from the peak areas. — oxygen-15 gas in the form of O2 : minimum 97 per cent of the total radioactivity, — disregard the first peak corresponding to components co-eluting from the inner column.

Purity : — minimum of 99 per cent of the total radioactivity corresponds to carbon-11, — minimum of 95 per cent of the total radioactivity corresponds to carbon-11 in the form of [methoxy-11C]raclopride. Content of raclopride : maximum of 10 µg per maximum recommended dose in millilitres.

PRODUCTION RADIONUCLIDE PRODUCTION Carbon-11 is a radioactive isotope of carbon most commonly produced by proton irradiation of nitrogen. Depending on the addition of either trace amounts of oxygen or small amounts of hydrogen, the radioactivity is obtained as [11C]carbon dioxide or [11C]methane, respectively. RADIOCHEMICAL SYNTHESIS [Methoxy-11C]raclopride may be prepared by O-alkylation of the corresponding phenolate anion (S)-3,5-dichloro-2,6-dihydroxy-N-[(1-ethylpyrrolidin-2yl)methyl]benzamide with iodo[11C]methane or [11C]methyl trifluoromethanesulphonate. Synthesis of iodo[11C]methane Iodo[11C]methane may be produced from [11C]carbon dioxide or from [11C]methane. The most frequently used method is reduction of [11C]carbon dioxide with lithium aluminium hydride. The lithium aluminium [11C]methanolate formed is reacted with hydroiodic acid to iodo[11C]methane via [11C]methanol. Alternatively [11C]methane, either obtained directly in the target or by on-line processes from [11C]carbon dioxide, is reacted with iodine. Synthesis of [11C]methyl trifluoromethanesulphonate [11C]Methyl trifluoromethanesulphonate may be prepared from iodo[11C]methane using a solid support such as graphitised carbon impregnated with silver RADIOACTIVITY trifluoromethanesulphonate. The radioactive concentration is determined before Synthesis of [methoxy-11C]raclopride administration. Methylation with iodo[11C]methane is performed Measure the radioactivity using suitable equipment by under alkaline conditions in a solvent such as comparison with a standardised fluorine-18 solution or by dimethyl sulphoxide. The methylation with [11C]methyl measurement in an instrument calibrated with the aid of trifluoromethanesulphonate is performed in a solvent such a solution. such as dimethylformamide or acetone. The resulting [methoxy-11C]raclopride may be purified by semi-preparative liquid chromatography using, for example, a column packed 01/2005:1924 with octadecylsilyl silica gel for chromatography eluted with a mixture of 25 volumes of acetonitrile and 75 volumes of 11 RACLOPRIDE ([ C]METHOXY) 0.01 M phosphoric acid. INJECTION PRECURSOR FOR SYNTHESIS (S)-3,5-Dichloro-2,6-dihydroxy-N-[(1-ethylpyrrolidin-2Raclopridi ([11C]methoxy) yl)methyl]benzamide hydrobromide solutio iniectabilis Melting point (2.2.14) : 211 °C to 213 °C. Specific optical rotation (2.2.7) : + 11.3 to + 11.5, determined on a 15.0 g/l solution in ethanol R at 22 °C. CHARACTERS Appearance : clear, colourless solution. Half-life and nature of radiation of carbon-11 : see Table of physical characteristics of radionuclides (5.7).

DEFINITION Sterile solution of 3,5-dichloro-N-[[(2S)-1-ethylpyrrolidin-2yl]methyl]-2-hydroxy-6-([11C]methoxy)benzamide. Content : 90 per cent to 110 per cent of the declared carbon-11 radioactivity at the date and time stated on the label. 838

IDENTIFICATION A. Gamma-ray spectrometry. Results : the only gamma photons have an energy of 0.511 MeV and, depending on the measurement geometry, a sum peak of 1.022 MeV may be observed. B. It complies with test B for radionuclidic purity (see Tests).



Sodium acetate ([1-11C]) injection

Residual solvents are limited according to the principles defined in the general chapter (5.4), using the general method (2.4.24). The preparation may be released for use before completion of the test. RADIONUCLIDIC PURITY Carbon-11 : minimum 99 per cent of the total radioactivity. The preparation may be released for use before completion TESTS of the test. pH (2.2.3) : 4.5 to 8.5. A. Gamma-ray spectrometry. Sterility. It complies with the test for sterility Comparison : standardised fluorine-18 solution, or by prescribed in the monograph on Radiopharmaceutical using a calibrated instrument. Standardised fluorine-18 preparations (0125). The injection may be released for use solutions and/or standardisation services are available before completion of the test. from the competent authority. Bacterial endotoxins (2.6.14) : less than 175/V IU/ml, Results : the spectrum obtained with the solution to be V being the maximum recommended dose in millilitres. The examined does not differ significantly from that obtained injection may be released for use before completion of the with a standardised fluorine-18 solution. test. B. Half-life. 19.9 min to 20.9 min. CHEMICAL PURITY Raclopride and impurity A. Liquid chromatography (2.2.29). RADIOCHEMICAL PURITY Liquid chromatography (2.2.29) as described in the test for Test solution. The preparation to be examined. raclopride and impurity A with the following modifications. Reference solution (a). Dissolve 7.2 mg of raclopride Injection : test solution and reference solution (d). tartrate R in water R and dilute to 50 ml with the same Limits : examine the chromatogram obtained with the solvent. radioactivity detector : Reference solution (b). Dissolve 1.2 mg of (S)-3,5-dichloro11 2,6-dihydroxy-N-[(1-ethylpyrrolidin-2-yl)methyl]benzamide — [Methoxy- C] raclopride : minimum of 95 per cent of the total radioactivity. hydrobromide R in methanol R and dilute to 100 ml with the same solvent. RADIOACTIVITY Reference solution (c). To 0.1 ml of reference solution (a) add 0.1 ml of reference solution (b) and dilute to V with water R, Mesure the radioactivity using suitable equipment by comparison with a standardised fluorine-18 solution or by V being the maximum recommended dose in millilitres. using a calibrated instrument. Reference solution (d). Dilute 1.0 ml of reference solution (a) to 10.0 ml with water R. LABELLING Column : The accompanying information specifies the maximum recommended dose in millilitres. — size : l = 0.05 m, Ø = 4.6 mm, — stationary phase: spherical end-capped octadecylsilyl IMPURITIES silica gel for chromatography R (3.5 µm) with a specific 2 surface area of 175 m /g, a pore size of 12.5 nm, a pore volume of 0.7 cm3/g and a carbon loading of 15 per cent, — temperature : 30 °C. Mobile phase : dissolve 2 g of sodium heptanesulphonate R in 700 ml of water R, adjust to pH 3.9 with phosphoric acid R and dilute to 1000 ml with acetonitrile R. Flow rate : 1 ml/min. A. 3,5-dichloro-N-[[(2S)-1-ethylpyrrolidin-2-yl]methyl]-2,6Detection : spectrophotometer at 220 nm and radioactivity dihydroxybenzamide. detector connected in series. Injection : loop injector ; inject the test solution and reference solutions (b) and (c). 01/2005:1920 Run time : 10 min. C. Examine the chromatograms obtained in the test for radiochemical purity. Results : the principal peak in the radiochromatogram obtained with the test solution is similar in retention time to the principal peak in the chromatogram obtained with reference solution (d).

Relative retention with reference to raclopride : impurity A = about 0.46. System suitability : reference solution (c) : — resolution : minimum of 5 between the peaks due to raclopride and to impurity A. Limits : examine the chromatogram obtained with the spectrophotometer : — raclopride : not more than the area of the corresponding peak in the chromatogram obtained with reference solution (c) (10 µg/V), — impurity A : not more than the area of the corresponding peak in the chromatogram obtained with reference solution (c) (1 µg/V). General Notices (1) apply to all monographs and other texts

SODIUM ACETATE ([1-11C]) INJECTION Natrii acetatis ([1-11C]) solutio iniectabilis CH311COONa DEFINITION Sterile solution of sodium [1-11C]acetate, in equilibrium with [1-11C]acetic acid. Content : 90 per cent to 110 per cent of the declared carbon-11 radioactivity at the date and time stated on the label. 839

Sodium chromate (51Cr) sterile solution


Mobile phase : 0.1 M sodium hydroxide protected from atmospheric carbon dioxide. Flow rate : 1 ml/min. Detection : spectrophotometer at 220 nm and radioactivity detector connected in series. Injection : loop injector. Run time : 10 min. System suitability : reference solution : — resolution : minimum 4.0 between the peaks due to hold-up volume and acetate. Limit : examine the chromatograms obtained with the spectrophotometer : — acetate : not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (20 mg per V). Residual solvents are limited according to the principles defined in the general chapter (5.4), using the general method (2.4.24). The preparation may be released for use before completion of the test. RADIONUCLIDIC PURITY Carbon-11 : minimum 99 per cent of the total radioactivity. The preparation may be released for use before completion of the tests. A. Gamma-ray spectrometry. CHARACTERS Comparison : standardised fluorine-18 solution, or by Appearance : clear, colourless solution. using a calibrated instrument. Standardised fluorine-18 Half-life and nature of radiation of carbon-11 : see Table of solutions and/or standardisation services are available physical characteristics of radionuclides (5.7). from laboratories recognised by the competent authority. Results : the spectrum obtained with the solution to be IDENTIFICATION examined does not differ significantly from that obtained A. Gamma-ray spectrometry. with a standardised fluorine-18 solution. Results : the only gamma photons have an energy of B. Half-life : 19.9 min to 20.9 min. 0.511 MeV and, depending on the measurement geometry, RADIOCHEMICAL PURITY a sum peak of 1.022 MeV may be observed. B. It complies with test B for radionuclidic purity (see Tests). [1-11C]Acetate. Liquid chromatography (2.2.29) as described in the test for acetate. C. Examine the chromatograms obtained in the test for Limit : examine the chromatograms obtained with the radiochemical purity. spectrophotometer and the radioactivity detector : Results : the principal peak in the radiochromatogram obtained with the test solution is similar in retention time — total of [1-11C]acetate : minimum 95 per cent of the total radioactivity. to the principal peak in the chromatogram obtained with the reference solution. RADIOACTIVITY TESTS Measure the radioactivity using suitable equipment by comparison with a standardised fluorine-18 solution or by pH (2.2.3) : 4.5 to 8.5. measurement with a calibrated instrument. Sterility. It complies with the test for sterility

PRODUCTION RADIONUCLIDE PRODUCTION Carbon-11 is a radioactive isotope of carbon which is most commonly produced by proton irradiation of nitrogen. By the addition of trace amounts of oxygen, the radioactivity is obtained as [11C]carbon dioxide. RADIOCHEMICAL SYNTHESIS [11C]Carbon dioxide may be separated from the target gas mixture by cryogenic trapping or by trapping on a molecular sieve at room temperature. [11C]Carbon dioxide is then released from the trap using an inert gas such as nitrogen at a temperature higher than the trapping temperature. [1-11C]Acetate is usually prepared by reaction of [11C]carbon dioxide with methylmagnesium bromide in organic solvents such as ether or tetrahydrofuran. Hydrolysis of the product yields [1-11C]acetic acid. It is purified by chromatographic procedures. The eluate is diluted with sodium chloride solution. PRECURSOR FOR SYNTHESIS Methylmagnesium bromide. The reactivity of methylmagnesium bromide is tested by decomposition of a defined amount with water. The amount of hydrogen released during this reaction is not less than 90 per cent of the theoretical value.

prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. Bacterial endotoxins (2.6.14) : less than 175/V IU/ml, V being the maximum recommended dose in millilitres. The injection may be released for use before completion of the test. CHEMICAL PURITY Acetate. Liquid chromatography (2.2.29). Test solution. The preparation to be examined. Reference solution. Dissolve 28 mg of sodium acetate R in water R and dilute to V, V being the maximum recommended dose in millilitres. Column : — size : l = 0.25 m, Ø = 4.0 mm, — stationary phase : strongly basic anion exchange resin for chromatography R (10 µm), — temperature : 25 °C. 840

LABELLING The accompanying information specifies the maximum recommended dose in millilitres. 01/2005:0279

SODIUM CHROMATE (51Cr) STERILE SOLUTION Natrii chromatis (51Cr) solutio sterilis DEFINITION Sodium chromate (51Cr) sterile solution is a sterile solution of sodium [51Cr]chromate made isotonic by the addition of sodium chloride. Chromium-51 is a radioactive isotope of chromium and may be prepared by neutron irradiation of chromium, either of natural isotopic composition or enriched in chromium-50. The solution contains not less than 90.0 per cent and not more than 110.0 per cent of



Sodium fluoride (18F) injection

Content : the declared chromium-51 radioactivity at the date and hour stated on the label. Not less than 90 per cent of the — fluorine-18 : 90 per cent to 110 per cent of the declared radioactivity corresponds to chromium-51 in the form of fluorine-18 radioactivity at the date and hour stated on chromate. The specific radioactivity is not less than 370 MBq the label, of chromium-51 per milligram of chromate ion. — fluoride : maximum 4.52 mg per maximum recommended dose in millilitres. CHARACTERS A clear, colourless or slightly yellow solution. PRODUCTION Chromium-51 has a half-life of 27.7 days and emits gamma The radionuclide fluorine-18 is most commonly produced radiation. by proton irradiation of water enriched in oxygen-18. Fluorine-18 in the form of fluoride is recovered from the IDENTIFICATION target water, generally by adsorption and desorption from A. Record the gamma-ray spectrum using a suitable anion-exchange resins or electrochemical deposition and instrument. The spectrum does not differ significantly redissolution. from that of a standardised chromium-51 solution. Standardised chromium-51 solution are available from CHARACTERS laboratories recognised by the competent authority. The Appearance : clear, colourless solution. gamma photon has an energy of 0.320 MeV. Half-life and nature of radiation of fluorine-18 : see Table of B. Examine the chromatogram obtained in the test for physical characteristics of radionuclides (5.7). radiochemical purity. The distribution of radioactivity contributes to the identification of the preparation. IDENTIFICATION A. Gamma-ray spectrometry. TESTS Results : the only gamma photons have an energy of pH (2.2.3). The pH of the solution is 6.0 to 8.5. 0.511 MeV and, depending on the measurement geometry, Total chromate. Not more than 2.7 µg of chromate ion a sum peak of 1.022 MeV may be observed. (CrO42-) per megabecquerel. Measure the absorbance of the B. It complies with test B for radionuclidic purity (see Tests). solution (2.2.25) at the absorption maximum at 370 nm. C. Examine the chromatograms obtained in the test for Calculate the content of chromate using the absorbance of radiochemical purity (see Tests). a standard consisting of a 1.7 mg/l solution of potassium Results : the principal peak in the radiochromatogram chromate R. If necessary, adjust the solution to be examined obtained with the test solution is similar in retention time and the standard to pH 8.0 by adding sodium hydrogen to the principal peak in the chromatogram obtained with carbonate solution R. the reference solution. In the chromatogram obtained Sterility. It complies with the test for sterility with the reference solution, the peak due to fluoride is prescribed in the monograph on Radiopharmaceutical negative. preparations (0125). The solution may be released for use before completion of the test. TESTS RADIONUCLIDIC PURITY pH (2.2.3) : 5.0 to 8.5. Record the gamma-ray spectrum using a suitable instrument. Fluoride. Liquid chromatography (2.2.29). The spectrum does not differ significantly from that of a Test solution. The preparation to be examined. standardised chromium-51 solution. Reference solution. Dissolve 10 mg of sodium fluoride R in RADIOCHEMICAL PURITY water R and dilute to V with the same solvent, V being the Examine by ascending paper chromatography (2.2.26). maximum recommended dose in millilitres. Apply to the paper a quantity of the solution sufficient for Column : the detection method. Begin the development immediately — size : l = 0.25 m, Ø = 4 mm, and develop for 2.5 h using a mixture of 25 volumes of ammonia R, 50 volumes of alcohol R and 125 volumes of — stationary phase : strongly basic anion-exchange resin water R. Chromic ions remain on the starting line. Determine for chromatography R (10 µm), the distribution of the radioactivity using a suitable detector. — temperature : constant, between 20 °C and 30 °C. Not less than 90 per cent of the total radioactivity of the chromatogram is found in the spot with an Rf value of about Mobile phase : 4 g/l solution of sodium hydroxide R, protected from atmospheric carbon dioxide. 0.9, corresponding to sodium chromate. Flow rate : 1 ml/min. RADIOACTIVITY Detection : spectrophotometer at 220 nm and a radioactivity Measure the radioactivity using suitable counting equipment detector connected in series. by comparison with a standardised chromium-51 solution or Injection : 20 µl. by measurement in an instrument calibrated with the aid of Run time : 15 min. such a solution. System suitability : examine the chromatogram obtained with the reference solution using the spectrophotometer : 01/2005:2100 — signal-to-noise ratio : minimum 10 for the principal peak, 18 SODIUM FLUORIDE ( F) INJECTION — retention time of fluoride : minimum 3 times the hold-up time. 18 Limit : examine the chromatogram obtained with the Natrii fluoridi ( F) solutio iniectabilis spectrophotometer : DEFINITION — fluoride : not more than the area of the corresponding peak in the chromatogram obtained with the reference Sterile solution containing fluorine-18 in the form of sodium solution (4.52 mg/V). fluoride. It may contain carrier fluoride and a suitable buffer. General Notices (1) apply to all monographs and other texts

841

Sodium iodide (123I) injection


Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. Bacterial endotoxins (2.6.14) : less than 175/V IU/ml, V being the maximum recommended dose in millilitres. The injection may be released for use before completion of the test. RADIONUCLIDIC PURITY Fluorine-18 : minimum 99.9 per cent of the total radioactivity. The preparation may be released for use before completion of the tests. A. Gamma-ray spectrometry. Determine the amount of fluorine-18 and radionuclidic impurities with a half-life longer than 2 h. For the detection and quantification of impurities, retain the preparation to be examined for a sufficient time to allow the fluorine-18 to decay to a level which permits the detection of impurities. Results : the spectrum obtained with the preparation to be examined does not differ significantly from that of a background spectrum. B. Half-life : 105 min to 115 min. RADIOCHEMICAL PURITY [18F]fluoride. Liquid chromatography (2.2.29) as described in the test for fluoride. If necessary, dilute the test solution with water R to obtain a radioactivity concentration suitable for the radioactivity detector. Limit : examine the chromatogram obtained with the radioactivity detector : — [18F]fluoride : minimum 98.5 per cent of the total radioactivity.

IDENTIFICATION A. Gamma-ray spectrometry. Results : the spectrum obtained with the preparation to be examined does not differ significantly from that of a standardised iodine-123 solution. The most prominent gamma photon has an energy of 0.159 MeV and is accompanied by the principal X-ray of 0.027 MeV. B. Examine the chromatograms obtained in the test for radiochemical purity. Results : the principal peak in the radiochromatogram obtained with the test solution is similar in retention time to the principal peak in the chromatogram obtained with reference solution (a).

TESTS pH (2.2.3) : 7.0 to 10.0. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The preparation may be released for use before completion of the test. RADIONUCLIDIC PURITY Iodine-123 : minimum 99.65 per cent of the total radioactivity. Gamma-ray spectrometry. Determine the relative amounts of iodine-123, iodine-125, tellurium-121 and other radionuclidic impurities present. For the detection of tellurium-121 and iodine-125, retain the preparation to be examined for a sufficient time to allow iodine-123 to decay to a level which permits the detection of radionuclidic impurities. No radionuclides with a half-life longer than that of iodine-125 are detected. The preparation may be released for use before completion of the test. RADIOCHEMICAL PURITY RADIOACTIVITY [123I]iodide. Liquid chromatography (2.2.29). Determine the radioactivity using a calibrated instrument. Test solution. Dilute the preparation to be examined with LABELLING a 2 g/l solution of sodium hydroxide R to a radioactive The label states the maximum recommended dose in concentration suitable for the detector. Add an equal volume millilitres. of a solution containing 1 g/l of potassium iodide R, 2 g/l of potassium iodate R and 10 g/l of sodium hydrogen 01/2005:0563 carbonate R and mix. Reference solution (a). Dilute 1 ml of a 26.2 mg/l solution of potassium iodide R to 10 ml with water R. SODIUM IODIDE (123I) INJECTION Reference solution (b). Dilute 1 ml of a 24.5 mg/l solution of potassium iodate R to 10 ml with water R. Mix equal Natrii iodidi (123I) solutio iniectabilis volumes of this solution and reference solution (a). DEFINITION Column : Sterile solution containing iodine-123 in the form of sodium — size : l = 0.25 m, Ø = 4.0 mm, iodide ; it may contain sodium thiosulphate or some other — stationary phase : octadecylsilyl silica gel for suitable reducing agent and a suitable buffer. chromatography R (5 µm), Content : 90 per cent to 110 per cent of the declared — temperature : constant between 20 °C and 30 °C. iodine-123 radioactivity at the date and hour stated on the Mobile phase : dissolve 5.85 g of sodium chloride R in label. 1000 ml of water R, add 0.65 ml of octylamine R and adjust PRODUCTION to pH 7.0 with dilute phosphoric acid R ; add 50 ml of acetonitrile R and mix. Iodine-123 is obtained by proton irradiation of xenon enriched in xenon-124 (minimum 98 per cent) followed by Flow rate : 1.5 ml/min. the decay of xenon-123 which is formed directly and by the Detection : spectrophotometer at 220 nm and a radioactivity decay of caesium-123. No carrier iodide is added. detector connected in series. Injection : 20 µl. CHARACTERS Run time : 12 min. Appearance : clear, colourless solution. Half-life and nature of radiation of iodine-123 : see Table of Relative retention with reference to iodide (retention time = about 5 min) : iodate = 0.2 to 0.3. physical characteristics of radionuclides (5.7). 842



Sodium iodide (131I) capsules for diagnostic use

TESTS Disintegration : the contents of the capsule dissolve completely within 15 min. In a water-bath at 37 °C, warm in a small beaker about 20 ml of a 2.0 g/l solution of potassium iodide R. Add a capsule to be examined. Stir magnetically at 20 r/min. Uniformity of content. Determine the radioactivity of each of not fewer than 10 capsules. Calculate the average radioactivity per capsule. The radioactivity of no capsule differs by more than 10 per cent from the average, the relative standard deviation is not greater than 3.5 per cent. RADIOACTIVITY Iodide. Liquid chromatography (2.2.29). Determine the radioactivity using a calibrated instrument. Test solution (a). Dissolve a capsule to be examined in 10 ml LABELLING of water R. Filter through a 0.2 µm filter. Test solution (b). Dissolve a capsule to be examined in The label states any substance added. water R. Filter through a 0.2 µm filter and dilute the filtrate IMPURITIES with a 2 g/l solution of sodium hydroxide R to a radioactive concentration suitable for the detector. Add an equal volume A. [123I]iodate ion. of a solution containing 1 g/l of potassium iodide R, 2 g/l of potassium iodate R and 10 g/l of sodium hydrogen carbonate R and mix. 01/2005:0938 Reference solution (a). Dilute 1 ml of a 26.2 mg/l solution of potassium iodide R to 10 ml with water R. SODIUM IODIDE (131I) CAPSULES FOR Reference solution (b). Dilute 1 ml of a 24.5 mg/l solution of potassium iodate R to 10 ml with water R. Mix equal DIAGNOSTIC USE volumes of this solution and reference solution (a). Blank solution. Prepare a solution containing 2 mg/ml of Natrii iodidi (131I) capsulae each constituent stated on the label, apart from iodide. ad usum diagnosticum Column : — size : l = 0.25 m, Ø = 4.0 mm, DEFINITION — stationary phase : octadecylsilyl silica gel for Capsules for diagnostic use containing iodine-131 in the chromatography R (5 µm), form of sodium iodide on a solid support ; they may contain sodium thiosulphate or some other suitable reducing agents — temperature : constant between 20 °C and 30 °C. and a suitable buffering substance. A package contains 1 or Mobile phase : dissolve 5.85 g of sodium chloride R in more capsules. 1000 ml of water R, add 0.65 ml of octylamine R and adjust to pH 7.0 with dilute phosphoric acid R ; add 50 ml of Content : acetonitrile R and mix. — iodine-131 : maximum 37 MBq per capsule ; the average Flow rate : 1.5 ml/min. radioactivity determined in the test for uniformity of content is 90 per cent to 110 per cent of the declared Detection : spectrophotometer at 220 nm and radioactivity iodine-131 radioactivity at the date and hour stated on detector connected in series. the label ; Injection : 20 µl of test solution (a), reference solutions (a) — iodide : maximum 20 µg per capsule. and (b) and the blank solution. Run time : 12 min. PRODUCTION Relative retention with reference to iodide (retention Iodine-131 is obtained by neutron irradiation of tellurium time = about 5 min) : iodate = 0.2 to 0.3. or by extraction from uranium fission products. No carrier System suitability : iodide is added. — in the chromatogram obtained with the blank solution, CHARACTERS none of the peaks has a retention time similar to that of the peak due to iodide, Half-life and nature of radiation of iodine-131 : see Table of physical characteristics of radionuclides (5.7). — resolution : minimum 2 between the peaks due to iodide and iodate in the chromatogram obtained with reference IDENTIFICATION solution (b) recorded with the spectrophotometer. A. Gamma-ray spectrometry. Limit : examine the chromatograms obtained with the Results : the spectrum obtained with the preparation to spectrophotometer : be examined does not differ significantly from that of a — iodide : not more than the area of the corresponding peak standardised iodine-131 solution. The most prominent in the chromatogram obtained with reference solution (a) gamma photon has an energy of 0.365 MeV. (20 µg/capsule). B. Examine the chromatograms obtained in the test for RADIONUCLIDIC PURITY radiochemical purity. Iodine-131 : minimum 99.9 per cent of the total radioactivity. Results : the principal peak in the radiochromatogram obtained with test solution (b) is similar in retention time Gamma-ray spectrometry. to the principal peak in the chromatogram obtained with Determine the relative amounts of iodine-131, iodine-133, iodine-135 and other radionuclidic impurities present. reference solution (a). System suitability : reference solution (b) : — resolution : minimum 2 between the peaks due to iodide and iodate in the chromatogram recorded with the spectrophotometer. Limit : examine the chromatogram obtained with the test solution using the radioactivity detector and locate the peak due to iodide by comparison with the chromatogram obtained with reference solution (a) using the spectrophotometer : — [123I]iodide : minimum 95 per cent of the total radioactivity.


843

Sodium iodide (131I) solution


Sterility. If intended for parenteral use, it complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The solution may be released for use before completion of the test. Iodide. Liquid chromatography (2.2.29). Test solution (a). The preparation to be examined. Test solution (b). Dilute the preparation to be examined with 0.05 M sodium hydroxide until the radioactivity is equivalent to about 74 MBq/ml. Add an equal volume of a solution containing 1 g/l of potassium iodide R, 2 g/l of potassium iodate R and 10 g/l of sodium hydrogen carbonate R and mix. RADIOACTIVITY Determine the radioactivity of the package using a calibrated Reference solution (a). Dilute 1 ml of a 26.2 mg/l solution of potassium iodide R to V with water R, V being the maximum instrument. recommended dose in millilitres. LABELLING Reference solution (b). Dilute 1 ml of a 24.5 mg/l solution of potassium iodate R to V with water R, V being the maximum The label states any substance added and the number of recommended dose in millilitres. Mix equal volumes of this capsules in the package. solution and of reference solution (a). IMPURITIES Blank solution. Prepare a solution containing 2 mg/ml of each of the components stated on the label, apart from 131 A. [ I]iodate ion. iodide. Column : — size : l = 0.25 m, Ø = 4.0 mm, 01/2005:0281 — stationary phase : octadecylsilyl silica gel for chromatography R (5 µm), 131 SODIUM IODIDE ( I) SOLUTION — temperature : maintain at a constant temperature between 20 °C and 30 °C. Natrii iodidi (131I) solutio Use stainless steel tubing. Mobile phase : dissolve 5.844 g of sodium chloride R in DEFINITION 1000 ml of water R, add 650 µl of octylamine R and adjust to Solution containing iodine-131 in the form of sodium pH 7.0 with phosphoric acid R ; add 50 ml of acetonitrile R iodide and also sodium thiosulphate or some other suitable and mix. reducing agent. It may contain a suitable buffer. Flow rate : 1.5 ml/min. Content : Detection : spectrophotometer at 220 nm and radioactivity — iodine-131 : 90 per cent to 110 per cent of the declared detector connected in series. radioactivity at the date and hour stated on the label, Injection : 25 µl ; inject test solution (a), the blank solution — iodide : maximum 20 µg in the maximum recommended and reference solutions (a) and (b). dose in millilitres. Run time : 12 min. Relative retention with reference to iodide (retention PRODUCTION time = about 5 min) : iodate = 0.2 to 0.3. Iodine-131 is a radioactive isotope of iodine and may be obtained by neutron irradiation of tellurium or by extraction System suitability : — in the chromatogram obtained with the blank solution, from uranium fission products. No carrier iodide is added. none of the peaks shows a retention time similar to that CHARACTERS of the peak due to iodide, — resolution : minimum 2 between the peaks due to iodide Appearance : clear, colourless solution. and iodate in the chromatogram obtained with reference Half-life and nature of radiation of iodine-131 : see Table of solution (b) recorded with the spectrophotometer. physical characteristics of radionuclides (5.7). Limit : examine the chromatogram obtained with the IDENTIFICATION spectrophotometer; locate the peak due to iodide by comparison with the chromatogram obtained with reference A. Gamma-ray spectrometry. solution (a) : Results : the spectrum obtained with the preparation to — iodide : not more than the area of the corresponding peak be examined does not differ significantly from that of a in the chromatogram obtained with reference solution (a). standardised iodine-131 solution. The most prominent gamma photon has an energy of 0.365 MeV. RADIONUCLIDIC PURITY B. Examine the chromatograms obtained in the test for Iodine-131 : minimum 99.9 per cent of the total radioactivity. iodide. Gamma-ray spectrometry. Results : the principal peak in the radiochromatogram Determine the relative amounts of iodine-131, iodine-133, obtained with test solution (a) is similar in retention time iodine-135 and other radionuclidic impurities present. to the principal peak in the chromatogram obtained with RADIOCHEMICAL PURITY reference solution (a). [131I]Iodide. Liquid chromatography (2.2.29) as described in TESTS the test for iodide with the following modification. Injection : test solution (b). pH (2.2.3) : 7.0 to 10.0. RADIOCHEMICAL PURITY [131I]iodide. Liquid chromatography (2.2.29) as described in the test for iodide with the following modifications. Injection : 20 µl of test solution (b) and reference solution (a). Limit : examine the chromatogram obtained with the test solution using the radioactivity detector and locate the peak due to iodide by comparison with the chromatogram obtained with reference solution (a) using the spectrophotometer : — [131I]iodide : minimum 95 per cent of the total radioactivity.

844



Sodium iodohippurate (123I) injection

Limit : examine the chromatogram obtained with the radioactivity detector : — [131I]iodide : minimum 95 per cent of the total radioactivity.

RADIOCHEMICAL PURITY [131I]iodide. Liquid chromatography (2.2.29). Test solution. Dilute the preparation to be examined with an equal volume of a solution containing 1 g/l of potassium iodide R, 2 g/l of potassium iodate R and 10 g/l of sodium RADIOACTIVITY hydrogen carbonate R and mix. If necessary, first dilute Measure the radioactivity using suitable equipment by the preparation to be examined with a 2 g/l solution of comparison with a standardised iodine-131 solution or by sodium hydroxide R to ensure that the final mixture has using a calibrated instrument. a radioactivity concentration suitable for the radioactivity detector. LABELLING Reference solution (a). Dissolve 10 mg of potassium The label states : iodide R in water R and dilute to 10 ml with the same solvent. — any substance added, Reference solution (b). Dissolve 20 mg of potassium — the maximum recommended dose, in millilitres, iodate R in water R and dilute to 10 ml with the same solvent. — where applicable, that the preparation is suitable for use Mix equal volumes of this solution and reference solution (a). in the manufacture of parenteral dosage forms. Column : IMPURITIES — size : l = 0.25 m, Ø = 4.0 mm, — stationary phase : octadecylsilyl silica gel for A. [131I]iodate ion. chromatography R (5 µm), 01/2005:2121 — temperature : constant, between 20 °C and 30 °C. Use stainless steel tubing. SODIUM IODIDE (131I) SOLUTION FOR Mobile phase : dissolve 5.85 g of sodium chloride R in 1000 ml of water R, add 0.65 ml of octylamine R and adjust RADIOLABELLING to pH 7.0 with dilute phosphoric acid R ; add 50 ml of acetonitrile R and mix. Natrii iodidi (131I) solutio Flow rate : 1.5 ml/min. ad radio-signandum Detection : spectrophotometer at 220 nm and a radioactivity detector connected in series. DEFINITION Strongly alkaline solution containing iodine-131 in the form Injection : 20 µl. of sodium iodide. It does not contain a reducing agent. Run time : 12 min. Content : 90 per cent to 110 per cent of the declared Relative retention with reference to iodide (retention iodine-131 radioactivity at the date and hour stated on the time = about 5 min) : iodate = 0.2 to 0.3. label. System suitability : reference solution (b) : — resolution : minimum 2 between the peaks due to iodide PRODUCTION and iodate in the chromatogram recorded with the Iodine-131 may be obtained by neutron irradiation of spectrophotometer. tellurium or by extraction from uranium fission products. No Limit : examine the chromatogram obtained with the carrier iodide is added. radioactivity detector : CHARACTERS — [131I]iodide : minimum 95 per cent of the total Appearance : clear, colourless solution. radioactivity. Half-life and nature of radiation of iodine-131 : see Table of RADIOACTIVITY physical characteristics of radionuclides (5.7). Determine the radioactivity using a calibrated instrument. IDENTIFICATION LABELLING A. Gamma-ray spectrometry. The label states : Results : the spectrum obtained with the preparation to be examined does not differ significantly from that of a — the method of production of iodine-131, standardised iodine-131 solution. The most prominent — the vehicle and any substance added, gamma photon of iodine-131 has an energy of 0.365 MeV. — that the preparation is not for direct human use. B. Examine the chromatograms obtained in the test for radiochemical purity (see Tests). IMPURITIES Results : the principal peak in the radiochromatogram A. [131I]iodate ion. obtained with the test solution is similar in retention time to the principal peak in the chromatogram obtained with 01/2005:0564 reference solution (a). TESTS Alkalinity (2.2.4). The preparation is strongly alkaline. RADIONUCLIDIC PURITY Iodine-131 : minimum 99.9 per cent of the total radioactivity. Gamma-ray spectrometry. Determine the relative amounts of iodine-130, iodine-131, iodine-133, iodine-135 and other radionuclidic impurities present. General Notices (1) apply to all monographs and other texts

SODIUM IODOHIPPURATE (123I) INJECTION Natrii iodohippurati (123I) solutio iniectabilis DEFINITION Sodium iodohippurate (123I) injection is a sterile solution of sodium (2-[123I]iodobenzamido)acetate. It may contain a suitable buffer and a suitable antimicrobial preservative 845

Sodium iodohippurate (131I) injection


such as benzyl alcohol. Iodine-123 is a radioactive isotope of iodine and may be obtained by proton irradiation of xenon enriched in xenon-124 (not less than 98 per cent) followed by the decay of caesium-123 formed via xenon-123. The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the declared iodine-123 radioactivity at the date and hour stated on the label. Not less than 96 per cent of the radioactivity corresponds to iodine-123 in the form of sodium 2-iodohippurate. The specific radioactivity is 0.74 GBq to 10.0 GBq of iodine-123 per gram of sodium 2-iodohippurate. Not more than 0.35 per cent of the total radioactivity is due to radionuclides other than iodine-123. CHARACTERS A clear, colourless liquid. Iodine-123 has a half-life of 13.2 h and emits gamma radiation and X-rays. IDENTIFICATION A. Record the gamma-ray and X-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised iodine-123 solution apart from any differences attributable to the presence of iodine-125, tellurium-121 and other radionuclidic impurities. Standardised iodine-123, iodine-125 and tellurium-121 solutions are available from laboratories recognised by the national authorities. The most prominent gamma photon of iodine-123 has an energy of 0.159 MeV and is accompanied by an X-ray of 0.027 MeV. Iodine-125 has a half-life of 59.4 days and emits an X-ray of 0.027 MeV and a photon of 0.035 MeV. Tellurium-121 has a half-life of 19.2 days and the most prominent photons have energies of 0.507 MeV and 0.573 MeV. B. Examine the chromatograms obtained in the test for radiochemical purity. The spot corresponding to the main peak of radioactivity in the chromatogram obtained with the test solution is similar in position to the spot corresponding to 2-iodohippuric acid in the chromatogram obtained with the reference solution. TESTS pH (2.2.3). The pH of the solution is 3.5 to 8.5. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. RADIONUCLIDIC PURITY Record the gamma-ray spectrum using a suitable instrument. Determine the relative amounts of iodine-125, tellurium-121 and other radionuclidic impurities present. No radionuclides with longer half lives than iodine-125 are detected. For the determination of iodine-125, tellurium-121 and other radionuclidic impurities, retain the solution to be examined for a sufficient time to allow the radioactivity of iodine-123 to decrease to a level which permits the detection of radionuclidic impurities. Record the gamma-ray spectrum and X-ray spectrum of the decayed material using a suitable instrument. Not more than 0.35 per cent of the total radioactivity is due to radionuclides other than iodine-123. The injection may be released for use before completion of the test. RADIOCHEMICAL PURITY Examine by thin-layer chromatography (2.2.27) using silica gel GF254 R as the coating substance. Test solution. Dissolve 1 g of potassium iodide R in 10 ml of water R, add 1 volume of this solution to 10 volumes of the injection to be examined and use within 10 min of mixing. 846

If necessary, dilute with the reference solution (carrier) to give a radioactive concentration sufficient for the detection method, for example 3.7 MBq per millilitre. Reference solution (carrier). Dissolve 40 mg of 2-iodohippuric acid R and 40 mg of 2-iodobenzoic acid R in 4 ml of 0.1 M sodium hydroxide, add 10 mg of potassium iodide R and dilute to 10 ml with water R. Apply separately to the plate 10 µl of each solution. Develop over a path of 12 cm (about 75 min) using a mixture of 1 volume of water R, 4 volumes of glacial acetic acid R, 20 volumes of butanol R and 80 volumes of toluene R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The chromatogram obtained with the reference solution shows a spot corresponding to 2-iodohippuric acid and nearer to the solvent front a spot corresponding to 2-iodobenzoic acid. Iodide ion remains near the starting-line. Determine the distribution of radioactivity using a suitable detector. In the chromatogram obtained with the test solution, not less than 96 per cent of the total radioactivity is found in the spot corresponding to 2-iodohippuric acid and not more than 2 per cent of the total radioactivity is found in either of the spots corresponding to 2-iodobenzoic acid and to iodide ion. RADIOACTIVITY Measure the radioactivity using suitable counting equipment by comparison with a standardised iodine-123 solution or by measurement in an instrument calibrated with the aid of such a solution. STORAGE Store protected from light. LABELLING The label states whether or not the preparation is suitable for renal plasma-flow studies. 01/2005:0282

SODIUM IODOHIPPURATE (131I) INJECTION Natrii iodohippurati (131I) solutio iniectabilis DEFINITION Sodium iodohippurate (131I) injection is a sterile solution of sodium 2-(2-[131I]iodobenzamido)acetate. It may contain a suitable buffer and a suitable antimicrobial preservative such as benzyl alcohol. Iodine-131 is a radioactive isotope of iodine and may be obtained by neutron irradiation of tellurium or by extraction from uranium fission products. The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the declared iodine-131 radioactivity at the date and hour stated on the label. Not less than 96 per cent of the iodine-131 is in the form of sodium 2-iodohippurate. The specific radioactivity is 0.74 GBq to 7.4 GBq of iodine-131 per gram of sodium 2-iodohippurate. CHARACTERS A clear, colourless liquid. Iodine-131 has a half-life of 8.04 days and emits beta and gamma radiation. IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised iodine-131 solution. Standardised iodine-131 solutions are available from


Sodium pertechnetate (99mTc) injection (fission)


laboratories recognised by the competent authority. The most prominent gamma photon of iodine-131 has an energy of 0.365 MeV. B. Examine the chromatograms obtained in the test for radiochemical purity. The main peak of radioactivity in the chromatogram obtained with the test solution is similar in position to the spot corresponding to 2-iodohippuric acid in the chromatogram obtained with the reference solution. TESTS pH (2.2.3). The pH of the injection is 6.0 to 8.5. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. RADIONUCLIDIC PURITY Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised iodine-131 solution. Determine the relative amounts of iodine-131, iodine-133, iodine-135 and other radionuclidic impurities present. Iodine-133 has a half-life of 20.8 h and its most prominent gamma photons have energies of 0.530 MeV and 0.875 MeV. Iodine-135 has a half-life of 6.55 h and its most prominent gamma photons have energies of 0.527 MeV, 1.132 MeV and 1.260 MeV. Not less than 99.9 per cent of the total radioactivity is due to iodine-131. RADIOCHEMICAL PURITY Examine by thin-layer chromatography (2.2.27) using silica gel GF254 R as the coating substance. Test solution. Dissolve 1 g of potassium iodide R in 10 ml of water R, add 1 volume of this solution to 10 volumes of the injection to be examined and use within 10 min of mixing. If necessary dilute with the reference solution (carrier) to give a radioactive concentration sufficient for the detection method, for example 3.7 MBq per millilitre.

LABELLING The label states that the preparation is not necessarily suitable for renal plasma-flow studies.

01/2005:0124

SODIUM PERTECHNETATE (99mTc) INJECTION (FISSION) Natrii pertechnetatis (99mTc) fissione formati solutio iniectabilis This monograph applies to sodium pertechnetate (99mTc) injection obtained from molybdenum-99 extracted from fission products of uranium. Sodium pertechnetate (99mTc) injection obtained from molybdenum-99 produced by the neutron irradiation of molybdenum is described in the monograph on Sodium pertechnetate (99mTc) injection (non-fission) (0283).

DEFINITION Sodium pertechnetate (99mTc) injection (fission) is a sterile solution containing technetium-99m in the form of pertechnetate ion and made isotonic by the addition of sodium chloride. Technetium-99m is a radionuclide formed by the decay of molybdenum-99. Molybdenum-99 is a radioactive isotope of molybdenum extracted from uranium fission products. The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the declared technetium-99m radioactivity at the date and hour stated on the label. Not less than 95 per cent of the radioactivity corresponds to technetium-99m in the form of pertechnetate ion. The radioactivity due to radionuclides other than technetium-99m, apart from that due to technetium-99 resulting from the decay of technetium-99m, is not greater Reference solution (carrier). Dissolve 40 mg of 2-iodohippuric acid R and 40 mg of 2-iodobenzoic acid R in than that shown below, expressed as a percentage of the total radioactivity and calculated with reference to the date 4 ml of 0.1 M sodium hydroxide, add 10 mg of potassium and hour of administration. iodide R and dilute to 10 ml with water R. Apply separately to the plate 10 µl of each solution. Develop over a path of 12 cm (about 75 min) using a mixture of 1 volume of water R, 4 volumes of glacial acetic acid R, 20 volumes of butanol R and 80 volumes of toluene R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The chromatogram obtained with the reference solution shows a spot corresponding to 2-iodohippuric acid and nearer to the solvent front a spot corresponding to 2-iodobenzoic acid. Iodide ion remains near the starting-line. Determine the distribution of radioactivity using a suitable detector. In the chromatogram obtained with the test solution, not less than 96 per cent of the total radioactivity is found in the spot corresponding to 2-iodohippuric acid and not more than 2 per cent of the total radioactivity is found in either of the spots corresponding to 2-iodobenzoic acid and to iodide ion. RADIOACTIVITY

molybdenum-99

0.1 per cent

iodine-131

5 × 10− 3 per cent

ruthenium-103

5 × 10− 3 per cent

strontium-89

6 × 10− 5 per cent

strontium-90

6 × 10− 6 per cent

alpha-emitting impurities

1 × 10− 7 per cent

other gamma-emitting impurities

0.01 per cent

The injection may be prepared from a sterile preparation of molybdenum-99 under aseptic conditions. CHARACTERS A clear, colourless solution. Technetium-99m has a half-life of 6.02 h and emits gamma radiation.

IDENTIFICATION Measure the radioactivity using suitable counting equipment Record the gamma-ray spectrum using a suitable by comparison with a standardised iodine-131 solution or by measurement in an instrument calibrated with the aid of instrument. The spectrum does not differ significantly from that of a standardised technetium-99m solution such a solution. either by direct comparison or by measurement in an instrument calibrated with the aid of such a solution. STORAGE Standardised technetium-99m, molybdenum-99, iodine-131, Store protected from light. ruthenium-103, strontium-89 and strontium/yttrium-90 General Notices (1) apply to all monographs and other texts

847

Sodium pertechnetate (99mTc) injection (non-fission)


solutions are available from laboratories recognised by the competent authority. The most prominent gamma photon of technetium-99m has an energy of 0.140 MeV. TESTS pH (2.2.3). The pH of the injection is 4.0 to 8.0. Aluminium. In a test tube about 12 mm in internal diameter, mix 1 ml of acetate buffer solution pH 4.6 R and 2 ml of a 1 in 2.5 dilution of the injection in water R. Add 0.05 ml of a 10 g/l solution of chromazurol S R. After 3 min, the colour of the solution is not more intense than that of a standard prepared at the same time and in the same manner using 2 ml of aluminium standard solution (2 ppm Al) R (5 ppm). Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. RADIONUCLIDIC PURITY Preliminary test. To obtain an approximate estimate before use of the injection, take a volume equivalent to 37 MBq and determine the gamma-ray spectrum using a sodium iodide detector with a shield of lead, of thickness 6 mm, interposed between the sample and the detector. The response in the region corresponding to the 0.740 MeV photon of molybdenum-99 does not exceed that obtained using 37 kBq of a standardised molybdenum-99 solution measured under the same conditions, when all measurements are expressed with reference to the date and hour of administration. Definitive test. Retain a sample of the injection for a sufficient time to allow the technetium-99m radioactivity to decay to a sufficiently low level to permit the detection of radionuclidic impurities. All measurements of radioactivity are expressed with reference to the date and hour of administration. — Molybdenum-99. Record the gamma-ray spectrum of the decayed material in a suitable instrument calibrated with the aid of a standardised molybdenum-99 solution. The most prominent photons have energies of 0.181 MeV, 0.740 MeV and 0.778 MeV. Molybdenum-99 has a half-life of 66.0 h. Not more than 0.1 per cent of the total radioactivity is due to molybdenum-99. — Iodine-131. Record the gamma-ray spectrum of the decayed material in a suitable instrument calibrated with the aid of a standardised iodine-131 solution. The most prominent photon has an energy of 0.365 MeV. Iodine-131 has a half-life of 8.04 days. Not more than 5 × 10− 3 per cent of the total radioactivity is due to iodine-131. — Ruthenium-103. Record the gamma-ray spectrum of the decayed material in a suitable instrument calibrated using a standardised ruthenium-103 solution. The most prominent photon has an energy of 0.497 MeV. Ruthenium-103 has a half-life of 39.3 days. Not more than 5 × 10− 3 per cent of the total radioactivity is due to ruthenium-103. — Strontium-89. Determine the presence of strontium-89 in the decayed material with an instrument suitable for the detection of beta rays, by comparison with a standardised strontium-89 solution. It is usually necessary first to carry out chemical separation of the strontium so that the standard and the sample may be compared in the same physical and chemical form. Strontium-89 decays with a beta emission of 1.492 MeV maximum energy and has a half-life of 50.5 days. Not more than 6 × 10− 5 per cent of the total radioactivity is due to strontium-89. — Strontium-90. Determine the presence of strontium-90 in the decayed material with an instrument suitable for the detection of beta rays. To distinguish strontium-90 from 848

strontium-89, compare the radioactivity of yttrium-90, the daughter nuclide of strontium-90, with an yttrium-90 standard after the chemical separation of the yttrium. If prior chemical separation of the strontium is necessary, the conditions of radioactive equilibrium must be ensured. The yttrium-90 standard and the sample must be compared in the same physical and chemical form. Strontium-90 and yttrium-90 decay with respective beta emissions of 0.546 MeV and 2.284 MeV maximum energy and half-lives of 29.1 years and 64.0 h. Not more than 6 × 10− 6 per cent of the total radioactivity is due to strontium-90. — Other gamma-emitting impurities. Examine the gamma-ray spectrum of the decayed material for the presence of other radionuclidic impurities, which should, where possible, be identified and quantified. The total gamma radioactivity due to these impurities does not exceed 0.01 per cent of the total radioactivity. — Alpha-emitting impurities. Measure the alpha radioactivity of the decayed material to detect any alpha-emitting radionuclidic impurities, which should, where possible, be identified and quantified. The total alpha radioactivity due to these impurities does not exceed 1 × 10− 7 per cent of the total radioactivity. RADIOCHEMICAL PURITY Examine by descending paper chromatography (2.2.26). Test solution. Dilute the preparation to be examined with water R to a suitable radioactive concentration. Apply 5 µl of the test solution. Develop for 2 h using a mixture of 20 volumes of water R and 80 volumes of methanol R. Allow the paper to dry. Determine the distribution of radioactivity using a suitable detector. Not less than 95 per cent of the total radioactivity is in the spot corresponding to pertechnetate ion, which has an Rf value of about 0.6. RADIOACTIVITY Measure the radioactivity using suitable counting equipment by comparison with a standardised technetium-99m solution or by measurement in an instrument calibrated with the aid of such a solution. 01/2005:0283

SODIUM PERTECHNETATE (99mTc) INJECTION (NON-FISSION) Natrii pertechnetatis (99mTc) sine fissione formati solutio iniectabilis This monograph applies to sodium pertechnetate (99mTc) injection obtained from molybdenum-99 produced by neutron irradiation of molybdenum. Sodium pertechnetate (99mTc) injection obtained from molybdenum-99 extracted from fission products of uranium is described in the monograph on Sodium pertechnetate(99mTc) injection (fission) (0124). DEFINITION Sodium pertechnetate (99mTc) injection (non-fission) is a sterile solution containing technetium-99m in the form of pertechnetate ion and made isotonic by the addition of sodium chloride. Technetium-99m is a radionuclide formed by the decay of molybdenum-99. Molybdenum-99 is a radioactive isotope of molybdenum produced by neutron irradiation of molybdenum. The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the declared technetium-99m radioactivity at the date and



hour stated on the label. Not less than 95 per cent of the radioactivity corresponds to technetium-99m in the form of pertechnetate ion. The radioactivity due to radionuclides other than technetium-99m, apart from that due to technetium-99 resulting from the decay of technetium-99m is not greater than that shown below, expressed as a percentage of the total radioactivity and calculated with reference to the date and hour of administration. Molybdenum-99

0.1 per cent

Other radionuclidic impurities

0.01 per cent

The injection may be prepared from a sterile preparation of molybdenum-99 under aseptic conditions. CHARACTERS A clear, colourless solution. Technetium-99m has a half-life of 6.02 h and emits gamma radiation. IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised technetium-99m solution either by direct comparison or by using an instrument calibrated with the aid of such a solution. Standardised technetium-99m and molybdenum-99 solutions are available from laboratories recognised by the competent authority. The most prominent gamma photon of technetium-99m has an energy of 0.140 MeV. B. Examine the chromatogram obtained in the test for radiochemical purity. The distribution of radioactivity contributes to the identification of the preparation. TESTS pH (2.2.3). The pH of the injection is 4.0 to 8.0. Aluminium. In a test tube about 12 mm in internal diameter, mix 1 ml of acetate buffer solution pH 4.6 R and 2 ml of a 1 in 2.5 dilution of the injection in water R. Add 0.05 ml of a 10 g/l solution of chromazurol S R. After 3 min, the colour of the solution is not more intense than that of a standard prepared at the same time in the same manner using 2 ml of aluminium standard solution (2 ppm Al) R (5 ppm). Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. RADIONUCLIDIC PURITY Preliminary test. To obtain an approximate estimate before use of the injection, take a volume equivalent to 37 MBq and record the gamma-ray spectrum using a sodium iodide detector with a shield of lead, of thickness 6 mm, interposed between the sample and the detector. The response in the region corresponding to the 0.740 MeV photon of molybdenum-99 does not exceed that obtained using 37 kBq of a standardised solution of molybdenum-99 measured under the same conditions, when all measurements are expressed with reference to the date and hour of administration. Definitive test. Retain a sample of the injection for a sufficient time to allow the technetium-99m radioactivity to decay to a sufficiently low level to permit the detection of radionuclidic impurities. All measurements of radioactivity are expressed with reference to the date and hour of administration. General Notices (1) apply to all monographs and other texts

Sodium phosphate (32P) injection

— Molybdenum-99. Record the gamma-ray spectrum of the decayed material in a suitable instrument calibrated using a standardised molybdenum-99 solution. The most prominent gamma photons have energies of 0.181 MeV, 0.740 MeV and 0.778 MeV. Molybdenum-99 has a half-life of 66.0 h. Not more than 0.1 per cent of the total radioactivity is due to molybdenum-99. — Other gamma-emitting impurities. Examine the gamma-ray spectrum of the decayed material for the presence of other radionuclidic impurities, which should, where possible, be identified and quantified. The total radioactivity due to other radionuclidic impurities does not exceed 0.01 per cent of the total radioactivity. RADIOCHEMICAL PURITY Examine by descending paper chromatography (2.2.26). Test solution. Dilute the injection with water R to a suitable radioactive concentration. Apply 5 µl of the test solution. Develop for 2 h using a mixture of 20 volumes of water R and 80 volumes of methanol R. Allow the paper to dry in air. Determine the distribution of radioactivity using a suitable detector. Not less than 95 per cent of the total radioactivity is found in the spot corresponding to pertechnetate ion, which has an Rf value of about 0.6. RADIOACTIVITY Measure the radioactivity using suitable counting equipment by comparison with a standardised technetium-99m solution or by measurement in an instrument calibrated with the aid of such a solution. 01/2005:0284

SODIUM PHOSPHATE (32P) INJECTION Natrii phosphatis (32P) solutio iniectabilis DEFINITION Sodium phosphate (32P) injection is a sterile solution of disodium and monosodium (32P) orthophosphates made isotonic by the addition of sodium chloride. Phosphorus-32 is a radioactive isotope of phosphorus and may be produced by neutron irradiation of sulphur. The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the declared phosphorus-32 radioactivity at the date and hour stated on the label. Not less than 95 per cent of the radioactivity corresponds to phosphorus-32 in the form of orthophosphate ion. The specific radioactivity is not less than 11.1 MBq of phosphorus-32 per milligram of orthophosphate ion. CHARACTERS A clear, colourless solution. Phosphorus-32 has a half-life of 14.3 days and emits beta radiation. IDENTIFICATION A. Record the beta-ray spectrum or the beta-ray absorption curve using a suitable method. The spectrum or curve does not differ significantly from that of a standardised phosphorus-32 solution obtained under the same conditions. Standardised phosphorus-32 solutions are available from laboratories recognised by the competent authority. The maximum energy of the beta radiation is 1.71 MeV. B. Examine the chromatogram obtained in the test for radiochemical purity. The distribution of radioactivity contributes to the identification of the preparation. 849

Strontium (89Sr) chloride injection


TESTS pH (2.2.3). The pH of the injection is 6.0 to 8.0. Phosphates. Dilute the injection with water R to give a radioactive concentration of 370 kBq of phosphorus-32 per millilitre. Mix in a volumetric flask, with shaking, 1.0 ml of the solution with a mixture of 0.5 ml of a 2.5 g/l solution of ammonium vanadate R, 0.5 ml of ammonium molybdate solution R and 1 ml of perchloric acid R and dilute to 5.0 ml with water R. After 30 min, the solution is not more intensely coloured than a standard prepared at the same time in the same manner using 1.0 ml of a solution containing 33 mg of orthophosphate ion per litre. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. RADIONUCLIDIC PURITY Record the beta-ray spectrum or the beta-ray absorption curve using a suitable method. The spectrum or curve does not differ significantly from that of a standardised phosphorus-32 solution obtained under the same conditions. RADIOCHEMICAL PURITY Examine by ascending paper chromatography (2.2.26). Test solution. Dilute the injection with water R until the radioactivity is equivalent to 10 000 to 20 000 counts per minute per 10 µl Reference solution. Prepare a solution of phosphoric acid R containing 2 mg of phosphorus per millilitre. Using a strip of paper 25 mm wide and about 300 mm long, apply 10 µl of the reference solution. Apply to the same starting-point 10 µl of the test solution. Develop for 16 h using a mixture of 0.3 ml of ammonia R, 5 g of trichloroacetic acid R, 25 ml of water R and 75 ml of 2-propanol R. Allow the paper to dry in air. Determine the position of the inactive phosphoric acid by spraying with a 50 g/l solution of perchloric acid R and then with a 10 g/l solution of ammonium molybdate R. Expose the paper to hydrogen sulphide R. A blue colour develops. Determine the position of the radioactive spot by autoradiography or by measuring the radioactivity over the whole length of the chromatogram. Not less than 95 per cent of the total radioactivity of the chromatogram is found in the spot corresponding to phosphoric acid.

the total radioactivity is due to radionuclides other than strontium-89. The specific radioactivity is not less than 1.8 MBq of strontium-89 per milligram of strontium. The injection contains 6.0 mg/ml to 12.5 mg/ml of strontium. CHARACTERS A clear, colourless solution. Strontium-89 has a half-life of 50.5 days and emits beta radiation with a maximum energy of 1.492 MeV. IDENTIFICATION A. Record the gamma-ray and X-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised strontium-89 solution, when measured either by direct comparison or by using an instrument calibrated with the aid of such a solution. Standardised strontium-89 solutions are available from laboratories recognised by the competent authority. The gamma photon detected has an energy of 0.909 MeV and is due to the short-lived daughter product, yttrium-89m (formed in 0.01 per cent of the disintegrations), in equilibrium with the strontium-89. B. To 0.1 ml of the injection to be examined, add 1 ml of a freshly prepared 1 g/l solution of sodium rhodizonate R. Mix and allow to stand for 1 min. A reddish-brown precipitate is formed. C. To 0.1 ml of silver nitrate solution R2 add 50 µl of the injection to be examined. A white precipitate is formed.

TESTS pH (2.2.3). The pH of the solution is 4.0 to 7.5. Note : the following tests for aluminium, iron and lead may be carried out simultaneously with the test for strontium. If this is not the case, the reference solutions are prepared such that they contain strontium at approximately the same concentration as in the test solution. Aluminium. Not more than 2 µg/ml, determined by atomic emission spectrometry (plasma or arc method) (2.2.22, Method I). Test solution. Dilute 0.2 ml of the injection to be examined to a suitable volume with dilute nitric acid R. Reference solutions. Prepare the reference solutions using aluminium standard solution (10 ppm Al) R diluted as necessary with dilute nitric acid R. Iron. Not more than 5 µg/ml, determined by atomic emission RADIOACTIVITY spectrometry (plasma or arc method) (2.2.22, Method I). Measure the radioactivity using suitable counting equipment Test solution. Dilute 0.2 ml of the injection to be examined by comparison with a standardised phosphorus-32 solution to a suitable volume with dilute nitric acid R. or by measurement in an instrument calibrated with the aid Reference solutions. Prepare the reference solutions using of such a solution. iron standard solution (20 ppm Fe) R diluted as necessary with dilute nitric acid R. 01/2005:1475 Lead. Not more than 5 µg/ml, determined by atomic emission spectrometry (plasma or arc method) (2.2.22, 89 Method I). STRONTIUM ( Sr) CHLORIDE Test solution. Dilute 0.2 ml of the injection to be examined INJECTION to a suitable volume with dilute nitric acid R. Reference solutions. Prepare the reference solutions using Strontii (89Sr) chloridi solutio iniectabilis lead standard solution (10 ppm Pb) R diluted as necessary with dilute nitric acid R. DEFINITION Strontium. 6.0 mg/ml to 12.5 mg/ml. Examine by atomic Strontium (89Sr) chloride injection is a sterile solution emission spectrometry (2.2.22, Method I). of [89Sr]strontium chloride. Strontium-89 is a radioactive isotope of strontium and is produced by neutron irradiation Test solution. Dilute 0.2 ml of the injection to be examined to a suitable volume with dilute nitric acid R. of strontium enriched in strontium-88. The injection Reference solutions. Prepare the reference solutions using contains not less than 90.0 per cent and not more than strontium standard solution (1.0 per cent Sr) R diluted as 110.0 per cent of the declared strontium-89 radioactivity at necessary with dilute nitric acid R. the date stated on the label. Not more than 0.6 per cent of 850



Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). RADIONUCLIDIC PURITY Gamma emitters. Record the gamma-ray and X-ray spectrum of the injection to be examined using a suitable instrument. Not more than 0.4 per cent of the total radioactivity in the preparation to be examined is due to gamma emitting radionuclides other than yttrium-89m. Beta emitters. Evaporate to dryness 100 µl of the injection to be examined under a radiant heat source. Dissolve the residue in 2 ml of 47 per cent hydrobromic acid R, evaporate to dryness under the radiant heat source and dissolve the residue in 2 ml of dilute hydrobromic acid R1. Transfer the solution to the top of a column, 5 mm to 6 mm in diameter, packed with approximately 2 ml of cationic exchange resin R1 (100 µm to 250 µm), previously conditioned with dilute hydrobromic acid R1 and elute the column with the same solvent until 10 ml of eluate has been collected into a container containing 50 µl of a 15 g/l solution of anhydrous sodium sulphate R in 1 M hydrochloric acid. To a liquid scintillation vial add an appropriate volume of scintillation liquid followed by 1 ml of water R, 0.1 ml of a 15 g/l solution of anhydrous sodium sulphate R in 1 M hydrochloric acid and 100 µl of eluate. Shake to obtain a clear solution. Using suitable counting equipment determine the radioactivity due to sulphur-35 and phosphorus-32 in the sample. Taking into account the recovery efficiency of the separation, counting efficiency and radioactive decay, determine the radioactive concentration of sulphur-35 and phosphorus-32 in the sample and hence the percentage of total beta emitting impurities in the injection to be examined. Not more than 0.2 per cent of the total radioactivity in the injection to be examined is due to the sum of sulphur-35 and phosphorus-32 radioactivities.

Technetium (99mTc) colloidal rhenium sulphide injection

It is prepared from sodium pertechnetate (99mTc) injection (fission or non-fission) using suitable sterile, apyrogenic ingredients and calculating the ratio of radionuclidic impurities with reference to the date and hour of administration. CHARACTERS A light-brown liquid. Technetium-99m has a half-life of 6.02 h and emits gamma radiation. IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised technetium-99m solution either by direct comparison or by using an instrument calibrated with the aid of such a solution. Standardised technetium-99m and molybdenum-99 solutions are available from laboratories recognised by the competent authority. The most prominent gamma photon of technetium-99m has an energy of 0.140 MeV. B. Examine the chromatogram obtained in the test for radiochemical purity. The distribution of radioactivity contributes to the identification of the injection. C. To 1 ml add 5 ml of hydrochloric acid R, 5 ml of a 50 g/l solution of thiourea R and 1 ml of a 200 g/l solution of stannous chloride R in hydrochloric acid R. A yellow colour is produced.

TESTS pH (2.2.3). The pH of the injection is 4.0 to 7.0. Rhenium Test solution. Use 1 ml of the injection to be examined. Reference solutions. Using a solution containing 100 µg of potassium perrhenate R (equivalent to 60 ppm of Re) and 240 µg of sodium thiosulphate R per millilitre, prepare a suitable range of solutions and dilute to the same final volume with water R. RADIOACTIVITY To the test solution and to 1 ml of each of the reference Measure the radioactivity using suitable equipment by solutions add 5 ml of hydrochloric acid R, 5 ml of a 50 g/l comparison with a standardised strontium-89 solution or by measurement in an instrument calibrated with the aid of solution of thiourea R and 1 ml of a 200 g/l solution of stannous chloride R in hydrochloric acid R and dilute such a solution. to 25.0 ml with water R. Allow to stand for 40 min and measure the absorbance (2.2.25) of each solution at 400 nm, 01/2005:0126 using a reagent blank as the compensation liquid. Using the absorbances obtained with the reference solutions, draw a calibration curve and calculate the concentration of rhenium 99m TECHNETIUM ( Tc) COLLOIDAL in the injection to be examined. RHENIUM SULPHIDE INJECTION Physiological distribution. Inject a volume not greater than 0.2 ml into the caudal vein of each of three mice each Rhenii sulfidi colloidalis et technetii (99mTc) weighing 20 g to 25 g. Sacrifice the mice 20 min after the injection, remove the liver, spleen and lungs and measure solutio iniectabilis the radioactivity in the organs using a suitable instrument. Measure the radioactivity in the rest of the body after having DEFINITION removed the tail. Determine the percentage of radioactivity 99m Technetium ( Tc) colloidal rhenium sulphide injection is a in the liver, the spleen and the lungs from the expression : sterile, apyrogenic colloidal dispersion of rhenium sulphide the micelles of which are labelled with technetium-99m. It is stabilised with gelatin. The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the declared technetium-99m radioactivity at the date and A = radioactivity of the organ concerned, hour stated on the label. Not less than 92 per cent of the B = total radioactivity in the liver, the spleen, the radioactivity corresponds to technetium-99m in colloidal lungs and the rest of the body. form. The pH of the injection may be adjusted by the addition of a suitable buffer such as a citrate buffer solution. In each of the three mice at least 80 per cent of the radioactivity is found in the liver and spleen and not The injection contains a variable amount of colloidal more than 5 per cent in the lungs. If the distribution of rhenium sulphide, not exceeding 0.22 mg of rhenium (Re) radioactivity in one of the three mice does not correspond per millilitre, according to the method of preparation. General Notices (1) apply to all monographs and other texts

851

Technetium (99mTc) colloidal sulphur injection


to the prescribed proportions, repeat the test on a further three mice. The preparation complies with the test if the prescribed distribution of radioactivity is found in five of the six mice used. The injection may be released for use before completion of the test. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. Pyrogens. It complies with the test for pyrogens prescribed in the monograph on Radiopharmaceutical preparations (0125). Inject not less than 0.1 ml per kilogram of the rabbit’s mass. The injection may be released for use before completion of the test. RADIOCHEMICAL PURITY Examine by ascending paper chromatography (2.2.26). Apply to the paper 10 µl of the injection. Develop immediately over a path of 10 cm to 15 cm using a 9 g/l solution of sodium chloride R. Allow the paper to dry. Determine the distribution of radioactivity using a suitable detector. Technetium-99m in colloidal form remains at the starting-point and pertechnetate ion migrates with an Rf of about 0.6. There may be other impurities with an Rf of 0.8 to 0.9. The radioactivity corresponding to technetium-99m in colloidal form represents not less than 92 per cent of the total radioactivity of the chromatogram.

Technetium-99m has a half-life of 6.02 h and emits gamma radiation. IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised technetium-99m solution either by direct comparison or by using an instrument calibrated with the aid of such a solution. Standardised technetium-99m and molybdenum-99 solutions are available from laboratories recognised by the competent authority. The most prominent gamma photon of technetium-99m has an energy of 0.140 MeV. B. Examine the chromatogram obtained in the test for radiochemical purity. The distribution of radioactivity contributes to the identification of the injection. C. In a test-tube 100 mm long and 16 mm in internal diameter, evaporate 0.2 ml of the injection to dryness. Dissolve the sulphur by shaking the residue with 0.2 ml of pyridine R and add about 20 mg of benzoin R. Cover the open end of the tube with a filter paper moistened with lead acetate solution R. Heat the test-tube in a bath containing glycerol at 150 °C. The paper slowly becomes brown.

TESTS pH (2.2.3). The pH of the injection is 4.0 to 7.0. Physiological distribution. Inject a volume not greater RADIOACTIVITY Measure the radioactivity using suitable counting equipment than 0.2 ml into the caudal vein of each of 3 mice, each by comparison with a standardised technetium-99m solution weighing 20 g to 25 g. Sacrifice the mice 20 min after the or by measurement in an instrument calibrated with the aid injection, remove the liver, spleen and lungs and measure the radioactivity in the organs using a suitable instrument. of such a solution. Measure the radioactivity in the rest of the body after having LABELLING removed the tail. Determine the percentage of radioactivity in the liver, the spleen and the lungs from the expression : The label states, in particular, the quantity of rhenium per millilitre. 01/2005:0131

TECHNETIUM (99mTc) COLLOIDAL SULPHUR INJECTION

A

=

radioactivity of the organ concerned,

B

=

total radioactivity in the liver, the spleen, the lungs and the rest of the body.

In each of the 3 mice at least 80 per cent of the radioactivity is found in the liver and spleen and not more than 5 per cent in the lungs. If the distribution of radioactivity in 1 of the 3 mice does not correspond to the prescribed proportions, DEFINITION repeat the test on a further 3 mice. The preparation complies with the test if the prescribed distribution of radioactivity is Technetium (99mTc) colloidal sulphur injection is a sterile, found in 5 of the 6 mice used. The injection may be released apyrogenic colloidal dispersion of sulphur, the micelles of which are labelled with technetium-99m. It may be stabilised for use before completion of the test. with a colloid-protecting substance based on gelatin. The Sterility. It complies with the test for sterility injection contains not less than 90.0 per cent and not prescribed in the monograph on Radiopharmaceutical more than 110.0 per cent of the declared technetium-99m preparations (0125). The injection may be released for use radioactivity at the date and hour stated on the label. Not before completion of the test. less than 92 per cent of the radioactivity corresponds to Pyrogens. It complies with the test for pyrogens technetium-99m in colloidal form. The pH of the injection prescribed in the monograph on Radiopharmaceutical may be adjusted by the addition of a suitable buffer, such preparations (0125). Inject not less than 0.1 ml per kilogram as an acetate, citrate or phosphate buffer solution. The of the rabbit’s mass. The injection may be released for use injection contains a variable amount of colloidal sulphur, before completion of the test. according to the method of preparation. 99m RADIOCHEMICAL PURITY It is prepared from sodium pertechnetate ( Tc) injection Examine by ascending paper chromatography (2.2.26). (fission or non-fission) using suitable sterile, apyrogenic Apply to the paper 10 µl of the injection. Develop ingredients and calculating the ratio of radionuclidic immediately over a path of 10 cm to 15 cm with a 9 g/l impurities with reference to the date and hour of solution of sodium chloride R. Allow the paper to dry. administration. Determine the distribution of radioactivity using a suitable CHARACTERS detector. Technetium-99m in colloidal form remains at the starting-point and pertechnetate ion migrates with an Rf A clear to opalescent, colourless to yellowish liquid.

Sulfuris colloidalis et technetii (99mTc) solutio iniectabilis

852


Technetium (99mTc) etifenin injection


of 0.6. There may be other impurities of Rf 0.8 to 0.9. The radioactivity corresponding to technetium-99m in colloidal form represents not less than 92 per cent of the total radioactivity of the chromatogram. RADIOACTIVITY Measure the radioactivity using suitable counting equipment by comparison with a standardised technetium-99m solution or by measurement in an instrument calibrated with the aid of such a solution. 01/2005:0689

TECHNETIUM (99mTc) COLLOIDAL TIN INJECTION Stanni colloidalis et technetii (99mTc) solutio iniectabilis DEFINITION Technetium (99mTc) colloidal tin injection is a sterile, colloidal dispersion of tin labelled with technetium-99m. The injection contains a variable quantity of tin not exceeding 1 mg of Sn per millilitre ; it contains fluoride ions, it may be stabilised with a suitable, apyrogenic colloid-protecting substance and it may contain a suitable buffer. The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the declared technetium-99m radioactivity at the date and hour stated on the label. Not less than 95 per cent of the radioactivity corresponds to technetium-99m in colloidal form. It is prepared from sodium pertechnetate (99mTc) injection (fission or non-fission) using suitable sterile ingredients and calculating the ratio of radionuclidic impurities with reference to the date and hour of administration. Syringes for handling the eluate intended for labelling of the final product, or the final product, should not contain rubber parts. CHARACTERS A clear or opalescent, colourless liquid. Technetium-99m has a half life of 6.02 h and emits gamma radiation. IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised technetium-99m solution either by direct comparison or by using an instrument calibrated with the aid of such a solution. Standardised technetium-99m and molybdenum-99 solutions are available from laboratories recognised by the competent authority. The most prominent gamma photon of technetium-99m has an energy of 0.140 MeV. B. Mix 0.05 ml of zirconyl nitrate solution R with 0.05 ml of alizarin S solution R. Add 0.05 ml of the injection to be examined. A yellow colour is produced.

To 1.0 ml of each solution add 0.4 ml of a 20 g/l solution of sodium laurilsulfate R, 0.05 ml of thioglycollic acid R, 0.1 ml of dithiol reagent R and 3.0 ml of 0.2 M hydrochloric acid. Mix. Measure the absorbance (2.2.25) of each solution at 540 nm, using 0.2 M hydrochloric acid as the compensation liquid. The absorbance of the test solution is not greater than that of the reference solution (1 mg of Sn per millilitre). Physiological distribution. Inject not more than 0.2 ml into a caudal vein of each of three mice, each weighing 20 g to 25 g. Kill the mice 20 min after the injection and remove the liver, spleen and lungs. Measure the radioactivity in the organs using a suitable instrument. Measure the radioactivity in the rest of the body, after having removed the tail. Determine the percentage of radioactivity in the liver, the spleen and the lungs with respect to the total radioactivity of all organs and the rest of the body excluding the tail. In each of the three mice at least 80 per cent of the radioactivity is found in the liver and spleen and not more than 5 per cent in the lungs. If the distribution of radioactivity in one of the three mice does not correspond to the prescribed proportions, repeat the test on a further three mice. The preparation complies with the test if the prescribed distribution of radioactivity is found in five of the six mice used. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. RADIOCHEMICAL PURITY Examine by thin-layer chromatography (2.2.27) using silica gel as the coating substance on a glass-fibre sheet. Heat the plate at 110 °C for 10 min. Use a plate such that during development the mobile phase migrates over a distance of 10 cm to 15 cm in about 10 min. Apply to the plate 5 µl to 10 µl of the injection to be examined. Develop immediately over a path of 10 cm to 15 cm using a 9 g/l solution of sodium chloride R purged with nitrogen R. Allow the plate to dry. Determine the distribution of radioactivity using a suitable detector. Technetium-99m in colloidal form remains at the starting point and pertechnetate ion migrates near to the solvent front. Not less than 95 per cent of the technetium-99m radioactivity corresponds to technetium in colloidal form. RADIOACTIVITY Measure the radioactivity using suitable counting equipment by comparison with a standardised technetium-99m solution or by measurement in an instrument calibrated with the aid of such a solution. 01/2005:0585

TECHNETIUM (99mTc) ETIFENIN INJECTION Technetii (99mTc) et etifenini solutio iniectabilis

TESTS pH (2.2.3). The pH of the injection to be examined is 4.0 to 7.0. Tin Test solution. Dilute 3.0 ml of the injection to be examined to 50.0 ml with 1 M hydrochloric acid. Reference solution. Dissolve 0.115 g of stannous chloride R in 1 M hydrochloric acid and dilute to 1000.0 ml with the same acid.

DEFINITION Technetium (99mTc) etifenin injection is a sterile solution which may be prepared by mixing sodium pertechnetate (99mTc) injection (fission or non-fission) with solutions of etifenin [[(2,6-diethylphenyl)carbamoylmethylimino]di-acetic acid ; C16H22N2O5] and stannous chloride. The injection contains a variable quantity of tin (Sn) not exceeding 0.2 mg/ml. The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the declared


853

Technetium (99mTc) exametazime injection


technetium-99m radioactivity at the date and hour stated on the label. Not less than 95.0 per cent of the radioactivity corresponds to technetium-99m complexed with etifenin. It is prepared from sodium pertechnetate (99mTc) injection (fission or non-fission) using suitable, sterile ingredients and calculating the ratio of radionuclidic impurities with reference to the date and hour of administration.

In not fewer than two mice the sum of the percentages of radioactivity in the gall-bladder and small and large intestine is not less than 80 per cent. Not more than 3 per cent of the radioactivity is present in the liver, and not more than 2 per cent in the kidneys Tin Test solution. Dilute 1.0 ml of the injection to be examined to 5.0 ml with 1 M hydrochloric acid. CHARACTERS Reference solution. Prepare a reference solution containing A clear, colourless solution. 0.075 mg of stannous chloride R per millilitre in 1 M hydrochloric acid. Technetium-99m has a half-life of 6.02 h and emits gamma radiation. To 1.0 ml of each solution add 0.4 ml of a 20 g/l solution of sodium laurilsulfate R, 0.05 ml of thioglycollic acid R, 0.l ml of dithiol reagent R and 3.0 ml of 0.2 M hydrochloric acid. IDENTIFICATION Mix. Measure the absorbance (2.2.25) of each solution at A. Record the gamma-ray spectrum using a suitable 540 nm, using 0.2 M hydrochloric acid as the compensation instrument. The spectrum does not differ significantly liquid. The absorbance of the test solution is not greater than from that of a standardised technetium-99m solution that of the reference solution (0.2 mg of Sn per millilitre). either by direct comparison or by using an instrument Sterility. It complies with the test for sterility calibrated with the aid of such a solution. Standardised prescribed in the monograph on Radiopharmaceutical technetium-99m and molybdenum-99 solutions are available from laboratories recognised by the competent preparations (0125). The injection may be released for use before completion of the test. authority. The most prominent gamma photon of technetium-99m has an energy of 0.140 MeV. RADIOCHEMICAL PURITY B. Examine by liquid chromatography (2.2.29). Examine by thin-layer chromatography (2.2.27) using silicic acid as the coating substance on a glass-fibre sheet. Heat the Test solution. Dilute the injection to be examined with methanol R to obtain a solution containing about l mg of plate at 110 °C for l0 min. The plate used should be such that during development the mobile phase moves over a etifenin per millilitre. distance of 10 cm to 15 cm in about 15 min. Reference solution. Dissolve 5.0 mg of etifenin CRS in Apply to the plate 5 µl to 10 µl of the injection to be methanol R and dilute to 5.0 ml with the same solvent. examined. Develop immediately over a path of 10 cm to 15 cm using a 9 g/l solution of sodium chloride R. Allow The chromatographic procedure may be carried out the plate to dry. Determine the distribution of radioactivity using : using a suitable detector. Technetium-99m complexed with — a column 0.25 m long and 4.6 mm in internal etifenin migrates almost to the middle of the chromatogram diameter packed with octadecylsilyl silica gel for and pertechnetate ion migrates with the solvent front. chromatography R (5 µm to 10 µm), Impurities in colloidal form remain at the starting point. The — as mobile phase at a flow rate of 1 ml/min a mixture radioactivity corresponding to technetium-99m complexed of 20 volumes of methanol R and 80 volumes of a with etifenin represents not less than 95.0 per cent of the 14 g/l solution of potassium dihydrogen phosphate R total radioactivity of the chromatogram. adjusted to pH 2.5 by the addition of phosphoric RADIOACTIVITY acid R, Measure the radioactivity using suitable counting equipment — a spectrophotometer set at 230 nm. by comparison with a standardised technetium-99m solution Inject 20 µl of each solution. The principal peak in or by measurement in an instrument calibrated with the aid the chromatogram obtained with the test solution has of such a solution. a similar retention time to the principal peak in the chromatogram obtained with the reference solution. TESTS pH (2.2.3). The pH of the injection is 4.0 to 6.0. Physiological distribution. Inject 0.1 ml (equivalent to about 3.7 MBq) into a caudal vein of each of three mice, each weighing 20 g to 25 g. Kill the mice l h after the injection. Remove the liver, gall-bladder, small intestine, large intestine and kidneys, collecting excreted urine. Measure the radioactivity in the organs using a suitable instrument. Measure the radioactivity of the rest of the body, after having removed the tail. Determine the percentage of radioactivity in each organ from the expression :

A

=


B

=

radioactivity of all organs and the rest of the body, excluding the tail.

854

01/2005:1925

TECHNETIUM (99mTC) EXAMETAZIME INJECTION Technetii (99mTc) exametazimi solutio iniectabilis



DEFINITION Sterile solution of lipophilic technetium-99m exametazime which may be prepared by dissolving a racemic mixture of (3RS,9RS)-4,8-diaza-3,6,6,9-tetramethylundecane-2,10-dione bisoxime in the presence of a stannous salt in Sodium pertechnetate (99mTc) injection (fission) (0124) or Sodium pertechnetate (99mTc) injection (non-fission) (0283). It may contain stabilisers and inert additives. Content : 90 per cent to 110 per cent of the declared technetium-99m radioactivity at the date and time stated on the label. Purity : minimum of 80 per cent of the total radioactivity corresponds to lipophilic technetium-99m exametazime and its meso isomer. CHARACTERS Appearance : clear solution. Half-life and nature of radiation of technetium-99m : see Table of physical characteristics of radionuclides (5.7). IDENTIFICATION A. Gamma-ray spectrometry. Comparison : standardised technetium-99m solution, or by using a calibrated instrument. Standardised technetium-99m solutions and/or standardisation services are available from the competent authority. Results : the spectrum obtained with the solution to be examined does not differ significantly from that obtained with a standardised technetium-99m solution. The most prominent gamma photon has an energy of 0.141 MeV. B. Examine the chromatograms obtained in the test Impurity A under Radiochemical purity. Results : the principal peak in the chromatogram obtained with the test solution is similar in retention time to the peak due to lipophilic technetium-99m exametazime in the chromatogram obtained with the reference solution. TESTS pH (2.2.3) : 5.0 to 10.0. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. RADIOCHEMICAL PURITY Impurity C. Thin-layer chromatography (2.2.27). Test solution. The preparation to be examined. Plate : TLC silica gel plate R ; use a glass-fibre plate. Mobile phase : 9 g/l solution of sodium chloride R. Application : about 5 µl. Development : immediate, over 2/3 of the plate. Drying : in air. Detection : determine the distribution of radioactivity using a suitable detector. Retention factors : impurity C = 0.8 to 1.0 ; lipophilic technetium-99m exametazime and impurities A, B, D and E do not migrate. Limits : — impurity C : maximum 10 per cent of the total radioactivity. Total of lipophilic technetium-99m exametazime and impurity A. Thin-layer chromatography (2.2.27). Test solution. The preparation to be examined. General Notices (1) apply to all monographs and other texts

Technetium (99mTc) exametazime injection

Plate : TLC silica gel plate R ; use a glass-fibre plate. Mobile phase : methyl ethyl ketone R. Application : about 5 µl. Development : immediate, over 2/3 of the plate. Drying : in air. Detection : determine the distribution of radioactivity using a suitable detector. Retention factors : lipophilic technetium-99m exametazime = 0.8 to 1.0, impurity A = 0.8 to 1.0, impurity C = 0.8 to 1.0 ; impurities B, D and E do not migrate. Limits : calculate the percentage of radioactivity due to impurities B, D and E from test B (B) and the percentage of the radioactivity due to impurity C from test A (A). Calculate the total percentage of lipophilic technetium-99m exametazime and impurity A from the expression :

— total of lipophilic technetium-99m exametazime and impurity A : minimum 80 per cent of the total radioactivity. Impurity A. Liquid chromatography (2.2.29). Test solution. The preparation to be examined. Reference solution. Dissolve the contents of a vial of meso-rich exametazime CRS in 0.5 ml of a 9 g/l solution of sodium chloride R and transfer to a lead-shielded, nitrogen-filled vial. Add 6 µl of a freshly prepared 1 g/l solution of stannous chloride R in 0.05 M hydrochloric acid and 2.5 ml of sodium pertechnetate (99mTc) injection (fission or non-fission) containing 370-740 MBq. Mix carefully and use within 30 min of preparation. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : spherical base-deactivated end-capped octadecylsilyl silica gel for chromatography R (5 µm) with a pore size of 13 nm and a carbon loading of 11 per cent. Mobile phase : mix 33 volumes of acetonitrile R and 67 volumes of 0.1 M phosphate buffer solution pH 3.0 R. Flow rate : 1.5 ml/min. Detection : radioactivity detector. Injection : loop injector. Run time : 20 min. Relative retention with reference to lipophilic technetium-99m exametazime : impurity A = about 1.2. System suitability : reference solution : — chromatogram similar to the chromatogram provided with meso-rich exametazime CRS, — resolution : minimum of 2 between the peaks due to lipophilic technetium-99m exametazime and to impurity A. Limits : — impurity A : maximum 5 per cent of the radioactivity due to lipophilic technetium-99m exametazime and impurity A. RADIOACTIVITY Measure the radioactivity using suitable equipment by comparison with a standardised technetium-99m solution or by using a calibrated instrument. 855

Technetium (99mTc) gluconate injection


Physiological distribution. Inject a volume not greater than 0.2 ml into the caudal vein of each of three rats weighing 150 g to 250 g. Measure the radioactivity of the syringe before and after injection. Sacrifice the rats 30 min after the injection. Remove at least 1 g of blood by a suitable method and remove the kidneys, the liver, the bladder plus voided urine and the tail. Weigh the sample of blood. Determine the radioactivity in the organs, the blood sample and the tail using a suitable instrument. Calculate the percentage of radioactivity in each organ and in 1 g of blood with respect to the total radioactivity calculated as A. meso isomer of lipophilic technetium-99m exametazime, the difference between the two measurements made on the syringe minus the activity in the tail. Correct the blood B. technetium-99m in colloidal form, concentration by multiplying by a factor of m/200 where m C. [99mTc]pertechnetate ion, is the body mass of the rat in grams. In not fewer than two of the three rats used, the radioactivity D. non lipophilic technetium-99m exametazime complex, in the kidneys is not less than 15 per cent, that in the bladder E. meso isomer of non lipophilic technetium-99m plus voided urine is not less than 20 per cent and that in the exametazime complex. liver is not more than 5 per cent. The radioactivity in the blood, after correction, is not more than 0.50 per cent. 01/2005:1047 Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use TECHNETIUM (99mTc) GLUCONATE before completion of the test. INJECTION RADIOCHEMICAL PURITY 99m Examine by thin-layer chromatography (2.2.27) using silica Technetii ( Tc) gluconatis gel as the coating substance on a glass-fibre sheet. Heat the solutio iniectabilis plate at 110 °C for 10 min. Use a plate such that during development the mobile phase migrates over a distance of DEFINITION 10 cm to 15 cm in about 10 min. m Technetium (99 Tc) gluconate injection is a sterile solution, a) Apply to the plate 5 µl to 10 µl of the solution to be which may be prepared by mixing solutions of calcium examined. Develop immediately over a path of 10 cm to gluconate and a stannous salt or other suitable reducing 15 cm using a 9 g/l solution of sodium chloride R. Allow m agent with sodium pertechnetate (99 Tc) injection (fission the plate to dry. Determine the distribution of radioactivity or non-fission). The injection contains not less than 90.0 per using a suitable detector. Impurities in colloidal form remain cent and not more than 110.0 per cent of the declared at the starting point. Technetium gluconate complex and technetium-99m radioactivity at the date and hour stated pertechnetate ion migrate near to the solvent front. on the label. Not less than 90 per cent of the radioactivity b) Apply to the plate 5 µl to 10 µl of the solution to be corresponds to technetium-99m gluconate complex. examined and allow to dry. Develop over a path of 10 cm It is prepared from sodium pertechnetate (99mTc) injection to 15 cm using methyl ethyl ketone R. Dry in a current of (fission or non-fission) using suitable sterile ingredients warm air. Determine the distribution of radioactivity using a and calculating the ratio of radionuclidic impurities with suitable detector. Pertechnetate ion impurity migrates near reference to the date and hour of administration. to the solvent front. Technetium gluconate complex and technetium in colloidal form remain at the starting point. CHARACTERS The sum of the percentages of radioactivity corresponding A slightly opalescent solution. to impurities in the chromatograms obtained in test (a) and Technetium-99m has a half-life of 6.02 h and emits gamma (b) does not exceed 10 per cent. radiation. RADIOACTIVITY IDENTIFICATION Measure the radioactivity using suitable counting equipment A. Record the gamma-ray spectrum using a suitable by comparison with a standardised technetium-99m solution instrument. The spectrum does not differ significantly or by measurement in an instrument calibrated with the aid from that of a standardised technetium-99m solution of such a solution. either by direct comparison or by using an instrument calibrated with the aid of such a solution. Standardised 01/2005:0640 technetium-99m and molybdenum-99 solutions are available from laboratories recognised by the competent TECHNETIUM (99mTc) HUMAN authority. The most prominent gamma photon of technetium-99m has an energy of 0.140 MeV. ALBUMIN INJECTION B. 5 µl of the solution complies with identification A Technetii (99mTc) humani albumini prescribed in the monograph on Calcium gluconate (0172). solutio iniectabilis C. Examine the chromatograms obtained in the test for radiochemical purity. The distribution of the radioactivity DEFINITION contributes to the identification of the preparation. Technetium (99mTc) human albumin injection is a sterile, apyrogenic solution of human albumin labelled with TESTS technetium-99m. It contains a reducing substance, such as a pH (2.2.3). The pH of the solution is 6.0 to 8.5. tin salt in an amount not exceeding 1 mg of Sn per millilitre ; IMPURITIES

856



Technetium (99mTc) human albumin injection

it may contain a suitable buffer and an antimicrobial preservative. Although, at present, no definite value for a maximum limit of tin can be fixed, available evidence tends to suggest the importance of keeping the ratio of tin to albumin as low as possible. The human albumin used complies with the requirements of the monograph on Human albumin solution (0255). The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the declared technetium-99m radioactivity at the date and hour stated on the label. The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the quantity of albumin stated on the label. Not less than 80 per cent of the radioactivity is associated with the albumin fractions II to V. Not more than 5.0 per cent of the radioactivity due to technetium-99m corresponds to free pertechnetate, as determined by the method described in the test for radiochemical purity. It is prepared from sodium pertechnetate (99mTc) injection (fission or non-fission) using suitable sterile and apyrogenic ingredients and calculating the ratio of radionuclidic impurities with reference to the date and hour of administration.

same manner. From the absorbances measured, calculate the content of albumin in the injection to be examined in milligrams per millilitre. Tin Test solution. To 1.0 ml of the injection to be examined add 1.0 ml of 2 M hydrochloric acid. Heat in a water-bath at 100 °C for 30 min. Cool and centrifuge at 300 g for 10 min. Dilute 1.0 ml of the supernatant liquid to 10 ml with 1 M hydrochloric acid. Reference solution. Dissolve 95 mg of stannous chloride R in 1 M hydrochloric acid and dilute to 1000.0 ml with the same acid. To 1.0 ml of each solution add 0.4 ml of a 20 g/l solution of sodium laurilsulfate R, 0.05 ml of thioglycollic acid R, 0.1 ml of dithiol reagent R and 3.0 ml of 0.2 M hydrochloric acid. Mix. Measure the absorbance (2.2.25) of each solution at 540 nm, using 0.2 M hydrochloric acid as the compensation liquid. The absorbance of the test solution is not greater than that of the reference solution (1 mg of Sn per millilitre). Physiological distribution. Inject a volume not greater than 0.5 ml and containing not more than 1.0 mg of albumin into a suitable vein such as a caudal vein or a saphenous vein CHARACTERS of each of three male rats, each weighing 150 g to 250 g. A clear, colourless or pale-yellow solution. Measure the radioactivity in the syringe before and after the injection. Kill the rats 30 min after the injection. Take one Technetium-99m has a half-life of 6.02 h and emits gamma millilitre of blood by a suitable method and remove the liver radiation. and, if a caudal vein has been used for the injection, the tail. Using a suitable instrument determine the radioactivity in IDENTIFICATION 1 ml of blood, in the liver and, if a caudal vein has been used A. Record the gamma-ray spectrum using a suitable for the injection, in the tail. Determine the percentage of instrument. The spectrum does not differ significantly radioactivity in the liver and in 1 ml of blood with respect to from that of a standardised technetium-99m solution the total radioactivity calculated as the difference between either by direct comparison or by using an instrument the measurements made on the syringe minus the activity calibrated with the aid of such a solution. Standardised in the tail (if a caudal vein has been used for the injection). technetium-99m and molybdenum-99 solutions are available from laboratories recognised by the competent Correct the blood concentration by multiplying by a factor of m/200 where m is the body mass of the rat in grams. In authority. The most prominent gamma photon of not fewer than two of the three rats used, the radioactivity in technetium-99m has an energy of 0.140 MeV. the liver is not more than 15 per cent and that in blood, after B. Using a suitable range of species-specific antisera, carry correction, is not less than 3.5 per cent. out precipitation tests on the preparation to be examined. Sterility. It complies with the test for sterility The test is to be carried out using antisera specific to prescribed in the monograph on Radiopharmaceutical the plasma proteins of each species of domestic animal currently used in the preparation of materials of biological preparations (0125). The injection may be released for use before completion of the test. origin in the country concerned. The injection is shown to contain proteins of human origin and gives negative Bacterial endotoxins (2.6.14) : less than 175/V IU/ml, V results with antisera specific to plasma proteins of other being the maximum recommended dose in millilitres. species. RADIOCHEMICAL PURITY C. Examine by a suitable immunoelectrophoresis technique. A. Examine by thin-layer chromatography (2.2.27) using Using antiserum to normal human serum, compare silica gel as the coating substance on a glass-fibre sheet. normal human serum and the injection to be examined, Heat the plate at 110 °C for 10 min. Use a plate such that both diluted if necessary. The main component of during development the mobile phase migrates over a the injection to be examined corresponds to the main distance of 10 cm to 15 cm in about 10 min. component of the normal human serum. The diluted Apply to the plate 5 µl to 10 µl of the injection to be solution may show the presence of small quantities of examined and allow to dry. Develop over a path of 10 cm other plasma proteins. to 15 cm using methyl ethyl ketone R. Allow the plate to dry. Determine the distribution of radioactivity using a TESTS suitable detector. The technetium-99m human albumin pH (2.2.3). The pH of the injection is 2.0 to 6.5. complex remains at the starting-point and pertechnetate Albumin ion migrates near to the solvent front. Not more than 5.0 per cent of the technetium-99m radioactivity Reference solution. Dilute human albumin solution R with corresponds to technetium in the form of pertechnetate a 9 g/l solution of sodium chloride R to a concentration of ion. 5 mg of albumin per millilitre. B. Examine by size-exclusion chromatography (2.2.30). To 1.0 ml of the injection to be examined and to 1.0 ml of Mobile phase (concentrated). Dissolve 1.124 g of the reference solution add 4.0 ml of biuret reagent R and potassium dihydrogen phosphate R, 4.210 g of disodium mix. After exactly 30 min, measure the absorbance (2.2.25) hydrogen phosphate R, 1.17 g of sodium chloride R and of each solution at 540 nm, using as the compensation 0.10 g of sodium azide R in 100 ml of water R. liquid a 9 g/l solution of sodium chloride R treated in the


857

Technetium (99mTc) macrosalb injection


Test solution. Mix 0.25 ml of the injection to be examined with 0.25 ml of the mobile phase (concentrated). Use immediately after dilution. The chromatographic procedure may be carried out using : — a stainless steel column 0.6 m long and 7.5 mm in internal diameter, packed with silica gel for size-exclusion chromatography R, — as the mobile phase at a flow rate of 0.6 ml/min a mixture of equal volumes of mobile phase (concentrated) and water R, — a radioactivity detector set for technetium-99m, — a loop injector. Inject 200 µl of the test solution. Continue the chromatography for at least 10 min after background level is reached. Peaks are eluted with the following retention times :

to the particles of the suspension as determined by the test for non-filterable radioactivity. The particles have a typical diameter between 10 µm and 100 µm. The specific radioactivity is not less than 37 MBq of technetium-99m per milligram of aggregated albumin at the date and hour of administration. It is prepared from sodium pertechnetate (99mTc) injection (fission or non-fission) using suitable sterile and apyrogenic ingredients and calculating the ratio of radionuclidic impurities with reference to the date and hour of administration. CHARACTERS A white suspension which may separate on standing. Technetium-99m has a half-life of 6.02 h and emits gamma radiation.

IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly II 23-24 min Poly III-albumin from that of a standardised technetium-99m solution III 25-27 min Poly II-albumin either by direct comparison or by using an instrument calibrated with the aid of such a solution. Standardised IV 28-29 min Poly I-albumin technetium-99m and molybdenum-99 solutions are V 32-33 min Human serum albumin available from laboratories recognised by the competent authority. The most prominent gamma photon of VI 40-47 min Tin colloid technetium-99m has an energy of 0.140 MeV. VII 48 min Pertechnetate B. The tests for non-filterable radioactivity and particle size contribute to the identification of the preparation. At least 80 per cent of the radioactivity applied to the column is associated with the albumin fractions II to V. C. Transfer 1 ml of the injection to a centrifuge tube and centrifuge at 2500 g for 5 min to 10 min. Decant RADIOACTIVITY the supernatant liquid. To the residue add 5 ml of Measure the radioactivity using suitable counting equipment cupri-tartaric solution R2, mix and allow to stand for by comparison with a standardised technetium-99m solution 10 min. If necessary, heat to dissolve the particles or by measurement in an instrument calibrated with the aid and allow to cool. Add rapidly 0.5 ml of dilute of such a solution. phosphomolybdotungstic reagent R, mixing immediately. A blue colour develops. LABELLING I

High molecular mass compound

19-20 min

The label states : — the amount of albumin, — the amount of tin, if any.

TESTS pH (2.2.3). The pH of the injection is 3.8 to 7.5. Non-filterable radioactivity. Use a polycarbonate membrane filter 13 mm to 25 mm in diameter, 10 µm thick and with 01/2005:0296 circular pores 3 µm in diameter. Fit the membrane into a suitable holder. Place 0.2 ml of the injection on the membrane and filter, adding 20 ml of a 9 g/l solution of TECHNETIUM (99mTc) MACROSALB sodium chloride R during the filtration. The radioactivity INJECTION remaining on the membrane represents not less than 90 per cent of the total radioactivity of the injection. Technetii (99mTc) macrosalbi Particle size. Examine using a microscope. Dilute the suspensio iniectabilis injection if necessary so that the number of particles is just low enough for individual particles to be distinguished. DEFINITION Using a syringe fitted with a needle having a calibre not less 99m Technetium ( Tc) macrosalb injection is a sterile, than 0.35 mm, place a suitable volume in a suitable counting apyrogenic suspension of human albumin in the form of chamber such as a haemocytometer cell, taking care not to irregular insoluble aggregates obtained by denaturing overfill the chamber. Allow the suspension to settle for 1 min human albumin in aqueous solution ; the particles are and carefully add a cover slide without squeezing the sample. labelled with technetium-99m. The injection contains Scan an area corresponding to at least 5000 particles. Not reducing substances, such as tin salts in an amount not more than 10 particles have a maximum dimension greater exceeding 3 mg of Sn per millilitre ; it may contain a suitable than 100 µm. No particle having a maximum dimension buffer such as acetate, citrate or phosphate buffer and greater than 150 µm is present. also non-denatured human albumin and an antimicrobial Aggregated albumin preservative such as benzyl alcohol. The human albumin Test solution. Transfer a volume of the injection expected to employed complies with the requirements prescribed in contain about 1 mg of aggregated albumin to a centrifuge the monograph on Human albumin solution (0255). The tube and centrifuge at about 2500 g for 5 min to 10 min. injection contains not less than 90.0 per cent and not Decant the supernatant liquid. Resuspend the sediment in more than 110.0 per cent of the declared technetium-99m 2.0 ml of a 9 g/l solution of sodium chloride R. Centrifuge radioactivity at the date and hour stated on the label. Not at 2500 g for 5 min to 10 min. Decant the supernatant liquid. less than 90 per cent of the technetium-99m is bound 858



Resuspend the sediment in 5.0 ml of sodium carbonate solution R1. Heat in a water-bath at 80 °C to 90 °C to dissolve the aggregated albumin. Allow to cool, transfer to a volumetric flask and dilute to 10.0 ml with sodium carbonate solution R1.

Technetium (99mTc) medronate injection

RADIOACTIVITY Measure the radioactivity using suitable counting equipment by comparison with a standardised technetium-99m solution or by measurement in an instrument calibrated with the aid of such a solution.

Reference solutions. Prepare a range of reference solutions LABELLING containing 0.05 mg to 0.2 mg of human albumin per millilitre The label states : in sodium carbonate solution R1. — that the preparation should be shaken before use, Introduce 3.0 ml of each solution separately into 25 ml flasks. — the quantity of tin per millilitre, if any, To each flask add 15.0 ml of cupri-tartaric solution R2, mix — that the preparation is not to be used if after shaking, the suspension does not appear homogeneous. and allow to stand for 10 min. Add rapidly 1.5 ml of dilute phosphomolybdotungstic reagent R and mix immediately. Allow to stand for 30 min and measure the absorbance 01/2005:0641 (2.2.25) at 750 nm using sodium carbonate solution R1 as the compensation liquid. Using the absorbances obtained TECHNETIUM (99mTc) MEDRONATE with the reference solutions, draw a calibration curve and INJECTION calculate the content of aggregated albumin in the injection. Tin Test solution. To 1.0 ml of the injection add 1.0 ml of 2 M hydrochloric acid. Heat in a water-bath for 30 min. Cool and centrifuge for 10 min at 300 g. Dilute 1.0 ml of the supernatant liquid to 25.0 ml with 1 M hydrochloric acid. Reference solution. Dissolve 0.115 g of stannous chloride R in 1 M hydrochloric acid and dilute to 1000.0 ml with the same acid. To 1.0 ml of each solution add 0.4 ml of a 20 g/l solution of sodium laurilsulfate R, 0.05 ml of thioglycollic acid R, 0.1 ml of dithiol reagent R and 3.0 ml of 0.2 M hydrochloric acid. Mix. Measure the absorbance (2.2.25) of each solution at 540 nm, using 0.2 M hydrochloric acid as the compensation liquid. The absorbance of the test solution is not greater than that of the reference solution (3 mg of Sn per millilitre). Physiological distribution. Inject a volume not greater than 0.2 ml into the caudal vein of each of three rats weighing 150 g to 250 g. Kill the rats 15 min after the injection, remove the liver, the spleen and the lungs and measure the radioactivity in the organs using a suitable instrument. Measure the radioactivity in the rest of the body, including the blood, after having removed the tail. Determine the percentage of radioactivity in the lungs, the liver and the spleen from the expression :

Technetii (99mTc) medronati solutio iniectabilis DEFINITION Technetium (99mTc) medronate injection is a sterile solution which may be prepared by mixing solutions of sodium methylenediphosphonate and a stannous salt with sodium pertechnetate (99mTc) injection (fission or non-fission). The injection contains a variable quantity of tin (Sn) not exceeding 3 mg/ml ; it may contain antimicrobial preservatives, antioxidants, stabilisers and buffers. The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the declared technetium-99m radioactivity at the date and hour stated on the label. Radioactivity present as chemical forms other than technetium-99m medronate complex is not greater than 5.0 per cent of the total radioactivity. It is prepared from sodium pertechnetate (99mTc) injection (fission or non-fission) using suitable sterile ingredients and calculating the ratio of radionuclidic impurities with reference to the date and hour of administration. CHARACTERS A clear, colourless solution. Technetium-99m has a half-life of 6.02 h and emits gamma radiation.

IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised technetium-99m solution A = radioactivity of the organ concerned, either by direct comparison or by using an instrument B = total radioactivity in the liver, the spleen, the calibrated with the aid of such a solution. Standardised lungs and the rest of the body. technetium-99m and molybdenum-99 solutions are available from laboratories recognised by the competent In not fewer than two of the three rats used, at least 80 per authority. The most prominent gamma photon of cent of the radioactivity is found in the lungs and not more technetium-99m has an energy of 0.140 MeV. than a total of 5 per cent in the liver and spleen. The injection B. Examine the chromatograms obtained in the test for may be released for use before completion of the test. radiochemical purity. The distribution of the radioactivity contributes to the identification of the preparation. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical C. Examine by thin-layer chromatography (2.2.27) using preparations (0125). The injection may be released for use cellulose as the coating substance. before completion of the test. Test solution. Dilute the injection to be examined with water R to obtain a solution containing about 0.1 mg to Pyrogens. It complies with the test for pyrogens 0.5 mg of sodium medronate per millilitre. prescribed in the monograph on Radiopharmaceutical Reference solution. Dissolve a suitable quantity (1 mg to preparations (0125). Inject into the animals not less than 5 mg) of medronic acid CRS in a mixture of a 9.0 g/l 0.1 ml per kilogram of the rabbit’s mass. The injection may solution of sodium chloride R and water R and dilute to be released for use before completion of the test. General Notices (1) apply to all monographs and other texts

859

Technetium (99mTc) mertiatide injection


10 ml with the same solvent so as to obtain a solution similar to the test solution with regard to medronate and sodium chloride concentrations. Apply separately to the plate 10 µl of each solution. Develop over a path of 12 cm to 14 cm (development time about 4 h) using a mixture of 20 volumes of 2-propanol R, 30 volumes of 1 M hydrochloric acid and 60 volumes of methyl ethyl ketone R. Allow the plate to dry in air and spray with ammonium molybdate solution R4. Expose the plate to ultraviolet light at 254 nm for about 10 min. The principal spot in the chromatogram obtained with the test solution is similar in position and colour to the spot in the chromatogram obtained with the reference solution.

10 cm to 15 cm in about 10 min. Determine hydrolysed technetium and technetium in colloidal form by test (a) and pertechnetate ion by test (b). (a) Apply to the plate 5 µl to 10 µl of the injection. Develop immediately over a path of 10 cm to 15 cm using a 136 g/l solution of sodium acetate R. Allow the plate to dry in air. Determine the distribution of radioactivity using a suitable detector. Hydrolysed technetium and technetium in colloidal form remain at the starting point. Technetium medronate complex and pertechnetate ion migrate near to the solvent front. (b) Apply to the plate 5 µl to 10 µl of the injection and dry quickly. Develop over a path of 10 cm to 15 cm using methyl ethyl ketone R. Allow the plate to dry. Determine TESTS the distribution of radioactivity using a suitable detector. Pertechnetate ion migrates near to the solvent front. pH (2.2.3). The pH of the solution is 3.5 to 7.5. Technetium medronate complex and technetium in colloidal Tin form remain at the starting-point. Test solution. Dilute 1.0 ml of the solution to 50.0 ml with The percentage of radioactivity corresponding to 1 M hydrochloric acid. pertechnetate ion in the chromatogram obtained in test Reference solution. Dissolve 0.115 g of stannous chloride R (b) is not greater than 2.0 per cent and the sum of the in 1 M hydrochloric acid and dilute to 1000.0 ml with the percentages of radioactivity corresponding to impurities in same acid. the chromatograms obtained in test (a) and test (b) (including To 1.0 ml of each solution add 0.4 ml of a 20 g/l solution pertechnetate ion) is not greater than 5.0 per cent. of sodium laurilsulfate R, 0.05 ml of thioglycollic acid R, 0.1 ml of dithiol reagent R and 3.0 ml of 0.2 M hydrochloric RADIOACTIVITY acid. Mix. Measure the absorbance (2.2.25) of each solution Measure the radioactivity using suitable counting equipment at 540 nm, using 0.2 M hydrochloric acid as compensation by comparison with a standardised technetium-99m solution or by measurement in an instrument calibrated with the aid liquid. The absorbance of the test solution is not greater than that of the reference solution (3 mg of Sn per millilitre). of such a solution. Physiological distribution. Inject a volume not greater than 0.2 ml, equivalent to not more than 0.05 mg of sodium medronate into a suitable vein such as a caudal vein or the saphenous vein of each of three rats, each weighing 150 g to 250 g. Measure the radioactivity in the syringe before and after injection. Kill the rats 2 h after the injection. Remove one femur, the liver, and some blood. Weigh the blood. Remove the tail if a caudal vein has been used for the injection. Using a suitable instrument measure the radioactivity in the femur, liver, and blood, and in the tail if a caudal vein has been used for the injection. Determine the percentage of radioactivity in each sample from the expression :

01/2005:1372

TECHNETIUM (99mTc) MERTIATIDE INJECTION Technetii (99mTc) mertiatidi solutio iniectabilis O O

2-

O N

N 99m

Tc

2 Na+ S

O

N

O

A

=

radioactivity of the sample concerned,

B

=

total radioactivity, which is equal to the difference between the two measurements made on the syringe minus the radioactivity in the tail if a caudal vein has been used for the injection.

O-

DEFINITION Technetium (99mTc) mertiatide injection is a sterile solution Calculate the radioactivity per unit mass in the blood. which may be prepared by either heating a mixture containing Correct the blood concentration by multiplying by a S-benzoylmercaptoacetyltriglycine (betiatide), a weak factor m/200 where m is the body mass of the rat in grams. chelating agent such as tartrate, a stannous salt and sodium In not fewer than two of the three rats : not less than 1.5 per pertechnetate (99mTc) injection (fission or non-fission), or by cent of the radioactivity is found in the femur ; not more than mixing solutions of mercaptoacetyltriglycine (mertiatide), 1.0 per cent is found in the liver and not more than 0.05 per a stannous salt and sodium pertechnetate (99mTc) injection (fission or non-fission) at alkaline pH. It may contain cent per gram is found in the blood. stabilisers and a buffer. The injection contains not less Sterility. It complies with the test for sterility than 90.0 per cent and not more than 110.0 per cent of prescribed in the monograph on Radiopharmaceutical the declared technetium-99m radioactivity at the date and preparations (0125). The injection may be released for use time stated on the label. Not less than 94 per cent of the before completion of the test. radioactivity corresponds to technetium-99m in the form of RADIOCHEMICAL PURITY [99mTc]technetium mertiatide. Examine by thin-layer chromatography (2.2.27) using silica gel as the coating substance on a glass-fibre sheet. Use plates CHARACTERS A clear, colourless solution. such that during development, the mobile phase migrates 860



Technetium-99m has a half-life of 6.02 h and emits gamma radiation. IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised technetium-99m solution either by direct comparison or by using an instrument calibrated with the aid of such a solution. Standardised technetium-99m solutions are available from laboratories recognised by the competent authority. The most prominent gamma photon of technetium-99m has an energy of 0.140 MeV. B. Examine the chromatogram obtained in test (b) for radiochemical purity. The principal peak in the chromatogram obtained with the test solution has approximately the same retention time as the principal peak in the chromatogram obtained with the reference solution. TESTS pH (2.2.3). The pH of the injection is 5.0 to 7.5. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. RADIOCHEMICAL PURITY (a) Examine by ascending paper chromatography (2.2.26) using a suitable paper as the stationary phase. Test solution. The solution to be examined. Apply 2 µl of the test solution to the paper. Develop over a path of 15 cm using a mixture of 40 volumes of water R and 60 volumes of acetonitrile R. Allow the paper to dry and determine the distribution of the radioactivity using a suitable detector. Not more than 2.0 per cent of the total radioactivity is retained at the origin (Rf value 0.0 to 0.1). (b) Examine by liquid chromatography (2.2.29). Test solution. The solution to be examined. Reference solution. Dissolve with heating on a water-bath 5 mg of S-benzylmercaptoacetyltriglycine CRS in 5 ml of water R. To 1 ml of this solution in a closed vial filled with nitrogen R, add 0.5 ml of a 40 g/l solution of sodium potassium tartrate R, 25 µl of a 4 g/l solution of stannous chloride R in 0.05 M hydrochloric acid R and 370 MBq to 740 MBq of sodium pertechnetate (99mTc) injection (fission or non-fission) in a volume not exceeding 3 ml. Heat the mixture on a water-bath for 10 min and allow to cool to room temperature. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.0 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.0 ml/min : Mobile phase A. A mixture of 7 volumes of ethanol R with 93 volumes of a solution of a 1.36 g/l solution of potassium dihydrogen phosphate R, adjusted to pH 6.0 with 0.1 M sodium hydroxide, Mobile phase B. A mixture of 10 volumes of water R with 90 volumes of methanol R, — a suitable radioactivity detector, — a 20 µl loop injector. Equilibrate the column with mobile phase A for 20 min. Inject the test solution and the reference solution. Switch 10 min after each injection to mobile phase B and continue the chromatographic procedure for 15 min. General Notices (1) apply to all monographs and other texts

Technetium (99mTc) microspheres injection

The test is not valid unless in the chromatogram obtained with the test solution the principal peak has approximately the same retention time as the principal peak in the chromatogram obtained with the reference solution. In the chromatogram obtained with the test solution the sum of the areas preceding the principal peak (corresponding to hydrophilic impurities, including [99mTc]pertechnetate) is not greater than 3.0 per cent of the sum of the areas of all peaks. The sum of the peaks following the principal peak (corresponding to lipophilic impurities) is not greater than 4.0 percent of the sum of the area of all peaks. Not less than 94 per cent of the radioactivity corresponds to [99mTc]technetium mertiatide. RADIOACTIVITY Measure the radioactivity using suitable counting equipment by comparison with a standardised technetium-99m solution or by measurement in an instrument calibrated with the aid of such a solution. 01/2005:0570

TECHNETIUM (99mTc) MICROSPHERES INJECTION Technetii (99mTc) microsphaerarum suspensio iniectabilis DEFINITION Technetium (99mTc) microspheres injection is a sterile, apyrogenic suspension of human albumin which has been denatured to form spherical insoluble particles ; the particles are labelled with technetium-99m. The injection contains reducing substances, such as tin salts in an amount not exceeding 3 mg of Sn per millilitre ; it may contain a suitable buffer such as acetate, citrate or phosphate and additives such as wetting agents. The human albumin used complies with the requirements of the monograph on Human albumin solution (0255). The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the declared technetium-99m radioactivity at the date and hour stated on the label. Not less than 95 per cent of the technetium-99m is bound to the particles of the suspension as determined by the test for non-filterable radioactivity. The particles have a typical diameter between 10 µm and 50 µm. The radioactivity is not less than 185 MBq of technetium-99m per million particles at the date and hour of administration. Technetium (99mTc) microspheres injection is prepared from sodium pertechnetate (99mTc) injection (fission or non-fission) using suitable sterile and apyrogenic ingredients and calculating the ratio of radionuclidic impurities with reference to the date and hour of administration. CHARACTERS A suspension of white, yellow or artificially coloured particles which may separate on standing. Technetium-99m has a half-life of 6.02 h and emits gamma radiation. IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised technetium-99m solution either by direct comparison or by using an instrument calibrated with the aid of such a solution. Standardised technetium-99m and molybdenum-99 solutions are 861

Technetium (99mTc) pentetate injection


available from laboratories recognised by the competent authority. The most prominent gamma photon of technetium-99m has an energy of 0.140 MeV. B. The tests for non-filterable radioactivity and particle size contribute to the identification of the preparation. C. Transfer 1 ml of the injection to a centrifuge tube and centrifuge at 2500 g for 5 min to 10 min. Decant the supernatant liquid. To the residue add 5 ml of cupri-tartaric solution R2, mix and allow to stand for 10 min. If necessary, heat to dissolve the particles and allow to cool. Add rapidly 0.5 ml of dilute phosphomolybdotungstic reagent R, mix immediately and allow to stand. A blue colour develops.

the blood and voided urine, after having discarded the tail. Determine the percentage of radioactivity in the liver, the spleen and the lungs from the expression :

A

=


B

=

total radioactivity in the liver, the spleen, the lungs and the rest of the body, including voided urine.

In not fewer than two of the three rats used, not less than 80 per cent of the radioactivity is found in the lungs and not more than a total of 5 per cent in the liver and spleen. The TESTS injection may be released for use before completion of the test. pH (2.2.3). The pH of the injection is 4.0 to 9.0. Non-filterable radioactivity. Use a polycarbonate membrane Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical filter 13 mm to 25 mm in diameter, 10 µm thick and with preparations (0125). The injection may be released for use circular pores 3 µm in diameter. Fit the membrane into before completion of the test. a suitable holder. Place 0.2 ml of the injection on the membrane and filter, adding 20 ml of a 9 g/l solution of Pyrogens. It complies with the test for pyrogens sodium chloride R during the filtration. The radioactivity prescribed in the monograph on Radiopharmaceutical remaining on the membrane represents not less than 95 per preparations (0125). Inject not less than 0.1 ml per kilogram cent of the total radioactivity of the injection. of the rabbit’s mass. The injection may be released for use before completion of the test. Particle size. Examine using a microscope. Dilute the injection if necessary so that the number of particles is just RADIOACTIVITY low enough for individual particles to be distinguished. Using a syringe fitted with a needle having a calibre not less Measure the radioactivity using suitable counting equipment than 0.35 mm, place a suitable volume in a suitable counting by comparison with a standardised technetium-99m solution or by measurement in an instrument calibrated with the aid chamber such as a haemocytometer cell, taking care not to overfill the chamber. Allow the suspension to settle for 1 min of such a solution. and carefully add a cover slide without squeezing the sample. LABELLING Scan an area corresponding to at least 5000 particles. The The label states : particles have a uniform spherical appearance. Not more — the quantity of tin per millilitre, if any, than 10 particles have a maximum dimension greater than 75 µm. No particle having a maximum dimension greater — that the preparation should be shaken before use. than 100 µm is present. Number of particles. Examine using a microscope. Fill a 01/2005:0642 suitable counting chamber such as a haemocytometer cell with a suitable dilution of the injection taking care that TECHNETIUM (99mTc) PENTETATE particles do not separate during the transfer. Count the INJECTION number of particles in the chamber. Repeat this procedure twice and calculate the number of particles per millilitre of the injection. Technetii (99mTc) pentetatis Tin solutio iniectabilis Test solution. To 1.0 ml of the injection add 0.5 ml of DEFINITION sulphuric acid R and 1.5 ml of nitric acid R. Heat and Technetium (99mTc) pentetate injection is a sterile solution evaporate to approximately 1 ml. Add 2 ml of water R and evaporate again to approximately 1 ml. Repeat this procedure which may be prepared by mixing solutions of sodium twice, cool and dilute to 25.0 ml with 1 M hydrochloric acid. diethylenetriaminepenta-acetate or calcium trisodium a stannous salt with Reference solution. Dissolve 0.115 g of stannous chloride R diethylenetriaminepenta-acetate and 99m a solution of sodium pertechnetate ( Tc). It contains a in 1 M hydrochloric acid and dilute to 1000.0 ml with the variable quantity of tin (Sn) not exceeding 1 mg/ml ; it may same acid. contain suitable antimicrobial preservatives, antioxidants, To 1.0 ml of each solution add 0.4 ml of a 20 g/l solution of stabilisers and buffers. The injection contains not less sodium laurilsulfate R, 0.05 ml of thioglycollic acid R, 0.1 ml than 90.0 per cent and not more than 110.0 per cent of of dithiol reagent R and 3.0 ml of 0.2 M hydrochloric acid. the declared technetium-99m radioactivity at the date and Mix. Measure the absorbance (2.2.25) of each solution at hour stated on the label. Not less than 95.0 per cent of the 540 nm, using 0.2 M hydrochloric acid as the compensation radioactivity corresponds to technetium-99m complexed liquid. The absorbance of the test solution is not greater with sodium pentetate or calcium trisodium pentetate. than that of the reference solution (3 mg of Sn per millilitre). It is prepared from sodium pertechnetate (99mTc) injection Physiological distribution. Inject a volume not greater than (fission or non-fission) using suitable, sterile ingredients 0.2 ml into a caudal vein of each of three rats weighing and calculating the ratio of radionuclidic impurities with 150 g to 250 g. Sacrifice the rats 15 min after the injection, reference to the date and hour of administration. remove the liver, the spleen and the lungs and measure CHARACTERS the radioactivity in the organs using a suitable instrument. Measure the radioactivity in the rest of the body, including A clear, colourless or slightly yellow solution. 862



Technetium-99m has a half life of 6.02 h and emits gamma radiation. IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised technetium-99m solution either by direct comparison or by using an instrument calibrated with the aid of such a solution. Standardised technetium-99m and molybdenum-99 solutions are available from laboratories recognised by the competent authority. The most prominent gamma photon of technetium-99m has an energy of 0.140 MeV. B. Examine the chromatograms obtained in the test for radiochemical purity. The distribution of radioactivity contributes to the identification of the preparation. C. Place in a clean, dry 10 ml glass tube a volume of the injection to be examined containing 2 mg of pentetate. Dilute, if necessary, to 1 ml with water R. Place in a second tube 1 ml of water R (blank). To each tube add 0.1 ml of a 1 g/l solution of nickel sulphate R, 0.5 ml of a 50 per cent V/V solution of glacial acetic acid R and 0.75 ml of a 50 g/l solution of sodium hydroxide R. Mix and verify that the pH is not above 5. To each tube add 0.1 ml of a 10 g/l solution of dimethylglyoxime R in alcohol R. Mix and allow to stand for 2 min. Adjust the pH in each tube to not less than 12 by adding a 100 g/l solution of sodium hydroxide R. Mix and check that the pH is not below 12. Allow to stand for 2 min. Heat the tubes gently on a water-bath for 2 min. The solution in the tube containing the injection to be examined remains clear and colourless throughout. The solution in the blank tube becomes red on addition of dimethylglyoxime solution and a red precipitate is formed when the tube is heated on a water-bath. TESTS pH (2.2.3). The pH of the injection is 4.0 to 7.5. Tin Test solution. Dilute 1.5 ml of the injection to 25.0 ml with 1 M hydrochloric acid. Reference solution. Dissolve 0.115 g of stannous chloride R in 1 M hydrochloric acid and dilute to 1000.0 ml with the same acid. To 1.0 ml of each solution add 0.4 ml of a 20 g/l solution of sodium laurilsulfate R, 0.05 ml of thioglycollic acid R, 0.1 ml of dithiol reagent R and 3.0 ml of 0.2 M hydrochloric acid. Mix. Measure the absorbance (2.2.25) of each solution at 540 nm, using 0.2 M hydrochloric acid as the compensation liquid. The absorbance of the test solution is not greater than that of the reference solution (1 mg of Sn per millilitre). Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. RADIOCHEMICAL PURITY Examine by thin-layer chromatography (2.2.27) using silica gel as the coating substance on a glass-fibre sheet. Heat the plate at 110 °C for 10 min. Use a plate such that during development the mobile phase migrates over a distance of 10 cm to 15 cm in about 10 min. (a) Apply to the plate 5 µl to 10 µl of the injection to be examined. Develop immediately over a path of 10 cm to 15 cm using a 9 g/l solution of sodium chloride R. Allow the plate to dry in air. Determine the distribution of radioactivity General Notices (1) apply to all monographs and other texts

Technetium (99mTc) sestamibi injection

using a suitable detector. Impurities in colloidal form remain at the starting point. Technetium pentetate complex and pertechnetate ion migrate near to the solvent front. (b) Apply to the plate 5 µl to 10 µl of the injection to be examined and allow to dry. Develop over a path of 10 cm to 15 cm using methyl ethyl ketone R. Allow the plate to dry. Determine the distribution of radioactivity using a suitable detector. Pertechnetate ion migrates near to the solvent front. Technetium pentetate complex and impurities in colloidal form remain at the starting point. The sum of the percentages of radioactivity corresponding to impurities in the chromatograms obtained in test (a) and (b) does not exceed 5.0 per cent. RADIOACTIVITY Measure the radioactivity using suitable counting equipment by comparison with a standardised technetium-99m solution or by measurement in an instrument calibrated with the aid of such a solution. 01/2005:1926

TECHNETIUM (99mTc) SESTAMIBI INJECTION Technetii (99mTc) sestamibi solutio iniectabilis

DEFINITION Sterile solution of (OC-6-11)-hexakis[1-(isocyano-κC)-2methoxy-2-methylpropane][99mTc]technetium(I) chloride, which may be prepared by heating a mixture containing [tetrakis(2-methoxy-2-methylpropyl-1-isocyanide)copper (1+)] tetrafluoroborate, a weak chelating agent, a stannous salt and Sodium pertechnetate (99mTc) injection (fission) (0124) or Sodium pertechnetate (99mTc) injection (non-fission) (0283). Content : 90 per cent to 110 per cent of the declared technetium-99m radioactivity at the date and hour stated on the label. CHARACTERS Appearance : clear, colourless solution. Half-life and nature of radiation of technetium-99m : see Table of physical characteristics of radionuclides (5.7). IDENTIFICATION A. Gamma-ray spectrometry. Results : the spectrum obtained with the solution to be examined does not differ significantly from that of a standardised technetium-99m solution. The most prominent gamma photon has an energy of 0.141 MeV. 863

Technetium (99mTc) sestamibi injection


B. Examine the chromatograms obtained in the test for impurity C under Radiochemical purity. Results : the principal peak in the radiochromatogram obtained with the test solution is similar in retention time to the principal peak in the radiochromatogram obtained with the reference solution. TESTS pH (2.2.3) : 5.0 to 6.0. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. RADIOCHEMICAL PURITY Impurity A and other polar impurities. Thin-layer chromatography (2.2.27). Test solution. The preparation to be examined.

— stationary phase : spherical base-deactivated end-capped octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : mix 20 volumes of acetonitrile R, 35 volumes of a 6.6 g/l solution of ammonium sulphate R and 45 volumes of methanol R. Flow rate : 1.5 ml/min. Detection : radioactivity detector. Injection : 25 µl. Run time : 25 min. Relative retention with reference to technetium-99m sestamibi : impurity C = about 1.3. System suitability : reference solution : — the chromatogram is similar to the chromatogram provided with sestamibi labelling kit CRS, — relative retention with reference to technetium-99m sestamibi : impurity C = minimum 1.2.

Plate : TLC octadecylsilyl silica gel plate R.

Limits : Mobile phase : mix 10 volumes of tetrahydrofuran R, — impurity C : not more than 3 per cent of the total 20 volumes of a 38.5 g/l solution of ammonium acetate R, radioactivity, 30 volumes of methanol R and 40 volumes of acetonitrile R. — technetium-99m sestamibi: minimum 94 per cent of the Application : about 5 µl. total radioactivity. Development : immediately over a path of 6 cm. Calculate the percentage of radioactivity due to technetium-99m sestamibi from the expression : Drying : in air. Detection : determine the distribution of radioactivity using a radioactivity detector. Retention factors : impurity B and apolar impurities = 0 to = percentage of radioactivity due to impurity B 0.1 ; impurity C and technetium-99m sestamibi = 0.3 to 0.6 ; B determined in the test for impurity B under impurity A and other polar impurities = 0.9 to 1.0. Radiochemical purity, Limit : see test for impurity B. T = area of the peak due to technetium-99m sestamibi Impurity B. Paper chromatography (2.2.26). If no activity is in the chromatogram obtained with the test found at retention factor 0 to 0.1 in the test for impurity A solution. and other polar impurities, impurity B is absent and the test for impurity B may be omitted. RADIOACTIVITY Test solution. The preparation to be examined. Determine the radioactivity using a calibrated instrument. Paper : paper for chromatography R. Mobile phase : mix equal volumes of acetonitrile R, 0.5 M IMPURITIES acetic acid and a 20 g/l solution of sodium chloride R . Application : about 5 µl.

A. [99mTc]O4– : (99mTc)pertechnetate ion,

Development : over a path of 10 cm. Drying : in air. Detection : determine the distribution of radioactivity using a radioactivity detector. Retention factors : impurity B = 0 to 0.1 ; impurity A, impurity C and technetium-99m sestamibi = 0.8 to 1.0.

B. technetium-99m in colloidal form,

Limit : — sum of impurity A and other polar impurities, and impurity B : maximum 5 per cent of the total radioactivity. Impurity C. Liquid chromatography (2.2.29). Test solution. The preparation to be examined. Reference solution. To a vial of sestamibi labelling kit CRS add 3 ml of a 9 g/l solution of sodium chloride R containing 700 MBq to 900 MBq of sodium pertechnetate (99mTc) injection (fission or non-fission). Heat the mixture in a water-bath for 10 min and allow to cool to room temperature. Column : — size : l = 0.25 m, Ø = 4.6 mm, 864

C. (OC-6-22)-pentakis[1-(isocyano-κC)-2-methoxy-2methylpropane][1-(isocyano-κC)-2-methylprop-1ene][99mTc]technetium (1+).



Technetium (99mTc) tin pyrophosphate injection

01/2005:0643 acid as the compensation liquid. The absorbance of the test solution is not greater than that of the reference solution 99m (1 mg of Sn per millilitre). TECHNETIUM ( Tc) SUCCIMER Physiological distribution. Inject a volume not greater INJECTION than 0.2 ml and containing not more than 0.1 mg of dimercaptosuccinic acid into a suitable vein, such as a 99m Technetii ( Tc) succimeri caudal vein or a saphenous vein, of each of three rats each weighing 150 g to 250 g. Measure the radioactivity solutio iniectabilis in the syringe before and after the injection. Kill the rats DEFINITION 1 h after the injection. Remove the kidneys, the liver, the stomach, the lungs and, if a caudal vein has been used for Technetium (99mTc) succimer injection is a sterile solution the injection, the tail. Using a suitable instrument determine of meso-2,3-dimercaptosuccinic acid labelled with the radioactivity in the organs and, if a caudal vein has been technetium-99m. It contains a reducing substance, such used for injection, in the tail. Determine the percentage as a tin salt in an amount not exceeding 1 mg of Sn per of radioactivity in each organ with respect to the total millilitre, and may contain stabilisers, antioxidants such as ascorbic acid, and inert additives. The injection contains not radioactivity calculated as the difference between the two measurements made on the syringe minus the activity in the less than 90.0 per cent and not more than 110.0 per cent tail (if a caudal vein has been used for the injection). of the declared technetium-99m radioactivity at the date and hour stated on the label. Not less than 95.0 per cent of In not fewer than two of the three rats used, the radioactivity the radioactivity corresponds to technetium-99m succimer in the kidneys is not less than 40 per cent, that in the liver complex. is not more than 10.0 per cent, that in the stomach is not more than 2.0 per cent and that in the lungs is not more It is prepared from sodium pertechnetate (99mTc) injection than 5.0 per cent. (fission or non-fission) using suitable sterile ingredients and calculating the ratio of radionuclidic impurities with Sterility. It complies with the test for sterility reference to the date and hour of administration. Syringes prescribed in the monograph on Radiopharmaceutical for handling the eluate intended for labelling of the final preparations (0125). The injection may be released for use product, or for handling the final product should not contain before completion of the test. rubber parts. RADIOCHEMICAL PURITY Examine by thin-layer chromatography (2.2.27) using silica CHARACTERS gel as the coating substance on a glass-fibre sheet. Heat the A clear, colourless solution. plate at 110 °C for 10 min. Use a plate such that during Technetium-99m has a half life of 6.02 h and emits gamma development the mobile phase migrates over a distance of radiation. 10 cm to 15 cm in about 10 min. IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised technetium-99m solution either by direct comparison or by using an instrument calibrated with the aid of such a solution. Standardised technetium-99m and molybdenum-99 solutions are available from laboratories recognised by the competent authority. The most prominent gamma photon of technetium-99m has an energy of 0.140 MeV. B. Examine the chromatogram obtained in the test for radiochemical purity. The distribution of the radioactivity contributes to the identification of the preparation. C. Place 1 ml of the injection to be examined in a test-tube and add 1 ml of a 20 g/l solution of sodium nitroprusside R and 0.1 ml of glacial acetic acid R. Mix. Place carefully at the top of the solution a layer of concentrated ammonia R. A violet ring develops between the layers. TESTS pH (2.2.3). The pH of the injection is 2.3 to 3.5. Tin Test solution. Dilute 1.5 ml of the injection to be examined to 25.0 ml with 1 M hydrochloric acid. Reference solution. Dissolve 0.115 g of stannous chloride R in 1 M hydrochloric acid and dilute to 1000.0 ml with the same acid. To 1.0 ml of each solution add 0.4 ml of a 20 g/l solution of sodium laurilsulfate R, 0.05 ml of thioglycollic acid R, 0.1 ml of dithiol reagent R and 3.0 ml of 0.2 M hydrochloric acid. Mix. Allow to stand for 60 min. Measure the absorbance (2.2.25) of each solution at 540 nm, using 0.2 M hydrochloric General Notices (1) apply to all monographs and other texts

Apply to the plate 5 µl to 10 µl of the injection to be examined. Develop immediately over a path of 10 cm to 15 cm using methyl ethyl ketone R. Allow the plate to dry. Determine the distribution of radioactivity using a suitable detector. Technetium succimer complex remains at the starting point. Pertechnetate ion migrates near to the solvent front. Not less than 95.0 per cent of the total radioactivity is found in the spot corresponding to technetium succimer complex. The radioactivity corresponding to pertechnetate ion represents not more than 2.0 per cent of the total radioactivity.

RADIOACTIVITY Measure the radioactivity using suitable counting equipment by comparison with a standardised technetium-99m solution or by measurement in an instrument calibrated with the aid of such a solution. STORAGE Store protected from light. 01/2005:0129

TECHNETIUM (99mTc) TIN PYROPHOSPHATE INJECTION Stanni pyrophosphatis et technetii (99mTc) solutio iniectabilis DEFINITION Technetium (99mTc) tin pyrophosphate injection is a sterile, apyrogenic solution which may be prepared by mixing solutions of sodium pyrophosphate and stannous chloride with sodium pertechnetate (99mTc) injection (fission or non-fission). The injection contains not less than 90.0 per 865

Technetium (99mTc) tin pyrophosphate injection


cent and not more than 110.0 per cent of the declared technetium-99m radioactivity at the date and hour stated on the label. Not less than 90 per cent of the radioactivity corresponds to technetium-99m complexed with tin pyrophosphate. The injection contains a quantity of sodium pyrophosphate (Na4P2O7,10H2O) that may vary from 1 mg to 50 mg per millilitre and a variable quantity of tin (Sn) not exceeding 3.0 mg per millilitre. It is prepared from sodium pertechnetate (99mTc) injection (fission or non-fission) using suitable sterile, apyrogenic ingredients and calculating the ratio of radionuclidic impurities with reference to the date and hour of administration. CHARACTERS A clear, colourless solution. Technetium-99m has a half-life of 6.02 h and emits gamma radiation. IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised technetium-99m solution either by direct comparison or by using an instrument calibrated with the aid of such a solution. Standardised technetium-99m and molybdenum-99 solutions are available from laboratories recognised by the competent authority. The most prominent gamma photon of technetium-99m has an energy of 0.140 MeV. B. Examine the chromatograms obtained in the test for radiochemical purity. The distribution of radioactivity contributes to the identification of the injection. C. To 1 ml add 1 ml of acetic acid R. Heat on a water-bath for 1 h. After cooling, add 10 ml of nitro-vanadomolybdic reagent R and allow to stand for 30 min. A yellow colour develops. D. To 1 ml add 2 ml of a 30 per cent V/V solution of sulphuric acid R, 1 ml of hydrochloric acid R, 0.05 ml of thioglycollic acid R, 0.4 ml of a 20 g/l solution of sodium laurilsulfate R and 0.1 ml of dithiol reagent R and allow to stand for 30 min. A pink colour develops. TESTS pH (2.2.3). The pH of the injection is 6.0 to 7.0. Sodium pyrophosphate Test solution. Use 1 ml of the injection to be examined or a suitable dilution of it. Reference solutions. Using a solution containing sodium pyrophosphate R and stannous chloride R in the same proportions as in the injection to be examined, prepare a range of solutions and dilute to the same final volume with water R. To the test solution and to 1 ml of each of the reference solutions add successively 10 ml of a 1 g/l solution of disodium hydrogen phosphate R, 10 ml of iron standard solution (8 ppm Fe) R, 5 ml of glacial acetic acid R and 5 ml of a 1 g/l solution of hydroxylamine hydrochloride R. Dilute each solution to 40 ml with water R and heat in a water-bath at 40 °C for 1 h. To each solution add 4 ml of a 1 g/l solution of phenanthroline hydrochloride R and dilute to 50.0 ml with water R. Measure the absorbance (2.2.25) of each solution at 515 nm using as the compensation liquid a reagent blank containing hydrochloric acid (1.1 g/l HCl) instead of the iron standard solution (8 ppm Fe) R. Using 866

the absorbances obtained with the reference solutions, draw a calibration curve and calculate the concentration of sodium pyrophosphate in the injection to be examined. Tin Test solution. Use 1 ml of the injection to be examined or a suitable dilution of it. Reference solutions. Using a solution in hydrochloric acid (6.2 g/l HCl) containing sodium pyrophosphate R and stannous chloride R in the same proportions as in the injection to be examined, prepare a range of solutions and dilute to the same volume with hydrochloric acid (6.2 g/l HCl). To the test solution and to 1 ml of each of the reference solutions add 2 ml of a 300 g/l solution of sulphuric acid R, 1 ml of hydrochloric acid R, 0.05 ml of thioglycollic acid R, 0.4 ml of a 20 g/l solution of sodium laurilsulfate R and 0.1 ml of dithiol reagent R and dilute to 15 ml with hydrochloric acid (6.2 g/l HCl). Allow the solutions to stand for 30 min and measure the absorbance (2.2.25) of each solution at 530 nm, using as the compensation liquid a reagent blank containing the same quantity of sodium pyrophosphate R as the injection to be examined. Using the absorbances obtained with the reference solutions, draw a calibration curve and calculate the concentration of tin in the injection to be examined. Sterility. lt complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. Pyrogens. It complies with the test for pyrogens prescribed in the monograph on Radiopharmaceutical preparations (0125). Inject not less than 0.1 ml per kilogram of the rabbit’s mass. The injection may be released for use before completion of the test. RADIOCHEMICAL PURITY (a) Examine by thin-layer chromatography (2.2.27) using silica gel as the coating substance on a glass-fibre sheet. Heat the plate at 110 °C for 10 min. The plate used should be such that during development the mobile phase migrates over a distance of 10 cm to 15 cm in about 10 min. Apply to the plate 5 µl to 10 µl of the injection and dry in a stream of nitrogen. Develop over a path of 10 cm to 15 cm using methyl ethyl ketone R through which nitrogen has been bubbled in the chromatography tank for 10 min immediately before the chromatography. Allow the plate to dry. Determine the distribution of radioactivity using a suitable detector. The technetium-99m tin pyrophosphate complex remains at the starting-point and pertechnetate ion migrates with an Rf of 0.95 to 1.0. (b) Examine by thin-layer chromatography (2.2.27) using silica gel as the coating substance on a glass-fibre sheet. Heat the plate at 110 °C for 10 min. The plate used should be such that during development the mobile phase migrates over a distance of 10 cm to 15 cm in about 10 min. Apply to the plate 5 µl to 10 µl of the injection. Develop immediately over a path of 10 cm to 15 cm using a 136 g/l solution of sodium acetate R. Allow the plate to dry and measure the distribution of radioactivity using a suitable detector. Impurities in colloidal form remain at the starting-point and technetium-99m tin pyrophosphate complex and pertechnetate ion migrate with an Rf of 0.9 to 1.0. Add together the percentages of radioactivity corresponding to impurities in the chromatograms obtained in test (a) and test (b). The sum does not exceed 10 per cent.


Tritiated (3H) water injection


RADIOACTIVITY Measure the radioactivity using suitable counting equipment by comparison with a standardised technetium-99m solution or by measurement in an instrument calibrated with the aid of such a solution. LABELLING The label states, in particular, the quantity of sodium pyrophosphate per millilitre and the quantity of tin per millilitre. 01/2005:0571

THALLOUS (201Tl) CHLORIDE INJECTION Thallosi (201Tl) chloridi solutio iniectabilis DEFINITION Thallous (201Tl) chloride injection is a sterile solution of thallium-201 in the form of thallous chloride. It may be made isotonic by the addition of Sodium chloride (0193) and may contain a suitable antimicrobial preservative such as Benzyl alcohol (0256). Thallium-201 is a radioactive isotope of thallium formed by the decay of lead-201. Lead-201 is a radioactive isotope of lead and may be obtained by irradiation, with protons of suitable energy, of thallium which may be enriched in thallium-203. Thallium-201 may be separated from lead-201 by passing through a column of an ion-exchange resin. The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the declared thallium-201 radioactivity at the date and hour stated on the label. Not more than 2.0 per cent of the total radioactivity is due to thallium-202 and not less than 97.0 per cent is due to thallium-201. Not less than 95.0 per cent of the radioactivity is due to thallium in the form of thallous ions. The specific radioactivity is not less than 3.7 GBq per milligram of thallium. CHARACTERS A clear, colourless solution. Thallium-201 has a half-life of 3.05 days and emits gamma radiation and X-rays.

layer is not more intensely coloured than the toluene layer of a standard prepared at the same time in the same manner using 0.5 ml of thallium standard solution (10 ppm Tl) R. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. RADIONUCLIDIC PURITY Record the gamma-ray and X-ray spectrum using a suitable instrument calibrated with the aid of standardised thallium-201 and thallium-202 solutions. The spectrum does not differ significantly from that of the standardised thallium-201 solution. Determine the relative amounts of thallium-201 and thallium-202 and other radionuclidic impurities present. Thallium-202 has a half-life of 12.2 days and its most prominent gamma photon has an energy of 0.440 MeV. Thallium-200 has a half-life of 1.09 days and its most prominent gamma photons have energies of 0.368 MeV, 0.579 MeV, 0.828 MeV and 1.206 MeV. Lead-201 has a half-life of 9.4 h and its most prominent gamma photon has an energy of 0.331 MeV. Lead-203 has a half-life of 2.17 days and its most prominent gamma photon has an energy of 0.279 MeV. Not more than 2.0 per cent of the total radioactivity is due to thallium-202 and not less than 97.0 per cent is due to thallium-201. RADIOCHEMICAL PURITY Examine by zone electrophoresis (2.2.31), using a suitable strip of cellulose acetate, as the support and a 18.6 g/l solution of sodium edetate R as the electrolyte solution. Soak the strip in the electrolyte solution for 45-60 min. Remove the strip with forceps taking care to handle the outer edges only. Place the strip between 2 absorbent pads and blot to remove excess solution. Test solution. Mix equal volumes of the injection to be examined and the electrolyte solution. Apply not less than 5 µl of the test solution to the centre of the strip and mark the point of application. Apply an electric field of 17 V/cm for at least 10 min. Allow the strip to dry in air. Determine the distribution of radioactivity using suitable equipment. Not less than 95.0 per cent of the radioactivity migrates towards the cathode.

RADIOACTIVITY Measure the radioactivity using suitable counting equipment by comparison with a standardised thallium-201 solution or IDENTIFICATION A. Record the gamma-ray and X-ray spectrum using a suitable by measurement in an instrument calibrated with the aid of such a solution. instrument. The spectrum does not differ significantly from that of a standardised thallium-201 solution when measured either by direct comparison or by use of an 01/2005:0112 instrument calibrated with the aid of such a solution. Standardised thallium-201 and thallium-202 solutions are available from laboratories recognised by the competent TRITIATED (3H) WATER INJECTION authority. The most prominent gamma photons have energies of 0.135 MeV, 0.166 MeV and 0.167 MeV. The Aquae tritiatae (3H) solutio iniectabilis X-rays have energies of 0.069 MeV to 0.083 MeV. DEFINITION B. Examine the electropherogram obtained in the test for radiochemical purity. The distribution of radioactivity Tritiated (3H) water injection is water for injections in which contributes to the identification of the preparation. some of the water molecules contain tritium atoms in place of protium atoms. It may be made isotonic by the addition TESTS of sodium chloride. Tritium (3H) may be obtained by the pH (2.2.3). The pH of the injection is 4.0 to 7.0. neutron irradiation of lithium. The injection contains not less than 90.0 per cent and not more than 110.0 per cent of Thallium. To 0.5 ml of the injection add 0.5 ml of the declared tritium activity at the date stated on the label. hydrochloric acid (220 g/l HCl) and 0.05 ml of bromine water R and mix. Add 0.1 ml of a 30 g/l solution of CHARACTERS sulphosalicylic acid R. After decolorisation add 1.0 ml of A clear, colourless liquid. a 1 g/l solution of rhodamine B R. Add 4 ml of toluene R and shake for 60 s. Separate the toluene layer. The toluene Tritium has a half-life of 12.3 years and emits beta radiation. General Notices (1) apply to all monographs and other texts

867

Water (15O) injection


IDENTIFICATION Record the beta-ray spectrum by the method prescribed in the test for radionuclidic purity. The spectrum does not differ significantly from that of a standardised tritiated (3H) water. Standardised tritiated (3H) water is available from laboratories recognised by the competent authority. The maximum energy of the beta radiation is 0.019 MeV. TESTS pH (2.2.3). The pH of the injection is 4.5 to 7.0. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). RADIONUCLIDIC PURITY (a) Mix 100 µl of a suitable dilution of the injection with 10 ml of a scintillation liquid consisting of 1000 ml of dioxan R, 100 g of naphthalene R, 7 g of diphenyloxazole R and 0.3 g of methylphenyloxazolylbenzene R, the reagents being of an analytical grade suitable for liquid scintillation. Measure the radioactivity of the mixture in a liquid scintillation counter fitted with a discriminator. The count should be about 5000 impulses per second at the lowest setting of the discriminator. Record the count at different discriminator settings. For each measurement count at least 10 000 impulses over a period of at least 1 min. Immediately determine in the same conditions the count for a standardised tritiated (3H) water having approximately the same activity. Plot the counts at each discriminator setting, correcting for background activity, on semi-logarithmic paper, the discriminator settings being in arbitrary units as the abscissae.The vertical distance between the two curves obtained is constant. They obey the mathematical relationship :

A1

=

B1

=

A2

=

B2

=

radioactivity recorded for the standardised preparation at the lowest discriminator setting, radioactivity recorded for the preparation to be examined at the lowest discriminator setting, radioactivity recorded for the standard at the , discriminator setting such that radioactivity recorded for the preparation to be examined at the latter discriminator setting.

(b) Record the gamma-ray spectrum. The instrument registers only background activity. RADIOCHEMICAL PURITY Place a quantity of the injection equivalent to about 2 µCi (74 kBq), diluted to 50 ml with water R, in an all-glass distillation apparatus of the type used for the determination of distillation range (2.2.11). Determine the radioactive concentration. Distil until about 25 ml of distillate has been collected. Precautions must be taken to avoid contamination of the air. If the test is carried out in a fume cupboard, the equipment must be protected from draughts. Determine the radioactive concentration of the distillate and of the liquid remaining in the distillation flask. Neither of the radioactive concentrations determined after distillation differs by more than 5 per cent from the value determined before distillation. RADIOACTIVITY Determine the radioactivity using a liquid scintillation counter. 868

01/2005:1582

WATER (15O) INJECTION Aquae (15O) solutio iniectabilis DEFINITION Water (15O) injection is a sterile solution of [15O]water for diagnostic use. The injection contains not less than 90.0 per cent and not more than 110.0 per cent of the declared oxygen-15 radioactivity at the date and time stated on the label. Not less than 99 per cent of the total radioactivity corresponds to oxygen-15 in the form of water. PRODUCTION RADIONUCLIDE PRODUCTION Oxygen-15 is a radioactive isotope of oxygen which may be produced by various nuclear reactions such as proton irradiation of nitrogen-15 or deuteron irradiation of nitrogen-14. RADIOCHEMICAL SYNTHESIS In order to recover oxygen-15 as molecular oxygen from the nitrogen target gas, carrier oxygen is added at concentrations generally ranging from 0.2 per cent V/V to 1.0 per cent V/V. [15O]Water can be prepared from [15O]oxygen by reaction with hydrogen using a suitable catalyst. An alternative method is to produce [15O]water “in-target” by adding hydrogen to the irradiated target gas at a concentration generally ranging from 2 per cent V/V to 5 per cent V/V. The [15O]water vapour contained in the gas-stream is either bubbled through a reservoir of a sterile 9 g/l solution of sodium chloride, or is exchanged by diffusion into such a solution through a membrane filter for dialysis. Ammonia is a possible chemical impurity in [15O]water. This may arise either from catalytic conversion of hydrogen and nitrogen on the catalyst or by radiolysis if “in-target” production is used. In addition, there is the possibility of contamination by oxygen-15-labelled oxides of nitrogen. Although these contaminants can be effectively removed from the gas phase by soda lime and charcoal adsorbers, they may break through and be present in the final preparation. CHARACTERS A clear, colourless solution. Oxygen-15 has a half-life of 2.04 min and emits positrons with a maximum energy of 1.732 MeV, followed by annihilation gamma radiation of 0.511 MeV. IDENTIFICATION A. Record the gamma-ray spectrum using a suitable instrument. The only gamma photons have an energy of 0.511 MeV and, depending on the measurement geometry, a sum peak of 1.022 MeV may be observed. B. It complies with the test for radionuclidic purity (see Tests). C. Examine the chromatogram obtained in the test for radiochemical purity. The retention time of the second peak is due to the radioactivity eluting in the void volume. TESTS pH (2.2.3). The pH of the injection is 5.5 to 8.5. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test.



Bacterial endotoxins (2.6.14) : less than 175/V IU/ml, V being the maximum administered volume in millilitres. The injection may be released for use before completion of the test. CHEMICAL PURITY (a) Ammonium (2.4.1). 1 ml complies with the limit test for ammonium (10 ppm). (b) Nitrates. To 1 ml add 49 ml of nitrate-free water R. Place 5 ml of this solution in a test-tube immersed in iced water, add 0.4 ml of a 100 g/l solution of potassium chloride R, 0.1 ml of diphenylamine solution R and, dropwise with shaking, 5 ml of sulphuric acid R. Transfer the tube to a water-bath at 50 °C. After 15 min, any blue colour in the solution is not more intense than that in a standard prepared at the same time in the same manner using a mixture of 4.5 ml of nitrate-free water R and 0.5 ml of nitrate standard solution (2 ppm NO3) R (10 ppm). The injection may be released for use before completion of tests (a) and (b). RADIONUCLIDIC PURITY Record the gamma-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised fluorine-18 solution. Standardised fluorine-18 solutions are available from the laboratories recognised by the competent authority. The half-life is between 1.9 min and 2.2 min. Not less than 99 per cent of total radioactivity corresponds to oxygen-15. The injection may be released for use before completion of the test. RADIOCHEMICAL PURITY Examine by liquid chromatography (2.2.29). Test solution. The preparation to be examined. The chromatographic procedure may be carried out using : — a column 0.25 m long and 4.0 mm in internal diameter packed with aminopropylsilyl silica gel for chromatography R (10 µm), — as mobile phase at a flow rate of 1 ml/min a 10 g/l solution of potassium dihydrogen phosphate R adjusted to pH 3 with phosphoric acid R, — a suitable radioactivity detector, — a loop injector, — an internal recovery detection system, consisting of a loop of the chromatographic tubing between the injector and the column through the radioactivity detector, which has been calibrated for count recovery, maintaining the column at a constant temperature between 20 °C and 30 °C. Inject the test solution. Continue the chromatography for 10 min. In the chromatogram obtained, the first peak corresponds to the injected radioactivity of the test solution, the second peak corresponds to the amount of radioactivity as [15O]water. Calculate the percentage content of [15O]water from the areas of the peaks in the chromatogram obtained with the test solution. Not less than 99 per cent of the total radioactivity injected corresponds to oxygen-15 in the form of water. The injection may be released for use before completion of the test. RADIOACTIVITY Measure the radioactivity using suitable equipment by comparison with a standardised fluorine-18 solution or by using an instrument calibrated with the aid of such a solution. General Notices (1) apply to all monographs and other texts

Xenon (133Xe) injection

01/2005:0133

XENON (133Xe) INJECTION Xenoni (133Xe) solutio iniectabilis DEFINITION Xenon (133Xe) injection is a sterile solution of xenon-133 that may be made isotonic by the addition of sodium chloride. Xenon-133 is a radioactive isotope of xenon and is obtained by separation from the other products of uranium fission. The injection contains not less than 80 per cent and not more than 130 per cent of the declared xenon-133 radioactivity at the date and hour stated on the label. The injection is presented in a container that allows the contents to be removed without introducing air bubbles. The container is filled as completely as possible and any gas bubble present does not occupy more than 1 per cent of the volume of the injection as judged by visual comparison with a suitable standard. CHARACTERS A clear, colourless solution. Xenon-133 has a half-life of 5.29 days and emits beta and gamma radiation and X-rays. IDENTIFICATION Record the gamma-ray and X-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised xenon-133 solution in a 9 g/l solution of sodium chloride R, apart from any differences attributable to the presence of xenon-131m and xenon-133m. If standardised xenon-133 solutions are not readily available, suitable standardised ionisation chambers are obtainable from laboratories recognised by the relevant competent authority. The most prominent gamma photon of xenon-133 has an energy of 0.081 MeV and there is an X-ray (resulting from internal conversion) of 0.030 MeV to 0.035 MeV. Xenon-131m has a half-life of 11.9 days and emits a gamma photon of 0.164 MeV. Xenon-133m has a half-life of 2.19 days and emits a gamma photon of 0.233 MeV. TESTS pH (2.2.3). The pH of the injection is 5.0 to 8.0. Sterility. It complies with the test for sterility prescribed in the monograph on Radiopharmaceutical preparations (0125). The injection may be released for use before completion of the test. RADIONUCLIDIC PURITY (a) Record the gamma-ray and X-ray spectrum using a suitable instrument. The spectrum does not differ significantly from that of a standardised xenon-133 solution in a 9 g/l solution of sodium chloride R, apart from any differences attributable to the presence of xenon-131m and xenon-133m. (b) Transfer 2 ml of the injection to an open flask and pass a current of air through the solution for 30 min, taking suitable precautions concerning the dispersion of radioactivity. Measure the residual beta and gamma activity of the solution. The activity does not differ significantly from the background activity detected by the instrument. RADIOACTIVITY Weigh the container with its contents. Determine its total radioactivity using suitable counting equipment by comparison with a standardised xenon-133 solution or by measurement in an instrument calibrated with the aid of such a solution, operating in strictly identical conditions. If an ionisation chamber is used its inner wall should be such 869

Xenon (133Xe) injection


that the radiation is not seriously attenuated. Remove at least half the contents and re-weigh the container. Measure the radioactivity of the container and the remaining contents as described above. From the measurements, calculate the radioactive concentration of xenon-133 in the injection.

870

CAUTION Significant amounts of xenon-133 may be present in the closures and on the walls of the container. This must be taken into account in applying the rules concerning the transport and storage of radioactive substances and in disposing of used containers


Catgut, sterile


01/2005:90004 TESTS If stored in a preserving liquid, remove the sutures from INTRODUCTION the sachet and measure promptly and in succession the length, diameter and breaking load. If stored in the dry The following monographs apply to sutures for state, immerse the sutures in alcohol R or a 90 per cent V/V human use : Catgut, sterile (0317), Sutures, sterile solution of 2-propanol R for 24 h and proceed with the non-absorbable (0324), Sutures, sterile synthetic measurements as indicated below. absorbable braided (0667) and Sutures, sterile synthetic absorbable monofilament (0666). They cover performance Length. Measure the length without applying to the suture more tension than is necessary to keep it straight. The characteristics of sutures and may include methods of length of each suture is not less than 90 per cent of the identification. Sutures are medical devices as defined in length stated on the label and does not exceed 350 cm. Directive 93/42/EEC. These monographs can be applied to show compliance Diameter. Carry out the test on 5 sutures. Use a suitable with essential requirements as defined in Article 3 of instrument capable of measuring with an accuracy of at Directive 93/42/EEC covering the following : least 0.002 mm and having a circular pressor foot 10 mm Physical performance characteristics : diameter, breaking to 15 mm in diameter. The pressor foot and the moving load, needle attachment, packaging, sterility, information parts attached to it are weighted so as to apply a total load supplied by the manufacturer (see Section 13 of Annex 1 of of 100 ± 10 g to the suture being tested. When making the measurement, lower the pressor foot slowly to avoid Directive 93/42/EEC), labelling. crushing the suture. Measure the diameter at intervals of To show compliance with other essential requirements, 30 cm over the whole length of the suture. For a suture less the application of appropriate harmonised standards than 90 cm in length, measure at 3 points approximately as defined in Article 5 of Directive 93/42/EEC may be evenly spaced along the suture. The suture is not subjected considered. to more tension than is necessary to keep it straight during 01/2005:0317 measurement. The average of the measurements carried out on the sutures being tested and not less than two-thirds of the measurements taken on each suture are within the limits CATGUT, STERILE given in the columns under A in Table 0317.-1 for the gauge number concerned. None of the measurements is outside Chorda resorbilis sterilis the limits given in the columns under B in Table 0317.-1 for the gauge number concerned. DEFINITION Sterile catgut consists of sutures prepared from collagen taken from the intestinal membranes of mammals. After cleaning, the membranes are split longitudinally into strips of varying width, which, when assembled in small numbers, according to the diameter required, are twisted under tension, dried, polished, selected and sterilised. The sutures may be treated with chemical substances such as chromium salts to prolong absorption and glycerol to make them supple, provided such substances do not reduce tissue acceptability. Appropriate harmonised standards may be considered when assessing compliance with respect to origin and processing of raw materials and with respect to biocompatibility. Sterile catgut is a surgical wound-closure device. Being an absorbable suture it serves to approximate tissue during the healing period and is subsequently metabolised by proteolytic activity.

Table 0317.-1.— Diameters and Breaking Loads Diameter (millimetres)

Gauge number

A

Breaking load (newtons) B

C

D

0.025

-

-

0.015

0.035

-

-

0.039

0.025

0.045

0.20

0.05

0.040

0.049

0.035

0.060

0.30

0.10

0.5

0.050

0.069

0.045

0.085

0.40

0.20

0.7

0.070

0.099

0.060

0.125

0.70

0.30

1

0.100

0.149

0.085

0.175

1.8

0.40

1.5

0.150

0.199

0.125

0.225

3.8

0.70

2

0.200

0.249

0.175

0.275

7.5

1.8

2.5

0.250

0.299

0.225

0.325

10

3.8

3

0.300

0.349

0.275

0.375

12.5

7.5

3.5

0.350

0.399

0.325

0.450

20

10

4

0.400

0.499

0.375

0.550

27.5

12.5

5

0.500

0.599

0.450

0.650

38.0

20.0

6

0.600

0.699

0.550

0.750

45.0

27.5

7

0.700

0.799

0.650

0.850

60.0

38.0

8

0.800

0.899

0.750

0.950

70.0

45.0

min.

max.

min.

max.

0.1

0.010

0.019

0.005

0.2

0.020

0.029

0.3

0.030

0.4

PRODUCTION Production complies with relevant regulations on the use of animal tissues in medical devices notably concerning the risk of transmission of animal spongiform encephalopathy agents. Appropriate harmonised standards may apply with respect to appropriate validated methods of sterilisation, environmental control during manufacturing, labelling and packaging. It is essential for the effectiveness and the performance characteristics during use and during the functional lifetime of catgut that the following physical properties are specified : consistent diameter, sufficient initial strength and firm needle attachment. The requirements outlined below have been established, taking into account stresses which occur during normal conditions of use. These requirements can be used to demonstrate that individual production batches of sterile catgut are suitable for wound closure according to usual surgical techniques.

Minimum breaking load. The minimum breaking load is determined over a simple knot formed by placing one end of a suture held in the right hand over the other end held in the left hand, passing one end over the suture and through the loop so formed (see Figure 0317.-1) and pulling the knot tight. Carry out the test on 5 sutures. Submit sutures of length greater than 75 cm to 2 measurements and shorter


873

Sutures, sterile non-absorbable


sutures to one measurement. Determine the breaking load using a suitable tensilometer. The apparatus has 2 clamps for holding the suture, one of which is mobile and is driven at a constant rate of 30 cm/min. The clamps are designed so that the suture being tested can be attached without any possibility of slipping. At the beginning of the test the length of suture between the clamps is 12.5 cm to 20 cm and the knot is midway between the clamps. Set the mobile clamp in motion and note the force required to break the suture. If the suture breaks in a clamp or within 1 cm of it, the result is discarded and the test repeated on another suture. The average of all the results, excluding those legitimately discarded, is equal to or greater than the value given in column C in Table 0317.-1 and no individual result is less than that given in column D for the gauge number concerned.

Sutures in their individual sachets (primary packaging) are kept in a protective cover (box) which maintains the physical and mechanical properties until the time of use. The application of appropriate harmonised standards for packaging of medical devices shall be considered. Table 0317.-2. – Minimum Strengths of Needle Attachment Gauge number

Mean value (newtons)

Individual values (newtons)

0.5

0.50

0.25

0.7

0.80

0.40

1

1.7

0.80

1.5

2.3

1.1

2

4.5

2.3

2.5

5.6

2.8

3

6.8

3.4

3.5

11.0

4.5

4

15.0

4.5

5

18.0

6.0

LABELLING Reference may be made to the appropriate harmonised standards for labelling of medical devices. The details strictly necessary for the user to identify the product properly are indicated on or in each sachet (primary packaging) and on the protective cover (box) and include at least : Figure 0317.-1. – Simple knot — gauge number, Soluble chromium compounds. Place 0.25 g in a conical — length in centimetres or metres, flask containing 1 ml of water R per 10 mg of catgut. Stopper — if appropriate, that the needle is detachable, the flask, allow to stand at 37 ± 0.5 °C for 24 h, cool and decant the liquid. Transfer 5 ml to a small test tube and add — name of the product, 2 ml of a 10 g/l solution of diphenylcarbazide R in alcohol R — intended use (surgical suture, absorbable). and 2 ml of dilute sulphuric acid R. The solution is not more intensely coloured than a standard prepared at the same time 01/2005:0324 using 5 ml of a solution containing 2.83 µg of potassium dichromate R per millilitre, 2 ml of dilute sulphuric acid R SUTURES, STERILE and 2 ml of a 10 g/l solution of diphenylcarbazide R in NON-ABSORBABLE alcohol R (1 ppm of Cr). Needle attachment. If the catgut is supplied with an eyeless Fila non resorbilia sterilia needle attached that is not stated to be detachable, it complies with the test for needle attachment. Carry out DEFINITION the test on 5 sutures. Use a suitable tensilometer, such Sterile non-absorbable sutures are sutures which, when as that described for the determination of the minimum introduced into a living organism, are not metabolised by breaking load. Fix the needle and suture (without knot) in that organism. Sterile non-absorbable sutures vary in origin, the clamps of the apparatus in such a way that the swaged which may be animal, vegetable, metallic or synthetic. They part of the needle is completely free of the clamp and in occur as cylindrical monofilaments or as multifilament line with the direction of pull on the suture. Set the mobile sutures consisting of elementary fibres which are assembled clamp in motion and note the force required to break the by twisting, cabling or braiding ; they may be sheathed ; they suture or to detach it from the needle. The average of the may be treated to render them non-capillary, and they may 5 determinations and all individual values are not less than be coloured. the respective values given in Table 0317.-2 for the gauge Appropriate harmonised standards may be considered when number concerned. If not more than one individual value assessing compliance with respect to origin and processing fails to meet the individual requirement, repeat the test on an additional 10 sutures. The catgut complies with the test if of raw materials and with respect to biocompatibility. Sterile non-absorbable surgical sutures serve to approximate none of these 10 values is less than the individual value in tissue during the healing period and provide continuing Table 0317.-2 for the gauge number concerned. wound support. Commonly used materials include the following : STORAGE (PACKAGING) Silk (Filum bombycis) Sterile catgut sutures are presented in individual sachets that maintain sterility and allow the withdrawal and use Sterile braided silk suture is obtained by braiding a number of the sutures in aseptic conditions. Sterile catgut may be of threads, according to the diameter required, of degummed stored dry or in a preserving liquid to which an antimicrobial silk obtained from the cocoons of the silkworm Bombyx agent but not an antibiotic may be added. mori L. 874



Linen (Filum lini) Sterile linen thread consists of the pericyclic fibres of the stem of Linum usitatissimum L. The elementary fibres, 2.5 cm to 5 cm long, are assembled in bundles 30 cm to 80 cm long and spun into continuous lengths of suitable diameter. Poly(ethylene terephthalate) (Filum ethyleni polyterephthalici) Sterile poly(ethylene terephthalate) suture is obtained by drawing poly(ethylene terephthalate) through a suitable die. The suture is prepared by braiding very fine filaments in suitable numbers, depending on the gauge required. Polyamide-6 (Filum polyamidicum-6) Sterile polyamide-6 suture is obtained by drawing through a suitable die a synthetic plastic material formed by the polymerisation of ε-caprolactam. It consists of smooth, cylindrical monofilaments or braided filaments, or lightly twisted sutures sheathed with the same material. Polyamide-6/6 (Filum polyamidicum-6/6) Sterile polyamide-6/6 suture is obtained by drawing through a suitable die a synthetic plastic material formed by the polycondensation of hexamethylenediamine and adipic acid. It consists of smooth, cylindrical monofilaments or braided filaments, or lightly twisted sutures sheathed with the same material. Polypropylene (Filum polypropylenicum) Polypropylene suture is obtained by drawing polypropylene through a suitable die. It consists of smooth cylindrical mono-filaments. Monofilament and multifilament stainless steel (Filum aciei irrubiginibilis monofilamentum/multifilamentum) Sterile stainless steel sutures have a chemical composition as specified in ISO 5832-1 - Metallic Materials for surgical implants - Part 1 : Specification for wrought stainless steel and comply with ISO 10334 - Implants for surgery - Malleable wires for use as sutures and other surgical applications. Stainless steel sutures consist of smooth, cylindrical monofilaments or twisted filaments or braided filaments. Poly(vinylidene difluoride) (PVDF) (Filum poly(vinylideni difluoridum)) Sterile PVDF suture is obtained by drawing through a suitable die a synthetic plastic material which is formed by polymerisation of 1,1-difluorethylene. It consists of smooth cylindrical monofilaments. IDENTIFICATION Non-absorbable sutures may be identified by chemical tests. Materials from natural origin may also be identified by microscopic examination of the morphology of these fibres. For synthetic materials, identification by infrared spectrophotometry (2.2.24) or by differential scanning calorimetry may be applied. Identification of silk A. Dissect the end of a suture, using a needle or fine tweezers, to isolate a few individual fibres. The fibres are sometimes marked with very fine longitudinal striations parallel to the axis of the suture. Examined under a microscope, a cross-section is more or less triangular to semi-circular, with rounded edges and without a lumen. B. Impregnate isolated fibres with iodinated potassium iodide solution R. The fibres are coloured pale yellow. Identification of linen A. Dissect the end of a suture, using a needle or fine tweezers, to isolate a few individual fibres. Examined under a microscope, the fibres are seen to be 12 µm General Notices (1) apply to all monographs and other texts


to 31 µm wide and, along the greater part of their length, have thick walls, sometimes marked with fine longitudinal striations, and a narrow lumen. The fibres gradually narrow to a long, fine point. Sometimes there are unilateral swellings with transverse lines. B. Impregnate isolated fibres with iodinated zinc chloride solution R. The fibres are coloured violet-blue. Identification of poly(ethyleneterephthalate) It is practically insoluble in most of the usual organic solvents, but is attacked by strong alkaline solutions. It is incompatible with phenols. A. 50 mg dissolves with difficulty when heated in 50 ml of dimethylformamide R. B. To about 50 mg add 10 ml of hydrochloric acid R1. The material remains intact even after immersion for 6 h. Identification of polyamide-6 It is practically insoluble in the usual organic solvents ; it is not attacked by dilute alkaline solutions (for example a 100 g/l solution of sodium hydroxide R) but is attacked by dilute mineral acids (for example a 20 g/l solution of sulphuric acid R), by hot glacial acetic acid R and by a 70 per cent m/m solution of anhydrous formic acid R. A. Heat about 50 mg with 0.5 ml of hydrochloric acid R1 in a sealed glass tube at 110 °C for 18 h and allow to stand for 6 h. No crystals appear. B. 50 mg dissolves in 20 ml of a 70 per cent m/m solution of anhydrous formic acid R. Identification of polyamide-6/6 It is practically insoluble in the usual organic solvents ; it is not attacked by dilute alkaline solutions (for example a 100 g/l solution of sodium hydroxide R) but is attacked by dilute mineral acids (for example a 20 g/l solution of sulphuric acid R), by hot glacial acetic acid R and by an 80 per cent m/m solution of anhydrous formic acid R. A. In contact with a flame it melts and burns, forming a hard globule of residue and gives off a characteristic odour resembling that of celery. B. Place about 50 mg in an ignition tube held vertically and heat gently until thick fumes are evolved. When the fumes fill the tube, withdraw it from the flame and insert a strip of nitrobenzaldehyde paper R. A violet-brown colour slowly appears on the paper and fades slowly in air ; it disappears almost immediately on washing with dilute sulphuric acid R. C. To about 50 mg add 10 ml of hydrochloride acid R1. The material disintegrates in the cold and dissolves within a few minutes. D. 50 mg does not dissolve in 20 ml of a 70 per cent m/m solution of anhydrous formic acid R but dissolves in 20 ml of an 80 per cent m/m solution of anhydrous formic acid R. Identification of polypropylene Polypropylene is soluble in decahydronaphthalene, 1-chloronaphthalene and trichloroethylene. It is not soluble in alcohol, in ether and in cyclohexanone. A. It softens at temperatures between 160 °C and 170 °C. It burns with a blue flame giving off an odour of burning paraffin wax and of octyl alcohol. B. To 0.25 g add 10 ml of toluene R and boil under a reflux condenser for about 15 min. Place a few drops of the solution on a disc of sodium chloride R slide and evaporate the solvent in an oven at 80 °C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with polypropylene CRS. 875



making the measurements, lower the pressor foot slowly to avoid crushing the suture. Measure the diameter at intervals of 30 cm over the whole length of the suture. For a suture less than 90 cm in length, measure at 3 points approximately evenly spaced along the suture. During the measurement submit monofilament sutures to a tension not greater than that required to keep them straight. Submit multifilament sutures to a tension not greater than one-fifth of the minimum breaking load shown in column C of Table 0324.-1 appropriate to the gauge number and type of material concerned or 10 N whichever is less. Stainless steel sutures do not require tension to be applied during the measurement of diameter. For multifilament sutures of gauge number above 1.5 make 2 measurements at each point, the second measurement being made after rotating the suture through 90°. The diameter of that point is the average of the 2 measurements. The average of the measurements carried out on the sutures being tested and not less than two-thirds of the measurements taken on each suture are within the limits given in the column under A in Table 0324.-1 for the gauge number concerned. None of the measurements are outside the limits given in the columns under B in Table 0324.-1 for the gauge number concerned. Minimum breaking load. Unless otherwise prescribed, determine the minimum breaking load by the following method using sutures in the condition in which they are presented. The minimum breaking load is determined over a simple knot formed by placing one end of a suture held in the right hand over the other end held in the left hand, passing one end over the suture and through the loop so formed PRODUCTION (see Figure 0324.-1) and pulling the knot tight. For stainless The appropriate harmonised standards may apply with steel sutures gauges 3.5 and above, the minimum breaking respect to appropriate validated methods of sterilisation, load is determined on a straight pull. Carry out the test on environmental control during manufacturing, labelling and 5 sutures. Submit sutures of length greater than 75 cm to packaging. 2 measurements and shorter sutures to 1 measurement. It is essential for the effectiveness and the performance Determine the breaking load using a suitable tensilometer. characteristics during use and during the functional lifetime The apparatus has 2 clamps for holding the suture, 1 of of these sutures that the following physical properties are which is mobile and is driven at a constant rate of 30 cm/min. specified : consistent diameter, sufficient initial strength and The clamps are designed so that the suture being tested firm needle attachment. can be attached without any possibility of slipping. At the beginning of the test the length of suture between the The requirements below have been established, taking into clamps is 12.5 cm to 20 cm and the knot is midway between account stresses which occur during normal conditions the clamps. Set the mobile clamp in motion and note the of use. These requirements can be used to demonstrate force required to break the suture. If the suture breaks in a that individual production batches of these sutures are suitable for wound closure in accordance with usual surgical clamp or within 1 cm of it, the result is discarded and the test repeated on another suture. The average of all the results, techniques. excluding those legitimately discarded, is equal to or greater TESTS than the value given in column C in Table 0324.-1 and no Remove the sutures from the sachet and measure promptly value is less than that given in column D for the gauge and in succession the length, diameter and minimum load. number and type of material concerned. If linen is tested the sutures are conditioned as follows : if stored in the dry state, expose to an atmosphere with a relative humidity of 65 ± 5 per cent at 20 ± 2 °C for 4 h immediately before measuring the diameter and for the determination of minimum breaking load immerse in water R at room temperature for 30 min immediately before carrying out the test. Length. Measure the length without applying more tension than is necessary to keep them straight. The length of the suture is not less than 95 per cent of the length stated on the label and does not exceed 400 cm. Diameter. Unless otherwise prescribed, measure the diameter by the following method using 5 sutures. Use a suitable mechanical instrument capable of measuring with Figure 0324.-1. – Simple knot an accuracy of at least 0.002 mm and having a circular pressor foot 10-15 mm in diameter. The pressor foot and the Needle attachment. If the sutures are supplied with an moving parts attached to it are weighted so as to apply a eyeless needle attached that is not stated to be detachable, total load of 100 ± 10 g to the suture being tested. When they comply with the test for needle attachment. Carry out

C. To 2 g add 100 ml of water R and boil under a reflux condenser for 2 h. Allow to cool. The relative density (2.2.5) of the material is 0.89 g/ml to 0.91 g/ml, determined using a hydrostatic balance. Identification of stainless steel Stainless steel sutures are identified by confirming that the composition is in accordance with ISO 5832 Part 1. Identification of poly(vinylidene difluoride) It is soluble in warm dimethylformamide. It is insoluble in ethanol, hot and cold isopropyl alcohol, ethyl acetate, tetrachlorethylene. A. The strand melts between 170 °C and 180 °C. It melts in a flame and does not burn after removal of the flame. Place a small piece of suture on an annealed copper wire or sheet. Heat in an oxidising flame. No green colour is produced. B. Dissolve 0.25 g of the suture in 10 ml of dimethylformamide R and boil under a reflux condenser for about 15 min. Place a few drops of the solution on a sodium chloride R slide and evaporate the solvent in an oven at 80 °C (1 h). Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of poly(vinylidene difluoride). C. To 2 g of suture add 100 ml of water R and boil under a reflux condenser for 2 h. Allow to cool. The relative density (2.2.5) of the material is 1.71 to 1.78.

876




Table 0324.-1. – Diameters and minimum breaking loads Diameter (millimetres) A

Gauge number

Minimum breaking load (newtons) B

All other non-absorbable strands

Linen thread

min.

max.

min.

max.

0.05

0.005

0.009

0.003

0.012

C -

D -

0.01

D -

0.1

0.010

0.019

0.005

0.025

-

-

0.03

-

0.025

-

-

0.06

0.01

-

0.1

-

0.15

0.015

0.019

0.012

C

Stainless steel D

C

0.2

0.020

0.029

0.015

0.035

-

0.3

0.030

0.039

0.025

0.045

-

-

0.35

0.06

0.4

0.040

0.049

0.035

0.060

-

-

0.60

0.15

1.1

0.045

0.085

-

-

1.0

0.35

1.6

0.5

0.050

0.069

0.7

0.070

0.099

0.060

0.125

1.0

0.3

1.5

0.60

2.7

1

0.100

0.149

0.085

0.175

2.5

0.6

3.0

1.0

5.3

4.0

1.5

0.150

0.199

0.125

0.225

5.0

1.0

5.0

1.5

8.0

6.0

2

0.200

0.249

0.175

0.275

8.0

2.5

9.0

3.0

13.3

10.0

2.5

0.250

0.299

0.225

0.325

9.0

5.0

13.0

5.0

15.5

11.6

3

0.300

0.349

0.275

0.375

11.0

8.0

15.0

9.0

17.7

13.3

3.5

0.350

0.399

0.325

0.450

15.0

9.0

22.0

13.0

33.4

25.0

4

0.400

0.499

0.375

0.550

18,0

11.0

27.0

15.0

46.7

35.0

5

0.500

0.599

0.450

0.650

26.0

15.0

35.0

22.0

57.9

43.4

6

0.600

0.699

0,.550

0.750

37.0

18.0

50.0

27.0

89.4

67.0

7

0.700

0.799

0.650

0.850

50.0

26.0

62.0

35.0

111.8

83.9

8

0.800

0.899

0.750

0.950

65.0

37.0

73.0

50.0

133.4

100.1

9

0.900

0.999

0.850

1.050

156.0

117.0

10

1.000

1.099

0.950

1.150

178.5

133.9

the test on 5 sutures. Use a suitable tensilometer, such as that described for the determination of the minimum breaking load. Fix the needle and suture (without knot) in the clamps of the apparatus in such a way that the swaged part of the needle is completely free of the clamp and in line with the direction of pull on the suture. Set the mobile clamp in motion and note the force required to break the suture or to detach it from the needle. The average of the 5 determinations and all individual values are not less than the respective values given in Table 0324.-2 for the gauge number concerned. If not more than 1 individual value fails to meet the individual requirement, repeat the test on an additional 10 sutures. The attachment complies with the test if none of these 10 values is less than the individual value in Table 0324.-2 for the gauge number concerned. Extractable colour. Sutures that are dyed and intended to remain so during use comply with the test for extractable colour. Place 0.25 g of the suture to be examined in a conical flask, add 25.0 ml of water R and cover the mouth of the flask with a short-stemmed funnel. Boil for 15 min, cool and adjust to the original volume with water R. Depending on the colour of the suture, prepare the appropriate reference solution as described in Table 0324.-3 using the primary colour solutions (2.2.2). The test solution is not more intensely coloured than the appropriate reference solution. General Notices (1) apply to all monographs and other texts

Table 0324.-2. – Minimum strengths of needle attachment Gauge number


Individual value (newtons)

0.4

0.50

0.25

0.5

0.80

0.40

0.7

1.7

0.80

1

2.3

1.1

1.5

4.5

2.3

2

6.8

3.4

2.5

9.0

4.5

3

11.0

4.5

3.5

15.0

4.5

4

18.0

6.0

5

18.0

7.0

6

25.0

12.5

7

25.0

12.5

8

50.0

25

9

50.0

25

10

75.0

37.5

877

Sutures, sterile synthetic absorbable braided


Table 0324.-3. – Colour reference solutions Colour of strand

Composition of reference solution (parts by volume) Water R

Red primary solution

Yellow primary solution

Yellow-brown

0.2

1.2

Blue primary solution -

Pink-red

1.0

-

-

9.0

-

-

2.0

8.0

8.4

-

Green-blue

8.6

are absorbed by that organism and cause no undue tissue irritation. They consist of completely polymerised material. They occur as multifilament sutures consisting of elementary fibres which are assembled by braiding. The sutures may be treated to facilitate handling and they may be coloured. Appropriate harmonised standards may be considered when assessing compliance with respect to origin and processing of raw materials and with respect to biocompatibility.

Sterile synthetic absorbable braided sutures are woundclosure devices. Being absorbable they serve to approximate tissue during the healing period and subsequently lose Monomer and oligomers. Polyamide-6 suture additionally complies with the following test for monomer and oligomers. tensile strength by hydrolysis. In a continuous-extraction apparatus, treat 1.00 g with 30 ml of methanol R at a rate of at least 3 extractions per hour for 7 h. Evaporate the extract to dryness, dry the residue at PRODUCTION 110 °C for 10 min, allow to cool in a desiccator and weigh. The residue weighs not more than 20 mg (2 per cent). Appropriate harmonised standards may apply with respect to appropriate validated methods of sterilisation, environmental STORAGE (PACKAGING) control during manufacturing, labelling and packaging. Sterile non-absorbable sutures are presented in a suitable sachet that maintains sterility and allows the withdrawal and It is essential for the effectiveness and the performance use of a suture in aseptic conditions. They may be stored dry characteristics during use and during the functional lifetime or in a preserving liquid to which an antimicrobial agent but of these sutures that the following physical properties are no antibiotic may be added. specified : consistent diameter, sufficient initial strength and Sterile non-absorbable sutures are intended to be used only firm needle attachment. on the occasion when the sachet is first opened. The requirements below have been established, taking into Sutures in their individual sachets (primary packaging) are account stresses which occur during normal conditions of kept in a protective cover (box) which maintains the physical use. These requirements can be used to demonstrate that and mechanical properties until the time of use. individual production batches of these sutures are suitable The application of appropriate harmonised standards for for wound closure according to usual surgical techniques. packaging of medical devices shall be considered in addition. Violet

1.6

-

LABELLING Reference may be made to the appropriate harmonised standards for the labelling of medical devices. The details strictly necessary for the user to identify the product properly are indicated on or in each sachet (primary packaging) and on the protective cover (box) and include at least : — gauge number, — length, in centimetres or metres, — if appropriate, that the needle is detachable, — name of the product, — intended use (surgical suture, non-absorbable), — if appropriate, that the suture is coloured, — if appropriate, the structure (braided, monofilament, sheathed).

TESTS Carry out the following tests on the sutures in the state in which they are removed from the sachet. Length. Measure the length of the suture without applying more tension than is necessary to keep it straight. The length of each suture is not less than 95 per cent of the length stated on the label and does not exceed 400 cm.

Diameter. Unless otherwise prescribed, measure the diameter by the following method, using five sutures in the condition in which they are presented. Use a suitable instrument capable of measuring with an accuracy of at least 0.002 mm and having a circular pressor foot 10 mm to 15 mm in diameter. The pressor foot and the moving parts attached to it are weighted so as to apply a total load of 100 ± 10 g to the suture being tested. When making the measurements, lower the pressor foot slowly to avoid crushing the suture. Measure the diameter at intervals of 30 cm over the whole length of the suture. For a suture less than 90 cm in length, 01/2005:0667 measure at three points approximately evenly spaced along the suture. During the measurement, submit the sutures to a tension not greater than one-fifth of the minimum breaking SUTURES, STERILE SYNTHETIC load shown in column C of Table 0667.-1 appropriate to the gauge number and type of material or 10 N whichever ABSORBABLE BRAIDED is less. For sutures of gauge number above 1.5 make two measurements at each point, the second measurement being Fila resorbilia synthetica torta sterilia made after rotating the suture through 90°. The diameter of that point is the average of the two measurements. DEFINITION The average of the measurements carried out on the sutures being tested and not less than two-thirds of the Sterile synthetic absorbable braided sutures consist of measurements taken on each suture are within the limits sutures prepared from a synthetic polymer, polymers or copolymers which, when introduced into a living organism, given in the columns under A in Table 0667.-1 for the gauge 878


Sutures, sterile synthetic absorbable braided


number concerned. None of the measurements is outside the limits given in the columns under B in Table 0667.-1 for the gauge number concerned. Table 0667.-1. – Diameters and breaking loads Diameter (millimetres)

Gauge number

A


C

D

0.005

-

-

0.003

0.012

-

-

0.019

0.005

0.025

-

-

0.020

0.029

0.015

0.035

-

-

0.3

0.030

0.039

0.025

0.045

0.45

0.23

0.4

0.040

0.049

0.035

0.060

0.70

0.35

0.5

0.050

0.069

0.045

0.085

1.4

0.7

0.7

0.070

0.099

0.060

0.125

2.5

1.3

1

0.100

0.149

0.085

0.175

6.8

3.4

min.

max.

min.

max.

0.01

0.001

0.004

0.0008

0.05

0.005

0.009

0.1

0.010

0.2

1.5

0.150

0.199

0.125

0.225

9.5

4.8

2

0.200

0.249

0.175

0.275

17.7

8.9

2.5

0.250

0.299

0.225

0.325

21.0

10.5

3

0.300

0.349

0.275

0.375

26.8

13.4

3.5

0.350

0.399

0.325

0.450

39.0

4

0.400

0.499

0.375

0.550

50.8

5

0.500

0.599

0.450

0.650

63.5

0.750

-

0.850

-

6 7

0.600 0.700

0.699 0.799

0.550 0.650

1 cm of it, the result is discarded and the test repeated on another suture. The average of all the results excluding those legitimately discarded is equal to or greater than the value given in column C in Table 0667.-1 and no individual result is less than that given in column D for the gauge number concerned. Needle attachment. If the suture is supplied with an eyeless needle attached that is not stated to be detachable the attachment, it complies with the test for needle attachment. Carry out the test on five sutures. Use a suitable tensilometer, such as that described for the determination of the minimum breaking load. Fix the needle and suture (without knot) in the clamps of the apparatus in such a way that the swaged part of the needle is completely free of the clamp and in line with the direction of pull on the suture. Set the mobile clamp in motion and note the force required to break the suture or to detach it from the needle. The average of the five deter-minations and all individual values are not less than the respective values given in Table 0667.-2 for the gauge number concerned. If not more than one individual value fails to meet the individual requirement, repeat the test on an additional ten sutures. The attachment complies with the test if none of the ten values is less than the individual value in Table 0667.-2 for the gauge number concerned. Table 0667.-2. – Minimum strengths of needle attachment Gauge number



18.5

0.4

0.50

0.25

25.4

0.5

0.80

0.40

31.8

0.7

1.7

0.80

-

1

2.3

1.1

-

1.5

4.5

2.3

2

6.8

3.4

2.5

9.0

4.5

3

11.0

4.5

3.5

15.0

4.5

4

18.0

6.0

5

18.0

7.0

Minimum breaking load. The minimum breaking load is determined over a simple knot formed by placing one end of a suture held in the right hand over the other end held in the left hand, passing one end over the suture and through the loop so formed (see Figure 0667.-1) and pulling the knot tight.

Figure 0667.-1. – Simple knot Carry out the test on five sutures. Submit sutures of length greater than 75 cm to two measurements and shorter sutures to one measurement. Determine the breaking load using a suitable tensilometer. The apparatus has two clamps for holding the suture, one of which is mobile and is driven at a constant rate of 25 cm to 30 cm per minute. The clamps are designed so that the suture being tested can be attached without any possibility of slipping. At the beginning of the test the length of suture between the clamps is 12.5 cm to 20 cm and the knot is midway between the clamps. Set the mobile clamp in motion and note the force required to break the suture. If the suture breaks in a clamp or within General Notices (1) apply to all monographs and other texts

STORAGE (PACKAGING) Sterile synthetic absorbable braided sutures are presented in a suitable sachet that maintains sterility and allows the withdrawal and use of the sutures in aseptic conditions. The sutures must be stored dry. They are intended to be used only on the occasion when the sachet is first opened. Sutures in their individual sachets (primary packaging) are kept in a protective cover (box) which maintains the physical and mechanical properties until the time of use. The application of appropriate harmonised standards for packaging of medical devices may be considered in addition. LABELLING Reference may be made to the appropriate harmonised standards for the labelling of medical devices. The details strictly necessary for the user to identify the product properly are indicated on or in each sachet (primary packaging) and on the protective cover (box) and include at least : — gauge number, — length in centimetres or metres, — if appropriate, that the needle is detachable, 879

Sutures, sterile synthetic absorbable monofilament

— — — —


the suture. During the measurement, submit the sutures to a tension not greater than that required to keep them straight. The average of the measurements carried out on the sutures being tested and not less than two-thirds of the measurements taken on each suture are within the limits given in the columns under A in Table 0666.-1 for the gauge number concerned. None of the measurements is outside 01/2005:0666 the limits given in the columns under B in Table 0666.-1 for the gauge number concerned.

name of the product, intended use (surgical absorbable suture), if appropriate, that the suture is coloured, the structure (braided).

SUTURES, STERILE SYNTHETIC ABSORBABLE MONOFILAMENT Fila resorbilia synthetica monofilamenta sterilia DEFINITION Sterile synthetic absorbable monofilament sutures consist of sutures prepared from a synthetic polymer, polymers or copolymers which, when introduced into a living organism, are absorbed by that organism and cause no undue tissue irritation. They consist of completely polymerised material. They occur as monofilament sutures. The sutures may be treated to facilitate handling and they may be coloured. Appropriate harmonised standards may be considered when assessing compliance with respect to origin and processing of raw materials and with respect to biocompatibility. Sterile synthetic absorbable monofilament sutures are woundclosure devices. Being absorbable they serve to approximate tissue during the healing period and subsequently lose tensile strength by hydrolysis. PRODUCTION The appropriate harmonised standards may apply with respect to appropriate validated methods of sterilisation, environmental control during manufacturing, labelling and packaging. It is essential for the effectiveness and the performance characteristics during use and during the functional lifetime of these sutures that the following physical properties are specified : consistent diameter, sufficient initial strength and firm needle attachment. The requirements below have been established, taking into account stresses which occur during normal conditions of use. These requirements can be used to demonstrate that individual production batches of these sutures are suitable for wound closure according to usual surgical techniques. TESTS Carry out the following tests on the sutures in the state in which they are removed from the sachet. Length. Measure the length of the suture without applying more tension than is necessary to keep it straight. The length of each suture is not less than 95 per cent of the length stated on the label and does not exceed 400 cm. Diameter. Unless otherwise prescribed, measure the diameter by the following method, using five sutures in the condition in which they are presented. Use a suitable instrument cap-able of measuring with an accuracy of at least 0.002 mm and having a circular pressor foot 10 mm to 15 mm in diameter. The pressor foot and the moving parts attached to it are weighted so as to apply a total load of 100 ± 10 g to the suture being tested. When making the measurements, lower the pressor foot slowly to avoid crushing the suture. Measure the diameter at intervals of 30 cm over the whole length of the suture. For a suture less than 90 cm in length, measure at three points approximately evenly spaced along 880

Table 0666.-1. – Diameters and breaking loads Diameter (millimetres)

Gauge number

A


C

D

0.125

1.4

0.7

0.175

2.5

1.3

0.125

0.225

6.8

3.4

0.249

0.175

0.275

9.5

4.7

0.250

0.339

0.225

0.375

17.5

8.9

0.340

0.399

0.325

0.450

26.8

13.4

3.5

0.400

0.499

0.375

0.550

39.0

18.5

4

0.500

0.570

0.450

0.600

50.8

25.4

5

0.571

0.610

0.500

0.700

63.5

31.8

min.

max.

min.

max.

0.5

0.050

0.094

0.045

0.7

0.095

0.149

0.075

1

0.150

0.199

1.5

0.200

2 3

Minimum breaking load. The minimum breaking load is determined over a simple knot formed by placing one end of a suture held in the right hand over the other end held in the left hand, passing one end over the suture and through the loop so formed (see Figure 0666.-1) and pulling the knot tight.

Figure 0666.-1. – Simple knot Carry out the test on five sutures. Submit sutures of length greater than 75 cm to two measurements and shorter sutures to one measurement. Determine the breaking load using a suitable tensilometer. The apparatus has two clamps for holding the suture, one of which is mobile and is driven at a constant rate of 25 cm to 30 cm per minute. The clamps are designed so that the suture being tested can be attached without any possibility of slipping. At the beginning of the test the length of suture between the clamps is 12.5 cm to 20 cm and the knot is midway between the clamps. Set the mobile clamp in motion and note the force required to break the suture. If the suture breaks in a clamp or within 1 cm of it, the result is discarded and the test repeated on another suture. The average of all the results excluding those legitimately discarded is equal to or greater than the value given in column C in Table 0666.-1 and no individual result is less than that given in column D for the gauge number concerned.


Sutures, sterile synthetic absorbable monofilament


Needle attachment. If the suture is supplied with an eyeless needle attached that is not stated to be detachable, the attachment complies with the test for needle attachment. Carry out the test on five sutures. Use a suitable tensilometer, such as that described for the determination of the minimum breaking load. Fix the needle and suture (without knot) in the clamps of the apparatus in such a way that the swaged part of the needle is completely free of the clamp and in line with the direction of pull on the suture. Set the mobile clamp in motion and note the force required to break the suture or to detach it from the needle. The average of the five determinations and all individual values are not less than the respective values given in Table 0666.-2 for the gauge number concerned. If not more than one individual value fails to meet the individual requirement, repeat the test on an additional ten sutures. The attachment complies with the test if none of the ten values is less than the individual value in Table 0666.-2 for the gauge number concerned. Table 0666.-2. – Minimum strengths of needle attachment

STORAGE (PACKAGING) Sterile synthetic absorbable monofilament sutures are presented in a suitable sachet that maintains sterility and allows the withdrawal and use of the sutures in aseptic conditions. The sutures must be stored dry. They are intended to be used only on the occasion when the sachet is first opened. Sutures in their individual sachets (primary packaging) are kept in a protective cover (box) which maintains the physical and mechanical properties until the time of use. The application of appropriate harmonised standards for packaging of medical devices may be considered in addition. LABELLING Reference may be made to appropriate harmonised standards for the labelling of medical devices.

Gauge number



0.5

0.80

0.40

0.7

1.7

0.80

1

2.3

1.1

1.5

4.5

2.3

2

6.8

3.4

2.5

9.0

4.5

— if appropriate, that the needle is detachable,

3

11.0

4.5

— name of the product,

3.5

15.0

4.5

— intended use (surgical absorbable suture),

4

18.0

6.0

— if appropriate, that the suture is coloured,

5

18.0

7.0

— the structure (monofilament).


The details strictly necessary for the user to identify the product properly are indicated on or in each sachet (primary packaging) and on the protective cover (box) and include at least : — gauge number, — length in centimetres or metres,

881

Catgut, sterile, in distributor for veterinary use


01/2005:0660 concerned. None of the measurements is outside the limits given in the columns under B in Table 0660.-1 for the gauge number concerned.

CATGUT, STERILE, IN DISTRIBUTOR FOR VETERINARY USE

Table 0660.-1. – Diameters and breaking loads Diameter (millimetres)

Gauge number

Chorda resorbilis sterilis in fuso ad usum veterinarium DEFINITION Sterile catgut in distributor for veterinary use consists of strands prepared from collagen taken from the intestinal membranes of mammals. After cleaning, the membranes are split longitudinally into strips of varying width, which, when assembled in small numbers, according to the diameter required, are twisted under tension, dried, polished, selected and sterilised. The strands may be treated with chemical substances such as chromium salts to prolong absorption and glycerol to make them supple, provided such substances do not reduce tissue acceptability. The strand is presented in a distributor that allows the withdrawal and use of all or part of it in aseptic conditions. The design of the distributor is such that with suitable handling the sterility of the content is maintained even when part of the strand has been withdrawn. It may be stored dry or in a preserving liquid to which an antimicrobial preservative but not an antibiotic may be added.

A


C

D

0.175

1.8

0.4

0.225

3.8

0.7

0.175

0.275

7.5

1.8

0.225

0.325

10

3.8

0.275

0.375

12.5

7.5

0.399

0.325

0.450

20

10

0.400

0.499

0.375

0.550

27.5

12.5

0.500

0.599

0.450

0.650

38.4

20.0

6

0.600

0.699

0.550

0.750

45.0

27.5

7

0.700

0.799

0.650

0.850

60.0

38.0

8

0.800

0.899

0.750

0.950

70.0

45.0

min.

max.

min.

max.

1

0.100

0.149

0.085

1.5

0.150

0.199

0.125

2

0.200

0.249

2.5

0.250

0.299

3

0.300

0.349

3.5

0.350

4 5

Minimum breaking load. The minimum breaking load is determined over a simple knot formed by placing one end of a strand held in the right hand over the other end held in the left hand, passing one end over the strand and through the loop so formed (see Figure 0660.-1) and pulling the knot tight.

TESTS If stored in a preserving liquid, remove the strand from the distributor and measure promptly and in succession the length, diameter and breaking load. If stored in the dry state, immerse the strand in alcohol R or a 90 per cent V/V solution of 2-propanol R for 24 h and proceed with the measurements as indicated above. Length. Measure the length without applying to the strand more tension than is necessary to keep it straight. The length is not less than 95 per cent of the length stated on the label. If the strand consists of several sections joined by knots, the length of each section is not less than 2.5 m.

Figure 0660.-1. – Simple knot Make not fewer than one measurement per 2 m of length. If the strand consists of several sections joined by knots, make Diameter. Carry out the test using a suitable instrument not fewer than three measurements per section and, in any capable of measuring with an accuracy of at least 0.002 mm case, not fewer than one measurement per 2 m of length at and having a circular pressor foot 10 mm to 15 mm in points evenly spaced along the strand or along each section. diameter. The pressor foot and the moving parts attached to Determine the breaking load using a suitable tensilometer. it are weighted so as to apply a total load of 100 ± 10 g to The apparatus has two clamps for holding the strand, one of the strand being tested. When making the measurements, which is mobile and is driven at a constant rate of 30 cm per lower the pressor foot slowly to avoid crushing the strand. minute. The clamps are designed so that the strand being Make not fewer than one measurement per 2 m of length. tested can be attached without any possibility of slipping. At If the strand consists of several sections joined by knots, the beginning of the test the length of strand between the make not fewer than three measurements per section. clamps is 12.5 cm to 20 cm and the knot is midway between In any case make not fewer than twelve measurements. the clamps. Set the mobile clamp in motion and note the Make the measurements at points evenly spaced along the force required to break the strand. If the strand breaks in a strand or along each section. The strand is not subjected to clamp or within 1 cm of it, the result is discarded and the more tension than is necessary to keep it straight during test repeated on another part of the strand. The average measurement. The average of the measurements carried out of all the results, excluding those legitimately discarded, is on the strand being tested and not less than two-thirds of equal to or greater than the value in column C and no value the individual measurements are within the limits given in is less than that given in column D in Table 0660.-1 for the the column under A in Table 0660.-1 for the gauge number gauge number concerned. General Notices (1) apply to all monographs and other texts

885

Linen thread, sterile, in distributor for veterinary use


Soluble chromium compounds. Place 0.25 g in a conical flask containing 1 ml of water R per 10 mg of catgut. Stopper the flask, allow to stand at 37 ± 0.5 °C for 24 h, cool and decant the liquid. Transfer 5 ml to a small test tube and add 2 ml of a 10 g/l solution of diphenylcarbazide R in alcohol R and 2 ml of dilute sulphuric acid R. The solution is not more intensely coloured than a standard prepared at the same time using 5 ml of a solution containing 2.83 µg of potassium dichromate R per millilitre, 2 ml of dilute sulphuric acid R and 2 ml of a 10 g/l solution of diphenylcarbazide R in alcohol R (1 ppm of Cr). Sterility (2.6.1). It complies with the test for sterility as applied to catgut and other surgical sutures. Carry out the test on three sections, each 30 cm long, cut off respectively from the beginning, the centre and the end of the strand. STORAGE Store protected from light and heat. LABELLING The label states : — the gauge number, — the length in centimetres or in metres.

STORAGE See the monograph on Strands, sterile non-absorbable, in distributor for veterinary use (0605). LABELLING See the monograph on Strands, sterile non-absorbable, in distributor for veterinary use (0605).

01/2005:0609

POLYAMIDE 6 SUTURE, STERILE, IN DISTRIBUTOR FOR VETERINARY USE Filum polyamidicum-6 sterile in fuso ad usum veterinarium

DEFINITION Sterile polyamide 6 suture in distributor for veterinary use is obtained by drawing through a suitable die a synthetic plastic material formed by the polymerisation of ε-caprolactam. It consists of smooth, cylindrical monofilaments or braided filaments, or lightly twisted strands sheathed with the same material. It may be coloured with colouring matter 01/2005:0608 authorised by the competent authority. The suture is sterilised.

LINEN THREAD, STERILE, IN DISTRIBUTOR FOR VETERINARY USE Filum lini sterile in fuso ad usum veterinarium

CHARACTERS It is practically insoluble in the usual organic solvents ; it is not attacked by dilute alkaline solutions (for example a 100 g/l solution of sodium hydroxide) but is attacked by dilute mineral acids (for example 20 g/l sulphuric acid), by hot glacial acetic acid and by 70 per cent m/m formic acid.

DEFINITION IDENTIFICATION Sterile linen thread in distributor for veterinary use consists A. Heat about 50 mg with 0.5 ml of hydrochloric acid R1 in of the pericyclic fibres of the stem of Linum usitatissimum L. a sealed glass tube at 110 °C for 18 h and allow to stand The elementary fibres, 2.5 cm to 5 cm long, are assembled for 6 h. No crystals appear. in bundles 30 cm to 80 cm long and spun into continuous B. To about 50 mg add 10 ml of hydrochloric acid R1. The lengths of suitable diameter. The thread may be creamy-white material disintegrates in the cold and dissolves completely or may be coloured with colouring matter authorised by the within a few minutes. competent authority. The thread is sterilised. C. It dissolves in a 70 per cent m/m solution of anhydrous IDENTIFICATION formic acid R. A. Dissect the end of a thread, using a needle or fine TESTS tweezers, to isolate a few individual fibres. Examined It complies with the tests prescribed in the monograph under a microscope, the fibres are seen to be 12 µm on Strands, sterile non-absorbable, in distributor for to 31 µm wide and, along the greater part of their veterinary use (0605) and with the following test : length, have thick walls, sometimes marked with fine longitudinal striations, and a narrow lumen. The fibres Monomer and oligomers. In a continuous-extraction gradually narrow to a long, fine point. Sometimes there apparatus, treat 1.00 g with 30 ml of methanol R at a rate are unilateral swellings with transverse lines. of at least three extractions per hour for 7 h. Evaporate the extract to dryness, dry the residue at 110 °C for 10 min, B. Impregnate isolated fibres with iodinated zinc chloride allow to cool in a desiccator and weigh. The residue weighs solution R. The fibres are coloured violet-blue. not more than 20 mg (2 per cent). TESTS STORAGE It complies with the tests prescribed in the monograph See the monograph on Strands, sterile non-absorbable, in on Strands, sterile non-absorbable, in distributor for distributor for veterinary use (0605). veterinary use (0605). If stored in a dry state, expose to an atmosphere with a LABELLING relative humidity of 65 ± 5 per cent at 20 ± 2 °C for 4 h See the monograph on Strands, sterile non-absorbable, in immediately before measuring the diameter and for the distributor for veterinary use (0605). determination of minimum breaking load immerse in water R at room temperature for 30 min immediately The label states whether the suture is braided, monofilament before carrying out the test. or sheathed. 886


Silk suture, sterile, braided, in distributor for veterinary use


01/2005:0610

POLYAMIDE 6/6 SUTURE, STERILE, IN DISTRIBUTOR FOR VETERINARY USE Filum polyamidicum-6/6 sterile in fuso ad usum veterinarium DEFINITION

01/2005:0607

POLY(ETHYLENE TEREPHTHALATE) SUTURE, STERILE, IN DISTRIBUTOR FOR VETERINARY USE Filum ethyleni polyterephthalici sterile in fuso ad usum veterinarium DEFINITION Sterile poly(ethylene terephthalate) suture in distributor for veterinary use is obtained by drawing poly(ethylene terephthalate) through a suitable die. The suture is prepared by braiding very fine filaments in suitable numbers, depending on the gauge required. It may be whitish in colour, or may be coloured with authorised colouring matter or pigments authorised by the competent authority. The suture is sterilised.

Sterile polyamide 6/6 suture in distributor for veterinary use is obtained by drawing through a suitable die a synthetic plastic material formed by the polycondensation of hexamethylene-diamine and adipic acid. It consists of smooth, cylindrical monofilaments or braided filaments, or lightly twisted strands sheathed with the same material. It may be coloured with authorised colouring matter or CHARACTERS pigments authorised by the competent authority. The suture It is practically insoluble in most of the usual organic is sterilised. solvents, but is attacked by strong alkaline solutions. It is incompatible with phenols. CHARACTERS IDENTIFICATION It is practically insoluble in the usual organic solvents ; it A. It dissolves with difficulty when heated in is not attacked by dilute alkaline solutions (for example a dimethylformamide R and in dichlorobenzene R. 100 g/l solution of sodium hydroxide) but is attacked by B. To about 50 mg add 10 ml of hydrochloric acid R1. The dilute mineral acids (for example 20 g/l sulphuric acid), by material remains intact even after immersion for 6 h. hot glacial acetic acid and by 80 per cent m/m formic acid. TESTS It complies with the tests prescribed in the monograph A. In contact with a flame it melts and burns, forming a hard on Strands, sterile non-absorbable, in distributor for globule of residue and gives off a characteristic odour veterinary use (0605). resembling that of celery. STORAGE B. Place about 50 mg in an ignition tube held vertically See the monograph on Strands, sterile non-absorbable, in and heat gently until thick fumes are evolved. When the distributor for veterinary use (0605). fumes fill the tube, withdraw it from the flame and insert a strip of nitrobenzaldehyde paper R. A violet-brown LABELLING colour slowly appears on the paper and fades slowly in See the monograph on Strands, sterile non-absorbable, in air ; it disappears immediately on washing with dilute distributor for veterinary use (0605). sulphuric acid R. IDENTIFICATION

C. To about 50 mg add 10 ml of hydrochloric acid R1. The material disintegrates in the cold and dissolves within a few minutes. D. It does not dissolve in a 70 per cent m/m solution of anhydrous formic acid R but dissolves in an 80 per cent m/m solution of anhydrous formic acid R. TESTS It complies with the tests prescribed in the monograph on Strands, sterile non-absorbable, in distributor for veterinary use (0605). STORAGE See the monograph on Strands, sterile non-absorbable, in distributor for veterinary use (0605).

01/2005:0606

SILK SUTURE, STERILE, BRAIDED, IN DISTRIBUTOR FOR VETERINARY USE Filum bombycis tortum sterile in fuso ad usum veterinarium DEFINITION Sterile braided silk suture in distributor for veterinary use is obtained by braiding a variable number of threads, according to the diameter required, of degummed silk obtained from the cocoons of the silkworm Bombyx mori L. It may be coloured with colouring matter authorised by the competent authority. The suture is sterilised.

IDENTIFICATION A. Dissect the end of a strand, using a needle or fine tweezers, to isolate a few individual fibres. The fibres are See the monograph on Strands, sterile non-absorbable, in sometimes marked with very fine longitudinal striations distributor for veterinary use (0605). parallel to the axis of the strand. Examined under a The label states whether the suture is braided, monofilament microscope, a cross-section is more or less triangular or or sheathed. semi-circular, with rounded edges and without a lumen. LABELLING


887

Strands, sterile non-absorbable, in distributor for veterinary use


the strand being tested. When making the measurements, lower the pressor foot slowly to avoid crushing the strand. Make not fewer than one measurement per 2 m of length TESTS and in any case not fewer than 12 measurements at points It complies with the tests prescribed in the monograph evenly spaced along the strand. During the measurement on Strands, sterile non-absorbable, in distributor for submit monofilament strands to a tension not greater than veterinary use (0605). that required to keep them straight. Submit multifilament strands to a tension not greater than one-fifth of the STORAGE minimum breaking load shown in column C of Table 0605.-1 See the monograph on Strands, sterile non-absorbable, in appropriate to the gauge number and type of material distributor for veterinary use (0605). concerned or 10 N whichever is less. For multifilament strands of gauge number above 1.5 make two measurements LABELLING at each point, the second measurement being made after See the monograph on Strands, sterile non-absorbable, in rotating the strand through 90°. The diameter of that point distributor for veterinary use (0605). is the average of the two measurements. The average of the measurements carried out on the strand being tested and 01/2005:0605 not less than two-thirds of the individual measurements are within the limits given in the columns under A in Table 0605.-1 for the gauge number concerned. None of the STRANDS, STERILE NONmeasurements is outside the limits given in the columns ABSORBABLE, IN DISTRIBUTOR under B in Table 0605.-1 for the gauge number concerned.

B. Impregnate isolated fibres with iodinated potassium iodide solution R. The fibres are coloured pale yellow.

FOR VETERINARY USE

Table 0605.-1. – Diameters and minimum breaking loads

Fila non resorbilia sterilia in fuso ad usum veterinarium DEFINITION The statements in this monograph are intended to be read in conjunction with the individual monographs on sterile non-absorbable strands in distributor for veterinary use in the Pharmacopoeia. The requirements do not necessarily apply to sterile non-absorbable strands which are not the subject of such monographs. Sterile non-absorbable strands in distributor for veterinary use are strands which, when introduced into a living organism, are not metabolised by that organism. Sterile non- absorbable strands vary in origin, which may be animal, vegetable or synthetic. They occur as cylindrical monofilaments or as multifilament strands. Multifilament strands consist of elementary fibres which are assembled by twisting, cabling or braiding. Such strands may be sheathed. Sterile non- absorbable strands may be treated to render them non-capillary, and they may be coloured with colouring matter or pigments authorised by the competent authority. The strands are sterilised. They are presented in a suitable distributor that allows the withdrawal and use of all or part of the strand in aseptic conditions. The design of the distributor is such that with suitable handling the sterility of the content is maintained even when part of the strand has been removed. They may be stored dry or in a preserving liquid to which an antimicrobial preservative but not an antibiotic may be added.

Diameter (millimetres) Gauge number

A

Minimum breaking load (newtons) All other Linen thread non-absorbable strands

B

min.

max.

min.

max.

0.069

0.045

0.085

D -

D

0.050

C -

C

0.5

1.0

0.35

0.7

0.070

0.099

0.060

0.125

1.0

0.3

1.5

0.60

1

0.100

0.149

0.085

0.175

2.5

0.6

3.0

1.0

1.5

0.150

0.199

0.125

0.225

5.0

1.0

5.0

1.5

2

0.200

0.249

0.175

0.275

8.0

2.5

9.0

3.0

2.5

0.250

0.299

0.225

0.325

9.0

5.0

13.0

5.0

3

0.300

0.349

0.275

0.375

11.0

8.0

15.0

9.0

3.5

0.350

0.399

0.325

0.450

15.0

9.0

22.0

13.0

4

0.400

0.499

0.375

0.550

18.0

11.0

27.0

15.0

5

0.500

0.599

0.450

0.650

26.0

15.0

35.0

22.0

6

0.600

0.699

0.550

0.750

37.0

18.0

50.0

27.0

7

0.700

0.799

0.650

0.850

50.0

26.0

62.0

35.0

8

0.800

0.899

0.750

0.950

65.0

37.0

73.0

50.0

Minimum breaking load. Unless otherwise prescribed, determine the minimum breaking load by the following method using the strand in the condition in which it is presented. The minimum breaking load is determined over a simple knot formed by placing one end of a strand held in the right hand over the other end held in the left hand, TESTS passing one end over the strand and through the loop so Remove the strand from the distributor and measure formed (see Figure 0605.-1) and pulling the knot tight. promptly and in succession the length, diameter and Make not fewer than one measurement per 2 m of length minimum breaking load. at points evenly spaced along the strand. Determine the Length. Measure the length in the condition in which the breaking load using a suitable tensilometer. The apparatus strand is presented and without applying more tension than has two clamps for holding the strand, one of which is is necessary to keep it straight. The length of the strand is mobile and is driven at a constant rate of 30 cm per minute. not less than 95 per cent of the length stated on the label. The clamps are designed so that the strand being tested Diameter. Unless otherwise prescribed, measure the can be attached with-out any possibility of slipping. At the diameter by the following method using the strand in the beginning of the test the length of strand between the condition in which it is presented. Use a suitable instrument clamps is 12.5 cm to 20 cm and the knot is midway between capable of measuring with an accuracy of at least 0.002 mm the clamps. Set the mobile clamp in motion and note the and having a circular pressor foot 10 mm to 15 mm in force required to break the strand. If the strand breaks in a diameter. The pressor foot and the moving parts attached to clamp or within 1 cm of it, the result is discarded and the test it are weighted so as to apply a total load of 100 ± 10 g to repeated on another part of the strand. The average of all 888



Strands, sterile non-absorbable, in distributor for veterinary use

the results, excluding those legitimately dis-carded, is equal to or greater than the value in column C and no value is less than that given in column D in Table 0605.-1 for the gauge number and type of material concerned.

adjust to the original volume with water R. Depending on the colour of the strand, prepare the appropriate reference solution as described in Table 0605.-2 using the primary colour solutions (2.2.2). Table 0605.-2. – Colour reference solutions Colour of strand

Yellow - brown Pink - red Green - blue Violet

Composition of reference solution (parts by volume) Water

1.2

Blue primary solution —

1.0

—

—

9.0

—

—

2.0

8.0

1.6

—

8.4

—

Red primary solution

Yellow primary solution

0.2

8.6

The test solution is not more intensely coloured than the appropriate reference solution. Figure 0605.-1. – Simple knot Sterility (2.6.1). They comply with the test for sterility as applied to catgut and other surgical sutures. Carry out the test on three sections each 30 cm long, cut off respectively from the beginning, the centre and the end of the strand. Extractable colour. Strands that are dyed and intended to remain so during use comply with the test for extractable colour. Place 0.25 g of the strand to be examined in a conical flask, add 25.0 ml of water R and cover the mouth of the flask with a short-stemmed funnel. Boil for 15 min, cool and


STORAGE Store protected from light and heat. LABELLING The label states : — the gauge number, — the length in centimetres or in metres, — where appropriate, that the strand is coloured and intended to remain so during use.

889


Homoeopathic preparations

01/2005:90006 starting material for the manufacture of homoeopathic preparations, a test for foreign matter (2.8.2) is carried out, unless otherwise prescribed in the individual monographs. INTRODUCTION A specific appropriate test may apply to herbal drugs for All general texts and other monographs of the European homoeopathic preparations liable to be falsified. Pharmacopoeia that are relevant to homoeopathy are If appropriate, the herbal drugs for homoeopathic applicable. preparations comply with other tests, for example, total ash The “Homoeopathy” chapter of the European (2.4.16) and bitterness value (2.8.15). Pharmacopoeia contains general monographs and The test for loss on drying (2.2.32) is carried out on individual monographs describing starting materials and dried herbal drugs for homoeopathic preparations. A preparations used virtually exclusively for homoeopathic determination of water (2.2.13) is carried out on herbal medicines. Reference to these monographs for other drugs for homoeopathic preparations with a high essential purposes may be authorised by licensing authorities. oil content. The water content of fresh herbal drugs for homoeopathic preparations is determined by an appropriate method. 01/2005:2045 Herbal drugs for homoeopathic preparations comply with the requirements for pesticide residues (2.8.13). The HERBAL DRUGS FOR requirements take into account the nature of the plant, where necessary the preparation in which the plant might HOMOEOPATHIC PREPARATIONS be used, and where available the knowledge of the complete record of treatment of the batch of the plant. The content Plantae medicinales of pesticide residues may be determined by the method ad praeparationes homoeopathicas described in the annex to the general method. The risk of contamination of herbal drugs for homoeopathic DEFINITION preparations by heavy metals must be considered. If an Herbal drugs for homoeopathic preparations are mainly individual monograph does not prescribe limits for heavy whole, fragmented or cut, plants, parts of plants including metals or specific elements, such limits may be required if algae, fungi or lichens in an unprocessed state, usually in justified. fresh form but sometimes dried. Certain exudates that have not been subjected to a specific treatment are also considered Limits for aflatoxins may be required. to be herbal drugs for homoeopathic preparations. Herbal In some specific circumstances, the risk of radioactive drugs for homoeopathic preparations are precisely defined by contamination is to be considered. the botanical scientific name of the source species according to the binomial system (genus, species, variety and author). ASSAY Where applicable, herbal drugs for homoeopathic PRODUCTION preparations are assayed by an appropriate method. Herbal drugs for homoeopathic preparations are obtained STORAGE from cultivated or wild plants. Suitable cultivation, harvesting, collection, sorting, drying, fragmentation and Fresh herbal drugs are processed as rapidly as possible after storage conditions are essential to guarantee the quality of harvesting ; they may also be stored deep-frozen or in ethanol herbal drugs for homoeopathic preparations. (96 per cent V/V) or in alcohol of a given concentration. Store dried herbal drugs protected from light. Herbal drugs for homoeopathic preparations are, as far as possible, free from impurities such as soil, dust, dirt and other contaminants such as fungal, insect and other animal contaminants. They do not present signs of decay. 01/2005:1038 If a decontaminating treatment has been used, it is necessary to demonstrate that the constituents of the plant are not HOMOEOPATHIC PREPARATIONS affected and that no harmful residues remain. The use of ethylene oxide is prohibited for the decontamination of Praeparationes homoeopathicas herbal drugs for homoeopathic preparations. Adequate measures have to be taken in order to ensure that DEFINITION the microbiological quality of homoeopathic preparations Homoeopathic preparations are prepared from substances, containing one or more herbal drugs comply with the products or preparations called stocks, in accordance with a recommendations given in the text on Microbiological homoeopathic manufacturing procedure. A homoeopathic quality of pharmaceutical preparations (5.1.4). preparation is usually designated by the Latin name of the IDENTIFICATION stock, followed by an indication of the degree of dilution. Herbal drugs for homoeopathic preparations are identified Raw materials using their macroscopic and, where necessary, microscopic Raw materials for the production of homoeopathic descriptions and any further tests that may be required (for preparations may be of natural or synthetic origin. example, thin-layer chromatography). For raw materials of zoological or human origin, adequate measures are taken to minimise the risk of agents of infection TESTS in the homoeopathic preparations. For this purpose, it is When a fresh plant is used as a starting material for the demonstrated that : manufacture of homoeopathic preparations, the content of foreign matter is as low as possible ; if necessary, the — the method of production includes a step or steps that maximum content of foreign matter is indicated in the have been shown to remove or inactivate agents of individual monographs. When a dried plant is used as a infection, General Notices (1) apply to all monographs and other texts

893

Mother tinctures for homoeopathic preparations


— where applicable, raw materials of zoological origin comply with the monograph on Products with risk of transmitting agents of animal spongiform encephalopathies (1483), — where applicable, the animals and the tissues used to obtain the raw materials comply with the health requirements of the competent authorities for animals for human consumption, — for materials of human origin, the donor follows the recommendations applicable to human blood donors and to donated blood (see Human plasma for fractionation (0853)), unless otherwise justified and authorised. A raw material of botanical, zoological or human origin may be used either in the fresh state or in the dried state. Where appropriate, fresh material may be kept deep-frozen. Raw materials of botanical origin comply with the requirements of the monograph on Herbal drugs for homoeopathic preparations (2045). Where justified and authorised for transportation or storage purposes, fresh plant material may be kept in ethanol (96 per cent V/V) or in alcohol of a suitable concentration, provided the whole material including the storage medium is used for processing. Raw materials comply with any requirements of the relevant monographs of the European Pharmacopoeia. Vehicles Vehicles are excipients used for the preparation of certain stocks or for the potentisation process. They may include for example : purified water, alcohol of a suitable concentration, glycerol and lactose. Vehicles comply with any requirements of the relevant monographs of the European Pharmacopoeia. Stocks Stocks are substances, products or preparations used as starting materials for the production of homoeopathic preparations. A stock is usually one of the following : a mother tincture or a glycerol macerate, for raw materials of botanical, zoological or human origin, or the substance itself, for raw materials of chemical or mineral origin. Mother tinctures comply with the requirements of the monograph on Mother tinctures for homoeopathic preparations (2029). Glycerol macerates are liquid preparations obtained from raw materials of botanical, zoological or human origin by using glycerol or a mixture of glycerol and either alcohol of a suitable concentration or a solution of sodium chloride of a suitable concentration. Potentisation Dilutions and triturations are obtained from stocks by a process of potentisation in accordance with a homoeopathic manufacturing procedure : this means successive dilutions and succussions, or successive appropriate triturations, or a combination of the 2 processes. The potentisation steps are usually one of the following : — 1 part of the stock plus 9 parts of the vehicle ; they may be designated as “D”, “DH” or “X” (decimal), — 1 part of the stock plus 99 parts of the vehicle ; they may be designated as “C” or “CH” (centesimal). The number of potentisation steps defines the degree of dilution ; for example, “D3”, “3 DH” or “3X” means 3 decimal potentisation steps, and “C3”, “3 CH” or “3C” means 3 centesimal potentisation steps. “LM-” (or “Q-”) potencies are manufactured according to a specific procedure. 894

Dosage forms A dosage form of a homoeopathic preparation complies with any relevant dosage form monograph in the European Pharmacopoeia and with the following : — for the purpose of dosage forms for homoeopathic use “active substances” are considered to be “dilutions or triturations of homoeopathic stocks”, — these dosage forms are prepared using appropriate excipients, — the test for uniformity of content is normally not appropriate. However, in certain circumstances, it is required. Homoeopathic dosage form “pillule” Pillules for homoeopathic use are solid preparations obtained from sucrose, lactose or other suitable excipients. They may be prepared by impregnation of preformed pillules with a dilution or dilutions of homoeopathic stocks or by progressive addition of these excipients and the addition of a dilution or dilutions of homoeopathic stocks. They are intended for oral or sublingual use. Homoeopathic dosage form “tablet” Tablets for homoeopathic use are solid preparations obtained from sucrose, lactose or other suitable excipients according to the monograph on Tablets (0478). They may either be prepared by compressing one or more solid active substances with the excipients or by impregnating preformed tablets with a dilution or dilutions of homoeopathic stocks. The preformed tablets for impregnation are obtained from sucrose, lactose or other suitable excipients according to the monograph on Tablets (0478). They are intended for oral or sublingual use.

01/2005:2029

MOTHER TINCTURES FOR HOMOEOPATHIC PREPARATIONS Tincturae maternae ad praeparationes homoeopathicas DEFINITION Mother tinctures for homoeopathic preparations are liquid preparations obtained by the solvent action of a suitable vehicle upon raw materials. The raw materials are usually in the fresh form but may be dried. Mother tinctures for homoeopathic preparations may also be obtained from plant juices, with, or without the addition of a vehicle. For some preparations, the matter to be extracted may undergo a preliminary treatment. PRODUCTION Mother tinctures for homoeopathic preparations are prepared by maceration, digestion, infusion, decoction, fermentation or as described in the individual monographs, usually using alcohol of suitable concentration. Mother tinctures for homoeopathic preparations are obtained using a fixed proportion of raw material to solvent, taking the moisture content of the raw material into account, unless otherwise justified and authorised. If fresh plants are used, suitable procedures are used to ensure freshness. The competent authorities may require that the freshness is demonstrated by means of a suitable test.



Mother tinctures for homoeopathic preparations are usually clear. A slight sediment may form on standing and that is acceptable as long as the composition of the tincture is not changed significantly. The manufacturing process is defined so that it is reproducible. Production by maceration. Unless otherwise prescribed, reduce the matter to be extracted to pieces of suitable size, mix thoroughly and extract according to the prescribed extraction method with the prescribed extraction solvent. Allow to stand in a closed vessel for the prescribed time. The residue is separated from the extraction solvent and, if necessary, pressed out. In the latter case, the 2 liquids obtained are combined. Adjustment of the contents. Adjustment of the content of constituents may be carried out if necessary, either by adding the extraction solvent of suitable concentration, or by adding another mother tincture for homoeopathic preparations of the vegetable or animal matter used for the preparation. IDENTIFICATION Where applicable, at least 1 chromatographic identification test is carried out. TESTS The limits in an individual monograph are set to include official methods of production. Specific limits will apply to each defined method of production. If the test for relative density is carried out, the test for ethanol need not be carried out, and vice versa. Relative density (2.2.5). The mother tincture for homoeopathic preparations complies with the limits prescribed in the monograph. Ethanol (2.9.10). The ethanol content complies with that prescribed in the monograph. Methanol and 2-propanol (2.9.11) : maximum 0.05 per cent V/V of methanol and maximum 0.05 per cent V/V of 2-propanol, unless otherwise prescribed. Dry residue (2.8.16). Where applicable, the mother tincture for homoeopathic preparations complies with the limits prescribed in the monograph. Pesticides (2.8.13). Where applicable, the mother tincture for homoeopathic preparations complies with the test. This requirement is met if the herbal drug has been shown to comply with the test. ASSAY Where applicable, an assay with quantitative limits is performed. STORAGE Protected from light. A maximum storage temperature may be specified. LABELLING The label states : — that the product is a mother tincture for homoeopathic preparations (designated as "TM" or "Ø"), — the name of the raw material using the Latin title of the European Pharmacopoeia monograph where one exists, — the method of preparation, — the ethanol content or other solvent content, in per cent V/V, in the mother tincture, — the ratio of raw material to mother tincture, — where applicable, the storage conditions. General Notices (1) apply to all monographs and other texts

Common stinging nettle for homoeopathic preparations

01/2005:1599

ARSENIOUS TRIOXIDE FOR HOMOEOPATHIC PREPARATIONS Arsenii trioxidum ad praeparationes homoeopathicas As2O3

Mr 197.8

DEFINITION Content : 99.5 per cent to 100.5 per cent of As2O3. CHARACTERS Appearance : white or almost white powder. Solubility : practically insoluble to sparingly soluble in water. It dissolves in solutions of alkali hydroxides and carbonates. IDENTIFICATION A. Dissolve 20 mg in 1 ml of dilute hydrochloric acid R, add 4 ml of water R and 0.1 ml of sodium sulphide solution R. The resulting yellow precipitate is soluble in dilute ammonia R1. B. Dissolve 20 mg in 1 ml of hydrochloric acid R1, add 5 ml of hypophosphorous reagent R and heat for 15 min on a water-bath. A black precipitate develops. TESTS Appearance of solution. A 100 g/l solution in dilute ammonia R1 is clear (2.2.1) and colourless (2.2.2, Method II). Sulphides. Dissolve 1.0 g in 10.0 ml of dilute sodium hydroxide solution R. Add 0.05 ml of lead acetate solution R. Any colour in the test solution is not more intense than that in a standard prepared at the same time and in the same manner using a mixture of 10.0 ml of a 0.015 g/l solution of sodium sulphide R in dilute sodium hydroxide solution R and 0.05 ml of lead acetate solution R (20 ppm). ASSAY Dissolve 40.0 mg in a mixture of 10 ml of water R and 10 ml of dilute sodium hydroxide solution R. Add 10 ml of dilute hydrochloric acid R and 3 g of sodium hydrogen carbonate R and mix. Add 1 ml of starch solution R and titrate with 0.05 M iodine. 1 ml of 0.05 M iodine is equivalent to 4.946 mg of As2O3. 01/2005:2030

COMMON STINGING NETTLE FOR HOMOEOPATHIC PREPARATIONS Urtica dioica ad praeparationes homoeopathicas DEFINITION Whole, fresh, flowering plant of Urtica dioica L. CHARACTERS Macroscopic characters described under Identification A. The plant causes an itching, burning sensation on the skin. IDENTIFICATION A. Common stinging nettle is perennial. The taproot sends out creeping subterranean rhizomes, more or less 4-angled in transverse section, from which extend 895

Copper for homoeopathic preparations


adventious secondary roots and very numerous brownish hairy rootlets. The stipes are erect, generally unbranched, 3 mm to 5 mm in diameter and 0.3 m to 1.5 m high, rarely up to 2.5 m high, 4-angled, greyish-green and covered in short hairs and stinging hairs. The decussate leaves are 30 mm to 150 mm long and 20 mm to 80 mm wide. The petiole is hispid and usually slightly less than one-third the length of the lamina. The leaf blade is ovate, acuminate, cordate or rounded at the base, and coarsely dentate ; the apical tooth is distinctly larger than the lateral teeth. The upper side of the leaves is dark green and usually matt, both sides bear short serried hairs intermingled with long stinging hairs. The 2 stipules are linear-subulate and free. The inflorescences growing from the leaf axils are complex, the flowers unisexual, and, particularly in male plants, generally distinctly longer than the petiole. After shedding their pollen, male inflorescences are erect at an oblique angle or horizontal ; female inflorescences are pendent when the fruit is ripe. All flowers have long stalks. The perianth of the male flowers is divided half-way down into equal green lobes, widest at their base, with short bristles and stinging hairs at the margins. The stamens are equal and opposite to the perianth segments, each with a long, whitish filament that curves inwards before pollen is shed and spreads out afterwards. The ovary is rudimentary, button or cup-shaped. The perianth of the female flowers is downy or bristly on the outside and consists of outer, and 2 inner segments ; the inner segments are about twice the length of the outer ones. The hypogynous, ovate, unilocular ovary bears a large capitate stigma with a brush-like shock of hair. As the one-seeded fruit grows ripe, the 2 inner segments of the perianth fold around it like wings. B. It complies with the test for Urtica urens (see Tests). TESTS Urtica urens. The margin of the lamina is not serrate with teeth twice as long as wide. The clusters of flowers in the axils are longer than the petiole of the leaf. Unisexual, apetalous flowers are not together on the same plant and in the same cluster. Foreign matter (2.8.2) : maximum 5 per cent. Loss on drying (2.2.32) : minimum 65.0 per cent, determined on 5.0 g of finely cut drug by drying in an oven at 100-105 °C for 2 h, if performed to demonstrate the freshness of the drug.

Mother tincture The mother tincture complies with the requirements of the general monograph on Mother tinctures for homoeopathic preparations (2029). PRODUCTION The mother tincture of Urtica dioica L. is prepared by maceration using alcohol of a suitable concentration. CHARACTERS Appearance : greenish-brown or orange-brown liquid. IDENTIFICATION Thin-layer chromatography (2.2.27). Test solution. The mother tincture to be examined. Reference solution. Dissolve 10 mg of phenylalanine R and 10 mg of serine R in a mixture of equal volumes of methanol R and water R and dilute to 10 ml with the same mixture of solvents. 896

Plate : TLC silica gel plate R. Mobile phase : glacial acetic acid R, water R, acetone R, butanol R (10:20:35:35 V/V/V/V). Application : 20 µl, as bands. Development : over a path of 10 cm. Drying : in air. Detection : spray with a 1 g/l solution of ninhydrin R in alcohol R. Heat the plate at 105-110 °C for 5-10 min then examine in daylight within 10 min. Results : see below the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. Top of the plate _______

_______

Phenylalanine : a violet to reddish-brown zone 4 red to violet zones _______ Serine : a reddish-violet zone

_______ A violet zone A violet zone

Reference solution

Test solution

TESTS Relative density (2.2.5) : 0.930 to 0.950. Ethanol (2.9.10) : 40 per cent V/V to 56 per cent V/V. Dry residue (2.8.16) : minimum 1.1 per cent. 01/2005:1610

COPPER FOR HOMOEOPATHIC PREPARATIONS Cuprum ad praeparationes homoeopathicas Cu

Ar 63.5

DEFINITION Content : 99.0 per cent to 101.0 per cent of Cu. CHARACTERS Appearance : reddish-brown powder. Solubility : practically insoluble in water, soluble in hydrochloric acid and in nitric acid, practically insoluble in alcohol. IDENTIFICATION A. To 2 ml of solution S (see Tests) add 0.5 ml of potassium ferrocyanide solution R. A reddish-brown precipitate is formed. B. To 5 ml of solution S add 0.6 ml of ammonia R. A blue precipitate is formed. Add 2 ml of ammonia R. The precipitate disappears ; the solution has an intense blue colour. TESTS Solution S. Dissolve 2.0 g in 10 ml of nitric acid R. After nitrous fumes are no longer evolved, dilute to 60 ml with distilled water R. Acidity or alkalinity. To 5.0 g add 20 ml of carbon dioxide-free water R. Boil for 1 min. Cool. Filter and dilute to 25.0 ml with carbon dioxide free water R. To 10 ml of the solution add 0.1 ml of bromothymol blue solution R1.



Not more than 0.5 ml of 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is required to change the colour of the indicator. Chlorides (2.4.4) : maximum 100 ppm. 15 ml of solution S complies with the limit test for chlorides. Sulphates (2.4.13) : maximum 300 ppm. 15 ml of solution S complies with the limit test for sulphates. Iron : maximum 50 ppm. Atomic absorption spectrometry (2.2.23, Method I). Test solution. Dissolve 1.00 g in 5 ml of nitric acid R and dilute to 50.0 ml with water R. Reference solutions. Prepare the reference solutions using iron standard solution (20 ppm Fe) R, diluted as necessary with a 1 per cent V/V solution of nitric acid R. Source : iron hollow-cathode lamp. Wavelength : 248.3 nm. Flame : air-acetylene. Lead : maximum 100 ppm. Atomic absorption spectrometry (2.2.23, Method I). Test solution. Use the test solution prepared for the test for iron. Reference solutions. Prepare the reference solutions using lead standard solution (0.1 per cent Pb) R, diluted as necessary with a 1 per cent V/V solution of nitric acid R. Source : lead hollow-cathode lamp. Wavelength : 283.3 nm. Flame : air-acetylene. Zinc : maximum 50 ppm. Atomic absorption spectrometry (2.2.23, Method I). Test solution. Use the test solution prepared for the test for iron. Reference solutions. Prepare the reference solutions using zinc standard solution (100 ppm Zn) R, diluted as necessary with a 1 per cent V/V solution of nitric acid R. Source : zinc hollow-cathode lamp. Wavelength : 213.9 nm. Flame : air-acetylene.

Garlic for homoeopathic preparations

CHARACTERS Macroscopic characters described under identification. It has a characteristic odour after cutting. IDENTIFICATION The bulb is generally 3 cm to 5 cm broad and almost spherical ; the flat base bears the remnants of numerous short greyish-brown adventitious roots. The bulb consists of about 10 daughter bulbs (cloves) arranged roughly in a circle around a central axis. Individual daughter bulbs are 1 cm to 3 cm long, laterally compressed and convex on the dorsal side. Each daughter bulb has a tough, white or reddish skin around a fleshy tubular leaf, investing a more or less rounded elongated cone of leaf primordia and vegetative apex. TESTS Foreign matter (2.8.2). It complies with the test for foreign matter. Water (2.2.13) : minimum 55.0 per cent, determined on 10.0 g of the finely cut drug, if performed to demonstrate the freshness of the drug.

Mother tincture The mother tincture complies with the requirements of the general monograph on Mother tinctures for homoeopathic preparations (2029). PRODUCTION The mother tincture of Allium sativum L. is prepared by maceration of the cut drug using alcohol of a suitable concentration. CHARACTERS Appearance : brownish-yellow liquid. It has a peculiar and unpleasant aromatic odour.

IDENTIFICATION A. To 2 ml of the mother tincture to be examined, add 0.2 ml of dilute sodium hydroxide solution R. A yellowish-white precipitate develops. B. Thin-layer chromatography (2.2.27). Test solution. Extract 5 ml of the mother tincture to be examined with 2 quantities, each of 10 ml, of ether R. Combine the ether layers and dry over anhydrous ASSAY sodium sulphate R. Filter and evaporate the filtrate in a water-bath at low temperature. Dissolve the residue in Dissolve 0.100 g in 5 ml of nitric acid R. Heat to expel the 0.4 ml of methanol R. nitrous fumes. Add 200 ml of water R and neutralise (2.2.3) with dilute ammonia R1. Add 1 g of ammonium chloride R Reference solution. Dissolve 10 mg of resorcinol R, and 3 mg of murexide R. Titrate with 0.1 M sodium edetate 10 mg of thymol R and 30 mg of gallic acid R in 10 ml until the colour changes from green to violet. of methanol R. Plate : TLC silica gel F254 plate R. 1 ml of 0.1 M sodium edetate is equivalent to 6.354 mg of Cu. Mobile phase : anhydrous formic acid R, toluene R, di-isopropyl ether R (10:40:50 V/V/V). Application : 40 µl of the test solution and 10 µl of the 01/2005:2023 reference solution. Development : over a path of 10 cm. GARLIC FOR HOMOEOPATHIC Drying : in air. Detection : examine in ultraviolet light at 254 nm and PREPARATIONS identify gallic acid ; spray with anisaldehyde solution R, heat to 105-110 °C for 5-10 min. Examine in daylight Allium sativum ad praeparationes within 10 min. homoeopathicas Results : see below the sequence of the zones present in the chromatograms obtained with the reference solution DEFINITION and the test solution. Other zones may also be visible in Fresh bulb of Allium sativum L. the chromatogram obtained with the test solution. General Notices (1) apply to all monographs and other texts

897

Honey bee for homoeopathic preparations


Top of the plate An intense reddish-violet zone Thymol: an orange-red zone An intense reddish-violet zone A violet zone A yellowish or greenish zone _______

_______

Resorcinol : an intense orange-red zone _______

_______

Gallic acid : a yellow zone

A violet zone

(UV at 254 nm : a fluorescent quenching zone)

A greenish-yellow zone A violet zone may be present

Reference solution

Test solution

TESTS Relative density (2.2.5) : 0.885 to 0.960. Ethanol (2.9.10) : 50 per cent V/V to 70 per cent V/V. Dry residue (2.8.16) : minimum 4.0 per cent. STORAGE In an airtight container, protected from light.

PRODUCTION The mother tincture of Apis mellifera L. is prepared by maceration using alcohol of a suitable concentration. CHARACTERS Pale yellow liquid that may darken on storage. IDENTIFICATION Thin-layer chromatography (2.2.27). Test solution. The mother tincture to be examined. Reference solution. Dissolve 12 mg of 4-aminobutanoic acid R, 12 mg of leucine R and 12 mg of proline R in 5 ml of water R and dilute to 50 ml with alcohol R. Plate : TLC silica gel plate R. Mobile phase : water R, ethanol R (17:63 V/V). Application : 20 µl, as bands. Development : over a path of 10 cm. Drying : in air. Detection : spray with ninhydrin solution R and heat at 100-105 °C for 10 min ; examine in daylight. Results : see below the sequence of the zones present in the chromatograms obtained with the reference and test solutions. Other zones may also be visible. Top of the plate _______

_______ A pink zone

01/2005:2024

HONEY BEE FOR HOMOEOPATHIC PREPARATIONS Apis mellifera ad praeparationes homoeopathicas

Leucine : a pink zone

A pink zone A pink zone A pink zone

_______

_______

Proline : an orange-yellow zone

An orange-yellow zone

4-Aminobutanoic acid : a pink zone

A pink zone

DEFINITION Live worker honey bee (Apis mellifera L.). Reference solution

CHARACTERS Characters described under Identification. PRODUCTION If the bee has been exposed to treatment to prevent or cure diseases, appropriate steps are taken to ensure that the levels of residues are as low as possible. IDENTIFICATION The body of a honey bee is about 15 mm long, black, with a silky sheen, and covered with red hairs with a touch of grey. The broad tibiae are without spines. The posterior margins of the segments and legs are brown, with gradual transition to orange-red. The claws are two-membered, the maxillary palps single-membered. On the hind legs are baskets or scoops invested with bristles. The wings have 3 complete cubital cells, with the radial cell twice as long as it is wide ; the 3 cells on the lower margin and the 3 middle cells are closed. A duct connects the barbed sting with the poison sac.

Test solution

TESTS Relative density (2.2.5) : 0.890 to 0.910. Ethanol (2.9.10) : 60 per cent V/V to 70 per cent V/V. Dry residue (2.8.16) : minimum 0.30 per cent.

01/2005:2028

HYPERICUM FOR HOMOEOPATHIC PREPARATIONS Hypericum perforatum ad praeparationes homoeopathicas

Mother tincture

DEFINITION Whole, fresh plant of Hypericum perforatum L., at the beginning of the flowering period.

The mother tincture complies with the requirements of the general monograph on Mother tinctures for homoeopathic preparations (2029).

CHARACTERS Macroscopic characters described under Identification.

898



Iron for homoeopathic preparations

IDENTIFICATION The perennial plant consists of a spindle-shaped root and a branched rhizome, giving rise to long, decumbent runners. The cylindrical, erect stem is woody at the base, 0.2 m to 1 m long, branched in the upper part, with 2 raised longitudinal lines. The leaves are opposite, sessile, exstipulate, oblong-oval and 15 mm to 30 mm long. The leaf margins show black glandular dots, and many small translucent oil glands are present on the entire surface and are visible by transmitted light. The flowers are regular and form corymbose clusters at the apex of the stem. They have 5 green, lanceolate sepals with acuminate apices, and black oil glands near the entire margins ; 5 orange-yellow petals, much longer than the sepals, with black oil glands near the terminal margins only ; 3 staminal blades, each divided into many orange-yellow stamens and 3 carpels surmounted by red styles. Each petal is asymmetrically linear-ovate in shape, with one of the margin entire and the other dentate.

Top of the plate A yellow to blue zone Hypericin : a red zone

2 red zones

_______

_______ Several zones

_______

_______

Hyperoside : a yellow to orange zone

Blue or yellow zones

Rutin : a yellow to orange zone

A yellow to orange zone

Reference solution

Test solution

TESTS Relative density (2.2.5) : 0.900 to 0.920. Ethanol (2.9.10) : 60 per cent V/V to 75 per cent V/V. Dry residue (2.8.16) : minimum 1.3 per cent.

TESTS Foreign matter (2.8.2) : maximum 4 per cent of fruits and maximum 1 per cent of other foreign matter. Loss on drying (2.2.32) : if performed to demonstrate the freshness of the drug, minimum 55 per cent, determined on 5.0 g of finely cut drug by drying in an oven at 100-105 °C.

IRON FOR HOMOEOPATHIC PREPARATIONS

Mother tincture

Ferrum ad praeparationes homoeopathicas

The mother tincture complies with the requirements of the general monograph on Mother tinctures for homoeopathic preparations (2029).


Fe

Ar 55.85

DEFINITION Obtained by reduction or sublimation as a fine blackish-grey PRODUCTION powder. The mother tincture of Hypericum perforatum L. is prepared Content : 97.5 per cent to 101.0 per cent. by maceration using alcohol of a suitable concentration. CHARACTERS CHARACTERS Appearance : fine, blackish-grey powder, without metallic Dark red to brownish red liquid. lustre. Solubility : practically insoluble in water and in alcohol. It IDENTIFICATION dissolves with heating in dilute mineral acids. Thin-layer chromatography (2.2.27). IDENTIFICATION Test solution. The mother tincture to be examined. Dissolve 50 mg in 2 ml of dilute sulphuric acid R and dilute Reference solution. Dissolve 5 mg of rutin R, 1 mg of to 10 ml with water R. The solution gives reaction (a) of iron hypericin R and 5 mg of hyperoside R in methanol R and (2.3.1). dilute to 5 ml with the same solvent. Plate : TLC silica gel plate R. TESTS Mobile phase : anhydrous formic acid R, water R, ethyl Solution S. To 10.0 g add 40 ml of water R. Boil for 1 min. acetate R (6:9:90 V/V/V). Cool, filter and dilute to 50.0 ml with water R. Application : 10 µl of the test solution and 5 µl of the Alkalinity. To 10 ml of solution S add 0.1 ml of bromothymol reference solution, as 10 mm bands. blue solution R1. Not more than 0.1 ml of 0.01 M Development : over a path of 10 cm. hydrochloric acid is required to change the colour of the Drying : at 100-105 °C for 10 min. indicator to yellow. Detection : spray with a 10 g/l solution of diphenylboric Substances insoluble in hydrochloric acid. Dissolve 2.00 g acid aminoethyl ester R in methanol R and then a 50 g/l in 40 ml of hydrochloric acid R. Heat on a water-bath. solution of macrogol 400 R in methanol R. Examine the As soon as fumes are no longer evolved, filter through a plates after 30 min in ultraviolet light at 365 nm. sintered-glass filter No. 16 (2.1.2). Rinse with water R. Dry Results : see below the sequence of the zones present in the the residue in an oven at 100-105 °C for 1 h. The residue chromatograms obtained with the reference solution and the weighs a maximum of 20 mg (1.0 per cent). test solution. In the chromatogram obtained with the test Substances soluble in water. Evaporate 10.0 ml of solution S solution, the zone due to rutin may be weak or even absent. on a water-bath and dry at 100-105 °C for 1 h. The residue The chromatogram obtained with the test solution shows a weighs a maximum of 2 mg (0.1 per cent). group of zones that may be blue or yellow, with a Rf similar Chlorides (2.4.4) : maximum 50 ppm. to that of the zone due to hyperoside in the chromatogram obtained with the reference solution. Other weak zones may Dilute 5 ml of solution S to 15 ml with water R. The solution complies with the limit test for chlorides. also be visible. General Notices (1) apply to all monographs and other texts

899

Saffron for homoeopathic preparations


Sulphides and phosphides. In a 100 ml conical flask carefully mix 1.0 g with 10 ml of dilute hydrochloric acid R. Within 30 s lead acetate paper R moistened with water R and placed over the mouth of the flask is not coloured more intensely than light brown by the resulting fumes. Arsenic (2.4.2) : maximum 5 ppm. Boil 0.2 g in 25 ml of dilute hydrochloric acid R until completely dissolved. The solution complies with limit test A. Copper : maximum 50 ppm. Atomic absorption spectrometry (2.2.23, Method I). Test solution. Dissolve 1.00 g in a mixture of 60 ml of dilute hydrochloric acid R and 10 ml of dilute hydrogen peroxide solution R. Reduce to a volume of 5 ml and dilute to 50.0 ml with water R. Reference solutions. Prepare the reference solutions using copper standard solution (0.1 per cent Cu) R, diluted as necessary with a 1 per cent V/V solution of hydrochloric acid R. Source : copper hollow-cathode lamp. Wavelength : 324.8 nm. Flame : air-acetylene. Lead : maximum 50 ppm. Atomic absorption spectrometry (2.2.23, Method I). Test solution. In a separating funnel, place 20 ml of the test solution prepared for the test for copper. Add 25 ml of lead-free hydrochloric acid R. Stir with 3 quantities, each of 25 ml, of di-isopropyl ether R. Collect the aqueous layer. Add 0.10 g of sodium sulphate decahydrate R. Evaporate to dryness. Take up the residue with 1 ml of lead-free nitric acid R and dilute to 20 ml with water R. Reference solutions. Prepare the reference solutions using lead standard solution (0.1 per cent Pb) R, diluted as necessary with a 10 per cent V/V solution of nitric acid R containing 5 g/l of sodium sulphate decahydrate R. Source : lead hollow-cathode lamp. Wavelength : 217 nm. Flame : air-acetylene. ASSAY Stir for 10 min 0.100 g in a hot solution of 1.25 g of copper sulphate R in 20 ml of water R in a 100 ml conical flask with a ground-glass stopper. Filter rapidly and wash the filter. Combine the filtrate and the washings, acidify with dilute sulphuric acid R and titrate with 0.02 M potassium permanganate until a pink colour is obtained. 1 ml of 0.02 M potassium permanganate is equivalent to 5.585 mg of Fe.

CHARACTERS Saffron has a characteristic, aromatic odour. It has the macroscopic and microscopic characters described under identification tests A and B. IDENTIFICATION A. The dark brick-red stigmas, when dry, are 20 mm to 40 mm long and after soaking with water, about 35 mm to 50 mm long. The tubes, gradually widening at the top, are incised on one side, the upper margin is open and finely crenated. The style connecting the 3 stigmas is pale yellow and not more than 5 mm long. B. Examine under a microscope using chloral hydrate solution R. It shows the following diagnostic characters : elongated epidermal cells, frequently with a short, central papilla ; in water they release a yellow colouring matter ; the upper border of the stigma has finger-shaped papillae, up to 150 µm long ; between them are single, globular pollen grains, about 100 µm wide, with a finely pitted exine, vascular bundles with small spirally thickened vessels and no fibres. C. Carefully crush pieces of the drug to coarse particles and moisten with 0.2 ml of phosphomolybdic acid solution R. The particles turn blue within 1-2 min or they have a blue areole around them. D. Examine by thin-layer chromatography (2.2.27). Test solution. Carefully crush 0.1 g of the drug with a glass rod and moisten with 0.2 ml of water R. After 3 min add 5 ml of methanol R, allow to stand for 20 min, protected from light, and filter through a plug of glass wool. Reference solution. Dissolve 5 mg of naphthol yellow R in 5 ml of methanol R and add a solution of 5 mg of Sudan red G R in 5 ml of methylene chloride R. Plate : TLC silica gel F254 plate R. Mobile phase : water R, 2-propanol R, ethyl acetate R (10:25:65 V/V/V). Application : 10 µl of the test solution and 5 µl of the reference solution as bands. Development : over a path of 10 cm. Drying : in air. Detection : examine in daylight. Results : see below the sequence of the zones present in the chromatograms obtained with the reference and test solutions. Top of the plate A red zone

LABELLING The label indicates whether the iron for homoeopathic preparations is obtained by reduction or sublimation.

A yellow zone 2 yellow zones An intense yellow zone (crocine)

01/2005:1624

SAFFRON FOR HOMOEOPATHIC PREPARATIONS Croci stigma ad praeparationes homoeopathicas

Test solution

Detection : in ultraviolet light at 254 nm. Results : see below the sequence of the zones present in the chromatograms obtained with the reference and test solutions. Top of the plate

DEFINITION Dried stigmas of Crocus sativus L. usually joined by the base to a short style. 900

Reference solution

A red zone

1 or 2 quenching zones

A yellow zone

A quenching zone

Reference solution

Test solution



Detection : spray with anisaldehyde solution R and examine in daylight while heating at 100-105 °C for 5-10 min. Results : see below the sequence of the zones present in the chromatograms obtained with the reference and test solutions.

Saffron for homoeopathic preparations

solution of diphenylboric acid aminoethyl ester R in methanol R. Examine in ultraviolet light at 365 nm. The spot shows an intense orange-yellow fluorescence.

TESTS Colouring power. Introduce 0.10 g into a 5 ml volumetric flask and add to 5.0 ml with distilled water R. Close the Top of the plate flask and shake every 30 min for 8 h. Then allow to stand for 16 h. Dilute 1.0 ml to 500.0 ml with distilled water R. A red zone 1 or 2 red to reddish-violet zones The absorbance (2.2.25) measured at 440 nm using distilled A blue to bluish-green zone A red to reddish-violet zone water R as the compensation liquid, is not less than 0.44. 2 blue to bluish-green zones Foreign matter. Examine the drug microscopically. No parts with rough walls, no crystals and no pollen grains containing An intense blue to bluish-green zone (crocine) 3 germinal pores are present. Reference solution Test solution Loss on drying (2.2.32) : maximum 10.0 per cent, determined E. Dilute 0.1 ml of the test solution (see Identification test D) on 0.200 g by drying in an oven at 100-105 °C. with 1 ml of methanol R. Deposit 0.1 ml of this solution Total ash (2.4.16) : maximum 7.0 per cent, determined on on a filter paper, allow to dry and spray with a 10 g/l the residue obtained in the test for loss on drying.


901


Acacia, spray-dried

01/2005:0307 of water R and evaporate the solution to dryness under reduced pressure. To the resulting clear film add 0.1 ml of water R and 0.9 ml of methanol R. Centrifuge to separate the ACACIA amorphous precipitate. Dilute the supernatant, if necessary, to 1 ml with methanol R. Acaciae gummi Reference solution. Dissolve 10 mg of arabinose R, 10 mg of galactose R, 10 mg of glucose R, 10 mg of rhamnose R DEFINITION and 10 mg of xylose R in 1 ml of water R and dilute to 10 ml Acacia is the air-hardened, gummy exudate flowing naturally with methanol R. from or obtained by incision of the trunk and branches of Apply to the plate as bands 10 µl of each solution. Develop Acacia senegal L. Willdenow, other species of Acacia of over a path of 10 cm using a mixture of 10 volumes of African origin and Acacia seyal Del. a 16 g/l solution of sodium dihydrogen phosphate R, 40 volumes of butanol R and 50 volumes of acetone R. CHARACTERS Dry the plate in a current of warm air for a few minutes Acacia is almost completely but very slowly soluble, after and develop again over a path of 15 cm using the same about 2 h, in twice its mass of water leaving only a very mobile phase. Dry the plate at 110 °C for 10 min, spray small residue of vegetable particles ; the liquid obtained is with anisaldehyde solution R and heat again at 110 °C for colourless or yellowish, dense, viscous, adhesive, translucent 10 min. The chromatogram obtained with the reference and weakly acid to blue litmus paper. Acacia is practically solution shows five clearly separated coloured zones due to insoluble in alcohol. galactose (greyish-green to green), glucose (grey), arabinose It has the macroscopic and microscopic characters described (yellowish-green) xylose (greenish-grey to yellowish-grey) under identification tests A and B. and rhamnose (yellowish-green), in order of increasing Rf value. The chromatogram obtained with the test solution IDENTIFICATION shows no grey zone and no greyish-green zone between A. Acacia occurs as yellowish-white, yellow or pale amber, the zones corresponding to galactose and arabinose in the sometimes with a pinkish tint, friable, opaque, spheroidal, chromatogram obtained with the reference solution. oval or reniform pieces (tears) of a diameter from about Starch, dextrin and agar. To 10 ml of solution S previously 1 cm to 3 cm, frequently with a cracked surface, easily boiled and cooled add 0.1 ml of 0.05 M iodine. No blue or broken into irregular, whitish or slightly yellowish angular fragments with conchoidal fracture and a glassy reddish-brown colour develops. and transparent appearance. In the centre of an unbroken Sterculia gum tear there is sometimes a small cavity. A. Place 0.2 g of the powdered drug (355) in a 10 ml ground-glass-stoppered cylinder graduated in 0.1 ml. Add B. Reduce to a powder (355). The powder is white or 10 ml of alcohol (60 per cent V/V) R and shake. Any gel yellowish-white. Examine under a microscope using formed occupies not more than 1.5 ml. glycerol R (50 per cent V/V). The powder presents angular, irregular, colourless, transparent fragments. B. To 1.0 g of the powdered drug (355) add 100 ml of water R Only traces of starch or vegetable tissues are visible. No and shake. Add 0.1 ml of methyl red solution R. Not stratified membrane is apparent. more than 5.0 ml of 0.01 M sodium hydroxide is required to change the colour of the indicator. C. Examine the chromatograms obtained in the test for glucose and fructose. The chromatogram obtained Tannins. To 10 ml of solution S add 0.1 ml of ferric chloride with the test solution shows three zones due to solution R1. A gelatinous precipitate is formed, but neither galactose, arabinose and rhamnose. No other important the precipitate nor the liquid shows a dark blue colour. zones are visible, particularly in the upper part of the Tragacantha. Examine the chromatograms obtained in chromatogram. the test for glucose and fructose. The chromatogram D. Dissolve 1 g of the powdered drug (355) in 2 ml of water R obtained with the test solution shows no greenish-grey to by stirring frequently for 2 h. Add 2 ml of alcohol R. After yellowish-grey zone corresponding to the zone of xylose in shaking, a white, gelatinous mucilage is formed which the chromatogram obtained with the reference solution. becomes fluid on adding 10 ml of water R. Loss on drying (2.2.32). Not more than 15.0 per cent, determined on 1.000 g of the powdered drug (355) by drying TESTS in an oven at 100-105 °C. Solution S. Dissolve 3.0 g of the powdered drug (355) in Total ash (2.4.16). Not more than 4.0 per cent. 25 ml of water R by stirring for 30 min. Allow to stand for 30 min and dilute to 30 ml with water R. Microbial contamination. Total viable aerobic count (2.6.12) Insoluble matter. To 5.0 g of the powdered drug (355) add not more than 104 micro-organisms per gram, determined 100 ml of water R and 14 ml of dilute hydrochloric acid R, by plate count. It complies with the test for Escherichia coli (2.6.13). boil gently for 15 min, shaking frequently, and filter while hot through a tared sintered-glass filter. Wash with hot water R and dry at 100-105 °C. The residue weighs not more than 25 mg (0.5 per cent). 01/2005:0308 Glucose and fructose. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R. ACACIA, SPRAY-DRIED Test solution. To 0.100 g of the powdered drug (355) in a thick-walled centrifuge tube add 2 ml of a 100 g/l solution Acaciae gummi dispersione desiccatum of trifluoroacetic acid R, shake vigorously to dissolve the forming gel, stopper the tube and heat the mixture at DEFINITION 120 °C for 1 h. Centrifuge the hydrolysate, transfer the clear supernatant carefully into a 50 ml flask, add 10 ml Spray-dried acacia is obtained from a solution of acacia. General Notices (1) apply to all monographs and other texts

905

Acamprosate calcium


Sterculia gum A. Place 0.2 g in a 10 ml ground-glass-stoppered cylinder graduated in 0.1 ml. Add 10 ml of alcohol (60 per cent V/V) R and shake. Any gel formed occupies not more than 1.5 ml. B. To 1.0 g add 100 ml of water R and shake. Add 0.1 ml of methyl red solution R. Not more than 5.0 ml of 0.01 M sodium hydroxide is required to change the IDENTIFICATION colour of the indicator. A. Examined under a microscope, in alcohol R, the powder Tannins. To 10 ml of solution S add 0.1 ml of ferric chloride is seen to consist predominantly of spheroidal particles solution R1. A gelatinous precipitate is formed, but neither about 4 µm to 40 µm in diameter, with a central cavity the precipitate nor the liquid shows a dark blue colour. containing one or several air-bubbles ; a few minute flat fragments are present. Only traces of starch granules are Tragacantha. Examine the chromatograms obtained in visible. No vegetable tissue is seen. the test for Glucose and fructose. The chromatogram obtained with the test solution shows no greenish-grey to B. Examine the chromatograms obtained in the test for yellowish-grey zone corresponding to the zone of xylose in glucose and fructose. The chromatogram obtained the chromatogram obtained with the reference solution. with the test solution shows 3 zones due to galactose, arabinose and rhamnose. No other important zones Loss on drying (2.2.32). Not more than 10.0 per cent, are visible, particularly in the upper part of the determined on 1.000 g by drying in an oven at 100-105 °C. chromatogram. Total ash (2.4.16). Not more than 4.0 per cent. C. Dissolve 1 g of the drug to be examined in 2 ml of water R Microbial contamination. Total viable aerobic count (2.6.12) by stirring frequently for 20 min. Add 2 ml of alcohol R. not more than 104 micro-organisms per gram, determined After shaking a white gelatinous mucilage is formed by plate count. It complies with the test for Escherichia which becomes fluid on adding 10 ml of water R. coli (2.6.13). CHARACTERS

It dissolves completely and rapidly, after about 20 min, in twice its mass of water. The liquid obtained is colourless or yellowish, dense, viscous, adhesive, translucent and weakly acid to blue litmus paper. Spray-dried acacia is practically insoluble in alcohol.

TESTS Solution S. Dissolve 3.0 g of the drug to be examined in 25 ml of water R by stirring for 10 min. Allow to stand for 20 min and dilute to 30 ml with water R. Glucose and fructose. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.

01/2005:1585

ACAMPROSATE CALCIUM Acamprosatum calcicum

Test solution. To 0.100 g in a thick-walled centrifuge tube add 2 ml of a 100 g/l solution of trifluoroacetic acid R, shake vigorously to dissolve the forming gel, stopper the tube and heat the mixture at 120 °C for 1 h. Centrifuge the hydrolysate, transfer the clear supernatant carefully into a 50 ml flask, add 10 ml of water R and evaporate the solution to dryness under reduced pressure. To the resulting clear film add 0.1 ml of water R and 0.9 ml of methanol R. Centrifuge to separate the amorphous precipitate. Dilute the supernatant, if necessary, to 1 ml with methanol R.

DEFINITION Calcium bis[3-(acetylamino)propane-1-sulphonate]. Content : 98.0 per cent to 102.0 per cent (dried substance).

Reference solution. Dissolve 10 mg of arabinose R, 10 mg of galactose R, 10 mg of glucose R, 10 mg of rhamnose R and 10 mg of xylose R in 1 ml of water R and dilute to 10 ml with methanol R.

CHARACTERS Appearance : white powder. Solubility : freely soluble in water, practically insoluble in alcohol and in methylene chloride.

Apply to the plate as bands 10 µl of each solution. Develop over a path of 10 cm using a mixture of 10 volumes of a 16 g/l solution of sodium dihydrogen phosphate R, 40 volumes of butanol R and 50 volumes of acetone R. Dry the plate in a current of warm air for a few minutes and develop again over a path of 15 cm using the same mobile phase. Dry the plate at 110 °C for 10 min, spray with anisaldehyde solution R and heat again at 110 °C for 10 min. The chromatogram obtained with the reference solution shows 5 clearly separated coloured zones due to galactose (greyish-green to green), glucose (grey), arabinose (yellowish-green) xylose (greenish-grey to yellowish-grey) and rhamnose (yellowish-green), in order of increasing Rf value. The chromatogram obtained with the test solution shows no grey zone and no greyish-green zone between the zones corresponding to galactose and arabinose in the chromatogram obtained with the reference solution. Starch, dextrin and agar. To 10 ml of solution S previously boiled and cooled add 0.1 ml of 0.05 M iodine. No blue or reddish-brown colour develops. 906

C10H20CaN2O8S2

Mr 400.5

IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of acamprosate calcium. B. It gives reaction (a) of calcium (2.3.1). TESTS Solution S. Dissolve 5.0 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). pH (2.2.3) : 5.5 to 7.0 for solution S. Impurity A. Liquid chromatography (2.2.29). Test solution. Dissolve 0.40 g of the substance to be examined in distilled water R and dilute to 20.0 ml with the same solvent. Dilute 10.0 ml of this solution to 100.0 ml with borate buffer solution pH 10.4 R. Place 3.0 ml of the solution obtained in a 25 ml ground-glass-stoppered


Acebutolol hydrochloride


tube. Add 0.15 ml of a freshly prepared 5 g/l solution of fluorescamine R in acetonitrile R. Shake immediately and vigorously for 30 s. Place in a water-bath at 50 °C for 30 min. Cool under a stream of cold water. Centrifuge and filter the supernatant through a suitable membrane filter (0.45 µm), 25 mm in diameter. Reference solution. Dissolve 50 mg of acamprosate impurity A CRS in distilled water R and dilute to 200.0 ml with the same solvent. Dilute 0.4 ml of the solution to 100.0 ml with borate buffer solution pH 10.4 R. Treat 3.0 ml of this solution in the same way as the test solution Column : — size : l = 0.15 m, Ø = 4.6 mm, — stationary phase : spherical octadecylsilyl silica gel for chromatography R (5 µm) with a specific surface area of 170 m2/g and a pore size of 12 nm. Mobile phase : acetonitrile R, methanol R, 0.1 M phosphate buffer solution pH 6.5 R (10:10:80 V/V/V). Flow rate : 1 ml/min. Detection : spectrophotometer at 261 nm. Injection : 20 µl. Run time : 6 times the retention time of impurity A Retention times : fluorescamine = about 4 min ; impurity A = about 8 min ; acamprosate is not detected by this system. Limits : — impurity A : not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.05 per cent). Heavy metals (2.4.8) : maximum 10 ppm. Dissolve 2.0 g in distilled water R and dilute to 20 ml with the same solvent. 12 ml of the solution complies with limit test A. Prepare the standard using 10 ml of lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32) : maximum 0.4 per cent, determined on 1.000 g by drying in an oven at 100-105 °C.

01/2005:0871

ACEBUTOLOL HYDROCHLORIDE Acebutololi hydrochloridum

C18H29ClN2O4

Mr 372.9

DEFINITION N-[3-Acetyl-4-[(2RS)-2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenyl]butanamide hydrochloride. Content : 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white or almost white, crystalline powder. Solubility : freely soluble in water and in alcohol, very slightly soluble in acetone and in methylene chloride. mp : about 143 °C.

IDENTIFICATION First identification : B, D. Second identification : A, C, D. A. Dissolve 20.0 mg in a 0.1 per cent V/V solution of hydrochloric acid R and dilute to 100.0 ml with the same acid solution. Dilute 5.0 ml to 100.0 ml with a 0.1 per cent V/V solution of hydrochloric acid R. Examined between 220 nm and 350 nm (2.2.25), the solution shows 2 absorption maxima, at 233 nm and 322 nm. The specific absorbance at the maximum at 233 nm is 555 to 605. B. Infrared absorption spectrophotometry (2.2.24). Preparation : discs. Comparison : acebutolol hydrochloride CRS. C. Thin-layer chromatography (2.2.27). ASSAY Test solution. Dissolve 20 mg of the substance to be examined in methanol R and dilute to 20 ml with the To 4 g of cation exchange resin R (75-150 µm) add 20 ml of same solvent. distilled water R and stir magnetically for 10 min. Introduce Reference solution (a). Dissolve 20 mg of acebutolol this suspension into a glass column, 45 cm long and 2.2 cm hydrochloride CRS in methanol R and dilute to 20 ml in internal diameter, equipped with a polytetrafluoroethylene with the same solvent. flow cap covered by a glass-wool plug. Allow a few millilitres of this solution to flow, then place a plug of glass wool over Reference solution (b). Dissolve 20 mg of pindolol CRS the resin. Pass 50 ml of 1 M hydrochloric acid through in methanol R and dilute to 20 ml with the same solvent. the column. The pH of the eluate is close to 1. Wash with To 1 ml add 1 ml of reference solution (a). 3 quantities, each of 200 ml, of distilled water R to obtain Plate : TLC silica gel F254 plate R. an eluate at pH 6. Dissolve 0.100 g of the substance to Mobile phase : perchloric acid R, methanol R, water R be examined in 15 ml of distilled water R. Pass through (5:395:600 V/V/V). the column and wash with 3 quantities, each of 25 ml, of Application : 10 µl. distilled water R, collecting the eluate. Allow to elute until an eluate at pH 6 is obtained. Titrate the solution obtained Development : over 3/4 of the plate. with 0.1 M sodium hydroxide, determining the end-point Drying : in air. potentiometrically (2.2.20). Detection : examine in ultraviolet light at 254 nm. 1 ml of 0.1 M sodium hydroxide corresponds to 20.02 mg of System suitability : the chromatogram obtained with C10H20CaN2O8S2. reference solution (b) shows 2 clearly separated principal spots. IMPURITIES Results : the principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). D. It gives reaction (a) of chlorides (2.3.1). A. 3-aminopropane-1-sulphonic acid (homotaurine). General Notices (1) apply to all monographs and other texts

907

Acebutolol hydrochloride


TESTS

Injection : 25 µl.

Appearance of solution. The solution is not more opalescent System suitability : reference solution (c) : than reference suspension II (2.2.1) and not more intensely — resolution : minimum 7.0 between the peaks due to coloured than reference solution BY5 (2.2.2, Method II). impurity I and acebutolol. Dissolve 0.5 g in water R and dilute to 10 ml with the same Limits : solvent. — impurity B : not more than the area of the principal peak pH (2.2.3) : 5.0 to 7.0. in the chromatogram obtained with reference solution (e) (0.2 per cent), Dissolve 0.20 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. — impurity C : not more than the area of the principal peak in the chromatogram obtained with reference solution (d) Related substances. Liquid chromatography (2.2.29). (0.1 per cent), Test solution. Dissolve 0.100 g of the substance to be — impurity I : not more than the area of the principal peak examined in mobile phase A and dilute to 50.0 ml with in the chromatogram obtained with reference solution (b) mobile phase A. (0.2 per cent), Reference solution (a). Dissolve 20.0 mg of the substance to — any other impurity : not more than the area of the be examined in mobile phase A and dilute to 100.0 ml with principal peak in the chromatogram obtained with mobile phase A. Dilute 0.5 ml to 50.0 ml with mobile phase A. reference solution (a) (0.1 per cent), Reference solution (b). Dissolve 5.0 mg of acebutolol impurity I CRS in 10.0 ml of acetonitrile R and dilute to 25.0 ml with mobile phase A. Dilute 1.0 ml to 50.0 ml with mobile phase A. Reference solution (c). Mix 2.0 ml of reference solution (a) and 1.0 ml of reference solution (b) and dilute to 10.0 ml with mobile phase A.

— total of impurities : not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent), — disregard limit : half the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). Heavy metals (2.4.8) : maximum 20 ppm.

Reference solution (d). Dissolve 5.0 mg of acebutolol impurity C CRS in 10 ml of acetonitrile R and dilute to 25.0 ml with mobile phase A. Dilute 0.5 ml to 50.0 ml with mobile phase A.

Dissolve 0.50 g of the substance to be examined in 20.0 ml of water R. The solution complies with limit test E. Prepare the standard by diluting 10.0 ml of lead standard solution (1 ppm Pb) R to 20.0 ml with water R.

Reference solution (e). Dissolve 5.0 mg of acebutolol impurity B CRS in 10.0 ml of acetonitrile R and dilute to 25.0 ml with mobile phase A. Dilute 1.0 ml to 50.0 ml with mobile phase A.

Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 3 h.

Column :

Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY

— size : l = 0.125 m, Ø = 4 mm, — stationary phase: end-capped octadecylsilyl silica gel for chromatography R (5 µm), — temperature : 40 °C.

Dissolve 0.300 g in 50 ml of alcohol R and add 1 ml of 0.1 M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the 2 points of inflexion.

Mobile phase :

1 ml of 0.1 M sodium hydroxide is equivalent to 37.29 mg of C18H29ClN2O4.

— mobile phase A : mix 2.0 ml of phosphoric acid R, and 3.0 ml of triethylamine R and dilute to 1000 ml with water R,

STORAGE Protected from light.

— mobile phase B : mix equal volumes of acetonitrile R and mobile phase A, IMPURITIES Time (min)

Mobile phase A (per cent V/V)

0-2

98

2

2 - 30.5

98 → 10

2 → 90

30.5 - 41

10

90

41 - 42

10 → 98

90 → 2

42 - 50

98

2

Flow rate : 1.2 ml/min. Detection : spectrophotometer at 240 nm. 908

Mobile phase B (per cent V/V)

Specified impurities : A, B, C, D, E, F, G, H, I, J, K.

A. N-[3-acetyl-4-[(2RS)-oxiran-2-ylmethoxy]phenyl]butanamide, See the information section on general monographs (cover pages)

Aceclofenac


01/2005:1281

ACECLOFENAC Aceclofenacum B. R1 = R2 = CO-CH3 : N-[3-acetyl-4-[(2RS)-2-hydroxy3-[(1-methylethyl)amino]propoxy]phenyl]acetamide (diacetolol), D. R1 = H, R2 = CO-CH3 : 1-[5-amino-2-[(2RS)-2-hydroxy-3-[(1methylethyl)amino]propoxy]phenyl]ethanone, E. R1 = CO-CH2-CH2-CH3, R2 = H : N-[4-[(2RS)-2-hydroxy-3[(1-methylethyl)amino]propoxy]phenyl]butanamide, J. R1 = CO-CH2-CH3, R2 = CO-CH3 : N-[3-acetyl-4-[(2RS)-2hydroxy-3-[(1-methylethyl)amino]propoxy]phenyl]propanamide, K. R1 = R2 = CO-CH2-CH2-CH3 : N-[3-butanoyl-4-[(2RS)-2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenyl]butanamide,

C. N-(3-acetyl-4-hydroxyphenyl)butanamide,

F. R = OH : N-[3-acetyl-4-[(2RS)-2,3-dihydroxypropoxy]phenyl]butanamide, I. R = NH-CH2-CH3 : N-[3-acetyl-4-[(2RS)-3-(ethylamino)-2hydroxypropoxy]phenyl]butanamide,

G. N,N′-[[(1-methylethyl)imino]bis[(2-hydroxypropane-1,3diyl)oxy(3-acetyl-1,4-phenylene)]]dibutanamide (biamine),

H. N,N′-[(2-hydroxypropane-1,3-diyl)bis[oxy(3-acetyl-1,4phenylene)]]dibutanamide. General Notices (1) apply to all monographs and other texts

C16H13Cl2NO4

Mr 354.2

DEFINITION [[[2-[(2,6-Dichlorophenyl)amino]phenyl]acetyl]oxy]acetic acid. Content : 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white or almost white, crystalline powder. Solubility : practically insoluble in water, freely soluble in acetone, soluble in alcohol. IDENTIFICATION First identification : B. Second identification : A, C. A. Dissolve 50.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the solution to 50.0 ml with methanol R. Examined between 220 nm and 370 nm (2.2.25), the solution shows an absorption maximum at 275 nm. The specific absorbance at the absorption maximum is 320 to 350. B. Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of aceclofenac. C. Dissolve about 10 mg in 10 ml of alcohol R. To 1 ml of the solution, add 0.2 ml of a mixture, prepared immediately before use, of equal volumes of a 6 g/l solution of potassium ferricyanide R and a 9 g/l solution of ferric chloride R. Allow to stand protected from light for 5 min. Add 3 ml of a 10.0 g/l solution of hydrochloric acid R. Allow to stand protected from light for 15 min. A blue colour develops and a precipitate is formed. TESTS Related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately before use. Test solution. Dissolve 50.0 mg of the substance to be examined in a mixture of 30 volumes of mobile phase A and 70 volumes of mobile phase B and dilute to 25.0 ml with the same mixture of solvents. Reference solution (a). Dissolve 21.6 mg of diclofenac sodium CRS in a mixture of 30 volumes of mobile phase A and 70 volumes of mobile phase B and dilute to 50.0 ml with the same mixture of solvents. Reference solution (b). Dilute 2.0 ml of the test solution to 10.0 ml with a mixture of 30 volumes of mobile phase A and 70 volumes of mobile phase B. Reference solution (c). Mix 1.0 ml of reference solution (a) and 1.0 ml of reference solution (b) and dilute to 100.0 ml with a mixture of 30 volumes of mobile phase A and 70 volumes of mobile phase B. 909

Aceclofenac


— impurity I : not more than the area of the corresponding Reference solution (d). Dissolve 4.0 mg of aceclofenac peak in the chromatogram obtained with reference impurity F CRS, 2.0 mg of aceclofenac impurity H CRS solution (e) (0.1 per cent), and 2.0 mg of diclofenac impurity A CRS (aceclofenac impurity I) in a mixture of 30 volumes of mobile phase A and — any other impurity : not more than half the area of the 70 volumes of mobile phase B and dilute to 10.0 ml with peak due to aceclofenac in the chromatogram obtained the same mixture of solvents. with reference solution (e) (0.1 per cent), Reference solution (e). Mix 1.0 ml of reference solution (b) — total : not more than 0.7 per cent, and 1.0 ml of reference solution (d) and dilute to 100.0 ml with a mixture of 30 volumes of mobile phase A and — disregard limit : 0.1 times the area of the peak due to 70 volumes of mobile phase B. aceclofenac in the chromatogram obtained with reference solution (e) (0.02 per cent). Column : Heavy metals (2.4.8) : maximum 10 ppm. — size : l = 0.25 m, Ø = 4.6 mm, To 2.0 g in a silica crucible, add 2 ml of sulphuric acid R — stationary phase: spherical end-capped octadecylsilyl to wet the substance. Heat progressively to ignition silica gel for chromatography R (5 µm) with a pore size and continue heating until an almost white or at most a of 10 nm and a carbon loading of 19 per cent, greyish residue is obtained. Carry out the ignition at a temperature not exceeding 800 °C. Allow to cool. Add 3 ml — temperature : 40 °C. of hydrochloric acid R and 1 ml of nitric acid R. Heat and Mobile phase : evaporate slowly to dryness. Cool and add 1 ml of a 100 g/l solution of hydrochloric acid R and 10.0 ml of distilled — mobile phase A : 1.12 g/l solution of phosphoric acid R water R. Neutralise with a 1.0 g/l solution of ammonia R adjusted to pH 7.0 using a 42 g/l solution of sodium using 0.1 ml of phenolphthalein solution R as indicator. Add hydroxide R, 2.0 ml of a 60 g/l solution of anhydrous acetic acid R and dilute to 20 ml with distilled water R. 12 ml of the solution — mobile phase B : water R, acetonitrile R (1:9 V/V), complies with limit test A. Prepare the standard using lead standard solution (1 ppm Pb) R. Time Mobile phase A Mobile phase B (min)

(per cent V/V)

(per cent V/V)

0 - 25

70 → 50

30 → 50

25 - 30

50 → 20

50 → 80

30 - 50

20

80

50 - 52

20 → 70

80 → 30

52 - 65

70

30

Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.300 g in 40 ml of methanol R. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20).

Flow rate : 1.0 ml/min. Detection : spectrophotometer at 275 nm.

1 ml of 0.1 M sodium hydroxide is equivalent to 35.42 mg of C16H13Cl2NO4.

Injection : 10 µl ; inject the test solution and reference solutions (c) and (e). Relative retention with reference to aceclofenac (retention time = about 14 min) : impurity A = about 0.8 ; impurity G = about 1.3 ; impurity H = about 1.5 ; impurity I = about 2.3 ; impurity D = about 2.6 ; impurity B = about 2.7 ; impurity E = about 2.8 ; impurity C = about 3.0 ; impurity F = about 3.2.

STORAGE In an airtight container, protected from light. IMPURITIES

System suitability : reference solution (c) : — resolution : minimum 5.0 between the peaks due to aceclofenac and to impurity A. Limits : — impurity A : not more than the area of the corresponding peak in the chromatogram obtained with reference solution (c) (0.2 per cent), — impurities B, C, D, E, G : for each impurity, not more than the area of the peak due to aceclofenac in the chromatogram obtained with reference solution (e) (0.2 per cent),

A. R = H : [2-[(2,6-dichlorophenyl)amino]phenyl]acetic acid (diclofenac),

— impurity F : not more than the area of the corresponding peak in the chromatogram obtained with reference solution (e) (0.2 per cent),

B. R = CH3 : methyl [2-[(2,6-dichlorophenyl)amino]phenyl]acetate (methyl ester of diclofenac),

— impurity H : not more than the area of the corresponding peak in the chromatogram obtained with reference C. R = C2H5 : ethyl [2-[(2,6-dichlorophenyl)amino]phenyl]acesolution (e) (0.1 per cent), tate (ethyl ester of diclofenac), 910



Acesulfame potassium

Test solution. Dissolve 5 mg of the substance to be examined in water R and dilute to 5 ml with the same solvent. Reference solution (a). Dissolve 5 mg of acesulfame potassium CRS in water R and dilute to 5 ml with the same solvent. Reference solution (b). Dissolve 5 mg of acesulfame potassium CRS and 5 mg of saccharin sodium R in water R and dilute to 5 ml with the same solvent. D. R = CH3 : methyl [[[2-[(2,6-dichlorophenyl)amino]pheApply to the plate as bands 5 µl of each solution. Develop nyl]acetyl]oxy]acetate (methyl ester of aceclofenac), twice over a path of 15 cm using a mixture of 10 volumes ofconcentrated ammonia R, 60 volumes of acetone R E. R = C2H5 : ethyl [[[2-[(2,6-dichlorophenyl)amino]pheand 60 volumes of ethyl acetate R. Dry the plate in a nyl]acetyl]oxy]acetate (ethyl ester of aceclofenac), current of warm air and examine in ultraviolet light at 254 nm. The principal band in the chromatogram F. R = CH2-C6H5 : benzyl [[[2-[(2,6-dichlorophenyl)amino]pheobtained with the test solution is similar in position and nyl]acetyl]oxy]acetate (benzyl ester of aceclofenac), size to the principal band in the chromatogram obtained G. R = CH2-CO2H : [[[[[2-[(2,6-dichlorophenyl)amino]phewith reference solution (a). The test is not valid unless nyl]acetyl]oxy]acetyl]oxy]acetic acid (acetic aceclofenac), the chromatogram obtained with reference solution (b) shows two clearly separated bands. H. R = CH2-CO-O-CH2-CO2H : [[[[[[[2-[(2,6-dichlorophenyl)amiC. 0.5 ml of solution S (see Tests) gives reaction (b) of no]phenyl]acetyl]oxy]acetyl]oxy]acetyl]oxy]acetic acid potassium (2.3.1). (diacetic aceclofenac), TESTS Solution S. Dissolve 10.0 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Acidity or alkalinity. To 20 ml of solution S add 0.1 ml of bromothymol blue solution R1. Not more than 0.2 ml of I. 1-(2,6-dichlorophenyl)-1,3-dihydro-2H-indol-2-one. 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is required to change the colour of the indicator. 01/2005:1282 Acetylacetamide. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating substance. Test solution. Dissolve 0.80 g of the substance to be ACESULFAME POTASSIUM examined in water R and dilute to 10 ml with the same solvent. Acesulfamum kalicum Reference solution (a). Dissolve 50 mg of acetylacetamide R in water R and dilute to 25 ml with the same solvent. To 5 ml of the solution add 45 ml of water R and dilute to 100 ml with methanol R. Reference solution (b). To 10 ml of reference solution (a) add 1 ml of the test solution and dilute to 20 ml with methanol R. Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 2 volumes of water R, C4H4KNO4S Mr 201.2 15 volumes of alcohol R and 74 volumes of ethyl acetate R. Allow the plate to dry in air until the solvents are completely DEFINITION removed. Spray the plate with vanillin phosphoric solution R and heat at 120 °C for about 10 min. Examine Acesulfame potassium contains not less than 99.0 per in daylight. Any spot corresponding to acetylacetamide cent and not more than the equivalent of 101.0 per cent in the chromatogram obtained with the test solution, of potassium 6-methyl-1,2,3-oxathiazin-4-olate 2,2-dioxide, is not more intense than the spot in the chromatogram calculated with reference to the dried substance. obtained with reference solution (a) (0.125 per cent). The CHARACTERS test is not valid unless the chromatogram obtained with A white, crystalline powder or colourless crystals, soluble in reference solution (a) shows a clearly visible spot and the chromatogram obtained with reference solution (b) shows water, very slightly soluble in acetone and in alcohol. two clearly separated spots. IDENTIFICATION Impurity B and related substances. Examine by liquid First identification : A, C. chromatography (2.2.29). Second identification : B, C. Test solution. Dissolve 0.100 g of the substance to be A. Examine by infrared absorption spectrophotometry examined in water R and dilute to 10.0 ml with the same (2.2.24), comparing with the spectrum obtained with solvent. acesulfame potassium CRS. Examine the substances Reference solution (a). Dissolve 20.0 mg of acesulfame prepared as discs. potassium impurity B CRS in water R and dilute to 500.0 ml with the same solvent. Dilute 0.5 ml of this solution to B. Examine by thin-layer chromatography (2.2.27), using cellulose for chromatography R as the coating substance. 100.0 ml with water R. General Notices (1) apply to all monographs and other texts

911

Acetazolamide


Reference solution (b). Dissolve 10.0 mg of acesulfame potassium impurity B CRS and 10.0 mg of acesulfame potassium CRS in water R and dilute to 500.0 ml with the same solvent. Dilute 5.0 ml of this solution to 100.0 ml with water R. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (3 µm),

B. 5-chloro-6-methyl-1,2,3-oxathiazin-4(3H)-one 2,2-dioxide.

— as mobile phase at a flow rate of 1 ml/min a mixture of 40 volumes of acetonitrile R and 60 volumes of a 3.3 g/l solution of tetrabutylammonium hydrogen sulphate R,

01/2005:0454

ACETAZOLAMIDE

— as detector a spectrophotometer set at 234 nm. Inject 20 µl of reference solution (b). Adjust the sensitivity of the system so that the height of the two principal peaks is at least 50 per cent of the full scale of the recorder. The test is not valid unless the resolution between the two peaks due to acesulfame and impurity B is at least 3.0. Inject 20 µl of the test solution and 20 µl of reference solution (a). Continue the chromatography of the test solution for at least three times the retention time of the principal peak. In the chromatogram obtained with the test solution the area of any peak apart from the principal peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (20 ppm). Fluorides. Not more than 3 ppm of F, determined potentiometrically (2.2.36, Method I) using as indicator electrode a fluoride selective-membrane electrode and as reference electrode a silver-silver chloride electrode. Test solution. Dissolve 3.000 g of the substance to be examined in distilled water R, add 15.0 ml of total ionic strength adjustment buffer R1 and dilute to 50.0 ml with distilled water R.

Acetazolamidum

C4 H 6 N 4 O 3 S 2

Mr 222.2

DEFINITION Acetazolamide contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of N-(5-sulphamoyl-1,3,4-thiadiazol-2-yl)acetamide, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, very slightly soluble in water, slightly soluble in alcohol. It dissolves in dilute solutions of alkali hydroxides.

IDENTIFICATION Reference solutions. To 0.5 ml, 1.0 ml, 1.5 ml and 3.0 ml of First identification : A, B. fluoride standard solution (10 ppm F) R add 15.0 ml of total Second identification : A, C, D. ionic strength adjustment buffer R1 and dilute to 50.0 ml with distilled water R. A. Dissolve 30.0 mg in 0.01 M sodium hydroxide and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of Carry out the measurements of each solution. the solution to 100.0 ml with 0.01 M sodium hydroxide Heavy metals (2.4.8). 12 ml of solution S complies with the (solution A). Examined between 230 nm and 260 nm limit test A for heavy metals (5 ppm). Prepare the standard (2.2.25), solution A shows an absorption maximum at using lead standard solution (1 ppm Pb) R. 240 nm. The specific absorbance at the maximum is 162 to 176. Dilute 25.0 ml of solution A to 100.0 ml with Loss on drying (2.2.32). Not more than 1.0 per cent, 0.01 M sodium hydroxide. Examined between 260 nm determined on 1.000 g by drying in an oven at 100 °C to and 350 nm, the solution shows an absorption maximum 105 °C for 3 h. at 292 nm. The specific absorbance at the maximum is 570 to 620. ASSAY B. Examine by infrared absorption spectrophotometry Dissolve 0.150 g in 50 ml of anhydrous acetic acid R. Titrate (2.2.24), comparing with the spectrum obtained with with 0.1 M perchloric acid, determining the end-point acetazolamide CRS. If the spectra obtained in the solid potentiometrically (2.2.20). state with the substance to be examined and the reference substance show differences, dissolve separately the 1 ml of 0.1 M perchloric acid is equivalent to 20.12 mg of substance to be examined and the reference substance in C4H4KNO4S. alcohol R, evaporate to dryness, prepare new discs and record the spectra. IMPURITIES C. Introduce about 20 mg into a test-tube and add 4 ml of dilute hydrochloric acid R and 0.2 g of zinc powder R. Immediately place a piece of lead acetate paper R over the mouth of the tube. The paper shows a brownish-black colour. D. Dissolve about 25 mg in a mixture of 0.1 ml of dilute sodium hydroxide solution R and 5 ml of water R. Add 0.1 ml of copper sulphate solution R. A greenish-blue precipitate is formed. A. 3-oxobutanamide (acetylacetamide), 912


Acetone


TESTS Appearance of solution. Dissolve 1.0 g in 10 ml of 1 M sodium hydroxide. The solution is not more opalescent than reference suspension II (2.2.1) and not more intensely coloured than reference solution Y5 or BY5 (2.2.2, Method II). Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 50 mg of the substance to be examined in a mixture of equal volumes of alcohol R and ethyl acetate R and dilute to 10 ml with the same mixture of solvents. Reference solution. Dilute 1 ml of the test solution to 100 ml with a mixture of equal volumes of alcohol R and ethyl acetate R. Apply separately to the plate 20 µl of each solution. Use the tank without lining the walls and allow to saturate for 1 h before development. Develop over a path of 15 cm using a freshly prepared mixture of 20 volumes of concentrated ammonia R, 30 volumes of ethyl acetate R and 50 volumes of 2-propanol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution (1 per cent). Sulphates (2.4.13). To 0.4 g add 20 ml of distilled water R and dissolve by heating to boiling. Allow to cool with frequent shaking and filter. 15 ml of the filtrate complies with the limit test for sulphates (500 ppm). Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.200 g in 25 ml of dimethylformamide R. Titrate with 0.1 M ethanolic sodium hydroxide, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M ethanolic sodium hydroxide is equivalent to 22.22 mg of C4H6N4O3S2.

IDENTIFICATION A. A 100 g/l solution is strongly acid (2.2.4). B. To 0.03 ml add 3 ml of water R and neutralise with dilute sodium hydroxide solution R. The solution gives reaction (b) of acetates (2.3.1). TESTS Solution S. Dilute 20 ml to 100 ml with distilled water R. Appearance. It is clear (2.2.1) and colourless (2.2.2, Method II). Freezing point (2.2.18). Not less than 14.8 °C. Reducing substances. To 5.0 ml add 10.0 ml of water R and mix. To 5.0 ml of the solution add 6 ml of sulphuric acid R, cool and add 2.0 ml of 0.0167 M potassium dichromate. Allow to stand for 1 min and add 25 ml of water R and 1 ml of a freshly prepared 100 g/l solution of potassium iodide R. Titrate with 0.1 M sodium thiosulphate, using 1.0 ml of starch solution R as indicator. Not less than 1.0 ml of 0.1 M sodium thiosulphate solution is required. Chlorides (2.4.4). 10 ml of solution S diluted to 15 ml with water R complies with the limit test for chlorides (25 mg/l). Sulphates (2.4.13). 15 ml of solution S complies with the limit test for sulphates (50 mg/l). Iron (2.4.9). 5.0 ml of solution (a) obtained in the test for heavy metals diluted to 10.0 ml with water R complies with the limit test for iron (5 ppm). Heavy metals (2.4.8). Dissolve the residue obtained in the test for residue on evaporation by heating with two quantities, each of 15 ml, of water R and dilute to 50.0 ml (solution (a)). 12 ml of solution (a) complies with limit test A for heavy metals (5 ppm). Prepare the standard using lead standard solution (2 ppm Pb) R. Residue on evaporation. Evaporate 20 g to dryness on a water-bath and dry at 100 °C to 105 °C. The residue weighs not more than 2.0 mg (0.01 per cent). ASSAY Weigh accurately a conical flask with a ground-glass stopper containing 25 ml of water R. Add 1.0 ml of the substance to be examined and weigh again accurately. Add 0.5 ml of phenolphthalein solution R and titrate with 1 M sodium hydroxide. 1 ml of 1 M sodium hydroxide is equivalent to 60.1 mg of C2 H 4 O 2 .

01/2005:0590 STORAGE Store in an airtight container.

ACETIC ACID, GLACIAL

01/2005:0872

Acidum aceticum glaciale

ACETONE Acetonum

C 2 H 4 O2

Mr 60.1

DEFINITION Glacial acetic acid contains not less than 99.0 per cent m/m and not more than the equivalent of 100.5 per cent m/m of C2H4O2. CHARACTERS A crystalline mass or a clear, colourless, volatile liquid, miscible with water, with alcohol and with methylene chloride. General Notices (1) apply to all monographs and other texts

C3 H 6 O

Mr 58.08

DEFINITION Acetone is propan-2-one. CHARACTERS A volatile clear, colourless liquid, miscible with water and with alcohol. The vapour is flammable. 913

Acetylcholine chloride


IDENTIFICATION A. To 1 ml, add 3 ml of dilute sodium hydroxide solution R and 0.3 ml of a 25 g/l solution of sodium nitroprusside R. An intense red colour is produced which becomes violet with the addition of 3.5 ml of acetic acid R. B. To 10 ml of a 0.1 per cent V/V solution in alcohol (50 per cent V/V) R, add 1 ml of a 10 g/l solution of nitrobenzaldehyde R in the same solvent and 0.5 ml of strong sodium hydroxide solution R. Allow to stand for about 2 min and acidify with acetic acid R. A greenish-blue colour is produced. TESTS Appearance of solution. To 10 ml add 10 ml of water R. The solution is clear (2.2.1) and colourless (2.2.2, Method II). Acidity or alkalinity. To 5 ml add 5 ml of carbon dioxide-free water R, 0.15 ml of phenolphthalein solution R and 0.5 ml of 0.01 M sodium hydroxide. The solution is pink. Add 0.7 ml of 0.01 M hydrochloric acid and 0.05 ml of methyl red solution R. The solution is red or orange. Relative density (2.2.5). 0.790 to 0.793. Related substances. Examine by gas chromatography (2.2.28). Test solution. The substance to be examined. Reference solution. To 0.5 ml of methanol R add 0.5 ml of 2-propanol R and dilute to 100.0 ml with the test solution. Dilute 1.0 ml to 10.0 ml with the test solution. The chromatographic procedure may be carried out using : — a fused-silica column 50 m long and 0.3 mm in internal diameter coated with a film (0.5 µm) of macrogol 20 000 R, — helium for chromatography R as the carrier gas with a split ratio of about 50:1 and at a linear flow of 21 cm/s, — a flame-ionisation detector, maintaining the temperature of the column at 45 °C until injection, then raising the temperature at a rate of 5 °C per minute to 100 °C and maintaining the temperature of the injection port at 150 °C and that of the detector at 250 °C. Inject 1 µl of the test solution and 1 µl of the reference solution. When the chromatograms are recorded in the conditions described above, the substances are eluted in the following order : acetone, methanol, 2-propanol. Continue the chromatography for three times the retention time of acetone (which is about 5.3 min). The test is not valid unless, in the chromatogram obtained with the reference solution, the resolution between the peaks corresponding to methanol and 2-propanol is at least 1.0. In the chromatogram obtained with the test solution : the area of any peak corresponding to methanol or 2-propanol is not greater than the difference between the areas of the corresponding peaks in the chromatogram obtained with the reference solution and the areas of the corresponding peaks in the chromatogram obtained with the test solution (0.05 per cent V/V for each impurity) ; the area of any peak, apart from the principal peak and any peaks corresponding to methanol and 2-propanol, is not greater than the difference between the area of the methanol peak in the chromatogram obtained with the reference solution and the area of the corresponding peak in the chromatogram obtained with the test solution (0.05 per cent V/V for each of the additional impurities). Matter insoluble in water. To 1 ml add 19 ml of water R. The solution is clear (2.2.1). 914

Reducing substances. To 30 ml add 0.1 ml of 0.02 M potassium permanganate and allow to stand in the dark for 2 h. The mixture is not completely decolourised. Residue on evaporation. Evaporate 20.0 g to dryness on a water-bath and dry at 100 °C to 105 °C. The residue weighs not more than 1 mg (50 ppm). Water (2.5.12). Not more than 3 g/l, determined on 10.0 ml by the semi-micro determination of water. Use 20 ml of anhydrous pyridine R as solvent. STORAGE Store protected from light. IMPURITIES A. CH3-OH : methanol, B. propan-2-ol. 01/2005:1485 corrected

ACETYLCHOLINE CHLORIDE Acetylcholini chloridum

C7H16ClNO2

Mr 181.7

DEFINITION 2-(Acetyloxy)-N,N,N-trimethylethanaminium chloride. Content : 98.5 per cent to 101.5 per cent (dried substance). CHARACTERS Appearance : white or almost white crystalline powder or colourless crystals, very hygroscopic. Solubility : very soluble in water, freely soluble in alcohol, slightly soluble in methylene chloride. IDENTIFICATION First identification : B, E. Second identification : A, C, D, E. A. Melting point (2.2.14) : 149 °C to 152 °C. Introduce the substance to be examined into a capillary tube. Dry in an oven at 100-105 °C for 3 h. Seal the tube and determine the melting point. B. Infrared absorption spectrophotometry (2.2.24). Comparison : acetylcholine chloride CRS. C. Examine the chromatograms obtained in the test for related substances. Results : the principal band in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal band in the chromatogram obtained with reference solution (b). D. To 15 mg add 10 ml of dilute sodium hydroxide solution R, 2 ml of 0.02 M potassium permanganate and heat. The vapours formed change the colour of red litmus paper R to blue. E. 0.5 ml of solution S (see Tests) gives reaction (a) of chlorides (2.3.1). TESTS Solution S. Dissolve 5.0 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent.


Acetylcysteine


Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y6 or BY6 (2.2.2, Method II). Acidity. Dilute 1 ml of solution S to 10 ml with carbon dioxide-free water R. Add 0.05 ml of phenolphthalein solution R. Not more than 0.4 ml of 0.01 M sodium hydroxide is required to change the colour of the indicator to pink. Related substances. Thin-layer chromatography (2.2.27). Prepare the solutions immediately before use. Test solution (a). Dissolve 0.30 g of the substance to be examined in methanol R and dilute to 3.0 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with methanol R. Reference solution (a). Dilute 1 ml of test solution (a) to 100 ml with methanol R. Reference solution (b). Dissolve 20.0 mg of acetylcholine chloride CRS in methanol R and dilute to 2.0 ml with the same solvent. Reference solution (c). Dissolve 20 mg of choline chloride R in methanol R, add 0.4 ml of test solution (a) and dilute to 2.0 ml with methanol R. Plate : TLC silica gel plate R. Mobile phase : mix 20 volumes of a 40 g/l solution of ammonium nitrate R, 20 volumes of methanol R and 60 volumes of acetonitrile R. Application : 5 µl as bands of 10 mm by 2 mm. Development : over 2/3 of the plate. Detection : spray with potassium iodobismuthate solution R3. System suitability : the chromatogram obtained with reference solution (c) shows 2 clearly separated bands. Limits : — any impurity : any bands in the chromatogram obtained with test solution (a), apart from the principal band, are not more intense than the principal band in the chromatogram obtained with reference solution (a) (1 per cent). Trimethylamine. Dissolve 0.1 g in 10 ml of sodium carbonate solution R and heat to boiling. No vapours appear which turn red litmus paper R blue. Heavy metals (2.4.8) : maximum 10 ppm. 12 ml of solution S complies with limit test A. Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32) : maximum 1.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 3 h. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on the residue obtained in the test for loss on drying.

IMPURITIES

A. 2-hydroxy-N,N,N-trimethylethanaminium chloride (choline chloride),

B. 2-(acetyloxy)-N,N-dimethylethanaminium chloride,

C. N,N-dimethylmethanamine. 01/2005:0967 corrected

ACETYLCYSTEINE Acetylcysteinum

C5H9NO3S

Mr 163.2

DEFINITION Acetylcysteine contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of (2R)-2-(acetylamino)-3-sulfanylpropanoic acid, calculated with reference to the dried substance. CHARACTERS A white, crystalline powder or colourless crystals, freely soluble in water and in alcohol, practically insoluble in methylene chloride.

IDENTIFICATION First identification : A, C. Second identification : A, B, D, E. A. It complies with the test for specific optical rotation (see Tests). B. Melting point (2.2.14) : 104 °C to 110 °C. C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with acetylcysteine CRS. Examine the substances prepared as discs using potassium bromide R. D. Examine the chromatograms obtained in the test for related substances. The retention time and size of the ASSAY principal peak in the chromatogram obtained with test Dissolve 0.200 g in 20 ml of carbon dioxide-free water R. solution (b) are approximately the same as those of Neutralise with 0.01 M sodium hydroxide using 0.15 ml the principal peak in the chromatogram obtained with of phenolphthalein solution R as indicator. Add 20.0 ml reference solution (b). of 0.1 M sodium hydroxide and allow to stand for 30 min. E. To 0.5 ml of solution S (see Tests) add 0.05 ml of a Titrate with 0.1 M hydrochloric acid. 50 g/l solution of sodium nitroprusside R and 0.05 ml of 1 ml of 0.1 M sodium hydroxide is equivalent to 18.17 mg of concentrated ammonia R. A dark violet colour develops. C7H16ClNO2. TESTS STORAGE Solution S. Dissolve 1.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. In ampoules, protected from light.


915

Acetylcysteine


Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). pH (2.2.3). To 2 ml of solution S add 8 ml of carbon dioxide-free water R and mix. The pH of the solution is 2.0 to 2.8. Specific optical rotation (2.2.7). In a 25 ml volumetric flask, mix 1.25 g with 1 ml of a 10 g/l solution of sodium edetate R. Add 7.5 ml of a 40 g/l solution of sodium hydroxide R, mix and dissolve. Dilute to 25.0 ml with phosphate buffer solution pH 7.0 R2. The specific optical rotation is + 21.0 to + 27.0, calculated with reference to the dried substance. Related substances. Examine by liquid chromatography (2.2.29). Except where otherwise prescribed, prepare the solutions immediately before use. Test solution (a). Suspend 0.80 g of the substance to be examined in 1 ml of 1 M hydrochloric acid and dilute to 100.0 ml with water R. Test solution (b). Dilute 5.0 ml of test solution (a) to 100.0 ml with water R. Dilute 5.0 ml of the solution to 50.0 ml with water R. Test solution (c). Use test solution (a) after storage for at least 1 h. Reference solution (a). Suspend 4.0 mg of acetylcysteine CRS, 4.0 mg of L-cystine R, 4.0 mg of L-cysteine R, 4.0 mg of acetylcysteine impurity C CRS and 4.0 mg of acetylcysteine impurity D CRS in 1 ml of 1 M hydrochloric acid and dilute to 100.0 ml with water R. Reference solution (b). Suspend 4.0 mg of acetylcysteine CRS in 1 ml of 1 M hydrochloric acid and dilute to 100.0 ml with water R. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate at 1.0 ml/min a mixture prepared as follows : stir 3 volumes of acetonitrile R and 97 volumes of water R in a beaker ; adjust to pH 3.0 with phosphoric acid R, — as detector a spectrophotometer set at 220 nm. When the chromatograms are recorded in the prescribed conditions, the retention times are : L-cystine, about 2.2 min ; L-cysteine, about 2.4 min ; 2-methyl-2-thiazoline-4-carboxylic acid, originating in test solution (c), about 3.3 min ; acetylcysteine, about 6.4 min ; acetylcysteine impurity C, about 12 min ; acetylcysteine impurity D, about 14 min. Inject 20 µl of reference solution (a). The test is not valid unless : — in the chromatogram obtained with reference solution (a), the resolution between the peaks due to L-cystine and L-cysteine is at least 1.5, — in the chromatogram obtained with reference solution (a), the resolution between the peaks due to acetylcysteine impurity C and acetylcysteine impurity D is at least 2.0. Inject 20 µl of 0.01 M hydrochloric acid as a blank. Inject three times 20 µl of reference solution (a), 20 µl of reference solution (b) and 20 µl of each test solution. Continue the chromatography for five times the retention time of acetylcysteine which is about 30 min. From the chromatogram obtained with test solution (a), calculate the percentage content of the known impurities and the unknown impurities using the following expressions :

A1

=

peak area of individual impurity (L-cystine, acetylcysteine impurity C and acetylcysteine impurity D) in the chromatogram obtained with test solution (a),

A2

=

A3

=

peak area of the corresponding individual impurity (L-cystine, L-cysteine, acetylcysteine impurity C and acetylcysteine impurity D) in the chromatogram obtained with reference solution (a), peak area of unknown impurity in the chromatogram obtained with test solution (a),

A4

=

peak area of acetylcysteine in the chromatogram obtained with reference solution (b),

m1

=

mass of the substance to be examined in test solution (a),

m2

=

mass of the individual impurity in reference solution (a),

m3

=

mass of acetylcysteine in reference solution (b).

L-cysteine,

The percentage content of each known impurity and of each unknown impurity is not greater than 0.5 per cent. The sum of the calculated percentage contents of known and unknown impurities is not greater than 0.5 per cent Disregard any peak due to the solvent, any peak appearing at retention time of about 3.3 min corresponding to 2-methyl-2-thiazoline-4-carboxylic acid and any peak with an area less than 0.1 times that of the principal peak in the chromatogram obtained with reference solution (b). Heavy metals (2.4.8). 2.0 g complies with limit test C for heavy metals (10 ppm). Prepare the standard using 2 ml of lead standard (10 ppm Pb) R. Zinc. Not more than 10 ppm of Zn, determined by atomic absorption spectrometry (2.2.23, Method II). Test solution. Dissolve 1.00 g in 0.001 M hydrochloric acid and dilute to 50.0 ml with the same acid. Reference solutions. Prepare the reference solutions using zinc standard solution (5 mg/ml Zn) R, diluted with 0.001 M hydrochloric acid. Measure the absorbance at 213.8 nm using a zinc hollow-cathode lamp as the source of radiation, an air-acetylene flame and a correction procedure for non-specific absorption. Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g by drying in an oven in vacuo at 70 °C for 3 h. Sulphated ash (2.4.14). Not more than 0.2 per cent, determined on 1.0 g. ASSAY Dissolve 0.140 g in 60 ml of water R and add 10 ml of dilute hydrochloric acid R. After cooling in iced water, add 10 ml of potassium iodide solution R and titrate with 0.05 M iodine, using 1 ml of starch solution R as indicator. 1 ml of 0.05 M iodine is equivalent to 16.32 mg of C5H9NO3S. STORAGE Store protected from light. IMPURITIES A. L-cystine, B. L-cysteine,

916



Acetylsalicylic acid

TESTS Appearance of solution. Dissolve 1.0 g in 9 ml of alcohol R. The solution is clear (2.2.1) and colourless (2.2.2, Method II). Related substances. Examine by liquid chromatography (2.2.29). Prepare the solutions immediately before use. Test solution. Dissolve 0.10 g of the substance to be examined in acetonitrile for chromatography R and dilute to 10.0 ml with the same solvent. C. N,N′-diacetyl-L-cystine, Reference solution (a). Dissolve 50.0 mg of salicylic acid R in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (b). Dissolve 10.0 mg of salicylic acid R in the mobile phase and dilute to 10.0 ml with the mobile phase. To 1.0 ml of this solution add 0.2 ml of the test solution and dilute to 100.0 ml with the mobile phase. D. N,S-diacetyl-L-cysteine. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for 01/2005:0309 chromatography R (5 µm), — as mobile phase at a flow rate of 1 ml/min a mixture ACETYLSALICYLIC ACID of 2 volumes of phosphoric acid R, 400 volumes of acetonitrile for chromatography R and 600 volumes of Acidum acetylsalicylicum water R, — as detector a spectrophotometer set at 237 nm. Inject 10 µl of each solution. Continue the chromatography of the test solution for seven times the retention time of acetylsalicylic acid. The test is not valid unless in the chromatogram obtained with reference solution (b), the resolution between the two principal peaks is at least 6.0. C 9 H 8 O4 Mr 180.2 In the chromatogram obtained with the test solution the area of any peak, apart from the principal peak, is not greater DEFINITION than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent) ; the sum Acetylsalicylic acid contains not less than 99.5 per cent of the areas of all the peaks is not greater than 2.5 times the and not more than the equivalent of 101.0 per cent of area of the principal peak in the chromatogram obtained 2-(acetyloxy)benzoic acid, calculated with reference to the with reference solution (a) (0.25 per cent). Disregard any dried substance. peak with an area less than 0.25 times the area of the CHARACTERS principal peak in the chromatogram obtained with reference A white, crystalline powder or colourless crystals, slightly solution (a). soluble in water, freely soluble in alcohol. Heavy metals (2.4.8). Dissolve 1.0 g in 12 ml of acetone R It melts at about 143 °C (instantaneous method). and dilute to 20 ml with water R. 12 ml of this solution complies with limit test B for heavy metals (20 ppm). Prepare IDENTIFICATION the standard using lead standard solution (1 ppm Pb) First identification : A, B. obtained by diluting lead standard solution (100 ppm Pb) R Second identification : B, C, D. with a mixture of 6 volumes of water R and 9 volumes of acetone R. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with Loss on drying (2.2.32). Not more than 0.5 per cent, acetylsalicylic acid CRS. determined on 1.000 g by drying in vacuo. B. To 0.2 g add 4 ml of dilute sodium hydroxide solution R Sulphated ash (2.4.14). Not more than 0.1 per cent, and boil for 3 min. Cool and add 5 ml of dilute sulphuric determined on 1.0 g. acid R. A crystalline precipitate is formed. Filter, wash the precipitate and dry at 100 °C to 105 °C. The melting ASSAY point (2.2.14) is 156 °C to 161 °C. In a flask with a ground-glass stopper, dissolve 1.000 g in C. In a test tube mix 0.1 g with 0.5 g of calcium hydroxide R. 10 ml of alcohol R. Add 50.0 ml of 0.5 M sodium hydroxide. Heat the mixture and expose to the fumes produced Close the flask and allow to stand for 1 h. Using 0.2 ml of a piece of filter paper impregnated with 0.05 ml of phenolphthalein solution R as indicator, titrate with 0.5 M nitrobenzaldehyde solution R. A greenish-blue or hydrochloric acid. Carry out a blank titration. greenish-yellow colour develops on the paper. Moisten 1 ml of 0.5 M sodium hydroxide is equivalent to 45.04 mg the paper with dilute hydrochloric acid R. The colour of C9H8O4. becomes blue. D. Dissolve with heating about 20 mg of the precipitate obtained in identification test B in 10 ml of water R and STORAGE cool. The solution gives reaction (a) of salicylates (2.3.1). Store in an airtight container. General Notices (1) apply to all monographs and other texts

917

N-Acetyltryptophan


IMPURITIES

A. R = H : 4-hydroxybenzoic acid, B. R = CO2H : 4-hydroxybenzene-1,3-dicarboxylic acid (4-hydroxyisophthalic acid), C. salicylic acid,

D. R = O-CO-CH3 : 2-[[2-(acetyloxy)benzoyl]oxy]benzoic acid (acetylsalicylsalicylic acid), E. R = OH : 2-[(2-hydroxybenzoyl)oxy]benzoic acid (salicylsalicylic acid),

F. 2-(acetyloxy)benzoic anhydride (acetylsalicylic anhydride).

Second identification : A, C, D, E. A. It complies with the test for optical rotation (see Tests). B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with N-acetyltryptophan CRS. C. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R. Test solution. Dissolve 50 mg of the substance to be examined in 0.2 ml of concentrated ammonia R and dilute to 10 ml with water R. Reference solution (a). Dissolve 50 mg of N-acetyltryptophan CRS in 0.2 ml of concentrated ammonia R and dilute to 10 ml with water R. Reference solution (b). Dissolve 10 mg of tryptophan R in the test solution and dilute to 2 ml with the same solution. Apply to the plate 2 µl of each solution. Develop over a path of 10 cm using a mixture of 25 volumes of glacial acetic acid R, 25 volumes of water R and 50 volumes of butanol R. Dry the plate in an oven at 100-105 °C for 15 min and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. D. Dissolve about 2 mg in 2 ml of water R. Add 2 ml of dimethylaminobenzaldehyde solution R6. Heat on a water-bath. A blue or greenish-blue colour develops. E. It gives the reaction of acetyl (2.3.1). Proceed as described for substances hydrolysable only with difficulty.

TESTS 01/2005:1383 Appearance of solution. Dissolve 1.0 g in a 40 g/l solution of sodium hydroxide R and dilute to 100 ml with the same alkaline solution. The solution is clear (2.2.1) and not more N-ACETYLTRYPTOPHAN intensely coloured than reference solution Y7 or GY7 (2.2.2, Method II). N-Acetyltryptophanum Optical rotation (2.2.7). Dissolve 2.50 g in a 40 g/l solution of sodium hydroxide R and dilute to 25.0 ml with the same alkaline solution. The angle of optical rotation is − 0.1° to + 0.1°. Related substances. Examine by liquid chromatography (2.2.29). C13H14N2O3 Mr 246.3 Buffer solution pH 2.3. Dissolve 3.90 g of sodium dihydrogen phosphate R in 1000 ml of water R. Add about DEFINITION 700 ml of a 2.9 g/l solution of phosphoric acid R and adjust N-Acetyltryptophan contains not less than 99.0 per cent the pH to 2.3 with the same acidic solution. and not more than the equivalent of 101.0 per cent Prepare the solutions immediately before use. of (RS)-2-acetylamino-3-(1H-indol-3-yl)propanoic acid, Test solution. Dissolve 0.10 g of the substance to be calculated with reference to the dried substance. examined in a mixture of 50 volumes of acetonitrile R and PRODUCTION 50 volumes of water R and dilute to 20.0 ml with the same Tryptophan used for the production of N-acetyltryptophan mixture of solvents. complies with the test for 1,1′-ethylidenebistryptophan Reference solution (a). Dilute 1.0 ml of the test solution to and other related substances in the monograph on 100.0 ml with a mixture of 10 volumes of acetonitrile R and Tryptophan (1272). 90 volumes of water R. Reference solution (b). Dissolve 1.0 mg of CHARACTERS 1,1′-ethylidenebis(tryptophan) CRS in a mixture of A white or almost white, crystalline powder, or colourless crystals, slightly soluble in water, very soluble in alcohol. It 10 volumes of acetonitrile R and 90 volumes of water R and dilute to 100.0 ml with the same mixture of solvents. dissolves in dilute solutions of alkali hydroxides. Reference solution (c). To 4.0 ml of reference solution (a), It melts at about 205 °C. add 20.0 ml of reference solution (b) and dilute to 100.0 ml IDENTIFICATION with a mixture of 10 volumes of acetonitrile R and 90 volumes of water R. First identification : A, B. 918


N-Acetyltryptophan


Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. chromatography R (5 µm), The chromatographic procedure may be carried out using :

— as mobile phase at a flow rate of 0.7 ml/min :

ASSAY Mobile phase A. A mixture of 115 volumes of Dissolve 0.200 g in 5 ml of methanol R. Add 50 ml of acetonitrile R and 885 volumes of buffer solution pH 2.3, ethanol R. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20). Mobile phase B. A mixture of 350 volumes of acetonitrile R and 650 volumes of buffer solution pH 2.3, 1 ml of 0.1 M sodium hydroxide is equivalent to 24.63 mg of C13H14N2O3.

Time (min)



Comment

equilibration

100

0

0 - 10

100

0

isocratic

10 - 45

100 → 0

0 → 100

linear gradient

45 - 65

0

100

isocratic

65 - 66

0 → 100

100 → 0

linear gradient

66 - 80

100

0

re-equilibration

STORAGE Store protected from light. IMPURITIES A. tryptophan,

— as detector a spectrophotometer set at 220 nm, maintaining the temperature of the column at 40 °C. When the chromatograms are recorded in the prescribed conditions, the retention times are about 29 min for N-acetyltryptophan and about 34 min for 1,1′-ethylidenebis(tryptophan). Adjust the sensitivity of the system so that the height of the peak due to N-acetyltryptophan in the chromatogram obtained with reference solution (a) is at least 50 per cent of the full scale of the recorder.

B. (S)-2-amino-3-[(3RS)-3-hydroxy-2-oxo-2,3-dihydro-1H-indol3-yl]propanoic acid (dioxyindolylalanine),

Inject 20 µl of reference solution (c). The test is not valid unless in the chromatogram obtained, the resolution C. R = H : (S)-2-amino-4-(2-aminophenyl)-4-oxobutanoic acid between the peaks corresponding to N-acetyltryptophan and (kynurenine), 1,1′-ethylidenebis(tryptophan) is at least 8.0. If necessary, adjust the time programme for the elution gradient. An E. R = CHO : (S)-2-amino-4-[2-(formylamino)phenyl]-4increase in the duration of elution with mobile phase A oxobutanoic acid (N-formylkynurenine), produces longer retention times and a better resolution. Inject 20 µl of the test solution and 20 µl of reference solution (a). Continue the chromatography of the test solution for 1.8 times the retention time of N-acetyltryptophan. In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.25 per cent) ; the sum of the areas of all the D. (S)-2-amino-3-(5-hydroxy-1H-indol-3-yl)propanoic acid peaks, apart from the principal peak, is not greater than (5-hydroxytryptophan), 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent). Disregard any peak due to the solvent and any peak with an area less than 0.01 times that of the peak due to N-acetyltryptophanin the chromatogram obtained with reference solution (a). Ammonium (2.4.1). 0.10 g complies with limit test B for ammonium (200 ppm). Prepare the standard using 0.2 ml of F. (S)-2-amino-3-(phenylamino)propanoic acid ammonium standard solution (100 ppm NH4) R. (3-phenylaminoalanine), Iron (2.4.9). Dissolve 1.0 g in 50 ml of hydrochloric acid R1, with heating at 50 °C. Allow to cool. In a separating funnel, shake with three quantities, each of 10 ml, of methyl isobutyl ketone R1, shaking for 3 min each time. To the combined organic layers add 10 ml of water R and shake for 3 min. The aqueous layer complies with the limit test for iron (10 ppm). Heavy metals (2.4.8). 2.0 g complies with limit test C for heavy metals (10 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. General Notices (1) apply to all monographs and other texts

G. (S)-2-amino-3-(2-hydroxy-1H-indol-3-yl)propanoic acid (2-hydroxytryptophan), 919

N-Acetyltyrosine


H. R = H : (3RS)-1,2,3,4-tetrahydro-9H-β-carboline-3carboxylic acid, I. R = CH3 : 1-methyl-1,2,3,4-tetrahydro-9H-β-carboline-3carboxylic acid,

B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with N-acetyltyrosine CRS. Examine the substances prepared as discs. C. Examine the chromatograms obtained in the test for related substances in ultraviolet light at 254 nm. The principal spot obtained with test solution (b) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). D. Solution S (see Tests) is strongly acid (2.2.4).

TESTS Solution S. Dissolve 2.50 g in water R and dilute to 100.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Specific optical rotation (2.2.7). Dilute 10.0 ml of solution S to 25.0 ml with water R. The specific optical rotation is + 46 to + 49, calculated with reference to the dried substance. J. R = CHOH-CH2-OH : (S)-2-amino-3-[2-[2,3-dihydroxy-1-(1H- Related substances. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R. indol-3-yl)propyl]-1H-indol-3-yl]propanoic acid, Test solution (a). Dissolve 0.80 g of the substance to be K. R = H : (S)-2-amino-3-[2-(1H-indol-3-ylmethyl)-1H-indol-3examined in 6 ml of a mixture of equal volumes of glacial yl]propanoic acid, acetic acid R and water R and dilute to 10 ml with ethanol R. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with ethanol R. Reference solution (a). Dissolve 80 mg of N-acetyltyrosine CRS in a mixture of 3 volumes of water R, 3 volumes of glacial acetic acid R and 94 volumes of ethanol R and dilute to 10 ml with the same mixture of solvents. Reference solution (b). Dilute 0.5 ml of test solution (b) to 10 ml with ethanol R. Reference solution (c). Dissolve 40 mg of tyrosine CRS in L. 1-(1H-indol-3-ylmethyl)-1,2,3,4-tetrahydro-9H-β-carboline- 20 ml of a mixture of equal volumes of water R and glacial 3-carboxylic acid. acetic acid R and dilute to 50 ml with ethanol R. Apply separately to the plate 5 µl of each solution. Develop 01/2005:1384 over a path of 10 cm using a mixture of 10 volumes of water R, 15 volumes of glacial acetic acid R and 75 volumes N-ACETYLTYROSINE of ethyl acetate R. Allow the plate to dry in air. Examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, N-Acetyltyrosinum is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). Spray with ninhydrin solution R and heat at 100 °C to 105 °C for 10 min. Examine in daylight. Any spot corresponding to tyrosine is not more intense that the spot in the chromatogram obtained with reference solution (c) (1 per cent). C11H13NO4 Mr 223.2 Chlorides (2.4.4). Dilute 10 ml of solution S to 15 ml with water R. The solution complies with the limit test for DEFINITION chlorides (200 ppm). N-Acetyltyrosine contains not less than 98.5 per cent Sulphates (2.4.13). Dissolve 1.0 g in distilled water R and and not more than the equivalent of 101.0 per cent of dilute to 20 ml with the same solvent. The solution complies (2S)-2-(acetylamino)-3-(4-hydroxyphenyl)propanoic acid, with the limit test for sulphates (200 ppm). calculated with reference to the dried substance. Ammonium. Prepare a cell consisting of two watch-glasses CHARACTERS 60 mm in diameter placed edge to edge. To the inner wall of A white, crystalline powder or colourless crystals, freely the upper watch-glass stick a piece of red litmus paper R soluble in water, practically insoluble in cyclohexane. 5 mm square and wetted with a few drops of water R. Finely powder the substance to be examined, place 50 mg IDENTIFICATION in the lower watch-glass and dissolve in 0.5 ml of water R. First identification : A, B. To the solution add 0.30 g of heavy magnesium oxide R. Second identification : A, C, D. Briefly triturate with a glass rod. Immediately close the cell A. It complies with the test for specific optical rotation (see by putting the two watch-glasses together. Heat at 40 °C for 15 min. The litmus paper is not more intensely blue Tests). 920


Aciclovir


coloured than a standard prepared at the same time and in the same manner using 0.1 ml of ammonium standard solution (100 ppm NH4) R, 0.5 ml of water R and 0.30 g of heavy magnesium oxide R (200 ppm). Iron (2.4.9). In a separating funnel, dissolve 0.5 g in 10 ml of dilute hydrochloric acid R. Shake with three quantities, each of 10 ml, of methyl isobutyl ketone R1, shaking for 3 min each time. To the combined organic layers add 10 ml of water R and shake for 3 min. The aqueous layer complies with the limit test for iron (20 ppm). Heavy metals (2.4.8). Dissolve 2.0 g in water R and dilute to 20 ml with the same solvent. 12 ml of the solution complies with limit test A for heavy metals (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. Pyrogens (2.6.8). If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of pyrogens, it complies with the test for pyrogens. Inject per kilogram of the rabbit’s mass 1.0 ml of a freshly prepared solution in water for injections R containing per millilitre 10.0 mg of the substance to be examined and 9.0 mg of pyrogen-free sodium chloride R. ASSAY Dissolve 0.180 g in 50 ml of carbon dioxide-free water R. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M sodium hydroxide is equivalent to 22.32 mg of C11H13NO4. STORAGE Store protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states, where applicable, that the substance is apyrogenic. IMPURITIES A. tyrosine,

DEFINITION Aciclovir contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of 2-amino9-[(2-hydroxyethoxy)methyl]-1,9-dihydro-6H-purin-6-one, calculated with reference to the anhydrous substance. CHARACTERS A white or almost white, crystalline powder, slightly soluble in water, freely soluble in dimethyl sulphoxide, very slightly soluble in alcohol. It dissolves in dilute solutions of mineral acids and alkali hydroxides. IDENTIFICATION Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with aciclovir CRS. TESTS Appearance of solution. Dissolve 0.25 g in 0.1 M sodium hydroxide and dilute to 25 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y7 (2.2.2, Method II). Related substances. A. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Prepare the solutions immediately before use. Test solution. Dissolve 0.1 g of the substance to be examined in dimethyl sulphoxide R and dilute to 10 ml with the same solvent. Reference solution. Dissolve 10 mg of aciclovir impurity A CRS in dimethyl sulphoxide R and dilute to 20 ml with the same solvent. Dilute 1 ml of the solution to 10 ml with dimethyl sulphoxide R. Apply to the plate 10 µl of each solution. Keep the spots compact by drying in a current of warm air. Allow the plate to cool and develop over a path of 10 cm with a mixture of 2 volumes of concentrated ammonia R, 20 volumes of methanol R and 80 volumes of methylene chloride R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. In the chromatogram obtained with the test solution, any spot with an Rf value greater than that of the principal spot is not more intense than the spot in the chromatogram obtained with the reference solution (0.5 per cent). B. Examine by liquid chromatography (2.2.29).

B. (2S)-2-(acetylamino)-3-[4-(acetoxy)phenyl]propanoic acid (diacetyltyrosine). 01/2005:0968

ACICLOVIR

Reference solution (a). Dilute 1.0 ml of the test solution to 200.0 ml with the mobile phase. Reference solution (b). Dissolve 20 mg of aciclovir CRS and 20 mg of aciclovir impurity A CRS in a mixture of 20 volumes of glacial acetic acid R and 80 volumes of water R and dilute to 100.0 ml with the same mixture of solvents. Dilute 1.0 ml of the solution to 10.0 ml with the mobile phase.

Aciclovirum

C8H11N5O3

Test solution. Dissolve 50.0 mg of the substance to be examined in 10 ml of a mixture of 20 volumes of glacial acetic acid R and 80 volumes of water R and dilute to 100.0 ml with the mobile phase.

Mr 225.2


Reference solution (c). Dissolve 7 mg of guanine R in 0.1 M sodium hydroxide and dilute to 100.0 ml with the same solution. Dilute 1.0 ml to 20.0 ml with the mobile phase. 921

Acitretin


The chromatographic procedure may be carried out using : — a stainless steel column 0.10 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (3 µm), — as mobile phase at a flow rate of 2 ml/min a mixture prepared as follows : dissolve 6.0 g of sodium dihydrogen phosphate R and 1.0 g of sodium decanesulphonate R in 900 ml of water R and adjust to pH 3 ± 0.1 with phosphoric acid R ; add 40 ml of acetonitrile R and dilute to 1 litre with water R, — as detector a spectrophotometer set at 254 nm, — a loop injector. Inject 20 µl of each solution. Record the chromatograms for seven times the retention time of aciclovir. The test is not valid unless in the chromatogram obtained with reference solution (b), the number of theoretical plates calculated for the peak due to aciclovir impurity A is at least 1500 and its mass distribution ratio is at least 7 (V0 can be calculated using dimethyl sulphoxide R). In the chromatogram obtained with the test solution : the area of any peak corresponding to guanine is not greater than that of the peak in the chromatogram obtained with reference solution (c) (0.7 per cent) ; the area of any peak apart from the principal peak and any peak corresponding to guanine is not greater than the area of the peak in the chromatogram obtained with reference solution (a) (0.5 per cent) and the sum of the areas of such peaks is not greater than twice the area of the peak in the chromatogram obtained with reference solution (a) (1 per cent). Disregard any peak with an area less than 0.05 times that of the principal peak in the chromatogram obtained with reference solution (a). Water (2.5.12). Not more than 6.0 per cent, determined on 0.500 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.150 g in 60 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 22.52 mg of C8H11N5O3. IMPURITIES

F. R = H : N-[9-[(2-hydroxyethoxy)methyl]-6-oxo-6,9-dihydro1H-purin-2-yl]acetamide, G. R = CO-CH3 : 2-[[2-(acetylamino)-6-oxo-1,6-dihydro-9Hpurin-9-yl]methoxy]ethyl acetate, H. R = CO-C6H5 : 2-[[2-(acetylamino)-6-oxo-1,6-dihydro-9Hpurin-9-yl]methoxy]ethyl benzoate. 01/2005:1385 corrected

ACITRETIN Acitretinum

C21H26O3

Mr 326.4

DEFINITION Acitretin contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of (all-E)-9-(4-methoxy2,3,6-trimethylphenyl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid, calculated with reference to the dried substance. CHARACTERS A yellow or greenish-yellow, crystalline powder, practically insoluble in water, sparingly soluble in tetrahydrofuran, slightly soluble in acetone and in alcohol, very slightly soluble in cyclohexane. It is sensitive to air, heat and light, especially in solution. Carry out all operations as rapidly as possible and avoid exposure to actinic light ; use freshly prepared solutions.

A. R = CH3 : 2-[(2-amino-6-oxo-1,6-dihydro-9H-purin-9yl)methoxy]ethyl acetate, D. R = C6H5 : 2-[(2-amino-6-oxo-1,6-dihydro-9H-purin-9yl)methoxy]ethyl benzoate,

B. R = H : 2-amino-1,7-dihydro-6H-purin-6-one (guanine), C. R = CH2-O-CH2-CH2-OH : 2-amino-7-[(2-hydroxyethoxy)methyl]-1,7-dihydro-6H-purin-6-one, 922

E. 6-amino-9-[(2-hydroxyethoxy)methyl]-1,9-dihydro-2Hpurin-2-one,

IDENTIFICATION First identification : B. Second identification : A, C. A. Dissolve 15.0 mg in 10 ml of tetrahydrofuran R and dilute immediately to 100.0 ml with the same solvent. Dilute 2.5 ml of this solution to 100.0 ml with tetrahydrofuran R. Examined between 300 nm and 400 nm (2.2.25), the solution shows an absorption maximum at 358 nm. The specific absorbance at the maximum is 1350 to 1475. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with acitretin CRS. Examine the substances prepared as discs.


Acitretin


C. Examine the chromatograms obtained in the assay. The principal peak in the chromatogram obtained with the test solution has a retention time similar to that of the principal peak in the chromatogram obtained with reference solution (a). TESTS Related substances. Examine by liquid chromatography (2.2.29) as described under Assay. Inject 10 µl each of reference solutions (b) and (c) and of the test solution (a). Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with reference solution (b) is not less than 40 per cent of the full scale of the recorder. The test is not valid unless the resolution between the peaks due to acitretin and tretinoin in the chromatogram obtained with reference solution (b) is at least 2.0. If necessary, adjust the concentration of ethanol R. Continue the chromatography of the test solution (a) for 2.5 times the retention time of the principal peak. In the chromatogram obtained with the test solution (a), the area of any peak, apart from the principal peak, is not greater than the area of the peak due to acitretin in the chromatogram obtained with reference solution (c) (0.3 per cent) ; the sum of the areas of any peaks, apart from the principal peak, is not greater than the area of the peak due to acitretin in the chromatogram obtained with reference solution (b) (1.0 per cent). Disregard any peak with an area less than 0.1 times that of the principal peak in the chromatogram obtained with reference solution (c). Heavy metals (2.4.8). 2.0 g complies with limit test C (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Palladium. Not more than 10 ppm of Pd, determined by atomic absorption spectrometry (2.2.23, Method I). Test solution. Introduce 2.0 g of the substance to be examined into a quartz beaker and add 3 ml of magnesium nitrate solution R. Heat in a muffle furnace to 350 °C at a rate of 40 °C/min to incinerate the content. Ignite at about 450 °C for 8 h and then at 550 °C for a further hour. Dissolve the residue in a mixture of 0.75 ml of hydrochloric acid R and 0.25 ml of nitric acid R warming gently. Cool, transfer the solution into a volumetric flask with water R and dilute to 50.0 ml with the same solvent. Reference solution. Dissolve 0.163 g of heavy magnesium oxide R in a mixture of 0.5 ml of nitric acid R, 1.5 ml of hydrochloric acid R and 50 ml of water R, add 2.0 ml of palladium standard solution (20 ppm Pd R) and dilute to 100.0 ml with water R.

Test solution (b). Dilute 10.0 ml of test solution (a) to 25.0 ml with ethanol R. Reference solution (a). Dissolve 25.0 mg of acitretin CRS in 5 ml of tetrahydrofuran R and dilute immediately to 100.0 ml with ethanol R. Dilute 10.0 ml to 25.0 ml with ethanol R. Reference solution (b). Dissolve 1.0 mg of tretinoin CRS in ethanol R and dilute to 20.0 ml with the same solvent. Mix 5.0 ml of the solution with 2.5 ml of reference solution (a) and dilute to 100.0 ml with ethanol R. Reference solution (c). Dilute 2.5 ml of reference solution (a) to 50.0 ml with ethanol R. Dilute 3.0 ml of the solution to 20.0 ml with ethanol R. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4 mm in internal diameter packed with microparticulate octadecylsilyl silica gel for chromatography R (5 µm), with a carbon loading of 20 per cent, a specific surface area of 200 m2/g and a pore size of 15 nm, — as mobile phase at a flow rate of 0.6 ml/min a 0.3 per cent V/V solution of glacial acetic acid R in a mixture of 8 volumes of water R and 92 volumes of ethanol R, — as detector a spectrophotometer set at 360 nm, — a sampler kept at 4 °C, maintaining the temperature of the column at 25 °C. When the chromatograms are recorded in the prescribed conditions, the retention times are : impurity A about 4.8 min, tretinoin about 5.2 min, acitretin about 6.2 min and impurity B about 10.2 min. Inject 10 µl of reference solution (a) 6 times. The test is not valid unless the relative standard deviation of the peak area for acitretin is at most 1.0 per cent. If necessary, adjust the integration parameters. Inject alternately the test solution (b) and reference solution (a). STORAGE In an airtight container, protected from light, at a temperature of 2 °C to 8 °C. It is recommended that the contents of an opened container be used as soon as possible and any unused part be protected by an atmosphere of inert gas. IMPURITIES

Measure the absorbance at 247.6 nm using a palladium hollow-cathode lamp as source of radiation and an air-acetylene flame. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in vacuo at 100 °C for 4 h. A. (2Z,4E,6E,8E)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7Sulphated ash (2.4.14). Not more than 0.1 per cent, dimethylnona-2,4,6,8-tetraenoic acid, determined on 1.0 g. ASSAY Carry out the assay protected from light, use amber volumetric flasks and prepare the solutions immediately before use. Examine by liquid chromatography (2.2.29). Test solution (a). Dissolve 25.0 mg of the substance to be examined in 5 ml of tetrahydrofuran R and dilute immediately to 100.0 ml with ethanol R. General Notices (1) apply to all monographs and other texts

B. ethyl (all-E)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7dimethylnona-2,4,6,8-tetraenoate. 923

Acriflavinium monochloride


01/2005:2043 Reference solution (b). Dilute 1.0 ml of reference solution (a) to 10.0 ml with the mobile phase. ACRIFLAVINIUM MONOCHLORIDE Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : octadecylsilyl silica gel for Acriflavinii monochloridum chromatography R (5 µm). Mobile phase : dissolve 1.0 g of sodium octanesulphonate R and 5 ml of triethylamine R in a mixture of 400 ml of acetonitrile R and 600 ml of phosphate buffer solution pH 2.8 R. Flow rate : 1 ml/min. Detection : spectrophotometer at 262 nm. Injection : 10 µl. Run time : 30 min. System suitability : reference solution (a) : — resolution : minimum 3.5 between the 2 principal peaks. DEFINITION Limits : Mixture of 3,6-diamino-10-methylacridinium chloride and 3,6-diaminoacridine hydrochloride. — first principal peak : 30.0 per cent to 40.0 per cent, Content : 95.0 per cent to 105.0 per cent (anhydrous — second principal peak : 50.0 per cent to 60.0 per cent, substance). — any other peak : maximum 6.0 per cent and not more than 2 such peaks have a peak area of more than 2.0 per cent, CHARACTERS — disregard limit : 0.5 times the area of the peak in the Appearance : reddish-brown powder, hygroscopic. chromatogram obtained with reference solution (b) Solubility : freely soluble in water, sparingly soluble in (0.05 per cent). alcohol, very slightly soluble in methylene chloride. Heavy metals (2.4.8) : maximum 40 ppm. IDENTIFICATION 0.5 g complies with limit test D. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. A. Thin-layer chromatography (2.2.27). Water (2.5.12) : maximum 10.0 per cent, determined on Test solution. Dilute 0.5 ml of solution S (see Tests) to 0.250 g. 10 ml with alcohol R. Reference solution. Dissolve 10 mg of acriflavinium Sulphated ash (2.4.14) : maximum 3.5 per cent, determined monochloride CRS in alcohol R and dilute to 10 ml with on 1.0 g. the same solvent. ASSAY Plate : TLC silica gel plate R. Mobile phase : glacial acetic acid R, water R, butanol R Prepare the solutions immediately before use and protected from light. (1:1:4 V/V/V). Determine the water content immediately before the assay. Application : 5 µl. Dissolve 0.100 g in 500.0 ml of water R. Dilute 5.0 ml of the Development : over 2/3 of the plate. solution to 250.0 ml with 0.1 M hydrochloric acid. Measure Drying : at 100-105 °C. the absorbance (2.2.25) at the maximum at 262 nm. Detection : examine in ultraviolet light at 365 nm. Calculate the content of the sum of C14H14ClN3 and C13H12ClN3 taking the specific absorbance to be 1820. Results : the 2 principal spots in the chromatogram obtained with the test solution are similar in position, STORAGE fluorescence and size to the principal spots in the chromatogram obtained with the reference solution. In an airtight container, protected from light. B. To 0.1 mg add 20 ml of water R. The solution obtained shows a yellowish-green fluorescence. Add 1 ml of 01/2005:0800 dilute sodium hydroxide solution R. The fluorescence disappears. ADENINE C. It complies with the test for pH (see Tests). TESTS Solution S. Dissolve with heating 0.500 g in 15 ml of carbon dioxide-free water R ; allow to cool and dilute to 25.0 ml with the same solvent. pH (2.2.3) : 4.5 to 7.5 for solution S. Composition. Liquid chromatography (2.2.29) : use the normalisation procedure. Test solution. Dissolve 10.0 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the mobile phase. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. 924

Adeninum

C5 H 5 N 5

Mr 135.1

DEFINITION Adenine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of 7H-purin-6-amine, calculated with reference to the dried substance.



Adenosine

CHARACTERS A white powder, very slightly soluble in water and in alcohol. It dissolves in dilute mineral acids and in dilute solutions of alkali hydroxides.

and dilute the filtrate to 15 ml with water R. The solution complies with the limit test for chlorides (100 ppm). When carrying out the test, add 2 ml of dilute nitric acid R instead of 1 ml of dilute nitric acid R. Sulphates (2.4.13). Dilute 10 ml of solution S to 15 ml with IDENTIFICATION distilled water R. The solution complies with the limit test First identification : A. for sulphates (300 ppm). Second identification : B, C. Ammonium. Prepare a cell consisting of two watch-glasses A. Examine by infrared absorption spectrophotometry 60 mm in diameter placed edge to edge. To the inner wall of (2.2.24), comparing with the spectrum obtained with the upper watch-glass stick a piece of red litmus paper R adenine CRS. Examine the substances prepared as discs. 5 mm square and wetted with a few drops of water R. Finely powder the substance to be examined, place 0.5 g in the B. Examine the chromatograms obtained in the test lower watch-glass and suspend in 0.5 ml of water R. To for related substances. The principal spot in the the suspension add 0.30 g of heavy magnesium oxide R. chromatogram obtained with test solution (b) is Briefly triturate with a glass rod. Immediately close the cell similar in position and size to the principal spot in the by putting the two watch-glasses together. Heat at 40 °C chromatogram obtained with reference solution (a). C. To 1 g add 3.5 ml of propionic anhydride R and boil for for 15 min. The litmus paper is not more intensely blue coloured than a standard prepared at the same time and in 15 min with stirring. Cool. To the resulting crystalline the same manner using 0.05 ml of ammonium standard mass add 15 ml of light petroleum R and heat to solution (100 ppm NH4) R, 0.5 ml of water R and 0.30 g of boiling with vigorous stirring. Cool and filter. Wash the heavy magnesium oxide R (10 ppm). precipitate with two quantities, each of 5 ml, of light petroleum R. Dissolve the precipitate in 10 ml of water R Heavy metals (2.4.8). 1.0 g complies with limit test C for and boil for 1 min. Filter the mixture at 30 °C to 40 °C. heavy metals (20 ppm). Prepare the standard using 2 ml of Allow to cool. Filter, and dry the precipitate at 100 °C lead standard solution (10 ppm Pb) R. to 105 °C for 1 h. The melting point (2.2.14) of the Loss on drying (2.2.32). Not more than 0.5 per cent, precipitate is 237 °C to 241 °C. determined on 1.000 g by drying in an oven at 100 °C to 105 °C. TESTS Sulphated ash (2.4.14). Not more than 0.1 per cent, Solution S. Suspend 2.5 g in 50 ml of distilled water R determined on 1.0 g. and boil for 3 min. Cool and dilute to 50 ml with distilled water R. Filter. Use the filtrate as solution S. ASSAY Appearance of solution. Dissolve 0.5 g in dilute hydrochloric Dissolve 0.100 g in a mixture of 20 ml of acetic anhydride R acid R and dilute to 50 ml with the same acid. The solution and 30 ml of anhydrous acetic acid R. Titrate with is clear (2.2.1) and colourless (2.2.2, Method II). 0.1 M perchloric acid, determining the end-point Acidity or alkalinity. To 10 ml of solution S add 0.1 ml of potentiometrically (2.2.20). bromothymol blue solution R1 and 0.2 ml of 0.01 M sodium 1 ml of 0.1 M perchloric acid is equivalent to 13.51 mg of hydroxide. The solution is blue. Add 0.4 ml of 0.01 M C5 H 5 N 5 . hydrochloric acid. The solution is yellow. Related substances. Examine by thin-layer chromatography 01/2005:1486 (2.2.27), using silica gel GF254 R as the coating substance. Test solution (a). Dissolve 0.10 g of the substance to be ADENOSINE examined in dilute acetic acid R, with heating if necessary, and dilute to 10 ml with the same acid. Adenosinum Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with dilute acetic acid R. Reference solution (a). Dissolve 10 mg of adenine CRS in dilute acetic acid R, with heating if necessary, and dilute to 10 ml with the same acid. Reference solution (b). Dilute 1 ml of test solution (b) to 20 ml with dilute acetic acid R. Reference solution (c). Dissolve 10 mg of adenine CRS and 10 mg of adenosine R in dilute acetic acid R, with heating if necessary, and dilute to 10 ml with the same acid. Apply to the plate 5 µl of each solution. Develop over a path C H N O Mr 267.2 10 13 5 4 of 12 cm using a mixture of 20 volumes of concentrated ammonia R, 40 volumes of ethyl acetate R and 40 volumes DEFINITION of propanol R. Dry the plate in a current of warm air and Adenosine contains not less than 99.0 per cent and examine in ultraviolet light at 254 nm. Any spot in the not more than the equivalent of 101.0 per cent of chromatogram obtained with test solution (a), apart from 9-β-D-ribofuranosyl-9H-purin-6-amine, calculated with the principal spot, is not more intense than the spot in the reference to the dried substance. chromatogram obtained with reference solution (b) (0.5 per cent). The test is not valid unless the chromatogram obtained CHARACTERS with reference solution (c) shows two clearly separated spots. A white, crystalline powder slightly soluble in water, soluble in hot water, practically insoluble in alcohol and in methylene Chlorides (2.4.4). To 10 ml of solution S add 1 ml of chloride. It dissolves in dilute mineral acids. concentrated ammonia R and 3 ml of silver nitrate solution R2. Filter. Wash the precipitate with a little water R It melts at about 234 °C. General Notices (1) apply to all monographs and other texts

925

Adipic acid


1 ml of 0.1 M perchloric acid is equivalent to 26.72 mg of IDENTIFICATION Examine by infrared absorption spectrophotometry (2.2.24), C10H13N5O4. comparing with the spectrum obtained with adenosine CRS. IMPURITIES TESTS A. adenine, Solution S. Suspend 5.0 g in 100 ml of distilled water R and heat to boiling. Allow to cool, filter with the aid of vacuum and dilute to 100 ml with distilled water R. Appearance of solution. Solution S is colourless (2.2.2, Method II). Acidity or alkalinity. To 10 ml of solution S, add 0.1 ml of bromocresol purple solution R and 0.1 ml of 0.01 M B. D-ribose, hydrochloric acid. The solution is yellow. Add 0.4 ml of 0.01 M sodium hydroxide, the solution is violet-blue. Specific optical rotation (2.2.7). Dissolve 1.25 g in 1 M hydrochloric acid and dilute to 50.0 ml with the same acid. Determined within 10 min and calculated with reference to the dried substance, the specific optical rotation is − 45 to − 49. Related substances. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R. Test solution. Dissolve 0.20 g of the substance to be examined in dilute acetic acid R with slight heating and dilute to 5 ml with the same acid. C. R = H : adenosine 3′-(dihydrogen phosphate), Reference solution (a). Dilute 1 ml of the test solution to D. R = PO3H2 : adenosine 3′-(trihydrogen diphosphate), 100 ml with water R. Reference solution (b). Dissolve 10 mg of adenosine CRS E. R = PO2H-O-PO3H2 : adenosine 3′-(tetrahydrogen and 10 mg of adenine CRS in dilute acetic acid R, with triphosphate). heating if necessary, and dilute to 10 ml with the same acid. Apply to the plate 5 µl of each solution. Develop over a 01/2005:1586 path of 12 cm using a mixture of 10 volumes of water R, 30 volumes of concentrated ammonia R and 60 volumes ADIPIC ACID of propanol R. Allow the plate to dry in a current of warm air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart Acidum adipicum from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (a) (1 per cent). Spray with a 5 g/l solution of potassium permanganate R in 1 M sodium hydroxide. Allow the plate C6H10O4 Mr 146.1 to dry in a current of warm air and examine in daylight. Any spot in the chromatogram obtained with the test solution, DEFINITION apart from the principal spot, is not more intense than Hexanedioic acid. the spot in the chromatogram obtained with reference Content : 99.0 per cent to 101.0 per cent (dried substance). solution (a) (1 per cent). The test is not valid unless the chromatogram obtained with reference solution (b) shows CHARACTERS two clearly separated spots. Appearance : white, crystalline powder. Chlorides (2.4.4). Dilute 10 ml of solution S to 15 ml Solubility : sparingly soluble in water, soluble in boiling with water R. The solution complies with the limit test for water, freely soluble in alcohol and in methanol, soluble in chlorides (100 ppm). acetone. Sulphates (2.4.13). 15 ml of solution S complies with the IDENTIFICATION limit test for sulphates (200 ppm). A. Melting point (2.2.14) : 151 °C to 154 °C. Ammonium (2.4.1). 0.5 g complies with limit test B for B. Infrared absorption spectrophotometry (2.2.24). ammonium (10 ppm). Prepare the standard using 5 ml of ammonium standard solution (1 ppm NH4) R. Comparison : adipic acid CRS. Loss on drying (2.2.32). Not more than 0.5 per cent, TESTS determined on 1.000 g by drying in an oven at 100 °C to Solution S. Dissolve 5.0 g with heating in distilled water R 105 °C. and dilute to 50 ml with the same solvent. Allow to cool Sulphated ash (2.4.14). Not more than 0.1 per cent, and to crystallise. Filter through a sintered-glass filter (40). determined on 1.0 g. Wash the filter with distilled water R. Collect the filtrate and the washings until a volume of 50 ml is obtained. ASSAY Dissolve 0.200 g, warming slightly if necessary, in a mixture Appearance of solution. The solution is clear (2.2.1) and of 20 ml of acetic anhydride R and 30 ml of anhydrous acetic colourless (2.2.2, Method II). acid R. Titrate with 0.1 M perchloric acid, determining the Dissolve 1.0 g in methanol R and dilute to 20 ml with the end-point potentiometrically (2.2.20). same solvent. 926


Adrenaline tartrate


Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 0.20 g of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (a). Dissolve 20 mg of glutaric acid R in 1.0 ml of the test solution and dilute to 10.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase, dilute 1.0 ml of the solution to 10.0 ml with the mobile phase. Column : — size : l = 0.125 m, Ø = 4.0 mm, — stationary phase: spherical octadecylsilyl silica gel for chromatography R (5 µm) with a specific surface area of 350 m2/g and a pore size of 10 nm, — temperature : 30 °C. Mobile phase : mix 3 volumes of acetonitrile R and 97 volumes of a 24.5 g/l solution of dilute phosphoric acid R. Flow rate : 1 ml/min. Detection : spectrophotometer at 209 nm. Injection : 20 µl. Run time : 3 times the retention time of adipic acid. System suitability : reference solution (a) : — resolution : minimum 9.0 between the peaks due to glutaric acid and adipic acid. Limits : — any impurity : not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent), — total: not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent), — disregard limit : 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.05 per cent). Chlorides (2.4.4) : maximum 200 ppm. 2.5 ml of solution S diluted to 15 ml with water R complies with the limit test for chlorides. Nitrates : maximum 30 ppm. To 1 ml of solution S add 2 ml of concentrated ammonia R, 0.5 ml of a 10 g/l solution of manganese sulphate R, 1 ml of a 10 g/l solution of sulfanilamide R and dilute to 20 ml with water R. Add 0.10 g of zinc powder R and cool in iced water for 30 min ; shake from time to time. Filter and cool 10 ml of the filtrate in iced water. Add 2.5 ml of hydrochloric acid R1 and 1 ml of a 10 g/l solution of naphthylethylenediamine dihydrochloride R. Allow to stand at room temperature. After 15 min the mixture is not more intensely coloured than a standard prepared at the same time and in the same manner, using 1.5 ml of nitrate standard solution (2 ppm NO3) R instead of 1 ml of solution S. The test is invalid if a blank solution prepared at the same time and in the same manner, using 1 ml of water R instead of 1 ml of solution S, is more intensely coloured than a 2 mg/l solution of potassium permanganate R. Sulphates (2.4.13) : maximum 500 ppm. 3 ml of solution S diluted to 15 ml with distilled water R complies with the limit test for sulphates. Iron (2.4.9) : maximum 10 ppm. 10 ml of solution S complies with the limit test for iron. General Notices (1) apply to all monographs and other texts

Heavy metals (2.4.8) : maximum 10 ppm. 12 ml of solution S complies with limit test A. Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32) : maximum 0.2 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14) : maximum 0.1 per cent. Melt 1.0 g completely over a gas burner, then ignite the melted substance with the burner. After ignition, lower or remove the flame in order to prevent the substance from boiling and keep it burning until completely carbonised. Carry out the test for sulphated ash using the residue. ASSAY Dissolve 60.0 mg in 50 ml of water R. Add 0.2 ml of phenolphthalein solution R and titrate with 0.1 M sodium hydroxide. 1 ml of 0.1 M sodium hydroxide is equivalent to 7.31 mg of C6H10O4. IMPURITIES

A. R = CH2-CO2H : pentanedioic acid (glutaric acid), B. R = CO2H : butanedioic acid (succinic acid), C. R = [CH2]3-CO2H : heptanedioic acid (pimelic acid). 01/2005:0254

ADRENALINE TARTRATE Adrenalini tartras

C13H19NO9

Mr 333.3

DEFINITION Adrenaline tartrate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of (1R)-1-(3,4-dihydroxyphenyl)-2-(methylamino)ethanol hydrogen (2R,3R)-2,3-dihydroxybutanedioate, calculated with reference to the dried substance. CHARACTERS A white or greyish-white, crystalline powder, freely soluble in water, slightly soluble in ethanol (96 per cent). IDENTIFICATION A. Dissolve 2 g in 20 ml of a 5 g/l solution of sodium metabisulphite R and make alkaline by addition of ammonia R. Keep the mixture in iced water for 1 h and filter. Reserve the filtrate for identification test C. Wash the precipitate with 3 quantities, each of 2 ml, of water R, with 5 ml of ethanol (96 per cent) R and finally with 5 ml of ether R. Dry in vacuo for 3 h. The specific optical rotation (2.2.7) of the precipitate (adrenaline base) is − 50 to − 54, determined using a 20.0 g/l solution in 0.5 M hydrochloric acid.

927

Agar


B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with adrenaline base prepared by the same method from a suitable amount of adrenaline tartrate CRS. Use adrenaline base prepared as described under identification test A. Examine the substances prepared as discs.

1 ml of 0.1 M perchloric acid is equivalent to 33.33 mg of C13H19NO9. STORAGE In an airtight container, or preferably in a sealed tube under vacuum or under an inert gas, protected from light.

C. 0.2 ml of the filtrate obtained in identification test A gives reaction (b) of tartrates (2.3.1). TESTS Appearance of solution. Dissolve 0.5 g in water R and dilute to 10 ml with the same solvent. Examine the solution immediately. The solution is not more opalescent than reference suspension II (2.2.1) and is not more intensely coloured than reference solution BY5 (2.2.2, Method II). Adrenalone. Dissolve 50.0 mg in 0.01 M hydrochloric acid and dilute to 25.0 ml with the same acid. The absorbance (2.2.25) of the solution measured at 310 nm is not greater than 0.10. Noradrenaline. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. Dissolve 0.25 g of the substance to be examined in water R and dilute to 10 ml with the same solvent. Prepare immediately before use. Reference solution (a). Dissolve 12.5 mg of noradrenaline tartrate CRS in water R and dilute to 10 ml with the same solvent. Prepare immediately before use. Reference solution (b). Dilute 2 ml of reference solution (a) to 10 ml with water R. Reference solution (c). Mix 2 ml of the test solution with 2 ml of reference solution (b). Apply separately to the plate as bands 20 mm by 2 mm 6 µl of the test solution, 6 µl of reference solution (a), 6 µl of reference solution (b) and 12 µl of reference solution (c). Allow to dry and spray the bands with a saturated solution of sodium bicarbonate R. Allow the plate to dry in air and spray the bands twice with acetic anhydride R, drying between the 2 sprayings. Heat the plate at 50 °C for 90 min. Develop over a path of 15 cm using a mixture of 0.5 volumes of anhydrous formic acid R, 50 volumes of acetone R and 50 volumes of methylene chloride R. Allow the plate to dry in air and spray with a solution freshly prepared by mixing 2 volumes of ethylenediamine R and 8 volumes of methanol R and adding 2 volumes of a 5 g/l solution of potassium ferricyanide R. Dry the plate at 60 °C for 10 min and examine in ultraviolet light at 254 nm and 365 nm. In the chromatogram obtained with the test solution, any zone situated between the 2 most intense zones is not more intense than the corresponding zone in the chromatogram obtained with reference solution (b) (1.0 per cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows between the 2 most intense zones a clearly separated zone corresponding to the most intense zone in the chromatogram obtained with reference solution (a). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.00 g by drying in vacuo for 18 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.300 g in 50 ml of anhydrous acetic acid R, heating gently if necessary. Titrate with 0.1 M perchloric acid until a bluish-green colour is obtained using 0.1 ml of crystal violet solution R as indicator. 928

01/2005:0310

AGAR Agar DEFINITION Agar consists of the polysaccharides from various species of Rhodophyceae mainly belonging to the genus Gelidium. It is prepared by treating the algae with boiling water ; the extract is filtered whilst hot, concentrated and dried. CHARACTERS Agar has a mucilaginous taste. It occurs in the form of a powder or in crumpled strips 2 mm to 5 mm wide or sometimes in flakes, colourless to pale yellow, translucent, somewhat tough and difficult to break, becoming more brittle on drying. It has the microscopic characters described under identification test A. IDENTIFICATION A. Examine under a microscope. When mounted in 0.005 M iodine, the strips or flakes are partly stained brownish-violet. Magnified 100 times, they show numerous minute, colourless, ovoid or rounded grains on an amorphous background ; occasional brown, round to ovoid spores with a reticulated surface, measuring up to 60 µm, may be present. Reduce to a powder, if necessary. The powder is yellowish-white. Examine under a microscope using 0.005 M iodine. The powder presents angular fragments with numerous grains similar to those seen in the strips and flakes ; some of the fragments are stained brownish-violet. B. Dissolve 0.1 g with heating in 50 ml of water R. Cool. To 1 ml of the mucilage carefully add 3 ml of water R so as to form two separate layers. Add 0.1 ml of 0.05 M iodine. A dark brownish-violet colour appears at the interface. Mix. The liquid becomes pale yellow. C. Heat 5 ml of the mucilage prepared for identification test B on a water-bath with 0.5 ml of hydrochloric acid R for 30 min. Add 1 ml of barium chloride solution R1. A white turbidity develops within 30 min. D. Heat 0.5 g with 50 ml of water R on a water-bath until dissolved. Only a few fragments remain insoluble. During cooling, the solution gels between 35 °C and 30 °C. Heat the gel thus obtained on a water-bath ; it does not liquefy below 80 °C. TESTS Swelling index (2.8.4). The swelling index, determined on the powdered drug (355), is not less than 10 and is within 10 per cent of the value stated on the label. Insoluble matter. To 5.00 g of the powdered drug (355) add 100 ml of water R and 14 ml of dilute hydrochloric acid R. Boil gently for 15 min with frequent stirring. Filter the hot liquid through a tared, sintered-glass filter (160), rinse the filter with hot water R and dry at 100 °C to 105 °C. The residue weighs not more than 50 mg (1.0 per cent).


Air, medicinal


Gelatin. To 1.00 g add 100 ml of water R and heat on a water-bath until dissolved. Allow to cool to 50 °C. To 5 ml of the solution add 5 ml of picric acid solution R. No turbidity appears within 10 min. Loss on drying (2.2.32). Not more than 20.0 per cent, determined on 1.000 g of the powdered drug (355) by drying in an oven at 100 °C to 105 °C. Total ash (2.4.16). Not more than 5.0 per cent. Microbial contamination. Total viable aerobic count (2.6.12) not more than 103 micro-organisms per gram, determined by plate-count. It complies with the tests for Escherichia coli and Salmonella (2.6.13). LABELLING The label states the swelling index.

Plate : TLC silica gel plate R. Mobile phase : anhydrous formic acid R, water R, ethyl acetate R (10:10:80 V/V/V). Application : 10 µl, as bands. Development : over a path of 12 cm. Drying : at 100-105 °C. Detection : spray the still warm plate with a 10 g/l solution of diphenylboric acid aminoethyl ester R in methanol R and then with a 50 g/l solution of macrogol 400 R in methanol R. Allow the plate to dry in air for 30 min. Examine in ultraviolet light at 365 nm Results : see below the sequence of the zones present in the chromatograms obtained with the reference and test solutions. Top of the plate An orange fluorescent zone may be present (quercitroside)

01/2005:1587

AGRIMONY Agrimoniae herba DEFINITION Dried flowering tops of Agrimonia eupatoria L. Content : minimum 2.0 per cent of tannins, expressed as pyrogallol (C6H6O3 ; Mr 126.1) (dried drug). CHARACTERS Macroscopic and microscopic characters described under identification tests A and B.

Isoquercitroside : an orange fluorescent zone

An orange fluorescent zone (isoquercitroside) An orange fluorescent zone (hyperoside)

Rutin : an orange fluorescent zone Reference solution

An orange fluorescent zone (rutin) Test solution

TESTS Foreign matter (2.8.2) : it complies with the test for foreign matter. Loss on drying (2.2.32) : maximum 10.0 per cent, determined on 1.000 g of the powdered drug (355) by drying in an oven at 100-105 °C for 2 h. Total ash (2.4.16) : maximum 10.0 per cent.

IDENTIFICATION A. The stem is green or, more usually, reddish, cylindrical and infrequently branched. It is covered with long, erect or tangled hairs. The leaves are compound imparipennate with 3 or 6 opposite pairs of leaflets, with 2 or 3 smaller leaflets between. The leaflets are deeply dentate to serrate, dark green on the upper surface, greyish and densely tormentose on the lower face. The flowers are small and form a terminal spike. They are pentamerous and borne in the axils of hairy bracts, the calyces closely surrounded by numerous terminal hooked spires, which occur on the rim of the hairy receptacle. The petals are free, yellow and deciduous. Fruit-bearing obconical receptacles, with deep furrows and hooked bristles, are usually present at the base of the inflorescence. B. Reduce to a powder (355). The powder is yellowish-green to grey. Examine under a microscope using chloral hydrate solution R. The powder shows numerous straight of bent, unicellular, long thick-walled (about 500 µm) trichomes finely warty and sometimes spiraly marked ; fragments of parenchyma with prisms and cluster crystals of calcium oxalate ; fragments of leaf epidermis with sinuous walls, those of the lower epidermis with abundant stomata, mostly anomocytic but occasionally anisocytic ; ovoid to subspherical-pollen grains, with 3 pores and a smooth exine ; fragments of glandular trichomes with a multicellular uniseriate stalk and a spherical unicellular or quadricellular head ; groups of fibres and spiral and bardered-fitted vessels from the stem. C. Thin-layer chromatography (2.2.27). Test solution. To 2.0 g of the powdered drug (355) add 20 ml of methanol R. Heat with shaking at 40 °C for 10 min. Filter. Reference solution. Dissolve 1.0 mg of rutin R and 1.0 mg of isoquercitroside R in 2 ml of methanol R.

PRODUCTION Carbon dioxide : maximum 500 ppm V/V, determined using an infrared analyser (2.5.24). Gas to be examined. Use the substance to be examined. It must be filtered to avoid stray light phenomena. Reference gas (a). Use a mixture of 79 per cent V/V of nitrogen R1 and 21 per cent V/V of oxygen R containing less than 1 ppm V/V of carbon dioxide R1. Reference gas (b). Use a mixture of 79 per cent V/V of nitrogen R1 and 21 per cent V/V of oxygen R containing 500 ppm V/V of carbon dioxide R1.


929

ASSAY Carry out the determination of tannins in herbal drugs (2.8.14). Use a 1.000 g of powdered drug (180). 01/2005:1238 corrected

AIR, MEDICINAL Aer medicinalis DEFINITION Compressed ambient air. Content : 20.4 per cent V/V to 21.4 per cent V/V of oxygen (O2). CHARACTERS A colourless, odourless gas. Solubility : at a temperature of 20 °C and a pressure of 101 kPa, 1 volume dissolves in about 50 volumes of water.

Air, medicinal


Calibrate the apparatus and set the sensitivity using reference gases (a) and (b). Measure the content of carbon dioxide in the gas to be examined. Carbon monoxide : maximum 5 ppm V/V, determined using an infrared analyser (2.5.25). Gas to be examined. Use the substance to be examined. It must be filtered to avoid stray light phenomena. Reference gas (a). Use a mixture of 79 per cent V/V of nitrogen R1 and 21 per cent V/V of oxygen R containing less than 1 ppm V/V of carbon monoxide R. Reference gas (b). Use a mixture of 79 per cent V/V of nitrogen R1 and 21 per cent V/V of oxygen R containing 5 ppm V/V of carbon monoxide R. Calibrate the apparatus and set the sensitivity using reference gases (a) and (b). Measure the content of carbon monoxide in the gas to be examined. Sulphur dioxide : maximum 1 ppm V/V, determined using an ultraviolet fluorescence analyser (Figure 1238.-1). The apparatus consists of the following : — a system generating ultraviolet radiation with a wavelength of 210 nm, made up of an ultraviolet lamp, a collimator, and a selective filter ; the beam is blocked periodically by a chopper rotating at high speeds ; — a reaction chamber, through which flows the gas to be examined ; — a system that detects radiation emitted at a wavelength of 350 nm, made up of a selective filter, a photomultiplier tube and an amplifier. Gas to be examined. Use the substance to be examined. It must be filtered.

Reference gas (a). Use a mixture of 79 per cent V/V of nitrogen R1 and 21 per cent V/V of oxygen R. Reference gas (b). Use a mixture of 79 per cent V/V of nitrogen R1 and 21 per cent V/V of oxygen R containing 0.5 ppm V/V to 2 ppm V/V of sulphur dioxide R1. Calibrate the apparatus and set the sensitivity using reference gases (a) and (b). Measure the content of sulphur dioxide in the gas to be examined. Oil : maximum 0.1 mg/m3 calculated for atmospheric pressure and at 0 °C, determined using a measuring system such as described in Figure 1238.-2. The apparatus consists of the following : — an on-off valve (1), — a three-way valve (2), — an oil cone (3), — a bypass line (4), — a pressure-regulator (5), — a flow-metering device (6). All of the apparatus is cleaned beforehand, using trichlorotrifluoroethane R free from oil and grease. Place a micro fibre-glass filter in the oil cone (3) ; this filter has the following characteristics : 100 per cent borosilicate glass without binder ; resistant to heat treatment at 500 °C (to eliminate organic traces) ; 99.999 per cent retention efficiency for NaCl particles with a diameter of 0.6 µm. Close the on-off valve (1) ; the substance to be examined enters the bypass line (4) and purges the three-way valve (2), the pressure-regulator (5) and the flow-metering device (6). Close the inlet valve of the compression and filtration system : open the on-off valve (1) and set the three-way valve (2) to the position allowing passage between the oil

Figure 1238.-1. – UV fluorescence analyser

Figure 1238.-2. – Measuring system for oil 930


Air, medicinal


cone and the pressure-regulator. Open the inlet valve and set the pressure-regulator (5) so that the flow indicated by the flow-metering device (6) is 20 litres/min. Pass 100.0 litres of the substance to be examined through the system. Test solution. Remove the micro fibre-glass filter and place in an airtight container. Carefully cut up the micro fibre-glass filter and place the pieces in 25.0 ml of trichlorotrifluoroethane R. Reference solutions. Prepare the reference solutions with quantities of oil (used for the lubrication of the compression system) ranging from 0.05 µg/ml to 0.5 µg/ml in trichlorotrifluoroethane R. Measure the absorbance of the test solution and the reference solutions, using an appropriate infrared spectrophotometer, at 2960.3 cm− 1, 2927.7 cm− 1 and 2855.0 cm− 1. The sum of the 3 absorbances gives the absorbance of the oil. Use potassium bromide cells with a pathlength of several centimetres. Plot the calibration curve from the absorbances obtained with the reference solutions and determine the quantity of oil from this curve. Nitrogen monoxide and nitrogen dioxide : maximum 2 ppm V/V in total, determined using a chemiluminescence analyser (2.5.26). Gas to be examined. Use the substance to be examined. Reference gas (a). Use a mixture of 79 per cent V/V of nitrogen R1 and 21 per cent V/V of oxygen R containing less than 0.05 ppm V/V of nitrogen monoxide and nitrogen dioxide. Reference gas (b). Use a mixture of 2 ppm V/V of nitrogen monoxide R in nitrogen R1. Calibrate the apparatus and set the sensitivity using reference gases (a) and (b). Measure the content of nitrogen monoxide and nitrogen dioxide in the gas to be examined. Water : maximum 67 ppm V/V, determined using an electrolytic hygrometer (2.5.28), except where the competent authority decides that the following limit applies to medicinal air generated on-site and distributed in pipe-line systems operating at a pressure not greater than 10 bars and a temperature not less than 5 °C : maximum 870 ppm V/V, determined using an electrolytic hygrometer (2.5.28). Assay. Determine the concentration of oxygen in air using a paramagnetic analyser (2.5.27).

pressure in the burette. Keeping the burette vertical, fill the funnel with a freshly prepared mixture of 21 ml of a 560 g/l solution of potassium hydroxide R and 130 ml of a 200 g/l solution of sodium dithionite R. Open the upper tap slowly. The solution absorbs the oxygen and enters the burette. Allow to stand for 10 min without shaking. Read the level of the liquid meniscus on the graduated part of the burette. This figure represents the percentage V/V of oxygen. The value read is 20.4 to 21.4.

IDENTIFICATION First identification : C. Second identification : A, B. A. In a conical flask containing the substance to be examined, place a glowing wood splinter. The splinter remains glowing. B. Use a gas burette (Figure 1238.-3) of 25 ml capacity in the form of a chamber in the middle of which is a tube graduated in 0.2 per cent between 19.0 per cent and 23.0 per cent, and isolated at each end by a tap with a conical barrel. The lower tap is joined to a tube with an olive-shaped nozzle and is used to introduce the gas into the apparatus. A cylindrical funnel above the upper tap is used to introduce the absorbent solution. Wash the burette with water R and dry. Open the 2 taps. Connect the nozzle to the source of the substance to be examined and set the flow rate to 1 litre/min. Flush the burette by passing the substance to be examined through it for 1 min. Close the lower tap of the burette and immediately afterwards the upper tap. Rapidly disconnect the burette from the source of the substance to be examined. Rapidly give a half turn to the upper tap to eliminate any excess

TESTS Carbon dioxide : maximum 500 ppm V/V, determined using a carbon dioxide detector tube (2.1.6). Sulphur dioxide : maximum 1 ppm V/V, determined using a sulphur dioxide detector tube (2.1.6). Oil : maximum 0.1 mg/m3, determined using an oil detector tube (2.1.6). Nitrogen monoxide and nitrogen dioxide : maximum 2 ppm V/V, determined using a nitrogen monoxide and nitrogen dioxide detector tube (2.1.6). Carbon monoxide : maximum 5 ppm V/V, determined using a carbon monoxide detector tube (2.1.6).


931

Figure 1238.-3. – Gas burette C. It complies with the limits of the assay.

Air, synthetic medicinal


Water vapour : maximum 67 ppm V/V, determined using a water vapour detector tube (2.1.6), except where the competent authority decides that the following limit applies to medicinal air generated on-site and distributed in pipe-line systems operating at a pressure not greater than 10 bars and a temperature not less than 5 °C : maximum 870 ppm V/V, determined using a water vapour detector tube (2.1.6). STORAGE As a gas, in suitable containers complying with the legal regulations or as a gas supplied by a pipe network. IMPURITIES A. carbon dioxide, B. sulphur dioxide, C. nitrogen monoxide, D. nitrogen dioxide, E. oil, F. carbon monoxide, G. water.

01/2005:1684

AIR, SYNTHETIC MEDICINAL Aer medicinalis artificiosus DEFINITION Mixture of Nitrogen (1247) and Oxygen (0417). Figure 1684.-1.– Gas burette Content : 95.0 per cent to 105.0 per cent of the nominal value which is between 21.0 per cent V/V to 22.5 per cent V/V of B. Use a gas burette (Figure 1684.-1) of 25 ml capacity in oxygen (O2). the form of a chamber, in the middle of which is a tube graduated in 0.2 per cent between 19.0 per cent and CHARACTERS 23.0 per cent, and isolated at each end by a tap with a conical barrel. The lower tap is joined to a tube with an Colourless and odourless gas. olive-shaped nozzle and is used to introduce the gas into Solubility : at a temperature of 20 °C and a pressure of the apparatus. A cylindrical funnel above the upper tap 101 kPa, 1 volume dissolves in about 50 volumes of water. is used to introduce the absorbent solution. Wash the burette with water R and dry. Open both taps. Connect PRODUCTION the nozzle to the source of the substance to be examined and set the flow rate to 1 litre/min. Flush the burette Water (2.5.28) : maximum 67 ppm V/V. by passing the substance to be examined through it for Assay (2.5.27). Carry out the determination of oxygen in 1 min. Close the lower tap of the burette and immediately gases. afterwards the upper tap. Rapidly disconnect the burette from the source of the substance to be examined. Rapidly IDENTIFICATION give a half turn of the upper tap to eliminate any excess pressure in the burette. Keeping the burette vertical, fill First identification : C. the funnel with a freshly prepared mixture of 21 ml of a Second identification : A, B. 560 g/l solution of potassium hydroxide R and 130 ml A. In a conical flask containing the substance to be of a 200 g/l solution of sodium dithionite R. Open the examined, place a glowing splinter of wood. The splinter upper tap slowly. The solution absorbs the oxygen and remains glowing. enters the burette. Allow to stand for 10 min without 932



Alanine

Appearance of solution. Dilute 10 ml of solution S to 20 ml with water R. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY6 (2.2.2, Method II). Specific optical rotation (2.2.7). Dissolve 2.50 g in hydrochloric acid R1 and dilute to 25.0 ml with the same TESTS acid. The specific optical rotation is + 13.5 to + 15.5, Water vapour : maximum 67 ppm V/V, determined using a calculated with reference to the dried substance. water vapour detector tube (2.1.6). Ninhydrin-positive substances. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R. STORAGE Test solution (a). Dissolve 0.10 g in water R and dilute to As a compressed gas in suitable containers complying with 10 ml with the same solvent. the legal regulations or as a compressed gas supplied by a Test solution (b). Dilute 1 ml of test solution (a) to 50 ml pipe network, after mixing of the components. with water R. LABELLING Reference solution (a). Dissolve 10 mg of alanine CRS in water R and dilute to 50 ml with the same solvent. The label states the nominal content of O2 in per cent V/V. Reference solution (b). Dilute 5 ml of test solution (b) to IMPURITIES 20 ml with water R. A. water. Reference solution (c). Dissolve 10 mg of alanine CRS and 10 mg of glycine CRS in water R and dilute to 25 ml with the same solvent. 01/2005:0752 Apply separately to the plate 5 µl of each solution. Allow the plate to dry in air. Develop over a path of 15 cm with a ALANINE mixture of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes of butanol R. Allow the plate to dry in air. Spray with ninhydrin solution R. Heat the plate at Alaninum 100 °C to 105 °C for 15 min. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). The test is not valid unless the chromatogram obtained with reference C3H7NO2 Mr 89.1 solution (c) shows two clearly separated spots. DEFINITION Chlorides (2.4.4). Dilute 5 ml of solution S to 15 ml with water R. The solution complies with the limit test for Alanine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of (S)-2-aminopropanoic chlorides (200 ppm). acid, calculated with reference to the dried substance. Sulphates (2.4.13). Dilute 10 ml of solution S to 15 ml with distilled water R. The solution complies with the limit test CHARACTERS for sulphates (300 ppm). White or almost white, crystalline powder or colourless Ammonium (2.4.1). 50 mg complies with limit test B for crystals, freely soluble in water, very slightly soluble in ammonium (200 ppm). Prepare the standard using 0.1 ml of alcohol. ammonium standard solution (100 ppm NH4) R. IDENTIFICATION Iron (2.4.9). In a separating funnel, dissolve 1.0 g in 10 ml First identification : A, B. of dilute hydrochloric acid R. Shake with three quantities, each of 10 ml, of methyl isobutyl ketone R1, shaking for Second identification : A, C, D. A. It complies with the test for specific optical rotation (see 3 min each time. To the combined organic layers add 10 ml of water R and shake for 3 min. The aqueous layer complies Tests). with the limit test for iron (10 ppm). B. Examine by infrared absorption spectrophotometry Heavy metals (2.4.8). Dissolve 2.0 g in water R and dilute to (2.2.24), comparing with the spectrum obtained with alanine CRS. Examine the substances prepared as discs. 20 ml with the same solvent. 12 ml of the solution complies with limit test A for heavy metals (10 ppm). Prepare the C. Examine the chromatograms obtained in the test for standard using lead standard solution (1 ppm Pb) R. ninhydrin-positive substances. The principal spot in the Loss on drying (2.2.32). Not more than 0.5 per cent, chromatogram obtained with test solution (b) is similar determined on 1.000 g by drying in an oven at 100 °C to in position, colour and size to the principal spot in the 105 °C. chromatogram obtained with reference solution (a). Sulphated ash (2.4.14). Not more than 0.1 per cent, D. Dissolve 0.5 g in a mixture of 1 ml of water R, 0.5 ml determined on 1.0 g. of a 100 g/l solution of sodium nitrite R and 0.25 ml of hydrochloric acid R1. Shake. Gas is given off. Add 2 ml of dilute sodium hydroxide solution R, followed by ASSAY 0.25 ml of iodinated potassium iodide solution R. After Dissolve 80.0 mg in 3 ml of anhydrous formic acid R. Add 30 ml of anhydrous acetic acid R. Using 0.1 ml of about 30 min, a yellow precipitate with a characteristic naphtholbenzein solution R as indicator, titrate with odour is formed. 0.1 M perchloric acid, until the colour changes from TESTS brownish-yellow to green. Solution S. Dissolve 2.5 g in distilled water R and dilute to 1 ml of 0.1 M perchloric acid is equivalent to 8.91 mg of C3H7NO2. 50 ml with the same solvent. shaking. Read the level of the liquid meniscus on the graduated part of the burette. This figure represents the percentage V/V of oxygen. The value read is 95.0 per cent to 105.0 per cent of the nominal value. C. It complies with the limits of the assay.


933

Albendazole


STORAGE Store protected from light.

Inject 20 µl of reference solution (a). Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is at least 50 per cent of the full scale of the recorder. Inject 20 µl of reference solution (b). The test is not valid unless the resolution between the 01/2005:1386 peaks corresponding to albendazole and oxybendazole is at least 3.0. ALBENDAZOLE Inject 20 µl of the test solution. Continue the chromatography for 1.5 times the retention time of albendazole. When the chromatograms are recorded in the prescribed conditions, Albendazolum the approximate relative retention times are : 0.80 for impurity A, 0.43 for impurities B and C, 0.40 for impurity D, 0.47 for impurity E and 0.57 for impurity F. In the chromatogram obtained with the test solution the area of any peak, apart from the principal peak, is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.75 per cent) and the C12H15N3O2S Mr 265.3 sum of the areas of any such peaks is not greater than 3 times the area of the principal peak in the chromatogram obtained DEFINITION with reference solution (a) (1.5 per cent). Disregard any peak Albendazole contains not less than 98.0 per cent and with an area less than 0.1 times the area of the principal peak not more than the equivalent of 102.0 per cent of methyl in the chromatogram obtained with reference solution (a). [5-(propylsulphanyl)-1H-benzimidazol-2-yl]carbamate, Loss on drying (2.2.32). Not more than 0.5 per cent, calculated with reference to the dried substance. determined on 1.000 g by drying in an oven at 100-105 °C for 4 h. CHARACTERS Sulphated ash (2.4.14). Not more than 0.2 per cent, A white or faintly yellowish powder, practically insoluble determined on 1.0 g. in water, freely soluble in anhydrous formic acid, very slightly soluble in methylene chloride, practically insoluble ASSAY in alcohol. In order to avoid overheating during the titration, mix IDENTIFICATION thoroughly throughout and stop the titration immediately after the end-point has been reached. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with Dissolve 0.250 g in 3 ml of anhydrous formic acid R albendazole CRS. Examine the substances prepared as discs. and add 40 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point TESTS potentiometrically (2.2.20). Appearance of solution. Dissolve 0.10 g in a mixture of 1 ml of 0.1 M perchloric acid is equivalent to 26.53 mg of 1 volume of anhydrous formic acid R and 9 volumes of C12H15N3O2S. methylene chloride R and dilute to 10 ml with the same mixture of solvents. The solution is clear (2.2.1) and not STORAGE more intensely coloured than reference solution BY6 (2.2.2, Store protected from light. Method II). IMPURITIES Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 25.0 mg of the substance to be examined in 5 ml of methanol R containing 1 per cent V/V of sulphuric acid R and dilute to 50.0 ml with the mobile phase. Reference solution (a). Dissolve 10.0 mg of the substance A. R = S-CH2-CH2-CH3 : 5-(propylsulphanyl)-1H-benzimidazolto be examined in 10 ml of methanol R containing 1 per 2-amine, cent V/V of sulphuric acid R and dilute to 100.0 ml with the mobile phase. Dilute 0.5 ml of this solution to 20.0 ml D. R = SO2-CH2-CH2-CH3 : 5-(propylsulphonyl)-1Hwith the mobile phase. benzimidazol-2-amine, Reference solution (b). Dissolve 50.0 mg of the substance to be examined and 50 mg of oxybendazole CRS in 5 ml of methanol R containing 1 per cent V/V of sulphuric acid R and dilute to 100.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in B. R = SO-CH2-CH2-CH3 : methyl [5-(propylsulphinyl)-1Hinternal diameter packed with spherical end-capped benzimidazol-2-yl]carbamate, octadecylsilyl silica gel for chromatography R (5 µm) with a pore size of 10 nm and a carbon loading of 19 per C. R = SO2-CH2-CH2-CH3 : methyl [5-(propylsulphonyl)-1Hcent, benzimidazol-2-yl]carbamate, — as the mobile phase at a flow rate of 0.7 ml/min a mixture E. R = H : methyl (1H-benzimidazol-2-yl)carbamate, of 300 volumes of a 1.67 g/l solution of ammonium dihydrogen phosphate R and 700 volumes of methanol R, F. R = S-CH : methyl [5-(methylsulphanyl)-1H-benzimidazol3 2-yl]carbamate. — as detector a spectrophotometer set at 254 nm. 934


Alcuronium chloride


01/2005:1387

ALCHEMILLA Alchemillae herba DEFINITION Alchemilla consists of the whole or cut, dried, flowering, aerial parts of Alchemilla vulgaris L. sensu latiore. It contains not less than 6.0 per cent of tannins, expressed as pyrogallol (C6H6O3, Mr 126.1), calculated with reference to the dried drug. CHARACTERS It has the macroscopic and microscopic characters described under identification tests A and B. IDENTIFICATION A. The greyish-green, partly brownish-green, radical leaves which are the main part of the drug are reniform to slightly semicircular with a diameter generally up to 8 cm, seldom up to 11 cm and have 7 to 9, or 11 lobes and a long petiole. The smaller, cauline leaves, which have a pair of large stipules at the base, have 5 to 9 lobes and a shorter petiole or they are sessile. The leaves are densely pubescent especially on the lower surface and have a coarsely serrated margin. Young leaves are folded with a whitish-silvery pubescence ; older leaves are slightly pubescent and have a finely meshed venation, prominent on the lower surface. The greyish-green to yellowish-green petiole is pubescent, about 1 mm in diameter, with an adaxial groove. The apetalous flowers are yellowish-green to light green and about 3 mm in diameter. The calyx is double with 4 small segments of the epicalyx alternating with 4 larger sepals, subacute to triangular. They are 4 short stamens and a single carpel with a capitate stigma. The greyish-green to yellowish-green stem is pubescent, more or less longitudinally wrinkled and hollow. B. Reduce to a powder (355). The powder is greyish-green. Examine under a microscope, using chloral hydrate solution R. The powder shows unicellular, narrow trichomes up to 1 mm long partly tortuous, acuminate, and bluntly pointed at the apex, with thick lignified walls, somewhat enlarged and pitted at the base ; fragments of leaves with 2 layers of palisade parenchyma, the upper layer of which is 2 to 3 times longer than the lower layer and with spongy parenchyma, containing scattered cluster crystals of calcium oxalate, up to 25 µm in diameter ; leaf fragments in surface view with sinuous to wavy epidermal cells, the anticlinal walls unevenly thickened and beaded, anomocytic stomata (2.8.3) ; groups of vascular tissue and lignified fibres from the petioles and stems, the vessels spirally thickened or with bordered pits ; occasional thin-walled conical trichomes, about 300 µm long ; thin-walled parenchyma containing cluster crystals of calcium oxalate ; spherical pollen grains, about 15 µm in diameter, with 3 distinct pores and a granular exine ; occasional fragments of the ovary wall with cells containing a single crystal of calcium oxalate. C. Thin-layer chromatography (2.2.27). Test solution. To 0.5 g of the powdered drug (355) add 5 ml of methanol R and heat in a water-bath at 70 °C under a reflux condenser for 5 min. Cool and filter. Reference solution. Dissolve 1.0 mg of caffeic acid R and 1.0 mg of chlorogenic acid R in 10 ml of methanol R. Plate : TLC silica gel plate R. Mobile phase : anhydrous formic acid R, water R, ethyl acetate R (8:8:84 V/V/V). General Notices (1) apply to all monographs and other texts

Application : 20 µl as bands for the test solution, 10 µl as bands for the reference solution. Development : over a path of 10 cm. Drying : at 100-105 °C for 5 min. Detection : spray with a 10 g/l solution of diphenylboric acid aminoethyl ester R in methanol R. Subsequently spray with a 50 g/l solution of macrogol 400 R in methanol R. Allow the plate to dry in air for about 30 min. Examine in ultraviolet light at 365 nm. Results : see below the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. Furthermore, other fluorescent zones may be present in the chromatogram obtained with the test solution. Top of the plate Two red fluorescent zones (chlorophyll) Caffeic acid : a light blue florescent zone

One or two intense light blue fluorescent zones One or several intense green to greenish yellow fluorescent zone _______

_______ Chlorogenic acid : a light blue fluorescent zone _______ Reference solution

An intense yellow to orange fluorescent zone _______ Test solution

TESTS Foreign matter (2.8.2) : maximum 2 per cent. Loss on drying (2.2.32) : maximum 10.0 per cent, determined on 1.000 g of powdered drug (355) by drying in an oven at 100-105 °C for 2 h. Total ash (2.4.16) : maximum 12.0 per cent. ASSAY Carry out the determination of tannins in herbal drugs (2.8.14). Use 0.50 g of the powdered drug (355). 01/2005:1285

ALCURONIUM CHLORIDE Alcuronii chloridum

C44H50Cl2N4O2

Mr 738

DEFINITION Alcuronium chloride contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of (1R,3aS,10S,11aS,12R,14aS,19aS,20bS,21S,22aS, 23E,26E)-23,26-bis(2-hydroxyethylidene)-1,12-bis(prop-2enyl)-2,3,11,11a,13,14,22,22a-octahydro-10H,21H-1,21:10, 12-diethano-19aH,20bH-[1,5]diazocino[1,2,3-lm:5,6,7935

Alcuronium chloride


l′m′]dipyrrolo[2′,3′-d:2′′,3′′ :d′]dicarbazolediium dichloride (4,4′-didesmethyl-4,4′-bis(prop-2-enyl)toxi- ferin I dichloride), calculated with reference to the anhydrous, propan-2-ol-free substance.

Test solution. Dissolve 0.20 g of the substance to be examined in the solvent mixture and dilute to 100.0 ml with the solvent mixture.

CHARACTERS

Reference solution (b). Dilute 4.0 ml of reference solution (a) to 10.0 ml with the solvent mixture.

A white or slightly greyish-white, crystalline powder, freely soluble in water and in methanol, soluble in alcohol, practically insoluble in cyclohexane. Carry out the identification, tests and assay as rapidly as possible avoiding exposure to actinic light. IDENTIFICATION

Reference solution (a). Dilute 0.5 ml of the test solution to 100.0 ml with the solvent mixture.

Reference solution (c). Dilute 1.0 ml of reference solution (a) to 10.0 ml with the solvent mixture. Reference solution (d). To 5.0 ml of the test solution add 5.0 mg of allylstrychnine bromide CRS, dissolve in the solvent mixture and dilute to 100.0 ml with the solvent mixture. The chromatographic procedure may be carried out using :

First identification : A, C. Second identification : B, C. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with alcuronium chloride CRS. B. Examine by thin-layer chromatography (2.2.27) using a TLC silica gel plate R. Test solution. Dissolve 10 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent.

— a stainless steel column 0.25 m long and 4 mm in internal diameter packed with octylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.2 ml/min a solution prepared as follows : mix 200 ml of methanol R, 400 ml of acetonitrile R and 400 ml of a 6.82 g/l solution of potassium dihydrogen phosphate R. Dissolve 2.18 g of sodium laurylsulphonate for chromatography R in the mixture and adjust the apparent pH to 5.4 with a 100 g/l solution of phosphoric acid R,

— as detector a spectrophotometer set at 254 nm. Reference solution. Dissolve 10 mg of alcuronium Inject 10 µl of reference solution (b). Adjust the sensitivity of chloride CRS in methanol R and dilute to 10 ml with the the system so that the height of the peak due to alcuronium same solvent. is at least 10 per cent of the full scale of the recorder. Apply to the plate 10 µl of each solution. Develop over a Inject 10 µl of reference solution (d). The test is not valid path of 15 cm using a mixture of 15 volumes of a 58.4 g/l unless the resolution between the peak corresponding to N-allylstrychnine and alcuronium is at least 4.0. Inject 10 µl solution of sodium chloride R, 35 volumes of dilute of the test solution, 10 µl of reference solution (a) and 10 µl ammonia R2 and 50 volumes of methanol R. Allow the of reference solution (c). Continue the chromatography plate to dry in air for 10 min and then spray with 0.1 M ammonium and cerium nitrate. The principal spot in the of the test solution for twice the retention time of the peak corresponding to alcuronium. In the chromatogram chromatogram obtained with the test solution is similar obtained with the test solution the area of any peak, apart in position, colour and size to the principal spot in the from the principal peak, is not greater than the area of the chromatogram obtained with the reference solution. principal peak in the chromatogram obtained with reference C. It gives reaction (a) of chlorides (2.3.1). solution (a) (0.5 per cent) and not more than one such peak has an area greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.2 per TESTS cent) ; the sum of the areas of all the peaks, apart from the Solution S. Dissolve 0.250 g in carbon dioxide-free water R principal peak, is not greater than twice the area of the and dilute to 25.0 ml with the same solvent. principal peak in the chromatogram obtained with reference Appearance of solution. Solution S is clear (2.2.1) and not solution (a) (1 per cent). Disregard any peak with an area less than the area of the principal peak in the chromatogram more intensely coloured than reference solution Y6, BY6 or obtained with reference solution (c) (0.05 per cent). B6 (2.2.2, Method I). Water (2.5.12). Not more than 5.0 per cent, determined on Acidity or alkalinity. To 10 ml of solution S add 0.1 ml of 0.500 g by the semi-micro determination of water. methyl red solution R and 0.2 ml of 0.01 M hydrochloric acid. The solution is red. Add 0.4 ml of 0.01 M sodium Sulphated ash (2.4.14). Not more than 0.1 per cent, hydroxide. The solution is yellow. determined on 1.0 g. Specific optical rotation (2.2.7) : − 430 to − 451, determined on solution S and calculated with reference to the anhydrous, ASSAY propan-2-ol-free substance. Dissolve 0.300 g by stirring in 70 ml of acetic anhydride R Propan-2-ol. Not more than 1.0 per cent (2.4.24, System A). for 1 min. Titrate with0.1 M perchloric acid until the colour changes from violet-blue to greenish-blue, using 0.1 ml of Related substances. Examine by liquid chromatography crystal violet solution R as indicator. (2.2.29). 1 ml of 0.1 M perchloric acid is equivalent to 36.9 mg of Solvent mixture. Mix 100 ml of methanol R, 200 ml of C44H50Cl2N4O2. acetonitrile R and 200 ml of a 6.82 g/l solution of potassium dihydrogen phosphate R. Dissolve 1.09 g of sodium laurylsulphonate for chromatography R in the mixture and STORAGE Store in an airtight container under nitrogen, protected from adjust the apparent pH to 8.0 with a 100 g/l solution of light, at a temperature of 2 °C to 8 °C. sodium hydroxide R. 936



IMPURITIES

A. (1R,3aS,9R,9aR,10R,11aS,12R,14aS,19aS,20R, 20aR,20bS,21R,22aS)-1,12-bis(prop-2-enyl)-2,3,9a,11,11a, 13,14,19a,20a,21,22,22a-dodecahydro-10H,20bH-1,23:12, 27-dimethano-9,10:20,21-bis(epoxyprop[2]eno)-9H,20H[1,5]diazocino[1,2,3-lm:5,6,7-l′m′]dipyrrolo[2′,3′-d:2′′, 3′′ :d′]dicarbazolediium dichloride (4,4′-diallylcaracurin V dichloride),

Alfacalcidol

IDENTIFICATION A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of alfacalcidol. B. Examine the chromatograms obtained in the assay. The principal peak in the chromatogram obtained with the test solution is similar in retention time and size to the principal peak in the chromatogram obtained with reference solution (a). TESTS Related substances. Examine by liquid chromatography (2.2.29) as described under Assay. Calculate the percentage content of related substances, apart from pre-alfacalcidol, that are eluted within twice the retention time of alfacalcidol from the areas of the peaks in the chromatogram obtained with the test solution by the normalisation procedure. The content of any individual related substance is not greater than 0.5 per cent and the sum of the related substances is not greater than 1.0 per cent. Disregard any peak below 0.1 per cent.

ASSAY Carry out the assay as rapidly as possible, avoiding exposure to actinic light and air. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 1.0 mg of the substance to be examined without heating in 10.0 ml of the mobile phase. B. (4bS,7R,7aS,8aR,13R,13aR,13bS)-13-hydroxy-7-(propReference solution (a). Dissolve 1.0 mg of alfacalcidol CRS 2-enyl)-5,6,7a,8,8a,11,13,13a,13b,14-decahydro-7,9without heating in 10.0 ml of the mobile phase. methano-7H-oxepino[3,4-a]pyrrolo[2,3-d]carbazolium Reference solution (b). Dilute reference solution (a) chloride ((4R,17R)-4-allyl-17,18-epoxy-17-hydroxy-19,20100 times with the mobile phase. didehydrocuranium chloride). Reference solution (c). Heat 2 ml of reference solution (a) in a water-bath at 80 °C under a reflux condenser for 2 h 01/2005:1286 and cool. The chromatographic procedure may be carried out using : ALFACALCIDOL — a column 0.25 m long and 4.0 mm in internal diameter packed with octadecylsilyl silica gel for Alfacalcidolum chromatography R2 (5 µm), — as mobile phase at a flow rate of 2.0 ml/min a mixture of 1 volume of ammonia R, 200 volumes of water R and 800 volumes of acetonitrile R, — as detector a spectrophotometer set at 265 nm, — a loop injector. Inject 100 µl of reference solution (c) and record the chromatogram. Make a total of six injections. When the chromatograms are recorded in the prescribed conditions, the retention time for pre-alfacalcidol, relative to alfacalcidol, is about 1.3. The assay is not valid unless the relative standard deviation of the response for alfacalcidol is at most 1 per cent and the resolution between the peaks due to pre-alfacalcidol and alfacalcidol is at least 4.0 ; adjust C27H44O2 Mr 400.6 the proportions of the constituents of the mobile phase, if necessary, to obtain this resolution. DEFINITION Inject 100 µl of reference solution (a) and 100 µl of reference Alfacalcidol contains not less that 97.0 per cent and solution (b) and record the chromatograms. Inject 100 µl not more than the equivalent of 102.0 per cent of of the test solution and record the chromatogram in the (5Z,7E)-9,10-secocholesta-5,7,10(19)-triene-1α,3β-diol. same manner, continuing the chromatography for twice the CHARACTERS retention time of the principal peak. White or almost white crystals, practically insoluble in water, STORAGE freely soluble in alcohol, soluble in fatty oils. It is sensitive Store under nitrogen, in an airtight container, protected to air, heat and light. from light, at a temperature of 2 °C to 8 °C. A reversible isomerisation to pre-alfacalcidol takes place in The contents of an opened container are to be used solution, depending on temperature and time. The activity immediately. is due to both compounds. General Notices (1) apply to all monographs and other texts

937

Alfadex


01/2005:1487

IMPURITIES

ALFADEX Alfadexum

A. (5E,7E)-9,10-secocholesta-5,7,10(19)-triene-1α,3β-diol (trans-alfacalcidol), [C6H10O5]6

Mr 973

DEFINITION Alfadex contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of cyclohexakis-(1→4)-(α-D-glucopyranosyl) (cyclomaltohexaose or α-cyclodextrin), calculated with reference to the dried substance. CHARACTERS A white or almost white, amorphous or crystalline powder, freely soluble in water and in propylene glycol, practically insoluble in ethanol and in methylene chloride.

B. (5Z,7E)-9,10-secocholesta-5,7,10(19)-triene-1β,3β-diol (1β-calcidol),

C. triazoline adduct of pre-alfacalcidol. 938

IDENTIFICATION A. It complies with the test for specific optical rotation (see Tests). B. Examine the chromatograms obtained in the assay. The retention time and size of the principal peak in the chromatogram obtained with test solution (b) are approximately the same as those of the principal peak in the chromatogram obtained with reference solution (c). C. Dissolve 0.2 g in 2 ml of iodine solution R4 by warming on a water-bath, and allow to stand at room temperature ; a yellowish-brown precipitate is formed. TESTS Solution S. Dissolve 1.000 g in carbon dioxide-free water R and dilute to 100.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1). pH (2.2.3). The pH of a mixture of 30 ml of solution S and 1 ml of a 223.6 g/l solution of potassium chloride R is 5.0 to 8.0. Specific optical rotation (2.2.7) : + 147 to + 152, determined on solution S and calculated with reference to the dried substance. Reducing sugars Test solution. To 1 ml of solution S add 1 ml of cupri-tartaric solution R4. Heat on a water-bath for 10 min, cool to room temperature. Add 10 ml of ammonium molybdate reagent R1 and allow to stand for 15 min.


Alfentanil hydrochloride


Reference solution. Prepare a reference solution at the same time and in the same manner as the test solution, using 1 ml of a 0.02 g/l solution of glucose R. Measure the absorbance of the test solution and the reference solution (2.2.25) at the maximum at 740 nm using water R as the compensation liquid. The absorbance of the test solution is not greater than that of the reference solution (0.2 per cent). Light-absorbing impurities. Examine solution S between 230 nm and 750 nm (2.2.25). Between 230 nm and 350 nm, the absorbance is not greater than 0.10. Between 350 nm and 750 nm, the absorbance is not greater than 0.05. Related substances. Examine by liquid chromatography (2.2.29) as described under Assay. Inject test solution (a) and reference solution (b). In the chromatogram obtained with test solution (a) : the areas of any peaks corresponding to betadex or gammacyclodextrin are not greater than half of the area of the corresponding peaks in the chromatogram obtained with reference solution (b) (0.25 per cent) ; the sum of the areas of all the peaks, apart from the principal peak and any peaks corresponding to betadex or gammacyclodextrin, is not greater than half of the area of the peak corresponding to alfadex in the chromatogram obtained with reference solution (b) (0.5 per cent). Heavy metals (2.4.8). 2.0 g complies with limit test C for heavy metals (10 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 11 per cent, determined on 1.000 g by drying in an oven at 120 °C for 2 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Examine by liquid chromatography (2.2.29). Test solution (a). Dissolve 0.25 g of the substance to be examined in water R with heating, cool and dilute to 25.0 ml with the same solvent. Test solution (b). Dilute 5.0 ml of test solution (a) to 50.0 ml with water R. Reference solution (a). Dissolve 25.0 mg of betadex CRS, 25.0 mg of gammacyclodextrin CRS and 50.0 mg of alfadex CRS in water R and dilute to 50.0 ml with the same solvent. Reference solution (b). Dilute 5.0 ml of reference solution (a) to 50.0 ml with water R. Reference solution (c). Dissolve 25.0 mg of alfadex CRS in water R and dilute to 25.0 ml with the same solvent. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (10 µm), — as mobile phase at a flow rate of 1.5 ml/min a mixture of 10 volumes of methanol R and 90 volumes of water R, — as detector a differential refractometer, — a 50 µl loop injector. Equilibrate the column with the mobile phase at a flow rate of 1.5 ml/min for about 3 h. Inject reference solution (a) 5 times and record the chromatograms for 3.5 times the retention time of alfadex. Adjust the sensitivity of the system so that the height of the peak corresponding to gammacyclodextrin is 55 per cent to 75 per cent of the full scale of the recorder. The retention time of alfadex is about 10 min, the relative retention of gammacyclodextrin is about 0.7 and that of betadex is about 2.2. The test is not valid unless the resolution between the peaks corresponding to General Notices (1) apply to all monographs and other texts

gammacyclodextrin and alfadex is at least 1.5 and the relative standard deviation for the area of the peak corresponding to alfadex is less than 2.0 per cent. If necessary, adjust the concentration of methanol in the mobile phase to achieve the required resolution. Inject alternately test solution (b) and reference solution (c). Calculate the percentage content of [C6H10O5]6 from the area of the principal peak in each of the chromatograms obtained and from the declared content of alfadex CRS. STORAGE Store in an airtight container. IMPURITIES A. betadex,

B. cyclooctakis-(1→4)-(α-D-glucopyranosyl) (cyclomaltooctaose or γ-cyclodextrin). 01/2005:1062

ALFENTANIL HYDROCHLORIDE Alfentanili hydrochloridum

C21H33ClN6O3

Mr 453.0

DEFINITION Alfentanil hydrochloride contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent of N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1H-tetrazol-1-yl)ethyl]4-(methoxymethyl)piperidin-4-yl]-N-phenylpropanamide hydrochloride, calculated with reference to the anhydrous substance. CHARACTERS A white or almost white powder, freely soluble in water, in alcohol and in methanol. It melts at about 140 °C, with decomposition. 939

Alfentanil hydrochloride


apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.25 per cent) ; the sum of the areas of all such peaks is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent). Disregard any peak obtained with the blank-run, and any peak with an area less than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.05 per cent). TESTS Water (2.5.12) : 3.0 per cent to 4.0 per cent, determined on Appearance of solution. Dissolve 0.2 g in water R and dilute 0.500 g by the semi-micro determination of water. to 20 ml with the same solvent. The solution is clear (2.2.1) ASSAY and colourless (2.2.2, Method II). Dissolve 0.350 g in 50 ml of a mixture of 1 volume of Related substances. Examine by liquid chromatography alcohol R and 4 volumes of water R and add 5.0 ml of 0.01 M (2.2.29). hydrochloric acid. Titrate with 0.1 M sodium hydroxide, Test solution. Dissolve 0.100 g of the substance to be determining the end-point potentiometrically (2.2.20). Read examined in methanol R and dilute to 10.0 ml with the same the volume added between the two points of inflexion. solvent. 1 ml of 0.1 M sodium hydroxide is equivalent to 45.30 mg Reference solution (a). In order to prepare the in situ of C21H33ClN6O3. degradation compound (alfentanil impurity E), dissolve 10 mg of the substance to be examined in 10.0 ml of dilute STORAGE hydrochloric acid R. Heat on a water-bath under a reflux condenser for 4 h. Neutralise with 10.0 ml of dilute sodium Store protected from light. hydroxide solution R. Evaporate to dryness on a water-bath. Cool and take up the residue in 10 ml of methanol R. Filter. IMPURITIES Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with methanol R. Dilute 5.0 ml of this solution to 20.0 ml with methanol R. The chromatographic procedure may be carried out using : — a stainless steel column 0.1 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica for chromatography R (3 µm), — as mobile phase at a flow rate of 1.5 ml/min, a gradient programme using the following conditions : Mobile phase A. A 5 g/l solution of ammonium carbonate R in a mixture of 10 volumes of tetrahydrofuran R and 90 volumes of water R, A. cis-N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1H-tetrazol1-yl)ethyl]-4-(methoxymethyl)piperidin-4-yl]-NMobile phase B. Acetonitrile R, phenylpropanamide N-oxide, IDENTIFICATION A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of alfentanil hydrochloride. B. Dissolve 50 mg in a mixture of 0.4 ml of ammonia R and 2 ml of water R. Mix, allow to stand for 5 min and filter. Acidify the filtrate with dilute nitric acid R. It gives reaction (a) of chlorides (2.3.1).

Time (min)



Comment

0 - 15

90 → 40

10 → 60

linear gradient

15 - 20

40

60

isocratic elution

20 - 25

90

10

switch to initial composition

25 = 0

90

10

restart gradient

B. trans-N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1H-tetrazol1-yl)ethyl]-4-(methoxymethyl)piperidin-4-yl]-N— as detector a spectrophotometer set at 220 nm. phenylpropanamide N-oxide, Equilibrate the column for at least 30 min with acetonitrile R and then equilibrate at the initial eluent composition for at least 5 min. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 10 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder. Inject 10 µl of reference solution (a). When the chromatograms are recorded in the prescribed conditions the C. N-[4-(methoxymethyl)piperidin-4-yl]-N-phenylpropanaretention times are : alfentanil impurity E about 6 min and mide, alfentanil hydrochloride about 7 min. Disregard any other peak. The test is not valid unless the resolution between the peaks corresponding to alfentanil hydrochloride and alfentanil impurity E is at least 4.0. If necessary, adjust the concentration of acetonitrile in the mobile phase or adjust the time programme for the linear-gradient elution. Inject 10 µl of methanol R as a blank, 10 µl of the test solution and 10 µl of reference solution (b). In the chromatogram D. N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1H-tetrazol-1-yl)ethyl]-4obtained with the test solution : the area of any peak, (methoxymethyl)piperidin-4-yl]-N-phenylacetamide, 940



Alfuzosin hydrochloride

B. To 1 ml of solution S (see Tests) add 1 ml of water R. The solution gives reaction (a) of chlorides (2.3.1). TESTS Solution S. Dissolve 0.500 g in carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. E. 1-ethyl-1,4-dihydro-4-[2-[[4-(methoxymethyl)-4pH (2.2.3). The pH of freshly prepared solution S is 4.0 phenylamino]piperidin-1-yl]ethyl]-5H-tetrazol-5-one, to 6.0. Optical rotation (2.2.7). The angle of optical rotation, determined on solution S, is − 0.10° to + 0.10°. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 20.0 mg of the substance to be F. N-[1-(2-hydroxyethyl)-4-(methoxymethyl)piperidin-4-yl]-Nexamined in the mobile phase and dilute to 100.0 ml with phenylpropanamide, the mobile phase. Reference solution (a). Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. Dilute 5.0 ml of the solution to 20.0 ml with the mobile phase. Reference solution (b). Dissolve 5 mg of alfuzosin impurity A CRS in the mobile phase and dilute to 25 ml with the mobile phase. To 1 ml of the solution, add 1 ml of the test solution and dilute to 100 ml with the mobile phase. G. N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1H-tetrazol-1The chromatographic procedure may be carried out using : yl)ethyl]-4-(propanoyloxymethyl)piperidin-4-yl]-N— a stainless steel column 0.15 m long and 4.6 mm phenylpropanamide, in internal diameter packed with microparticulate octadecylsilyl silica gel for chromatography R (5 µm), with a carbon loading of 18.5 per cent, a specific surface area of 320 m2/g, a pore size of 15 nm, and end-capped with hexamethyldisilane, — as mobile phase at a flow rate of 1.5 ml/min a mixture of 1 volume of tetrahydrofuran R, 20 volumes of H. N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1H-tetrazol-1-yl)ethyl]-4acetonitrile R and 80 volumes of a solution of sodium (methoxymethyl)piperidin-4-yl]-N-phenylbutanamide. perchlorate prepared as follows : dilute 5.0 ml of perchloric acid R in 900 ml of water R, adjust to pH 3.5 01/2005:1287 with dilute sodium hydroxide solution R and dilute to 1000 ml with water R, ALFUZOSIN HYDROCHLORIDE — as detector a spectrophotometer set at 254 nm. Inject 20 µl of reference solution (b). Adjust the sensitivity Alfuzosini hydrochloridum of the system so that the height of the two peaks in the chromatogram obtained is at least 50 per cent of the full scale of the recorder. The test is not valid unless the resolution between the peaks corresponding to alfuzosin and alfuzosin impurity A is at least 3.0. Inject 20 µl of the test solution and 20 µl of reference solution (a). In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than 0.6 times the area of the C19H28ClN5O4 Mr 425.9 principal peak in the chromatogram obtained with reference solution (a) (0.3 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than the DEFINITION area of the principal peak in the chromatogram obtained Alfuzosin hydrochloride contains not less than 98.5 per with reference solution (a) (0.5 per cent). Disregard any peak cent and not more than the equivalent of 101.0 per with an area less than 0.025 times that of the principal peak cent of (RS)-N-[3-[(4-amino-6,7-dimethoxyquinazolin-2in the chromatogram obtained with reference solution (a). yl)(methyl)amino]propyl]tetrahydrofuran-2-carboxamide hydrochloride, calculated with reference to the anhydrous Water (2.5.12). Not more than 2.0 per cent, determined on substance. 0.500 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.1 per cent, CHARACTERS determined on 1.0 g. A white or almost white, crystalline powder, slightly hygroscopic, freely soluble in water, sparingly soluble in ASSAY alcohol, practically insoluble in methylene chloride. Dissolve 0.300 g in a mixture of 40 ml of anhydrous acetic acid R and 40 ml of acetic anhydride R. Titrate IDENTIFICATION with 0.1 M perchloric acid, determining the end-point A. Examine by infrared absorption spectrophotometry potentiometrically (2.2.20). (2.2.24), comparing with the spectrum obtained with 1 ml of 0.1 M perchloric acid is equivalent to 42.59 mg of alfuzosin hydrochloride CRS. Examine the substances C19H28ClN5O4. prepared as discs. General Notices (1) apply to all monographs and other texts

941

Alginic acid


B. To 5 ml of the filtrate obtained in identification test A add 0.5 ml of a 123 g/l solution of magnesium sulphate R. No voluminous gelatinous mass is formed. C. To 5 mg add 5 ml of water R, 1 ml of a freshly prepared 10 g/l solution of 1,3-dihydroxynaphthalene R in alcohol R and 5 ml of hydrochloric acid R. Boil gently for 3 min, cool, add 5 ml of water R, and shake with 15 ml of di-isopropyl ether R. Carry out a blank test. The upper layer obtained with the substance to be examined exhibits a deeper bluish-red colour than that obtained with the blank.

STORAGE Store in an airtight container, protected from light. IMPURITIES

TESTS Chlorides. Not more than 1.0 per cent. To 2.50 g add 50 ml of dilute nitric acid R, shake for 1 h and dilute to 100.0 ml with dilute nitric acid R. Filter. To 50.0 ml of the filtrate add 10.0 ml of 0.1 M silver nitrate and 5 ml of toluene R. Titrate with 0.1 M ammonium thiocyanate, using 2 ml of ferric ammonium sulphate solution R2 as indicator and shaking vigorously towards the end point. 1 ml of 0.1 M silver nitrate is equivalent to 3.545 mg of Cl. Heavy metals (2.4.8). 1.0 g complies with the limit test F for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 15.0 per cent, determined on 0.1000 g by drying in an oven at 100 °C to 105 °C for 4 h. Sulphated ash (2.4.14). Not more than 8.0 per cent, determined on 0.100 g and calculated with reference to the dried substance. Microbial contamination. Total viable aerobic count (2.6.12) not more than 102 micro-organisms per gram, determined by plate-count. It complies with the tests for Escherichia coli and Salmonella (2.6.13).

A. N-[3-[(4-amino-6,7-dimethoxyquinazolin-2yl)(methyl)amino]propyl]furan-2-carboxamide, B. R-Cl : 2-chloro-6,7-dimethoxyquinazolin-4-amine,

C. (RS)-N-[3-[(4-amino-6,7-dimethoxyquinazolin2-yl)amino]propyl]-N-methyltetrahydrofuran2-carboxamide,

D. N-(4-amino-6,7-dimethoxyquinazolin-2-yl)-Nmethylpropane-1,3-diamine,

ASSAY To 0.2500 g add 25 ml of water R, 25.0 ml of 0.1 M sodium E. N-[3-[(4-amino-6,7-dimethoxyquinazolin-2hydroxide and 0.2 ml of phenolphthalein solution R. Titrate yl)(methyl)amino]propyl]formamide. with 0.1 M hydrochloric acid. 1 ml of 0.1 M sodium hydroxide is equivalent to 4.502 mg 01/2005:0591 of carboxyl groups (COOH).

ALGINIC ACID 01/2005:1288

Acidum alginicum

ALLANTOIN

DEFINITION Alginic acid is a mixture of polyuronic acids [(C6H8O6)n] composed of residues of D-mannuronic and L-guluronic acid and is obtained mainly from algae belonging to the Phaeophyceae. A small proportion of the carboxyl groups may be neutralised. It contains not less than 19.0 per cent and not more than 25.0 per cent of carboxyl groups (COOH), calculated with reference to the dried substance. CHARACTERS A white or pale yellowish-brown, crystalline or amorphous powder, very slightly soluble or practically insoluble in alcohol, practically insoluble in organic solvents. It swells in water but does not dissolve ; it dissolves in solutions of alkali hydroxides. IDENTIFICATION A. To 0.2 g add 20 ml of water R and 0.5 ml of sodium carbonate solution R. Shake and filter. To 5 ml of the filtrate add 1 ml of calcium chloride solution R. A voluminous gelatinous mass is formed. 942

Allantoinum

C4 H 6 N 4 O 3

Mr 158.1

DEFINITION Allantoin contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of (RS)-(2,5-dioxoimidazolidin-4-yl)urea. CHARACTERS A white, crystalline powder, slightly soluble in water, very slightly soluble in alcohol. It melts at about 225 °C, with decomposition.


Allopurinol


IDENTIFICATION First identification : A. Second identification : B, C, D. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with allantoin CRS. B. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). C. Boil 20 mg with a mixture of 1 ml of dilute sodium hydroxide solution R and 1 ml of water R. Allow to cool. Add 1 ml of dilute hydrochloric acid R. To 0.1 ml of the solution add 0.1 ml of a 100 g/l solution of potassium bromide R, 0.1 ml of a 20 g/l solution of resorcinol R and 3 ml of sulphuric acid R. Heat for 5 min to 10 min on a water-bath. A dark blue colour develops, which becomes red after cooling and pouring into about 10 ml of water R. D. Heat about 0.5 g. Ammonia vapour is evolved, which turns red litmus paper R blue.

solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows two clearly separated principal spots. Loss on drying (2.2.32). Not more than 0.1 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

TESTS Solution S. Dissolve 0.5 g in carbon dioxide-free water R, with heating if necessary, and dilute to 100 ml with the same solvent. Acidity or alkalinity. To 5 ml of solution S add 5 ml of carbon dioxide-free water R, 0.1 ml of methyl red solution R and 0.2 ml of 0.01 M sodium hydroxide. The solution is yellow. Add 0.4 ml of 0.01 M hydrochloric acid. The solution is red. Optical rotation (2.2.7). The angle of optical rotation, determined on solution S, is − 0.10° to + 0.10°. Reducing substances. Shake 1.0 g with 10 ml of water R for 2 min. Filter. Add 1.5 ml of 0.02 M potassium permanganate. The solution must remain violet for at least 10 min. Related substances. Examine by thin-layer chromatography (2.2.27), using a suitable cellulose for chromatography R as the coating substance. Test solution (a). Dissolve 0.10 g of the substance to be examined in 5.0 ml of water R with heating. Allow to cool. Dilute to 10 ml with methanol R. Use the solution immediately after preparation. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of 1 volume of methanol R and 1 volume of water R. Reference solution (a). Dissolve 10 mg of allantoin CRS in a mixture of 1 volume of methanol R and 1 volume of water R and dilute to 10 ml with the same mixture of solvents. Reference solution (b). Dissolve 10 mg of urea R in 10 ml of water R. Dilute 1 ml of this solution to 10 ml with methanol R. Reference solution (c). Mix 1 ml of reference solution (a) and 1 ml of reference solution (b). Apply to the plate 10 µl of test solution (a) and 5 µl each of test solution (b), reference solution (a), reference solution (b) and reference solution (c). Develop over a path of 10 cm using a mixture of 15 volumes of glacial acetic acid R, 25 volumes of water R and 60 volumes of butanol R. Allow the plate to dry in air. Spray the plate with a 5 g/l solution of dimethylaminobenzaldehyde R in a mixture of 1 volume of hydrochloric acid R and 3 volumes of methanol R. Dry the plate in a current of hot air. Examine in daylight after 30 min. Any spot in the chromatogram obtained with test

A. glyoxylic acid,


ASSAY Dissolve 120.0 mg in 40 ml of water R. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M sodium hydroxide is equivalent to 15.81 mg of C4H6N4O3. IMPURITIES

B. urea. 01/2005:0576

ALLOPURINOL Allopurinolum

C5 H 4 N 4 O

Mr 136.1

DEFINITION 1,5-Dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one. Content : 98.0 per cent to 102.0 per cent (dried substance). CHARACTERS Appearance : white or almost white powder. Solubility : very slightly soluble in water and in alcohol. It dissolves in dilute solutions of alkali hydroxides. IDENTIFICATION First identification : B. Second identification : A, C, D. A. Dissolve 10 mg in 1 ml of a 4 g/l solution of sodium hydroxide R and dilute to 100.0 ml with a 10.3 g/l solution of hydrochloric acid R. Dilute 10.0 ml of this solution to 100.0 ml with a 10.3 g/l solution of hydrochloric acid R. Examined between 220 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 250 nm and an absorption minimum at 231 nm. The ratio of the absorbance measured at the absorption minimum at 231 nm to that measured at the absorption maximum at 250 nm is 0.52 to 0.62. B. Infrared absorption spectrophotometry (2.2.24). Preparation : discs. Comparison : allopurinol CRS. 943

Allopurinol


C. Dissolve 0.3 g in 2.5 ml of dilute sodium hydroxide solution R and add 50 ml of water R. Add slowly and with shaking 5 ml of silver nitrate solution R1. A white precipitate is formed which does not dissolve on the addition of 5 ml of ammonia R. D. Thin-layer chromatography (2.2.27). Test solution. Dissolve 20 mg of the substance to be examined in concentrated ammonia R and dilute to 10 ml with the same solvent. Reference solution. Dissolve 20 mg of allopurinol CRS in concentrated ammonia R and dilute to 10 ml with the same solvent. Plate : TLC silica gel F254 plate R. Mobile phase : ethanol R, methylene chloride R (40:60 V/V). Application : 10 µl. Development : over 2/3 of the plate. Drying : in air. Detection : examine in ultraviolet light at 254 nm. Results : the principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. TESTS Related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately before use. Store them and inject them at 8 °C using a cooled autosampler. Test solution (a). Dissolve 25.0 mg of the substance to be examined in 2.5 ml of a 4 g/l solution of sodium hydroxide R and dilute immediately to 50.0 ml with the mobile phase. Test solution (b). Dissolve 20.0 mg of the substance to be examined in 5.0 ml of a 4 g/l solution of sodium hydroxide R and dilute immediately to 250.0 ml with the mobile phase. Reference solution (a). Dilute 2.0 ml of test solution (a) to 100.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (b). Dissolve 5.0 mg of allopurinol impurity A CRS, 5.0 mg of allopurinol impurity B CRS and 5.0 mg of allopurinol impurity C CRS in 5.0 ml of a 4 g/l solution of sodium hydroxide R and dilute immediately to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (c). Dissolve 20.0 mg of allopurinol CRS in 5.0 ml of a 4 g/l solution of sodium hydroxide R and dilute immediately to 250.0 ml with the mobile phase. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase: octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : 1.25 g/l solution of potassium dihydrogen phosphate R. Flow rate : 1.4 ml/min. Detection : spectrophotometer at 230 nm. Injection : 20 µl of test solution (a) and reference solutions (a) and (b). Run time : twice the retention time of allopurinol. Elution order : impurity A, impurity B, impurity C, allopurinol. Retention time : allopurinol = about 10 min. System suitability : reference solution (b) : — resolution : minimum 1.1 between the peaks due to impurity B and impurity C. 944

Limits : — impurity A : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent), — impurity B : not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), — impurity C : not more than the area of the corresponding peak in the chromatogram obtained with reference solution (b) (0.1 per cent), — any other impurity : for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), — sum of impurities other than A, B and C : not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.3 per cent), — disregard limit : 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). Impurities D and E. Liquid chromatography (2.2.29). Prepare the solutions immediately before use. Store them and inject them at 8 °C using a cooled autosampler. Solution A : 1.25 g/l solution of potassium dihydrogen phosphate R. Test solution. Dissolve 50.0 mg of the substance to be examined in 5.0 ml of a 4 g/l solution of sodium hydroxide R and dilute immediately to 100.0 ml with solution A. Reference solution. Dissolve 5.0 mg of allopurinol impurity D CRS and 5.0 mg of allopurinol impurity E CRS in 5.0 ml of a 4 g/l solution of sodium hydroxide R and dilute immediately to 100.0 ml with solution A. Dilute 1.0 ml of this solution to 100.0 ml with solution A. Column : — size : l = 0.05 m, Ø = 4.6 mm, — stationary phase : base-deactivated octadecylsilyl silica gel for chromatography R (3 µm). Mobile phase : methanol R, 1.25 g/l solution of potassium dihydrogen phosphate R (10:90 V/V). Flow rate : 2 ml/min. Detection : spectrophotometer at 230 nm. Injection : 20 µl. Run time : 1.5 times the retention time of impurity E. Retention times : impurity D = about 3.6 min ; impurity E = about 4.5 min. System suitability : reference solution : — resolution : minimum 2.0 between the peaks due to impurity D and impurity E. Limits : — impurity D : not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.1 per cent), — impurity E : not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.1 per cent). Heavy metals (2.4.8) : maximum 20 ppm. 1.0 g complies with limit test C. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g.



ASSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modification. Injection : test solution (b) and reference solution (c). Calculate the percentage content of C5H4N4O from the areas of the peaks and the declared content of allopurinol CRS.

Almagate

C. 2 ml of the solution prepared under identification test B gives the reaction of magnesium (2.3.1).

TESTS pH (2.2.3) : 9.1 to 9.7. Disperse 4.0 g in 100 ml of carbon dioxide-free water R, stir for 2 min and filter. IMPURITIES Neutralising capacity. Carry out the test at 37 °C. Disperse Specified impurities : A, B, C, D, E. 0.5 g in 100 ml of water R, heat, add 100.0 ml of 0.1 M hydrochloric acid, previously heated and stir continuously ; the pH (2.2.3) of the solution between 5 min and 20 min is not less than 3.0 and not greater than 4.5. Add 10.0 ml of 0.5 M hydrochloric acid, previously heated, stir continuously for 1 h and titrate with 0.1 M sodium hydroxide to pH 3.5 ; not more than 20.0 ml of 0.1 M sodium hydroxide is required. A. R1 = NH2, R2 = H : 5-amino-1H-pyrazole-4-carboxamide, Chlorides (2.4.4) : maximum 0.1 per cent. B. R1 = NH2, R2 = CHO : 5-(formylamino)-1H-pyrazole-4Dissolve 0.33 g in 5 ml of dilute nitric acid R and dilute to carboxamide, 100 ml with water R. 15 ml of the solution complies with the limit test for chlorides. Prepare simultaneously the standard D. R1 = O-CH2-CH3, R2 = H : ethyl 5-amino-1H-pyrazole-4by diluting 0.7 ml of dilute nitric acid R to 5 ml with water R carboxylate, and adding 10 ml of chloride standard solution (5 ppm Cl) R. E. R1 = O-CH2-CH3, R2 = CHO : ethyl 5-(formylamino)-1HSulphates (2.4.13) : maximum 0.4 per cent. pyrazole-4-carboxylate, Dissolve 0.25 g in 5 ml of dilute hydrochloric acid R and dilute to 100 ml with distilled water R. 15 ml of the solution complies with the limit test for sulphates. Prepare simultaneously the standard by adding 0.8 ml of dilute hydrochloric acid R to 15 ml of sulphate standard solution (10 ppm SO4) R. Sodium : maximum 150 ppm. Atomic absorption spectrometry (2.2.23, Method I). C. 5-(4H-1,2,4-triazol-4-yl)-1H-pyrazole-4-carboxamide. Test solution. Dissolve 0.25 g in 50 ml of a 103 g/l solution of hydrochloric acid R. 01/2005:2010 corrected Reference solutions. Prepare the reference solutions using sodium standard solution (200 ppm Na) R, diluted as necessary with a 103 g/l solution of hydrochloric acid R. ALMAGATE Heavy metals (2.4.8) : maximum 20 ppm. Dissolve 1.0 g in dilute hydrochloric acid R and dilute to Almagatum 20.0 ml with the same acid. 12 ml of the solution complies Al2Mg6C2O20H14,4H2O Mr 630 with limit test A. Prepare the standard using lead standard solution (1 ppm Pb) R. DEFINITION Loss on ignition : 43.0 per cent to 49.0 per cent, determined on 1.000 g by ignition at 900 °C. Hydrated aluminium magnesium hydroxycarbonate. Content : Microbial contamination. Total viable aerobic count (2.6.12) not more than 103 micro-organisms per gram determined by — aluminium : 15.0 per cent to 17.0 per cent (calculated plate count. It complies with the tests for Escherichia coli as Al2O3), and Pseudomonas aeruginosa (2.6.13). — magnesium : 36.0 per cent to 40.0 per cent (calculated as MgO), ASSAY — carbonic acid : 12.5 per cent to 14.5 per cent (calculated Aluminium. Dissolve 1.000 g in 5 ml of hydrochloric acid R, as CO2). heating if necessary. Allow to cool to room temperature CHARACTERS and dilute to 100.0 ml with water R (solution A). Introduce Appearance : white or almost white, fine crystalline powder. 10.0 ml of solution A into a 250 ml conical flask, add 25.0 ml of 0.05 M sodium edetate, 20 ml of buffer solution pH 3.5 R, Solubility : practically insoluble in water, in alcohol and 40 ml of ethanol R and 2 ml of a freshly prepared 0.25 g/l in methylene chloride. It dissolves with effervescence and solution of dithizone R in ethanol R. Titrate the excess of heating in dilute mineral acids. sodium edetate with 0.05 M zinc sulphate until the colour changes from greenish-violet to pink. IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). 1 ml of 0.05 M sodium edetate is equivalent to 2.549 mg Comparison : Ph. Eur. reference spectrum of almagate. of Al2O3. B. Dissolve 150 mg in dilute hydrochloric acid R and dilute Magnesium. Introduce 10.0 ml of solution A prepared in the to 20 ml with the same acid. 2 ml of the solution gives the assay of aluminium into a 500 ml conical flask, add 200 ml of water R, 20 ml of triethanolamine R with shaking, 10 ml of reaction of aluminium (2.3.1). General Notices (1) apply to all monographs and other texts

945

all-rac-α-Tocopherol


IMPURITIES

A. 4-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-2-deoxy-6-O-(2, 6-diamino-2,6-dideoxy-α-D-glucopyranosyl)-L-streptamine (kanamycin B),

C. Thin-layer chromatography (2.2.27). Test solution. Dissolve 10 mg of the substance to be examined in 2 ml of cyclohexane R. Reference solution. Dissolve 10 mg of α-tocopherol CRS in 2 ml of cyclohexane R. Plate : TLC silica gel F254 plate R. Mobile phase : ether R, cyclohexane R (20:80 V/V). Application : 10 µl. Development : over 2/3 of the plate. Drying : in a current of air. Detection : examine in ultraviolet light at 254 nm. Results : the principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.

TESTS Related substances. Gas chromatography (2.2.28) : use the normalisation procedure. Internal standard solution. Dissolve 1.0 g of squalane R in cyclohexane R and dilute to 100.0 ml with the same solvent. Test solution (a). Dissolve 0.100 g of the substance to be examined in 10.0 ml of the internal standard solution. Test solution (b). Dissolve 0.100 g of the substance to be examined in 10 ml of cyclohexane R. Reference solution (a). Dissolve 0.100 g of α-tocopherol CRS B. R = H : 2-deoxy-4-O-(2,6-diamino-2,3,6-trideoxy-α-D-riboin 10.0 ml of the internal standard solution. hexopyranosyl)-D-streptamine (nebramine), Reference solution (b). Dissolve 10 mg of the substance C. R = OH : 2-deoxy-4-O-(2,6-diamino-2,6-dideoxy-α-Dto be examined and 10 mg of α-tocopheryl acetate R in glucopyranosyl)-D-streptamine (neamine). cyclohexane R and dilute to 100.0 ml with the same solvent. 01/2005:0692 Reference solution (c). Dissolve 10 mg of all-rac-α-tocopherol for peak identification CRS in cyclohexane R and dilute to 1 ml with the same solvent. all-rac-α-TOCOPHEROL Reference solution (d). Dilute 1.0 ml of test solution (b) to 100.0 ml with cyclohexane R. Dilute 1.0 ml of this solution int-rac-α-Tocopherolum to 10.0 ml with cyclohexane R. Column : — material : fused silica, — size : l = 30 m, Ø = 0.25 mm, — stationary phase : poly(dimethyl)siloxane R (film thickness 0.25 µm). Carrier gas : helium for chromatography R. C29H50O2 Mr 430.7 Flow rate : 1 ml/min. Split ratio : 1:100. DEFINITION Temperature : all-rac-2,5,7,8-Tetramethyl-2-(4,8,12-trimethyltridecyl)-3,4— column : 280 °C, dihydro-2H-1-benzopyran-6-ol. — injection port and detector : 290 °C. Content : 96.0 per cent to 101.5 per cent. Detection : flame ionisation. Injection : 1 µl of test solution (b) and reference solutions (b), CHARACTERS (c) and (d). Appearance : clear, colourless or yellowish-brown, viscous, Run time : twice the retention time of all-rac-α-tocopherol. oily liquid. Solubility : practically insoluble in water, freely soluble in Relative retention with reference to all-rac-α-tocopherol acetone, in ethanol, in methylene chloride and in fatty oils. (retention time = about 13 min) : squalane = about 0.5 ; impurity A = about 0.7 ; impurity B = about 0.8. Use the IDENTIFICATION chromatogram supplied with the CRS and the chromatogram First identification : A, B. obtained with reference solution (c) to identify the peaks due to impurities A and B. Second identification : A, C. System suitability : reference solution (b) : A. Optical rotation (2.2.7) : − 0.01° to + 0.01°. — resolution : minimum 3.5 between the peaks due to Dissolve 2.50 g in ethanol R and dilute to 25.0 ml with all-rac-α-tocopherol and α-tocopheryl acetate. the same solvent. Limits : B. Infrared absorption spectrophotometry (2.2.24). Comparison : α-tocopherol CRS. — impurity A : maximum 0.5 per cent, 2590



RRR-α-Tocopherol

IDENTIFICATION First identification : B, D. Second identification : A, C, D. A. It complies with the test for absorbance (see Tests). B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with α-tocopherol CRS. ASSAY C. Examine by thin-layer chromatography (2.2.27), using a Gas chromatography (2.2.28) as described in the test for TLC silica gel F254 plate R. related substances with the following modification. Test solution. Dissolve 10 mg of the substance to be Injection : test solution (a) and reference solution (a). examined in 2 ml of cyclohexane R. Calculate the percentage content of C29H50O2 taking into Reference solution. Dissolve 10 mg of α-tocopherol CRS account the declared content of α-tocopherol CRS. in 2 ml of cyclohexane R. STORAGE Apply separately to the plate 10 µl of each solution. Under an inert gas, protected from light. Develop over a path of 15 cm using a mixture of 20 volumes of ether R and 80 volumes of cyclohexane R. IMPURITIES Dry the plate in a current of air and examine in ultraviolet Specified impurities : A, B. light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. Spray the plate with a mixture of 10 volumes of hydrochloric acid R, 40 volumes of a 2.5 g/l solution of ferric chloride R in alcohol R and 40 volumes of a 10 g/l solution of phenanthroline hydrochloride R in alcohol R. After 1 h to 2 h, the principal spots are orange. D. RRR-α-tocopherol is dextrorotatory (2.2.7). The specific A. all-rac-trans-2,3,4,6,7-pentamethyl-2-(4,8,12optical rotation after oxidation to the quinone form is not trimethyltridecyl)-2,3-dihydrobenzofuran-5-ol, less than + 24. Dissolve 1.0 g of the substance to be examined in 50 ml of ether R. To the solution add 20 ml of a 100 g/l solution of potassium ferricyanide R in an 8 g/l solution of sodium hydroxide R and shake for 3 min. Wash the ether solution with four quantities, each of 50 ml, of water R, discard the washings and dry the ether layer over anhydrous sodium sulphate R. Evaporate the ether on a water-bath under reduced pressure or in an atmosphere of nitrogen until a few millilitres remain, then complete the evaporation B. all-rac-cis-2,3,4,6,7-pentamethyl-2-(4,8,12removing the last traces of ether without the application trimethyltridecyl)-2,3-dihydrobenzofuran-5-ol. of heat. Immediately dissolve the residue in 5.0 ml of trimethylpentane R and determine the optical rotation. 01/2005:1256 Calculate the specific optical rotation of the substance in the test solution using as c the number of grams of RRR-α-TOCOPHEROL RRR-α-tocopherol in 1000 ml. — impurity B : maximum 1.5 per cent, — any other impurity : for each impurity, maximum 0.25 per cent, — total: maximum 2.5 per cent, — disregard limit: the area of the principal peak in the chromatogram obtained with reference solution (d) (0.1 per cent).

RRR-α-Tocopherolum

TESTS Absorbance (2.2.25). Dissolve 0.100 g in ethanol R and dilute to 100 ml with the same solvent (solution A). Dilute 10.0 ml of solution A to 100.0 ml with ethanol R (solution B). Measure the absorbance of solution B at the maximum at 292 nm and of solution A at the minimum at 255 nm. The specific absorbance at the maximum is 72.0 to 76.0 and that at the minimum is 5.5 to 8.0. C29H50O2 Mr 430.7 Acid value (2.5.1). Not more than 2.0, determined on 2.00 g. Heavy metals (2.4.8). 0.50 g complies with limit test D for DEFINITION heavy metals (20 ppm). Prepare the standard using 1 ml of RRR-α-Tocopherol contains not less than 96.0 per cent lead standard solution (10 ppm Pb) R. and not more than the equivalent of 102.0 per cent of (2R)-2,5,7,8-tetramethyl-2-[(4R,8R)-4,8,12-trimethyltridecyl]- Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. Use sulphuric acid R instead of dilute 3,4-dihydro-2H-1-benzopyran-6-ol. sulphuric acid R. CHARACTERS ASSAY A clear, colourless or yellowish-brown, viscous, oily liquid, Examine by gas chromatography (2.2.28), using practically insoluble in water, freely soluble in acetone, in dotriacontane R as the internal standard. ethanol, in methylene chloride and in fatty oils. General Notices (1) apply to all monographs and other texts

2591

all-rac-α-Tocopheryl acetate


Test solution (a). Dissolve 0.100 g of the substance to be examined in 10.0 ml of the internal standard solution. Test solution (b). Dissolve 0.100 g of the substance to be examined in 10 ml of cyclohexane R. LABELLING Reference solution (a). Dissolve 0.100 g of α-tocopheryl The label states the content of α-tocopherol acetate, acetate CRS in 10.0 ml of the internal standard solution. expressed in grams per 100 g of concentrate. Reference solution (b). Dissolve 10 mg of the substance to be examined and 10 mg of α-tocopherol R in cyclohexane R 01/2005:0439 and dilute to 100.0 ml with the same solvent. Reference solution (c). Dissolve 10 mg of all-rac-α-tocopheryl acetate for peak identification CRS in cyclohexane R and all-rac-α-TOCOPHERYL ACETATE dilute to 1 ml with the same solvent. Reference solution (d). Dilute 1.0 ml of test solution (b) to int-rac-α-Tocopherylis acetas 100.0 ml with cyclohexane R. Dilute 1.0 ml of this solution to 10.0 ml with cyclohexane R. Column : — material : fused silica, — size : l = 30 m, Ø = 0.25 mm, — stationary phase : poly(dimethyl)siloxane R (film thickness 0.25 µm). Carrier gas : helium for chromatography R. C31H52O3 Mr 472.7 Flow rate : 1 ml/min. Split ratio : 1:100. DEFINITION Temperature : all-rac-2,5,7,8-Tetramethyl-2-(4,8,12-trimethyltridecyl)-3,4— column : 280 °C, dihydro-2H-1-benzopyran-6-yl acetate. — injection port and detector : 290 °C. Content : 96.5 per cent to 101.0 per cent. Detection : flame ionisation. Injection : 1 µl of test solution (b) and reference solutions (a), CHARACTERS (b), (c) and (d). Appearance : clear, colourless or slightly greenish-yellow, Run time : twice the retention time of all-rac-α-tocopheryl viscous, oily liquid. acetate. Solubility : practically insoluble in water, freely soluble in Relative retention with reference to all-rac-α-tocopheryl acetone, in ethanol and in fatty oils. acetate (retention time = about 15 min) : squalane = about 0.4 ; IDENTIFICATION impurity A = about 0.7 ; impurity B = about 0.8 ; impurity C = about 0.9 ; use the chromatogram supplied First identification : A, B. with the CRS to identify the peaks due to impurities A and B Second identification : A, C. in the chromatogram obtained with reference solution (c). A. Optical rotation (2.2.7) : − 0.01° to + 0.01°. System suitability : Dissolve 2.50 g in ethanol R and dilute to 25.0 ml with — resolution : minimum 3.5 between the peaks due to the same solvent. impurity C and all-rac-α-tocopheryl acetate in the B. Infrared absorption spectrophotometry (2.2.24). chromatogram obtained with reference solution (b), Comparison : α-tocopheryl acetate CRS. — in the chromatogram obtained with reference solution (a), C. Thin-layer chromatography (2.2.27). the area of the peak due to impurity C is not greater than 0.2 per cent of the area of the peak due to Test solution. Dissolve about 10 mg of the substance to all-rac-α-tocopheryl acetate. be examined in 2 ml of cyclohexane R. Limits : Reference solution. Dissolve about 10 mg of α-tocopheryl acetate CRS in 2 ml of cyclohexane R. — impurities A, C : for each impurity, maximum 0.5 per cent, — impurity B : maximum 1.5 per cent, Plate : TLC silica gel F254 plate R. — any other impurity : for each impurity, maximum 0.25 per Mobile phase : ether R, cyclohexane R (20:80 V/V). cent, Application : 10 µl. — total : maximum 2.5 per cent, Development : over 2/3 of the plate. — disregard limit : the area of the principal peak in the Drying : in a current of air. chromatogram obtained with reference solution (d) Detection : examine in ultraviolet light at 254 nm. (0.1 per cent). Results : the principal spot in the chromatogram obtained ASSAY with the test solution is similar in position and size to the principal spot in the chromatogram obtained with Gas chromatography (2.2.28) as described in the test for the reference solution. related substances with the following modification. Injection : test solution (a) and reference solution (a). TESTS Calculate the percentage content of C31H52O3 taking into Related substances. Gas chromatography (2.2.28) : use the account the declared content of α-tocopheryl acetate CRS. normalisation procedure. Internal standard solution. Dissolve 1.0 g of squalane R in STORAGE cyclohexane R and dilute to 100.0 ml with the same solvent. Protected from light. STORAGE Store in an airtight, well-filled container, protected from light.

2594


RRR-α-Tocopheryl acetate


Test solution (b). In a ground glass-stoppered tube, dissolve 10 mg of the substance to be examined in 2 ml of 2.5 M alcoholic sulphuric acid R. Heat on a water-bath for 5 min. Cool and add 2 ml of water R and 2 ml of cyclohexane R. Shake for 1 min. Use the upper layer. Reference solution (a). Dissolve 10 mg of α-tocopheryl acetate CRS in 2 ml of cyclohexane R.

IMPURITIES Specified impurities : A, B, C.

A. all-rac-trans-2,3,4,6,7-pentamethyl-2-(4,8,12trimethyltridecyl)-2,3-dihydrobenzofuran-5-yl acetate,

B. all-rac-cis-2,3,4,6,7-pentamethyl-2-(4,8,12trimethyltridecyl)-2,3-dihydrobenzofuran-5-yl acetate, C. all-rac-α-tocopherol. 01/2005:1257

RRR-α-TOCOPHERYL ACETATE RRR-α-Tocopherylis acetas

Reference solution (b). Prepare as described for test solution (b), using α-tocopheryl acetate CRS instead of the substance to be examined. Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm using a mixture of 20 volumes of ether R and 80 volumes of cyclohexane R. Dry the plate in a current of air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with test solution (a) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). In the chromatograms obtained with test solution (b) and reference solution (b), there are two spots : the spot with the higher Rf value is due to α-tocopheryl acetate and corresponds to the spot in the chromatogram obtained with reference solution (a) ; the spot with the lower Rf value is due to α-tocopheryl acetate and corresponds to the spot in the chromatogram obtained with reference solution (a) ; the spot with the lower Rf value is due to α-tocopherol. Spray the plate with a mixture of 10 volumes of hydrochloric acid R, 40 volumes of a 2.5 g/l solution of ferric chloride R in alcohol R and 40 volumes of a 10 g/l solution of phenanthroline hydrochloride R in alcohol R. In the chromatograms obtained with test solution (b) and reference solution (b), the spot due to α-tocopherol is orange.

D. After saponification, the resulting RRR-α-tocopherol is dextrorotatory (2.2.7). The specific optical rotation after oxidation to the quinone form is at least + 24. Carry out the test avoiding exposure to actinic light. Transfer 1.0 g of the substance to be examined to a 250 ml round-bottomed flask with a ground-glass stopper ; dissolve in 30 ml of ethanol R and heat under reflux for 3 min. While the solution is boiling, add, through the condenser, 20 ml of 2 M alcoholic potassium hydroxide solution R. Continue heating under reflux for C31H52O3 Mr 472.7 20 min and, without cooling, add 4.0 ml of hydrochloric DEFINITION acid R dropwise through the condenser. Cool, rinse the condenser with 10 ml of ethanol R, transfer the contents RRR-α-Tocopheryl acetate contains not less than 96.0 per of the flask to a 500 ml separating funnel. Rinse the flask cent and not more than the equivalent of 102.0 per cent of with four quantities, each of 25 ml, of water R and four (2R)-2,5,7,8-tetramethyl-2-[(4R,8R)-4,8,12-trimethyltridecyl]quantities, each of 25 ml, of ether R. Add the rinsings to 3,4-dihydro-2H-1-benzopyran-6-yl acetate. the separating funnel. Shake vigorously for 2 min, allow CHARACTERS the layers to separate and collect each of the two layers in individual separating funnels. Shake the aqueous A clear, pale greenish-yellow, viscous, oily liquid, practically layer with two quantities, each of 50 ml, of ether R and insoluble in water, freely soluble in acetone, in ethanol and add these extracts to the main ether extract. Wash the in fatty oils, soluble in alcohol. combined ether extracts with four quantities, each of IDENTIFICATION 100 ml, of water R and discard the washings. First identification : B, D. To the ether solution add 40 ml of a 100 g/l solution of potassium ferricyanide R in an 8 g/l solution of sodium Second identification : A, C, D. hydroxide R and shake for 3 min. Wash the ether solution A. It complies with the test for absorbance (see Tests). with four quantities, each of 50 ml, of water R, discard the B. Examine by infrared absorption spectrophotometry washings and dry the ether layer over anhydrous sodium (2.2.24), comparing with the spectrum obtained with sulphate R. Evaporate the ether on a water-bath under α-tocopheryl acetate CRS. reduced pressure or in an atmosphere of nitrogen until C. Examine by thin-layer chromatography (2.2.27), using a a few millilitres remain, then complete the evaporation TLC silica gel F254 plate R. removing the last traces of ether without the application of heat. Immediately dissolve the residue in 25.0 ml of Test solution (a). Dissolve 10 mg of the substance to be trimethylpentane R and determine the optical rotation. examined in 2 ml of cyclohexane R. General Notices (1) apply to all monographs and other texts

2595


ASSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modification. Injection : test solution (b) and reference solution (c). Calculate the percentage content of C5H4N4O from the areas of the peaks and the declared content of allopurinol CRS.

Almagate

C. 2 ml of the solution prepared under identification test B gives the reaction of magnesium (2.3.1).

TESTS pH (2.2.3) : 9.1 to 9.7. Disperse 4.0 g in 100 ml of carbon dioxide-free water R, stir for 2 min and filter. IMPURITIES Neutralising capacity. Carry out the test at 37 °C. Disperse Specified impurities : A, B, C, D, E. 0.5 g in 100 ml of water R, heat, add 100.0 ml of 0.1 M hydrochloric acid, previously heated and stir continuously ; the pH (2.2.3) of the solution between 5 min and 20 min is not less than 3.0 and not greater than 4.5. Add 10.0 ml of 0.5 M hydrochloric acid, previously heated, stir continuously for 1 h and titrate with 0.1 M sodium hydroxide to pH 3.5 ; not more than 20.0 ml of 0.1 M sodium hydroxide is required. A. R1 = NH2, R2 = H : 5-amino-1H-pyrazole-4-carboxamide, Chlorides (2.4.4) : maximum 0.1 per cent. B. R1 = NH2, R2 = CHO : 5-(formylamino)-1H-pyrazole-4Dissolve 0.33 g in 5 ml of dilute nitric acid R and dilute to carboxamide, 100 ml with water R. 15 ml of the solution complies with the limit test for chlorides. Prepare simultaneously the standard D. R1 = O-CH2-CH3, R2 = H : ethyl 5-amino-1H-pyrazole-4by diluting 0.7 ml of dilute nitric acid R to 5 ml with water R carboxylate, and adding 10 ml of chloride standard solution (5 ppm Cl) R. E. R1 = O-CH2-CH3, R2 = CHO : ethyl 5-(formylamino)-1HSulphates (2.4.13) : maximum 0.4 per cent. pyrazole-4-carboxylate, Dissolve 0.25 g in 5 ml of dilute hydrochloric acid R and dilute to 100 ml with distilled water R. 15 ml of the solution complies with the limit test for sulphates. Prepare simultaneously the standard by adding 0.8 ml of dilute hydrochloric acid R to 15 ml of sulphate standard solution (10 ppm SO4) R. Sodium : maximum 150 ppm. Atomic absorption spectrometry (2.2.23, Method I). C. 5-(4H-1,2,4-triazol-4-yl)-1H-pyrazole-4-carboxamide. Test solution. Dissolve 0.25 g in 50 ml of a 103 g/l solution of hydrochloric acid R. 01/2005:2010 corrected Reference solutions. Prepare the reference solutions using sodium standard solution (200 ppm Na) R, diluted as necessary with a 103 g/l solution of hydrochloric acid R. ALMAGATE Heavy metals (2.4.8) : maximum 20 ppm. Dissolve 1.0 g in dilute hydrochloric acid R and dilute to Almagatum 20.0 ml with the same acid. 12 ml of the solution complies Al2Mg6C2O20H14,4H2O Mr 630 with limit test A. Prepare the standard using lead standard solution (1 ppm Pb) R. DEFINITION Loss on ignition : 43.0 per cent to 49.0 per cent, determined on 1.000 g by ignition at 900 °C. Hydrated aluminium magnesium hydroxycarbonate. Content : Microbial contamination. Total viable aerobic count (2.6.12) not more than 103 micro-organisms per gram determined by — aluminium : 15.0 per cent to 17.0 per cent (calculated plate count. It complies with the tests for Escherichia coli as Al2O3), and Pseudomonas aeruginosa (2.6.13). — magnesium : 36.0 per cent to 40.0 per cent (calculated as MgO), ASSAY — carbonic acid : 12.5 per cent to 14.5 per cent (calculated Aluminium. Dissolve 1.000 g in 5 ml of hydrochloric acid R, as CO2). heating if necessary. Allow to cool to room temperature CHARACTERS and dilute to 100.0 ml with water R (solution A). Introduce Appearance : white or almost white, fine crystalline powder. 10.0 ml of solution A into a 250 ml conical flask, add 25.0 ml of 0.05 M sodium edetate, 20 ml of buffer solution pH 3.5 R, Solubility : practically insoluble in water, in alcohol and 40 ml of ethanol R and 2 ml of a freshly prepared 0.25 g/l in methylene chloride. It dissolves with effervescence and solution of dithizone R in ethanol R. Titrate the excess of heating in dilute mineral acids. sodium edetate with 0.05 M zinc sulphate until the colour changes from greenish-violet to pink. IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). 1 ml of 0.05 M sodium edetate is equivalent to 2.549 mg Comparison : Ph. Eur. reference spectrum of almagate. of Al2O3. B. Dissolve 150 mg in dilute hydrochloric acid R and dilute Magnesium. Introduce 10.0 ml of solution A prepared in the to 20 ml with the same acid. 2 ml of the solution gives the assay of aluminium into a 500 ml conical flask, add 200 ml of water R, 20 ml of triethanolamine R with shaking, 10 ml of reaction of aluminium (2.3.1). General Notices (1) apply to all monographs and other texts

945

Almond oil, refined


ammonium chloride buffer solution pH 10.0 R and 50 mg of mordant black 11 triturate R. Titrate with 0.05 M sodium edetate until the colour changes from violet to pure blue. 1 ml of 0.05 M sodium edetate is equivalent to 2.015 mg of MgO.

01/2005:1064

ALMOND OIL, REFINED Amygdalae oleum raffinatum

Carbonic acid : 12.5 per cent to 14.5 per cent.

DEFINITION Refined almond oil is the fatty oil from the ripe seeds of Test sample. Place 7.00 mg of the substance to be examined Prunus dulcis (Miller) D.A. Webb var. dulcis or Prunus in a tin capsule. Seal the capsule. dulcis (Miller) D.A. Webb var. amara (D.C.) Buchheim or Reference sample. Place 7.00 mg of almagate CRS in a tin a mixture of both varieties by cold expression. It is then refined. A suitable antioxidant may be added. capsule. Seal the capsule. Introduce separately the test sample and the reference sample into a combustion chamber of a CHN analyser purged with helium for chromatography R and maintained at a temperature of 1020 °C. Simultaneously, introduce oxygen R at a pressure of 276 kPa and a flow rate of 20 ml/min and allow complete combustion of the sample. Sweep the combustion gases through a reduction reactor and separate the gases formed by gas chromatography (2.2.28). Column : — size : l = 2 m, Ø = 4 mm, — stationary phase: ethylvinylbenzene-divinylbenzene copolymer R1. Carrier gas : helium for chromatography R. Flow rate : 100 ml/min. Temperature : — column : 65 °C, — detector : 190 °C. Detection : thermal conductivity. Run time : 16 min. System suitability : — average percentage of carbon in 5 reference samples must be within ± 0.2 per cent of the value assigned to the CRS. The difference between the upper and the lower values of the percentage of carbon in these samples must be below 0.2 per cent. Calculate the percentage content of carbonic acid in the test sample according to the following formula :

C

=

percentage content of carbonic acid in the reference sample,

K

=

mean value for the 5 reference samples of the ratio of the mass in milligrams to the area of the peak due to carbonic acid,

A

=

area of the peak due to carbonic acid in the chromatogram obtained with the test sample,

m

=

sample mass, in milligrams.

STORAGE In an airtight container. 946

CHARACTERS A pale yellow, clear liquid, slightly soluble in alcohol, miscible with light petroleum. It solidifies at about − 18 °C and has a relative density of about 0.916. IDENTIFICATION A. Carry out the identification of fatty oils by thin-layer chromatography (2.3.2). The chromatogram obtained is comparable with the type chromatogram for almond oil. B. It complies with the test for composition of fatty acids (see Tests). TESTS Absorbance (2.2.25). Dissolve 0.100 g in cyclohexane R and dilute to 10.0 ml with the same solvent. The absorbance measured at the maximum between 264 nm and 276 nm is 0.2 to 6.0. Acid value (2.5.1). Not more than 0.5, determined on 5.0 g. Peroxide value (2.5.5). Not more than 5.0. Unsaponifiable matter (2.5.7). Not more than 0.7 per cent, determined on 5.0 g. Composition of fatty acids (2.4.22, Method A). The fatty acid fraction of the oil has the following composition : — saturated fatty acids of chain length less than C16 : not more than 0.1 per cent, — palmitic acid : 4.0 per cent to 9.0 per cent, — palmitoleic acid (equivalent chain length on polyethyleneglycol adipate 16.3) : not more than 0.6 per cent, — margaric acid : not more than 0.2 per cent, — stearic acid : not more than 3.0 per cent, — oleic acid (equivalent chain length on polyethyleneglycol adipate 18.3) : 62.0 per cent to 86.0 per cent, — linoleic acid (equivalent chain length on polyethyleneglycol adipate 18.9) : 20.0 per cent to 30.0 per cent, — linolenic acid (equivalent chain length on polyethyleneglycol adipate 19.7) : not more than 0.4 per cent, — arachidic acid : not more than 0.2 per cent, — eicosenoic acid (equivalent chain length on polyethyleneglycol adipate 20.3) : not more than 0.3 per cent, — behenic acid : not more than 0.2 per cent, — erucic acid (equivalent chain length on polyethyleneglycol adipate 22.3) : not more than 0.1 per cent. Sterols. Carry out the test for sterols in fatty oils (2.4.23). The sterol fraction of the oil has the following composition : — cholesterol : not more than 0.7 per cent, — campesterol : not more than 5.0 per cent,


Aloes, barbados


— margaric acid : not more than 0.2 per cent, — stearic acid : not more than 3.0 per cent, — oleic acid (equivalent chain length on polyethyleneglycol adipate 18.3) : 62.0 per cent to 86.0 per cent, — linoleic acid (equivalent chain length on polyethyleneglycol adipate 18.9) : 20.0 per cent to 30.0 per cent, — linolenic acid (equivalent chain length on polyethyleneglycol adipate 19.7) : not more than 0.4 per cent, STORAGE — arachidic acid : not more than 0.2 per cent, Store in a well-filled container, protected from light. — eicosenoic acid (equivalent chain length on polyethyleneglycol adipate 20.3) : not more than 0.3 per LABELLING cent, The label states : — behenic acid : not more than 0.2 per cent, — where applicable, that the substance is suitable for use in — erucic acid (equivalent chain length on polyethyleneglycol the manufacture of parenteral dosage forms, adipate 22.3) : not more than 0.1 per cent. — the name and concentration of any added antioxidant. Sterols. Carry out the test for sterols in fatty oils (2.4.23). The sterol fraction of the oil has the following composition : 01/2005:0261 — cholesterol : not more than 0.7 per cent, — campesterol : not more than 4.0 per cent, ALMOND OIL, VIRGIN — stigmasterol : not more than 3.0 per cent, — β-sitosterol : 73.0 per cent to 87.0 per cent, Amygdalae oleum virginale — ∆5-avenasterol: at least 10.0 per cent, DEFINITION — ∆7-avenasterol: not more than 3.0 per cent, Virgin almond oil is the fatty oil obtained by cold expression from the ripe seeds of Prunus dulcis (Miller) D.A. Webb var. — ∆7-stigmastenol: not more than 3.0 per cent, dulcis or Prunus dulcis (Miller) D.A. Webb var. amara (D.C.) — brassicasterol : not more than 0.3 per cent. Buchheim or a mixture of both varieties. STORAGE CHARACTERS Store in a well-filled container, protected from light. A yellow, clear, liquid, slightly soluble in alcohol, miscible with light petroleum. 01/2005:0257 It solidifies at about − 18 °C and has a relative density of about 0.916. ALOES, BARBADOS — stigmasterol : not more than 4.0 per cent, — β-sitosterol : 73.0 per cent to 87.0 per cent, — ∆5-avenasterol: at least 5.0 per cent, — ∆7-avenasterol: not more than 3.0 per cent, — ∆7-stigmastenol : not more than 3.0 per cent, — brassicasterol : not more than 0.3 per cent. Water (2.5.32). If intended for use in the manufacture of parenteral dosage forms, not more than 0.1 per cent, determined on 5.00 g by the micro-determination of water.

IDENTIFICATION First identification : A, C. Second identification : A, B. A. It complies with the test for absorbance (see Tests). B. Carry out the identification of fatty oils by thin-layer chromatography (2.3.2). The chromatogram obtained is comparable with the type chromatogram for almond oil. C. It complies with the test for composition of fatty acids (see Tests).

Aloe barbadensis DEFINITION Barbados aloes consists of the concentrated and dried juice of the leaves of Aloe barbadensis Miller. It contains not less than 28.0 per cent of hydroxyanthracene derivatives, expressed as barbaloin (C21H22O9 ; Mr 418.4) and calculated with reference to the dried drug.

CHARACTERS Dark brown masses, slightly shiny or opaque with a TESTS conchoidal fracture, or a brown powder, soluble in hot Absorbance (2.2.25). Dissolve 0.100 g in cyclohexane R and alcohol, partly soluble in boiling water. dilute to 10.0 ml with the same solvent. The absorbance measured at the maximum between 264 nm and 276 nm is IDENTIFICATION not greater than 0.2. The ratio of the absorbance at 232 nm A. Examine by thin-layer chromatography (2.2.27), using to that at 270 nm is greater than 7. silica gel G R as the coating substance. Acid value (2.5.1). Not more than 2.0, determined on 5.0 g. Test solution. To 0.25 g of the powdered drug add 20 ml of methanol R and heat to boiling in a water-bath. Shake Peroxide value (2.5.5). Not more than 15.0. for a few minutes and decant the solution. Store at about Unsaponifiable matter (2.5.7). Not more than 0.7 per cent, 4 °C and use within 24 h. determined on 5.0 g. Reference solution. Dissolve 25 mg of barbaloin R in Composition of fatty acids (2.4.22, Method A). The fatty acid methanol R and dilute to 10 ml with the same solvent. fraction of the oil has the following composition : Apply separately to the plate as bands 20 mm by not — saturated fatty acids of chain length less than C16 : not more than 3 mm 10 µl of each solution. Develop over more than 0.1 per cent, a path of 10 cm using a mixture of 13 volumes of — palmitic acid : 4.0 per cent to 9.0 per cent, water R, 17 volumes of methanol R and 100 volumes of ethyl acetate R. Allow the plate to dry in air, spray — palmitoleic acid (equivalent chain length on with a 100 g/l solution of potassium hydroxide R in polyethyleneglycol adipate 16.3) : not more than 0.6 per methanol R and examine in ultraviolet light at 365 nm. cent, General Notices (1) apply to all monographs and other texts

947

Aloes, Cape


The chromatogram obtained with the test solution 01/2005:0258 shows in the central part a zone of yellow fluorescence (barbaloin) similar in position to the zone corresponding ALOES, CAPE to barbaloin in the chromatogram obtained with the reference solution and in the lower part a zone of light-blue fluorescence (aloesine). Heat the plate at Aloe capensis 110 °C for 5 min. In the chromatogram obtained with the test solution, a zone of violet fluorescence appears just DEFINITION below the zone corresponding to barbaloin. Cape aloes consists of the concentrated and dried juice B. Shake 1 g of the powdered drug with 100 ml of boiling of the leaves of various species of Aloe, mainly Aloe ferox water R. Cool, add 1 g of talc R and filter. To 10 ml of Miller and its hybrids. It contains not less than 18.0 per cent the filtrate add 0.25 g of disodium tetraborate R and of hydroxyanthracene derivatives, expressed as barbaloin heat to dissolve. Pour 2 ml of the solution into 20 ml of (C21H22O9 ; Mr 418.4) and calculated with reference to the water R. Yellowish-green fluorescence appears which is dried drug. particularly marked in ultraviolet light at 365 nm. C. To 5 ml of the filtrate obtained in identification test B CHARACTERS add 1 ml of freshly prepared bromine water R. A brownish-yellow precipitate is formed and the supernatant Dark brown masses tinged with green and having a shiny liquid is violet. conchoidal fracture, or a greenish-brown powder, soluble in hot alcohol, partly soluble in boiling water. TESTS IDENTIFICATION Loss on drying (2.2.32). Not more than 12.0 per cent, determined on 1.000 g of the powdered drug by drying in an oven at 100 °C to 105 °C. Total ash (2.4.16). Not more than 2.0 per cent. ASSAY Carry out the assay protected from bright light. Introduce 0.300 g of powdered drug (180) into a 250 ml conical flask. Moisten with 2 ml of methanol R, add 5 ml of water R warmed to about 60 °C, mix, add a further 75 ml of water R at about 60 °C and shake for 30 min. Cool, filter into a volumetric flask, rinse the conical flask and filter with 20 ml of water R, add the rinsings to the volumetric flask and dilute to 1000.0 ml with water R. Transfer 10.0 ml of this solution to a 100 ml round-bottomed flask containing 1 ml of a 600 g/l solution of ferric chloride R and 6 ml of hydrochloric acid R. Heat in a water-bath under a reflux condenser for 4 h, with the water level above that of the liquid in the flask. Allow to cool, transfer the solution to a separating funnel, rinse the flask successively with 4 ml of water R, 4 ml of 1 M sodium hydroxide and 4 ml of water R and add the rinsings to the separating funnel. Shake the contents of the separating funnel with three quantities, each of 20 ml, of ether R. Wash the combined ether layers with two quantities, each of 10 ml, of water R. Discard the washings and dilute the organic phase to 100.0 ml with ether R. Evaporate 20.0 ml carefully to dryness on a water-bath and dissolve the residue in 10.0 ml of a 5 g/l solution of magnesium acetate R in methanol R. Measure the absorbance (2.2.25) at 512 nm using methanol R as the compensation liquid. Calculate the percentage content of hydroxyanthracene derivatives, as barbaloin, from the expression :

i.e. taking the specific absorbance of barbaloin to be 255. A m

=

absorbance at 512 nm,

=

mass of the substance to be examined in grams.

STORAGE Store in an airtight container, protected from light. 948

A. Examine the chromatograms obtained in the test for Barbados aloes. The chromatogram obtained with the test solution shows in the central part a zone of yellow fluorescence (barbaloin) similar in position to the zone corresponding to barbaloin in the chromatogram obtained with the reference solution and in the lower part two zones of yellow fluorescence (aloinosides A and B) and a zone of blue fluorescence (aloesine). B. Shake 1 g of the powdered drug with 100 ml of boiling water R. Cool, add 1 g of talc R and filter. To 10 ml of the filtrate add 0.25 g of disodium tetraborate R and heat to dissolve. Pour 2 ml of the solution into 20 ml of water R. A yellowish-green fluorescence appears which is particularly marked in ultraviolet light at 365 nm.

C. To 5 ml of the filtrate obtained in identification test B add 1 ml of freshly prepared bromine water R. A yellow precipitate is formed. The supernatant liquid is not coloured violet. TESTS Barbados aloes. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. To 0.25 g of the powdered drug add 20 ml of methanol R and heat to boiling in a water-bath. Shake for a few minutes and decant the solution. Store at about 4 °C and use within 24 h. Reference solution. Dissolve 25 mg of barbaloin R in methanol R and dilute to 10 ml with the same solvent. Apply separately to the plate as bands 20 mm by not more than 3 mm 10 µl of each solution. Develop over a path of 10 cm using a mixture of 13 volumes of water R, 17 volumes of methanol R and 100 volumes of ethyl acetate R. Allow the plate to dry in air, spray with a 100 g/l solution of potassium hydroxide R in methanol R. Heat the plate at 110 °C for 5 min and examine in ultraviolet light at 365 nm. The chromatogram obtained with the test solution shows no zone of violet fluorescence just below the zone corresponding to barbaloin. Loss on drying (2.2.32). Not more than 10.0 per cent, determined on 1.000 g of the powdered drug by drying in an oven at 100 °C to 105 °C. Total ash (2.4.16). Not more than 2.0 per cent.



Aloes dry extract, standardised

ASSAY Reference solution. Dissolve 25 mg of barbaloin R in methanol R and dilute to 10 ml with the same solvent. Carry out the assay protected from bright light. Introduce 0.400 g of powdered drug (180) into a 250 ml Apply separately to the plate as bands 20 mm by not conical flask. Moisten with 2 ml of methanol R, add 5 ml of more than 3 mm 10 µl of each solution. Develop over water R warmed to about 60 °C, mix, add a further 75 ml of a path of 10 cm using a mixture of 13 volumes of water R at about 60 °C and shake for 30 min. Cool, filter water R, 17 volumes of methanol R and 100 volumes into a volumetric flask, rinse the conical flask and filter with of ethyl acetate R. Allow the plate to dry in air, spray 20 ml of water R, add the rinsings to the volumetric flask with a 100 g/l solution of potassium hydroxide R in and dilute to 1000.0 ml with water R. Transfer 10.0 ml of methanol R and examine in ultraviolet light at 365 nm. this solution to a 100 ml round-bottomed flask containing The chromatogram obtained with the test solution 1 ml of a 600 g/l solution of ferric chloride R and 6 ml of shows in the central part a zone of yellow fluorescence hydrochloric acid R. Heat in a water-bath under a reflux (barbaloin) similar in position to the zone corresponding condenser for 4 h, with the water level above that of the to barbaloin in the chromatogram obtained with the liquid in the flask. Allow to cool, transfer the solution to a reference solution and in the lower part there is a zone separating funnel, rinse the flask successively with 4 ml of of light blue fluorescence (aloesine). In the lower part of water R, 4 ml of 1 M sodium hydroxide and 4 ml of water R the chromatogram obtained with the test solution two and add the rinsings to the separating funnel. Shake the zones of yellow fluorescence (aloinosides A and B)(Cape contents of the separating funnel with three quantities, aloes) and one zone of violet fluorescence just below the each of 20 ml, of ether R. Wash the combined ether layers zone corresponding to barbaloin (Barbados aloes) may with two quantities, each of 10 ml, of water R. Discard be present. the washings and dilute the organic phase to 100.0 ml B. Shake 1 g of the preparation to be examined with 100 ml with ether R. Evaporate 20.0 ml carefully to dryness on a of boiling water R. Cool, add 1 g of talc R and filter. To water-bath and dissolve the residue in 10.0 ml of a 5 g/l 10 ml of the filtrate add 0.25 g of disodium tetraborate R solution of magnesium acetate R in methanol R. Measure and heat to dissolve. Pour 2 ml of the solution into 20 ml the absorbance (2.2.25) at 512 nm using methanol R as the of water R. A yellowish-green fluorescence appears which compensation liquid. is particularly marked in ultraviolet light at 365 nm. Calculate the percentage content of barbaloin from the expression : TESTS Loss on drying (2.8.17) : maximum 4.0 per cent m/m. Total ash (2.4.16). Not more than 2.0 per cent. i.e. taking the specific absorbance of hydroxyanthracene derivatives, as barbaloin, to be 255. ASSAY A m

=


=


Carry out the assay protected from bright light.

Introduce 0.400 g into a 250 ml conical flask. Moisten with 2 ml of methanol R, add 5 ml of water R warmed to about STORAGE 60 °C, mix, add a further 75 ml of water R at about 60 °C Store in an airtight container, protected from light. and shake for 30 min. Cool, filter into a volumetric flask, rinse the conical flask and filter with 20 ml of water R, add 01/2005:0259 the rinsings to the volumetric flask and dilute to 1000.0 ml with water R. Transfer 10.0 ml of this solution to a 100 ml round-bottomed flask containing 1 ml of a 600 g/l solution ALOES DRY EXTRACT, of ferric chloride R and 6 ml of hydrochloric acid R. Heat in a water-bath under a reflux condenser for 4 h, with the STANDARDISED water level above that of the liquid in the flask. Allow to cool, transfer the solution to a separating funnel, rinse the Aloes extractum siccum normatum flask successively with 4 ml of water R, 4 ml of 1 M sodium hydroxide and 4 ml of water R, and add the rinsings to the DEFINITION separating funnel. Shake the contents of the separating Standardised aloes dry extract is prepared from Barbados funnel with three quantities, each of 20 ml, of ether R. aloes or Cape aloes, or a mixture of the two, by treatment Wash the combined ether layers with two quantities, each with boiling water. It is adjusted, if necessary, to contain not less than 19.0 per cent and not more than 21.0 per cent of 10 ml, of water R. Discard the washings and dilute the organic phase to 100.0 ml with ether R. Evaporate 20.0 ml of hydroxyanthracene derivatives, expressed as barbaloin carefully to dryness on a water-bath and dissolve the residue (C21H22O9 ; Mr 418.4) and calculated with reference to the in 10.0 ml of a 5 g/l solution of magnesium acetate R in dried extract. methanol R. Measure the absorbance (2.2.25) at 512 nm CHARACTERS using methanol R as the compensation liquid. A brown or yellowish-brown powder, sparingly soluble in Calculate the percentage content of hydroxyanthracene boiling water. derivatives, as barbaloin, from the expression : IDENTIFICATION A. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. i.e. taking the specific absorbance of barbaloin to be 255. Test solution. To 0.25 g of the preparation to be examined add 20 ml of methanol R and heat to boiling A = absorbance at 512 nm, in a water-bath. Shake for a few minutes and decant the m = mass of the substance to be examined in grams. solution. Store at about 4 °C and use within 24 h. General Notices (1) apply to all monographs and other texts

949

Alprazolam


01/2005:1065

ALPRAZOLAM

chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separately spots.

TESTS Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 0.100 g of the substance to be examined in dimethylformamide R and dilute to 10.0 ml with the same solvent. Reference solution (a). Dissolve 2 mg of alprazolam CRS and 2 mg of triazolam CRS in dimethylformamide R and dilute to 100.0 ml with the same solvent. Reference solution (b). Dilute 5.0 ml of the test solution to 100.0 ml with dimethylformamide R. Dilute 0.5 ml of this solution to 10.0 ml with dimethylformamide R. C17H13ClN4 Mr 308.8 The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in DEFINITION internal diameter packed with phenylsilyl silica gel for Alprazolam contains not less than 99.0 per cent and not more chromatography R1 (5 µm), than the equivalent of 101.0 per cent of 8-chloro-1-methyl— as mobile phase at a flow rate of 2 ml/min and at a 6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine, temperature of 40 °C a gradient programme using the calculated with reference to the dried substance. following conditions : CHARACTERS Mobile phase A. A mixture of 44 volumes of buffer solution and 56 volumes of methanol R. A white, crystalline powder, practically insoluble in water, freely soluble in methylene chloride sparingly soluble in Mobile phase B. A mixture of 5 volumes of buffer solution acetone and in alcohol. and 95 volumes of methanol R. It shows polymorphism. Buffer solution. Dissolve 7.7 g of ammonium acetate R in 1000 ml of water R and adjust to pH 4.2 with glacial IDENTIFICATION acetic acid R, First identification : B. Time Mobile phase A Mobile phase B Comment Second identification : A, C. (min) (per cent V/V) (per cent V/V) A. Dissolve the substance to be examined in the smallest 0 98 2 isocratic necessary quantity of ethyl acetate R and evaporate to 98 2 15 begin linear gradient dryness on a water-bath. Thoroughly mix 5.0 mg of the substance to be examined with 5.0 mg of alprazolam CRS. 99 35 1 begin isocratic The melting point (2.2.14) of the mixture does not deviate 40 99 1 end chromatogram from the melting point of the substance to be examined and switch to initial by more than 2 °C. equilibration B. Examine by infrared absorption spectrophotometry 98 2 41 begin equilibration (2.2.24), comparing with the spectrum obtained with 98 2 50 = 0 end equilibration alprazolam CRS. If the spectra obtained in the solid state show differences, dissolve the substance to be — as detector a spectrophotometer set at 254 nm. examined and the reference substance separately in Equilibrate the column for at least 30 min with the initial the minimum volume of ethyl acetate R, evaporate to dryness on a water-bath and record new spectra using the eluent composition. For subsequent chromatograms, use the conditions described from 40 min to 50 min. Adjust residues. Examine the substances prepared as discs. the sensitivity of the system so that the height of the C. Examine by thin-layer chromatography (2.2.27), using principal peak in the chromatogram obtained with reference silica gel GF254 R as the coating substance. solution (b) is at least 50 per cent of the full scale of the Test solution. Dissolve 10 mg of the substance to be recorder. examined in methanol R and dilute to 10 ml with the Inject 10 µl of reference solution (a). When the chromatogram same solvent. is recorded in the prescribed conditions the retention times Reference solution (a). Dissolve 10 mg of alprazolam CRS are : triazolam, about 9 min and alprazolam, about 10 min. in methanol R and dilute to 10 ml with the same solvent. The test is not valid unless the resolution between the peaks Reference solution (b). Dissolve 10 mg of alprazolam CRS corresponding to alprazolam and triazolam is at least 1.5. and 10 mg of midazolam CRS in methanol R and dilute Inject separately 10 µl of dimethylformamide R as a blank, to 10 ml with the same solvent. 10 µl of the test solution and 10 µl of reference solution (b). In the chromatogram obtained with the test solution, the Apply separately to the plate 5 µl of each solution. sum of the areas of the peaks, apart from the principal peak, Develop over a path or 12 cm using a mixture of 2 volumes of glacial acetic acid R, 15 volumes of water R, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.25 per 20 volumes of methanol R and 80 volumes of ethyl cent). Disregard any peak in the chromatogram obtained acetate R. Allow the plate to dry in air and examine with the blank and any peak with an area less than 0.2 times in ultraviolet light at 254 nm. The principal spot in the area of the principal peak in the chromatogram obtained the chromatogram obtained with the test solution is with reference solution (b). similar in position and size to the principal spot in the

Alprazolamum

950


Alprazolam


Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.140 g in 50 ml of a mixture of 3 volumes E. (2-amino-5-chlorophenyl)phenylmethanone, of anhydrous acetic acid R and 2 volumes of acetic anhydride R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). Titrate to the second point of inflexion. 1 ml of 0.1 M perchloric acid is equivalent to 15.44 mg of C17H13CIN4. STORAGE Store protected from light. IMPURITIES G. 7-chloro-1-methyl-5-phenyl[1,2,4]triazolo[4,3-a]quinolin4-amine,

A. (4RS)-3-amino-6-chloro-2-methyl-4-phenyl-3,4dihydroquinazolin-4-ol, H. bis[[4-(2-benzoyl-4-chlorophenyl)-5-methyl-4H-1,2,4triazol-3-yl]methyl]amine,

B. R = CH2OH : [5-chloro-2-[3-(hydroxymethyl)-5-methyl-4H-1, 2,4-triazol-4-yl]phenyl]phenylmethanone, C. R = H : [5-chloro-2-[3-methyl-4H-1,2,4-triazol-4yl]phenyl]phenylmethanone, F. R = CH2Cl : [5-chloro-2-[3-(chloromethyl)-5-methyl-4H-1,2, 4-triazol-4-yl]phenyl]phenylmethanone,

D. 8-chloro-1-ethenyl-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1, 4]benzodiazepine, General Notices (1) apply to all monographs and other texts

I. [5-chloro-2-[3-[[(6RS)-8-chloro-6-hydroxy-1-methyl-6phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin5(6H)-yl]methyl]-5-methyl-4H-1,2,4-triazol-4yl]phenyl]phenylmethanone,

J. 2,17-dichloro-6,13-dimethyl-18b,19a-diphenyl-8b, 19a-dihydro-10H,18bH-[1,2,4]triazolo[4′′′,3′′′:1″, 2″]quinolo[3″,4″:4′,5′]oxazolo[3′,2′-d]-1,2,4-triazolo[4,3a][1,4]benzodiazepine. 951

Alprenolol hydrochloride


01/2005:0876

ALPRENOLOL HYDROCHLORIDE Alprenololi hydrochloridum

C15H24ClNO2

Reference solution (c). Dilute 5 ml of test solution (b) to 50 ml with methanol R.

Mr 285.8

DEFINITION Alprenolol hydrochloride contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of (2RS)-1[(1-methylethyl)amino]-3-[2-(prop-2-enyl)phenoxy]propan-2-ol hydrochloride, calculated with reference to the dried substance. CHARACTERS A white, crystalline powder or colourless crystals, very soluble in water, freely soluble in alcohol and in methylene chloride. IDENTIFICATION First identification : B, D. Second identification : A, C, D. A. Melting point (2.2.14) : 108 °C to 112 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with alprenolol hydrochloride CRS. C. Examine the chromatograms obtained in test A for related substances in daylight, after exposure to iodine vapour for 30 min. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). D. It gives reaction (a) of chlorides (2.3.1). TESTS Solution S. Dissolve 1.0 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution B9 (2.2.2, Method II). Acidity or alkalinity. To 10 ml of solution S add 0.2 ml of methyl red solution R and 0.2 ml of 0.01 M hydrochloric acid. The solution is red. Add 0.4 ml of 0.01 M sodium hydroxide. The solution is yellow. 1-Isopropylamino-3-(2-prop-1-enylphenoxy)propan-2-ol. Dissolve 0.25 g in alcohol R and dilute to 25 ml with the same solvent. The absorbance (2.2.25) measured at 297 nm is not greater than 0.20 (0.1 per cent). Related substances A. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution (a). Dissolve 0.50 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 50 ml with methanol R. Reference solution (a). Dissolve 10 mg of alprenolol hydrochloride CRS in methanol R and dilute to 10 ml with the same solvent. 952

Reference solution (b). Dissolve 10 mg of alprenolol hydrochloride CRS and 10 mg of oxprenolol hydrochloride CRS in methanol R and dilute to 10 ml with the same solvent.

Place two beakers each containing 30 ml of ammonia R at the bottom of the chamber which is saturated with the mobile phase and ammonia R for at least 1 h before use. Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 5 volumes of methanol R and 95 volumes of ethyl acetate R. Dry the plate at 100 °C for 15 min. Expose the plate to iodine vapour for up to 6 h. In the chromatogram obtained with test solution (a) any spot with an Rf value higher than that of the principal spot is not more intense than the spot in the chromatogram obtained with reference solution (c). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. B. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 20.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (a). Dissolve 4.0 mg of alprenolol hydrochloride CRS and 0.8 mg of 4-isopropylphenol R in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (b). Dilute 4.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 10.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.15 m long and 4 mm in internal diameter packed with octylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows : mix 0.656 g of sodium octanesulphonate R with 150 ml of acetonitrile R and dilute to 500 ml with phosphate buffer pH 2.8 (prepared from 1.78 g of phosphoric acid R and 15.6 g of sodium dihydrogen phosphate R diluted to 2000 ml with water R), — as detector a spectrophotometer set at 280 nm. Equilibrate the column with the mobile phase at a flow rate of 1 ml/min for about 1 h. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 20 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder. Inject 20 µl of reference solution (a). When the chromatograms are recorded under the prescribed conditions, the retention times are : alprenolol hydrochloride about 11 min ; 4-isopropylphenol about 18 min. The test is not valid unless, in the chromatogram obtained with reference solution (a), the resolution between the peaks corresponding to alprenolol hydrochloride and 4-isopropylphenol is at least 5 ; if necessary, adjust the concentration of sodium octanesulphonate and/or acetonitrile in the mobile phase (increase the concentration of sodium octanesulphonate to increase the retention time of alprenolol hydrochloride and increase the concentration of acetonitrile to decrease the retention times of both compounds).


Alprostadil


Inject separately 20 µl of the test solution and 20 µl of reference solution (b). Continue the chromatography for twice the retention time of the principal peak. In the chromatogram obtained with the test solution, the sum of the areas of any peaks apart from the principal peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.4 per cent). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b). Heavy metals (2.4.8). Dissolve 2.0 g in 20 ml of water R. 12 ml of the solution complies with limit test A for heavy metals (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying over diphosphorus pentoxide R at a pressure not exceeding 2.7 kPa. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.400 g in 25 ml of a mixture of equal volumes of ethanol R and water R. Add 10 ml of 0.01 M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the two points of inflexion.

01/2005:1488

ALPROSTADIL Alprostadilum

C20H34O5

Mr 354.5

DEFINITION 7-[(1R,2R,3R)-3-Hydroxy-2-[(1E,3S)-3-hydroxyoct-1-enyl]-5oxocyclopentyl]heptanoic acid. Content : 95.0 per cent to 102.5 per cent (anhydrous substance). CHARACTERS Appearance : white or slightly yellowish, crystalline powder. Solubility : practically insoluble in water, freely soluble in alcohol, soluble in acetone, slightly soluble in ethyl acetate.

IDENTIFICATION 1 ml of 0.1 M sodium hydroxide is equivalent to 28.58 mg of A. Specific optical rotation (2.2.7) : − 60 to − 70 (anhydrous substance). C15H24ClNO2. Immediately before use, dissolve 50 mg in alcohol R and dilute to 10.0 ml with the same solvent. STORAGE B. Infrared absorption spectrophotometry (2.2.24). Store protected from light. Preparation : discs. Comparison : alprostadil CRS. IMPURITIES C. Examine the chromatograms obtained in the assay. Results : the principal peak in the chromatogram obtained with the test solution is similar in retention time and size to the principal peak in the chromatogram obtained with the reference solution. TESTS Related substances. Liquid chromatography (2.2.29). A. R1 = OH, R2 = CH2-CH=CH2 : (2RS)-3-[2-(prop-2Prepare the solutions protected from light. enyl)phenoxy]propan-1,2-diol, Test solution. Dissolve 10.0 mg of the substance to be examined in a mixture of equal volumes of acetonitrile R1 and water R and dilute to 10.0 ml with the same mixture C. R1 = NH-CH(CH3)2, R2 = CH=CH-CH3 : (2RS)-1-[(1methylethyl)amino]-3-[2-(prop-1-enyl)phenoxy]propan-2-ol, of solvents. Reference solution (a). Dilute 100 µl of the test solution to 20.0 ml with a mixture of equal volumes of acetonitrile R1 and water R. Reference solution (b). Dissolve 1.0 mg of dinoprostone impurity C CRS (alprostadil impurity H) and 1.0 mg of alprostadil CRS in a mixture of equal volumes of acetonitrile R1 and water R and dilute to 20.0 ml with the B. 2-(prop-2-enyl)phenol, same mixture of solvents. Reference solution (c). In order to prepare in situ the degradation compounds (impurity A and impurity B), dissolve 1 mg of the substance to be examined in 100 µl of 1 M sodium hydroxide (the solution becomes brownish-red), wait for 3 min and add 100 µl of 1 M phosphoric acid (yellowish-white opalescent solution) ; dilute to 5.0 ml with a mixture of equal volumes of acetonitrile R1 and water R. System A Column : D. 1,1′-[(1-methylethyl)imino]bis[3-[2-(prop-2— size : l = 0.25 m, Ø = 4.0 mm, enyl)phenoxy]propan-2-ol]. General Notices (1) apply to all monographs and other texts

953

Alprostadil


— stationary phase : octylsilyl base-deactivated silica gel for chromatography R (4 µm) with a pore size of 6 nm, — temperature : 35 °C. Mobile phase : — mobile phase A. Dissolve 3.9 g of sodium dihydrogen phosphate R in water R and dilute to 1000.0 ml with the same solvent ; adjust to pH 2.5 with a 2.9 g/l solution of phosphoric acid R (approximately 600 ml is required) ; to 740 ml of the buffer solution add 260 ml of acetonitrile R1; — mobile phase B. Dissolve 3.9 g of sodium dihydrogen phosphate R in water R and dilute to 1000.0 ml with the same solvent ; adjust to pH 2.5 with a 2.9 g/l solution of phosphoric acid R (approximately 600 ml is required) ; to 200 ml of the buffer solution add 800 ml of acetonitrile R1; Time (min)



0 - 75

100

0

75 - 76

100 → 0

0 → 100

76 - 86

0

100

86 - 87

0 → 100

100 → 0

87 - 102

100

0

Time (min)



Carry out the test according to system A and B. Limits : — correction factors : multiply the areas of the corresponding peaks using the correction factors in Table 1488.-1 to obtain the corrected areas, Table 1488.-1 Impurity impurity G

Relative retention Relative retention Correction factor (system A) (system B) 0.80 0.7

impurity F

0.88

-

0.8

impurity D

0.90

-

1.0

impurity H

0.96

-

0.7

impurity E

1.10

-

0.7

impurity C

-

1.36

1.9

impurity K

-

1.85

0.06

impurity A

-

2.32

0.7

impurity B

-

2.45

1.5

impurity I

-

4.00

1.0

impurity J

-

5.89

1.0

— impurity A (corrected area) : not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (a) (1.5 per cent), Flow rate : 1 ml/min. — impurity B (corrected area) : not more than the area of Detection : spectrophotometer at 200 nm. the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent), Injection : 20 µl loop injector. — any other impurity (corrected area) : not more System suitability : than 1.8 times the area of the principal peak in the — retention time : alprostadil = about 63 min, chromatogram obtained with reference solution (a) (0.9 per cent), and not more than 1 such peak has an — resolution : minimum of 1.5 between the peaks due to area greater than the area of the principal peak in the impurity H and alprostadil in the chromatogram obtained chromatogram obtained with reference solution (a) with reference solution (b). (0.5 per cent). Evaluate impurities appearing at relative System B retentions less than 1.2 by system A and impurities Use the same conditions as for system A with the following appearing at relative retentions greater than 1.2 by mobile phase and elution programme : system B, — total (corrected area) : not more than 3 times the area of — mobile phase A. Dissolve 3.9 g of sodium dihydrogen the principal peak in the chromatogram obtained with phosphate R in water R and dilute to 1000.0 ml with reference solution (a) (1.5 per cent), the same solvent ; adjust to pH 2.5 with a 2.9 g/l solution of phosphoric acid R (approximately 600 ml is — disregard limit : 0.1 times the area of the principal peak required) ; to 600 ml of the buffer solution add 400 ml of in the chromatogram obtained with reference solution (a) acetonitrile R1; (0.05 per cent). — mobile phase B. Use mobile phase B as described under Water (2.5.32) : maximum 0.5 per cent, determined on system A; 50 mg. ASSAY Liquid chromatography (2.2.29) as described in the test 0 0 - 50 100 for related substances, system A. Prepare the solutions protected from light. 50 - 51 100 → 0 0 → 100 Test solution. Dissolve 10.0 mg of the substance to be 0 51 - 61 100 examined in a mixture of equal volumes of acetonitrile R1 61 - 62 0 → 100 100 → 0 and water R and dilute to 25.0 ml with the same mixture of solvents. Dilute 3.0 ml of the solution to 20.0 ml with a 62 - 72 0 100 mixture of equal volumes of acetonitrile R1 and water R. System suitability : Reference solution. Dissolve 10.0 mg of alprostadil CRS in a mixture of equal volumes of acetonitrile R1 and water R — relative retentions with reference to alprostadil and dilute to 25.0 ml with the same mixture of solvents. (retention time = about 7 min) : impurity A = about 2.4 ; Dilute 3.0 ml of the solution to 20.0 ml with a mixture of impurity B = about 2.6, equal volumes of acetonitrile R1 and water R. — resolution : minimum of 1.5 between the peaks due to impurity A and impurity B in the chromatogram obtained Injection : 20 µl. Calculate the percentage content of C20H34O5. with reference solution (c). 954


Alprostadil


STORAGE At a temperature of 2 °C to 8 °C. IMPURITIES

F. 7-[(1S,2R,3R)-3-hydroxy-2-[(1E,3S)-3-hydroxyoct-1-enyl]-5oxocyclopentyl]heptanoic acid (8-epiprostaglandin E1),

A. 7-[(1R,2S)-2-[(1E,3S)-3-hydroxyoct-1-enyl]-5-oxocyclopent3-enyl]heptanoic acid (prostaglandin A1),

G. (5Z)-7-[(1R,2R,3R)-3-hydroxy-2-[(1E,3S)-3-hydroxyoct-1enyl]-5-oxocyclopentyl]hept-5-enoic acid (dinoprostone),

B. 7-[2-[(1E,3S)-3-hydroxyoct-1-enyl]-5-oxocyclopent-1enyl]heptanoic acid (prostaglandin B1),

H. (5E)-7-[(1R,2R,3R)-3-hydroxy-2-[(1E,3S)-3hydroxyoct-1-enyl]-5-oxocyclopentyl]hept-5-enoic acid ((5E)-prostaglandin E2),

C. 7-[(1R,2R,3R)-3-hydroxy-2-[(1E)-3-oxooct-1-enyl]-5oxocyclopentyl]heptanoic acid (15-oxoprostaglandin E1),

I. R = CH2-CH3 : ethyl 7-[(1R,2R,3R)-3-hydroxy-2-[(1E, 3S)-3-hydroxyoct-1-enyl]-5-oxocyclopentyl]heptanoate (prostaglandin E1, ethyl ester),

D. 7-[(1R,2R,3R)-3-hydroxy-2-[(1E,3R)-3-hydroxyoct-1-enyl]-5oxocyclopentyl]heptanoic acid (15-epiprostaglandin E1),

J. R = CH(CH3)2 : 1-methylethyl 7-[(1R,2R,3R)-3-hydroxy-2[(1E,3S)-3-hydroxyoct-1-enyl]-5-oxocyclopentyl]heptanoate (prostaglandin E1, isopropyl ester),

E. 7-[(1R,2R,3S)-3-hydroxy-2-[(1E,3S)-3-hydroxyoct-1-enyl]-5oxocyclopentyl]heptanoic acid (11-epiprostaglandin E1),

K. triphenylphosphine oxide.


955

Alteplase for injection

ALTEPLASE FOR


01/2005:1170 dilution of alteplase samples is necessary, the samples are corrected diluted in formulation buffer. For the calculation of the alteplase concentration the absorbance value (A280 − A320) is divided by the specific absorption coefficient for alteplase INJECTION of 1.9. Potency. The potency of alteplase is determined in an in-vitro clot-lysis assay as described under Assay. The specific activity of bulk alteplase is approximately 580 000 IU per milligram of alteplase.

Alteplasum ad iniectabile

N-terminal sequence. N-terminal sequencing is applied to determine the correct N-terminal sequence and to determine semiquantitatively additional cleavage sites in the alteplase molecule, for example at position AA 275-276 or at position AA 27-28. The N-terminal sequence must conform with the sequence of human tissue plasminogen activator. Isoelectric focusing. The consistency in the microheterogeneity of glycosylation of the alteplase molecule can be demonstrated by isoelectric focusing (IEF). A complex banding pattern with ten major and several minor bands in the pH range 6.5-8.5 is observed. Denaturing conditions are applied to achieve a good separation of differently charged variants of alteplase. The broad charge distribution observed is due to a population of molecules, which differ in the fine structure of biantenary and triantenary complex-type carbohydrate residues, with different degrees of substitution with sialic acids. The banding pattern of alteplase test samples must be consistent with the pattern of alteplase reference standard.

DEFINITION Alteplase for injection is a sterile, freeze-dried preparation of alteplase, a tissue plasminogen activator produced by recombinant DNA technology. It has a potency of not less than 500 000 IU per milligram of protein. Tissue plasminogen activator binds to fibrin clots and activates plasminogen, leading to the generation of plasmin and to the degradation of fibrin clots or blood coagulates. Alteplase consists of 527 amino acids with a calculated relative molecular mass of 59 050 without consideration of the carbohydrate moieties attached at positions Asn 117, Asn 184 and Asn 448. The total relative molecular mass is approximately 65 000. Alteplase is cleaved by plasmin between amino-acids 275 and 276 into a two-chain form (A chain and B chain) that are connected by a disulphide bridge between Cys 264 and Cys 395. The single-chain form and the two-chain form show comparable fibrinolytic activity in vitro. PRODUCTION Alteplase is produced by recombinant DNA synthesis in cell culture ; the fermentation takes place in serum-free medium. The purification process is designed to remove efficiently potential impurities, such as antibiotics, DNA and protein contaminants derived both from the host cell and from the production medium, and potential viral contaminants. If alteplase is stored in bulk form, stability (maintenance of potency) in the intended storage conditions must be demonstrated. The production, purification and product consistency are checked by a number of analytical methods described below, carried out routinely as in-process controls. Protein content. The protein concentration of alteplase solutions is determined by measuring the absorbance (2.2.25) of the protein solution at 280 nm and at 320 nm, using formulation buffer as the compensation liquid. If 956

Single-chain alteplase content. The alteplase produced by CHO (Chinese hamster ovary) cells in serum-free medium is predominantly single-chain alteplase. The single-chain form can be separated from the two-chain form by gel-permeation liquid chromatography under reducing conditions as described under Single-chain content (see Tests). The single-chain alteplase content in bulk samples must be higher than 60 per cent. Tryptic-peptide mapping. The primary structure of the alteplase molecule is verified by tryptic-peptide mapping as described under Identification B. The reduced and carboxymethylated molecule is cleaved by trypsin into about fifty peptides, which are separated by reverse-phase liquid chromatography. A characteristic chromatogram (fingerprint) is obtained. The identity of the tryptic-peptide map of a given alteplase sample with the profile of a well-characterised reference standard is an indirect confirmation of the amino-acid sequence, because even single amino-acid exchanges in individual peptides can be detected by this sensitive technique. In addition, complex peaks of the glycopeptides can be isolated from the tryptic-peptide map and separated in a second dimension, either by reverse-phase liquid chromatography under modified conditions or by capillary electrophoresis. By this two-dimensional separation of glycopeptide variants, lot-to-lot consistency of the microheterogeneity of glycosylation can be demonstrated. The tryptic-peptide map of alteplase samples must be consistent with the tryptic-peptide map of alteplase reference standard. Monomer content. The monomer content of alteplase is measured by gel-permeation liquid chromatography under non-reduced conditions as described under Monomer content (see Tests). The monomer content of alteplase bulk samples must be higher than 95 per cent. Type I/Type II alteplase content. CHO cells produce two glycosylation variants of alteplase. Type I alteplase contains one polymannose-type glycosylation at position Asn 117 and




two complex-type glycosylation sites at positions Asn 184 and and reference standard, as well as 2 ml of each mannose concentration in duplicate in reagent tubes. Add 50 µl of Asn 448. Type II alteplase is only glycosylated at positions phenol R, followed by 5 ml of sulphuric acid R in each Asn 117 and Asn 448. reagent tube. Incubate the mixture for 30 min at room The ratio of Type I/Type II alteplase is constant in the temperature. Measure the absorbance at 492 nm for each range of 45 to 65 per cent of Type I and 35 to 55 per cent of tube. Read the content of neutral sugars from the mannose Type II. The content of alteplase Type I and Type II can be calibration curve. The neutral sugar content is expressed determined by a densitometric scan of SDS-PAGE (sodium in moles of neutral sugar per mole of alteplase, taking dodecyl sulphate polyacrylamide gel electrophoresis) gel. Plasmin-treated samples of alteplase, which are reduced and into account the dilution factor for alteplase samples and carboxymethylated before loading on the gel, are separated reference standard and using a relative molecular mass of 180.2 for mannose and a relative molecular mass of into three bands : Type I alteplase A-chain (AA 1-275), 59 050 for the alteplase protein moiety. The neutral sugar Type II alteplase A-chain (AA 1-275) and alteplase B-chain content of the alteplase samples must be in the range of 70 (AA 276-527). The ratio of Type I/Type II alteplase is determined from a calibration curve, which is obtained by a to 130 per cent compared to alteplase reference standard, which contains about 12 moles of neutral sugar per mole densitometric scan of defined mixtures of purified Type I of alteplase. alteplase and Type II alteplase standards. SDS-PAGE. SDS-PAGE (silver staining) is used to demonstrate purity of the alteplase bulk material and the integrity of the alteplase molecule. For alteplase bulk samples, no additional protein bands compared to reference standard or degradation products must occur in SDS-PAGE gels at a loading amount of 2.5 µg alteplase protein per lane and a limit of detection of 5 ng per protein (BSA) band. Bacterial endotoxins (2.6.14) : less than 1 IU per milligram of alteplase. Sialic acids. Dialyse samples and alteplase reference standard against enzyme buffer (8.9 g/l sodium chloride R, 4.1 g/l sodium acetate R, pH 5.5) using a membrane with a cut-off point corresponding to a relative molecular mass of 10 000 for globular proteins. After dialysis, determine the protein concentration. Add 5 µl of calcium chloride solution (19.98 per cent m/m calcium chloride R) to 1 ml of protein solution. Add 10 milliunits of neuraminidase per milligram of protein. Incubate this solution at 37 °C for about 17 h.

CHARACTERS A white or slightly yellow powder or solid friable mass. Reconstitute the preparation as stated on the label immediately before carrying out the Identification, Tests (except those for solubility and water) and Assay.

Prepare standard dilutions between 1.56 mg/ml and 25.0 mg/ml from an N-acetylneuraminic acid R reference stock solution with a concentration of 50 mg/ml. Pipette 0.2 ml of the sample and of the protein reference standard in duplicate into reagent tubes. Pipette also 0.2 ml of the standard dilutions into reagent tubes. Add 0.25 ml of periodate reagent (5.4 g/l solution of sodium periodate R in a 1.25 per cent V/V solution of sulphuric acid R), mix and incubate for 30 min at 37 °C. Add 0.2 ml of arsenite reagent (20 g/l solution of sodium arsenite R in a 1.55 per cent V/V solution of hydrochloric acid R) and mix. A yellowish-brown colour develops and disappears. Add 2.0 ml of a 28.9 g/l solution of thiobarbituric acid R and mix. Heat the closed tubes in boiling water for 7.5 min and then cool them in an ice-bath for 5 min. Add 2.0 ml of a mixture of butanol R and hydrochloric acid R (95:5) and mix. Centrifuge the tubes at 3000 r/min for 3 min. Measure the absorbance of the butanol-HCl layer at 552 nm within 30 min using the butanol-HCl mixture as the compensation solution. Perform a linear-regression analysis for the N-acetylneuraminic acid standard. The molar content of N-acetylneuraminic acid for the samples and for alteplase reference standard is calculated from the calibration curve. The sialic acids content for the test samples must be within the range 70 to 130 per cent of alteplase reference standard, which contains about 3 moles of sialic acids per mole of alteplase. Neutral sugars. Dilute alteplase samples and the reference standard in the assay buffer, containing 34.8 g/l of arginine R, 0.1 g/l of polysorbate 80 R and adjusted to pH 7.4 with phosphoric acid R, to a protein concentration of 50 µg/ml. Prepare the following concentrations of mannose in the same assay buffer for a calibration curve : 20, 30, 40, 50 and 60 µg/ml. Pipette 2 ml of alteplase samples

IDENTIFICATION A. The assay serves also to identify the preparation. B. Tryptic-peptide mapping. Examine by liquid chromatography (2.2.29). Test solution. Dilute the preparation to be examined with water R to obtain a solution containing about 1 mg of alteplase per millilitre. Dialyse about 2.5 ml of the solution for at least 12 h into a solution containing 480 g/l of urea R, 44 g/l of tris(hydroxymethyl)aminomethane R and 1.5 g/l of disodium edetate R and adjusted to pH 8.6, using a membrane with a cut-off point corresponding to a relative molecular mass of 10 000 for globular proteins. Measure the volume of the solution, transfer it to a clean test-tube and add per millilitre 10 µl of a 156 g/l solution of dithiothreitol R. Allow to stand for 4 h, cool in iced water and add per millilitre of solution 25 µl of a freshly prepared 190 g/l solution of iodoacetic acid R. Allow to stand in the dark for 30 min. Add per millilitre 50 µl of dithiothreitol solution to stop the reaction. Dialyse for 24 h against an 8 g/l solution of ammonium hydrogen carbonate R. Add 1 part of trypsin for peptide mapping R to 100 parts of the protein and allow to stand for 6 h to 8 h. Repeat the addition of trypsin and allow to stand for a total of 24 h. Reference solution. Prepare as for the test solution using alteplase CRS instead of the preparation to be examined. The chromatographic procedure may be carried out using : — a column 0.1 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm to 10 µm), Mobile phase A. A 8 g/l solution of sodium dihydrogen phosphate R, adjusted to pH 2.85 with phosphoric acid R, filtered and degassed, Mobile phase B. Acetonitrile R 75 per cent V/V in mobile phase A, — as detector a spectrophotometer set at 210 nm. Equilibrate the system with mobile phase A at a flow rate of 1 ml/min. After injection of the solution, increase the proportion of mobile phase B at a rate of 0.44 per cent per minute until the ratio of mobile phase A to mobile phase B is 60:40 and then increase the proportion of mobile phase B at a rate of 1.33 per cent per minute until the ratio of mobile phase A to mobile phase B is 20:80


957



a tube, add 3 ml of a 3 g/l solution of dithiothreitol R in the mobile phase, place a cap on the tube and heat at about 80 °C for 3 min to 5 min. The chromatographic procedure may be carried out using : — a column 0.6 m long and 7.5 mm in internal diameter packed with silica-based rigid, hydrophilic gel with spherical particles 10 µm to 13 µm in diameter, suitable for size-exclusion chromatography, — as mobile phase at a flow rate of 0.5 ml/min a solution containing 30 g/l of sodium dihydrogen phosphate R and 1 g/l of sodium dodecyl sulphate R, adjusted to pH 6.8 with dilute sodium hydroxide solution R, — as detector a spectrophotometer set at 214 nm. Inject about 50 µl of the test solution and record the TESTS chromatogram. The chromatogram shows two major peaks Appearance of solution. The reconstituted preparation is corresponding to single-chain and two-chain alteplase. clear (2.2.1) and not more intensely coloured than reference Calculate the relative amount of single-chain alteplase from solution Y7 (2.2.2, Method II). the peak area values. The test is not valid unless : the number of theoretical plates pH (2.2.3) : 7.1 to 7.5. Solubility. Add the volume of the liquid stated on the label. calculated on the basis of the single-chain alteplase peak is The preparation dissolves completely within 2 min at 20 °C at least 1000. The content of single-chain alteplase is not less than 60 per cent of the total amount of alteplase-related to 25 °C. substances found. Protein content. Prepare a solution of the substance to be Monomer content. Examine by liquid chromatography examined with an accurately known concentration of about (2.2.29). 1 g/l. Using a 34.8 g/l solution of arginine R adjusted Test solution. Reconstitute the preparation to be examined to pH 7.3 with phosphoric acid R, dilute an accurately to obtain a solution containing about 1 mg per millilitre. measured volume of the solution of the substance to be examined so that the absorbance measured at the maximum The chromatographic procedure may be carried out using : at about 280 nm is 0.5 to 1.0 (test solution). Measure the — a column 0.6 m long and 7.5 mm in internal diameter absorbance (2.2.25) of the solution at the maximum at about packed with silica-based rigid, hydrophilic gel with 280 nm and at 320 nm using the arginine solution as the spherical particles 10 µm to 13 µm in diameter, suitable compensation liquid. Calculate the protein content in the for size-exclusion chromatography, portion of alteplase taken from the expression : — as mobile phase at a flow rate of 0.5 ml/min a solution containing 30 g/l of sodium dihydrogen phosphate R and 1 g/l of sodium dodecyl sulphate R, adjusted to pH 6.8 with dilute sodium hydroxide solution R, in which V is the volume of arginine solution required to — as detector a spectrophotometer set at 214 nm. prepare the test solution, A280 is the absorbance at the Inject the test solution and record the chromatogram. The maximum at about 280 nm and A320 is the absorbance at test is not valid unless the number of theoretical plates 320 nm. calculated for the alteplase monomer peak is at least 1000. Single-chain content. Examine by liquid chromaMeasure the response for all peaks, i.e. peaks corresponding tography(2.2.29). to alteplase species of different molecular masses. Calculate the relative content of monomer from the area values of Test solution. Dissolve the preparation to be examined these peaks. The monomer content for alteplase must be at in water R to obtain a solution containing about 1 mg of least 95 per cent. alteplase per millilitre. Place about 1 ml of the solution in and then continue elution with this mixture for a further 10 min. Record the chromatogram for the reference solution : the test is not valid unless the resolution of peaks 6 (peptides 268-275) and 7 (peptides 1-7) is at least 1.5 ; b0.5a and b0.5b are not more than 0.4 min. Inject about 100 µl of the test solution and record the chromatogram. Verify the identity of the peaks by comparison with the chromatograms of the reference solution. There should not be any additional significant peaks or shoulders, a significant peak or shoulder being defined as one with an area response equal to or greater than 5 per cent of peak 19 (peptides 278-296) ; no significant peak is missing. A type chromatogram for identification of the peaks cited is given at the end of the monograph (see Figure 1170.-1).

Figure 1170.-1. – Chromatogram for tryptic-peptide mapping of alteplase 958


Alum


Water (2.5.12). Not more than 4.0 per cent, determined by the semi-micro determination of water. Bacterial endotoxins (2.6.14) : less than 1 IU per milligram of protein. Sterility (2.6.1). It complies with the test for sterility. ASSAY The potency of alteplase is determined by comparing its ability to activate plasminogen to form plasmin with the same capacity of a reference preparation calibrated in International Units. The formation of plasmin is measured by the determination of the lysis time of a fibrin clot in given conditions. The International Unit is the activity of a stated quantity of the International Standard of alteplase. The equivalence in International Units of the International Standard is stated by the World Health Organisation. Solvent buffer. A solution containing 1.38 g/l of sodium dihydrogen phosphate monohydrate R, 7.10 g/l of anhydrous disodium hydrogen phosphate R, 0.20 g/l of sodium azide R and 0.10 g/l of polysorbate 80 R. Human thrombin solution. A solution of human thrombin R containing 33 IU/ml in solvent buffer. Human fibrinogen solution. A 2 g/l solution of fibrinogen R in solvent buffer. Human plasminogen solution. A 1 g/l solution of human plasminogen R in solvent buffer. Test solutions. Using a solution of the substance to be examined containing 1 g/l, prepare serial dilutions using solvent buffer, for example 1:5000, 1:10 000, 1:20 000. Reference solutions. Using a solution of alteplase CRS having an accurately known concentration of about 1 g/l (580 000 IU of alteplase per millilitre), prepare five serial dilutions using water R to obtain reference solutions having known concentrations in the range 9.0 IU/ml to 145 IU/ml. To each of a set of labelled glass test-tubes, add 0.5 ml of human thrombin solution. Allocate each test and reference solution to a separate tube and add to each tube 0.5 ml of the solution allocated to it. To each of a second set of labelled glass tubes, add 20 µl of human plasminogen solution, and 1 ml of human fibrinogen solution, mix and store on ice. Beginning with the reference/thrombin mixture containing the lowest number of International Units per millilitre, record the time and separately add 200 µl of each of the thrombin mixtures to the test tubes containing the plasminogen-fibrinogen mixture. Using a vortex mixture, intermittently mix the contents of each tube for a total of 15 s and carefully place in a rack in a circulating water-bath at 37 °C. A visibly turbid clot forms within 30 s and bubbles subsequently form within the clot. Record the clot-lysis time as the time between the first addition of alteplase solution and the moment when the last bubble rises to the surface. Using a least-squares fit, determine the equation of the line using the logarithms of the concentrations of the reference preparation in International Units per millilitre versus the logarithms of the values of their clot-lysis times in seconds, according to the equation :

Calculate the alteplase activity in International Units per millilitre from the expression :

in which D is the dilution factor for the test solution. Calculate the specific activity in the portion of the substance to be examined from the expression :

in which P is the concentration of protein obtained in the test for protein content. The estimated potency is not less than 90 per cent and not more than 110 per cent of the stated potency. STORAGE Store in a colourless, glass container, under vacuum or under an inert gas, protected from light, at a temperature between 2 °C and 30 °C. LABELLING The label states : — the number of International Units per container, — the amount of protein per container. 01/2005:0006

ALUM Alumen AlK(SO4)2,12H2O

Mr 474.4

DEFINITION Alum contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent of AlK(SO4)2,12H2O. CHARACTERS A granular powder or colourless, transparent, crystalline masses, freely soluble in water, very soluble in boiling water, soluble in glycerol, practically insoluble in alcohol. IDENTIFICATION A. Solution S (see Tests) gives the reactions of sulphates (2.3.1). B. Solution S gives the reaction of aluminium (2.3.1). C. Shake 10 ml of solution S with 0.5 g of sodium bicarbonate R and filter. The filtrate gives reaction (a) of potassium (2.3.1).

TESTS Solution S. Dissolve 2.5 g in water R and dilute to 50 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). pH (2.2.3). Dissolve 1.0 g in carbon dioxide-free water R in which t is the clot-lysis time, US the activity in International and dilute to 10 ml with the same solvent. The pH of the Units per millilitre of the reference preparation, b is the slope solution is 3.0 to 3.5. and a the y-intercept of the line. The test is not valid unless Ammonium (2.4.1). To 1 ml of solution S add 4 ml of the correlation coefficient is − 0.9900 to − 1.0000. From the water R. Dilute 0.5 ml of the solution to 14 ml with the line equation and the clot-lysis time for the test solution, same solvent. The solution complies with the limit test for calculate the logarithm of the activity UA from the equation : ammonium (0.2 per cent). General Notices (1) apply to all monographs and other texts

959

Aluminium chloride hexahydrate


Iron (2.4.9). 2 ml of solution S diluted to 10 ml with water R complies with the limit test for iron (100 ppm). Use in this test 0.3 ml of thioglycollic acid R. Heavy metals (2.4.8). 12 ml of solution S complies with limit test A for heavy metals (20 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. ASSAY Dissolve 0.900 g in 20 ml of water R and carry out the complexometric titration of aluminium (2.5.11). 1 ml of 0.1 M sodium edetate is equivalent to 47.44 mg of AIK(SO4)2,12H2O. 01/2005:0971

ALUMINIUM CHLORIDE HEXAHYDRATE

STORAGE Store in an airtight container. 01/2005:1664

ALUMINIUM HYDROXIDE, HYDRATED, FOR ADSORPTION Aluminii hydroxidum hydricum ad adsorptionem

Aluminii chloridum hexahydricum AlCl3,6H2O

ASSAY Dissolve 0.500 g in 25.0 ml of water R. Carry out the complexometric titration of aluminium (2.5.11). Titrate with 0.1 M zinc sulphate until the colour of the indicator changes from greyish-green to pink. Carry out a blank titration. 1 ml of 0.1 M sodium edetate is equivalent to 24.14 mg of AlCl3,6H2O.

Mr 241.4 [AlO(OH)],nH2O

DEFINITION Aluminium chloride hexahydrate contains not less than 95.0 per cent and not more than the equivalent of 101.0 per cent of AlCl3,6H2O.

DEFINITION Content : 90.0 per cent to 110.0 per cent of the content of aluminium stated on the label. NOTE : shake the gel vigorously for at least 30 s immediately before examining.

CHARACTERS CHARACTERS A white or slightly yellow, crystalline powder or colourless crystals, deliquescent, very soluble in water, freely soluble in Appearance : white or almost white, translucent, viscous, alcohol, soluble in glycerol. colloidal gel. A supernatant may be formed upon standing. Solubility : a clear or almost clear solution is obtained with IDENTIFICATION alkali hydroxide solutions and mineral acids. A. Dilute 0.1 ml of solution S2 (see Tests) to 2 ml with water R. The solution gives reaction (a) of chlorides IDENTIFICATION (2.3.1). Solution S (see Tests) gives the reaction of aluminium. B. Dilute 0.3 ml of solution S2 to 2 ml with water R. The To 10 ml of solution S add about 0.5 ml of dilute hydrochloric solution gives the reaction of aluminium (2.3.1). acid R and about 0.5 ml of thioacetamide reagent R. No precipitate is formed. Add dropwise 5 ml of dilute sodium TESTS hydroxide solution R. Allow to stand for 1 h. A gelatinous Solution S1. Dissolve 10.0 g in distilled water R and dilute white precipitate is formed which dissolves upon addition of to 100 ml with the same solvent. 5 ml of dilute sodium hydroxide solution R. Gradually add Solution S2. Dilute 50 ml of solution S1 to 100 ml with 5 ml of ammonium chloride solution R and allow to stand water R. for 30 min. The gelatinous white precipitate is re-formed. Appearance of solution. Solution S2 is clear (2.2.1) and not TESTS more intensely coloured than reference solution B7 (2.2.2, Solution S. Add 1 g to 4 ml of hydrochloric acid R. Heat at Method II). 60 °C for 1 h, cool, dilute to 50 ml with distilled water R Sulphates (2.4.13). 15 ml of solution S1 complies with the and filter if necessary. limit test for sulphates (100 ppm). pH (2.2.3) : 5.5 to 8.5. Iron (2.4.9). 10 ml of solution S1 complies with the limit Adsorption power. Dilute the substance to be examined test for iron (10 ppm). with distilled water R to obtain an aluminium concentration Alkali and alkaline-earth metals. To 20 ml of solution S2 of 5 mg/ml. Prepare bovine albumin R solutions with the add 100 ml of water R and heat to boiling. To the hot following concentrations of bovine albumin : 0.5 mg/ml, solution add 0.2 ml of methyl red solution R. Add dilute 1 mg/ml, 2 mg/ml, 3 mg/ml, 5 mg/ml and 10 mg/ml. If ammonia R1 until the colour of the indicator changes to necessary, adjust the gel and the bovine albumin R solutions yellow and dilute to 150 ml with water R. Heat to boiling to pH 6.0 with dilute hydrochloric acid R or dilute sodium and filter. Evaporate 75 ml of the filtrate to dryness on a hydroxide solution R. water-bath and ignite to constant mass. The residue weighs For adsorption, mix 1 part of the diluted gel with 4 parts not more than 2.5 mg (0.5 per cent). of each of the solutions of bovine albumin R and allow Heavy metals (2.4.8). 12 ml of solution S1 complies with to stand at room temperature for 1 h. During this time limit test A for heavy metals (20 ppm). Prepare the standard shake the mixture vigorously at least 5 times. Centrifuge using lead standard solution (2 ppm Pb) R. or filter through a non-protein-retaining filter. Immediately determine the protein content (2.5.33, Method 2) of either Water (2.5.12) : 42.0 per cent to 48.0 per cent, determined the supernatant or the filtrate. on 50.0 mg by the semi-micro determination of water. 960


Aluminium chloride hexahydrate


Iron (2.4.9). 2 ml of solution S diluted to 10 ml with water R complies with the limit test for iron (100 ppm). Use in this test 0.3 ml of thioglycollic acid R. Heavy metals (2.4.8). 12 ml of solution S complies with limit test A for heavy metals (20 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. ASSAY Dissolve 0.900 g in 20 ml of water R and carry out the complexometric titration of aluminium (2.5.11). 1 ml of 0.1 M sodium edetate is equivalent to 47.44 mg of AIK(SO4)2,12H2O. 01/2005:0971

ALUMINIUM CHLORIDE HEXAHYDRATE


ALUMINIUM HYDROXIDE, HYDRATED, FOR ADSORPTION Aluminii hydroxidum hydricum ad adsorptionem

Aluminii chloridum hexahydricum AlCl3,6H2O

ASSAY Dissolve 0.500 g in 25.0 ml of water R. Carry out the complexometric titration of aluminium (2.5.11). Titrate with 0.1 M zinc sulphate until the colour of the indicator changes from greyish-green to pink. Carry out a blank titration. 1 ml of 0.1 M sodium edetate is equivalent to 24.14 mg of AlCl3,6H2O.

Mr 241.4 [AlO(OH)],nH2O

DEFINITION Aluminium chloride hexahydrate contains not less than 95.0 per cent and not more than the equivalent of 101.0 per cent of AlCl3,6H2O.

DEFINITION Content : 90.0 per cent to 110.0 per cent of the content of aluminium stated on the label. NOTE : shake the gel vigorously for at least 30 s immediately before examining.

CHARACTERS CHARACTERS A white or slightly yellow, crystalline powder or colourless crystals, deliquescent, very soluble in water, freely soluble in Appearance : white or almost white, translucent, viscous, alcohol, soluble in glycerol. colloidal gel. A supernatant may be formed upon standing. Solubility : a clear or almost clear solution is obtained with IDENTIFICATION alkali hydroxide solutions and mineral acids. A. Dilute 0.1 ml of solution S2 (see Tests) to 2 ml with water R. The solution gives reaction (a) of chlorides IDENTIFICATION (2.3.1). Solution S (see Tests) gives the reaction of aluminium. B. Dilute 0.3 ml of solution S2 to 2 ml with water R. The To 10 ml of solution S add about 0.5 ml of dilute hydrochloric solution gives the reaction of aluminium (2.3.1). acid R and about 0.5 ml of thioacetamide reagent R. No precipitate is formed. Add dropwise 5 ml of dilute sodium TESTS hydroxide solution R. Allow to stand for 1 h. A gelatinous Solution S1. Dissolve 10.0 g in distilled water R and dilute white precipitate is formed which dissolves upon addition of to 100 ml with the same solvent. 5 ml of dilute sodium hydroxide solution R. Gradually add Solution S2. Dilute 50 ml of solution S1 to 100 ml with 5 ml of ammonium chloride solution R and allow to stand water R. for 30 min. The gelatinous white precipitate is re-formed. Appearance of solution. Solution S2 is clear (2.2.1) and not TESTS more intensely coloured than reference solution B7 (2.2.2, Solution S. Add 1 g to 4 ml of hydrochloric acid R. Heat at Method II). 60 °C for 1 h, cool, dilute to 50 ml with distilled water R Sulphates (2.4.13). 15 ml of solution S1 complies with the and filter if necessary. limit test for sulphates (100 ppm). pH (2.2.3) : 5.5 to 8.5. Iron (2.4.9). 10 ml of solution S1 complies with the limit Adsorption power. Dilute the substance to be examined test for iron (10 ppm). with distilled water R to obtain an aluminium concentration Alkali and alkaline-earth metals. To 20 ml of solution S2 of 5 mg/ml. Prepare bovine albumin R solutions with the add 100 ml of water R and heat to boiling. To the hot following concentrations of bovine albumin : 0.5 mg/ml, solution add 0.2 ml of methyl red solution R. Add dilute 1 mg/ml, 2 mg/ml, 3 mg/ml, 5 mg/ml and 10 mg/ml. If ammonia R1 until the colour of the indicator changes to necessary, adjust the gel and the bovine albumin R solutions yellow and dilute to 150 ml with water R. Heat to boiling to pH 6.0 with dilute hydrochloric acid R or dilute sodium and filter. Evaporate 75 ml of the filtrate to dryness on a hydroxide solution R. water-bath and ignite to constant mass. The residue weighs For adsorption, mix 1 part of the diluted gel with 4 parts not more than 2.5 mg (0.5 per cent). of each of the solutions of bovine albumin R and allow Heavy metals (2.4.8). 12 ml of solution S1 complies with to stand at room temperature for 1 h. During this time limit test A for heavy metals (20 ppm). Prepare the standard shake the mixture vigorously at least 5 times. Centrifuge using lead standard solution (2 ppm Pb) R. or filter through a non-protein-retaining filter. Immediately determine the protein content (2.5.33, Method 2) of either Water (2.5.12) : 42.0 per cent to 48.0 per cent, determined the supernatant or the filtrate. on 50.0 mg by the semi-micro determination of water. 960



It complies with the test if no bovine albumin is detectable in the supernatant or filtrate of the 2 mg/ml bovine albumin R solution (maximum level of adsorption) and in the supernatant or filtrate of bovine albumin R solutions of lower concentrations. Those containing 3 mg/ml, 5 mg/ml and 10 mg/ml bovine albumin R solutions may show bovine albumin in the supernatant or filtrate, proportional to the amount of bovine albumin in the solutions. Sedimentation. If necessary, adjust the substance to be examined to pH 6.0 using dilute hydrochloric acid R or dilute sodium hydroxide solution R. Dilute with distilled water R to obtain an aluminium concentration of approximately 5 mg/ml. If the aluminium content of the substance to be examined is lower than 5 mg/ml, adjust to pH 6.0 and dilute with a 9 g/l solution of sodium chloride R to obtain an aluminium concentration of about 1 mg/ml. After shaking for at least 30 s, place 25 ml of the preparation in a 25 ml graduated cylinder and allow to stand for 24 h. It complies with the test if the volume of the clear supernatant is less than 5 ml for the gel with an aluminium content of about 5 mg/ml. It complies with the test if the volume of the clear supernatant is less than 20 ml for the gel with an aluminium content of about 1 mg/ml. Chlorides (2.4.4) : maximum 0.33 per cent. Dissolve 0.5 g in 10 ml of dilute nitric acid R and dilute to 500 ml with water R. Nitrates : maximum 100 ppm. Place 5 g in a test-tube immersed in ice-water, add 0.4 ml of a 100 g/l solution of potassium chloride R, 0.1 ml of diphenylamine solution R and, dropwise with shaking, 5 ml of sulphuric acid R. Transfer the tube to a water-bath at 50 °C. After 15 min, any blue colour in the solution is not more intense than that in a standard prepared at the same time and in the same manner using 5 ml of nitrate standard solution (100 ppm NO3) R. Sulphates (2.4.13) : maximum 0.5 per cent. Dilute 2 ml of solution S to 20 ml with water R. Ammonium (2.4.1) : maximum 50 ppm. 1.0 g complies with limit test B. Prepare the standard using 0.5 ml of ammonium standard solution (100 ppm NH4) R. Arsenic (2.4.2, Method A) : maximum 1 ppm, determined on 1 g. Iron (2.4.9) : maximum 10 ppm, determined on 1 g. Heavy metals (2.4.8) : maximum 20 ppm. Dissolve 2.0 g in 10 ml of dilute nitric acid R and dilute to 20 ml with water R. The solution complies with limit test A. Prepare the standard using lead standard solution (2 ppm Pb) R. Bacterial endotoxins (2.6.14) : less than 5 IU of endotoxin per milligram of aluminium, if intended for use in the manufacture of an adsorbed product without a further appropriate procedure for the removal of bacterial endotoxins. ASSAY Dissolve 10.00 g in 10 ml of hydrochloric acid R1, heating on a water-bath. Cool and dilute to 20 ml with water R. To 10 ml of the solution, add dilute ammonia R1 until a precipitate is obtained. Add the smallest quantity of dilute hydrochloric acid R needed to dissolve the precipitate and dilute to 20 ml with water R. Carry out the complexometric titration of aluminium (2.5.11). Carry out a blank titration. General Notices (1) apply to all monographs and other texts

Aluminium magnesium silicate

STORAGE At a temperature not exceeding 30 °C. Do not allow to freeze. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states : — where applicable, that the substance is free from bacterial endotoxins, — the declared content of aluminium. 01/2005:1388

ALUMINIUM MAGNESIUM SILICATE Aluminii magnesii silicas DEFINITION Aluminium magnesium silicate is a mixture of particles with colloidal particle size of montmorillonite and saponite, free from grit and nonswellable ore. It contains not less than 95.0 per cent and not more than 105.0 per cent of the amount of aluminium and magnesium stated on the label. CHARACTERS Almost white powder, granules or plates, practically insoluble in water and in organic solvents. It swells in water to produce a colloidal dispersion. IDENTIFICATION A. Fuse 1 g with 2 g of anhydrous sodium carbonate R. Warm the residue with water R and filter. Acidify the filtrate with hydrochloric acid R and evaporate to dryness on a water-bath. 0.25 g of the residue gives the reaction of silicates (2.3.1). B. Dissolve the remainder of the residue obtained in identification test A in a mixture of 5 ml of dilute hydrochloric acid R and 10 ml of water R. Filter and add ammonium chloride buffer solution pH 10.0 R. A white, gelatinous precipitate is formed. Centrifuge, keep the supernatant for identification C. Dissolve the remaining precipitate in dilute hydrochloric acid R. The solution gives the reaction of aluminium (2.3.1). C. The supernatant liquid obtained after centrifugation in identification test B gives the reaction of magnesium (2.3.1). TESTS pH (2.2.3). Disperse 5.0 g in 100 ml of carbon dioxide-free water R. The pH of the dispersion is 9.0 to 10.0. Arsenic (2.4.2). Transfer 16.6 g to a 250 ml beaker containing 100 ml of dilute hydrochloric acid R. Mix, cover with a watch glass and boil gently, with occasional stirring, for 15 min. Allow the insoluble matter to settle and decant the supernatant liquid through a rapid-flow filter paper into a 250 ml volumetric flask, retaining as much sediment as possible in the beaker. To the residue in the beaker add 25 ml of hot dilute hydrochloric acid R, stir, heat to boiling, allow the insoluble matter to settle and decant the supernatant liquid through the filter into the volumetric flask. Repeat the extraction with four additional quantities, each of 25 ml, of hot dilute hydrochloric acid R, decanting each supernatant liquid through the filter into the volumetric flask. At the last extraction, transfer as much of the insoluble matter as possible onto the filter. Allow to cool the combined filtrates to room temperature and dilute to 250.0 ml with dilute 961

Aluminium oxide, hydrated


hydrochloric acid R. Dilute 5.0 ml of the solution to 25.0 ml with dilute hydrochloric acid R. The solution complies with limit test A for arsenic (3 ppm). Lead. Not more than 15 ppm of Pb, determined by atomic absorption spectrometry (2.2.23, Method I). Test solution. Transfer 10.0 g to a 250 ml beaker containing 100 ml of dilute hydrochloric acid R. Mix, cover with a watch glass and boil for 15 min. Allow to cool to room temperature and allow the insoluble matter to settle. Decant the supernatant liquid through a rapid-flow filter paper into a 400 ml beaker. To the insoluble matter in the 250 ml beaker add 25 ml of hot water R. Stir, allow the insoluble matter to settle and decant the supernatant liquid through the filter into the 400 ml beaker. Repeat the extraction with two additional quantities, each of 25 ml, of water R, decanting each supernatant liquid through the filter into the 400 ml beaker. Wash the filter with 25 ml of hot water R, collecting this filtrate in the 400 ml beaker. Concentrate the combined filtrates by gently boiling to about 20 ml. If a precipitate appears, add about 0.1 ml of nitric acid R, heat to boiling and allow to cool to room temperature. Filter the concentrated extracts through a rapid-flow filter paper into a 50 ml volumetric flask. Transfer the remaining contents of the 400 ml beaker through the filter paper and into the flask with water R. Dilute to 50.0 ml with water R. Reference solutions. Prepare the reference solutions using lead standard solution (10 ppm Pb) R, diluted if necessary with water R. Measure the absorbance at 217 nm, using a lead hollow-cathode lamp as source of radiation and an oxidising air-acetylene flame. Loss on drying (2.2.32). Not more than 8.0 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Microbial contamination. Total viable aerobic count (2.6.12) not more than 103 micro-organisms per gram determined by plate count. It complies with the test for Escherichia coli (2.6.13). ASSAY Aluminium. Determine by atomic absorption spectrometry (2.2.23, Method I). Test solution. In a platinum crucible mix 0.200 g with 1.0 g of lithium metaborate R. Heat slowly at first and ignite at 1000 °C to 1200 °C for 15 min. Cool, place the crucible in a 100 ml beaker containing 25 ml of dilute nitric acid R and add an additional 50 ml of dilute nitric acid R, filling and submerging the crucible. Place a polytetrafluoroethylene coated magnetic stirring bar in the crucible and stir gently with a magnetic stirrer until dissolution is complete. Pour the contents into a 250 ml beaker and remove the crucible. Warm the solution and transfer through a rapid-flow filter paper into a 250 ml volumetric flask, wash the filter and beaker with water R and dilute to 250.0 ml with water R (solution A). To 20.0 ml of this solution add 20 ml of a 10 g/l solution of sodium chloride R and dilute to 100.0 ml with water R. Reference solutions. Dissolve 1.000 g of aluminium R in a mixture of 10 ml of hydrochloric acid R and 10 ml of water R by gentle heating. Cool and dilute to 1000.0 ml with water R (1 mg of aluminium per millilitre). Into three identical volumetric flasks, each containing 0.20 g of sodium chloride R, introduce 2.0 ml, 5.0 ml and 10.0 ml of the solution, respectively and dilute to 100.0 ml with water R. Measure the absorbance at 309 nm using an aluminium hollow-cathode lamp as the source of radiation and an oxidising acetylene-nitrous oxide flame. 962

Calculate the content of aluminium in the substance to be examined. Magnesium. Determine by atomic absorption spectrometry (2.2.23, Method I). Test solution. Dilute 25.0 ml of solution A, prepared in the assay for aluminium, to 50.0 ml with water R. To 5.0 ml of this solution add 20.0 ml of lanthanum nitrate solution R and dilute to 100.0 ml with water R. Reference solutions. Place 1.000 g of magnesium R in a 250 ml beaker containing 20 ml of water R and carefully add 20 ml of hydrochloric acid R, warming if necessary to dissolve. Transfer the solution to a volumetric flask and dilute to 1000.0 ml with water R (1 mg of magnesium per millilitre). Dilute 5.0 ml of the solution to 250.0 ml with water R. Into four identical volumetric flasks, introduce 5.0 ml, 10.0 ml, 15.0 ml and 20.0 ml of the solution, respectively. To each flask add 20.0 ml of lanthanum nitrate solution R and dilute to 100.0 ml with water R. Measure the absorbance at 285 nm using a magnesium hollow-cathode lamp as source of radiation and a reducing air-acetylene flame. Calculate the content of magnesium in the substance to be examined. LABELLING The label states the content of aluminium and magnesium. 01/2005:0311

ALUMINIUM OXIDE, HYDRATED Aluminii oxidum hydricum DEFINITION Hydrated aluminium oxide contains the equivalent of not less than 47.0 per cent and not more than 60.0 per cent of Al2O3 (Mr 102.0). CHARACTERS A white, amorphous powder, practically insoluble in water. It dissolves in dilute mineral acids and in solutions of alkali hydroxides. IDENTIFICATION Solution S (see Tests) gives the reaction of aluminium (2.3.1). TESTS Solution S. Dissolve 2.5 g in 15 ml of hydrochloric acid R, heating on a water-bath. Dilute to 100 ml with distilled water R. Appearance of solution. Solution S is not more opalescent than reference suspension II (2.2.1) and not more intensely coloured than reference solution GY6 (2.2.2, Method II). Alkaline impurities. Shake 1.0 g with 20 ml of carbon dioxide-free water R for 1 min and filter. To 10 ml of the filtrate add 0.1 ml of phenolphthalein solution R. Any pink colour disappears on the addition of 0.3 ml of 0.1 M hydrochloric acid. Neutralising capacity. Carry out the test at 37 °C. Disperse 0.5 g in 100 ml of water R, heat, add 100.0 ml of 0.1 M hydrochloric acid, previously heated, and stir continuously ; the pH (2.2.3) of the solution after 10 min, 15 min and 20 min is not less than 1.8, 2.3 and 3.0 respectively and is at no time greater than 4.5. Add 10.0 ml of 0.5 M hydrochloric acid, previously heated, stir continuously for 1 h and titrate with 0.1 M sodium hydroxide to pH 3.5 ; not more than 35.0 ml of 0.1 M sodium hydroxide is required.


Aluminium phosphate, hydrated


Chlorides (2.4.4). Dissolve 0.1 g with heating in 10 ml of dilute nitric acid R and dilute to 100 ml with water R. 5 ml of the solution diluted to 15 ml with water R complies with the limit test for chlorides (1 per cent). Sulphates (2.4.13). Dilute 4 ml of solution S to 100 ml with distilled water R. 15 ml of the solution complies with the limit test for sulphates (1 per cent). Arsenic (2.4.2). 10 ml of solution S complies with limit test A for arsenic (4 ppm). Heavy metals (2.4.8). Neutralise 20 ml of solution S with concentrated ammonia R, using metanil yellow solution R as an external indicator. Filter, if necessary, and dilute to 30 ml with water R. 12 ml of the solution complies with limit test A for heavy metals (60 ppm). Prepare the standard using 10 ml of lead standard solution (1 ppm Pb) R. Microbial contamination. Total viable aerobic count (2.6.12) not more than 103 micro-organisms per gram, determined by plate count. It complies with the tests for enterobacteria and certain other gram-negative bacteria and with the test for Escherichia coli (2.6.13). ASSAY Dissolve 0.800 g in 10 ml of hydrochloric acid R1, heating on a water-bath. Cool and dilute to 50.0 ml with water R. To 10.0 ml of the solution, add dilute ammonia R1 until a precipitate begins to appear. Add the smallest quantity of dilute hydrochloric acid R needed to dissolve the precipitate and dilute to 20 ml with water R. Carry out the complexometric titration of aluminium (2.5.11). 1 ml of 0.1 M sodium edetate is equivalent to 5.098 mg of Al2O3. STORAGE Store in an airtight container, at a temperature below 30 °C.

01/2005:1598

ALUMINIUM PHOSPHATE, HYDRATED Aluminii phosphas hydricus AlPO4,x H2O

Mr 122.0 (anhydrous substance)

DEFINITION Content : 94.0 per cent to 102.0 per cent of AlPO4 (Mr 122.0) (ignited substance). CHARACTERS Appearance : white or almost white powder. Solubility : very slightly soluble in water, practically insoluble in alcohol. It dissolves in dilute solutions of mineral acids and alkali hydroxides. IDENTIFICATION A. Solution S (see Tests) gives reaction (b) of phosphates (2.3.1). B. Solution S gives the reaction of aluminium (2.3.1). TESTS Solution S. Dissolve 2.00 g in dilute hydrochloric acid R and dilute to 100 ml with the same acid. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). General Notices (1) apply to all monographs and other texts

pH (2.2.3) : 5.5 to 7.2 Shake 4.0 g with carbon dioxide-free water R and dilute to 100 ml with the same solvent. Chlorides (2.4.4) : maximum 1.3 per cent. Dissolve 50.0 mg in 10 ml of dilute nitric acid R and dilute to 200 ml with water R. 15 ml of the solution complies with the limit test for chlorides. Soluble phosphates : maximum 1.0 per cent, calculated as PO43-. Test solution. Stir 5.0 g with 150 ml of water R for 2 h. Filter and wash the filter with 50 ml of water R. Combine the filtrate and the washings and dilute to 250.0 ml with water R. Dilute 10.0 ml of this solution to 100.0 ml with water R. Reference solution (a). Dissolve 2.86 g of potassium dihydrogen phosphate R in water R and dilute to 100 ml with the same solvent. Reference solution (b). Dilute 1 ml of reference solution (a) to 5 ml with water R. Reference solution (c). Dilute 3 ml of reference solution (a) to 5 ml with water R. Treat each solution as follows. To 5.0 ml add 4 ml of dilute sulphuric acid R, 1 ml of ammonium molybdate solution R, 5 ml of water R and 2 ml of a solution containing 0.10 g of 4-methylaminophenol sulphate R, 0.5 g of anhydrous sodium sulphite R and 20.0 g of sodium metabisulphite R in 100 ml of water R. Shake and allow to stand for 15 min. Dilute to 25.0 ml with water R and allow to stand for a further 15 min. Measure the absorbance (2.2.25) at 730 nm. Calculate the content of soluble phosphates from a calibration curve prepared using reference solutions (a), (b) and (c) after treatment. Sulphates (2.4.13) : maximum 0.6 per cent. Dilute 8 ml of solution S to 100 ml with distilled water R. 15 ml of the solution complies with the limit test for sulphates. Arsenic (2.4.2) : maximum 1 ppm. 1.0 g complies with limit test A. Heavy metals (2.4.8) : maximum 20 ppm. Dissolve 1.0 g in dilute hydrochloric acid R and dilute to 20 ml with the same acid. 12 ml of the solution complies with limit test A. Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on ignition. 10.0 per cent to 20.0 per cent, determined on 1.000 g at 800 °C. Neutralising capacity. Add 0.50 g to 30 ml of 0.1 M hydrochloric acid previously heated to 37 °C and maintain at this temperature for 15 min while stirring. The pH (2.2.3) of the mixture after 15 min at 37 °C is 2.0 to 2.5. ASSAY Dissolve 0.400 g in 10 ml of dilute hydrochloric acid R and dilute to 100.0 ml with water R. To 10.0 ml of the solution, add 10.0 ml of 0.1 M sodium edetate and 30 ml of a mixture of equal volumes of ammonium acetate solution R and dilute acetic acid R. Boil for 3 min, then cool. Add 25 ml of alcohol R and 1 ml of a freshly prepared 0.25 g/l solution of dithizone R in alcohol R. Titrate the excess of sodium edetate with 0.1 M zinc sulphate until the colour changes to pink. 1 ml of 0.1 M sodium edetate is equivalent to 12.20 mg of AlPO4. STORAGE In an airtight container. 963

Aluminium sulphate


01/2005:0165


ALUMINIUM SULPHATE

AMANTADINE HYDROCHLORIDE

Aluminii sulfas

Amantadini hydrochloridum

DEFINITION Aluminium sulphate contains not less than 51.0 per cent and not more than the equivalent of 59.0 per cent of Al2(SO4)3 C10H18ClN Mr 187.7 (Mr 342.1). It contains a variable quantity of water of crystallisation. DEFINITION Amantadine hydrochloride contains not less than 98.5 per CHARACTERS cent and not more than the equivalent of 101.0 per cent of 3,7 Colourless, lustrous crystals or crystalline masses, soluble in tricyclo[3.3.1.1 ]decan-1-amine hydrochloride, calculated with reference to the anhydrous substance. cold water, freely soluble in hot water, practically insoluble in alcohol.

IDENTIFICATION

CHARACTERS A white or almost white, crystalline powder, freely soluble in water and in alcohol. It sublimes on being heated.

A. Solution S (see Tests) gives reaction (a) of sulphates (2.3.1).

IDENTIFICATION First identification : A, D. Second identification : B, C, D. B. Solution S gives the reaction of aluminium (2.3.1). A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with TESTS amantadine hydrochloride CRS. Examine the substances Solution S. Dissolve 2.5 g in water R and dilute to 50 ml prepared as discs. with the same solvent. B. To 0.1 g add 1 ml of pyridine R, mix and add 0.1 ml of acetic anhydride R. Heat to boiling for about 10 s. Appearance of solution. Solution S is not more opalescent Pour the hot solution into 10 ml of dilute hydrochloric than reference suspension III (2.2.1) and is colourless (2.2.2, acid R, cool to 5 °C and filter. The precipitate, washed Method II). with water R and dried in vacuo at 60 °C for 1 h, melts pH (2.2.3). Dissolve 0.5 g in carbon dioxide-free water R (2.2.14) at 147 °C to 151 °C. and dilute to 25 ml with the same solvent. The pH of the C. Dissolve 0.2 g in 1 ml of 0.1 M hydrochloric acid. Add solution is 2.5 to 4.0. 1 ml of a 500 g/l solution of sodium nitrite R. A white Alkali and alkaline-earth metals. To 20 ml of solution S precipitate is formed. add 100 ml of water R, heat and add 0.1 ml of methyl red D. 1 ml of solution S (see Tests) gives reaction (a) of chlorides solution R. Add dilute ammonia R1 until the colour of the (2.3.1). indicator changes to yellow. Dilute to 150 ml with water R, heat to boiling and filter. Evaporate 75 ml of the filtrate to TESTS dryness on a water-bath and ignite. The residue weighs not Solution S. Dissolve 2.5 g in carbon dioxide-free water R more than 2 mg (0.4 per cent). and dilute to 25 ml with the same solvent. Ammonium (2.4.1). 0.4 ml of solution S diluted to 14 ml Appearance of solution. Solution S is clear (2.2.1) and not with water R complies with the limit test for ammonium more intensely coloured than reference solution Y7 (2.2.2, (500 ppm). Method II). Iron (2.4.9). 2 ml of solution S diluted to 10 ml with water R Acidity or alkalinity. Dilute 2 ml of solution S to 10 ml complies with the limit test for iron (100 ppm). Use 0.3 ml of with carbon dioxide-free water R. Add 0.1 ml of methyl red thioglycollic acid R in this test. solution R and 0.2 ml of 0.01 M sodium hydroxide. The Heavy metals (2.4.8). Dilute 8 ml of solution S to 20 ml with solution is yellow. Add 0.4 ml of 0.01 M hydrochloric acid. water R. 12 ml of the solution complies with limit test A for The solution is red. heavy metals (50 ppm). Prepare the standard using lead Related substances. Examine by gas chromatography standard solution (1 ppm Pb) R. (2.2.28). Test solution. Dissolve 0.10 g of the substance to be examined in 2 ml of water R. Add 2 ml of a 200 g/l solution ASSAY of sodium hydroxide R and 2 ml of chloroform R. Shake for Dissolve 0.500 g in 20 ml of water R. Carry out the 10 min. Separate the chloroform layer, dry over anhydrous complexometric titration of aluminium (2.5.11). sodium sulphate R and filter. The chromatographic procedure may be carried out using : 1 ml of 0.1 M sodium edetate is equivalent to 17.11 mg of Al2(SO4)3. — a glass column 1.8 m long and 2 mm in internal diameter with a packing prepared as follows : mix 19.5 g of silanised diatomaceous earth for gas chromatography R STORAGE with 60 ml of a 3.3 g/l solution of potassium hydroxide R in methanol R and evaporate the solvent under reduced Store in an airtight container. 964



Ambroxol hydrochloride

A. Dissolve 20.0 mg in 0.05 M sulphuric acid and dilute to pressure while rotating the mixture slowly (support) ; 100.0 ml with the same acid. Dilute 2.0 ml of the solution dissolve 0.4 g of low-vapour-pressure hydrocarbons to 10.0 ml with 0.05 M sulphuric acid. Examined between (type L) R in 60 ml of toluene R (dissolution requires up 200 nm and 350 nm (2.2.25), the solution shows two to 5 h), add this solution to the support and evaporate absorption maxima at 245 nm and 310 nm. The ratio of the solvent under reduced pressure while rotating the the absorbance measured at 245 nm to that measured at mixture slowly, 310 nm is 3.2 to 3.4. — nitrogen for chromatography R as the carrier gas at a B. Infrared absorption spectrophotometry (2.2.24). flow rate of 30 ml/min, Comparison : ambroxol hydrochloride CRS. — a flame-ionisation detector. Programme the temperature of the column linearly at a rate C. Examine by thin-layer chromatography (2.2.27). of 6 °C per minute from 100 °C to 200 °C. Maintain the Test solution. Dissolve 50 mg of the substance to be temperature of the injection port at 220 °C and that of the examined in methanol R and dilute to 5 ml with the same detector at 300 °C. Inject 1 µl or the chosen volume of the solvent. test solution. Continue the chromatography for a period at Reference solution. Dissolve 50 mg of ambroxol least 2.5 times the retention time of the principal peak. In the hydrochloride CRS in methanol R and dilute to 5 ml chromatogram the sum of the area of any peaks apart from with the same solvent. that corresponding to amantadine does not exceed 1 per Plate : TLC silica gel F254 plate R. cent of the total area of the peaks ; no peak, apart from that Mobile phase : concentrated ammonia R, 1-propanol R, corresponding to amantadine, has an area exceeding 0.3 per ethyl acetate R, hexane R (1:10:20:70 V/V/V/V). cent of the total area. Disregard the peak corresponding to the solvent during the evaluation. Application : 10 µl. Development : over 2/3 of the plate. Heavy metals (2.4.8). 12 ml of solution S complies with limit test A for heavy metals (20 ppm). Prepare the standard using Drying : in air. lead standard solution (2 ppm Pb) R. Detection : examine in ultraviolet light at 254 nm. Water (2.5.12). Not more than 0.5 per cent, determined on Results : the principal spot in the chromatogram obtained 2.000 g by the semi-micro determination of water. with the test solution is similar in position and size to the principal spot in the chromatogram obtained with Sulphated ash (2.4.14). Not more than 0.1 per cent, the reference solution. determined on 1.0 g. D. Dissolve 25 mg in 2.5 ml of water R, mix with 1.0 ml of ASSAY dilute ammonia R1 and allow to stand for 5 min. Filter Dissolve 0.150 g in a mixture of 5.0 ml of 0.01 M hydrochloric and acidify the filtrate with dilute nitric acid R. The acid and 50 ml of alcohol R. Carry out a potentiometric filtrate gives reaction (a) of chlorides (2.3.1). titration (2.2.20), using 0.1 M sodium hydroxide. Read TESTS the volume added between the two points of inflexion. Solution S. Dissolve 0.75 g in methanol R and dilute to 1 ml of 0.1 M sodium hydroxide is equivalent to 18.77 mg 15 ml with the same solvent. of C10H18ClN. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, 01/2005:1489 Method II). pH (2.2.3) : 4.5 to 6.0. AMBROXOL HYDROCHLORIDE Dissolve 0.2 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Ambroxoli hydrochloridum Related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately before use. Test solution. Dissolve 50.0 mg of the substance to be examined in water R and dilute to 50.0 ml with the same solvent. Reference solution (a). Dilute 5.0 ml of the test solution to 250.0 ml with water R. Dilute 1.0 ml of this solution to 20.0 ml with the mobile phase. C13H19Br2ClN2O Mr 414.6 Reference solution (b). Dissolve 5 mg of the substance to be examined in 0.2 ml of methanol R and add 0.04 ml of a mixture of 1 volume of formaldehyde solution R and DEFINITION 99 volumes of water R. Heat at 60 °C for 5 min. Evaporate trans-4-[(2-Amino-3,5-dibromobenzyl)amino]cyclohexanol to dryness under a current of nitrogen. Dissolve the residue hydrochloride. in 5 ml of water R and dilute to 20 ml with the mobile phase. Content : 99.0 per cent to 101.0 per cent (dried substance). Column : CHARACTERS — size : l = 0.25 m, Ø = 4.0 mm, Appearance : white or yellowish crystalline powder. — stationary phase : octadecylsilyl silica gel for chromatography R (5 µm). Solubility : sparingly soluble in water, soluble in methanol, practically insoluble in methylene chloride. Mobile phase : a mixture of equal volumes of acetonitrile R and a solution prepared as follows : dissolve 1.32 g of IDENTIFICATION ammonium phosphate R in 900 ml of water R, adjust to First identification : B, D. pH 7.0 with phosphoric acid R and dilute to 1000 ml with water R. Second identification : A, C, D. General Notices (1) apply to all monographs and other texts

965

Amfetamine sulphate


Flow rate : 1 ml/min. Detection : spectrophotometer at 248 nm. Injection : 20 µl. Sensitivity : reference solution (a). Run time : 3 times the retention time of the principal peak in the chromatogram obtained with the test solution. System suitability : — resolution : minimum of 4.0 between the peaks due to impurity B and ambroxol in the chromatogram obtained with reference solution (b). Limits : — any impurity : not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), — total: not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.3 per cent), — disregard limit : 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (a). Heavy metals (2.4.8) : maximum 20 ppm. 1.0 g complies with limit test C. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.300 g in 70 ml of alcohol R and add 5 ml of 0.01 M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the two points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 41.46 mg of C13H19Br2ClN2O. STORAGE Store protected from light. IMPURITIES

A. Ar-CH2OH : (2-amino-3,5-dibromophenyl)methanol,

B. trans-4-(6,8-dibromo-1,4-dihydroquinazolin-3(2H)yl)cyclohexanol,

C. trans-4-[[(E)-2-amino-3,5-dibromobenzyliden]amino]cyclohexanol, 966

D. cis-4-[(2-amino-3,5-dibromobenzyl)amino]cyclohexanol, E. Ar-CH=O : 2-amino-3,5-dibromobenzaldehyde. 01/2005:0368

AMFETAMINE SULPHATE Amfetamini sulfas

C18H28N2O4S

Mr 368.5

DEFINITION Amfetamine sulphate contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent of bis[(2RS)-1-phenylpropan-2-amine] sulphate, calculated with reference to the dried substance. CHARACTERS A white powder, freely soluble in water, slightly soluble in alcohol. IDENTIFICATION First identification : A, B, E. Second identification : A, C, D, E. A. The angle of optical rotation (2.2.7) of solution S (see Tests), measured in a 2 dm tube, is − 0.04 to + 0.04. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of amfetamine sulphate. Examine as a mull in liquid paraffin R. C. To 50 ml of solution S add 5 ml of strong sodium hydroxide solution R and 0.5 ml of benzoyl chloride R and shake. Continue to add benzoyl chloride R in portions of 0.5 ml until no further precipitate is formed. Filter, wash the precipitate with water R and recrystallise twice from a mixture of equal volumes of alcohol R and water R and dry at 100 °C to 105 °C. The crystals melt (2.2.14) at 131 °C to 135 °C. D. To about 2 mg add 1 ml of sulphuric acid-formaldehyde reagent R. An orange colour develops and quickly becomes dark-brown. E. Solution S gives reaction (a) of sulphates (2.3.1). TESTS Solution S. Dissolve 2.0 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Acidity or alkalinity. To 25 ml of solution S add 0.1 ml of methyl red solution R. Not more than 0.1 ml of 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is required to change the colour of the indicator. Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.00 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.



ASSAY Dissolve 0.300 g in 30 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 36.85 mg of C18H28N2O4S.

Amidotrizoic acid dihydrate

Reference solution (a). Dilute 1 ml of test solution (b) to 50 ml with a 3 per cent V/V solution of ammonia R in methanol R. Reference solution (b). Dissolve 50 mg of amidotrizoic acid dihydrate CRS in a 3 per cent V/V solution of ammonia R in methanol R and dilute to 10 ml with the same solvent.

Apply separately to the plate 2 µl of each solution. Develop over a path of 15 cm using a mixture of 20 volumes of anhydrous formic acid R, 25 volumes of methyl ethyl ketone R and 60 volumes of toluene R. Allow the plate to dry until the solvents have evaporated and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained 01/2005:0873 with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained AMIDOTRIZOIC ACID DIHYDRATE with reference solution (a) (0.2 per cent). Halides. Dissolve 0.55 g in a mixture of 4 ml of dilute sodium hydroxide solution R and 15 ml of water R. Add Acidum amidotrizoicum dihydricum 6 ml of dilute nitric acid R and filter. 15 ml of the filtrate complies with the limit test for chlorides (2.4.4) (150 ppm expressed as chloride). Free aromatic amines. Maintain the solutions and reagents in iced water protected from bright light. To 0.50 g in a 50 ml volumetric flask add 15 ml of water R. Shake and add 1 ml of dilute sodium hydroxide solution R. Cool in iced water, add 5 ml of a freshly prepared 5 g/l solution of sodium nitrite R and 12 ml of dilute hydrochloric C11H9I3N2O4,2H2O Mr 650 acid R. Shake gently and allow to stand for exactly 2 min after adding the hydrochloric acid. Add 10 ml of a 20 g/l DEFINITION solution of ammonium sulphamate R. Allow to stand for Amidotrizoic acid dihydrate contains not less than 98.5 per 5 min, shaking frequently, and add 0.15 ml of a 100 g/l cent and not more than the equivalent of 101.0 per cent of solution of α-naphthol R in alcohol R. Shake and allow to 3,5-bis(acetylamino)-2,4,6-triiodobenzoic acid, calculated stand for 5 min. Add 3.5 ml of buffer solution pH 10.9 R, with reference to the dried substance. mix and dilute to 50.0 ml with water R. The absorbance (2.2.25), measured within 20 min at 485 nm using as the CHARACTERS compensation liquid a solution prepared at the same time and in the same manner but omitting the substance to be A white or almost white, crystalline powder, very slightly examined, is not greater than 0.30. soluble in water and in alcohol. It dissolves in dilute solutions of alkali hydroxides. Heavy metals (2.4.8). Dissolve 2.0 g in 4 ml of dilute sodium hydroxide solution R and dilute to 20 ml with water R. IDENTIFICATION 12 ml of this solution complies with limit test A for heavy First identification : A. metals (20 ppm). Prepare the standard using lead standard solution (2 ppm Pb) R. Second identification : B, C. Loss on drying (2.2.32) : 4.5 per cent to 7.0 per cent, A. Examine by infrared absorption spectrophotometry determined on 0.500 g by drying in an oven at 100 °C to (2.2.24), comparing with the spectrum obtained with 105 °C. amidotrizoic acid dihydrate CRS. Sulphated ash (2.4.14). Not more than 0.1 per cent, B. Examine the chromatograms obtained in the test for determined on 1.0 g. related substances (see Tests). The principal spot in the chromatogram obtained with test solution (b) is similar in position and size to the principal spot in the ASSAY chromatogram obtained with reference solution (b). To 0.150 g in a 250 ml round-bottomed flask add 5 ml of C. Heat 50 mg gently in a small porcelain dish over a naked strong sodium hydroxide solution R, 20 ml of water R, flame. Violet vapour is evolved. 1 g of zinc powder R and a few glass beads. Boil under a reflux condenser for 30 min. Allow to cool and rinse the TESTS condenser with 20 ml of water R, adding the rinsings to Appearance of solution. Dissolve 1.0 g in dilute sodium the flask. Filter through a sintered-glass filter and wash the hydroxide solution R and dilute to 20 ml with the same filter with several quantities of water R. Collect the filtrate solvent. The solution is clear (2.2.1) and colourless (2.2.2, and washings. Add 40 ml of dilute sulphuric acid R and Method II). titrate immediately with 0.1 M silver nitrate. Determine Related substances. Examine by thin-layer chromatography the end-point potentiometrically (2.2.20), using a suitable electrode system such as silver-mercurous sulphate. (2.2.27), using silica gel GF254 R as the coating substance. 1 ml of 0.1 M silver nitrate is equivalent to 20.47 mg of Test solution (a). Dissolve 0.50 g of the substance to be C11H9I3N2O4. examined in a 3 per cent V/V solution of ammonia R in methanol R and dilute to 10 ml with the same solvent. STORAGE Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a 3 per cent V/V solution of ammonia R in methanol R. Store protected from light. STORAGE Store protected from light.


967

Amikacin


IMPURITIES

A. 3-(acetylamino)-5-amino-2,4,6-triiodobenzoic acid. 01/2005:1289

TESTS pH (2.2.3). Dissolve 0.1 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. The pH of the solution is 9.5 to 11.5. Specific optical rotation (2.2.7). Dissolve 0.50 g in water R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 97 to + 105, calculated with reference to the anhydrous substance. Related substances. Examine by liquid chromatography (2.2.29), as prescribed under Assay.

Inject 20 µl of reference solution (a). Adjust the sensitivity of the system so that the height of the principal peak in the AMIKACIN chromatogram obtained is at least 50 per cent of the full scale of the recorder. Inject 20 µl of reference solution (c). Amikacinum The test is not valid unless, in the chromatogram obtained, the resolution between the peaks corresponding to amikacin and impurity A is at least 3.5 (see Figure 1289.-1). Inject 20 µl of test solution (a). Continue the chromatography for four times the retention time of amikacin. In the chromatogram obtained with test solution (a) : the area of any peak corresponding to impurity A is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (1 per cent) ; the area of any peak, apart from the principal peak and any peak corresponding to impurity A, is not greater than half the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent) ; the sum of the areas of C22H43N5O13 Mr 585.6 any such peaks is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference DEFINITION solution (a) (1.5 per cent). Disregard any peak due to the Amikacin is 6-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-4-Oblank and any peak with an area less than 0.1 times that (6-amino-6-deoxy-α-D-glucopyranosyl)-1-N-[(2S)-4-amino-2of the principal peak in the chromatogram obtained with hydroxybutanoyl]-2-deoxy-D-streptamine, an antimicrobial reference solution (a). substance obtained from kanamycin A. It contains not less Water (2.5.12). Not more than 8.5 per cent, determined on than 96.5 per cent and not more than the equivalent of 0.200 g by the semi-micro determination of water. 102.5 per cent of C22H43N5O13, calculated with reference to Sulphated ash (2.4.14). Not more than 0.5 per cent, the anhydrous substance. determined on 1.0 g. CHARACTERS A white or almost white powder, sparingly soluble in water, ASSAY slightly soluble in methanol, practically insoluble in acetone Examine by liquid chromatography (2.2.29). and in alcohol. Test solution (a). Dissolve 0.100 g of the substance IDENTIFICATION to be examined in water R and dilute to 10.0 ml with the same solvent. In a round-glass-stoppered vial, add A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum of amikacin CRS. 0.2 ml of this solution to 2.0 ml of a 10 g/l solution of 2,4,6-trinitrobenzene sulphonic acid R. Then add 3.0 ml of B. Examine by thin-layer chromatography (2.2.27) using a pyridine R and close the vial tightly. Shake vigorously for TLC silica gel plate R. 30 s and heat in a water-bath at 75 °C for 45 min. Cool in Test solution. Dissolve 25 mg of the substance to be cold water for 2 min and add 2 ml of glacial acetic acid R. examined in water R and dilute to 10 ml with the same Shake vigorously for 30 s. solvent. Reference solution (a). Dissolve 25 mg of amikacin CRS in water R and dilute to 10 ml with the same solvent. Reference solution (b). Dissolve 5 mg of kanamycin monosulphate CRS in 1 ml of the test solution and dilute to 10 ml with water R. Apply to the plate 5 µl of each solution. Develop over a path of 15 cm using the lower layer of a mixture of equal volumes of concentrated ammonia R, methanol R and methylene chloride R. Allow the plate to dry in air and spray with ninhydrin solution R1. Heat at 110 °C for 5 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution (a). The identification test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. 968

Test solution (b). Dissolve 50.0 mg of the substance to be examined in water R and dilute to 50.0 ml with the same solvent. Then prepare as prescribed for test solution (a).

Reference solution (a). Dissolve 10.0 mg of amikacin impurity A CRS in water R and dilute to 100.0 ml with the same solvent. Then prepare as prescribed for test solution (a). Reference solution (b). Dissolve 50.0 mg of amikacin CRS in water R and dilute to 50.0 ml with the same solvent. Then prepare as prescribed for test solution (a). Reference solution (c). Dissolve 5 mg of amikacin CRS and 5 mg of amikacin impurity A CRS in water R and dilute to 50 ml with the same solvent. Then prepare as prescribed for test solution (a). Blank solution. Prepare as described for test solution (a) using 0.2 ml of water R.


Amikacin


IMPURITIES The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1 ml/min a mixture of 30 volumes of a 2.7 g/l solution of potassium dihydrogen phosphate R adjusted to pH 6.5 with a 22 g/l solution of potassium hydroxide R and 70 volumes of methanol R, — as detector a spectrophotometer set at 340 nm, maintaining the temperature of the column at 30 °C and that of the solutions to be examined at 10 °C. Inject 20 µl of reference solution (b). Adjust the sensitivity A. R1 = R3 = H, R2 = acyl : 4-O-(3-amino-3-deoxyof the system so that the height of the principal peak is at α-D-glucopyranosyl)-6-O-(6-amino-6-deoxy-α-Dleast 50 per cent of the full scale of the recorder. Inject glucopyranosyl)-1-N-[(2S)-4-amino-2-hydroxybutanoyl]-2reference solution (b) six times. The assay is not valid deoxy-L-streptamine, unless the relative standard deviation of the peak area for amikacin is 2.0 per cent. Inject test solution (b) and reference B. R1 = R2 = acyl, R3 = H : 4-O-(3-amino-3-deoxysolution (b). α-D-glucopyranosyl)-6-O-(6-amino-6-deoxyα-D-glucopyranosyl)-1,3-N-bis[(2S)-4-amino-2hydroxybutanoyl]-2-deoxy-L-streptamine, C. R1 = R2 = H, R3 = acyl : 4-O-(6-amino-6-deoxyα-D-glucopyranosyl)-6-O-[3-[[(2S)-4-amino-2hydroxybutanoyl]amino]-3-deoxy-α-D-glucopyranosyl]-2deoxy-D-streptamine, D. R1 = R2 = R3 = H : kanamycin.

Figure 1289.-1. – Chromatogram for the test for related substances of amikacin General Notices (1) apply to all monographs and other texts

969

Amikacin sulphate


01/2005:1290 Specific optical rotation (2.2.7). Dissolve 0.50 g in water R corrected and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 76 to + 84, calculated with reference to the dried substance. AMIKACIN SULPHATE Related substances. Examine by liquid chromatography (2.2.29), as prescribed under Assay. Amikacini sulfas Inject 20 µl of reference solution (a). Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is at least 50 per cent of the full scale of the recorder. Inject 20 µl of reference solution (c). The test is not valid unless in the chromatogram obtained, the resolution between the peaks corresponding to amikacin and impurity A is at least 3.5 (see Figure 1290.-1). Inject 20 µl of test solution (a). Continue the chromatography for four times the retention time of amikacin. In the chromatogram obtained with test solution (a) : the area of any peak corresponding to impurity A is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (1 per cent) ; the area C22H47N5O21S2 Mr 782 of any peak, apart from the principal peak and any peak corresponding to impurity A, is not greater than half the area of the principal peak in the chromatogram obtained DEFINITION with reference solution (a) (0.5 per cent) ; the sum of the Amikacin sulphate is 6-O-(3-amino-3-deoxy-α-Dglucopyranosyl)-4-O-(6-amino-6-deoxy-α-D-glucopyranosyl)-1- areas of any such peaks is not greater than 1.5 times the area of the principal peak in the chromatogram obtained N-[(2S)-4-amino-2-hydroxybutanoyl]-2-deoxy-D-streptamine with reference solution (a) (1.5 per cent). Disregard any peak sulphate, an antimicrobial substance obtained from kanamycin A. It contains not less than 72.3 per cent and not due to the blank, any peak eluting before the principal peak and any peak with an area less than 0.1 times that of the more than the equivalent of 76.8 per cent of C22H43N5O13, principal peak in the chromatogram obtained with reference calculated with reference to the dried substance. solution (a). CHARACTERS Sulphate : 23.3 per cent to 25.8 per cent of sulphate (SO4), calculated with reference to the dried substance. Dissolve A white or almost white powder, freely soluble in water, 0.250 g in 100 ml of water R and adjust the solution to practically insoluble in acetone and in alcohol. pH 11 using concentrated ammonia R. Add 10.0 ml of 0.1 M barium chloride and about 0.5 mg of phthalein purple R. IDENTIFICATION Titrate with 0.1 M sodium edetate adding 50 ml of alcohol R A. Examine by infrared absorption spectrophotometry when the colour of the solution begins to change and (2.2.24), comparing with the spectrum obtained with continue the titration until the violet-blue colour disappears. amikacin sulphate CRS. 1 ml of 0.1 M barium chloride is equivalent to 9.606 mg of B. Examine by thin-layer chromatography (2.2.27), using a sulphate (SO4). TLC silica gel plate R. Loss on drying (2.2.32). Not more than 13.0 per cent, Test solution. Dissolve 25 mg of the substance to be determined on 0.500 g by drying in an oven at 100-105 °C at examined in water R and dilute to 10 ml with the same a pressure not exceeding 0.7 kPa for 3 h. solvent. Pyrogens (2.6.8). If intended for use in the manufacture Reference solution (a). Dissolve 25 mg of amikacin of parenteral dosage forms without a further appropriate sulphate CRS in water R and dilute to 10 ml with the procedure for the removal of pyrogens, it complies with the same solvent. test for pyrogens. Inject per kilogram of the rabbit’s mass Reference solution (b). Dissolve 5 mg of kanamycin 5 ml of a solution containing 25 mg of the substance to be monosulphate CRS in 1 ml of the test solution and dilute examined in water for injections R. to 10 ml with water R. ASSAY Apply to the plate 5 µl of each solution. Develop over Examine by liquid chromatography (2.2.29). a path of 15 cm using the lower layer of a mixture of equal volumes of concentrated ammonia R, methanol R Test solution (a). Dissolve 0.100 g of the substance and methylene chloride R. Allow the plate to dry in air to be examined in water R and dilute to 10.0 ml with and spray with ninhydrin solution R1. Heat at 110 °C for the same solvent. In a round-glass-stoppered vial, add 5 min. The principal spot in the chromatogram obtained 0.2 ml of this solution to 2.0 ml of a 10 g/l solution of with the test solution is similar in position, colour and 2,4,6-trinitrobenzene sulphonic acid R. Then add 3.0 ml of size to the principal spot in the chromatogram obtained pyridine R and close the vial tightly. Shake vigorously for with the reference solution (a). The identification test 30 s and heat on a water-bath at 75 °C for 2 h. Cool in cold is not valid unless the chromatogram obtained with water for 2 min and add 2 ml of glacial acetic acid R. Shake reference solution (b) shows two clearly separated spots. vigorously for 30 s. C. It gives reaction (a) of sulphates (2.3.1). Test solution (b). Dissolve 50.0 mg of the substance to be examined in water R and dilute to 50.0 ml with the same TESTS solvent. Then prepare as prescribed for test solution (a). Reference solution (a). Dissolve 10.0 mg of amikacin pH (2.2.3). Dissolve 0.1 g in carbon dioxide-free water R impurity A CRS in water R and dilute to 100.0 ml with the and dilute to 10 ml with the same solvent. The pH of the same solvent. Then prepare as prescribed for test solution (a). solution is 2.0 to 4.0. 970



Amikacin sulphate

Figure 1290.-1. – Chromatogram for the test for related substances of amikacin sulphate

— as detector a spectrophotometer set at 340 nm, Reference solution (b). Dissolve 50.0 mg of amikacin sulphate CRS in water R and dilute to 50.0 ml with the same maintaining the temperature of the column at 30 °C and solvent. Then prepare as prescribed for test solution (a). that of the solutions to be examined at 10 °C. Reference solution (c). Dissolve 5 mg of amikacin Inject 20 µl of reference solution (b). Adjust the sensitivity sulphate CRS and 5 mg of amikacin impurity A CRS in of the system so that the height of the principal peak is at water R and dilute to 50 ml with the same solvent. Then least 50 per cent of the full scale of the recorder. Inject prepare as prescribed for test solution (a). reference solution (b) six times. The assay is not valid unless the relative standard deviation of the peak area for Blank solution. Prepare as described for test solution (a) amikacin is 2.0 per cent. Inject test solution (b) and reference using 0.2 ml of water R. solution (b). The chromatographic procedure may be carried out using :

STORAGE — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for If the substance is sterile, store in a sterile, airtight, tamper-proof container. chromatography R (5 µm), — as mobile phase at a flow rate of 1 ml/min a mixture of LABELLING 30 volumes of a 2.7 g/l solution of potassium dihydrogen phosphate R adjusted to pH 6.5 with a 22 g/l solution of The label states, where applicable, that the substance is apyrogenic. potassium hydroxide R and 70 volumes of methanol R, General Notices (1) apply to all monographs and other texts

971

Amiloride hydrochloride


IMPURITIES

A. R1 = R3 = H, R2 = acyl : 4-O-(3-amino-3-deoxyα-D-glucopyranosyl)-6-O-(6-amino-6-deoxy-α-Dglucopyranosyl)-1-N-[(2S)-4-amino-2-hydroxybutanoyl]-2deoxy-L-streptamine,

Apply to the plate 5 µl of each solution. Develop over a path of 12 cm using a freshly prepared mixture of 6 volumes of dilute ammonia R1, 6 volumes of water R and 88 volumes of dioxan R. Allow the plate to dry in air and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, fluorescence and size to the principal spot in the chromatogram obtained with the reference solution. C. Dissolve about 10 mg in 10 ml of water R. Add 10 ml of a 200 g/l solution of cetrimide R, 0.25 ml of dilute sodium hydroxide solution R and 1 ml of bromine water R. A greenish-yellow colour is produced. Add 2 ml of dilute hydrochloric acid R. The solution becomes deep yellow and shows blue fluorescence in ultraviolet light at 365 nm. D. It gives reaction (b) of chlorides (2.3.1).

TESTS Free acid. Dissolve 1.0 g in a mixture of 50 ml of methanol R and 50 ml of water R and titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20). Not more than 0.3 ml of 0.1 M sodium hydroxide is C. R1 = R2 = H, R3 = acyl : 4-O-(6-amino-6-deoxyrequired to reach the end-point. α-D-glucopyranosyl)-6-O-[3-[[(2S)-4-amino-2Related substances. Examine by liquid chromatography hydroxybutanoyl]amino]-3-deoxy-α-D-glucopyranosyl]-2(2.2.29). deoxy-D-streptamine, Test solution. Dissolve 20.0 mg of the substance to be D. R1 = R2 = R3 = H : kanamycin. examined in a mixture of 1 volume of acetonitrile R and 3 volumes of water R and dilute to 10.0 ml with the same mixture of solvents. 01/2005:0651 Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with a mixture of 1 volume of acetonitrile R and AMILORIDE HYDROCHLORIDE 3 volumes of water R. Reference solution (b). Dilute 1.0 ml of reference solution (a) Amiloridi hydrochloridum to 10.0 ml with a mixture of 1 volume of acetonitrile R and 3 volumes of water R. Reference solution (c). Dissolve 20.0 mg of amiloride impurity A CRS in a mixture of 1 volume of acetonitrile R and 3 volumes of water R and dilute to 20.0 ml with the same mixture of solvents. Dilute 1.0 ml of the solution to 100.0 ml with a mixture of 1 volume of acetonitrile R and C6H9Cl2N7O,2H2O Mr 302.1 3 volumes of water R. The chromatographic procedure may be carried out using : DEFINITION — a stainless steel column 0.25 m long and 4.6 mm in Amiloride hydrochloride contains not less than 98.0 per cent internal diameter packed with octadecylsilyl silica gel for and not more than the equivalent of 101.0 per cent of 3,5chromatography R (5 µm), diamino-N-carbamimidoyl-6-chloropyrazine-2-carboxamide, — as mobile phase at a flow rate of 1 ml/min a mixture calculated with reference to the anhydrous substance. of 5 volumes of tetramethylammonium hydroxide solution R, 250 volumes of acetonitrile R and 745 volumes CHARACTERS of water R ; adjust to pH 7.0 using a mixture of 1 volume A pale-yellow to greenish-yellow powder, slightly soluble in of phosphoric acid R and 9 volumes of water R, water and in ethanol. — as detector a spectrophotometer set at 254 nm. IDENTIFICATION Inject 20 µl of reference solution (c). Adjust the concentration of acetonitrile in the mobile phase so that the retention First identification : A, D. time of impurity A is 5 min to 6 min (an increase in the Second identification : B, C, D. concentration of acetonitrile results in a shorter retention A. Examine by infrared absorption spectrophotometry time). Inject 20 µl of reference solution (a). Adjust the (2.2.24), comparing with the spectrum obtained with concentration of tetramethylammonium hydroxide and amiloride hydrochloride CRS. of phosphoric acid keeping the pH at 7.0 so that the B. Examine by thin-layer chromatography (2.2.27), using a retention time of amiloride is 9 min to 12 min (an increase suitable silica gel as the coating substance. in the concentration results in a shorter retention time for amiloride). Inject 20 µl of reference solution (b). The test is Test solution. Dissolve 40 mg of the substance to be not valid unless the signal-to-noise ratio for the peak due to examined in methanol R and dilute to 10 ml with the amiloride is at least 5.0. same solvent. Inject 20 µl of the test solution and 20 µl of reference Reference solution. Dissolve 40 mg of amiloride solution (c). Record the chromatograms for five times the hydrochloride CRS in methanol R and dilute to 10 ml retention time of amiloride. In the chromatogram obtained with the same solvent. B. R1 = R2 = acyl, R3 = H : 4-O-(3-amino-3-deoxyα-D-glucopyranosyl)-6-O-(6-amino-6-deoxyα-D-glucopyranosyl)-1,3-N-bis[(2S)-4-amino-2hydroxybutanoyl]-2-deoxy-L-streptamine,

972


4-Aminobenzoic acid


with the test solution, the sum of the areas of the peaks, apart from the peak corresponding to amiloride, is not greater than the area of the peak corresponding to impurity A in the chromatogram obtained with reference solution (c) (0.5 per cent). Disregard any peak with an area less than 10 per cent of the area of the peak corresponding to impurity A in the chromatogram obtained with reference solution (c). Water (2.5.12) : 11.0 per cent to 13.0 per cent, determined on 0.200 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.200 g in a mixture of 5.0 ml of 0.01 M hydrochloric acid and 50 ml of alcohol R. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the two points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 26.61 mg of C6H9Cl2N7O.

Reference solution (b). Dissolve 10 mg of 4-nitrobenzoic acid R in 10 ml of reference solution (a). Plate : suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm as the coating substance. Mobile phase : glacial acetic acid R, hexane R, methylene chloride R (5:20:75 V/V/V). Application : 1 µl. Development : over a path of 10 cm. Drying : in air. Detection : examine in ultraviolet light at 254 nm. System suitability : the chromatogram obtained with reference solution (b) shows 2 clearly separated spots. Results : the principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a).

IDENTIFICATION First identification : B. Second identification : A, C. A. Melting point (2.2.14) : 186 °C to 189 °C. B. Infrared absorption spectrophotometry (2.2.24). Comparison : 4-aminobenzoic acid CRS. C. Thin-layer chromatography (2.2.27). Test solution. Dissolve 20 mg of the substance to be examined in methanol R and dilute to 20 ml with the same solvent. Reference solution (a). Dissolve 20 mg of 4-aminobenzoic acid CRS in methanol R and dilute to 20 ml with the same solvent.

TESTS Appearance of solution. The solution is clear (2.2.1) and not more intensely coloured than reference solution B5 (2.2.2, Method II). Dissolve 1.0 g in alcohol R and dilute to 20 ml with the same solvent. Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution. Dissolve 25.0 mg of 4-nitrobenzoic acid R and 25.0 mg of benzocaine R in methanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml to 50.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Column : — size : l = 0.12 m, Ø = 4.0 mm, — stationary phase : octylsilyl silica gel for chromatography R (5 µm). Mobile phase : mix 20 volumes of a mixture of 70 volumes of acetonitrile R and 80 volumes of methanol R, and 80 volumes of a solution containing 1.5 g/l of potassium dihydrogen phosphate R and 2.5 g/l of sodium octanesulphonate R adjusted to pH 2.2 with phosphoric acid R. Flow rate : 1.0 ml/min. Detection : spectrophotometer at 270 nm. Injection : 20 µl. Run time : 11 times the retention time of 4-aminobenzoic acid. Relative retention with reference to 4-aminobenzoic acid (retention time = about 3 min) : impurity A = about 4 ; impurity B = about 9. Limits : — impurity A : not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.2 per cent), — impurity B : not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.2 per cent), — any other impurity : not more than 0.5 times the area of the peak due to impurity A in the chromatogram obtained with the reference solution (0.1 per cent), — total : not more than 2.5 times the area of the peak due to impurity A in the chromatogram obtained with the reference solution (0.5 per cent),


973

STORAGE Store protected from light. IMPURITIES

A. methyl 3,5-diamino-6-chloropyrazine-2-carboxylate. 01/2005:1687

4-AMINOBENZOIC ACID Acidum 4-aminobenzoicum

C7H7NO2

Mr 137.1

DEFINITION 4-Aminobenzoic acid. Content : 99.0 per cent to 101.0 per cent (anhydrous substance). CHARACTERS Appearance : white or slightly yellow, crystalline powder. Solubility : slightly soluble in water, freely soluble in alcohol. It dissolves in dilute solutions of alkali hydroxides.

Aminocaproic acid


— disregard limit: 0.1 times the area of the peak due to impurity A in the chromatogram obtained with the reference solution (0.02 per cent). Impurity C and impurity D. Gas chromatography (2.2.28). Internal standard solution. Dissolve 20.0 mg of lauric acid R in methylene chloride R and dilute to 100.0 ml with the same solvent. Test solution. Dissolve 1.000 g of the substance to be examined in 10.0 ml of an 84 g/l solution of sodium hydroxide R and extract with 2 quantities, each of 10 ml, of methylene chloride R. Combine and wash with 5 ml of water R ; filter through anhydrous sodium sulphate R. Wash the filter with methylene chloride R. Evaporate in a water-bath at 50-60 °C to obtain a volume of about 1-5 ml. Add 1.0 ml of the internal standard solution and dilute to 10.0 ml with methylene chloride R. Reference solution (a). Dissolve 20.0 mg of aniline R in methylene chloride R and dilute to 100.0 ml with the same solvent. Reference solution (b). Dissolve 20.0 mg of p-toluidine R in methylene chloride R and dilute to 100.0 ml with the same solvent. Reference solution (c). Dilute 0.50 ml of reference solution (a), 0.50 ml of reference solution (b) and 10.0 ml of the internal standard solution to 100.0 ml with methylene chloride R. Column : — material : fused silica, — size : l = 30 m, Ø = 0.32 mm, — stationary phase: poly[methyl(95)phenyl(5)] siloxane R (film thickness 0.5 µm). Carrier gas : helium for chromatography R. Flow rate : 1.0 ml/min. Split ratio : 1:10. Temperature :

Column

Time (min)

Temperature (°C)

0-4

130

4 - 6.5

130 → 180

6.5 - 11.5

180

Injection port

280

Detector

300

ASSAY Dissolve 0.100 g with heating in 50 ml of carbon dioxide-free water R. Titrate with 0.1 M sodium hydroxide determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M sodium hydroxide is equivalent to 13.71 mg of C7H7NO2. STORAGE Protected from light. IMPURITIES

A. R = CO2H, R′ = NO2 : 4-nitrobenzoic acid, B. R = CO-O-C2H5, R′ = NH2 : benzocaine, C. R = H, R′ = NH2 : aniline, D. R = CH3, R′ = NH2 : 4-methylaniline (p-toluidine). 01/2005:0874

AMINOCAPROIC ACID Acidum aminocaproicum C6H13NO2

Detection : flame ionisation. Injection : 2 µl ; inject the test solution and reference solution (c). Retention time : internal standard = about 9.5 min. Limits : — impurity C : calculate the ratio (R) of the area of the peak due to impurity C to the area of the peak due to the internal standard from the chromatogram obtained with reference solution (c) ; calculate the ratio of the area of the peak due to impurity C to the area of the peak due to the internal standard from the chromatogram obtained with the test solution : this ratio is not greater than R (10 ppm), — impurity D : calculate the ratio (R) of the area of the peak due to impurity D to the area of the peak due to the internal standard from the chromatogram obtained with reference solution (c) ; calculate the ratio of the area of the peak due to impurity D to the area of the peak due to the internal standard from the chromatogram obtained with the test solution : this ratio is not greater than R (10 ppm). 974

Iron (2.4.9) : maximum 40 ppm. Dissolve 0.250 g in 3 ml of alcohol R and dilute to 10.0 ml with water R. Heavy metals (2.4.8) : maximum 20 ppm. 1.0 g complies with limit test C. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Water (2.5.12) : maximum 0.2 per cent, determined on 1.00 g. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g.

Mr 131.2

DEFINITION Aminocaproic acid contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of 6-aminohexanoic acid, calculated with reference to the dried substance. CHARACTERS A white, crystalline powder or colourless crystals, freely soluble in water, slightly soluble in alcohol. It melts at about 205 °C with decomposition. IDENTIFICATION First identification : A. Second identification : B, C, D. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with aminocaproic acid CRS. Examine the substances prepared as discs. B. Examine the chromatograms obtained in the test for ninhydrin-positive substances. The principal spot in the chromatogram obtained with the test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a).


Aminoglutethimide


C. Dissolve 0.5 g in 4 ml of a mixture of equal volumes of dilute hydrochloric acid R and water R. Evaporate to dryness by heating on a water-bath. Dry the residue in a desiccator. Dissolve the residue in about 2 ml of boiling ethanol R. Allow to cool and maintain at 4 °C to 8 °C for 3 h. Filter under reduced pressure. The residue washed with about 10 ml of acetone R and dried at 60 °C for 30 min, melts (2.2.14) at 131 °C to 133 °C. D. Dissolve about 5 mg in 0.5 ml of distilled water R. Add 3 ml of dimethylformamide R and 2 ml of ascorbic acid solution R. Heat on a water-bath. An orange colour develops.

ASSAY Dissolve 0.100 g in 20 ml of anhydrous acetic acid R. Using 0.1 ml of crystal violet solution R as indicator, titrate with 0.1 M perchloric acid until the colour changes from bluish-violet to bluish-green. 1 ml of 0.1 M perchloric acid is equivalent to 13.12 mg of C6H13NO2.

TESTS Solution S. dissolve 10.0 g in carbon dioxide-free water R and dilute to 50.0 ml with the same solvent. Appearance of solution. Solution S is colourless (2.2.2, Method II) and remains clear (2.2.1) on standing for 24 h. pH (2.2.3). The pH of solution S is 7.5 to 8.0. Absorbance (2.2.25). A. The absorbance of solution S at 287 nm is not more than 0.10 and at 450 nm is not more than 0.03. B. Place 2.0 g in an even layer in a shallow dish 9 cm in diameter, cover and allow to stand at 98 °C to 102 °C for 72 h. Dissolve in water R and dilute to 10.0 ml with the same solvent. The absorbance of the solution at 287 nm is not more than 0.15 and at 450 nm is not more than 0.03. Ninhydrin-positive substances. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating substance. Test solution (a). Dissolve 0.10 g of the substance to be examined in water R and dilute to 10 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 50 ml with water R. Reference solution (a). Dissolve 10 mg of aminocaproic acid CRS in water R and dilute to 50 ml with the same solvent. Reference solution (b). Dilute 5 ml of test solution (b) to 20 ml with water R. Reference solution (c). Dissolve 10 mg of aminocaproic acid CRS and 10 mg of leucine CRS in water R and dilute to 25 ml with the same solvent. Apply separately to the plate 5 µl of each solution. Allow the plate to dry in air. Develop over a path of 15 cm using a mixture of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes of butanol R. Dry the plate in a current of warm air. Spray with ninhydrin solution R and heat at 100 °C to 105 °C for 15 min. Any spot in the chromatogram obtained with the test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows two clearly separated principal spots. Heavy metals (2.4.8). 12 ml of solution S complies with limit test A for heavy metals (10 ppm). Prepare the standard using lead standard solution (2 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

Aminoglutethimidum

IDENTIFICATION First identification : B. Second identification : A, C. A. Melting point (2.2.14). 150 °C to 154 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with aminoglutethimide CRS. Examine the substances prepared as discs. C. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm. Test solution. Dissolve 25 mg of the substance to be examined in acetone R and dilute to 5 ml with the same solvent. Reference solution (a). Dissolve 25 mg of aminoglutethimide CRS in acetone R and dilute to 5 ml with the same solvent. Reference solution (b). Dissolve 25 mg of aminoglutethimide CRS and 25 mg of glutethimide CRS in acetone R and dilute to 5 ml with the same solvent. Apply to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 0.5 volumes of glacial acetic acid R, 15 volumes of methanol R and 85 volumes of ethyl acetate R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The identification is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots.


975

01/2005:1291

AMINOGLUTETHIMIDE

C13H16N2O2

Mr 232.3

DEFINITION Aminoglutethimide contains not less than 98.0 per cent and not more than the equivalent of 101.5 per cent of (3RS)-3-(4-aminophenyl)-3-ethylpiperidine-2,6-dione, calculated with reference to the dried substance. CHARACTERS A white or slightly yellow, crystalline powder, practically insoluble in water, freely soluble in acetone, soluble in methanol.

Aminoglutethimide


TESTS Solution S. Dissolve 1.0 g in methanol R and dilute to 20.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y7 (2.2.2, Method II). Optical rotation (2.2.7). The angle of optical rotation, determined on solution S, is − 0.10° to + 0.10°. 3-Aminoglutethimide and other related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 0.10 g of the substance to be examined in a mixture of equal volumes of methanol R and acetate buffer solution pH 5.0 R and dilute to 50.0 ml with the same mixture of solvents. Reference solution (a). Dissolve 5.0 mg of aminoglutethimide impurity A CRS in a mixture of equal volumes of methanol R and acetate buffer solution pH 5.0 R and dilute to 25.0 ml with the same mixture of solvents.

chromatogram obtained with reference solution (c) (1 per cent) ; the sum of the contents of all the impurities is not greater than 2.0 per cent. Disregard any peak with an area less than 0.05 times that of the principal peak in the chromatogram obtained with reference solution (c). Azoglutethimide. Not more than 300 ppm, determined by liquid chromatography (2.2.29). Carry out the test protected from light. Use shaking, not sonication or heat, to dissolve the reference substance and the substance to be examined. Test solution. Dissolve 0.100 g of the substance to be examined in dimethyl sulphoxide R and dilute to 100.0 ml with the same solvent. Reference solution. Dissolve 3.0 mg of aminoglutethimide impurity D CRS in dimethyl sulphoxide R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this solution to 100.0 ml with dimethyl sulphoxide R. The chromatographic procedure may be carried out using : — a stainless steel column 0.12 m long and 4 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm),

Reference solution (b). Dilute 1.0 ml of reference solution (a) to 10.0 ml with a mixture of equal volumes of methanol R — as mobile phase at a flow of 1.0 ml/min a mixture and acetate buffer solution pH 5.0 R. prepared as follows : dissolve 0.285 g of sodium edetate R in water R, add 7.5 ml of dilute acetic acid R and 50 ml Reference solution (c). Dilute 1.0 ml of the test solution to of 0.1 M potassium hydroxide and dilute to 1000 ml with 100.0 ml with a mixture of equal volumes of methanol R and water R ; adjust to pH 5.0 with glacial acetic acid R ; mix acetate buffer solution pH 5.0 R. 350 ml of this solution with 650 ml of methanol R, Reference solution (d). Dilute 1.0 ml of the test solution to 10.0 ml with reference solution (a).

— as detector a spectrophotometer set at 328 nm.

Inject 10 µl of each solution. The test is not valid unless : in the chromatogram obtained with the test solution the number of theoretical plates calculated for the principal peak — a stainless steel column 0.15 m long and 3.9 mm in is at least 3300 ; the mass distribution ratio of the principal internal diameter packed with octadecylsilyl silica gel for peak is 2.0 to 5.0 and the symmetry factor of the principal chromatography R (4 µm), peak is less than 1.2. The area of the peak due to impurity D in the chromatogram obtained with the test solution is not — as mobile phase at a flow rate of 1.3 ml/min a mixture greater than that of the principal peak in the chromatogram of 27 volumes of methanol R and 73 volumes of acetate obtained with the reference solution. buffer solution pH 5.0 R, Sulphates (2.4.13). Dilute 6 ml of solution S to 15 ml with distilled water R. The solution complies with the limit test — as detector a spectrophotometer set at 240 nm, for sulphates (500 ppm). maintaining the temperature of the column at 40 °C. Heavy metals (2.4.8). Dissolve 2.0 g in 15 ml of acetone R and dilute to 20 ml with water R. 12 ml of the solution Inject 10 µl of reference solution (d). When the complies with limit test B for heavy metals (10 ppm). Prepare chromatograms are recorded in the prescribed conditions, the standard using lead standard solution (1 ppm Pb) the retention times are : aminoglutethimide about 9 min obtained by diluting lead standard solution (100 ppm Pb) R and impurity A about 12 min. Adjust the sensitivity of with a mixture of 15 ml of acetone R and 5 ml of water R. the system so that the height of the principal peak in the The chromatographic procedure may be carried out using :

chromatogram obtained with reference solution (d) is at least 60 per cent of the full scale of the recorder. The test is not valid unless in the chromatogram obtained with reference solution (d), the resolution between the peaks corresponding to aminoglutethimide and impurity A is at least 2.0. Inject 10 µl of the test solution, 10 µl of reference solution (b) and 10 µl of reference solution (c). Continue the chromatography of the test solution for four times the retention time of the principal peak. In the chromatogram obtained with the test solution : the area of any peak due to impurity A is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (2 per cent) ; the sum of the areas of any peaks apart from the principal peak and the peak due to impurity A is not greater than the area of the principal peak in the 976

Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C.

Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

ASSAY Dissolve 0.180 g in 50 ml of anhydrous acetic acid R and titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 23.23 mg of C13H16N2O2.



Amiodarone hydrochloride

Impurity H. Thin-layer chromatography (2.2.27). Prepare the solutions immediately before use and keep protected from bright light. Test solution. Dissolve 0.5 g of the substance to be examined in methylene chloride R and dilute to 5.0 ml with the same solvent. Reference solution. Dissolve 10 mg of (2-chloroethyl)diethylamine hydrochloride R in methylene chloride R and A. R3 = NH2, R4 = H : (3RS)-3-(3-aminophenyl)-3dilute to 50.0 ml with the same solvent. ethylpiperidine-2,6-dione (3-aminoglutethimide), Plate : TLC silica gel F254 plate R. B. R3 = NO2, R4 = H : (3RS)-3-ethyl-3-(3-nitrophenyl)pipeMobile phase : anhydrous formic acid R, methanol R, ridine-2,6-dione, methylene chloride R (5:10:85 V/V/V). Application : 5 µl. C. R3 = H, R4 = NO2 : (3RS)-3-ethyl-3-(4-nitrophenyl)piperidine-2,6-dione, Development : over a path of 15 cm. Drying : in a current of cold air until the odour of solvents is no longer perceptible. Detection : spray with potassium iodobismuthate solution R1 and then with dilute hydrogen peroxide solution R. Examine immediately in daylight. Limit : — impurity H : any spot corresponding to impurity H is not more intense than the spot in the chromatogram obtained D. 3,3′-[diazenediylbis(4,1-phenylene)]bis(3-ethylpiperidinewith the reference solution (0.2 per cent). 2,6-dione) (azoglutethimide). Related substances. Liquid chromatography (2.2.29). 01/2005:0803 Buffer solution pH 4.9. To 800 ml of water R, add 3.0 ml of glacial acetic acid R, adjust to pH 4.9 with dilute ammonia R1 and dilute to 1000 ml with water R. AMIODARONE HYDROCHLORIDE Test solution. Dissolve 0.125 g of the substance to be examined in a mixture of equal volumes of acetonitrile R Amiodaroni hydrochloridum and water R and dilute to 25.0 ml with the same mixture of solvents. Reference solution. Dissolve 10 mg of amiodarone impurity D CRS, 10 mg of amiodarone impurity E CRS and 10.0 mg of amiodarone hydrochloride CRS in methanol R and dilute to 50.0 ml with the same solvent. Dilute 1.0 ml of the solution to 20.0 ml with a mixture of equal volumes of acetonitrile R and water R. Column : C25H30ClI2NO3 Mr 682 — size : l = 0.15 m, Ø = 4.6 mm, DEFINITION — stationary phase : octadecylsilyl silica gel for chromatography R (5 µm), (2-Butylbenzofuran-3-yl)[4-[2-(diethylamino)ethoxy]-3,5diiodophenyl]methanone hydrochloride. — temperature : 30 °C. Content : 98.5 per cent to 101.0 per cent (dried substance). Mobile phase : buffer solution pH 4.9, methanol R, acetonitrile R (30:30:40 V/V/V). CHARACTERS Flow rate : 1 ml/min. Appearance : white or almost white, fine crystalline powder. Detection : spectrophotometer at 240 nm. Solubility : very slightly soluble in water, freely soluble in Injection : 10 µl. methylene chloride, soluble in methanol, sparingly soluble Run time : twice the retention time of amiodarone. in alcohol. Relative retention with reference to amiodarone IDENTIFICATION (retention time = about 24 min) : impurity A = about 0.26 ; A. Infrared absorption spectrophotometry (2.2.24). impurity D = about 0.29 ; impurity E = about 0.37 ; impurity B = about 0.49 ; impurity C = about 0.55 ; Comparison : amiodarone hydrochloride CRS. impurity G = about 0.62 ; impurity F = about 0.69. B. It gives reaction (b) of chlorides (2.3.1). System suitability : reference solution : TESTS — resolution : minimum 3.5 between the peaks due to Appearance of solution. The solution is clear (2.2.1) and not impurity D and impurity E. more intensely coloured than reference solution GY5 or BY5 Limits : (2.2.2, Method II). — any impurity : not more than the area of the peak due Dissolve 1.0 g in methanol R and dilute to 20 ml with the to amiodarone in the chromatogram obtained with the same solvent. reference solution (0.2 per cent), pH (2.2.3) : 3.2 to 3.8. — total : not more than 2.5 times the area of the peak due Dissolve 1.0 g in carbon dioxide-free water R, heating at to amiodarone in the chromatogram obtained with the 80 °C, cool and dilute to 20 ml with the same solvent. reference solution (0.5 per cent), IMPURITIES


977

Amisulpride


— disregard limit : 0.1 times the area of the peak due to amiodarone in the chromatogram obtained with the reference solution (0.02 per cent). Iodides : maximum 150 ppm. Prepare the test and reference solutions simultaneously. Solution A. Add 1.50 g of the substance to be examined to 40 ml of water R at 80 °C and shake until completely dissolved. Cool and dilute to 50.0 ml with water R. Test solution. To 15.0 ml of solution A add 1.0 ml of 0.1 M hydrochloric acid and 1.0 ml of 0.05 M potassium iodate. Dilute to 20.0 ml with water R. Allow to stand protected from light for 4 h. Reference solution. To 15.0 ml of solution A add 1.0 ml of 0.1 M hydrochloric acid, 1.0 ml of a 88.2 mg/l solution of potassium iodide R and 1.0 ml of 0.05 M potassium iodate. Dilute to 20.0 ml with water R. Allow to stand protected from light for 4 h. Measure the absorbances (2.2.25) of the solutions at 420 nm, using as the compensation liquid a mixture of 15.0 ml of solution A and 1.0 ml of 0.1 M hydrochloric acid diluted to 20.0 ml with water R. The absorbance of the test solution is not greater than half the absorbance of the reference solution. Heavy metals (2.4.8) : maximum 20 ppm. 1.0 g complies with limit test C. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying at 50 °C at a pressure not exceeding 0.3 kPa for 4 h. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.600 g in a mixture of 5.0 ml of 0.01 M hydrochloric acid and 75 ml of alcohol R. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the 2 points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 68.18 mg of C25H30ClI2NO3. STORAGE Protected from light, at a temperature not exceeding 30 °C. IMPURITIES

D. R1 = R2 = I : (2-butylbenzofuran-3-yl)(4-hydroxy-3,5diiodophenyl)methanone, E. R1 = R2 = H : (2-butylbenzofuran-3-yl)(4hydroxyphenyl)methanone, F. R1 = I, R2 = H : (2-butylbenzofuran-3-yl)(4-hydroxy-3iodophenyl)methanone,

H. 2-chloro-N,N-diethylethanamine (2-chlorotriethylamine,(2chloroethyl)diethylamine). 01/2005:1490

AMISULPRIDE Amisulpridum

C17H27N3O4S

Mr 369.5

DEFINITION 4-Amino-N-[[(2RS)-1-ethylpyrrolidin-2-yl]methyl]-5(ethylsulphonyl)-2-methoxybenzamide. Content : 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white or almost white, crystalline powder. Solubility : practically insoluble in water, freely soluble in methylene chloride, sparingly soluble in ethanol. mp : about 126 °C. IDENTIFICATION Infrared absorption spectrophotometry (2.2.24). Comparison : amisulpride CRS.

A. R1 = R2 = R4 = H, R3 = C2H5 : (2-butylbenzofuran-3-yl)[4[2-(diethylamino)ethoxy]phenyl]methanone, B. R1 = R2 = I, R3 = R4 = H : (2-butylbenzofuran-3-yl)[4-[2(ethylamino)ethoxy]-3,5-diiodophenyl]methanone, C. R1 = I, R2 = R4 = H, R3 = C2H5 : (2-butylbenzofuran-3yl)[4-[2-(diethylamino)ethoxy]-3-iodophenyl]methanone, G. R1 = R2 = I, R3 = C2H5, R4 = OCH3 : [2-[(1RS)-1-methoxybutyl]benzofuran-3-yl][4-[2-(diethylamino)ethoxy]-3,5-diiodophenyl]methanone, 978

TESTS Appearance of solution. The solution is not more opalescent than reference suspension II (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). Dissolve 1.0 g in 3 ml of a mixture of 1 volume of acetic acid R and 4 volumes of water R and dilute to 20 ml with water R. Optical rotation (2.2.7) : − 0.10° to + 0.10°. Dissolve 5.0 g in dimethylformamide R and dilute to 50.0 ml with the same solvent. Impurity A. Thin-layer chromatography (2.2.27). Test solution. Dissolve 0.20 g in methanol R and dilute to 10 ml with the same solvent.


Amisulpride


Reference solution (a). Dissolve 20 mg of amisulpride impurity A CRS in methanol R and dilute to 100 ml with the same solvent. Dilute 2 ml of the solution to 20 ml with methanol R. Reference solution (b). Dilute 1 ml of the test solution to 10 ml with reference solution (a). Plate : TLC silica gel G plate R. Mobile phase : the upper layer obtained after shaking a mixture of a 50 per cent V/V solution of concentrated ammonia R, ethanol R and di-isopropyl ether R (10:25:65 V/V/V). Application : 10 µl. Development : over a path of 12 cm. Drying : in air. Detection : spray with ninhydrin solution R and heat at 100-105 °C for 15 min. System suitability : the chromatogram obtained with reference solution (b) shows 2 clearly separated spots. Limit : — impurity A : any spot corresponding to impurity A is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.1 per cent). Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 0.10 g in 30 ml of methanol R and dilute to 100.0 ml with mobile phase B. Reference solution (a). Dilute 5.0 ml of the test solution to 100.0 ml with a mixture of 30 volumes of mobile phase A and 70 volumes of mobile phase B. Dilute 1.0 ml of the solution to 25.0 ml with a mixture of 30 volumes of mobile phase A and 70 volumes of mobile phase B. Reference solution (b). Dissolve 5 mg of amisulpride impurity B CRS in 5 ml of the test solution and dilute to 50 ml with a mixture of 30 volumes of mobile phase A and 70 volumes of mobile phase B. Dilute 1 ml of the solution to 10 ml with a mixture of 30 volumes of mobile phase A and 70 volumes of mobile phase B. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase: octylsilyl silica gel for chromatography R (5 µm) with a carbon loading of 16 per cent, a specific surface area of 330 m2/g and a pore size of 7.5 nm. Mobile phase : — mobile phase A : methanol R, — mobile phase B : a 0.7 g/l solution of sodium octanesulphonate R in a 0.25 per cent V/V solution of dilute sulphuric acid R, Time (min)



0 - 18

30 → 36

70 → 64

18 - 35

36 → 52

64 → 48

35 - 45

52

48

45 - 46

52 → 30

48 → 70

46 - 56

30

70

Flow rate : 1.5 ml/min. Detection : spectrophotometer at 225 nm. Injection : 10 µl. System suitability : reference solution (b) : — resolution : minimum 2.0 between the peaks due to amisulpride and impurity B. General Notices (1) apply to all monographs and other texts

Limits : — any impurity : not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), — total : not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.3 per cent), — disregard limit : 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.02 per cent). Chlorides (2.4.4) : maximum 200 ppm. Shake 0.5 g with 30 ml of water R for 10 min. Filter. 15 ml of the filtrate complies with the limit test for chlorides. Heavy metals (2.4.8) : maximum 10 ppm. Dissolve 4.0 g by gently heating in 5 ml of dilute acetic acid R. Allow to cool and dilute to 20 ml with water R. 12 ml of the solution complies with limit test A. Prepare the standard using lead standard solution (2 ppm Pb) R. Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 3 h. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.300 g with shaking in a mixture of 5 ml of acetic anhydride R and 50 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 36.95 mg of C17H27N3O4S. IMPURITIES

A. [(2RS)-1-ethylpyrrolidin-2-yl]methanamine,

B. R1 = OH, R2 = SO2-CH2-CH3 : 4-amino-N-[[(2RS)1-ethylpyrrolidin-2-yl]methyl]-5-(ethylsulphonyl)-2hydroxybenzamide, C. R1 = OCH3, R2 = I : 4-amino-N-[[(2RS)-1-ethylpyrrolidin-2yl]methyl]-5-iodo-2-methoxybenzamide, D. R1 = OCH3, R2 = SO2-CH3 : 4-amino-N-[[(2RS)1-ethylpyrrolidin-2-yl]methyl]-2-methoxy-5(methylsulphonyl)benzamide,

E. 4-amino-5-(ethylsulphonyl)-2-methoxybenzoic acid. 979

Amitriptyline hydrochloride


01/2005:0464 Related substances. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R. Prepare the solutions in subdued light and develop the chromatograms AMITRIPTYLINE HYDROCHLORIDE protected from light. Test solution. Dissolve 0.20 g of the substance to be examined in alcohol R and dilute to 10 ml with the same Amitriptylini hydrochloridum solvent. Reference solution (a). Dissolve 10 mg of dibenzosuberone CRS in alcohol R and dilute to 10 ml with the same solvent. Dilute 1 ml of the solution to 100 ml with alcohol R. Reference solution (b). Dissolve 10 mg of cyclobenzaprine hydrochloride CRS in alcohol R and dilute to 10 ml with the same solvent. Dilute 2 ml of the solution to 50 ml with alcohol R. C20H24ClN Mr 313.9 Apply to the plate 5 µl of each solution. Develop in an unsaturated tank over a path of 14 cm using a mixture DEFINITION of 3 volumes of diethylamine R, 15 volumes of ethyl Amitriptyline hydrochloride contains not less than 99.0 per acetate R and 85 volumes of cyclohexane R. Heat the plate at 100-105 °C for 10 min and spray with a freshly cent and not more than the equivalent of 101.0 per cent of prepared mixture of 4 volumes of formaldehyde solution R 3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ylidene)-N, and 96 volumes of sulphuric acid R. Heat at 100-105 °C N-dimethylpropan-1-amine hydrochloride, calculated with for 10 min and examine immediately in ultraviolet light at reference to the dried substance. 365 nm and at 254 nm. In the chromatogram obtained with the test solution : any spots corresponding to CHARACTERS dibenzosuberone and cyclobenzaprine hydrochloride are A white or almost white powder or colourless crystals, freely not more intense than the spots in the chromatograms soluble in water, in alcohol and in methylene chloride. obtained with reference solutions (a) (0.05 per cent) and (b) (0.2 per cent), respectively ; any spot, apart from the principal IDENTIFICATION spot and any spots corresponding to dibenzosuberone and cyclobenzaprine hydrochloride, is not more intense than First identification : C, E. the spot in the chromatogram obtained with reference Second identification : A, B, D, E. solution (b) (0.2 per cent). A. Melting point (2.2.14) : 195 °C to 199 °C. Heavy metals (2.4.8). 1.0 g complies with limit test F (20 ppm). Prepare the standard using 2 ml of lead standard B. Dissolve 25.0 mg in methanol R and dilute to 100.0 ml solution (10 ppm Pb) R. with the same solvent. Dilute 5.0 ml of the solution to 100.0 ml with methanol R. Examined between 230 nm Loss on drying (2.2.32). Not more than 0.5 per cent, and 350 nm (2.2.25), the solution shows an absorption determined on 1.000 g by drying in an oven at 100-105 °C maximum at 239 nm. The specific absorbance at the for 2 h. maximum is 435 to 475. Sulphated ash (2.4.14). Not more than 0.1 per cent, C. Examine by infrared absorption spectrophotometry determined on 1.0 g. (2.2.24), comparing with the Ph. Eur. reference spectrum of amitriptyline hydrochloride. ASSAY D. Dissolve 0.1 g in 10 ml of dilute sulphuric acid R. Add 2 ml Dissolve 0.250 g in 30 ml of alcohol R. Titrate with of a saturated solution of potassium permanganate R. 0.1 M sodium hydroxide, determining the end-point The violet colour of the solution disappears quickly. Heat potentiometrically (2.2.20). on a water-bath until the brown precipitate is almost completely dissolved. Cool, shake with 15 ml of ether R to 1 ml of 0.1 M sodium hydroxide is equivalent to 31.39 mg remove the white turbidity and discard the ether layer. To of C20H24ClN. the aqueous layer add 5 ml of concentrated ammonia R STORAGE and shake for 2 min. Add 3 ml of methylene chloride R and shake again. A violet-red colour is produced in the Store protected from light. lower layer. IMPURITIES E. 50 mg gives reaction (b) of chlorides (2.3.1). TESTS Appearance of solution. Dissolve 1.25 g in water R and dilute to 25 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution B7 (2.2.2, Method II). Acidity or alkalinity. Dissolve 0.20 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. Add 0.1 ml of methyl red solution R and 0.2 ml of 0.01 M sodium hydroxide. The solution is yellow. Add 0.4 ml of 0.01 M A. 10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-one hydrochloric acid. The solution is red. (dibenzosuberone), 980


Amlodipine besilate


01/2005:1491

AMLODIPINE BESILATE Amlodipini besilas B. 3-(5H-dibenzo[a,d][7]annulen-5-ylidene)-N,Ndimethylpropan-1-amine (cyclobenzaprine),

C26H31ClN2O8S

C. 3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ylidene)-Nmethylpropan-1-amine,

Mr 567.1

DEFINITION 3-Ethyl 5-methyl (4RS)-2-[(2-aminoethoxy)methyl]-4-(2chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate benzenesulphonate. Content : 97.0 per cent to 102.0 per cent (anhydrous substance). CHARACTERS Appearance : white or almost white powder. Solubility : slightly soluble in water, freely soluble in methanol, sparingly soluble in ethanol, slightly soluble in 2-propanol.

D. 5-[3-(dimethylamino)propyl]-10,11-dihydro-5H-dibenzo[a, d][7]annulen-5-ol,

E. 3-(1,2,3,4,4a,10,11,11a-octahydro-5H-dibenzo[a, d][7]annulen-5-ylidene)-N,N-dimethylpropan-1-amine,

F. (10RS)-5-[3-(dimethylamino)propylidene]-10,11-dihydro5H-dibenzo[a,d][7]annulen-10-ol. General Notices (1) apply to all monographs and other texts

IDENTIFICATION First identification : A. Second identification : B, C. A. Infrared absorption spectrophotometry (2.2.24). Comparison : amlodipine besilate CRS. Preparation : mulls. B. Examine the chromatograms obtained in test A for related substances in ultraviolet light at 366 nm. Results : the principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (b). C. Dissolve 5.0 mg in a 1 per cent V/V solution of 0.1 M hydrochloric acid in methanol R and dilute to 100.0 ml with the same acid solution. Examined between 300 nm and 400 nm (2.2.25), the solution shows an absorption maximum at 360 nm. The specific absorbance at the maximum is 113 to 121. TESTS Optical rotation (2.2.7) : − 0.10° to + 0.10°. Dissolve 0.250 g in methanol R and dilute to 25.0 ml with the same solvent. Related substances A. Thin-layer chromatography (2.2.27). Test solution (a). Dissolve 0.140 g of the substance to be examined in methanol R and dilute to 2.0 ml with the same solvent. Test solution (b). Dilute 1.0 ml of test solution (a) to 10.0 ml with methanol R. Reference solution (a). Dissolve 70.0 mg of amlodipine besilate CRS in 1.0 ml of methanol R. Reference solution (b). Dilute 1 ml of reference solution (a) to 10 ml with methanol R. 981

Amlodipine besilate


Reference solution (c). Dilute 3.0 ml of test solution (b) to 100.0 ml with methanol R. Reference solution (d). Dilute 1.0 ml of test solution (b) to 100.0 ml with methanol R. Plate : TLC silica gel F254 plate R. Mobile phase : the upper layer of a mixture of glacial acetic acid R, water R and methyl isobutyl ketone R (25:25:50 V/V/V). Application : 10 µl. Development : over a path of 15 cm. Drying : for 15 min at 80 °C. Detection : examine in ultraviolet light at 254 nm and 366 nm. System suitability : the chromatogram obtained with reference solution (a) shows 2 clearly separated minor spots with Rf values of about 0.18 and 0.22. Limits : in the chromatogram obtained with test solution (a) : — any impurity : any spot, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (c) (0.3 per cent) and at most 2 spots are more intense than the spot in the chromatogram obtained with reference solution (d) (0.1 per cent). B. Liquid chromatography (2.2.29). Test solution (a). Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Test solution (b). Dilute 5.0 ml of test solution (a) to 100.0 ml with the mobile phase. Reference solution (a). Dissolve 50.0 mg of amlodipine besilate CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 5.0 ml of the solution to 100.0 ml with the mobile phase. Reference solution (b). Dilute 3.0 ml of test solution (a) to 100.0 ml with the mobile phase and dilute 5.0 ml of the solution to 50.0 ml with the mobile phase. Reference solution (c). Dissolve 5 mg of the substance to be examined in 5 ml of strong hydrogen peroxide solution R. Heat at 70 °C for 45 min. Column : — size : l = 0.15 m, Ø = 3.9 mm, — stationary phase: octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : mix 15 volumes of acetonitrile R, 35 volumes of methanol R and 50 volumes of a solution prepared as follows : dissolve 7.0 ml of triethylamine R in 1 litre of water R and adjust to pH 3.0 ± 0.1 with phosphoric acid R. Flow rate : 1.0 ml/min. Detection : spectrophotometer at 237 nm. Injection : 10 µl ; inject test solution (a) and reference solutions (b) and (c). Run time : 3 times the retention time of amlodipine. Relative retention with reference to amlodipine (retention time = about 7 min) : impurity D = about 0.5. System suitability : reference solution (c) : — resolution : minimum 4.5 between the peaks corresponding to amlodipine and impurity D. Limits : — correction factor : for the calculation of content, multiply the peak area of impurity D by 2, 982

— impurity D : not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.3 per cent), — total of other impurities : not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.3 per cent) ; disregard any peak due to benzene sulphonate (relative retention = about 0.2), — disregard limit : 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.03 per cent). Water (2.5.12) : maximum 0.5 per cent, determined on 3.000 g. Sulphated ash (2.4.14) : maximum 0.2 per cent, determined on 1.0 g. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modification. Injection : test solution (b), reference solution (a). Calculate the percentage content of amlodipine besilate from the areas of the peaks and the declared content of C26H31ClN208S in amlodipine besilate CRS. STORAGE In an airtight container, protected from light. IMPURITIES

A. 3-ethyl 5-methyl (4RS)-4-(2-chlorophenyl)-2-[[2-(1,3-dioxo1,3-dihydro-2H-isoindol-2-yl)ethoxy]methyl]-6-methyl-1,4dihydropyridine-3,5-dicarboxylate,

B. 3-ethyl 5-methyl (4RS)-4-(2-chlorophenyl)-6-methyl-2-[[2[[2-(methylcarbamoyl)benzoyl]amino]ethoxy]methyl]-1,4dihydropyridine-3,5-dicarboxylate,

C. ethyl methyl (4RS)-2,6-bis[(2-aminoethoxy)methyl]-4-(2chlorophenyl)-1,4-dihydropyridine-3,5-dicarboxylate,



D. 3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4-(2chlorophenyl)-6-methylpyridine-3,5-dicarboxylate.

Ammonio methacrylate copolymer (type A)

Iron (2.4.9). Dilute 4 ml of solution S to 10 ml with water R. The solution complies with the limit test for iron (0.25 ppm). Heavy metals (2.4.8). Dilute 4 ml of solution S to 20 ml with water R. 12 ml of the solution complies with limit test A for heavy metals (1 ppm). Prepare the standard using lead standard solution (2 ppm Pb) R. Residue on evaporation. Evaporate 50 ml to dryness on a water-bath and dry at 100 °C to 105 °C for 1 h. The residue weighs not more than 1 mg (0.02 g/l).

ASSAY Weigh accurately a flask with a ground-glass neck containing 01/2005:0877 50.0 ml of 1 M hydrochloric acid. Add 2 ml of the substance to be examined and re-weigh. Add 0.1 ml of methyl red solution R as indicator. Titrate with 1 M sodium hydroxide AMMONIA SOLUTION, until the colour changes from red to yellow. CONCENTRATED 1 ml of 1 M hydrochloric acid is equivalent to 17.03 mg of NH3.

Ammoniae solutio concentrata

DEFINITION Concentrated ammonia solution contains not less than 25.0 per cent m/m and not more than 30.0 per cent m/m of ammonia (NH3 ; Mr 17.03).

STORAGE Store protected from air, at a temperature not exceeding 20 °C. 01/2005:2081

CHARACTERS A clear, colourless liquid, very caustic, miscible with water and with alcohol.

AMMONIO METHACRYLATE COPOLYMER (TYPE A)

IDENTIFICATION A. Relative density (2.2.5) : 0.892 to 0.910. B. It is strongly alkaline (2.2.4). C. To 0.5 ml add 5 ml of water R. Bubble air through the solution and lead the gaseous mixture obtained over the surface of a solution containing 1 ml of 0.1 M hydrochloric acid and 0.05 ml of methyl red solution R. The colour changes from red to yellow. Add 1 ml of sodium cobaltinitrite solution R. A yellow precipitate is formed.

Ammonio methacrylatis copolymerum A

TESTS Solution S. Evaporate 220 ml almost to dryness on a water-bath. Cool, add 1 ml of dilute acetic acid R and dilute to 20 ml with distilled water R. Appearance of solution. To 2 ml add 8 ml of water R. The solution is clear (2.2.1) and colourless (2.2.2, Method II). Oxidisable substances. Cautiously add, whilst cooling, 8.8 ml to 100 ml of dilute sulphuric acid R. Add 0.75 ml of 0.002 M potassium permanganate. Allow to stand for 5 min. The solution remains faintly pink. Pyridine and related substances. Measure the absorbance (2.2.25) at 252 nm using water R as the compensation liquid. The absorbance is not greater than 0.06 (2 ppm calculated as pyridine). Carbonates. To 10 ml in a test-tube with a ground-glass neck add 10 ml of calcium hydroxide solution R. Stopper immediately and mix. Any opalescence in the solution is not more intense than that in a standard prepared at the same time and in the same manner using 10 ml of a 0.1 g/l solution of anhydrous sodium carbonate R (60 ppm). Chlorides (2.4.4). Dilute 5 ml of solution S to 15 ml with water R. The solution complies with the limit test for chlorides (1 ppm). Sulphates (2.4.13). Dilute 3 ml of solution S to 15 ml with distilled water R. The solution complies with the limit test for sulphates (5 ppm). General Notices (1) apply to all monographs and other texts

DEFINITION Poly(ethyl propenoate-co-methyl 2-methylpropenoate-co2-(trimethylammonio)ethyl 2-methylpropenoate) chloride having a mean relative molecular mass of about 150 000. The ratio of ethyl propenoate groups to methyl 2-methylpropenoate groups to 2-(trimethylammonio)ethyl 2-methylpropenoate groups is about 1:2:0.2. Content of ammonio methacrylate groups : 8.9 per cent to 12.3 per cent (dried substance). CHARACTERS Appearance : colourless to white or almost white granules or powder. Solubility : practically insoluble in water, freely soluble in ethanol and in methylene chloride giving clear to cloudy solutions. Due to the polymeric nature of the substance, a stirring time of up to 5 h may be necessary. IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Preparation : drop solution S (see Tests) on a disc of potassium bromide R and dry in vacuo at 70 °C for 2 h. Comparison : Ph. Eur. reference spectrum of ammonio methacrylate copolymer (type A). B. It complies with the test for viscosity (see Tests). 983

Ammonio methacrylate copolymer (type B)


ASSAY Dissolve 1.000 g in a mixture of 3 ml of anhydrous formic TESTS acid R and 30 ml of anhydrous acetic acid R and heat to dissolve. Add 20 ml of acetic anhydride R. Titrate Solution S. Dissolve a quantity of the substance to be examined corresponding to 12.5 g of the dried substance in with 0.1 M perchloric acid, determining the end-point a mixture of 35.0 g of acetone R and 52.5 g of 2-propanol R. potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 20.77 mg Viscosity (2.2.10) : maximum 15 mPa·s, determined on of C9H18O2NCl (ammonio methacrylate groups). solution S. C. It complies with the limits of the assay.

Apparatus : rotating viscosimeter. Dimensions : — spindle : diameter = 25.15 mm ; height = 90.74 mm ; shaft diameter = 4.0 mm ; — cylinder : diameter = 27.62 mm ; height = 0.135 m. Stirring speed : 30 r/min. Volume of solution : 16 ml of solution S. Temperature : 20 °C. Appearance of a film. Spread 2 ml of solution S evenly on a glass plate. Upon drying a clear film is formed. Monomers. Liquid chromatography (2.2.29). Solution A. Dissolve 3.5 g of sodium perchlorate R in water for chromatography R and dilute to 100 ml with the same solvent. Test solution. Dissolve 5.00 g of the substance to be examined in methanol R and dilute to 50.0 ml with the same solvent. To 10.0 ml of this solution add 5.0 ml of solution A, dropwise while continuously stirring. Remove the precipitated polymer by centrifugation. Use the clear supernatant solution. Reference solution. Dissolve 50.0 mg of ethyl acrylate R and 10.0 mg of methyl methacrylate R in methanol R and dilute to 50.0 ml with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with methanol R. Add 10 ml of this solution to 5 ml of solution A. Column : — size : l = 0.12 m, Ø = 4.6 mm, — stationary phase: octadecylsilyl silica gel for chromatography R (7 µm). Mobile phase : dilute phosphoric acid R with water for chromatography R to obtain a solution at pH 2.0 ; mix 800 ml of this solution and 200 ml of methanol R, filter and degas. Flow rate : 2.0 ml/min. Detection : spectrophotometer at 202 nm. Injection : 50 µl. System suitability : reference solution : — resolution : minimum 1.5 between the peaks due to impurity A and impurity B. Limits : — impurity A : not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (100 ppm), — impurity B : not more than 2.5 times the area of the corresponding peak in the chromatogram obtained with the reference solution (50 ppm). Methanol (2.4.24, System A) : maximum 1.5 per cent. Heavy metals (2.4.8) : maximum 20 ppm. 1.0 g complies with limit test C. Prepare the standard using 2.0 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32) : maximum 3.0 per cent, determined on 1.000 g by drying in vacuo at 80 °C for 5 h. 984

IMPURITIES Specified impurities : A, B.

A. R = H, R′ = C2H5 : ethyl propenoate (ethyl acrylate), B. R = R′ = CH3 : methyl 2-methylpropenoate (methyl methacrylate). 01/2005:2082

AMMONIO METHACRYLATE COPOLYMER (TYPE B) Ammonio methacrylatis copolymerum B

DEFINITION Poly(ethyl propenoate-co-methyl 2-methylpropenoate-co2-(trimethylammonio)ethyl 2-methylpropenoate) chloride having a mean relative molecular mass of about 150 000. The ratio of ethyl propenoate groups to methyl 2-methylpropenoate groups to 2-(trimethylammonio)ethyl 2-methylpropenoate groups is about 1:2:0.1. Content of ammonio methacrylate groups : 4.5 per cent to 7.0 per cent (dried substance). CHARACTERS Appearance : colourless to white or almost white granules or powder. Solubility : practically insoluble in water, freely soluble in ethanol and in methylene chloride giving clear to cloudy solutions. Due to the polymeric nature of the substance, a stirring time of up to 5 h may be necessary. IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Preparation : drop solution S (see Tests) on a disc of potassium bromide R and dry in vacuo at 70 °C for 2 h. Comparison : Ph. Eur. reference spectrum of ammonio methacrylate copolymer (type B). B. It complies with the test for viscosity (see Tests). C. It complies with the limits of the assay.


Ammonium bromide


TESTS Solution S. Dissolve a quantity of the substance to be examined corresponding to 12.5 g of the dried substance in a mixture of 35.0 g of acetone R and 52.5 g of 2-propanol R. Viscosity (2.2.10) : maximum 15 mPa·s, determined on solution S. Apparatus : rotating viscosimeter. Dimensions : — spindle : diameter = 25.15 mm ; height = 90.74 mm ; shaft diameter = 4.0 mm ;

ASSAY Dissolve 2.000 g in a mixture of 3 ml of anhydrous formic acid R and 30 ml of anhydrous acetic acid R and heat to dissolve. Add 20 ml of acetic anhydride R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 20.77 mg of C9H18O2NCl (ammonio methacrylate groups). IMPURITIES Specified impurities : A, B.

— cylinder : diameter = 27.62 mm ; height = 0.135 m. Stirring speed : 30 r/min. Volume of solution : 16 ml of solution S. Temperature : 20 °C. A. R = H, R′ = C2H5 : ethyl propenoate (ethyl acrylate), Appearance of a film. Spread 2 ml of solution S evenly on a B. R = R′ = CH3 : methyl 2-methylpropenoate (methyl glass plate. Upon drying a clear film is formed. methacrylate). Monomers. Liquid chromatography (2.2.29). 01/2005:1389 Solution A. Dissolve 3.5 g of sodium perchlorate R in water for chromatography R and dilute to 100 ml with the same AMMONIUM BROMIDE solvent. Test solution. Dissolve 5.00 g of the substance to be examined in methanol R and dilute to 50.0 ml with the same solvent. To 10.0 ml of this solution add 5.0 ml of solution A, dropwise while continuously stirring. Remove the precipitated polymer by centrifugation. Use the clear supernatant solution. Reference solution. Dissolve 50.0 mg of ethyl acrylate R and 10.0 mg of methyl methacrylate R in methanol R and dilute to 50.0 ml with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with methanol R. Add 10 ml of this solution to 5 ml of solution A. Column : — size : l = 0.12 m, Ø = 4.6 mm, — stationary phase: octadecylsilyl silica gel for chromatography R (7 µm).

Ammonii bromidum NH4Br

Mr 97.9

DEFINITION Content : 98.5 per cent to 100.5 per cent (dried substance). CHARACTERS Appearance : white or almost white, crystalline powder or colourless crystals, hygroscopic. Solubility : freely soluble in water, sparingly soluble in alcohol. It becomes yellow when exposed to light or air. IDENTIFICATION A. It gives reaction (a) of bromides (2.3.1). B. 10 ml of solution S (see Tests) gives the reaction of ammonium salts (2.3.1).

Mobile phase : dilute phosphoric acid R with water for chromatography R to obtain a solution at pH 2.0 ; mix 800 ml of this solution and 200 ml of methanol R, filter and degas. TESTS Flow rate : 2.0 ml/min. Solution S. Dissolve 10.0 g in carbon dioxide-free water R prepared from distilled water R and dilute to 100 ml with Detection : spectrophotometer at 202 nm. the same solvent. Injection : 50 µl. Appearance of solution. Solution S is clear (2.2.1) and System suitability : reference solution : colourless (2.2.2, Method II). — resolution : minimum 1.5 between the peaks due to Acidity or alkalinity. To 10 ml of solution S add 0.05 ml impurity A and impurity B. of methyl red solution R. Not more than 0.5 ml of 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is required Limits : to change the colour of the indicator. — impurity A : not more than the area of the corresponding Bromates. To 10 ml of solution S add 1 ml of starch peak in the chromatogram obtained with the reference solution R, 0.1 ml of a 100 g/l solution of potassium solution (100 ppm), iodide R and 0.25 ml of 0.5 M sulphuric acid and allow to — impurity B : not more than 2.5 times the area of the stand protected from light for 5 min. No blue or violet colour corresponding peak in the chromatogram obtained with develops. the reference solution (50 ppm). Chlorides : maximum 0.6 per cent. Methanol (2.4.24, System A) : maximum 1.5 per cent. In a conical flask, dissolve 1.000 g in 20 ml of dilute nitric acid R. Add 5 ml of strong hydrogen peroxide solution R Heavy metals (2.4.8) : maximum 20 ppm. and heat on a water-bath until the solution is completely 1.0 g complies with limit test C. Prepare the standard using decolorised. Wash down the sides of the flask with a little 2.0 ml of lead standard solution (10 ppm Pb) R. water R and heat on a water-bath for 15 min. Allow to cool, Loss on drying (2.2.32) : maximum 3.0 per cent, determined dilute to 50 ml with water R and add 5.0 ml of 0.1 M silver on 1.000 g by drying in vacuo at 80 °C for 5 h. nitrate and 1 ml of dibutyl phthalate R. Shake and titrate General Notices (1) apply to all monographs and other texts

985

Ammonium chloride


with 0.1 M ammonium thiocyanate using 5 ml of ferric ammonium sulphate solution R2 as indicator. Not more than 1.7 ml of 0.1 M silver nitrate is used. Note the volume of 0.1 M silver nitrate used (see Assay). Carry out a blank test. Iodides. To 5 ml of solution S add 0.15 ml of ferric chloride solution R1 and 2 ml of methylene chloride R. Shake and allow to separate. The lower layer is colourless (2.2.2, Method I). Sulphates (2.4.13) : maximum 100 ppm. 15 ml of solution S complies with the limit test for sulphates. Iron (2.4.9) : maximum 20 ppm. 5 ml of solution S diluted to 10 ml with water R complies with the limit test for iron. Magnesium and alkaline-earth metals (2.4.7) : maximum 200 ppm, calculated as Ca. 10.0 g complies with the limit test for magnesium and alkaline-earth metals. The volume of 0.01 M sodium edetate used does not exceed 5.0 ml. Heavy metals (2.4.8) : maximum 10 ppm. 12 ml of solution S complies with limit test A. Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32) : maximum 1.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 1.500 g in water R and dilute to 100.0 ml with the same solvent. To 10.0 ml of the solution add 50 ml of water R, 5 ml of dilute nitric acid R, 25.0 ml of 0.1 M silver nitrate and 2 ml of dibutyl phthalate R. Shake. Titrate with 0.1 M ammonium thiocyanate using 2 ml of ferric ammonium sulphate solution R2 as indicator and shaking vigorously towards the end-point. 1 ml of 0.1 M silver nitrate is equivalent to 9.794 mg of NH4Br.

b

=

percentage content of NH4Br and NH4Cl obtained in the assay and calculated as NH4Br,

=

percentage content of Cl obtained in the test for chlorides.

STORAGE In an airtight container, protected from light.

Ammonium chloride contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent of NH4Cl, calculated with reference to the dried substance.

CHARACTERS A white, crystalline powder or colourless crystals, freely soluble in water.

IDENTIFICATION A. It gives the reactions of chlorides (2.3.1). B. 10 ml of solution S (see Tests) gives the reaction of ammonium salts (2.3.1).

TESTS Solution S. Dissolve 10.0 g in carbon dioxide-free water R prepared from distilled water R and dilute to 100 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Acidity or alkalinity. To 10 ml of solution S add 0.05 ml of methyl red solution R. Not more than 0.5 ml of 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is required to change the colour of the indicator. Bromides and iodides. To 10 ml of solution S add 0.1 ml of dilute hydrochloric acid R and 0.05 ml of chloramine solution R. After 1 min, add 2 ml of chloroform R and shake vigorously. The chloroform layer remains colourless (2.2.2, Method I). Sulphates (2.4.13). 10 ml of solution S diluted to 15 ml with distilled water R complies with the limit test for sulphates (150 ppm). Calcium (2.4.3). 5 ml of solution S diluted to 15 ml with distilled water R complies with the limit test for calcium (200 ppm).

Calculate the percentage content of NH4Br from the expression :

a

DEFINITION

Iron (2.4.9). 5 ml of solution S diluted to 10 ml with water R complies with the limit test for iron (20 ppm). Heavy metals (2.4.8). 12 ml of solution S complies with limit test A (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.00 g by drying in an oven at 100-105 °C for 2 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 2.0 g.

01/2005:0007 ASSAY

AMMONIUM CHLORIDE Ammonii chloridum NH4Cl 986

Dissolve 1.000 g in 20 ml of water R and add a mixture of 5 ml of formaldehyde solution R, previously neutralised to phenolphthalein solution R, and 20 ml of water R. After 1 min to 2 min, titrate slowly with 1 M sodium hydroxide, using a further 0.2 ml of the same indicator. 1 ml of 1 M sodium hydroxide is equivalent to 53.49 mg of Mr 53.49 NH4Cl. See the information section on general monographs (cover pages)

Ammonium glycyrrhizate


01/2005:1772 Reference solution (a). Dilute 1.0 ml of the test solution to 20.0 ml with the mobile phase.

AMMONIUM GLYCYRRHIZATE Ammonii glycyrrhizas

Reference solution (b). Dissolve 50 mg of ammonium glycyrrhizate CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 20.0 ml with the mobile phase. Column : — size : l = 0.25 m, Ø = 4.0 mm, — stationary phase : octadecylsilyl silica gel for chromatography R (5-10 µm). Mobile phase : glacial acetic acid R, acetonitrile R, water R (6:380:614 V/V/V). Flow rate : 1.2 ml/min. Detection : spectrophotometer at 254 nm. Injection : 10 µl. Run time : 3 times the retention time of 18β-glycyrrhizic acid.

C42H65NO16

Relative retention with reference to 18β-glycyrrhizic acid (retention time = about 8 min) : impurity A = about 0.8 ; Mr 840 18α-glycyrrhizic acid = about 1.2.

DEFINITION Mixture of ammonium 18α- and 18β-glycyrrhizate (ammonium salt of (20β)-3β-[[2-O-(β-D-glucopyranosyluronic acid)-α-D-glucopyranosyluronic acid]oxy]-11-oxoolean-12-en29-oic acid), the 18β-isomer being the main component. Content : 98.0 per cent to 102.0 per cent (anhydrous substance). CHARACTERS Appearance : white or yellowish-white, hygroscopic powder.

System suitability : reference solution (b) : — resolution : minimum 2.0 between the peaks due to 18β-glycyrrhizic acid and 18α-glycyrrhizic acid. Limits : — 18α-glycyrrhizic acid : not more than twice the sum of the areas of the peaks in the chromatogram obtained with reference solution (a) (10.0 per cent), — impurity A : not more than the sum of the areas of the peaks in the chromatogram obtained with reference solution (a) (5.0 per cent),

Solubility : slightly soluble in water, very slightly soluble — any other impurity : for each impurity, not more than in anhydrous ethanol, practically insoluble in acetone. It 0.4 times the sum of the areas of the peaks in the dissolves in dilute solutions of acids and of alkali hydroxides. chromatogram obtained with reference solution (a) (2.0 per cent), IDENTIFICATION — total of other impurities : not more than 1.4 times the A. Infrared absorption spectrophotometry (2.2.24). sum of the areas of the peaks in the chromatogram obtained with reference solution (a) (7.0 per cent), Comparison : ammonium glycyrrhizate CRS. B. Dissolve 0.1 g in 20 ml of water R, add 2 ml of dilute sodium hydroxide solution R and heat cautiously. On heating, the solution gives off vapours that may be identified by the alkaline reaction of wet litmus paper (2.3.1). TESTS Appearance of solution. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY7 (2.2.2, Method I).

— disregard limit : 0.04 times the sum of the areas of the peaks in the chromatogram obtained with reference solution (a) (0.2 per cent). Heavy metals (2.4.8) : maximum 20 ppm. 1.0 g complies with limit test C. Prepare the reference solution using 2 ml of lead standard solution (10 ppm Pb) R. Water (2.5.12) : maximum 6.0 per cent, determined on 0.250 g. Sulphated ash (2.4.14) : maximum 0.2 per cent, determined on 1.0 g.

Dissolve 1.0 g in ethanol (20 per cent V/V) R and dilute to ASSAY 100.0 ml with the same solvent. Specific optical rotation (2.2.7) : + 49.0 to + 54.0 (anhydrous Dissolve 0.600 g in 60 ml of acetic acid R heating at 80 °C if necessary. Cool. Titrate with 0.1 M perchloric acid, substance). determining the end-point potentiometrically (2.2.20). Dissolve 0.5 g in ethanol (50 per cent V/V) R and dilute to 1 ml of 0.1 M perchloric acid is equivalent to 84.0 mg 50.0 ml with the same solvent. of C42H65NO16. Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 0.100 g of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. General Notices (1) apply to all monographs and other texts

STORAGE In an airtight container. 987

Ammonium hydrogen carbonate


ASSAY Dissolve cautiously 1.0 g in 20.0 ml of 0.5 M sulphuric acid and dilute to 50 ml with water R. Boil, cool and titrate the excess of acid with 1 M sodium hydroxide, using 0.1 ml of methyl red solution R as indicator. 1 ml of 0.5 M sulphuric acid is equivalent to 79.1 mg of NH4HCO3.

IMPURITIES


AMOBARBITAL Amobarbitalum

A. (4β,20β)-3β-[[2-O-(β-D-glucopyranosyluronic acid)-α-D-glucopyranosyluronic acid]oxy]-23-hydroxy11-oxoolean-12-en-29-oic acid (24-hydroxyglycyrrhizinic acid). 01/2005:1390

AMMONIUM HYDROGEN CARBONATE Ammonii hydrogenocarbonas

C11H18N2O3

Mr 226.3

DEFINITION Amobarbital contains not less than 99.0 per cent and NH4HCO3 Mr 79.1 not more than the equivalent of 101.0 per cent of 5-ethyl-5-(3-methylbutyl)pyrimidin-2,4,6(1H,3H,5H)-trione, DEFINITION calculated with reference to the dried substance. Ammonium hydrogen carbonate contains not less than 98.0 per cent and not more than 101.0 per cent of the CHARACTERS equivalent of ammonium hydrogen carbonate. A white, crystalline powder, very slightly soluble in water, freely soluble in alcohol, soluble in methylene chloride. It CHARACTERS forms water-soluble compounds with alkali hydroxides and A fine, white, crystalline powder or white crystals, slightly carbonates and with ammonia. hygroscopic, freely soluble in water, practically insoluble in alcohol. IDENTIFICATION It volatilises rapidly at 60 °C. The volatilisation takes First identification : A, B. place slowly at ambient temperatures if the substance is Second identification : A, C, D. slightly moist. It is in a state of equilibrium with ammonium A. Determine the melting point (2.2.14) of the substance carbamate. to be examined. Mix equal parts of the substance to be examined and amobarbital CRS and determine the IDENTIFICATION melting point of the mixture. The difference between the A. It gives the reaction of carbonates and bicarbonates melting points (which are about 157 °C) is not greater (2.3.1). than 2 °C. B. Dissolve 50 mg in 2 ml of water R. The solution gives the B. Examine by infrared absorption spectrophotometry reaction of ammonium salts (2.3.1). (2.2.24), comparing with the spectrum obtained with amobarbital CRS. TESTS C. Examine by thin-layer chromatography (2.2.27), using Solution S. Dissolve 14.0 g in 100 ml of distilled water R. silica gel GF254 R as the coating substance. Boil to remove the ammonia, allow to cool and dilute to Test solution. Dissolve 0.1 g of the substance to be 100.0 ml with distilled water R. examined in alcohol R and dilute to 100 ml with the same Chlorides (2.4.4). Dilute 5 ml of solution S to 15 ml with solvent. water R. The solution complies with the limit test for Reference solution. Dissolve 0.1 g of amobarbital CRS in chlorides (70 ppm). alcohol R and dilute to 100 ml with the same solvent. Sulphates (2.4.13). 15 ml of solution S complies with the Apply separately to the plate 10 µl of each solution. limit test for sulphates (70 ppm). Develop over a path of 18 cm using the lower layer from Iron (2.4.9). Dilute 1.8 ml of solution S to 10 ml with a mixture of 5 volumes of concentrated ammonia R, water R. The solution complies with the limit test for iron 15 volumes of alcohol R and 80 volumes of chloroform R. (40 ppm). Examine immediately in ultraviolet light at 254 nm. The Heavy metals (2.4.8). Dissolve cautiously 2.5 g in 25 ml of principal spot in the chromatogram obtained with the test 1 M hydrochloric acid. 12 ml of the solution complies with solution is similar in position and size to the principal limit test A for heavy metals (10 ppm). Prepare the standard spot in the chromatogram obtained with the reference using lead standard solution (1 ppm Pb) R. solution. 988



D. It gives the reaction of non-nitrogen substituted barbiturates (2.3.1). TESTS Appearance of solution. Dissolve 1.0 g in a mixture of 4 ml of dilute sodium hydroxide solution R and 6 ml of water R. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). Acidity or alkalinity. To 1.0 g add 50 ml of water R and boil for 2 min. Allow to cool and filter. To 10 ml of the filtrate add 0.15 ml of methyl red solution R and 0.1 ml of 0.01 M sodium hydroxide. The solution is yellow. Add 0.2 ml of 0.01 M hydrochloric acid. The solution is red. Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 1.0 g of the substance to be examined in alcohol R and dilute to 100 ml with the same solvent. Reference solution. Dilute 0.5 ml of the test solution to 100 ml with alcohol R. Apply separately to the plate 20 µl of each solution. Develop over a path of 15 cm using the lower layer from a mixture of 5 volumes of concentrated ammonia R, 15 volumes of alcohol R and 80 volumes of chloroform R. Examine the plate immediately in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution. Spray with diphenylcarbazone mercuric reagent R. Allow the plate to dry in air and spray with freshly prepared alcoholic potassium hydroxide solution R diluted 1 in 5 with aldehyde-free alcohol R. Heat at 100 °C to 105 °C for 5 min and examine immediately. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution (0.5 per cent). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

Amobarbital sodium

DEFINITION Amobarbital sodium contains not less than 98.5 per cent and not more than the equivalent of 102.0 per cent of sodium derivative of 5-ethyl-5-(3-methylbutyl)pyrimidin-2,4,6(1H,3H, 5H)-trione, calculated with reference to the dried substance. CHARACTERS A white, granular powder, hygroscopic, very soluble in carbon dioxide-free water (a small fraction may be insoluble), freely soluble in alcohol.

IDENTIFICATION First identification : A, B, E. Second identification : A, C, D, E. A. Acidify 10 ml of solution S (see Tests) with dilute hydrochloric acid R and shake with 20 ml of ether R. Separate the ether layer, wash with 10 ml of water R, dry over anhydrous sodium sulphate R and filter. Evaporate the filtrate to dryness and dry the residue at 100 °C to 105 °C (test residue). Repeat the operations using 0.1 g of amobarbital sodium CRS (reference residue). Determine the melting point (2.2.14) of the test residue. Mix equal parts of the test residue and the reference residue and determine the melting point of the mixture. The difference between the melting points (which are about 157 °C) is not greater than 2 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing the spectrum obtained with the reference residue prepared from amobarbital sodium CRS with that obtained with the test residue (see identification test A). C. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 0.1 g of the substance to be examined in alcohol R and dilute to 100 ml with the same solvent. Reference solution. Dissolve 0.1 g of amobarbital sodium CRS in alcohol R and dilute to 100 ml with the same solvent. Apply separately to the plate 10 µl of each solution. Develop over a path of 18 cm using the lower layer of ASSAY a mixture of 5 volumes of concentrated ammonia R, 15 volumes of alcohol R and 80 volumes of chloroform R. Dissolve 0.100 g in 5 ml of pyridine R. Add 0.5 ml of Examine immediately in ultraviolet light at 254 nm. The thymolphthalein solution R and 10 ml of silver nitrate principal spot in the chromatogram obtained with the test solution in pyridine R. Titrate with 0.1 M ethanolic sodium solution is similar in position and size to the principal hydroxide until a pure blue colour is obtained. Carry out spot in the chromatogram obtained with the reference a blank titration. solution. 1 ml of 0.1 M ethanolic sodium hydroxide is equivalent to 11.31 mg of C11H18N2O3. D. It gives the reaction of non-nitrogen substituted barbiturates (2.3.1). E. It gives reaction (a) of sodium (2.3.1). 01/2005:0166 TESTS Solution S. Dissolve 5.0 g in alcohol (50 per cent V/V) R AMOBARBITAL SODIUM and dilute to 50 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not Amobarbitalum natricum more intensely coloured than reference solution Y7 (2.2.2, Method II). pH (2.2.3). Dissolve 5.0 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent. Disregard any slight residue. The pH of the solution is not more than 11.0. Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 1.0 g of the substance to be examined C11H17N2NaO3 Mr 248.3 in alcohol R and dilute to 100 ml with the same solvent. General Notices (1) apply to all monographs and other texts

989

Amoxicillin sodium


of a mixture of 1 volume of water R and 9 volumes of Reference solution. Dilute 0.5 ml of the test solution to acetone R, then dry in an oven at 60 °C for 30 min. 100 ml with alcohol R. Examine by infrared absorption spectrophotometry Apply separately to the plate 20 µl of each solution. Develop (2.2.24), comparing with the spectrum obtained with over a path of 15 cm using the lower layer of a mixture amoxicillin trihydrate CRS. of 5 volumes of concentrated ammonia R, 15 volumes B. Examine by thin-layer chromatography (2.2.27), using a of alcohol R and 80 volumes of chloroform R. Examine TLC silanised silica gel plate R. the plate immediately in ultraviolet light at 254 nm. Spray with diphenylcarbazone mercuric reagent R. Allow the Test solution. Dissolve 25 mg of the substance to be plate to dry in air and spray with freshly prepared alcoholic examined in 10 ml of sodium hydrogen carbonate potassium hydroxide solution R diluted 1 in 5 with solution R. aldehyde-free alcohol R. Heat at 100 °C to 105 °C for 5 min Reference solution (a). Dissolve 25 mg of amoxicillin and examine immediately. When examined in ultraviolet trihydrate CRS in 10 ml of sodium hydrogen carbonate light and after spraying, any spot in the chromatogram solution R. obtained with the test solution, apart from the principal Reference solution (b). Dissolve 25 mg of amoxicillin spot, is not more intense than the spot in the chromatogram trihydrate CRS and 25 mg of ampicillin trihydrate CRS obtained with the reference solution (0.5 per cent). Disregard in 10 ml of sodium hydrogen carbonate solution R. any spot at the starting-point. Apply to the plate 1 µl of each solution. Develop over a Loss on drying (2.2.32). Not more than 3.0 per cent, path of 15 cm using a mixture of 10 volumes of acetone R determined on 0.50 g by drying in an oven at 130 °C. and 90 volumes of a 154 g/l solution of ammonium acetate R, the pH of which has been adjusted to 5.0 with ASSAY glacial acetic acid R. Allow the plate to dry in air and Dissolve 0.200 g in 5 ml of ethanol R. Add 0.5 ml of expose it to iodine vapour until the spots appear. Examine thymolphthalein solution R and 10 ml of silver nitrate in daylight. The principal spot in the chromatogram solution in pyridine R. Titrate with 0.1 M ethanolic sodium obtained with the test solution is similar in position, hydroxide until a pure blue colour is obtained. Carry out colour and size to the principal spot in the chromatogram a blank titration. obtained with reference solution (a). The test is not 1 ml of 0.1 M ethanolic sodium hydroxide is equivalent to valid unless the chromatogram obtained with reference 24.83 mg of C11H17N2NaO3. solution (b) shows 2 clearly separated spots. C. Place about 2 mg in a test-tube about 150 mm long and STORAGE 15 mm in diameter. Moisten with 0.05 ml of water R and Store in an airtight container. add 2 ml of sulphuric acid-formaldehyde reagent R. Mix the contents of the tube by swirling ; the solution is practically colourless. Place the test-tube in a water-bath 01/2005:0577 for 1 min ; a dark yellow colour develops. corrected D. It gives reaction (a) of sodium (2.3.1).

AMOXICILLIN SODIUM

TESTS Appearance of solution. Dissolve 1.0 g in water R and Amoxicillinum natricum dilute to 10.0 ml with the same solvent. Immediately after dissolution, the solution is not more opalescent than reference suspension II (2.2.1). The solution may show an initial, but transient, pink colour. After 5 min, the absorbance measured at 430 nm (2.2.25) is not greater than 0.20. pH (2.2.3). Dissolve 2.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. The pH of the solution is 8.0 to 10.0. C16H18N3NaO5S Mr 387.4 Specific optical rotation (2.2.7). Dissolve 62.5 mg in a 4 g/l solution of potassium hydrogen phthalate R and dilute to DEFINITION 25.0 ml with the same solution. The specific optical rotation Amoxicillin sodium contains not less than 89.0 per is + 240 to + 290, calculated with reference to the anhydrous cent and not more than the equivalent of 102.0 per substance. cent of sodium (2S,5R,6R)-6-[[(2R)-2-amino-2-(4Related substances. Examine by liquid chromatography hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1(2.2.29) as described under Assay. Inject 50 µl of reference azabicyclo[3.2.0]heptane-2-carboxylate, calculated with solution (d) and elute isocratically until elution of the reference to the anhydrous substance. amoxicillin peak. Inject 50 µl of test solution (b) and start the elution isocratically. Immediately after elution of the CHARACTERS amoxicillin peak start the following linear gradient. If the A white or almost white powder, very hygroscopic, very mobile phase composition has been adjusted to achieve the soluble in water, sparingly soluble in ethanol, very slightly required resolution, the adjusted composition will apply at soluble in acetone. time zero in the gradient. IDENTIFICATION Time Comment Mobile phase A Mobile phase B (min) (per cent V/V) (per cent V/V) First identification : A, D. 92 → 0 0 - 25 8 → 100 linear gradient Second identification : B, C, D. A. Dissolve 0.250 g in 5 ml of water R, add 0.5 ml of dilute 0 isocratic 25 - 40 100 acetic acid R, swirl and allow to stand for 10 min in iced 92 8 40 - 55 re-equilibration water. Filter the crystals and wash with 2 ml to 3 ml 990



Amoxicillin sodium

Inject mobile phase A and use the same elution pattern to obtain a blank. Inject reference solution (e). The 3 principal peaks eluted after the main peak correspond to amoxicillin diketopiperazine, amoxicillin dimer (impurity J ; n = 1) and amoxicillin trimer (impurity J ; n = 2) and, compared to the principal peak they have a relative retention time of about 3.4, 4.1 and 4.5, respectively. In the chromatogram obtained with test solution (b) : the area of any peak corresponding to amoxicillin dimer is not greater than 3 times the area of the principal peak in the chromatogram obtained with reference solution (d) (3 per cent) ; the area of any peak, apart from the principal peak and any peak corresponding to amoxicillin dimer, is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (d) (2 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than 9 times the area of the principal peak in the chromatogram obtained with reference solution (d) (9 per cent). Disregard any peak with an area less than 0.1 times that of the principal peak in the chromatogram obtained with reference solution (d). N,N-Dimethylaniline (2.4.26, Method A or B). Not more than 20 ppm. 2-Ethylhexanoic acid (2.4.28). Not more than 0.8 per cent m/m. Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Water (2.5.12). Not more than 3.0 per cent, determined on 0.400 g by the semi-micro determination of water. Bacterial endotoxins (2.6.14) : less than 0.25 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins.

— as mobile phase at a flow rate of 1.0 ml/min : Mobile phase A. Mix 1 volume of acetonitrile R and 99 volumes of a 25 per cent V/V solution of 0.2 M potassium dihydrogen phosphate R adjusted to pH 5.0 with dilute sodium hydroxide solution R, Mobile phase B. Mix 20 volumes of acetonitrile R and 80 volumes of a 25 per cent V/V solution of 0.2 M potassium dihydrogen phosphate R adjusted to pH 5.0 with dilute sodium hydroxide solution R, — as detector a spectrophotometer set at 254 nm. Equilibrate the column with a mobile phase ratio A:B of 92:8. Inject 50 µl of reference solution (b). The test is not valid unless in the chromatogram obtained, the resolution between the peaks corresponding to amoxicillin and cefadroxil is at least 2.0 (if necessary, adjust the ratio A:B of the mobile phase) and the mass distribution ratio for the first peak (amoxicillin) is 1.3 to 2.5. Inject reference solution (c). Adjust the system to obtain a peak with a signal-to-noise ratio of at least 3. Inject reference solution (a) 6 times. The test is not valid unless the relative standard deviation for the area of the principal peak is at most 1.0 per cent. Inject test solution (a) and reference solution (a). Calculate the percentage content of amoxicillin sodium by multiplying the percentage content of amoxicillin by 1.060.


991

STORAGE Store in an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins.

IMPURITIES ASSAY Examine by liquid chromatography (2.2.29). Test solution (a). Dissolve 30.0 mg of the substance to be examined in mobile phase A and dilute to 50.0 ml with the same mobile phase. Test solution (b). Prepare immediately before use. Dissolve 30.0 mg of the substance to be examined in mobile phase A A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylic acid and dilute to 20.0 ml with the same mobile phase. (6-aminopenicillanic acid), Reference solution (a). Dissolve 30.0 mg of amoxicillin trihydrate CRS in mobile phase A and dilute to 50.0 ml with the same mobile phase. Reference solution (b). Dissolve 4.0 mg of cefadroxil CRS in mobile phase A and dilute to 50 ml with the same mobile phase. To 5.0 ml of this solution add 5.0 ml of reference solution (a) and dilute to 100 ml with mobile phase A. Reference solution (c). Dilute 1.0 ml of reference solution (a) to 20.0 ml with mobile phase A. Dilute 1.0 ml of the solution B. (2S,5R,6R)-6-[[(2S)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicycto 50.0 ml with mobile phase A. lo[3.2.0]heptane-2-carboxylic acid (L-amoxicillin), Reference solution (d). Dilute 2.0 ml of reference solution (a) to 20.0 ml with mobile phase A. Dilute 5.0 ml of the solution to 20.0 ml with mobile phase A. Reference solution (e). To 0.20 g of amoxicillin trihydrate R add 1.0 ml of water R. Shake and add dropwise dilute sodium hydroxide solution R to obtain a solution. The pH of the solution is about 8.5. Store the solution at room temperature for 4 h. Dilute 0.5 ml of the solution to 50.0 ml with mobile phase A. The chromatographic procedure may be carried out using : — a column 0.25 m long and 4.6 mm in internal C. (4S)-2-[5-(4-hydroxyphenyl)-3,6-dioxopiperazin-2-yl]-5, diameter packed with octadecylsilyl silica gel for 5-dimethylthiazolidine-4-carboxylic acid (amoxicillin chromatography R (5 µm), diketopiperazines),

Amoxicillin trihydrate


D. (4S)-2-[[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]carboxymethyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penicilloic acids of amoxicillin),

J. co-oligomers of amoxicillin and penicilloic acids of amoxicillin, E. (2RS,4S)-2-[[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penilloic acids of amoxicillin),

K. oligomers of penicilloic acids of amoxicillin. F. 3-(4-hydroxyphenyl)pyrazin-2-ol,

01/2005:0260

AMOXICILLIN TRIHYDRATE Amoxicillinum trihydricum

G. (2S,5R,6R)-6-[[(2R)-2-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-2-(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (D-(4-hydroxyphenyl)glycylamoxicillin),

H. (2R)-2-[(2,2-dimethylpropanoyl)amino]-2-(4hydroxyphenyl)acetic acid,

I. (2R)-2-amino-2-(4-hydroxyphenyl)acetic acid, 992

C16H19N3O5S,3H2O

Mr 419.4

DEFINITION Amoxicillin trihydrate contains not less than 95.0 per cent and not more than the equivalent of 102.0 per cent of (2S,5R, 6R)-6-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-3,3dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, calculated with reference to the anhydrous substance. CHARACTERS A white or almost white, crystalline powder, slightly soluble in water, very slightly soluble in alcohol, practically insoluble in fatty oils. It dissolves in dilute acids and dilute solutions of alkali hydroxides. IDENTIFICATION First identification : A. Second identification : B, C. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with amoxicillin trihydrate CRS. B. Examine by thin-layer chromatography (2.2.27), using silanised silica gel H R as the coating substance.




Test solution. Dissolve 25 mg of the substance to be examined in 10 ml of sodium hydrogen carbonate solution R. Reference solution (a). Dissolve 25 mg of amoxicillin trihydrate CRS in 10 ml of sodium hydrogen carbonate solution R. Reference solution (b). Dissolve 25 mg of amoxicillin trihydrate CRS and 25 mg of ampicillin trihydrate CRS in 10 ml of sodium hydrogen carbonate solution R. Apply to the plate 1 µl of each solution. Develop over a path of 15 cm using a mixture of 10 volumes of acetone R and 90 volumes of a 154 g/l solution of ammonium acetate R, the pH of which has been adjusted to 5.0 with glacial acetic acid R. Allow the plate to dry in air and expose it to iodine vapour until the spots appear. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows 2 clearly separated spots. C. Place about 2 mg in a test-tube about 150 mm long and 15 mm in diameter. Moisten with 0.05 ml of water R and add 2 ml of sulphuric acid-formaldehyde reagent R. Mix the contents of the tube by swirling ; the solution is practically colourless. Place the test-tube in a water-bath for 1 min ; a dark yellow colour develops. TESTS Solution S. With the aid of ultrasound or gentle heating, dissolve 0.100 g in carbon dioxide-free water R and dilute to 50.0 ml with the same solvent. Appearance of solution. Dissolve 1.0 g in 10 ml of 0.5 M hydrochloric acid. Dissolve separately 1.0 g in 10 ml of dilute ammonia R2. Immediately after dissolution, the solutions are not more opalescent than reference suspension II (2.2.1). pH (2.2.3). The pH of solution S is 3.5 to 5.5. Specific optical rotation (2.2.7) : + 290 to + 315, determined on solution S and calculated with reference to the anhydrous substance.

ASSAY Examine by liquid chromatography (2.2.29). Test solution (a). Dissolve 30.0 mg of the substance to be examined in mobile phase A and dilute to 50.0 ml with the same mobile phase. Test solution (b). Dissolve 30.0 mg of the substance to be examined in mobile phase A and dilute to 20.0 ml with the same mobile phase. Reference solution (a). Dissolve 30.0 mg of amoxicillin trihydrate CRS in mobile phase A and dilute to 50.0 ml with the same mobile phase. Reference solution (b). Dissolve 4.0 mg of cefadroxil CRS in mobile phase A and dilute to 50 ml with the same mobile phase. To 5.0 ml of this solution add 5.0 ml of reference solution (a) and dilute to 100 ml with mobile phase A. Reference solution (c). Dilute 1.0 ml of reference solution (a) to 20.0 ml with mobile phase A. Dilute 1.0 ml of this solution to 50.0 ml with mobile phase A. Reference solution (d). Dilute 2.0 ml of reference solution (a) to 20.0 ml with mobile phase A. Dilute 5.0 ml of this solution to 20.0 ml with mobile phase A. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.0 ml/min : Mobile phase A. A mixture of 1 volume of acetonitrile R and 99 volumes of buffer solution pH 5.0, Mobile phase B. A mixture of 20 volumes of acetonitrile R and 80 volumes of buffer solution pH 5.0, Prepare the buffer solution as follows : to 250 ml of 0.2 M potassium dihydrogen phosphate R add dilute sodium hydroxide solution R to pH 5.0 and dilute to 1000.0 ml with water R, — as detector a spectrophotometer set at 254 nm, — a 50 µl loop injector. Equilibrate the column with a mobile phase with ratio A:B of 92:8. Inject reference solution (b). The assay is not valid unless the resolution between the 2 principal peaks is at least 2.0. If necessary, adjust the ratio A:B of the mobile phase. The mass distribution ratio for the first peak (amoxicillin) is 1.3 to 2.5. Inject reference solution (c). Adjust the system to obtain a peak with a signal-to-noise ratio of at least 3. Inject reference solution (a) 6 times. The test is not valid unless the relative standard deviation for the area of the principal peak is at most 1.0 per cent. Inject alternately test solution (a) and reference solution (a).

Related substances. Examine by liquid chromatography (2.2.29) adjusting the ratio A:B of the mobile phase and the attenuation as described under Assay. Inject reference solution (d). Inject a freshly prepared test solution (b) and start the elution isocratically with the chosen mobile phase. Immediately after elution of the amoxicillin peak start a linear gradient elution to reach a mobile phase A:B of 0:100 over a period of 25 min. Continue the chromatography with mobile phase B for 15 min, then equilibrate the column STORAGE for 15 min with the mobile phase chosen originally. Inject Store in an airtight container. mobile phase A and use the same elution gradient to obtain a blank. In the chromatogram obtained with test solution (b), the area of any peak, apart from the principal peak and any IMPURITIES peak observed in the blank chromatogram, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (d) (1 per cent). N,N-Dimethylaniline (2.4.26, Method A or B). Not more than 20 ppm. Water (2.5.12) : 11.5 per cent to 14.5 per cent, determined on 0.100 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 1.0 per cent, determined on 1.0 g. General Notices (1) apply to all monographs and other texts

A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylic acid (6-aminopenicillanic acid), 993



B. (2S,5R,6R)-6-[[(2S)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (L-amoxicillin),

H. (2R)-2-[(2,2-dimethylpropanoyl)amino]-2-(4hydroxyphenyl)acetic acid,

I. (2R)-2-amino-2-(4-hydroxyphenyl)acetic acid,

C. (4S)-2-[5-(4-hydroxyphenyl)-3,6-dioxopiperazin-2-yl]-5, 5-dimethylthiazolidine-4-carboxylic acid (amoxicillin diketopiperazines),

D. R = CO2H : (4S)-2-[[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]carboxymethyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penicilloic acids of amoxicillin),

J. co-oligomers of amoxicillin and of penicilloic acids of amoxicillin,

E. R = H : (2RS,4S)-2-[[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penilloic acids of amoxicillin),

K. oligomers of penicilloic acids of amoxicillin, F. 3-(4-hydroxyphenyl)pyrazin-2-ol,

G. (2S,5R,6R)-6-[[(2R)-2-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-2-(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (D-(4-hydroxyphenyl)glycylamoxicillin), 994

L. (2S,5R,6R)-6-[[(2S,5R,6R)-6-[[(2R)-2-amino-2-(4hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carbonyl]amino]-3,3-dimethyl7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (6-APA amoxicillin amide).


Amphotericin B


01/2005:1292

AMPHOTERICIN B

produced at the junction of the liquids. Mix, an intense blue colour is produced. Add 15 ml of water R and mix ; the solution becomes pale yellow.

TESTS Content of tetraenes. Not more than 10.0 per cent and not Amphotericinum B more than 5.0 per cent if intended for use in the manufacture of parenteral dosage forms, determined by the following method. Test solution. Dissolve 50.0 mg in 5 ml of dimethyl sulphoxide R and dilute to 50.0 ml with methanol R. Dilute 4.0 ml of the solution to 50.0 ml with methanol R. Reference solution (a). Dissolve 50.0 mg of amphotericin B CRS in 5 ml of dimethyl sulphoxide R and dilute to 50.0 ml with methanol R. Dilute 4.0 ml of the solution to 50.0 ml with methanol R. Reference solution (b). Dissolve 25.0 mg of nystatin CRS in 25 ml of dimethyl sulphoxide R and dilute to 250.0 ml with methanol R. Dilute 4.0 ml of the solution to 50.0 ml with methanol R. Measure the absorbances (2.2.25) of the test solution and of reference solutions (a) and (b) at the maxima at 282 nm and 304 nm respectively, using a 0.8 per cent V/V solution of C47H73NO17 Mr 924 dimethyl sulphoxide R in methanol R as the compensation liquid. Calculate the specific absorbance of the substance being examined, of nystatin CRS and of amphotericin B CRS DEFINITION at both wavelengths, each with reference to the dried Amphotericin B is a mixture of antifungal polyenes substance, and calculate the percentage content of tetraenes produced by the growth of certain strains of using the following expression : Streptomyces nodosus or by any other means. It consists mainly of amphotericin B which is (1R,3S,5R,6R,9R,11R,15S,16R,17R,18S,19E,21E,23E, 25E, 27E,29E,31E,33R,35S,36R,37S)-33-[(3-amino-3,6-dideoxy-βD-mannopyranosyl)oxy]-1,3,5,6,9,11,17,37-octahydroxy-15,16, S and S = specific absorbances of the substance 1 2 18-trimethyl-13-oxo-14,39-dioxabicyclo[33.3.1]nonatriacontato be examined at 282 nm and 304 nm 19,21,23,25,27,29,31-heptaene-36-carboxylic acid. The respectively, potency is not less than 750 IU/mg, calculated with reference N1 and N2 = specific absorbance of nystatin CRS at to the dried substance. 282 nm and 304 nm respectively, CHARACTERS A yellow or orange powder, practically insoluble in water, soluble in dimethyl sulphoxide and in propylene glycol, slightly soluble in dimethylformamide, very slightly soluble in methanol, practically insoluble in alcohol.

B1 and B2 = specific absorbance of amphotericin B CRS at 282 nm and 304 nm respectively, F = declared content of tetraenes in amphotericin B CRS.

Loss on drying (2.2.32). Not more than 5.0 per cent, determined on 1.000 g by drying in an oven at 60 °C at a pressure not exceeding 0.7 kPa. Sulphated ash (2.4.14). Not more than 3.0 per cent and not IDENTIFICATION more than 0.5 per cent if intended for use in the manufacture of parenteral dosage forms, determined on 1.0 g. A. Dissolve 25 mg in 5 ml of dimethyl sulphoxide R Bacterial endotoxins (2.6.14) : less than 1.0 IU/mg, if and dilute to 50 ml with methanol R. Dilute 2 ml of intended for use in the manufacture of parenteral dosage the solution to 200 ml with methanol R. Examined between 300 nm and 450 nm (2.2.25), the solution shows forms without a further appropriate procedure for the removal of bacterial endotoxins. 3 absorption maxima at 362 nm, 381 nm and 405 nm. The ratio of the absorbance measured at 362 nm to that ASSAY measured at 381 nm is 0.57 to 0.61. The ratio of the Protect all solutions from light throughout the assay. absorbance measured at 381 nm to that measured at Dissolve 25.0 mg in dimethyl sulphoxide R and dilute, 405 nm is 0.87 to 0.93. with shaking, to 25.0 ml with the same solvent. Under B. Examine by infrared absorption spectrophotometry constant stirring of this stock solution, dilute with (2.2.24), comparing with the spectrum obtained with dimethyl sulphoxide R to obtain solutions of appropriate amphotericin B CRS. If the spectra obtained show concentrations (the following concentrations have been differences dry the substance to be examined at 60 °C at found suitable : 44.4, 66.7 and 100 IU/ml). Final solutions a pressure not exceeding 0.7 kPa for 1 h and prepare a are prepared by diluting 1 to 20 with 0.2 M phosphate buffer new spectrum. solution pH 10.5 so that they all contain 5 per cent V/V of C. To 1 ml of a 0.5 g/l solution in dimethyl sulphoxide R, dimethyl sulphoxide. Prepare the reference and the test add 5 ml of phosphoric acid R to form a lower layer, solutions simultaneously. Carry out the microbiological avoiding mixing the 2 liquids. A blue ring is immediately assay of antibiotics (2.7.2). It is sensitive to light in dilute solutions and is inactivated at low pH values.


995

Ampicillin, anhydrous


STORAGE Store protected from light, at a temperature of 2 °C to 8 °C. If the substance is sterile, store in a sterile, airtight, tamper-proof container.

Test solution. Dissolve 25 mg of the substance to be examined in 10 ml of sodium hydrogen carbonate solution R. Reference solution (a). Dissolve 25 mg of anhydrous ampicillin CRS in 10 ml of sodium hydrogen carbonate LABELLING solution R. The label states : Reference solution (b). Dissolve 25 mg of amoxicillin — where applicable, that the substance is free from bacterial trihydrate CRS and 25 mg of anhydrous ampicillin CRS endotoxins, in 10 ml of sodium hydrogen carbonate solution R. — where applicable, that the substance is suitable for use in Apply separately to the plate 1 µl of each solution. the manufacture of parenteral dosage forms. Develop over a path of 15 cm using a mixture of 10 volumes of acetone R and 90 volumes of a 154 g/l IMPURITIES solution of ammonium acetate R, the pH of which has been adjusted to 5.0 with glacial acetic acid R. Allow the plate to dry in air and expose it to iodine vapour until the spots appear. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows 2 clearly separated spots. C. Place about 2 mg in a test-tube about 150 mm long and 15 mm in diameter. Moisten with 0.05 ml of water R and add 2 ml of sulphuric acid-formaldehyde reagent R. Mix the contents of the tube by swirling ; the solution is practically colourless. Place the test-tube in a water-bath for 1 min ; a dark yellow colour develops. D. It complies with the test for water (see Tests). A. amphotericin A (tetraene). TESTS 01/2005:0167 Appearance of solution. Dissolve 1.0 g in 10 ml of 1 M hydrochloric acid. Separately dissolve 1.0 g in 10 ml of dilute AMPICILLIN, ANHYDROUS ammonia R2. Immediately after dissolution, the solutions are not more opalescent than reference suspension II (2.2.1). Ampicillinum anhydricum pH (2.2.3). Dissolve 0.1 g in carbon dioxide-free water R and dilute to 40 ml with the same solvent. The pH of the solution is 3.5 to 5.5. Specific optical rotation (2.2.7). Dissolve 62.5 mg in water R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 280 to + 305, calculated with reference to the anhydrous substance. Related substances. Examine by liquid chromatography C16H19N3O4S Mr 349.4 (2.2.29) as described under Assay. Inject reference solution (c) and elute isocratically. Inject freshly prepared DEFINITION test solution (b) and start the elution isocratically. Anhydrous ampicillin contains not less than 96.0 per Immediately after elution of the ampicillin peak start the cent and not more than the equivalent of 100.5 per cent following linear gradient. If the mobile phase composition of (2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]amino]-3,3has been adjusted to achieve the required resolution, the dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic adjusted composition will apply at time zero in the gradient. acid, calculated with reference to the anhydrous substance. Time CHARACTERS A white, crystalline powder, sparingly soluble in water, practically insoluble in acetone, in alcohol and in fatty oils. It dissolves in dilute solutions of acids and of alkali hydroxides. It shows polymorphism. IDENTIFICATION First identification : A, D. Second identification : B, C, D. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with anhydrous ampicillin CRS. Examine the substances prepared as discs using potassium bromide R. B. Examine by thin-layer chromatography (2.2.27), using silanised silica gel H R as the coating substance. 996

(min)



0

85

15

30

0

100

45

0

100

Equilibrate the column with the originally chosen mobile phase for 15 min. Inject mobile phase A and use the same elution gradient to obtain a blank. In the chromatogram obtained with test solution (b), the area of any peak, apart from the principal peak and any peak observed in the blank chromatogram, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (c) (1.0 per cent). N,N-Dimethylaniline (2.4.26, Method B). Not more than 20 ppm.


Ampicillin, anhydrous


Water (2.5.12). Not more than 2.0 per cent, determined on 0.300 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.5 per cent, determined on 1.0 g. ASSAY Examine by liquid chromatography (2.2.29). Test solution (a). Dissolve 27.0 mg of the substance to be examined in mobile phase A and dilute to 50.0 ml with the same solvent. Test solution (b). Dissolve 27.0 mg of the substance to be examined in mobile phase A and dilute to 10.0 ml with the same solvent. Reference solution (a). Dissolve 27.0 mg of anhydrous ampicillin CRS in mobile phase A and dilute to 50.0 ml with the same solvent. Reference solution (b). Dissolve 2.0 mg of cefradine CRS in mobile phase A and dilute to 50 ml with the same solvent. To 5.0 ml of this solution add 5.0 ml of reference solution (a). Reference solution (c). Dilute 1.0 ml of reference solution (a) to 20.0 ml with mobile phase A. Reference solution (d). Dilute 1.0 ml of reference solution (c) to 25.0 ml with mobile phase A. The chromatographic procedure may be carried out using : — a column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.0 ml/min : Mobile phase A. A mixture of 0.5 ml of dilute acetic acid R, 50 ml of 0.2 M potassium dihydrogen phosphate R and 50 ml of acetonitrile R, diluted to 1000 ml with water R, Mobile phase B. A mixture of 0.5 ml of dilute acetic acid R, 50 ml of 0.2 M potassium dihydrogen phosphate R and 400 ml of acetonitrile R, diluted to 1000 ml with water R, — as detector a spectrophotometer set at 254 nm, — a 50 µl loop injector. Equilibrate the column with a mobile phase with ratio A:B of 85:15. Inject reference solution (b). The test is not valid unless the resolution between the two principal peaks is at least 3.0. If necessary, adjust the ratio A:B of the mobile phase. The mass distribution ratio for the first peak (ampicillin) is 2.0 to 2.5. Inject reference solution (d). Adjust the system to obtain a peak with a signal-to-noise ratio of at least 3. Inject reference solution (a) 6 times. The test is not valid unless the relative standard deviation for the area of the principal peak is at most 1.0 per cent. Inject alternately test solution (a) and reference solution (a). Calculate the percentage content of ampicillin.

B. (2S,5R,6R)-6-[[(2S)-2-amino-2-phenylacetyl]amino]-3, 3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2carboxylic acid (L-ampicillin),

C. (4S)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5dimethylthiazolidine-4-carboxylic acid (diketopiperazines of ampicillin),

D. R = CO2H : (4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]carboxymethyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penicilloic acids of ampicillin), F. R = H : (2RS,4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penilloic acids of ampicillin),

E. (2R)-2-[[[(2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-yl]carbonyl]amino]-2-phenylacetic acid (ampicillinyl-D-phenylglycine),

STORAGE Store in an airtight container, at a temperature not exceeding 30 °C. IMPURITIES G. (3R,6R)-3,6-diphenylpiperazine-2,5-dione,

A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylic acid (6-aminopenicillanic acid), General Notices (1) apply to all monographs and other texts

H. 3-phenylpyrazin-2-ol, 997

Ampicillin sodium



AMPICILLIN SODIUM Ampicillinum natricum I. (2S,5R,6R)-6-[[(2R)-2-[[(2R)-2-amino-2-phenylacetyl]amino]-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (D-phenylglycylampicillin), C16H18N3NaO4S

Mr 371.4

DEFINITION Ampicillin sodium contains not less than 91.0 per cent and not more than the equivalent of 100.5 per cent of sodium (2S, 5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]amino]-3,3-dimethyl7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, calculated with reference to the anhydrous substance. J. (2S,5R,6R)-6-[(2,2-dimethylpropanoyl)amino]-3,3dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2carboxylic acid,

CHARACTERS A white or almost white powder, hygroscopic, freely soluble in water, sparingly soluble in acetone, practically insoluble in fatty oils and in liquid paraffin.

K. (2R)-2-[(2,2-dimethylpropanoyl)amino]-2-phenylacetic acid,

L. (2R)-2-amino-2-phenylacetic acid (D-phenylglycine),

M. co-oligomers of ampicillin and of penicilloic acids of ampicillin. 998

IDENTIFICATION First identification : A, D. Second identification : B, C, D. A. Dissolve 0.250 g in 5 ml of water R, add 0.5 ml of dilute acetic acid R, swirl and allow to stand for 10 min in iced water. Filter the crystals through a small sintered-glass filter (40), applying suction, wash with 2 ml to 3 ml of a mixture of 1 volume of water R and 9 volumes of acetone R and dry in an oven at 60 °C for 30 min. Examine the crystals by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with ampicillin trihydrate CRS. B. Examine by thin-layer chromatography (2.2.27), using silanised silica gel H R as the coating substance. Test solution. Dissolve 25 mg of the substance to be examined in 10 ml of sodium hydrogen carbonate solution R. Reference solution (a). Dissolve 25 mg of ampicillin trihydrate CRS in 10 ml of sodium hydrogen carbonate solution R. Reference solution (b). Dissolve 25 mg of amoxicillin trihydrate CRS and 25 mg of ampicillin trihydrate CRS in 10 ml of sodium hydrogen carbonate solution R. Apply separately to the plate 1 µl of each solution. Develop over a path of 15 cm using a mixture of 10 volumes of acetone R and 90 volumes of a 154 g/l solution of ammonium acetate R, the pH of which has been adjusted to 5.0 with glacial acetic acid R. Allow the plate to dry in air and expose it to iodine vapour until the spots appear. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. C. Place about 2 mg in a test-tube about 150 mm long and 15 mm in diameter. Moisten with 0.05 ml of water R and add 2 ml of sulphuric acid-formaldehyde reagent R.



Ampicillin sodium

Mix the contents of the tube by swirling ; the solution is practically colourless. Place the test-tube in a water-bath for 1 min ; a dark yellow colour develops. D. It gives reaction (a) of sodium (2.3.1).

Test solution (b). Dissolve 1.0 g of the substance to be examined in water R, add 1.0 ml of the internal standard solution and dilute to 10.0 ml with water R. Reference solution. Dissolve 1.0 ml of methylene chloride R in water R and dilute to 500.0 ml with the same solvent. To 1.0 ml of the solution add 1.0 ml of the internal standard TESTS solution and dilute to 10.0 ml with water R. Appearance of solution. Place 1.0 g in a conical flask The chromatographic procedure may be carried out using : and add slowly and with continuous swirling 10 ml of 1 M — a glass column 1.5 m long and 4 mm in internal hydrochloric acid. Separately dissolve 1.0 g in water R diameter packed with diatomaceous earth for gas and dilute to 10.0 ml with the same solvent. Immediately chromatography R impregnated with 10 per cent m/m after dissolution, the solutions are not more opalescent than of macrogol 1000 R, reference suspension II (2.2.1). The absorbance (2.2.25) of the solution in watermeasured at 430 nm is not greater — nitrogen for chromatography R as the carrier gas at a than 0.15. flow rate of 40 ml/min, pH (2.2.3). Dissolve 2.0 g in carbon dioxide-free water R — a flame-ionisation detector, and dilute to 20 ml with the same solvent. The pH of the maintaining the temperature of the column at 60 °C, that solution, measured 10 min after dissolution, is 8.0 to 10.0. of the injection port at 100 °C and that of the detector at Specific optical rotation (2.2.7). Dissolve 62.5 mg in a 4 g/l 150 °C. Calculate the content of methylene chloride taking its density at 20 °C to be 1.325 g/ml. solution of potassium hydrogen phthalate R and dilute to 25.0 ml with the same solvent. The specific optical rotation Heavy metals (2.4.8). 1.0 g complies with limit test C for is + 258 to + 287, calculated with reference to the anhydrous heavy metals (20 ppm). Prepare the standard using 2 ml of substance. lead standard solution (10 ppm Pb) R. Related substances. Examine by liquid chromatography Water (2.5.12). Not more than 2.0 per cent, determined on (2.2.29) as described under Assay. Inject 50 µl of reference 0.300 g by the semi-micro determination of water. solution (d) and elute isocratically until elution of the Bacterial endotoxins (2.6.14) : less than 0.15 IU/mg, if ampicillin peak. Inject 50 µl of test solution (b) and start intended for use in the manufacture of parenteral dosage the elution isocratically. Immediately after elution of the forms without a further appropriate procedure for the ampicillin peak start the following linear gradient. If the removal of bacterial endotoxins. mobile phase composition has been adjusted to achieve the required resolution, the adjusted composition will apply at ASSAY time zero in the gradient. Examine by liquid chromatography (2.2.29). Time Comment Mobile phase A Mobile phase B Test solution (a). Dissolve 31.0 mg of the substance to be (min) examined in mobile phase A and dilute to 50.0 ml with (per cent V/V) (per cent V/V) mobile phase A. 0 - 30 85 → 0 15 → 100 linear gradient Test solution (b). Prepare immediately before use. Dissolve 100 0 isocratic 30 - 45 31.0 mg of the substance to be examined in mobile phase A 45 - 60 85 15 re-equilibration and dilute to 10.0 ml with mobile phase A. Reference solution (a). Dissolve 27.0 mg of anhydrous Inject mobile phase A and use the same elution pattern to ampicillin CRS in mobile phase A and dilute to 50.0 ml with obtain a blank. Inject reference solution (e) and use the same elution pattern. The chromatogram obtained with reference mobile phase A. Reference solution (b). Dissolve 2.0 mg of cefradine CRS in solution (e), shows an ampicillin peak and a dimer peak, mobile phase A and dilute to 50 ml with mobile phase A. To with a retention time of 2.8 relative to ampicillin. In the chromatogram obtained with test solution (b), the area of any 5.0 ml of the solution add 5.0 ml of reference solution (a). peak corresponding to ampicillin dimer is not greater than Reference solution (c). Dilute 1.0 ml of reference solution (a) 4.5 times the area of the principal peak in the chromatogram to 20.0 ml with mobile phase A. Dilute 1.0 ml of the solution obtained with reference solution (d) (4.5 per cent) ; the area to 25.0 ml with mobile phase A. of any peak, apart from the principal peak and any peak Reference solution (d). Dilute 1.0 ml of reference solution (a) corresponding to ampicillin dimer, is not greater than twice to 20.0 ml with mobile phase A. the area of the principal peak in the chromatogram obtained Reference solution (e). To 0.20 g of the substance to be with reference solution (d) (2 per cent). Disregard any peak examined add 1.0 ml of water R. Heat the solution at 60 °C due to the blank. for 1 h. Dilute 0.5 ml of the solution to 50.0 ml with mobile N,N-Dimethylaniline (2.4.26, Method B). Not more than phase A. 20 ppm. The chromatographic procedure may be carried out using : 2-Ethylhexanoic acid (2.4.28). Not more than 0.8 per — a column 0.25 m long and 4.6 mm in internal cent m/m. diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), Methylene chloride. Not more than 0.2 per cent m/m, determined by gas chromatography (2.2.28), using ethylene — as mobile phase at a flow rate of 1.0 ml/min : chloride R as the internal standard. Mobile phase A. A mixture of 0.5 ml of dilute acetic acid R, 50 ml of 0.2 M potassium dihydrogen phosphate R and Internal standard solution. Dissolve 1.0 ml of ethylene 50 ml of acetonitrile R, diluted to 1000 ml with water R, chloride R in water R and dilute to 500.0 ml with the same solvent. Mobile phase B. A mixture of 0.5 ml of dilute acetic acid R, 50 ml of 0.2 M potassium dihydrogen phosphate R and Test solution (a). Dissolve 1.0 g of the substance to be 400 ml of acetonitrile R, diluted to 1000 ml with water R, examined in water R and dilute to 10.0 ml with the same solvent. — as detector a spectrophotometer set at 254 nm. General Notices (1) apply to all monographs and other texts

999

Ampicillin sodium


F. R = H : (2RS,4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amiEquilibrate the column with a mobile phase with A:B ratio no]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid of 85:15. Inject 50 µl of reference solution (b). The assay (penilloic acids of ampicillin), is not valid unless in the chromatogram obtained, the resolution between the two principal peaks is at least 3.0. If necessary, adjust the A:B ratio of the mobile phase. The mass distribution ratio for the first peak (ampicillin) is 2.0 to 2.5. Inject 50 µl of reference solution (c). Adjust the system to obtain a peak with a signal-to-noise ratio of at least 3. Inject reference solution (a) six times. The test is not valid unless the relative standard deviation for the area of the principal peak is at most 1.0 per cent. Inject alternately test solution (a) and reference solution (a). Calculate the percentage content of ampicillin sodium by E. (2R)-2-[[[(2S,5R,6R)-6-[[(2R)-2-amino-2-phenymultiplying the percentage content of ampicillin by 1.063. lacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-yl]carbonyl]amino]-2-phenylacetic acid STORAGE (ampicillinyl-D-phenylglycine), Store in an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins. IMPURITIES G. (3R,6R)-3,6-diphenylpiperazine-2,5-dione,

A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylic acid (6-aminopenicillanic acid),

H. 3-phenylpyrazin-2-ol,


I. (2S,5R,6R)-6-[[(2R)-2-[[(2R)-2-amino-2-phenylacetyl]amino]-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (D-phenylglycylampicillin),


J. (2S,5R,6R)-6-[(2,2-dimethylpropanoyl)amino]-3,3dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2carboxylic acid,

D. R = CO2H : (4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]carboxymethyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penicilloic acids of ampicillin),

K. (2R)-2-[(2,2-dimethylpropanoyl)amino]-2-phenylacetic acid,

1000


Ampicillin trihydrate



M. co-oligomers of ampicillin and of penicilloic acids of ampicillin,

B. Examine by thin-layer chromatography (2.2.27), using silanised silica gel H R as the coating substance. Test solution. Dissolve 25 mg of the substance to be examined in 10 ml of sodium hydrogen carbonate solution R. Reference solution (a). Dissolve 25 mg of ampicillin trihydrate CRS in 10 ml of sodium hydrogen carbonate solution R. Reference solution (b). Dissolve 25 mg of amoxicillin trihydrate CRS and 25 mg of ampicillin trihydrate CRS in 10 ml of sodium hydrogen carbonate solution R. Apply separately to the plate 1 µl of each solution. Develop over a path of 15 cm using a mixture of 10 volumes of acetone R and 90 volumes of a 154 g/l solution of ammonium acetate R, the pH of which has been adjusted to 5.0 with glacial acetic acid R. Allow the plate to dry in air and expose it to iodine vapour until the spots appear. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows 2 clearly separated spots. C. Place about 2 mg in a test-tube about 150 mm long and 15 mm in diameter. Moisten with 0.05 ml of water R and add 2 ml of sulphuric acid-formaldehyde reagent R. Mix the contents of the tube by swirling ; the solution is practically colourless. Place the test-tube in a water-bath for 1 min ; a dark yellow colour develops. D. It complies with the test for water (see Tests).

TESTS Appearance of solution. Dissolve 1.0 g in 10 ml of 1 M hydrochloric acid. Separately dissolve 1.0 g in 10 ml of dilute 01/2005:0168 ammonia R2. Immediately after dissolution, the solutions are not more opalescent than reference suspension II (2.2.1). AMPICILLIN TRIHYDRATE pH (2.2.3). Dissolve 0.1 g in carbon dioxide-free water R and dilute to 40 ml with the same solvent. The pH of the Ampicillinum trihydricum solution is 3.5 to 5.5. Specific optical rotation (2.2.7). Dissolve 62.5 mg in water R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 280 to + 305, calculated with reference to the anhydrous substance. Related substances. Examine by liquid chromatography (2.2.29) as described under Assay. Inject reference solution (c) and elute isocratically. Inject freshly prepared C16H19N3O4S,3H2O Mr 403.5 test solution (b) and start the elution isocratically. Immediately after elution of the ampicillin peak start the DEFINITION following linear gradient. If the mobile phase composition has been adjusted to achieve the required resolution, the Ampicillin trihydrate contains not less than 96.0 per cent adjusted composition will apply at time zero in the gradient. and not more than the equivalent of 100.5 per cent of (2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]amino]-3,3Time Mobile phase A Mobile phase B dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic (min) (per cent V/V) (per cent V/V) acid, calculated with reference to the anhydrous substance. N. oligomers of penicilloic acids of ampicillin.

CHARACTERS A white, crystalline powder, slightly soluble in water, practically insoluble in alcohol and in fatty oils. It dissolves in dilute solutions of acids and of alkali hydroxides. IDENTIFICATION First identification : A, D. Second identification : B, C, D. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with ampicillin trihydrate CRS. General Notices (1) apply to all monographs and other texts

0

85

15

30

0

100

45

0

100

Equilibrate the column with the originally chosen mobile phase for 15 min. Inject mobile phase A and use the same elution gradient to obtain a blank. In the chromatogram obtained with test solution (b), the area of any peak, apart from the principal peak and any peak observed in the blank chromatogram, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (c) (1.0 per cent). 1001

Ampicillin trihydrate


N,N-Dimethylaniline (2.4.26, Method B). Not more than 20 ppm. Water (2.5.12). 12.0 per cent to 15.0 per cent, determined on 0.100 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.5 per cent, determined on 1.0 g. ASSAY Examine by liquid chromatography (2.2.29). Test solution (a). Dissolve 31.0 mg of the substance to be examined in mobile phase A and dilute to 50.0 ml with the same solvent. Test solution (b). Dissolve 31.0 mg of the substance to be examined in mobile phase A and dilute to 10.0 ml with the same solvent. Reference solution (a). Dissolve 27.0 mg of anhydrous ampicillin CRS in mobile phase A and dilute to 50.0 ml with the same solvent. Reference solution (b). Dissolve 2.0 mg of cefradine CRS in mobile phase A and dilute to 50 ml with the same solvent. To 5.0 ml of this solution add 5.0 ml of reference solution (a). Reference solution (c). Dilute 1.0 ml of reference solution (a) to 20.0 ml with mobile phase A. Reference solution (d). Dilute 1.0 ml of reference solution (c) to 25.0 ml with mobile phase A. The chromatographic procedure may be carried out using : — a column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.0 ml/min : Mobile phase A. A mixture of 0.5 ml of dilute acetic acid R, 50 ml of 0.2 M potassium dihydrogen phosphate R, 50 ml of acetonitrile R diluted to 1000 ml with water R, Mobile phase B. A mixture of 0.5 ml of dilute acetic acid R, 50 ml of 0.2 M potassium dihydrogen phosphate R and 400 ml of acetonitrile R diluted to 1000 ml with water R, — as detector a spectrophotometer set at 254 nm, — a 50 µl loop injector. Equilibrate the column with a mobile phase with ratio A:B of 85:15. Inject reference solution (b). The test is not valid unless the resolution between the two principal peaks is at least 3.0. If necessary, adjust the ratio A:B of the mobile phase. The mass distribution ratio for the first peak (ampicillin) is 2.0 to 2.5. Inject reference solution (d). Adjust the system to obtain a peak with a signal-to-noise ratio of at least 3. Inject reference solution (a) 6 times. The test is not valid unless the relative standard deviation for the area of the principal peak is at most 1.0 per cent. Inject alternately test solution (a) and reference solution (a). Calculate the percentage content of ampicillin.



D. R = CO2H : (4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]carboxymethyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penicilloic acids of ampicillin), F. R = H : (2RS,4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penilloic acids of ampicillin),

E. (2R)-2-[[[(2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-yl]carbonyl]amino]-2-phenylacetic acid (ampicillinyl-D-phenylglycine),

STORAGE Store in an airtight container, at a temperature not exceeding 30 °C. IMPURITIES G. (3R,6R)-3,6-diphenylpiperazine-2,5-dione,


H. 3-phenylpyrazin-2-ol, See the information section on general monographs (cover pages)

Angelica root


Content : minimum 2.0 ml/kg of essential oil (dried drug). CHARACTERS Bitter taste. Macroscopic and microscopic characters described under identification tests A and B. IDENTIFICATION A. The rhizome is greyish-brown or reddish-brown, I. (2S,5R,6R)-6-[[(2R)-2-[[(2R)-2-amino-2-phenytransversely annulated. The base bears greyish-brown lacetyl]amino]-2-phenylacetyl]amino]-3,3-dimethor reddish-brown, cylindrical, longitudinally furrowed, yl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid occasionally branched roots often with incompletely (D-phenylglycylampicillin), encircling, transverse ridges. The apex sometimes shows remnants of stem and leaf bases. The fracture is uneven. The transversely cut surface shows a greyish-white, spongy, distinctly radiate bark, in which the secretory channels are visible as brown spots, and a bright yellow to greyish-yellow wood which, in the rhizome, surrounds the greyish or brownish-white pith. B. Reduce to a powder (355). The powder is brownish-white. J. (2S,5R,6R)-6-[(2,2-dimethylpropanoyl)amino]-3,3Examine under a microscope using chloral hydrate dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2solution R. The powder shows : fragments of cork carboxylic acid, consisting of several layers of thin-walled greyish-brown or reddish-brown cells ; fragments of large, yellowish-brown secretory channels ; fragments of medullary rays 2 to 4 cells wide ; fragments of xylem with medullary rays and radially arranged, lignified vessels with reticulate thickening. Examine under a microscope using a 50 per cent V/V solution of glycerol R. The powder shows numerous, simple starch granules 2 µm to 4 µm in diameter. K. (2R)-2-[(2,2-dimethylpropanoyl)amino]-2-phenylacetic C. Examine the chromatograms obtained in the test for acid, lovage root. Results : see below the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. Top of the plate


_______

_______

Eugenol: (marked at 254 nm) Coumarin : (marked at 254 nm) _______

_______ An intense blue fluorescent zone Yellow fluorescent zones A blue fluorescent zone A yellow fluorescent zone An intense blue fluorescent zone

Reference solution

Test solution

TESTS Lovage root. Thin-layer chromatography (2.2.27). Test solution. To 1.0 g of the freshly powdered drug (355) add 5 ml of methanol R and boil for 30 s. Cool and filter. M. co-oligomers of ampicillin and of penicilloic acids of Reference solution. Dissolve 5 mg of coumarin R and 25 µl ampicillin. of eugenol R in 10 ml of methanol R. Plate : TLC silica gel F254 plate R. 01/2005:1857 Mobile phase : methylene chloride R, toluene R (50:50 V/V). Application : 20 µl, as bands. ANGELICA ROOT Development : twice over a path of 10 cm. Drying : in air. Angelicae radix Detection : examine in ultraviolet light at 254 nm. Mark DEFINITION the quenching zones due to coumarin and eugenol on Whole or cut, carefully dried rhizome and root of Angelica the chromatogram obtained with the reference solution. archangelica L. (Archangelica officinalis Haffm.). Examine in ultraviolet light at 365 nm. General Notices (1) apply to all monographs and other texts

1003

Anise oil


Results : the chromatogram obtained with the test solution shows no pale blue to white fluorescent zone between the zones of coumarin and eugenol in the chromatogram obtained with the reference solution. Foreign matter (2.8.2) : maximum 5 per cent of leaf bases and stem bases, maximum 5 per cent of discoloured pieces and maximum 1 per cent of other foreign matter. Loss on drying (2.2.32) : maximum 10.0 per cent, determined on 1.000 g of the powdered drug (355) by drying in an oven at 100-105 °C for 2 h. Total ash (2.4.16) : maximum 10.0 per cent. Ash insoluble in hydrochloric acid (2.8.1) : maximum 2.0 per cent. ASSAY Carry out the determination of essential oils in vegetable drugs (2.8.12). Reduce the drug to a powder (500) and immediately use 40.0 g for the determination. Use a 2 litre round-bottomed flask, 10 drops of liquid paraffin R, 500 ml of water R as distillation liquid and 0.50 ml of xylene R in the graduated tube. Distil at a rate of 2-3 ml/min for 4 h.

Top of the plate Anethole : a quenching zone _______

A quenching zone Anisaldehyde : a quenching zone

ANISE OIL

_______

Reference solution

Test solution

Detection B : spray with methyl 4-acetylbenzoate reagent R and heat at 100-105 °C for 10 min ; examine the still hot plate in daylight within 5 min. Results B : see below the sequence of zones present in the chromatograms obtained with the reference solution and the test solution. Furthermore, other zones may be present in the chromatogram obtained with the test solution. Top of the plate A violet-brown zone (monoterpene hydrocarbons) (solvent front)

_______

A very strong brown zone (anethole), distinctly separated _______ A grey zone

Anisaldehyde : a yellow zone

A yellow zone (anisaldehyde)

_______

Anisi aetheroleum

Linalol: a grey zone

DEFINITION Essential oil obtained by steam distillation from the dry ripe fruits of Pimpinella anisum L. CHARACTERS Appearance : clear, colourless or pale yellow liquid. IDENTIFICATION First identification : B. Second identification : A. A. Thin-layer chromatography (2.2.27). Test solution. Dissolve 1 g of the substance to be examined in toluene R and dilute to 10 ml with the same solvent. Reference solution. Dissolve 10 µl of linalol R, 30 µl of anisaldehyde R and 200 µl of anethole R in toluene R and dilute to 15 ml with the same solvent. Dilute 1 ml of this solution to 5 ml with toluene R. Plate : TLC silica gel F254 plate R. Mobile phase : ethyl acetate R, toluene R (7:93 V/V). Application : 5 µl as bands of 10 mm (for normal TLC plates) or 2 µl as bands of 10 mm (for fine particle size plates). Development : over a path of 15 cm (for normal TLC plates) or over a path of 6 cm (for fine particle size plates). Drying : in air. Detection A : examine in ultraviolet light at 254 nm. Results A : see below the sequence of zones present in the chromatograms obtained with the reference solution and the test solution. Furthermore, other zones may be present in the chromatogram obtained with the test solution. 1004

A quenching zone (anisaldehyde)

_______

Anethole : a brown zone


A very strong quenching zone (anethole) _______

_______ A grey zone (linalol) A grey zone

Reference solution

Test solution

B. Examine the chromatograms obtained in the test for chromatographic profile. Results : the characteristic peaks in the chromatogram obtained with the test solution are similar in retention time to those in the chromatogram obtained with the reference solution. TESTS Relative density (2.2.5) : 0.980 to 0.990. Refractive index (2.2.6) : 1.552 to 1.561. Freezing point (2.2.18) : 15 °C to 19 °C. Fenchone. Gas chromatography (2.2.28) as described in the test for chromatographic profile with the following modifications. Test solution. Dissolve 400 µl of the substance to be examined in 2.0 ml of hexane R. Reference solution (a). Dilute 10 µl of fenchone R to 1.2 g with hexane R. Reference solution (b). Dilute 100 µl of reference solution (a) to 100 ml with hexane R. System suitability : reference solution (b) : — signal-to-noise ratio : minimum 10 for the principal peak. Limit : — fenchone : maximum 0.01 per cent. Foeniculin. Gas chromatography (2.2.28) as described in the test for chromatographic profile with the following modifications. Test solution. The substance to be examined.


Anise oil


Reference solution (a). Dilute 10 mg of the test solution to 1.000 g with hexane R. Dilute 0.5 ml of this solution to 100 ml with hexane R. Reference solution (b). Foeniculin for peak identification CRS. System suitability : — the chromatogram obtained with reference solution (b) is similar to the chromatogram provided with foeniculin for peak identification CRS, — signal-to-noise ratio : minimum 10 for the principal peak in the chromatogram obtained with reference solution (a). Limit : locate the peak due to foeniculin by comparison with the chromatogram provided with foeniculin for peak identification CRS. — foeniculin : maximum 0.01 per cent. Fatty oils and resinified essential oils (2.8.7). It complies with the test for fatty oils and resinified essential oils. Chromatographic profile. Gas chromatography (2.2.28) : use the normalisation procedure. Test solution. Dissolve 200 µl of the substance to be examined in 1.0 ml of hexane R. Reference solution. To 1.0 ml of hexane R, add 20 µl of linalol R, 20 µl of estragole R, 20 µl of α-terpineol R, 60 µl of anethole R and 30 µl of anisaldehyde R. Column : — material : fused silica, — size : l = 30 m, Ø = 0.25 mm, — stationary phase: macrogol 20 000 R (film thickness 0.25 µm). Carrier gas : helium for chromatography R. Flow rate : 1.0 ml/min. Split ratio : 1:100.

Temperature :

Column

Time (min)

Temperature ( °C)

0–5

60

5 – 80

60 → 210

80 – 95

210

Injection port

200

Detector

220

Detection : flame ionisation. Injection : 0.2 µl. Elution order : order indicated in the composition of the reference solution. Record the retention times of these substances. System suitability : reference solution : — resolution : minimum 1.5 between the peaks due to estragole and α-terpineol. Using the retention times determined from the chromatogram obtained with the reference solution, locate the components of the reference solution in the chromatogram obtained with the test solution and locate cis-anethole and pseudoisoeugenyl 2-methylbutyrate using the chromatogram shown in Figure 0804.-1 (disregard any peak due to hexane). Determine the percentage content of these components. The percentages are within the following ranges : — linalol : less than 1.5 per cent, — estragole : 0.5 per cent to 5.0 per cent, — α-terpineol: less than 1.2 per cent, — cis-anethole : 0.1 per cent to 0.4 per cent, — trans-anethole : 87 per cent to 94 per cent, — anisaldehyde : 0.1 per cent to 1.4 per cent, — pseudoisoeugenyl 2-methylbutyrate : 0.3 per cent to 2.0 per cent.

1. linalol

3. α-terpineol

5. trans-anethole

2. estragole

4. cis-anethole

6. anisaldehyde

7. pseudoisoeugenyl 2-methylbutyrate

Figure 0804.-1. – Chromatogram for the test for chromatographic profile of anise oil General Notices (1) apply to all monographs and other texts

1005

Aniseed


STORAGE In a well-filled, airtight container, protected from light and at a temperature not exceeding 25 °C. 01/2005:0262

ANISEED

corresponding to anethole in the chromatograms obtained with 1 µl and 3 µl of the reference solution. The chromatograms obtained with the test solution show in the lower third a blue spot (triglycerides) similar in position to the spot in the lower third of the chromatograms obtained with the reference solution (triglycerides of olive oil).

TESTS Foreign matter (2.8.2). It complies with the test for foreign Anisi fructus matter. DEFINITION Water (2.2.13). Not more than 70 ml/kg, determined by Aniseed consists of the whole dry cremocarp of Pimpinella distillation on 20.0 g of the powdered drug. anisum L. It contains not less than 20 ml/kg of essential oil. Total ash (2.4.16). Not more than 12.0 per cent. CHARACTERS Ash insoluble in hydrochloric acid (2.8.1). Not more than Aniseed has an odour reminiscent of anethole. 2.5 per cent. The fruit is a cremocarp and generally entire ; a small ASSAY fragment of the thin, rigid, slightly curved pedicel is Carry out the determination of essential oils in vegetable frequently attached. drugs (2.8.12). Use a 250 ml round-bottomed flask, 100 ml It has the macroscopic and microscopic characters described of water R as the distillation liquid and 0.50 ml of xylene R under identification tests A and B. in the graduated tube. Reduce the drug to a coarse powder and immediately use 10.0 g for the determination. Distil at a IDENTIFICATION rate of 2.5-3.5 ml/min for 2 h. A. The cremocarp is ovoid or pyriform and slightly compressed laterally, yellowish-green or greenish-grey, STORAGE 3 mm to 5 mm long and up to 3 mm wide, surmounted Store protected from light. by a stylopod with 2 short, reflexed stylar points. The mericarps are attached by their tops to the carpophore with a plane commissural surface and a convex dorsal 01/2005:0972 surface, the latter being covered with short, warty trichomes visible using a lens ; the fruit shows 5 primary ANTAZOLINE HYDROCHLORIDE ridges, running longitudinally, comprising 3 dorsal ridges and 2 lateral ridges, non-prominent, and lighter in colour. Antazolini hydrochloridum B. Reduce to a powder (355). The powder is greenish-yellow to brownish-green. Examine under a microscope using chloral hydrate solution R. The powder shows the following diagnostic characters : whole or broken trichomes, mostly unicellular, sometimes curved, with blunt apex and warty cuticle ; fragments of epidermis with striated cuticle, occasional anomocytic stomata ; fragments of numerous narrow, branched vittae, fragments of endosperm containing aleurone grains and micro-rosettes of calcium oxalate ; oblong sclereids from C17H20ClN3 Mr 301.8 the commissural zone and bundles of sclerenchymatous DEFINITION fibres from the carpophore and the pedicel. Starch is absent. Antazoline hydrochloride contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent C. Examine by thin-layer chromatography (2.2.27), using of N-benzyl-N-[(4,5-dihydro-1H-imidazol-2-yl)methyl]aniline silica gel GF254 R as the coating substance. Test solution. Shake 0.10 g of the powdered drug (1500) hydrochloride, calculated with reference to the dried with 2 ml of methylene chloride R for 15 min. Filter and substance. carefully evaporate the filtrate to dryness on a water-bath CHARACTERS at 60 °C. Dissolve the residue in 0.5 ml of toluene R. A white or almost white, crystalline powder, sparingly Reference solution. Dissolve 3 µl of anethole R and 40 µl soluble in water, soluble in alcohol, slightly soluble in of olive oil R in 1 ml of toluene R. methylene chloride. Apply to the plate at 2 cm intervals 2 µl and 3 µl of the test It melts at about 240 °C, with decomposition. solution and 1 µl, 2 µl and 3 µl of the reference solution. Develop over a path of 10 cm using toluene R. Allow IDENTIFICATION the plate to dry in air and examine in ultraviolet light at First identification : A, D. 254 nm. The chromatograms show a quenching zone (anethole) in the central part against a light background. Second identification : B, C, D. Spray the plate with a freshly prepared 200 g/l solution A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with of phosphomolybdic acid R in alcohol R, using 10 ml antazoline hydrochloride CRS. Examine the substances for a plate 200 mm square, and heat at 120 °C for 5 min. as discs prepared using potassium chloride R. The spots corresponding to anethole appear blue against B. Examine the chromatograms obtained in the test for a yellow background. In the chromatogram obtained related substances in daylight after spraying. The with 2 µl of the test solution, the spot corresponding principal spot in the chromatogram obtained with test to anethole is intermediate in size between the spots 1006



Anticoagulant and preservative solutions for human blood

solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (b). C. To 5 ml of solution S (see Tests) add, drop by drop, dilute sodium hydroxide solution R until an alkaline reaction is produced. Filter. The precipitate, washed with two quantities, each of 10 ml, of water R and dried in a desicator under reduced pressure, melts (2.2.14) at 119 °C to 123 °C. D. It gives reaction (a) of chlorides (2.3.1).

1 ml of 0.1 M alcoholic potassium hydroxide is equivalent to 30.18 mg of C17H20ClN3.

TESTS Solution S. Dissolve 2.0 g in carbon dioxide-free water R prepared from distilled water R, heating at 60 °C if necessary. Allow to cool and dilute to 100 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y7 (2.2.2, Method II). Acidity or alkalinity. To 10 ml of solution S add 0.2 ml of methyl red solution R. Not more than 0.1 ml of 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is required to change the colour of the indicator. Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Heat the plate at 110 °C for 15 min before using. Test solution (a). Dissolve 0.10 g of the substance to be examined in methanol R and dilute to 5 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 5 ml with methanol R. Reference solution (a). Dilute 0.5 ml of test solution (a) to 100 ml with methanol R. Reference solution (b). Dissolve 20 mg of antazoline hydrochloride CRS in methanol R and dilute to 5 ml with the same solvent. Reference solution (c). Dissolve 20 mg of xylometazoline hydrochloride CRS in 1 ml of test solution (a) and dilute to 5 ml with methanol R. Apply to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 5 volumes of diethylamine R, 10 volumes of methanol R and 85 volumes of ethyl acetate R. Dry the plate in a current of warm air for 15 min. Examine in ultraviolet light at 254 nm. The test is not valid unless the chromatogram obtained with reference solution (c) shows two clearly separated principal spots. Spray with a mixture of equal volumes of a 200 g/l solution of ferric chloride R and a 5 g/l solution of potassium ferricyanide R. Examine immediately in daylight. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.5 per cent). Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C for 3 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on the residue obtained in the test for loss on drying. ASSAY Dissolve 0.250 g in 100 ml of alcohol R. Add 0.1 ml of phenolphthalein solution R1. Titrate with 0.1 M alcoholic potassium hydroxide. General Notices (1) apply to all monographs and other texts

IMPURITIES

A. N-(2-aminoethyl)-2-(benzylphenylamino)acetamide.

01/2005:0209

ANTICOAGULANT AND PRESERVATIVE SOLUTIONS FOR HUMAN BLOOD Solutiones anticoagulantes et sanguinem humanum conservantes DEFINITION Anticoagulant and preservative solutions for human blood are sterile and pyrogen-free solutions prepared with water for injections, filtered, distributed in the final containers and sterilised. The content of sodium citrate (C6H5Na3O7,2H2O), glucose monohydrate (C6H12O6,H2O) or anhydrous glucose (C6H12O6) and sodium dihydrogen phosphate dihydrate (NaH2PO4,2H2O) is not less than 95.0 per cent and not more than 105.0 per cent of that stated in the formulae below. The content of citric acid monohydrate (C6H8O7,H2O) or anhydrous citric acid (C6H8O7) is not less than 90.0 per cent and not more than 110.0 per cent of that stated in the formulae below. Subject to agreement by the competent authority, other substances, such as red-cell preservatives, may be included in the formula provided that their name and concentration are stated on the label. Anticoagulant and preservative solutions for human blood are presented in airtight, tamper-proof containers of glass (3.2.1) or plastic (3.2.3).

Anticoagulant acid-citrate-glucose solutions (ACD) A

B

Sodium citrate (0412)

22.0 g

13.2 g

Citric acid monohydrate (0456)

8.0 g

4.8 g

or Citric acid, anhydrous (0455)

7.3 g

4.4 g

24.5 g

14.7 g

Glucose monohydrate (0178)

*

22.3 g

13.4 g

Water for injections (0169) to

1000.0 ml

1000.0 ml

Volume to be used per 100 ml of blood

15.0 ml

25.0 ml

or Glucose, anhydrous (0177)

*

*

The competent authority may require that the substances comply with the test for pyrogens given in the monographs on Glucose monohydrate (0178) and Glucose, anhydrous (0177), respectively.

CHARACTERS A colourless or faintly yellow, clear liquid, practically free from particles. 1007


IDENTIFICATION A. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. Dilute 2 ml of the solution to be examined (for formula A) or 3 ml (for formula B) to 100 ml with a mixture of 2 volumes of water R and 3 volumes of methanol R. Reference solution (a). Dissolve 10 mg of glucose CRS in a mixture of 2 volumes of water R and 3 volumes of methanol R and dilute to 20 ml with the same mixture of solvents. Reference solution (b). Dissolve 10 mg each of glucose CRS, lactose CRS, fructose CRS and sucrose CRS in a mixture of 2 volumes of water R and 3 volumes of methanol R and dilute to 20 ml with the same mixture of solvents. Apply separately to the plate 2 µl of each solution and thoroughly dry the starting points. Develop over a path of 15 cm using a mixture of 10 volumes of water R, 15 volumes of methanol R, 25 volumes of anhydrous acetic acid R and 50 volumes of ethylene chloride R. The volumes of solvents have to be measured accurately since a slight excess of water produces cloudiness. Dry the plate in a current of warm air. Repeat the development immediately, after renewing the mobile phase. Dry the plate in a current of warm air and spray evenly with a solution of 0.5 g of thymol R in a mixture of 5 ml of sulphuric acid R and 95 ml of alcohol R. Heat at 130 °C for 10 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows 4 clearly separated spots. B. To 2 ml add 5 ml of cupri-citric solution R. Heat to boiling. An orange precipitate is formed and the solution becomes yellow. C. To 2 ml (for formula A) add 3 ml of water R or to 4 ml (for formula B) add 1 ml of water R. The solution gives the reaction of citrates (2.3.1). D. 0.5 ml gives reaction (b) of sodium (2.3.1). TESTS pH (2.2.3). The pH of the solution to be examined is 4.7 to 5.3. Hydroxymethylfurfural. To 2.0 ml add 5.0 ml of a 100 g/l solution of p-toluidine R in 2-propanol R containing 10 per cent V/V of glacial acetic acid R and 1.0 ml of a 5 g/l solution of barbituric acid R. The absorbance (2.2.25), determined at 550 nm after allowing the mixture to stand for 2 min to 3 min, is not greater than that of a standard prepared at the same time in the same manner using 2.0 ml of a solution containing 5 ppm of hydroxymethylfurfural R for formula A or 3 ppm of hydroxymethylfurfural R for formula B. Sterility (2.6.1). They comply with the test for sterility. Pyrogens (2.6.8). They comply with the test for pyrogens. Dilute with a pyrogen-free, 9 g/l solution of sodium chloride R to obtain a solution containing approximately 5 g/l of sodium citrate. Inject 10 ml of the diluted solution per kilogram of the rabbit’s mass.

1008


ASSAY Citric acid. To 10.0 ml (for formula A) or to 20.0 ml (for formula B) add 0.1 ml of phenolphthalein solution R1. Titrate with 0.2 M sodium hydroxide until a pink colour is obtained. 1 ml of 0.2 M sodium hydroxide is equivalent to 14.01 mg of C6H8O7,H2O or to 12.81 mg of C6H8O7. Sodium citrate. Prepare a chromatography column 0.10 m long and 10 mm in internal diameter and filled with strongly acidic ion-exchange resin R (300 µm to 840 µm). Maintain a 1 cm layer of liquid above the resin at all times. Wash the column with 50 ml of de-ionised water R at a flow rate of 12-14 ml/min. Dilute 10.0 ml of the solution to be examined (for formula A) or 15.0 ml (for formula B) to about 40 ml with de-ionised water R in a beaker and transfer to the column reservoir, washing the beaker 3 times with a few millilitres of de-ionised water R. Allow the solution to run through the column at a flow rate of 12-14 ml/min and collect the eluate. Wash the column with 2 quantities, each of 30 ml, and with one quantity of 50 ml, of de-ionised water R. The column can be used for 3 successive determinations before regeneration with 3 times its volume of dilute hydrochloric acid R. Titrate the combined eluate and washings (about 150 ml) with 0.2 M sodium hydroxide, using 0.1 ml of phenolphthalein solution R1 as indicator. Calculate the content of sodium citrate in grams per litre from the following expressions : For formula A : or For formula B : or n

=

number of millilitres of 0.2 M sodium hydroxide used in the titration,

C

=

content of citric acid monohydrate in grams per litre determined as prescribed above,

C′

=

content of anhydrous citric acid in grams per litre determined as prescribed above.

Reducing sugars. Dilute 5.0 ml (for formula A) or 10.0 ml (for formula B) to 100.0 ml with water R. Introduce 25.0 ml of the solution into a 250 ml conical flask with ground-glass neck and add 25.0 ml of cupri-citric solution R1. Add a few pieces of porous material, attach a reflux condenser, heat so that boiling begins within 2 min and boil for exactly 10 min. Cool and add 3 g of potassium iodide R dissolved in 3 ml of water R. Add 25 ml of a 25 per cent m/m solution of sulphuric acid R with caution and in small quantities. Titrate with 0.1 M sodium thiosulphate using 0.5 ml of starch solution R, added towards the end of the titration, as indicator (n1 ml). Carry out a blank titration using 25.0 ml of water R (n2 ml). Calculate the content of reducing sugars as anhydrous glucose or as glucose monohydrate, as appropriate, from the Table 0209.-1.




Table 0209.-1

Apply separately to the plate 2 µl of each solution and thoroughly dry the starting points. Develop over a path of 15 cm using a mixture of 10 volumes of water R, 15 volumes of methanol R, 25 volumes of anhydrous acetic acid R and 50 volumes of ethylene chloride R. The volumes of solvents have to be measured accurately since a slight excess of water produces cloudiness. Dry the plate in a current of warm air. Repeat the development immediately, after renewing the mobile phase. Dry the plate in a current of warm air and spray evenly with a solution of 0.5 g of thymol R in a mixture of 5 ml of sulphuric acid R and 95 ml of alcohol R. Heat at 130 °C for 10 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows 4 clearly separated spots. To 2 ml add 5 ml of cupri-citric solution R. Heat to boiling. An orange precipitate is formed and the solution becomes yellow. To 2 ml add 3 ml of water R. The solution gives the reaction of citrates (2.3.1). 1 ml gives reaction (b) of phosphates (2.3.1). 0.5 ml gives reaction (b) of sodium (2.3.1).

Anhydrous glucose in milligrams

Volume of 0.1 M sodium thiosulphate (n2−n1 ml)

Glucose monohydrate in milligrams

8

19.8

21.6

9

22.4

24.5

10

25.0

27.2

11

27.6

30.2

12

30.3

33.1

13

33.0

36.1

14

35.7

39.0

15

38.3

42.1

16

41.3

45.2

STORAGE Store in an airtight, tamper-proof container, protected from light. LABELLING The label states : — the composition and volume of the solution, — the maximum amount of blood to be collected in the container.

Anticoagulant citrate-phosphate-glucose solution (CPD) Sodium citrate (0412)

26.3 g

Citric acid monohydrate (0456)

3.27 g

or Citric acid, anhydrous (0455)

2.99 g

Glucose monohydrate (0178)*

25.5 g

or Glucose, anhydrous (0177)

*

23.2 g

Sodium dihydrogen phosphate dihydrate (0194)

2.51 g

Water for injections (0169) to

1000.0 ml

Volume to be used per 100 ml of blood

14.0 ml

The competent authority may require that the substances comply with the test for pyrogens given in the monographs on Glucose monohydrate (0178) and Glucose, anhydrous (0177), respectively.

B. C. D. E.

TESTS pH (2.2.3). The pH of the solution is 5.3 to 5.9. Hydroxymethylfurfural. To 2.0 ml add 5.0 ml of a 100 g/l solution of p-toluidine R in 2-propanol R containing 10 per cent V/V of glacial acetic acid R and 1.0 ml of a 5 g/l solution of barbituric acid R. The absorbance (2.2.25), determined at 550 nm after allowing the mixture to stand for 2 min to 3 min, is not greater than that of a standard prepared at the same time in the same manner using 2.0 ml of a solution containing 5 ppm of hydroxymethylfurfural R. Sterility (2.6.1). They comply with the test for sterility. Pyrogens (2.6.8). They comply with the test for pyrogens. Dilute with a pyrogen-free, 9 g/l solution of sodium chloride R to obtain a solution containing approximately 5 g/l of sodium citrate. Inject 10 ml of the diluted solution per kilogram of the rabbit’s mass.

*

CHARACTERS A colourless or faintly yellow, clear liquid, practically free from particles. IDENTIFICATION A. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. Dilute 2 ml of the solution to be examined to 100 ml with a mixture of 2 volumes of water R and 3 volumes of methanol R. Reference solution (a). Dissolve 10 mg of glucose CRS in a mixture of 2 volumes of water R and 3 volumes of methanol R and dilute to 20 ml with the same mixture of solvents. Reference solution (b). Dissolve 10 mg each of glucose CRS, lactose CRS, fructose CRS and sucrose CRS in a mixture of 2 volumes of water R and 3 volumes of methanol R and dilute to 20 ml with the same mixture of solvents. General Notices (1) apply to all monographs and other texts

ASSAY Sodium dihydrogen phosphate. Dilute 10.0 ml to 100.0 ml with water R. To 10.0 ml of this solution add 10.0 ml of nitro-vanado-molybdic reagent R. Mix and allow to stand at 20 °C to 25 °C for 30 min. At the same time and in the same manner, prepare a reference solution using 10.0 ml of a standard solution containing 0.219 g of potassium dihydrogen phosphate R per litre. Measure the absorbance (2.2.25) of the 2 solutions at 450 nm using as the compensation liquid a solution prepared in the same manner using 10 ml of water R. Calculate the content of sodium dihydrogen phosphate dihydrate (P) in grams per litre from the expression :

C

=

concentration of potassium dihydrogen phosphate R in the standard solution in grams per litre,

A1

=

absorbance of the test solution,

A2

=

absorbance of the reference solution. 1009

Anti-T lymphocyte immunoglobulin for human use, animal

Citric acid. To 20.0 ml add 0.1 ml of phenolphthalein solution R1 and titrate with 0.2 M sodium hydroxide.

STORAGE Store in an airtight, tamper-proof container, protected from light.

Calculate the content of citric acid monohydrate (C), or anhydrous citric acid (C′), in grams per litre from the equations :

n

=

P

=


LABELLING The label states : — the composition and volume of the solution, — the maximum amount of blood to be collected in the container.

number of millilitres of 0.2 M sodium hydroxide used in the titration, content of sodium dihydrogen phosphate dihydrate in grams per litre determined as prescribed above.

01/2005:1928

ANTI-T LYMPHOCYTE IMMUNOGLOBULIN FOR HUMAN USE, ANIMAL

Sodium citrate. Prepare a chromatography column 0.10 m long and 10 mm in internal diameter and filled with strongly acidic ion-exchange resin R (300 µm to 840 µm). Maintain Immunoglobulinum anti-T lymphocytorum a 1 cm layer of liquid above the resin at all times. Wash the ex animale ad usum humanum column with 50 ml of de-ionised water R at a flow rate of 12-14 ml/min. DEFINITION Dilute 10.0 ml of the solution to be examined to about 40 ml Anti-T lymphocyte animal immunoglobulin for human with de-ionised water R in a beaker and transfer to the use is a liquid or freeze-dried preparation containing column reservoir, washing the beaker 3 times with a few immunoglobulins, obtained from serum or plasma of millilitres of de-ionised water R. Allow the solution to run animals, mainly rabbits or horses, immunised with human through the column at a flow rate of 12-14 ml/min and collect lymphocytic antigens. the eluate. Wash the column with 2 quantities, each of 30 ml, The immunoglobulin has the property of diminishing and with one quantity of 50 ml, of de-ionised water R. The the number and function of immunocompetent cells, column can be used for 3 successive determinations before regeneration with 3 times its volume of dilute hydrochloric in particular T-lymphocytes. The preparation contains principally immunoglobulin G. It may contain antibodies acid R. Titrate the combined eluate and washings (about against other lymphocyte subpopulations and against 150 ml) with 0.2 M sodium hydroxide, using 0.1 ml of other cells. The preparation is intended for intravenous phenolphthalein solution R1 as indicator. administration, after dilution with a suitable diluent where Calculate the content of sodium citrate in grams per litre applicable. from the following expressions : Applicable provisions of the monograph on Immunosera for human use, animal (0084) are stated below.

n

=

P

=

C

=

C′

=

number of millilitres of 0.2 M sodium hydroxide used in the titration, content of sodium dihydrogen phosphate dihydrate in grams per litre determined as prescribed above, content of citric acid monohydrate in grams per litre determined as prescribed above, content of anhydrous citric acid in grams per litre determined as prescribed above.

Reducing sugars. Dilute 5.0 ml to 100.0 ml with water R. Introduce 25.0 ml of the solution into a 250 ml conical flask with ground-glass neck and add 25.0 ml of cupri-citric solution R1. Add a few pieces of porous material, attach a reflux condenser, heat so that boiling begins within 2 min and boil for exactly 10 min. Cool and add 3 g of potassium iodide R dissolved in 3 ml of water R. Add 25 ml of a 25 per cent m/m solution of sulphuric acid R with caution and in small quantities. Titrate with 0.1 M sodium thiosulphate using 0.5 ml of starch solution R, added towards the end of the titration, as indicator (n1 ml). Carry out a blank titration using 25.0 ml of water R (n2 ml). Calculate the content of reducing sugars as anhydrous glucose or as glucose monohydrate, as appropriate, from the Table 0209.-1. 1010

PRODUCTION GENERAL PROVISIONS The production method has been shown to yield consistently immunoglobulins of acceptable safety, potency in man and stability. Any reagent of biological origin used in production shall be free of contamination with bacteria, fungi and viruses. The method of preparation includes a step or steps that have been shown to remove or inactivate known agents of infection. During development studies, it shall be demonstrated that the production method yields a product that : — does not transmit infectious agents, — is characterised by a defined pattern of immunological activity, notably : antigen binding, complement-dependent and independent cytotoxicity, cytokine release, induction of T-cell activation, cell death, — does not contain antibodies that cross-react with human tissues to a degree that would impair clinical safety, — has a defined maximum content of anti-thrombocyte antibody activity, — has a defined maximum content of haemoglobin. The product has been shown, by suitable tests in animals and evaluation during clinical trials, to be well tolerated. Reference preparation. A batch shown to be suitable for checking the validity of the assay and whose efficacy has been demonstrated in clinical trials, or a batch representative thereof.




ANIMALS The animals used are of a species approved by the competent authority, are healthy and exclusively reserved for production of anti-T lymphocyte immunoglobulin. They are tested and shown to be free from a defined list of infectious agents. The introduction of animals into a closed herd follows specified procedures, including definition of quarantine measures. Where appropriate, tests for additional specific agents are considered depending on the geographical localisation of the establishment used for the breeding and production of the animals. The feed originates from a controlled source and no animal proteins are added. The suppliers of animals are certified by the competent authority. If the animals are treated with antibiotics, a suitable withdrawal period is allowed before collection of blood or plasma. The animals are not treated with penicillin antibiotics. If a live vaccine is administered, a suitable waiting period is imposed between vaccination and collection of serum or plasma for immunoglobulin production. The species, origin and identification number of the animals are specified. IMMUNISATION The antigens used are identified and characterised, where appropriate. They are identified by their names and a batch number ; information on the source and preparation are recorded. The selected animals are isolated for at least 1 week before being immunised according to a defined schedule with booster injections at suitable intervals. Adjuvants may be used. Animals are kept under general health surveillance and specific antibody production is controlled at each cycle of immunisation. Animals are thoroughly examined before collection of blood or plasma. If an animal shows any pathological lesion not related to the immunisation process, it is not used, nor are any other of the animals in the group concerned, unless it is evident that their use will not impair the safety of the product. Human antigens such as continuously growing T-lymphocyte cell lines or thymocytes are used to immunise the animals. Cells may be subjected to a sorting procedure. The immunising antigens are shown to be free from infectious agents by validated methods for relevant blood-borne pathogens, notably hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) and other relevant adventitious agents originating from the preparation of the antigen. The cells used comply with defined requirements for purity of the cell population and freedom from adventitious agents. COLLECTION OF BLOOD OR PLASMA Collection of blood is made by venepuncture or plasmapheresis. The puncture area is shaved, cleaned and disinfected. The animals may be anaesthetised under conditions that do not influence the quality of the product. No antimicrobial preservative is added to the plasma and serum samples. The blood or plasma is collected in such a manner as to maintain sterility of the product. The blood or plasma collection is conducted at a site separate from the area where the animals are kept or bred and the area where the immunoglobulin is purified. If the serum or plasma is stored before further processing, precautions are taken to avoid microbial contamination. Several single plasma or serum samples may be pooled before purification. The single or pooled samples are tested before purification for the following tests.

Tests for contaminating viruses. Each pool is tested for contaminating viruses by suitable in vitro tests including inoculation to cell cultures capable of detecting a wide range of viruses relevant for the particular product. Where applicable, in vitro tests for contaminating viruses are carried out on the adsorbed pool, after the last production stage that may introduce viral contaminants. PURIFICATION AND VIRAL INACTIVATION The immunoglobulins are concentrated and purified by fractional precipitation, chromatography, immuno-adsorption or by other suitable chemical or physical methods. The methods are selected and validated to avoid contamination at all steps of processing and to avoid formation of protein aggregates that effect immunobiological characteristics of the product. Unless otherwise justified and authorised, validated procedures are applied for removal and/or inactivation of viruses. After purification and treatment for removal and/or inactivation of viruses, a stabiliser may be added to the intermediate product, which may be stored for a period defined in the light of stability data. Only an intermediate product that complies with the following requirements may be used in the preparation of the final bulk. If the method of preparation includes a step for adsorption of cross-reacting anti-human antibodies using material from human tissues and/or red blood cells, the human materials are submitted to a validated procedure for inactivation of infectious agents, unless otherwise justified and authorised. If erythrocytes are used for adsorption, the donors for such materials comply with the requirements for donors of blood and plasma of the monograph on Human plasma for fractionation (0853). If other human material is used, it is shown by validated methods to be free from relevant blood-borne pathogens, notably HBV, HCV and HIV. If substances are used for inactivation or removal of viruses, it shall have been shown that any residues present in the final product have no adverse effects on the patients treated with the anti-T lymphocyte immunoglobulin. FINAL BULK The final bulk is prepared from a single intermediate product or from a pool of intermediate products obtained from animals of the same species. A stabiliser may be added. No antimicrobial preservative is added either during the manufacturing procedure or for preparation of the final bulk solution. During manufacturing, the solution is passed through a bacteria-retentive filter. FINAL LOT The final bulk of anti-T-lymphocyte immunoglobulin is distributed aseptically into sterile, tamper-proof containers. The containers are closed as to prevent contamination. Only a final lot that complies with the requirements prescribed below under Identification, Tests and Assay may be released for use.


1011

CHARACTERS The liquid preparation is clear or slightly opalescent and colourless or pale yellow. The freeze-dried preparation is a white or slightly yellow powder or solid friable mass, which after reconstitution gives a liquid preparation corresponding to the description above. IDENTIFICATION A. Using a suitable range of species-specific antisera, carry out precipitation tests on the preparation to be examined. It is recommended that the test be carried



System suitability : — reference solution : the principal peak corresponds to IgG monomer and there is a peak corresponding to dimer with a retention time relative to monomer of 0.85 ± 0.05, — test solution : the relative retentions of monomer and dimer are 1 ± 0.05 with reference to the corresponding peaks in the chromatogram obtained with the reference B. Examine by a suitable immunoelectrophoresis technique. solution. Using antiserum to normal serum of the animal used for Limits : production, compare this serum and the preparation to be examined, both diluted to a concentration that will allow — total monomer and dimer : at least 95 per cent of the total area of the peaks, a clear gammaglobulin precipitation arc to be obtained on the gel. The main component of the preparation to be — total polymers and aggregates : maximum 5 per cent of the total area of the peaks. examined corresponds to the IgG component of normal serum of the animal used for production. Purity. Polyacrylamide gel electrophoresis (2.2.31), under non-reducing and reducing conditions. C. The preparation complies with the assay. Resolving gel. Non-reducing conditions : 8 per cent acrylamide ; reducing conditions : 12 per cent acrylamide. TESTS Solubility. To a container of the preparation to be examined, Test solution. Dilute the preparation to be examined to a add the volume of the liquid for reconstitution stated on the protein concentration of 0.5-2 mg/ml. label. The preparation dissolves completely within the time Reference solution. Dilute the reference preparation to the stated on the label. same protein concentration as the test solution. Application : 10 µl. Extractable volume (2.9.17). It complies with the requirement for extractable volume. Detection : Coomassie staining. pH (2.2.3). The pH is within the limits approved for the Results : compared with the electropherogram of the particular product. reference solution, no additional bands are found in the electropherogram of the test solution. Osmolality (2.2.35) : minimum 240 mosmol/kg after dilution, where applicable. Anti-A and anti-B haemagglutinins (2.6.20). The 1 to 64 Proteins (2.5.33) : 90 per cent to 110 per cent of the amount dilution does not show agglutination. Where applicable, dilute the preparation to be examined as stated on the label. prescribed for use before preparing the dilutions for the test. Stabiliser. Determine the amount of stabiliser by a suitable physico-chemical method. The preparation contains not less Haemolysins. Prepare a 1 to 64 dilution of the preparation to be examined, diluted if necessary as stated on the label. than 80 per cent and not more than 120 per cent of the Take 6 aliquots of the 1 to 64 dilution. To 1 volume of 3 of quantity stated on the label. the aliquots, add 1 volume of a 10 per cent V/V suspension Distribution of molecular size. Size exclusion of group A1, group B and group O erythrocytes in a 9 g/l chromatography (2.2.30). solution of sodium chloride R, respectively. To 1 volume of the remaining 3 aliquots, add 1 volume of a 10 per cent V/V Test solution. Dilute the preparation to be examined with suspension of group A1, group B and group O erythrocytes a 9 g/l solution of sodium chloride R to a concentration in a 9 g/l solution of sodium chloride R, respectively, and suitable for the chromatographic system used. A to each aliquot 1 volume of fresh group AB serum (as a concentration in the range 2-20 g/l is usually suitable. source of complement). Mix and incubate at 37 °C for 1 h. Reference solution. Dilute human immunoglobulin BRP Examine the supernatant liquids for haemolysis. No signs of with a 9 g/l solution of sodium chloride R to the same haemolysis are present. protein concentration as the test solution. Thrombocyte antibodies. Examined by a suitable method, Column : the level of thrombocyte antibodies is shown to be below — size : l = 0.6 m, Ø = 7.5 mm, that approved for the specific product. Water (2.5.12) : maximum 3 per cent. — stationary phase : silica gel for size-exclusion chromatography R, a grade suitable for fractionation of Sterility (2.6.1). It complies with the test for sterility. globular proteins in the molecular mass range of 20 000 Pyrogens (2.6.8). Unless otherwise justified and authorised, to 200 000. it complies with the test for pyrogens. Unless otherwise Mobile phase : dissolve 4.873 g of disodium hydrogen prescribed, inject 1 ml per kilogram of the rabbit’s body mass. phosphate dihydrate R, 1.741 g of sodium dihydrogen ASSAY phosphate monohydrate R and 11.688 g of sodium chloride R in 1 litre of water R. The biological activity is determined by measuring the complement-dependent cytotoxicity on target cells. Flow Flow rate : 0.5 ml/min. cytometry is performed with read-out of dead cells stained Detection : spectrophotometer at 280 nm. using propidium iodide. The activity is expressed as the concentration of anti-T lymphocyte immunoglobulin Injection : 50-600 µg of protein. in milligrams per millilitre which mediates 50 per cent Retention time : identify the peaks in the chromatogram cytotoxicity. obtained with the test solution by comparison with the Lymphocyte separation medium. Commercial separation chromatogram obtained with the reference solution ; any media with low viscosity and a density of 1.077 g/ml. peak with a retention time shorter than that of dimer Complement. Commercial complement is suitable. corresponds to polymers and aggregates. out using antisera specific to the plasma proteins of each species of domestic animal commonly used in the preparation of materials of biological origin in the country concerned and antisera specific to human plasma proteins. The preparation is shown to contain proteins originating from the animal used for the anti-T lymphocyte immunoglobulin production.

1012




Buffered salt solution pH 7.2. Dissolve 8.0 g of sodium chloride R, 0.2 g of potassium chloride R, 3.18 g of disodium hydrogen phosphate R and 0.2 g of potassium dihydrogen phosphate R in water R and dilute to 1000.0 ml with the same solvent. Buffer solution for flow cytometry. Add 40 ml of 0.1 per cent V/V sodium azide R and 10 ml of foetal calf serum to 440 ml of buffered salt solution pH 7.2. The foetal calf serum is inactivated at 56 °C for 30 min prior to use. Store at 4 °C. Propidium iodide solution. Dissolve propidium iodide R in buffered salt solution pH 7.2, to a concentration of 1 mg/ml. Store this stock solution at 2-8 °C and use within 1 month. For the assay, dilute this solution with buffer solution for flow cytometry, to obtain a concentration of 5 µg/ml. Store at 2-8 °C and use within 3 h. Microtitre plates. Plates used to prepare immunoglobulin dilutions are U- or V-bottomed polystyrene or poly(vinyl chloride) plates without surface treatment. Micronic tubes. Suitable for flow cytometry measurement. Cell suspension. Collect blood in anticoagulant from at least one healthy donor. Immediately isolate the peripheral blood mononuclear cells (PBMC) by gradient centrifugation in lymphocyte separation medium so that the PBMC form a visible clean interface between the plasma and the separation medium. Collect the layer containing the cells and dispense into centrifuge tubes containing buffered salt solution pH 7.2. Centrifuge at 400 g at 2-8 °C for 10 min. Discard the supernatant. Suspend the cell pellet in buffer solution for flow cytometry. Repeat the centrifugation and resuspension procedure of the cells twice. After the third centrifugation, resuspend the cell pellet in 1 ml of buffer solution for flow cytometry. Determine the number and vitality of the cells using a haemocytometer. Cell viability of at least 90 per cent is required. Adjust the cell number to 7 × 106/ml by adding buffer solution for flow cytometry. Store the cell suspension at 4 °C and use within 12 h. If necessary, the first PBMC pellet may be resuspended in buffered salt solution pH 7.2 containing 20 per cent fœtal calf serum and stored overnight at 2 °C. Centrifuge at 400 g at 2-8 °C for 10 min. Discard the supernatant. Suspend the cell pellet in buffer solution for flow cytometry. Determine the number and vitality of the cells using a haemocytometer. Cell viability of at least 90 per cent is required. Adjust the cell number to 7 × 106/ml by adding buffer solution for flow cytometry. It is also possible for cells to be immediately frozen and stored in nitrogen using the following method. Buffer solution for freezing. To 20 ml of cell culture medium, add 25 ml of fœtal calf serum and 5 ml of dimethyl sulphoxide (DMSO). Store this solution at 2-8 °C and use within 3 h. 20 × 106 cells per ampoule are frozen. These ampoules are stored in liquid nitrogen. Buffer solution for thawing. To 450 ml of cell culture medium, add 50 ml of foetal calf serum. Store this solution at 2-8 °C and use within 3 h. Each ampoule is thawed in a water-bath at 37 °C with shaking. Cell suspension is repeated in a buffer solution for thawing. Centrifuge at 200 g at 2-8 °C for 10 min. Discard the supernatant. Suspend the cell pellet in buffer solution for flow cytometry. Repeat the procedure for centrifugation and resuspension of cells once. After the second centrifugation, resuspend the cells pellet in 1 ml of buffer solution for flow cytometry. Determine the number and vitality of the cells using a haemocytometer. Cell viability of at least 90 per cent

is required. Adjust the cell number to 7 × 106/ml by adding buffer solution for flow cytometry. Store the cell suspension at 4 °C and use within 3 h. Test solutions. For freeze-dried preparations, reconstitute as stated on the label. Prepare 3 independent series of not fewer than 7 dilutions using buffer solution for flow cytometry as diluent. Reference solutions. For freeze-dried preparations, reconstitute according to the instructions for use. Prepare 3 independent dilution series of not fewer than 7 dilutions using buffer solution for flow cytometry as diluent. Distribute 75 µl of each of the dilutions of the test solution or reference solution to each of a series of wells of a microtitre plate. Add 25 µl of the cell suspension of PBMC into each well. Add 25 µl of rabbit complement to each of the wells. Incubate at 37 °C for 30 min. Centrifuge the plates at 200 g at 4 °C for 8 min, discard the supernatant and keep the plate on ice. Preparation for flow cytometry measurement is done step-wise by using a certain number of wells in order to allow labelling with propidium iodide R solution and measurement within a defined time period. Resuspend carefully the cell pellet of a certain number of wells with 200 µl of propidium iodide solution. Transfer the suspension into tubes. Incubate at 25 °C for 10 min then place immediately on ice. Proceed with fluorescence measurement in a flow cytometer. Define a region including all propidium iodide-positive cells on the basis of Forward-Scattered, light (FSC) and flourescence (FL2 or FL3 for propidium iodide). Measure the percentage of propidium iodide-positive cells, without gating but excluding debris. Analyse at least 3000 cells for each of the test and reference solutions. Use the percentages of dead cells to estimate the potency as the concentration in milligrams per millilitre of the preparation to be examined necessary to induce 50 per cent of cytotoxicity by fitting a sigmoidal dose response curve to the data obtained with the test and the reference preparations and by using a 4-parameter logistic model (see, for example, chapter 5.3) and suitable software. The test is not valid unless the percentage of propidium iodide-positive cells at the lower asymptote of the curve is less then 15 per cent and the percentage of propidium iodide-positive cells at the upper asymptote of the curve is at least 80 per cent. The estimated activity is 70 per cent to 130 per cent of the activity approved for the particular product. The confidence limits (P = 0.95) are not less than 80 per cent and not more than 125 per cent of the estimated potency.


1013

STORAGE Protected from light at the temperature stated on the label. Expiry date. The expiry date is calculated from the beginning of the assay. LABELLING The label states : — for liquid preparations, the volume of the preparation in the container and the protein content, — for freeze-dried preparations : — the name and the volume of the reconstitution liquid to be added, — the quantity of protein in the container, — that the immunoserum is to be used immediately after reconstitution, — the time required for complete dissolution, — the animal species of origin,

Apomorphine hydrochloride


Specific optical rotation (2.2.7) : − 48 to − 52 (dried substance). Dissolve 0.25 g in 0.02 M hydrochloric acid and dilute to 25.0 ml with the same acid. 01/2005:0136 Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 0.25 g of the substance to be APOMORPHINE HYDROCHLORIDE examined in a 1 per cent V/V solution of glacial acetic acid R and dilute to 100.0 ml with the same solution. Apomorphini hydrochloridum Reference solution (a). Dilute 1.0 ml of the test solution to 10.0 ml with a 1 per cent V/V solution of glacial acetic acid R. Dilute 1.0 ml to 100.0 ml with a 1 per cent V/V solution of glacial acetic acid R. Reference solution (b). Dissolve 25 mg of boldine R in a 1 per cent V/V solution of glacial acetic acid R and dilute to 10.0 ml with the same solvent. To 1 ml of this solution, add 1 ml of the test solution and dilute to 10.0 ml with a 1 per cent V/V solution of glacial acetic acid R. C17H18ClNO2,1/2H2O Mr 312.8 Column : — size : l = 0.15 m, Ø = 4.6 mm, DEFINITION — stationary phase : octadecylsilyl silica gel for (6aR)-6-Methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de, chromatography R (5 µm), g]quinoline-10,11-diol hydrochloride hemihydrate. — temperature : 35 °C. Content : 99.0 per cent to 101.0 per cent (dried substance). Mobile phase : CHARACTERS — mobile phase A : 1.1 g/l solution of sodium octanesulphonate R, adjusted to pH 2.2 using a 50 per Appearance : white or slightly yellowish-brown or cent m/m solution of phosphoric acid R, green-tinged greyish, crystalline powder or crystals ; on exposure to air and light, the green tinge becomes more — mobile phase B : acetonitrile R, pronounced. Time Mobile phase A Mobile phase B Solubility : sparingly soluble in water and in alcohol, (min) (per cent V/V) (per cent V/V) practically insoluble in toluene. 0 - 30 85 → 68 15 → 32 — the name and amount of stabiliser, where applicable, — the dilution to be made before use of the product.

IDENTIFICATION First identification : B, D. Second identification : A, C, D. A. Dissolve 10.0 mg in 0.1 M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute 10.0 ml of the solution to 100.0 ml with 0.1 M hydrochloric acid. Examined between 230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 273 nm and a shoulder at 300 nm to 310 nm. The specific absorbance at the maximum is 530 to 570. B. Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of apomorphine hydrochloride. C. To 5 ml of solution S (see Tests) add a few millilitres of sodium hydrogen carbonate solution R until a permanent, white precipitate is formed. The precipitate slowly becomes greenish. Add 0.25 ml of 0.05 M iodine and shake. The precipitate becomes greyish-green. Collect the precipitate. The precipitate dissolves in ether R giving a purple solution, in methylene chloride R giving a violet-blue solution and in alcohol R giving a blue solution. D. To 2 ml of solution S add 0.1 ml of nitric acid R. Mix and filter. The filtrate gives reaction (a) of chlorides (2.3.1). TESTS Solution S. Dissolve 0.25 g without heating in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution BY5 or GY5(2.2.2, Method II). pH (2.2.3) : 4.0 to 5.0 for solution S. 1014

30 - 35

68

32

35 - 45

68 → 85

32 → 15

Flow rate : 1.5 ml/min. Detection : spectrophotometer at 280 nm. Injection : 10 µl. System suitability : reference solution (b) : — resolution : minimum 2.5 between the peaks due to boldine and apomorphine. Limits : — any impurity : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent), — total : not more than 8 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.8 per cent), — disregard limit : 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.02 per cent). Heavy metals (2.4.8) : maximum 20 ppm. 1.0 g complies with limit test C. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32) : 2.5 per cent to 4.2 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.250 g in a mixture of 5.0 ml of 0.01 M hydrochloric acid and 50 ml of alcohol R. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the first 2 points of inflexion.



Aprotinin

Development : over a path of 12 cm. Drying : in air. Detection : spray with a solution of 0.1 g of ninhydrin R STORAGE in a mixture of 6 ml of a 10 g/l solution of cupric In an airtight container, protected from light. chloride R, 21 ml of glacial acetic acid R and 70 ml of ethanol R. Dry the plate at 60 °C. IMPURITIES Results : the principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. B. Determine the ability of the substance to be examined to inhibit trypsin activity using the method described below. Test solution. Dilute 1 ml of solution S to 50 ml with buffer solution pH 7.2 R. A. (6aR)-10-methoxy-6-methyl-5,6,6a,7-tetrahydro-4HTrypsin solution. Dissolve 10 mg of trypsin BRP in dibenzo[de,g]quinolin-11-ol (apocodeine), 0.002 M hydrochloric acid and dilute to 100 ml with the same acid. B. morphine. Casein solution. Dissolve 0.2 g of casein R in buffer solution pH 7.2 R and dilute to 100 ml with the same buffer solution. 01/2005:0580 Precipitating solution. Mix 1 volume of glacial acetic acid R, 49 volumes of water R and 50 volumes of APROTININ ethanol R. Mix 1 ml of the test solution with 1 ml of the trypsin Aprotininum solution. Allow to stand for 10 min and add 1 ml of the casein solution. Incubate at 35 °C for 30 min. Cool in DEFINITION iced water and add 0.5 ml of the precipitating solution. Aprotinin is a polypeptide consisting of a chain of 58 amino Shake and allow to stand at room temperature for 15 min. acids. It inhibits stoichiometrically the activity of several The solution is cloudy. Carry out a blank test under the proteolytic enzymes such as chymotrypsin, kallikrein, same conditions using buffer solution pH 7.2 R instead of plasmin and trypsin. It contains not less than 3.0 Ph. Eur. U. the test solution. The solution is not cloudy. of aprotinin activity per milligram, calculated with reference to the dried substance. TESTS Solution S. Prepare a solution of the substance to be PRODUCTION examined containing 15 Ph. Eur. U./ml, calculated from the The animals from which aprotinin is derived must fulfil the activity stated on the label. requirements for the health of animals suitable for human consumption to the satisfaction of the competent authority. Appearance of solution. Solution S is clear (2.2.1). Absorbance (2.2.25) : maximum 0.80 by measuring at the The manufacturing process is validated to demonstrate absorption maximum at 277 nm. suitable inactivation or removal of any contamination by viruses or other infectious agents. Prepare a solution of the substance to be examined The method of manufacture is validated to demonstrate that containing 3.0 Ph. Eur. U./ml. the product, if tested, would comply with the following tests. Protein impurities of higher molecular mass. Size-exclusion Abnormal toxicity (2.6.9). Inject into each mouse a quantity chromatography (2.2.30). of the substance to be examined containing 2 Ph. Eur. U. Use cross-linked dextran for chromatography R2. Use a dissolved in a sufficient quantity of water for injections R to 180 g/l solution of anhydrous acetic acid R to swell the give a volume of 0.5 ml. gel and as the eluent. Prepare a column of gel 0.8 m to Histamine (2.6.10) : maximum 0.2 µg of histamine base per 1.0 m long and 25 mm in diameter, taking care to avoid the 3 Ph. Eur. U. introduction of air bubbles. Place at the top of the column a quantity of the substance to be examined containing CHARACTERS 300 Ph. Eur. U. dissolved in 1 ml of a 180 g/l solution of Appearance : almost white powder, hygroscopic. anhydrous acetic acid R and allow to elute. Collect the eluate in fractions of 2 ml. Measure the absorbance (2.2.25) Solubility : soluble in water and in isotonic solutions, of each fraction at the absorption maximum at 277 nm and practically insoluble in organic solvents. plot the values on a graph. The chromatogram obtained does not present an absorption maximum before the elution IDENTIFICATION of the aprotinin. A. Thin-layer chromatography (2.2.27). Loss on drying (2.2.32) : maximum 6.0 per cent, determined Test solution. Solution S (see Tests). on 0.100 g by drying in vacuo. Reference solution. Aprotinin solution BRP. Bacterial endotoxins (2.6.14) : less than 0.14 IU per Plate : TLC silica gel G plate R. European Pharmacopoeia Unit of aprotinin, if intended for Mobile phase : water R, glacial acetic acid R (80:100 V/V) use in the manufacture of parenteral dosage forms without a containing 100 g/l of sodium acetate R. further appropriate procedure for the removal of bacterial endotoxins. Application : 10 µl. 1 ml of 0.1 M sodium hydroxide is equivalent to 30.38 mg of C17H18ClNO2.


1015

Aprotinin concentrated solution


ASSAY The activity of aprotinin is determined by measuring its inhibitory action on a solution of trypsin of known activity. The inhibiting activity of the aprotinin is calculated from the difference between the initial activity and the residual activity of the trypsin. The inhibiting activity of aprotinin is expressed in European Pharmacopoeia Units. 1 Ph. Eur. U. inhibits 50 per cent of the enzymatic activity of 2 microkatals of trypsin. Use a reaction vessel with a capacity of about 30 ml and provided with : — a device that will maintain a temperature of 25 ± 0.1 °C ; — a stirring device, such as a magnetic stirrer ; — a lid with 5 holes for accommodating the electrodes, the tip of a burette, a tube for the admission of nitrogen and the introduction of the reagents. An automatic or manual titration apparatus may be used. In the latter case the burette is graduated in 0.05 ml and the pH-meter is provided with a wide reading scale and glass and calomel electrodes. Test solution. Prepare a solution of the substance to be examined in 0.0015 M borate buffer solution pH 8.0 R expected to contain 1.67 Ph. Eur. U./ml (about 0.6 mg (m mg) per millilitre). Trypsin solution. Prepare a solution of trypsin BRP containing about 0.8 microkatals per millilitre (about 1 mg/ml), using 0.001 M hydrochloric acid as the solvent. Use a freshly prepared solution and keep in iced water. Trypsin and aprotinin solution. To 4.0 ml of the trypsin solution add 1.0 ml of the test solution. Dilute immediately to 40.0 ml with 0.0015 M borate buffer solution pH 8.0 R. Allow to stand at room temperature for 10 min and then keep in iced water. Use within 6 h of preparation. Dilute trypsin solution. Dilute 0.5 ml of the trypsin solution to 10.0 ml with 0.0015 M borate buffer solution pH 8.0 R. Allow to stand at room temperature for 10 min and then keep in iced water. Maintain an atmosphere of nitrogen in the reaction flask and stir continuously ; introduce 9.0 ml of 0.0015 M borate buffer solution pH 8.0 R and 1.0 ml of a freshly prepared 6.9 g/l solution of benzoylarginine ethyl ester hydrochloride R. Adjust to pH 8.0 with 0.1 M sodium hydroxide. When the temperature has reached equilibrium at 25 ± 0.1 °C, add 1.0 ml of the trypsin and aprotinin solution and start a timer. Maintain at pH 8.0 by the addition of 0.1 M sodium hydroxide and note the volume added every 30 s. Continue the reaction for 6 min. Determine the number of millilitres of 0.1 M sodium hydroxide used per second (n1 ml). Carry out, under the same conditions, a titration using 1.0 ml of the dilute trypsin solution. Determine the number of millilitres of 0.1 M sodium hydroxide used per second (n2 ml). Calculate the aprotinin activity in European Pharmacopoeia Units per milligram from the expression :

The estimated activity is not less than 90 per cent and not more than 110 per cent of the activity stated on the label. STORAGE In an airtight, tamper-proof container, protected from light. LABELLING The label states : — the number of European Pharmacopoeia Units of aprotinin activity per milligram, 1016

— where applicable, that the substance is free from bacterial endotoxins. 01/2005:0579

APROTININ CONCENTRATED SOLUTION Aprotinini solutio concentrata DEFINITION Aprotinin concentrated solution is a solution of aprotinin, a polypeptide consisting of a chain of 58 amino acids, which inhibits stoichiometrically the activity of several proteolytic enzymes such as chymotrypsin, kallikrein, plasmin and trypsin. It contains not less than 15.0 Ph. Eur. U. of aprotinin activity per millilitre. PRODUCTION The animals from which aprotinin is derived must fulfil the requirements for the health of animals suitable for human consumption to the satisfaction of the competent authority. The manufacturing process is validated to demonstrate suitable inactivation or removal of any contamination by viruses or other infectious agents. The method of manufacture is validated to demonstrate that the product, if tested, would comply with the following tests. Abnormal toxicity (2.6.9). Inject into each mouse a quantity of the preparation to be examined containing 2 Ph. Eur. U. diluted with a sufficient quantity of water for injections R to give a volume of 0.5 ml. Histamine (2.6.10) : maximum 0.2 µg of histamine base per 3 Ph. Eur. U. CHARACTERS Appearance : clear and colourless liquid. IDENTIFICATION A. Thin-layer chromatography (2.2.27). Test solution. Solution S (see Tests). Reference solution. Aprotinin solution BRP. Plate : TLC silica gel G plate R. Mobile phase : water R, glacial acetic acid R (80:100 V/V) containing 100 g/l of sodium acetate R). Application : 10 µl. Development : over a path of 12 cm. Drying : in air. Detection : spray with a solution of 0.1 g of ninhydrin R in a mixture of 6 ml of a 10 g/l solution of cupric chloride R, 21 ml of glacial acetic acid R and 70 ml of ethanol R. Dry the plate at 60 °C. Results : the principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. B. Determine the ability of the preparation to be examined to inhibit trypsin activity using the method described below. Test solution. Dilute 1 ml of solution S to 50 ml with buffer solution pH 7.2 R. Trypsin solution. Dissolve 10 mg of trypsin BRP in 0.002 M hydrochloric acid and dilute to 100 ml with the same acid. Casein solution. Dissolve 0.2 g of casein R in buffer solution pH 7.2 R and dilute to 100 ml with the same buffer solution.


Aprotinin concentrated solution


Precipitating solution. Mix 1 volume of glacial acetic acid R, 49 volumes of water R and 50 volumes of ethanol R.

Use a reaction vessel with a capacity of about 30 ml and provided with :

Mix 1 ml of the test solution with 1 ml of the trypsin solution. Allow to stand for 10 min and add 1 ml of the casein solution. Incubate at 35 °C for 30 min. Cool in iced water and add 0.5 ml of the precipitating solution. Shake and allow to stand at room temperature for 15 min. The solution is cloudy. Carry out a blank test under the same conditions using buffer solution pH 7.2 R instead of the test solution. The solution is not cloudy.

— a stirring device, such as a magnetic stirrer ;

— a device that will maintain a temperature of 25 ± 0.1 °C ; — a lid with 5 holes for accommodating the electrodes, the tip of a burette, a tube for the admission of nitrogen and the introduction of the reagents. An automatic or manual titration apparatus may be used. In the latter case the burette is graduated in 0.05 ml and the pH-meter is provided with a wide reading scale and glass and calomel electrodes.

TESTS

Test solution. With 0.0015 M borate buffer solution pH 8.0 R Solution S. Prepare a solution containing 15 Ph. Eur. U./ml, prepare an appropriate dilution (D) of the concentrated solution expected on the basis of the stated potency to if necessary by dilution on the basis of the activity stated contain 1.67 Ph. Eur. U./ml. on the label. Appearance of solution. Solution S is clear (2.2.1). Absorbance (2.2.25) : maximum 0.80 by measuring at the absorption maximum at 277 nm.

Trypsin solution. Prepare a solution of trypsin BRP containing about 0.8 microkatals per millilitre (about 1 mg/ml), using 0.001 M hydrochloric acid as the solvent. Use a freshly prepared solution and keep in iced water.

Prepare from the concentrated solution a dilution containing Trypsin and aprotinin solution. To 4.0 ml of the trypsin 3.0 Ph. Eur. U./ml. solution add 1.0 ml of the test solution. Dilute immediately Protein impurities of higher molecular mass. Size-exclusion to 40.0 ml with 0.0015 M borate buffer solution pH 8.0 R. Allow to stand at room temperature for 10 min and then chromatography (2.2.30). keep in iced water. Use within 6 h of preparation. Freeze-dry the preparation to be examined using a pressure Dilute trypsin solution. Dilute 0.5 ml of the trypsin solution of 2.7 Pa and a temperature of − 30 °C ; the operation, to 10.0 ml with 0.0015 M borate buffer solution pH 8.0 R. including freeze-drying and a period of drying at 15-25 °C, Allow to stand at room temperature for 10 min and then takes 6-12 h. keep in iced water. Use cross-linked dextran for chromatography R2. Use a Maintain an atmosphere of nitrogen in the reaction flask and 180 g/l solution of anhydrous acetic acid R to swell the stir continuously ; introduce 9.0 ml of 0.0015 M borate buffer gel and as the eluent. Prepare a column of gel 0.8 m to solution pH 8.0 R and 1.0 ml of a freshly prepared 6.9 g/l 1.0 m long and 25 mm in diameter, taking care to avoid the solution of benzoylarginine ethyl ester hydrochloride R. introduction of air bubbles. Place at the top of the column Adjust to pH 8.0 with 0.1 M sodium hydroxide. When the a quantity of the preparation to be examined containing temperature has reached equilibrium at 25 ± 0.1 °C, add 300 Ph. Eur. U. dissolved in 1 ml of a 180 g/l solution of 1.0 ml of the trypsin and aprotinin solution and start a anhydrous acetic acid R and allow to elute. Collect the eluate in fractions of 2 ml. Measure the absorbance (2.2.25) timer. Maintain at pH 8.0 by the addition of 0.1 M sodium of each fraction at the absorption maximum at 277 nm and hydroxide and note the volume added every 30 s. Continue the reaction for 6 min. Determine the number of millilitres of plot the values on a graph. The chromatogram obtained does not present an absorption maximum before the elution 0.1 M sodium hydroxide used per second (n1 ml). Carry out, under the same conditions, a titration using 1.0 ml of the of the aprotinin. dilute trypsin solution. Determine the number of millilitres Specific activity of the dry residue : minimum 3.0 Ph. Eur. U. of 0.1 M sodium hydroxide used per second (n2 ml). of aprotinin activity per milligram of dry residue. Calculate the aprotinin activity in European Pharmacopoeia Evaporate 25.0 ml to dryness in a water-bath, dry the residue Units per millilitre from the expression : at 110 °C for 15 h and weigh. From the mass of the residue and the activity determined as described below, calculate the number of European Pharmacopoeia Units per milligram of dry residue. D = dilution factor of the aprotinin concentrated solution to be examined in order to obtain a Bacterial endotoxins (2.6.14) : less than 0.14 IU per solution containing 1.67 Ph. Eur. U./ml. European Pharmacopoeia Unit of aprotinin, if intended for use in the manufacture of parenteral dosage forms without a The estimated activity is not less than 90 per cent and not further appropriate procedure for the removal of bacterial more than 110 per cent of the activity stated on the label. endotoxins. ASSAY The activity of aprotinin is determined by measuring its inhibitory action on a solution of trypsin of known activity. The inhibiting activity of the aprotinin is calculated from the difference between the initial activity and the residual activity of the trypsin. The inhibiting activity of aprotinin is expressed in European Pharmacopoeia Units. 1 Ph. Eur. U. inhibits 50 per cent of the enzymatic activity of 2 microkatals of trypsin. General Notices (1) apply to all monographs and other texts

STORAGE In an airtight, tamper-proof container, protected from light. LABELLING The label states : — the number of European Pharmacopoeia Units of aprotinin activity per millilitre. — where applicable, that the solution is free from bacterial endotoxins. 1017

Arachis oil, hydrogenated


01/2005:1171 — linoleic acid and isomers (C18:2, equivalent chain length on poly(cyanopropyl)siloxane 19.4 to 19.8) : not more than 10.0 per cent, ARACHIS OIL, HYDROGENATED — arachidic acid : 1.0 per cent to 3.0 per cent, — eicosenoic acids (C20:1, equivalent chain length on Arachidis oleum hydrogenatum poly(cyanopropyl)siloxane 20.4 to 20.7): not more than 2.1 per cent, DEFINITION Hydrogenated arachis oil is the product obtained by refining, — behenic acid : 1.0 per cent to 5.0 per cent, — erucic acid and isomers (C22:1, equivalent chain length bleaching, hydrogenating and deodorising oil obtained on poly(cyanopropyl)siloxane 22.4 to 22.6) : not more from the shelled seeds of Arachis hypogaea L. Each type than 0.5 per cent, of hydrogenated arachis oil is characterised by its nominal drop point. — lignoceric acid : 0.5 per cent to 3.0 per cent. Nickel. Not more than 1 ppm of Ni, determined by atomic CHARACTERS absorption spectrometry (2.2.23, Method II). A white or faintly yellowish, soft mass which melts to a clear, pale yellow liquid when heated, practically insoluble in water, Test solution. Into a platinum or silica crucible previously tared after ignition introduce 5.0 g. Cautiously heat and freely soluble in methylene chloride and in light petroleum introduce into the substance a wick formed from twisted (bp : 65 °C to 70 °C), very slightly soluble in alcohol. ashless filter paper. Ignite the wick. When the substance has ignited stop heating. After combustion, ignite in a muffle IDENTIFICATION furnace at about 600 °C. Continue ignition until white ash First identification : A, B. is obtained. After cooling, take up the residue with two Second identification : A, C. quantities, each of 2 ml, of dilute hydrochloric acid R and A. It complies with the test for drop point (see Tests). transfer into a 25 ml graduated flask. Add 0.3 ml of nitric acid R and dilute to 25.0 ml with water R. B. Carry out the identification of fatty oils by thin-layer Reference solutions. Prepare three reference solutions by chromatography (2.3.2). The chromatogram obtained is adding 1.0 ml, 2.0 ml and 4.0 ml of nickel standard solution similar to the typical chromatogram for arachis oil. (0.2 ppm Ni) R to 2.0 ml of the test solution and diluting to C. It complies with the test for composition of fatty acids. 10.0 ml with water R. TESTS Measure the absorbance at 232 nm using a nickel hollow-cathode lamp as a source of radiation, a graphite Drop point (2.2.17) : 32 °C to 43 °C. Within this range furnace as an atomic generator and argon R as the carrier the drop point does not differ by more than 3 °C from the gas. nominal value. Water (2.5.12). Not more than 0.3 per cent, determined on Acid value (2.5.1). Not more than 0.5. Dissolve 10.0 g in 1.000 g by the semi-micro determination of water. 50 ml of the prescribed solvent by heating on a water-bath. Peroxide value (2.5.5). Not more than 5.0. Dissolve 5.0 g in STORAGE 30 ml of the prescribed solvent by heating on a water-bath. Store protected from light. Unsaponifiable matter (2.5.7). Not more than 1.0 per cent. LABELLING Alkaline impurities (2.4.19). It complies with the test for The label states the nominal drop point. alkaline impurities in fatty oils. Composition of fatty acids (2.4.22, Method A). 01/2005:0263 The chromatographic procedure may be carried out using : ARACHIS OIL, REFINED — a capillary fused-silica column 25 m long and 0.25 mm in internal diameter coated with poly(cyanopropyl)siloxane R (film thickness Arachidis oleum raffinatum 0.2 µm), — helium for chromatography R as the carrier gas at a flow DEFINITION The refined fatty oil obtained from the shelled seeds of rate of 0.7 ml/min, Arachis hypogaea L. A suitable antioxidant may be added. — a flame-ionisation detector, — a split injector (1:100), CHARACTERS Appearance : clear, yellowish, viscous liquid. maintaining the temperature of the column at 180 °C for 20 min and that of the injection port and of the detector at Solubility : very slightly soluble in alcohol, miscible with 250 °C. light petroleum. The fatty acid fraction of the oil has the following Relative density : about 0.915. composition : It solidifies at about 2 °C. — saturated fatty acids of chain length less than C14 : not IDENTIFICATION more than 0.5 per cent, It complies with the identification test for fatty oils by — myristic acid : not more than 0.5 per cent, thin-layer chromatography (2.3.2). The chromatogram — palmitic acid : 7.0 per cent to 16.0 per cent, obtained is similar to the typical chromatogram for arachis — stearic acid : 3.0 per cent to 19.0 per cent, oil. — oleic acid and isomers (C18:1, equivalent chain length on TESTS poly(cyanopropyl)siloxane 18.5 to 18.8): 54.0 per cent to 78.0 per cent, Acid value (2.5.1) : maximum 0.5, determined on 10.0 g. 1018


Arginine


01/2005:0806

Peroxide value (2.5.5) : maximum 5.0. Unsaponifiable matter (2.5.7) : maximum 1.0 per cent, determined on 5.0 g. Alkaline impurities (2.4.19). It complies with the test for alkaline impurities in fatty oils. Composition of fatty acids. Gas chromatography (2.4.22, Method A). Reference solution (a). Prepare 0.50 g of a mixture of the calibrating substances as indicated in Table 0263-1. Dissolve in heptane R and dilute to 50 ml with the same solvent.

Argininum

C6H14N4O2

Table 0263.-1 Calibrating substances

ARGININE

Composition (per cent m/m)

Methyl palmitate R

10

Methyl stearate R

5

Methyl oleate R

40

Methyl linoleate R

25

Methyl linolenate R

2

Methyl arachidate R

5

Methyl eicosenoate R

3

Methyl behenate R

5

Methyl erucate R

2

Methyl lignocerate R

3

Composition of the fatty acid fraction of the oil : — saturated fatty acids of chain length less than C16 : maximum 0.4 per cent, — palmitic acid : 7.0 per cent to 16.0 per cent, — stearic acid : 1.3 per cent to 6.5 per cent, — oleic acid (equivalent chain length on polyethyleneglycol adipate 18.3) : 35.0 per cent to 72.0 per cent, — linoleic acid (equivalent chain length on polyethyleneglycol adipate 18.9) : 13.0 per cent to 43.0 per cent, — linolenic acid (equivalent chain length on polyethyleneglycol adipate 19.7) : maximum 0.6 per cent, — arachidic acid : 0.5 per cent to 3.0 per cent, — eicosenoic acid (equivalent chain length on polyethyleneglycol adipate 20.3) : 0.5 per cent to 2.1 per cent, — behenic acid : 1.0 per cent to 5.0 per cent, — erucic acid (equivalent chain length on polyethyleneglycol adipate 22.3) : maximum 0.5 per cent, — lignoceric acid : 0.5 per cent to 3.0 per cent. Water (2.5.12) : maximum 0.3 per cent, determined on 3.00 g, if intended for use in the manufacture of parenteral dosage forms.

Mr 174.2

DEFINITION Arginine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of (S)-2-amino-5-guanidinopentanoic acid, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder or colourless crystals, freely soluble in water, very slightly soluble in alcohol. IDENTIFICATION First identification : A, C. Second identification : A, B, D, E. A. It complies with the test for specific optical rotation (see Tests). B. Solution S (see Tests) is strongly alkaline (2.2.4). C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with arginine CRS. Examine the substances prepared as discs. D. Examine the chromatograms obtained in the test for ninhydrin-positive substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). E. Dissolve about 25 mg in 2 ml of water R. Add 1 ml of α-naphthol solution R and 2 ml of a mixture of equal volumes of strong sodium hypochlorite solution R and water. A red colour develops.

TESTS Solution S. Dissolve 2.5 g in distilled water R and dilute to 50 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution BY6 (2.2.2, Method II). Specific optical rotation (2.2.7). Dissolve 2.00 g in hydrochloric acid R1 and dilute to 25.0 ml with the same acid. The specific optical rotation is + 25.5 to + 28.5, calculated with reference to the dried substance. Ninhydrin-positive substances. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R. Test solution (a). Dissolve 0.10 g of the substance to be examined in dilute hydrochloric acid R and dilute to 10 ml STORAGE with the same acid. In a well-filled container, protected from light. Test solution (b). Dilute 1 ml of test solution (a) to 50 ml with water R. LABELLING Reference solution (a). Dissolve 10 mg of arginine CRS in The label states : 0.1 M hydrochloric acid and dilute to 50 ml with the same — where applicable, that the substance is suitable for use in acid. the manufacture of parenteral dosage forms, Reference solution (b). Dilute 5 ml of test solution (b) to — the name and concentration of any added antioxidant. 20 ml with water R.


1019

Arginine aspartate


Reference solution (c). Dissolve 10 mg of arginine CRS and 10 mg of lysine hydrochloride CRS in 0.1 M hydrochloric acid and dilute to 25 ml with the same acid. Apply to the plate 5 µl of each solution. Allow the plate to dry in air. Develop over a path of 15 cm using a mixture of 30 volumes of concentrated ammonia R and 70 volumes of 2-propanol R. Dry the plate at 100 °C to 105 °C until the ammonia disappears completely. Spray with ninhydrin solution R and heat at 100 °C to 105 °C for 15 min. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows two clearly separated spots. Chlorides (2.4.4). To 5 ml of solution S add 0.5 ml of dilute nitric acid R and dilute to 15 ml with water R. The solution complies with the limit test for chlorides (200 ppm). Sulphates (2.4.13). To 10 ml of solution S, add 1.7 ml of dilute hydrochloric acid R and dilute to 15 ml with distilled water R. The solution complies with the limit test for sulphates (300 ppm). Ammonium (2.4.1). 50 mg complies with limit test B for ammonium (200 ppm). Prepare the standard using 0.1 ml of ammonium standard solution (100 ppm NH4) R. Iron (2.4.9). In a separating funnel, dissolve 1.0 g in 10 ml of dilute hydrochloric acid R. Shake with three quantities, each of 10 ml, of methyl isobutyl ketone R1, shaking for 3 min each time. To the combined organic layers add 10 ml of water R and shake for 3 min. The aqueous layer complies with the limit test for iron (10 ppm). Heavy metals (2.4.8). Dissolve 2.0 g in water R and dilute to 20 ml with the same solvent. 12 ml of the solution complies with limit test A for heavy metals (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

DEFINITION (2S)-2-Amino-5-guanidinopentanoic acid (2S)-2aminobutanedioate. Content : 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white granules or powder. Solubility : very soluble in water, practically insoluble in alcohol and in methylene chloride. IDENTIFICATION A. It complies with the test for specific optical rotation (see Tests). B. Infrared absorption spectrophotometry (2.2.24). Comparison : arginine aspartate CRS. C. Examine the chromatograms obtained in the test for ninhydrin-positive substances. Results : the 2 principal spots in the chromatogram obtained with test solution (b) are similar in position, colour and size to the 2 principal spots in the chromatogram obtained with reference solution (a).

TESTS Solution S. Dissolve 5.0 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y7 (2.2.2, Method II). pH (2.2.3) : 6.0 to 7.0 for solution S. Specific optical rotation (2.2.7) : + 25 to + 27 (dried substance). Dissolve 2.50 g in dilute hydrochloric acid R and dilute to 25.0 ml with the same acid. Ninhydrin-positive substances. Thin-layer chromatography (2.2.27). Test solution (a). Dissolve 0.20 g of the substance to be examined in water R and dilute to 10 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with water R. ASSAY Reference solution (a). Dissolve 25 mg of arginine R and Dissolve 0.150 g in 50 ml of water R. Using 0.2 ml of 25 mg of aspartic acid R in water R and dilute to 25 ml with methyl red mixed solution R as indicator, titrate with 0.1 M the same solvent. hydrochloric acid until the colour changes from green to Reference solution (b). Dilute 2 ml of reference solution (a) violet-red. to 50 ml with water R. 1 ml of 0.1 M hydrochloric acid is equivalent to 17.42 mg Plate : TLC silica gel G plate R. of C6H14N4O2. Mobile phase : ammonia R, propanol R (36:64 V/V). STORAGE Application : 5 µl. Store protected from light. Development : over 2/3 of the plate. Drying : at 100-105 °C for 10 min. Detection : spray with ninhydrin solution R and heat at 01/2005:2096 100-105 °C for 10 min. System suitability : reference solution (b) : ARGININE ASPARTATE — the chromatogram shows 2 clearly separated principal spots. Limit : test solution (a) : Arginini aspartas — any impurity : any spots, apart from the 2 principal spots, are not more intense than each of the 2 principal spots in the chromatogram obtained with reference solution (b) (0.2 per cent). Chlorides (2.4.4) : maximum 200 ppm. C10H21N5O6 Mr 307.3 Dilute 2.5 ml of solution S to 15 ml with water R. 1020



Arginine hydrochloride

Sulphates (2.4.13) : maximum 300 ppm. To 0.5 g add 2.5 ml of dilute hydrochloric acid R and dilute to 15 ml with distilled water R. Examine after 30 min. Ammonium (2.4.1) : maximum 100 ppm, determined on 100 mg. Heavy metals (2.4.8) : maximum 20 ppm. 12 ml of solution S complies with limit test A. Prepare the standard using lead standard solution (2 ppm Pb) R. Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 60 °C for 24 h. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g.

TESTS Solution S. Dissolve 2.5 g in distilled water R and dilute to 50 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution BY6 (2.2.2, Method II). Specific optical rotation (2.2.7). Dissolve 2.00 g in hydrochloric acid R1 and dilute to 25.0 ml with the same acid. The specific optical rotation is + 21.0 to + 23.5, calculated with reference to the dried substance. Ninhydrin-positive substances. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R. Test solution (a). Dissolve 0.10 g of the substance to be examined in water R and dilute to 10 ml with the same ASSAY solvent. Dissolve 80.0 mg in 2 ml of anhydrous formic acid R. Test solution (b). Dilute 1 ml of test solution (a) to 50 ml Add 50 ml of anhydrous acetic acid R. Titrate with with water R. 0.1 M perchloric acid, determining the end-point Reference solution (a). Dissolve 10 mg of arginine potentiometrically (2.2.20). hydrochloride CRS in water R and dilute to 50 ml with the 1 ml of 0.1 M perchloric acid is equivalent to 10.24 mg same solvent. of C10H21N5O6. Reference solution (b). Dilute 5 ml of test solution (b) to 20 ml with water R. 01/2005:0805 Reference solution (c). Dissolve 10 mg of arginine hydrochloride CRS and 10 mg of lysine hydrochloride CRS in water R and dilute to 25 ml with the same solvent. ARGININE HYDROCHLORIDE Apply to the plate 5 µl of each solution. Allow the plate to dry in air. Develop over a path of 15 cm using a mixture of Arginini hydrochloridum 30 volumes of concentrated ammonia R and 70 volumes of 2-propanol R. Dry the plate at 100 °C to 105 °C until the ammonia disappears completely. Spray with ninhydrin solution R and heat at 100 °C to 105 °C for 15 min. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot C6H15ClN4O2 Mr 210.7 in the chromatogram obtained with reference solution (b) (0.5 per cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows two clearly DEFINITION Arginine hydrochloride contains not less than 98.5 per cent separated spots. Sulphates (2.4.13). Dilute 10 ml of solution S to 15 ml with and not more than the equivalent of 101.0 per cent of the distilled water R. The solution complies with the limit test hydrochloride of (S)-2-amino-5-guanidinopentanoic acid, for sulphates (300 ppm). calculated with reference to the dried substance. Ammonium (2.4.1). 50 mg complies with limit test B for CHARACTERS ammonium (200 ppm). Prepare the standard using 0.1 ml of A white or almost white, crystalline powder or colourless ammonium standard solution (100 ppm NH4) R. crystals, freely soluble in water, very slightly soluble in Iron (2.4.9). In a separating funnel, dissolve 1.0 g in 10 ml alcohol. of dilute hydrochloric acid R. Shake with three quantities, each of 10 ml, of methyl isobutyl ketone R1, shaking for IDENTIFICATION 3 min each time. To the combined organic layers add 10 ml First identification : A, B, E. of water R and shake for 3 min. The aqueous layer complies Second identification : A, C, D, E. with the limit test for iron (10 ppm). A. It complies with the test for specific optical rotation (see Heavy metals (2.4.8). Dissolve 2.0 g in water R and dilute to Tests). 20 ml with the same solvent. 12 ml of the solution complies B. Examine by infrared absorption spectrophotometry with limit test A for heavy metals (10 ppm). Prepare the (2.2.24), comparing with the spectrum obtained with standard using lead standard solution (1 ppm Pb) R. arginine hydrochloride CRS. Examine the substances Loss on drying (2.2.32). Not more than 0.5 per cent, prepared as discs. determined on 1.000 g by drying in an oven at 100 °C to C. Examine the chromatograms obtained in the test for 105 °C. ninhydrin-positive substances. The principal spot in the Sulphated ash (2.4.14). Not more than 0.1 per cent, chromatogram obtained with test solution (b) is similar determined on 1.0 g. in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). ASSAY D. Dissolve about 25 mg in 2 ml of water R. Add 1 ml Dissolve 0.180 g in 3 ml of anhydrous formic acid R. of α-naphthol solution R and 2 ml of a mixture of Add 30 ml of anhydrous acetic acid R. Using 0.1 ml of equal volumes of strong sodium hypochlorite solution R naphtholbenzein solution R as indicator, titrate with and water R. A red colour develops. 0.1 M perchloric acid until the colour changes from brownish-yellow to green. E. About 20 mg gives reaction (a) of chlorides (2.3.1). General Notices (1) apply to all monographs and other texts

1021

Arnica flower


of lobed or elongated cells, a few stomata and trichomes of different types : covering trichomes, with very sharp ends, whose length may exceed 500 µm, consisting of one STORAGE to three proximal cells with thickened walls and two to four distal cells with thin walls ; secretory trichomes with Store protected from light. biseriate multicellular heads ; secretory trichomes with multicellular stalks and multicellular globular heads. The ligule ends in rounded papillose cells. The epidermis of 01/2005:1391 the ovary is covered with trichomes : secretory trichomes with short stalks and multicellular globular heads ; twinned covering trichomes usually consisting of two ARNICA FLOWER longitudinally united cells, with common punctuated walls ; their ends are sharp and sometimes bifid. The Arnicae flos epidermises of the calyx consist of elongated cells bearing short, unicellular, covering trichomes pointing towards DEFINITION the upper end of the bristle. The pollen grains have a Arnica flower consists of the whole or partially broken, dried diameter of about 30 µm and are rounded, with a spiny flower-heads of Arnica montana L. It contains not less than exine ; they have three germinal pores. 0.40 per cent m/m of total sesquiterpene lactones expressed as dihydrohelenalin tiglate, calculated with reference to the C. Examine the chromatograms obtained in the test for Calendula officinalis L. - Heterotheca inuloides. The dried drug. chromatogram obtained with the test solution shows, in the middle, a fluorescent blue zone corresponding to CHARACTERS the zone due to chlorogenic acid in the chromatogram It has an aromatic odour. obtained with the reference solution ; it shows, above this The capitulum when spread out, is about 20 mm in diameter zone, three fluorescent yellowish-brown to orange-yellow and about 15 mm deep, and has a peduncle of 2 cm to 3 cm zones, and above these three zones a fluorescent in length. The involucre consists of eighteen to twenty-four greenish-yellow zone corresponding to astragalin. The elongated lanceolate bracts, with acute apices, arranged in zone located below the astragalin zone corresponds one or two rows : the bracts, 8 mm to about 10 mm long, to isoquercitroside ; the zone located just below this are green with yellowish-green external hairs visible under zone corresponds to luteolin-7-glucoside. It also shows a lens. The receptacle, about 6 mm in diameter, is convex, a fluorescent greenish-blue zone below the zone due alveolate and covered with hairs. Its periphery bears about to caffeic acid in the chromatogram obtained with the twenty ligulate florets 20 mm to 30 mm long ; the disc bears reference solution. a greater number of tubular florets about 15 mm long. The ovary, 4 mm to 8 mm long, is crowned by a pappus of whitish TESTS bristles 4 mm to 8 mm long. Some brown achenes, crowned Foreign matter (2.8.2). Not more than 5.0 per cent. or not by a pappus, may be present. It has the macroscopic and microscopic characters described Calendula officinalis L. - Heterotheca inuloides. Examine by thin-layer chromatography (2.2.27), using a TLC silica under identification tests A and B. gel plate R. IDENTIFICATION Test solution. To 2.00 g of the powdered drug (710) add A. The involucre consists of elongated oval bracts with acute 10 ml of methanol R. Heat in a water-bath at 60 °C for 5 min with shaking. Cool and filter. apices ; the margin is ciliated. The ligulate floret has a reduced calyx crowned by fine, shiny, whitish bristles, Reference solution. Dissolve 2.0 mg of caffeic acid R, 2.0 mg bearing small coarse trichomes. The orange-yellow corolla of chlorogenic acid R and 5.0 mg of rutin R in methanol R bears seven to ten parallel veins and ends in three small and dilute to 30 ml with the same solvent. lobes. The stamens, with free anthers, are incompletely Apply separately to the plate, as bands, 15 µl of each developed. The narrow, brown ovary bears a stigma solution. Develop over a path of 15 cm using a mixture divided into two branches curving outwards. The tubular of 10 volumes of anhydrous formic acid R, 10 volumes floret is actinomorphic. The ovary and the calyx are of water R, 30 volumes of methyl ethyl ketone R and similar to those of the ligulate floret. The short corolla 50 volumes of ethyl acetate R. Allow the plate to dry in air for has five reflexed triangular lobes ; the five fertile stamens a few minutes. Spray with a 10 g/l solution of diphenylboric are fused at the anthers. acid aminoethyl ester R in methanol R and then with a B. Separate the capitulum into its different parts. Reduce 50 g/l solution of macrogol 400 R in methanol R. Heat for to a powder (355). Examine under a microscope using 5 min at 100 °C to 105 °C. Allow the plate to dry in air and chloral hydrate solution R. The powder shows the examine in ultraviolet light at 365 nm. The chromatogram following characteristics. The epidermises of the bracts obtained with the reference solution shows in the lower part of the involucre have stomata and trichomes, which are an orange-yellow fluorescent zone (rutin), in the middle more abundant on the outer (abaxial) surface. There part a fluorescent zone due to chlorogenic acid and in the are several different types of trichomes : uniseriate upper part a light bluish fluorescent zone (caffeic acid). The multicellular covering trichomes, varying in length from chromatogram obtained with the test solution does not show 50 µm to 500 µm, particularly abundant on the margins a fluorescent orange-yellow zone corresponding to rutin in of the bract ; secretory trichomes with uni- or biseriate the chromatogram obtained with the reference solution, nor multicellular stalks and with multicellular, globular does it show a zone below the zone corresponding to rutin. heads, about 300 µm long, abundant on the outer surface of the bract ; secretory trichomes with uniseriate Loss on drying (2.2.32). Not more than 10.0 per cent, determined on 1.000 g of the powdered drug (355) by drying multicellular stalks and with multicellular, globular heads, about 80 µm long, abundant on the inner surface in an oven at 100 °C to 105 °C for 2 h. of the bract. The epidermis of the ligulate corolla consists Total ash (2.4.16). Not more than 10.0 per cent. 1 ml of 0.1 M perchloric acid is equivalent to 21.07 mg of C6H15ClN4O2.

1022


Articaine hydrochloride


ASSAY Examine by liquid chromatography (2.2.29), using santonin R as the internal standard. Internal standard solution. Dissolve immediately before use 0.010 g of santonin R accurately weighed in 10.0 ml of methanol R. Test solution. In a 250 ml round-bottomed flask introduce 1.00 g of the powdered drug (355), add 50 ml of a mixture of equal volumes of methanol R and water R and heat under a reflux condenser in a water-bath at 50 °C to 60 °C for 30 min, shaking frequently. Allow to cool and filter through a paper filter. Add the paper filter, cut in pieces, to the residue in the round-bottomed flask, add 50 ml of a mixture of equal volumes of methanol R and water R and heat under a reflux condenser in a water-bath at 50 °C to 60 °C for 30 min, shaking frequently. Repeat this procedure twice. To the combined filtrate add 3.00 ml of the internal standard solution and evaporate to 18 ml under reduced pressure. Rinse the round-bottomed flask with water R and dilute, with the washings, to 20.0 ml. Transfer the solution to a chromatography column about 0.15 m long and about 30 mm in internal diameter containing 15 g of kieselguhr for chromatography R. Allow to stand for 20 min. Elute with 200 ml of a mixture of equal volumes of ethyl acetate R and methylene chloride R. Evaporate the eluate to dryness in a 250 ml round-bottomed flask. Dissolve the residue in 10.0 ml of methanol R and add 10.0 ml of water R. Add 7.0 g of neutral aluminium oxide R, shake for 120 s, centrifuge (at 5000 g for 10 min) and filter through a paper filter. Evaporate 10.0 ml of the filtrate to dryness. Dissolve the residue in 3.0 ml of a mixture of equal volumes of methanol R and water R and filter. The chromatographic procedure may be carried out using : — a stainless steel column 0.12 m long and 4 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (4 µm), — as mobile phase at a flow rate of 1.2 ml/min : Mobile phase A. Water R, Mobile phase B. Methanol R, Time (min)



Comment

0-3

62

38

isocratic

3 - 20

62 → 55

38 → 45

linear gradient

20 - 30

55

45

isocratic

30 - 55

55 → 45

45 → 55

linear gradient

55 - 57

45 → 0

55 → 100

linear gradient

57 - 70

0

100

isocratic

70 - 90

62

38

isocratic

— as detector a spectrophotometer set at 225 nm, — a 20 µl loop injector. Calculate the percentage of total sesquiterpene lactones, expressed as dihydrohelenalin tiglate, from the expression :

SLS SS

= area of all peaks corresponding to sesquiterpene lactones appearing after the santonin peak in the chromatogram obtained with the test solution, = area of the peak corresponding to santonin in the chromatogram obtained with the test solution,


m C V

= mass of the drug to be examined, in grams, = concentration of santonin in the internal standard solution used for the test solution, in milligrams per millilitre, = volume of the internal standard solution used for the test solution, in millilitres,

1.187 = peak correlation factor between dihydrohelenalin tiglate and santonin. STORAGE Store protected from light. 01/2005:1688

ARTICAINE HYDROCHLORIDE Articaini hydrochloridum

C13H21ClN2O3S

Mr 320.8

DEFINITION Methyl 4-methyl-3-[[(2RS)-2-(propylamino)propanoyl]amino]thiophene-2-carboxylate hydrochloride. Content : 98.5 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white or almost white, crystalline powder. Solubility : freely soluble in water and in alcohol. IDENTIFICATION First identification : B, D. Second identification : A, C, D. A. Dissolve 50.0 mg in a 1 g/l solution of hydrochloric acid R and dilute to 100.0 ml with the same acid. Dilute 5.0 ml of the solution to 100.0 ml with a 1 g/l solution of hydrochloric acid R. Examined between 200 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 272 nm. The specific absorbance at the maximum is 290 to 320. B. Infrared absorption spectrophotometry (2.2.24). Preparation : place dropwise 20 µl of the test solution on 300 mg discs. Test solution. Dissolve 0.1 g in 5 ml of water R, add 3 ml of a saturated solution of sodium hydrogen carbonate R and shake twice with 2 ml of methylene chloride R. Combine the methylene chloride layers, dilute to 5.0 ml with methylene chloride R and dry over anhydrous sodium sulphate R. Comparison : articaine hydrochloride CRS. C. Thin-layer chromatography (2.2.27). Test solution. Dissolve 20 mg of the substance to be examined in 5 ml of alcohol R. Reference solution. Dissolve 20 mg of articaine hydrochloride CRS in 5 ml of alcohol R. Plate : TLC silica gel F254 plate R. Mobile phase : triethylamine R, ethyl acetate R, heptane R (10:35:65 V/V/V). Application : 5 µl. 1023

Articaine hydrochloride


impurity G = about 1.7 ; impurity H = about 2.1 ; impurity I = about 2.6 ; impurity C = about 3.6 ; impurity J = about 4.0.

Development : over a path of 15 cm. Drying : in air. Detection : examine in ultraviolet light at 254 nm.

System suitability : reference solution (c) : Results : the principal spot in the chromatogram obtained — resolution : minimum 1.2 between the peaks due to with the test solution is similar in position and size to impurity A and impurity E. the principal spot in the chromatogram obtained with Limits : the reference solution. — impurity A : not more than the area of the corresponding D. It gives reaction (a) of chlorides (2.3.1). peak in the chromatogram obtained with reference solution (d) (0.2 per cent), TESTS — any other impurity : not more than the area of the principal peak in the chromatogram obtained with Solution S. Dissolve 0.50 g in water R and dilute to 10 ml reference solution (a) (0.1 per cent), with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution BY6 (2.2.2, Method I).

— total of other impurities : not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent),

pH (2.2.3) : 4.2 to 5.2.

— disregard limit : half the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). Heavy metals (2.4.8) : maximum 5 ppm.

Dissolve 0.20 g in carbon dioxide-free water R and dilute to 20.0 ml with the same solvent. Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 10.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (b). Dissolve 10.0 mg of articaine impurity A CRS and 5.0 mg of articaine impurity E CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (c). Add 1.0 ml of reference solution (b) to 50.0 mg of articaine hydrochloride CRS and dilute to 50 ml with the mobile phase.

Dissolve 4.0 g in 20.0 ml of water R. 12 ml of the solution complies with limit test A. Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 5 h. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.250 g in a mixture of 5.0 ml of 0.01 M hydrochloric acid and 50 ml of alcohol R. Carry out a potentiometric titration (2.2.20) using 0.1 M sodium hydroxide. Read the volume added between the 2 points of inflexion.

1 ml of 0.1 M sodium hydroxide is equivalent to 32.08 mg Reference solution (d). Dilute 1.0 ml of reference solution (b) of C13H21ClN2O3S. to 50.0 ml with the mobile phase. STORAGE Column : Protected from light. — size : l = 0.25 m, Ø = 4.6 mm, IMPURITIES — stationary phase: spherical end-capped octadecylsilyl silica gel for chromatography R (5 µm) with a specific Specified impurities : A, B, C. surface area of 335 m2/g and a carbon loading of 19 per Other detectable impurities : D, E, F, G, H, I, J. cent, — temperature : 45 °C. Mobile phase : mix 25 volumes of acetonitrile R and 75 volumes of a solution prepared as follows : dissolve 2.02 g of sodium heptanesulphonate R and 4.08 g of potassium dihydrogen phosphate R in water R and dilute to 1000 ml with the same solvent. Adjust to pH 2.0 with phosphoric acid R. A. R = CH3, R′ = H : methyl 3-[[2-(propylamino)acetyl]amino]4-methylthiophene-2-carboxylate (acetamidoarticaine),

Flow rate : 1 ml/min. Detection : spectrophotometer at 276 nm. Injection : 10 µl ; inject the test solution and reference solutions (a), (c) and (d). Run time : 5 times the retention time of articaine.

B. R = H, R′ = CH3 : 4-methyl-3-[[(2RS)-2(propylamino)propanoyl]amino]thiophene-2-carboxylic acid (articaine acid),

Relative retentions with reference to articaine (retention time = about 9.3 min) : impurity B = about 0.6 ; impurity D = about 0.7 ; impurity A = about 0.8 ; impurity E = about 0.86 ; impurity F = about 0.9 ;

C. R = CH(CH3)2, R′ = CH3 : 1-methylethyl 4-methyl-3-[[(2RS)2-(propylamino)propanoyl]amino]thiophene-2-carboxylate (articaine isopropyl ester),

1024



D. R1 = CH2-CH3, R2 = H, R3 = OCH3 : methyl 3-[[(2RS)-2-(ethylamino)propanoyl]amino]-4methylthiophene-2-carboxylate (ethylarticaine), E. R1 = CH(CH3)2, R2 = H, R3 = OCH3 : methyl 4-methyl-3-[[(2RS)-2-[(1-methylethyl)amino]propanoyl]amino]thiophene-2-carboxylate (isopropylarticaine), F. R1 = CH2-CH2-CH3, R2 = H, R3 = NH-CH2CH2-CH3 : 4-methyl-N-propyl-3-[[(2RS)-2(propylamino)propanoyl]amino]thiophene-2-carboxamide (articaine acid propionamide), G. R1 = (CH2)3-CH3, R2 = H, R3 = OCH3 : methyl 3-[[(2RS)-2-(butylamino)propanoyl]amino]-4methylthiophene-2-carboxylate (butylarticaine),

Ascorbic acid

IDENTIFICATION First identification : B, C. Second identification : A, C, D. A. Dissolve 0.10 g in water R and dilute immediately to 100.0 ml with the same solvent. To 10 ml of 0.1 M hydrochloric acid, add 1.0 ml of the solution and dilute to 100.0 ml with water R. Measure the absorbance (2.2.25) at the maximum at 243 nm immediately after dissolution. The specific absorbance at the maximum is 545 to 585. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with ascorbic acid CRS. Examine the substance prepared as discs containing 1 mg. C. The pH (2.2.3) of solution S (see Tests) is 2.1 to 2.6. D. To 1 ml of solution S add 0.2 ml of dilute nitric acid R and 0.2 ml of silver nitrate solution R2. A grey precipitate is formed.

TESTS Solution S. Dissolve 1.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. H. R1 = R2 = CH2-CH2-CH3, R3 = OCH3 : methyl 3-[[(2RS)-2-(dipropylamino)propanoyl]amino]-4Appearance of solution. Solution S is clear (2.2.1) and not methylthiophene-2-carboxylate (dipropylarticaine), more intensely coloured than reference solution BY7 (2.2.2, Method II). Specific optical rotation (2.2.7). Dissolve 2.50 g in water R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 20.5 to + 21.5. Oxalic acid. Dissolve 0.25 g in 5 ml of water R. Neutralise to red litmus paper R using dilute sodium hydroxide solution R and add 1 ml of dilute acetic acid R and 0.5 ml I. methyl 3-amino-4-methylthiophene-2-carboxylate of calcium chloride solution R (test solution). Prepare a (3-aminoarticaine), reference solution as follows : dissolve 70 mg of oxalic acid R in water R and dilute to 500 ml with the same solvent ; to 5 ml of this solution add 1 ml of dilute acetic acid R and 0.5 ml of calcium chloride solution R (reference solution). Allow the solutions to stand for 1 h. Any opalescence in the test solution is not more intense than that in the reference solution (0.2 per cent). Copper. Not more than 5 ppm of Cu, determined by atomic J. methyl 3-[[(2RS)-2-bromopropanoyl]amino]-4absorption spectrometry (2.2.23, Method I). methylthiophene-2-carboxylate (bromo compound). Test solution. Dissolve 2.0 g of the substance to be examined 01/2005:0253 in 0.1 M nitric acid and dilute to 25.0 ml with the same acid. Reference solutions. Prepare reference solutions containing 0.2 ppm, 0.4 ppm and 0.6 ppm of Cu by diluting copper ASCORBIC ACID standard solution (10 ppm Cu) R with 0.1 M nitric acid. Measure the absorbance at 324.8 nm using a copper Acidum ascorbicum hollow-cathode lamp as a source of radiation and an air-acetylene flame. Adjust the zero of the apparatus using 0.1 M nitric acid. Iron. Not more than 2 ppm of Fe, determined by atomic absorption spectrometry (2.2.23, Method I). Test solution. Dissolve 5.0 g of the substance to be examined in 0.1 M nitric acid and dilute to 25.0 ml with the same acid. C 6 H 8 O6 Mr 176.1 Reference solutions. Prepare reference solutions containing 0.2 ppm, 0.4 ppm and 0.6 ppm of Fe by diluting iron DEFINITION standard solution (20 ppm Fe) R with 0.1 M nitric acid. Ascorbic acid contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent of (5R)-5-[(1S)-1, Measure the absorbance at 248.3 nm using an iron hollow-cathode lamp as a source of radiation and an 2-dihydroxyethyl]-3,4-dihydroxyfuran-2(5H)-one. air-acetylene flame. Adjust the zero of the apparatus using CHARACTERS 0.1 M nitric acid. A white or almost white, crystalline powder or colourless Heavy metals (2.4.8). Dissolve 2.0 g in water R and dilute to crystals, becoming discoloured on exposure to air and 20 ml with the same solvent. 12 ml of the solution complies moisture, freely soluble in water, soluble in alcohol. with limit test A for heavy metals (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. It melts at about 190 °C, with decomposition. General Notices (1) apply to all monographs and other texts

1025

Ascorbyl palmitate




ASSAY Dissolve 0.150 g in a mixture of 10 ml of dilute sulphuric acid R and 80 ml of carbon dioxide-free water R. Add 1 ml of starch solution R. Titrate with 0.05 M iodine until a persistent violet-blue colour is obtained. 1 ml of 0.05 M iodine is equivalent to 8.81 mg of C6H8O6.

ASSAY Dissolve 0.160 g in 50 ml of methanol R. Add 30 ml of water R and 1 ml of starch solution R. Titrate with 0.05 M iodine until a persistent violet-blue colour is obtained. 1 ml of 0.05 M iodine is equivalent to 20.73 mg of C22H38O7.

STORAGE Store in a non-metallic container, protected from light.

STORAGE Store in an airtight container, protected from light, at a temperature of 8 °C to 15 °C. 01/2005:1600

01/2005:0807

ASCORBYL PALMITATE

Fraxini folium

Ascorbylis palmitas

C22H38O7

ASH LEAF DEFINITION Dried leaf of Fraxinus excelsior L. or Fraxinus oxyphylla M. Bieb. Content : minimum 2.5 per cent of total hydroxycinnamic acid derivatives expressed as chlorogenic acid (C16H18O9 ; Mr 354.3) (dried drug).

CHARACTERS Macroscopic and microscopic characters described under Mr 414.5 identification tests A and B.

IDENTIFICATION A. The leaf consists of leaflets which are sometimes detached and separated from the rachis. The leaflet is about 6 cm long and 3 cm wide. Each leaflet is subsessile or shortly petiolate, oblong, lanceolate, somewhat unequal at the base, acuminate at the apex, with fine acute teeth on the margins, the upper surface is dark green and the lower CHARACTERS surface is greyish-green. The midrib and secondary veins are whitish and prominent on the lower surface. A white or yellowish-white powder, practically insoluble in water, freely soluble in alcohol and in methanol, practically B. Reduce to a powder (355). The powder is greyish-green. insoluble in methylene chloride and in fatty oils. Examined under a microscope, using chloral hydrate solution R. The powder shows fragments of the lamina in IDENTIFICATION surface view, with the lower epidermis showing numerous anomocytic stomata (2.8.3.) and some of the cells of the A. It complies with the test for specific optical rotation (see upper epidermis with cuticular striations ; occasional Tests). uniseriate, conical covering trichomes composed of 1 or 2 B. Examine by infrared absorption spectrophotometry cells with thick walls and a striated cuticle ; rare peltate (2.2.24), comparing with the Ph. Eur. reference spectrum glands with a unicellular stalk and a shield-like glandular of ascorbyl palmitate. head composed of 8 radiating cells ; groups of fibres and C. Dissolve about 10 mg in 5 ml of methanol R. The solution fragments of vascular tissue from the veins. decolourises dichlorophenolindophenol standard C. Examine the chromatograms obtained in the test for solution R. Fraxinus ornus L. TESTS Results : see below the sequence of the zones present in the chromatograms obtained with the reference and Solution S. Dissolve 2.50 g in methanol R and dilute to test solutions. Furthermore, other fluorescent zones 25.0 ml with the same solvent. are present in the chromatogram obtained with the test Appearance of solution. Solution S is clear (2.2.1) and not solution. more intensely coloured than reference solution BY4 (2.2.2, Top of the plate Method I). A light blue fluorescence zone Specific optical rotation (2.2.7) : + 21 to + 24, determined on solution S and calculated with reference to the dried An intense blue fluorescent zone substance. (acteoside) Chlorogenic acid : a blue A blue fluorescent zone Heavy metals (2.4.8). 2.0 g complies with limit test C for fluorescent zone (chlorogenic acid) heavy metals (10 ppm). Prepare the standard using 2 ml of Rutin : an orange fluorescent An orange fluorescent zone lead standard solution (10 ppm Pb) R. zone (rutin) Loss on drying (2.2.32). Not more than 1.0 per cent, Reference solution Test solution determined on 1.000 g by drying in vacuo at 60 °C for 5 h. DEFINITION Ascorbyl palmitate contains not less than 98.0 per cent and not more than the equivalent of 100.5 per cent of (2S)-2-[(5R)-3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl]-2hydroxyethyl hexadecanoate, calculated with reference to the dried substance.

1026


Asparagine monohydrate


01/2005:2086

TESTS Foreign matter (2.8.2) : maximum 3.0 per cent of stems and 2.0 per cent of other foreign matter. Fraxinus ornus L. Thin-layer chromatography (2.2.27). Test solution. To 1 g of the powdered drug (355) add 20 ml of methanol R. Heat with shaking at 40 °C for 10 min. Filter. Reference solution. Dissolve 5.0 mg of rutin R and 5.0 mg of chlorogenic acid R in 10 ml of methanol R. Plate : TLC silica gel plate R. Mobile phase : anhydrous formic acid R, water R, ethyl acetate R (10:10:80 V/V/V). Application : 10 µl as bands. Development : over a path of 10 cm. Drying : in air.

ASPARAGINE MONOHYDRATE Asparaginum monohydricum

C4H8N2O3,H2O

Mr 150.1

DEFINITION (2S)-2,4-Diamino-4-oxobutanoic acid monohydrate. Content : 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white, crystalline powder or colourless crystals. Solubility : slightly soluble in water, practically insoluble in alcohol and in methylene chloride.

Detection : spray with a solution containing 10 g/l of diphenylboric acid aminoethyl ester R and 50 g/l of macrogol 400 R in methanol R. Examine in ultraviolet light at 365 nm.

IDENTIFICATION First identification : A, B. Second identification : A, C. A. It complies with the test for specific optical rotation (see Results : in the case of a substitution by F. ornus, the Tests). chromatogram obtained with the test solution does not show B. Infrared absorption spectrophotometry (2.2.24). the zones of acteoside, chlorogenic acid and rutin. Comparison : asparagine monohydrate CRS. Loss on drying (2.2.32) : maximum 10.0 per cent, determined C. Examine the chromatograms obtained in the test for on 1.000 g of the powdered drug (355) by drying in an oven ninhydrin-positive substances. at 100-105 °C for 2 h. Results : the principal spot in the chromatogram obtained Total ash (2.4.16) : maximum 12.0 per cent. with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (c). ASSAY TESTS Test solution (a). To 0.300 g of the powdered drug (355) add Solution S. Dissolve with heating 2.0 g in carbon dioxide-free 95 ml of alcohol (50 per cent V/V) R. Boil in a water-bath water R and dilute to 100 ml with the same solvent. under a reflux condenser for 30 min. Allow to cool and filter. Appearance of solution. Solution S is clear (2.2.1) and Rinse the filter with 5 ml of alcohol (50 per cent V/V) R. colourless (2.2.2, Method II). Combine the filtrate and the rinsings in a volumetric flask and dilute to 100.0 ml with alcohol (50 per cent V/V) R. pH (2.2.3) : 4.0 to 6.0 for solution S. Test solution (b). To 1.0 ml of test solution (a) in a test tube, Specific optical rotation (2.2.7) : + 33.7 to + 36.0 (dried substance). add 2 ml of 0.5 M hydrochloric acid, 2 ml of a solution prepared by dissolving 10 g of sodium nitrite R and 10 g of Dissolve 2.50 g in a 309.0 g/l solution of hydrochloric sodium molybdate R in 100 ml of water R, then add 2 ml of acid R and dilute to 25.0 ml with the same acid. dilute sodium hydroxide solution R and dilute to 10.0 ml Ninhydrin-positive substances. Thin-layer chromatography with water R ; mix. (2.2.27). Test solution (a). Dissolve 0.25 g of the substance to be Immediately measure the absorbance (2.2.25) of test examined in water R, heating to not more than 40 °C, and solution (b) at 525 nm, using as compensation liquid a dilute to 10 ml with the same solvent. solution prepared as follows : mix 1.0 ml of test solution (a), Test solution (b). Dilute 1 ml of test solution (a) to 10 ml 2 ml of 0.5 M hydrochloric acid and 2 ml of dilute sodium with water R. hydroxide solution R and dilute to 10.0 ml with water R. Reference solution (a). Dilute 1.0 ml of test solution (a) to Calculate the percentage content of total hydroxycinnamic 200 ml with water R. acid derivatives expressed as chlorogenic acid, from the Reference solution (b). Dissolve 25 mg of glutamic acid R expression : in water R, add 1 ml of test solution (a) and dilute to 10 ml with water R. Reference solution (c). Dissolve 25 mg of asparagine monohydrate CRS in water R and dilute to 10 ml with the taking the specific absorbance of chlorogenic acid at 525 nm same solvent. Plate : TLC silica gel G plate R. to be 188. Mobile phase : glacial acetic acid R, water R, butanol R A = absorbance at 525 nm, (25:25:50 V/V/V). m = mass of the test sample in grams. Application : 5 µl. General Notices (1) apply to all monographs and other texts

1027

Aspartame



Development : over half of the plate. Drying : at 110 °C for 15 min.

ASPARTAME

Detection : spray with ninhydrin solution R and heat at 110 °C for 10 min.

Aspartamum

System suitability : reference solution (b) : — the chromatogram shows 2 clearly separated principal spots. Limit : test solution (a) : — any impurity : any spot, apart from the principal spot, is not more intense than the principal spot in the chromatogram obtained with reference solution (a) (0.5 per cent). Chlorides (2.4.4) : maximum 200 ppm.

C14H18N2O5

Mr 294.3

DEFINITION Aspartame contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of (3S)-3-amino-4Sulphates (2.4.13) : maximum 200 ppm. [[(1S)-1-benzyl-2-methoxy-2-oxoethyl]amino]-4-oxobutanoic To 0.75 g add 2.5 ml of dilute hydrochloric acid R and dilute acid, calculated with reference to the dried substance. to 15 ml with distilled water R. Examine after 30 min. CHARACTERS Ammonium (2.4.1, Method B) : maximum 0.1 per cent, A white, crystalline powder, slightly hygroscopic, sparingly determined on 10 mg. soluble or slightly soluble in water and in alcohol, practically Iron (2.4.9) : maximum 10 ppm. insoluble in hexane and in methylene chloride. Dilute 12.5 ml of solution S to 15 ml with water R.

IDENTIFICATION First identification : B. Second identification : A, C, D. A. Dissolve 0.1 g in alcohol R and dilute to 100 ml with the same solvent. Examined between 230 nm and 300 nm Heavy metals (2.4.8) : maximum 10 ppm. (2.2.25), the solution shows absorption maxima at 247 nm, 252 nm, 258 nm and 264 nm. Dissolve 2.0 g in a mixture of 3 ml of dilute hydrochloric B. Examine by infrared absorption spectrophotometry acid R and 15 ml of water R with gentle warming if necessary. (2.2.24), comparing with the spectrum obtained with Dilute to 20 ml with water R. 12 ml of the solution complies aspartame CRS. Examine the substances prepared as with limit test A. Prepare the standard using lead standard discs. solution (1 ppm Pb) R. C. Examine by thin-layer chromatography (2.2.27), using Loss on drying (2.2.32) : 10.5 per cent to 12.5 per cent, silica gel G R as the coating substance. determined on 1.000 g by drying in an oven at 60 °C for 24 h. Test solution. Dissolve 15 mg of the substance to be Sulphated ash (2.4.14) : maximum 0.1 per cent, determined examined in 2.5 ml of water R and dilute to 10 ml with on 1.0 g. acetic acid R. Reference solution. Dissolve 15 mg of aspartame CRS in 2.5 ml of water R and dilute to 10 ml with acetic acid R. ASSAY Apply to the plate 20 µl of each solution. Develop over a Dissolve 0.110 g in 5 ml of anhydrous formic acid R. path of 15 cm using a mixture of 2 volumes of water R, Add 50 ml of anhydrous acetic acid R. Titrate with 4 volumes of anhydrous formic acid R, 30 volumes of 0.1 M perchloric acid, determining the end-point methanol R and 64 volumes of methylene chloride R. potentiometrically (2.2.20). Allow the plate to dry in air. Spray with ninhydrin solution R and heat at 100 °C to 105 °C for 15 min. The 1 ml of 0.1 M perchloric acid is equivalent to 13.21 mg spot in the chromatogram obtained with the test solution of C4H8N2O3. is similar in position, colour and size to the spot in the chromatogram obtained with the reference solution. D. Dissolve about 20 mg in 5 ml of methanol R and add IMPURITIES 1 ml of alkaline hydroxylamine solution R1. Heat on a Specified impurities : A, B. water-bath for 15 min. Allow to cool and adjust to about pH 2 with dilute hydrochloric acid R. Add 0.1 ml of ferric chloride solution R1. A brownish-red colour is produced. Dissolve 1.0 g in dilute hydrochloric acid R and dilute to 10 ml with the same acid. Shake 3 times with 10 ml of methyl isobutyl ketone R1 for 3 min. Wash the combined organic phases with 10 ml of water R for 3 min. The aqueous phase complies with the limit test for iron.

A. aspartic acid,

B. glutamic acid. 1028

TESTS Solution S. Dissolve 0.8 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution GY6 (2.2.2, Method II). See the information section on general monographs (cover pages)


Aspartic acid

Conductivity (2.2.38). Not more than 30 µS·cm− 1. Dissolve 0.80 g in carbon dioxide-free water R prepared from distilled water R and dilute to 100.0 ml with the same solvent. Measure the conductivity of the solution (C1) and that of the water used for preparing the solution (C2). The readings must be stable within 1 per cent over a period of 30 s. Calculate the conductivity of the solution of the substance to be examined from the expression :

is not greater than the area of the principal peak in the chromatogram obtained with reference solution (c) (1.5 per cent). Disregard any peak due to the solvent. Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (10 ppm). Prepare the standard using 1 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 4.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.2 per cent, Specific optical rotation (2.2.7). Dissolve 2.00 g in a 690 g/l determined on 1.0 g. solution of anhydrous formic acid R and dilute to 50.0 ml with the same solution. The specific optical rotation is + 14.5 ASSAY to + 16.5, measured within 30 min of preparation of the Dissolve 0.250 g in 1.5 ml of anhydrous formic acid R solution and calculated with reference to the dried substance. and 60 ml of anhydrous acetic acid R. Titrate immediately with 0.1 M perchloric acid, determining the end-point Related substances. Examine by liquid chromatography potentiometrically (2.2.20). (2.2.29). 1 ml of 0.1 M perchloric acid is equivalent to 29.43 mg of Test solution. Dissolve 0.60 g of the substance to be examined in a mixture of 1.5 volumes of glacial acetic acid R C14H18N2O5. and 98.5 volumes of water R and dilute to 100.0 ml with the STORAGE same mixture of solvents. Store in an airtight container. Reference solution (a). Dissolve 4.5 mg of aspartame impurity A CRS in a mixture of 1.5 volumes of glacial acetic IMPURITIES acid R and 98.5 volumes of water R and dilute to 50.0 ml A. 2-(5-benzyl-3,6-dioxopiperazin-2-yl)acetic acid with the same mixture of solvents. (diketopiperazine), Reference solution (b). Dissolve 30.0 mg of phenylalanine R in a mixture of 15 volumes of glacial acetic acid R and B. L-aspartyl-L-phenylalanine, 85 volumes of water R and dilute to 100.0 ml with the same mixture of solvents. Dilute 1.0 ml of the solution to 10.0 ml C. phenylalanine. with water R. Reference solution (c). Dilute 5.0 ml of the test solution 01/2005:0797 to 10.0 ml with water R. Dilute 3.0 ml of the solution to 100.0 ml with water R. ASPARTIC ACID Reference solution (d). Dissolve 30.0 mg of L-aspartyl-L-phenylalanine R in a mixture of 15 volumes of Acidum asparticum glacial acetic acid R and 85 volumes of water R and dilute to 100.0 ml with the same mixture of solvents. Dilute 1.0 ml of the solution to 10.0 ml with water R. Mix 1.0 ml of this solution with 1.0 ml of reference solution (b). The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.0 mm in C4H7NO4 Mr 133.1 internal diameter packed with octadecylsilyl silica gel for DEFINITION chromatography R (5 µm to 10 µm), Aspartic acid contains not less than 98.5 per cent — as mobile phase at a flow rate of 1 ml/min a mixture and not more than the equivalent of 101.5 per cent of of 10 volumes of acetonitrile R and 90 volumes of a (2S)-2-aminobutanedioic acid, calculated with reference to 6.8 g/l solution of potassium dihydrogen phosphate R the dried substance. previously adjusted to pH 3.7 with phosphoric acid R, — as detector a spectrophotometer set at 220 nm. Inject 20 µl of reference solution (c). Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is not less than 50 per cent of the full scale of the recorder. Inject 20 µl of reference solution (d). The test is not valid unless in the chromatogram obtained, the resolution between the peaks due to phenylalanine and L-aspartyl-L-phenylalanine is at least 3.5. Inject separately 20 µl of each solution. Continue the chromatography for twice the retention time of aspartame. In the chromatogram obtained with the test solution : the area of any peak corresponding to aspartame impurity A is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (1.5 per cent) ; the area of any peak corresponding to phenylalanine is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent) ; the sum of the areas of any other peaks, apart from the principal peak, General Notices (1) apply to all monographs and other texts

CHARACTERS A white or almost white, crystalline powder or colourless crystals, slightly soluble in water, practically insoluble in alcohol. It dissolves in dilute mineral acids and in dilute solutions of alkali hydroxides. IDENTIFICATION First identification : A, C. Second identification : A, B, D. A. It complies with the test for specific optical rotation (see Tests). B. A suspension of 1 g in 10 ml of water R is strongly acid (2.2.4). C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with aspartic acid CRS. Examine the substances prepared as discs. 1029

Astemizole


D. Examine the chromatograms obtained in the test for ninhydrin-positive substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). TESTS Appearance of solution. Dissolve 0.5 g in 1 M hydrochloric acid and dilute to 10 ml with the same acid. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY6 (2.2.2, Method II). Specific optical rotation (2.2.7). Dissolve 2.000 g in hydrochloric acid R1 and dilute to 25.0 ml with the same acid. The specific optical rotation is + 24.0 to + 26.0, calculated with reference to the dried substance. Ninhydrin-positive substances. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R. Test solution (a). Dissolve 0.10 g of the substance to be examined in 2 ml of ammonia R and dilute to 10 ml with water R. Test solution (b). Dilute 1 ml of test solution (a) to 50 ml with water R. Reference solution (a). Dissolve 10 mg of aspartic acid CRS in 2 ml of dilute ammonia R1 and dilute to 50 ml with water R. Reference solution (b). Dilute 5 ml of test solution (b) to 20 ml with water R. Reference solution (c). Dissolve 10 mg of aspartic acid CRS and 10 mg of glutamic acid CRS in 2 ml of dilute ammonia R1 and dilute to 25 ml with water R. Apply separately to the plate 5 µl of each solution. Allow the plate to dry in air. Develop over a path of 15 cm using a mixture of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes of butanol R. Allow the plate to dry in air, spray with ninhydrin solution R. Heat at 100-105 °C for 15 min. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows 2 clearly separated principal spots. Chlorides (2.4.4). Dissolve 0.25 g in 3 ml of dilute nitric acid R and dilute to 15 ml with water R. The solution, to which 1 ml of water R is added instead of dilute nitric acid R, complies with the limit test for chlorides (200 ppm). Sulphates (2.4.13). Dissolve 0.5 g in 4 ml of hydrochloric acid R and dilute to 15 ml with distilled water R. The solution complies with the limit test for sulphates (300 ppm). Carry out the evaluation of the test after 30 min. Ammonium.(2.4.1) 50 mg complies with limit test B (200 ppm). Prepare the standard using 0.1 ml of ammonium standard solution (100 ppm NH4) R. Iron (2.4.9). In a separating funnel, dissolve 1.0 g in 10 ml of dilute hydrochloric acid R. Shake with 3 quantities, each of 10 ml, of methyl isobutyl ketone R1, shaking for 3 min each time. To the combined organic layers add 10 ml of water R and shake for 3 min. The aqueous layer complies with the limit test for iron (10 ppm). Heavy metals (2.4.8). 2.0 g complies with limit test D (10 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. 1030

ASSAY Dissolve 0.100 g in 50 ml of carbon dioxide-free water R, with slight heating if necessary. Cool and add 0.1 ml of bromothymol blue solution R1. Titrate with 0.1 M sodium hydroxide until the colour changes from yellow to blue. 1 ml of 0.1 M sodium hydroxide is equivalent to 13.31 mg of C4H7NO4. STORAGE Protected from light. 01/2005:1067

ASTEMIZOLE Astemizolum

C28H31FN4O

Mr 458.6

DEFINITION Astemizole contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of 1-(4-fluorobenzyl)-N-[1-[2-(4-methoxyphenyl)ethyl]piperidin-4yl]-1H-benzimidazol-2-amine, calculated with reference to the dried substance. CHARACTERS A white or almost white powder, practically insoluble in water, freely soluble in methylene chloride and in methanol, soluble in alcohol. IDENTIFICATION First identification : A, B. Second identification : A, C, D. A. Melting point (2.2.14) : 175 °C to 178 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with astemizole CRS. Examine the substances prepared as discs. C. Examine by thin layer chromatography (2.2.27), using a suitable octadecylsilyl silica gel as the coating substance. Test solution. Dissolve 30 mg of the substance to be examined in methanol R and dilute to 5 ml with the same solvent. Reference solution (a). Dissolve 30 mg of astemizole CRS in methanol R and dilute to 5 ml with the same solvent. Reference solution (b). Dissolve 30 mg of astemizole CRS and 30 mg of ketoconazole CRS in methanol R and dilute to 5 ml with the same solvent. Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 20 volumes of ammonium acetate solution R, 40 volumes of dioxan R and 40 volumes of methanol R. Dry the plate in a current of warm air for 15 min and expose it to iodine vapour until the spots appear. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the


Astemizole


chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. D. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is obtained (usually less than 5 min). Allow to cool, add 1 ml of water R, 0.05 ml of phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the solution colourless. Filter. To a freshly prepared mixture of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R, add 1 ml of the filtrate. Mix, allow to stand for 5 min and compare the colour of the solution with that of a blank prepared in the same manner. The test solution is yellow and the blank is red. TESTS Appearance of solution. Dissolve 0.2 g in methanol R and dilute to 20 ml with the same solvent. The solution is clear (2.2.1) and is not more intensely coloured than reference solution Y7 (2.2.2, Method II). Related substances. Examine by liquid chromatography (2.2.29). Prepare the solutions immediately before use. Test solution. Dissolve 0.100 g of the substance to be examined in methanol R and dilute to 10.0 ml with the same solvent. Reference solution (a). Dissolve 2.5 mg of astemizole CRS and 25 mg of ketoconazole CRS in methanol R and dilute to 100.0 ml with the same solvent. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with methanol R. Dilute 5.0 ml of this solution to 20.0 ml with methanol R. The chromatographic procedure may be carried out using : — a stainless steel column 0.1 m long and 4.6 mm in internal diameter packed with base-deactivated octadecylsilyl silica gel for chromatography R (3 µm), — as mobile phase at a flow rate of 1.5 ml/min, a linear gradient programme as follows : Mobile phase A. A 17 g/l solution of tetrabutylammonium hydrogen sulphate R, Mobile phase B. Acetonitrile R, Time (min)



Comment

0

95

5

start gradient

15

80

20

end gradient

18

0

100

purge time

23

95

5

switch to initial equilibration

28 = 0

95

5

start of next chromatogram

final concentration of acetonitrile or the percentage of tetrabutylammonium hydrogen sulphate in the mobile phase or adjust the time programme for the linear gradient elution. Inject separately 10 µl of methanol R as a blank, 10 µl of the test solution and 10 µl of reference solution (b). In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.25 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent). Disregard any peak obtained with the blank and any peak with an area less than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (b). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.200 g in 50 ml of a mixture of 1 volume of anhydrous acetic acid R and 7 volumes of methyl ethyl ketone R. Titrate with 0.1 M perchloric acid, using 0.2 ml of naphtholbenzein solution R as indicator. 1 ml of 0.1 M perchloric acid is equivalent to 22.93 mg of C28H31FN4O. STORAGE Store protected from light. IMPURITIES

A. 1-(4-fluorobenzyl)-N-(piperidin-4-yl)-1H-benzimidazol-2amine,

— as detector a spectrophotometer set at 278 nm. Equilibrate the column for at least 30 min with acetonitrile R and then equilibrate the initial elution composition for at B. 1-(2-fluorobenzyl)-N-[1-[2-(4-methoxyphenyl)ethyl]piperileast 5 min. din-4-yl]-1H-benzimidazol-2-amine, Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 10 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder. Inject 10 µl of reference solution (a). When the chromatograms are recorded in the prescribed conditions the retention times are : ketoconazole, about 8 min and astemizole, about 9 min. The test is not valid unless the resolution between the peaks corresponding to ketoconazole C. 1-benzyl-N-[1-[2-(4-methoxyphenyl)ethyl]piperidin-4-yl]1H-benzimidazol-2-amine, and astemizole is a least 1.5. If necessary, adjust the General Notices (1) apply to all monographs and other texts

1031

Atenolol


DEFINITION Atenolol contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of 2-[4-[(2RS)-2hydroxy-3-[(1-methylethyl)amino]propoxy]phenyl]acetamide, calculated with reference to the dried substance.

D. 1-(3-fluorobenzyl)-N-[1-[2-(4-methoxyphenyl)ethyl]piperidin-4-yl]-1H-benzimidazol-2-amine,

CHARACTERS A white or almost white powder, sparingly soluble in water, soluble in ethanol, slightly soluble in methylene chloride.

IDENTIFICATION First identification : C. Second identification : A, B, D. A. Melting point (2.2.14) : 152 °C to 155 °C. B. Dissolve 0.100 g in methanol R and dilute to 100 ml with the same solvent. Dilute 10.0 ml of this solution to 100 ml with methanol R. Examined between 230 nm and 350 nm (2.2.25), the solution shows two absorption maxima, E. 1-(4-fluorobenzyl)-N-[cis-1-[2-(4-methoxyphenyl)ethyl]pipeat 275 nm and 282 nm. The ratio of the absorbance ridin-4-yl 1-oxide]-1H-benzimidazol-2-amine, measured at the maximum at 275 nm to that measured at the maximum at 282 nm is 1.15 to 1.20. C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with atenolol CRS. D. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 10 mg of the substance to be examined in 1 ml of methanol R. F. 1-(4-fluorobenzyl)-N-[trans-1-[2-(4-methoxyphenyl)ethyl]piReference solution. Dissolve 10 mg of atenolol CRS in peridin-4-yl 1-oxide]-1H-benzimidazol-2-amine, 1 ml of methanol R. Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm using a mixture of 1 volume of concentrated ammonia R1 and 99 volumes of methanol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. G. N-[1-[2-(4-methoxyphenyl)ethyl]piperidin-4-yl]-1-[4-(1TESTS methylethoxy)benzyl]-1H-benzimidazol-2-amine, Solution S. Dissolve 0.10 g in water R and dilute to 10 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than degree 6 of the range of reference solutions of the most appropriate colour (2.2.2, Method II). Optical rotation (2.2.7). Determined on solution S, the angle of optical rotation is + 0.10° to − 0.10°. Related substances. Examine by liquid chromatography H. 2-(4-methoxyphenyl)ethyl 4-[[1-(4-fluorobenzyl)-1H(2.2.29). benzimidazol-2-yl]amino]piperidin-1-carboxylate. Test solution (a). Dissolve 50.0 mg of the substance to be examined in 20 ml of the mobile phase and dilute to 25.0 ml 01/2005:0703 with the mobile phase. Test solution (b). Dissolve 50.0 mg of the substance to be examined in 0.1 ml of dimethyl sulphoxide R, if necessary ATENOLOL applying gentle heat by placing the containing vessel in a water-bath for a few seconds, and dilute to 25.0 ml with the Atenololum mobile phase. Reference solution (a). Dilute 0.5 ml of test solution (a) to 100.0 ml with the mobile phase. Reference solution (b). Dissolve 50.0 mg of atenolol for column validation CRS in 0.1 ml of dimethyl sulphoxide R, if necessary applying gentle heat by placing the containing vessel in a water-bath for a few seconds, and dilute to 25.0 ml C14H22N2O3 Mr 266.3 with the mobile phase. 1032


Atropine


The chromatographic procedure may be carried out using : — a stainless steel column 0.15 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.0 ml/min a mixture prepared as follows : dissolve 1.0 g of sodium octanesulphonate R and 0.4 g of tetrabutylammonium hydrogen sulphate R in 1 litre of a mixture of 20 volumes of tetrahydrofuran R, 180 volumes of methanol R and 800 volumes of a 3.4 g/l solution of potassium dihydrogen phosphate R ; adjust to pH 3.0 with phosphoric acid R, — as detector a spectrophotometer set at 226 nm. Equilibrate the column with the mobile phase at a flow rate of 1.0 ml/min for about 30 min. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 10 µl of reference solution (a) is at least 50 per cent of the full scale of the recorder. Inject 10 µl of reference solution (b). The resulting chromatogram is similar to that of the specimen chromatogram provided with atenolol for column validation CRS in that the peak due to bis-ether precedes and is separated from that due to tertiary amine which normally appears as a doublet. If necessary, adjust the concentration of sodium octanesulphonate in the mobile phase. Increasing the concentration of sodium octanesulphonate increases the retention time of the tertiary amine. Inject separately 10 µl of test solution (a) and reference solution (a). Continue the chromatography for four times the retention time of the principal peak. In the chromatogram obtained with test solution (a) : the area of any peak apart from the principal peak is not greater than half the area of the principal peak in the chromatogram obtained with reference solution (a) (0.25 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent). Disregard any peak with an area less than 0.1 times of that of the principal peak in the chromatogram obtained with reference solution (a). If the substance to be examined is found to contain more than 0.15 per cent of bis-ether, its compliance is confirmed by repeating the chromatography using 10 µl of test solution (b). Chlorides (2.4.4). Dissolve 50 mg in a mixture of 1 ml of dilute nitric acid R and 15 ml of water R. The solution, without further addition of dilute nitric acid R, complies with the limit test for chlorides (0.1 per cent). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.200 g in 80 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 26.63 mg of C14H22N2O3. IMPURITIES


A. R-H : 2-(4-hydroxyphenyl)acetamide,

B. 2-[4-[(2RS)-2,3-dihydroxypropoxy]phenyl]acetamide,

C. 2-[4-[[(2RS)-oxiran-2-yl]methoxy]phenyl]acetamide,

D. 2-[4-[(2RS)-3-chloro-2-hydroxypropoxy]phenyl]acetamide,

E. 2,2′-[2-hydroxypropan-1,3-diylbis(oxy-4,1phenylene)]diacetamide,

F. 2,2′-[(1-methylethyl)iminobis(2-hydroxypropan-3,1-diyloxy4,1-phenylene)]diacetamide,

G. 2-[4-[(2RS)-2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenyl]acetic acid,

H. 2-[4-[(2RS)-2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenyl]acetonitrile. 01/2005:2056

ATROPINE Atropinum

C17H23NO3

Mr 289.4

DEFINITION (1R,3r,5S)-8-Methyl-8-azabicyclo[3.2.1]oct-3-yl (2RS)-3-hydroxy-2-phenylpropanoate. Content : 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white, crystalline powder or colourless crystals. Solubility : very slightly soluble in water, freely soluble in alcohol and in methylene chloride. 1033

Atropine


IDENTIFICATION First identification : A, B, E. Second identification : A, C, D, E. A. Melting point (2.2.14) : 115 °C to 119 °C. B. Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of atropine.

Time (min)



0 - 12

100

0

12 - 25

100 → 70

0 → 30

25 - 26

70 → 100

30 → 0

26 - 30

100

0

Flow rate : 1 ml/min. Detection : spectrophotometer at 210 nm. Test solution. Dissolve 10 mg of the substance to be examined in methanol R and dilute to 10 ml with the Injection : 10 µl. same solvent. System suitability : reference solution (b) : Reference solution. Dissolve 10 mg of atropine — the chromatogram obtained is similar to the sulphate CRS in methanol R and dilute to 10 ml with chromatogram supplied with atropine for system the same solvent. suitability CRS, Plate : TLC silica gel plate R. — peak-to-valley ratio : minimum 20, where Hp = height above the baseline of the peak due to impurity B and Mobile phase : concentrated ammonia R, water R, Hv = height above the baseline of the lowest point of the acetone R (3:7:90 V/V/V). curve separating this peak from the peak due to atropine. Application : 10 µl. Limits : locate the impurities by comparing with the Development : over half of the plate. chromatogram obtained with reference solution (b) and Drying : at 100-105 °C for 15 min. with the chromatogram supplied with atropine for system suitability CRS : Detection : after cooling, spray with dilute potassium iodobismuthate solution R. — impurity B : not more than 3 times the area of the principal peak in the chromatogram obtained with Results : the principal spot in the chromatogram obtained reference solution (a) (0.3 per cent), with the test solution is similar in position, colour and — impurity A : not more than 4 times the area of the size to the principal spot in the chromatogram obtained principal peak in the chromatogram obtained with with the reference solution. reference solution (a) (0.4 per cent), D. Place about 3 mg in a porcelain crucible and add 0.2 ml — any other impurity : not more than twice the area of of fuming nitric acid R. Evaporate to dryness on a the principal peak in the chromatogram obtained with water-bath. Dissolve the residue in 0.5 ml of a 30 g/l reference solution (a) (0.2 per cent), solution of potassium hydroxide R in methanol R. A violet colour develops. — total : not more than 15 times the area of the principal peak in the chromatogram obtained with reference E. It complies with the test for optical rotation (see Tests). solution (a) (1.5 per cent), TESTS — disregard limit : the area of the principal peak in the chromatogram obtained with reference solution (a) Optical rotation (2.2.7) : − 0.70° to + 0.05°. (0.1 per cent) ; disregard peaks due to the blank. Dissolve 1.25 g in alcohol R and dilute to 25.0 ml with the Loss on drying (2.2.32) : maximum 0.2 per cent, determined same solvent. Measure in a 2 dm tube. on 1.000 g by drying in an oven at 100-105 °C for 2 h. Related substances. Liquid chromatography (2.2.29). ASSAY Test solution. Dissolve 50 mg of the substance to be examined in mobile phase A and dilute to 50 ml with mobile Dissolve 0.250 g in 40 ml of anhydrous acetic acid R phase A. Dilute 10 ml of the solution to 50 ml with mobile and heat if necessary. Allow the solution to cool. Titrate phase A. with 0.1 M perchloric acid, determining the end-point Reference solution (a). Dilute 1.0 ml of the test solution to potentiometrically (2.2.20). 100.0 ml with mobile phase A. Dilute 1.0 ml of this solution 1 ml of 0.1 M perchloric acid is equivalent to 28.94 mg to 10.0 ml with mobile phase A. of C17H23NO3. Reference solution (b). Dissolve 5 mg of atropine for system STORAGE suitability CRS in mobile phase A and dilute to 25 ml with mobile phase A. Protected from light. Column : IMPURITIES — size : l = 0.125 m, Ø = 4 mm,

C. Thin-layer chromatography (2.2.27).

— stationary phase: octylsilyl silica gel for chromatography R (4 µm). Mobile phase : — mobile phase A : to 606 ml of a 7.0 g/l solution of potassium dihydrogen phosphate R adjusted to pH 3.3 with 0.05 M phosphoric acid, add 3.5 g of sodium dodecyl sulphate R and 320 ml of acetonitrile R, — mobile phase B : acetonitrile R, 1034

A. (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl 2-phenylpropenoate (apoatropine),



Atropine sulphate

It melts at about 190 °C with decomposition, determined on the substance dried at 135 °C for 15 min.

B. (1R,3r,5S)-8-azabicyclo[3.2.1]oct-3-yl (2RS)-3-hydroxy-2phenylpropanoate (noratropine),

C. (2RS)-3-hydroxy-2-phenylpropanoic acid (tropic acid),

IDENTIFICATION First identification : A, B, E. Second identification : C, D, E, F. A. An aqueous solution shows almost no optical rotation (see Tests). B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with atropine sulphate CRS. C. Dissolve about 50 mg in 5 ml of water R and add 5 ml of picric acid solution R. The precipitate, washed with water R and dried at 100 °C to 105 °C for 2 h, melts (2.2.14) at 174 °C to 179 °C. D. To about 1 mg add 0.2 ml of fuming nitric acid R and evaporate to dryness in a water-bath. Dissolve the residue in 2 ml of acetone R and add 0.1 ml of a 30 g/l solution of potassium hydroxide R in methanol R. A violet colour develops. E. It gives the reactions of sulphates (2.3.1). F. It gives the reaction of alkaloids (2.3.1).

TESTS pH (2.2.3). Dissolve 0.6 g in carbon dioxide-free water R and dilute to 30 ml with the same solvent. The pH of the solution is 4.5 to 6.2. E. R1 = H, R2 = OH : (1S,3R,5S,6RS)-6-hydroxy-8Optical rotation (2.2.7). Dissolve 2.50 g in water R and methyl-8-azabicyclo[3.2.1]oct-3-yl (2S)-3-hydroxy-2dilute to 25.0 ml with the same solvent. The angle of optical phenylpropanoate (7-hydroxyhyoscyamine), rotation measured in a 2 dm tube is − 0.50° to + 0.05°. F. hyoscine, Foreign alkaloids and decomposition products. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. Dissolve 0.2 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution (a). Dilute 1 ml of the test solution to 100 ml with methanol R. Reference solution (b). Dilute 5 ml of reference solution (a) G. (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl to 10 ml with methanol R. (3RS)-3-hydroxy-3-phenylpropanoate (isolittorine). Apply separately to the plate 10 µl of each solution. Develop over a path of 10 cm using a mixture of 90 volumes 01/2005:0068 of acetone R, 7 volumes of water R and 3 volumes of concentrated ammonia R. Dry the plate at 100 °C to 105 °C for 15 min. Allow to cool and spray with dilute potassium ATROPINE SULPHATE iodobismuthate solution R until the spots appear. Any spot in the chromatogram obtained with the test solution, apart Atropini sulfas from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (a) (1.0 per cent) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). Apoatropine. Dissolve 0.10 g in 0.01 M hydrochloric acid and dilute to 100.0 ml with the same acid. Determine the absorbance (2.2.25) at 245 nm. The specific absorbance is not greater than 4.0, calculated with reference to the C34H48N2O10S,H2O Mr 695 anhydrous substance (about 0.5 per cent). Water (2.5.12). 2.0 per cent to 4.0 per cent, determined on DEFINITION 0.50 g by the semi-micro determination of water. Atropine sulphate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent Sulphated ash (2.4.14). Not more than 0.1 per cent, of bis[(1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl determined on 1.0 g. (2RS)-3-hydroxy-2-phenylpropanoate] sulphate, calculated ASSAY with reference to the anhydrous substance. Dissolve 0.500 g in 30 ml of anhydrous acetic acid R, CHARACTERS warming if necessary. Cool the solution. Titrate with 0.1 M perchloric acid and determining the end-point A white, crystalline powder or colourless crystals, very potentiometrically (2.2.20). soluble in water, freely soluble in alcohol. D. R1 = OH, R2 = H : (1R,3S,5R,6RS)-6-hydroxy-8methyl-8-azabicyclo[3.2.1]oct-3-yl (2S)-3-hydroxy-2phenylpropanoate (6-hydroxyhyoscyamine),


1035

Azaperone for veterinary use


1 ml of 0.1 M perchloric acid is equivalent to 67.68 mg of C34H48N2O10S. STORAGE Store protected from light.

01/2005:1708

AZAPERONE FOR VETERINARY USE

Mobile phase : — mobile phase A : dissolve 1.4 g of anhydrous sodium sulphate R in 900 ml of water R, add 16.0 ml of 0.01 M sulphuric acid and dilute to 1000 ml with water R, — mobile phase B : methanol R. Time (min)



0 - 15

95 → 20

5 → 80

15 - 20

20

80

20 - 21

20 → 95

80 → 5

Azaperonum ad usum veterinarium

Flow rate : 1.5 ml/min. Detection : spectrophotometer at 230 nm. Equilibration : at least 4 min with the mobile phase at the initial composition. Injection : 10 µl. Relative retention with reference to azaperone (retention time = about 9 min) : impurity A = about 0.9 ; impurity B = about 1.1 ; impurity C = about 1.15. System suitability : reference solution (a) : C19H22FN3O Mr 327.4 — resolution : minimum 8.0 between the peaks due to azaperone and to benperidol. DEFINITION Limits : 1-(4-Fluorophenyl)-4-[4-(pyridin-2-yl)piperazin-1-yl]butan-1— impurity A : not more than the area of the principal peak one. in the chromatogram obtained with reference solution (b) (0.25 per cent), Content : 99.0 per cent to 101.0 per cent (dried substance). — total of impurities B and C : not more than 3 times the CHARACTERS area of the principal peak in the chromatogram obtained Appearance : white or almost white powder. with reference solution (b) (0.75 per cent), — total : not more than 4 times the area of the principal peak Solubility : practically insoluble in water, freely soluble in in the chromatogram obtained with reference solution (b) acetone and in methylene chloride, soluble in alcohol. (1.0 per cent), It shows polymorphism. — disregard limit : 0.2 times the area of the principal peak IDENTIFICATION in the chromatogram obtained with reference solution (b) (0.05 per cent). Infrared absorption spectrophotometry (2.2.24). Loss on drying (2.2.32) : maximum 0.5 per cent, determined Preparation : discs. on 1.000 g by drying in vacuo at 60 °C for 4 h. Comparison : azaperone CRS. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined If the spectra obtained show differences, dissolve the on 1.0 g. substance to be examined and the reference substance separately in acetone R, evaporate to dryness and record ASSAY new spectra using the residues. Dissolve 0.130 g in 70 ml of a mixture of 1 volume of anhydrous acetic acid R and 7 volumes of methyl ethyl TESTS ketone R. Titrate with 0.1 M perchloric acid, using 0.2 ml of Appearance of solution. The solution is clear (2.2.1) and not naphtholbenzein solution R as indicator. more intensely coloured than reference solution Y6 (2.2.2, 1 ml of 0.1 M perchloric acid is equivalent to 16.37 mg of Method II). C H FN O. Dissolve 1.0 g in 25 ml of a 14 g/l solution of tartaric acid R. 19 22 3 STORAGE Related substances. Liquid chromatography (2.2.29). Protected from light. Test solution. Dissolve 0.100 g of the substance to be examined in methanol R and dilute to 10.0 ml with the same IMPURITIES solvent. Reference solution (a). Dissolve 5.0 mg of azaperone CRS and 6.0 mg of benperidol CRS in methanol R and dilute to 200.0 ml with the same solvent. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with methanol R. Dilute 5.0 ml of the solution to 20.0 ml with methanol R. Column : — size : l = 0.10 m, Ø = 4.6 mm, — stationary phase: base-deactivated octadecylsilyl silica gel for chromatography R (3 µm), A. 1-(2-fluorophenyl)-4-[4-(pyridin-2-yl)piperazin-1-yl]butan— temperature : 25 °C. 1-one, 1036


Azelastine hydrochloride


TESTS Acidity or alkalinity. To 0.5 g add 25 ml of carbon dioxide-free water R, shake for 15 min and filter. To 20 ml of the filtrate add 0.1 ml of methyl red solution R. Not more than 0.2 ml of 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is required to change the colour of the indicator. B. 4-[4-(pyridin-2-yl)piperazin-1-yl]-1-[4-[4-(pyridin-2Chloromethylnitroimidazole and mercaptopurine. Examine yl)piperazin-1-yl]phenyl]butan-1-one, by thin-layer chromatography (2.2.27), using cellulose for chromatography F254 R as the coating substance. Test solution. Dissolve 0.2 g of the substance to be examined in dilute ammonia R1 and dilute to 10 ml with the same solvent. Prepare immediately before use. Reference solution (a). Dissolve 10 mg of chloromethylnitroimidazole CRS in dilute ammonia R1 and C. 4-hydroxy-1-[4-[4-(pyridin-2-yl)piperazin-1-yl]phenyl]butan- dilute to 50 ml with the same solvent. Prepare immediately 1-one. before use. Reference solution (b). Dissolve 10 mg of mercaptopurine R in dilute ammonia R1 and dilute to 50 ml with the same solvent. Prepare immediately before use. 01/2005:0369 Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm using butanol R saturated with dilute ammonia R1. Dry the plate at 50 °C and examine AZATHIOPRINE in ultraviolet light at 254 nm. In the chromatogram obtained with the test solution, any spots corresponding to Azathioprinum chloromethyl-nitroimidazole and mercaptopurine are not more intense than the spots in the chromatograms obtained with reference solution (a) (1.0 per cent) and reference solution (b) (1.0 per cent), respectively. Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 0.50 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. Mr 277.3 ASSAY Dissolve 0.250 g in 25 ml of dimethylformamide R. Titrate with 0.1 M tetrabutylammonium hydroxide, determining DEFINITION the end-point potentiometrically (2.2.20). Azathioprine contains not less than 98.5 per cent 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent and not more than the equivalent of 101.0 per cent of to 27.73 mg of C9H7N7O2S. 6-[(1-methyl-4-nitro-1H-imidazol-5-yl)sulfanyl]-7H-purine, calculated with reference to the dried substance. STORAGE C 9 H 7 N7 O 2 S

CHARACTERS A pale-yellow powder, practically insoluble in water and in alcohol. It is soluble in dilute solutions of alkali hydroxides and sparingly soluble in dilute mineral acids. IDENTIFICATION A. Dissolve 0.150 g in 30 ml of dimethyl sulphoxide R and dilute to 500.0 ml with 0.1 M hydrochloric acid. Dilute 25.0 ml of the solution to 1000.0 ml with 0.1 M hydrochloric acid. Examined between 230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 280 nm. The specific absorbance at the maximum is 600 to 660. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with azathioprine CRS. C. To about 20 mg add 100 ml of water R, heat and filter. To 5 ml of the filtrate add 1 ml of hydrochloric acid R and about 10 mg of zinc powder R. Allow to stand for 5 min. The solution becomes yellow. Filter, cool in iced water, add 0.1 ml of sodium nitrite solution R and 0.1 g of sulphamic acid R and shake until the bubbles disappear. Add 1 ml of β-naphthol solution R. A pale-pink precipitate is formed. General Notices (1) apply to all monographs and other texts

Store protected from light. 01/2005:1633 corrected

AZELASTINE HYDROCHLORIDE Azelastini hydrochloridum

C22H25Cl2N3O

Mr 418.4

DEFINITION 4-(4-Chlorobenzyl)-2-[(4RS)-1-methylhexahydro-1H-azepin-4yl]phthalazin-1(2H)-one hydrochloride. Content : 99.0 per cent to 101.0 per cent (dried substance). 1037

Azelastine hydrochloride


CHARACTERS — impurities A, B, C, D, E : for each impurity, not more than the area of the principal peak in the chromatogram Appearance : white or almost white, crystalline powder. obtained with reference solution (a) (0.1 per cent) ; Solubility : sparingly soluble in water, soluble in ethanol and — any other impurity : for each impurity, not more than the in methylene chloride. area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent) ; IDENTIFICATION — total : not more than twice the area of the principal peak A. Infrared absorption spectrophotometry (2.2.24). in the chromatogram obtained with reference solution (a) Comparison : azelastine hydrochloride CRS. (0.2 per cent) ; B. Solution S (see Tests) gives reaction (a) of chlorides — disregard limit : 0.5 times the area of the principal peak (2.3.1). in the chromatogram obtained with reference solution (a) (0.05 per cent). TESTS Loss on drying (2.2.32) : maximum 0.5 per cent, determined Solution S. Dissolve 1.0 g in carbon dioxide-free water R on 1.000 g by drying in an oven at 100-105 °C. and dilute to 100 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and ASSAY colourless (2.2.2, Method II). In order to avoid overheating in the reaction medium, mix Acidity or alkalinity. To 10 ml of solution S add 0.2 ml of thoroughly throughout and stop the titration immediately bromothymol blue solution R1. Not more than 0.1 ml of after the end-point has been reached. 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is Dissolve 0.300 g in 5 ml of anhydrous formic acid R. required to change the colour of the solution. Add 30 ml of acetic anhydride R. Titrate quickly with Related substances. Liquid chromatography (2.2.29). 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). Solvent mixture : acetonitrile for chromatography R, water R (45:55 V/V). 1.0 ml of 0.1 M perchloric acid is equivalent to 41.84 mg of C22H25Cl2N3O. Test solution. Dissolve 0.125 g of the substance to be examined in the solvent mixture and dilute to 50.0 ml with IMPURITIES the solvent mixture. Specified impurities : A, B, C, D, E. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Reference solution (b). Dissolve 1 mg of azelastine impurity B CRS, 1 mg of azelastine impurity D CRS and 1 mg of azelastine impurity E CRS in the test solution and dilute to 20 ml with the test solution. A. benzoyldiazane (benzohydrazide), Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : nitrile silica gel for chromatography R (10 µm), — temperature : 30°C. Mobile phase : dissolve 2.16 g of sodium octanesulphonate R and 0.68 g of potassium dihydrogen phosphate R in 740 ml B. 1-benzoyl-2-[(4RS)-1-methylhexahydro-1H-azepin-4of water for chromatography R, adjust to pH 3.0-3.1 with yl]diazane, dilute phosphoric acid R, add 260 ml of acetonitrile for chromatography R and mix. Flow rate : 2.0 ml/min. Detection : spectrophotometer at 210 nm. Injection : 10 µl. Run time : twice the retention time of azelastine. Relative retention with reference to azelastine (retention time = about 8-9 min) : impurity A = about 0.2 ; impurity B = about 0.3 ; impurity C = about 0.4 ; C. 2-[(4-chlorophenyl)acetyl]benzoic acid, impurity D = about 0.6 ; impurity E = about 1.4. System suitability : reference solution (b) : — resolution : minimum 4.0 between the peaks due to impurity B and impurity D, — the peaks due to impurity D and impurity E are baseline separated from the principal peak. Limits : — correction factors : for the calculation of contents, multiply the peak areas of the following impurities by the corresponding correction factor : impurity B = 3.6 ; impurity D = 0.7 ; impurity E = 2.1 ; D. 4-(4-chlorobenzyl)phthalazin-1(2H)-one, 1038


Azithromycin


E. 3-(4-chlorobenzylidene)isobenzofuran-1(3H)-one. 01/2005:1649 corrected

AZITHROMYCIN Azithromycinum

C38H72N2O12

Mr 749

DEFINITION (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-2-ethyl3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6trideoxy-3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]-1oxa-6-azacyclopentadecan-15-one. Content : 94.0 per cent to 102.0 per cent (anhydrous substance). CHARACTERS Appearance : white or almost white powder. Solubility : practically insoluble in water, freely soluble in ethanol and in methylene chloride. IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Comparison : azithromycin CRS. If the spectra obtained show differences, prepare further spectra using 90 g/l solutions in methylene chloride R. B. Examine the chromatograms obtained in the assay. Results : the principal peak in the chromatogram obtained with test solution (b) is similar in retention time and size to the principal peak in the chromatogram obtained with reference solution (a). TESTS Solution S. Dissolve 0.500 g in ethanol R and dilute to 50.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). General Notices (1) apply to all monographs and other texts

pH (2.2.3) : 9.0 to 11.0. Dissolve 0.100 g in 25.0 ml of methanol R and dilute to 50.0 ml with carbon dioxide-free water R. Specific optical rotation (2.2.7) : − 45 to − 49 (anhydrous substance), determined on solution S. Related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately before use. Solvent mixture : acetonitrile R, water R (40:60 V/V). Test solution (a). Dissolve 0.100 g of the substance to be examined in the solvent mixture and dilute to 25.0 ml with the solvent mixture. Test solution (b). Dilute 5.0 ml of test solution (a) to 20.0 ml with the solvent mixture. Reference solution (a). Dissolve 50.0 mg of azithromycin CRS in the solvent mixture and dilute to 50.0 ml with the solvent mixture. Reference solution (b). Dilute 1.0 ml of test solution (a) to 100.0 ml with the solvent mixture. Reference solution (c). Dissolve 5.0 mg of azithromycin CRS and 5.0 mg of azithromycin impurity A CRS in the solvent mixture and dilute to 50 ml with the solvent mixture. Reference solution (d). Dissolve 2 mg of azithromycin impurity B CRS in the solvent mixture and dilute to 50 ml with the solvent mixture. Use this solution only for identification of the peak due to impurity B. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : end-capped polar-embedded octadecylsilyl amorphous organosilica polymer R (5 µm), — temperature : 70 °C. Mobile phase : mix 10 volumes of a 34.84 g/l solution of dipotassium hydrogen phosphate R adjusted to pH 6.5 with phosphoric acid R, 35 volumes of acetonitrile R and 55 volumes of water R. Flow rate : 1.0 ml/min. Detection : spectrophotometer at 215 nm. Injection : 100 µl ; inject test solution (a) and reference solutions (b), (c) and (d). Run time : 4.5 times the retention time of azithromycin. Relative retention with reference to azithromycin (retention time = about 26 min) : impurity D = about 0.37 ; impurity J = about 0.39 ; impurity A = about 0.42 ; impurity I = about 0.5 ; impurity C = about 0.65 ; impurity K = about 0.9 ; impurity F = about 1.6 ; impurity B = about 1.7 ; impurity G = about 2.8. System suitability : reference solution (c) : — resolution : minimum 7.0 between the peaks due to impurity A and azithromycin. Limits : — impurity B : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (2.0 per cent), — any other impurity : for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent), — total : not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (5.0 per cent), — disregard limit : 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent). 1039

Azithromycin


Heavy metals (2.4.8) : maximum 25 ppm. Dissolve 2.0 g in a mixture of 15 volumes of water R and 85 volumes of ethanol R and dilute to 20 ml with the same mixture of solvents. 12 ml of the solution complies with limit test B. Prepare the standard using lead standard solution (2.5 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a mixture of 15 volumes of water R and 85 volumes of ethanol R. Water (2.5.12) : 1.8 per cent to 6.5 per cent, determined on 0.20 g. Sulphated ash (2.4.14) : maximum 0.2 per cent, determined on 1.0 g. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances, with the following modifications. Injection : 25 µl ; inject test solution (b) and reference solution (a). Calculate the percentage content of C38H72N2O12 using the declared content of azithromycin CRS.

E. 3′-(N,N-didemethyl)azithromycin (aminoazithromycin),

STORAGE In an airtight container. IMPURITIES Specified impurities : A, B, C, D, E, F, G, H, I, J, K. H. 3′-de(dimethylamino)-3′,4′-didehydroazithromycin,

J. decladinosylazithromycin,

A. R1 = OH, R2 = H, R3 = R4 = R5 = CH3 : 6-demethylazithromycin, B. R1 = H, R2 = R3 = R4 = R5 = CH3 : 3-deoxyazithromycin (azithromycin B), C. R1 = OH, R2 = R3 = R5 = CH3, R4 = H : 3′-O-demethylazithromycin (azithromycin C), D. R1 = OH, R2 = R3 = R4 = CH3, R5 = CH2OH : 14-demethyl-14-(hydroxymethyl)azithromycin (azithromycin F), F. R1 = OH, R2 = R4 = R5 = CH3, R3 = CHO : 3′-N-demethyl-3′-N-formylazithromycin, G. R1 = OH, R2 = R4 = R5 = CH3, R3 = SO2-C6H4-CH3 : 3′-Ndemethyl-3′-N-[(4-methylphenyl)sulphonyl]azithromycin, I. R1 = OH, R2 = R4 = R5 = CH3, R3 = H : 3′-N-demethylazithromycin,

1040

K. (2S,4′R,4aR,5′S,6′S,7R,8S,9R,10R,13R,15R, 16R,17S,17aS)-7-ethyl-5′,8,9,15-tetrahydroxy4′-methoxy-4′,6′,8,10,11,13,15,17-octamethyl16-[[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylohexopyranosyl]oxy]octadecahydro-5H-spiro[1,3-dioxino[4, 5-m][1,6]oxazacyclopentadecine-2,2′-[2H]pyran]-5-one (azithromycin E).


Bacampicillin hydrochloride


01/2005:0808

BACAMPICILLIN HYDROCHLORIDE Bacampicillini hydrochloridum

C21H28ClN3O7S

Mr 502.0

The test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly separated spots. C. Place about 2 mg in a test-tube about 150 mm long and 15 mm in diameter. Moisten with 0.05 ml of water R and add 2 ml of sulphuric acid-formaldehyde reagent R. Mix the contents of the tube by swirling ; the solution is practically colourless. Place the test-tube on a water-bath for 1 min ; a dark yellow colour develops. D. Dissolve about 25 mg in 2 ml of water R. Add 2 ml of dilute sodium hydroxide solution R and shake. Wait a few minutes and add 3 ml of dilute nitric acid R and 0.5 ml of silver nitrate solution R1. A white precipitate is formed. Add 0.5 ml of concentrated ammonia R. The precipitate dissolves.

IDENTIFICATION First identification : A, D. Second identification : B, C, D. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with bacampicillin hydrochloride CRS. B. Examine by thin-layer chromatography (2.2.27), using a TLC silanised silica gel plate R. Test solution. Dissolve 10 mg of the substance to be examined in 2 ml of methanol R. Reference solution (a). Dissolve 10 mg of bacampicillin hydrochloride CRS in 2 ml of methanol R. Reference solution (b). Dissolve 10 mg each of bacampicillin hydrochloride CRS, talampicillin hydrochloride CRS and pivampicillin CRS in 2 ml of methanol R. Apply to the plate 1 µl of each solution. Develop over a path of 15 cm using a mixture of 10 volumes of a 272 g/l solution of sodium acetate R, the pH of which has been adjusted to 5.0 with glacial acetic acid R, 40 volumes of water R and 50 volumes of alcohol R. Dry the plate in a current of warm air, then spray with ninhydrin solution R1 and heat at 60 °C for 10 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a).

TESTS Appearance of solution. Dissolve 0.200 g in 20 ml of water R ; the solution is not more opalescent than reference suspension II (2.2.1). Dissolve 0.500 g in 10 ml of water R ; the absorbance (2.2.25) of the solution measured at 430 nm is not greater than 0.10. pH (2.2.3). Dissolve 1.0 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent. The pH of the solution is 3.0 to 4.5. Specific optical rotation (2.2.7). Dissolve 0.250 g in water R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 175 to + 195, calculated with reference to the anhydrous, solvent-free substance. Butyl acetate and ethyl acetate. Not more than 2.0 per cent m/m of butyl acetate, not more than 4.0 per cent m/m of ethyl acetate and not more than 5.0 per cent m/m for the sum determined by head-space gas chromatography (2.2.28), using the method of standard additions. Sample solution. Dissolve 50.0 mg of the substance to be examined in water R and dilute to 10.0 ml with the same solvent. The chromatographic procedure may be carried out using System A of the test for residual solvents (2.4.24) and the following static head-space injection conditions : — equilibration temperature : 60 °C, — equilibration time : 20 min. Related substances. Examine by liquid chromatography (2.2.29) as prescribed in the Assay. Inject 20 µl of reference solution (b). Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is at least 50 per cent of the full scale of the recorder. Inject 20 µl of reference solution (d). The test is not valid unless in the chromatogram obtained, the peak corresponding to ampicillin is separated from the peaks due to the solvent. Inject 20 µl of the test solution and continue the chromatography for 3.5 times the retention time of the principal peak. In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (1.5 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than three times the area of the principal peak in the chromatogram obtained with reference solution (b) (3 per cent). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b). Water (2.5.12). Not more than 0.8 per cent, determined on 0.300 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 1.5 per cent, determined on 1.0 g.


1043

DEFINITION Bacampicillin hydrochloride contains not less than 95.0 per cent and not more than the equivalent of 102.0 per cent of (1RS)-1-[(ethoxycarbonyl)oxy]ethyl (2S,5R,6R)-6-[[(2R)2-amino-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylate hydrochloride, calculated with reference to the anhydrous, solvent-free substance. PRODUCTION If manufactured by a process that may leave residues of dimethylaniline in the substance and/or by a process using starting materials or intermediates which contain residues of dimethylaniline, it complies with the following test : N,N-Dimethylaniline (2.4.26, Method A). Not more than 20 ppm. CHARACTERS A white or almost white powder or granules, hygroscopic, soluble in water, freely soluble in alcohol, soluble in methylene chloride.

Bacampicillin hydrochloride


ASSAY Examine by liquid chromatography (2.2.29). Prepare the test solution and reference solutions (a), (b) and (d) immediately before use. Phosphate buffer A. Dissolve 1.4 g of sodium dihydrogen phosphate monohydrate R in water R and dilute to about 800 ml with the same solvent. Adjust to pH 3.0 with dilute phosphoric acid R and dilute to 1000.0 ml with water R. Phosphate buffer B. Dissolve 2.75 g of sodium dihydrogen phosphate monohydrate R and 2.3 g of disodium hydrogen phosphate dihydrate R in water R and dilute to about 1800 ml with the same solvent. Adjust to pH 6.8, if necessary, using dilute phosphoric acid R or dilute sodium hydroxide solution R and dilute to 2000.0 ml with water R. Test solution. Dissolve 30.0 mg of the substance to be examined in phosphate buffer A and dilute to 100.0 ml with the same solution. Reference solution (a). Dissolve 30.0 mg of bacampicillin hydrochloride CRS in phosphate buffer A and dilute to 100.0 ml with the same solution. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 100.0 ml with phosphate buffer A. Reference solution (c). Dissolve 30 mg of the substance to be examined in phosphate buffer B and dilute to 100 ml with the same solution. Heat the solution at 80 °C for about 30 min. Reference solution (d). Dissolve 20 mg of ampicillin trihydrate CRS in phosphate buffer A and dilute to 250 ml with the same solution. Dilute 5 ml of the solution to 100 ml with phosphate buffer A. The chromatographic procedure may be carried out using : — a column 0.05 m long and 3.9 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.0 ml/min a mixture of 30 volumes of acetonitrile R and 70 volumes of a 0.06 per cent m/m solution of tetrahexylammonium hydrogen sulphate R in phosphate buffer B, — as detector a spectrophotometer set at 220 nm. Inject 20 µl of reference solution (a). Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is at least 50 per cent of the full scale of the recorder. Inject 20 µl of reference solution (c). The test is not valid unless, in the chromatogram obtained, the retention time of a degradation product eluting just after bacampicillin is 1.12 to 1.38 relative to bacampicillin. If necessary adjust the concentration of tetrahexylammonium hydrogen sulphate in the mobile phase. Inject reference solution (a) six times. The test is not valid unless the relative standard deviation of the peak area for bacampicillin is at most 1.0 per cent. Inject alternately the test solution and reference solution (a). Calculate the percentage content of bacampicillin hydrochloride.

B. R = H : (2R)-2-amino-2-phenylacetic acid (D-phenylglycine), G. R = CH3 : methyl (2R)-2-amino-2-phenylacetate (methyl D-phenylglycinate),

C. R = H : (2RS,4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penilloic acids of ampicillin), D. R = CO2H : (4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]carboxymethyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penicilloic acids of ampicillin),

E. (4S)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5dimethylthiazolidine-4-carboxylic acid (diketopiperazines of ampicillin),

F. (2RS)-2-amino-3-methyl-3-sulphanylbutanoic acid (DL-penicillamine),

STORAGE Store in an airtight container. IMPURITIES


H. (1RS)-1-[(ethoxycarbonyl)oxy]ethyl (2S,5R,6R)-6-[[(2R)2-(acetylamino)-2-phenylacetyl]amino]-3,3-dimethyl7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate (N-acetylbacampicillin), I. ampicillin. See the information section on general monographs (cover pages)


Bacitracin

01/2005:0465 pH (2.2.3) : 6.0 to 7.0 for solution S. Composition. Liquid chromatography (2.2.29) : use the BACITRACIN normalisation procedure. Prepare the solutions immediately before use. Bacitracinum Test solution. Dissolve 0.100 g of the substance to be examined in 50.0 ml of the mobile phase. Reference solution (a). Suspend 20.0 mg of bacitracin zinc CRS in water R, add 0.2 ml of dilute hydrochloric acid R and dilute to 10.0 ml with water R. Reference solution (b). Dilute 5.0 ml of the test solution to 100.0 ml with the mobile phase. Reference solution (c). Dilute 1.0 ml of reference solution (b) to 10.0 ml with the mobile phase. Reference solution (d). Dissolve 50.0 mg of the substance to be examined in 25.0 ml of a 40 g/l solution of sodium edetate R adjusted to pH 7.0 with dilute sodium hydroxide R. Heat in a boiling water-bath for 30 min. Cool to room temperature. Blank solution. A 40 g/l solution of sodium edetate R adjusted to pH 7.0 with dilute sodium hydroxide R. Column : DEFINITION — size : l = 0.25 m, Ø = 4.6 mm, Mixture of antimicrobial polypeptides produced by certain — stationary phase : end-capped octadecylsilyl silica gel strains of Bacillus licheniformis or Bacillus subtilis, the for chromatography R (5 µm). main components being bacitracins A, B1, B2 and B3. Mobile phase : add 520 volumes of methanol R1, 40 volumes Content : minimum 60 IU/mg (dried substance). of acetonitrile R and 300 volumes of water R to 100 volumes of a 34.8 g/l solution of dipotassium hydrogen phosphate R CHARACTERS adjusted to pH 6.0 with a 27.2 g/l solution of potassium Appearance : white or almost white powder, hygroscopic. dihydrogen phosphate R. Solubility : freely soluble in water and in alcohol. Flow rate : 1.0 ml/min. Detection : spectrophotometer at 254 nm. IDENTIFICATION Injection : 100 µl ; inject the blank, the test solution and First identification : B, C. reference solutions (a) and (c). Second identification : A, C. Run time : 3 times the retention time of bacitracin A. A. Thin-layer chromatography (2.2.27). Relative retention with reference to bacitracin A (retention Test solution. Dissolve 10 mg of the substance to be examined in a 3.4 g/l solution of hydrochloric acid R and time = 15 min to 25 min) : bacitracin B1 = about 0.6 ; bacitracin B3 = about 0.8 ; impurity E = about 2.5. dilute to 1.0 ml with the same solution. If necessary, adjust the composition of the mobile phase by Reference solution. Dissolve 10 mg of bacitracin zinc CRS in a 3.4 g/l solution of hydrochloric acid R and changing the amount of organic modifier whilst keeping the ratio constant between methanol and acetonitrile. dilute to 1.0 ml with the same solution. System suitability : reference solution (a) : Plate : TLC silica gel plate R. — peak-to-valley ratio : minimum of 1.2, where Hp = height Mobile phase : glacial acetic acid R, water R, butanol R above the baseline of the peak due to bacitracin B1 and (1:2:4 V/V/V). Hv = height above the baseline of the lowest point of Application : 10 µl. the curve separating this peak from the peak due to Development : over half of the plate. bacitracin B2. Drying : at 100-105 °C. Limits : Detection : spray with ninhydrin solution R1 and heat — bacitracin A : minimum 40.0 per cent, at 110 °C for 5 min. — sum of bacitracins A, B1, B2 and B3: minimum 70.0 per Results : the spots in the chromatogram obtained with cent, the test solution are similar in position, size and colour — disregard limit : the area of the peak due to bacitracin A to the spots in the chromatogram obtained with the in the chromatogram obtained with reference solution (c) reference solution. (0.5 per cent) ; disregard any peak observed in the blank B. It complies with the test for composition (see Tests). run. C. Ignite 0.2 g. An insignificant residue remains which is Related peptides. Liquid chromatography (2.2.29) as not yellow at high temperature. Allow to cool. Dissolve described in the test for composition. the residue in 0.1 ml of dilute hydrochloric acid R. Add See Figure 0465.-1. 5 ml of water R and 0.2 ml of strong sodium hydroxide Limit : solution R. No white precipitate is formed. — sum of the areas of all peaks eluting before the peak due TESTS to bacitracin B1 : maximum 20.0 per cent. Solution S. Dissolve 0.25 g in carbon dioxide-free water R Impurity E. Liquid chromatography (2.2.29) as described and dilute to 25 ml with the same solvent. in the test for composition. See Figure 0465.-2. Appearance of solution. Solution S is clear (2.2.1). General Notices (1) apply to all monographs and other texts

1045

Bacitracin


1. impurity A

5. bacitracin B2

2. impurity B

6. bacitracin B3

3. impurity C

7. bacitracin A

4. bacitracin B1

Figure 0465.-1. — Chromatogram of the test for composition in bacitracin obtained with the test solution at 254 nm

1. bacitracin B1

3. bacitracin A

2. bacitracin B3

4. impurity E

Figure 0465.-2. — Chromatogram of the test for impurity E in bacitracin obtained with reference solution (d) at 300 nm 1046


Bacitracin zinc


Detection : spectrophotometer at 254 nm ; spectrophotometer at 300 nm for reference solution (d). Injection : test solution and reference solutions (b) and (d). Limit : — impurity E : not more than 1.2 times the area of the principal peak in the chromatogram obtained with reference solution (b) (6.0 per cent). Loss on drying (2.2.32) : maximum 5.0 per cent, determined on 1.000 g by drying at 60 °C over diphosphorus pentoxide R at a pressure not exceeding 0.1 kPa for 3 h. Sulphated ash (2.4.14) : maximum 1.0 per cent, determined on 1.0 g. Sterility (2.6.1). If intended for the preparation of ophthalmic dosage forms without a further appropriate sterilisation procedure, it complies with the test for sterility. Bacterial endotoxins (2.6.14) : less than 0.8 IU/mg, if intended for use in the manufacture of ophthalmic dosage forms without a further appropriate procedure for the removal of bacterial endotoxins.

K. X = Y = L-Val, R = CH3 : bacitracin I3. 01/2005:0466

BACITRACIN ZINC Bacitracinum zincum DEFINITION Zinc complex of bacitracin, which consists of a mixture of antimicrobial polypeptides produced by certain strains of Bacillus licheniformis or Bacillus subtilis, the main components being bacitracins A, B1, B2 and B3. Content : minimum 60 IU/mg (dried substance). CHARACTERS Appearance : white or light yellowish-grey powder, hygroscopic. Solubility : slightly soluble in water and in alcohol.

IDENTIFICATION ASSAY First identification : B, C. Carry out the microbiological assay of antibiotics (2.7.2). Use Second identification : A, C. bacitracin zinc CRS as the reference substance. A. Thin-layer chromatography (2.2.27). STORAGE Test solution. Dissolve 10 mg of the substance to be In an airtight container at 2 °C to 8 °C. If the substance is examined in 0.5 ml of dilute hydrochloride acid R and sterile, store in a sterile, airtight, tamper-proof container. dilute to 1.0 ml with water R. Reference solution. Dissolve 10 mg of bacitracin LABELLING zinc CRS in 0.5 ml of dilute hydrochloric acid R and The label states, where applicable : dilute to 1.0 ml with water R. — that the substance is sterile, Plate : TLC silica gel plate R. — that the substance is free from bacterial endotoxins. Mobile phase : glacial acetic acid R, water R, butanol R (1:2:4 V/V/V). IMPURITIES Application : 10 µl. Development : over half of the plate. Drying : at 100-105 °C. Detection : spray with ninhydrin solution R1 and heat at 110 °C for 5 min. Results : the spots in the chromatogram obtained with the test solution are similar in position, size and colour to the spots in the chromatogram obtained with the reference solution. B. It complies with the test for composition (see Tests). A. X = L-Val, Y = L-Ile, R = H : bacitracin C1, C. Ignite about 0.15 g, allow to cool and dissolve the residue B. X = L-Ile, Y = L-Val, R = H : bacitracin C2, in 1 ml of dilute hydrochloric acid R. Add 4 ml of water R. C. X = Y = L-Val, R = CH3 : bacitracin C3, The solution gives the reaction of zinc (2.3.1). D. X = Y = L-Val, R = H : bacitracin E, TESTS pH (2.2.3) : 6.0 to 7.5. Shake 1.0 g for about 1 min with 10 ml of carbon dioxide-free water R and filter. Composition. Liquid chromatography (2.2.29) : use the normalisation procedure. Prepare the solutions immediately before use. Test solution. Dissolve 0.100 g of the substance to be examined in 50.0 ml of a 40 g/l solution of sodium edetate R adjusted to pH 7.0 with dilute sodium hydroxide R. E. X = Y = L-Ile, R = CH3 : bacitracin F, Reference solution (a). Dissolve 20.0 mg of bacitracin F. X = Y = L-Ile, R = H : bacitracin H1, zinc CRS in 10.0 ml of a 40 g/l solution of sodium edetate R adjusted to pH 7.0 with dilute sodium hydroxide R. G. X = L-Val, Y = L-Ile, R = CH3 : bacitracin H2, Reference solution (b). Dilute 5.0 ml of the test solution to H. X = L-Ile, Y = L-Val, R = CH3 : bacitracin H3, 100.0 ml with water R. I. X = L-Val, Y = L-Ile, R = H : bacitracin I1, Reference solution (c). Dilute 1.0 ml of reference solution (b) to 10.0 ml with water R. J. X = L-Ile, Y = L-Val, R = H : bacitracin I2, General Notices (1) apply to all monographs and other texts

1047

Bacitracin zinc


1. impurity A

5. bacitracin B2

2. impurity B

6. bacitracin B3

3. impurity C

7. bacitracin A

4. bacitracin B1

Figure 0466.-1. — Chromatogram of the test for composition in bacitracin zinc obtained with the test solution at 254 nm Reference solution (d). Dissolve 50.0 mg of the substance System suitability : reference solution (a) : to be examined in 25.0 ml of a 40 g/l solution of sodium — peak-to-valley ratio : minimum of 1.2, where Hp = height edetate R adjusted to pH 7.0 with dilute sodium hydroxide R. above the baseline of the peak due to bacitracin B1 and Heat in a boiling water-bath for 30 min. Cool to room Hv = height above the baseline of the lowest point of temperature. the curve separating this peak from the peak due to bacitracin B2. Blank solution. A 40 g/l solution of sodium edetate R Limits : adjusted to pH 7.0 with dilute sodium hydroxide R. — bacitracin A : minimum 40.0 per cent, Column : — sum of bacitracins A, B1, B2 and B3: minimum 70.0 per — size : l = 0.25 m, Ø = 4.6 mm, cent. — disregard limit : the area of the peak due to bacitracin A — stationary phase: end-capped octadecylsilyl silica gel in the chromatogram obtained with reference solution (c) for chromatography R (5 µm). (0.5 per cent) ; disregard any peak observed in the blank Mobile phase : add 520 volumes of methanol R1, 40 volumes run. of acetonitrile R and 300 volumes of water R to 100 volumes Related peptides. Liquid chromatography (2.2.29) as of a 34.8 g/l solution of dipotassium hydrogen phosphate R, described in the test for composition. adjusted to pH 6.0 with a 27.2 g/l solution of potassium See Figure 0466.-1. dihydrogen phosphate R. Limit : Flow rate : 1.0 ml/min. — sum of the areas of all peaks eluting before the peak due to bacitracin B1 : maximum 20.0 per cent. Detection : spectrophotometer at 254 nm. Impurity E. Liquid chromatography (2.2.29) as described Injection : 100 µl ; inject the blank, the test solution and in the test for composition. reference solutions (a) and (c). See Figure 0466.-2. Run time : 3 times the retention time of bacitracin A. Detection : spectrophotometer at 254 nm ; spectrophotometer Relative retention with reference to bacitracin A (retention at 300 nm for reference solution (d). time = 15 min to 25 min) : bacitracin B1 = about 0.6 ; Injection : test solution and reference solutions (b) and (d). bacitracin B3 = about 0.8 ; impurity E = about 2.5. Limit : If necessary, adjust the composition of the mobile phase by — impurity E : not more than 1.2 times the area of the principal peak in the chromatogram obtained with changing the amount of organic modifier whilst keeping the reference solution (b) (6.0 per cent). ratio constant between methanol and acetonitrile. 1048


Bacitracin zinc


1. bacitracin B1

3. bacitracin A

2. bacitracin B3

4. impurity E

Figure 0466.-2. — Chromatogram of the test for impurity E in bacitracin zinc obtained with reference solution (d) at 300 nm

Zinc : 4.0 per cent to 6.0 per cent (dried substance).

STORAGE In an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container.

Dissolve 0.200 g in a mixture of 2.5 ml of dilute acetic acid R and 2.5 ml of water. Add 50 ml of water R, 50 mg of xylenol orange triturate R and sufficient hexamethylenetetramine R to produce a red colour. Add 2 g LABELLING of hexamethylenetetramine R in excess. Titrate with 0.01 M The label states, where applicable : sodium edetate until a yellow colour is obtained. — that the substance is sterile, 1 ml of 0.01 M sodium edetate is equivalent to 0.654 mg — that the substance is apyrogenic. of Zn. Loss on drying (2.2.32) : maximum 5.0 per cent, determined IMPURITIES on 1.000 g by drying at 60 °C over diphosphorus pentoxide R at a pressure not exceeding 0.1 kPa for 3 h. Sterility (2.6.1). If intended for administration by spraying into internal body cavities without a further appropriate sterilisation procedure, it complies with the test for sterility.

Pyrogens (2.6.8). If intended for administration by spraying into internal body cavities without a further appropriate procedure for the removal of pyrogens, it complies with the test for pyrogens. Inject per kilogram of the rabbit’s mass 1 ml of the supernatant liquid obtained by centrifuging a suspension containing 11 mg per millilitre in a 9 g/l solution A. X = L-Val, Y = L-Ile, R = H : bacitracin C1, of sodium chloride R. ASSAY Suspend 50.0 mg in 5 ml of water R, add 0.5 ml of dilute hydrochloric acid R and dilute to 100.0 ml with water R. Allow the solution to stand for 30 min. Carry out the microbiological assay of antibiotics (2.7.2). General Notices (1) apply to all monographs and other texts

B. X = L-Ile, Y = L-Val, R = H : bacitracin C2, C. X = Y = L-Val, R = CH3 : bacitracin C3, D. X = Y = L-Val, R = H : bacitracin E, 1049

Baclofen

E. F. G. H. I. J. K.


X = Y = L-Ile, R = CH3 : bacitracin F, X = Y = L-Ile, R = H : bacitracin H1, X = L-Val, Y = L-Ile, R = CH3 : bacitracin H2, X = L-Ile, Y = L-Val, R = CH3 : bacitracin H3, X = L-Val, Y = L-Ile, R = H : bacitracin I1, X = L-Ile, Y = L-Val, R = H : bacitracin I2, X = Y = L-Val, R = CH3 : bacitracin I3. 01/2005:0653

BACLOFEN Baclofenum

C10H12ClNO2

Mr 213.7

DEFINITION Baclofen contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of (3RS)-4-amino-3-(4-chlorophenyl)butanoic acid, calculated with reference to the anhydrous substance. CHARACTERS A white or almost white powder, slightly soluble in water, very slightly soluble in alcohol, practically insoluble in acetone. It dissolves in dilute mineral acids and in dilute solutions of alkali hydroxides. It shows polymorphism. IDENTIFICATION First identification : B. Second identification : A, C. A. Dissolve 70 mg in water R and dilute to 100.0 ml with the same solvent. Examined between 220 nm and 320 nm (2.2.25), the solution shows three absorption maxima, at 259 nm, 266 nm and 275 nm. The specific absorbances at these maxima are 9.8 to 10.8, 11.5 to 12.7 and 8.4 to 9.3, respectively. The test is not valid unless in the test for resolution (2.2.25) the ratio of absorbances is at least 1.5. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with baclofen CRS. Examine as discs prepared using 3 mg of substance and 300 mg of potassium bromide R. If the spectra obtained with the substance to be examined and the reference substance show differences, dissolve 0.1 g of each of the substances separately in 1 ml of dilute sodium hydroxide solution R and add 10 ml of alcohol R and 1 ml of dilute acetic acid R. Allow to stand for 1 h. Filter, wash the precipitate with alcohol R and dry in vacuo. Prepare new discs and record the spectra. 1050

C. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. Dissolve 10 mg of the substance to be examined in the mobile phase (see below) and dilute to 10 ml with the mobile phase. Reference solution. Dissolve 10 mg of baclofen CRS in the mobile phase and dilute to 10 ml with the mobile phase. Apply separately to the plate 5 µl of each solution. Develop over a path of 12 cm using a mixture of 5 volumes of anhydrous formic acid R, 5 volumes of water R, 20 volumes of methanol R, 30 volumes of chloroform R and 40 volumes of ethyl acetate R. Allow the solvents to evaporate. Spray with ninhydrin solution R3 until the plate is slightly wet. Place the plate in an oven maintained at 100 °C for 10 min. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. TESTS Appearance of solution. Dissolve 0.50 g in 1 M sodium hydroxide and dilute to 25 ml with the same solvent. The solution is not more opalescent than reference suspension II (2.2.1) and not more intensely coloured than reference solution BY5 (2.2.2, Method II). Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (a). Dissolve 25.0 mg of baclofen impurity A CRS in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 100.0 ml with the mobile phase. Reference solution (c). Dilute 2.0 ml of the test solution to 100.0 ml with the mobile phase. Reference solution (d). Dilute 2.0 ml of the test solution and 2.0 ml of reference solution (a) to 100.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.0 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (10 µm), — as mobile phase at a flow-rate of 2.0 ml/min a mixture prepared as follows : dissolve 1.822 g of sodium hexanesulphonate R in 1 litre of a mixture of 560 volumes of water R, 440 volumes of methanol R and 5 volumes of glacial acetic acid R, — as detector a spectrophotometer set at 266 nm. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 20 µl of reference solution (c) is at least 50 per cent of the full scale of the recorder. Inject 20 µl of reference solution (d). The test is not valid unless the resolution between the peaks corresponding to baclofen and baclofen impurity A is at least 2.0. Inject separately 20 µl of the test solution, reference solution (b) and reference solution (c). Continue the chromatography for five times the retention time of the principal peak. In the chromatogram obtained with the test solution : the area of the peak corresponding to baclofen impurity A is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent) ; the sum of the areas of all the peaks, apart



Bambuterol hydrochloride

show differences, dissolve the substance to be examined from the principal peak, is not greater than the area of the and the reference substance in a mixture of 1 volume principal peak in the chromatogram obtained with reference of water R and 6 volumes of acetone R, cool in ice to solution (c) (2.0 per cent). precipitate and dry both precipitates in vacuo at 50 °C to Water (2.5.12). Not more than 1.0 per cent, determined on constant weight. Record new spectra using the residues. 1.000 g by the semi-micro determination of water. B. It gives reaction (a) of chlorides (2.3.1). Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. TESTS Solution S. Dissolve 4.0 g in carbon dioxide-free water R ASSAY and dilute to 20.0 ml with the same solvent. Dissolve 0.1500 g in 50 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point Acidity or alkalinity. To 10 ml of solution S add 0.2 ml of potentiometrically (2.2.20). methyl red solution R and 0.2 ml of 0.01 M hydrochloric acid. The solution is red. Add 0.4 ml of 0.01 M sodium 1 ml of 0.1 M perchloric acid is equivalent to 21.37 mg of hydroxide. The solution is yellow. C10H12ClNO2. Optical rotation (2.2.7). Dilute 1 ml of solution S to 10 ml IMPURITIES with carbon dioxide-free water R. The angle of optical rotation is − 0.10° to + 0.10°. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 5.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. A. (4RS)-4-(4-chlorophenyl)pyrrolidin-2-one, Reference solution (a). Dissolve 1.0 mg of formoterol fumarate dihydrate CRS in the mobile phase and dilute to 10.0 ml with the mobile phase. Mix 0.8 ml of the solution with 0.4 ml of the test solution and dilute to 100.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. Dilute 2.0 ml of the solution to 20.0 ml with the mobile phase. The chromatographic procedure may be carried out using : B. (3RS)-5-amino-3-(4-chlorophenyl)-5-oxopentanoic acid. — a stainless steel column 0.15 m long and 4.6 mm in internal diameter packed with base-deactivated 01/2005:1293 octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.5 ml/min a BAMBUTEROL HYDROCHLORIDE mixture prepared as follows : dissolve 1.3 g of sodium octanesulphonate R in 430 ml of a mixture of 25 volumes of acetonitrile R1 and 75 volumes of methanol R ; then Bambuteroli hydrochloridum mix this solution with 570 ml of 0.050 M phosphate buffer pH 3.0 (dissolve 6.90 g of sodium dihydrogen phosphate monohydrate R in water R and dilute to 1000 ml with water R, adjust the pH to 3.0 with a 50 g/l solution of dilute phosphoric acid R), — as detector a spectrophotometer set at 214 nm. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 20 µl of reference solution (b) is about 50 per cent of the full scale of the recorder. C18H30ClN3O5 Mr 403.9 Inject 20 µl of reference solution (a). When the chromatograms are recorded under the prescribed DEFINITION conditions, the retention times are : formoterol about 7 min Bambuterol hydrochloride contains not less than 98.5 per and bambuterol about 9 min. The test is not valid unless the cent and not more than the equivalent of 101.5 per cent of resolution between the peaks corresponding to bambuterol 5-[(1RS)-2-[(1,1-dimethylethyl)amino]-1-hydroxyethyl]-1,3phenylene bis(dimethylcarbamate) hydrochloride, calculated and formoterol is at least 5.0. Continue the chromatography of the test solution for 1.5 times the retention time of with reference to the anhydrous substance. bambuterol. If necessary adjust the composition of the CHARACTERS mobile phase. Increase the content of phosphate buffer to increase the retention time. A white or almost white, crystalline powder, freely soluble in water, soluble in alcohol. Inject separately 20 µl of the mobile phase, 20 µl of the test solution and 20 µl of reference solution (b). In the It shows polymorphism. chromatogram obtained with the test solution the area IDENTIFICATION of any peak, apart from the principal peak, is not greater than the area of the principal peak obtained with reference A. Examine by infrared absorption spectrophotometry solution (b) (0.2 per cent) ; the sum of the areas of all (2.2.24), comparing with the spectrum obtained the peaks, apart from the principal peak, is not greater with bambuterol hydrochloride CRS. Examine the than three times the area of the principal peak in the substances prepared as discs. If the spectra obtained General Notices (1) apply to all monographs and other texts

1051

Barbital


chromatogram obtained with reference solution (b) (0.6 per cent). Disregard any peak due to the mobile phase and any peak with an area less than 0.25 times the area of the principal peak obtained with reference solution (b). Water (2.5.12). Not more than 0.5 per cent, determined on 0.500 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.320 g in 50 ml of alcohol R and add 5 ml of 0.01 M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the two points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 40.39 mg of C18H30ClN3O5.

DEFINITION Barbital contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of 5,5-diethylpyrimidine-2,4,6(1H,3H,5H)-trione, calculated with reference to the dried substance. CHARACTERS A white, crystalline powder or colourless crystals, slightly soluble in water, soluble in boiling water and in alcohol. It forms water-soluble compounds with alkali hydroxides and carbonates and with ammonia.

IDENTIFICATION First identification : A, B. Second identification : A, C, D. A. Determine the melting point (2.2.14) of the substance to be examined. Mix equal parts of the substance to be examined and barbital CRS and determine the melting IMPURITIES point of the mixture. The difference between the melting points (which are about 190 °C) is not greater than 2 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with barbital CRS. C. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 75 mg of the substance to be A. R1 = NH-C(CH3)3, R2 = R3 = H : (1RS)-1-(3,5examined in alcohol R and dilute to 25 ml with the same dihydroxyphenyl)-2-[(1,1-dimethylethyl)amino]ethanol solvent. (terbutaline), Reference solution. Dissolve 75 mg of barbital CRS in alcohol R and dilute to 25 ml with the same solvent. B. R1 = OH, R2 = R3 = CO-N(CH3)2 : 5-[(1RS)-1,2Apply separately to the plate 10 µl of each solution. dihydroxyethyl]-1,3-phenylene bis(dimethylcarbamate), Develop over a path of 18 cm using the lower layer of C. R1 = NH-C(CH3)3, R2 = H, R3 = CO-N(CH3)2 : 3-[(1RS)-2-[(1, a mixture of 5 volumes of concentrated ammonia R, 1-dimethylethyl)amino]-1-hydroxyethyl]-5-hydroxyphenyl 15 volumes of alcohol R and 80 volumes of chloroform R. dimethylcarbamate, Examine immediately in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test D. R1 = H, R2 = R3 = CO-N(CH3)2 : 5-[(1RS)-1-hydroxyethyl]solution is similar in position and size to the principal 1,3-phenylene bis(dimethylcarbamate), spot in the chromatogram obtained with the reference solution. D. It gives the reaction of non-nitrogen substituted barbiturates (2.3.1). TESTS Appearance of solution. Dissolve 1.0 g in a mixture of 4 ml of dilute sodium hydroxide solution R and 6 ml of water R. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). E. R = H : 5-acetyl-1,3-phenylene bis(dimethylcarbamate), Acidity. Boil 1.0 g with 50 ml of water R for 2 min, allow to cool and filter. To 10 ml of the filtrate add 0.15 ml of methyl F. R = NH-C(CH3)3 : 5-[[(1,1-dimethylethyl)amino]acetyl]-1, red solution R. The solution is orange-yellow. Not more than 3-phenylene bis(dimethylcarbamate). 0.1 ml of 0.1 M sodium hydroxide is required to produce a pure yellow colour. Related substances. Examine by thin-layer chromatography 01/2005:0170 (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 1.0 g of the substance to be examined BARBITAL in alcohol R and dilute to 100 ml with the same solvent. Reference solution. Dilute 0.5 ml of the test solution to 100 ml with alcohol R. Barbitalum Apply separately to the plate 20 µl of each solution. Develop over a path of 15 cm using the lower layer of a mixture of 5 volumes of concentrated ammonia R, 15 volumes of alcohol R and 80 volumes of chloroform R. Examine immediately in ultraviolet light at 254 nm. Spray with diphenylcarbazone mercuric reagent R. Allow the plate to dry in air and spray with freshly prepared alcoholic C8H12N2O3 Mr 184.2 potassium hydroxide solution R diluted 1 in 5 with 1052



Basic butylated methacrylate copolymer

and shake well. The colour of the solution is more intense than that of a standard prepared at the same time and in the same manner omitting the potassium iodate. Soluble barium salts. To 10 ml of solution S add 1 ml of dilute sulphuric acid R. After 1 h, any opalescence in the solution is not more intense than that in a mixture of 10 ml of solution S and 1 ml of distilled water R. Phosphates. To 1.0 g add a mixture of 3 ml of dilute nitric acid R and 7 ml of water R. Heat on a water-bath for 5 min, filter and dilute the filtrate to 10 ml with water R. Add 5 ml of molybdovanadic reagent R. After 5 min, any yellow colour in the test solution is not more intense than that in a ASSAY standard prepared at the same time and in the same manner Dissolve 85.0 mg in 5 ml of pyridine R. Add 0.5 ml of using 10 ml of phosphate standard solution (5 ppm PO4) R thymolphthalein solution R and 10 ml of silver nitrate (50 ppm). solution in pyridine R. Titrate with 0.1 M ethanolic sodium Arsenic (2.4.2). In a small long-necked combustion flask, hydroxide until a pure blue colour is obtained. Carry out shake 0.5 g with 2 ml of nitric acid R and 30 ml of water R, a blank titration. insert a small funnel in the neck of the flask, heat in an 1 ml of 0.1 M ethanolic sodium hydroxide is equivalent to inclined position on a water-bath for 2 h, allow to cool, adjust 9.21 mg of C8H12N2O3. to the original volume with water R and filter. Wash the residue by decantation with three quantities, each of 5 ml, 01/2005:0010 of water R. Combine the filtrate and washings, add 1 ml of sulphuric acid R, evaporate to dryness on a water-bath, and heat until copious white fumes are evolved. Dissolve the BARIUM SULPHATE residue in 10 ml of dilute sulphuric acid R and add 10 ml of water R. The solution complies with limit test A for arsenic Barii sulfas (2 ppm). BaSO4 Mr 233.4 Heavy metals (2.4.8). Dilute 10 ml of solution S to 20 ml with water R, 12 ml of this solution complies with limit test A CHARACTERS for heavy metals (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. A fine, heavy, white powder, free from gritty particles, practically insoluble in water and in organic solvents. It Loss on ignition. Not more than 2.0 per cent, determined on is very slightly soluble in acids and in solutions of alkali 1.0 g at 600 °C. hydroxides. Sedimentation. Place 5.0 g in a glass-stoppered, 50 ml graduated cylinder having the 50 ml graduation mark 14 cm IDENTIFICATION from the base. Add water R to 50 ml, shake the mixture for A. Boil 0.2 g with 5 ml of a 500 g/l solution of sodium 5 min and allow to stand for 15 min. The barium sulphate carbonate R for 5 min, add 10 ml of water R, filter and does not settle below the 15 ml mark. acidify a part of the filtrate with dilute hydrochloric acid R. The solution gives the reactions of sulphates 01/2005:1975 (2.3.1). B. Wash the residue collected in the preceding test with BASIC BUTYLATED METHACRYLATE three successive small quantities of water R. To the residue add 5 ml of dilute hydrochloric acid R, filter and COPOLYMER add to the filtrate 0.3 ml of dilute sulphuric acid R. A white precipitate is formed that is insoluble in dilute Copolymerum methacrylatis butylati sodium hydroxide solution R. aldehyde-free alcohol R. Heat at 100 °C to 105 °C for 5 min and examine immediately. When examined in ultraviolet light and after spraying, any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution (0.5 per cent). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.00 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

TESTS Solution S. To 20.0 g add 40 ml of distilled water R and 60 ml of dilute acetic acid R. Boil for 5 min, filter and dilute the cooled filtrate to 100 ml with distilled water R. Acidity or alkalinity. Heat 5.0 g with 20 ml of carbon dioxide-free water R on a water-bath for 5 min and filter. To 10 ml of the filtrate add 0.05 ml of bromothymol blue solution R1. Not more than 0.5 ml of 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is required to change the colour of the indicator. Acid-soluble substances. Evaporate 25 ml of solution S to dryness on a water-bath and dry to constant mass at 100-105 °C. The residue weighs not more than 15 mg (0.3 per cent). Oxidisable sulphur compounds. Shake 1.0 g with 5 ml of water R for 30 s and filter. To the filtrate add 0.1 ml of starch solution R, dissolve 0.1 g of potassium iodide R in the mixture, add 1.0 ml of a freshly prepared 3.6 mg/l solution of potassium iodate R and 1 ml of 1 M hydrochloric acid General Notices (1) apply to all monographs and other texts

basicum

DEFINITION Copolymer of 2-(dimethylamino)ethyl methacrylate, butyl methacrylate and methyl methacrylate having a mean relative molecular mass of about 150 000. The ratio of 2-dimethylaminoethyl methacrylate groups to butyl methacrylate and methyl methacrylate groups is about 2:1:1. Content of dimethylaminoethyl groups: 20.8 per cent to 25.5 per cent (dried substance). CHARACTERS Appearance : colourless or yellowish granules or white powder, slightly hygroscopic. Solubility : practically insoluble in water, freely soluble in methylene chloride. It dissolves slowly in alcohol. IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of basic butylated methacrylate copolymer. 1053

Bearberry leaf


B. It complies with the limits of the assay. TESTS Solution S. Dissolve 12.5 g in a mixture of 35.0 g of acetone R and 52.5 g of 2-propanol R. Viscosity (2.2.10) : 3 mPa·s to 6 mPa·s, determined on solution S. Apparatus : rotating viscosimeter. Dimensions : — spindle : diameter = 25.15 mm, height = 90.74 mm, shaft diameter = 4 mm, — cylinder : diameter = 27.62 mm, height = 0.135 m. Stirring speed : 30 r/min. Volume of solution : 16 ml of solution S. Temperature : 20 °C. Absorbance (2.2.25) : maximum 0.30 at 420 nm, determined on solution S. Appearance of film. Spread evenly 1.0 ml of solution S on a glass plate. Upon drying a clear film is formed. Monomers : maximum 0.3 per cent, for the sum of contents of butyl methacrylate, methyl methacrylate and 2-dimethylaminoethyl methacrylate calculated by procedures A and B. A. Butyl methacrylate and methyl methacrylate. Liquid chromatography (2.2.29). Test solution. Dissolve 1.00 g of the substance to be examined in phosphate buffer solution pH 2.0 R and dilute to 50.0 ml with the same buffer solution. Reference solution. Dissolve 10.0 mg of butyl methacrylate R and 10.0 mg of methyl methacrylate R in 10.0 ml of acetonitrile R and dilute to 50.0 ml with water R. Dilute 1.0 ml of the solution to 50.0 ml with water R. Column : — size : l = 0.125 m, Ø = 4.6 mm, — stationary phase: octadecylsilyl silica gel for chromatography R (7 µm). Mobile phase : phosphate buffer solution pH 2.0 R, methanol R (45:55 V/V). Flow rate : 2.0 ml/min. Detection : spectrophotometer at 205 nm. Injection : 50 µl. B. 2-Dimethylaminoethyl methacrylate. Liquid chromatography (2.2.29). Test solution. Dissolve 1.00 g of the substance to be examined in tetrahydrofuran R and dilute to 50.0 ml with the same solvent. Reference solution. Dissolve 10.0 mg of 2-(dimethylamino)ethyl methacrylate R in tetrahydrofuran R and dilute to 50.0 ml with the same solvent. Dilute 2.0 ml of the solution to 50.0 ml with tetrahydrofuran R. Column : — size : l = 0.125 m, Ø = 4.6 mm, — stationary phase: aminopropylsilyl silica gel for chromatography R (10 µm). Mobile phase : mix 25 volumes of a 3.404 g/l solution of potassium dihydrogen phosphate R and 75 volumes of tetrahydrofuran R. Flow rate : 2.0 ml/min. Detection : spectrophotometer at 215 nm. Injection : 50 µl. 1054

Heavy metals (2.4.8) : maximum 20 ppm. 2.0 g complies with limit test C. Prepare the standard using 4.0 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32) : maximum 2.0 per cent, determined on 1.000 g by drying in an oven at 110 °C for 3 h. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.200 g in a mixture of 4 ml of water R and 96 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 7.21 mg of C4H10N. STORAGE In an airtight container. IMPURITIES

A. R = [CH2]3-CH3 : butyl methacrylate, B. R = CH3 : methyl methacrylate, C. R = CH2-CH2-N(CH3)2 : 2-(dimethylamino)ethyl methacrylate. 01/2005:1054

BEARBERRY LEAF Uvae ursi folium DEFINITION Whole or cut, dried leaf of Arctostaphylos uva-ursi (L.) Spreng. Content : minimum 7.0 per cent of anhydrous arbutin (C12H16O7 ; Mr 272.3) (dried drug). CHARACTERS Macroscopic and microscopic characters described under identification tests A and B. IDENTIFICATION A. The leaf, shiny and dark green on the adaxial surface, lighter on the abaxial surface, is normally 7 mm to 30 mm long and 5 mm to 12 mm wide. The entire leaf is obovate with smooth margins, somewhat reflexed downwards, narrowing at the base into a short petiole. The leaf is obtuse or retuse at its apex. The lamina is thick and coriaceous. The venation, pinnate and finely reticulate, is clearly visible on both surfaces. The adaxial surface is marked with sunken veinlets, giving it a characteristic grainy appearance. Only the young leaf has ciliated margins. Old leaves are glabrous. B. Reduce to a powder (355). The powder is green to greenish-grey or yellowish-green. Examine under a microscope using chloral hydrate solution R. The powder consists of fragments of epidermises which, seen in surface view, show polygonal cells covered by a thick smooth cuticle, and with straight, thick and irregularly pitted walls ; anomocytic stomata (2.8.3), surrounded by 5 to 11 subsidiary cells and scars of hair bases only on the abaxial epidermis ; fragments of palisade parenchyma, with 3 or 4 layers of cells of unequal lengths, and spongy


Beclometasone dipropionate


parenchyma ; groups of lignified fibres from the pericycle, with rows of cells containing prisms of calcium oxalate ; occasional conical, unicellular covering trichomes. C. Thin-layer chromatography (2.2.27). Test solution. To 0.5 g of the powdered drug (355) add 5 ml of a mixture of equal volumes of methanol R and water R, and heat under a reflux condenser for 10 min. Filter whilst hot. Wash the flask and the filter with a mixture of equal volumes of methanol R and water R and dilute to 5 ml with the same mixture of solvents. Reference solution. Dissolve 25 mg of arbutin R, 25 mg of gallic acid R and 25 mg of hydroquinone R in methanol R and dilute to 10.0 ml with the same solvent. Plate : TLC silica gel G plate R. Mobile phase : anhydrous formic acid R, water R, ethyl acetate R (6:6:88 V/V/V). Application : 10 µl of the reference solution and 20 µl of the test solution, as bands. Development : over a path of 15 cm. Drying : at 105-110 °C until the mobile phase has evaporated. Detection : spray with a 10 g/l solution of dichloroquinonechlorimide R in methanol R. Next spray with a 20 g/l solution of anhydrous sodium carbonate R. Results : see below the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. Furthermore, 2 or 3 blue bands and several brown or brownish-grey bands may be present in the chromatogram obtained with the test solution.

— size : l = 0.25 m, Ø = 4 mm, — stationary phase : base-deactivated octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : methanol R, water R (10:90 V/V). Flow rate : 1.2 ml/min. Detection : spectrophotometer at 280 nm. Calculate the percentage content of arbutin using the following expression :

F1

=

area of the peak due to arbutin in the chromatogram obtained with the test solution,

F2

=

area of the peak due to arbutin in the chromatogram obtained with the reference solution,

m1

=

mass of the drug to be examined, in grams,

m2

=

mass of arbutin R in the reference solution, in grams. 01/2005:0654

BECLOMETASONE DIPROPIONATE Beclometasoni dipropionas

Top of the plate Hydroquinone : a blue zone

A blue zone

Gallic acid : a brownish zone

A brownish zone

_______

_______

_______

_______

Arbutin : a light blue zone Reference solution

A light blue zone (arbutin) Test solution

TESTS Foreign matter (2.8.2) : maximum 5 per cent of stems and maximum 3 per cent of other foreign matter. Leaves of different colour : maximum 10 per cent, determined in the same manner as foreign matter (2.8.2). Loss on drying (2.2.32) : maximum 10.0 per cent, determined on 1.000 g of the powdered drug (355) by drying in an oven at 100-105 °C for 2 h. Total ash (2.4.16) : maximum 5.0 per cent. ASSAY Liquid chromatography (2.2.29). Test solution. In a 100 ml flask with a ground-glass neck, place 0.800 g of the powdered drug (250). Add 20 ml of water R and heat under a reflux condenser on a water-bath for 30 min. Allow to cool and filter the liquid through a plug of absorbent cotton. Add the absorbent cotton to the residue in the 100 ml flask and extract with 20 ml of water R under a reflux condenser on a water-bath for 30 min. Allow to cool and filter through a paper filter. Combine the filtrates and dilute to 50.0 ml with water R. Filter the liquid through a paper filter. Discard the first 10 ml of the filtrate. Reference solution. Dissolve 50.0 mg of arbutin R in the mobile phase and dilute to 50.0 ml with the mobile phase. Column : General Notices (1) apply to all monographs and other texts

C28H37ClO7

Mr 521.1

DEFINITION Beclometasone dipropionate contains not less than 96.0 per cent and not more than the equivalent of 103.0 per cent of 9-chloro-11β,17,21-trihydroxy-16β-methylpregna-1,4-diene-3, 20-dione 17,21-dipropionate, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, practically insoluble in water, freely soluble in acetone, sparingly soluble in alcohol. It melts at about 210 °C, with decomposition. IDENTIFICATION First identification : A, D. Second identification : B, C, D. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with beclometasone dipropionate CRS. If the spectra obtained in the solid state with the substance to be examined and the reference substance show differences, record further spectra using 50 g/l solutions in chloroform R. B. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm. 1055

Beclometasone dipropionate


Test solution (a). Dissolve 25 mg of the substance to be examined in methanol R with gentle heating and dilute to 5 ml with the same solvent. (This solution is also used to prepare test solution (b)). Dilute 2 ml of the solution to 10 ml with chloroform R. Test solution (b). Transfer 2 ml of the solution obtained during preparation of test solution (a) to a 15 ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10 ml of saturated methanolic potassium hydrogen carbonate solution R and immediately pass a stream of nitrogen R briskly through the solution for 5 min. Stopper the tube. Heat in a water-bath at 45 °C, protected from light, for 3 h. Allow to cool. Reference solution (a). Dissolve 25 mg of beclometasone dipropionate CRS in methanol R with gentle heating and dilute to 5 ml with the same solvent. (This solution is also used to prepare reference solution (b)). Dilute 2 ml of the solution to 10 ml with chloroform R. Reference solution (b). Transfer 2 ml of the solution obtained during preparation of reference solution (a) to a 15 ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10 ml of saturated methanolic potassium hydrogen carbonate solution R and immediately pass a stream of nitrogen R briskly through the solution for 5 min. Stopper the tube. Heat in a water-bath at 45 °C, protected from light, for 3 h. Allow to cool. Apply separately to the plate 5 µl of each solution. Prepare the mobile phase by mixing 1.2 volumes of water R and 8 volumes of methanol R and adding the mixture to a mixture of 15 volumes of ether R and 77 volumes of methylene chloride R. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The chromatogram obtained with test solution (b) shows not fewer than two distinct spots which are similar in position and size to the spots in the chromatogram obtained with reference solution (b). The principal spot in the chromatogram obtained with test solution (a) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). Spray with alcoholic solution of sulphuric acid R. Heat at 120 °C for 10 min or until the spots appear. Allow to cool. Examine the plate in daylight and in ultraviolet light at 365 nm. The chromatogram obtained with test solution (b) shows not fewer than two distinct spots, similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the spots in the chromatogram obtained with reference solution (b). The principal spot in the chromatogram obtained with test solution (a) is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (a). The principal spots in the chromatograms obtained with test solution (b) and reference solution (b) have Rf values distinctly lower than the principal spots in the chromatograms obtained with test solution (a) and reference solution (a). C. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a deep reddish-brown colour develops. Add the solution to 10 ml of water R and mix. The colour is discharged and a clear solution remains. D. Treat 25 mg by the oxygen-flask method (2.5.10). Use a mixture of 1 ml of 1 M sodium hydroxide and 20 ml of water R to absorb the combustion products. The solution gives reaction (a) of chlorides (2.3.1). 1056

TESTS Specific optical rotation (2.2.7). Dissolve 0.250 g in dioxan R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 88 to + 94, calculated with reference to the dried substance. Absorbance (2.2.25). Dissolve 50.0 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the solution to 50.0 ml with alcohol R. The specific absorbance at the maximum at 238 nm is 284 to 302, calculated with reference to the dried substance. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 62.5 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the mobile phase. Reference solution (a). Dissolve 2 mg of beclometasone 17-propionate CRS and 2 mg of beclometasone 21-propionate CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows : mix 600 ml of acetonitrile R and 350 ml of water R, allow to cool, dilute to 1000 ml with water R and mix again, — as detector a spectrophotometer set at 254 nm and with the sensitivity adjusted so that the height of the principal peak in the chromatogram obtained with reference solution (b) is 70 per cent to 90 per cent of the full scale. Equilibrate the column with the mobile phase at a flow rate of 1 ml/min for about 45 min. When the chromatograms are recorded in the conditions described above, the retention times are : beclometasone 17-propionate, about 6.2 min ; beclometasone 21-propionate, about 6.7 min ; beclometasone dipropionate, 13 min to 14 min. Inject 20 µl of reference solution (a). The system is not suitable unless the resolution between the peaks corresponding to beclometasone 17-propionate and beclometasone 21-propionate is at least 1.4 ; if this resolution is not achieved, adjust the concentration of acetonitrile in the mobile phase. Verify the repeatability by making five separate injections of 20 µl of reference solution (b) ; the system is not suitable unless the relative standard deviation for the area of the principal peak in the chromatograms obtained with reference solution (b) is less than 2.0 per cent. Inject separately 20 µl of the test solution and 20 µl of reference solution (b). Continue the chromatography for twice the retention time of the principal peak. In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (2.0 per cent) and not more than one such peak has an area greater than half the area of the principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than 1.25 times the area of the principal peak in the chromatogram obtained with reference solution (b) (2.5 per cent). Disregard any peak due to the solvent and any peak with an area less than 0.025 times the area of the principal peak in the chromatogram obtained with reference solution (b).


Beeswax, white


Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C for 3 h. ASSAY Protect the solutions from light throughout the assay. The coloured reaction products tend to adsorb to the surface of glassware. To avoid low results, it is recommended that the glassware be treated with the coloured reaction products before use and that the treated glassware be reserved for this assay and washed only with water R between assays. Dissolve an accurately weighed quantity in sufficient aldehyde-free alcohol R to produce a solution containing 340 µg to 360 µg in 10.0 ml. Prepare a reference solution at the same time and in the same manner, using beclometasone dipropionate CRS. Into two 25 ml volumetric flasks, introduce respectively 10.0 ml of each solution. Into a third identical flask, introduce 10 ml of aldehyde-free alcohol R. To each flask add 2.0 ml of triphenyltetrazolium chloride solution R. Displace the air from the flasks using oxygen-free nitrogen R, immediately add 2.0 ml of dilute tetramethylammonium hydroxide solution R and again displace the air with oxygen-free nitrogen R. Stopper the flasks, mix the contents by gentle swirling and allow to stand in a water-bath at 35 °C for 2 h. Cool rapidly and dilute to 25.0 ml with aldehyde-free alcohol R. Immediately measure the absorbance (2.2.25) of the test solution and of the reference solution at the maximum at 485 nm, using a closed 1 cm cell and as the compensation liquid the solution prepared from 10 ml of aldehyde-free alcohol R. Treat the test solution and the reference solution in such a manner that the period of time that elapses between the addition of the dilute tetramethylammonium hydroxide solution R and measurement of the absorbance is exactly the same for each solution. Calculate the content of C28H37ClO7 from the absorbances measured and the concentrations of the solutions.

TESTS Drop point (2.2.17) : 61 °C to 66 °C. Melt the beeswax by heating on a water-bath, pour onto a glass plate and allow to cool to a semi-solid mass. Fill the metal cup by inserting the wider end into the beeswax and repeating the procedure until beeswax extrudes from the narrow opening. Remove the excess with a spatula and insert the thermometer immediately. Remove the beeswax displaced. Allow to stand at room temperature for at least 12 h before determining the drop point. Acid value : 17.0 to 24.0. To 2.00 g (m g), in a 250 ml conical flask fitted with a reflux condenser, add 40 ml of xylene R and a few glass beads. Heat until the substance is dissolved. Add 20 ml of alcohol R and 0.5 ml of phenolphthalein solution R1 and titrate the hot solution with 0.5 M alcoholic potassium hydroxide until a red colour persists for at least 10 s (n1 ml). Carry out a blank test (n2 ml).

Ester value (2.5.2) : 70 to 80. Saponification value : 87 to 104. To 2.00 g (m g), in a 250 ml conical flask fitted with a reflux condenser, add 30 ml of a mixture of equal volumes of alcohol R and xylene R and a few glass beads. Heat until the substance is dissolved. Add 25.0 ml of 0.5 M alcoholic potassium hydroxide and heat under a reflux condenser for 3 h. Titrate the hot solution immediately with 0.5 M hydrochloric acid, using 1 ml of phenolphthalein solution R1 as indicator (n1 ml). Reheat the solution to boiling several times during the course of the titration. Carry out a blank test (n2 ml).


Ceresin, paraffins and certain other waxes. To 3.0 g, in a 100 ml round-bottomed flask, add 30 ml of a 40 g/l solution of potassium hydroxide R in aldehyde-free alcohol R and boil gently under a reflux condenser for 2 h. Remove the 01/2005:0069 condenser and immediately insert a thermometer. Place the flask in a water-bath at 80 °C and allow to cool, swirling the solution continuously. No precipitate is formed until 65 °C, BEESWAX, WHITE although the solution may be slightly opalescent. Above 65 °C, the solution may become cloudy and precipitates may be formed. At 59 °C, the solution is cloudy. Cera alba

DEFINITION Wax obtained by bleaching yellow beeswax. CHARACTERS Appearance : white or yellowish-white pieces or plates, translucent when thin, with a fine-grained, matt and non-crystalline fracture ; when warmed in the hand they become soft and malleable. It has an odour similar to that of yellow beeswax, though fainter and never rancid. It is tasteless and does not stick to the teeth. Solubility : practically insoluble in water, partially soluble in hot alcohol (90 per cent V/V) and completely soluble in fatty and essential oils. Relative density : about 0.960. General Notices (1) apply to all monographs and other texts

Glycerol and other polyols : maximum 0.5 per cent m/m, calculated as glycerol. To 0.20 g add 10 ml of alcoholic potassium hydroxide solution R and heat on a water-bath under a reflux condenser for 30 min. Add 50 ml of dilute sulphuric acid R, cool and filter. Rinse the flask and the filter with dilute sulphuric acid R. Combine the filtrate and washings and dilute to 100.0 ml with dilute sulphuric acid R. Place 1.0 ml of the solution in a test-tube, add 0.5 ml of a 10.7 g/l solution of sodium periodate R, mix and allow to stand for 5 min. Add 1.0 ml of decolorised fuchsin solution R and mix. Any precipitate disappears. Place the tube in a beaker containing water at 40 °C. During cooling observe for 10-15 min. Any violet-blue colour in the solution is not more intense than that in a standard prepared at the same time and in the same manner using 1.0 ml of a 10 mg/l solution of glycerol R in dilute sulphuric acid R. 1057

Beeswax, yellow


01/2005:0070 although the solution may be slightly opalescent. Above 65 °C, the solution may become cloudy and precipitates may be formed. At 59 °C, the solution is cloudy. BEESWAX, YELLOW Glycerol and other polyols : maximum 0.5 per cent m/m, calculated as glycerol. Cera flava To 0.20 g add 10 ml of alcoholic potassium hydroxide solution R and heat on a water-bath under a reflux condenser DEFINITION Wax obtained by melting the walls of the honeycomb made for 30 min. Add 50 ml of dilute sulphuric acid R, cool and filter. Rinse the flask and the filter with dilute sulphuric by the honey-bee, Apis mellifera L., with hot water and acid R. Combine the filtrate and washings and dilute to removing foreign matter. 100.0 ml with dilute sulphuric acid R. Place 1.0 ml of the solution in a test-tube, add 0.5 ml of a 10.7 g/l solution CHARACTERS of sodium periodate R, mix and allow to stand for 5 min. Appearance : yellow or light brown pieces or plates with Add 1.0 ml of decolorised fuchsin solution R and mix. Any a fine-grained, matt and non-crystalline fracture ; when precipitate disappears. Place the tube in a beaker containing warmed in the hand they become soft and malleable. water at 40 °C. During cooling observe for 10-15 min. Any It has a faint odour, characteristic of honey. It is tasteless violet-blue colour in the solution is not more intense than and does not stick to the teeth. that in a standard prepared at the same time and in the same manner using 1.0 ml of a 10 mg/l solution of glycerol R in Solubility : practically insoluble in water, partially soluble dilute sulphuric acid R. in hot alcohol (90 per cent V/V) and completely soluble in fatty and essential oils. 01/2005:0221 Relative density : about 0.960. TESTS Drop point (2.2.17) : 61 °C to 66 °C. Melt the beeswax by heating on a water-bath, pour onto a glass plate and allow to cool to a semi-solid mass. Fill the metal cup by inserting the wider end into the beeswax and repeating the procedure until beeswax extrudes from the narrow opening. Remove the excess with a spatula and insert the thermometer immediately. Remove the beeswax displaced. Allow to stand at room temperature for at least 12 h before determining the drop point. Acid value : 17.0 to 22.0. To 2.00 g (m g), in a 250 ml conical flask fitted with a reflux condenser, add 40 ml of xylene R and a few glass beads. Heat until the substance is dissolved. Add 20 ml of alcohol R and 0.5 ml of phenolphthalein solution R1 and titrate the hot solution with 0.5 M alcoholic potassium hydroxide until a red colour persists for at least 10 s (n1 ml). Carry out a blank test (n2 ml).

BELLADONNA LEAF Belladonnae folium DEFINITION Belladonna leaf consists of the dried leaf or of the dried leaf and flowering, and occasionally fruit-bearing, tops of Atropa belladonna L. It contains not less than 0.30 per cent of total alkaloids, calculated as hyoscyamine (Mr 289.4) with reference to the drug dried at 100 °C to 105 °C. The alkaloids consist mainly of hyoscyamine together with small quantities of hyoscine (scopolamine). CHARACTERS Belladonna leaf has a slightly nauseous odour. It has the macroscopic and microscopic characters described under identification tests A and B.

IDENTIFICATION A. The leaves are green to brownish-green, slightly darker on the upper surface, often crumpled and rolled and partly matted together in the drug. The leaf is petiolate and the base of the lamina is acute and decurrent and Ester value (2.5.2) : 70 to 80. the margin entire. The flowering stems are flattened and Saponification value : 87 to 102. bear at each node a pair of leaves unequal in size, in the To 2.00 g (m g), in a 250 ml conical flask fitted with a axils of which occur singly the flowers or occasionally reflux condenser, add 30 ml of a mixture of equal volumes fruits. The flowers have a gamosepalous calyx and of alcohol R and xylene R and a few glass beads. Heat campanulate corolla. The fruits are globular berries, until the substance is dissolved. Add 25.0 ml of 0.5 M green to brownish-black and surrounded by the persistent alcoholic potassium hydroxide and heat under a reflux calyx with widely spread lobes. condenser for 3 h. Titrate the hot solution immediately with B. Reduce to a powder (355). The powder is dark 0.5 M hydrochloric acid, using 1 ml of phenolphthalein green. Examine under a microscope, using chloral solution R1 as indicator (n1 ml). Reheat the solution to hydrate solution R. The powder shows the following boiling several times during the course of the titration. diagnostic characters : fragments of leaf lamina showing Carry out a blank test (n2 ml). sinuous-walled epidermal cells, a striated cuticle and numerous stomata predominantly present on the lower epidermis (anisocytic and also some anomocytic) ; multicellular uniseriate covering trichomes with smooth cuticle, glandular trichomes with unicellular heads and Ceresin, paraffins and certain other waxes. To 3.0 g, in a multicellular, uniseriate stalks or with multicellular 100 ml round-bottomed flask, add 30 ml of a 40 g/l solution heads and unicellular stalks ; parenchyma cells including of potassium hydroxide R in aldehyde-free alcohol R and rounded cells containing microsphenoidal crystals of boil gently under a reflux condenser for 2 h. Remove the calcium oxalate ; annular and spirally thickened vessels. condenser and immediately insert a thermometer. Place the The powdered drug may also show the following : flask in a water-bath at 80 °C and allow to cool, swirling the fibres and reticulately thickened vessels from the solution continuously. No precipitate is formed until 65 °C, 1058


Belladonna leaf


stems ; subspherical pollen grains, 40 µm to 50 µm in diameter, with three germinal pores, three furrows and an extensively pitted exine ; fragments of the corolla, with a papillose epidermis or bearing numerous covering or glandular trichomes of the types previously described ; brownish-yellow seed fragments containing irregularly sclerified and pitted cells of the testa. C. Shake 1 g of powdered drug with 10 ml of 0.05 M sulphuric acid for 2 min. Filter and add to the filtrate 1 ml of concentrated ammonia R and 5 ml of water R. Shake cautiously with 15 ml of ether R, avoiding formation of an emulsion. Separate the ether layer and dry over anhydrous sodium sulphate R. Filter and evaporate the ether in a porcelain dish. Add 0.5 ml of fuming nitric acid R and evaporate to dryness on a water-bath. Add 10 ml of acetone R and, dropwise, a 30 g/l solution of potassium hydroxide R in alcohol R. A deep violet colour develops. D. Examine the chromatogram obtained in the chromatography test. The principal zones in the chromatogram obtained with the test solution are similar in position, colour and size to the principal zones in the chromatogram obtained with the same volume of the reference solution. TESTS Chromatography. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. To 0.6 g of powdered drug (180) add 15 ml of 0.05 M sulphuric acid, shake for 15 min and filter. Wash the filter with 0.05 M sulphuric acid until 20 ml of filtrate is obtained. To the filtrate add 1 ml of concentrated ammonia R and shake with two quantities, each of 10 ml, of peroxide-free ether R. If necessary, separate by centrifugation. Dry the combined ether layers over anhydrous sodium sulphate R, filter and evaporate to dryness on a water-bath. Dissolve the residue in 0.5 ml of methanol R. Reference solution. Dissolve 50 mg of hyoscyamine sulphate R in 9 ml of methanol R. Dissolve 15 mg of hyoscine hydrobromide R in 10 ml of methanol R. Mix 1.8 ml of the hyoscine hydrobromide solution and 8 ml of the hyoscyamine sulphate solution. Apply separately to the plate as bands 20 mm by 3 mm 10 µl and 20 µl of each solution, leaving 1 cm between the bands. Develop over a path of 10 cm using a mixture of 3 volumes of concentrated ammonia R, 7 volumes of water R and 90 volumes of acetone R. Dry the plate at 100 °C to 105 °C for 15 min, allow to cool and spray with potassium iodobismuthate solution R2, using about 10 ml for a plate 200 mm square, until the orange or brown zones become visible against a yellow background. The zones in the chromatograms obtained with the test solution are similar to those in the chromatograms obtained with the reference solution with respect to their position (hyoscyamine in the lower third, hyoscine in the upper third of the chromatogram) and their colour. The zones in the chromatograms obtained with the test solution are at least equal in size to the corresponding zones in the chromatogram obtained with the same volume of the reference solution. Faint secondary zones may appear, particularly in the middle of the chromatogram obtained with 20 µl of the test solution or near the starting-point in the chromatogram obtained with 10 µl of the test solution. General Notices (1) apply to all monographs and other texts

Spray the plate with sodium nitrite solution R until the coating is transparent. Examine after 15 min. The zones corresponding to hyoscyamine in the chromatograms obtained with the test solution and the reference solution change from brown to reddish-brown but not to greyish-blue (atropine) and any secondary zones disappear. Foreign matter (2.8.2). Not more than 3 per cent of stem having a diameter exceeding 5 mm. Total ash (2.4.16). Not more than 16.0 per cent. Ash insoluble in hydrochloric acid (2.8.1). Not more than 4.0 per cent ASSAY Take about 50 g of drug and completely reduce it to a powder (180). Using the powder, determine the loss on drying and the total alkaloids. a) Determine the loss on drying (2.2.32) on 2.000 g, by drying in an oven at 100 °C to 105 °C. b) Moisten 10.00 g of powder with a mixture of 5 ml of ammonia R, 10 ml of alcohol R and 30 ml of peroxide-free ether R and mix thoroughly. Transfer the mixture to a suitable percolator, if necessary with the aid of the extracting mixture. Allow to macerate for 4 h and percolate with a mixture of 1 volume of chloroform R and 3 volumes of peroxide-free ether R until the alkaloids are completely extracted. Evaporate to dryness a few millilitres of the liquid flowing from the percolator, dissolve the residue in 0.25 M sulphuric acid and verify the absence of alkaloids using potassium tetraiodomercurate solution R. Concentrate the percolate to about 50 ml by distilling on a water-bath and transfer it to a separating funnel, rinsing with peroxide-free ether R. Add a quantity of peroxide-free ether R equal to at least 2.1 times the volume of the percolate to produce a liquid of a density well below that of water. Shake the solution with no fewer than three quantities, each of 20 ml, of 0.25 M sulphuric acid, separate the two layers by centrifugation if necessary and transfer the acid layers to a second separating funnel. Make the acid layer alkaline with ammonia R and shake with three quantities, each of 30 ml, of chloroform R. Combine the chloroform layers, add 4 g of anhydrous sodium sulphate R and allow to stand for 30 min with occasional shaking. Decant the chloroform and wash the sodium sulphate with three quantities, each of 10 ml, of chloroform R. Add the washings to the chloroform extract, evaporate to dryness on a water-bath and heat in an oven at 100 °C to 105 °C for 15 min. Dissolve the residue in a few millilitres of chloroform R, add 20.0 ml of 0.01 M sulphuric acid and remove the chloroform by evaporation on a water-bath. Titrate the excess of acid with 0.02 M sodium hydroxide using methyl red mixed solution R as indicator. Calculate the percentage content of total alkaloids, expressed as hyoscyamine, from the expression :

d n

=

loss on drying as a percentage,

=

m

=

volume of 0.02 M sodium hydroxide used in millilitres, mass of drug used in grams.

STORAGE Store protected from light. 1059

Belladonna leaf dry extract, standardised


01/2005:1294 Reference solution. Dissolve 50 mg of hyoscyamine sulphate R in 9 ml of methanol R. Dissolve 15 mg of hydrobromide R in 10 ml of methanol R. Mix BELLADONNA LEAF DRY EXTRACT, hyoscine 1.8 ml of the hyoscine hydrobromide solution and 8 ml of STANDARDISED the hyoscyamine sulphate solution. Apply to the plate, as bands, 20 µl of each solution. Develop over a path of 10 cm using a mixture of 3 volumes of Belladonnae folii extractum siccum concentrated ammonia R, 7 volumes of water R and normatum 90 volumes of acetone R. Dry the plate at 100 °C to 105 °C for 15 min, allow to cool and spray with potassium DEFINITION iodobismuthate solution R2, until the orange or brown Standardised belladonna leaf dry extract is produced from zones become visible against a yellow background. The Belladonna leaf (0221). It contains not less than 0.95 per zones in the chromatogram obtained with the test solution cent and not more than 1.05 per cent of total alkaloids, are similar to those in the chromatogram obtained with calculated as hyoscyamine (C17H23NO3, Mr 289.4), with the reference solution with respect to their position reference to the dried extract. (hyoscyamine in the lower third, hyoscine in the upper third of the chromatogram) and their colour. Other faint zones PRODUCTION may be present in the chromatogram obtained with the test The extract is produced from the drug and ethanol (70 per solution. Spray the plate with sodium nitrite solution R cent V/V) using an appropriate procedure. until the coating is transparent. Examine after 15 min. The zones corresponding to hyoscyamine in the chromatograms CHARACTERS obtained with the test solution and the reference solution A hygroscopic brown or greenish powder. change from orange or brown to reddish-brown but not to IDENTIFICATION A. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating substance. Test solution. To 1 g of the extract to be examined add 5.0 ml of methanol R. Shake for 2 min and filter. Reference solution. Dissolve 1.0 mg of chlorogenic acid R and 2.5 mg of rutin R in 10 ml of methanol R. Apply to the plate, as bands, 20 µl of each solution. Develop over a path of 15 cm using a mixture of 10 volumes of anhydrous formic acid R, 10 volumes of water R, 30 volumes of methyl ethyl ketone R and 50 volumes of ethyl acetate R. Dry the plate at 100 °C to 105 °C and spray the warm plate with a 10 g/l solution of diphenylboric acid aminoethyl ester R in methanol R. Subsequently spray the plate with a 50 g/l solution of macrogol 400 R in methanol R. Allow the plate to dry in air for 30 min and examine in ultraviolet light at 365 nm. The chromatograms obtained with the reference solution and the test solution show in the central part a light blue fluorescent zone (chlorogenic acid) and in the lower part a yellowish-brown fluorescent zone (rutin). Furthermore, the chromatogram obtained with the test solution shows a little above the start a yellowish-brown fluorescent zone and directly above a yellow fluorescent zone, a yellow or yellowish-brown fluorescent zone between the zone due to rutin and the zone due to chlorogenic acid. Further zones may be present. B. Examine the chromatogram obtained in the test for atropine. The principal zones in the chromatogram obtained with the test solution are similar in position and colour to the principal zones in the chromatogram obtained with the reference solution.

greyish-blue (atropine). Loss on drying (2.8.17) : maximum 5.0 per cent. Microbial contamination. Total viable aerobic count (2.6.12) not more than 104 micro-organisms per gram of which not more than 102 fungi per gram, determined by plate count. It complies with the tests for Escherichia coli and for Salmonella (2.6.13).

ASSAY At each extraction stage it is necessary to check that the alkaloids have been completely extracted. If the extraction is into the organic phase this is done by evaporating to dryness a few millilitres of the last organic layer, dissolving the residue in 0.25 M sulphuric acid and verifying the absence of alkaloids using potassium tetraiodomercurate solution R. If the extraction is into the acid aqueous phase, this is done by taking a few millilitres of the last acid aqueous phase and verifying the absence of alkaloids using potassium tetraiodomercurate solution R. Disperse 3.00 g in a mixture of 5 ml of ammonia R and 15 ml of water R. Shake with no fewer than three quantities, each of 40 ml of a mixture of 1 volume of methylene chloride R and 3 volumes of peroxide-free ether R until the alkaloids are completely extracted. Concentrate the combined organic layers to about 50 ml by distilling on a water-bath and transfer the resulting liquid to a separating funnel, rinsing with peroxide-free ether R. Add a quantity of peroxide-free ether R equal to at least 2.1 times the volume of the liquid to produce a layer having a density well below that of water. Shake the resulting solution with no fewer than three quantities, each of 20 ml, of 0.25 M sulphuric acid until the alkaloids are completely extracted. Separate the layers by centrifugation, if necessary and transfer the acid layers to a second separating funnel. Make the combined acid layers TESTS alkaline with ammonia R and shake with no fewer than Atropine. Examine by thin-layer chromatography (2.2.27), three quantities, each of 30 ml, of methylene chloride R using a suitable silica gel as the coating substance. until the alkaloids are completely extracted. Combine the Test solution. To 0.20 g of the extract to be examined add organic layers, add 4 g of anhydrous sodium sulphate R and 10.0 ml of 0.05 M sulphuric acid, shake for 2 min and filter. allow to stand for 30 min with occasional shaking. Decant Add 1.0 ml of concentrated ammonia R and shake with the methylene chloride and wash the sodium sulphate with two quantities, each of 10 ml, of peroxide-free ether R. If three quantities, each of 10 ml, of methylene chloride R. necessary, separate by centrifugation. Dry the combined Combine the organic extracts, evaporate to dryness on ether layers over about 2 g of anhydrous sodium sulphate R, a water-bath. Heat the residue in an oven at 100 °C to filter and evaporate to dryness on a water-bath. Dissolve the 105 °C for 15 min. Dissolve the residue in a few millilitres of residue in 0.5 ml of methanol R. methylene chloride R, evaporate to dryness on a water-bath 1060


Belladonna leaf tincture, standardised


and again heat the residue in an oven at 100 °C to 105 °C for 15 min. Dissolve the residue in a few millilitres of methylene chloride R, add 20.0 ml of 0.01 M sulphuric acid and remove the methylene chloride by evaporation on a water-bath. Titrate the excess of acid with 0.02 M sodium hydroxide using methyl red mixed solution R as indicator. Calculate the percentage content of total alkaloids, expressed as hyoscyamine, from the expression :

Top of the plate _______

_______

Chlorogenic acid : a light blue fluorescent zone

A light blue fluorescent zone (chlorogenic acid)

_______

A yellow or yellowish-brown fluorescent zone _______

Rutin : a yellowish-brown fluorescent zone

A bluish-grey fluorescent zone A yellow fluorescent zone

n

=

volume of 0.02 M sodium hydroxide used, in millilitres,

m

=

mass of the substance to be examined, in grams.

STORAGE Store in an airtight container, protected from light.

01/2005:1812

BELLADONNA LEAF TINCTURE, STANDARDISED Belladonnae folii tinctura normata DEFINITION Tincture produced from Belladonna leaf (0221). Content : 0.027 per cent to 0.033 per cent of total alkaloids, calculated as hyoscyamine (C17H23NO3 ; Mr 289.4). The alkaloids consist mainly of hyoscyamine together with small quantities of hyoscine. PRODUCTION The tincture is produced from 1 part of the powdered drug (355) and 10 parts of ethanol (70 per cent V/V) by a suitable procedure. IDENTIFICATION A. Thin-layer chromatography (2.2.27). Test solution. Evaporate to dryness 10.0 ml of the tincture to be examined in a water-bath at 40 °C under reduced pressure. Dissolve the residue in 1.0 ml of methanol R. Reference solution. Dissolve 1.0 mg of chlorogenic acid R and 2.5 mg of rutin R in 10 ml of methanol R. Plate : TLC silica gel plate R. Mobile phase : anhydrous formic acid R, water R, methyl ethyl ketone R, ethyl acetate R (10:10:30:50 V/V/V/V). Application : 40 µl, as bands. Development : over a path of 15 cm. Drying : at 100-105 °C. Detection : spray the warm plate with a 10 g/l solution of diphenylboric acid aminoethyl ester R in methanol R ; subsequently spray the plate with a 50 g/l solution of macrogol 400 R in methanol R ; allow the plate to dry in air for 30 min and examine in ultraviolet light at 365 nm. Results : see below the sequence of zones present in the chromatograms obtained with the reference solution and the test solution. Furthermore, other fluorescent zones may be present in the chromatogram obtained with the test solution. General Notices (1) apply to all monographs and other texts

A yellowish-brown fluorescent zone Reference solution

Test solution

B. Examine the chromatograms obtained in the test for atropine, detection A. Results A : see below the sequence of zones present in the chromatograms obtained with the reference solution and the test solution. Faint secondary zones may appear, particularly in the middle of the chromatogram obtained with 40 µl of the test solution or near the starting point in the chromatogram obtained with 20 µl of the test solution. Top of the plate Hyoscine : a brownish-orange zone _______

A brownish-orange zone (hyoscine) _______ Faint secondary zones

_______ Hyoscyamine : a brownish-orange zone

_______ A brownish-orange zone (hyoscyamine) Faint secondary zones

Reference solution

Test solution

TESTS Atropine. Thin-layer chromatography (2.2.27). Test solution. To 15.0 ml of the tincture to be examined add 15 ml of 0.05 M sulphuric acid. Filter. Add 1 ml of concentrated ammonia R to the filtrate and shake with 2 quantities, each of 10 ml, of peroxide-free ether R. Separate by centrifugation if necessary. Dry the combined ether layers over anhydrous sodium sulphate R. Filter and evaporate to dryness on a water-bath. Dissolve the residue in 0.5 ml of methanol R. Reference solution. Dissolve 50 mg of hyoscyamine sulphate R in 9 ml of methanol R. Dissolve 15 mg of hyoscine hydrobromide R in 10 ml of methanol R. Mix 1.8 ml of the hyoscine hydrobromide solution and 8 ml of the hyoscyamine sulphate solution. Plate : TLC silica gel plate R. Mobile phase : concentrated ammonia R, water R, acetone R (3:7:90 V/V/V). Application : 20 µl and 40 µl of each solution, as bands. Development : over a path of 10 cm. Drying : at 100-105 °C for 15 min. Detection A : spray with potassium iodobismuthate solution R2. Detection B : spray with sodium nitrite solution R until the plate is transparent. Examine after 15 min. 1061

Belladonna, prepared


Results B : the zones due to hyoscyamine in the chromatograms obtained with the test solution and the reference solution change from brownish-orange to reddish-brown but not to greyish-blue (atropine) and any secondary zones disappear. Ethanol (2.9.10) : 64 per cent V/V to 69 per cent V/V.

CHARACTERS Slightly nauseous odour. IDENTIFICATION A. The powder is dark green. Examine under a microscope, using chloral hydrate solution R. The powder shows the following diagnostic characters : fragments of leaf lamina showing sinuous-walled epidermal cells, a striated cuticle and numerous stomata predominantly present on the lower epidermis (anisocytic and also some anomocytic) ; multicellular uniseriate covering trichomes with smooth cuticle, glandular trichomes with unicellular heads and multicellular, uniseriate stalks or with multicellular heads and unicellular stalks ; parenchyma cells including rounded cells containing microsphenoidal crystals of calcium oxalate ; annular and spirally thickened vessels. The powdered drug may also show the following : fibres and reticulately thickened vessels from the stems ; subspherical pollen grains, 40 µm to 50 µm in diameter, with three germinal pores, three furrows and an extensively pitted exine ; fragments of the corolla, with a papillose epidermis or bearing numerous covering or glandular trichomes of the types previously described ; brownish-yellow seed fragments containing irregularly sclerified and pitted cells of the testa. Examined in glycerol (85 per cent) R, it may be seen to contain lactose crystals. B. Shake 1 g with 10 ml of 0.05 M sulphuric acid for 2 min. Filter and add to the filtrate 1 ml of concentrated ammonia R and 5 ml of water R. Shake cautiously with 15 ml of ether R, avoiding formation of an emulsion. Separate the ether layer and dry over anhydrous sodium sulphate R. Filter and evaporate the ether in a porcelain dish. Add 0.5 ml of fuming nitric acid R and evaporate to dryness on a water-bath. Add 10 ml of acetone R and, dropwise, a 30 g/l solution of potassium hydroxide R in alcohol R. A deep violet colour develops. C. Examine the chromatogram obtained in the chromatography test. The principal zones in the chromatogram obtained with the test solution are similar in position, colour and size to the principal zones in the chromatogram obtained with the same volume of the reference solution.

ASSAY Evaporate 50.0 g of the tincture to be examined to a volume of about 10 ml. Transfer quantitatively to a separating funnel, with the minimum volume of alcohol (70 per cent V/V) R. Add 5 ml of ammonia R and 15 ml of water R. Shake with not fewer than 3 quantities each of 40 ml of a mixture of 1 volume of methylene chloride R and 3 volumes of peroxide-free ether R, carefully to avoid emulsion, until the alkaloids are completely extracted. Combine the organic layers and concentrate the solution to a volume of about 50 ml by distilling on a water-bath. Transfer the resulting solution quantitatively to a separating funnel, rinsing with peroxide-free ether R. Add a quantity of peroxide-free ether R equal to at least 2.1 times the volume of the solution to produce a layer having a density well below that of water. Shake the resulting solution with not fewer than 3 quantities each of 20 ml of 0.25 M sulphuric acid until the alkaloids are completely extracted. Separate the layers by centrifugation if necessary and transfer the layers to a separating funnel. Make the combined layers alkaline with ammonia R and shake with not fewer than 3 quantities each of 30 ml of methylene chloride R until the alkaloids are completely extracted. Combine the organic layers, add 4 g of anhydrous sodium sulphate R and allow to stand for 30 min with occasional shaking. Decant the methylene chloride and filter. Wash the sodium sulphate with 3 quantities each of 10 ml of methylene chloride R. Combine the organic extracts, evaporate to dryness on a water-bath. Heat the residue in an oven at 100-105 °C for 15 min. Dissolve the residue in a few millilitres of methylene chloride R, evaporate to dryness on a water-bath and heat the residue in an oven at 100-105 °C for 15 min again. Dissolve the residue in a few millilitres of methylene chloride R. Add 20.0 ml of 0.01 M sulphuric acid and remove the methylene chloride by evaporation on a water-bath. Titrate the excess of acid with 0.02 M sodium hydroxide using methyl red mixed solution R as indicator. Calculate the percentage content of total alkaloids, expressed TESTS as hyoscyamine, from the expression : Chromatography. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. To 0.6 g add 15 ml of 0.05 M sulphuric acid, shake for 15 min and filter. Wash the filter with 0.05 M n = volume of 0.02 M sodium hydroxide used, in sulphuric acid until 20 ml of filtrate is obtained. To the millilitres, filtrate add 1 ml of concentrated ammonia R and shake m with two quantities, each of 10 ml, of peroxide-free ether R. = mass of drug used, in grams. If necessary, separate by centrifugation. Dry the combined ether layers over anhydrous sodium sulphate R, filter and evaporate to dryness on a water-bath. Dissolve the residue 01/2005:0222 in 0.5 ml of methanol R. Reference solution. Dissolve 50 mg of hyoscyamine BELLADONNA, PREPARED sulphate R in 9 ml of methanol R. Dissolve 15 mg of hyoscine hydrobromide R in 10 ml of methanol R. Mix 1.8 ml of the hyoscine hydrobromide solution and 8 ml of Belladonnae pulvis normatus the hyoscyamine sulphate solution. DEFINITION Apply separately to the plate as bands 20 mm by 3 mm 10 µl and 20 µl of each solution, leaving 1 cm between Prepared belladonna is belladonna leaf powder (180) the bands. Develop over a path of 10 cm using a mixture adjusted if necessary by adding powdered lactose or of 3 volumes of concentrated ammonia R, 7 volumes belladonna leaf powder with a lower alkaloidal content to of water R and 90 volumes of acetone R. Dry the plate contain 0.28 per cent to 0.32 per cent of total alkaloids, at 100 °C to 105 °C for 15 min, allow to cool and spray calculated as hyoscyamine (Mr 289.4) with reference to the with potassium iodobismuthate solution R2, using about dried drug. 1062


Bendroflumethiazide


10 ml for a plate 200 mm square, until the orange or brown zones become visible against a yellow background. The zones in the chromatograms obtained with the test solution are similar to those in the chromatograms obtained with the reference solution with respect to their position (hyoscyamine in the lower third, hyoscine in the upper third of the chromatogram) and their colour. The zones in the chromatograms obtained with the test solution are at least equal in size to the corresponding zones in the chromatogram obtained with the same volume of the reference solution. Faint secondary zones may appear, particularly in the middle of the chromatogram obtained with 20 µl of the test solution or near the starting-point in the chromatogram obtained with 10 µl of the test solution. Spray the plate with sodium nitrite solution R until the coating is transparent. Examine after 15 min. The zones corresponding to hyoscyamine in the chromatograms obtained with the test solution and the reference solution change from brown to reddish-brown but not to greyish-blue (atropine) and any secondary zones disappear. Loss on drying (2.2.32). Not more than 5.0 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C.

Calculate the percentage content of total alkaloids, expressed as hyoscyamine, from the expression :

d n

=


=

m

=

volume of 0.02 M sodium hydroxide used in millilitres, mass of drug used in grams.

STORAGE Store in an airtight container, protected from light. 01/2005:0370

BENDROFLUMETHIAZIDE Bendroflumethiazidum

Total ash (2.4.16). Not more than 16.0 per cent. Ash insoluble in hydrochloric acid (2.8.1). Not more than 4.0 per cent.

C15H14F3N3O4S2

Mr 421.4

ASSAY

DEFINITION Bendroflumethiazide contains not less than 98.0 per a) Determine the loss on drying (2.2.32) on 2.000 g, by cent and not more than the equivalent of 102.0 per drying in an oven at 100 °C to 105 °C. cent of 3-benzyl-3,4-dihydro-6-(trifluoromethyl)-2H-1,2,4b) Moisten 10.00 g with a mixture of 5 ml of ammonia R, benzothiadiazine-7-sulphonamide 1,1-dioxide, calculated 10 ml of alcohol R and 30 ml of peroxide-free ether R and mix with reference to the dried substance. thoroughly. Transfer the mixture to a suitable percolator, if necessary with the aid of the extracting mixture. Allow to CHARACTERS macerate for 4 h and percolate with a mixture of 1 volume of A white or almost white, crystalline powder, practically chloroform R and 3 volumes of peroxide-free ether R until insoluble in water, freely soluble in acetone, soluble in the alkaloids are completely extracted. Evaporate to dryness alcohol. a few millilitres of the liquid flowing from the percolator, dissolve the residue in 0.25 M sulphuric acid and verify the IDENTIFICATION absence of alkaloids using potassium tetraiodomercurate First identification : A. solution R. Concentrate the percolate to about 50 ml by Second identification : B, C, D. distilling on a water-bath and transfer it to a separating A. Examine by infrared absorption spectrophotometry funnel, rinsing with peroxide-free ether R. Add a quantity of (2.2.24), comparing with the spectrum obtained with peroxide-free ether R equal to at least 2.1 times the volume bendroflumethiazide CRS. Examine the substances of the percolate to produce a liquid of a density well below prepared in the form of discs. that of water. Shake the solution with no fewer than three B. Examine the chromatograms obtained in the test quantities, each of 20 ml, of 0.25 M sulphuric acid, separate for related substances. The principal spot in the the two layers by centrifugation if necessary and transfer chromatogram obtained with 4 µl of the test solution is the acid layers to a second separating funnel. Make the similar in position, colour and size to the principal spot in acid layer alkaline with ammonia R and shake with three the chromatogram obtained with reference solution (a). quantities, each of 30 ml, of chloroform R. Combine the C. In a test-tube, heat 0.5 ml of a saturated solution of chloroform layers, add 4 g of anhydrous sodium sulphate R chromic acid cleansing mixture R in a naked flame and allow to stand for 30 min with occasional shaking. until white fumes appear in the upper part of the tube. Decant the chloroform and wash the sodium sulphate with The solution wets the side of the tube and there is three quantities, each of 10 ml, of chloroform R. Add the no appearance of greasiness. Add about 2 mg of the washings to the chloroform extract, evaporate to dryness substance to be examined and heat again in a naked flame on a water-bath and heat in an oven at 100 °C to 105 °C until white fumes appear. The solution does not wet the for 15 min. Dissolve the residue in a few millilitres of side of the tube and does not pour easily from the tube. chloroform R, add 20.0 ml of 0.01 M sulphuric acid and remove the chloroform by evaporation on a water-bath. D. Heat about 5 mg with a mixture of 5 ml of potassium Titrate the excess of acid with 0.02 M sodium hydroxide permanganate solution R and 1 ml of sulphuric acid R. using methyl red mixed solution R as indicator. An odour of benzaldehyde is produced.


1063

Benfluorex hydrochloride


TESTS Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. Dissolve 25 mg of the substance to be examined in acetone R and dilute to 5 ml with the same solvent. Reference solution (a). Dissolve 25 mg of bendroflumethiazide CRS in acetone R and dilute to 5 ml with the same solvent. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with acetone R. Apply separately to the plate 4 µl and 20 µl of the test solution, 4 µl of reference solution (a) and 20 µl of reference solution (b). Develop over a path of 15 cm using ethyl acetate R. Dry the plate in a current of air for 10 min and spray with a mixture of equal volumes of alcoholic solution of sulphuric acid R and alcohol R ; use about 10 ml for a plate 200 mm square and spray in small portions, allowing the solvent to evaporate each time to avoid excessive wetting. Heat at 100 °C to 105 °C for 30 min and immediately place the plate above, but not in contact with, 10 ml of a saturated solution of sodium nitrite R in a glass tank. Carefully add 0.5 ml of sulphuric acid R to the sodium nitrite solution, close the tank, and allow to stand for 15 min. Remove the plate, heat in a ventilated oven at 40 °C for 15 min and spray with three quantities, each of 5 ml, of a freshly prepared 5 g/l solution of naphthylethylenediamine dihydrochloride R in alcohol R. Examine the plate by transmitted light. Any spot in the chromatogram obtained with 20 µl of the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (1.0 per cent). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.00 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g

Content : 98.5 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white or almost white powder. Solubility : slightly soluble in water, freely soluble in methanol, soluble in methylene chloride, sparingly soluble or soluble in alcohol. It shows polymorphism. IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Preparation : mulls in liquid paraffin R. Comparison : benfluorex hydrochloride CRS. If the spectra obtained show differences, heat the substance to be examined and the reference substance separately in an oven at 150 °C for 3 h and record new spectra. B. It gives reaction (a) of chlorides (2.3.1).

TESTS Optical rotation (2.2.7) : − 0.10° to + 0.10°. Dissolve 0.2 g in ethanol R and dilute to 20.0 ml with the same solvent. Impurity B. Gas chromatography (2.2.28) : use the normalisation procedure. Test solution. Dissolve 0.30 g of the substance to be examined in methylene chloride R and dilute to 20 ml with the same solvent. Transfer to a separating funnel, add 10 ml of a 40 g/l solution of sodium hydroxide R. Shake the flask vigorously and allow the phases to separate. Collect the organic layer. Reference solution. Dissolve 0.30 g of benfluorex hydrochloride for system suitability CRS in methylene chloride R and dilute to 20 ml with the same solvent. Transfer to a separating funnel and add 10 ml of a 40 g/l solution of sodium hydroxide R. Shake the flask vigorously and allow the phases to separate. Collect the organic layer. ASSAY Dissolve 0.200 g in 50 ml of anhydrous pyridine R. Titrate Column : — material : fused silica, with 0.1 M tetrabutylammonium hydroxide, determining the end-point potentiometrically at the second point of — size : l = 25 m, Ø = 0.32 mm, inflexion (2.2.20). Carry out a blank titration. — stationary phase : macrogol 20 000 R (film thickness 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent 0.2 µm). to 21.07 mg of C15H14F3N3O4S2. Carrier gas : hydrogen for chromatography R. Linear velocity : 75 cm/s. 01/2005:1601 Split ratio : 1:35. corrected Temperature : — column : 220 °C, BENFLUOREX HYDROCHLORIDE — injection port and detector : 250 °C. Detection : flame ionisation. Benfluorexi hydrochloridum Injection : 1 µl. Run time : 1.5 times the retention time of benfluorex. Relative retention with reference to benfluorex (retention time = about 4.5 min) : impurity B = about 1.1. System suitability : — peak-to-valley ratio : minimum 2.5, where Hp = height above the baseline of the peak due to impurity B, and Hv = height above the baseline of the lowest point of C19H21ClF3NO2 Mr 387.8 the curve separating this peak from the peak due to benfluorex. DEFINITION Limit : 2-[[(1RS)-1-Methyl-2-[3-(trifluoromethyl)phenyl]ethyl]ami— impurity B : maximum 0.1 per cent. no]ethyl benzoate hydrochloride. 1064


Benperidol


Related substances, other than impurity B. Liquid chromatography (2.2.29). Test solution. Dissolve 60.0 mg of the substance to be examined in 50 ml of acetonitrile R and dilute to 100.0 ml with water R. Reference solution (a). Dissolve 60.0 mg of benfluorex hydrochloride for system suitability CRS in 50 ml of acetonitrile R and dilute to 100.0 ml with water R. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with a mixture of equal volumes of acetonitrile R and water R. Dilute 5.0 ml of this solution to 50.0 ml with the same mixture of solvents. Column : — size : l = 0.15 m, Ø = 4.6 mm, — stationary phase : silica gel bonded with alkylamide groups (5 µm), — temperature : 60 °C. Mobile phase : a mixture of equal volumes of acetonitrile R and a solution containing 2.18 g/l of potassium dihydrogen phosphate R adjusted to pH 2.5 with phosphoric acid R and 6.5 g/l of sodium decyl sulphate R. Flow rate : 1.4 ml/min. Detection : spectrophotometer at 210 nm. Injection : 10 µl. Run time : 3 times the retention time of benfluorex. Relative retention with reference to benfluorex (retention time = about 5 min) : impurity A = about 0.9. System suitability: — signal-to-noise ratio : minimum 20 for the principal peak in the chromatogram obtained with reference solution (b), — peak-to-valley ratio : minimum 2.5, where Hp = height above the baseline of the peak due to impurity A, and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to benfluorex in the chromatogram obtained with reference solution (a). Limits : — any impurity : not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent), — total: not more than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (0.2 per cent), — disregard limit : 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.05 per cent). Heavy metals (2.4.8) : maximum 20 ppm. 1.0 g complies with limit test C. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY In order to avoid overheating in the reaction medium, mix thoroughly throughout and stop the titration immediately after the end-point has been reached. Dissolve 0.250 g rapidly in 2.0 ml of anhydrous formic acid R and add 50.0 ml of acetic anhydride R. Titrate immediately with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 38.78 mg of C19H21ClF3NO2. General Notices (1) apply to all monographs and other texts

IMPURITIES

A. R = CF3, R′ = H : 2-[[(1RS)-1-methyl-2-[2(trifluoromethyl)phenyl]ethyl]amino]ethyl benzoate, B. R = H, R′ = CF3 : 2-[[(1RS)-1-methyl-2-[4(trifluoromethyl)phenyl]ethyl]amino]ethyl benzoate, C. benzoic acid,

D. R = CH2-CH2-OH, R′ = H : 2-[[(1RS)-1-methyl-2-[3(trifluoromethyl)phenyl]ethyl]amino]ethanol, E. R = CH2-CH2-OH, R′ = CO-C6H5 : N-(2-hydroxyethyl)-N-[(1RS)-1-methyl-2-[3-(trifluoromethyl)phenyl]ethyl]benzamide, F. R = CH2-CH2-O-CO-C6H5, R′ = CO-C6H5 : 2-[benzoyl[(1RS)1-methyl-2-[3-(trifluoromethyl)phenyl]ethyl]amino]ethyl benzoate. 01/2005:1172

BENPERIDOL Benperidolum

C22H24FN3O2

Mr 381.4

DEFINITION Benperidol contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of 1-[1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl]-1,3-dihydro2H-benzimidazol-2-one, calculated with reference to the dried substance. CHARACTERS A white or almost white powder, practically insoluble in water, freely soluble in dimethylformamide, soluble in methylene chloride, slightly soluble in alcohol. It shows polymorphism. IDENTIFICATION First identification : A. Second identification : B, C, D. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with benperidol CRS. Examine the substances prepared as discs. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the 1065

Benperidol


reference substance separately in the minimum volume of methyl isobutyl ketone R, evaporate to dryness and record new spectra using the residues. B. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm. Test solution. Dissolve 30 mg of the substance to be examined in a mixture of 1 volume of acetone R and 9 volumes of methanol R and dilute to 10 ml with the same mixture of solvents. Reference solution (a). Dissolve 30 mg of benperidol CRS in a mixture of 1 volume of acetone R and 9 volumes of methanol R and dilute to 10 ml with the same mixture of solvents. Reference solution (b). Dissolve 30 mg of benperidol CRS and 30 mg of droperidol CRS in a mixture of 1 volume of acetone R and 9 volumes of methanol R and dilute to 10 ml with the same mixture of solvents. Apply to the plate 10 µl of each solution. Develop over a path of 15 cm using a mixture of 1 volume of acetone R and 9 volumes of methanol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. C. Dissolve about 10 mg in 5 ml of ethanol R. Add 0.5 ml of dinitrobenzene solution R and 0.5 ml of 2 M alcoholic potassium hydroxide R. A violet colour is produced which becomes brownish-red after 20 min. D. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is obtained (usually less than 5 min). Allow to cool, add 1 ml of water R, 0.05 ml of phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the solution colourless. Filter. To a freshly prepared mixture of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R, add 1.0 ml of the filtrate. Mix, allow to stand for 5 min and compare the colour of the solution with that of a blank prepared in the same manner. The test solution is yellow and the blank is red. TESTS Related substances. Examine by liquid chromatography (2.2.29). Prepare the solutions immediately before use. Test solution. Dissolve 0.10 g of the substance to be examined in dimethylformamide R and dilute to 10.0 ml with the same solvent. Reference solution (a). Dissolve 2.5 mg of benperidol CRS and 2.5 mg of droperidol CRS in dimethylformamide R and dilute to 100.0 ml with the same solvent. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with dimethylformamide R. Dilute 5.0 ml of this solution to 20.0 ml with dimethylformamide R. The chromatographic procedure may be carried out using : — a stainless steel column 0.1 m long and 4.6 mm in internal diameter packed with base-deactivated octadecylsilyl silica gel for chromatography R (3 µm), — as mobile phase at a flow rate of 1.5 ml/min : Mobile phase A. A 10 g/l solution of tetrabutylammonium hydrogen sulphate R, Mobile phase B. Acetonitrile R, 1066

Time (min)



Comment

0 - 15

100 → 60

0 → 40

linear gradient

15 - 20

60

40

isocratic elution

20 - 25

100

0

switch to initial eluent composition

25 = 0

100

0

restart gradient

— as detector a spectrophotometer set at 275 nm. Equilibrate the column for at least 30 min with acetonitrile R and then equilibrate with the initial eluent composition for at least 5 min. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 10 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder. Inject 10 µl of reference solution (a). When the chromatogram is recorded in the prescribed conditions, the retention times are : benperidol about 6.5 min and droperidol about 7 min. The test is not valid unless the resolution between the peaks corresponding to benperidol and droperidol is at least 2.0. If necessary, adjust the concentration of acetonitrile in the mobile phase or adjust the time programme for the linear gradient. Inject 10 µl of dimethylformamide R as a blank, 10 µl of the test solution and 10 µl of reference solution (b). In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.25 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent). Disregard any peak obtained with the blank and any peak with an area less than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (b). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g in a platinum crucible. ASSAY Dissolve 0.300 g in 50 ml of a mixture of 1 volume of anhydrous acetic acid R and 7 volumes of methyl ethyl ketone R and titrate with 0.1 M perchloric acid, using 0.2 ml of naphtholbenzein solution R as indicator. 1 ml of 0.1 M perchloric acid is equivalent to 38.14 mg of C22H24FN3O2. STORAGE Store protected from light. IMPURITIES

A. 1-(piperidin-4-yl)-1,3-dihydro-2H-benzimidazol-2-one,



Benserazide hydrochloride

differences, dissolve the substance to be examined and the reference substance separately in hot methanol R, evaporate to dryness and record new spectra using the residues. B. Solution S (see Tests) gives reaction (b) of chlorides (2.3.1). B. 1-[1-[4-(2-fluorophenyl)-4-oxobutyl]piperidin-4-yl]-1,3dihydro-2H-benzimidazol-2-one,

TESTS Solution S. Dissolve 1.0 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution BY6 (2.2.2, Method II). pH (2.2.3). The pH of solution S is 4.0 to 5.0. Optical rotation (2.2.7). The angle of optical rotation, determined on solution S, is − 0.05 to + 0.05. C. 1-[1-[4-oxo-4-[4-[4-(2-oxo-2,3-dihydro-1H-benzimidazol-1Related substances. Examine by liquid chromatography yl)piperidin-1-yl]phenyl]butyl]piperidin-4-yl]-1,3-dihydro(2.2.29). 2H-benzimidazol-2-one, Prepare the solutions using the mobile phase cooled to 4 °C and inject immediately. Test solution. Dissolve 0.10 g of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution. Dissolve 5.0 mg of benserazide D. cis-1-[1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl impurity A CRS and 5.0 mg of benserazide 1-oxide]-1,3-dihydro-2H-benzimidazol-2-one, hydrochloride CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 5.0 ml of the solution to 100.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.125 m long and4 mm in internal diameter packed with octylsilyl silica gel for E. trans-1-[1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl chromatography R (5 µm), 1-oxide]-1,3-dihydro-2H-benzimidazol-2-one. — as mobile phase at a flow rate of 1.2 ml/min a mixture prepared as follows : dissolve 4.76 g of potassium 01/2005:1173 dihydrogen phosphate R in 800 ml of water R ; add 200 ml of acetonitrile R and 1.22 g of sodium BENSERAZIDE HYDROCHLORIDE decanesulphonate R ; adjust to pH 3.5 with phosphoric acid R, Benserazidi hydrochloridum — as detector a spectrophotometer set at 220 nm. Inject 20 µl of the reference solution. The test is not valid unless the resolution between the peaks corresponding to impurity A (first peak) and benserazide (second peak) is at least 2.0. Inject 20 µl of the test solution. Continue the chromatography for nine times the retention time of benserazide. In the chromatogram obtained with the test solution : the area of C10H16ClN3O5 Mr 293.7 any peak due to impurity A is not greater than the area of the corresponding peak in the chromatogram obtained DEFINITION with the reference solution (0.5 per cent) ; the area of any peak, apart from the principal peak and any peak due to Benserazide hydrochloride contains not less than impurity A, is not greater than the area of the peak due 98.5 per cent and not more than the equivalent of to benserazide in the chromatogram obtained with the 101.0 per cent of (RS)-2-amino-3-hydroxy-2′-(2,3,4reference solution (0.5 per cent) ; the sum of the areas of any trihydroxybenzyl)propanohydrazide hydrochloride, such peaks is not greater than twice the area of the peak calculated with reference to the anhydrous substance. due to benserazide in the chromatogram obtained with the CHARACTERS reference solution (1 per cent). Disregard any peak with an A white or yellowish-white or orange-white, crystalline area less than 0.1 times that of the peak due to benserazide powder, freely soluble in water, very slightly soluble in in the chromatogram obtained with the reference solution. ethanol, practically insoluble in acetone. Heavy metals (2.4.8). 1.0 g complies with limit test C for It shows polymorphism. heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. IDENTIFICATION Water (2.5.12). Not more than 1.0 per cent, determined on A. Examine by infrared absorption spectrophotometry 0.500 g by the semi-micro determination of water. (2.2.24), comparing with the spectrum obtained with benserazide hydrochloride CRS. Examine the substances Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. prepared as discs. If the spectra obtained show General Notices (1) apply to all monographs and other texts

1067

Bentonite


ASSAY In order to avoid overheating during the titration, mix thoroughly throughout and stop the titration immediately after the end-point has been reached. Dissolve 0.250 g in 5 ml of anhydrous formic acid R. Add 70 ml of anhydrous acetic acid R. Titrate immediately with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 29.37 mg of C10H16ClN3O5.

and 5 ml of water R. Filter. To the filtrate add 1 ml of strong sodium hydroxide solution R and filter. To the filtrate add 3 ml of ammonium chloride solution R. A gelatinous white precipitate is formed. B. It complies with the test for swelling power with water (see Tests). C. 0.25 g gives the reaction of silicates (2.3.1).

TESTS Alkalinity. To 2 g add 100 ml of carbon dioxide-free water R and shake for 5 min. To 5 ml of the suspension add 0.1 ml of STORAGE thymolphthalein solution R. The liquid becomes bluish. Add 0.1 ml of 0.1 M hydrochloric acid. The liquid is decolourised Store protected from light. within 5 min. IMPURITIES Coarse particles. To 20 g add 1000 ml of water R and mix for 15 min using a high-speed mixer capable of operating at not less than 5000 r/min. Transfer to a wet sieve (75), tared after drying at 100 °C to 105 °C. Wash with three quantities, each of 500 ml, of water R, ensuring that any agglomerates have been dispersed. Dry at 100 °C to 105 °C and weigh. The particles on the sieve weigh not more than 0.1 g (0.5 per cent). Sedimentation volume. To 6.0 g add 200 ml of water R and mix for 20 min using a high-speed mixer capable of operating at 10 000 r/min. Transfer 100 ml of the suspension to a A. (RS)-2-amino-3-hydroxypropanohydrazide, graduated cylinder. Allow to stand for 24 h. The volume of the clear supernatant liquid is not greater than 2 ml. Swelling power with water. Add 2.0 g in twenty portions to 100 ml of a 10 g/l solution of sodium laurilsulfate R in a 100 ml graduated cylinder about 30 mm in diameter. Allow 2 min between additions for each portion to settle. Allow to stand for 2 h. The apparent volume of the sediment is not B. (RS)-2-amino-3-hydroxy-2′,2′-bis(2,3,4-trihydroxybenless than 22 ml. zyl)propanohydrazide, Heavy metals (2.4.8). To 5.0 g add 7.5 ml of dilute hydrochloric acid R and 27.5 ml of water R. Boil for 5 min. Centrifuge and filter the supernatant liquid. Wash the centrifugation residue with water R and filter. Dilute the combined filtrates to 50.0 ml with water R. To 5 ml of the solution add 5 ml of water R, 10 ml of hydrochloric acid R C. (RS)-2-amino-3-hydroxy-2′-(2,3,4-trihydroxybenand 25 ml of methyl isobutyl ketone R and shake for 2 min. zylidene)propanohydrazide. Separate the layers. Evaporate the aqueous layer to dryness on a water-bath. Dissolve the residue in 1 ml of acetic acid R, dilute to 25 ml with water R and filter. 12 ml of the filtrate 01/2005:0467 complies with limit test A for heavy metals (50 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32). Not more than 15 per cent, BENTONITE determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Bentonitum Microbial contamination. Total viable aerobic count (2.6.12) DEFINITION not more than 103 micro-organisms per gram, determined Bentonite is a natural clay containing a high proportion of by plate-count. montmorillonite, a native hydrated aluminium silicate in which some aluminium and silicon atoms may be replaced 01/2005:0372 by other atoms such as magnesium and iron. CHARACTERS BENZALKONIUM CHLORIDE A very fine, homogeneous, greyish-white powder with a more or less yellowish or pinkish tint, practically insoluble in Benzalkonii chloridum water and in aqueous solutions. It swells with a little water DEFINITION forming a malleable mass. Benzalkonium chloride is a mixture of alkylbenzyldiIDENTIFICATION methylammonium chlorides, the alkyl groups having chain A. To 0.5 g in a metal crucible add 1 g of potassium nitrate R lengths of C8 to C18. It contains not less than 95.0 per and 3 g of sodium carbonate R and heat until the mixture cent and not more than the equivalent of 104.0 per cent melts. Allow to cool. To the residue add 20 ml of boiling of alkylbenzyldimethylammonium chlorides, calculated water R, mix and filter. Wash the residue with 50 ml of as C22H40ClN (Mr 354.0) with reference to the anhydrous water R. To the residue add 1 ml of hydrochloric acid R substance. 1068



Benzalkonium chloride solution

ASSAY Dissolve 2.00 g in water R and dilute to 100.0 ml with the same solvent. Transfer 25.0 ml of the solution to a separating funnel, add 25 ml of chloroform R, 10 ml of 0.1 M sodium hydroxide and 10.0 ml of a freshly prepared 50 g/l solution of potassium iodide R. Shake well, allow to separate and discard the chloroform layer. Shake the aqueous layer with three quantities, each of 10 ml, of chloroform R and discard IDENTIFICATION the chloroform layers. To the aqueous layer add 40 ml of A. Dissolve 80 mg in water R and dilute to 100 ml with the hydrochloric acid R, allow to cool and titrate with 0.05 M same solvent. Examined between 220 nm and 350 nm potassium iodate until the deep-brown colour is almost (2.2.25), the solution shows three absorption maxima, at discharged. Add 2 ml of chloroform R and continue the 257 nm, 263 nm and 269 nm, and a shoulder at about titration, shaking vigorously, until the chloroform layer no 250 nm. longer changes colour. Carry out a blank titration on a B. To 2 ml of solution S (see Tests) add 0.1 ml of glacial acetic mixture of 10.0 ml of the freshly prepared 50 g/l solution acid R and, dropwise, 1 ml of sodium tetraphenylborate of potassium iodide R, 20 ml of water R and 40 ml of solution R. A white precipitate is formed. Filter. Dissolve hydrochloric acid R. the precipitate in a mixture of 1 ml of acetone R and 1 ml of 0.05 M potassium iodate is equivalent to 35.4 mg 5 ml of alcohol R, heating to not more than 70 °C. Add of C22H40ClN. water R dropwise to the warm solution until a slight opalescence forms. Heat gently until the solution is clear and allow to cool. White crystals separate. Filter, wash 01/2005:0371 with three quantities, each of 10 ml, of water R and dry in vacuo over diphosphorus pentoxide R or anhydrous BENZALKONIUM CHLORIDE silica gel R at a temperature not exceeding 50 °C. The crystals melt (2.2.14) at 127 °C to 133 °C. SOLUTION C. To 5 ml of dilute sodium hydroxide solution R add 0.1 ml of bromophenol blue solution R1 and 5 ml Benzalkonii chloridi solutio of chloroform R and shake. The chloroform layer is DEFINITION colourless. Add 0.1 ml of solution S and shake. The chloroform layer becomes blue. Benzalkonium chloride solution is an aqueous solution of a mixture of alkylbenzyldimethylammonium chlorides, the D. To 2 ml of solution S add 1 ml of dilute nitric acid R. alkyl groups having chain lengths of C8 to C18. Benzalkonium A white precipitate is formed which dissolves on the chloride solution contains not less than 475 g/l and not addition of 5 ml of alcohol R. The solution gives more than 525 g/l of alkylbenzyldimethylammonium reaction (a) of chlorides (2.3.1). chlorides, calculated as C22H40ClN (Mr 354.0). The solution may contain alcohol. TESTS Solution S. Dissolve 1.0 g in carbon dioxide-free water R CHARACTERS and dilute to 100 ml with the same solvent. A clear, colourless or slightly yellowish liquid, miscible with Appearance of solution. Solution S is clear (2.2.1) and not water and with alcohol. It froths copiously when shaken. more intensely coloured than reference solution Y6 (2.2.2, IDENTIFICATION Method II). A. Dilute 0.3 ml to 100 ml with water R. Examined between Acidity or alkalinity. To 50 ml of solution S add 0.1 ml 220 nm and 350 nm (2.2.25), the solution shows three of bromocresol purple solution R. Not more than 0.1 ml absorption maxima, at 257 nm, 263 nm and 269 nm, and of 0.1 M hydrochloric acid or 0.1 M sodium hydroxide is a shoulder at about 250 nm. required to change the colour of the indicator. B. To 0.05 ml add 2 ml of water R, 0.1 ml of glacial acetic Amines and amine salts. Dissolve 5.0 g with heating in acid R and, dropwise, 1 ml of sodium tetraphenylborate 20 ml of a mixture of 3 volumes of 1 M hydrochloric acid and solution R. A white precipitate is formed. Filter. Dissolve 97 volumes of methanol R and add 100 ml of 2-propanol R. the precipitate in a mixture of 1 ml of acetone R and Pass a stream of nitrogen R slowly through the solution. 5 ml of alcohol R, heating to not more than 70 °C. Add Gradually add 12.0 ml of 0.1 M tetrabutylammonium water R dropwise to the warm solution until a slight hydroxide and record the potentiometric titration curve opalescence forms. Heat gently until the solution is clear (2.2.20). If the curve shows two points of inflexion, and allow to cool. White crystals separate. Filter, wash the volume of titrant added between the two points is not with three quantities, each of 10 ml, of water R and dry greater than 5.0 ml. If the curve shows no point of inflexion, in vacuo over diphosphorus pentoxide R or anhydrous the substance to be examined does not comply with the test. silica gel R at a temperature not exceeding 50 °C. The If the curve shows one point of inflexion, repeat the test but crystals melt (2.2.14) at 127 °C to 133 °C. add 3.0 ml of a 25.0 g/l solution of dimethyldecylamine R C. To 5 ml of dilute sodium hydroxide solution R add in 2-propanol R before the titration. If the titration curve 0.1 ml of bromophenol blue solution R1 and 5 ml after addition of 12.0 ml of the titrant shows only one point of chloroform R and shake. The chloroform layer is of inflexion, the substance to be examined does not comply colourless. Add 0.05 ml of the solution to be examined with the test. and shake. The chloroform layer becomes blue. Water (2.5.12). Not more than 10 per cent, determined on D. To 0.05 ml add 1 ml of dilute nitric acid R. A white 0.300 g by the semi-micro determination of water. precipitate is formed which dissolves on the addition Sulphated ash (2.4.14). Not more than 0.1 per cent, of 5 ml of alcohol R. The solution gives reaction (a) of determined on 1.0 g. chlorides (2.3.1). CHARACTERS A white or yellowish-white powder or gelatinous, yellowish-white fragments, hygroscopic, soapy to the touch, very soluble in water and in alcohol. On heating it forms a clear molten mass. An aqueous solution froths copiously when shaken.


1069

Benzbromarone


TESTS Solution S. Dilute 2.0 g to 100 ml with carbon dioxide-free water R. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). Acidity or alkalinity. To 50 ml of solution S add 0.1 ml of bromocresol purple solution R. Not more than 0.1 ml of 0.1 M hydrochloric acid or 0.1 M sodium hydroxide is required to change the colour of the indicator. Amines and amine salts. Mix 10.0 g, while heating, with 20 ml of a mixture of 3 volumes of 1 M hydrochloric acid and 97 volumes of methanol R and add 100 ml of 2-propanol R. Pass a stream of nitrogen R slowly through the solution. Gradually add 12.0 ml of 0.1 M tetrabutylammonium hydroxide and record the potentiometric titration curve (2.2.20). If the curve shows two points of inflexion, the volume of titrant added between the two points is not greater than 5.0 ml. If the curve shows no point of inflexion, the solution to be examined does not comply with the test. If the curve shows one point of inflexion, repeat the test but add 3.0 ml of a 25.0 g/l solution of dimethyldecylamine R in 2-propanol R before the titration. If the titration curve after the addition of 12.0 ml of the titrant shows only one point of inflexion, the solution to be examined does not comply with the test. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Determine the density (2.2.5) of the solution to be examined. Dilute 4.00 g to 100.0 ml with water R. Transfer 25.0 ml of the solution to a separating funnel, add 25 ml of chloroform R, 10 ml of 0.1 M sodium hydroxide and 10.0 ml of a freshly prepared 50 g/l solution of potassium iodide R. Shake well, allow to separate and discard the chloroform layer. Shake the aqueous layer with three quantities, each of 10 ml, of chloroform R and discard the chloroform layers. To the aqueous layer add 40 ml of hydrochloric acid R, allow to cool and titrate with 0.05 M potassium iodate until the deep-brown colour is almost discharged. Add 2 ml of chloroform R and continue the titration, shaking vigorously, until the chloroform layer no longer changes colour. Carry out a blank titration on a mixture of 10.0 ml of the freshly prepared 50 g/l solution of potassium iodide R, 20 ml of water R and 40 ml of hydrochloric acid R. 1 ml of 0.05 M potassium iodate is equivalent to 35.4 mg of C22H40ClN. LABELLING The label states the content of alcohol, if any. 01/2005:1393

BENZBROMARONE Benzbromaronum

C17H12Br2O3 1070

Mr 424.1

DEFINITION Benzbromarone contains not less than 98.0 and not more than the equivalent of 101.0 per cent of (3,5-dibromo4-hydroxyphenyl)(2-ethylbenzofuran-3-yl)methanone, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, practically insoluble in water, freely soluble in acetone and in methylene chloride, sparingly soluble in alcohol. It melts at about 152 °C. IDENTIFICATION A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of benzbromarone. B. By means of a copper wire, previously ignited, introduce a small amount of the substance into the non-illuminated part of a flame. The colour of the flame becomes green. TESTS Appearance of solution. Dissolve 1.25 g in dimethylformamide R and dilute to 25 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y5 (2.2.2, Method II). Acidity or alkalinity. Shake 0.5 g with 10 ml of carbon dioxide-free water R for 1 min and filter. To 2.0 ml of the filtrate add 0.1 ml of methyl red solution R and 0.1 ml of 0.01 M hydrochloric acid. The solution is red. Add 0.3 ml of 0.01 M sodium hydroxide. The solution is yellow. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 0.125 g of the substance in 30 ml of methanol R and dilute to 50.0 ml with the mobile phase. Reference solution (a). Dilute 1.0 ml of the test solution to 100 ml with the mobile phase. Dilute 1 ml of this solution to 10 ml with the mobile phase. Reference solution (b). Dissolve 10 mg of benzarone CRS in the mobile phase and dilute to 20 ml with the mobile phase. Reference solution (c). To 5 ml of reference solution (b) add 1 ml of the test solution and dilute to 100 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.5 ml/min a mixture of 5 volumes of glacial acetic acid R, 25 volumes of acetonitrile R, 300 volumes of water R and 990 volumes of methanol R, — as detector a spectrophotometer set at 231 nm. Inject 20 µl of reference solution (c). Adjust the sensitivity of the system so that the heights of the principal peaks in the chromatogram obtained are at least 50 per cent of the full scale of the recorder. The test is not valid unless the resolution between the first peak (impurity C) and the second peak (benzbromarone) is at least 10.0. Inject 20 µl of the test solution and 20 µl of reference solution (a). Continue the chromatography of the test solution for 2.5 times the retention time of benzbromarone. If peaks other than the principal peak are observed in the chromatogram obtained with the test solution, these may be due to impurity A or to impurity B. When the


Benzethonium chloride


chromatograms are recorded in the prescribed conditions, the relative retention times are : impurity A about 0.6 and impurity B about 2. In the chromatogram obtained with the test solution : the area of the peak corresponding to impurity A is not greater than four times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.4 per cent) ; the area of the peak corresponding to impurity B is not greater than ten times the area of the principal peak in the chromatogram obtained with reference solution (a) (1 per cent) ; the area of any peak, apart from the principal peak and the peaks corresponding to impurity A and impurity B, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent) ; the sum of the areas of any such peaks is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent). Disregard any peak with an area less than 0.2 times that of the principal peak in the chromatogram obtained with reference solution (a). Halides expressed as chlorides (2.4.4). Shake 1.25 g of the substance to be examined with a mixture of 5 ml of dilute nitric acid R and 15 ml of water R. Filter. Rinse the filter with water R and dilute the filtrate to 25 ml with the same solvent. Dilute 2.5 ml to 15 ml with water R. The solution obtained complies with the limit test for chlorides (400 ppm). Iron (2.4.9). Moisten the residue obtained in the test for sulphated ash with 2 ml of hydrochloric acid R and evaporate to dryness on a water-bath. Add 0.05 ml of hydrochloric acid R and 10 ml of water R and heat until boiling for 1 min. Allow to cool. Rinse the crucible with water R, collect the rinsings and dilute to 25 ml with water R. Dilute 2 ml of this solution to 10 ml with water R. The solution complies with the limit test for iron (125 ppm). Heavy metals (2.4.8). 0.5 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 1 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven in vacuo at 50 °C for 4 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

B. R1 = R2 = R3 = Br : (6-bromo-2-ethylbenzofuran-3-yl)(3,5dibromo-4-hydroxyphenyl)methanone, C. R1 = R2 = R3 = H : (2-ethylbenzofuran-3-yl)(4hydroxyphenyl)methanone (benzarone). 01/2005:0974

BENZETHONIUM CHLORIDE Benzethonii chloridum

C27H42ClNO2

Mr 448.1

DEFINITION Benzethonium chloride contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent of N-benzyl-N,N-dimethyl-2-[2-[4-(1,1,3,3tetramethylbutyl)phenoxy]ethoxy]ethanaminium chloride, calculated with reference to the dried substance. CHARACTERS A white or yellowish-white powder, very soluble in water and in alcohol, freely soluble in methylene chloride. An aqueous solution froths copiously when shaken.

IDENTIFICATION A. Melting point (2.2.14) : 158 °C to 164 °C, after drying at 105 °C for 4 h. B. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm. Test solution. Dissolve 25 mg of the substance to be examined in water R and dilute to 5 ml with the same solvent. Reference solution. Dissolve 25 mg of benzethonium ASSAY chloride CRS in water R and dilute to 5 ml with the same Dissolve 0.300 g in 60 ml of methanol R. Stir until solvent. completely dissolved and add 10 ml of water R. Titrate Apply to the plate 20 µl of each solution. Develop over a with 0.1 M sodium hydroxide, determining the end-point path of 12 cm using a mixture of 5 volumes of water R, potentiometrically (2.2.20). 5 volumes of glacial acetic acid R and 100 volumes of 1 ml of 0.1 M sodium hydroxide is equivalent to 42.41 mg of methanol R. Dry the plate in a current of warm air and C17H12Br2O3. examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is STORAGE similar in position and size to the principal spot in the Store protected from light. chromatogram obtained with the reference solution. C. To 5 ml of dilute sodium hydroxide solution R add 0.1 ml IMPURITIES of bromophenol blue solution R1 and 5 ml of methylene chloride R and shake. The lower layer is colourless. Add 0.1 ml of solution S (see Tests) and shake. A blue colour develops in the lower layer. D. To 2 ml of solution S add 1 ml of dilute nitric acid R. A white precipitate is formed which dissolves upon addition of 5 ml of alcohol R. The solution gives reaction (a) of chlorides (2.3.1).

A. R1 = R2 = H, R3 = Br : (3-bromo-4-hydroxyphenyl)(2ethylbenzofuran-3-yl)methanone, General Notices (1) apply to all monographs and other texts

TESTS Solution S. Dissolve 5.0 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent. 1071

Benzocaine


Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). Acidity or alkalinity. To 25 ml of solution S add 0.1 ml of phenolphthalein solution R. The solution is colourless. Add 0.3 ml of 0.01 M sodium hydroxide. The solution is pink. Add 0.1 ml of methyl red solution R and 0.5 ml of 0.01 M hydrochloric acid. The solution is orange-red. Volatile bases and salts of volatile bases (2.4.1). 0.20 g complies with limit test B for ammonium (50 ppm). Prepare the standard using 0.1 ml of ammonium standard solution (100 ppm NH4) R. Replace heavy magnesium oxide by 2.0 ml of strong sodium hydroxide solution R. Loss on drying (2.2.32). Not more than 5.0 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C for 4 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

Second identification : A, C, D. A. Melting point (2.2.14) : 89 °C to 92 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with benzocaine CRS. C. To about 50 mg in a test-tube add 0.2 ml of a 500 g/l solution of chromium trioxide R. Cover the mouth of the tube with a piece of filter paper moistened with a freshly prepared mixture of equal volumes of a 50 g/l solution of sodium nitroprusside R and a 200 g/l solution of piperazine hydrate R. Boil gently for at least 30 s. A blue colour develops on the filter paper. D. Dissolve about 50 mg in alcohol R and dilute to 100 ml with the same solvent. 2 ml of the solution gives the reaction of primary aromatic amines (2.3.1).



TESTS Appearance of solution. Dissolve 1.0 g in alcohol R and dilute to 20 ml with the same solvent. The solution is clear (2.2.1) and colourless (2.2.2, Method II). ASSAY Dissolve 2.000 g in water R and dilute to 100.0 ml with Acidity or alkalinity. Dissolve 0.5 g in 10 ml of alcohol R the same solvent. Transfer 25.0 ml of the solution to a previously neutralised to 0.05 ml of phenolphthalein separating funnel, add 10 ml of a 4 g/l solution of sodium solution R. Add 10 ml of carbon dioxide-free water R. The hydroxide R, 10.0 ml of a freshly prepared 50 g/l solution solution remains colourless and not more than 0.5 ml of of potassium iodide R and 25 ml of methylene chloride R. 0.01 M sodium hydroxide is required to change the colour Shake vigorously, allow to separate and discard the lower of the indicator. layer. Shake the upper layer with three quantities, each of Loss on drying (2.2.32). Not more than 0.5 per cent, 10 ml, of methylene chloride R and discard the lower layers. determined on 1.00 g by drying in vacuo. To the upper layer add 40 ml of hydrochloric acid R, allow to cool and titrate with 0.05 M potassium iodate until the deep Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. brown colour is almost discharged. Add 4 ml of methylene chloride R and continue the titration, shaking vigorously, ASSAY until the lower layer is no longer brown. Carry out a blank Dissolve 0.400 g in a mixture of 25 ml of hydrochloric titration using a mixture of 10.0 ml of a freshly prepared 50 g/l solution of potassium iodide R, 20 ml of water R and acid R and 50 ml of water R. Carry out the determination of primary aromatic amino-nitrogen (2.5.8). 40 ml of hydrochloric acid R. 1 ml of 0.1 M sodium nitrite is equivalent to 16.52 mg 1 ml of 0.05 M potassium iodate is equivalent to 44.81 mg of C9H11NO2. of C27H42ClNO2.

01/2005:0011

01/2005:0066

BENZOCAINE

BENZOIC ACID

Benzocainum

C9H11NO2

Acidum benzoicum

Mr 165.2

DEFINITION Benzocaine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of ethyl 4-aminobenzoate, calculated with reference to the dried substance.

C7 H 6 O 2

Mr 122.1

DEFINITION Benzoic acid contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent of benzenecarboxylic acid.

CHARACTERS A white, crystalline powder or colourless crystals, very slightly soluble in water, freely soluble in alcohol.

CHARACTERS A white, crystalline powder or colourless crystals, odourless or with a very slight characteristic odour, slightly soluble in water, soluble in boiling water, freely soluble in alcohol and in fatty oils.

IDENTIFICATION First identification : A, B.

IDENTIFICATION A. Melting point (2.2.14) : 121 °C to 124 °C.

1072


Benzoyl peroxide, hydrous


01/2005:0704

B. Solution S (see Tests) gives reaction (a) of benzoates (2.3.1). TESTS Solution S. Dissolve 5.0 g in alcohol R and dilute to 100 ml with the same solvent.

BENZOYL PEROXIDE, HYDROUS Benzoylis peroxidum cum aqua

Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Carbonisable substances. Dissolve 0.5 g with shaking in 5 ml of sulphuric acid R. After 5 min, the solution is not more intensely coloured than reference solution Y5 (2.2.2, Method I). Oxidisable substances. Dissolve 0.2 g in 10 ml of boiling water R. Cool, shake and filter. To the filtrate add 1 ml of dilute sulphuric acid R and 0.2 ml of 0.02 M potassium permanganate. After 5 min, the solution is still coloured pink. Halogenated compounds and halides.

C14H10O4

Mr 242.2

DEFINITION Content : — dibenzoyl peroxide : 70.0 per cent to 77.0 per cent, — water : minimum 20.0 per cent.

CHARACTERS Appearance : white, amorphous or granular powder. Solubility : practically insoluble in water, soluble in acetone, soluble in methylene chloride with the separation of water, Solution (a). Dissolve 6.7 g of the substance to be examined slightly soluble in alcohol. in a mixture of 40 ml of 1 M sodium hydroxide and 50 ml It loses water rapidly on exposure to air with a risk of of alcohol R and dilute to 100.0 ml with water R. To 10.0 ml explosion. of this solution add 7.5 ml of dilute sodium hydroxide Mix the entire sample thoroughly before carrying out the solution R and 0.125 g of nickel-aluminium alloy R and following tests. heat on a water-bath for 10 min. Allow to cool to room temperature, filter into a 25 ml volumetric flask and wash IDENTIFICATION with three quantities, each of 2 ml, of alcohol R. Dilute the filtrate and washings to 25.0 ml with water R. This solution First identification : B Second identification : A, C, D. is used to prepare solution A. Solution (b). In the same manner, prepare a similar solution A. Dissolve 80.0 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of the solution without the substance to be examined. This solution is used to 100.0 ml with alcohol R (solution A). Dilute 10.0 ml to prepare solution B. of solution A to 100.0 ml with alcohol R (solution B). In four 25 ml volumetric flasks, place separately 10 ml Examined between 250 nm and 300 nm (2.2.25), of solution (a), 10 ml of solution (b), 10 ml of chloride solution A shows an absorption maximum at 274 nm and standard solution (8 ppm Cl) R (used to prepare solution C) a shoulder at about 282 nm. Examined between 220 nm and 10 ml of water R. To each flask add 5 ml of ferric and 250 nm, solution B shows an absorption maximum at ammonium sulphate solution R5, mix and add dropwise 235 nm. The ratio of the absorbance at the maximum at and with swirling 2 ml of nitric acid R and 5 ml of mercuric 235 nm (solution B) to that at the maximum at 274 nm thiocyanate solution R. Shake. Dilute the contents of each (solution A) is 1.17 to 1.21. flask to 25.0 ml with water R and allow the solutions to B. Infrared absorption spectrophotometry (2.2.24). stand in a water-bath at 20 °C for 15 min. Measure at 460 nm Comparison : Ph. Eur. reference spectrum of hydrous the absorbance (2.2.25) of solution A using solution B as benzoyl peroxide. the compensation liquid, and the absorbance of solution C C. Dissolve about 25 mg in 2 ml of acetone R. Add 1 ml of a using the solution obtained with 10 ml of water R as the 10 g/l solution of diethylphenylenediamine sulphate R compensation liquid. The absorbance of solution A is not and mix. A red colour develops which quickly darkens greater than that of solution C (300 ppm). and becomes dark violet within 5 min. Heavy metals (2.4.8). 12 ml of solution S complies with limit test B for heavy metals (10 ppm). Prepare the standard using D. To 1 g add 5 ml of alcohol R, 5 ml of dilute sodium hydroxide solution R and 10 ml of water R. Boil the a mixture of 5 ml of lead standard solution (1 ppm Pb) R mixture under reflux for 20 min. Cool. The solution gives and 5 ml of alcohol R. reaction (c) of benzoates (2.3.1). Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. TESTS Acidity. Dissolve a quantity of the substance to be examined ASSAY containing the equivalent of 1.0 g of dibenzoyl peroxide in Dissolve 0.200 g in 20 ml of alcohol R and titrate with 0.1 M 25 ml of acetone R, add 75 ml of water R and filter. Wash sodium hydroxide, using 0.1 ml of phenol red solution R as the residue with two quantities, each of 10 ml, of water R. indicator, until the colour changes from yellow to violet-red. Combine the filtrate and the washings and add 0.25 ml of phenolphthalein solution R1. Not more than 1.25 ml of 0.1 M sodium hydroxide is required to change the colour of 1 ml of 0.1 M sodium hydroxide is equivalent to 12.21 mg the indicator. Carry out a blank test. of C7H6O2. All glassware used must be chloride-free and may be prepared by soaking overnight in a 500 g/l solution of nitric acid R, rinsed with water R and stored full of water R. It is recommended that glassware be reserved for this test.


1073

Benzoyl peroxide, hydrous


Related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately before use.

Chlorides (2.4.4) : maximum 0.4 per cent. Dissolve a quantity of the substance to be examined containing the equivalent of 0.5 g of dibenzoyl peroxide in Test solution. Dissolve a quantity of the substance to be 15 ml of acetone R. Add, while stirring, 50 ml of 0.05 M examined containing the equivalent of 0.10 g of dibenzoyl peroxide in acetonitrile R and dilute to 50 ml with the same nitric acid. Allow to stand for 10 min and filter. Wash the residue with 2 quantities, each of 10 ml, of 0.05 M nitric solvent. acid. Combine the filtrate and the washings and dilute to Reference solution (a). Dilute 1.0 ml of the test solution to 100 ml with 0.05 M nitric acid. 2.5 ml of the solution diluted 100.0 ml with acetonitrile R. Dilute 1.0 ml of this solution to to 15.0 ml with water R complies with the limit test for 10.0 ml with acetonitrile R. chlorides. Reference solution (b). Dissolve 30.0 mg of benzoic acid R ASSAY in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 10.0 ml with the Solution (a). Dissolve 2.500 g immediately before use in mobile phase. 75 ml of dimethylformamide R and dilute to 100.0 ml with Reference solution (c). Dissolve 50.0 mg of ethyl benzoate R the same solvent. Dibenzoyl peroxide. To 5.0 ml of solution (a) add 20 ml in the mobile phase and dilute to 100.0 ml with the mobile of acetone R and 3 ml of a 500 g/l solution of potassium phase. Dilute 1.0 ml of the solution to 100.0 ml with the iodide R and mix. Allow to stand for 1 min. Titrate with mobile phase. 0.1 M sodium thiosulphate using 1 ml of starch solution R, Reference solution (d). Dissolve 50.0 mg of benzaldehyde R added towards the end of the titration, as indicator. Carry in the mobile phase and dilute to 100.0 ml with the mobile out a blank titration. phase. Dilute 1.0 ml of the solution to 100.0 ml with the 1 ml of 0.1 M sodium thiosulphate is equivalent to 12.11 mg mobile phase. of C14H10O4. Reference solution (e). Dissolve 30.0 mg of benzoic acid R Water (2.5.12). Carry out the semi-micro determination and 30.0 mg of benzaldehyde R in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of the of water, using 5.0 ml of solution (a). Use as the solvent a mixture of 20.0 ml of anhydrous methanol R and solution to 10.0 ml with the mobile phase. 3.0 ml of a 100 g/l solution of potassium iodide R in Column : dimethylformamide R. After adding solution (a), stir for 5 min before starting the titration. Carry out a blank — size : l = 0.25 m, Ø = 4.6 mm, determination. — stationary phase: octadecylsilyl silica gel for Calculate the percentage content of water using the chromatography R (10 µm), expression : Mobile phase : glacial acetic acid R, acetonitrile R, water R (1:500:500 V/V/V). Flow rate : 1 ml/min. Detection : spectrophotometer at 235 nm.

n1

=

Injection : 20 µl loop injector.

number of millilitres of iodosulphurous reagent R used in the sample determination,

n2

=

number of millilitres of iodosulphurous reagent R used in the blank determination,

w

=

water equivalent of iodosulphurous reagent R in milligrams of water per millilitre of reagent,

m

=

mass of the substance to be examined used for the preparation of solution (a) in grams,

Run time : 2 times the retention time of dibenzoyl peroxide. Relative retention with reference to dibenzoyl peroxide (retention time = about 28.4 min) : impurity B = about 0.15 ; impurity A = about 0.2 ; impurity C = about 0.4. System suitability : reference solution (e) :

= percentage content of dibenzoyl peroxide. — resolution : minimum 6 between the peaks corresponding p to benzoic acid and benzaldehyde. STORAGE Limits : In a container that has been treated to reduce static — impurity A : not more than the area of the principal peak discharge and that has a device for release of excess pressure, in the chromatogram obtained with reference solution (d) at a temperature of 2 °C to 8 °C, protected from light. (0.25 per cent), — impurity B : not more than the area of the principal peak IMPURITIES in the chromatogram obtained with reference solution (b) (1.5 per cent), — impurity C : not more than the area of the principal peak in the chromatogram obtained with reference solution (c) (0.25 per cent), — any other impurity : not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent),

A. R = H : benzaldehyde,

— disregard limit : 0.2 times the area of the principal peak B. R = OH : benzoic acid, in the chromatogram obtained with reference solution (a) C. R = O-CH2-CH3 : ethyl benzoate. (0.02 per cent). 1074


Benzyl alcohol


BENZYL

01/2005:0256 — stationary phase : macrogol 20 000 R (film thickness corrected 0.5 µm). Carrier gas : helium for chromatography R. ALCOHOL Linear velocity : 25 cm/s. Temperature :

Alcohol benzylicus

Column

C7H 8O

Mr 108.1

DEFINITION Phenylmethanol. Content : 98.0 per cent to 100.5 per cent.

Time (min)

Temperature (°C)

0 - 34

50 → 220

34 - 69

220

Injection port

200

Detector

310

Detection : flame ionisation. Benzyl alcohol not intended for parenteral use. Injection : without air-plug, 0.1 µl of the test solution and CHARACTERS 0.1 µl of reference solution (a). Appearance : clear, colourless, oily liquid. Relative retention with reference to benzyl alcohol Solubility : soluble in water, miscible with alcohol and with (retention time = about 26 min) : ethylbenzene = about 0.28 ; fatty and essential oils. dicyclohexyl = about 0.59 ; benzaldehyde = about 0.68 ; cyclohexylmethanol = about 0.71. Relative density : 1.043 to 1.049. System suitability : reference solution (a) : IDENTIFICATION — resolution : minimum 3.0 between the peaks Infrared absorption spectrophotometry (2.2.24). corresponding to benzaldehyde and to Comparison : Ph. Eur. reference spectrum of benzyl cyclohexylmethanol. alcohol. In the chromatogram obtained with the test solution, verify that there are no peaks with the same retention time as the TESTS standards. Appearance of solution. Shake 2.0 ml with 60 ml of water R. Limits : It dissolves completely. The solution is clear (2.2.1) and — benzaldehyde : not more than the difference between colourless (2.2.2, Method II). the area of the peak due to benzaldehyde in the Acidity. To 10 ml add 10 ml of alcohol R and 1 ml of chromatogram obtained with reference solution (a) phenolphthalein solution R. Not more than 1 ml of 0.1 M and the area of the peak due to benzaldehyde in the sodium hydroxide is required to change the colour of the chromatogram obtained with the test solution (0.15 per indicator to pink. cent). Refractive index (2.2.6) : 1.538 to 1.541. — cyclohexylmethanol : not more than the difference Peroxide value (2.5.5) : maximum 5. between the area of the peak due to cyclohexylmethanol in the chromatogram obtained with reference solution (a) Benzaldehyde and other related substances. Gas and the area of the peak due to cyclohexylmethanol in the chromatography (2.2.28). chromatogram obtained with the test solution (0.10 per Test solution. The substance to be examined. cent). Standard solution (a). Dissolve 0.100 g of ethylbenzene R — total of other peaks with a relative retention less than in the test solution and dilute to 10.0 ml with the same that of benzyl alcohol : not more than 4 times the area solution. Dilute 2.0 ml of this solution to 20.0 ml with the of the peak due to ethylbenzene in the chromatogram test solution. obtained with reference solution (a) (0.04 per cent). Standard solution (b). Dissolve 2.000 g of dicyclohexyl R — total of peaks with a relative retention greater than that in the test solution and dilute to 10.0 ml with the same of benzyl alcohol : not more than the area of the peak solution. Dilute 2.0 ml of this solution to 20.0 ml with the due to dicyclohexyl in the chromatogram obtained with test solution. reference solution (a) (0.3 per cent). Reference solution (a). Dissolve 0.750 g of benzaldehyde R — disregard limit : 0.01 times the area of the peak due and 0.500 g of cyclohexylmethanol R in the test solution to ethylbenzene in the chromatogram obtained with and dilute to 25.0 ml with the test solution. Add 1.0 ml of reference solution (a) (0.0001 per cent). this solution to a mixture of 2.0 ml of standard solution (a) Benzyl alcohol intended for parenteral use. and 3.0 ml of standard solution (b) and dilute to 20.0 ml with the test solution. Injection : without air-plug, 0.1 µl of the test solution and Reference solution (b). Dissolve 0.250 g of benzaldehyde R 0.1 µl of reference solution (b). and 0.500 g of cyclohexylmethanol R in the test solution Relative retention with reference to benzyl alcohol and dilute to 25.0 ml with the test solution. Add 1.0 ml of (retention time = about 26 min) : ethylbenzene = about 0.28 ; this solution to a mixture of 2.0 ml of standard solution (a) dicyclohexyl = about 0.59 ; benzaldehyde = about 0.68 ; and 2.0 ml of standard solution (b) and dilute to 20.0 ml cyclohexylmethanol = about 0.71. with the test solution. System suitability : reference solution (b) : Column : — resolution : minimum 3.0 between the peaks — material : fused silica, corresponding to benzaldehyde and to cyclohexylmethanol. — size : l = 30 m, Ø = 0.32 mm, General Notices (1) apply to all monographs and other texts

1075

Benzyl benzoate


01/2005:0705

In the chromatogram obtained with the test solution, verify that there are no peaks with the same retention time as the standards.

BENZYL BENZOATE

Limits :

Benzylis benzoas

— benzaldehyde : not more than the difference between the area of the peak due to benzaldehyde in the chromatogram obtained with reference solution (b) and the area of the peak due to benzaldehyde in the chromatogram obtained with the test solution (0.05 per cent). — cyclohexylmethanol : not more than the difference between the area of the peak due to cyclohexylmethanol in the chromatogram obtained with reference solution (b) and the area of the peak due to cyclohexylmethanol in the chromatogram obtained with the test solution (0.10 per cent). — total of other peaks with a relative retention less than that of benzyl alcohol : not more than twice the area of the peak due to ethylbenzene in the chromatogram obtained with reference solution (b) (0.02 per cent). — total of peaks with a relative retention greater than that of benzyl alcohol: not more than the area of the peak due to dicyclohexyl in the chromatogram obtained with reference solution (b) (0.2 per cent).

C14H12O2

Mr 212.2

DEFINITION Benzyl benzoate contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent of phenylmethyl benzoate. CHARACTERS Colourless or almost colourless crystals or a colourless or almost colourless, oily liquid, practically insoluble in water, miscible with alcohol, with methylene chloride and with fatty and essential oils. It boils at about 320 °C.

IDENTIFICATION First identification : A. Second identification : B, C. — disregard limit : 0.01 times the area of the peak due A. Examine by infrared absorption spectrophotometry to ethylbenzene in the chromatogram obtained with (2.2.24), comparing with the Ph. Eur. reference spectrum reference solution (b) (0.0001 per cent). of benzyl benzoate. Residue on evaporation : maximum 0.05 per cent. B. To 2 g add 25 ml of alcoholic potassium hydroxide solution R and boil under a reflux condenser for 2 h. After ensuring that the substance to be examined complies Remove the ethanol on a water-bath, add 50 ml of water R with the test for peroxide value, evaporate 10.0 g to dryness and distil ; collect about 25 ml of distillate and use it for on a water-bath, dry at 100-105 °C for 1 h and allow to cool identification test C. Acidify the liquid remaining in the in a desiccator. The residue weighs a maximum of 5 mg. distillation flask with dilute hydrochloric acid R ; a white precipitate is formed, which, when washed with water R and dried in vacuo, melts (2.2.14) at 121 °C to 124 °C. C. To the distillate obtained in identification test B add 2.5 g ASSAY of potassium permanganate R and 5 ml of dilute sodium To 0.900 g (m g) add 15.0 ml of a freshly prepared mixture of hydroxide solution R. Boil under a reflux condenser for 1 volume of acetic anhydride R and 7 volumes of pyridine R 15 min, cool and filter. Acidify the filtrate with dilute and boil under a reflux condenser for 30 min. Cool and hydrochloric acid R ; a white precipitate is formed which, add 25 ml of water R. Using 0.25 ml of phenolphthalein when washed with water R and dried in vacuo, melts solution R as indicator, titrate with 1 M sodium hydroxide (2.2.14) at 121 °C to 124 °C. (n1 ml). Carry out a blank titration (n2 ml). TESTS Calculate the percentage content of C7H8O from the Acidity. Dissolve 2.0 g in alcohol R and dilute to 10 ml with expression : the same solvent. Titrate with 0.1 M sodium hydroxide using phenolphthalein solution R as indicator. Not more than 0.2 ml is required to change the colour of the indicator to pink. Relative density (2.2.5) : 1.118 to 1.122. Refractive index (2.2.6) : 1.568 to 1.570. STORAGE Freezing point (2.2.18). Not less than 17.0 °C. In an airtight container, under nitrogen, protected from light Sulphated ash (2.4.14). Not more than 0.1 per cent, at a temperature between 2 °C and 8 °C. determined on 1.0 g.

LABELLING The label states, where applicable, that the substance is suitable for use in the manufacture of parenteral dosage forms. 1076

ASSAY To 2.000 g add 50.0 ml of 0.5 M alcoholic potassium hydroxide and boil gently under a reflux condenser for 1 h. Titrate the hot solution with 0.5 M hydrochloric acid using 1 ml of phenolphthalein solution R as indicator. Carry out a blank determination.



Benzylpenicillin, benzathine

C. Place about 2 mg in a test-tube about 150 mm long and 15 mm in diameter. Moisten with 0.05 ml of water R and add 2 ml of sulphuric acid-formaldehyde reagent R. STORAGE Mix the contents of the tube by swirling ; the solution is practically colourless. Place the test-tube on a water-bath Store in an airtight, well-filled container, protected from for 1 min ; a reddish-brown colour develops. light. D. To 0.1 g add 2 ml of 1 M sodium hydroxide and shake for 2 min. Shake the mixture with two quantities, each of 3 ml, of ether R. Evaporate the combined ether layers 01/2005:0373 to dryness and dissolve the residue in 1 ml of alcohol (50 per cent V/V) R. Add 5 ml of picric acid solution R, BENZYLPENICILLIN, BENZATHINE heat at 90 °C for 5 min and allow to cool slowly. Separate the crystals and recrystallise from alcohol (25 per cent V/V) R containing 10 g/l of picric acid R. The Benzylpenicillinum benzathinum crystals melt (2.2.14) at about 214 °C. 1 ml of 0.5 M alcoholic potassium hydroxide is equivalent to 106.1 mg of C14H12O2.

TESTS Acidity or alkalinity. To 0.50 g add 100 ml of carbon dioxide-free water R and shake for 5 min. Filter through a sintered-glass filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution R1. The solution is green or yellow. Not more than 0.2 ml of 0.02 M sodium hydroxide is required to change the colour of the indicator to blue. C48H56N6O8S2 Mr 909 Related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately before use, using DEFINITION sonication (for about 2 min) to dissolve the samples. Avoid any over heating during the sample preparation. Benzathine benzylpenicillin is N,N′-dibenzylethane-1,2diamine compound (1:2) with (2S,5R,6R)-3,3-dimethyl-7-oxo- Test solution. Dissolve 70.0 mg of the substance to be 6-[(phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2examined in 25 ml of methanol R and dilute to 50.0 ml with carboxylic acid. It contains not less than 96.0 per cent and a solution containing 6.8 g/l of potassium dihydrogen not more than the equivalent of 102.0 per cent of benzathine phosphate R and 1.02 g/l of disodium hydrogen benzylpenicillin and not less than 24.0 per cent and not phosphate R. more than 27.0 per cent of N,N′-dibenzylethylenediamine Reference solution (a). Dissolve 70.0 mg of benzathine (benzathine C16H20N2 ; Mr 240.3), both calculated with benzylpenicillin CRS in 25 ml of methanol R and dilute reference to the anhydrous substance. It contains a variable to 50.0 ml with a solution containing 6.8 g/l of potassium quantity of water. Dispersing or suspending agents may be dihydrogen phosphate R and 1.02 g/l of disodium hydrogen added. phosphate R. Reference solution (b). Dilute 1.0 ml of reference solution (a) CHARACTERS A white powder, very slightly soluble in water, freely soluble to 100.0 ml with mobile phase A. in dimethylformamide and in formamide, slightly soluble in Column : alcohol. — size : l = 0.25 m, Ø = 4.0 mm, — stationary phase : end-capped octadecylsilyl silica gel IDENTIFICATION for chromatography R (5 µm), First identification : A. — temperature : 40 °C. Second identification : B, C, D. Mobile phase : A. Examine by infrared absorption spectrophotometry — mobile phase A : mix 10 volumes of a 34 g/l solution of (2.2.24), comparing with the spectrum obtained with potassium dihydrogen phosphate R adjusted to pH 3.5 benzathine benzylpenicillin CRS. with phosphoric acid R, 30 volumes of methanol R and B. Examine by thin-layer chromatography (2.2.27), using a 60 volumes of water R, TLC silanised silica gel plate R. — mobile phase B : mix 10 volumes of a 34 g/l solution of Test solution. Dissolve 25 mg of the substance to be potassium dihydrogen phosphate R adjusted to pH 3.5 examined in 5 ml of methanol R. with phosphoric acid R, 30 volumes of water R and Reference solution. Dissolve 25 mg of benzathine 60 volumes of methanol R, benzylpenicillin CRS in 5 ml of methanol R. Time Mobile phase A Mobile phase B Apply to the plate 1 µl of each solution. Develop over a (min) (per cent V/V) (per cent V/V) path of 15 cm using a mixture of 30 volumes of acetone R 0 - 10 75 25 and 70 volumes of a 154 g/l solution of ammonium 10 - 20 75 → 0 25 → 100 acetate R, the pH of which has been adjusted to 7.0 with ammonia R. Allow the plate to dry in air and expose it to 0 20 - 55 100 iodine vapour until the spots appear. Examine in daylight. 55 - 70 75 25 The two principal spots in the chromatogram obtained with the test solution are similar in position, colour and Flow rate : 1 ml/min. size to the two principal spots in the chromatogram obtained with the reference solution. The test is not valid Detection : spectrophotometer at 220 nm. unless the chromatogram obtained with the reference Injection : 20 µl ; inject the test solution and the reference solution shows two clearly separated spots. solutions. General Notices (1) apply to all monographs and other texts

1077

Benzylpenicillin potassium


System suitability : reference solution (a) — relative retention with reference to benzylpenicillin : benzathine = 0.3 to 0.4 ; impurity C = about 2.4. If necessary, adjust the concentration of methanol in the mobile phase. Limits : — impurity C : not more than twice the sum of the areas of the 2 principal peaks in the chromatogram obtained with reference solution (b) (2 per cent), — any other impurity : not more than the sum of the areas C. benzylpenicilloic acids benzathide, of the 2 principal peaks in the chromatogram obtained with reference solution (b) (1 per cent), — disregard limit: 0.05 times the sum of the areas of the 2 principal peaks in the chromatogram obtained with reference solution (b) (0.05 per cent). Water (2.5.12) : 5.0 per cent to 8.0 per cent, determined on 0.300 g by the semi-micro determination of water. Bacterial endotoxins (2.6.14, Method E). Suspend 20 mg of the substance to be examined in 20 ml of a solution of 0.1 M D. (3S,7R,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7asodium hydroxide diluted 1 to 100, shake thoroughly and tetrahydroimidazo[5,1-b]thiazole-3,7-dicarboxylic centrifuge. The supernatant contains less than 0.13 IU/ml, acid (penillic acid of benzylpenicillin), if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances. Mobile phase : phosphate buffer solution pH 3.5 R, methanol R, water R (10:35:55 V/V/V). Injection : 20 µl ; inject the test solution and reference solution (a). Calculate the percentage contents of benzathine and of benzathine benzylpenicillin. Calculate the latter by multiplying the percentage content of benzylpenicillin by 1.36. STORAGE Store in an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states : — where applicable, the name and quantity of any added dispersing or suspending agent, — where applicable, that the substance is free from bacterial endotoxins. IMPURITIES

E. (4S)-2-[carboxy[(phenylacetyl)amino]methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penicilloic acids of benzylpenicillin),

F. (2RS,4S)-2-[[(phenylacetyl)amino]methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penilloic acids of benzylpenicillin). 01/2005:0113

BENZYLPENICILLIN POTASSIUM Benzylpenicillinum kalicum

A. monobenzylethylenediamine,

C16H17KN2O4S

B. phenylacetic acid,

DEFINITION Benzylpenicillin potassium is potassium (2S,5R,6R)3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylate, a substance produced by the growth of certain strains of Penicillium notatum or related organisms, or obtained by any other

1078

Mr 372.5


Benzylpenicillin potassium


means. It contains not less than 96.0 per cent and not more than the equivalent of 102.0 per cent of benzylpenicillin potassium, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, very soluble in water, practically insoluble in fatty oils and in liquid paraffin. IDENTIFICATION First identification : A, D.

phase. Inject 20 µl of test solution (b) and start the elution isocratically. Immediately after elution of the benzylpenicillin peak start the following linear gradient : Time (min)



Comment

0 - 20

70 → 0

30 → 100

linear gradient

20 - 35

0

100

isocratic

35 - 50

70

30

re-equilibration

Inject water R and use the same elution pattern to obtain a blank. In the chromatogram obtained with test solution (b), the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (d) (1 per cent). B. Examine by thin-layer chromatography (2.2.27), using a Loss on drying (2.2.32). Not more than 1.0 per cent, TLC silanised silica gel plate R. determined on 1.000 g by drying in an oven at 100-105 °C. Test solution. Dissolve 25 mg of the substance to be Bacterial endotoxins (2.6.14, Method E) : less than examined in 5 ml of water R. 0.16 IU/mg, if intended for use in the manufacture of Reference solution (a). Dissolve 25 mg of benzylpenicillin parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. potassium CRS in 5 ml of water R. Second identification : B, C, D. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with benzylpenicillin potassium CRS.

Reference solution (b). Dissolve 25 mg of benzylpenicillin ASSAY potassium CRS and 25 mg of phenoxymethylpenicillin Examine by liquid chromatography (2.2.29). Prepare the potassium CRS in 5 ml of water R. solutions immediately before use. Apply to the plate 1 µl of each solution. Develop over a Test solution (a). Dissolve 50.0 mg of the substance to be path of 15 cm using a mixture of 30 volumes of acetone R examined in water R and dilute to 50.0 ml with the same and 70 volumes of a 154 g/l solution of ammonium solvent. acetate R, the pH of which has been adjusted to 5.0 with Test solution (b). Dissolve 80.0 mg of the substance to be glacial acetic acid R. Allow the plate to dry in air and expose it to iodine vapour until the spots appear. Examine examined in water R and dilute to 20.0 ml with the same solvent. in daylight. The principal spot in the chromatogram Reference solution (a). Dissolve 50.0 mg of benzylpenicillin obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram sodium CRS in water R and dilute to 50.0 ml with the same solvent. obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference Reference solution (b). Dissolve 10 mg of benzylpenicillin solution (b) shows 2 clearly separated spots. sodium CRS and 10 mg of phenylacetic acid CRS in water R and dilute to 50 ml with the same solvent. C. Place about 2 mg in a test-tube about 150 mm long and 15 mm in diameter. Moisten with 0.05 ml of water R and Reference solution (c). Dilute 1.0 ml of reference solution (a) add 2 ml of sulphuric acid-formaldehyde reagent R. to 20.0 ml with water R. Dilute 1.0 ml of the solution to Mix the contents of the tube by swirling ; the solution is 50.0 ml with water R. practically colourless. Place the test-tube on a water-bath Reference solution (d). Dilute 4.0 ml of reference solution (a) for 1 min ; a reddish-brown colour develops. to 100.0 ml with water R. D. It gives reaction (a) of potassium (2.3.1). The chromatographic procedure may be carried out using : — a column 0.25 m long and 4.6 mm in internal TESTS diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), pH (2.2.3). Dissolve 2.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. The pH of the — as mobile phase at a flow rate of 1.0 ml/min : solution is 5.5 to 7.5. Mobile phase A. Mix 10 volumes of a 68 g/l solution of Specific optical rotation (2.2.7). Dissolve 0.500 g in carbon potassium dihydrogen phosphate R adjusted to pH 3.5 dioxide-free water R and dilute to 25.0 ml with the same with a 500 g/l solution of dilute phosphoric acid R, solvent. The specific optical rotation is + 270 to + 300, 30 volumes of methanol R and 60 volumes of water R, calculated with reference to the dried substance. Mobile phase B. Mix 10 volumes of a 68 g/l solution of potassium dihydrogen phosphate R adjusted to pH 3.5 Absorbance (2.2.25). Dissolve 94.0 mg in water R and with a 500 g/l solution of dilute phosphoric acid R, dilute to 50.0 ml with the same solvent. Measure the 40 volumes of water R and 50 volumes of methanol R, absorbance of the solution at 325 nm, 280 nm and at the maximum at 264 nm, diluting the solution, if necessary, for — as detector a spectrophotometer set at 225 nm. the measurement at 264 nm. The absorbances at 325 nm Equilibrate the column with a mobile phase ratio A:B and 280 nm do not exceed 0.10 and that at the maximum of 70:30. Inject 20 µl of reference solution (b). The test at 264 nm is 0.80 to 0.88, calculated on the basis of the is not valid unless the resolution between the 2 principal undiluted (1.88 g/l) solution. Verify the resolution of the peaks is at least 6.0 (if necessary, adjust the ratio A:B of apparatus (2.2.25) ; the ratio of the absorbances is at least 1.7. the mobile phase) and the mass distribution ratio for the Related substances. Examine by liquid chromatography second peak (benzylpenicillin) is 4.0 to 6.0. Inject 20 µl of (2.2.29) as described under Assay. Inject 20 µl of reference reference solution (c). Adjust the system to obtain a peak solution (d) and elute isocratically with the chosen mobile with a signal-to-noise ratio of at least 3. General Notices (1) apply to all monographs and other texts

1079

Benzylpenicillin, procaine


Calculate the percentage content of benzylpenicillin potassium by multiplying the percentage content of benzylpenicillin sodium by 1.045. STORAGE Store in an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins. IMPURITIES

F. (2RS,4S)-2-[[(phenylacetyl)amino]methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penilloic acids of benzylpenicillin). 01/2005:0115

BENZYLPENICILLIN, PROCAINE Benzylpenicillinum procainum

A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylic acid (6-aminopenicillanic acid), C29H38N4O6S,H2O

DEFINITION Procaine benzylpenicillin is the monohydrate of the (2S,5R,6R)-3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylic acid compound with 2-(diethylamino)ethyl 4-aminobenzoate. It contains not less than 96.0 per cent and not more than the equivalent of 102.0 per cent of procaine benzylpenicillin and not less than 39.0 per cent and not more than 42.0 per cent of procaine (C13H20N2O2 ; Mr 236.3), both calculated with reference to the anhydrous substance. Dispersing or suspending agents (for example, lecithin and polysorbate 80) may be added.

B. phenylacetic acid,

CHARACTERS A white, crystalline powder, slightly soluble in water, sparingly soluble in alcohol.

C. (2S,5R,6R)-6-[[(4-hydroxyphenyl)acetyl]amino]-3,3dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2carboxylic acid,

D. (3S,7R,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7atetrahydroimidazo[5,1-b]thiazole-3,7-dicarboxylic acid (penillic acid of benzylpenicillin),

E. (4S)-2-[carboxy[(phenylacetyl)amino]methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penicilloic acids of benzylpenicillin), 1080

Mr 588.7

IDENTIFICATION First identification : A. Second identification : B, C, D. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with procaine benzylpenicillin CRS. B. Examine by thin-layer chromatography (2.2.27), using a TLC silanised silica gel plate R. Test solution. Dissolve 25 mg of the substance to be examined in 5 ml of acetone R. Reference solution. Dissolve 25 mg of procaine benzylpenicillin CRS in 5 ml of acetone R. Apply to the plate 1 µl of each solution. Develop over a path of 15 cm using a mixture of 30 volumes of acetone R and 70 volumes of a 154 g/l solution of ammonium acetate R, the pH of which has been adjusted to 7.0 with ammonia R. Allow the plate to dry in air and expose it to iodine vapour until the spots appear. Examine in daylight. The 2 principal spots in the chromatogram obtained with the test solution are similar in position, colour and size to the 2 principal spots in the chromatogram obtained with the reference solution. The test is not valid unless the chromatogram obtained with the reference solution shows 2 clearly separated spots.



Benzylpenicillin, procaine

— as mobile phase at a flow rate of 1.75 ml/min a mixture prepared as follows : mix 250 ml of acetonitrile R, 250 ml of water R and 500 ml of a solution containing 14 g/l of potassium dihydrogen phosphate R and 6.5 g/l of tetrabutylammonium hydroxide solution (400 g/l) R adjusted to pH 7.0 with 1 M potassium hydroxide ; adjust the mixture to pH 7.2 with dilute phosphoric acid R, if necessary, — as detector a spectrophotometer set at 225 nm. Inject 10 µl of reference solution (b). When the chromatogram TESTS is recorded in the prescribed conditions, the substances pH (2.2.3). Dissolve 50 mg in carbon dioxide-free water R elute in the following order : 4-aminobenzoic acid, procaine, and dilute to 15 ml with the same solvent. Shake until benzylpenicillin. Adjust the sensitivity of the system so that dissolution is complete. The pH of the solution is 5.0 to 7.5. the height of the peak corresponding to 4-aminobenzoic Specific optical rotation (2.2.7). Dissolve 0.250 g in a acid is at least 50 per cent of the full scale of the recorder. mixture of 2 volumes of water R and 3 volumes of acetone R The test is not valid unless, the resolution between the first and dilute to 25.0 ml with the same mixture of solvents. The peak (4-aminobenzoic acid) and the second peak (procaine) specific optical rotation is + 165 to + 180, calculated with is at least 2.0. If necessary, adjust the concentration of reference to the anhydrous substance. acetonitrile in the mobile phase. Inject reference solution (a) 6 times. The test is not valid unless the relative standard Related substances. Examine by liquid chromatography (2.2.29) as prescribed under Assay. Inject 10 µl of reference deviation for the areas of the 2 peaks is at most 1.0 per cent. Inject alternately test solution (b) and reference solution (a). solution (c). Adjust the sensitivity of the system so that Calculate the percentage contents of procaine and procaine the height of the peak due to benzylpenicillin is at least benzylpenicillin. 50 per cent of the full scale of the recorder. Inject 10 µl of test solution (a) and continue the chromatography for STORAGE 1.5 times the retention time of the benzylpenicillin peak. In the chromatogram obtained with test solution (a) : Store in an airtight container. If the substance is sterile, the area of any peak corresponding to 4-aminobenzoic store in a sterile, airtight, tamper-proof container. acid is not greater than the area of the corresponding peak in the chromatogram obtained with reference LABELLING solution (c) (0.024 per cent) ; the area of any peak, apart The label states : from the 2 principal peaks and any peak corresponding to — where applicable, the name and quantity of any added 4-aminobenzoic acid, is not greater than the area of the dispersing or suspending agents, peak corresponding to benzylpenicillin in the chromatogram obtained with reference solution (c) (1 per cent). — where applicable, that the substance is free from bacterial endotoxins. Water (2.5.12). 2.8 per cent to 4.2 per cent, determined on 0.500 g by the semi-micro determination of water. IMPURITIES Bacterial endotoxins (2.6.14, Method E) : less than 0.10 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. C. Place about 2 mg in a test-tube about 150 mm long and 15 mm in diameter. Moisten with 0.05 ml of water R and add 2 ml of sulphuric acid-formaldehyde reagent R. Mix the contents of the tube by swirling ; the solution is practically colourless. Place the test-tube on a water-bath for 1 min ; a reddish-brown colour develops. D. Dissolve 0.1 g in 2 ml of dilute hydrochloric acid R and use the solution which may be turbid. The solution gives the reaction of primary aromatic amines (2.3.1).

ASSAY Examine by liquid chromatography (2.2.29). Prepare the A. 4-aminobenzoic acid, solutions immediately before use. Test solution (a). Dissolve 70.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Test solution (b). Dissolve 70.0 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (a). Dissolve 70.0 mg of procaine benzylpenicillin CRS in the mobile phase and dilute to B. (4S)-2-[carboxy[(phenylacetyl)amino]methyl]-5,5100.0 ml with the mobile phase. dimethylthiazolidine-4-carboxylic acid (penicilloic acids Reference solution (b). Dissolve 4 mg of 4-aminobenzoic of benzylpenicillin), acid R in reference solution (a) and dilute to 25 ml with the same solution. Reference solution (c). Dissolve 16.8 mg of 4-aminobenzoic acid R in water R and dilute to 50.0 ml with the same solvent. Dilute 1.0 ml of the solution to 10.0 ml with water R. To 1.0 ml of this solution, add 1.0 ml of test solution (a) and dilute to 100.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in C. (2RS,4S)-2-[[(phenylacetyl)amino]methyl]-5,5internal diameter packed with octadecylsilyl silica gel for dimethylthiazolidine-4-carboxylic acid (penilloic acids of chromatography R (5 µm), benzylpenicillin), General Notices (1) apply to all monographs and other texts

1081

Benzylpenicillin sodium


in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows 2 clearly separated spots. C. Place about 2 mg in a test-tube about 150 mm long and 15 mm in diameter. Moisten with 0.05 ml of water R and add 2 ml of sulphuric acid-formaldehyde reagent R. Mix the contents of the tube by swirling ; the solution is practically colourless. Place the test-tube on a water-bath for 1 min ; a reddish-brown colour develops. D. It gives reaction (a) of sodium (2.3.1).

D. (3S,7R,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7atetrahydroimidazo[5,1-b]thiazole-3,7-dicarboxylic acid (penillic acid of benzylpenicillin),

TESTS pH (2.2.3). Dissolve 2.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. The pH of the 01/2005:0114 solution is 5.5 to 7.5. Specific optical rotation (2.2.7). Dissolve 0.500 g in carbon dioxide-free water R and dilute to 25.0 ml with the same BENZYLPENICILLIN SODIUM solvent. The specific optical rotation is + 285 to + 310, calculated with reference to the dried substance. Benzylpenicillinum natricum Absorbance (2.2.25). Dissolve 90.0 mg in water R and dilute to 50.0 ml with the same solvent. Measure the absorbance of the solution at 325 nm, at 280 nm and at the maximum at 264 nm, diluting the solution, if necessary, for the measurement at 264 nm. The absorbances at 325 nm and 280 nm are not greater than 0.10 and the absorbance at the maximum at 264 nm is 0.80 to 0.88, calculated on the basis of the undiluted (1.80 g/l) solution. Verify the resolution C16H17N2NaO4S Mr 356.4 of the apparatus (2.2.25) ; the ratio of the absorbances is at least 1.7. DEFINITION Related substances. Liquid chromatography (2.2.29) as Benzylpenicillin sodium is sodium (2S,5R,6R)-3, described under Assay. Inject 20 µl of reference solution (d) 3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-1and elute isocratically with the chosen mobile phase. Inject azabicyclo[3.2.0]heptane-2-carboxylate, a substance 20 µl of test solution (b) and start the elution isocratically. produced by the growth of certain strains of Penicillium Immediately after elution of the benzylpenicillin peak start notatum or related organisms, or obtained by any other means. It contains not less than 96.0 per cent and not more the following linear gradient : than the equivalent of 102.0 per cent of benzylpenicillin Time Mobile phase A Mobile phase B Comment sodium, calculated with reference to the dried substance. (min) (per cent V/V) (per cent V/V) E. phenylacetic acid.

CHARACTERS A white or almost white, crystalline powder, very soluble in water, practically insoluble in fatty oils and in liquid paraffin. IDENTIFICATION First identification : A, D. Second identification : B, C, D. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with benzylpenicillin sodium CRS. B. Examine by thin-layer chromatography (2.2.27), using a TLC silanised silica gel plate R. Test solution. Dissolve 25 mg of the substance to be examined in 5 ml of water R. Reference solution (a). Dissolve 25 mg of benzylpenicillin sodium CRS in 5 ml of water R. Reference solution (b). Dissolve 25 mg of benzylpenicillin sodium CRS and 25 mg of phenoxymethylpenicillin potassium CRS in 5 ml of water R. Apply to the plate 1 µl of each solution. Develop over a path of 15 cm using a mixture of 30 volumes of acetone R and 70 volumes of a 154 g/l solution of ammonium acetate R, the pH of which has been adjusted to 5.0 with glacial acetic acid R. Allow the plate to dry in air and expose it to iodine vapour until the spots appear. Examine 1082

0 - 20

70 → 0

30 → 100

linear gradient

20 - 35

0

100

isocratic

35 - 50

70

30

re-equilibration

Inject water R and use the same elution pattern to obtain a blank. In the chromatogram obtained with test solution (b), the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (d) (1 per cent). 2-Ethylhexanoic acid (2.4.28). Not more than 0.5 per cent m/m. Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Bacterial endotoxins (2.6.14, Method E) : less than 0.16 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. ASSAY Liquid chromatography (2.2.29). Prepare the solutions immediately before use. Test solution (a). Dissolve 50.0 mg of the substance to be examined in water R and dilute to 50.0 ml with the same solvent.


Betacarotene


Test solution (b). Dissolve 80.0 mg of the substance to be examined in water R and dilute to 20.0 ml with the same solvent. Reference solution (a). Dissolve 50.0 mg of benzylpenicillin sodium CRS in water R and dilute to 50.0 ml with the same solvent. Reference solution (b). Dissolve 10 mg of benzylpenicillin sodium CRS and 10 mg of phenylacetic acid CRS in water R C. (2S,5R,6R)-6-[[(4-hydroxyphenyl)acetyl]amino]-3,3dimethyl-7-oxo-4-thia- 1-azabicyclo[3.2.0]heptane-2and dilute to 50 ml with the same solvent. carboxylic acid, Reference solution (c). Dilute 1.0 ml of reference solution (a) to 20.0 ml with water R. Dilute 1.0 ml of the solution to 50.0 ml with water R. Reference solution (d). Dilute 4.0 ml of reference solution (a) to 100.0 ml with water R. The chromatographic procedure may be carried out using : — a column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for D. (3S,7R,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7achromatography R (5 µm), tetrahydroimidazo[5,1-b]thiazole-3,7-dicarboxylic — as mobile phase at a flow rate of 1.0 ml/min : acid (penillic acid of benzylpenicillin), Mobile phase A. Mix 10 volumes of a 68 g/l solution of potassium dihydrogen phosphate R adjusted to pH 3.5 with a 500 g/l solution of dilute phosphoric acid R, 30 volumes of methanol R and 60 volumes of water R. Mobile phase B. Mix 10 volumes of a 68 g/l solution of potassium dihydrogen phosphate R adjusted to pH 3.5 with a 500 g/l solution of dilute phosphoric acid R, 40 volumes of water R and 50 volumes of methanol R. E. (4S)-2-[carboxy[(phenylacetyl)amino]methyl]-5,5— as detector a spectrophotometer set at 225 nm. dimethylthiazolidine-4-carboxylic acid (penicilloic acids of benzylpenicillin), Equilibrate the column with a mobile phase ratio A:B of 70:30. Inject 20 µl of reference solution (b). The test is not valid unless the resolution between the 2 principal peaks is at least 6.0 (if necessary, adjust the ratio A:B of the mobile phase) and the mass distribution ratio for the second peak (benzylpenicillin) is 4.0 to 6.0. Inject 20 µl of reference solution (c). Adjust the system to obtain a peak with a signal-to-noise ratio of at least 3. STORAGE Store in an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container.

F. (2RS,4S)-2-[[(phenylacetyl)amino]methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penilloic acids of benzylpenicillin).

LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins.

01/2005:1069

BETACAROTENE

IMPURITIES

Betacarotenum

A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylic acid (6-aminopenicillanic acid),

B. phenylacetic acid, General Notices (1) apply to all monographs and other texts

C40H56

Mr 536.9 1083

Betadex


01/2005:1070

DEFINITION Betacarotene contains not less than 96.0 per cent and not more than the equivalent of 101.0 per cent of (all-E)-3,7,12,16-Tetramethyl-1,18-bis(2,6,6-trimethylcyclohex1-enyl)octadeca-1,3,5,7,9,11,13,15,17-nonaene, calculated with reference to the dried substance.

BETADEX Betadexum

CHARACTERS A brown-red or brownish-red, crystalline powder, practically insoluble in water, slightly soluble in cyclohexane, practically insoluble in ethanol. It is sensitive to air, heat and light, especially in solution. Carry out all operations as rapidly as possible avoiding exposure to actinic light ; use freshly prepared solutions.

IDENTIFICATION Dissolve 50.0 mg in 10 ml of chloroform R and dilute immediately to 100.0 ml with cyclohexane R. Dilute 5.0 ml of this solution to 100.0 ml with cyclohexane R (solution A ; use solution A also for the test for related substances). Dilute 5.0 ml of solution A to 50.0 ml with cyclohexane R. (Solution B ; use solution B also for the test for related substances and for the assay). Determine the absorbance (2.2.25) of solution B at 455 nm and at 483 nm using cyclohexane R as the compensation liquid. The ratio of the absorbance at 455 nm to that at 483 nm is between 1.14 and 1.18.

TESTS

[C6H10O5]7

Mr 1135

DEFINITION Betadex (betacyclodextrin) contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of cyclo-α-(1→4)-D-heptaglucopyranoside, calculated with reference to the dried substance. CHARACTERS A white or almost white, amorphous or crystalline powder, sparingly soluble in water, freely soluble in propylene glycol, practically insoluble in ethanol and in methylene chloride.

Related substances. Determine the absorbance (2.2.25) of solution B at 455 nm and that of solution A at 340 nm, used IDENTIFICATION in Identification. The ratio of the absorbance at 455 nm to A. It complies with the test for specific optical rotation (see that at 340 nm is not less than 1.5. Tests). B. Examine the chromatograms obtained in the assay. Heavy metals (2.4.8). 2.0 g complies with limit test D for The retention time and size of the principal peak in heavy metals (10 ppm). Prepare the standard using 2 ml of the chromatogram obtained with test solution (b) are lead standard solution (10 ppm Pb) R. approximately the same as those of the principal peak in Loss on drying (2.2.32). Not more than 0.2 per cent, the chromatogram obtained with reference solution (c). determined on 1.000 g by drying in vacuo over diphosphorus C. Dissolve 0.2 g in 2 ml of iodine solution R4 by warming pentoxide R at 40 °C for 4 h. on a water-bath, and allow to stand at room temperature. Sulphated ash (2.4.14). Not more than 0.2 per cent, A yellowish-brown precipitate is formed. determined on 1.0 g, moistened with a mixture of 2 ml of TESTS dilute sulphuric acid R and 5 ml of alcohol R. Solution S. Dissolve 1.000 g in carbon dioxide-free water R with heating, allow to cool and dilute to 100.0 ml with the same solvent. ASSAY Appearance of solution. Solution S is clear (2.2.1). Measure the absorbance (2.2.25) of solution B used pH (2.2.3). To 10 ml of solution S add 0.1 ml of a saturated in Identification at the maximum at 455 nm, using solution of potassium chloride R. The pH of the solution cyclohexane R as the compensation liquid. is 5.0 to 8.0. Calculate the content of C40H56 taking the specific absorbance Specific optical rotation (2.2.7) : + 160 to + 164, determined on solution S and calculated with reference to the dried to be 2500. substance. Reducing sugars Test solution. To 1 ml of solution S add 1 ml of cupri-tartaric STORAGE solution R4. Heat on a water-bath for 10 min, cool to room temperature. Add 10 ml of ammonium molybdate Store in an airtight container, protected from light, at a reagent R1 and allow to stand for 15 min. temperature not exceeding 25 °C. 1084


Betadex


Reference solution. Prepare a reference solution at the same time and in the same manner as the test solution, using 1 ml of a 0.02 g/l solution of glucose R. Measure the absorbance of the test solution and the reference solution (2.2.25) at the maximum at 740 nm using water R as the compensation liquid. The absorbance of the test solution is not greater than that of the reference solution (0.2 per cent). Light-absorbing impurities. Examine solution S between 230 nm and 750 nm (2.2.25). Between 230 nm and 350 nm, the absorbance is not greater than 0.10. Between 350 nm and 750 nm, the absorbance is not greater than 0.05. Related substances. Examine by liquid chromatography (2.2.29), as described under Assay. Inject separately test solution (a) and reference solution (b). In the chromatogram obtained with test solution (a) : the areas of any peaks corresponding to gammacyclodextrin and alphacyclodextrin are not greater than half of the area of the corresponding peaks in the chromatogram obtained with reference solution (b) (0.25 per cent) ; the sum of the areas of all the peaks, apart from the principal peak and any peaks corresponding to alphacyclodextrin and gammacyclodextrin, is not greater than half of the area of the peak corresponding to betadex in the chromatogram obtained with reference solution (b) (0.5 per cent). Residual solvents. Not more than 10 ppm of trichloroethylene and not more than 10 ppm of toluene. Examine by head-space gas chromatography (2.2.28), using the standard additions method and ethylene chloride R as the internal standard. Test solutions. In each of four identical 20 ml flasks, dissolve 500 mg of the substance to be examined in water R and add 0.10 g of calcium chloride R and 30 µl of α-amylase solution R. Add 1 ml of reference solutions (a), (b), (c) and (d), adding a different solution to each flask. Dilute to 10 ml with water R. Reference solutions. Prepare reference solution (a) containing 10 µl of ethylene chloride R per litre. From reference solution (a), prepare reference solutions (b), (c) and (d) containing per litre : 5 µl, 10 µl and 15 µl each of trichloroethylene R and of toluene R. The chromatographic procedure may be carried out using : — a fused-silica column 25 m long and 0.32 mm in internal diameter coated with a layer about 1 µm thick of macrogol 20 000 R, — helium for chromatography R as the carrier gas, — a flame-ionisation detector, maintaining the temperature of the column at 50 °C, that of the injection port at 140 °C and that of the detector at 280 °C. Place the samples in a thermostated chamber at 45 °C for 2 h. Inject 200 µl of the head-space of each flask and repeat each test at least three times. The retention time of toluene is about 10 min. The test is not valid unless : the resolutions between the peaks corresponding to trichloroethylene and toluene and between the peaks corresponding to toluene and ethylene chloride are greater than 1.1 and the relative standard deviations of the ratios of the areas of the peaks corresponding to trichloroethylene and toluene to that of the peak corresponding to ethylene chloride are less than 5 per cent. Calculate the content of trichloroethylene and of toluene taking their relative densities to be 1.46 and 0.87, respectively. Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (10 ppm). Prepare the standard using 1 ml of lead standard solution (10 ppm Pb) R.

Loss on drying (2.2.32). Not more than 16.0 per cent, determined on 1.000 g by drying in an oven at 120 °C for 2 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.


1085

ASSAY Examine by liquid chromatography (2.2.29). Test solution (a). Dissolve 0.25 g of the substance to be examined in water R with heating, cool and dilute to 25.0 ml with the same solvent. Test solution (b). Dilute 5.0 ml of test solution (a) to 50.0 ml with water R. Reference solution (a). Dissolve 25.0 mg of alfadex CRS, 25.0 mg of gammacyclodextrin CRS and 50.0 mg of betadex CRS in water R and dilute to 50.0 ml with the same solvent. Reference solution (b). Dilute 5.0 ml of reference solution (a) to 50.0 ml with water R. Reference solution (c). Dissolve 25.0 mg of betadex CRS in water R and dilute to 25.0 ml with the same solvent. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (10 µm), — as mobile phase at a flow rate of 1.5 ml/min a mixture of 10 volumes of methanol R and 90 volumes of water R, — as detector a differential refractometer, — a 50 µl loop injector. Equilibrate the column with the mobile phase at a flow rate of 1.5 ml/min for about 3 h. Inject each solution. Record the chromatograms for 1.5 times the retention time of betadex. Adjust the sensitivity of the detector so that the height of the peak corresponding to gammacyclodextrin, in the chromatogram obtained with reference solution (a), is 55 per cent to 75 per cent of the full scale of the recorder. The retention time of betadex is about 10 min, the relative retention time of gammacyclodextrin is about 0.3 and that of alfadex is about 0.45. The test is not valid unless the resolution between the peaks corresponding to gammacyclodextrin and alfadex is not less than 1.5, and the relative standard deviation of the area of the peak corresponding to betadex is less than 2.0 per cent. If necessary, adjust the concentration of methanol in the mobile phase to achieve the required resolution. Calculate the percentage content of [C6H10O5]7 from the area of the principal peak in each of the chromatograms obtained with test solution (b) and reference solution (c) and the declared content of betadex CRS. STORAGE Store in an airtight container. IMPURITIES

A. n = 6 : alfadex, B. n = 8 : gammacyclodextrin.

Betahistine mesilate


01/2005:1071 TESTS Solution S. Dissolve 5.0 g in carbon dioxide-free water R prepared from distilled water R, and dilute to 50 ml with BETAHISTINE MESILATE the same solvent. Appearance of solution. Solution S is clear (2.2.1) and Betahistini mesilas colourless (2.2.2, Method II). pH (2.2.3). The pH of solution S is 2.0 to 3.0. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 50 mg of the substance to be C10H20N2O6S2 Mr 328.4 examined in the mobile phase and dilute to 10.0 ml with the mobile phase. DEFINITION Reference solution (a). Dissolve 10 mg of betahistine Betahistine mesilate contains not less than 98.0 per cent mesilate CRS and 10 mg of 2-vinylpyridine R in the mobile and not more than the equivalent of 101.0 per cent of N-methyl-2-(pyridin-2-yl)ethanamine bis(methanesulphonate), phase and dilute to 50.0 ml with the mobile phase. Dilute 2.0 ml of the solution to 50.0 ml with the mobile phase. calculated with reference to the anhydrous, 2-propanol-free Reference solution (b). Dilute 1.0 ml of the test solution to substance. 100.0 ml with the mobile phase. PRODUCTION Reference solution (c). Dilute 2.0 ml of reference solution (b) The production method must be evaluated to determine to 10.0 ml with the mobile phase. the potential for formation of alkyl mesilates, which is The chromatographic procedure may be carried out using : particularly likely to occur if the reaction medium contains — a stainless steel column 0.25 m long and 4.6 mm in lower alcohols. Where necessary, the production method internal diameter packed with octadecylsilyl silica gel for is validated to demonstrate that alkyl mesilates are not chromatography R (5 µm), detectable in the final product. — as mobile phase at a flow rate of 1 ml/min a mixture CHARACTERS prepared as follows : dissolve 2.0 g of sodium dodecyl A white, crystalline powder, very hygroscopic, very soluble sulphate R in a mixture of 15 volumes of a 10 per cent V/V in water, freely soluble in alcohol, very slightly soluble in solution of sulphuric acid R, 35 volumes of a 17 g/l 2-propanol. solution of tetrabutylammoniumhydrogen sulphate R and 650 volumes of water R ; adjust to pH 3.3 using dilute IDENTIFICATION sodium hydroxide solution R and mix with 300 volumes of acetonitrile R, First identification : B. — as detector a spectrophotometer set at 260 nm. Second identification : A, C, D. Inject 20 µl of reference solution (a). When using a recorder, A. Melting point (2.2.14) : 108 °C to 112 °C. adjust the sensitivity of the system so that the height of the B. Examine by infrared absorption spectrophotometry first peak in the chromatogram obtained with reference (2.2.24), comparing with the spectrum obtained with solution (a) is not less than 70 per cent of the full scale of the betahistine mesilate CRS. Examine the substances recorder. The test is not valid unless : in the chromatogram prepared as discs. obtained with reference solution (a), the resolution between C. Examine by thin-layer chromatography (2.2.27), using a the peaks corresponding to 2-vinylpyridine and betahistine suitable silica gel with a fluorescent indicator having an mesilate is at least 3.5. optimal intensity at 254 nm as the coating substance. Inject 20 µl of the test solution and of reference solutions (b) Test solution. Dissolve 10 mg of the substance to be and (c). Continue the chromatography for 3 times the examined in alcohol R and dilute to 2 ml with the same retention time of betahistine mesilate (which is about 8 min). solvent. In the chromatogram obtained with the test solution the Reference solution. Dissolve 10 mg of betahistine area of any peak, apart from the principal peak, is not greater mesilate CRS, in alcohol R and dilute to 2 ml with the than the area of the principal peak in the chromatogram same solvent. obtained with reference solution (c) (0.2 per cent) ; the sum of the areas of any peaks, apart from the principal peak, is Apply to the plate 2 µl of each solution. Develop over not greater than half of the area of the principal peak in the a path of 15 cm using a mixture of 0.75 volumes of concentrated ammonia R, 15 volumes of ethyl acetate R chromatogram obtained with reference solution (b) (0.5 per cent). and 30 volumes of methanol R. Dry the plate at 110 °C for 10 min and examine in ultraviolet light at 254 nm. Disregard any peak with an area less than 0.025 times that The principal spot in the chromatogram obtained with of the principal peak in the chromatogram obtained with the test solution is similar in position and size to the reference solution (b). principal spot in the chromatogram obtained with the 2-Propanol. Not more than 0.5 per cent, determined by the reference solution. test for residual solvents (2.4.24). D. To 0.1 g add 5 ml of dilute hydrochloric acid R and Chlorides (2.4.4). To 14 ml of solution S add 1 ml of water R. shake for about 5 min. Add 1 ml of barium chloride The solution complies with the limit test for chlorides solution R1. The solution remains clear. To a further (35 ppm). 0.1 g add 0.5 g of anhydrous sodium carbonate R, mix and ignite until a white residue is obtained. Allow to cool Sulphates (2.4.13). Dilute 6 ml of solution S to 15 ml with and dissolve the residue in 7 ml of water R. The solution distilled water R. The solution complies with the limit test for sulphates (250 ppm). gives reaction (a) of sulphates (2.3.1). 1086



Betamethasone

Heavy metals (2.4.8). 12 ml of solution S complies with limit test A for heavy metals (20 ppm). Prepare the standard using lead standard solution (2 ppm Pb) R. Water (2.5.12). Not more than 2.0 per cent, determined on C. 0.50 g by the semi-micro determination of water.

examined and the reference substance separately in the smallest necessary quantity of methylene chloride R and evaporate to dryness on a water-bath. Using the residues, record the spectra again. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a ASSAY fluorescent indicator having an optimal intensity at 254 nm. Dissolve 0.140 g in 50 ml of a mixture of 1 volume of anhydrous acetic acid R and 7 volumes of acetic Test solution. Dissolve 10 mg of the substance to be anhydride R. Titrate with 0.1 M perchloric acid, determining examined in a mixture of 1 volume of methanol R and the end-point potentiometrically (2.2.20). 9 volumes of methylene chloride R and dilute to 10 ml with the same mixture of solvents. 1 ml of 0.1 M perchloric acid is equivalent to 16.42 mg of C10H20N2O6S2. Reference solution (a). Dissolve 20 mg of betamethasone CRS in a mixture of 1 volume of STORAGE methanol R and 9 volumes of methylene chloride R and Store in an airtight container. dilute to 20 ml with the same mixture of solvents. Reference solution (b). Dissolve 10 mg of IMPURITIES dexamethasone CRS in reference solution (a) and dilute to 10 ml with the same solution. Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 5 volumes of butanol R saturated with water R, 10 volumes of A. 2-ethenylpyridine. toluene R and 85 volumes of ether R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal 01/2005:0312 spot in the chromatogram obtained with reference solution (a). Spray with alcoholic solution of sulphuric BETAMETHASONE acid R. Heat at 120 °C for 10 min or until the spots appear. Allow to cool. Examine the chromatograms in daylight and in ultraviolet light at 365 nm. The principal Betamethasonum spot in the chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two spots which may however not be completely separated. D. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is obtained (usually less than 5 min). Allow to cool, add C22H29FO5 Mr 392.5 1 ml of water R, 0.05 ml of phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render DEFINITION the solution colourless. Filter. Add 1.0 ml of the filtrate Betamethasone contains not less than 97.0 per cent and to a freshly prepared mixture of 0.1 ml of alizarin S not more than the equivalent of 103.0 per cent of 9-fluorosolution R and 0.1 ml of zirconyl nitrate solution R. 11β,17,21-trihydroxy-16β-methylpregna-1,4-diene-3,20-dione, Mix, allow to stand for 5 min and compare the colour of calculated with reference to the dried substance. the solution with that of a blank prepared in the same manner. The test solution is yellow and the blank is red. CHARACTERS E. Add about 2 mg to 2 ml of sulphuric acid R and shake A white or almost white, crystalline powder, practically to dissolve. Within 5 min, a deep reddish-brown colour insoluble in water, sparingly soluble in ethanol, very slightly develops. Add the solution to 10 ml of water R and mix. soluble in methylene chloride. The colour is discharged and a clear solution remains. IDENTIFICATION TESTS First identification : B, C. Specific optical rotation (2.2.7). Dissolve 0.125 g in Second identification : A, C, D, E. methanol R and dilute to 25.0 ml with the same solvent. The A. Dissolve 10.0 mg in ethanol R and dilute to 100.0 ml specific optical rotation is + 118 to + 126, calculated with with the same solvent. Place 2.0 ml of the solution in a reference to the dried substance. stoppered tube, add 10.0 ml of phenylhydrazine-sulphuric acid solution R, mix and heat in a water-bath at 60 °C for Related substances. Examine by liquid chromatography 20 min. Cool immediately. The absorbance (2.2.25) of the (2.2.29). solution measured at 419 nm is not greater than 0.10. Test solution. Dissolve 25.0 mg of the substance to be examined in a mixture of equal volumes of acetonitrile R B. Examine by infrared absorption spectrophotometry and methanol R and dilute to 10.0 ml with the same solvent. (2.2.24), comparing with the spectrum obtained with betamethasone CRS. If the spectra obtained in the solid Reference solution (a). Dissolve 2 mg of betamethasone CRS state with the substance to be examined and the reference and 2 mg of methylprednisolone CRS in mobile phase A and substance show differences, dissolve the substance to be dilute to 100.0 ml with the same mobile phase. General Notices (1) apply to all monographs and other texts

1087

Betamethasone


ASSAY Dissolve 0.100 g in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the solution to 100.0 ml with The chromatographic procedure may be carried out using : alcohol R. Measure the absorbance (2.2.25) at the maximum — a stainless steel column 0.25 m long and 4.6 mm in at 238.5 nm. internal diameter packed with octadecylsilyl silica gel for Calculate the content of C22H29FO5 taking the specific chromatography R (5 µm), absorbance to be 395. — as mobile phase at a flow rate of 2.5 ml/min, a STORAGE linear-gradient programme using the following Store protected from light. conditions : Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A.

Mobile phase A. In a 1000 ml volumetric flask mix 250 ml IMPURITIES of acetonitrile R with 700 ml of water R and allow to A. dexamethasone, equilibrate ; adjust the volume to 1000 ml with water R and mix again, Mobile phase B. Acetonitrile R, Time (min)



Comment

0

100

0

isocratic

15

100

0

begin linear gradient

40

0

100

end chromatogram, return to 100A

41

100

0

begin equilibration with A

46 = 0

100

0

end equilibration, begin next chromatogram

B. 21-chloro-9-fluoro-11β,17-dihydroxy-16β-methylpregna1,4-diene-3,20-dione,

— as detector a spectrophotometer set at 254 nm, maintaining the temperature of the column at 45 °C. Equilibrate the column with mobile phase B at a flow rate of 2.5 ml/min for at least 30 min and then with mobile phase A for 5 min. For subsequent chromatograms, use the conditions described from 40 min to 46 min.

C. 17,21-dihydroxy-16β-methylpregna-1,4,9(11)-triene-3,20dione,

Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 20 µl of reference solution (b) is not less than 50 per cent of the full scale of the recorder. Inject 20 µl of reference solution (a). When the chromatograms are recorded in the conditions described above, the retention times are : methylprednisolone, about 11.5 minutes and betamethasone, about 12.5 minutes. The test is not valid unless the resolution between the peaks corresponding to methylprednisolone and betamethasone is at least 1.5 ; if necessary, adjust the concentration of acetonitrile in mobile phase A.

D. 9-fluoro-11β,17-dihydroxy-16β-methyl-3,20-dioxopregna-1, 4-dien-21-yl ethoxycarboxylate,

Inject separately 20 µl of the mixture of equal volumes of acetonitrile R and methanol R as a blank, 20 µl of the test solution and 20 µl of reference solution (b). In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent) and not more than E. 9,11β-epoxy-17,21-dihydroxy-16β-methyl-9β-pregna-1,4one such peak has an area greater than half the area of the diene-3,20-dione, principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (2.0 per cent). Disregard any peak due to the blank and any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (b). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 0.500 g by drying in an oven at 100 °C to F. 17,21-dihydroxy-16β-methylpregna-1,4,11-triene-3,20105 °C. dione, 1088



Betamethasone acetate

CHARACTERS A white or almost white, crystalline powder, practically insoluble in water, freely soluble in acetone, soluble in alcohol and in methylene chloride. It shows polymorphism. IDENTIFICATION First identification : B, C. G. 11α,17,21-trihydroxy-16β-methylpregna-1,4-diene-3,20Second identification : A, C, D, E, F. dione, A. Dissolve 10.0 mg in ethanol R and dilute to 100.0 ml with the same solvent. Place 2.0 ml of this solution in a ground-glass-stoppered tube, add 10.0 ml of phenylhydrazine-sulphuric acid solution R, mix and heat in a water-bath at 60 °C for 20 min. Cool immediately. The absorbance (2.2.25) of the solution measured at 419 nm is not more than 0.10. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with betamethasone acetate CRS. If the spectra obtained in H. 14-fluoro-11β,17,21-trihydroxy-16β-methyl-8α,9β,14βthe solid state show differences, dissolve the substance to pregna-1,4-diene-3,20-dione, be examined and the reference substance separately in the minimum volume of methanol R, evaporate to dryness on a water-bath and record new spectra using the residues. C. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm. Test solution. Dissolve 10 mg of the substance to be examined in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 10 ml I. 8-fluoro-11β,17,21-trihydroxy-16β-methyl-8α,9β-pregnawith the same mixture of solvents. 1,4-diene-3,20-dione, Reference solution (a). Dissolve 20 mg of betamethasone acetate CRS in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 20 ml with the same mixture of solvents. Reference solution (b). Dissolve 10 mg of prednisolone acetate CRS in reference solution (a) and dilute to 10 ml with the same solution. Apply to the plate 5 µl of each solution. Prepare the mobile phase by adding a mixture of 1.2 volumes of water R and 8 volumes of methanol R to a mixture of J. 17,21-dihydroxy-16β-methylpregna-1,4-diene-3,20-dione. 15 volumes of ether R and 77 volumes of methylene chloride R. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the 01/2005:0975 test solution is similar in position and size to the principal spot in the chromatogram obtained with reference BETAMETHASONE ACETATE solution (a). Spray with alcoholic solution of sulphuric acid R. Heat at 120 °C for 10 min or until the spots Betamethasoni acetas appear. Allow to cool. Examine the plate in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. D. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a deep reddish-brown colour develops. Add the solution to 10 ml of water R and mix. C24H31FO6 Mr 434.5 The colour is discharged and a clear solution remains. DEFINITION E. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is Betamethasone acetate contains not less than 97.0 per obtained (usually less than 5 min). Allow to cool, add 1 ml cent and not more than the equivalent of 103.0 per cent of water R, 0.05 ml of phenolphthalein solution R1 and of 9-fluoro-11β,17-dihydroxy-16β-methyl-3,20-dioxopregnaabout 1 ml of dilute hydrochloric acid R to render the 1,4-diene-21-yl acetate, calculated with reference to the solution colourless. Filter. To a freshly prepared mixture anhydrous substance. General Notices (1) apply to all monographs and other texts

1089

Betamethasone dipropionate


of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R, add 1.0 ml of the filtrate. Mix, allow to stand for 5 min and compare the colour of the solution with that of a blank prepared in the same manner. The test solution is yellow and the blank is red. F. About 10 mg gives the reaction of acetyl (2.3.1). TESTS Specific optical rotation (2.2.7). Dissolve 0.250 g in dioxan R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 120 to + 128, calculated with reference to the anhydrous substance. Related substances. Examine by liquid chromatography (2.2.29).

ASSAY Dissolve 0.100 g in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the solution to 100.0 ml with alcohol R. Measure the absorbance (2.2.25) at the maximum at 240 nm. Calculate the content of C24H31FO6 taking the specific absorbance to be 350. STORAGE Store protected from light. IMPURITIES A. betamethasone, B. dexamethasone acetate,

Test solution. Dissolve 25.0 mg of the substance to be examined in 4 ml of acetonitrile R and dilute to 10.0 ml with the same solvent. Reference solution (a). Dissolve 2 mg of betamethasone acetate CRS and 2 mg of dexamethasone acetate CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase.

C. betamethasone 11,21-diacetate,

The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows : in a 1000 ml volumetric flask mix 380 ml of acetonitrile R with 550 ml of water R and allow to equilibrate ; dilute to 1000 ml with water R and mix again,

D. 9,11β-epoxy-17-hydroxy-16β-methyl-3,20-dioxo-9β-pregna1,4-diene-21-yl acetate.

— as detector a spectrophotometer set at 254 nm. Equilibrate the column with the mobile phase at a flow rate of 1 ml/min for about 30 min. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 20 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder.

01/2005:0809

BETAMETHASONE DIPROPIONATE Betamethasoni dipropionas

Inject 20 µl of reference solution (a).When the chromatograms are recorded in the prescribed conditions, the retention times are : betamethasone acetate about 19 min and dexamethasone acetate about 22 min. The test is not valid unless the resolution between the peaks due to betamethasone acetate and dexamethasone acetate is at least 3.3 ; if necessary, adjust slightly the concentration of acetonitrile in the mobile phase. Inject 20 µl of the test solution and 20 µl of reference solution (b). Continue the chromatography for 2.5 times the retention time of the principal peak in the chromatogram obtained with the test solution. In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than half the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than 1.25 times the area of the principal peak in the chromatogram obtained with reference solution (b) (1.25 per cent). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (b). Water (2.5.12). Not more than 4.0 per cent, determined on 0.100 g by the semi-micro determination of water. 1090

C28H37FO7

Mr 504.6

DEFINITION Betamethasone dipropionate contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent of 9-fluoro-11β-hydroxy-16β-methyl-3,20-dioxopregna-1,4-diene17,21-diyl dipropanoate, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, practically insoluble in water, freely soluble in acetone and in methylene chloride, sparingly soluble in alcohol. IDENTIFICATION First identification : B, C.



Betamethasone dipropionate

Reference solution (a). Dissolve 25 mg of betamethasone dipropionate CRS in methanol R with gentle heating and Dissolve 10.0 mg in ethanol R and dilute to 100.0 ml dilute to 5 ml with the same solvent. This solution is also with the same solvent. Place 2.0 ml of this solution used to prepare reference solution (b). Dilute 2 ml of the in a ground-glass-stoppered tube, add 10.0 ml of solution to 10 ml with methylene chloride R. phenylhydrazine-sulphuric acid solution R, mix and heat Reference solution (b). Transfer 2 ml of the solution in a water-bath at 60 °C for 20 min. Cool immediately. obtained during preparation of reference solution (a) The absorbance (2.2.25) of the solution measured at to a 15 ml glass tube with a ground-glass stopper or a 419 nm is not more than 0.10. polytetrafluoroethylene cap. Add 10 ml of saturated Examine by infrared absorption spectrophotometry methanolic potassium hydrogen carbonate solution R (2.2.24), comparing with the spectrum obtained with and immediately pass a current of nitrogen R briskly betamethasone dipropionate CRS. through the solution for 5 min. Stopper the tube. Heat in a water-bath at 45 °C, protected from light, for 2 h. Examine by thin-layer chromatography (2.2.27), using Allow to cool. as the coating substance a suitable silica gel with a Apply to the plate 5 µl of each solution. Prepare the fluorescent indicator having an optimal intensity at mobile phase by adding a mixture of 1.2 volumes of 254 nm. water R and 8 volumes of methanol R to a mixture of Test solution. Dissolve 10 mg of the substance to be 15 volumes of ether R and 77 volumes of methylene examined in a mixture of 1 volume of methanol R and chloride R. Develop over a path of 15 cm. Allow the plate 9 volumes of methylene chloride R and dilute to 10 ml to dry in air and examine in ultraviolet light at 254 nm. with the same mixture of solvents. The principal spot in each of the chromatograms obtained with the test solutions is similar in position and size to Reference solution (a). Dissolve 10 mg of betamethasone the principal spot in the chromatogram obtained with dipropionate CRS in a mixture of 1 volume of methanol R the corresponding reference solution. Spray the plate and 9 volumes of methylene chloride R and dilute to with alcoholic solution of sulphuric acid R. Heat at 10 ml with the same mixture of solvents. 120 °C for 10 min or until the spots appear. Allow to cool. Reference solution (b). Dissolve 10 mg of desoxycortone Examine in daylight and in ultraviolet light at 365 nm. acetate CRS in a mixture of 1 volume of methanol R and The principal spot in each of the chromatograms obtained 9 volumes of methylene chloride R and dilute to 10 ml with the test solutions is similar in position, colour in with the same mixture of solvents. Dilute 5 ml of this daylight, fluorescence in ultraviolet light at 365 nm and solution to 10 ml with reference solution (a). size to the principal spot in the chromatogram obtained with the corresponding reference solution. The principal Apply to the plate 5 µl of each solution. Prepare the spot in each of the chromatograms obtained with test mobile phase by adding a mixture of 1.2 volumes of solution (b) and reference solution (b) has an Rf value water R and 8 volumes of methanol R to a mixture of distinctly lower than that of the principal spots in each of 15 volumes of ether R and 77 volumes of methylene the chromatograms obtained with test solution (a) and chloride R. Develop over a path of 15 cm. Allow the plate reference solution (a). to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the E. Add about 2 mg to 2 ml of sulphuric acid R and shake test solution is similar in position and size to the principal to dissolve. Within 5 min, a deep reddish-brown colour spot in the chromatogram obtained with reference develops. Add the solution to 10 ml of water R and mix. solution (a). Spray the plate with alcoholic solution of The colour is discharged and a clear solution remains. sulphuric acid R. Heat at 120 °C for 10 min or until the F. Mix about 5 mg with 45 mg of heavy magnesium oxide R spots appear. Allow to cool. Examine in daylight and and ignite in a crucible until an almost white residue is in ultraviolet light at 365 nm. The principal spot in the obtained (usually less than 5 min). Allow to cool, add chromatogram obtained with the test solution is similar 1 ml of water R, 0.05 ml of phenolphthalein solution R1 in position, colour in daylight, fluorescence in ultraviolet and about 1 ml of dilute hydrochloric acid R to render light at 365 nm and size to the principal spot in the the solution colourless. Filter. Add 1.0 ml of the filtrate chromatogram obtained with reference solution (a). The to a freshly prepared mixture of 0.1 ml of alizarin S test is not valid unless the chromatogram obtained with solution R and 0.1 ml of zirconyl nitrate solution R. reference solution (b) shows two clearly separated spots. Mix, allow to stand for 5 min and compare the colour of the solution with that of a blank prepared in the same Examine by thin-layer chromatography (2.2.27), using manner. The test solution is yellow and the blank is red. as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at TESTS 254 nm. Specific optical rotation (2.2.7). Dissolve 0.250 g in Test solution (a). Dissolve 25 mg of the substance to be dioxan R and dilute to 25.0 ml with the same solvent. The examined in methanol R with gentle heating and dilute to 5 ml with the same solvent. This solution is also used specific optical rotation is + 63 to + 70, calculated with to prepare test solution (b). Dilute 2 ml of the solution to reference to the dried substance. Related substances. Examine by liquid chromatography 10 ml with methylene chloride R. (2.2.29). Test solution (b). Transfer 2 ml of the solution obtained during preparation of test solution (a) to a 15 ml glass tube Test solution. Dissolve 62.5 mg of the substance to be with a ground-glass stopper or a polytetrafluoroethylene examined in the mobile phase and dilute to 25.0 ml with the mobile phase. cap. Add 10 ml of saturated methanolic potassium hydrogen carbonate solution R and immediately pass a Reference solution (a). Dissolve 2.5 mg of betamethasone current of nitrogen R briskly through the solution for dipropionate CRS and 2.5 mg of beclometasone 5 min. Stopper the tube. Heat in a water-bath at 45 °C, dipropionate CRS in the mobile phase and dilute to 50.0 ml protected from light, for 2 h. Allow to cool. with the same solvent.

Second identification : A, D, E, F. A.

B.

C.

D.


1091

Betamethasone sodium phosphate


Reference solution (b). Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm),

01/2005:0810

BETAMETHASONE SODIUM PHOSPHATE Betamethasoni natrii phosphas

— as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows : mix carefully 350 ml of water R with 600 ml of acetonitrile R and allow to equilibrate ; adjust the volume to 1000 ml with water R and mix again, — as detector a spectrophotometer set at 254 nm. C H FNa2O8P Mr 516.4 Adjust the sensitivity so that the height of the principal peak 22 28 in the chromatogram obtained with reference solution (b) is DEFINITION 70 per cent to 90 per cent of the full scale of the recorder. Betamethasone sodium phosphate contains not less than 96.0 per cent and not more than the equivalent of Equilibrate the column with the mobile phase at a flow rate 103.0 per cent of 9-fluoro-11β,17-dihydroxy-16β-methyl-3,20of 1 ml/min for about 45 min. Inject 20 µl of reference dioxopregna-1,4-diene-21-yl disodium phosphate, calculated solution (a). When the chromatograms are recorded with reference to the anhydrous substance. in the prescribed conditions, the retention times are : CHARACTERS betamethasone dipropionate, about 9 min ; beclometasone A white or almost white powder, very hygroscopic, freely dipropionate, about 10.7 min. The test is not valid soluble in water, slightly soluble in alcohol, practically unless the resolution between the peaks corresponding insoluble in methylene chloride. to betamethasone dipropionate and beclometasone dipropionate is at least 2.5 ; if necessary, adjust the IDENTIFICATION concentration of acetonitrile in the mobile phase. First identification : B, C. Second identification : A, C, D, E, F. Inject separately 20 µl of the test solution and 20 µl of reference solution (b). Continue the chromatography for A. Dissolve 10.0 mg in 5 ml of water R and dilute to 2.5 times the retention time of the principal peak. In 100.0 ml with ethanol R. Place 2.0 ml of this solution the chromatogram obtained with the test solution : the in a ground-glass-stoppered tube, add 10.0 ml of area of any peak apart from the principal peak is not phenylhydrazine-sulphuric acid solution R, mix and heat greater than 0.75 times the area of the principal peak in in a water-bath at 60 °C for 20 min. Cool immediately. the chromatogram obtained with reference solution (b) The absorbance (2.2.25) of the solution measured at the (1.5 per cent) and not more than one such peak has an maximum at 450 nm is not more than 0.10. area greater than half the area of the principal peak in the B. Examine by infrared absorption spectrophotometry chromatogram obtained with reference solution (b) (1 per (2.2.24), comparing with the spectrum obtained with cent) ; the sum of the areas of all the peaks, apart from the betamethasone sodium phosphate CRS. If the spectra principal peak, is not greater than 1.25 times the area of the obtained in the solid state show differences, dissolve the principal peak in the chromatogram obtained with reference substance to be examined and the reference substance solution (b) (2.5 per cent). Disregard any peak with an area separately in the minimum volume of alcohol R, evaporate less than 0.025 times the area of the principal peak in the to dryness on a water-bath and record new spectra using chromatogram obtained with reference solution (b). the residues. C. Examine by thin-layer chromatography (2.2.27), using Loss on drying (2.2.32). Not more than 1.0 per cent, as the coating substance a suitable silica gel with a determined on 0.500 g by drying in an oven at 100-105 °C. fluorescent indicator having an optimal intensity at 254 nm. Test solution. Dissolve 10 mg of the substance to be ASSAY examined in methanol R and dilute to 10 ml with the same solvent. Dissolve 50.0 mg in alcohol R and dilute to 100.0 ml with Reference solution (a). Dissolve 10 mg of betamethasone the same solvent. Dilute 2.0 ml of the solution to 50.0 ml sodium phosphate CRS in methanol R and dilute to with alcohol R. Measure the absorbance (2.2.25) at the 10 ml with the same solvent. maximum at 240 nm. Reference solution (b). Dissolve 10 mg of prednisolone sodium phosphate CRS in methanol R and dilute to Calculate the content of C28H37FO7 taking the specific 10 ml with the same solvent. Dilute 5 ml of this solution absorbance to be 305. to 10 ml with reference solution (a). Apply to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes STORAGE of butanol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is Store protected from light. 1092



similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). Spray the plate with alcoholic solution of sulphuric acid R. Heat at 120 °C for 10 min or until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two spots which may however not be completely separated.

Betamethasone sodium phosphate

The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows : in a 250 ml conical flask, weigh 1.360 g of potassium dihydrogen phosphate R and 0.600 g of hexylamine R, mix and allow to stand for 10 min and then dissolve in 185 ml of water R ; add 65 ml of acetonitrile R, mix and filter (0.45 µm), — as detector a spectrophotometer set at 254 nm.

D. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, an intense reddish-brown colour develops. Add the solution to 10 ml of water R and mix. The colour is discharged and a clear solution remains.

Equilibrate the column with the mobile phase at a flow rate of 1 ml/min for about 45 min.

F. To about 40 mg add 2 ml of sulphuric acid R and heat gently until white fumes are evolved. Add nitric acid R dropwise, continue the heating until the solution is almost colourless and cool. Add 2 ml of water R, heat until white fumes are again evolved, cool, add 10 ml of water R and neutralise to red litmus paper R with dilute ammonia R1. The solution gives reaction (a) of sodium (2.3.1) and reaction (b) of phosphates (2.3.1).

Inject 20 µl of the test solution and 20 µl of reference solution (b). Continue the chromatography for twice the retention time of the principal peak. In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (2 per cent) and not more than one such peak has an area greater than half the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (3 per cent). Disregard any peak with an area less than 0.025 times the area of the principal peak in the chromatogram obtained with reference solution (b).

Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with reference solution (b) is 70 per cent to 90 per cent of the full scale of E. Mix about 5 mg with 45 mg of heavy magnesium oxide R the recorder. and ignite in a crucible until an almost white residue is obtained (usually less than 5 min). Allow to cool, add Inject 20 µl of reference solution (a). When the 1 ml of water R, 0.05 ml of phenolphthalein solution R1 chromatograms are recorded in the conditions described and about 1 ml of dilute hydrochloric acid R to render above, the retention times are : betamethasone sodium the solution colourless. Filter. Add 1.0 ml of the filtrate phosphate about 14 min ; dexamethasone sodium phosphate to a freshly prepared mixture of 0.1 ml of alizarin S about 15.5 min. The test is not valid unless the resolution solution R and 0.1 ml of zirconyl nitrate solution R. between the peaks corresponding to betamethasone sodium Mix, allow to stand for 5 min and compare the colour of phosphate and dexamethasone sodium phosphate is at least the solution with that of a blank prepared in the same 2.0 ; if necessary, increase the concentration of acetonitrile manner. The test solution is yellow and the blank is red. or increase the concentration of water in the mobile phase.

TESTS Solution S. Dissolve 1.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution B7 (2.2.2, Method II).

Inorganic phosphate. Dissolve 50 mg in water R and dilute to 100 ml with the same solvent. To 10 ml of this solution add 5 ml of molybdovanadic reagent R, mix and allow to pH (2.2.3). Dilute 1 ml of solution S to 5 ml with carbon stand for 5 min. Any yellow colour in the solution is not dioxide-free water R. The pH of the solution is 7.5 to 9.0. more intense than that in a standard prepared at the same Specific optical rotation(2.2.7). Dissolve 0.250 g in water R time and in the same manner using 10 ml of phosphate and dilute to 25.0 ml with the same solvent. The specific standard solution (5 ppm PO4) R (1 per cent). optical rotation is + 98 to + 104, calculated with reference Water (2.5.12). Not more than 8.0 per cent, determined on to the anhydrous substance. 0.200 g by the semi-micro determination of water. Related substances. Examine by liquid chromatography (2.2.29). ASSAY Test solution. Dissolve 62.5 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the Dissolve 0.100 g in water R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of the solution to 250.0 ml with mobile phase. water R. Measure the absorbance (2.2.25) at the maximum at 241 nm. Reference solution (a). Dissolve 25 mg of betamethasone sodium phosphate CRS and 25 mg of dexamethasone Calculate the content of C22H28FNa2O8P taking the specific sodium phosphate CRS in the mobile phase and dilute to absorbance to be 297. 25.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 25.0 ml with the mobile phase. STORAGE Reference solution (b). Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. Store in an airtight container, protected from light. General Notices (1) apply to all monographs and other texts

1093

Betamethasone valerate


01/2005:0811

Apply to the plate 5 µl of each solution. Prepare the mobile phase by adding a mixture of 1.2 volumes of water R and 8 volumes of methanol R to a mixture of BETAMETHASONE VALERATE 15 volumes of ether R and 77 volumes of methylene chloride R. Develop over a path of 15 cm. Allow the plate Betamethasoni valeras to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). Spray the plate with alcoholic solution of sulphuric acid R. Heat at 120 °C for 10 min or until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the C27H37FO6 Mr 476.6 chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with DEFINITION reference solution (b) shows two clearly separated spots. Betamethasone valerate contains not less than 97.0 per E. Examine by thin-layer chromatography (2.2.27), using cent and not more than the equivalent of 103.0 per cent of as the coating substance a suitable silica gel with a 9-fluoro-11β,21-dihydroxy-16β-methyl-3,20-dioxopregna-1,4fluorescent indicator having an optimal intensity at dien-17-yl pentanoate, calculated with reference to the dried 254 nm. substance. Test solution (a). Dissolve 25 mg of the substance to be CHARACTERS examined in methanol R with gentle heating and dilute A white or almost white, crystalline powder, practically to 5 ml with the same solvent. This solution is also used insoluble in water, freely soluble in acetone and in methylene to prepare test solution (b). Dilute 2 ml of the solution to chloride, soluble in alcohol. 10 ml with methylene chloride R. It melts at about 192 °C, with decomposition. Test solution (b). Transfer 2 ml of the solution obtained during preparation of test solution (a) to a 15 ml glass tube IDENTIFICATION with a ground-glass stopper or a polytetrafluoroethylene First identification : C, D. cap. Add 10 ml of saturated methanolic potassium hydrogen carbonate solution R and immediately pass a Second identification : A, B, E, F, G. current of nitrogen R briskly through the solution for A. It complies with the test for specific optical rotation (see 5 min. Stopper the tube. Heat in a water-bath at 45 °C, Tests). protected from light, for 3 h. Allow to cool. B. Dissolve 10.0 mg in ethanol R and dilute to 100.0 ml Reference solution (a). Dissolve 25 mg of betamethasone with the same solvent. Place 2.0 ml of this solution 17-valerate CRS in methanol R with gentle heating and in a ground-glass-stoppered tube, add 10.0 ml of dilute to 5 ml with the same solvent. This solution is also phenylhydrazine-sulphuric acid solution R, mix and heat used to prepare reference solution (b). Dilute 2 ml of the in a water-bath at 60 °C for 20 min. Cool immediately. solution to 10 ml with methylene chloride R. The absorbance (2.2.25) of the solution measured at Reference solution (b). Transfer 2 ml of the solution 419 nm is not more than 0.10. obtained during preparation of reference solution (a) C. Examine by infrared absorption spectrophotometry to a 15 ml glass tube with a ground glass-stopper or a (2.2.24), comparing with the spectrum obtained with polytetrafluoroethylene cap. Add 10 ml of saturated betamethasone 17-valerate CRS. If the spectra obtained methanolic potassium hydrogen carbonate solution R in the solid state show differences, dissolve the substance and immediately pass a current of nitrogen R briskly to be examined and the reference substance separately through the solution for 5 min. Stopper the tube. Heat in the minimum volume of chloroform R, evaporate to in a water-bath at 45 °C, protected from light, for 3 h. dryness on a water-bath and record new spectra using Allow to cool. the residues. Apply to the plate 5 µl of each solution. Prepare the D. Examine by thin-layer chromatography (2.2.27), using mobile phase by adding a mixture of 1.2 volumes of as the coating substance a suitable silica gel with a water R and 8 volumes of methanol R to a mixture of fluorescent indicator having an optimal intensity at 15 volumes of ether R and 77 volumes of methylene 254 nm. chloride R. Develop over a path of 15 cm. Allow the Test solution. Dissolve 10 mg of the substance to be plate to dry in air and examine under ultraviolet light at examined in a mixture of 1 volume of methanol R and 254 nm. The principal spot in each of the chromatograms 9 volumes of methylene chloride R and dilute to 10 ml obtained with the test solutions is similar in position and with the same mixture of solvents. size to the principal spot in the chromatogram obtained Reference solution (a). Dissolve 10 mg of betamethasone with the corresponding reference solution. Spray with 17-valerate CRS in a mixture of 1 volume of methanol R alcoholic solution of sulphuric acid R. Heat at 120 °C and 9 volumes of methylene chloride R and dilute to for 10 min or until the spots appear. Allow to cool. 10 ml with the same mixture of solvents. Examine in daylight and in ultraviolet light at 365 nm. The principal spot in each of the chromatograms obtained Reference solution (b). Dissolve 10 mg of betamethasone with the test solutions is similar in position, colour in 21-valerate CRS in a mixture of 1 volume of methanol R daylight, fluorescence in ultraviolet light at 365 nm and and 9 volumes of methylene chloride R and dilute to size to the principal spot in the chromatograms obtained 10 ml with the same mixture of solvents. Dilute 5 ml of with the corresponding reference solution. The principal this solution to 10 ml with reference solution (a). 1094


Betaxolol hydrochloride


spot in each of the chromatograms obtained with test solution (b) and reference solution (b) has an Rf value distinctly lower than that of the principal spots in each of the chromatograms obtained with test solution (a) and reference solution (a). F. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a deep reddish-brown colour develops. Add the solution to 10 ml of water R and mix. The colour is discharged and a clear solution remains. G. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is obtained (usually less than 5 min). Allow to cool, add 1 ml of water R, 0.05 ml of phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the solution colourless. Filter. Add 1.0 ml of the filtrate to a freshly prepared mixture of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R. Mix, allow to stand for 5 min and compare the colour of the solution with that of a blank prepared in the same manner. The test solution is yellow and the blank is red.

one such peak has an area greater than half the area of the principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (3.0 per cent). Disregard any peak with an area less than 0.025 times the area of the principal peak in the chromatogram obtained with reference solution (b). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. ASSAY Dissolve 50.0 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the solution to 50.0 ml with alcohol R. Measure the absorbance (2.2.25) at the maximum at 240 nm. Calculate the content of C27H37FO6 taking the specific absorbance to be 325. STORAGE Store protected from light.

TESTS Specific optical rotation (2.2.7). Dissolve 0.250 g in dioxan R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 75 to + 82, calculated with reference to the dried substance. Related substances. Examine by liquid chromatography (2.2.29). Solution A. To 1000 ml of the mobile phase add 1 ml of glacial acetic acid R and mix carefully. Test solution. Dissolve 62.5 mg of the substance to be examined in solution A and dilute to 25.0 ml with solution A. Reference solution (a). Dissolve 2 mg of betamethasone 17-valerate CRS and 2 mg of betamethasone 21-valerate CRS in solution A and dilute to 50.0 ml with solution A. Reference solution (b). Dilute 1.0 ml of the test solution to 50.0 ml with solution A. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows : mix 350 ml of water R with 600 ml of acetonitrile R and allow to equilibrate ; adjust the volume to 1000 ml with water R and mix again, — as detector a spectrophotometer set at 254 nm. Equilibrate the column with the mobile phase for about 45 min. Adjust the sensitivity so that the height of the principal peak in the chromatogram obtained with reference solution (b) is 70 per cent to 90 per cent of the full scale of the recorder. Inject 20 µl of reference solution (a). When the chromatograms are recorded in the prescribed conditions, the retention times are : betamethasone 17-valerate, about 7 min ; betamethasone 21-valerate, about 9 min. The test is not valid unless the resolution between the peaks corresponding to betamethasone 17-valerate and betamethasone 21-valerate is at least 5.0 ; if necessary, adjust the concentration of acetonitrile in the mobile phase. Inject 20 µl of the test solution and 20 µl of reference solution (b). Continue the chromatography for 2.5 times the retention time of the principal peak. In the chromatogram obtained with the test solution : the area of any peak apart from the principal peak is not greater than 0.75 times the area of the principal peak in the chromatogram obtained with reference solution (b) (1.5 per cent) and not more than

IDENTIFICATION First identification : B, D. Second identification : A, C, D. A. Melting point (2.2.14) : 113 °C to 117 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with betaxolol hydrochloride CRS. C. Examine by thin-layer chromatography (2.2.27), using as the coating substance octadecylsilyl silica gel for chromatography R with a fluorescent indicator having an optimal intensity at 254 nm. Test solution. Dissolve 10 mg of the substance to be examined in 1 ml of methanol R. Reference solution (a). Dissolve 20 mg of betaxolol hydrochloride CRS in 2 ml of methanol R. Reference solution (b). Dissolve 10 mg of oxprenolol hydrochloride CRS in 1 ml of reference solution (a).


1095

01/2005:1072

BETAXOLOL HYDROCHLORIDE Betaxololi hydrochloridum

C18H30ClNO3

Mr 343.9

DEFINITION Betaxolol hydrochloride contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent of (2RS)-1-[4-[2-(cyclopropylmethoxy)ethyl]phenoxy]-3-[(1methylethyl)amino]propan-2-ol hydrochloride, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, very soluble in water, freely soluble in alcohol, soluble in methylene chloride.

Bezafibrate


Apply separately to the plate 2 µl of each solution. Develop over a path of 10 cm using a mixture of 0.5 volumes of perchloric acid R, 50 volumes of methanol R and 50 volumes of water R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). Spray the plate with a 50 g/l solution of vanillin R in a mixture of 5 volumes of sulphuric acid R, 10 volumes of glacial acetic acid R and 85 volumes of methanol R. Heat the plate at 100 °C to 105 °C until the colour of the spots reaches maximum intensity (10 min to 15 min). Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. D. It gives reaction (a) of chlorides (2.3.1). TESTS Appearance of solution. Dissolve 0.5 g in water R and dilute to 25 ml with the same solvent. The solution is clear (2.2.1) and colourless (2.2.2, Method II). Acidity or alkalinity. Dissolve 0.20 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Add 0.2 ml of methyl red solution R and 0.2 ml of 0.01 M hydrochloric acid. The solution is red. Add 0.4 ml of 0.01 M sodium hydroxide. The solution is yellow. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 10.0 mg of the substance to be examined in the mobile phase and dilute to 5.0 ml with the mobile phase. Reference solution (a). Dissolve 8 mg of the substance to be examined and 4 mg of betaxolol impurity A CRS in 20.0 ml of the mobile phase. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4 mm in internal diameter packed with octylsilyl silica gel for chromatography R (5 µm), — as the mobile phase at a flow rate of 1.5 ml/min a mixture prepared as follows : mix 175 ml of acetonitrile R with 175 ml of methanol R and dilute the mixture to 1 litre with a 3.4 g/l solution of potassium dihydrogen phosphate R, previously adjusted to pH 3.0 with phosphoric acid R, — as the detector a spectrophotometer set at 273 nm, — a loop injector. Inject 20 µl of each solution. Continue the chromatography for at least four times the retention time of the principal peak in the chromatogram obtained with the test solution. In the chromatogram obtained with the test solution : the area of any peak apart from the principal peak is not greater than 0.3 times the area of the peak in the chromatogram obtained with reference solution (b) (0.3 per cent) and the sum of the areas of such peaks is not greater than the area of the peak in the chromatogram obtained with reference solution (b) (1 per cent). The test is not valid unless the resolution between the peaks due to betaxolol impurity A and betaxolol in the chromatogram obtained with reference solution (a) is at least 2.0. Disregard any peak with an area less than 0.025 times that of the peak in the chromatogram obtained with reference solution (b). 1096

Heavy metals (2.4.8). Dissolve 2.0 g in 20 ml of water R. 12 ml of the solution complies with limit test A for heavy metals (10 ppm). Prepare the standard using 10 ml of lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.300 g in a mixture of 10.0 ml of 0.01 M hydrochloric acid and 50 ml of alcohol R. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the two points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 34.39 mg of C18H30ClNO3. STORAGE Store protected from light. IMPURITIES

A. R = H : (2RS)-1-(4-ethylphenoxy)-3-[(1-methylethyl)amino]propan-2-ol, B. R = OH : (2RS)-1-[4-(2-hydroxyethyl)phenoxy]-3-[(1methylethyl)amino]propan-2-ol, E. R = O-CH2-CH2-CH2-CH3 : (2RS)-1-[4-(2butoxyethyl)phenoxy]-3-[(1-methylethyl)amino]propan-2ol,

C. 2-[[4-[2-(cyclopropylmethoxy)ethyl]phenoxy]methyl]oxirane,

D. 4-[2-(cyclopropylmethoxy)ethyl]phenol. 01/2005:1394

BEZAFIBRATE Bezafibratum

C19H20ClNO4

Mr 361.8


Bezafibrate


— as mobile phase at a flow rate of 1 ml/min a mixture of 40 volumes of a 2.72 g/l solution of potassium dihydrogen phosphate R adjusted to pH 2.3 with phosphoric acid R and 60 volumes of methanol R, — as detector a spectrophotometer set at 228 nm. Inject separately 20 µl of the test solution and 20 µl of CHARACTERS reference solutions (a), (b) and (c). When the chromatogram A white or almost white crystalline powder, practically is recorded in the prescribed conditions, the retention times insoluble in water, freely soluble in dimethylformamide, are : impurity A about 3 min, impurity B about 3.5 min, sparingly soluble in acetone and in alcohol. It dissolves in bezafibrate about 6.0 min, impurity C about 9 min, dilute solutions of alkali hydroxides. impurity D about 14 min and impurity E about 37 min. It shows polymorphism. Continue the chromatography for the time necessary to detect the ester, which, depending on the route of synthesis, IDENTIFICATION may be impurity C, D or E. The test is not valid unless : in First identification : A, B. the chromatogram obtained with reference solution (c) the resolution between the two principal peaks is at least 5.0 Second identification : A, C. and the principal peak in the chromatogram obtained with A. Melting point (2.2.14) : 181 °C to 185 °C. reference solution (b) has a signal-to-noise ratio of at least 5. B. Examine by infrared absorption spectrophotometry In the chromatogram obtained with the test solution : the (2.2.24), comparing with the spectrum obtained with area of any peak, apart from the principal peak, is not greater bezafibrate CRS. Examine the substances prepared as than the area of the principal peak in the chromatogram discs. If the spectra obtained show differences, dissolve obtained with reference solution (a) (0.5 per cent) ; the sum the substance to be examined and the reference substance of the areas of all the peaks, apart from the principal peak, separately in methanol R and evaporate to dryness. Dry is not greater than 1.5 times the area of the principal peak the residues in vacuo at 80 °C for 1 h and record new in the chromatogram obtained with reference solution (a) spectra using the residues. (0.75 per cent). Disregard any peak with an area less than C. Examine by thin-layer chromatography (2.2.27), using a 0.1 times the area of the principal peak in the chromatogram TLC silica gel F254 plate R. obtained with reference solution (a). Test solution. Dissolve 10 mg of the substance to be Chlorides (2.4.4). Dilute 10 ml of solution S to 50 ml with examined in methanol R and dilute to 5 ml with the same water R. Filter the resultant suspension through a wet filter solvent. previously washed with water R until free from chlorides. 15 ml of the filtrate complies with the limit test for chlorides Reference solution. Dissolve 10 mg of bezafibrate CRS (300 ppm). Prepare the standard using 9 ml of chloride in methanol R and dilute to 5 ml with the same solvent. standard solution (5 ppm Cl) R and 6 ml of water R. Apply to the plate 5 µl of each solution. Develop over a path of 10 cm using a mixture of 2.7 volumes of glacial Heavy metals (2.4.8). 2.0 g complies with limit test C for acetic acid R, 30 volumes of methyl ethyl ketone R and heavy metals (10 ppm). Prepare the standard using 2 ml of 60 volumes of xylene R. Dry the plate at 120 °C for at lead standard solution (10 ppm Pb) R. least 15 min and examine in ultraviolet light at 254 nm. Loss on drying (2.2.32). Not more than 0.5 per cent, The principal spot in the chromatogram obtained with determined on 1.000 g by drying in an oven at 100-105 °C. the test solution is similar in position and size to the Sulphated ash (2.4.14). Not more than 0.1 per cent, principal spot in the chromatogram obtained with the determined on 1.0 g. reference solution. DEFINITION Bezafibrate contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of 2-[4-[2-[(4chlorobenzoyl)amino]ethyl]phenoxy]-2-methylpropanoic acid, calculated with reference to the dried substance.

TESTS Solution S. Dissolve 1.0 g in dimethylformamide R and dilute to 20 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution BY5 (2.2.2, Method II). Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (a). Dilute 10.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (b). Dilute 5.0 ml of reference solution (a) to 50.0 ml with the mobile phase. Reference solution (c). To 1 ml of the test solution, add 1 ml of 0.1 M hydrochloric acid and evaporate to dryness on a hot plate. Dissolve the residue in 20 ml of the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.125 m long and 4 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), General Notices (1) apply to all monographs and other texts

ASSAY Dissolve 0.300 g in 50 ml of a mixture of 25 volumes of water R and 75 volumes of alcohol R. Using 0.1 ml of phenolphthalein solution R as indicator, titrate with 0.1 M sodium hydroxide until a pink colour is obtained. Carry out a blank titration. 1 ml of 0.1 M sodium hydroxide is equivalent to 36.18 mg of C19H20ClNO4. IMPURITIES

A. 4-chloro-N-[2-(4-hydroxyphenyl)ethyl]benzamide (chlorobenzoyltyramine),

B. 4-chlorobenzoic acid, 1097

Bifonazole


Reference solution (b). Dissolve 25.0 mg of imidazole R (impurity C) in acetonitrile R and dilute to 25.0 ml with the same solvent. Dilute 0.25 ml of the solution to 100.0 ml with buffer solution pH 3.2. Reference solution (c). Dissolve 34.2 mg of 4-[(RS)-(biphenyl-4-yl)phenylmethyl]-1H-imidazole trifluoroacetate CRS (corresponding to 25.0 mg of impurity B base) in acetonitrile R and dilute to 25.0 ml with C. R = CH3 : methyl 2-[4-[2-[(4-chlorobenzoyl)amino]eththe same solvent. yl]phenoxy]-2 methylpropanoate, Reference solution (d). Dilute 0.25 ml of reference D. R = CH2-CH3 : ethyl 2-[4-[2-[(4-chlorobenzoyl)amino]ethsolution (c) to 50.0 ml with buffer solution pH 3.2. yl]phenoxy]-2-methylpropanoate, Reference solution (e). Mix 0.25 ml of the test solution and E. R = CH2-CH2-CH2-CH3 : butyl 2-[4-[2-[(40.25 ml of reference solution (c) and dilute to 50.0 ml with chlorobenzoyl)amino]ethyl]phenoxy]-2buffer solution pH 3.2. methylpropanoate. The chromatographic procedure may be carried out using : — a stainless steel column 0.125 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for 01/2005:1395 chromatography R (5 µm), — as mobile phase at a flow rate of 1 ml/min a gradient BIFONAZOLE programme using the following conditions : Mobile phase A. A mixture of 20 volumes of acetonitrile R Bifonazolum and 80 volumes of buffer solution pH 3.2, Mobile phase B. A mixture of 20 volumes of buffer solution pH 3.2 and 80 volumes of acetonitrile R,

C22H18N2

Mr 310.4

DEFINITION Bifonazole contains not less than 98.0 per cent and not more than the equivalent of 100.5 per cent of 1-[(RS)-(biphenyl-4-yl)phenylmethyl]-1H-imidazole, calculated with reference to the dried substance.

Time (min)



Comment

0-8

60

40

isocratic elution

8 - 12

60 → 10

40 → 90

linear gradient

12 - 30

10

90

isocratic elution

30 - 32

10 → 60

90 → 40


32 - 40

60

40

equilibration

40 = 0

60

40

restart isocratic elution

— as detector a spectrophotometer set at 210 nm, maintaining the column temperature at 40 °C. Adjust the sensitivity of the system so that the height of the bifonazole peak in the chromatogram obtained with 50 µl of reference solution (e) is at least 50 per cent of the full scale of the recorder. IDENTIFICATION Inject 50 µl of reference solution (e). When the chromatogram Examine by infrared absorption spectrophotometry is recorded in the prescribed conditions the retention times (2.2.24), comparing with the spectrum obtained with are : impurity B about 4 min and bifonazole about 4.5 min. bifonazole CRS. If the spectra obtained in the solid state The test is not valid unless the resolution between the peaks show differences, dissolve the substance to be examined corresponding to impurity B and bifonazole is at least 2.5. and the reference substance separately in the minimum volume of 2-propanol R, evaporate to dryness and record Inject 50 µl of the test solution and 50 µl each of reference new spectra using the residues. solutions (a), (b) and (d). In the chromatogram obtained with the test solution : the area of any peak corresponding to TESTS impurity C is not greater than the corresponding peak in the Optical rotation (2.2.7). Dissolve 0.20 g in 20.0 ml of chromatogram obtained with reference solution (b) (0.25 per methanol R. The angle of optical rotation is − 0.10° to cent) ; the area of any peak corresponding to impurity B is + 0.10°. not greater than 3 times the area of the corresponding peak in the chromatogram obtained with reference solution (d) Related substances. Examine by liquid chromatography (1.5 per cent) ; none of the peaks, apart from the principal (2.2.29). peak and the peaks corresponding to impurities B and C, Buffer solution pH 3.2. Mix 2.0 ml of phosphoric acid R has an area greater than the area of the peak in the with water R and dilute to 1000.0 ml with the same solvent. chromatogram obtained with reference solution (a) (0.5 per Adjust to pH 3.2 (2.2.3) with triethylamine R. cent) ; the sum of the areas of all the peaks, apart from the Test solution. Dissolve 50.0 mg of the substance to be principal peak, is not greater than 4 times the area of the examined in 25 ml of acetonitrile R and dilute to 50.0 ml principal peak in the chromatogram obtained with reference with buffer solution pH 3.2. solution (a) (2 per cent). Disregard any peak whose area Reference solution (a). Dilute 0.25 ml of the test solution to is less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (a). 50.0 ml with buffer solution pH 3.2. CHARACTERS A white or almost white, crystalline powder, practically insoluble in water, sparingly soluble in ethanol. It shows polymorphism.

1098


Bilberry fruit, fresh


Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

epicarp consisting of polygonal cells with moderately thickened walls ; brownish-yellow fragments of the outer seed testa made up of elongated cells with U-shaped thickened walls ; clusters and prisms crystals of various size of calcium oxalate. ASSAY C. Thin-layer chromatography (2.2.27) Dissolve 0.250 g in 80 ml of anhydrous acetic acid R. Titrate Test solution. To 2 g of the powdered drug (355) add with 0.1 M perchloric acid, determining the end-point 20 ml of methanol R. Shake for 15 min and filter. potentiometrically (2.2.20). Reference solution. Dissolve 5 mg of chrysanthemin R 1 ml of 0.1 M perchloric acid is equivalent to 31.04 mg of in 10 ml of methanol R. C22H18N2. Plate : TLC silica gel plate R. Mobile phase : anhydrous formic acid R, water R, IMPURITIES butanol R (16:19:65 V/V/V). Application : 10 µl, as bands. Development : over a path of 10 cm. Drying : in air. Detection : examine in daylight. A. R-OH : (RS)-(biphenyl-4-yl)phenylmethanol, Results : see below the sequence of the zones present in the chromatograms obtained with the reference and test solutions. Top of the plate A violet-red zone of low intensity

B. 4-[(RS)-(biphenyl-4-yl)phenylmethyl]-1H-imidazole,

Chrysanthemin : a violet-red zone

A principal violet-red zone A compact set of other principal zones : — a violet-red zone — several violet-blue zones

C. 1H-imidazole, Reference solution

Test solution

TESTS Foreign matter (2.8.2) : it complies with the test for foreign matter. Loss on drying (2.2.32) : maximum 12.0 per cent, determined D. 1,3-bis[(biphenyl-4-yl)phenylmethyl]-1H-imidazolium ion. on 1.000 g of the powdered drug by drying in an oven at 100-105 °C for 2 h. 01/2005:1588 Total ash (2.4.16) : maximum 5.0 per cent.

BILBERRY FRUIT, DRIED Myrtilli fructus siccus DEFINITION Dried ripe fruit of Vaccinium myrtillus L. Content : minimum 1.0 per cent of tannins, expressed as pyrogallol (C6H6O3 ; Mr 126.1) (dried drug). CHARACTERS Dried bilberry has a sweet and slightly astringent taste. Macroscopic and microscopic characters described under identification tests A and B. IDENTIFICATION A. Dried bilberry is a dark blue, subglobular, shrunken berry about 5 mm in diameter, with a scar at the lower end and surmounted by the persistent calyx, which appears as a circular fold and the remains of the style. The deep violet, fleshy mesocarp contains numerous small, brown, ovoid seeds. B. Reduce to a powder (355). The powder is violet-brown. Examine under a microscope using chloral hydrate solution R. The powder shows : violet-pink sclereids from the endocarp and the mesocarp, usually aggregated, with thick, channelled walls ; reddish-brown fragments of the General Notices (1) apply to all monographs and other texts

ASSAY Carry out the determination of tannins in herbal drugs (2.8.14). Use 1.500 g of the powdered drug (355). 01/2005:1602

BILBERRY FRUIT, FRESH Myrtilli fructus recens DEFINITION Fresh or frozen ripe fruit of Vaccinium myrtillus L. Content : minimum 0.30 per cent of anthocyanins, expressed as cyanidin-3-glucoside chloride (chrysanthemin, C21H21ClO11 ; Mr 485.5) (dried drug). CHARACTERS Sweet and slightly astringent taste. Macroscopic and microscopic characters described under identification tests A and B. IDENTIFICATION A. The fresh fruit is a blackish-blue globular berry about 5 mm in diameter. Its lower end shows a scar or, rarely, a fragment of the pedicel. The upper end is flattened and surmounted by the remains of the persistent style 1099

Biotin


and of the calyx, which appears as a circular fold. The violet, fleshy mesocarp includes 4 to 5 locules containing numerous small, brown, ovoid seeds. B. The crushed fresh fruit is violet-red. Examine under a microscope using chloral hydrate solution R. It shows violet-pink sclereids from the endocarp and the mesocarp, usually aggregated, with thick, channelled walls ; reddish-brown fragments of the epicarp consisting of polygonal cells with moderately thickened walls ; brownish-yellow fragments of the outer layer of the testa composed of elongated cells with U-shaped thickened walls ; cluster crystals of calcium oxalate. C. Thin-layer chromatography (2.2.27).

Calculate the percentage content of anthocyanins, expressed as cyanidin-3-glucoside chloride, from the expression :

718 = A m

specific absorbance of cyanidin-3-glucoside chloride at 528 nm,

=


=


STORAGE When frozen, store at or below − 18 °C.

Test solution. To 5 g of the freshly crushed drug, add 20 ml of methanol R. Stir for 15 min and filter.

01/2005:1073

Reference solution. Dissolve 5 mg of chrysanthemin R in 10 ml of methanol R.

BIOTIN

Plate : TLC silica gel plate R.

Biotinum

Mobile phase : anhydrous formic acid R, water R, butanol R (16:19:65 V/V/V). Application : 10 µl, as bands. Development : over a path of 10 cm. Drying : in air. Detection : examine in daylight. Results : see below the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. Top of the plate A violet-red zone Chrysanthemin : a violet-red zone

A principal violet-red zone A compact set of other principal zones : — a violet-red zone

C10H16N2O3S

Mr 244.3

DEFINITION Biotin contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of 5-[(3aS,4S,6aR)2-oxohexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid, calculated with reference to the dried substance. CHARACTERS A white, crystalline powder or colourless crystals, very slightly soluble in water and in alcohol, practically insoluble in acetone. It dissolves in dilute solutions of alkali hydroxides.

— several violet-blue zones

IDENTIFICATION First identification : A. Second identification : B, C. A. Examine by infrared absorption spectrophotometry TESTS (2.2.24), comparing with the spectrum obtained with Total ash (2.4.16) : maximum 0.6 per cent. biotin CRS. Foreign matter (2.8.2) : it complies with the test for foreign B. Examine the chromatograms obtained in the test for matter. related substances (see Tests). The principal spot in the chromatogram obtained with test solution (b) is Loss on drying (2.2.32) : 80.0 per cent to 90.0 per cent, similar in position and size to the principal spot in the determined on 5.000 g of the freshly crushed drug by drying chromatogram obtained with reference solution (a). in an oven at 100-105 °C. C. Dissolve about 10 mg in 20 ml of water R with heating. Allow to cool. Add 0.1 ml of bromine water R. The ASSAY bromine water is decolourised. Crush 50 g extemporaneously. To about 5.00 g of the TESTS crushed, accurately weighed drug, add 95 ml of methanol R. Solution S. Dissolve 0.250 g in a 4 g/l solution of sodium Stir mechanically for 30 min. Filter into a 100.0 ml hydroxide R and dilute to 25.0 ml with the same alkaline volumetric flask. Rinse the filter and dilute to 100.0 ml with methanol R. Prepare a 50-fold dilution of this solution solution. in a 0.1 per cent V/V solution of hydrochloric acid R in Appearance of solution. Solution S is clear (2.2.1) and methanol R. colourless (2.2.2, Method II). Measure the absorbance (2.2.25) of the solution at 528 nm, Specific optical rotation (2.2.7). The specific optical rotation using a 0.1 per cent V/V solution of hydrochloric acid R in is + 89 to + 93, determined on solution S and calculated with methanol R as the compensation liquid. reference to the dried substance. Reference solution

1100

Test solution


Biperiden hydrochloride


Related substances. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel (5 µm). Prepare the solutions immediately before use and keep protected from bright light. Test solution (a). Dissolve 50 mg of the substance to be examined in glacial acetic acid R and dilute to 10 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with glacial acetic acid R. Reference solution (a). Dissolve 5 mg of biotin CRS in glacial acetic acid R and dilute to 10 ml with the same solvent. Reference solution (b). Dilute 1 ml of test solution (b) to 20 ml with glacial acetic acid R. Reference solution (c). Dilute 1 ml of test solution (b) to 40 ml with glacial acetic acid R. Apply to the plate 10 µl of each solution. Develop over a path of 15 cm using a mixture of 5 volumes of methanol R, 25 volumes of glacial acetic acid R and 75 volumes of toluene R. Dry the plate in a current of warm air. Allow to cool and spray with 4-dimethylaminocinnamaldehyde solution R. Examine immediately in daylight. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent) and at most one such spot is more intense than the spot in the chromatogram obtained with reference solution (c) (0.25 per cent). Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (10 ppm). Prepare the standard using 10 ml of lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

C. 5-(3,4-diamino-2-thienyl)pentanoic acid,

D. 2-methyl-5-[(3aS,4S,6aR)-2-oxohexahydrothieno[3,4d]imidazol-4-yl]pentanoic acid,

E. 5-[(3aS,4S,6aR)-3-benzyl-2-oxohexahydrothieno[3,4d]imidazol-4-yl]pentanoic acid and 5-[(3aS,4S,6aR)1-benzyl-2-oxohexahydrothieno[3,4-d]imidazol-4yl]pentanoic acid. 01/2005:1074

BIPERIDEN HYDROCHLORIDE Biperideni hydrochloridum

ASSAY Suspend 0.200 g in 5 ml of dimethylformamide R. Heat until the substance has dissolved completely. Add 50 ml of ethanol R and titrate with 0.1 M tetrabutylammonium hydroxide, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to 24.43 mg of C10H16N2O3S. STORAGE Store protected from light. IMPURITIES

A. di[3-[(3aS,4S,6aR)-2-oxohexahydrothieno[3,4-d]imidazol4-yl]propyl]acetic acid,

B. 4-[(3aS,4S,6aR)-2-oxohexahydrothieno[3,4-d]imidazol-4yl]butane-1,1-dicarboxylic acid, General Notices (1) apply to all monographs and other texts

C21H30ClNO

Mr 347.9

DEFINITION (1RS)-1-[(1RS,2SR,4RS)-Bicyclo[2.2.1]hept-5-en-2-yl]-1phenyl-3-(piperidin-1-yl)propan-1-ol hydrochloride. Content : 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white, crystalline powder. Solubility : slightly soluble in water and in alcohol, very slightly soluble in methylene chloride. mp : about 280 °C, with decomposition. IDENTIFICATION First identification : A, D. Second identification : B, C, D. A. Infrared absorption spectrophotometry (2.2.24). Comparison : biperiden hydrochloride CRS. B. Thin-layer chromatography (2.2.27). 1101

Biperiden hydrochloride


Carrier gas : nitrogen for chromatography R. Test solution. Dissolve 25 mg of the substance to be examined in methanol R and dilute to 5 ml with the same Flow rate : 0.4 ml/min. solvent. Split ratio : 1:250. Reference solution (a). Dissolve 25 mg of biperiden Temperature : hydrochloride CRS in methanol R and dilute to 5 ml with the same solvent. Reference solution (b). Dissolve 5 mg of biperiden impurity A CRS in reference solution (a) and dilute to 2 ml with the same solution. Plate : TLC silica gel F254 plate R. Mobile phase : diethylamine R, methanol R, toluene R (1:1:20 V/V/V).

Column

Time (min)

Temperature (°C)

0-5

200

5 - 40

200 → 270

Injection port

250

Detector

300

Application : 5 µl.

Detection : flame ionisation.

Development : over a path of 15 cm.

Injection : 2 µl.

Drying : in air.

Run time : twice the retention time of biperiden.

Detection A : examine in ultraviolet light at 254 nm.

Relative retention with reference to biperiden : impurities A, B and C = between 0.95 and 1.05.

Results A : the principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a).

System suitability :

— resolution : minimum 2.5 between the peak due to biperiden (1st peak) and the peak due to impurity A nd (2 peak) in the chromatogram obtained with reference Detection B : spray with dilute potassium iodobismuthate solution (b), solution R and then with sodium nitrite solution R and examine in daylight. — signal-to-noise ratio : minimum 6 for the principal peak in the chromatogram obtained with reference solution (a). Results B : the principal spot in the chromatogram obtained with the test solution is similar in position, Limits : colour and size to the principal spot in the chromatogram — impurities A, B, C : for each impurity, maximum 0.50 per obtained with reference solution (a). cent of the area of the principal peak, System suitability : reference solution (b) : — any other impurity : for each impurity, maximum 0.10 per — the chromatogram shows 2 clearly separated spots. cent of the area of the principal peak, C. To about 20 mg add 5 ml of phosphoric acid R. A green — total of impurities A, B and C : maximum 1.0 per cent of colour develops. the area of the principal peak, D. It gives reaction (a) of chlorides (2.3.1). — total of impurities other than A, B and C: maximum 0.50 per cent of the area of the principal peak, TESTS — disregard limit : 0.05 per cent of the area of the principal peak. Solution S. Dissolve 0.10 g in carbon dioxide-free water R, heating gently if necessary, and dilute to 50 ml with the Impurity F (2.4.24) : maximum 2 ppm. same solvent. Heavy metals (2.4.8) : maximum 20 ppm. Appearance of solution. Solution S is not more opalescent than reference suspension II (2.2.1) and is colourless (2.2.2, 1.0 g complies with limit test D. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Method II). Loss on drying (2.2.32) : maximum 0.5 per cent, determined pH (2.2.3) : 5.0 to 6.5 for solution S. on 1.000 g by drying in an oven at 100-105 °C for 2 h. Related substances. Gas chromatography (2.2.28). Sulphated ash (2.4.14) : maximum 0.1 per cent, determined Test solution. Dissolve 0.10 g of the substance to be on 1.0 g. examined in methanol R and dilute to 10 ml with the same solvent. ASSAY Reference solution (a). Dilute 0.5 ml of the test solution to Dissolve 0.200 g in 60 ml of alcohol R. In a closed 100 ml with methanol R. Dilute 10 ml of this solution to vessel, titrate with 0.1 M alcoholic potassium hydroxide, 50 ml with methanol R. determining the end-point potentiometrically (2.2.20). Reference solution (b). Dissolve 5 mg of the substance 1 ml of 0.1 M alcoholic potassium hydroxide is equivalent to be examined and 5 mg of biperiden impurity A CRS in to 34.79 mg of C21H30ClNO. methanol R and dilute to 5 ml with the same solvent. Dilute 1 ml of the solution to 10 ml with methanol R. STORAGE

Column :

In an airtight container, protected from light.

— material : fused silica, — size : l = 50 m, Ø = 0.25 mm,

IMPURITIES

— stationary phase : poly(dimethyl)(diphenyl)(divinyl)siloxane R (film thickness 0.25 µm).

Other detectable impurities : D, E.

1102

Specified impurities : A, B, C, F.



Birch leaf

CHARACTERS It has the macroscopic and microscopic characters described under identification tests A and B. IDENTIFICATION A. The leaves of both species are dark green on the adaxial surface and a lighter greenish-grey colour on the abaxial surface ; they show a characteristic dense reticulate venation. The veins are light brown to almost white. A. (1RS)-1-[(1SR,2SR,4SR)-bicyclo[2.2.1]hept-5-en-2-yl]-1The leaves of Betula pendula are glabrous and show phenyl-3-(piperidin-1-yl)propan-1-ol (endo form), closely spaced glandular pits on both surfaces. The leaves of Betula pendula are 3 cm to 7 cm long and 2 cm to 5 cm wide ; the petiole is long and the doubly dentate lamina is triangular to rhomboid and broadly cuneate or truncate at the base. The angle on each side is unrounded or slightly rounded, and the apex is long and acuminate. The leaves of Betula pubescens show few glandular trichomes and are slightly pubescent on both surfaces. The abaxial surface shows small bundles of yellowish-grey trichomes at the branch points of the veins. The leaves of B. (1RS)-1-[(1SR,2RS,4SR)-bicyclo[2.2.1]hept-5-en-2-yl]-1Betula pubescens are slightly smaller, oval to rhomboid phenyl-3-(piperidin-1-yl)propan-1-ol, and more rounded. They are more roughly and more regularly dentate. The apex is neither long nor acuminate. B. Reduce to a powder (355). The powder is greenish-grey. Examine under a microscope using chloral hydrate solution R. The powder shows numerous fragments of lamina with straight-walled epidermal cells and cells of the lower epidermis surrounding anomocytic stomata (2.8.3). Peltate large glands usually measuring 100 µm to 120 µm are found on the upper and lower epidermises. The mesophyll fragments contain calcium oxalate crystals. Fragments of radial vascular bundles and sclerenchyma C. (1RS)-1-[(1RS,2RS,4RS)-bicyclo[2.2.1]hept-5-en-2-yl]-1fibres are accompanied by crystal sheaths. If Betula phenyl-3-(piperidin-1-yl)propan-1-ol, pubescens is present, the powder also contains unicellular covering trichomes with very thick walls, about 80 µm to 600 µm long, usually between 100 µm and 200 µm. C. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel. Test solution. To 1 g of the powdered drug (355) add 10 ml of methanol R. Heat in a water-bath at 60 °C for 5 min. Cool and filter the solution. D. 1-[(1RS,2SR,4RS)-bicyclo[2.2.1]hept-5-en-2-yl]-3Reference solution. Dissolve 1 mg of caffeic acid R and (piperidin-1-yl)propan-1-one, 1 mg of chlorogenic acid R, 2.5 mg of hyperoside R and 2.5 mg of rutin R in 10 ml of methanol R. Apply separately to the plates as bands, 10 µl of each solution. Develop over a path of 10 cm using a mixture of 10 volumes of anhydrous formic acid R, 10 volumes of water R, 30 volumes of methyl ethyl ketone R and 50 volumes of ethyl acetate R. Dry the plate in a current of warm air. Spray with a 10 g/l solution of diphenylboric E. 1-[(1RS,2RS,4RS)-bicyclo[2.2.1]hept-5-en-2-yl]-3acid aminoethyl ester R in methanol R. Subsequently (piperidin-1-yl)propan-1-one, spray the plate with a 50 g/l solution of macrogol 400 R in methanol R. Allow the plate to dry in air for F. benzene. 30 min and examine in ultraviolet light at 365 nm. The chromatogram obtained with the reference solution 01/2005:1174 shows three zones in its lower half : in increasing order of Rf a yellowish-brown fluorescent zone (rutin), a light blue fluorescent zone (chlorogenic acid) and a yellowish-brown BIRCH LEAF fluorescent zone (hyperoside), and in its upper third, a light blue fluorescent zone (caffeic acid). Betulae folium The chromatogram obtained with the test solution shows DEFINITION three zones similar in position and fluorescence to the zones due to rutin, chlorogenic acid and hyperoside in Birch leaf consists of the whole or fragmented dried leaves the chromatogram obtained with the reference solution. of Betula pendula Roth and/or Betula pubescens Ehrh. The zone corresponding to rutin is very faint and the as well as hybrids of both species. It contains not less zone corresponding to hyperoside is intense. It also than 1.5 per cent of flavonoids, calculated as hyperoside shows other yellowish-brown faint fluorescence zones (C21H20O12 ; Mr 464.4) with reference to the dried drug. General Notices (1) apply to all monographs and other texts

1103

Bisacodyl


01/2005:0595

between the zones corresponding to caffeic acid and chlorogenic acid in the chromatogram obtained with the reference solution. Near the solvent front, the red fluorescent zone due to chlorophylls is visible. In the chromatogram obtained with the test solution, between this zone and the zone corresponding to caffeic acid in the chromatogram obtained with the reference solution, there is a brownish-yellow zone corresponding to quercetin.

BISACODYL Bisacodylum

TESTS Foreign matter (2.8.2). Not more than 3 per cent of fragments of female catkins and not more than 3 per cent of other foreign matter. Loss on drying (2.2.32). Not more than 10.0 per cent, determined on 1.000 g of powered drug (355) by drying in an oven at 100 °C to 105 °C for 2 h. Total ash (2.4.16). Not more than 5.0 per cent. ASSAY Stock solution. In a 100 ml round-bottomed flask introduce 0.200 g of the powdered drug (355), 1 ml of a 5 g/l solution of hexamethylenetetramine R, 20 ml of acetone R and 2 ml of hydrochloric acid R1. Boil the mixture under a reflux condenser for 30 min. Filter the liquid through a plug of absorbent cotton in a 100 ml flask. Add the absorbent cotton to the residue in the round-bottomed flask and extract with two quantities, each of 20 ml, of acetone R, each time boiling under a reflux condenser for 10 min. Allow to cool to room temperature, filter the liquid through a plug of absorbent cotton then filter the solution through a filter-paper in the volumetric flask, and dilute to 100.0 ml with acetone R by rinsing of the flask and filter. Introduce 20.0 ml of the solution into a separating funnel, add 20 ml of water R and extract the mixture with one quantity of 15 ml and then three quantities, each of 10 ml, of ethyl acetate R. Combine the ethyl acetate extracts in a separating funnel, rinse with two quantities, each of 50 ml, of water R, and filter the extract over 10 g of anhydrous sodium sulphate R into a 50 ml volumetric flask and dilute to 50.0 ml with ethyl acetate R.

C22H19NO4

Mr 361.4

DEFINITION Bisacodyl contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of 4,4′-(pyridin-2-ylmethylene)diphenyl diacetate, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, practically insoluble in water, soluble in acetone, sparingly soluble in alcohol. It dissolves in dilute mineral acids.

IDENTIFICATION First identification : C. Second identification : A, B, D. A. Melting point (2.2.14) : 131 °C to 135 °C. B. Dissolve 10.0 mg in a 6 g/l solution of potassium hydroxide R in methanol R and dilute to 100.0 ml with the same alkaline solvent. Dilute 10.0 ml of this solution to 100.0 ml with a 6 g/l solution of potassium hydroxide R in methanol R. Examined between 220 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 248 nm and a shoulder at about 290 nm. The specific absorbance at the maximum is 632 to 672. C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with Test solution. To 10.0 ml of the stock solution add 1 ml bisacodyl CRS. If the spectra obtained with the substance of aluminium chloride reagent R and dilute to 25.0 ml to be examined and the reference substance show with a 5 per cent V/V solution of glacial acetic acid R in differences, dissolve separately the substance to be methanol R. examined and the reference substance in chloroform R, evaporate to dryness and record the spectra again. Compensation solution. Dilute 10.0 ml of the stock solution to 25.0 ml with a 5 per cent V/V solution of glacial acetic D. Spray the chromatograms obtained in the test for related acid R in methanol R. substances with a mixture of equal volumes of 0.05 M iodine and dilute sulphuric acid R. Examine in daylight. Measure the absorbance (2.2.25) of the test solution after The principal spot in the chromatogram obtained with 30 min, by comparison with the compensation solution at test solution (b) is similar in position and size to the 425 nm. principal spot in the chromatogram obtained with reference solution (a). Calculate the percentage content of flavonoids, calculated as hyperoside, from the expression : TESTS Acidity or alkalinity. To 1.0 g add 20 ml of carbon dioxide-free water R, shake, heat to boiling, cool and filter. Add 0.2 ml of 0.01 M sodium hydroxide and 0.1 ml of methyl i.e. taking the specific absorbance of hyperoside to be 500. red solution R. The solution is yellow. Not more than 0.4 ml of 0.01 M hydrochloric acid is required to change the colour A = absorbance at 425 nm, of the indicator to red. m = mass of the substance to be examined, in grams. Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution (a). Dissolve 0.20 g of the substance to be STORAGE examined in acetone R and dilute to 10 ml with the same solvent. Store protected from light. 1104



Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with acetone R. Reference solution (a). Dissolve 20 mg of bisacodyl CRS in acetone R and dilute to 10 ml with the same solvent. Reference solution (b). Dilute 1 ml of test solution (a) to 100 ml with acetone R. Reference solution (c). Dilute 5 ml of reference solution (b) to 10 ml with acetone R. Apply separately to the plate 10 µl of each solution. Develop over a path of 10 cm using a mixture of 50 volumes of xylene R and 50 volumes of methyl ethyl ketone R. Allow the plate to dry in air, if necessary heating at 100 °C to 105 °C, and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (1.0 per cent) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (c) (0.5 per cent). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 0.500 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

Bismuth subcarbonate

Nitrates. To 0.25 g in a 125 ml conical flask, add 20 ml of water R, 0.05 ml of indigo carmine solution R1 and then, as a single addition but with caution, 30 ml of sulphuric acid R. Titrate immediately with indigo carmine solution R1 until a stable blue colour is obtained. Not more than n ml of the titrant is required, n ml being the volume corresponding to 1 mg of NO3 (0.4 per cent). Alkali and alkaline-earth metals. To 1.0 g add 10 ml of water R and 10 ml of acetic acid R. Boil for 2 min, cool and filter. Wash the residue with 20 ml of water R. To the combined filtrate and washings add 2 ml of dilute hydrochloric acid R and 20 ml of water R. Boil and pass hydrogen sulphide R through the boiling solution until no further precipitate is formed. Filter, wash the residue with water R, evaporate the combined filtrate and washings to dryness on a water-bath and add 0.5 ml of sulphuric acid R. Ignite gently and allow to cool. The residue weighs not more than 10 mg (1.0 per cent).

Arsenic (2.4.2). To 0.5 g in a distillation flask add 5 ml of water R and 7 ml of sulphuric acid R, allow to cool and add 5 g of reducing mixture R and 10 ml of hydrochloric acid R. Heat the contents of the flask to boiling gradually over 15 min to 30 min and continue heating at such a rate that the distillation proceeds steadily until the volume in the flask is reduced by half or until 5 min after the air-condenser has become full of steam. It is important that distillation ASSAY be discontinued before fumes of sulphur trioxide appear. Dissolve 0.300 g in 60 ml of anhydrous acetic acid R. Collect the distillate in a tube containing 15 ml of water R Titrate with 0.1 M perchloric acid determining the end-point cooled in ice-water. Wash down the condenser with water R potentiometrically (2.2.20). and dilute the distillate to 25 ml with the same solvent. 1 ml of 0.1 M perchloric acid is equivalent to 36.14 mg of The solution complies with limit test A for arsenic (5 ppm). C22H19NO4. Prepare the standard using a mixture of 2.5 ml of arsenic standard solution (1 ppm As) R and 22.5 ml of water R. STORAGE Copper. To 5 ml of solution S, add 2 ml of ammonia R Store protected from light. and dilute to 50 ml with water R. Filter. To 10 ml of the filtrate add 1 ml of a 1 g/l solution of sodium 01/2005:0012 diethyldithiocarbamate R. The solution is not more intensely coloured than a standard prepared at the same time in the same manner using a mixture of 0.25 ml of copper standard BISMUTH SUBCARBONATE solution (10 ppm Cu) R and 9.75 ml of water R instead of 10 ml of the filtrate (50 ppm).

Bismuthi subcarbonas

DEFINITION Bismuth subcarbonate contains not less than 80.0 per cent and not more than 82.5 per cent of Bi (Ar 209.0), calculated with reference to the dried substance. CHARACTERS A white or almost white powder, practically insoluble in water and in alcohol. It dissolves with effervescence in mineral acids. IDENTIFICATION A. It gives the reaction of carbonates (2.3.1). B. It gives the reactions of bismuth (2.3.1). TESTS Solution S. Shake 5.0 g with 10 ml of water R and add 20 ml of nitric acid R. Heat to dissolve, cool and dilute to 100 ml with water R. Appearance of solution. Solution S is not more opalescent than reference suspension II (2.2.1) and is colourless (2.2.2, Method II). Chlorides (2.4.4). To 6.6 ml of solution S add 4 ml of nitric acid R and dilute to 50 ml with water R. 15 ml of the solution complies with the limit test for chlorides (500 ppm). General Notices (1) apply to all monographs and other texts

Lead. Not more than 20 ppm of Pb, determined by atomic absorption spectrometry (2.2.23, Method II). Test solution. Dissolve 12.5 g of the substance to be examined in 75 ml of a mixture of equal volumes of lead-free nitric acid R and water R. Boil for 1 min, cool and dilute to 100.0 ml with water R. Reference solutions. Prepare the reference solutions using appropriate quantities of lead standard solution and a 37 per cent V/V solution of lead-free nitric acid R. Measure the absorbance at 283.3 nm, using a hollow-cathode lamp as source of radiation and an air-acetylene flame. Depending on the apparatus, the line at 217.0 nm may be used. Silver. To 2.0 g add 1 ml of water R and 4 ml of nitric acid R. Heat gently until dissolved and dilute to 11 ml with water R. Cool and add 2 ml of 1 M hydrochloric acid. Allow to stand for 5 min, protected from light. Any opalescence in the solution is not more intense than that in a standard prepared at the same time in the same manner using a mixture of 10 ml of silver standard solution (5 ppm Ag) R, 1 ml of nitric acid R and 2 ml of 1 M hydrochloric acid (25 ppm). Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. 1105

Bismuth subgallate


ASSAY Dissolve 0.500 g in 3 ml of nitric acid R and dilute to 250 ml with water R. Carry out the complexometric titration of bismuth (2.5.11). 1 ml of 0.1 M sodium edetate is equivalent to 20.90 mg of Bi. STORAGE Store protected from light.

01/2005:1493

BISMUTH SUBGALLATE Bismuthi subgallas

C7H5BiO6

Mr 394.1

DEFINITION Complex of bismuth and gallic acid. Content : 48.0 per cent to 51.0 per cent of Bi (Ar 209.0) (dried substance). CHARACTERS Appearance : yellow powder. Solubility : practically insoluble in water and in alcohol. It dissolves in mineral acids with decomposition and in solutions of alkali hydroxides, producing a reddish-brown liquid. IDENTIFICATION A. Mix 0.1 g with 5 ml of water R and 0.1 ml of phosphoric acid R. Heat to boiling and maintain boiling for 2 min. Cool and filter. To the filtrate, add 1.5 ml of ferric chloride solution R1, a blackish-blue colour develops. B. It gives reaction (b) of bismuth (2.3.1). TESTS Solution S. In a porcelain or quartz dish, ignite 1.0 g, increasing the temperature very gradually. Heat in a muffle furnace at 600 ± 25 °C for 2 h. Cool and dissolve the residue with warming in 4 ml of a mixture of equal volumes of lead-free nitric acid R and water R and dilute to 20 ml with water R. Acidity. Shake 1.0 g with 20 ml of water R for 1 min and filter. To the filtrate add 0.1 ml of methyl red solution R. Not more than 0.15 ml of 0.1 M sodium hydroxide is required to change the colour of the indicator to yellow. Chlorides (2.4.4) : maximum 200 ppm. To 0.5 g add 10 ml of dilute nitric acid R. Heat on a water-bath for 5 min and filter. Dilute 5 ml of the filtrate to 15 ml with water R. Nitrates : maximum 0.2 per cent. To 1.0 g add 25 ml of water R then 25 ml of a mixture of 2 volumes of sulphuric acid R and 9 volumes of water R. Heat at about 50 °C for 1 min with stirring and filter. To 10 ml of the filtrate, carefully add 30 ml of sulphuric acid R. The solution is not more intensely brownish-yellow coloured than a reference solution prepared at the same 1106

time as follows : to 0.4 g of gallic acid R, add 20 ml of nitrate standard solution (100 ppm NO3) R and 30 ml of a mixture of 2 volumes of sulphuric acid R and 9 volumes of water R, then filter ; to 10 ml of the filtrate, carefully add 30 ml of sulphuric acid R. Copper : maximum 50 ppm. Atomic absorption spectrometry (2.2.23, Method I). Test solution. Solution S. Reference solutions. Prepare the reference solutions using copper standard solution (10 ppm Cu) R and diluting with a 6.5 per cent V/V solution of lead-free nitric acid R. Source : copper hollow-cathode lamp. Wavelength : 324.7 nm. Atomisation device : air-acetylene flame. Lead : maximum 20 ppm. Atomic absorption spectrometry (2.2.23, Method II). Test solution. Solution S. Reference solutions. Prepare the reference solutions using lead standard solution (10 ppm Pb) R and diluting with a 6.5 per cent V/V solution of lead-free nitric acid R. Source : lead hollow-cathode lamp. Wavelength : 283.3 nm (depending on the apparatus, the line at 217.0 nm may be used). Atomisation device : air-acetylene flame. Silver : maximum 25 ppm. Atomic absorption spectrometry (2.2.23, Method I). Test solution. Solution S. Reference solutions. Prepare the reference solutions using silver standard solution (5 ppm Ag) R and diluting with a 6.5 per cent V/V solution of lead-free nitric acid R. Source : silver hollow-cathode lamp. Wavelength : 328.1 nm. Atomisation device : air-acetylene flame. Substances not precipitated by ammonia: maximum 1.0 per cent. In a porcelain or quartz dish, ignite 2.0 g, increasing the temperature very gradually to 600 °C ; allow to cool. Moisten the residue with 2 ml of nitric acid R, evaporate to dryness on a water-bath and carefully heat and ignite once more at 600 °C. After cooling, dissolve the residue in 5 ml of nitric acid R and dilute to 20 ml with water R. To 10 ml of this solution, add concentrated ammonia R until alkaline and filter. Wash the residue with water R, evaporate the combined filtrate and washings to dryness on a water-bath and add 0.3 ml of dilute sulphuric acid R. Ignite, the residue weighs a maximum of 10 mg. Loss on drying (2.2.32) : maximum 7.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 3 h. ASSAY To 0.300 g add 10 ml of a mixture of equal volumes of nitric acid R and water R, heat to boiling and maintain boiling for 2 min. Add 0.1 g of potassium chlorate R, heat to boiling and maintain boiling for 1 min. Add 10 ml of water R and heat until the solution becomes colourless. To the hot solution, add 200 ml of water R and 50 mg of xylenol orange triturate R. Titrate with 0.1 M sodium edetate until a yellow colour is obtained. 1 ml of 0.1 M sodium edetate is equivalent to 20.90 mg of Bi. STORAGE Protected from light.


Bismuth subsalicylate


01/2005:1494 Measure the absorbance at 283.3 nm, using a lead hollow-cathode lamp as source of radiation and an air-acetylene flame. Depending on the apparatus, the line at BISMUTH SUBNITRATE, HEAVY 217.0 nm may be used. Silver. Not more than 25 ppm of Ag, determined by atomic Bismuthi subnitras ponderosum absorption spectrometry (2.2.23, Method I). Test solution. Solution S2. 4[BiNO3(OH)2],BiO(OH) Mr 1462 Reference solutions. Prepare the reference solutions using silver standard solution (5 ppm Ag) R and diluting with a DEFINITION 37 per cent V/V solution of lead-free nitric acid R. Heavy bismuth subnitrate contains not less than 71.0 per Measure the absorbance at 328.1 nm using a silver cent and not more than 74.0 per cent of Bi (Ar 209.0), hollow-cathode lamp as source of radiation and an calculated with reference to the dried substance. air-acetylene flame. CHARACTERS Substances not precipitated by ammonia. To 20 ml of A white powder, practically insoluble in water and in alcohol. solution S1, add concentrated ammonia R until alkaline and filter. Wash the residue with water R, and evaporate the It dissolves in mineral acids with decomposition. combined filtrate and washings to dryness on a water-bath. IDENTIFICATION Add 0.3 ml of dilute sulphuric acid R and ignite. The residue weighs not more than 10 mg (1.0 per cent). A. Dilute 1 ml of solution S1 (see Tests) to 5 ml with water R, add 0.3 ml of potassium iodide solution R. A Loss on drying (2.2.32). Not more than 3.0 per cent, black precipitate is formed which dissolves into an orange determined on 1.000 g by drying in an oven at 100-105 °C. solution with the addition of 2 ml of potassium iodide ASSAY solution R. Dissolve with heating 0.250 g in 10 ml of a mixture of B. It gives reaction (b) of bismuth (2.3.1). 2 volumes of perchloric acid R and 5 volumes of water R. To C. It gives the reaction of nitrates (2.3.1). the hot solution, add 200 ml of water R and 50 mg of xylenol D. The pH (2.2.3) of solution S2 (see Tests) is not more orange triturate R. Titrate with 0.1 M sodium edetate until than 2.0. a yellow colour is obtained. 1 ml of 0.1 M sodium edetate is equivalent to 20.90 mg of Bi. TESTS Solution S1. Shake 5.0 g by gently heating in 10 ml of water R, add 20 ml of nitric acid R. Heat until dissolution, cool and dilute to 100 ml with water R. Solution S2. Place 1.00 g in a 20 ml volumetric flask and add 2.0 ml of lead-free nitric acid R. Allow acid attack to take place without heating and if necessary warm slightly at the end to dissolve the test sample completely. Add 10 ml of water R, shake and add, in small fractions, 4.5 ml of lead-free ammonia R ; shake, allow to cool, dilute to 20.0 ml with water R, shake again and allow the solids to settle. The clear supernatant solution is solution S2. Acidity. Suspend 1.0 g in 15 ml of water R and shake several times. Allow to stand for 5 min and filter. To 10 ml of the filtrate, add 0.5 ml of phenolphthalein solution R1. Not more than 0.5 ml of 0.1 M sodium hydroxide is required to change the colour of the indicator to pink. Chlorides (2.4.4). To 5.0 ml of solution S1, add 3 ml of nitric acid R and dilute to 15 ml with water R. The solution complies with the limit test for chlorides (200 ppm). Copper. Not more than 50 ppm of Cu, determined by atomic absorption spectrometry (2.2.23, Method I). Test solution. Solution S2. Reference solutions. Prepare the reference solutions using copper standard solution (10 ppm Cu) R and diluting with a 37 per cent V/V solution of lead-free nitric acid R. Measure the absorbance at 324.7 nm using a copper hollow-cathode lamp as source of radiation and an air-acetylene flame. Lead. Not more than 20 ppm of Pb, determined by atomic absorption spectrometry (2.2.23, Method II). Test solution. Solution S2. Reference solutions. Prepare the reference solutions using appropriate quantities of lead standard solution (10 ppm Pb) R and diluting with a 37 per cent V/V solution of lead-free nitric acid R.

TESTS Solution S. In a porcelain or quartz dish, ignite 1.0 g, increasing the temperature very gradually. Heat in a muffle furnace at 600 ± 25 °C for 2 h. Cool and dissolve the residue with warming in 4 ml of a mixture of equal volumes of lead-free nitric acid R and water R and dilute to 20 ml with water R.


1107

01/2005:1495

BISMUTH SUBSALICYLATE Bismuthi subsalicylas C7H5BiO4

Mr 362.1

DEFINITION Complex of bismuth and salicylic acid. Content : 56.0 per cent to 59.4 per cent of Bi (Ar 209.0) (dried substance). CHARACTERS Appearance : white powder. Solubility : practically insoluble in water and in alcohol. It dissolves in mineral acids with decomposition. IDENTIFICATION A. To 0.5 g add 10 ml of hydrochloric acid R1. Heat on a boiling water-bath for 5 min. Cool and filter. Retain the filtrate for identification test B. Wash the residue with dilute hydrochloric acid R and then with water R. Dissolve the residue in 0.5-1 ml of dilute sodium hydroxide solution R. Add 15 ml of water R. Neutralise with dilute hydrochloric acid R. The solution gives reaction (a) of salicylates (2.3.1). B. The filtrate obtained in identification test A gives reaction (b) of bismuth (2.3.1).

Bitter-fennel fruit oil


Acidity. Shake 2.0 g with 30 ml of ether R for 1 min and filter. To the filtrate add 30 ml of alcohol R and 0.1 ml of thymol blue solution R. Not more than 0.35 ml of 0.1 M sodium hydroxide is required to change the colour of the indicator to blue. Chlorides (2.4.4) : maximum 200 ppm. Dissolve 0.250 g in a mixture of 2 ml of nitric acid R, 5 ml of water R and 8 ml of methanol R. Nitrates : maximum 0.4 per cent. To 0.1 g add 10 ml of water R and, with caution, 20 ml of sulphuric acid R and stir. The solution is not more intensely yellow coloured than a reference solution prepared at the same time using 0.1 g of salicylic acid R, 6 ml of water R, 4 ml of nitrate standard solution (100 ppm NO3) R and 20 ml of sulphuric acid R. Copper : maximum 50 ppm. Atomic absorption spectrometry (2.2.23, Method I). Test solution. Solution S. Reference solutions. Prepare the reference solutions using copper standard solution (10 ppm Cu) R and diluting with a 6.5 per cent V/V solution of lead-free nitric acid R. Source : copper hollow-cathode lamp. Wavelength : 324.7 nm. Atomisation device : air-acetylene flame. Lead : maximum 20 ppm. Atomic absorption spectrometry (2.2.23, Method II). Test solution. Solution S. Reference solutions. Prepare the reference solutions using lead standard solution (10 ppm Pb) R and diluting with a 6.5 per cent V/V solution of lead-free nitric acid R. Source : lead hollow-cathode lamp. Wavelength : 283.3 nm (depending on the apparatus, the line at 217.0 nm may be used). Atomisation device : air-acetylene flame. Silver: maximum 25 ppm. Atomic absorption spectrometry (2.2.23, Method I). Test solution. Solution S. Reference solutions. Prepare the reference solutions using silver standard solution (5 ppm Ag) R and diluting with a 6.5 per cent V/V solution of lead-free nitric acid R. Source : silver hollow-cathode lamp. Wavelength : 328.1 nm. Atomisation device : air-acetylene flame. Soluble bismuth : maximum 40 ppm. Atomic absorption spectrometry (2.2.23, Method I). Test solution. Suspend 5.0 g in 100 ml of water R. Stir constantly for 2 h at 20-23 °C. Filter through filter paper (slow filtration) then through a cellulose micropore membrane filter (0.1 µm). To 10.0 ml of clear filtrate, add 0.1 ml of nitric acid R. Reference solutions. Prepare the reference solutions using bismuth standard solution (100 ppm Bi) R and diluting with a mixture of equal volumes of dilute nitric acid R and water R. Source : bismuth hollow-cathode lamp. Wavelength : 223.06 nm. Atomisation device : air-acetylene flame. Loss on drying (2.2.32) : maximum 1.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. 1108

ASSAY Dissolve with heating 0.300 g in 10 ml of a mixture of 2 volumes of perchloric acid R and 5 volumes of water R. To the hot solution, add 200 ml of water R and 50 mg of xylenol orange triturate R. Titrate with 0.1 M sodium edetate until a yellow colour is obtained. 1 ml of 0.1 M sodium edetate is equivalent to 20.90 mg of Bi. STORAGE Protected from light. 01/2005:1826

BITTER-FENNEL FRUIT OIL Foeniculi amari fructus aetheroleum DEFINITION Essential oil obtained by steam distillation from the ripe fruits of Foeniculum vulgare Miller, ssp. vulgare var. vulgare. Content : — fenchone : 12.0 per cent to 25.0 per cent, — trans-anethole : 55.0 per cent to 75.0 per cent. CHARACTERS Appearance : clear, colourless or pale yellow liquid. It has a characteristic odour. IDENTIFICATION First identification : B. Second identification : A. A. Thin-layer chromatography (2.2.27). Test solution. Dissolve 0.1 ml of the oil to be examined in 5 ml of toluene R. Reference solution. Dissolve 10 µl of fenchone R and 80 µl of anethole R in 5 ml of toluene R. Plate : TLC silica gel plate R. Mobile phase : ethyl acetate R, toluene R (5:95 V/V). Application : 10 µl as bands. Development : over a path of 15 cm. Drying : in air. Detection : spray with a freshly prepared 200 g/l solution of phosphomolybdic acid R in ethanol (96 per cent) R and heat at 150 °C for 15 min ; examine in daylight. Results : see below the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. Furthermore, other zones may be present in the chromatogram obtained with the test solution. Top of the plate Anethole : a dark blue to dark violet zone

A dark blue to dark violet zone (anethole)

_______ Fenchone : a blue or bluish-grey zone

_______ A blue or bluish-grey zone (fenchone)

_______ Reference solution

_______ Test solution

B. Examine the chromatograms obtained in the test for chromatographic profile.


Bitter-fennel fruit oil


Results : the characteristic peaks in the chromatogram obtained with the test solution are similar in retention time to those in the chromatogram obtained with the reference solution.

Temperature :

Column

TESTS

Time (min)

Temperature (°C)

0–4

60

4 – 26

60 → 170

26 – 41

Relative density (2.2.5) : 0.961 to 0.975. Refractive index (2.2.6) : 1.528 to 1.539. Optical rotation (2.2.7) : + 10.0° to + 24.0°.

170

Injection port

220

Detector

270

Detection : flame ionisation. Injection : 1.0 µl. Elution order : order indicated in the composition of the Test solution. Dissolve 0.20 ml of the oil to be examined in reference solution. Record the retention times of these heptane R and dilute to 10.0 ml with the same solvent. substances. System suitability : reference solution : Reference solution. Dissolve 20 µl of α-pinene R, 20 µl of limonene R, 50 µl of fenchone R, 20 µl of estragole R, 100 µl — resolution : minimum 5.0 between the peaks due to of anethole R and 20 µl of anisaldehyde R in heptane R and estragole and trans-anethole. dilute to 10.0 ml with the same solvent. Using the retention times determined from the chromatogram obtained with the reference solution, locate the components Column : of the reference solution on the chromatogram obtained with — material : fused silica, the test solution and locate cis-anethole using Figure 1826.-1. (Disregard the peak due to heptane). — size : l = 60 m, Ø = 0.25 mm, Determine the percentage content of each of these — stationary phase: macrogol 20 000 R (film thickness components. The percentages are within the following 0.25 µm). ranges : — α-pinene : 1.0 per cent to 10.0 per cent, Carrier gas : helium for chromatography R. — limonene : 0.9 per cent to 5.0 per cent, Flow rate : 1 ml/min. — fenchone : 12.0 per cent to 25.0 per cent, Split ratio : 1:200. — estragole : maximum 6.0 per cent, Chromatographic profile. Gas chromatography (2.2.28) : use the normalisation procedure.

1. α-pinene

3. fenchone

5. cis-anethole

2. limonene

4. estragole

6. trans-anethole

7. anisaldehyde

Figure 1826.-1. – Chromatogram for the test for chromatographic profile of bitter-fennel fruit oil General Notices (1) apply to all monographs and other texts

1109

Bitter-orange epicarp and mesocarp


— cis-anethole : maximum 0.5 per cent, — trans-anethole : 55.0 per cent to 75.0 per cent, — anisaldehyde : maximum 2.0 per cent. The ratio of α-pinene content to limonene content is greater than 1.0.

Results : see below the sequence of the zones present in the chromatograms obtained with the reference and test solutions. Furthermore, other fluorescent zones are present in the chromatogram obtained with the test solution. Top of the plate

STORAGE In a well-filled, airtight container, protected from light and at a temperature not exceeding 25 °C. 01/2005:1603

BITTER-ORANGE EPICARP AND MESOCARP

A light blue fluorescent zone A light blue fluorescent zone Caffeic acid : a light blue fluorescent zone A light blue fluorescent zone A light blue fluorescent zone Naringin : a dark green fluorescent zone

A dark green fluorescent zone (naringin)

Aurantii amari epicarpium et mesocarpium

A red fluorescent zone (neoeriocitrin)

DEFINITION Dried epicarp and mesocarp of the ripe fruit of Citrus aurantium L. ssp. aurantium (C. aurantium L. ssp. amara Engl.) partly freed from the white spongy tissue of the mesocarp and endocarp. Content : minimum 20 ml/kg of essential oil (anhydrous drug).

An orange fluorescent zone Reference solution

Test solution

TESTS Foreign matter (2.8.2) : it complies with the test for foreign matter. Water (2.2.13) : maximum 10.0 per cent, determined on 20.0 g of powdered drug (355) by distillation. CHARACTERS Aromatic odour and spicy bitter taste. Total ash (2.4.16) : maximum 7.0 per cent Macroscopic and microscopic characters described under Extractable matter : minimum 6.0 per cent. identification A and B. To 2.000 g of the powdered drug (250) add a mixture of 3 ml of water R and 7 ml of alcohol R and extract for 2 h, shaking IDENTIFICATION frequently. Filter, evaporate 2.000 g of the filtrate to dryness A. The drug consists of elliptical to irregular pieces 5 cm to on a water-bath and dry in an oven at 100-105 °C for 3 h. 8 cm long, 3 cm to 5 cm broad and about 3 mm thick. The Allow to cool in a dessicator over diphosphorus pentoxide R outer surface is yellowish to reddish-brown and distinctly and weigh. The residue weighs a minimum of 120 mg. punctate, the inner surface is yellowish to brownish-white. ASSAY B. Reduce to a powder (355). The powder is light brown. Examine under a microscope using chloral hydrate Carry out the determination of essential oil in vegetable solution R. The powder shows small polygonal cells with drugs (2.8.12). Use a 500 ml round-bottomed flask, 200 ml slightly thickened anticlinal walls, filled with orange-red of water R as the distillation liquid and 0.5 ml of xylene R chromatophores, and very occasional anomocytic in the graduated tube. Reduce the drug to a powder (710) stomata (2.8.3) ; fragments of the hypodermic showing and immediately use 15.0 g for the determination. Distil at a collenchymatous thickening ; groups of parenchyma rate of 2-3 ml/min for 90 min. with each cell containing a prism crystal of calcium oxalate ; fragments of lysigenous oil glands ; parenchyma 01/2005:1604 containing crystals of hesperidin which dissolve in a 20 g/l potassium hydroxide R solution giving a yellow BITTER-ORANGE-EPICARP AND colour. MESOCARP TINCTURE C. Thin-layer chromatography (2.2.27). Test solution. To 1.0 g of the powdered drug (710) add 10 ml of methanol R and heat in a water-bath at 65 °C for Aurantii amari epicarpii et mesocarpii 5 min shaking frequently. Allow to cool and filter. tinctura Reference solution. Dissolve 1.0 mg of naringin R and DEFINITION 1.0 mg of caffeic acid R in 1 ml of methanol R. Tincture produced from Bitter-orange epicarp and Plate : TLC silica gel plate R. Mobile phase : water R, anhydrous formic acid R, ethyl mesocarp (1603). acetate R (10:15:75 V/V/V). PRODUCTION Application : 20 µl, as bands. The tincture is produced from 1 part of the freshly powdered Development : over a path of 10 cm. drug (2000) and 5 parts of alcohol (70 per cent V/V/V) by Drying : in air, and heat in an oven at 110-120 °C for an appropriate procedure. 5 min. CHARACTERS Detection : spray the warm plate with a 10 g/l solution of diphenylboric acid aminoethyl ester R in methanol R Liquid with a bitter taste. and then with a 50 g/l solution of macrogol 400 R in methanol R. After at least 1 h, examine in ultraviolet light IDENTIFICATION Examine by thin-layer chromatography (2.2.27). at 365 nm. 1110


Bitter-orange flower


peduncle which is about 5 mm to 10 mm long. The flower Test solution. The tincture to be examined. buds contain at least 20 stamens with yellow anthers and Reference solution. Dissolve 1.0 mg of naringin R and with filaments fused at the base into groups of 4 or 5 ; the 1.0 mg of caffeic acid R in 1 ml of methanol R. ovary is superior, brownish-black and spherical, consists Plate : TLC silica gel plate R. of 8 to 10 multi-ovular loculi and is surrounded at the Mobile phase : water R, anhydrous formic acid R, ethyl base by an annular granular hypogynous disc ; the thick, acetate R (10:15:75 V/V/V). cylindrical style ends in a capitate stigma. Application : 20 µl, as bands. B. Reduce to a powder (355). The powder is brownish-yellow. Development : over a path of 10 cm. Examine under a microscope using chloral hydrate solution R. The powder shows numerous spherical pollen Drying : in air, and heat in an oven at 110-120 °C for 5 min. grains, with a finely pitted exine and 3 to 5 germinal Detection : spray the warm plate with a 10 g/l solution pores ; fragments of the epidermis of the sepals with of diphenylboric acid aminoethyl ester R in methanol R unicellular trichomes and with large prism crystals of and then with a 50 g/l solution of macrogol 400 R in calcium oxalate in the underlying mesophyll ; fragments methanol R. After 1 h, examine in ultraviolet light at 365 nm. of the epidermis of the petals with a distinctly striated Results : see below the sequence of the zones present in cuticle ; fragments of large schizolysigenous oil glands the chromatograms obtained with the reference and test which measure up to 100 µm in diameter, numerous solutions. Furthermore, other zones are present in the anomocytic stomata (2.8.3). Examine under a microscope chromatogram obtained with the test solution. using a 300 g/l solution of potassium hydroxide R. The Top of the plate powder shows yellow crystals of hesperidin. C. Examine the chromatograms obtained in the test for A light blue fluorescent zone sweet-orange flower. A light blue fluorescent zone Results : see below the sequence of the zones present in Caffeic acid : a light blue the chromatograms obtained with the reference solution fluorescent zone and the test solution. A light blue fluorescent zone A light blue fluorescent zone Naringin : a dark green fluorescent zone

Reference solution

A dark green fluorescent zone (naringin) A red fluorescent zone (neoeriocitrin) An orange fluorescent zone Test solution

TESTS Ethanol content (2.9.10) : 63 per cent to 67 per cent V/V. Methanol and 2-propanol (2.9.11) : maximum 0.05 per cent V/V of methanol and maximum 0.05 per cent V/V of 2-propanol. Dry residue : minimum 6.0 per cent m/m, determined on 2.00 g of tincture to be examined.

Top of the plate A weak yellow fluorescent zone A weak yellow fluorescent zone Hesperidin : a greenish-yellow fluorescent zone

A greenish-yellow fluorescent zone (hesperidin)

Naringin : a yellow fluorescent zone

A yellow fluorescent zone (naringin) A red fluorescent zone (neoeriocitrin)

Reference solution

A yellow fluorescent zone (diosmin and neodiosmin) Test solution

TESTS Sweet-orange flower. Thin-layer chromatography (2.2.27). Test solution. To 0.5 g of the powdered drug (355), add 5 ml of methanol R. Heat with stirring at 40 °C for 10 min. Filter. 01/2005:1810 Reference solution. Dissolve 3.0 mg of naringin R and 3.0 mg of hesperidin R in 10 ml of methanol R. BITTER-ORANGE FLOWER Plate : TLC silica gel plate R. Mobile phase : water R, anhydrous formic acid R, ethyl acetate R (10:15:75 V/V/V). Aurantii amari flos Application : 10 µl, as bands. DEFINITION Development : over a path of 10 cm. Whole, dried, unopened flower of Citrus aurantium L. Drying : in air, then heat in an oven at 110-120 °C for 5 min. ssp. aurantium (C. aurantium L. ssp. amara Engl.). Content : minimum 8.0 per cent of total flavonoids, expressed Detection : spray the hot plate with a 10 g/l solution of diphenylboric acid aminoethyl ester R in methanol R as naringin (C27H32O14 ; Mr 580.5) (dried drug). and then with a 50 g/l solution of macrogol 400 R in methanol R. After at least 1 h, examine in ultraviolet light CHARACTERS at 365 nm. Macroscopic and microscopic characters described under Results : the chromatogram obtained with the test solution identification tests A and B. shows a yellow zone similar in position to the zone of IDENTIFICATION naringin in the chromatogram obtained with the reference solution and immediately below it a red zone (neoeriocitrin). A. The flower buds are white or yellowish-white and may reach up to 25 mm in length. The dialypetalous corolla Foreign matter (2.8.2) : maximum 2 per cent. is composed of 5 thick, oblong and concave petals Loss on drying (2.2.32) : maximum 11.0 per cent, determined dotted with oil glands visible under a hand lens ; the short, yellowish-green persistent gamosepalous calyx has on 1.000 g of the powdered drug (355) by drying in an oven at 100-105 °C. 5 spreading sepals, connate at the base and forming a star-shaped structure attached to the yellowish-green Total ash (2.4.16) : maximum 10.0 per cent. General Notices (1) apply to all monographs and other texts

1111

Bitter-orange-flower oil


ASSAY Stock solution. To 0.175 g of the powdered drug (355) add 95 ml of alcohol (50 per cent V/V) R. Heat on a water-bath under a reflux condenser for 30 min. Allow to cool and filter through a sintered-glass filter. Rinse the filter with 5 ml of alcohol (50 per cent V/V) R. Combine the filtrate and the rinsings in a volumetric flask and dilute to 100.0 ml with alcohol (50 per cent V/V) R. Test solution. Into a test tube (10 mm × 180 mm) introduce 0.150 g of powdered (250) magnesium R, a magnetic stirring bar 25 mm long and 2.00 ml of the stock solution. Maintain the test tube upright, centrifuge at 125 g and carefully add dropwise, especially at the beginning, 2.0 ml of hydrochloric acid R, and then 6.0 ml of alcohol (50 per cent V/V) R. Stopper the tube and mix by inverting. Compensation solution. Into a second tube, introduce 2.00 ml of the stock solution and carefully add dropwise, especially at the beginning, 2.0 ml of hydrochloric acid R and then 6.0 ml of alcohol (50 per cent V/V) R. After 10 min, measure the absorbance (2.2.25) of the test solution at 530 nm. Calculate the percentage content of total flavonoids, expressed as naringin, from the expression :

i.e. taking the value of the specific absorbance of the reaction product of naringin to be 52. A m

=


=

mass of the substance to be examined, in grams. 01/2005:1175

BITTER-ORANGE-FLOWER OIL Aurantii amari floris aetheroleum DEFINITION Bitter-orange-flower oil is obtained by steam distillation from the fresh flowers of Citrus aurantium L. subsp. aurantium (C. aurantium L. subsp. amara Engl.). CHARACTERS A clear, pale-yellow or dark-yellow liquid, with a characteristic odour reminiscent of bitter-orange flowers, miscible with alcohol, with light petroleum, with fatty oils and with liquid paraffin. IDENTIFICATION First identification : B. Second identification : A. A. Examine in ultraviolet light at 365 nm the chromatograms obtained in the test for bergapten. Before spraying with the reagent, the chromatogram obtained with the test solution shows a band similar in position and fluorescence to that corresponding to methyl anthranilate in the chromatogram obtained with the reference solution. Other bands may be visible. Examine in ultraviolet light at 365 nm after spraying with the reagent. The chromatogram obtained with the reference solution shows in the upper half a band of brownish-orange fluorescence corresponding to linalyl acetate, in the lower half a band of brownish-orange fluorescence corresponding to linalol and immediately below, a band of yellow-greenish fluorescence corresponding to bergapten. 1112

The chromatogram obtained with the test solution shows two bands similar in position and fluorescence to the bands corresponding to linalyl acetate and to linalol in the chromatogram obtained with the reference solution. Other bands may also be present. B. Examine the chromatograms obtained in the test for chromatographic profile. The retention times of the principal peaks in the chromatogram obtained with the test solution are approximately the same as those of the peaks in the chromatogram obtained with the reference solution. TESTS Relative density (2.2.5) : 0.866 to 0.880. Refractive index (2.2.6) : 1.468 to 1.474. Optical rotation (2.2.7) : + 1.5° to + 11.5°. Acid value (2.5.1). Not more than 2.0. Bergapten. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating substance. Test solution. Dissolve 0.1 g of the substance to be examined in alcohol R and dilute to 5.0 ml with the same solvent. Reference solution. Dissolve 5 µl of methyl anthranilate R, 10 µl of linalol R, 20 µl of linalyl acetate R and 10 mg of bergapten R in alcohol R and dilute to 10.0 ml with the same solvent. Apply separately to the plate, as bands, 10 µl of each solution. Develop over a path of 15 cm using a mixture of 15 volumes of ethyl acetate R and 85 volumes of toluene R. Allow the plate to dry in air and examine in ultraviolet light at 365 nm. The chromatogram obtained with the reference solution shows in the middle a band of blue fluorescence (methyl anthranilate) and below a band of greenish-yellow fluorescence (bergapten). Spray with anisaldehyde solution R. Heat the plate at 100 °C to 105 °C for 10 min. Examine in ultraviolet light at 365 nm. The chromatogram obtained with the test solution does not show a band corresponding to that due to bergapten (essential oil of bitter-orange peel) in the chromatogram obtained with the reference solution. Chromatographic profile. Examine by gas chromatography (2.2.28). Test solution. The substance to be examined. Reference solution. Dissolve 20 µl of β-pinene R, 5 µl of sabinene R, 40 µl of limonene R, 40 µl of linalol R, 20 µl of linalyl acetate R, 5 µl of α-terpineol R, 5 µl of neryl acetate R, 5 µl of geranyl acetate R, 5 µl of trans-nerolidol R and 5 µl of methyl anthranilate R in 1 ml of hexane R. The chromatographic procedure may be carried out using : — a fused-silica capillary column 25 m to 60 m long and about 0.25 mm in internal diameter, impregnated with macrogol 20 000 R as the bonded phase, — helium for chromatography R as the carrier gas at a flow rate of 1.5 ml/min, — a flame-ionisation detector, — a split ratio of 1:100, maintaining the temperature of the column at 75 °C for 4 min, then raising the temperature at a rate of 4 °C/min to 230 °C and maintaining at 230 °C for 20 min, maintaining the temperature of the injection port and of the detector at 270 °C. Inject about 0.1 µl of the reference solution. When the chromatograms are recorded in the prescribed conditions, the components elute in the order indicated in the composition of the reference solution. Record the retention times of these substances.


Black horehound


head ; fragments of the leaf epidermis with sinuous walls, The test is not valid unless : the number of theoretical plates those from the lower epidermis with numerous stomata, is not less than 30 000, calculated from the limonene peak at some diacytic (2.8.3) but the majority anomocytic ; 110 °C ; the resolution between the peaks due to β-pinene epidermis of the corolla composed of polygonal cells, and to sabinene is not less than 1.5. those of the inner epidermis papillose ; pollen grains Inject about 0.2 µl of the substance to be examined. Using subspherical with 3 pores and a smooth exine ; groups the retention times determined from the chromatogram of collenchyma and lignified, spirally thickened and obtained with the reference solution, locate the components bordered pitted vessels, from the stem. of the reference solution on the chromatogram obtained C. Thin-layer chromatography (2.2.27). with the test solution (disregard the peak due to hexane). Test solution. To 2 g of the powdered drug (355) add Determine the percentage content of each of these 100 ml of methanol R. Heat on a water-bath under components by the normalisation procedure. a reflux condenser for 30 min. Allow to cool. Filter. The percentages are within the following ranges : Evaporate the filtrate under reduced pressure until a — β-pinene : 7.0 per cent to 17.0 per cent, volume of about 10 ml is obtained. — limonene : 9.0 per cent to 18.0 per cent, Reference solution. Dissolve 2.5 mg of rutin R and 1 mg — linalol : 18.0 per cent to 42.0 per cent, of chlorogenic acid R in 10 ml of methanol R. — linalyl acetate : 3.0 per cent to 16.0 per cent, Plate : TLC silica gel plate R. — α-terpineol: 2.0 per cent to 7.0 per cent, Mobile phase : anhydrous formic acid R, glacial acetic acid R, water R, ethyl acetate R (7.5:7.5:18:67 V/V/V/V). — neryl acetate : 1.0 per cent to 3.0 per cent, Application : 20 µl, as bands. — geranyl acetate : 1.5 per cent to 4.0 per cent, Development : over a path of 15 cm. — trans-nerolidol : 1.0 per cent to 9.0 per cent, Drying : in air. — methyl anthranilate : 0.1 per cent to 1.0 per cent. Detection : spray with a solution containing 10 g/l of STORAGE diphenylboric acid aminoethyl ester R and 50 g/l of Store in a well-filled, airtight container, protected from light macrogol 400 R in methanol R. Allow to dry in a current and heat. of warm air. Examine in ultraviolet light at 365 nm after 30 min. Results : see below the sequence of the zones present in 01/2005:1858 the chromatogram obtained with the reference solution and the test solution. Furthermore, other fluorescent BLACK HOREHOUND zones may be present in the chromatogram obtained with the test solution.

Ballotae nigrae herba

Top of the plate

DEFINITION Dried flowering tops of Ballota nigra L. Content : minimum 1.5 per cent of total orthodihydroxycinnamic acid derivatives, expressed as acteoside (C29H36O15 ; Mr 624.6) (dried drug). CHARACTERS Macroscopic and microscopic characters described under identification tests A and B. IDENTIFICATION A. Stems conspicuously four-angled, longitudinally striated, dark green or reddish-brown and more or less pubescent. Leaves greyish-green, petiolate, lamina ovate to orbicular, 2 cm to 4 cm wide, margin irregularly crenate, cuneate to cordate at the base ; both surfaces covered with abundant whitish hairs ; venation pinnate, prominent on the lower surface, slightly depressed on the upper. Flowers sessile or very shortly pedicellate, calyx infundibuliform, densely pubescent, with 10 prominent ribs and 5 subequal, broadly ovate teeth ; corolla purple, tube slightly shorter than the calyx tube, bilabiate, the upper lip pubescent on the outer surface, the lower lip with 3 lobes, the middle lobe notched. B. Reduce to a powder (355). The powder is greyish-green and slightly flocculent. Examine under a microscope using chloral hydrate solution R. The powder shows numerous long, uniseriate, multicellular covering trichomes consisting of 4 or more cells, thickened and swollen at the junctions, with slightly lignified and pitted walls ; fewer glandular trichomes, some with a unicellular or multicellular stalk and a globose, uni- or bicellular head, others with a unicellular stalk and a multicellular General Notices (1) apply to all monographs and other texts

A reddish fluorescent zone A faint yellow fluorescent zone A light blue fluorescent zone (caffeoylmalic acid) A greenish-blue fluorescent zone (acteoside) A yellow-brown fluorescent zone (luteolin 7-lactate) Chlorogenic acid : a light blue fluorescent zone Rutin : an orange-yellow fluorescent zone

A greenish-blue fluorescent zone (forsythoside B) 2 greenish-blue fluorescent zones (arenarioside) A yellow fluorescent zone (luteolin 7-lactate glucoside). A faint greenish-blue fluorescent zone (ballotetroside).

Reference solution

Test solution

TESTS Foreign matter (2.8.2) : maximum 2 per cent m/m. Loss on drying (2.2.32) : maximum 12.0 per cent, determined on 1.000 g of the powdered drug (355) in an oven at 100-105 °C for 2 h. Total ash (2.4.16) : maximum 13.0 per cent. ASSAY Stock solution. Place 1.000 g of the powdered drug (355) in a flask. Add 90 ml of alcohol (50 per cent V/V) R. Heat under a reflux condenser on a water-bath for 30 min. Allow to cool and filter collecting the filtrate into a 100 ml 1113

Bleomycin sulphate


volumetric flask. Rinse the flask and the filter with 10 ml of alcohol (50 per cent V/V) R. Add the rinsings to the filtrate and dilute to 100.0 ml with alcohol (50 per cent V/V) R. Test solution. In a 10 ml volumetric flask add successively, with shaking after each addition, 1.0 ml of the stock solution, 2 ml of 0.5 M hydrochloric acid, 2 ml of a solution containing 100 g/l of sodium nitrite R and 100 g/l of sodium molybdate R, 2 ml of dilute sodium hydroxide solution R and dilute to 10.0 ml with water R. Compensation liquid. In a 10 ml volumetric flask, add 1.0 ml of the stock solution, 2 ml of 0.5 M hydrochloric acid, 2 ml of dilute sodium hydroxide solution R and dilute to 10.0 ml with water R. Measure immediately the absorbance (2.2.25) of the test solution, by comparison with the compensation liquid at 525 nm. Calculate the percentage content of total ortho-dihydroxycinnamic acid derivatives, calculated as acteoside, from the expression :

CHARACTERS A white or yellowish-white powder, very hygroscopic, very soluble in water, slightly soluble in ethanol, practically insoluble in acetone. IDENTIFICATION A. Examine the chromatograms obtained in the test for composition. The retention times and sizes of the two principal peaks in the chromatogram obtained with the test solution are approximately the same as those of the two principal peaks in the chromatogram obtained with reference solution (a). B. It gives the reactions of sulphates (2.3.1).

TESTS Appearance of solution. Dissolve 0.200 g in water R and dilute to 10.0 ml with the same solvent. The solution is clear (2.2.1). The absorbance (2.2.25) measured at 430 nm is not greater than 0.10. pH (2.2.3). Dissolve 50 mg in carbon dioxide-free water R and dilute to 10 ml with the same solvent. The pH of the solution is 4.5 to 6.0. i.e. taking the specific absorbance of acteoside to be 185 Composition. Examine by liquid chromatography (2.2.29). at 525 nm. Test solution. Dissolve 25.0 mg of the substance to be examined in water R and dilute to 50.0 ml with the same A = absorbance of the test solution at 525 nm, solvent. m = mass of the substance to be examined, in grams. Reference solution (a). Dissolve 25.0 mg of bleomycin sulphate CRS in water R and dilute to 50.0 ml with the same solvent. 01/2005:0976 Reference solution (b). Dilute 1.5 ml of reference solution (a) to 100.0 ml with water R. BLEOMYCIN SULPHATE The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in Bleomycini sulfas internal diameter packed with octadecylsilyl silica gel for chromatography R (7 µm), — gradient elution at a flow rate of 1.2 ml/min with a mobile phase initially composed of 10 per cent V/V of methanol R and 90 per cent V/V of a mixture prepared as follows : dissolve 0.960 g of sodium pentanesulphonate R in 900 ml of acetic acid (4.8 g/l C2H4O2), add 1.86 g of sodium edetate R, dilute to 1000 ml with the same solvent and adjust to pH 4.3 using ammonia R ; increasing the proportion of methanol R to 40 per cent V/V over 60 min and continuing with the final mixture for about 20 min, until demethylbleomycin A2 is eluted (retention time 1.5 to 2.5, relative to bleomycin A2), — as detector a spectrophotometer set at 254 nm, — a 20 µl loop injector. Inject reference solution (a). The test is not valid unless the resolution between the two principal peaks is at least 5. Inject reference solution (b). The test is not valid unless the signal-to-noise ratio calculated for the principal peak is at least 20. Inject reference solution (a) six times. The test is not valid unless the relative standard deviation of the area of the principal peak is at most 2 per cent. Inject the test solution. The composition, calculated by the DEFINITION normalisation procedure and disregarding any peak with an area less than 0.1 per cent of the total, is : bleomycin A2 Bleomycin sulphate is the sulphate of a mixture of (first principal peak) 55 per cent to 70 per cent ; bleomycin glycopeptides produced by Streptomyces verticillus or by any other means ; the two principal components of the mixture B2 (second principal peak) 25 per cent to 32 per cent ; sum of bleomycin A2 and bleomycin B2 not less than 85 per cent ; are N1-[3-(dimethylsulphonio)propyl]bleomycinamide (bleomycin A2) and N1-4-(guanidobutyl)bleomycinamide demethylbleomycin A2 (retention time relative to bleomycin (bleomycin B2). The potency is not less than 1500 IU/mg, A2 1.5 to 2.5) not more than 5.5 per cent ; other related calculated with reference to the dried substance. substances not more than 9.5 per cent. 1114


Boldo leaf


anomocytic stomata (2.8.3), on both surfaces, with Copper. Not more than 200 ppm of Cu, determined by the subsidiary cells showing radiating striations ; atomic absorption spectrometry (2.2.23, Method I). epidermal cells from the veins straight walled and Test solution. Dissolve 50 mg in water R and dilute to papillose ; fragments of mesophyll parenchyma with large 10.0 ml with the same solvent. intercellular spaces (aerenchyma) ; irregular cells with Reference solution. Dilute 1.0 ml of copper standard rare sclereids ; fragments of spiral or annular vessels. solution (10 ppm Cu) R to 10.0 ml with water R. C. Thin-layer chromatography (2.2.27). Measure the absorbance at 324.7 nm using a copper Test solution. To 1.0 g of the powdered drug (355) add hollow-cathode lamp as source of radiation and an 10 ml of methanol R. Heat, with stirring, in a water-bath air-acetylene flame. at 60 °C for 5 min. Allow to cool and filter. Evaporate to Loss on drying (2.2.32). Not more than 3.0 per cent, dryness under reduced pressure in a water-bath at 60 °C. determined on 50 mg by drying at 60 °C at a pressure not Dissolve the residue in 2.0 ml of methanol R. exceeding 670 Pa for 3 h. Reference solution. Dissolve 5 mg of loganin R in 15 ml Bacterial endotoxins (2.6.14) : less than 5 IU/mg, if intended of methanol R. for use in the manufacture of parenteral dosage forms Plate : TLC silica gel plate R. without a further appropriate procedure for the removal of Mobile phase : water R, methanol R, ethyl acetate R bacterial endotoxins. (8:15:77 V/V/V). ASSAY Application : 30 µl, as bands. Carry out the microbiological assay of antibiotics (2.7.2), Development : over a path of 15 cm. using the diffusion method. Use bleomycin sulphate CRS Drying : in air. as the reference substance. Detection : spray with vanillin reagent R. Heat in an oven STORAGE at 100-105 °C for 10 min. Examine in daylight. Store in an airtight container, at a temperature of 2 °C to Results : see below the sequence of the zones present in 8 °C. If the substance is sterile, store in a sterile, aritight, the chromatograms obtained with the reference and test tamper-proof container. solutions. Furthermore, other zones are present in the chromatogram obtained with the test solution. LABELLING Top of the plate The label states, where applicable, that the substance is free from bacterial endotoxins. A violet zone IMPURITIES A. R = OH : bleomycinic acid, B. R = NH-[CH2]3-NH-[CH2]4-NH2 : bleomycin A5, C. R = NH-[CH2]4-NH-C(=NH)-NH-[CH2]4-NH-C(=NH)-NH2 : bleomycin B4, D. R = NH-[CH2]3-S-CH3 : demethylbleomycin A2. 01/2005:1605

BOGBEAN LEAF Menyanthidis trifoliatae folium DEFINITION Dried, entire or fragmented leaf of Menyanthes trifoliata L. CHARACTERS Very bitter and persistant taste. Macroscopic and microscopic characters described under identification tests A and B. IDENTIFICATION A. The leaf is long-petiolated, trifoliate, with long sheaths from the base ; the petiole is up to 5 mm in diameter and strongly striated longitudinally. The lamina is divided into equal leaflets, sessile, obovate up to 10 cm long and up to 5 cm wide, with an entire, occasionally sinuous margin with brownish or reddish hydathodes and a spathulate base ; it is glabrous, dark green on the upper surface and paler green on the lower surface, with a wide, whitish, finely striated prominent midrib. B. Reduce to a powder (355). The powder is yellowish-green. Examine under a microscope using chloral hydrate solution R. The powder shows fragments of upper epidermis with polyhedral cells and thin wavy walls ; fragments of lower epidermis with sinuous walls ; General Notices (1) apply to all monographs and other texts

An intense blue zone Loganine : a greyish-violet zone

A violet to greyish-violet zone A grey to greyish-blue zone A brownish zone

Reference solution

Test solution

TESTS Foreign matter (2.8.2) : it complies with the test for foreign matter. Loss on drying (2.2.32) : maximum 10.0 per cent, determined on 1.000 g of the powdered drug (355) by drying in an oven at 100-105 °C for 2 h. Total ash (2.4.16) : maximum 10.0 per cent. Bitterness value (2.8.15) : minimum 3000. 01/2005:1396

BOLDO LEAF Boldi folium DEFINITION Boldo leaf consists of the whole or fragmented dried leaf of Peumus boldus Molina. The whole drug contains not less than 20.0 ml/kg and not more than 40.0 ml/kg and the fragmented drug not less than 15.0 ml/kg of essential oil. It contains not less than 0.1 per cent of total alkaloids, expressed as boldine, (C19H21NO4 ; Mr 327.4), calculated with reference to the anhydrous drug. CHARACTERS Boldo leaf has an aromatic odour especially when rubbed. It has the microscopic and macroscopic characters described under Identification tests A and B. 1115

Borax


IDENTIFICATION A. The leaf is oval or elliptical usually 5 cm long with a short petiole, an obtuse or slightly emarginate or mucronate apex and an equal and rounded base ; the margin is entire and slightly undulate and the thickened edges are more or less revolute. The lamina is greyish-green, thick, tough and brittle. The upper surface is rough with numerous prominent small protuberances and a depressed venation. The lower surface is finely pubescent, with the protuberances less well-marked, and a prominent, pinnate venation. B. Reduce to a powder (355). The powder is greyish-green. Examine under a microscope, using chloral hydrate solution R. The powder shows fragments of the upper epidermis and underlying hypodermis with straight or slightly sinuous thickened and beaded walls, those of the lower epidermis with numerous stomata surrounded by four to seven subsidiary cells ; solitary, bifurcated or stellate clustered unicellular covering trichomes with more or less thickened and lignified wall ; fragments of the lamina showing a two-layered palisade ; debris of the spongy mesophyll including numerous, large rounded oil cells and parenchyma containing fine needle-shaped crystals ; thick walled fibres and lignified, pitted parenchymatous cells associated with vascular tissue from the veins. C. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R. Test solution. Add to 0.5 g of the powdered drug (355) a mixture of 1 ml of dilute hydrochloric acid R and 20 ml of water R and heat on a water-bath under reflux for 10 min. Cool and filter. Add to the filtrate 2 ml of dilute ammonia R1 and extract with two quantities, each of 20 ml of ether R avoiding emulsifying. Combine the organic layers and evaporate the solvent on a water-bath. Dissolve the residue in 1.0 ml of methanol R. Reference solution. Dissolve 2 mg of boldine R in 5 ml of methanol R. Apply to the plate as bands 20 µl of the test solution and 10 µl of the reference solution. Develop over a path of 15 cm using a mixture of 10 volumes of methanol R, 10 volumes of diethylamine R and 80 volumes of toluene R. Allow the plate to dry in air. Spray the plate with potassium iodobismuthate solution R2. Allow the plate to dry in air for 5 min and then spray the plate with sodium nitrite solution R. Examine in daylight. The chromatograms show in the lower third the brown to reddish-brown zone of boldine. The chromatogram obtained with the test solution shows several brownish zones above and below the zone corresponding to boldine. TESTS Foreign matter (2.8.2). Not more than 4 per cent of twigs and 2 per cent of other foreign matter. Water (2.2.13). Not more than 100 ml/kg determined by distillation of 20.0 g of the powdered drug (355). Total ash (2.4.16). Not more than 13.0 per cent. ASSAY Essential oil. Carry out the determination of essential oils in vegetable drugs (2.8.12). Use 10.0 g of the freshly crushed drug, a 1000 ml flask and 300 ml of water R as the distillation liquid. Distil at a rate of 2 ml/min to 3 ml/min for 3 h. Alkaloids. Examine by liquid chromatography (2.2.29). Test solution. To 1.000 g (m1) of the powdered drug (355) add 50 ml of dilute hydrochloric acid R. Shake in a 1116

water-bath at 80 °C for 30 min. Filter, take up the residue with 50 ml of dilute hydrochloric acid R and shake in a water-bath at 80 °C for 30 min. Filter and repeat the operation once on the residue obtained. Filter. Combine the cooled filtrates and shake with 100 ml of a mixture of equal volumes of ethyl acetate R and hexane R. Adjust the aqueous layer to pH 9.5 with dilute ammonia R1. Shake successively with 100 ml, 50 ml and 50 ml of methylene chloride R and combine the lower layers and evaporate to dryness under reduced pressure. In a 10.0 ml volumetric flask dilute the residue to 10.0 ml with the mobile phase. Reference solution. In a 100.0 ml volumetric flask dissolve 12 mg (m2) of boldine R in 100.0 ml of the mobile phase. Dilute 1.0 ml of the solution to 10.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.5 ml/min a mixture of 16 volumes of solution A and 84 volumes of solution B, Solution A. Mix 99.8 ml of acetonitrile R and 0.2 ml of diethylamine R, Solution B. Mix 99.8 ml of water R and 0.2 ml of diethylamine R, adjusted to pH 3 with formic acid R, — as detector a spectrophotometer set at 304 nm. Inject 20 µl of each solution. When the chromatograms are recorded in the prescribed conditions, the retention times relative to boldine are : isoboldine about 0.9 ; isocorydine N-oxide about 1.8 ; laurotetanine about 2.2 ; isocorydine about 2.8 and N-methyllaurotetanine about 3.2. Additional peaks may be present. Calculate the percentage content of total alkaloids expressed as boldine from the expression :

m1

=

mass of the substance to be examined, in grams,

m2

=

mass of boldine R, in grams,

A1 = A2

=

sum of the areas of the peaks due to the six alkaloids identified in the chromatogram obtained with the test solution, area of the peak due to boldine in the chromatogram obtained with the reference solution.

STORAGE Store protected from light. 01/2005:0013

BORAX Borax Na2B4O7,10H2O

Mr 381.4

DEFINITION Borax contains not less than 99.0 per cent and not more than the equivalent of 103.0 per cent of disodium tetraborate decahydrate. CHARACTERS A white, crystalline powder, colourless crystals or crystalline masses, efflorescent, soluble in water, very soluble in boiling water, freely soluble in glycerol.



IDENTIFICATION A. To 1 ml of solution S (see Tests) add 0.1 ml of sulphuric acid R and 5 ml of methanol R and ignite. The flame has a green border. B. To 5 ml of solution S add 0.1 ml of phenolphthalein solution R. The solution is red. On the addition of 5 ml of glycerol (85 per cent) R the colour disappears. C. Solution S gives the reactions of sodium (2.3.1).

Botulinum toxin type A for injection

IDENTIFICATION A. Dissolve 0.1 g by gently heating in 5 ml of methanol R, add 0.1 ml of sulphuric acid R and ignite the solution. The flame has a green border. B. Solution S (see Tests) is acid (2.2.4).

TESTS Solution S. Dissolve 3.3 g in 80 ml of boiling distilled water R, cool and dilute to 100 ml with carbon dioxide-free water R prepared from distilled water R. TESTS Appearance of solution. Solution S is clear (2.2.1) and Solution S. Dissolve 4.0 g in carbon dioxide-free water R colourless (2.2.2, Method II). prepared from distilled water R and dilute to 100 ml with the same solvent. pH (2.2.3). The pH of solution S is 3.8 to 4.8. Appearance of solution. Solution S is clear (2.2.1) and Solubility in alcohol. Dissolve 1.0 g in 10 ml of boiling colourless (2.2.2, Method II). alcohol R. The solution is not more opalescent than reference suspension II (2.2.1) and is colourless (2.2.2, Method II). pH (2.2.3). The pH of solution S is 9.0 to 9.6. Organic matter. It does not darken on progressive heating Sulphates (2.4.13). 15 ml of solution S complies with the limit test for sulphates (50 ppm). Use 1.0 ml of acetic acid R to dull redness. instead of the 0.5 ml prescribed. Prepare the standard using Sulphates (2.4.13). 10 ml of solution S diluted to 15 ml with a mixture of 3 ml of sulphate standard solution (10 ppm distilled water R complies with the limit test for sulphates SO4) R and 12 ml of distilled water R. (450 ppm). Ammonium (2.4.1). 6 ml of solution S diluted to 14 ml with Heavy metals (2.4.8). 12 ml of solution S complies with limit water R complies with the limit test for ammonium (10 ppm). test A for heavy metals (15 ppm). Prepare the standard using Prepare the standard using a mixture of 2.5 ml of ammonium a mixture of 2.5 ml of lead standard solution (2 ppm Pb) R and 7.5 ml of water R. standard solution (1 ppm NH4) R and 7.5 ml of water R. Arsenic (2.4.2). 5 ml of solution S complies with limit test A ASSAY for arsenic (5 ppm). Dissolve 1.000 g with heating in 100 ml of water R containing Calcium (2.4.3). 15 ml of solution S complies with the limit 15 g of mannitol R. Titrate with 1 M sodium hydroxide, test for calcium (100 ppm). Prepare the standard using a using 0.5 ml of phenolphthalein solution R as indicator, mixture of 6 ml of calcium standard solution (10 ppm Ca) R until a pink colour is obtained. and 9 ml of distilled water R. 1 ml of 1 M sodium hydroxide is equivalent to 61.8 mg of Heavy metals (2.4.8). 12 ml of solution S complies with limit H3BO3. test A for heavy metals (25 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. 01/2005:2113 ASSAY BOTULINUM TOXIN TYPE A Dissolve 20 g of mannitol R in 100 ml of water R, heating if necessary, cool, add 0.5 ml of phenolphthalein solution R FOR INJECTION and neutralise with 0.1 M sodium hydroxide until a pink colour is obtained. To this solution add 3.00 g of the Toxinum botulinicum typum A ad iniectabile substance to be examined, heat until dissolution is complete, cool, and titrate with 1 M sodium hydroxide until the pink DEFINITION colour reappears. Botulinum toxin type A for injection is a dried preparation containing purified botulinum neurotoxin type A which may 1 ml of 1 M sodium hydroxide is equivalent to 0.1907 g of be present in the form of a complex with haemagglutinins Na2B4O7,10H2O. and non-toxic proteins. Botulinum neurotoxin type A or its haemagglutinin complex is prepared by a suitable purification process of the liquid supernatant from a 01/2005:0001 broth-culture of a suitable strain of Clostridium botulinum type A. The purified complexes consist of several proteins and can BORIC ACID be of various sizes. The largest complex (relative molecular mass of about 900 000) consists of a 150 000 relative Acidum boricum molecular mass neurotoxin, a 130 000 relative molecular mass non-toxic protein and various haemagglutinins ranging H3BO3 Mr 61.8 between relative molecular mass 14 000 and 43 000. The purified toxin moiety is composed of only the same DEFINITION 150 000 relative molecular mass neurotoxin as is found in Boric acid contains not less than 99.0 per cent and not more the 900 000 relative molecular mass neurotoxin complex, which is initially produced as a single chain and further than the equivalent of 100.5 per cent of H3BO3. cleaved (nicked) by endogenous proteases into a fully active, CHARACTERS disulphide-linked, 54 000 relative molecular mass light chain A white, crystalline powder, colourless, shiny plates greasy to and a 97 000 relative molecular mass heavy chain. the touch, or white crystals, soluble in water and in alcohol, The preparation is reconstituted before use, as stated on freely soluble in boiling water and in glycerol (85 per cent). the label. General Notices (1) apply to all monographs and other texts

1117

Botulinum toxin type A for injection


PRODUCTION GENERAL PROVISIONS Production of the toxin is based on seed cultures, managed in a defined seed-lot system in which the ability to produce toxin is conserved. The production method must be shown to yield consistently product of activity and profile comparable to that of lots shown in clinical studies to be of adequate safety and efficacy. The production method is validated to demonstrate that the product, if tested, would comply with the general test of abnormal toxicity (2.6.9) using not less than the maximum human clinical dose, in the presence of a suitable amount of specific botulinum type A antitoxin used for neutralisation. The production method and stability of the finished product and relevant intermediates are evaluated using the tests below. Such tests include the specific toxin activity per milligram of protein of purified toxin in an appropriate functional model of toxin activity and may be supported by tests confirming the presence of botulinum toxin type A, and, if appropriate, associated non-toxic proteins. BACTERIAL SEED LOTS A highly toxigenic strain of C. botulinum of known toxin type A and confirmed absence of genes encoding other botulinum toxins (particularly botulinum toxin type B), with known origin and history, is grown using suitable media. The bacterial strain, used for the master seed lot, shall be identified by historical records that include information on its origin and the tests used to characterise the strain. These will include morphological, cultural, biochemical, genetic and serological properties of the strain. The master seed lot and the working seed lot, where applicable, must be demonstrated to have identical profiles. Only a seed lot that complies with the following requirements may be used.

BULK PURIFIED TOXIN C. botulinum type A strain is grown anaerobically, in suitable media, from which cultures are selected for step-up incubations under a suitably controlled anaerobic atmosphere through the seed culture and bulk fermentation stages to allow maximum production of toxin. The toxin is purified by suitable methods to remove nucleic acids and components likely to cause adverse reactions. Only a purified toxin that complies with the following requirements may be used in the preparation of the final bulk. For each test and for each product, limits of acceptance are established and each new purified toxin must comply with these limits. Residual reagents. Removal of residual reagents used in purification steps is confirmed by suitable limit tests or by validation of the process. Nucleic acids. Removal of nucleic acids is confirmed by suitable limit tests or by validation of the process. Immunological identity. The presence of specific type A toxin is confirmed by a suitable immunochemical method (2.7.1). Specific activity. The specific activity is confirmed in a mouse model of toxicity or by in vivo/ex vivo methods validated with respect to the LD50 assay and expressed in mouse LD50 units per milligram of protein. Specific activity must not be less than 1 × 108 mouse LD50 units per milligram of protein for the 150 000 relative molecular mass neurotoxin and must not be less than 1 × 107 mouse LD50 units per milligram of protein for the 900 000 relative molecular mass neurotoxin complex.

Protein. The total protein concentration is determined by a suitable method. An acceptable value is established for the product and each batch must be shown to comply with the Identification. Each seed lot is identified as containing pure limits. cultures of C. botulinum type A bacteria with no extraneous Protein profile. Identity and protein composition are bacterial or fungal contamination. determined by polyacrylamide gel electrophoresis (2.2.31) under reducing or non-reducing conditions or by other Microbial purity. Each seed lot complies with the requirements for absence of contaminating micro-organisms. suitable physicochemical methods such as size-exclusion chromatography (2.2.30), comparing with suitable reference The purity of bacterial cultures is verified by methods of standards. suitable sensitivity. These may include inoculation into suitable media and examination of colony morphology. Total viable count. It complies with the limits approved for the particular product. Phenotypic parameters. Each seed lot must have a known fatty acid profile, sugar fermentation profile (glucose, lactose, FINAL BULK mannose, etc.) and proteolytic activity and must demonstrate The final bulk is prepared by adding approved excipients relevant lipase, lecithinase and gelatinase activity. to the bulk purified toxin. The solution is filtered through Genetic purity. Each seed lot must have information on the a bacteria-retentive filter. If human albumin is added, it complies with the monograph on Human albumin toxin gene sequence and comply with requirements for the solution (0255). absence of other genes encoding other toxin serotypes. Production of active toxin. A bacterial strain producing a high yield of active toxin, as determined by an acute toxicity assay, is suitable. Seed lots should demonstrate a capability of producing at least a minimum toxicity level appropriate for the manufacturing process and scale. MANUFACTURER’S REFERENCE PREPARATIONS During development, reference preparations are established for subsequent verification of batch consistency during production and for control of the bulk purified toxin and finished product. They are derived from representative batches of botulinum toxin type A that are characterised as described under Bulk Purified Toxin. The reference preparations are suitably characterised for their intended purpose and are stored in suitably sized aliquots under conditions ensuring their suitability. 1118

FINAL LOT The final bulk is distributed aseptically into sterile, tamper-proof containers. Uniformity of fill is verified during filling and the test for uniformity of content (2.9.6) is not required. The containers are closed so as to prevent contamination. Only a final lot that is within the limits approved for the particular product and is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. pH (2.2.3). The pH of the reconstituted product is within ± 0.5 pH units of the limit approved for the particular product. Water : not more than the limit approved for the particular product.


Bromazepam


IDENTIFICATION The presence of botulinum toxin type A is confirmed by a suitable immunochemical method (2.7.1). TESTS Sterility (2.6.1). It complies with the test for sterility. Bacterial endotoxins (2.6.14) : less than 10 IU per vial. ASSAY The potency of the reconstituted product is determined by an LD50 assay in mice or by a method validated with respect to the LD50 assay. The potency is expressed in terms of the LD50 for mice or relative to the reference preparation. For determination of the LD50, graded doses of the product are injected intraperitoneally into groups of mice and the LD50 is calculated by the usual statistical methods (5.3) from the mouse lethality in each group. A suitable reference preparation is assayed in parallel ; the potency of the toxin is expressed relative to the reference or the value found for the reference is within suitable limits defined in terms of the assigned potency. After validation with respect to the LD50 assay (reference method), the product may also be assayed by other methods that are preferable in terms of animal welfare, including 1 of the following : 1. endopeptidase assay in vitro ; 2. ex vivo assay using the mouse phrenic nerve diaphragm ; 3. mouse bioassay using paralysis as the end-point. For these other methods, the potency is calculated with respect to a suitable reference preparation calibrated in mouse LD50 units. The estimated potency is not less than 80 per cent and not more than 125 per cent of the stated potency. The confidence limits (P = 0.95) are not less than 80 per cent and not more than 125 per cent of the estimated potency. The test may be repeated but when more than 1 test is performed, the results of all valid tests must be combined in the estimate of potency. LABELLING The label states : — the number of units of toxin per vial with a statement that units are product specific and not applicable to other preparations containing botulinum toxin type A, — the name and the volume of the diluent to be added for reconstitution of a dried product.

01/2005:0879

BROMAZEPAM Bromazepamum

DEFINITION Bromazepam contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of 7-bromo5-(pyridin-2-yl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one, calculated with reference to the dried substance. CHARACTERS A white or yellowish, crystalline powder, practically insoluble in water, sparingly soluble in alcohol and in methylene chloride. IDENTIFICATION First identification : B. Second identification : A, C, D, E. A. Dissolve 50.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with methanol R. Examined between 220 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 233 nm and a shoulder at about 260 nm and may show a broad absorption maximum at about 325 nm. The specific absorbance at the maximum at 233 nm is 980 to 1080. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with bromazepam CRS. Examine the substances prepared as discs. C. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm. Test solution. Dissolve 10 mg of the substance to be examined in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 10 ml with the same mixture of solvents. Reference solution (a). Dissolve 10 mg of bromazepam CRS in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 10 ml with the same mixture of solvents. Reference solution (b). Dissolve 10 mg of bromazepam CRS and 10 mg of temazepam CRS in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 10 ml with the same mixture of solvents. Apply separately to the plate 5 µl of each solution. Develop over a path of 10 cm using a mixture of 30 volumes of diethylamine R and 70 volumes of ether R. Dry the plate in a current of air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated principal spots. D. Dissolve about 20 mg in 5 ml of methanol R. Add 5 ml of water R and 1 ml of a 10 g/l solution of ferrous ammonium sulphate R. A violet colour develops.

C14H10BrN3O

E. To 0.15 g in a porcelain crucible add 0.5 g of anhydrous sodium carbonate R. Heat over an open flame for 10 min. Allow to cool. Take up the residue in 10 ml of dilute nitric acid R and filter. To 1 ml of the filtrate add 1 ml of water R. The solution gives reaction (a) of bromides (2.3.1). Mr 316.2


1119

Bromhexine hydrochloride


TESTS Appearance of solution. Dissolve 0.5 g in a mixture of 1 volume of methanol R and 4 volumes of tetrahydrofuran R and dilute to 20 ml with the same mixture of solvents. The solution is clear (2.2.1). Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Prepare the solutions immediately before use and carry out the test protected from light. Test solution. Dissolve 50 mg of the substance to be examined in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 5 ml with the same mixture of solvents. Reference solution. Dilute 1 ml of the test solution to 20 ml with a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R. Dilute 2 ml of the solution to 50 ml with a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R. Apply separately to the plate 5 µl of each solution. Develop over a path of 7.5 cm using a mixture of 5 volumes of alcohol R, 5 volumes of triethylamine R, 20 volumes of methylene chloride R and 70 volumes of light petroleum R1. Dry the plate in a current of air for 20 min and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution (0.2 per cent). Loss on drying (2.2.32). Not more than 0.2 per cent, determined on 1.000 g by drying at 80 °C at a pressure not exceeding 2.7 kPa for 4 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.250 g in 20 ml of anhydrous acetic acid R. Add 50 ml of acetic anhydride R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 31.62 mg of C14H10BrN3O. STORAGE Store protected from light. IMPURITIES

A. R = H : (2-amino-5-bromophenyl)(pyridin-2-yl)methanone, B. R = CO-CH2-Cl : N-[4-bromo-2-(pyridin-2ylcarbonyl)phenyl]-2-chloroacetamide,

C. 7-bromo-5-(6-methylpyridin-2-yl)-1,3-dihydro-2H-1,4benzodiazepin-2-one, 1120

D. 3-amino-6-bromo-4-(pyridin-2-yl)quinolin-2(1H)-one. 01/2005:0706

BROMHEXINE HYDROCHLORIDE Bromhexini hydrochloridum

C14H21Br2ClN2

Mr 412.6

DEFINITION N-(2-Amino-3,5-dibromobenzyl)-N-methylcyclohexanamine hydrochloride. Content : 98.5 per cent to 101.5 per cent (dried substance). CHARACTERS Appearance : white or almost white, crystalline powder. Solubility : very slightly soluble in water, slightly soluble in alcohol and in methylene chloride. It shows polymorphism. IDENTIFICATION First identification : A, E. Second identification : B, C, D, E. A. Infrared absorption spectrophotometry (2.2.24). Comparison : bromhexine hydrochloride CRS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in methanol R, evaporate to dryness and record new spectra using the residues. B. Thin-layer chromatography (2.2.27). Test solution. Dissolve 20 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution. Dissolve 20 mg of bromhexine hydrochloride CRS in methanol R and dilute to 10 ml with the same solvent. Plate : TLC silica gel F254 plate R. Mobile phase : glacial acetic acid R, water R, butanol R (17:17:66 V/V/V). Application : 20 µl. Development : over 3/4 of the plate. Drying : in air. Detection : examine in ultraviolet light at 254 nm. Results : the principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.


Bromocriptine mesilate


C. Dissolve about 25 mg in a mixture of 1 ml of dilute sulphuric acid R and 50 ml of water R. Add 2 ml of methylene chloride R and 5 ml of chloramine solution R and shake. A brownish-yellow colour develops in the lower layer. D. Dissolve about 1 mg in 3 ml of 0.1 M hydrochloric acid. The solution gives the reaction of primary aromatic amines (2.3.1). E. Dissolve about 20 mg in 1 ml of methanol R and add 1 ml of water R. The solution gives reaction (a) of chlorides (2.3.1). TESTS Appearance of solution. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). Dissolve 0.6 g in methanol R and dilute to 20 ml with the same solvent. Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 50 mg of the substance to be examined in methanol R and dilute to 10.0 ml with the same solvent. Reference solution (a). Dissolve 5 mg of bromhexine impurity C CRS in methanol R, add 1.0 ml of the test solution and dilute to 10.0 ml with the same solvent. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with methanol R. Dilute 1.0 ml of this solution to 10.0 ml with methanol R. Column : — size : l = 0.12 m, Ø = 4.6 mm, — stationary phase: end-capped octadecylsilyl silica gel for chromatography R (3 µm). Mobile phase : mix 0.50 ml of phosphoric acid R in 950 ml of water R, adjust to pH 7.0 with triethylamine R (about 1.5 ml) and dilute to 1000 ml with water R ; mix 20 volumes of this solution with 80 volumes of acetonitrile R. Flow rate : 1.0 ml/min. Detection : spectrophotometer at 248 nm. Injection : 10 µl. Run time : 2.5 times the retention time of bromhexine. Relative retention with reference to bromhexine (retention time = about 11 min) : impurity A = about 0.1 ; impurity B = about 0.2 ; impurity C = about 0.4 ; impurity D = about 0.5. System suitability : reference solution (a) : — resolution : minimum 12.0 between the peaks due to impurity C and bromhexine. Limits : — any impurity : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (0.2 per cent), and not more than 1 such peak has an area greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent), — total: not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.3 per cent), — disregard limit : 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.05 per cent). Loss on drying (2.2.32) : maximum 1.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. General Notices (1) apply to all monographs and other texts

ASSAY Dissolve 0.300 g in 70 ml of alcohol R and add 1 ml of 0.1 M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume between the 2 points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 41.26 mg of C14H21Br2ClN2. STORAGE Protected from light. IMPURITIES Specified impurities : A, B, C, D. Other detectable impurities : E.

A. R = CH2OH : (2-amino-3,5-dibromophenyl)methanol, B. R = CHO : 2-amino-3,5-dibromobenzaldehyde,

C. R = H : N-(2-aminobenzyl)-N-methylcyclohexanamine, D. R = Br : N-(2-amino-5-bromobenzyl)-N-methylcyclohexanamine,

E. (3RS)-6,8-dibromo-3-cyclohexyl-3-methyl-1,2,3,4tetrahydroquinazolin-3-ium. 01/2005:0596

BROMOCRIPTINE MESILATE Bromocriptini mesilas

C33H44BrN5O8S

Mr 751

DEFINITION Bromocriptine mesilate contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of (6aR,9R)-5-bromo-N-[(2R,5S,10aS,10bS)-10b-hydroxy1121



TESTS Appearance of solution. Dissolve 0.25 g in methanol R and dilute to 25 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution B5, BY5 or Y5 (2.2.2, Method II). PRODUCTION pH (2.2.3). Dissolve 0.2 g in a mixture of 2 volumes of methanol R and 8 volumes of carbon dioxide-free water R The production method must be evaluated to determine and dilute to 20 ml with the same mixture of solvents. The the potential for formation of alkyl mesilates, which is pH of the solution is 3.1 to 3.8. particularly likely to occur if the reaction medium contains lower alcohols. Where necessary, the production method Specific optical rotation (2.2.7). Dissolve 0.100 g in a is validated to demonstrate that alkyl mesilates are not mixture of equal volumes of methanol R and methylene detectable in the final product. chloride R and dilute to 10.0 ml with the same mixture of solvents. The specific optical rotation is + 95 to + 105, CHARACTERS calculated with reference to the dried substance. A white or slightly coloured, fine crystalline powder, very Related substances. Examine by liquid chromatography sensitive to light, practically insoluble in water, freely (2.2.29). soluble in methanol, soluble in alcohol, sparingly soluble Test solution. Dissolve 0.500 g of the substance to be in methylene chloride. The identification, tests and assay are to be carried out as examined in 5.0 ml of methanol R and dilute to 10.0 ml with buffer solution pH 2.0 R. rapidly as possible, protected from light. Reference solution (a). Dilute 1.0 ml of the test solution to IDENTIFICATION 100.0 ml with a mixture of equal volumes of buffer solution First identification : B. pH 2.0 R and methanol R. Second identification : A, C, D, E. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 10.0 ml with a mixture of equal volumes of buffer solution A. Dissolve 10.0 mg in 10 ml of methanol R and dilute to pH 2.0 R and methanol R. 200.0 ml with 0.01 M hydrochloric acid. Examined between 250 nm and 380 nm (2.2.25), the solution shows Reference solution (c). Dissolve 5.0 mg of bromocriptine an absorption maximum at 305 nm and a minimum at impurity A CRS in a mixture of equal volumes of buffer 270 nm. The specific absorbance at the maximum is 120 solution pH 2.0 R and methanol R and dilute to 5.0 ml with to 135, calculated with reference to the dried substance. the same mixture of solvents. B. Examine by infrared absorption spectrophotometry Reference solution (d). Dissolve 5.0 mg of bromocriptine (2.2.24), comparing with the spectrum obtained with impurity B CRS in a mixture of equal volumes of buffer bromocriptine mesilate CRS. solution pH 2.0 R and methanol R and dilute to 5.0 ml with C. Examine by thin-layer chromatography (2.2.27), using the same mixture of solvents. a TLC silica gel G plate R. Prepare the solutions Reference solution (e). Mix 0.5 ml of reference solution (c) immediately before use. and 0.5 ml of reference solution (d) and dilute to 10.0 ml Test solution. Dissolve 10 mg of the substance to with a mixture of equal volumes of buffer solution pH 2.0 R be examined in a mixture of 3 volumes of alcohol R, and methanol R. 3 volumes of methanol R and 4 volumes of methylene Reference solution (f). Dilute 1.0 ml of reference solution (c) chloride R and dilute to 10 ml with the same mixture of to 100.0 ml with a mixture of equal volumes of buffer solvents. solution pH 2.0 R and methanol R. Reference solution. Dissolve 10 mg of bromocriptine The chromatographic procedure may be carried out using : mesilate CRS in a mixture of 3 volumes of alcohol R, — a stainless steel column 0.12 m long and 4 mm in 3 volumes of methanol R and 4 volumes of methylene internal diameter packed with octadecylsilyl silica gel for chloride R and dilute to 10 ml with the same mixture of chromatography R (5 µm), solvents. — as mobile phase at a flow rate of 2 ml/min : Apply to the plate 10 µl of each solution. Develop Mobile phase A. A 0.791 g/l solution of ammonium immediately in an unsaturated tank over a path of carbonate R, 15 cm using a mixture of 0.1 volumes of concentrated ammonia R, 1.5 volumes of water R, 3 volumes of Mobile phase B. Acetonitrile R, 2-propanol R, 88 volumes of methylene chloride R and Time Comment Mobile phase A Mobile phase B 100 volumes of ether R. Dry the plate in a current of (min) (per cent V/V) (per cent V/V) cold air for 2 min. Spray with ammonium molybdate 0 - 30 90 → 40 10 → 60 linear gradient solution R3. Dry the plate at 100 °C until the spots 60 appear (about 10 min). The principal spot in the 40 isocratic 30 - 45 chromatogram obtained with the test solution is similar — as detector a spectrophotometer set at 300 nm. in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. Inject 20 µl of reference solution (e) and adjust the sensitivity D. To 0.1 g add 5 ml of dilute hydrochloric acid R and shake of the system so that the heights of the 2 peaks are about for about 5 min. Filter and add 1 ml of barium chloride 20 per cent of the full scale of the recorder. The test is not solution R1. The filtrate remains clear. To a further 0.1 g valid unless the resolution between the peaks corresponding to impurity A and impurity B is at least 1.1. add 0.5 g of anhydrous sodium carbonate R, mix and ignite until a white residue is obtained. Allow to cool Inject 20 µl of all other solutions. In the chromatogram and dissolve the residue in 7 ml of water R (solution A). obtained with the test solution : the area of the peak Solution A gives reaction (a) of sulphates (2.3.1). corresponding to impurity A is not greater than the area E. Solution A obtained in identification test D gives of the principal peak in the chromatogram obtained with reaction (a) of bromides (2.3.1). reference solution (f) (0.02 per cent) and the area of the 2-(1-methylethyl)-5-(2-methylpropyl)-3,6-dioxooctahydro8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6, 6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide monomethanesulphonate, calculated with reference to the dried substance.

1122




peak corresponding to impurity C, with a relative retention of about 1.2, is not greater than 4 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.4 per cent) ; the area of any peak, apart from the principal peak and the peak corresponding to impurity C, is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (0.2 per cent) and not more than one such peak has an area greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, C. (6aR,9S)-5-bromo-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2-(1is not greater than 1.5 times the area of the principal peak methylethyl)-5-(2-methylpropyl)-3,6-dioxooctahydro-8Hin the chromatogram obtained with reference solution (a) oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a, (1.5 per cent). Disregard any peak, apart from the peak due 7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide to impurity A, with an area less than half the area of the ((9S)-2-bromo-α-ergocriptine), principal peak in the chromatogram obtained with reference solution (b) (0.05 per cent). Loss on drying (2.2.32). Not more than 3.0 per cent, determined on 0.500 g by drying in vacuo at 80 °C for 5 h. ASSAY Dissolve 0.500 g in 80 ml of a mixture of 10 volumes of anhydrous acetic acid R and 70 volumes of acetic anhydride R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 75.1 mg of C33H44BrN5O8S.

D. R = OH : (6aR,9R)-5-bromo-7-methyl-4,6,6a,7,8,9hexahydroindolo[4,3-fg]quinoline-9-carboxylic acid,

STORAGE Store in an airtight container, protected from light, at a temperature not exceeding − 15 °C.

E. R = NH2 : (6aR,9R)-5-bromo-7-methyl-4,6,6a,7,8,9hexahydroindolo[4,3-fg]quinoline-9-carboxamide,

IMPURITIES

A. (6aR,9R)-5-bromo-N-[(2R,5S)-2-(1-methylethyl)-5-(2methylpropyl)-3,6-dioxo-2,3,5,6,9,10-hexahydro-8Hoxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a, 7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (2-bromodehydro-α-ergocriptine),

B. (6aR,9R)-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2-(1methylethyl)-5-(2-methylpropyl)-3,6-dioxooctahydro-8Hoxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a, 7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (α-ergocriptine), General Notices (1) apply to all monographs and other texts

F. (6aR,9R)-5-bromo-N-[(2S,5S,10aS,10bS)-10b-hydroxy-2-(1methylethyl)-5-(2-methylpropyl)-3,6-dioxooctahydro-8Hoxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a, 7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide ((2′S)-2-bromo-α-ergocriptine),

G. (6aR,9R)-5-bromo-N-[(2R,5S,10aS,10bS)-10b-methoxy-2(1-methylethyl)-5-(2-methylpropyl)-3,6-dioxooctahydro-8Hoxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a, 7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (2-bromo-10′b-O-methyl-α-ergocriptine). 1123

Bromperidol


01/2005:1178

BROMPERIDOL

nitric acid R and filter. To 1 ml of the filtrate add 1 ml of water R. The solution gives reaction (a) of bromides (2.3.1).

TESTS Bromperidolum Appearance of solution. Dissolve 0.2 g in 20 ml of a 1 per cent V/V solution of lactic acid R. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y7 (2.2.2, Method II). Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 0.100 g of the substance to be examined in methanol R and dilute to 10.0 ml with the same solvent. C21H23BrFNO2 Mr 420.3 Reference solution (a). Dissolve 2.5 mg of bromperidol CRS and 5.0 mg of haloperidol CRS in methanol R and dilute to DEFINITION 50.0 ml with the same solvent. Bromperidol contains not less than 99.0 per cent Reference solution (b). Dilute 5.0 ml of the test solution to and not more than the equivalent of 101.0 per cent 100.0 ml with methanol R. Dilute 1.0 ml of this solution to of 4-[4-(4-bromophenyl)-4-hydroxypiperidin-1-yl]-1-(410.0 ml with methanol R. fluorophenyl)butan-1-one, calculated with reference to the The chromatographic procedure may be carried out using : dried substance. — a stainless steel column 0.1 m long and 4.0 mm in internal CHARACTERS diameter packed with base-deactivated octadecylsilyl A white or almost white powder, practically insoluble in silica gel for chromatography R (3 µm), water, sparingly soluble in methanol and in methylene — as mobile phase at a flow rate of 1.5 ml/min : chloride, slightly soluble in alcohol. Mobile phase A. A 17 g/l solution of tetrabutylammonium hydrogen sulphate R, IDENTIFICATION Mobile phase B. Acetonitrile R, First identification : B, E. Second identification : A, C, D, E. A. Melting point (2.2.14) : 156 °C to 159 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with bromperidol CRS. Examine the substances prepared as discs. C. Examine by thin layer chromatography (2.2.27), using as the coating substance a suitable octadecylsilyl silica gel. Test solution. Dissolve 10 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution (a). Dissolve 10 mg of bromperidol CRS in methanol R and dilute to 10 ml with the same solvent. Reference solution (b). Dissolve 10 mg of bromperidol CRS and 10 mg of haloperidol CRS in methanol R and dilute to 10 ml with the same solvent. Apply separately to the plate 1 µl of each solution. Develop in an unsaturated tank over a path of 15 cm using a mixture of 10 volumes of tetrahydrofuran R, 45 volumes of methanol R and 45 volumes of a 58 g/l solution of sodium chloride R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two spots which may, however, not be completely separated. D. Dissolve about 10 mg in 5 ml of ethanol R. Add 0.5 ml of dinitrobenzene solution R and 0.5 ml of 2 M alcoholic potassium hydroxide solution R. A violet colour is produced that becomes brownish-red after 20 min. E. To 0.1 g in a porcelain crucible add 0.5 g of anhydrous sodium carbonate R. Heat over an open flame for 10 min. Allow to cool. Take up the residue with 5 ml of dilute 1124

Time (min)



Comment

0 - 15

90 → 50

10 → 50

linear gradient

15 - 20

50

50

isocratic elution

20 - 25

90

10


25 = 0

90

10

restart gradient

— as detector a spectrophotometer set at 230 nm. Equilibrate the column for at least 30 min with acetonitrile R and then equilibrate with the initial eluent composition for at least 5 min. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 10 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder. Inject 10 µl of reference solution (a). When the chromatogram is recorded in the prescribed conditions, the retention times are : haloperidol, about 5.5 min and bromperidol about 6 min. The test is not valid unless the resolution between the peaks due to haloperidol and bromperidol is at least 3.0. If necessary, adjust the concentration of acetonitrile in the mobile phase or adjust the time programme for the linear gradient elution. Inject separately 10 µl of methanol R as a blank, 10 µl of the test solution and 10 µl of reference solution (b). In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent) ; the sum of the areas of all peaks, apart from the principal peak, is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent). Disregard any peak obtained with the blank and any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b).


Bromperidol decanoate


Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g in a platinum crucible.

01/2005:1397

BROMPERIDOL DECANOATE Bromperidoli decanoas

ASSAY Dissolve 0.300 g in 50 ml of a mixture of 1 volume of anhydrous acetic acid R and 7 volumes of methyl ethyl ketone R. Titrate with 0.1 M perchloric acid, using 0.2 ml of naphtholbenzein solution R as indicator. 1 ml of 0.1 M perchloric acid is equivalent to 42.03 mg of C21H23BrFNO2. STORAGE Store protected from light. IMPURITIES

C31H41BrFNO3

Mr 574.6

DEFINITION Bromperidol decanoate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of 4-(4bromophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl decanoate, calculated with reference to the dried substance.

CHARACTERS A white or almost white powder, practically insoluble in water, very soluble in methylene chloride, soluble in alcohol. A. R1 = R2 = R3 = H, R4 = F : 1-(4-fluorophenyl)-4-(4-hydroxyIt melts at about 60 °C. 4-phenylpiperidin-1-yl)butan-1-one, IDENTIFICATION A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with bromperidol decanoate CRS. Examine the substances C. R1 = C6H5, R2 = R3 = H, R4 = F : 4-[4-(biphenyl-4-yl)-4prepared as mulls in liquid paraffin R. hydroxypiperidin-1-yl]-1-(4-fluorophenyl)butan-1-one, B. To 0.1 g in a porcelain crucible add 0.5 g of anhydrous sodium carbonate R. Heat over an open flame for 10 min. D. R1 = Br, R2 = H, R3 = C2H5, R4 = F : 4-[4-(4-bromophenyl)Allow to cool. Take up the residue with 5 ml of dilute 4-hydroxypiperidin-1-yl]-1-(3-ethyl-4-fluorophenyl)butannitric acid R and filter. To 1 ml of the filtrate add 1 ml 1-one, of water R. The solution gives reaction (a) of bromides (2.3.1). B. R1 = Br, R2 = F, R3 = R4 = H : 4-[4-(4-bromophenyl)-4hydroxypiperidin-1-yl]-1-(2-fluorophenyl)butan-1-one,

E. 4-[4-(4-bromophenyl)-4-hydroxypiperidin-1-yl]-1-[4-[4-(4bromophenyl)-4-hydroxypiperidin-1-yl]phenyl]butan-1one,

F. 4-[4-(4′-bromobiphenyl-4-yl)-4-hydroxypiperidin-1-yl]-1-(4fluorophenyl)butan-1-one. General Notices (1) apply to all monographs and other texts

TESTS Appearance of solution. Dissolve 2.0 g in methylene chloride R and dilute to 20 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution B5 (2.2.2, Method II). Related substances. Examine by liquid chromatography (2.2.29). Prepare the solutions immediately before use and protect from light. Test solution. Dissolve 0.100 g of the substance to be examined in methanol R and dilute to 10.0 ml with the same solvent. Reference solution (a). Dissolve 2.5 mg of bromperidol decanoate CRS and 2.5 mg of haloperidol decanoate CRS in methanol R and dilute to 50.0 ml with the same solvent. Reference solution (b). Dilute 5.0 ml of the test solution to 100.0 ml with methanol R. Dilute 1.0 ml of this solution to 10.0 ml with methanol R. The chromatographic procedure may be carried out using : — a stainless steel column 0.1 m long and 4.0 mm in internal diameter packed with base-deactivated octadecylsilyl silica gel for chromatography R (3 µm), — as mobile phase at a flow rate of 1.5 ml/min : Mobile phase A. A 27 g/l solution of tetrabutylammonium hydrogen sulphate R, Mobile phase B. Acetonitrile R, 1125

Bromperidol decanoate


Time (min)



Comment

B. R1 = Br, R2 = F, R3 = R4 = H : 4-(4-bromophenyl)-1-[4-(2fluorophenyl)-4-oxobutyl]-piperidin-4-yl decanoate,

0 - 30

80 → 40

20 → 60

linear gradient

30 - 35

40

60

isocratic elution

35 - 40

40 → 80

60 → 20


C. R1 = Br, R2 = H, R3 = C2H5, R4 = F : 4-(4-bromophenyl)1-[4-(3-ethyl-4-fluorophenyl)-4-oxobutyl]-piperidin-4-yl decanoate,

40 = 0

80

20

restart gradient

F. R1 = C6H5, R2 = R3 = H, R4 = F : 4-(biphenyl-4-yl)-1-[4-(4fluorophenyl)-4-oxobutyl]piperidin-4-yl decanoate,

— as detector a spectrophotometer set at 230 nm. Equilibrate the column for at least 30 min with acetonitrile R and then equilibrate at the initial eluent composition for at least 5 min. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 10 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder. Inject 10 µl of reference solution (a). When the chromatogram is recorded in the prescribed conditions, the retention times are : haloperidol decanoate about 24 min ; bromperidol decanoate about 24.5 min. The test is not valid unless the resolution between the peaks due to haloperidol decanoate and bromperidol decanoate is at least 1.5. If necessary, adjust the gradient or the time programme for the linear gradient elution. D. 4-(4-bromophenyl)-1-[4-[4-[4-(4-bromophenyl)-4Inject separately 10 µl of methanol R as a blank, 10 µl of hydroxypiperidin-1-yl]phenyl]-4-oxobutyl]piperidin-4-yl the test solution and 10 µl of reference solution (b). In the decanoate, chromatogram obtained with the test solution : the area of any peak apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent) ; the sum of the areas of all the peaks apart from the principal peak is not greater than three times the area of the principal peak in the chromatogram obtained with reference solution (b) (1.5 per cent). Disregard any peak due to the blank and any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b). Loss on drying (2.2.32). Not more than 0.5 per cent, E. 4-(4′-bromobiphenyl-4-yl)-1-[4-(4-fluorophenyl)-4determined on 1.000 g by drying in vacuo at 30 °C. oxobutyl]piperidin-4-yl decanoate, Sulphated ash (2.4.14). Not more than 0.1 per cent, G. bromperidol, determined on 1.0 g in a platinum crucible. ASSAY Dissolve 0.450 g in 50 ml of a mixture of 1 volume of anhydrous acetic acid R and 7 volumes of methyl ethyl ketone R. Titrate with 0.1 M perchloric acid using 0.2 ml of naphtholbenzein solution R as indicator. 1 ml of 0.1 M perchloric acid is equivalent to 57.46 mg of C31H41BrFNO3. STORAGE Store at a temperature below 25 °C, protected from light. IMPURITIES Specified impurities : A, B, C, D, E, F, G, H, I, J, K. Other detectable impurities : L.

H. n = 5 : 4-(4-bromophenyl)-1-[4-(4-fluorophenyl)-4oxobutyl]piperidin-4-yl octanoate, I. n = 6 : 4-(4-bromophenyl)-1-[4-(4-fluorophenyl)-4oxobutyl]piperidin-4-yl nonanoate, J. n = 8 : 4-(4-bromophenyl)-1-[4-(4-fluorophenyl)-4oxobutyl]piperidin-4-yl undecanoate, K. n = 9 : 4-(4-bromophenyl)-1-[4-(4-fluorophenyl)-4oxobutyl]piperidin-4-yl dodecanoate,

A. R1 = R2 = R3 = H, R4 = F : 1-[4-(4-fluorophenyl)-4oxobutyl]-4-phenylpiperidin-4-yl decanoate, 1126

L. 1-(4-fluorophenyl)ethanone. See the information section on general monographs (cover pages)

Brompheniramine maleate


01/2005:0977

BROMPHENIRAMINE MALEATE Brompheniramini maleas

two clearly separated spots. The upper spot is similar in position and size to the spot in the chromatogram obtained with the reference solution. F. To 0.15 g in a porcelain crucible add 0.5 g of anhydrous sodium carbonate R. Heat over an open flame for 10 min. Allow to cool. Take up the residue in 10 ml of dilute nitric acid R and filter. To 1 ml of the filtrate add 1 ml of water R. The solution gives reaction (a) of bromides (2.3.1).

IDENTIFICATION First identification : A, B, C, D, E. Second identification : A, B, E, F. A. Melting point (2.2.14) : 130 °C to 135 °C. B. Dissolve 65 mg in 0.1 M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute 5.0 ml of this solution to 100.0 ml with 0.1 M hydrochloric acid. Examined between 220 nm and 320 nm (2.2.25), the solution shows an absorption maximum at 265 nm. The specific absorbance at the maximum is 190 to 210. C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with brompheniramine maleate CRS. Examine the substances prepared as discs using potassium bromide R. D. Examine the chromatograms obtained in the test for related substances. The retention time and size of the principal peak in the chromatogram obtained with the test solution are approximately the same as those of the principal peak in the chromatogram obtained with reference solution (a). The chromatogram obtained with reference solution (c) shows two principal peaks with retention times corresponding to the retention times of the peaks obtained with reference solutions (a) and (b). E. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm. Test solution. Dissolve 0.10 g of the substance to be examined in methanol R and dilute to 5.0 ml with the same solvent. Reference solution. Dissolve 56 mg of maleic acid R in methanol R and dilute to 10 ml with the same solvent. Apply to the plate 5 µl of each solution. Develop over a path of 12 cm using a mixture of 3 volumes of water R, 7 volumes of anhydrous formic acid R, 20 volumes of methanol R and 70 volumes of di-isopropyl ether R. Allow the plate to dry in a current of air for a few minutes and examine in ultraviolet light at 254 nm. The chromatogram obtained with the test solution shows

TESTS Appearance of solution. Dissolve 2.0 g in methanol R and dilute to 20 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY6 (2.2.2, Method II). pH (2.2.3). Dissolve 0.20 g in 20 ml of carbon dioxide-free water R. The pH of the solution is 4.0 to 5.0. Optical rotation (2.2.7). Dissolve 2.5 g in water R and dilute to 25.0 ml with the same solvent. The angle of optical rotation measured in a 2 dm tube is − 0.2° to + 0.2°. Related substances. Examine by gas chromatography (2.2.28). Test solution. Dissolve 0.10 g of the substance to be examined in 10 ml of methylene chloride R. Reference solution (a). Dissolve 10 mg of brompheniramine maleate CRS in methylene chloride R and dilute to 1 ml with the same solvent. Reference solution (b). Dissolve 5 mg of chlorphenamine maleate CRS in methylene chloride R and dilute to 1 ml with the same solvent. Reference solution (c). To 0.5 ml of the test solution add 0.5 ml of reference solution (b). The chromatographic procedure may be carried out using : — a glass column 2.3 m long and 2 mm in internal diameter packed with acid- and base-washed silanised diatomaceous earth for gas chromatography R (135-175 µm) impregnated with 3 per cent m/m of polymethylphenylsiloxane R, — nitrogen for chromatography R as the carrier gas at a flow rate of 20 ml/min, — a flame-ionisation detector, maintaining the temperature of the column at 205 °C and that of the injection port and of the detector at 250 °C. Inject 1 µl of each solution. The test is not valid unless, in the chromatogram obtained with reference solution (c), the resolution between the peaks corresponding to brompheniramine and chlorphenamine is at least 1.5. After injecting the test solution, continue the chromatography for at least 2.5 times the retention time of the principal peak. In the chromatogram obtained with the test solution : the sum of the areas of the peaks, apart from the principal peak, is not greater than 1 per cent of the area of the principal peak ; none of the peaks, apart from the principal peak, has an area greater than 0.4 per cent of the area of the principal peak. Disregard any peak with an area less than 0.1 per cent of that of the peak corresponding to brompheniramine in the chromatogram obtained with the test solution. Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 3 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.


1127

C20H23BrN2O4

Mr 435.3

DEFINITION Brompheniramine maleate contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of (3RS)-3-(4-bromophenyl)-N,N-dimethyl-3-(pyridin-2-yl)propan1-amine (Z)-butenedioate, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, soluble in water, freely soluble in alcohol, in methanol and in methylene chloride.

Budesonide


ASSAY Dissolve 0.260 g in 50 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 21.77 mg of C20H23BrN2O4. STORAGE Store protected from light. A. chlorphenamine, B. dexchlorpheniramine,

C. (3RS)-N,N-dimethyl-3-phenyl-3-(pyridin-2-yl)propan-1amine (pheniramine). 01/2005:1075

Budesonidum

Reference solution (a). Dissolve 25 mg budesonide CRS in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 10 ml with the same mixture of solvents. Reference solution (b). Dissolve 12.5 mg triamcinolone acetonide CRS in reference solution (a) and dilute to 5 ml with the same solution.

IMPURITIES

BUDESONIDE

Test solution. Dissolve 25 mg of the substance to be examined in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 10 ml with the same mixture of solvents.

Apply separately to the plate 5 µl of each solution. Prepare the mobile phase by adding a mixture of 1.2 volumes of water R and 8 volumes of methanol R to a mixture of 15 volumes of ether R and 77 volumes of methylene chloride R. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). Spray with alcoholic solution of sulphuric acid R. Heat at 120 °C for 10 min or until spots appear. Allow to cool. Examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with the reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. C. Dissolve about 2 mg in 2 ml of sulphuric acid R. Within 5 min, a yellow colour develops. Within 30 min, the colour changes to brown or reddish-brown. Add cautiously the solution to 10 ml of water R and mix. The colour fades and a clear solution remains.

D. Dissolve about 1 mg in 2 ml of a solution containing 2 g of phosphomolybdic acid R dissolved in a mixture of 10 ml of dilute sodium hydroxide solution R, 15 ml of water R and 25 ml of glacial acetic acid R. Heat for 5 min on a C25H34O6 Mr 430.5 water-bath. Cool in iced water for 10 min and add 3 ml of dilute sodium hydroxide solution R. The solution is blue. DEFINITION Budesonide contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of a mixture of TESTS the C-22S (epimer A) and the C-22R (epimer B) epimers of 16α,17-[(1RS)-butylidenebis(oxy)]-11β,21-dihydroxypregnaRelated substances. Examine by liquid chromatography 1,4-diene-3,20-dione, calculated with reference to the dried (2.2.29) as described under Assay. substance. Inject 20 µl of reference solution (a). Adjust the sensitivity CHARACTERS of the system so that the height of the peak corresponding to epimer B (the first of the two principal peaks) in the A white or almost white, crystalline powder, practically chromatogram obtained is not less than 50 per cent of the insoluble in water, freely soluble in methylene chloride, full scale of the recorder. Inject 20 µl of the test solution, sparingly soluble in alcohol. 20 µl of reference solution (a) and 20 µl of reference IDENTIFICATION solution (b). Continue the chromatography for 1.5 times the retention time of epimer B. In the chromatogram obtained First identification : A. with the test solution : the area of any peak, apart from Second identification : B, C, D. the peaks corresponding to epimer A and epimer B, is not A. Examine by infrared absorption spectrophotometry greater than the sum of the areas of the epimer peaks in (2.2.24), comparing with the spectrum obtained with the chromatogram obtained with reference solution (b) budesonide CRS. Examine the substances prepared as (0.5 per cent) ; the sum of the areas of any such peaks is discs. not greater than the sum of the areas of the epimer peaks in the chromatogram obtained with reference solution (a) B. Examine by thin-layer chromatography (2.2.27), using (1.5 per cent). Disregard any peak with an area less than as the coating substance a suitable silica gel with a 0.1 times the sum of the areas of the epimer peaks in the fluorescent indicator having an optimal intensity at chromatogram obtained with reference solution (b). 254 nm. 1128


Budesonide


Epimer A. Examine by liquid chromatography (2.2.29) as described under Assay. Inject 20 µl of the test solution. The content of epimer A (second peak) is 40.0 per cent to 51.0 per cent of the sum of the areas of the two epimer peaks of budesonide. Methanol. Not more than 0.1 per cent, determined by head-space gas chromatography using the standard addition method (2.2.28). Test solution. Dissolve 2.000 g of the substance to be examined in dimethylacetamide R and dilute to 20.0 ml with the same solvent. Reference solution. Dilute 0.500 g of methanol R in dimethylacetamide R and dilute to 100.0 ml with the same solvent. The following head-space conditions may be used : — equilibration temperature : 80 °C, — equilibration time : 30 min, — transfer-line temperature : 85 °C, — pressurisation time : 10 s, — injection time : 10 s. The chromatographic procedure may be carried out using : — a fused-silica capillary column 30 m long and 0.32 mm in internal diameter coated with macrogol 20 000 R (film thickness 1 µm), — nitrogen for chromatography R as the carrier gas at a pressure of 55 kPa, — a flame-ionisation detector, maintaining the temperature of the column at 50 °C for 5 min, then raising the temperature at a rate of 30 °C per minute to 220 °C and maintaining at 220 °C for 2 min, and maintaining the temperature of the injection port at 250 °C and that of the detector at 300 °C. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. ASSAY Examine by liquid chromatography (2.2.29). Protect the solutions from light throughout the assay. Test solution. Dissolve 25.0 mg of the substance to be examined in 15 ml of acetonitrile R and dilute to 50.0 ml with phosphate buffer solution pH 3.2 R. Allow to stand for at least 15 min before use. Reference solution (a). Dilute 15.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 10.0 ml with the mobile phase. Reference solution (b). Dilute 5.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 10.0 ml with the mobile phase. Reference solution (c). Dissolve 25.0 mg of budesonide CRS in 15 ml of acetonitrile R and dilute to 50.0 ml with phosphate buffer solution pH 3.2 R. The chromatographic procedure may be carried out using : — a stainless steel column 0.12 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.5 ml/min a mixture of 32 volumes of acetonitrile R and 68 volumes of phosphate buffer solution pH 3.2 R, — as detector a spectrophotometer set at 240 nm. Inject 20 µl of reference solution (c). Adjust the sensitivity of the system so that the height of the peak corresponding to epimer B (the first of the two principal peaks) in the General Notices (1) apply to all monographs and other texts

chromatogram obtained is not less than 50 per cent of the full scale of the recorder. When the chromatograms are recorded in the prescribed conditions, the retention time for epimer B is about 16 min. If necessary, adjust the concentration of acetonitrile in the mobile phase (increasing the concentration to decrease the retention time). The test is not valid unless : in the chromatogram obtained with reference solution (c), the resolution between the peaks corresponding to epimer A and epimer B is not less than 1.5 ; the number of theoretical plates determined from the epimer B peak is at least 4000 ; the symmetry factor for the same peak is less than 1.5. Inject 20 µl of reference solution (c) six times. The assay is not valid unless the relative standard deviation of the sum of the peak areas of the two epimers is at most 1.0 per cent. Inject alternatively the test solution and reference solution (c). Calculate the percentage content of C25H34O6 from the sum of the areas of the two epimer peaks. IMPURITIES

A. 11β,16α,17,21-tetrahydroxypregna-1,4-diene-3,20-dione,

B. R = CH3, R′ = H : 16α,17-[(1RS)-ethylidenebis(oxy)]-11β, 21-dihydroxypregna-1,4-diene-3,20-dione, F. R = R′ = CH3 : 16α,17-[1-methylethylidenebis(oxy)]-11β, 21-dihydroxypregna-1,4-diene-3,20-dione,

C. 16α,17-[(1RS)-butylidenebis(oxy)]-11β-hydroxy-17(hydroxymethyl)-D-homoandrosta-1,4-diene-3,17a-dione,

D. 16α,17-[(1RS)-butylidenebis(oxy)]-11β-hydroxy-3,20dioxopregna-1,4-dien-21-al, 1129

Bufexamac

E. 16α,17-[(1RS)-butylidenebis(oxy)]-11β,21dihydroxypregna-1,4,14-triene-3,20-dione,


Reference solution (b). Dissolve 10 mg of salicylic acid R in reference solution (a) and dilute to 5 ml with the same solution. Apply to the plate 10 µl of each solution. Develop over a path of 15 cm using a mixture of 4 volumes of glacial acetic acid R, 20 volumes of dioxan R and 90 volumes of toluene R. Dry the plate in a current of warm air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots.

TESTS Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 20.0 ml with the same solvent. G. 16α,17-[(1RS)-butylidenebis(oxy)]-11β,21-dihydroxypregnReference solution (a). Dilute 5.0 ml of the test solution to 4-ene-3,20-dione. 25.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 100.0 ml with the mobile phase. 01/2005:1179 Reference solution (b). Dissolve 5 mg of bufexamac CRS and 5 mg of salicylic acid R in the mobile phase and dilute BUFEXAMAC to 10 ml with the mobile phase. Dilute 1 ml of the solution to 10 ml with the mobile phase. Bufexamacum The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm) having a specific surface area of 350 m2/g and a pore size of 10 nm, — as mobile phase at a flow rate of 1 ml/min a mixture of C12H17NO3 Mr 223.3 30 volumes of a 1.4 g/l solution of dipotassium hydrogen phosphate R and 70 volumes of methanol R adjusted to DEFINITION pH 3.6 with dilute phosphoric acid R, Bufexamac contains not less than 98.5 per cent and — as detector a spectrophotometer set at 275 nm. not more than the equivalent of 101.5 per cent of Inject 20 µl of reference solution (a) and 20 µl of reference 2-(4-butoxyphenyl)-N-hydroxyacetamide, calculated with solution (b). Adjust the sensitivity of the system so that the reference to the dried substance. height of the principal peak in the chromatogram obtained CHARACTERS with reference solution (a) is at least 50 per cent of the full A white or almost white, crystalline powder, practically scale of the recorder. The test is not valid unless in the insoluble in water, soluble in dimethylformamide, slightly chromatogram obtained with reference solution (b) the soluble in ethyl acetate and in methanol. resolution between the peaks corresponding to salicylic acid and to bufexamac is at least 2.0. IDENTIFICATION Inject 20 µl of the test solution. Continue the chromatography First identification : B. for four times the retention time of bufexamac. In the Second identification : A, C. chromatogram obtained with the test solution : the area of A. Dissolve 20 mg in methanol R and dilute to 20 ml with any peak, apart from the principal peak is not greater than the same solvent. Dilute 1 ml of the solution to 50 ml the area of the principal peak in the chromatogram obtained with methanol R. Examined between 210 nm and 360 nm with reference solution (a) (0.2 per cent) ; the sum of the (2.2.25), the solution shows three absorption maxima, at areas of all the peaks, apart from the principal peak, is not 228 nm, 277 nm and 284 nm. greater than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per B. Examine by infrared absorption spectrophotometry cent). Disregard any peak due to the solvent and any peak (2.2.24), comparing with the spectrum obtained with with an area less than 0.05 times that of the principal peak bufexamac CRS. Examine the substances prepared as in the chromatogram obtained with reference solution (a). discs. C. Examine by thin-layer chromatography (2.2.27), using Loss on drying (2.2.32). Not more than 0.5 per cent, as the coating substance a suitable silica gel with a determined on 1.000 g by drying in vacuo at 80 °C for 3 h. fluorescent indicator having an optimal intensity at Sulphated ash (2.4.14). Not more than 0.1 per cent, 254 nm. determined on 1.0 g. Test solution. Dissolve 10 mg of the substance to be examined in methanol R and dilute to 5 ml with the same ASSAY solvent. Dissolve 0.200 g in 50 ml of dimethylformamide R. Titrate Reference solution (a). Dissolve 20 mg of bufexamac CRS with 0.1 M lithium methoxide, determining the end-point in methanol R and dilute to 10 ml with the same solvent. potentiometrically (2.2.20). 1130


Buflomedil hydrochloride


1 ml of 0.1 M lithium methoxide is equivalent to 22.33 mg of C12H17NO3. STORAGE Store protected from light. IMPURITIES

A. R = OH : 2-(4-butoxyphenyl)acetic acid, B. R = OCH3 : methyl 2-(4-butoxyphenyl)acetate, C. R = OC4H9 : butyl 2-(4-butoxyphenyl)acetate, D. R = NH2 : 2-(4-butoxyphenyl)acetamide. 01/2005:1398

BUFLOMEDIL HYDROCHLORIDE Buflomedili hydrochloridum

C17H26ClNO4

Mr 343.9

DEFINITION 4-(Pyrrolidin-1-yl)-1-(2,4,6-trimethoxyphenyl)butan-1-one hydrochloride. Content : 98.5 per cent to 101.5 per cent (dried substance). CHARACTERS Appearance : white or almost white, microcrystalline powder. Solubility : freely soluble in water, soluble in alcohol, very slightly soluble in acetone. mp : about 195 °C, with decomposition. IDENTIFICATION First identification : B, D. Second identification : A, C, D. A. Dissolve 25.0 mg in alcohol R and dilute to 50.0 ml with the same solvent. Dilute 2.0 ml to 20.0 ml with alcohol R. Examined between 220 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 275 nm. The specific absorbance at the maximum is 143 to 149. B. Infrared absorption spectrophotometry (2.2.24). Preparation : discs. Comparison : buflomedil hydrochloride CRS. C. Thin-layer chromatography (2.2.27). Test solution. Dissolve 40 mg of the substance to be examined in methanol R and dilute to 2 ml with the same solvent. Reference solution. Dissolve 40 mg of buflomedil hydrochloride CRS in methanol R and dilute to 2 ml with the same solvent. Plate : TLC silica gel F254 plate R. Mobile phase : triethylamine R, 2-propanol R, toluene R (5:50:50 V/V/V). Application : 10 µl. Development : over a path of 15 cm. General Notices (1) apply to all monographs and other texts

Drying : in air. Detection : examine in ultraviolet light at 254 nm. Results : the principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. D. It gives reaction (a) of chlorides (2.3.1). TESTS Solution S. Dissolve 2.5 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). pH (2.2.3) : 5.0 to 6.5 for solution S. Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 0.10 g of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (a). Dilute 0.5 ml of the test solution to 100.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (b). Dissolve 2 mg of buflomedil impurity B CRS in the mobile phase. Add 0.5 ml of the test solution and dilute to 100 ml with the mobile phase. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : end-capped octadecylsilyl silica gel for chromatography R (5 µm), — temperature : 40 °C. Mobile phase : mix 45 volumes of acetonitrile R and 55 volumes of a 9.25 g/l solution of potassium dihydrogen phosphate R adjusted to pH 2.5 with phosphoric acid R. Flow rate : 1 ml/min. Detection : spectrophotometer at 210 nm. Injection : 10 µl. Run time : twice the retention time of buflomedil. Retention time : buflomedil = about 5 min. System suitability : reference solution (b) : — resolution : minimum 5.0 between the peaks due to buflomedil and impurity B. Limits : — any impurity : not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.25 per cent), — total : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent), — disregard limit : 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). Heavy metals (2.4.8) : maximum 10 ppm. 2.0 g complies with limit test C. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 2 h. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.300 g in 15 ml of anhydrous acetic acid R and add 35 ml of acetic anhydride R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1131

Bumetanide


Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 50 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the IMPURITIES mobile phase. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (b). Dissolve 2 mg of bumetanide impurity A CRS and 2 mg of bumetanide impurity B CRS in the mobile phase and dilute to 10.0 ml with the mobile A. R1 = OH, R2 = OCH3 : 4-(pyrrolidin-1-yl)-1-(2-hydroxy-4,6- phase. Dilute 1.0 ml of this solution to 100.0 ml with the dimethoxyphenyl)butan-1-one, mobile phase. Column : B. R1 = OCH3, R2 = OH : 4-(pyrrolidin-1-yl)-1-(4-hydroxy-2,6— size : l = 0.15 m, Ø = 4.6 mm, dimethoxyphenyl)butan-1-one, — stationary phase : end-capped octylsilyl silica gel for C. R1 = R2 = OH : 4-(pyrrolidin-1-yl)-1-(2,4-dihydroxy-6chromatography R (3.5 µm). methoxyphenyl)butan-1-one. Mobile phase : mix 70 volumes of methanol R, 25 volumes of water for chromatography R and 5 volumes of a 27.2 g/l solution of potassium dihydrogen phosphate R previously adjusted to pH 7.0 with a 280 g/l solution of potassium 01/2005:1076 hydroxide R ; add tetrahexylammonium bromide R to this mixture to obtain a concentration of 2.17 g/l. BUMETANIDE Flow rate : 1.0 ml/min. Detection : spectrophotometer at 254 nm. Bumetanidum Injection : 10 µl. Run time : 5 times the retention time of bumetanide. Relative retention with reference to bumetanide (retention time = about 6 min) : impurity B = about 0.4 ; impurity A = about 0.6 ; impurity D = about 2.5 ; impurity C = about 4.4. System suitability : reference solution (b) : — resolution : minimum 2.0 between the peaks due to impurity A and impurity B. C17H20N2O5S Mr 364.4 Limits : — impurities A, B, C, D : for each impurity, not more than DEFINITION the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), 3-(Butylamino)-4-phenoxy-5-sulphamoylbenzoic acid. — other impurities : for each impurity, not more than the Content : 99.0 per cent to 101.0 per cent (dried substance). area of the principal peak in the chromatogram obtained CHARACTERS with reference solution (a) (0.1 per cent), Appearance : white, crystalline powder. — total : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (a) Solubility : practically insoluble in water, soluble in acetone (0.2 per cent), and in alcohol, slightly soluble in methylene chloride. It dissolves in dilute solutions of alkali hydroxides. — disregard limit : 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) It shows polymorphism. (0.05 per cent). mp : about 233 °C. Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 4 h. IDENTIFICATION Infrared absorption spectrophotometry (2.2.24). Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. Comparison : bumetanide CRS. If the spectra obtained in the solid state show differences, ASSAY dissolve the substance to be examined and the reference substance separately in acetone R, evaporate to dryness and Dissolve 0.300 g in 50 ml of alcohol R. Add 0.1 ml of phenol red solution R. Titrate with 0.1 M sodium hydroxide until a record new spectra using the residues. violet-red colour is obtained. Carry out a blank titration. TESTS 1 ml of 0.1 M sodium hydroxide is equivalent to 36.44 mg of C17H20N2O5S. Appearance of solution. The solution is clear (2.2.1) and colourless (2.2.2, Method II). Dissolve 0.1 g in a 6 g/l solution of potassium hydroxide R STORAGE and dilute to 20 ml with the same solution. Protected from light. 1 ml of 0.1 M perchloric acid is equivalent to 34.39 mg of C17H26ClNO4.

1132


Bupivacaine hydrochloride


Reference solution. Dissolve 25 mg of bupivacaine hydrochloride CRS in methanol R and dilute to 5 ml with the same solvent. Plate : TLC silica gel G plate R. Mobile phase : concentrated ammonia R, methanol R (0.1:100 V/V). Application : 5 µl. Development : over a path of 10 cm. Drying : in air. Detection : spray with dilute potassium iodobismuthate solution R. A. R1 = H, R2 = NO2 : 3-nitro-4-phenoxy-5-sulphamoylbenzoic Results : the principal spot in the chromatogram obtained acid, with the test solution is similar in position, colour and B. R1 = H, R2 = NH2 : 3-amino-4-phenoxy-5size to the principal spot in the chromatogram obtained sulphamoylbenzoic acid, with the reference solution. C. R1 = C4H9, R2 = NH-C4H9 : butyl 3-(butylamino)-4-phenoxy- C. Dissolve 0.1 g in 10 ml of water R, add 2 ml of dilute sodium hydroxide solution R and shake with 2 quantities, 5-sulphamoylbenzoate, each of 15 ml, of ether R. Dry the combined ether layers over anhydrous sodium sulphate R and filter. Evaporate the ether, recrystallise the residue from alcohol (90 per cent V/V) R and dry under reduced pressure. The crystals melt (2.2.14) at 105 °C to 108 °C. D. It gives reaction (a) of chlorides (2.3.1). IMPURITIES Specified impurities : A, B, C, D.

IDENTIFICATION First identification : A, D. Second identification : B, C, D. A. Infrared absorption spectrophotometry (2.2.24). Preparation : discs of potassium bromide R. Comparison : bupivacaine hydrochloride CRS. B. Thin-layer chromatography (2.2.27). Test solution. Dissolve 25 mg of the substance to be examined in methanol R and dilute to 5 ml with the same solvent.

TESTS Solution S. Dissolve 1.0 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Acidity or alkalinity. To 10 ml of solution S add 0.2 ml of 0.01 M sodium hydroxide ; the pH (2.2.3) is not less than 4.7. Add 0.4 ml of 0.01 M hydrochloric acid ; the pH is not greater than 4.7. Related substances. Gas chromatography (2.2.28). Internal standard solution. Dissolve 25 mg of methyl behenate R in methylene chloride R and dilute to 500 ml with the same solvent. Test solution. Dissolve 50.0 mg of the substance to be examined in 2.5 ml of water R, add 2.5 ml of dilute sodium hydroxide solution R and extract with 2 quantities, each of 5 ml, of the internal standard solution. Filter the lower layer. Reference solution (a). Dissolve 10 mg of the substance to be examined, 10 mg of bupivacaine impurity B CRS and 10 mg of bupivacaine impurity E CRS in 2.5 ml of water R, add 2.5 ml of dilute sodium hydroxide solution R and extract with 2 quantities, each of 5 ml, of the internal standard solution. Filter the lower layer and dilute to 20 ml with the internal standard solution. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with the internal standard solution. Reference solution (c). Dilute 5.0 ml of reference solution (b) to 10.0 ml with the internal standard solution. Reference solution (d). Dilute 1.0 ml of reference solution (b) to 10.0 ml with the internal standard solution. Column : — material : fused silica, — size : l = 30 m, Ø = 0.32 mm, — stationary phase : poly(dimethyl)(diphenyl)siloxane R (film thickness 0.25 µm). Carrier gas : helium for chromatography R. Flow rate : 2.5 ml/min. Split ratio : 1:12.


1133

D. 3-[[(2RS)-2-ethylhexyl]amino]-4-phenoxy-5sulphamoylbenzoic acid. 01/2005:0541

BUPIVACAINE HYDROCHLORIDE Bupivacaini hydrochloridum

C18H29ClN2O,H2O

Mr 342.9

DEFINITION (2RS)-1-Butyl-N-(2,6-dimethylphenyl)piperidine-2carboxamide hydrochloride monohydrate. Content : 98.5 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white, crystalline powder or colourless crystals. Solubility : soluble in water, freely soluble in alcohol. mp : about 254 °C, with decomposition.

Bupivacaine hydrochloride


Temperature :

Column

Time (min)

Temperature (°C)

0

180

0 - 10

180 → 230

10 - 15

230

Injection port

250

Detector

250

Detection : flame ionisation. Injection : 1 µl. Relative retention with reference to bupivacaine (retention time = about 10 min) : impurity C = about 0.5 ; impurity A = about 0.6 ; impurity B = about 0.7 ; impurity D = about 0.8 ; impurity E = about 1.1 ; internal standard = about 1.4. System suitability : reference solution (a) : — resolution : minimum 3.0 between the peaks due to bupivacaine and impurity E.

test B. Prepare the standard using lead standard solution (1 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a mixture of 15 volumes of water R and 85 volumes of methanol R. Loss on drying (2.2.32) : 4.5 per cent to 6.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.250 g in a mixture of 20 ml of water R and 25 ml of alcohol R. Add 5.0 ml of 0.01 M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1 M ethanolic sodium hydroxide. Read the volume added between the 2 points of inflexion. 1 ml of 0.1 M ethanolic sodium hydroxide is equivalent to 32.49 mg of C18H29ClN2O. STORAGE Protected from light. IMPURITIES

Limits : — impurity B : calculate the ratio (R) of the area of the principal peak to the area of the peak due to the internal standard from the chromatogram obtained with reference solution (c) ; from the chromatogram obtained with the test solution, calculate the ratio of the area of the peak due to impurity B to the area of the peak due to the A. N-(2,6-dimethylphenyl)pyridine-2-carboxamide, internal standard : this ratio is not greater than R (0.5 per cent), — any other impurity : calculate the ratio (R) of the area of the principal peak to the area of the peak due to the internal standard from the chromatogram obtained with reference solution (d) ; from the chromatogram obtained with the test solution, calculate the ratio of the area of any peak, apart from the principal peak, the peak due to impurity B and the peak due to the internal standard, to the area of the peak due to the internal standard : this ratio is not greater than R (0.1 per cent), — total: calculate the ratio (R) of the area of the principal peak to the area of the peak due to the internal standard from the chromatogram obtained with reference solution (b) ; from the chromatogram obtained with the test solution, calculate the ratio of the sum of the areas of any peaks, apart from the principal peak and the peak due to the internal standard, to the area of the peak due to the internal standard : this ratio is not greater than R (1.0 per cent),

B. (2RS)-N-(2,6-dimethylphenyl)piperidine-2-carboxamide,

C. 1-(2,6-dimethylphenyl)-1,5,6,7-tetrahydro-2H-azepin-2-one,

— disregard limit : ratio less than 0.01 times R (0.01 per cent). 2,6-Dimethylaniline : maximum 100 ppm.

D. R1 = R2 = Cl : (2RS)-2,6-dichloro-N-(2,6Dissolve 0.50 g in methanol R and dilute to 10 ml with the dimethylphenyl)hexanamide, same solvent. To 2 ml of the solution add 1 ml of a freshly prepared 10 g/l solution of dimethylaminobenzaldehyde R E. R1 = H, R2 = NH-(CH2)3-CH3 : 6-(butylamino)-N-(2,6in methanol R and 2 ml of glacial acetic acid R and allow dimethylphenyl)hexanamide, to stand for 10 min. Any yellow colour in the solution is not more intense than that in a standard prepared at the same time and in the same manner using 2 ml of a 5 mg/l solution of 2,6-dimethylaniline R in methanol R. Heavy metals (2.4.8) : maximum 10 ppm. Dissolve 2.0 g in a mixture of 15 volumes of water R and 85 volumes of methanol R and dilute to 20 ml with the same mixture of solvents. 12 ml of the solution complies with limit F. 2,6-dimethylaniline. 1134



Buprenorphine

01/2005:1180 — as detector a spectrophotometer set at 288 nm, maintaining the temperature of the column at 40 °C. BUPRENORPHINE Inject 20 µl of reference solution (a). Adjust the flow rate so that the retention time of the peak corresponding to buprenorphine is about 15 min. The test is not valid unless Buprenorphinum the chromatogram obtained with reference solution (a) presents 2 peaks, the first having a retention time of 0.93 relative to the second peak (buprenorphine). Inject 20 µl of each solution. Record the chromatogram of the test solution for 2.5 times the retention time of the principal peak. In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.25 per cent) ; the sum of the areas of such peaks is not greater than the area of the peak C29H41NO4 Mr 467.6 in the chromatogram obtained with reference solution (c) (0.65 per cent). Disregard any peak with an area less than DEFINITION that of the principal peak in the chromatogram obtained with reference solution (d). Buprenorphine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of (2S)-2Loss on drying (2.2.32). Not more than 1.0 per cent, [17-(cyclopropylmethyl)-4,5α-epoxy-3-hydroxy-6-methoxydetermined on 1.000 g by drying in an oven at 100-105 °C. 6α,14-ethano-14α-morphinan-7α-yl]-3,3-dimethylbutan-2-ol, calculated with reference to the dried substance. ASSAY Dissolve 0.400 g in 40 ml of anhydrous acetic acid R. Titrate CHARACTERS with 0.1 M perchloric acid until the colour changes from A white or almost white, crystalline powder, very slightly violet-blue to green, using 0.1 ml of crystal violet solution R soluble in water, freely soluble in acetone, soluble in as indicator. methanol, slightly soluble in cyclohexane. It dissolves in 1 ml of 0.1 M perchloric acid is equivalent to 46.76 mg of dilute solutions of acids. C29H41NO4. It melts at about 217 °C. STORAGE IDENTIFICATION Store protected from light. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of IMPURITIES buprenorphine. TESTS Solution S. Dissolve 0.250 g in ethanol R and dilute to 25.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Specific optical rotation (2.2.7) : − 103 to − 107, determined on solution S and calculated with reference to the dried substance. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (a). Dissolve 5.0 mg of the substance to be examined in 2.0 ml of methanol R. Add 0.25 ml of 2 M hydrochloric acid. Reference solution (b). Dilute 0.5 ml of the test solution to 200.0 ml with the mobile phase. Reference solution (c). Dilute 0.65 ml of the test solution to 100.0 ml with the mobile phase. Reference solution (d). Dilute 4.0 ml of reference solution (b) to 10.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of about 1 ml/min, a mixture of 10 volumes of a 10 g/l solution of ammonium acetate R and 60 volumes of methanol R, General Notices (1) apply to all monographs and other texts

A. R1 = H, R2 = CH2-CH2-CH=CH2 : (2S)-2-[17-(but-3-enyl)4,5α-epoxy-3-hydroxy-6-methoxy-6α,14-ethano-14αmorphinan-7α-yl]-3,3-dimethylbutan-2-ol, B. R1 = R2 = H : (2S)-2-(4,5α-epoxy-3-hydroxy-6-methoxy-6α, 14-ethano-14α-morphinan-7α-yl)-3,3-dimethylbutan-2-ol, C. R1 = CH3, R2 = CN : 4,5α-epoxy-7α-[(1S)-1-hydroxy-1, 2,2-trimethylpropyl]-3,6-dimethoxy-6α,14-ethano-14αmorphinan-17-carbonitrile,

D. R1 = R2 = CH3 : (2S)-2-[17-(cyclopropylmethyl)-4,5αepoxy-3,6-dimethoxy-6α,14-ethano-14α-morphinan-7α-yl]3,3-dimethylbutan-2-ol (3-O-methylbuprenorphine), E. R1 = R2 = H : (2S)-2-[17-(cyclopropylmethyl)-4,5α-epoxy3,6-dihydroxy-6α,14-ethano-14α-morphinan-7α-yl]-3,3dimethylbutan-2-ol (6-O-desmethylbuprenorphine). 1135

Buprenorphine hydrochloride


01/2005:1181 Reference solution (c). Dilute 0.65 ml of the test solution to 100.0 ml with the mobile phase. solution (d). Dilute 4.0 ml of reference solution (b) BUPRENORPHINE HYDROCHLORIDE Reference to 10.0 ml with the mobile phase. The chromatographic procedure may be carried out using : Buprenorphini hydrochloridum — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as the mobile phase at a flow rate of about 1 ml/min, a mixture of 10 volumes of a 10 g/l solution of ammonium acetate R and 60 volumes of methanol R, — as detector a spectrophotometer set at 288 nm, maintaining the temperature of the column at 40 °C. Inject 20 µl of reference solution (a). Adjust the flow rate C29H42ClNO4 Mr 504.1 so that the retention time of the peak corresponding to buprenorphine is about 15 min. The test is not valid unless the chromatogram obtained with reference solution (a) DEFINITION presents two peaks, the first having a retention time of 0.93 relative to the second peak (buprenorphine). Inject Buprenorphine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of 101.0 per separately 20 µl of each solution. Record the chromatogram of the test solution for 2.5 times the retention time of the cent of (2S)-2-[17-(cyclopropylmethyl)-4,5α-epoxy-3main peak. In the chromatogram obtained with the test hydroxy-6-methoxy-6α,14-ethano-14α-morphinan-7α-yl]-3,3dimethylbutan-2-ol hydrochloride, calculated with reference solution : the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the to the dried substance. chromatogram obtained with reference solution (b) (0.25 per cent) and the sum of the areas of such peaks is not greater CHARACTERS than the area of the peak in the chromatogram obtained A white or almost white, crystalline powder, sparingly with reference solution (c) (0.65 per cent). Disregard any soluble in water, freely soluble in methanol, soluble in peak with an area less than that of the principal peak in the alcohol, practically insoluble in cyclohexane. chromatogram obtained with reference solution (d). Loss on drying (2.2.32). Not more than 1.0 per cent, IDENTIFICATION determined on 1.000 g by heating in an oven at 115-120 °C. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum ASSAY of buprenorphine hydrochloride. Dissolve 0.400 g in 40 ml of anhydrous acetic acid R and add B. 3 ml of solution S (see Tests) gives reaction (a) of chlorides 10 ml of acetic anhydride R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). (2.3.1). 1 ml of 0.1 M perchloric acid is equivalent to 50.41 mg of C29H42ClNO4. TESTS Solution S. Dissolve 0.250 g in 5.0 ml of methanol R and while stirring dilute to 25.0 ml with carbon dioxide-free water R.


Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Acidity or alkalinity. To 10.0 ml of solution S add 0.05 ml of methyl red solution R. Not more than 0.2 ml of 0.02 M sodium hydroxide or 0.02 M hydrochloric acid is required to change the colour of the indicator.

IMPURITIES

Specific optical rotation (2.2.7). Dissolve 0.100 g in methanol R and dilute to 10.0 ml with the same solvent. The specific optical rotation is − 92 to − 98, calculated with reference to the dried substance. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (a). Dissolve 5 mg of the substance to be examined in 2.0 ml of methanol R. Add 0.25 ml of 2 M hydrochloric acid. Reference solution (b). Dilute 0.5 ml of the test solution to 200.0 ml with the mobile phase. 1136

A. R1 = H, R2 = CH2-CH2-CH=CH2 : (2S)-2-[17-(but-3-enyl)4,5α-epoxy-3-hydroxy-6-methoxy-6α,14-ethano-14αmorphinan-7α-yl]-3,3-dimethylbutan-2-ol, B. R1 = H, R2 = H : (2S)-2-(4,5α-epoxy-3-hydroxy-6-methoxy6α,14-ethano-14α-morphinan-7α-yl)-3,3-dimethylbutan-2ol, C. R1 = CH3, R2 = CN : 4,5α-epoxy-7α-[(1S)-1-hydroxy-1, 2,2-trimethylpropyl]-3,6-dimethoxy-6α,14-ethano-14αmorphinan-17-carbonitrile.



Buserelin

01/2005:1077 Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 5.0 mg of the substance to be examined in 5.0 ml of the mobile phase. BUSERELIN Reference solution (a). Dissolve the contents of a vial of D-His-buserelin CRS in the mobile phase. Dilute an Buserelinum appropriate volume of this solution in the mobile phase to obtain a final concentration of 1 mg/ml. Add 1.0 ml of the test solution to 1.0 ml of this solution. Reference solution (b). Dissolve the contents of a vial of buserelin CRS in the mobile phase. Dilute an appropriate volume of this solution in the mobile phase to obtain a final concentration of 1.0 mg/ml. Reference solution (c). Dilute 1.0 ml of the test solution to C60H86N16O13 Mr 1239 100.0 ml with the mobile phase. Column : DEFINITION — size : l = 0.25 m, Ø = 4 mm, 5-Oxo-L-prolyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-O-(1,1dimethylethyl)-D-seryl-L-leucyl-L-arginyl-L-prolylethylamide. — stationary phase : octadecylsilyl silica gel for chromatography R (5 µm). Synthetic nonapeptide analogue of human gonadotrophin-releasing hormone GnRH with agonistic Mobile phase : mix 200 ml of acetonitrile R and 700 ml of an activity to gonadorelin. It is obtained by chemical synthesis 11.2 g/l solution of phosphoric acid R and adjust to pH 2.5 and is available as an acetate. with triethylamine R. Content : 95.0 per cent to 102.0 per cent (anhydrous, acetic Flow rate : 0.8 ml/min. acid-free substance). Detection : spectrophotometer at 220 nm. Injection : 10 µl of the test solution, reference solution (a) CHARACTERS and reference solution (c). Appearance : white or slightly yellowish powder, Relative retention with reference to buserelin (retention hygroscopic. time = about 36 min) : impurity B = about 0.76 ; Solubility : sparingly soluble in water and in dilute acids. impurity C = about 0.83 ; impurity A = about 0.90 ; impurity D = about 0.94 ; impurity E = about 0.94. IDENTIFICATION System suitability : reference solution (a) : A. Examine the chromatograms obtained in the assay. Results : the principal peak in the chromatogram obtained — resolution : minimum 1.5 between peaks due to impurity A and buserelin. with the test solution is similar in retention time and size to the principal peak in the chromatogram obtained with Limits : reference solution (b). — sum of impurities D and E : not more than 3 times the B. Nuclear magnetic resonance spectrometry (2.2.33). area of the principal peak in the chromatogram obtained with reference solution (c) (3 per cent), Preparation : 4 mg/ml solution in a mixture of 20 volumes of deuterated acetic acid R and 80 volumes of deuterium — any other impurity : for each impurity, not more than oxide R. 3 times the area of the principal peak in the chromatogram obtained with reference solution (c) (3 per cent), Results : the 1H NMR spectrum obtained is qualitatively similar to the Ph. Eur. reference spectrum of buserelin. — total : not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (c) C. Amino acid analysis (2.2.56). For protein hydrolysis use (5 per cent), Method 1 and for analysis use Method 1. — disregard limit : 0.1 times the area of the principal peak Express the content of each amino acid in moles. in the chromatogram obtained with reference solution (c) Calculate the relative proportions of the amino acids, (0.1 per cent). taking one-sixth of the sum of the number of moles of glutamic acid, histidine, tyrosine, leucine, arginine, Acetic acid (2.5.34) : 3.0 per cent to 7.0 per cent. proline as equal to one. The values fall within the Test solution. Dissolve 20.0 mg of the substance to be following limits : serine 1.4 to 2.0 ; proline 0.8 to 1.2 ; examined in a mixture of 5 volumes of mobile phase B and glutamic acid 0.9 to 1.1 ; leucine 0.9 to 1.1 ; tyrosine 0.9 95 volumes of mobile phase A and dilute to 10.0 ml with the to 1.1 ; histidine 0.9 to 1.1 and arginine 0.9 to 1.1. Not same mixture of solvents. more than traces of other amino acids are present, with Water (2.5.12) : maximum 4.0 per cent, determined on the exception of tryptophan. 80.0 mg. TESTS Bacterial endotoxins (2.6.14) : less than 55.5 IU/mg, if intended for use in the manufacture of parenteral dosage Appearance of solution. A 10 g/l solution is clear (2.2.1) and not more intensely coloured than reference solution Y7 forms without a further appropriate procedure for the removal of bacterial endotoxins. (2.2.2, Method II). Specific optical rotation (2.2.7) : − 49 to − 58 (anhydrous, ASSAY acetic acid-free substance), determined on a 10 g/l solution. Liquid chromatography (2.2.29) as described in the test for Specific absorbance (2.2.25) : 49 to 56 by measuring at the related substances with the following modification. absorption maximum at 278 nm (anhydrous, acetic acid-free Injection : 10 µl of the test solution and reference solution (b). substance). Calculate the content of buserelin (C60H86N16O13) using the Dissolve 10.0 mg in 100.0 ml of 0.01 M hydrochloric acid. areas of the peaks of the chromatograms obtained and the stated content of C60H86N16O13 in buserelin CRS. General Notices (1) apply to all monographs and other texts

1137

Busulfan


STORAGE In an airtight container, protected from light, at a temperature of 2 °C to 8 °C. If the substance is sterile, store in an airtight, sterile, tamper-proof container.

A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with busulfan CRS. B. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. Dissolve 20 mg of the substance to be LABELLING examined in 2 ml of acetone R. The label states : Reference solution. Dissolve 20 mg of busulfan CRS in — the mass of peptide in the container, 2 ml of acetone R. — where applicable, that the substance is free from bacterial Apply separately to the plate 5 µl of each solution. endotoxins. Develop over a path of 15 cm using a mixture of equal volumes of acetone R and toluene R. Dry the IMPURITIES plate in a stream of hot air and spray with anisaldehyde Specified impurities : A, B, C, D, E. solution R. Heat the plate at 120 °C. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. C. To 0.1 g add 5 ml of 1 M sodium hydroxide. Heat until a clear solution is obtained. Allow to cool. To 2 ml of the solution add 0.1 ml of potassium permanganate solution R. The colour changes from purple through A. X2 = D-His, X4 = L-Ser, X5 = L-Tyr : [2-D-histidine]buserelin, violet to blue and finally to green. Filter and add 1 ml of ammoniacal silver nitrate solution R. A precipitate B. X2 = L-His, X4 = D-Ser, X5 = L-Tyr : [4-D-serine]buserelin, is formed. D. X2 = L-His, X4 = L-Ser, X5 = D-Tyr : [5-D-tyrosine]buserelin, D. To 0.1 g add 0.1 g of potassium nitrate R and 0.25 g of sodium hydroxide R, mix and heat to fusion. Allow to cool and dissolve the residue in 5 ml of water R. Adjust to pH 1 to 2 using dilute hydrochloric acid R. The solution gives reaction (a) of sulphates (2.3.1). C. buserelin-(3-9)-peptide,

E. [1-(5-oxo-D-proline)]buserelin.

BUSULFAN Busulfanum

TESTS Appearance of solution. Dissolve 0.25 g in 20 ml of acetonitrile R, dilute to 25 ml with water R and examine immediately. The solution is clear (2.2.1) and not more intensely coloured than reference solution B7 (2.2.2, Method II). Acidity. Dissolve 0.20 g with heating in 50 ml of ethanol R. Add 0.1 ml of methyl red solution R. Not more than 0.05 ml of 0.1 M sodium hydroxide is required to change the colour of the indicator. 01/2005:0542 Loss on drying (2.2.32). Not more than 2.0 per cent, determined on 1.000 g by drying in vacuo at 60 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

ASSAY To 0.250 g add 50 ml of water R. Shake. Boil under a reflux condenser for 30 min and, if necessary, make up to the initial volume with water R. Allow to cool. Using 0.3 ml of phenolphthalein solution R as indicator, titrate with 0.1 M C6H14O6S2 Mr 246.3 sodium hydroxide until a pink colour is obtained. 1 ml of 0.1 M sodium hydroxide is equivalent to 12.32 mg DEFINITION of C6H14O6S2. Busulfan contains not less than 99.0 per cent and not more STORAGE than the equivalent of 100.5 per cent of butane-1,4-diyl di(methanesulphonate), calculated with reference to the Store in an airtight container, protected from light. dried substance. CHARACTERS A white or almost white, crystalline powder, very slightly soluble in water, freely soluble in acetone and in acetonitrile, very slightly soluble in alcohol. It melts at about 116 °C. IDENTIFICATION First identification : A. Second identification : B, C, D. 1138

01/2005:1847

BUTCHER’S BROOM Rusci rhizoma DEFINITION Dried, whole or fragmented underground parts of Ruscus aculeatus L.


Butcher’s broom


Content : minimum 1.0 per cent of total sapogenins, expressed as ruscogenins [mixture of neoruscogenin (C27H40O4 ; Mr 428.6) and ruscogenin (C27H42O4 ; Mr 430.6)] (dried drug).

Top of the plate Several zones of various colours Stigmasterol: a violet zone

A violet zone

_______

_______

CHARACTERS Macroscopic and microscopic characters described under identification tests A and B.

A violet zone Ruscogenins : a yellow zone

A yellow zone (ruscogenins) Several zones of various colours

_______

IDENTIFICATION A. The rhizome consists of yellowish, branched, articulated, somewhat knotty pieces, cylindrical or subconical, about 5 cm to 10 cm long and about 5 mm thick. The surface is marked with thin annulations about 1 mm to 3 mm wide, separated from one another ; rounded scars of the aerial stems are present on the upper surface. On the lower surface numerous roots, or their scars, occur ; the roots are about 2 mm in diameter and similar in colour to the rhizome. The outer layer is easily detached, revealing a yellowish-white, very hard central cylinder.

_______

Reference solution

Test solution

TESTS Foreign matter (2.8.2) : maximum 5 per cent. Loss on drying (2.2.32) : maximum 12.0 per cent, determined on 1.000 g of the powdered drug (355) by drying in an oven at 100-105 °C for 2 h. Total ash (2.4.16) : maximum 12.0 per cent. Ash insoluble in hydrochloric acid (2.8.1) : maximum 5.0 per cent.

B. Reduce to a powder (355). The powder is yellowish. Examine under a microscope using chloral hydrate solution R. The powder shows groups of sclereids from the ground tissue of the rhizome, variously-shaped cells, ranging from rounded to elongated or rectangular ; the walls are moderately thickened and distinctly beaded, with large, rounded to oval pits. Fragments of the endodermis composed of a single layer of irregularly-thickened cells. Groups of rounded parenchymatous cells, thickened at the corners, with small, triangular intercellular spaces ; thin-walled parenchyma containing raphides of calcium oxalate. Groups of thick-walled fibres and small vessels, up to about 50 µm in diameter, the walls showing numerous small, slit-shaped pits.

ASSAY Liquid chromatography (2.2.29). Test solution. To 2.000 g of the powdered drug (355), add 60 ml of ethanol R, 15 ml of water R and 0.2 g of potassium hydroxide R. Extract under reflux on a water-bath for 4 h. Allow to cool and filter into a 100 ml volumetric flask. Rinse the extraction flask and the residue in the filter with 3 quantities, each of 10 ml, of ethanol R and add the rinsings to the volumetric flask. Dilute to 100.0 ml with ethanol R. Introduce 25.0 ml of the solution into a round-bottomed flask fitted to a rotary evaporator and evaporate to dryness. Dissolve the residue in 10 ml of butanol R, add 3 ml of hydrochloric acid R1 and 8 ml of water R. Heat under reflux on a water-bath for 1 h. Allow to cool and transfer the liquid C. Thin-layer chromatography (2.2.27) into a separating funnel, rinse the round-bottomed flask with Test solution. Introduce 1.0 g of the powdered drug (355) 2 quantities, each of 10 ml, of butanol R. Add the rinsings to the separating funnel. Extract with 3 quantities, each of and 50 ml of dilute hydrochloric acid R into a 100 ml 20 ml, of butanol R saturated with water R. Combine the flask with a ground-glass neck. Heat on a water-bath butanolic extracts and evaporate to dryness using a rotary under a reflux condenser for 40 min. Allow to cool and evaporator. Dissolve the residue in 20 ml of methanol R and extract the unfiltered mixture with 3 quantities, each of transfer to a 100 ml volumetric flask. Rinse the extraction 25 ml, of methylene chloride R. Combine the organic flask with 20 ml, 20 ml and 10 ml of methanol R and add solutions and dry over anhydrous sodium sulphate R. the rinsings to the volumetric flask. Dilute to 100 ml with Filter and evaporate to dryness. Dissolve the residue in methanol R. 5 ml of methanol R. Reference solution. Dissolve 5.0 mg of ruscogenins R in Reference solution. Dissolve 1 mg of ruscogenins R and 100 ml of methanol R. 1 mg of stigmasterol R in methanol R and dilute to 5 ml Column : with the same solvent. — size : l = 0.25 m, Ø = 4.6 mm, Plate : TLC silica gel plate R. — stationary phase : octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : methanol R, methylene chloride R Mobile phase : (7:93 V/V). — mobile phase A : water R, Application : 10 µl, as bands. — mobile phase B : acetonitrile for chromatography R, Development : over a path of 15 cm. Time Mobile phase A Mobile phase B Drying : in air. Detection : spray with vanillin reagent R, dry the plate in an oven at 100-105 °C for 1 min and examine in daylight. Results : see below the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. Furthermore, other weak zones may be present in the chromatogram obtained with the test solution. General Notices (1) apply to all monographs and other texts

(min)

(per cent V/V)

(per cent V/V)

0 - 25

40

60

25 - 27

40 → 0

60 → 100

27 - 37

0

100

37 - 39

0 → 40

100 → 60

39 - 42

40

60

Flow rate : 1.2 ml/min. 1139

Butyl parahydroxybenzoate


Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution BY6 (2.2.2, Method II). Acidity. To 2 ml of solution S add 3 ml of alcohol R, 5 ml of carbon dioxide-free water R and 0.1 ml of bromocresol green solution R. Not more than 0.1 ml of 0.1 M sodium hydroxide is required to change the colour of the indicator to blue. Related substances. Thin-layer chromatography (2.2.27). Test solution (a). Dissolve 0.10 g of the substance to be examined in acetone R and dilute to 10 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with acetone R. Reference solution (a). Dilute 0.5 ml of test solution (a) to 100 ml with acetone R. 01/2005:0881 Reference solution (b). Dissolve 10 mg of butyl parahydroxybenzoate CRS in acetone R and dilute to BUTYL PARAHYDROXYBENZOATE 10 ml with the same solvent. Reference solution (c). Dissolve 10 mg of propyl Butylis parahydroxybenzoas parahydroxybenzoate R in 1 ml of test solution (a) and dilute to 10 ml with acetone R. Plate : suitable octadecylsilyl silica gel with a fluorescent indicator having an optimal intensity at 254 nm as the coating substance. Mobile phase : glacial acetic acid R, water R, methanol R (1:30:70 V/V/V). C11H14O3 Mr 194.2 Application : 2 µl. Development : over a path of 15 cm. DEFINITION Drying : in air. Butyl 4-hydroxybenzoate. Detection : examine in ultraviolet light at 254 nm. Content : 98.0 per cent to 102.0 per cent. System suitability : the chromatogram obtained with reference solution (c) shows 2 clearly separated principal CHARACTERS spots. Appearance : white or almost white, crystalline powder or Limits : colourless crystals. — any impurity : any spot in the chromatogram obtained Solubility : very slightly soluble in water, freely soluble in with test solution (a), apart from the principal spot, is not alcohol and in methanol. more intense than the spot in the chromatogram obtained with reference solution (a) (0.5 per cent). IDENTIFICATION First identification : A, B. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. Second identification : A, C, D. Detection : spectrophotometer at 203 nm. Injection : 20 µl. Retention time with reference to neoruscogenin (retention time = about 16 min) : ruscogenin = about 1.2. System suitability : reference solution : — resolution : minimum 1.5 between the peaks due to neoruscogenin and ruscogenin. Calculate the percentage content of sapogenins expressed as ruscogenins (neoruscogenin and ruscogenin) in the test solution by comparing the areas of the peaks in the chromatograms obtained with the test solution and the reference solution.

A. Melting point (2.2.14) : 68 °C to 71 °C. B. Infrared absorption spectrophotometry (2.2.24). Comparison : butyl parahydroxybenzoate CRS. C. Examine the chromatograms obtained in the test for related substances. Results : the principal spot in the chromatogram obtained with test solution (b) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (b). D. To about 10 mg in a test-tube add 1 ml of sodium carbonate solution R, boil for 30 s and cool (solution A). To a further 10 mg in a similar test-tube add 1 ml of sodium carbonate solution R ; the substance partly dissolves (solution B). Add at the same time to solution A and solution B 5 ml of aminopyrazolone solution R and 1 ml of potassium ferricyanide solution R and mix. Solution B is yellow to orange-brown. Solution A is orange to red, the colour being clearly more intense than any similar colour which may be obtained with solution B. TESTS Solution S. Dissolve 1.0 g in alcohol R and dilute to 10 ml with the same solvent. 1140

ASSAY To 1.000 g add 20.0 ml of 1 M sodium hydroxide. Heat at about 70 °C for 1 h. Cool rapidly in an ice bath. Prepare a blank in the same manner. Carry out the titration on the solutions at room temperature. Titrate the excess sodium hydroxide with 0.5 M sulphuric acid, continuing the titration until the second point of inflexion and determining the end-point potentiometrically (2.2.20). 1 ml of 1 M sodium hydroxide is equivalent to 194.2 mg of C11H14O3. IMPURITIES

A. R = H : 4-hydroxybenzoic acid, B. R = CH3 : methyl 4-hydroxybenzoate, C. R = CH2-CH3 : ethyl 4-hydroxybenzoate, D. R = CH2-CH2-CH3 : propyl 4-hydroxybenzoate.


Butylhydroxytoluene


01/2005:0880 Apply separately to the plate 5 µl of each solution. Develop over a path of 10 cm using methylene chloride R. Allow the plate to dry in air and spray with a freshly prepared mixture of BUTYLHYDROXYANISOLE 10 volumes of potassium ferricyanide solution R, 20 volumes of ferric chloride solution R1 and 70 volumes of water R. In the chromatogram obtained with test solution (a) : any Butylhydroxyanisolum violet-blue spot with an Rf value of about 0.35 (corresponding to 3-(1,1-dimethylethyl)-4-methoxyphenol) is not more intense than the principal spot in the chromatogram obtained with reference solution (a) (10 per cent) ; any spot corresponding to hydroquinone is not more intense than the principal spot in the chromatogram obtained with reference solution (c) (0.2 per cent) ; any spot, apart C11H16O2 Mr 180.3 from the principal spot and any spots corresponding to 3-(1,1-dimethylethyl)-4-methoxyphenol and hydroquinone, is DEFINITION not more intense than the principal spot in the chromatogram obtained with reference solution (b) (0.5 per cent). Butylhydroxyanisole is 2-(1,1-dimethylethyl)-4methoxyphenol containing not more than 10 per cent of Heavy metals (2.4.8). 1.0 g complies with limit test C for 3-(1,1-dimethylethyl)-4-methoxyphenol. heavy metals (10 ppm). Prepare the standard using 1 ml of lead standard solution (10 ppm Pb) R. CHARACTERS Sulphated ash (2.4.14). Not more than 0.1 per cent, A white, yellowish or slightly pinkish, crystalline powder, determined on 1.0 g. practically insoluble in water, very soluble in methylene chloride, freely soluble in alcohol and in fatty oils. It STORAGE dissolves in dilute solutions of alkali hydroxides. Store protected from light. IDENTIFICATION IMPURITIES A. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). A. benzene-1,4-diol (hydroquinone). B. To 0.5 ml of solution S (see Tests) add 10 ml of aminopyrazolone solution R and 1 ml of potassium 01/2005:0581 ferricyanide solution R. Mix and add 10 ml of methylene chloride R. Shake vigorously. After separation, the BUTYLHYDROXYTOLUENE organic layer is red. C. Dissolve about 10 mg in 2 ml of alcohol R. Add 1 ml of a Butylhydroxytoluenum 1 g/l solution of testosterone propionate R in alcohol R and 2 ml of dilute sodium hydroxide solution R. Heat in a water-bath at 80 °C for 10 min and allow to cool. A red colour develops. TESTS Solution S. Dissolve 2.5 g in alcohol R and dilute to 25 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than intensity 5 of the range of reference solutions of the most appropriate colour (2.2.2, Method II). Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution (a). Dissolve 0.25 g of the substance to be examined in methylene chloride R and dilute to 10 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with methylene chloride R. Reference solution (a). Dissolve 25 mg of butylhydroxyanisole CRS in methylene chloride R and dilute to 10 ml with the same solvent. Reference solution (b). Dilute 1 ml of reference solution (a) to 20 ml with methylene chloride R. Reference solution (c). Dissolve 50 mg of hydroquinone R in 5 ml of alcohol R and dilute to 100 ml with methylene chloride R. Dilute 1 ml of this solution to 10 ml with methylene chloride R. General Notices (1) apply to all monographs and other texts

C15H24O

Mr 220.4

DEFINITION Butylhydroxytoluene is 2,6-bis(1,1-dimethylethyl)-4methylphenol. CHARACTERS A white or yellowish-white, crystalline powder, practically insoluble in water, very soluble in acetone, freely soluble in alcohol and in vegetable oils. IDENTIFICATION First identification : A, C. Second identification : A, B, D. A. It complies with the test for the freezing-point (see Tests). B. Dissolve 0.500 g in ethanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this solution to 100.0 ml with ethanol R. Examined between 230 nm and 300 nm (2.2.25), the solution shows an absorption maximum at 278 nm. The specific absorbance at the maximum is 80 to 90. 1141

Butylhydroxytoluene


C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with butylhydroxytoluene CRS. D. Dissolve about 10 mg in 2 ml of alcohol R. Add 1 ml of a 1 g/l solution of testosterone propionate R in alcohol R and 2 ml of dilute sodium hydroxide solution R. Heat in a water-bath at 80 °C for 10 min and allow to cool. A blue colour develops. TESTS Appearance of solution. Dissolve 1.0 g in methanol R and dilute to 10 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y5 or BY5 (2.2.2, Method II). Freezing-point (2.2.18) : 69 °C to 70 °C. Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. Dissolve 0.2 g of the substance to be examined in methanol R and dilute to 10.0 ml with the same solvent.

1142

Reference solution. Dilute 1 ml of the test solution to 200 ml with methanol R. Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm using methylene chloride R. Dry the plate in air and spray with a freshly prepared mixture of 10 volumes of potassium ferricyanide solution R, 20 volumes of ferric chloride solution R1 and 70 volumes of water R. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution (0.5 per cent). Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.


Caffeine


01/2005:0267 Related substances. Examine by thin-layer chromatography (2.2.27) using silica gel GF254 R as the coating substance. Test solution. Dissolve 0.2 g of the substance to be examined CAFFEINE in a mixture of 4 volumes of methanol R and 6 volumes of methylene chloride R and dilute to 10 ml with the same mixture of solvents. Coffeinum Reference solution. Dilute 0.5 ml of the test solution to 100 ml with a mixture of 4 volumes of methanol R and 6 volumes of methylene chloride R. Apply to the plate 10 µl of each solution. Develop over a path of 15 cm using a mixture of 10 volumes of concentrated ammonia R, 30 volumes of acetone R, 30 volumes of methylene chloride R and 40 volumes of butanol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the C8H10N4O2 Mr 194.2 test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with DEFINITION the reference solution (0.5 per cent). Sulphates (2.4.13). 15 ml of solution S complies with the Caffeine contains not less than 98.5 per cent and limit test for sulphates (500 ppm). Prepare the standard not more than the equivalent of 101.5 per cent of using a mixture of 7.5 ml of sulphate standard solution 1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione, calculated (10 ppm SO4) R and 7.5 ml of distilled water R. with reference to the dried substance. Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of CHARACTERS lead standard solution (10 ppm Pb) R. A white, crystalline powder or silky, white crystals, sublimes Loss on drying (2.2.32). Not more than 0.5 per cent, readily, sparingly soluble in water, freely soluble in boiling water, slightly soluble in ethanol. It dissolves in concentrated determined on 1.000 g by drying in an oven at 100-105 °C for 1 h. solutions of alkali benzoates or salicylates. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. IDENTIFICATION First identification : A, B, E. Second identification : A, C, D, E, F. A. Melting point (2.2.14) : 234 °C to 239 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with caffeine CRS. C. To 2 ml of a saturated solution add 0.05 ml of iodinated potassium iodide solution R. The solution remains clear. Add 0.1 ml of dilute hydrochloric acid R. A brown precipitate is formed. Neutralise with dilute sodium hydroxide solution R. The precipitate dissolves. D. In a glass-stoppered tube, dissolve about 10 mg in 0.25 ml of a mixture of 0.5 ml of acetylacetone R and 5 ml of dilute sodium hydroxide solution R. Heat in a water-bath at 80 °C for 7 min. Cool and add 0.5 ml of dimethylaminobenzaldehyde solution R2. Heat again in a water-bath at 80 °C for 7 min. Allow to cool and add 10 ml of water R. An intense blue colour develops.

ASSAY Dissolve 0.170 g with heating in 5 ml of anhydrous acetic acid R. Allow to cool, add 10 ml of acetic anhydride R and 20 ml of toluene R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 19.42 mg of C8H10N4O2. IMPURITIES Specified impurities : A. Other detectable impurities : B, C. A. theophylline,

E. It complies with the test for loss on drying (see Tests). F. It gives the reaction of xanthines (2.3.1). TESTS

B. N-[6-amino-1,3-dimethyl-2,4(1H,3H)-dioxopyrimidin-5yl]formamide,

Solution S. Dissolve 0.5 g with heating in 50 ml of carbon dioxide-free water R prepared from distilled water R, cool and dilute to 50 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Acidity. To 10 ml of solution S add 0.05 ml of bromothymol blue solution R1. The solution is green or yellow. Not more than 0.2 ml of 0.01 M sodium hydroxide is required to change the colour of the indicator to blue. General Notices (1) apply to all monographs and other texts

C. 1,3,9-trimethyl-3,9-dihydro-1H-purine-2,6-dione (isocaffeine). 1145

Caffeine monohydrate


01/2005:0268 Related substances. Examine by thin-layer chromatography (2.2.27) using silica gel GF254 R as the coating substance. Test solution. Dissolve 0.2 g of the substance to be examined CAFFEINE MONOHYDRATE in a mixture of 4 volumes of methanol R and 6 volumes of methylene chloride R and dilute to 10 ml with the same mixture of solvents. Coffeinum monohydricum Reference solution. Dilute 0.5 ml of the test solution to 100 ml with a mixture of 4 volumes of methanol R and 6 volumes of methylene chloride R. Apply to the plate 10 µl of each solution. Develop over a path of 15 cm using a mixture of 10 volumes of concentrated ammonia R, 30 volumes of acetone R, 30 volumes of methylene chloride R and 40 volumes of butanol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the C8H10N4O2,H2O Mr 212.2 test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with DEFINITION the reference solution (0.5 per cent). Caffeine monohydrate contains not less than 98.5 per cent Sulphates (2.4.13). 15 ml of solution S complies with the and not more than the equivalent of 101.5 per cent of limit test for sulphates (500 ppm). Prepare the standard 1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione, calculated using a mixture of 7.5 ml of sulphate standard solution with reference to the dried substance. (10 ppm SO4) R and 7.5 ml of distilled water R. Heavy metals (2.4.8). 1.0 g complies with limit test C for CHARACTERS heavy metals (20 ppm). Prepare the standard using 2 ml of A white, crystalline powder or silky, white crystals, sublimes lead standard solution (10 ppm Pb) R. readily, sparingly soluble in water, freely soluble in boiling Loss on drying (2.2.32). 5.0 per cent to 9.0 per cent, water, slightly soluble in ethanol. It dissolves in concentrated determined on 1.000 g by drying in an oven at 100-105 °C solutions of alkali benzoates or salicylates. for 1 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, IDENTIFICATION determined on 1.0 g. First identification : A, B, E. ASSAY Second identification : A, C, D, E, F. Dissolve 0.170 g, previously dried at 100-105 °C, with heating in 5 ml of anhydrous acetic acid R. Allow to cool, A. Melting point (2.2.14) : 234 °C to 239 °C, determined add 10 ml of acetic anhydride R and 20 ml of toluene R. after drying at 100-105 °C. Titrate with 0.1 M perchloric acid, determining the end-point B. Dry the substance to be examined at 100-105 °C. Examine potentiometrically (2.2.20). by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with caffeine CRS. 1 ml of 0.1 M perchloric acid is equivalent to 19.42 mg of C8H10N4O2. C. To 2 ml of a saturated solution add 0.05 ml of iodinated potassium iodide solution R. The solution remains IMPURITIES clear. Add 0.1 ml of dilute hydrochloric acid R. A brown Specified impurities : A. precipitate is formed. Neutralise with dilute sodium Other detectable impurities : B, C. hydroxide solution R. The precipitate dissolves. D. In a glass-stoppered tube, dissolve about 10 mg in 0.25 ml of a mixture of 0.5 ml of acetylacetone R and 5 ml of dilute sodium hydroxide solution R. Heat in a water-bath at 80 °C for 7 min. Cool and add 0.5 ml of dimethylaminobenzaldehyde solution R2. Heat again in a water-bath at 80 °C for 7 min. Allow to cool and add 10 ml of water R. An intense blue colour develops.

A. theophylline,

E. It complies with the test for loss on drying (see Tests). F. It gives the reaction of xanthines (2.3.1). TESTS Solution S. Dissolve 0.5 g with heating in 50 ml of carbon dioxide-free water R prepared from distilled water R, cool and dilute to 50 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Acidity. To 10 ml of solution S add 0.05 ml of bromothymol blue solution R1. The solution is green or yellow. Not more than 0.2 ml of 0.01 M sodium hydroxide is required to change the colour of the indicator to blue. 1146

B. N-[6-amino-1,3-dimethyl-2,4(1H, 3H)-dioxopyrimidin-5yl]formamide,

C. 1,3,9-trimethyl-3,9-dihydro-1H-purine-2,6-dione (isocaffeine).


Calcifediol


01/2005:1295 Examine by liquid chromatography (2.2.29). Test solution. Dissolve 1.0 mg of the substance to be examined without heating in 10.0 ml of the mobile phase. CALCIFEDIOL Reference solution (a). Dissolve 1.0 mg of calcifediol CRS without heating in 10.0 ml of the mobile phase. Calcifediolum Reference solution (b). Dilute reference solution (a) 100 times with the mobile phase. Reference solution (c). Heat 2 ml of reference solution (a) in a water-bath at 80 °C under a reflux condenser for 2 h and cool. The chromatographic procedure may be carried out using : — a column 0.15 m long and 4.6 mm in internal diameter packed with octylsilyl silica gel for chromatography R1 (5 µm), — as mobile phase at a flow rate of 1.5 ml/min a mixture of 200 volumes of water R and 800 volumes of methanol R, — as detector a spectrophotometer set at 265 nm, — a loop injector. C27H44O2,H2O Mr 418.7 Inject 50 µl of reference solution (c) and record the chromatogram. Make a total of six injections. When the DEFINITION chromatograms are recorded in the prescribed conditions, Calcifediol is the monohydrate of (5Z,7E)-9,10-secocholesta- the retention time for pre-calcifediol, relative to calcifediol, is 5,7,10(19)-triene-3β,25-diol. It contains not less than 97.0 per about 1.3. The assay is not valid unless the relative standard cent and not more than the equivalent of 102.0 per cent deviation of the response for calcifediol is at most 1 per cent of C27H44O2, calculated with reference to the anhydrous and the resolution between the peaks due to pre-calcifediol substance. and calcifediol is at least 5.0 ; adjust the proportions of the constituents of the mobile phase, if necessary, to obtain this CHARACTERS resolution. White or almost white crystals, practically insoluble in water, Inject 50 µl of reference solution (a) and 50 µl of reference freely soluble in alcohol, soluble in fatty oils. It is sensitive solution (b) and record the chromatograms. Inject 50 µl to air, heat and light. of the test solution and record the chromatogram in the same manner, continuing the chromatography for twice the A reversible isomerisation to pre-calcifediol takes place in retention time of the principal peak. solution, depending on temperature and time. The activity is due to both compounds. STORAGE IDENTIFICATION Store under nitrogen, in an airtight container, protected from light, at a temperature of 2 °C to 8 °C. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum The contents of an opened container are to be used of calcifediol, prepared using 2 mg of the substance to be immediately. examined and 225 mg of potassium bromide R. IMPURITIES B. Examine the chromatograms obtained in the assay. The principal peak in the chromatogram obtained with the test solution is similar in retention time and approximate size to the principal peak in the chromatogram obtained with reference solution (a). TESTS Related substances. Examine by liquid chromatography (2.2.29) as described under Assay. Calculate the percentage content of related substances, apart from pre-calcifediol, that are eluted within twice the retention time of calcifediol from the areas of the peaks in the chromatogram obtained with the test solution by the normalisation procedure. The content of any individual related substance is not greater than 0.5 per cent and the sum of the related substances is not greater than 1.0 per cent. Disregard any peak below 0.1 per cent. Water (2.5.32) : 3.8 per cent to 5.0 per cent, determined on 10.0 mg by the micro determination of water. ASSAY Carry out the assay as rapidly as possible, avoiding exposure to actinic light and air. General Notices (1) apply to all monographs and other texts

A. 9β,10α-cholesta-5,7-diene-3β,25-diol,

B. cholesta-5,7-diene-3β,25-diol, 1147

Calcitonin (salmon)


Test solution. Dissolve 4 mg of the substance to be examined in 2 ml of a mixture of 2 volumes of acetic acid R and 98 volumes of water R. Prepare immediately before use. Reference solution. Dissolve the contents of a vial of calcitonin (salmon) CRS in a mixture of 2 volumes of acetic acid R and 98 volumes of water R to obtain a concentration of 2.0 mg/ml. Prepare immediately before use. Apply to the plate 1 µl of each solution. Develop over a path of 15 cm using a mixture of 6 volumes of glacial acetic acid R, 20 volumes of pyridine R, 24 volumes of water R and 30 volumes of butanol R. Allow the plate to dry in air for 1 h, heat at 110 °C for 10 min and spray the hot plate with strong sodium hypochlorite solution R diluted with water R immediately before use to contain 5 g/l of available chlorine. Dry in a current of cold air until a sprayed area of the plate below the line of application gives at most a very faint blue colour with a drop of potassium iodide and starch solution R ; avoid prolonged exposure to cold air. Spray with potassium iodide and starch solution R until the spots are clearly visible. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution.

C. (6E)-9,10-secocholesta-5(10),6,8-triene-3β,25-diol,

B. Examine the chromatograms obtained in the Assay. The retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the principal peak in the chromatogram obtained with the reference solution. 01/2005:0471

D. (5E,7E)-9,10-secocholesta-5,7,10(19)-triene-3β,25-diol.

CALCITONIN (SALMON) Calcitoninum salmonis

TESTS Acetic acid (2.5.34) : 4.0 per cent to 15.0 per cent. Test solution. Dissolve 10.0 mg of the substance to be examined in a mixture of 5 volumes of mobile phase B and 95 volumes of mobile phase A and dilute to 10.0 ml with the same mixture of solvents. Amino acids. Examine by means of an amino-acid analyser. Standardise the apparatus with a mixture containing equimolar amounts of ammonia, glycine and the L-form of the following amino acids :

C145H240N44O48S2

Mr 3432 lysine arginine histidine serine methionine glutamic acid DEFINITION Calcitonin (salmon) is a synthetic polypeptide having the isoleucine proline leucine structure determined for salmon calcitonin I. It lowers the tyrosine aspartic acid alanine calcium concentration in plasma of mammals by diminishing threonine the rate of bone resorption. It is obtained by chemical valine phenylalanine synthesis and is available as an acetate. Calcitonin (salmon) contains not less than 90.0 per cent and not more than the together with half the equimolar amount of L-cystine. For the equivalent of 105.0 per cent of the peptide C145H240N44O48S2, validation of the method, an appropriate internal standard, such as DL-norleucine R, is used. calculated with reference to the anhydrous, acetic acid-free substance. Test solution. Place 1.0 mg of the substance to be examined in a rigorously cleaned hard-glass tube 100 mm long and By convention, for the purpose of labelling calcitonin 6 mm in internal diameter. Add a suitable amount of a (salmon) preparations, 1 mg of calcitonin (salmon) 50 per cent V/V solution of hydrochloric acid R. Immerse (C145H240N44O48S2) is equivalent to 6000 I.U of biological the tube in a freezing mixture at − 5 °C, reduce the pressure activity. to below 133 Pa and seal. Heat at 110 °C to 115 °C for 16 h. CHARACTERS Cool, open the tube, transfer the contents to a 10 ml flask with the aid of five quantities, each of 0.2 ml, of water R and A white or almost white powder, freely soluble in water. evaporate to dryness over potassium hydroxide R under IDENTIFICATION reduced pressure. Take up the residue in water R and evaporate to dryness over potassium hydroxide R under A. Examine by thin-layer chromatography (2.2.27), using reduced pressure ; repeat these operations once. Take up cellulose for chromatography R1 as the coating the residue in a buffer solution suitable for the amino-acid substance.

1148


Calcitriol


analyser used and dilute to a suitable volume with the same buffer solution. Apply a suitable volume to the amino-acid analyser. Express the content of each amino acid in moles. Calculate the relative proportions of the amino acids taking as equivalent to 1 the sum, divided by 20, of the number of moles of aspartic acid, glutamic acid, proline, glycine, valine, leucine, histidine, arginine and lysine. The values fall within the following limits : aspartic acid 1.8 to 2.2 ; glutamic acid 2.7 to 3.3 ; proline 1.7 to 2.3 ; glycine 2.7 to 3.3 ; valine 0.9 to 1.1 ; leucine 4.5 to 5.3 ; histidine 0.9 to 1.1 ; arginine 0.9 to 1.1 ; lysine 1.8 to 2.2 ; serine 3.2 to 4.2 ; threonine 4.2 to 5.2 ; tyrosine 0.7 to 1.1 ; half-cystine 1.4 to 2.1. Related peptides. Examine by liquid chromatography (2.2.29) as described under Assay. Inject 20 µl of the test solution. In the chromatogram obtained, the area of any peak apart from the principal peak is not greater than 3.0 per cent of the total area of the peaks. The sum of the areas of all the peaks, apart from the principal peak, is not greater than 5.0 per cent of the total area of the peaks. Disregard any peak due to the solvent and any peak with an area less than 0.1 per cent of that of the principal peak. Water (2.5.32). Not more than 10.0 per cent, determined by the micro determination of water. Acetic acid and water. Not more than 20 per cent, calculated by adding together the percentage contents of acetic acid and water determined by the methods described above. Bacterial endotoxins (2.6.14) : less than 1000 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. ASSAY Examine by liquid chromatography (2.2.29). Test solution. Prepare a 1.0 mg/ml solution of the substance to be examined in mobile phase A. Reference solution. Dissolve the contents of a vial of calcitonin (salmon) CRS in mobile phase A to obtain a concentration of 1.0 mg/ml. Resolution solution. Dissolve the contents of a vial of N-acetyl-cys1calcitonin CRS in 400 µl of mobile phase A and add 100 µl of the test solution. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.0 ml/min : Mobile phase A. Dissolve 3.26 g of tetramethylammonium hydroxide R in 900 ml of water R, adjust the pH to 2.5 with phosphoric acid R and mix with 100 ml of acetonitrile for chromatography R ; filter and degas, Mobile phase B. Dissolve 1.45 g of tetramethylammonium hydroxide R in 400 ml of water R, adjust the pH to 2.5 with phosphoric acid R and mix with 600 ml of acetonitrile for chromatography R ; filter and degas, Time (min)



Comment

0 - 30

72 → 48

28 → 52

linear gradient

30 - 32

48 → 72

52 → 28


32 - 55

72

28

re-equilibration

— as detector a spectrophotometer set at 220 nm, maintaining the temperature of the column at 65 °C. General Notices (1) apply to all monographs and other texts

Equilibrate the column with a mixture of 72 volumes of mobile phase A and 28 volumes of mobile phase B. Inject 20 µl of the resolution solution. When the chromatogram is recorded in the prescribed conditions, the relative retention of N-acetyl-cys1calcitonin is about 1.15 relative to the principal peak. The test is not valid unless the resolution between the peaks corresponding to calcitonin and N-acetyl-cys1calcitonin is at least 5.0 and the symmetry factor for the N-acetyl-cys1calcitonin peak is not greater than 2.5. If necessary, adjust the initial A:B ratio of the mobile phase. Inject 20 µl of the test solution and 20 µl of the reference solution. Calculate the content of calcitonin (salmon) (C145H240N44O48S2) from the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C145H240N44O48S2 in calcitonin (salmon) CRS. Proceed with tangential integration of the peak areas. STORAGE Store protected from light at a temperature between 2 °C and 8 °C. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states : — the calcitonin peptide content (C145H240N44O48S2), — where applicable, that the substance is free from bacterial endotoxins. 01/2005:0883

CALCITRIOL Calcitriolum

C27H44O3

Mr 416.6

DEFINITION Calcitriol contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent of (5Z,7E)-9,10-secocholesta-5,7,10(19)-triene-1α,3β,25-triol. CHARACTERS White or almost white crystals, practically insoluble in water, freely soluble in alcohol, soluble in fatty oils. It is sensitive to air, heat and light. A reversible isomerisation to pre-calcitriol takes place in solution, depending on temperature and time. The activity is due to both compounds. IDENTIFICATION A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of calcitriol. 1149

Calcium ascorbate


B. Examine the chromatograms obtained in the assay. The retention time and size of the principal peak in the chromatogram obtained with the test solution are approximately the same as those of the principal peak in the chromatogram obtained with reference solution (a).

IMPURITIES

TESTS Related substances. Examine by liquid chromatography (2.2.29) as described under Assay. Calculate the percentage content of related substances, apart from pre-calcitriol, that are eluted within twice the retention time of calcitriol from the areas of the peaks in the chromatogram obtained with the test solution by the normalisation procedure. The content of any individual related substance is not greater than 0.5 per cent and the sum of the related substances is not A. (5E,7E)-9,10-secocholesta-5,7,10(19)-triene-1α,3β,25-triol greater than 1.0 per cent. Disregard any peak with an area (trans-calcitriol), less than 0.1 times that of the peak in the chromatogram obtained with reference solution (b) (0.1 per cent). ASSAY Carry out the assay as rapidly as possible, avoiding exposure to actinic light and air. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 1.000 mg of the substance to be examined without heating in 10.0 ml of the mobile phase. Reference solution (a). Dissolve 1.000 mg of calcitriol CRS without heating in 10.0 ml of the mobile phase. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 100.0 ml with the mobile phase. B. (5Z,7E)-9,10-secocholesta-5,7,10(19)-triene-1β,3β,25-triol Reference solution (c). Keep 2 ml of reference solution (a) (1β-calcitriol), for 30 min at 80 °C. The chromatographic procedure may be carried out using : — a column 0.25 m long and 4.6 mm in internal diameter packed with octylsilyl silica gel for chromatography R1 (5 µm), — as mobile phase at a flow rate of 1.0 ml/min, a mixture of 450 volumes of a solution containing 1.0 g/l of tris(hydroxymethyl)aminomethane R adjusted to pH 7.0 to 7.5 with phosphoric acid R, and of 550 volumes of acetonitrile R, — as detector a spectrophotometer set at 230 nm, — a loop injector, maintaining the temperature of the column at 40 °C. C. (6aR,7R,9aR)-11-[(3S,5R)-3,5-dihydroxy-2-methylcyclohex1-enyl]-7-[(1R)-5-hydroxy-1,5-dimethylhexyl]-6aInject 50 µl of reference solution (c) and six times 50 µl methyl-2-phenyl-5,6,6a,7,8,9,9a,11-octahydro-1H,4aHof reference solution (a). When the chromatograms are cyclopenta[f]1,2,4]triazolo[1,2-a]cinnoline-1,3(2H)-dione recorded in the prescribed conditions, the retention time for (triazoline adduct of pre-calcitriol). pre-calcitriol, relative to calcitriol, is about 0.9. The assay is not valid unless the number of theoretical plates calculated for the peak due to calcitriol in the chromatogram obtained 01/2005:1182 with reference solution (a) is at least 10 000, the relative standard deviation of the peak area due to calcitriol is at most 1 per cent and the resolution between the peaks due to CALCIUM ASCORBATE calcitriol and pre-calcitriol is at least 3.5. Inject 50 µl of reference solution (b) and 50 µl of the test Calcii ascorbas solution. Continue the chromatography for twice the retention time of calcitriol. Calculate the percentage content of calcitriol. STORAGE Store under nitrogen, in an airtight container, protected from light, at a temperature of 2 °C to 8 °C. The contents of an opened container are to be used immediately. 1150

C12H14CaO12,2H2O

Mr 426.3



Calcium carbonate

Measure the absorbance at 324.8 nm using a copper hollow-cathode lamp as source of radiation and an air-acetylene flame. Iron. Not more than 2 ppm of Fe, determined by atomic absorption spectrometry (2.2.23, Method I). Test solution. Dissolve 5.0 g of the substance to be examined CHARACTERS in a 9.7 g/l solution of nitric acid R and dilute to 25.0 ml A white or slightly yellowish, crystalline powder, freely with the same acid solution. soluble in water, practically insoluble in alcohol. Reference solutions. Prepare the reference solutions using iron standard solution (10 ppm Fe) R, diluting with a 9.7 g/l IDENTIFICATION solution of nitric acid R. First identification : A, B, E. Measure the absorbance at 248.3 nm using an iron Second identification : A, C, D, E. hollow-cathode lamp as the radiation source and an A. It complies with the test for specific optical rotation (see air-acetylene flame. Tests). Heavy metals (2.4.8). 2.0 g complies with limit test D for B. Examine by infrared absorption spectrophotometry heavy metals (10 ppm). Prepare the standard using 2.0 ml of (2.2.24), comparing with the Ph. Eur. reference spectrum lead standard solution (10 ppm Pb) R. of calcium ascorbate. Loss on drying (2.2.32). Not more than 0.1 per cent, C. Dilute 1 ml of solution S (see Tests) to 10 ml with water R. determined on 1.000 g by drying in an oven at 100-105 °C To 2 ml of the solution add 0.2 ml of a 100 g/l solution of for 2 h. ferrous sulphate R. A deep violet colour develops. ASSAY D. To 1 ml of solution S add 0.2 ml of dilute nitric acid R and 0.2 ml of silver nitrate solution R2. A grey precipitate Dissolve 80.0 mg in a mixture of 10 ml of dilute sulphuric acid R and 80 ml of carbon dioxide-free water R. Add 1 ml is formed. of starch solution R. Titrate with 0.05 M iodine until a E. The substance gives reaction (b) of calcium (2.3.1). persistent violet-blue colour is obtained. TESTS 1 ml of 0.05 M iodine is equivalent to 10.66 mg of C12H14CaO12,2H2O. Solution S. Dissolve 5.00 g in carbon dioxide-free water R and dilute to 50.0 ml with the same solvent. STORAGE Appearance of solution. Solution S is clear (2.2.1) and not Store in a non-metallic container, protected from light. more intensely coloured than reference solution Y6 (2.2.2, Method II). Examine the colour of the solution immediately after preparation of the solution. 01/2005:0014 pH (2.2.3). The pH of solution S is 6.8 to 7.4. CALCIUM CARBONATE Specific optical rotation (2.2.7) : + 95 to + 97, determined using freshly prepared solution S and calculated with reference to the dried substance. Calcii carbonas Fluorides. Not more than 10 ppm of F, determined CaCO3 Mr 100.1 potentiometrically (2.2.36, Method I) using as indicator electrode a fluoride-selective electrode and as reference DEFINITION electrode a silver-silver chloride electrode. Calcium carbonate contains not less than 98.5 per cent and Test solution. In a 50 ml volumetric flask, dissolve 1.000 g in a 10.3 g/l solution of hydrochloric acid R, add 5.0 ml of not more than the equivalent of 100.5 per cent of CaCO3, fluoride standard solution (1 ppm F) R and dilute to 50.0 ml calculated with reference to the dried substance. with a 10.3 g/l solution of hydrochloric acid R. To 20.0 ml of CHARACTERS the solution add 20.0 ml of total-ionic-strength-adjustment buffer R and 3 ml of an 82 g/l solution of anhydrous sodium A white powder, practically insoluble in water. DEFINITION Calcium ascorbate contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent of calcium di[(R)-2-[(S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3olate] dihydrate.

acetate R. Adjust to pH 5.2 with ammonia R and dilute to 50.0 ml with distilled water R. Reference solutions. To 0.25 ml, 0.5 ml, 1.0 ml, 2.0 ml and 5.0 ml of fluoride standard solution (10 ppm F) R add 20.0 ml of total-ionic-strength-adjustment buffer R and dilute to 50.0 ml with distilled water R. Carry out the measurements for each solution. Calculate the concentration of fluorides using the calibration curve, taking into account the addition of fluoride to the test solution. Copper. Not more than 5 ppm of Cu, determined by atomic absorption spectrometry (2.2.23, Method I). Test solution. Dissolve 2.0 g of the substance to be examined in a 9.7 g/l solution of nitric acid R and dilute to 25.0 ml with the same acid solution. Reference solutions. Prepare the reference solutions using copper standard solution (10 ppm Cu) R, diluting with a 9.7 g/l solution of nitric acid R.

TESTS Solution S. Dissolve 5.0 g in 80 ml of dilute acetic acid R. When the effervescence ceases, boil the solution for 2 min, allow to cool, dilute to 100 ml with dilute acetic acid R and filter, if necessary, through a sintered-glass filter. Substances insoluble in acetic acid. Wash any residue obtained during the preparation of solution S with four quantities, each of 5 ml, of hot water R and dry at 100-105 °C for 1 h. The residue weighs not more than 10 mg (0.2 per cent). Chlorides (2.4.4). 3 ml of solution S diluted to 15 ml with water R complies with the limit test for chlorides (330 ppm).


1151

IDENTIFICATION A. It gives the reaction of carbonates (2.3.1). B. 0.2 ml of solution S (see Tests) gives the reactions of calcium (2.3.1).

Calcium chloride dihydrate


Sulphates (2.4.13). 1.2 ml of solution S diluted to 15 ml with distilled water R complies with the limit test for sulphates (0.25 per cent). Arsenic (2.4.2). 5 ml of solution S complies with limit test A for arsenic (4 ppm). Barium. To 10 ml of solution S add 10 ml of calcium sulphate solution R. After at least 15 min, any opalescence in the solution is not more intense than that in a mixture of 10 ml of solution S and 10 ml of distilled water R. Iron (2.4.9). Dissolve 50 mg in 5 ml of dilute hydrochloric acid R and dilute to 10 ml with water R. The solution complies with the limit test for iron (200 ppm). Magnesium and alkali metals. Dissolve 1.0 g in 12 ml of dilute hydrochloric acid R. Boil the solution for about 2 min and add 20 ml of water R, 1 g of ammonium chloride R and 0.1 ml of methyl red solution R. Add dilute ammonia R1 until the colour of the indicator changes and then 2 ml in excess. Heat to boiling and add 50 ml of hot ammonium oxalate solution R. Allow to stand for 4 h, dilute to 100 ml with water R and filter through a suitable filter. To 50 ml of the filtrate add 0.25 ml of sulphuric acid R. Evaporate to dryness on a water-bath and ignite to constant mass at 600 °C. The residue weighs not more than 7.5 mg (1.5 per cent). Heavy metals (2.4.8). 12 ml of solution S complies with limit test A for heavy metals (20 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32). Not more than 2.0 per cent, determined on 1.000 g by drying in an oven at 200 °C.

TESTS Solution S. Dissolve 10.0 g in carbon dioxide-free water R prepared from distilled water R and dilute to 100 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). Acidity or alkalinity. To 10 ml of freshly prepared solution S add 0.1 ml of phenolphthalein solution R. If the solution is red, not more than 0.2 ml of 0.01 M hydrochloric acid is required to discharge the colour and if the solution is colourless, not more than 0.2 ml of 0.01 M sodium hydroxide is required to turn it red. Sulphates (2.4.13) : maximum 300 ppm. 5 ml of solution S diluted to 15 ml with distilled water R complies with the limit test for sulphates. Aluminium. To 10 ml of solution S add 2 ml of ammonium chloride solution R and 1 ml of dilute ammonia R1 and boil the solution. No turbidity or precipitate is formed. If intended for use in the manufacture of dialysis solutions, it complies with the following test for aluminium (2.4.17) which replaces the test prescribed above : maximum 1 ppm. Prescribed solution. Dissolve 4 g in 100 ml of water R and add 10 ml of acetate buffer solution pH 6.0 R. Reference solution. Mix 2 ml of aluminium standard solution (2 ppm Al) R, 10 ml of acetate buffer solution pH 6.0 R and 98 ml of water R. Blank solution. Mix 10 ml of acetate buffer solution pH 6.0 R and 100 ml of water R. ASSAY Barium. To 10 ml of solution S add 1 ml of calcium sulphate Dissolve 0.150 g in a mixture of 3 ml of dilute hydrochloric solution R. After at least 15 min, any opalescence in the acid R and 20 ml of water R. Boil for 2 min, allow to cool and solution is not more intense than that in a mixture of 10 ml dilute to 50 ml with water R. Carry out the complexometric of solution S and 1 ml of distilled water R. titration of calcium (2.5.11). Iron (2.4.9) : maximum 10 ppm. 1 ml of 0.1 M sodium edetate is equivalent to 10.01 mg of 10 ml of solution S complies with the limit test for iron. CaCO3. Magnesium and alkali metals : maximum 0.5 per cent. To a mixture of 20 ml of solution S and 80 ml of water R add 2 g of ammonium chloride R and 2 ml of dilute 01/2005:0015 ammonia R1, heat to boiling and pour into the boiling solution a hot solution of 5 g of ammonium oxalate R in 75 ml of water R. Allow to stand for 4 h, dilute to 200 ml with water R and filter through a suitable filter. To 100 ml CALCIUM CHLORIDE DIHYDRATE of the filtrate add 0.5 ml of sulphuric acid R. Evaporate to dryness on a water-bath and ignite to constant mass at Calcii chloridum dihydricum 600 °C. The residue weighs a maximum of 5 mg. Heavy metals (2.4.8) : maximum 20 ppm. CaCl2,2H2O Mr 147.0 12 ml of solution S complies with limit test A. Prepare the standard using lead standard solution (2 ppm Pb) R. DEFINITION ASSAY Content : 97.0 per cent to 103.0 per cent of CaCl2,2H2O. Dissolve 0.280 g in 100 ml of water R and carry out the CHARACTERS complexometric titration of calcium (2.5.11). 1 ml of 0.1 M sodium edetate is equivalent to 14.70 mg of Appearance : white, crystalline powder, hygroscopic. CaCl2,2H2O. Solubility : freely soluble in water, soluble in alcohol. IDENTIFICATION A. Solution S (see Tests) gives reaction (a) of chlorides (2.3.1). B. It gives the reactions of calcium (2.3.1). C. It complies with the limits of the assay. 1152

LABELLING The label states, where applicable, that the substance is suitable for use in the manufacture of dialysis solutions. STORAGE In an airtight container. See the information section on general monographs (cover pages)

Calcium dobesilate monohydrate


01/2005:0707 a suitable filter. To 100 ml of the filtrate add 0.5 ml of sulphuric acid R. Evapor-ate to dryness on a water-bath and CALCIUM CHLORIDE HEXAHYDRATE ignite to constant mass at 600 °C. The residue weighs not more than 5 mg (0.3 per cent). Heavy metals (2.4.8). 12 ml of solution S complies with limit Calcii chloridum hexahydricum test A for heavy metals (15 ppm). Prepare the standard using lead standard solution (2 ppm Pb) R. CaCl2,6H2O Mr 219.1 ASSAY DEFINITION Calcium chloride hexahydrate contains not less than 97.0 per Dissolve 0.200 g in 100 ml of water R. Carry out the complexometric titration of calcium (2.5.11). cent and not more than the equivalent of 103.0 per cent of 1 ml of 0.1 M sodium edetate is equivalent to 21.91 mg of CaCl2,6H2O. CaCl2,6H2O. CHARACTERS A white, crystalline mass or colourless crystals, very soluble LABELLING The label states, where applicable, that the substance is in water, freely soluble in alcohol. suitable for use in the manufacture of dialysis solutions. It freezes at about 29 °C. IDENTIFICATION A. Solution S (see Tests) gives reaction (a) of chlorides (2.3.1). B. It gives the reactions of calcium (2.3.1). C. It complies with the limits of the assay. TESTS Solution S. Dissolve 15.0 g in carbon dioxide-free water R prepared from distilled water R and dilute to 100 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). Acidity or alkalinity. To 10 ml of freshly prepared solution S add 0.1 ml of phenolphthalein solution R. If the solution is red, not more than 0.2 ml of 0.01 M hydrochloric acid is required to discharge the colour and if the solution is colourless, not more than 0.2 ml of 0.01 M sodium hydroxide is required to turn it red. Sulphates (2.4.13). 5 ml of solution S diluted to 15 ml with distilled water R complies with the limit test for sulphates (200 ppm). Aluminium. To 10 ml of solution S add 2 ml of ammonium chloride solution R and 1 ml of dilute ammonia R1. Heat to boiling. No turbidity or precipitate is formed. If intended for use in the manufacture of dialysis solutions, it complies with the following test which replaces the test for aluminium prescribed above. Dissolve 6 g in 100 ml of water R and add 10 ml of acetate buffer solution pH 6.0 R. The solution complies with the limit test for aluminium (1 ppm). Use as the reference solution a mixture of 2 ml of aluminium standard solution (2 ppm Al) R, 10 ml of acetate buffer solution pH 6.0 R and 98 ml of water R. To prepare the blank use a mixture of 10 ml of acetate buffer solution pH 6.0 R and 100 ml of water R. Barium. To 10 ml of solution S add 1 ml of calcium sulphate solution R. After at least 15 min, any opalescence in the solution is not more intense than that in a mixture of 10 ml of solution S and 1 ml of distilled water R. Iron (2.4.9). 10 ml of solution S complies with the limit test for iron (7 ppm). Magnesium and alkali metals. To a mixture of 20 ml of solution S and 80 ml of water R add 2 g of ammonium chloride R and 2 ml of dilute ammonia R1, heat to boiling and pour into the boiling solution a hot solution of 5 g of ammonium oxalate R in 75 ml of water R. Allow to stand for 4 h, dilute to 200 ml with water R and filter through General Notices (1) apply to all monographs and other texts

01/2005:1183

CALCIUM DOBESILATE MONOHYDRATE Calcii dobesilas monohydricum

C12H10CaO10S2,H2O

Mr 436.4

DEFINITION Calcium dobesilate monohydrate contains not less than 99.0 per cent and not more than the equivalent of 102.0 per cent of calcium di(2,5-dihydroxybenzenesulphonate), calculated with reference to the anhydrous substance. CHARACTERS A white or almost white, hygroscopic powder, very soluble in water, freely soluble in ethanol, very slightly soluble in 2-propanol, practically insoluble in methylene chloride. IDENTIFICATION A. Dissolve 0.100 g in water R and dilute to 200.0 ml with the same solvent. Dilute 5.0 ml of the solution to 100.0 ml with water R. Examined between 210 nm and 350 nm (2.2.25), the solution shows two absorption maxima, at 221 nm and 301 nm. The specific absorbance at the maximum at 301 nm is 174 to 181. B. Mix 1 ml of ferric chloride solution R2, 1 ml of a freshly prepared 10 g/l solution of potassium ferricyanide R and 0.1 ml of nitric acid R. To this mixture add 5 ml of freshly prepared solution S (see Tests) : a blue colour and a precipitate are immediately produced. C. 2 ml of freshly prepared solution S (see Tests) gives reaction (b) of calcium (2.3.1). TESTS Solution S. Dissolve 10.0 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Appearance of solution. Solution S, when freshly prepared, is clear (2.2.1) and colourless (2.2.2, Method II). pH (2.2.3). The pH of solution S is 4.5 to 6.0. 1153

Calcium folinate


Hydroquinone. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm.

DEFINITION Calcium folinate contains not less than 97.0 per cent and not more than the equivalent of 102.0 per cent of calcium (2S)-2-[[4-[[[(6RS)-2Test solution. Dissolve 2.0 g of the substance to be examined amino-5-formyl-4-oxo-1,4,5,6,7,8-hexahydropteridin6-yl]methyl]amino]benzoyl]amino]pentanedioate and not in water R and dilute to 10 ml with the same solvent. less than 7.54 per cent and not more than the equivalent of Reference solution. Dissolve 10 mg of hydroquinone R in 8.14 per cent of Ca, both calculated with reference to the water R and dilute to 50 ml with the same solvent. anhydrous and solvent-free substance. Apply to the plate 10 µl of each solution and dry the starting points in a current of cool air. Develop over a path of 15 cm CHARACTERS using a mixture of 20 volumes of methylene chloride R, A white or light yellow, amorphous or crystalline powder, 30 volumes of methyl acetate R and 50 volumes of ethyl sparingly soluble in water, practically insoluble in acetone acetate R. Dry the plate in a current of hot air and examine and in alcohol. The amorphous form may produce in ultraviolet light at 254 nm. Any spot corresponding to supersaturated solutions in water. hydroquinone in the chromatogram obtained with the test IDENTIFICATION solution is not more intense than the principal spot in the chromatogram obtained with the reference solution (0.1 per First identification : A, B, D. cent). Second identification : A, C, D. Heavy metals (2.4.8). 1.0 g complies with limit test C for A. It complies with the test for specific optical rotation (see heavy metals (15 ppm). Prepare the standard using 1.5 ml of Tests). lead standard solution (10 ppm Pb) R. B. Examine by infrared absorption spectrophotometry Iron (2.4.9). 10 ml of solution S complies with the limit test (2.2.24), comparing with the spectrum obtained with for iron (10 ppm). calcium folinate CRS. Examine the substances prepared as discs. If the spectra obtained show differences, Water (2.5.12) : 4.0 per cent to 6.0 per cent, determined on dissolve the substance to be examined and the reference 0.500 g by the semi-micro determination of water. substance separately in the minimum quantity of water R and add dropwise sufficient acetone R to produce ASSAY sufficient precipitate. Allow to stand for 15 min, collect Dissolve 0.200 g in a mixture of 10 ml of water R and40 ml of the precipitate by centrifugation, wash the precipitate dilute sulphuric acid R. Titrate with 0.1 M cerium sulphate, with two small quantities of acetone R and dry. Record determining the end-point potentiometrically (2.2.20). new spectra using the residues. 1 ml of 0.1 M cerium sulphate is equivalent to 10.45 mg of C. Examine by thin-layer chromatography (2.2.27), using C12H10CaO10S2. cellulose for chromatography F254 R as the coating substance. STORAGE Test solution. Dissolve 15 mg of the substance to be Store in an airtight container, protected from light. examined in a 3 per cent V/V solution of ammonia R and dilute to 5 ml with the same solvent. IMPURITIES Reference solution. Dissolve 15 mg of calcium folinate CRS in a 3 per cent V/V solution of ammonia R and dilute to 5 ml with the same solvent. Apply to the plate 5 µl of each solution. Develop over a path of 15 cm using the lower layer of a mixture of 1 volume of isoamyl alcohol R and 10 volumes of a 50 g/l solution of citric acid R previously adjusted to A. benzene-1,4-diol (hydroquinone). pH 8 with ammonia R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. 01/2005:0978 D. It gives reaction (b) of calcium (2.3.1). Carry out the tests and the assay as rapidly as possible, CALCIUM FOLINATE protected from actinic light. TESTS Solution S. Dissolve 1.25 g in carbon dioxide-free water R, heating at 40 °C if necessary, and dilute to 50.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1). The absorbance (2.2.25) of solution S, measured at 420 nm using water R as the compensation liquid, is not greater than 0.60. pH (2.2.3). The pH of solution S is 6.8 to 8.0. Specific optical rotation (2.2.7). The specific optical rotation is + 14.4 to + 18.0, determined on solution S and calculated Mr 511.5 (anhydrous substance) with reference to the anhydrous and solvent-free substance.

Calcii folinas

C20H21CaN7O7,xH2O 1154


Calcium folinate


Acetone, ethanol and methanol. Not more than 0.5 per cent of acetone, not more than 3.0 per cent of ethanol and not more than 0.5 per cent of methanol. Examine by head-space gas chromatography (2.2.28) using the standard additions method. Test solution. Dissolve 0.25 g of the substance to be examined in water R and dilute to 10.0 ml with the same solvent. Reference solution. Dilute 0.125 g of acetone R, 0.750 g of ethanol R and 0.125 g of methanol R in water R and dilute to 1000.0 ml with water R. The chromatographic procedure may be carried out using : — a fused-silica column 10 m long and 0.32 mm in internal diameter coated with styrene-divinylbenzene copolymer R, — nitrogen for chromatography R as the carrier gas at a flow rate of 4 ml/min, — a flame-ionisation detector, raising the temperature of the column from 125 °C to 185 °C at a rate of 10 °C/min and maintaining at 185 °C until the total run time is 15 min. Maintain the temperature of the injection port at 250 °C and that of the detector at 250 °C. Place the samples in a thermostatically controlled chamber at 80 °C for 20 min and pressurise them for 30 s. Repeat the injections at least three times. Related substances. Examine the chromatograms obtained in the Assay. In the chromatogram obtained with the test solution : the area of any peak corresponding to formylfolic acid is not greater than the area of the principal peak in the chromatogram obtained with reference solution (c) (1 per cent) ; the area of any peak, apart from the principal peak and any peak corresponding to formylfolic acid, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (2.5 per cent). Disregard any peak with an area less than that of the principal peak in the chromatogram obtained with reference solution (d). Chlorides (2.4.4). Dissolve 67 mg in 10 ml of water R and add 3 ml of acetic acid R. Filter and wash the precipitate with five quantities, each of 5 ml, of water R. Collect the filtrate and washings and dilute to 100 ml with water R. 15 ml of this solution complies with the limit test for chlorides (0.5 per cent). Heavy metals (2.4.8). 1.0 g complies with limit test F for heavy metals (50 ppm). Prepare the standard using 5 ml of lead standard solution (10 ppm Pb) R. Platinum. Not more than 20 ppm of Pt, determined by atomic absorption spectrometry (2.2.23, Method II). Test solution. Dissolve 1.00 g in water R and dilute to 100.0 ml with the same solvent. Reference solutions. Prepare the reference solutions using platinum standard solution (30 ppm Pt) R, diluted as necessary with a mixture of 1 volume of nitric acid R and 99 volumes of water R. Measure the absorbance at 265.9 nm using a platinum hollow-cathode lamp as source of radiation. Water (2.5.12). Not more than 17.0 per cent, determined on 0.200 g (ground to a very fine powder) by the semi-micro determination of water. Stir the substance to be examined in the titration solvent for about 6 min before titrating and use a suitable titrant that does not contain pyridine. General Notices (1) apply to all monographs and other texts

Bacterial endotoxins (2.6.14) : less than 0.5 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. ASSAY Calcium. Dissolve 0.400 g in 150 ml of water R and dilute to 300 ml with the same solvent. Carry out the complexometric titration of calcium (2.5.11). 1 ml of 0.1 M sodium edetate is equivalent to 4.008 mg of Ca. Calcium folinate. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 10.0 mg of the substance to be examined in water R and dilute to 10.0 ml with the same solvent. Reference solution (a). Dissolve 10.0 mg of calcium folinate CRS in water R and dilute to 10.0 ml with the same solvent. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 100.0 ml with water R. Reference solution (c). Dissolve 10.0 mg of formylfolic acid CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with water R. Reference solution (d). Dilute 1.0 ml of reference solution (b) to 10.0 ml with water R. Reference solution (e). Dilute 5.0 ml of reference solution (c) to 10.0 ml with reference solution (b). The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows : mix 220 ml of methanol R and 780 ml of a solution containing 2.0 ml of tetrabutylammonium hydroxide solution (400 g/l) R and 2.2 g of disodium hydrogen phosphate R previously adjusted to pH 7.8 with phosphoric acid R, — as detector a spectrophotometer set at 280 nm, maintaining the temperature of the column at 40 °C. Inject 10 µl of each solution. Continue the chromatography for 2.5 times the retention time of the principal peak in the chromatogram obtained with the test solution. The assay is not valid unless, in the chromatogram obtained with reference solution (e), the resolution between the peaks corresponding to calcium folinate and formylfolic acid is at least 2.2 and the relative standard deviation of the area of the principal peak for six replicate injections of reference solution (a) is at most 2.0 per cent. Calculate the percentage content of C20H21CaN7O7 from the peak areas and the declared content of calcium folinate CRS. STORAGE Store in an airtight container, protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins. 1155

Calcium glucoheptonate


01/2005:1399

IMPURITIES

CALCIUM GLUCOHEPTONATE Calcii glucoheptonas A. (2S)-2[(4-aminobenzoyl)amino]pentanedioic acid,

C14H26CaO16

B. (2S)-2-[[4-[[[(6RS)-2-amino-5-formyl-4-oxo-1,4,5,6,7,8hexahydropteridin-6-yl]methyl]formylamino]benzoyl]amino]pentanedioic acid (5,10-diformyltetrahydrofolic acid),

DEFINITION Calcium glucoheptonate is a mixture, in variable proportions, of calcium di(D-glycero-D-gulo-heptonate) and calcium di(D-glycero-D-ido-heptonate). It contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of calcium 2,3,4,5,6,7-hexahydroxyheptanoate, calculated with reference to the dried substance. CHARACTERS A white or very slightly yellow, amorphous powder, hygroscopic, very soluble in water, practically insoluble in acetone and in alcohol.

C. folic acid,

D. (2S)-2-[[4-[[(2-amino-4-oxo-1,4-dihydropteridin-6yl)methyl]formylamino]benzoyl]amino]pentanedioic acid (10-formylfolic acid),

E. 4-[[[(6RS)-2-amino-5-formyl-4-oxo-1,4,5,6,7,8hexahydropteridin-6-yl]methyl]amino]benzoic acid (5-formyltetrahydropteroic acid),

Mr 490.4

IDENTIFICATION A. Examine by thin-layer chromatography (2.2.27), using cellulose for chromatography R1 as the coating substance. Test solution. Dissolve 20 mg of the substance to be examined in 1 ml of water R. Reference solution (a). Dissolve 20 mg of calcium glucoheptonate CRS in 1 ml of water R. Reference solution (b). Dissolve 10 mg of calcium gluconate CRS in 0.5 ml of the test solution and dilute to 1 ml with water R. Apply separately to the plate, as bands 20 mm by 2 mm, 10 µl of each solution. Allow the tank to saturate for 10 min. Develop over a path of 12 cm using a freshly prepared mixture of 20 volumes of anhydrous formic acid R, 20 volumes of water R, 30 volumes of acetone R and 30 volumes of butanol R. Allow the plate to dry in air and spray with 0.02 M potassium permanganate. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. B. 0.2 ml of solution S (see Tests) gives reaction (b) of calcium (2.3.1).

TESTS Solution S. Dissolve 10.0 g in carbon dioxide-free water R prepared from distilled water R and dilute to 100 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not F. R = CHO : (2S)-2-[[4-[[(2-amino-4-oxo-1,4,7,8-tetrahydropte- more intensely coloured than reference solution Y6 (2.2.2, ridin-6-yl)methyl]formylamino]benzoyl]amino]pentanedio- Method II). ic acid (10-formyldihydrofolic acid), pH (2.2.3). The pH of solution S is 6.0 to 8.0. Reducing sugars. Dissolve 1.0 g in 5 ml of water R with G. R = H : (2S)-2-[[4-[[(2-amino-4-oxo-1,4,7,8-tetrahydropteri- the aid of gentle heat. Cool and add 20 ml of cupri-citric din-6-yl)methyl]amino]benzoyl]amino]pentanedioic acid solution R and a few glass beads. Heat so that boiling (dihydrofolic acid). begins after 4 min and maintain boiling for 3 min. Cool 1156


Calcium gluconate


rapidly and add 100 ml of a 2.4 per cent V/V solution of glacial acetic acid R and 20.0 ml of 0.025 M iodine. With continuous shaking, add 25 ml of a mixture of 6 volumes of hydrochloric acid R and 94 volumes of water R until the precipitate dissolves, titrate the excess of iodine with 0.05 M sodium thiosulphate using 1 ml of starch solution R added towards the end of the titration, as indicator. Not less than 12.6 ml of 0.05 M sodium thiosulphate is required (1 per cent expressed as glucose). Cyanide. Dissolve 5.0 g in 50 ml of water R and add 2.0 g of tartaric acid R. Place this solution in a distillation apparatus (2.2.11). The plain bend adapter attached to the end of the condenser has a vertical part that is long enough to extend to 1 cm from the bottom of a 50 ml test-tube used as a receiver. Place 10 ml of water R and 2 ml of 0.1 M sodium hydroxide into the receiver. Distil, collect 25 ml of distillate and dilute to 50 ml with water R. To 25 ml of this solution add 25 mg of ferrous sulphate R and boil for a short time. After cooling to about 70 °C add 10 ml of hydrochloric acid R1. After 30 min, filter the solution and wash the filter. A yellow spot appears on the filter ; there is no blue or green spot. Chlorides (2.4.4). To 5 ml of solution S, add 10 ml of water R. The solution complies with the limit test for chlorides (100 ppm).

01/2005:0172

CALCIUM GLUCONATE Calcii gluconas

C12H22CaO14,H2O

Mr 448.4

DEFINITION Calcium gluconate contains not less than 98.5 per cent and not more than the equivalent of 102.0 per cent of calcium D-gluconate monohydrate. CHARACTERS A white, crystalline or granular powder, sparingly soluble in water, freely soluble in boiling water.

IDENTIFICATION A. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Iron (2.4.9). Dilute 2.5 ml of solution S to 10 ml with Test solution. Dissolve 20 mg of the substance to be water R. The solution complies with the limit test for iron examined in 1 ml of water R, heating if necessary in a (40 ppm). water-bath at 60 °C. Heavy metals (2.4.8). Dissolve 2.0 g in 10 ml of buffer Reference solution. Dissolve 20 mg of calcium solution pH 3.5 R and dilute to 20 ml with water R. 12 ml gluconate CRS in 1 ml of water R, heating if necessary in of the solution complies with limit test A for heavy metals a water-bath at 60 °C. (10 ppm). Prepare the standard using lead standard solution Apply separately to the plate 5 µl of each solution. Develop (1 ppm Pb) R. over a path of 10 cm using a mixture of 10 volumes of Loss on drying (2.2.32). Not more than 5.0 per cent, ethyl acetate R, 10 volumes of concentrated ammonia R, determined on 1.000 g by drying in an oven at 100 °C to 30 volumes of water R and 50 volumes of alcohol R. Dry 105 °C for 3 h. the plate at 100 °C for 20 min. Allow to cool and spray with a 50 g/l solution of potassium dichromate R in Bacterial endotoxins (2.6.14) : less than 167 IU/g, if a 40 per cent m/m solution of sulphuric acid R. After intended for use in the manufacture of parenteral dosage 5 min, the principal spot in the chromatogram obtained forms without a further appropriate procedure for the with the test solution is similar in position, colour and removal of bacterial endotoxins. size to the principal spot in the chromatogram obtained with the reference solution. B. Solution S (see Tests) gives the reactions of calcium ASSAY (2.3.1). Dissolve 0.800 g in a mixture of 150 ml of water R and 2 ml TESTS of 3 M hydrochloric acid. While stirring, add 12.5 ml of 0.1 M sodium edetate, 15 ml of 1 M sodium hydroxide and Solution S. Dissolve 1.0 g in water R heated to 60 °C and dilute to 50 ml with the same solvent. 0.3 g of hydroxynaphthol blue, sodium salt R. Titrate with 0.1 M sodium edetate until the colour changes from violet Appearance of solution. At 60 °C, solution S is not to pure blue. more intensely coloured than reference solution Y6 (2.2.2, Method II). After cooling, it is not more opalescent than 1 ml of 0.1 M sodium edetate is equivalent to 49.04 mg of reference suspension II (2.2.1). C14H26CaO16. Organic impurities and boric acid. Introduce 0.5 g into a porcelain dish previously rinsed with sulphuric acid R and placed in a bath of iced water. Add 2 ml of cooled sulphuric STORAGE acid R and mix. No yellow or brown colour develops. Add In an airtight container. If the substance is sterile, store in a 1 ml of chromotrope II B solution R. A violet colour develops and does not become dark blue. Compare the colour sterile, airtight, tamper-proof container. obtained with that of a mixture of 1 ml of chromotrope II B solution R and 2 ml of cooled sulphuric acid R. Sucrose and reducing sugars. Dissolve 0.5 g in a mixture LABELLING of 2 ml of hydrochloric acid R1 and 10 ml of water R. Boil The label states, where applicable, that the substance is free for 5 min, allow to cool, add 10 ml of sodium carbonate from bacterial endotoxins. solution R and allow to stand. Dilute to 25 ml with water R Sulphates (2.4.13). 15 ml of solution S complies with the limit test for sulphates (100 ppm).


1157

Calcium gluconate for injection


and filter. To 5 ml of the filtrate add 2 ml of cupri-tartaric solution R and boil for 1 min. Allow to stand for 2 min. No red precipitate is formed. Chlorides (2.4.4). 12.5 ml of solution S diluted to 15 ml with water R complies with the limit test for chlorides (200 ppm). Sulphates (2.4.13). Dissolve 10.0 g with heating in a mixture of 10 ml of acetic acid R and 90 ml of distilled water R. 15 ml of the solution complies with the limit test for sulphates (100 ppm). Heavy metals (2.4.8). 2.0 g complies with limit test D for heavy metals (10 ppm). Heat the substance to be examined gradually and with care until it is almost completely transformed into a white mass and then ignite. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Magnesium and alkali metals. Dissolve 1.00 g in 100 ml of boiling water R, add 10 ml of ammonium chloride solution R, 1 ml of ammonia R and, dropwise, 50 ml of hot ammonium oxalate solution R. Allow to stand for 4 h, dilute to 200 ml with water R and filter. Evaporate 100 ml of the filtrate to dryness and ignite. The residue weighs not more than 2 mg (0.4 per cent). Microbial contamination. Total viable aerobic count (2.6.12) not more than 103 micro-organisms per gram, determined by plate count. ASSAY Dissolve 0.8000 g in 20 ml of hot water R, allow to cool and dilute to 300 ml with water R. Carry out the complexometric titration of calcium (2.5.11). 1 ml of 0.1 M sodium edetate is equivalent to 44.84 mg of C12H22CaO14,H2O. 01/2005:0979

CALCIUM GLUCONATE FOR INJECTION Calcii gluconas ad iniectabile

C12H22CaO14,H2O

Mr 448.4

DEFINITION Calcium gluconate for injection contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of calcium D-gluconate monohydrate. CHARACTERS A white, crystalline or granular powder, sparingly soluble in water, freely soluble in boiling water. IDENTIFICATION A. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. Dissolve 20 mg of the substance to be examined in 1 ml of water R, heating if necessary in a water-bath at 60 °C. Reference solution. Dissolve 20 mg of calcium gluconate CRS in 1 ml of water R, heating if necessary in a water-bath at 60 °C. 1158

Apply to the plate 5 µl of each solution. Develop over a path of 10 cm using a mixture of 10 volumes of concentrated ammonia R, 10 volumes of ethyl acetate R, 30 volumes of water R and 50 volumes of alcohol R. Dry the plate at 100 °C for 20 min. Allow to cool and spray with a 50 g/l solution of potassium dichromate R in a 40 per cent m/m solution of sulphuric acid R. After 5 min, the principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. B. About 20 mg gives reaction (b) of calcium (2.3.1). TESTS Solution S. To 10.0 g add 90 ml of boiling distilled water R and boil with stirring, for not more than 10 s, until completely dissolved. Dilute to 100.0 ml with the same solvent. Appearance of solution. Solution S at 60 °C is not more intensely coloured than reference solution B7 (2.2.2, Method II). After cooling to 20 °C, it is not more opalescent than reference suspension II (2.2.1). pH (2.2.3). Dissolve 1.0 g in 20 ml of carbon dioxide-free water R, heating on a water-bath. The pH of the solution is 6.4 to 8.3. Organic impurities and boric acid. Introduce 0.5 g into a porcelain dish previously rinsed with sulphuric acid R and placed in a bath of iced water. Add 2 ml of cooled sulphuric acid R and mix. No yellow or brown colour develops. Add 1 ml of chromotrope II B solution R. A violet colour develops and does not become dark blue. The solution is not more intensely coloured than that of a mixture of 1 ml of chromotrope II B solution R and 2 ml of cooled sulphuric acid R. Oxalate. Not more than 100 ppm, determined by liquid chromatography (2.2.29). Test solution. Dissolve 1.00 g of the substance to be examined in water for chromatography R and dilute to 100.0 ml with the same solvent. Reference solution. Dissolve 1.00 g of the substance to be examined in water for chromatography R, add 0.5 ml of a 0.152 g/l solution of sodium oxalate R in water for chromatography R and dilute to 100.0 ml with the same solvent. The chromatographic procedure may be carried out using : — a guard column 30 mm long and 4 mm in internal diameter packed with a suitable strong anion exchange resin (30 µm to 50 µm), — two columns each 0.25 m long and 4 mm in internal diameter packed with a suitable strong anion exchange resin (30 µm to 50 µm), — a micromembrane anion-suppressor column, connected in series with the guard and analytical columns ; the anion-suppressor column is equipped with a micromembrane that separates the mobile phase from the suppressor regeneration solution, flowing countercurrent to the mobile phase at a rate of 4 ml/min, — as mobile phase at a flow rate of 2 ml/min a solution prepared as follows : dissolve 0.212 g of anhydrous sodium carbonate R and 63 mg of sodium hydrogen carbonate R in water for chromatography R and dilute to 1000.0 ml with the same solvent, — as suppressor regeneration solution a 1.23 g/l solution of sulphuric acid R in water for chromatography R, — a conductance detector, — a loop injector.


Calcium glycerophosphate


Inject 50 µl of the reference solution five times. The test is not valid unless the relative standard deviation of the peak area for oxalate in the chromatogram obtained with the reference solution is at most 2.0 per cent. Inject 50 µl of each solution three times. Calculate the content of oxalate in parts per million using the following expression :

ST

=

area of the oxalate peak in the chromatogram obtained with the test solution,

SR

=

area of the oxalate peak in the chromatogram obtained with the reference solution.

Sucrose and reducing sugars. Dissolve 0.5 g in a mixture of 2 ml of hydrochloric acid R1 and 10 ml of water R. Boil for 5 min, allow to cool, add 10 ml of sodium carbonate solution R and allow to stand for 10 min. Dilute to 25 ml with water R and filter. To 5 ml of the filtrate add 2 ml of cupri-tartaric solution R and boil for 1 min. Allow to stand for 2 min. No red precipitate is formed. Chlorides (2.4.4). To 10 ml of previously filtered solution S add 5 ml of water R. The solution complies with the limit test for chlorides (50 ppm). Phosphates (2.4.11). Dilute 1 ml of solution S to 100 ml with water R. The solution complies with the limit test for phosphates (100 ppm). Sulphates (2.4.13). 15 ml of previously filtered solution S complies with the limit test for sulphates (50 ppm). Prepare the standard using a mixture of 7.5 ml of sulphate standard solution (10 ppm SO4) R and 7.5 ml of distilled water R.

Microbial contamination. Total viable aerobic count (2.6.12) not more than 102 micro-organisms per gram, determined by plate count. It complies with the tests for Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus (2.6.13). ASSAY Dissolve 0.350 g in 20 ml of hot water R, allow to cool and dilute to 300 ml with water R. Carry out the complexometric titration of calcium (2.5.11). Use 50 mg of calconecarboxylic acid triturate R. 1 ml of 0.1 M sodium edetate is equivalent to 44.84 mg of C12H22CaO14,H2O. 01/2005:0980

CALCIUM GLYCEROPHOSPHATE Calcii glycerophosphas C3H7CaO6P

Mr 210.1

DEFINITION Calcium glycerophosphate is a mixture in variable proportions of calcium (RS)-2,3-dihydroxypropyl phosphate, and of calcium 2-hydroxy-1-(hydroxymethyl)ethyl phosphate, which may be hydrated. Calcium glycerophosphate contains not less than 18.6 per cent and not more than 19.4 per cent of Ca, calculated with reference to the dried substance. CHARACTERS A white powder, hygroscopic, sparingly soluble in water, practically insoluble in alcohol.

IDENTIFICATION A. Mix 1 g with 1 g of potassium hydrogen sulphate R in a test tube fitted with a glass tube. Heat strongly and direct the white vapour towards a piece of filter paper Test solution. Introduce 2.0 g into a 100 ml impregnated with a freshly prepared 10 g/l solution of polytetrafluoroethylene beaker and add 5 ml of sodium nitroprusside R. The filter paper develops a blue nitric acid R. Boil, evaporating almost to dryness. Add 1 ml colour in contact with piperidine R. of strong hydrogen peroxide solution R and evaporate again almost to dryness. Repeat the hydrogen peroxide treatment B. Ignite 0.1 g in a crucible. Take up the residue with 5 ml of nitric acid R and heat on a water-bath for 1 min. Filter. until a clear solution is obtained. Using 2 ml of nitric acid R, The filtrate gives reaction (b) of phosphates (2.3.1). transfer the solution into a 25 ml volumetric flask. Dilute to 25.0 ml with dilute hydrochloric acid R. In the same manner, C. It gives reaction (b) of calcium (2.3.1). prepare a compensation solution using 0.65 g of calcium TESTS chloride R1 instead of the substance to be examined. Solution S. Dissolve 1.5 g at room temperature in carbon Reference solutions. Prepare the reference solutions from dioxide-free water R prepared from distilled water R and iron solution (20 ppm Fe) R diluted with dilute hydrochloric dilute to 150 ml with the same solvent. acid R. Appearance of solution. Solution S is not more opalescent Measure the absorbance at 248.3 nm, using an iron than reference suspension III (2.2.1). hollow-cathode lamp as source of radiation and an Acidity or alkalinity. To 100 ml of solution S add 0.1 ml of air-acetylene flame. Carry out a basic correction using a phenolphthalein solution R. Not more than 1.5 ml of 0.1 M deuterium lamp. hydrochloric acid or 0.5 ml of 0.1 M sodium hydroxide is Magnesium and alkali metals. To 0.50 g add a mixture required to change the colour of the indicator. of 1.0 ml of dilute acetic acid R and 10.0 ml of water R Citric acid. Shake 5.0 g with 20 ml of carbon dioxide-free and rapidly boil, whilst shaking, until completely dissolved. water R and filter. To the filtrate add 0.15 ml of sulphuric To the boiling solution add 5.0 ml of ammonium oxalate acid R and filter again. To the filtrate add 5 ml of mercuric solution R and allow to stand for at least 6 h. Filter through sulphate solution R and heat to boiling. Add 0.5 ml of a a sintered-glass filter (1.6) into a porcelain crucible. Carefully 3.2 g/l solution of potassium permanganate R and again evaporate the filtrate to dryness and ignite. The residue heat to boiling. No precipitate is formed. weighs not more than 2 mg (0.4 per cent). Glycerol and alcohol-soluble substances. Shake 1.000 g Heavy metals (2.4.8). 12 ml of solution S complies with limit with 25 ml of alcohol R for 1 min. Filter. Evaporate the test A for heavy metals (10 ppm). Prepare the standard using filtrate to dryness on a water-bath and dry the residue at lead standard solution (1 ppm Pb) R. 70 °C for 1 h. The residue weighs not more than 5 mg (0.5 per cent). Bacterial endotoxins (2.6.14) : less than 167 IU/g.

Iron. Not more than 5 ppm of Fe, determined by atomic absorption spectrometry (2.2.23, Method I).


1159

Calcium hydrogen phosphate, anhydrous


Chlorides (2.4.4). Dissolve 0.1 g in a mixture of 2 ml of acetic acid R and 8 ml of water R and dilute to 15 ml with water R. The solution complies with the limit test for chlorides (500 ppm). Phosphates (2.4.11). Dilute 2.5 ml of solution S to 100 ml with water R. The solution complies with the limit test for phosphates (400 ppm). Sulphates (2.4.13). 15 ml of solution S complies with the limit test for sulphates (0.1 per cent). Arsenic (2.4.2). Dissolve 0.33 g in water R and dilute to 25 ml with the same solvent. The solution complies with limit test A for arsenic (3 ppm). Iron (2.4.9). 0.20 g complies with the limit test for iron (50 ppm). Heavy metals (2.4.8). Dissolve 2.0 g in 10 ml of buffer solution pH 3.5 R and dilute to 20 ml with water R. 12 ml of the solution complies with limit test A for heavy metals (20 ppm). Prepare the standard using lead standard solution (2 ppm Pb) R. Loss on drying (2.2.32). Not more than 12.0 per cent, determined on 1.000 g by drying in an oven at 150 °C for 4 h.

Chlorides (2.4.4) : maximum 330 ppm. Dissolve 0.5 g in a mixture of 1 ml of nitric acid R and 10 ml of water R and dilute to 50 ml with water R. 15 ml of the solution complies with the limit test for chlorides. Fluorides (2.4.5) : maximum 100 ppm. 0.5 g complies with the limit test for fluorides. Sulphates (2.4.13) : maximum 0.5 per cent. To 1 ml of solution S add 2 ml of dilute hydrochloric acid R and dilute to 25 ml with distilled water R. 15 ml of the solution complies with the limit test for sulphates. Arsenic (2.4.2) : maximum 10 ppm. 2 ml of solution S complies with limit test A. Barium. To 10 ml of filtered solution S add 0.5 ml of dilute sulphuric acid R. Allow to stand for 15 min. Any opalescence in the solution is not more intense than that in a mixture of 10 ml of solution S and 0.5 ml of distilled water R. Iron (2.4.9) : maximum 400 ppm. Dilute 0.5 ml of solution S to 10 ml with water R. The solution complies with the limit test for iron. Heavy metals (2.4.8) : maximum 40 ppm. Dilute 10 ml of solution S to 20 ml with water R. 12 ml of ASSAY the solution complies with limit test A. Prepare the standard Dissolve 0.200 g in water R. Carry out the complexometric using lead standard solution (1 ppm Pb) R. titration of calcium (2.5.11). 1 ml of 0.1 M sodium edetate is equivalent to 4.008 mg of Ca. Loss on drying (2.2.32) : maximum 2.0 per cent, determined on 1.000 g by drying in an oven at 150 °C for 2 h.

ASSAY 01/2005:0981 Dissolve 0.250 g in a mixture of 1 ml of hydrochloric corrected acid R1 and 5 ml of water R. Add 25.0 ml of 0.1 M sodium edetate and dilute to 200 ml with water R. Neutralise with CALCIUM HYDROGEN PHOSPHATE, concentrated ammonia R, add 10 ml of ammonium chloride buffer solution pH 10.0 R and about 50 mg of mordant ANHYDROUS black 11 triturate R. Titrate the excess of sodium edetate with 0.1 M zinc sulphate until the colour changes from blue Calcii hydrogenophosphas anhydricus to violet. Carry out a blank titration. 1 ml of 0.1 M sodium edetate is equivalent to 13.61 mg of CaHPO4 Mr 136.1 CaHPO4. DEFINITION Content : 98.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white, crystalline powder, or colourless crystals. Solubility : practically insoluble in water and in alcohol. It dissolves in dilute hydrochloric acid and in dilute nitric acid. IDENTIFICATION A. It complies with the limits of the assay. B. Dissolve 0.1 g in a mixture of 5 ml of dilute nitric acid R and 5 ml of water R. The solution gives reaction (b) of phosphates (2.3.1). C. Dissolve 5 mg in 5 ml of acetic acid R. The solution gives reaction (b) of calcium (2.3.1). TESTS Solution S. Dissolve 2.5 g in 20 ml of dilute hydrochloric acid R. Filter if necessary. Add dilute ammonia R1 until a precipitate is formed. Dissolve by adding the minimum quantity needed of dilute hydrochloric acid R and dilute to 50 ml with distilled water R. Carbonates. Shake 0.5 g with 5 ml of carbon dioxide-free water R and add 1 ml of hydrochloric acid R. No effervescence is produced. 1160

01/2005:0116

CALCIUM HYDROGEN PHOSPHATE DIHYDRATE Calcii hydrogenophosphas dihydricus CaHPO4,2H2O

Mr 172.1

DEFINITION Content : 98.0 per cent to 105.0 per cent. CHARACTERS Appearance : white, crystalline powder. Solubility : practically insoluble in cold water and in alcohol. It dissolves in dilute hydrochloric acid and in dilute nitric acid. IDENTIFICATION A. Dissolve 0.1 g in a mixture of 5 ml of dilute nitric acid R and 5 ml of water R. The solution gives reaction (b) of phosphates (2.3.1). B. 5 mg gives reaction (b) of calcium (2.3.1). C. It complies with the limits of the assay.


Calcium hydroxide


TESTS Solution S. Dissolve 2.5 g in 20 ml of dilute hydrochloric acid R, filter if necessary and add dilute ammonia R1 until a precipitate is formed. Add just sufficient dilute hydrochloric acid R to dissolve the precipitate and dilute to 50 ml with distilled water R. Carbonates. Shake 0.5 g with 5 ml of carbon dioxide-free water R and add 1 ml of hydrochloric acid R. No effervescence is produced. Chlorides (2.4.4) : maximum 330 ppm.

CHARACTERS

Fluorides (2.4.5) : maximum 100 ppm.

B. Dissolve about 0.1 g in dilute hydrochloric acid R and dilute to 10 ml with water R. 5 ml of the solution give reaction (b) of calcium (2.3.1).

A white or almost white, fine powder, practically insoluble in water. IDENTIFICATION

A. To 0.80 g in a mortar, add 10 ml of water R and 0.5 ml of phenolphthalein solution R and mix. The suspension turns red. On addition of 17.5 ml of 1 M hydrochloric acid, the suspension becomes colourless without effervescing. The red colour occurs again when the mixture is triturated for 1 min. On addition of a further Dissolve 0.5 g in a mixture of 1 ml of nitric acid R and 10 ml 6 ml of 1 M hydrochloric acid and triturating, the of water R and dilute to 50 ml with water R. 15 ml of the solution becomes colourless. solution complies with the limit test for chlorides. 0.5 g complies with the limit test for fluorides. Sulphates (2.4.13) : maximum 0.5 per cent. To 1 ml of solution S add 2 ml of dilute hydrochloric acid R and dilute to 25 ml with distilled water R. 15 ml of the solution complies with the limit test for sulphates. Arsenic (2.4.2) : maximum 10 ppm.

2 ml of solution S complies with limit test A. Barium. To 10 ml of solution S add 0.5 ml of dilute sulphuric acid R. After 15 min, any opalescence in the solution is not more intense than that in a mixture of 10 ml of solution S and 0.5 ml of distilled water R. Iron (2.4.9) : maximum 400 ppm. 0.5 ml of solution S diluted to 10 ml with water R complies with the limit test for iron. Heavy metals (2.4.8) : maximum 40 ppm.

TESTS Matter insoluble in hydrochloric acid. Dissolve 2.0 g in 30 ml of hydrochloric acid R. Boil the solution and filter. Wash the residue with hot water R. The residue weighs not more than 10 mg (0.5 per cent). Carbonates. Not more than 5.0 per cent of CaCO3. Add 5.0 ml of 1 M hydrochloric acid to the titrated solution obtained under Assay and titrate with 1 M sodium hydroxide using 0.5 ml of methyl orange solution R as indicator. 1 ml of 1 M hydrochloric acid is equivalent to 50.05 mg of CaCO3. Chlorides (2.4.4). Dissolve 0.30 g in a mixture of 2 ml of nitric acid R and 10 ml of water R and dilute to 30 ml with water R. 15 ml of the solution complies with the limit test for chlorides (330 ppm).

Dilute 10 ml of solution S to 20 ml with water R. 12 ml of the solution complies with limit test A. Prepare the standard Sulphates (2.4.13). Dissolve 0.15 g in a mixture of 5 ml of using lead standard solution (1 ppm Pb) R. dilute hydrochloric acid R and 10 ml of distilled water R and dilute to 60 ml with distilled water R. 15 ml of the solution ASSAY complies with the limit test for sulphates (0.4 per cent). Dissolve 0.300 g in a mixture of 1 ml of hydrochloric Arsenic (2.4.2). Dissolve 0.50 g in 5 ml of brominated acid R1 and 5 ml of water R. Add 25.0 ml of 0.1 M sodium hydrochloric acid R and dilute to 50 ml with water R. 25 ml edetate and dilute to 200 ml with water R. Neutralise with concentrated ammonia R, add 10 ml of ammonium chloride of the solution complies with the limit test A for arsenic (4 ppm). buffer solution pH 10.0 R and about 50 mg of mordant black 11 triturate R. Titrate the excess of sodium edetate Magnesium and alkali metals. Dissolve 1.0 g in a mixture of with 0.1 M zinc sulphate until the colour changes from blue 10 ml of hydrochloric acid R and 40 ml of water R. Boil and to violet. Carry out a blank titration. add 50 ml of a 63 g/l solution of oxalic acid R. Neutralise with ammonia R and dilute to 200 ml with water R. Allow to 1 ml of 0.1 M sodium edetate is equivalent to 17.21 mg of stand for 1 h and filter through a suitable filter. To 100 ml CaHPO4,2H2O. of the filtrate, add 0.5 ml of sulphuric acid R. Cautiously evaporate to dryness and ignite. The residue weighs not more than 20 mg (4.0 per cent calculated as sulphates).

CALCIUM

Heavy metals (2.4.8). Dissolve 1.0 g in 10 ml of hydrochloric 01/2005:1078 acid R1 and evaporate to dryness on a water-bath. Dissolve corrected the residue in 20 ml of water R and filter. 12 ml of the filtrate complies with limit test A for heavy metals (20 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. HYDROXIDE

Calcii hydroxidum Ca(OH)2 DEFINITION

ASSAY To 1.500 g in a mortar, add 20 ml to 30 ml of water R and 0.5 ml of phenolphthalein solution R. Titrate with 1 M Mr 74.1 hydrochloric acid by triturating the substance until the red colour disappears. The final solution is used in the tests for carbonates.

Calcium hydroxide contains not less than 95.0 per cent and not more than the equivalent of 100.5 per cent of Ca(OH)2. General Notices (1) apply to all monographs and other texts

1 ml of 1 M hydrochloric acid is equivalent to 37.05 mg of Ca(OH)2. 1161

Calcium lactate pentahydrate


01/2005:0468 4 h, dilute to 100.0 ml with water R and filter. To 50.0 ml of the filtrate add 0.5 ml of sulphuric acid R, evaporate to dryness and ignite the residue to constant mass at 600 °C. CALCIUM LACTATE PENTAHYDRATE The residue weighs not more than 5 mg (1 per cent). Loss on drying (2.2.32) : 22.0 per cent to 27.0 per cent, Calcii lactas pentahydricus determined on 0.500 g by drying in an oven at 125 °C. ASSAY Dissolve 0.200 g in water R and dilute to 300 ml with the same solvent. Carry out the complexometric titration of C6H10CaO6,xH2O with x 5 Mr 218.2 (anhydrous substance) calcium (2.5.11). 1 ml of 0.1 M sodium edetate is equivalent to 21.82 mg of DEFINITION C6H10CaO6. Calcium lactate pentahydrate contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of 01/2005:0469 calcium bis(2-hydroxypropanoate) or of a mixture of calcium (R)-, (S)- and (RS)-2-hydroxypropionates, calculated with CALCIUM LACTATE TRIHYDRATE reference to the dried substance. It contains not less than 22.0 per cent and not more than 27.0 per cent of water, Calcii lactas trihydricus determined by the loss on drying. CHARACTERS A white or almost white, crystalline or granular powder, slightly efflorescent, soluble in water, freely soluble in boiling water, very slightly soluble in alcohol. IDENTIFICATION A. It complies with the test for loss on drying (see Tests). B. It gives the reaction of lactates (2.3.1). C. It gives reaction (b) of calcium (2.3.1).

C6H10CaO6,xH2O with x

3 Mr 218.2 (anhydrous substance)

DEFINITION Calcium lactate trihydrate contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of calcium bis(2-hydroxypropanoate) or of a mixture of calcium (R)-, (S)- and (RS)-2-hydroxypropionates, calculated with TESTS reference to the dried substance. It contains not less than Solution S. Dissolve 5.0 g with heating in carbon dioxide-free 15.0 per cent and not more than 20.0 per cent of water, water R prepared from distilled water R, allow to cool and determined by the loss on drying. dilute to 100 ml with the same solvent. Appearance of solution. Solution S is not more opalescent CHARACTERS than reference suspension II (2.2.1) and not more intensely A white or almost white, crystalline or granular powder, soluble in water, freely soluble in boiling water, very slightly coloured than reference solution BY6 (2.2.2, Method II). soluble in alcohol. Acidity or alkalinity. To 10 ml of solution S add 0.1 ml of phenolphthalein solution R and 0.5 ml of 0.01 M IDENTIFICATION hydrochloric acid. The solution is colourless. Not more than A. It complies with the test for loss on drying (see Tests). 2.0 ml of 0.01 M sodium hydroxide is required to change the B. It gives the reaction of lactates (2.3.1). colour of the indicator to pink. C. It gives reaction (b) of calcium (2.3.1). Volatile fatty acids. In a 100 ml ground-glass-stoppered flask stir 0.5 g with 1 ml of phosphoric acid R. Close the TESTS flask. Heat cautiously at 50 °C for 10 min. No unpleasant Solution S. Dissolve 5.0 g with heating in carbon dioxide-free odour resembling that of the lower fatty acids is recognisable water R prepared from distilled water R, allow to cool and immediately after opening the flask. dilute to 100 ml with the same solvent. Chlorides (2.4.4). 5 ml of solution S diluted to 15 ml with Appearance of solution. Solution S is not more opalescent water R complies with the limit test for chlorides (200 ppm). than reference suspension II (2.2.1) and not more intensely Sulphates (2.4.13). 7.5 ml of solution S diluted to 15 ml with coloured than reference solution BY6 (2.2.2, Method II). distilled water R complies with the limit test for sulphates Acidity or alkalinity. To 10 ml of solution S add 0.1 ml (400 ppm). of phenolphthalein solution R and 0.5 ml of 0.01 M Barium. To 10 ml of solution S add 1 ml of calcium sulphate hydrochloric acid. The solution is colourless. Not more than solution R. Allow to stand for 15 min. Any opalescence in 2.0 ml of 0.01 M sodium hydroxide is required to change the the solution is not more intense than that in a mixture of colour of the indicator to pink. 1 ml of distilled water R and 10 ml of solution S. Volatile fatty acids. In a 100 ml ground-glass-stoppered Heavy metals (2.4.8). 12 ml of solution S complies with limit flask stir 0.5 g with 1 ml of phosphoric acid R. Close the test A for heavy metals (20 ppm). Prepare the standard using flask. Heat cautiously at 50 °C for 10 min. No unpleasant lead standard solution (1 ppm Pb) R. odour resembling that of the lower fatty acids is recognisable Iron (2.4.9). 4 ml of solution S diluted to 10 ml with water R immediately after opening the flask. complies with the limit test for iron (50 ppm). Chlorides (2.4.4). 5 ml of solution S diluted to 15 ml with water R complies with the limit test for chlorides (200 ppm). Magnesium and alkali salts. To 20 ml of solution S add 20 ml of water R, 2 g of ammonium chloride R and 2 ml of Sulphates (2.4.13). 7.5 ml of solution S diluted to 15 ml with dilute ammonia R1. Heat to boiling and rapidly add 40 ml distilled water R complies with the limit test for sulphates of hot ammonium oxalate solution R. Allow to stand for (400 ppm). 1162



Calcium levofolinate pentahydrate

Barium. To 10 ml of solution S add 1 ml of calcium sulphate B. Infrared absorption spectrophotometry (2.2.24). solution R. Allow to stand for 15 min. Any opalescence in Preparation : discs. the solution is not more intense than that in a mixture of Comparison : calcium folinate CRS. 1 ml of distilled water R and 10 ml of solution S. If the spectra obtained show differences, dissolve the Heavy metals (2.4.8). 12 ml of solution S complies with limit substance to be examined and the reference substance test A for heavy metals (20 ppm). Prepare the standard using separately in the minimum quantity of water R and add lead standard solution (1 ppm Pb) R. dropwise sufficient acetone R to produce a precipitate. Allow to stand for 15 min, collect the precipitate by Iron (2.4.9). 4 ml of solution S diluted to 10 ml with water R centrifugation, wash the precipitate twice with a minimum complies with the limit test for iron (50 ppm). quantity of acetone R and dry. Record new spectra using Magnesium and alkali salts. To 20 ml of solution S add the residues. 20 ml of water R, 2 g of ammonium chloride R and 2 ml of C. Thin-layer chromatography (2.2.27). dilute ammonia R1. Heat to boiling and rapidly add 40 ml Test solution. Dissolve 15 mg of the substance to be of hot ammonium oxalate solution R. Allow to stand for examined in a 3 per cent V/V solution of ammonia R and 4 h, dilute to 100.0 ml with water R and filter. To 50.0 ml dilute to 5 ml with the same solvent. of the filtrate add 0.5 ml of sulphuric acid R, evaporate to dryness and ignite the residue to constant mass at 600 °C. Reference solution. Dissolve 15 mg of calcium The residue weighs not more than 5 mg (1 per cent). folinate CRS in a 3 per cent V/V solution of ammonia R and dilute to 5 ml with the same solvent. Loss on drying (2.2.32) : 15.0 per cent to 20.0 per cent, determined on 0.500 g by drying in an oven at 125 °C. Plate : cellulose for chromatography F254 R. Mobile phase : the lower layer of a mixture of 1 volume of ASSAY isoamyl alcohol R and 10 volumes of a 50 g/l solution of Dissolve 0.200 g in water R and dilute to 300 ml with the citric acid R previously adjusted to pH 8 with ammonia R. same solvent. Carry out the complexometric titration of Application : 5 µl. calcium (2.5.11). Development : over a path of 15 cm. 1 ml of 0.1 M sodium edetate is equivalent to 21.82 mg of Drying : in air. C6H10CaO6. Detection : examine in ultraviolet light at 254 nm. 01/2005:1606 Results : the principal spot in the chromatogram obtained with the test solution is similar in position and size to CALCIUM LEVOFOLINATE the principal spot in the chromatogram obtained with the reference solution. PENTAHYDRATE D. It gives reaction (b) of calcium (2.3.1). Carry out the tests and the assay as rapidly as possible, Calcii levofolinas pentahydricus protected from bright light. TESTS Solution S. Dissolve 0.40 g in carbon dioxide-free water R, heating at 40 °C if necessary, and dilute to 50.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and its absorbance (2.2.25) at 420 nm has a maximum of 0.25. pH (2.2.3) : 7.5 to 8.5 for solution S. C20H21CaN7O7,5H2O Mr 511.5 (anhydrous substance) Specific optical rotation (2.2.7) : − 10 to − 15 (anhydrous and solvent-free substance). DEFINITION Dissolve 0.200 g in tris(hydroxymethyl)aminomethane Calcium (2S)-2-[[4-[[[(6S)-2-amino-5-formyl-4-oxo-1,4,5,6,7,8solution R previously adjusted to pH 8.1 with sodium hexahydropteridin-6-yl]methyl]amino]benzoyl]amino]penhydroxide solution R or hydrochloric acid R1 and dilute to tanedioate pentahydrate. 20.0 ml with the same solvent. Content : Acetone and ethanol. Head-space gas chromatography — calcium levofolinate (C20H21CaN7O7 ; Mr 511.5) : 97.0 per (2.2.28) : use the standard additions method. cent to 102.0 per cent (anhydrous and solvent-free Test solution. Dissolve 0.25 g of the substance to be substance). examined in water R and dilute to 10.0 ml with the same — calcium (Ca ; Ar 40.08) : 7.54 per cent to 8.14 per cent solvent. (anhydrous and solvent-free substance). Reference solution. Dissolve 0.125 g of acetone R and CHARACTERS 0.750 g of ethanol R in water R and dilute to 1000.0 ml Appearance : white or light yellow, amorphous or crystalline with water R. powder, hygroscopic. Column : Solubility : slightly soluble in water, practically insoluble in — material : fused silica, acetone and in alcohol. — size : l = 10 m, Ø = 0.32 mm, — stationary phase : styrene-divinylbenzene copolymer R. IDENTIFICATION Carrier gas : nitrogen for chromatography R. First identification : A, B, D. Flow rate : 4 ml/min. Second identification : A, C, D. A. It complies with the test for specific optical rotation (see Static head-space conditions which may be used : Tests). — equilibration temperature : 80 °C, General Notices (1) apply to all monographs and other texts

1163

Calcium levofolinate pentahydrate


— equilibration time : 20 min, — pressurisation time : 30 s. Temperature :

Column

Time (min)

Temperature (°C)

0 - 14

80 → 220

Injection port

110

Detector

270

Detection : flame ionisation. Injection : at least 3 times. Limits : — acetone : maximum 0.5 per cent, — ethanol: maximum 3.0 per cent. Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 10.0 mg of the substance to be examined in water R and dilute to 10.0 ml with the same solvent. Reference solution (a). Dissolve 10.0 mg of calcium folinate CRS in water R and dilute to 10.0 ml with the same solvent. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 100.0 ml with water R. Reference solution (c). Dissolve 10.0 mg of formylfolic acid CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with water R. Reference solution (d). Dilute 1.0 ml of reference solution (b) to 20.0 ml with water R. Reference solution (e). Dilute 5.0 ml of reference solution (c) to 10.0 ml with reference solution (b). Column : — size : l = 0.25 m, Ø = 4 mm, — stationary phase: octadecylsilyl silica gel for chromatography R (5 µm), — temperature : 40 °C. Mobile phase : mix 220 ml of methanol R and 780 ml of a solution containing 2.0 ml of tetrabutylammonium hydroxide solution (400 g/l) R and 2.2 g of disodium hydrogen phosphate R previously adjusted to pH 7.8 with phosphoric acid R. If necessary adjust the concentration of methanol R to achieve the prescribed resolution. Flow rate : 1 ml/min. Detection : spectrophotometer at 280 nm. Injection : 10 µl. Run time : 2.5 times the retention time of the principal peak in the chromatogram obtained with the test solution. System suitability : reference solution (e) : — resolution : minimum of 2.2 between the peaks due to folinate and to impurity D. Limits : — impurity D : not more than 0.8 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.8 per cent), — any other impurity : not more than 0.8 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.8 per cent), — total of other impurities : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (2.0 per cent), 1164

— disregard limit : area of the principal peak in the chromatogram obtained with reference solution (d) (0.05 per cent). Impurity H. Liquid chromatography (2.2.29) : use the normalisation procedure. Test solution. Dissolve 50.0 mg of the substance to be examined in water R and dilute to 100.0 ml with the same solvent. Reference solution (a). Dissolve 10.0 mg of calcium folinate CRS in water R and dilute to 20.0 ml with the same solvent. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 100.0 ml with water R. Column : — size : l = 0.15 m, Ø = 4 mm, — stationary phase : human albumin coated silica gel for chromatography R (5 µm), — temperature : 40 °C. Mobile phase : dissolve 9.72 g of sodium dihydrogen phosphate R in 890 ml of water R and adjust to pH 5.0 with sodium hydroxide solution R. Add 100 ml of 2-propanol R and 10 ml of acetonitrile R. Flow rate : 1 ml/min. Detection : spectrophotometer at 286 nm. Injection : 10 µl. Retention times : levofolinate = about 9 min ; impurity H = about 19 min. System suitability : — resolution : minimum of 5.0 between the peaks due to levofolinate and to impurity H in the chromatogram obtained with reference solution (a). The sum of the areas of the 2 peaks is 100 per cent. The peak area of impurity H is 48 per cent to 52 per cent. In the chromatogram obtained with reference solution (b) 2 clearly visible peaks are obtained. Limits : — impurity H : maximum 0.5 per cent. Chlorides : maximum 0.5 per cent. Dissolve 0.300 g in 50 ml of water R heating at 40 °C if necessary. Add 10 ml of 2 M nitric acid and titrate with 0.005 M silver nitrate determining the end-point potentiometrically (2.2.20). 1 ml of 0.005 M silver nitrate is equivalent to 0.177 mg of Cl. Platinum : maximum 10 ppm. Atomic absorption spectrometry (2.2.23, Method II). Test solution. Dissolve 1.0 g in water R and dilute to 100.0 ml with the same solvent. Reference solutions. Prepare the reference solutions using platinum standard solution (30 ppm Pt) R, diluted as necessary with a mixture of 1 volume of nitric acid R and 99 volumes of water R. Source : platinum hollow-cathode lamp. Wavelength : 265.9 nm. Heavy metals (2.4.8) : maximum 50 ppm. 1.0 g complies with limit test F. Prepare the standard using 5 ml of lead standard solution (10 ppm Pb) R. Water (2.5.12) : 10.0 per cent to 17.0 per cent, determined on 0.200 g (ground to a very fine powder). Stir the substance to be examined in the titration solvent for about 15 min before titrating and use iodosulphurous reagent R as titrant.


Calcium levulinate dihydrate


Bacterial endotoxins (2.6.14) : less than 0.5 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. ASSAY Calcium. Dissolve 0.400 g in 150 ml of water R and dilute to 300 ml with the same solvent. Carry out the complexometric F. R = CHO : (2S)-2-[[4-[[(2-amino-4-oxo-1,4,7,8-tetrahydroptetitration of calcium (2.5.11). ridin-6-yl)methyl]formylamino]benzoyl]amino]pentanedio1 ml of 0.1 M sodium edetate is equivalent to 4.008 mg of Ca. ic acid (10-formyldihydrofolic acid), Calcium folinate. Liquid chromatography (2.2.29) as G. R = H : (2S)-2-[[4-[[(2-amino-4-oxo-1,4,7,8-tetrahydropteridescribed in the test for related substances. din-6-yl)methyl]amino]benzoyl]amino]pentanedioic acid Calculate the percentage content of C20H21CaN7O7 from the (dihydrofolic acid), areas of the peaks in the chromatograms obtained with the test solution and reference solution (a) and the declared content of calcium folinate CRS. STORAGE In an airtight container, protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins.

H. (2S)-2-[[4-[[[(6R)-2-amino-5-formyl-4-oxo-1,4,5,6,7,8hexahydropteridin-6-yl]methyl]amino]benzoyl]amino]pentanedioic acid.

IMPURITIES

01/2005:1296

CALCIUM LEVULINATE DIHYDRATE Calcii laevulinas dihydricum A. (2S)-2-[(4-aminobenzoyl)amino]pentanedioic acid,

C10H14CaO6,2H2O

B. (2S)-2-[[4-[[[(6R)-2-amino-5-formyl-4-oxo-1,4,5,6,7,8hexahydropteridin-6-yl]methyl]formylamino]benzoyl]amino]pentanedioic acid (5,10-diformyltetrahydrofolic acid), C. folic acid,

D. (2S)-2-[[4-[[(2-amino-4-oxo-1,4-dihydropteridin-6yl)methyl]formylamino]benzoyl]amino]pentanedioic acid (10-formylfolic acid),

E. 4-[[[(6S)-2-amino-5-formyl-4-oxo-1,4,5,6,7,8hexahydropteridin-6-yl]methyl]amino]benzoic acid (5-formyltetrahydropteroic acid), General Notices (1) apply to all monographs and other texts

Mr 306.3

DEFINITION Calcium levulinate dihydrate contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of calcium di(4-oxopentanoate), calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, freely soluble in water, very slightly soluble in alcohol, practically insoluble in methylene chloride. IDENTIFICATION First identification : A, D, E. Second identification : B, C, D, E. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with calcium levulinate dihydrate CRS. B. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating substance. Test solution. Dissolve 60 mg of the substance to be examined in water R and dilute to 1 ml with the same solvent. Reference solution. Dissolve 60 mg of calcium levulinate dihydrate CRS in water R and dilute to 1 ml with the same solvent. Apply separately to the plate 10 µl of each solution. Develop over a path of 10 cm using a mixture of 10 volumes of concentrated ammonia R, 10 volumes of ethyl acetate R, 30 volumes of water R and 50 volumes 1165

Calcium pantothenate


of alcohol R. Dry the plate at 100 °C to 105 °C for 20 min and allow to cool. Spray with a 30 g/l solution of potassium permanganate R. Dry the plate in a current of warm air for about 5 min or until the spots become yellow. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. C. To 1 ml of solution S (see Tests), add 20 ml of a 2.5 g/l solution of dinitrophenylhydrazine R in dilute hydrochloric acid R. Allow to stand for 15 min. Filter, wash the precipitate with water R. Dry the precipitate in an oven at 100 °C to 105 °C. The melting point (2.2.14) is 203 °C to 210 °C. D. It gives reaction (b) of calcium (2.3.1). E. It complies with the test for loss on drying (see Tests).

ASSAY Dissolve 0.240 g in 50 ml of water R. Carry out the complexometric titration of calcium (2.5.11). 1 ml of 0.1 M sodium edetate is equivalent to 27.03 mg of C10H14CaO6. STORAGE Store protected from light. LABELLING The label states, where applicable, that the substance is apyrogenic. 01/2005:0470

CALCIUM PANTOTHENATE Calcii pantothenas

TESTS Solution S. Dissolve 10.0 g in carbon dioxide-free water R prepared from distilled water R and dilute to 100.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). pH (2.2.3). The pH of solution S is 6.8 to 7.8. Oxidisable substances. To 1 ml of solution S, add 10 ml of water R, 1 ml of dilute sulphuric acid R and 0.25 ml of a 3.0 g/l solution of potassium permanganate R. Mix. After 5 min, the violet colour of the mixture is still visible. Sucrose and reducing sugars. To 5 ml of solution S, add 2 ml of hydrochloric acid R1 and dilute to 10 ml with water R. Heat to boiling for 5 min and allow to cool. Add 10 ml of sodium carbonate solution R. Allow to stand for 5 min, dilute to 25 ml with water R and filter. To 5 ml of the filtrate, add 2 ml of cupri-tartaric solution R and heat to boiling for 1 min. No red precipitate is formed. Chlorides (2.4.4). Dilute 10 ml of solution S to 15 ml with water R. The solution complies with the limit test for chlorides (50 ppm). Sulphates (2.4.13). Dilute 7.5 ml of solution S to 15 ml with distilled water R. The solution complies with the limit test for sulphates (200 ppm). Magnesium and alkali metals. To 10 ml of solution S, add 80 ml of water R, 10 ml of ammonium chloride solution R and 1 ml of ammonia R. Heat to boiling. To the boiling solution, add dropwise 50 ml of warm ammonium oxalate solution R. Allow to stand for 4 h, then dilute to 200 ml with water R and filter. To 100 ml of the filtrate, add 0.5 ml of sulphuric acid R. Evaporate to dryness on a water-bath and ignite to constant mass at 600 °C. The residue weighs not more than 5.0 mg (1.0 per cent).

C18H32CaN2O10

Mr 476.5

DEFINITION Calcium pantothenate contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of calcium bis[3-[[(2R)-2,4-dihydroxy-3,3dimethylbutanoyl]amino]propanoate], calculated with reference to the dried substance. CHARACTERS A white powder, slightly hygroscopic, freely soluble in water, slightly soluble in alcohol. IDENTIFICATION A. It complies with the test for specific optical rotation (see Tests). B. Examine the chromatograms obtained in the test for 3-aminopropionic acid. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). C. To 1 ml of solution S (see Tests) add 1 ml of dilute sodium hydroxide solution R and 0.1 ml of copper sulphate solution R. A blue colour develops. D. It gives reaction (a) of calcium (2.3.1).

TESTS Solution S. Dissolve 2.50 g in carbon dioxide-free water R and dilute to 50.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Heavy metals (2.4.8). 12 ml of solution S complies with limit pH (2.2.3). The pH of solution S is 6.8 to 8.0. test A for heavy metals (10 ppm). Prepare the standard using Specific optical rotation (2.2.7) : + 25.5 to + 27.5, determined lead standard solution (1 ppm Pb) R. on solution S and calculated with reference to the dried substance. Loss on drying (2.2.32) : 11.0 per cent to 12.5 per cent, determined on 0.200 g by drying at 100 °C to 105 °C. 3-Aminopropionic acid. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating Pyrogens (2.6.8). If intended for use in the manufacture substance. of parenteral dosage forms without a further appropriate procedure for the removal of pyrogens, it complies with the Test solution (a). Dissolve 0.2 g of the substance to be examined in water R and dilute to 5 ml with the same solvent. test for pyrogens. Inject per kilogram of the rabbit’s mass Test solution (b). Dilute 1 ml of test solution (a) to 10 ml 4 ml of a solution containing per millilitre 50 mg of the with water R. substance to be examined.

1166


Calcium stearate


Fluorides. Not more than 75 ppm of F, determined potentiometrically (2.2.36, Method I) using a fluoride-selective indicator electrode and a silver-silver chloride reference electrode. Test solution. In a 50 ml volumetric flask, dissolve 0.250 g in 0.1 M hydrochloric acid, add 5.0 ml of fluoride standard solution (1 ppm F) R and dilute to 50.0 ml with 0.1 M hydrochloric acid. To 20.0 ml of the solution add 20.0 ml of total-ionic-strength-adjustment buffer R and 3 ml of an 82 g/l solution of anhydrous sodium acetate R. Adjust to pH 5.2 with ammonia R and dilute to 50.0 ml with distilled water R. Reference solutions. To 5.0 ml, 2.0 ml, 1.0 ml, 0.5 ml and 0.25 ml of fluoride standard solution (10 ppm F) R add 20.0 ml of total-ionic-strength-adjustment buffer R and dilute to 50.0 ml with distilled water R. Carry out the measurements on 20.0 ml of each solution. Calculate the concentration of fluorides using the calibration curve, taking into account the addition of fluoride to the test solution. Sulphates (2.4.13). Dilute 1 ml of solution S to 25 ml with distilled water R. 15 ml of the solution complies with the limit test for sulphates (0.5 per cent). ASSAY Dissolve 0.180 g in 50 ml of anhydrous acetic acid R. Arsenic (2.4.2). 5 ml of solution S complies with limit test A Titrate with 0.1 M perchloric acid determining the end-point for arsenic (4 ppm). potentiometrically (2.2.20). Iron (2.4.9). Dilute 0.5 ml of solution S to 10 ml with 1 ml of 0.1 M perchloric acid is equivalent to 23.83 mg of water R. The solution complies with the limit test for iron C18H32CaN2O10. (400 ppm). Heavy metals (2.4.8). Dilute 13 ml of solution S to 20 ml STORAGE with water R. 12 ml of the solution complies with limit test A Store in an airtight container. for heavy metals (30 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. 01/2005:1052 Acid-insoluble matter. Dissolve 5.0 g in a mixture of 10 ml of hydrochloric acid R and 30 ml of water R. Filter, wash CALCIUM PHOSPHATE the residue with water R and dry to constant mass at 100 °C to 105 °C. The residue weighs not more than 10 mg (0.2 per cent). Tricalcii phosphas Loss on ignition. Not more than 8.0 per cent, determined on DEFINITION 1.000 g by ignition at 800 °C for 30 min. Calcium phosphate consists of a mixture of calcium phosphates. It contains not less than 35.0 per cent and not ASSAY more than the equivalent of 40.0 per cent of Ca (Ar 40.08). Dissolve 0.200 g in a mixture of 1 ml of hydrochloric acid R1 and 5 ml of water R. Add 25.0 ml of 0.1 M sodium edetate CHARACTERS and dilute to 200 ml with water R. Adjust to about pH 10 A white or almost white powder, practically insoluble in with concentrated ammonia R. Add 10 ml of ammonium water. It dissolves in dilute hydrochloric acid and in dilute chloride buffer solution pH 10.0 R and a few milligrams nitric acid. of mordant black 11 triturate R. Titrate the excess sodium edetate with 0.1 M zinc sulphate until the colour changes IDENTIFICATION from blue to violet. A. Dissolve 0.1 g in 5 ml of a 25 per cent V/V solution 1 ml of 0.1 M sodium edetate is equivalent to 4.008 mg of Ca. of nitric acid R. The solution gives reaction (b) of phosphates (2.3.1). B. It gives reaction (b) of calcium (2.3.1). Filter before 01/2005:0882 adding potassium ferrocyanide solution R. Reference solution (a). Dissolve 20 mg of calcium pantothenate CRS in water R and dilute to 5 ml with the same solvent. Reference solution (b). Dissolve 10 mg of 3-aminopropionic acid R in water R and dilute to 50 ml with the same solvent. Apply separately to the plate 5 µl of each solution. Develop over a path of 12 cm using a mixture of 35 volumes of water R and 65 volumes of ethanol R. Dry the plate in a current of air and spray with ninhydrin solution R1. Heat at 110 °C for 10 min. Any spot corresponding to 3-aminopropionic acid in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). Chlorides (2.4.4). 5 ml of solution S diluted to 15 ml with water R complies with the limit test for chlorides (200 ppm). Heavy metals (2.4.8). 12 ml of solution S complies with limit test A for heavy metals (20 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32). Not more than 3.0 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C.

C. It complies with the limits of the assay.

CALCIUM STEARATE

TESTS Solution S. Dissolve 2.50 g in 20 ml of dilute hydrochloric acid R. If the solution is not clear, filter it. Add dilute ammonia R1 dropwise until a precipitate is formed. Dissolve the precipitate by adding dilute hydrochloric acid R and dilute to 50 ml with distilled water R. Chlorides (2.4.4). Dissolve 0.22 g in a mixture of 1 ml of nitric acid R and 10 ml of water R and dilute to 100 ml with water R. 15 ml of the solution complies with the limit test for chlorides (0.15 per cent).

DEFINITION Calcium stearate is a mixture of calcium salts of different fatty acids consisting mainly of stearic acid [(C17H35COO)2Ca ; Mr 607] and palmitic acid [(C15H31COO)2Ca ; Mr 550.9] with minor proportions of other fatty acids. It contains not less than 6.4 per cent and not more than 7.4 per cent of Ca (Ar 40.08), calculated with reference to the dried substance. The


1167

Calcii stearas

Calcium stearate


Measure the absorbance at 228.8 nm using a cadmium hollow-cathode lamp as source of radiation and a graphite furnace as atomic generator. Lead. Not more than 10 ppm of Pb, determined by atomic CHARACTERS absorption spectrometry (2.2.23, Method II). A fine, white or almost white, crystalline powder, practically Test solution. Use the solution described in the test for cadmium. insoluble in water and in alcohol. Reference solutions. Prepare the reference solutions using lead standard solution (10 ppm Pb) R, diluted if necessary IDENTIFICATION with water R. First identification : C, D. Measure the absorbance at 283.3 nm using a lead Second identification : A, B, D. hollow-cathode lamp as source of radiation and a graphite A. The residue obtained in the preparation of solution S (see furnace as atomic generator. Depending on the apparatus, Tests) has a freezing point (2.2.18) not lower than 53 °C. the line at 217.0 nm may be used. Nickel. Not more than 5 ppm of Ni, determined by atomic B. The acid value of the fatty acids (2.5.1) is 195 to 210, absorption spectrometry (2.2.23, Method II). determined on 0.200 g of the residue obtained in the preparation of solution S dissolved in 25 ml of the Test solution. Use the solution described in the test for prescribed mixture of solvents. cadmium. Reference solutions. Prepare the reference solutions using C. Examine the chromatograms obtained in the test for nickel standard solution (10 ppm Ni) R, diluted if necessary fatty acid composition. The retention times of the with water R. principal peaks in the chromatogram obtained with the test solution are approximately the same as those of the Measure the absorbance at 232.0 nm using a nickel principal peaks in the chromatogram obtained with the hollow-cathode lamp as source of radiation and a graphite reference solution. furnace as atomic generator. D. Neutralise 5 ml of solution S to red litmus paper R using Loss on drying (2.2.32). Not more than 6.0 per cent, strong sodium hydroxide solution R. The solution gives determined on 1.000 g by drying in an oven at 100-105 °C. reaction (b) of calcium (2.3.1). Microbial contamination. Total viable aerobic count (2.6.12) not more than 103 micro-organisms per gram, determined TESTS by plate count. It complies with the test for Escherichia coli (2.6.13). Solution S. To 5.0 g add 50 ml of peroxide-free ether R, 20 ml of dilute nitric acid R and 20 ml of distilled water R. Boil under a reflux condenser until dissolution is complete. ASSAY Allow to cool. In a separating funnel, separate the aqueous Calcium. To 0.500 g in a 250 ml conical flask add 50 ml of a layer and shake the ether layer with 2 quantities, each of mixture of equal volumes of butanol R and ethanol R, 5 ml 5 ml, of distilled water R. Combine the aqueous layers, wash of concentrated ammonia R, 3 ml of ammonium chloride with 15 ml of peroxide-free ether R and dilute the aqueous buffer solution pH 10.0 R, 30.0 ml of 0.1 M sodium edetate layer to 50 ml with distilled water R (solution S). Evaporate and 15 mg of mordant black 11 triturate R. Heat to 45-50 °C the ether layer to dryness and dry the residue at 100-105 °C. until the solution is clear. Cool and titrate with 0.1 M zinc Keep the residue for identification tests A and B. sulphate until the colour changes from blue to violet. Carry out a blank titration. Acidity or alkalinity. To 1.0 g add 20 ml of carbon dioxide-free water R and boil for 1 min with continuous 1 ml of 0.1 M sodium edetate is equivalent to 4.008 mg of Ca. shaking. Cool and filter. To 10 ml of the filtrate add 0.05 ml Fatty acid composition. Examine by gas chromatography of bromothymol blue solution R1. Not more than 0.5 ml of (2.2.28). 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is Test solution. In a conical flask fitted with a reflux required to change the colour of the indicator. condenser, dissolve 0.10 g of the substance to be examined in Chlorides (2.4.4). Dilute 0.5 ml of solution S to 15 ml 5 ml of boron trifluoride-methanol solution R. Boil under a with water R. The solution complies with the limit test for reflux condenser for 10 min. Add 4 ml of heptane R through chlorides (0.1 per cent). the condenser and boil again under a reflux condenser for Sulphates (2.4.13). Dilute 0.5 ml of solution S to 15 ml with 10 min. Allow to cool. Add 20 ml of a saturated sodium distilled water R. The solution complies with the limit test chloride solution R. Shake and allow the layers to separate. for sulphates (0.3 per cent). Remove about 2 ml of the organic layer and dry over 0.2 g of anhydrous sodium sulphate R. Dilute 1.0 ml of the solution Cadmium. Not more than 3 ppm of Cd, determined by to 10.0 ml with heptane R. atomic absorption spectrometry (2.2.23, Method II). Reference solution. Prepare the reference solution in the Test solution. Place 50.0 mg of the substance to be examined same manner as the test solution using 50.0 mg of palmitic in a polytetrafluoroethylene digestion bomb and add acid CRS and 50.0 mg of stearic acid CRS instead of calcium 0.5 ml of a mixture of 1 volume of hydrochloric acid R and stearate. 5 volumes of cadmium- and lead-free nitric acid R. Allow to digest at 170 °C for 5 h. Allow to cool. Dissolve the residue The chromatographic procedure may be carried out using : in water R and dilute to 5.0 ml with the same solvent. — a fused-silica column 30 m long and 0.32 mm in internal diameter coated with macrogol 20 000 R (film thickness Reference solutions. Prepare the reference solutions using 0.5 µm), cadmium standard solution (10 ppm Cd) R, diluted if necessary with a 1 per cent V/V solution of hydrochloric — helium for chromatography R as the carrier gas at a flow acid R. rate of 2.4 ml/min, fatty acid fraction contains not less than 40.0 per cent of stearic acid and the sum of stearic acid and palmitic acid is not less than 90.0 per cent.

1168


Calendula flower


Iron (2.4.9). To 0.25 g add a mixture of 5 ml of hydrochloric acid R and 20 ml of water R. Heat to boiling, cool and filter. 10 ml of the filtrate complies with the limit test for iron Time Temperature Rate Comment (100 ppm). (min) (°C) (°C/min) Heavy metals (2.4.8). To 2.5 g add a mixture of 2 ml of 0-2 70 isothermal Column hydrochloric acid R and 15 ml of water R. Heat to boiling. 2 - 36 70 → 240 5 Cool and then add 0.5 ml of phenolphthalein solution R. linear gradient Cautiously add concentrated ammonia R until the colour 240 36 - 41 isothermal changes to pink. Add 0.5 ml of glacial acetic acid R and Injection port 220 dilute to 25 ml with water R. Filter. 12 ml of the filtrate complies with limit test A for heavy metals (20 ppm). Prepare Detector 260 the standard using lead standard solution (2 ppm Pb) R. Inject 1 µl of the reference solution. When the chromatogram Loss on ignition : 18.0 per cent to 22.0 per cent, determined is recorded in the prescribed conditions, the retention time on 1.000 g by ignition to constant mass at 800 °C. of methyl palmitate relative to that of methyl stearate is about 0.88. The test is not valid unless, in the chromatogram ASSAY obtained with the reference solution, the resolution between Dissolve 0.150 g in 120 ml of water R. Carry out the the peaks corresponding to methyl stearate and methyl complexometric titration of calcium (2.5.11). palmitate is at least 5.0. 1 ml of 0.1 M sodium edetate is equivalent to 17.22 mg of Inject 1 µl of the test solution. Calculate the percentage CaSO4,2H2O. content of stearic acid and palmitic acid from the areas of the peaks in the chromatogram obtained with the test solution by the normalisation procedure, disregarding the peak due 01/2005:1297 to the solvent. — a flame-ionisation detector, with the following temperature programme :

CALENDULA FLOWER 01/2005:0982

CALCIUM SULPHATE DIHYDRATE

Calendulae flos

DEFINITION Calendula flower consists of the whole or cut, dried, and Calcii sulfas dihydricus fully opened flowers which have been detached from the receptacle of the cultivated, double-flowered varieties of CaSO4,2H2O Mr 172.2 Calendula officinalis L. It contains not less than 0.4 per cent of flavonoids, calculated as hyperoside (C21H20O12, Mr 464.4) DEFINITION with reference to the dried drug. Calcium sulphate dihydrate contains not less than 98.0 per CHARACTERS cent and not more than the equivalent of 102.0 per cent of CaSO4,2H2O. It has the macroscopic and microscopic characters described under identification tests A and B. CHARACTERS IDENTIFICATION A white, fine powder, very slightly soluble in water, practically insoluble in alcohol. A. The ligulate florets consist of a yellow or orange-yellow ligule, about 3 mm to 5 mm wide and about 7 mm in the IDENTIFICATION middle part, with a three toothed apex and a hairy, partly A. It complies with the test for loss on ignition (see Tests). sickle-shaped yellowish-brown to orange-brown tube with a projecting style and a bifid stigma occasionally with a B. Solution S (see Tests) gives reaction (a) of sulphates partly bent yellowish-brown to orange-brown ovary. The (2.3.1). tubular florets, about 5 mm long, are present and consist C. Solution S gives reaction (a) of calcium (2.3.1). of the yellow, orange-red or red-violet five lobed corolla and the yellowish-brown or orange-brown tube, hairy in TESTS its lower part, mostly with a partly bent yellowish-brown Solution S. Dissolve 1.0 g in 50 ml of a 10 per cent V/V to orange-brown ovary. solution of hydrochloric acid R by heating at 50 °C for B. Reduce to a powder (355). The powder is yellowish-brown. 5 min. Allow the solution to cool. Examine under a microscope using chloral hydrate Acidity or alkalinity. Shake 1.5 g with 15 ml of carbon solution R. The powder shows fragments of the corollas dioxide-free water R for 5 min. Allow to stand for 5 min and containing light yellow oil droplets, some with fairly filter. To 10 ml of the filtrate, add 0.1 ml of phenolphthalein large anomocytic stomata (2.8.3), others containing solution R and 0.25 ml of 0.01 M sodium hydroxide. prisms and very small cluster crystals of calcium oxalate ; The solution is red. Add 0.30 ml of 0.01 M hydrochloric covering trichomes biseriate, multicellular and conical, acid. The solution is colourless. Add 0.2 ml of methyl red glandular trichomes with a uniseriate or biseriate, solution R. The solution is reddish-orange. multicellular biseriate stalk and a large, ovoid, biseriate Chlorides (2.4.4). Shake 0.5 g with 15 ml of water R for and multicellular head ; spherical pollen grains up to 5 min. Allow to stand for 15 min and filter. Dilute 5 ml of the about 40 µm in diameter with a sharply spiny exine and filtrate to 15 ml with water R. The solution complies with three germinal pores ; occasional fragments of the stigmas the limit test for chlorides (300 ppm). with short, bulbous papillae. Arsenic (2.4.2). 5 ml of solution S complies with limit test A C. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating substance. for arsenic (10 ppm). General Notices (1) apply to all monographs and other texts

1169

D-Camphor


Test solution. To 1.0 g of the powdered drug (500) add 10 ml of methanol R and heat on a water-bath under a reflux condenser for 10 min. Cool and filter. Reference solution. Dissolve 1.0 mg of caffeic acid R, 1.0 mg of chlorogenic acid R and 2.5 mg of rutin R in 10 ml of methanol R. Apply to the plate, as bands, 20 µl of the test solution and 10 µl of the reference solution. Develop over a path of 10 cm using a mixture of 10 volumes of anhydrous formic acid R, 10 volumes of water R and 80 volumes of ethyl acetate R. Allow the plate to dry at 100 °C to 105 °C and spray the still warm plate with a 10 g/l solution of diphenylboric acid aminoethyl ester R in methanol R and then spray with a 50 g/l solution of macrogol 400 R in methanol R. Allow the plate to dry in air for 30 min and examine in ultraviolet light at 365 nm. The chromatogram obtained with the reference solution shows in the lower part a yellowish-brown fluorescent zone (rutin), in the middle part a light bluish fluorescent zone (chlorogenic acid) and in the upper part a light bluish fluorescent zone (caffeic acid). The chromatogram obtained with the test solution shows a yellowish-brown fluorescent zone corresponding in position to the zone due to rutin in the chromatogram obtained with the reference solution, below and directly above it, it shows a yellowish-green fluorescent zone and a light bluish fluorescent zone corresponding to the zone due to chlorogenic acid in the chromatogram obtained with the reference solution, a yellowish-green fluorescent zone above it and a light bluish fluorescent zone shortly below the zone due to caffeic acid in the chromatogram obtained with the reference solution. Further zones are present. TESTS Foreign matter (2.8.2). Not more than 5 per cent of bracts and not more than 2 per cent of other foreign matter. Loss on drying (2.2.32). Not more than 12.0 per cent, determined on 1.000 g of the powdered drug (500) by drying in an oven at 100 °C to 105 °C for 2 h. Total ash (2.4.16). Not more than 10.0 per cent. ASSAY Stock solution. In a 100 ml round-bottomed flask introduce 0.800 g of the powdered drug (500), 1 ml of a 5 g/l solution of hexamethylenetetramine R, 20 ml of acetone R and 7 ml of hydrochloric acid R1. Boil the mixture under a reflux condenser for 30 min. Filter the liquid through a plug of absorbent cotton in a 100 ml flask. Add the absorbent cotton to the residue in the round-bottomed flask and extract with two quantities, each of 20 ml, of acetone R, each time boiling under a reflux condenser for 10 min. Allow to cool to room temperature, filter the liquid through a plug of absorbent cotton then filter the combined acetone solution through a filter-paper in the volumetric flask and dilute to 100.0 ml with acetone R by rinsing of the flask and the filter. Introduce 20.0 ml of the solution into a separating funnel, add 20 ml of water R and extract the mixture with one quantity of 15 ml and then three quantities, each of 10 ml, of ethyl acetate R. Combine the ethyl acetate extracts in a separating funnel, rinse with two quantities, each of 50 ml, of water R, filter the extract over 10 g of anhydrous sodium sulphate R in to a 50 ml volumetric flask and dilute to 50.0 ml with ethyl acetate R. Test solution. To 10.0 ml of the stock solution add 1 ml of aluminium chloride reagent R and dilute to 25.0 ml with a 5 per cent V/V solution of glacial acetic acid R in methanol R. 1170

Compensation solution. Dilute 10.0 ml of the stock solution to 25.0 ml with a 5 per cent V/V solution of glacial acetic acid R in methanol R. Measure the absorbance (2.2.25) of the test solution after 30 min, by comparison with the compensation solution at 425 nm. Calculate the percentage content of flavonoids, calculated as hyperoside, from the expression :

i.e. taking the specific absorbance of hyperoside to be 500. A m

=


=



D-CAMPHOR D-Camphora

C10H16O

Mr 152.2

DEFINITION (1R,4R)-1,7,7-Trimethylbicyclo[2.2.1]heptan-2-one. CHARACTERS Appearance : white, crystalline powder or friable, crystalline masses. Highly volatile even at room temperature. Solubility : slightly soluble in water, very soluble in alcohol and in light petroleum, freely soluble in fatty oils, very slightly soluble in glycerol. IDENTIFICATION First identification : A, C. Second identification : A, B, D. A. Specific optical rotation (see Tests). B. Melting point (2.2.14) : 175 °C to 179 °C. C. Infrared absorption spectrophotometry (2.2.24). Comparison : racemic camphor CRS. D. Dissolve 1.0 g in 30 ml of methanol R. Add 1.0 g of hydroxylamine hydrochloride R and 1.0 g of anhydrous sodium acetate R. Boil under a reflux condenser for 2 h. Allow to cool and add 100 ml of water R. Filter, wash the precipitate obtained with 10 ml of water R and recrystallise from 10 ml of a mixture of 4 volumes of alcohol R and 6 volumes of water R. The crystals, dried in vacuo, melt (2.2.14) at 118 °C to 121 °C. TESTS Carry out the weighings and dissolution rapidly. Solution S. Dissolve 2.50 g in 10 ml of alcohol R and dilute to 25.0 ml with the same solvent.


D-Camphor


Test solution. To 1.0 g of the powdered drug (500) add 10 ml of methanol R and heat on a water-bath under a reflux condenser for 10 min. Cool and filter. Reference solution. Dissolve 1.0 mg of caffeic acid R, 1.0 mg of chlorogenic acid R and 2.5 mg of rutin R in 10 ml of methanol R. Apply to the plate, as bands, 20 µl of the test solution and 10 µl of the reference solution. Develop over a path of 10 cm using a mixture of 10 volumes of anhydrous formic acid R, 10 volumes of water R and 80 volumes of ethyl acetate R. Allow the plate to dry at 100 °C to 105 °C and spray the still warm plate with a 10 g/l solution of diphenylboric acid aminoethyl ester R in methanol R and then spray with a 50 g/l solution of macrogol 400 R in methanol R. Allow the plate to dry in air for 30 min and examine in ultraviolet light at 365 nm. The chromatogram obtained with the reference solution shows in the lower part a yellowish-brown fluorescent zone (rutin), in the middle part a light bluish fluorescent zone (chlorogenic acid) and in the upper part a light bluish fluorescent zone (caffeic acid). The chromatogram obtained with the test solution shows a yellowish-brown fluorescent zone corresponding in position to the zone due to rutin in the chromatogram obtained with the reference solution, below and directly above it, it shows a yellowish-green fluorescent zone and a light bluish fluorescent zone corresponding to the zone due to chlorogenic acid in the chromatogram obtained with the reference solution, a yellowish-green fluorescent zone above it and a light bluish fluorescent zone shortly below the zone due to caffeic acid in the chromatogram obtained with the reference solution. Further zones are present. TESTS Foreign matter (2.8.2). Not more than 5 per cent of bracts and not more than 2 per cent of other foreign matter. Loss on drying (2.2.32). Not more than 12.0 per cent, determined on 1.000 g of the powdered drug (500) by drying in an oven at 100 °C to 105 °C for 2 h. Total ash (2.4.16). Not more than 10.0 per cent. ASSAY Stock solution. In a 100 ml round-bottomed flask introduce 0.800 g of the powdered drug (500), 1 ml of a 5 g/l solution of hexamethylenetetramine R, 20 ml of acetone R and 7 ml of hydrochloric acid R1. Boil the mixture under a reflux condenser for 30 min. Filter the liquid through a plug of absorbent cotton in a 100 ml flask. Add the absorbent cotton to the residue in the round-bottomed flask and extract with two quantities, each of 20 ml, of acetone R, each time boiling under a reflux condenser for 10 min. Allow to cool to room temperature, filter the liquid through a plug of absorbent cotton then filter the combined acetone solution through a filter-paper in the volumetric flask and dilute to 100.0 ml with acetone R by rinsing of the flask and the filter. Introduce 20.0 ml of the solution into a separating funnel, add 20 ml of water R and extract the mixture with one quantity of 15 ml and then three quantities, each of 10 ml, of ethyl acetate R. Combine the ethyl acetate extracts in a separating funnel, rinse with two quantities, each of 50 ml, of water R, filter the extract over 10 g of anhydrous sodium sulphate R in to a 50 ml volumetric flask and dilute to 50.0 ml with ethyl acetate R. Test solution. To 10.0 ml of the stock solution add 1 ml of aluminium chloride reagent R and dilute to 25.0 ml with a 5 per cent V/V solution of glacial acetic acid R in methanol R. 1170

Compensation solution. Dilute 10.0 ml of the stock solution to 25.0 ml with a 5 per cent V/V solution of glacial acetic acid R in methanol R. Measure the absorbance (2.2.25) of the test solution after 30 min, by comparison with the compensation solution at 425 nm. Calculate the percentage content of flavonoids, calculated as hyperoside, from the expression :

i.e. taking the specific absorbance of hyperoside to be 500. A m

=


=



D-CAMPHOR D-Camphora

C10H16O

Mr 152.2

DEFINITION (1R,4R)-1,7,7-Trimethylbicyclo[2.2.1]heptan-2-one. CHARACTERS Appearance : white, crystalline powder or friable, crystalline masses. Highly volatile even at room temperature. Solubility : slightly soluble in water, very soluble in alcohol and in light petroleum, freely soluble in fatty oils, very slightly soluble in glycerol. IDENTIFICATION First identification : A, C. Second identification : A, B, D. A. Specific optical rotation (see Tests). B. Melting point (2.2.14) : 175 °C to 179 °C. C. Infrared absorption spectrophotometry (2.2.24). Comparison : racemic camphor CRS. D. Dissolve 1.0 g in 30 ml of methanol R. Add 1.0 g of hydroxylamine hydrochloride R and 1.0 g of anhydrous sodium acetate R. Boil under a reflux condenser for 2 h. Allow to cool and add 100 ml of water R. Filter, wash the precipitate obtained with 10 ml of water R and recrystallise from 10 ml of a mixture of 4 volumes of alcohol R and 6 volumes of water R. The crystals, dried in vacuo, melt (2.2.14) at 118 °C to 121 °C. TESTS Carry out the weighings and dissolution rapidly. Solution S. Dissolve 2.50 g in 10 ml of alcohol R and dilute to 25.0 ml with the same solvent.


D-Camphor


Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Acidity or alkalinity. To 10 ml of solution S add 0.1 ml of phenolphthalein solution R1. The solution is colourless. Not more than 0.2 ml of 0.1 M sodium hydroxide is required to change the colour of the indicator. Specific optical rotation (2.2.7) : + 40.0 to + 43.0, determined on solution S. Related substances. Gas chromatography (2.2.28). Test solution. Dissolve 2.50 g of the substance to be examined in heptane R and dilute to 25.0 ml with the same solvent. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with heptane R. Reference solution (b). Dilute 10.0 ml of reference solution (a) to 20.0 ml with heptane R. Reference solution (c). Dissolve 0.50 g of borneol R in heptane R and dilute to 25.0 ml with the same solvent. Dilute 5.0 ml of the solution to 50.0 ml with heptane R. Reference solution (d). Dissolve 50 mg of linalol R and 50 mg of bornyl acetate R in heptane R and dilute to 100.0 ml with the same solvent. Column : — size : l = 30 m, Ø = 0.25 mm, — stationary phase: macrogol 20 000 R (0.25 µm). Carrier gas : helium for chromatography R. Split ratio : 1:70. Flow rate : 45 cm/s. Temperature :

Column

Time (min)

Temperature (°C)

0 - 10

50

10 - 35

50 → 100

35 - 45

100 → 200

45 - 55

200

Injection port

220

Detector

250

Detection : flame ionisation. Injection : 1 µl. System suitability : reference solution (d). — resolution : minimum 3.0 between the peaks due to bornyl acetate and to linalol. Limits : — borneol: not more than the area of the principal peak in the chromatogram obtained with reference solution (c) (2.0 per cent), — any other impurity : not more than half of the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent), — total of other impurities : not more than 4 times the area of the principal peak in the chromatogram obtained with reference solution (a) (4.0 per cent), — disregard limit : 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.05 per cent). Halogens : maximum 100 ppm. Dissolve 1.0 g in 10 ml of 2-propanol R in a distillation flask. Add 1.5 ml of dilute sodium hydroxide solution R and 50 mg of nickel-aluminium alloy R. Heat on a water-bath until the General Notices (1) apply to all monographs and other texts

2-propanol R has evaporated. Allow to cool and add 5 ml of water R. Mix and filter through a wet filter previously washed with water R until free from chlorides. Dilute the filtrate to 10.0 ml with water R. To 5.0 ml of the solution, add nitric acid R dropwise until the precipitate which forms is redissolved and dilute to 15 ml with water R. The solution complies with the limit test for chlorides (2.4.4). Residue on evaporation (2.8.9) : maximum 0.05 per cent. Evaporate 2.0 g on a water-bath and dry in an oven at 100-105 °C for 1 h. The residue weighs a maximum of 1 mg. Water. Dissolve 1 g in 10 ml of light petroleum R. The solution is clear (2.2.1). IMPURITIES

A. 2,6,6-trimethylbicyclo[3.1.1]hept-2-ene (α-pinene),

B. 2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane (camphene),

C. 6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane (β-pinene),

D. 3,3-dimethyl-2-oxabicyclo[2.2.2]octane (cineole),

E. R1 = CH3, R2 + R3 = O : 1,3,3-trimethylbicyclo[2.2.1]heptan-2-one (fenchone), F. R1 = CH3, R2 = OH, R3 = H : exo-1,3,3trimethylbicyclo[2.2.1]heptan-2-ol (fenchol), G. R1 = H, R2 = OH, R3 = CH3 : exo-2,3,3trimethylbicyclo[2.2.1]heptan-2-ol (camphene hydrate), H. R1 = H, R2 = CH3, R3 = OH : endo-2,3,3trimethylbicyclo[2.2.1]heptan-2-ol (methylcamphenilol), 1171

Camphor, racemic


I. R = OH, R′ = H : exo-1,7,7-trimethylbicyclo[2.2.1]heptan-2ol (exo-borneol), J. R = H, R′ = OH : endo-1,7,7-trimethylbicyclo[2.2.1]heptan2-ol (endo-borneol). 01/2005:0655

CAMPHOR, RACEMIC Camphora racemica

C10H16O

Mr 152.2

DEFINITION Racemic camphor is (1RS,4RS)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one. CHARACTERS A white, crystalline powder or friable, crystalline masses, highly volatile even at room temperature, slightly soluble in water, very soluble in alcohol and in light petroleum, freely soluble in fatty oils, very slightly soluble in glycerol. IDENTIFICATION First identification : A, C. Second identification : A, B, D. A. It complies with the test for optical rotation (see Tests). B. Melting point (2.2.14) : 172 °C to 180 °C. C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with racemic camphor CRS. Examine the substances as mulls in liquid paraffin R. D. Dissolve 1.0 g in 30 ml of methanol R. Add 1.0 g of hydroxylamine hydrochloride R and 1.0 g of anhydrous sodium acetate R. Boil under a reflux condenser for 2 h. Allow to cool and add 100 ml of water R. A precipitate is formed. Filter, wash with 10 ml of water R and recrystallize from 10 ml of a mixture of 4 volumes of alcohol R and 6 volumes of water R. The crystals, dried in vacuo, melt (2.2.14) at 118 °C to 121 °C.

Related substances. Examine by gas chromatography (2.2.28). Test solution. Dissolve 50 mg of the substance to be examined in hexane R and dilute to 50.0 ml with the same solvent. Reference solution (a). Dissolve 50 mg of the substance to be examined and 50 mg of bornyl acetate R in hexane R and dilute to 50.0 ml with the same solvent. Reference solution (b). Dilute 1.0 ml of the test solution to 200.0 ml with hexane R. The chromatographic procedure may be carried out using : — a column 2 m long and 2 mm in internal diameter packed with diatomaceous earth for gas chromatography R impregnated with 10 per cent m/m of macrogol 20 000 R, — nitrogen for chromatography R as the carrier gas at a flow rate of 30 ml/min, — a flame-ionisation detector, maintaining the temperature of the column at 130 °C and that of the injection port and the detector at 200 °C. Inject 1 µl of each solution and adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with the test solution is about 80 per cent of the full scale of the recorder. Record the chromatograms for three times the retention time of camphor. The test is not valid unless in the chromatogram obtained with reference solution (a), the resolution between the peaks corresponding to camphor and bornyl acetate is not less than 1.5 ; in the chromatogram obtained with reference solution (b), the principal peak has a signal-to-noise ratio of at least 5. In the chromatogram obtained with the test solution : the sum of the areas of the peaks, apart from the principal peak, is not greater than 4 per cent of the area of the principal peak ; none of the peaks, apart from the principal peak, has an area greater than 2 per cent of the area of the principal peak. Disregard any peak with an area less than that of the peak in the chromatogram obtained with reference solution (b). Halogens. Dissolve 1.0 g in 10 ml of 2-propanol R in a distillation flask. Add 1.5 ml of dilute sodium hydroxide solution R and 50 mg of nickel-aluminium alloy R. Heat on a water-bath until the 2-propanol R has evaporated. Allow to cool and add 5 ml of water R. Mix and filter through a wet filter previously washed with water R until free from chlorides. Dilute the filtrate to 10.0 ml with water R. To 5.0 ml of the solution, add nitric acid R dropwise until the precipitate which forms is redissolved and dilute to 15 ml with water R. The solution complies with the limit test for chlorides (2.4.4) (100 ppm). Water. Dissolve 1 g in 10 ml of light petroleum R. The solution is clear (2.2.1). Residue on evaporation. Evaporate 2.0 g on a water-bath and dry at 100 °C to 105 °C for 1 h. The residue weighs not more than 1 mg (0.05 per cent).

TESTS Carry out the weighings rapidly. Solution S. Dissolve 2.50 g in 10 ml of alcohol R and dilute to 25.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Acidity or alkalinity. Dissolve 1.0 g in 10 ml of alcohol R and add 0.1 ml of phenolphthalein solution R1. The solution is colourless. Not more than 0.2 ml of 0.1 M sodium hydroxide is required to change the colour of the indicator. Optical rotation (2.2.7) : + 0.15° to − 0.15°, determined on C8H16O2 solution S. 1172

01/2005:1401

CAPRYLIC ACID Acidum caprylicum

Mr 144.2


Caprylocaproyl macrogolglycerides


DEFINITION Caprylic acid contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent of octanoic acid, calculated with reference to the anhydrous substance. CHARACTERS A clear, colourless or slightly yellowish, oily liquid, very slightly soluble in water, very soluble in acetone and in alcohol. It dissolves in dilute solutions of alkali hydroxides. IDENTIFICATION A. It complies with the test for relative density (see Tests). B. Examine the chromatograms obtained in the test for related substances. The retention time and size of the principal peak in the chromatogram obtained with the test solution are approximately the same as those of the principal peak in the chromatogram obtained with reference solution (a). TESTS Appearance. The substance to be examined is clear (2.2.1) and not more intensely coloured than reference solution Y5 (2.2.2, Method II). Relative density (2.2.5) : 0.909 to 0.912. Related substances. Examine by gas chromatography (2.2.28). Test solution. Dissolve 0.10 g of the substance to be examined in ethyl acetate R and dilute to 10.0 ml with the same solvent. Reference solution (a). Dissolve 0.10 g of caprylic acid CRS in ethyl acetate R and dilute to 10.0 ml with the same solvent. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with ethyl acetate R. Dilute 5.0 ml of the solution to 50.0 ml with ethyl acetate R. The chromatographic procedure may be carried out using : — a fused-silica column 30 m long and 0.25 mm in internal diameter coated with macrogol 20 000 2-nitroterephthalate R (film thickness 0.25 µm), — helium for chromatography R as the carrier gas at a flow rate of 1.5 ml/min, — a flame-ionisation detector, — a split ratio of 1:100, with the following temperature programme : Time (min) Column

0-1 1 - 25

Temperature Rate (°C) (°C/min) – 100 100 → 220

5

Comment isothermal linear gradient

Heavy metals (2.4.8). Dissolve 2.0 g in alcohol R and dilute to 20 ml with the same solvent. 12 ml of the solution complies with limit test B for heavy metals (10 ppm). Prepare the standard using 1 ml of lead standard solution (10 ppm Pb) R and 9 ml of alcohol R. Water (2.5.12). Not more than 0.7 per cent, determined on 1.000 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.125 g in 25 ml of alcohol R. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M sodium hydroxide is equivalent to 14.42 mg of C8H16O2. IMPURITIES

A. n = 4 : hexanoic acid, B. n = 5 : heptanoic acid, C. n = 7 : nonanoic acid, D. n = 8 : decanoic acid, E. valproic acid,

F. R = OCH3, n = 6 : methyl octanoate, G. R = OC2H5, n = 6 : ethyl octanoate, H. R = OCH3, n = 8 : methyl decanoate, I. R = CH3, n = 8 : undecan-2-one,

J. 5-butyltetrahydrofuran-2-one (γ-hydroxyoctanoic acid lactone). 01/2005:1184

CAPRYLOCAPROYL MACROGOLGLYCERIDES Macrogolglyceridorum caprylocaprates

DEFINITION Caprylocaproyl macrogolglycerides are mixtures of Injection port 250 monoesters, diesters and triesters of glycerol and monoesters and diesters of macrogols with a mean relative molecular Detector 250 mass between 200 and 400. They are obtained by partial Inject 1 µl of reference solution (b). The test is not valid alcoholysis of medium-chain triglycerides using macrogol unless in the chromatogram obtained the principal peak has or by esterification of glycerol and macrogol with caprylic a signal-to-noise ratio of at least 5. acid and capric acid or a mixture of glycerol esters and condensates of ethylene oxide with caprylic acid (octanoic Inject 1 µl of the test solution and 1 µl of reference acid) and capric acid (decanoic acid). They may contain free solution (a). Calculate the percentage content of related substances from the areas of the peaks in the chromatogram macrogols. obtained with the test solution by the normalisation CHARACTERS procedure, disregarding any peaks with an area less than 0.5 times the area of the peak in the chromatogram obtained Pale-yellow oily liquids, dispersible in hot water, freely soluble in methylene chloride. with reference solution (b). The content of any related substance is not greater than 0.3 per cent and the sum of the The relative density at 20 °C is about 1.0, the refractive index at 20 °C is about 1.4. contents is not greater than 0.5 per cent. 25 - 35

220

isothermal


1173

Capsicum


IDENTIFICATION A. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating substance. Test solution. Dissolve 1.0 g of the substance to be examined in methylene chloride R and dilute to 20 ml with the same solvent. Apply to the plate 50 µl of the test solution. Develop over a path of 15 cm using a mixture of 30 volumes of hexane R and 70 volumes of ether R. Allow the plate to dry in air. Spray with a 0.1 g/l solution of rhodamine B R in alcohol R and examine in ultraviolet light at 365 nm. The chromatogram shows a spot corresponding to triglycerides with an Rf value of about 0.9 (Rst 1) and spots corresponding to 1,3-diglycerides (Rst 0.7), to 1,2-diglycerides (Rst 0.6), to monoglycerides (Rst 0.1) and to esters of macrogol (Rst 0). B. They comply with the test for hydroxyl value (see Tests). C. They comply with the test for saponification value (see Tests). D. They comply with the test for fatty acid composition (see Tests). TESTS Viscosity (2.2.9). The ranges are presented in Table 1184.-1, determined at 20 ± 0.5 °C. Table 1184.-1 Ethylene oxide units per molecule (nominal value)

Type of macrogol

Viscosity (mPa·s)

4

200

30 to 50

6

300

60 to 80

8

400

80 to 110

Acid value (2.5.1). Not more than 2.0, determined on 2.0 g. Hydroxyl value (2.5.3, Method A). The ranges are presented in Table 1184.-2, determined on 1.0 g. Table 1184.-2 Ethylene oxide units per molecule (nominal value)

Type of macrogol

Hydroxyl value

4

200

80 to 120

6

300

140 to 180

8

400

170 to 205

Peroxide value (2.5.5). Not more than 6.0, determined on 2.0 g. Saponification value (2.5.6). The ranges are presented in Table 1184.-3, determined on 2.0 g. Table 1184.-3 Ethylene oxide units per molecule (nominal value)

Type of macrogol

Saponification value

4

200

265 to 285

6

300

170 to 190

8

400

85 to 105

Alkaline impurities. Introduce into a test-tube 5.0 g and carefully add a mixture, neutralised if necessary with 0.01 M hydrochloric acid or with 0.01 M sodium hydroxide, of 0.05 ml of a 0.4 g/l solution of bromophenol blue R in alcohol R, 0.3 ml of water R and 10 ml of alcohol R. Shake and allow to stand. Not more than 1.0 ml of 0.01 M hydrochloric acid is required to change the colour of the upper layer to yellow. 1174

Free glycerol. Not more than 5.0 per cent. Dissolve 1.20 g in 25.0 ml of methylene chloride R. Heat if necessary. After cooling, add 100 ml of water R. Shake and add 25.0 ml of a 6 g/l solution of periodic acid R. Shake and allow to stand for 30 min. Add 40 ml of a 75 g/l solution of potassium iodide R. Allow to stand for 1 min. Add 1 ml of starch solution R. Titrate the iodine with 0.1 M sodium thiosulphate. Carry out a blank titration. 1 ml of 0.1 M sodium thiosulphate is equivalent to 2.3 mg of glycerol. Fatty acid composition (2.4.22, Method A). The fatty acid fraction has the following composition : — caproic acid : not more than 2.0 per cent, — caprylic acid : 50.0 per cent to 80.0 per cent, — capric acid : 20.0 per cent to 50.0 per cent, — lauric acid : not more than 3.0 per cent, — myristic acid : not more than 1.0 per cent. Ethylene oxide and dioxan (2.4.25). Not more than 1 ppm of ethylene oxide and not more than 10 ppm of dioxan. Heavy metals (2.4.8). 2.0 g complies with limit test C for heavy metals (10 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Water (2.5.12). Not more than 1.0 per cent, determined on 1.0 g by the semi-micro determination of water. Use a mixture of 30 volumes of anhydrous methanol R and 70 volumes of methylene chloride R as solvent. Total ash (2.4.16). Not more than 0.1 per cent, determined on 1.0 g. LABELLING The label states the type of macrogol used (mean relative molecular mass) or the number of ethylene oxide units per molecule (nominal value). 01/2005:1859

CAPSICUM Capsici fructus DEFINITION Dried ripe fruits of Capsicum annuum L. var. minimum (Miller) Heiser and small-fruited varieties of Capsicum frutescens L. Content : minimum 0.4 per cent of total capsaicinoids expressed as capsaicin (C18H27NO3 ; Mr 305.4) (dried drug). CHARACTERS Extremely pungent taste. Macroscopic and microscopic characters described under identification tests A and B. IDENTIFICATION A. The fruit is yellowish-orange to reddish-brown, oblong conical with an obtuse apex, about 1 cm to 3 cm long and up to 1 cm in diameter at the widest part, occasionally attached to a 5-toothed inferior calyx and a straight pedicel. Pericarp somewhat shrivelled, glabrous, enclosing about 10 to 20 flat, reniform seeds 3 mm to 4 mm long, either loose or attached to a reddish dissepiment. B. Reduce to a powder (355). The powder is orange. Examine under a microscope using chloral hydrate solution R. The powder shows the following diagnostic characters : fragments of the pericarp having an outer epicarp with cells often arranged in rows of 5 to


Capsicum


7, cuticle uniformly striated ; parenchymatous cells frequently containing droplets of red oil, occasionally containing microsphenoidal crystals of calcium oxalate ; endocarp with characteristic island groups of sclerenchymatous cells, the groups being separated by thin-walled parenchymatous cells. Fragments of the seeds having an episperm composed of large, greenish-yellow, sinuous-walled sclereids with thin outer walls and strongly and unevenly thickened radial and inner walls which are conspicuously pitted ; endosperm parenchymatous cells with drops of fixed oil and aleurone grains 3 µm to 6 µm in diameter. Occasional fragments from the calyx having an outer epidermis with anisocytic stomata (2.8.3), inner epidermis with many trichomes but no stomata ; trichomes glandular, with uniseriate stalks and multicellular heads ; mesophyll with many idioblasts containing microsphenoidal crystals of calcium oxalate. C. Thin-layer chromatography (2.2.27). Test solution. To 0.50 g of the powdered drug (500) add 5.0 ml of ether R, shake for 5 min and filter. Reference solution. Dissolve 2 mg of capsaicin R and 2 mg of dihydrocapsaicin R in 5.0 ml of ether R. Plate : TLC octadecylsilyl silica gel plate R. Mobile phase : water R, methanol R (20:80 V/V). Application : 20 µl, as bands. Development : over a path of 12 cm. Drying : in air. Detection : spray with a 5 g/l solution of dichloroquinonechlorimide R in methanol R. Expose the plate to ammonia vapour until blue zones appear. Examine in daylight. Results : see below the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. Furthermore, other zones may be present in the chromatogram obtained with the test solution. Top of the plate _______

_______

Capsaicin : a blue zone

A blue zone (capsaicin)

Dihydrocapsaicin : a blue zone

A blue zone (dihydrocapsaicin)

_______

_______

Reference solution

Test solution

TESTS Nonivamide. Liquid chromatography (2.2.29). Test solution. To 2.5 g of the powdered drug (500) add 100 ml of methanol R. Allow to macerate for 30 min.

Place in an ultrasonic bath for 15 min. Filter into a 100 ml volumetric flask, rinse the flask and filter with methanol R. Dilute to 100.0 ml with methanol R. Reference solution. Dissolve 20.0 mg of capsaicin R and 4.0 mg of nonivamide R in 100.0 ml of methanol R. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : phenylsilyl silica gel for chromatography R (5 µm), — temperature : 30 °C. Mobile phase : mixture of 40 volumes of acetonitrile R and 60 volumes of a 1 g/l solution of phosphoric acid R. Flow rate : 1.0 ml/min. Detection : spectrophotometer at 225 nm. Injection : 10 µl. Elution order : elution order similar to that obtained in Figure 1859.-1. System suitability : reference solution : — resolution : minimum 3.0 between the peaks due to capsaicin and nonivamide. Limit : calculate the percentage content of nonivamide from the expression :

F1

=

F2

=

area of the peak corresponding to nonivamide in the chromatogram obtained with the test solution, area of the peak corresponding to nonivamide in the chromatogram obtained with the reference solution,

m1

=

mass of the drug to be examined in grams,

m2

=

p1

=

mass of nonivamide used to prepare the reference solution in grams, percentage content of nonivamide in the reagent.

— nonivamide : maximum 5.0 per cent of the total capsaicinoid content. Foreign matter (2.8.2) : maximum 2 per cent m/m. Fruits of C. annuum L. var. longum (Sendtn.) are absent. Loss on drying (2.2.32) : maximum 11.0 per cent, determined on 1.000 g of the powdered drug (500) by drying in an oven at 100-105 °C for 2 h. Total ash (2.4.16) : maximum 10.0 per cent. ASSAY Liquid chromatography (2.2.29) as described in the test for nonivamide.

1. nordihydrocapsaicin

3. capsaicin

2. nonivamide

4. dihydrocapsaicin

Figure 1859.-1. – Chromatogram for the test for nonivamide and the assay of capsicum General Notices (1) apply to all monographs and other texts

1175

Captopril


Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 50 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (a). Dilute 2.0 ml of the test solution to F3 = area of the peak corresponding to capsaicin in the 100.0 ml with the mobile phase. Reference solution (b). Dissolve 10 mg of the substance to chromatogram obtained with the test solution, be examined in the mobile phase, add 1 ml of 0.05 M iodine F4 = area of the peak corresponding to capsaicin in and dilute to 100.0 ml with the mobile phase. Dilute 10.0 ml the chromatogram obtained with the reference of the solution to 100.0 ml with the mobile phase. solution, Column : F5 = area of the peak corresponding to — size : l = 0.125 m, Ø = 4 mm, dihydrocapsaicin in the chromatogram — stationary phase : octylsilyl silica gel for obtained with the test solution, chromatography R (5 µm). F6 = area of the peak corresponding to Mobile phase : phosphoric acid R, methanol R, water R nordihydrocapsaicin in the chromatogram (0.05:50:50 V/V/V). obtained with the test solution, Flow rate : 1 ml/min. m3 = mass of the drug to be examined in grams, Detection : spectrophotometer at 220 nm. m4 = mass of capsaicin used to prepare the reference Injection : 20 µl. solution in grams, Run time : 3 times the retention time of captopril. p2 = percentage content of capsaicin in the reagent. System suitability : reference solution (b) : — the chromatogram shows 3 peaks, — resolution : minimum of 2.0 between the last 2 eluting 01/2005:1079 principal peaks, Limits : CAPTOPRIL — any impurity : not more than half the area of the principal peak in the chromatogram obtained with reference Captoprilum solution (a) (1.0 per cent), — total : not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (2.0 per cent), — disregard limit : 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent). Disregard any peak with a retention time C9H15NO3S Mr 217.3 less than 1.4 min. Heavy metals (2.4.8) : maximum 20 ppm. DEFINITION 1.0 g complies with limit test C. Prepare the standard using (2S)-1-[(2S)-2-Methyl-3-sulphanylpropanoyl]pyrrolidine-22 ml of lead standard solution (10 ppm Pb) R. carboxylic acid. Loss on drying (2.2.32) : maximum 1.0 per cent, determined Content : 98.0 per cent to 101.5 per cent (dried substance). on 1.000 g by drying under high vacuum at 60 °C for 3 h. CHARACTERS Sulphated ash (2.4.14) : maximum 0.2 per cent, determined Appearance : white or almost white crystalline powder. on 1.0 g. Solubility : freely soluble in water, in methylene chloride ASSAY and in methanol. It dissolves in dilute solutions of alkali Dissolve 0.150 g in 30 ml of water R. Titrate with 0.05 M hydroxides. iodine, determining the end-point potentiometrically (2.2.20). Use a combined platinum electrode. IDENTIFICATION 1 ml of 0.05 M iodine is equivalent to 21.73 mg of C9H15NO3S. Infrared absorption spectrophotometry (2.2.24). Comparison : captopril CRS. STORAGE In an airtight container. TESTS Calculate the percentage content of capsaicinoids from the expression :

Solution S. Dissolve 0.5 g in carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). pH (2.2.3) : 2.0 to 2.6 for solution S. Specific optical rotation(2.2.7) : − 127 to − 132 (dried substance). Dissolve 0.250 g in ethanol R and dilute to 25.0 ml with the same solvent. 1176

IMPURITIES

A. (2S,2′S)-1,1′-[disulphanediylbis[(2S)-2-methyl-1oxopropane-3,1-diyl]-bis[pyrrolidine-2-carboxylic] acid (captopril-disulphide).


Carbachol


01/2005:1080

CARAWAY FRUIT Carvi fructus DEFINITION Caraway fruit consists of the whole, dry mericarp of Carum carvi L. It contains not less than 30 ml/kg of essential oil, calculated with reference to the anhydrous drug. CHARACTERS Caraway fruit has an odour reminiscent of carvone. It has the macroscopic and microscopic characters described under identification tests A and B. IDENTIFICATION A. The fruit is a cremocarp of almost cylindrical shape. It is generally 3 mm to 6.5 mm long and 1 mm to 1.5 mm wide. The mericarps, usually free, are greyish-brown to brown, glabrous, mostly sickle-shaped, with both ends sharply terminated. Each bears five prominent narrow ridges. When cut transversely the profile shows an almost regular pentagon and four vittae on the dorsal surface and two on the commissural surface may be seen with a lens. B. Reduce to a powder (355). The powder is yellowish-brown. Examine under a microscope using chloral hydrate solution R. The powder shows the following diagnostic characters : fragments of the secretory cells composed of yellowish-brown to brown, thin-walled, polygonal secretory cells, frequently associated with a layer of thin-walled transversely elongated cells, 8-12 µm wide ; fragments of the epicarp with thick-walled cells and occasional anomocytic stomata (2.8.3) ; numerous endosperm fragments containing aleurone grains, droplets of fatty oil and microcrystals of calcium oxalate in rosette formation ; spiral vessels accompanied by sclerenchymatous fibres ; rarely some fibre bundles from the carpophore ; groups of rectangular to sub-rectangular sclereids from the mesocarp with moderately thickened and pitted walls may be present. C. Examine by thin-layer chromatography (2.2.27) using a suitable silica gel as the coating substance. Test solution. Shake 0.5 g of the powdered drug (710) with 5.0 ml of ethyl acetate R for 2 min to 3 min. Filter over 2 g of anhydrous sodium sulphate R. Use the filtrate as the test solution. Reference solution. Dissolve 2 µl of carvone R and 5 µl of olive oil R in 1.0 ml of ethyl acetate R. Apply to the plate as bands 20 µl of the test solution and 10 µl of the reference solution. Develop over a path of 10 cm using a mixture of 5 volumes of ethyl acetate R and 95 volumes of toluene R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The chromatograms of the test solution and of the reference solution show a quenching zone (carvone) in the central part against a light background. Spray with anisaldehyde solution R and heat under observation at 100 °C to 105 °C for 2 to 4 min. Examine in daylight. The zones corresponding to carvone appear strong orange-brown. The chromatogram obtained with the test solution shows above the zone corresponding to carvone a violet zone corresponding to triglycerides similar in position and colour to the zone in the chromatogram obtained with the reference solution (triglycerides of olive oil). The chromatogram obtained with the test solution shows close General Notices (1) apply to all monographs and other texts

to the solvent front a weak violet zone corresponding to terpene hydrocarbons and in the lower part some weak, mostly violet-greyish and brownish zones. TESTS Foreign matter (2.8.2). It complies with the test for foreign matter. Water (2.2.13). Not more than 100 ml/kg, determined on 10.0 g of powdered drug by distillation. Total ash (2.4.16). Not more than 7.0 per cent. ASSAY Carry out the determination of essential oils in vegetable drugs (2.8.12). Use a 500 ml round-bottomed flask, 200 ml of water R as the distillation liquid and 0.50 ml of xylene R in the graduated tube. Reduce the drug to a powder (710) and immediately use 10.0 g for the determination. Distil at a rate of 2-3 ml/min for 90 min. STORAGE Store protected from light. 01/2005:1971

CARBACHOL Carbacholum

C6H15ClN2O2

Mr 182.7

DEFINITION 2-(Carbamoyloxy)-N,N,N-trimethylethanaminium chloride. Content : 99.0 per cent to 101.5 per cent (dried substance). CHARACTERS Appearance : white, crystalline, hygroscopic powder. Solubility : very soluble in water, sparingly soluble in alcohol, practically insoluble in acetone. IDENTIFICATION First identification : A, C. Second identification : B, C. A. Infrared absorption spectrophotometry (2.2.24). Comparison : carbachol CRS. B. Examine the chromatograms obtained in the test for related substances. Results : the principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). C. 0.5 ml of solution S (see Tests) gives reaction (a) of chlorides (2.3.1). TESTS Solution S. Dissolve 2.5 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Acidity or alkalinity. To 2.0 ml of solution S, add 0.05 ml of methyl red mixed solution R. Not more than 0.2 ml of 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is required to change the colour of the indicator. 1177

Carbamazepine


Related substances. Thin-layer chromatography (2.2.27).

01/2005:0543

Prepare the solutions immediately before use.

CARBAMAZEPINE

Test solution (a). Dissolve 0.20 g of the substance to be examined in methanol R and dilute to 5.0 ml with the same solvent.

Carbamazepinum

Test solution (b). Dilute 2.0 ml of test solution (a) to 20.0 ml with methanol R. Reference solution (a). Dissolve 20 mg of carbachol CRS in methanol R and dilute to 5.0 ml with the same solvent. Reference solution (b). Dissolve 8 mg of choline chloride R and 8 mg of acetylcholine chloride CRS in methanol R and dilute to 10.0 ml with the same solvent. Dilute 5.0 ml to 10.0 ml with methanol R. Plate : cellulose for chromatography R as the coating substance.

C15H12N2O

Mr 236.3

DEFINITION 5H-dibenzo[b,f]azepine-5-carboxamide. Content : 98.0 per cent to 102.0 per cent (dried substance).

Mobile phase : water R, methanol R (10:90 V/V). Application : 10 µl.

CHARACTERS Appearance : white or almost white crystalline powder. Solubility : very slightly soluble in water, freely soluble in methylene chloride, sparingly soluble in acetone and in alcohol. It shows polymorphism ; the acceptable crystalline form corresponds to carbamazepine CRS.

Development : over 2/3 of the plate. Detection : spray with potassium iodobismuthate solution R3. System suitability : the chromatogram obtained with reference solution (b) shows 2 clearly separated spots. Limits : in the chromatogram obtained with test solution (a) :

IDENTIFICATION — any impurity : any spot, apart from the principal spot, A. Melting point (2.2.14) : 189 °C to 193 °C. is not more intense than one or other of the 2 principal B. Infrared absorption spectrophotometry (2.2.24). spots in the chromatogram obtained with reference Comparison : carbamazepine CRS. solution (b) (1 per cent). Compare the spots with the spot of the most appropriate colour in the chromatogram Preparation : examine the substances as discs without obtained with reference solution (b). prior treatment. Heavy metals (2.4.8) : maximum 20 ppm. TESTS 12 ml of solution S complies with limit test A. Prepare the standard using lead standard solution (2 ppm Pb) R.

Acidity or alkalinity. To 1.0 g add 20 ml of carbon dioxide-free water R, shake for 15 min and filter. To 10 ml Loss on drying (2.2.32) : maximum 1.0 per cent, determined of the filtrate add 0.05 ml of phenolphthalein solution R1 and 0.5 ml of 0.01 M sodium hydroxide ; the solution is on 1.000 g by drying in an oven at 100-105 °C for 2 h. red. Add 1.0 ml of 0.01 M hydrochloric acid ; the solution Sulphated ash (2.4.14) : maximum 0.1 per cent, determined is colourless. Add 0.15 ml of methyl red solution R ; the on 1.0 g of the residue obtained in the test for loss on drying. solution is red. Related substances. Liquid chromatography (2.2.29). ASSAY Test solution (a). Dissolve 0.150 g of the substance to be Dissolve 0.150 g in a mixture of 10 ml of anhydrous acetic examined in methanol R2 and dilute to 50.0 ml with the acid R and 40 ml of acetic anhydride R. Titrate with 0.1 M same solvent. Sonicate. Dilute 10.0 ml of this solution to perchloric acid. Determine the end-point potentiometrically 20.0 ml with water R. (2.2.20). Test solution (b). Dilute 10.0 ml of test solution (a) to 50.0 ml with a mixture of equal volumes of methanol R2 and water R. 1 ml of 0.1 M perchloric acid is equivalent to 18.27 mg of C6H15ClN2O2. Reference solution (a). Dissolve 7.5 mg of carbamazepine CRS, 7.5 mg of carbamazepine impurity A CRS and 7.5 mg of iminodibenzyl R (impurity E) STORAGE in methanol R2 and dilute to 100.0 ml with the same solvent. In an airtight container, protected from light. Dilute 1.0 ml of this solution to 50.0 ml with a mixture of equal volumes of methanol R2 and water R. Reference solution (b). Dissolve 0.150 g of IMPURITIES carbamazepine CRS in methanol R2 and dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of this solution to 50.0 ml with a mixture of equal volumes of methanol R2 and water R. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : nitrile silica gel for A. 2-hydroxy-N,N,N-trimethylethanaminium chloride chromatography R1 (10 µm). (choline chloride). 1178


Carbasalate calcium


Mobile phase : tetrahydrofuran R, methanol R2, water R (3:12:85 V/V/V). To 1000 ml of this solution add 0.2 ml of anhydrous formic acid R and 0.5 ml of triethylamine R. Flow rate : 2.0 ml/min. Detection : a spectrophotometer at 230 nm. Injection : 20 µl ; inject test solution (a) and reference solution (a). Run time : 6 times the retention time of carbamazepine which is about 10 min. Relative retention with reference to carbamazepine : impurity B = about 0.7 ; impurity A = about 0.9 ; impurity C = about 1.6 ; impurity D = about 3.5 ; impurity E = about 5.1. System suitability : — resolution : minimum of 1.7 between the peaks due to carbamazepine and impurity A in the chromatogram obtained with reference solution (a). Limits : — impurity A : not more than the area of the corresponding peak in the chromatogram obtained with reference solution (a) (0.1 per cent), — impurity E : not more than the area of the corresponding peak in the chromatogram obtained with reference solution (a) (0.1 per cent), — any other impurity : not more than the area of the peak due to carbamazepine in the chromatogram obtained with reference solution (a) (0.1 per cent), — total: not more than 5 times the area of the peak due to carbamazepine in the chromatogram obtained with reference solution (a) (0.5 per cent), — disregard limit : 0.5 times the area of the peak due to carbamazepine in the chromatogram obtained with reference solution (a) (0.05 per cent). Chlorides (2.4.4) : maximum 140 ppm. Suspend 0.715 g in 20 ml of water R and boil for 10 min. Cool and dilute to 20 ml with water R. Filter through a membrane filter (nominal pore size : 0.8 µm). Dilute 10 ml of the filtrate to 15 ml with water R. This solution complies with the limit test for chlorides. Heavy metals (2.4.8) : maximum 20 ppm. 1.0 g complies with limit test C. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 2h. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances. Injection : test solution (b) and reference solution (b). System suitability : — repeatability : reference solution (b). Calculate the percentage content m/m of dried substance. STORAGE In an airtight container. IMPURITIES Specified impurities : A, B, C, D, E. Other detectable impurities : F General Notices (1) apply to all monographs and other texts

A. R = CO-NH2 : 10,11-dihydro-5H-dibenzo[b,f]azepine-5carboxamide (10,11-dihydrocarbamazepine), E. R = H : 10,11-dihydro-5H-dibenzo[b,f]azepine (iminodibenzyl),

B. 9-methylacridine,

C. R = CO-NH-CO-NH2 : (5H-dibenzo[b,f]azepin-5ylcarbonyl)urea (N-carbamoylcarbamazepine), D. R = H : 5H-dibenzo[b,f]azepine (iminostilbene), F. R = CO-Cl : 5H-dibenzo[b,f]azepine-5-carbonyl chloride (5-chlorocarbonyliminostilbene). 01/2005:1185

CARBASALATE CALCIUM Carbasalatum calcicum

C19H18CaN2O9

Mr 458.4

DEFINITION Carbasalate calcium contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of an equimolecular compound of calcium di[2-(acetyloxy)benzoate] and urea, calculated with reference to the anhydrous substance. CHARACTERS A white, crystalline powder, freely soluble in water and dimethylformamide, practically insoluble in acetone and in anhydrous methanol. Protect the substance from moisture during handling. Examination in aqueous solutions has to be performed immediately after preparation. IDENTIFICATION First identification : B, E. Second identification : A, C, D, E. 1179

Carbidopa


A. Dissolve 0.250 g in water R and dilute to 100.0 ml with IMPURITIES the same solvent. To 1.0 ml of the solution add 75 ml of water R and 5 ml of dilute hydrochloric acid R, mix and dilute with water R to 100.0 ml. Examined immediately after preparation between 220 nm and 350 nm (2.2.25), the solution shows two absorption maxima, at 228 nm and 276 nm. The specific absorbances at the maxima are 363 to 379 and 49 to 53, respectively. B. Examine by infrared spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of carbasalate calcium. C. Dissolve 0.1 g in 10 ml of water R, boil for 2 min and cool. A. 2-(acetyloxy)benzoic anhydride, The solution gives reaction (a) of salicylates (2.3.1). D. Heat 0.2 g with 0.2 g of sodium hydroxide R ; a yellow to yellowish-brown colour is produced and the vapour turns red litmus paper R blue. E. It gives reaction (a) of calcium (2.3.1). TESTS Appearance of solution. Dissolve 2.5 g in 50 ml ofwater R. The solution is not more opalescent than reference suspension II (2.2.1) and is colourless (2.2.2, Method II). Related substances. In a 100 ml volumetric flask, dissolve 0.150 g in 10 ml of 0.1 M tetrabutylammonium hydroxide in 2-propanol. Allow to stand for 10 min shaking occasionally. Add 8.0 ml of 0.1 M hydrochloric acid and 20.0 ml of a 19 g/l solution of disodium tetraborate R and mix. While swirling continuously, add 2.0 ml of a 10 g/l solution of aminopyrazolone R and 2.0 ml of a 10 g/l solution of potassium ferricyanide R. Allow to stand for 2 min, dilute to 100.0 ml with water R, mix and allow to stand for 20 min. Measure the absorbance (2.2.25) of the solution at the maximum at 505 nm using water R as the compensation liquid. The absorbance is not greater than 0.125 (0.1 per cent, expressed as acetylsalicylsalicylic acid). Salicylic acid. In a 100 ml volumetric flask, dissolve 0.200 g in 80 ml of water R and add 10 ml of a 10 g/l solution of ferric nitrate R in a 80 g/l solution of dilute nitric acid R. Dilute to 100.0 ml with water R. Immediately after preparation, measure the absorbance (2.2.25) of the solution at the maximum of 525 nm using water R as the compensation liquid. The absorbance is not greater than 0.115 (0.5 per cent, expressed as salicylic acid). Sodium. Not more than 0.1 per cent, determined by atomic emission spectrometry (2.2.22, Method I) using 1.0 g dissolved in 500.0 ml of water R. Heavy metals (2.4.8). Dissolve 2.0 g in 8 ml of water R, with heating, cool and add 12 ml of acetone R. 12 ml of the solution complies with limit test B for heavy metals (10 ppm). Prepare the standard using 10 ml of lead standard solution (1 ppm Pb) R. Water (2.5.12). Not more than 0.1 per cent, determined on 1.000 g by the semi-micro determination of water. Use a mixture of 15 ml of anhydrous methanol R and 15 ml of dimethylformamide R as the solvent. ASSAY In a flask with a ground-glass stopper, dissolve 0.400 g in 25 ml of water R. Add 25.0 ml of 0.1 M sodium hydroxide. Close the flask and allow to stand for 2 h. Titrate the excess of alkali with 0.1 M hydrochloric acid, using 0.2 ml of phenolphthalein solution R. Carry out a blank titration. 1 ml of 0.1 M sodium hydroxide is equivalent to 22.92 mg of C19H18CaN2O9. STORAGE Store in an airtight container. 1180

B. 2-[[2-(acetyloxy)benzoyl]oxy]benzoic acid (acetylsalicylsalicylic acid), C. 2-hydroxybenzoic acid (salicylic acid). 01/2005:0755 corrected

CARBIDOPA Carbidopum

C10H14N2O4,H2O

Mr 244.2

DEFINITION Carbidopa contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of (2S)-3-(3,4-dihydroxyphenyl)-2-hydrazino-2-methylpropanoic acid, calculated with reference to the dried substance. CHARACTERS A white or yellowish-white powder, slightly soluble in water, very slightly soluble in alcohol, practically insoluble in methylene chloride. It dissolves in dilute solutions of mineral acids. IDENTIFICATION First identification : A, C. Second identification : A, B, D, E. A. It complies with the test for specific optical rotation (see Tests). B. Dissolve 50.0 mg in a 8.5 g/l solution of hydrochloric acid R in methanol R and dilute to 100.0 ml with the same acid. Dilute 10.0 ml of this solution to 100.0 ml with a 8.5 g/l solution of hydrochloric acid R in methanol R. Examined between 230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 283 nm. The specific absorbance at the maximum is 135 to 150, calculated with reference to the dried substance. C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with carbidopa CRS. Examine the substances prepared as discs.


Carbidopa


D. Shake vigorously about 5 mg with 10 ml of water R for 1 min and add 0.3 ml of ferric chloride solution R2. An intense green colour is produced, which quickly turns to reddish-brown.

Reference solution (b). Prepare the solution at the same time and in the same manner as described for test solution (b) using 1.0 ml of reference solution (a) in place of 1.0 ml of test solution (a).

E. Suspend about 20 mg in 5 ml of water R and add 5 ml of cupri-tartaric solution R. On heating, the colour of the solution changes to dark brown and a red precipitate is formed.

Apply separately to the plate 10 µl of test solution (b) and 10 µl of reference solution (b). Develop over a path of 10 cm using a mixture of 1 volume of water R and 2 volumes of methanol R. Allow the plate to dry in air. Examine in ultraviolet light at 365 nm. Any spot in the chromatogram obtained with test solution (b) showing a yellow fluorescence is not more intense than the corresponding spot in the chromatogram obtained with reference solution (b) (20 ppm of hydrazine).

TESTS

Appearance of solution. Dissolve 0.25 g in 25 ml of 1 M hydrochloric acid. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY6 or B6 (2.2.2, Methyldopa and methylcarbidopa. Examine by liquid chromatography (2.2.29). Method II). Test solution. Dissolve 0.100 g of the substance to be Specific optical rotation (2.2.7). With the aid of an examined in 0.1 M hydrochloric acid and dilute to 10.0 ml ultrasonic bath, dissolve completely 0.250 g in aluminium with the same acid. chloride solution R and dilute to 25.0 ml with the same solution. The specific optical rotation is − 22.5 to − 26.5, Reference solution (a). Dissolve the contents of a vial of calculated with reference to the dried substance. methylcarbidopa CRS in 0.1 M hydrochloric acid, add 1 mg Hydrazine. Examine by thin-layer chromatography (2.2.27), of methyldopa CRS and dilute to 20.0 ml with the same acid. using silanised silica gel H R as the coating substance. Reference solution (b). Dissolve 5 mg of carbidopa CRS and 5 mg of methyldopa CRS in 0.1 M hydrochloric acid and Test solution (a). Dissolve 0.50 g in dilute hydrochloric dilute to 10.0 ml with the same acid. acid R and dilute to 2.0 ml with the same acid. Test solution (b). Place 25 g of strongly basic anion exchange resin R into each of two conical flasks with ground-glass stoppers. To each, add 150 ml of carbon dioxide-free water R and shake from time to time during 30 min. Decant the liquid from both flasks and repeat the process with further quantities, each of 150 ml, of carbon dioxide-free water R.

The chromatographic procedure may be carried out using :

Take two 100 ml measuring cylinders 3.5 cm to 4.5 cm in internal diameter and label these A and B. Into cylinder A, transfer as completely as possible the resin from one conical flask using 60 ml of carbon dioxide-free water R ; into cylinder B, transfer the second quantity of resin, this time using 20 ml of carbon dioxide-free water R.

— as detector a spectrophotometer set at 282 nm,

Separate the toluene layer in a 100 ml separating funnel and shake vigorously with two quantities, each of 20 ml, of a 200 g/l solution of sodium metabisulphite R and finally with two quantities, each of 50 ml, of water R. Separate the toluene layer.

Dissolve 0.150 g with gentle heating in 75 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20).

— a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1 ml/min a mixture of 2 volumes of methanol R and 98 volumes of a 14 g/l solution of potassium dihydrogen phosphate R, — a loop injector.

Inject 20 µl of each solution. The test is not valid unless the resolution between the peaks corresponding to methyldopa and carbidopa in the chromatogram obtained with reference solution (b) is greater than 4.0. In the chromatogram Into each cylinder, insert a gas-inlet tube, the end of which obtained with the test solution, the areas of any peaks has an internal diameter of 2 mm to 3 mm and which corresponding to methyldopa and methylcarbidopa are not reaches almost to the bottom of the cylinder. Pass a rapid greater than the areas of the corresponding peaks in the stream of nitrogen for chromatography R through each mixture so that homogeneous suspensions are formed. After chromatogram obtained with reference solution (a) (0.5 per 30 min, without interrupting the gas flow, add 1.0 ml of test cent). solution (a) to cylinder A ; after 1 min stop the gas flow into Heavy metals (2.4.8). 1.0 g complies with limit test C for cylinder A and transfer the contents, through a moistened heavy metals (20 ppm). Prepare the standard using 2 ml of filter paper, into cylinder B. After 1 min, stop the gas flow lead standard solution (10 ppm Pb) R. to cylinder B and pour the solution immediately through a moistened filter paper into a freshly prepared mixture of 1 ml Loss on drying (2.2.32) : 6.9 per cent to 7.9 per cent, determined on 1.000 g by drying in an oven at 100 °C to of a 200 g/l solution of salicylaldehyde R in methanol R and 20 ml of phosphate buffer solution pH 5.5 R in a conical 105 °C. flask, shake thoroughly for 1 min and heat in a water-bath at Sulphated ash (2.4.14). Not more than 0.1 per cent, 60 °C for 15 min. The liquid becomes clear. Allow to cool, determined on 1.0 g. add 2.0 ml of toluene R and shake vigorously for 2 min. Transfer the mixture into a centrifuge tube and centrifuge. ASSAY

1 ml of 0.1 M perchloric acid is equivalent to 22.62 mg of C10H14N2O4.

Reference solution (a). Dissolve 10 mg of hydrazine sulphate R in dilute hydrochloric acid R and dilute to 50 ml STORAGE with the same acid. Dilute 1.0 ml of this solution to 10.0 ml with dilute hydrochloric acid R. Store protected from light. General Notices (1) apply to all monographs and other texts

1181

Carbimazole


01/2005:0884 Reference solution (b). Dissolve 5.0 mg of thiamazole R in a mixture of 20 volumes of acetonitrile R and 80 volumes of water R and dilute to 10.0 ml with the same mixture of CARBIMAZOLE solvents. Dilute 1.0 ml of this solution to 100.0 ml with a mixture of 20 volumes of acetonitrile R and 80 volumes of Carbimazolum water R. Column : — size : l = 0.15 m, Ø = 3.9 mm, — stationary phase : octadecylsilyl silica gel for chromatography R (5 µm). C7H10N2O2S Mr 186.2 Mobile phase : acetonitrile R, water R (10:90 V/V). Flow rate : 1 ml/min. DEFINITION Detection : spectrophotometer at 254 nm. Ethyl 3-methyl-2-thioxo-2,3-dihydro-1H-imidazole-1Injection : 10 µl. carboxylate. Run time : 1.5 times the retention time of carbimazole. Content : 98.0 per cent to 102.0 per cent (dried substance). Retention time : carbimazole = about 6 min. CHARACTERS System suitability : reference solution (a) : Appearance : white or yellowish-white, crystalline powder. — resolution : minimum 5.0 between the peaks due to Solubility : slightly soluble in water, soluble in acetone and impurity A and carbimazole. in alcohol. Limits : IDENTIFICATION — impurity A : not more than half the area of the principal peak in the chromatogram obtained with reference First identification : B. solution (b) (0.5 per cent), Second identification : A, C, D. — any other impurity : not more than 0.1 times the area of A. Melting point (2.2.14) : 122 °C to 125 °C. the principal peak in the chromatogram obtained with B. Infrared absorption spectrophotometry (2.2.24). reference solution (b) (0.1 per cent). Preparation : discs. Loss on drying (2.2.32) : maximum 0.5 per cent, determined Comparison : carbimazole CRS. on 1.000 g by drying in a desiccator over diphosphorus C. Thin-layer chromatography (2.2.27). pentoxide R at a pressure not exceeding 0.7 kPa for 24 h. Test solution. Dissolve 10 mg of the substance to be Sulphated ash (2.4.14) : maximum 0.1 per cent, determined examined in methylene chloride R and dilute to 10 ml on 1.0 g. with the same solvent. Reference solution. Dissolve 10 mg of carbimazole CRS ASSAY in methylene chloride R and dilute to 10 ml with the Dissolve 50.0 mg in water R and dilute to 500.0 ml with the same solvent. same solvent. To 10.0 ml add 10 ml of dilute hydrochloric Plate : TLC silica gel GF254 plate R. acid R and dilute to 100.0 ml with water R. Measure the absorbance (2.2.25) at the maximum at 291 nm. Calculate Mobile phase : acetone R, methylene chloride R the content of C7H10N2O2S taking the specific absorbance (20:80 V/V). to be 557. Application : 10 µl. Development : over a path of 15 cm. IMPURITIES Drying : in air for 30 min. Detection : examine in ultraviolet light at 254 nm. Results : the principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. A. 1-methyl-1H-imidazole-2-thiol (thiamazole). D. Dissolve about 10 mg in a mixture of 50 ml of water R and 0.05 ml of dilute hydrochloric acid R. Add 1 ml of 01/2005:0885 potassium iodobismuthate solution R. A red precipitate is formed. TESTS Impurity A and other related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 5.0 mg of the substance to be examined in 10.0 ml of a mixture of 20 volumes of acetonitrile R and 80 volumes of water R. Use this solution within 5 min of preparation. Reference solution (a). Dissolve 5 mg of thiamazole R and 0.10 g of carbimazole CRS in a mixture of 20 volumes of acetonitrile R and 80 volumes of water R and dilute to 100.0 ml with the same mixture of solvents. Dilute 1.0 ml of this solution to 10.0 ml with a mixture of 20 volumes of acetonitrile R and 80 volumes of water R. 1182

CARBOCISTEINE Carbocisteinum

C5H9NO4S

Mr 179.2

DEFINITION Carbocisteine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of (2R)-2-amino-3-[(carboxymethyl)sulphanyl]propanoic acid, calculated with reference to the dried substance.


Carbomers


CHARACTERS A white, crystalline powder, practically insoluble in water and in alcohol. It dissolves in dilute mineral acids and in dilute solutions of alkali hydroxides. IDENTIFICATION First identification : A, B. Second identification : A, C, D. A. It complies with the test for specific optical rotation (see Tests). B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with carbocisteine CRS. Examine the substances prepared as discs. C. Examine the chromatograms obtained in the test for ninhydrin-positive substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). D. Dissolve 0.1 g in 4.5 ml of dilute sodium hydroxide solution R. Heat on a water-bath for 10 min. Cool and add 1 ml of a 25 g/l solution of sodium nitroprusside R. A dark red colour is produced, which changes to brown and then to yellow within a few minutes. TESTS Solution S. Disperse 5.00 g in 20 ml of water R and add dropwise with shaking 2.5 ml of strong sodium hydroxide solution R. Adjust to pH 6.3 with 1 M sodium hydroxide and dilute to 50.0 ml with water R. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). pH (2.2.3). Shake 0.2 g with 20 ml of carbon dioxide-free water R. The pH of the suspension is 2.8 to 3.0. Specific optical rotation (2.2.7) : − 32.5 to − 35.5, determined on solution S and calculated with reference to the dried substance. Ninhydrin-positive substances. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating substance. Test solution (a). Dissolve 0.10 g of the substance to be examined in dilute ammonia R2 and dilute to 10 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 50 ml with water R. Reference solution (a). Dissolve 10 mg of carbocisteine CRS in dilute ammonia R2 and dilute to 50 ml with the same solvent. Reference solution (b). Dilute 5 ml of test solution (b) to 20 ml with water R. Reference solution (c). Dissolve 10 mg of carbocisteine CRS and 10 mg of arginine hydrochloride CRS in 5 ml of dilute ammonia R2 and dilute to 25 ml with water R. Apply separately to the plate 5 µl of each solution. Allow the plate to dry in air. Develop over a path of 15 cm using a mixture of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes of butanol R. Dry the plate in a current of warm air. Spray with ninhydrin solution R and heat at 100 °C to 105 °C for 15 min. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows two clearly separated principal spots. General Notices (1) apply to all monographs and other texts

Chlorides (2.4.4). Dissolve 33 mg in 5 ml of dilute nitric acid R and dilute to 15 ml with water R. The solution, without further addition of nitric acid, complies with the limit test for chlorides (0.15 per cent). Sulphates (2.4.13). Dissolve 0.5 g in 5 ml of dilute hydrochloric acid R and dilute to 15 ml with distilled water R. The solution complies with the limit test for sulphates (300 ppm). Heavy metals (2.4.8). 2.0 g complies with limit test D for heavy metals (10 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C for 2 h. Sulphated ash (2.4.14). Not more than 0.3 per cent, determined on 1.0 g. ASSAY Dissolve 0.150 g in 10 ml of anhydrous formic acid R with slight heating and shake until dissolution is complete. Add 50 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 17.92 mg of C5H9NO4S. STORAGE Store protected from light. 01/2005:1299

CARBOMERS Carbomera DEFINITION High molecular mass polymers of acrylic acid cross-linked with polyalkenyl ethers of sugars or polyalcohols. Content : 56.0 per cent to 68.0 per cent of carboxylic acid (-COOH) groups (dried substance). CHARACTERS Appearance : white, fluffy powder, hygroscopic. Solubility : swells in water and in other polar solvents after dispersion and neutralisation with sodium hydroxide solution. IDENTIFICATION First identification : A, E. Second identification : B, C, D, E. A. Infrared absorption spectrophotometry (2.2.24). Main bands : at 2960 cm− 1, 1720 cm− 1, 1455 cm− 1, 1415 cm− 1, 1250 cm− 1, 1175 cm− 1 and 800 cm− 1, with the strongest band at 1720 cm− 1. B. Adjust a 10 g/l dispersion to about pH 7.5 with 1 M sodium hydroxide. A highly viscous gel is formed. C. Add 2 ml of a 100 g/l solution of calcium chloride R with continuous stirring to 10 ml of the gel from test B. A white precipitate is immediately produced. D. Add 0.5 ml of thymol blue solution R to 10 ml of a 10 g/l dispersion. An orange colour is produced. Add 0.5 ml of cresol red solution R to 10 ml of a 10 g/l dispersion. A yellow colour is produced. E. It complies with the apparent nominal viscosity indicated on the label. 1183

Carbomers


TESTS Apparent viscosity : the nominal apparent viscosity is in the range 300 mPa·s to 115 000 mPa·s. For a product with a nominal apparent viscosity of 20 000 mPa·s or greater, the apparent viscosity is 70.0 per cent to 130.0 per cent of the value stated on the label ; for a product with a nominal apparent viscosity less than 20 000 mPa·s, the apparent viscosity is 50.0 per cent to 150.0 per cent of the value stated on the label.

Time (min)



0-8

100

0

8-9

100 → 0

0 → 100

9 - 20

0

100

20 - 21

0 → 100

100 → 0

21 - 30

100

0

Flow rate : 1 ml/min. Detection : spectrophotometer at 205 nm. Injection : 20 µl. Retention time : acrylic acid = about 6.0 min. Limit : — acrylic acid : not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.25 per cent). Benzene. Gas chromatography (2.4.24, System A). Solvent solution. Dissolve 0.100 g of benzene R in dimethyl sulphoxide R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with water R. Dilute 1.0 ml of this solution to 100.0 ml with water R. Test solution. Weigh 50.0 mg of the substance to be examined into an injection vial and add 5.0 ml of water R and 1.0 ml of dimethyl sulphoxide R. Reference solution. Weigh 50.0 mg of the substance to be examined into an injection vial and add 4.0 ml of water R, 1.0 ml of dimethyl sulphoxide R and 1.0 ml of the solvent solution. Close the vials with a tight rubber membrane stopper coated with polytetrafluoroethylene and secure with an aluminium crimped cap. Shake to obtain a homogeneous dispersion. Stratic head-space conditions which may be used : Free acrylic acid. Liquid chromatography (2.2.29). — equilibration temperature : 80 °C, — equilibration time : 60 min, Test solution. Mix 0.125 g of the substance to be examined with a 25 g/l solution of aluminium potassium sulphate R — transfer line temperature : 90 °C. Injection : 1 ml of the gaseous phase of the test solution and and dilute to 25.0 ml with the same solution. Heat the 1 ml of the gaseous phase of the reference solution ; repeat suspension at 50 °C for 20 min with shaking. Then shake the suspension at room temperature for 60 min. Centrifuge these injections twice more. and use the clear supernatant solution as the test solution. System suitability : — repeatability : maximum relative standard deviation of the Reference solution. Dissolve 62.5 mg of acrylic acid R in a differences in area between the analyte peaks obtained 25 g/l solution of aluminium potassium sulphate R and from the 3 replicate pair injections of the reference dilute to 100.0 ml with the same solution. Dilute 1.0 ml of solution and the test solution is 15 per cent. this solution to 50.0 ml with a 25 g/l solution of aluminium Limit : potassium sulphate R. — benzene : the mean area of the peak corresponding to benzene in the chromatograms obtained with the test Column : solution is not greater than half the mean area of the peak corresponding to benzene in the chromatograms — size : l = 0.12 m, Ø = 4.6 mm, obtained with the reference solution (2 ppm). Heavy metals (2.4.8) : maximum 20 ppm. — stationary phase: octadecylsilyl silica gel for 1.0 g complies with limit test C. Prepare the standard using chromatography R (5 µm). 2 ml of lead standard solution (10 ppm Pb) R. Mobile phase : Loss on drying (2.2.32) : maximum 3.0 per cent, determined on 1.000 g by drying in vacuo at 80 °C for 60 min. — mobile phase A : a 1.361 g/l solution of potassium Sulphated ash (2.4.14) : maximum 4.0 per cent, determined dihydrogen phosphate R, adjusted to pH 2.5 using dilute on 1.0 g. phosphoric acid R, ASSAY — mobile phase B : equal volumes of a 1.361 g/l solution of Slowly add 50 ml of water R to 0.120 g whilst stirring and potassium dihydrogen phosphate R and acetonitrile for heating at 60 °C for 15 min. Stop heating, add 150 ml chromatography R. of water R and continue stirring for 30 min. Add 2 g Dry the substance to be examined in vacuo at 80 °C for 1 h. Carefully add 2.50 g of the previously dried substance to be examined to 500 ml of water R in a 1000 ml beaker while stirring continuously at 1000 ± 50 r/min, with the stirrer shaft set at an angle of 60° to one side of the beaker. Add the previously dried substance over a period of 45-90 s, at a uniform rate, ensuring that loose aggregates of powder are broken up and continue stirring at 1000 ± 50 r/min for 15 min. Remove the stirrer, and place the beaker containing the dispersion in a water-bath at 25 ± 0.2 °C for 30 min. Insert the stirrer to a depth necessary to ensure that air is not drawn into the dispersion, and while stirring at 300 ± 25 r/min, titrate with a glass-calomel electrode system to pH 7.3-7.8 by adding a 180 g/l solution of sodium hydroxide R below the surface, determining the end-point potentiometrically (2.2.20). The total volume of the 180 g/l solution of sodium hydroxide R used is about 6.2 ml. Allow 2-3 min before the final pH determination. If the final pH exceeds 7.8, discard the preparation, and prepare another using a smaller amount of sodium hydroxide for titration. Return the neutralised preparation to the water-bath at 25 °C for 1 h, then perform the viscosity determination without delay to avoid slight viscosity changes that occur 75 min after neutralisation. Determine the viscosity (2.2.10) with a rotating viscometer with a spindle rotating at 20 r/min, using a spindle suitable for the expected apparent viscosity.

1184


Carbon dioxide


of potassium chloride R and titrate with 0.2 M sodium hydroxide, determining the end-point potentiometrically (2.2.20). 1 ml of 0.2 M sodium hydroxide is equivalent to 9.0 mg of carboxylic acid (-COOH) groups.

— size : l = 2 m, Ø = 4 mm, — stationary phase : an appropriate molecular sieve for chromatography (0.5 nm). Carrier gas : helium for chromatography R. Flow rate : 60 ml/min. STORAGE Temperature : In an airtight container. — column : 50 °C, — injection port and detector : 130 °C. LABELLING Detection : flame ionisation with methaniser. The label states the nominal apparent viscosity. Injection : loop injector. 01/2005:0375 Adjust the injected volumes and the operating conditions so that the height of the peak due to carbon monoxide in the chromatogram obtained with the reference gas is at least CARBON DIOXIDE 35 per cent of the full scale of the recorder. Limit : Carbonei dioxidum — carbon monoxide : not more than the area of the corresponding peak in the chromatogram obtained with CO2 Mr 44.01 the reference gas (5 ppm V/V). DEFINITION Nitrogen monoxide and nitrogen dioxide : maximum Content : minimum 99.5 per cent V/V of CO2 in the gaseous 2 ppm V/V in total, determined using a chemiluminescence analyser (2.5.26). phase. This monograph applies to carbon dioxide for medicinal use. Gas to be examined. The substance to be examined. Reference gas (a). Carbon dioxide R1. CHARACTERS Reference gas (b). A mixture containing 2 ppm V/V Appearance : colourless gas. of nitrogen monoxide R in carbon dioxide R1 or in Solubility : at 20 °C and at a pressure of 101 kPa, 1 volume nitrogen R1. dissolves in about 1 volume of water. Calibrate the apparatus and set the sensitivity using reference gases (a) and (b). Measure the content of nitrogen PRODUCTION monoxide and nitrogen dioxide in the gas to be examined. Examine the gaseous phase. If nitrogen is used instead of carbon dioxide in reference If the test is performed on a cylinder of gas, keep the gas (b), multiply the result obtained by the quenching cylinder of the substance to be examined at room correction factor in order to correct the quenching effect on temperature for not less than 6 h before carrying out the the analyser response caused by the carbon dioxide matrix tests. Keep the cylinder in the vertical position with the effect. outlet valve uppermost. The quenching correction factor is determined by applying Carbon monoxide. Gas chromatography (2.2.28). a known reference mixture of nitrogen monoxide in carbon Gas to be examined. The substance to be examined. dioxide and comparing the actual content with the content Reference gas. A mixture containing 5 ppm V/V of carbon indicated by the analyser which has been calibrated with monoxide R in nitrogen R1. a NO/N2 reference mixture. Column : = — material : stainless steel,

Figure 0375.-1.— UV Fluorescence Analyser General Notices (1) apply to all monographs and other texts

1185

Carboplatin


Total sulphur : maximum 1 ppm V/V, determined using an ultraviolet fluorescence analyser after oxidation of the sulphur compounds by heating at 1000 °C (Figure 0375.-1). The apparatus consists of the following : — a system generating ultraviolet radiation with a wavelength of 210 nm, made up of an ultraviolet lamp, a collimator, and a selective filter ; the beam is blocked periodically by a chopper rotating at high speed, — a reaction chamber through which flows the previously filtered gas to be examined, — a system that detects radiation emitted at a wavelength of 350 nm, made up of a selective filter, a photomultiplier tube and an amplifier. Gas to be examined. The substance to be examined. Reference gas (a). Carbon dioxide R1. Reference gas (b). A mixture containing between 0.5 ppm V/V and 2 ppm V/V of hydrogen sulphide R1 in carbon dioxide R1. Calibrate the apparatus and set the sensitivity using reference gases (a) and (b). Pass the gas to be examined through a quartz oven heated to 1000 °C. Oxygen R is circulated in the oven at a tenth of the flow rate of the gas to be examined. Measure the sulphur dioxide content in the gaseous mixture leaving the oven. Water : maximum 67 ppm V/V, determined using an electrolytic hygrometer (2.5.28). Assay. Infrared analyser (2.5.24). Gas to be examined. The substance to be examined. It must be filtered to avoid stray light phenomena. Reference gas (a). Carbon dioxide R1. Reference gas (b). A mixture containing 95.0 per cent V/V of carbon dioxide R1 and 5.0 per cent V/V of nitrogen R1. Calibrate the apparatus and set the sensitivity using reference gases (a) and (b). Measure the content of carbon dioxide in the gas to be examined. IDENTIFICATION First identification : A. Second identification : B, C. A. Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of carbon dioxide. B. Place a glowing splinter of wood in an atmosphere of the substance to be examined. It is extinguished. C. Pass a stream of the substance to be examined through barium hydroxide solution R. A white precipitate is formed which dissolves with effervescence in dilute acetic acid R. TESTS Examine the gaseous phase. If the test is performed on a cylinder of gas, keep the cylinder of the substance to be examined at room temperature for not less than 6 h before carrying out the tests. Keep the cylinder in the vertical position with the outlet valve uppermost. Carbon monoxide : maximum 5 ppm V/V, determined using a carbon monoxide detector tube (2.1.6). Hydrogen sulphide : maximum 1 ppm V/V, determined using a hydrogen sulphide detector tube (2.1.6). Nitrogen monoxide and nitrogen dioxide : maximum 2 ppm V/V in total, determined using a nitrogen monoxide and nitrogen dioxide detector tube (2.1.6). 1186

Sulphur dioxide : maximum 2 ppm V/V, determined using a sulphur dioxide detector tube (2.1.6). Water vapour : maximum 67 ppm V/V, determined using a water vapour detector tube (2.1.6). STORAGE Store liquefied under pressure in suitable containers complying with the legal regulations. IMPURITIES A. nitrogen monoxide, B. nitrogen dioxide, C. carbon monoxide, D. total sulphur, E. water. 01/2005:1081

CARBOPLATIN Carboplatinum

C6H12N2O4Pt

Mr 371.3

DEFINITION Carboplatin contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of (SP-4-2)diammine[cyclobutan-1,1-dicarboxylato(2-)-O,O′]platin, calculated with reference to the dried substance. CHARACTERS A colourless, crystalline powder, sparingly soluble in water, very slightly soluble in acetone and in alcohol. It melts at about 200 °C, with decomposition. IDENTIFICATION Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of carboplatin. TESTS Solution S1. Dissolve 0.25 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Solution S2. Dissolve 1.0 g in water R, heating slightly if necessary, and dilute to 40 ml with the same solvent. Filter if necessary. Appearance of solution. Solution S1 is clear (2.2.1) and colourless (2.2.2, Method II). Impurity B and acidity. To 10 ml of solution S1 add 0.1 ml of phenolphthalein solution R1. The solution is colourless. Not more than 0.7 ml of 0.01 M sodium hydroxide is required to change the colour of the indicator to pink (0.5 per cent calculated as impurity B). Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 20.0 mg of the substance to be examined in a mixture of equal volumes of acetonitrile R and water R and dilute to 20.0 ml with the same mixture.


Carisoprodol


Reference solution. Dilute 0.5 ml of the test solution to 200.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with aminopropylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 2 ml/min a mixture of 130 volumes of water R and 870 volumes of acetonitrile R, — a 10 µl loop injector, — as detector a spectrophotometer set at 230 nm. Inject the test solution. The test is not valid unless : — the mass distribution ratio, Dm, is not less than 4.0, — the number of theoretical plates, n, is not less than 5000, — the symmetry factor is not more than 2.0. If necessary adjust the concentration of acetonitrile in the mobile phase. Inject the test solution and the reference solution. Continue the chromatography for 2.5 times the retention time of the principal peak. In the chromatogram obtained with the test solution ; the area of any peak apart from the principal peak is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (0.25 per cent) ; the sum of the areas of all the peaks apart from the principal peak is not greater than twice the area of the principal peak in the chromatogram obtained with the reference solution (0.5 per cent). Disregard any peak with an area less than 0.2 times the area of the principal peak in the chromatogram obtained with the reference solution. Chlorides (2.4.4). Dilute 10 ml of solution S2 to 15 ml with water R. The solution complies with the limit test for chlorides (100 ppm). Prepare the standard using 5 ml of chloride standard solution (5 ppm Cl) R. Ammonium (2.4.1). 0.20 g complies with the limit test B for ammonium (100 ppm). Prepare the standard using 0.2 ml of ammonium standard solution (100 ppm NH4) R. Silver. Not more than 10 ppm of Ag, determined by atomic emission spectrometry (2.2.22, Method I). Test solution. Dissolve 0.50 g in a 1 per cent V/V solution of nitric acid R and dilute to 50.0 ml with the same acid. Reference solutions. Prepare the reference solutions using silver standard solution (5 ppm Ag) R, diluted with a 1 per cent V/V solution of nitric acid R. Measure the intensity emitted at 328.1 nm. Soluble barium. Not more than 10 ppm of Ba, determined by atomic emission spectrometry (2.2.22, Method I). Test solution. Use the solution described in the test for silver. Reference solutions. Prepare the reference solutions using barium standard solution (50 ppm Ba) R, diluted with a 1 per cent V/V solution of nitric acid R. Measure the intensity emitted at 455.4 nm. Heavy metals (2.4.8). 12 ml of solution S2 complies with limit test A for heavy metals (20 ppm). Prepare the standard using 5 ml of lead standard solution (1 ppm Pb R). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C.

IMPURITIES A. cisplatin,

B. cyclobutane-1,1-dicarboxylic acid. 01/2005:1689

CARISOPRODOL Carisoprodolum

C12H24N2O4

Mr 260.3

DEFINITION (2RS)-2-[(Carbamoyloxy)methyl]-2-methylpentyl (1-methylethyl)carbamate. Content : 98.0 per cent to 102.0 per cent (dried substance). CHARACTERS Appearance : white or almost white, fine powder. Solubility : very slightly soluble in water, freely soluble in acetone, in alcohol and in methylene chloride. IDENTIFICATION First identification : A, B. Second identification : A, C, D. A. Melting point (2.2.14) : 92 °C to 95 °C. B. Infrared absorption spectrophotometry (2.2.24). Comparison : carisoprodol CRS. C. Examine the chromatograms obtained in the test for related substances. Results : the principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (d). D. Dissolve 0.2 g in 15 ml of a 28 g/l solution of potassium hydroxide R in alcohol R and boil under a reflux condenser for 15 min. Add 0.5 ml of glacial acetic acid R and 1 ml of a 50 g/l solution of cobalt nitrate R in ethanol R. An intense blue colour develops.

TESTS Optical rotation (2.2.7) : − 0.10° to + 0.10°. Dissolve 2.5 g in alcohol R and dilute to 25.0 ml with the same solvent. Related substances. Thin-layer chromatography (2.2.27). Test solution (a). Dissolve 0.20 g of the substance to be examined in methylene chloride R and dilute to 10 ml with the same solvent. ASSAY Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with methylene chloride R. Use the residue obtained in the test for loss on drying. Reference solution (a). Dissolve 5.0 mg of meprobamate CRS Ignite 0.200 g of the residue to constant mass at 800 °C. 1 mg of the residue is equivalent to 1.903 mg of C6H12N2O4Pt. in methylene chloride R and dilute to 50 ml with the same solvent. STORAGE Reference solution (b). Dilute 1 ml of test solution (b) to 50 ml with methylene chloride R. Store protected from light. General Notices (1) apply to all monographs and other texts

1187

Carmellose calcium


Reference solution (c). Dilute 5 ml of reference solution (b) to 10 ml with methylene chloride R. Reference solution (d). Dissolve 20 mg of carisoprodol CRS in methylene chloride R and dilute to 10 ml with the same solvent. Reference solution (e). Dissolve 10 mg of carisoprodol impurity A CRS in 5 ml of reference solution (d) and dilute to 50 ml with methylene chloride R. Plate : TLC silica gel plate R. Mobile phase : acetone R, methylene chloride R (20:80 V/V). Application : 5 µl. Development : over a path of 15 cm. Drying : in air for 15 min. Detection : spray with a solution prepared as follows : dissolve 5 g of phosphomolybdic acid R in a mixture of 50 ml of glacial acetic acid R and 10 ml of sulphuric acid R, and dilute to 100 ml with glacial acetic acid R. Heat the plate at 100-105 °C for 30 min. System suitability : — the chromatogram obtained with reference solution (c) shows 1 clearly visible spot, — the chromatogram obtained with reference solution (e) shows 2 clearly separated spots. Limits : in the chromatogram obtained with test solution (a) : — impurity D : any spot due to impurity D is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.5 per cent), — any other impurity : any spot, apart from the principal spot and any spot due to impurity D, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.2 per cent). Heavy metals (2.4.8) : maximum 10 ppm. 2.0 g complies with limit test C. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g in vacuo at 60 °C for 3 h. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.100 g in 15 ml of a 25 per cent V/V solution of sulphuric acid R and boil under a reflux condenser for 3 h. Cool, dissolve by cautiously adding 30 ml of water R, cool again and place in a steam-distillation apparatus. Add 40 ml of strong sodium hydroxide solution R and distil immediately by passing steam through the mixture. Collect the distillate into 40 ml of a 40 g/l solution of boric acid R until the total volume in the receiver reaches about 200 ml. Add 0.25 ml of methyl red mixed solution R. Titrate with 0.1 M hydrochloric acid, until the colour changes from green to violet. Carry out a blank titration. 1 ml of 0.1 M hydrochloric acid is equivalent to 13.02 mg of C12H24N2O4. IMPURITIES

A. (2RS)-2-(hydroxymethyl)-2-methylpentyl (1-methylethyl)carbamate, 1188

B. 5-methyl-5-propyl-1,3-dioxan-2-one,

C. 2-methyl-2-propylpropane-1,3-diol, D. meprobamate. 01/2005:0886

CARMELLOSE CALCIUM Carmellosum calcicum DEFINITION Calcium salt of a partly O-carboxymethylated cellulose. CHARACTERS Appearance : white or yellowish-white powder, hygroscopic after drying. Solubility : practically insoluble in acetone, in alcohol and in toluene. It swells with water to form a suspension. IDENTIFICATION A. Shake 0.1 g thoroughly with 10 ml of water R. Add 2 ml of dilute sodium hydroxide solution R and allow to stand for 10 min (solution A). Dilute 1 ml of solution A to 5 ml with water R. To 0.05 ml add 0.5 ml of a 0.5 g/l solution of chromotropic acid, sodium salt R in a 75 per cent m/m solution of sulphuric acid R and heat on a water-bath for 10 min. A reddish-violet colour develops. B. Shake 5 ml of solution A obtained in identification test A with 10 ml of acetone R. A white, flocculent precipitate is produced. C. Shake 5 ml of solution A obtained in identification test A with 1 ml of ferric chloride solution R1. A brown, flocculent precipitate is formed. D. Ignite 1 g and dissolve the residue in a mixture of 5 ml of acetic acid R and 10 ml of water R. Filter if necessary and boil the filtrate for a few minutes. Cool and neutralise with dilute ammonia R1. The solution gives reaction (a) of calcium (2.3.1). TESTS Solution S. Shake 1.0 g with 50 ml of distilled water R, add 5 ml of dilute sodium hydroxide solution R and dilute to 100 ml with distilled water R. Alkalinity. Shake 1.0 g thoroughly with 50 ml of carbon dioxide-free water R and add 0.05 ml of phenolphthalein solution R. No red colour develops. Chlorides (2.4.4) : maximum 0.36 per cent. Heat 28 ml of solution S with 10 ml of dilute nitric acid R on a water-bath until a flocculent precipitate is produced. Cool, centrifuge and separate the supernatant liquid. Wash the precipitate with 3 quantities, each of 10 ml, of water R, centrifuging each time. Combine the supernatant liquid and the washings and dilute to 100 ml with water R. To 25 ml


Carmellose sodium


add 6 ml of dilute nitric acid R and dilute to 50 ml with water R. Dilute 10 ml of the solution to 15 ml with water R. Sulphates (2.4.13) : maximum 1 per cent. Heat 20 ml of solution S with 1 ml of hydrochloric acid R on a water-bath until a flocculent precipitate is produced. Cool, centrifuge and separate the supernatant liquid. Wash the precipitate with 3 quantities, each of 10 ml, of distilled water R, centrifuging each time. Combine the supernatant liquid and the washings and dilute to 100 ml with distilled water R. To 25 ml add 1 ml of dilute hydrochloric acid R and dilute to 50 ml with distilled water R. Heavy metals (2.4.8) : maximum 20 ppm. 1.0 g complies with limit test D. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32) : maximum 10.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 4 h. Sulphated ash (2.4.14) : 10.0 per cent to 20.0 per cent, determined on 1.0 g in a platinum crucible. STORAGE In an airtight container.

CARMELLOSE

Apparent viscosity. While stirring, introduce a quantity of the substance to be examined equivalent to 2.00 g of the dried substance into 50 ml of water R heated to 90 °C. For a product of low viscosity, use if necessary, the quantity required to give the concentration indicated on the label. Allow to cool, dilute to 100.0 ml with water R and stir until dissolution is complete. Determine the viscosity (2.2.10) using a rotating viscometer at 20 °C and a shear rate of 10 s− 1. If it is impossible to obtain a shear rate of exactly 10 s− 1, use a shear rate slightly higher and a rate slightly lower and interpolate. The apparent viscosity is not less than 75 per cent and not more than 140 per cent of the value stated on the label. Sodium glycollate. Place a quantity of the substance to be examined equivalent to 0.500 g of dried substance in a beaker. Add 5 ml of acetic acid R and 5 ml of water R. Stir until dissolution is complete (about 30 min). Add 80 ml of acetone R and 2 g of sodium chloride R. Filter through a fast filter paper impregnated with acetone R into a volumetric flask, rinse the beaker and filter with acetone R and dilute the filtrate to 100.0 ml with the same solvent. Allow to stand for 24 h without shaking. Use the clear supernatant liquid to prepare the test solution.

In a volumetric flask, dissolve 0.310 g of glycollic acid R, previously dried in vacuo over diphosphorus pentoxide R, in water R and dilute to 1000.0 ml with the same solvent. 01/2005:0472 Place 5.0 ml of this solution in a volumetric flask, add 5 ml of acetic acid R and allow to stand for about 30 min. Add 80 ml of acetone R and 2 g of sodium chloride R and dilute SODIUM to 100.0 ml with acetone R. Use this solution to prepare the reference solution.

Carmellosum natricum

DEFINITION Carmellose sodium (carboxymethylcellulose sodium) is the sodium salt of a partly O-carboxymethylated cellulose. It contains not less than 6.5 per cent and not more than 10.8 per cent of sodium (Na), calculated with reference to the dried substance. CHARACTERS A white or almost white, granular powder, hygroscopic after drying, practically insoluble in acetone, in ethanol and in toluene. It is easily dispersed in water giving colloidal solutions.

Place 2.0 ml of each solution in a separate 25 ml volumetric flask. Heat on a water-bath to eliminate acetone. Cool to room temperature and add 5.0 ml of 2,7-dihydroxynaphthalene solution R to each flask. Shake and add 15.0 ml of 2,7-dihydroxynaphthalene solution R. Close the flasks with aluminium foil and heat on a water-bath for 20 min. Cool under running water and dilute to 25.0 ml with sulphuric acid R. Within 10 min, transfer 10.0 ml of each solution to a flat-bottomed tube. Examine the solutions viewing vertically. The test solution is not more intensely coloured than the reference solution (0.4 per cent). Chlorides (2.4.4). Dilute 2 ml of solution S to 15 ml with water R. The solution complies with the limit test for chlorides (0.25 per cent).

IDENTIFICATION A. To 10 ml of solution S (see Tests) add 1 ml of copper sulphate solution R. A blue, cotton-like precipitate is formed. B. Boil 5 ml of solution S for a few minutes. No precipitate is formed. C. The solution prepared from the sulphated ash in the test for heavy metals gives the reactions of sodium (2.3.1).

Heavy metals (2.4.8). To the residue obtained in the determination of the sulphated ash, add 1 ml of hydrochloric acid R and evaporate on a water-bath. Take up the residue in 20 ml of water R. 12 ml of the solution complies with limit test A for heavy metals (20 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R.

TESTS Solution S. Sprinkle a quantity of the substance to be examined equivalent to 1.0 g of the dried substance onto 90 ml of carbon dioxide-free water R at 40 °C to 50 °C stirring vigorously. Continue stirring until a colloidal solution is obtained, cool and dilute to 100 ml with carbon dioxide-free water R. Appearance of solution. Solution S is not more opalescent than reference suspension III (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). pH (2.2.3). The pH of solution S is 6.0 to 8.0.

Sulphated ash (2.4.14) : 20.0 per cent to 33.3 per cent, determined on 1.0 g using a mixture of equal volumes of sulphuric acid R and water R and calculated with reference to the dried substance. These limits correspond to a content of 6.5 per cent to 10.8 per cent of sodium (Na).



LABELLING The label states the apparent viscosity in millipascal seconds for a 20 g/l solution ; for a product of low viscosity, the label states the concentration of the solution to be used and the apparent viscosity in millipascal seconds. 1189

Carmellose sodium, low-substituted


01/2005:1186 Prepare the reference solutions as follows : in a 100 ml volumetric flask, dissolve 0.100 g of glycollic acid R, previously dried in vacuo over diphosphorus pentoxide R, CARMELLOSE SODIUM, in water R and dilute to 100.0 ml with the same solvent. LOW-SUBSTITUTED Transfer 0.5 ml, 1.0 ml, 1.5 ml and 2.0 ml of the solution to separate volumetric flasks ; dilute the contents of each flask to 5.0 ml with water R, add 5 ml of glacial acetic acid R, Carmellosum natricum, substitutum humile dilute to 100.0 ml with acetone R and mix. DEFINITION Transfer 2.0 ml of the test solution and 2.0 ml of each of the Low-substituted carmellose sodium (sodium carboxymethyl- reference solutions to separate 25 ml volumetric flasks. Heat cellulose) is the sodium salt of a partly O-(carboxymethylated) the uncovered flasks in a water-bath to eliminate the acetone. cellulose. It contains not less than 2.0 per cent and not more Allow to cool and add 5.0 ml of 2,7-dihydroxynaphthalene solution R to each flask. Mix, add a further 15.0 ml of than 4.5 per cent of sodium (Na), calculated with reference 2,7-dihydroxynaphthalene solution R and mix again. Close to the dried substance. the flasks with aluminium foil and heat in a water-bath for CHARACTERS 20 min. Cool and dilute to 25.0 ml with sulphuric acid R. A white or almost white powder or short fibres, practically Measure the absorbance (2.2.25) of each solution at 540 nm. insoluble in acetone, in ethanol and in toluene. It swells in Prepare a blank using 2.0 ml of a solution containing 5 per water to form a gel. cent V/V each of glacial acetic acid R and water R in IDENTIFICATION A. Shake 1 g with 100 ml of a 100 g/l solution of sodium hydroxide R. A suspension is produced. B. Shake 1 g with 50 ml of water R. Transfer 1 ml of the mixture to a test tube, add 1 ml of water R and 0.05 ml of a freshly prepared 40 g/l solution of α-naphthol R in methanol R. Incline the test tube and add carefully 2 ml of sulphuric acid R down the side so that it forms a lower layer. A reddish-purple colour develops at the interface. C. It complies with the test for sulphated ash (2.4.14) (see Tests). D. The solution prepared from the sulphated ash for the test for heavy metals gives reaction (a) of sodium (2.3.1). TESTS pH (2.2.3). Shake 1 g with 100 ml of carbon dioxide-free water R for 5 min. Centrifuge. The pH of the suspension is 6.0 to 8.5. Sodium chloride and sodium glycollate. The sum of the percentages of sodium chloride and sodium glycollate is not more than 0.5 per cent, calculated with reference to the dried substance. Sodium chloride. Place 5.00 g in a 250 ml conical flask, add 50 ml of water R and 5 ml of strong hydrogen peroxide solution R and heat on a water bath for 20 min, stirring occasionally to ensure total hydration. Cool, add 100 ml of water R and 10 ml of nitric acid R. Titrate with 0.05 M silver nitrate determining the end-point potentiometrically (2.2.20) using a silver-based indicator electrode and a double-junction reference electrode containing a 100 g/l solution of potassium nitrate R in the outer jacket and a standard filling solution in the inner jacket. 1 ml of 0.05 M silver nitrate is equivalent to 2.922 mg of NaCl. Sodium glycollate. Place a quantity of the substance to be examined equivalent to 0.500 g of the dried substance in a beaker. Add 5 ml of glacial acetic acid R and 5 ml of water R and stir to ensure total hydration (about 30 min). Add 80 ml of acetone R and 2 g of sodium chloride R. Stir for several minutes to ensure complete precipitation of the carboxymethylcellulose. Filter through a fast filter paper impregnated with acetone R into a volumetric flask, rinse the beaker and filter with acetone R and dilute the filtrate to 100.0 ml with the same solvent. Allow to stand for 24 h without shaking. Use the clear supernatant as the test solution. 1190

acetone R. Prepare a standard curve using the absorbances obtained with the reference solutions. From the standard curve and the absorbance of the test solution, determine the mass a, in milligrams, of glycollic acid in the substance to be examined and calculate the content of sodium glycollate from the formula :

1.29 =

the factor converting glycollic acid to sodium glycollate,

b m

=

the loss on drying as a percentage,

=

the mass of the substance to be examined, in grams.

Water-soluble substances. Not more than 70.0 per cent. Disperse 5.00 g in 400.0 ml of water R and stir for 1 min every 10 min during the first 30 min. Allow to stand for 1 h and centrifuge, if necessary. Decant 100.0 ml of the supernatant liquid onto a fast filter paper in a vacuum filtration funnel, apply vacuum and collect 75.0 ml of the filtrate. Evaporate to dryness and dry the residue at 100-105 °C for 4 h. Heavy metals (2.4.8). To the residue obtained in the determination of the sulphated ash add 1 ml of hydrochloric acid R and evaporate on a water-bath. Take up the residue in 20 ml of water R. 12 ml of the solution complies with limit test A for heavy metals (20 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32). Not more than 10.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14) : 6.5 per cent to 13.5 per cent, determined on 1.0 g using a mixture of equal volumes of sulphuric acid R and water R and calculated with reference to the dried substance. These limits correspond to a content of 2.0 per cent to 4.5 per cent of Na. The following test concerning the pharmaco-technological properties may be carried out depending on the intended formulation. It is not a mandatory requirement. Settling volume. In a 100 ml graduated cylinder, place 20 ml of 2-propanol R, add 5.0 g of the substance to be examined and shake vigorously. Dilute to 30 ml with 2-propanol R then to 50 ml with water R and shake vigorously. Within 15 min, repeat the shaking three times. Allow to stand for 4 h and determine the volume of the settled mass (15.0 ml to 35.0 ml).


Carnauba wax


CARMUSTINE

01/2005:1187 STORAGE Store in an airtight container, protected from light, at a temperature of 2 °C to 8 °C. IMPURITIES

Carmustinum

A. 1,3-bis(2-chloroethyl)urea. C5H9Cl2N3O2

Mr 214.1

DEFINITION Carmustine contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of 1,3-bis(2-chloroethyl)-1-nitrosourea, calculated with reference to the anhydrous substance.

01/2005:0597

CARNAUBA WAX Cera carnauba

CHARACTERS A yellowish, granular powder, very slightly soluble in water, DEFINITION very soluble in methylene chloride, freely soluble in ethanol. Purified wax obtained from the leaves of Copernicia cerifera Mart. It melts at about 31 °C with decomposition. IDENTIFICATION Examine by infrared absorption spectrophotometry(2.2.24), comparing with the Ph. Eur. reference spectrum of carmustine. Examine the melted substances prepared as films. TESTS 1,3-bis(2-chloroethyl)urea (impurity A). Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating substance. Test solution. Dissolve 0.10 g of the substance to be examined in methylene chloride R and dilute to 5 ml with the same solvent. Reference solution (a). Dissolve 2 mg of carmustine impurity A CRS in methylene chloride R and dilute to 10 ml with the same solvent. Reference solution (b). Dilute 1 ml of the test solution to 10 ml with methylene chloride R. To 5 ml of this solution, add 5 ml of reference solution (a). Apply separately to the plate 2 µl of each solution. Develop over a path of 10 cm using a mixture of 10 volumes of methanol R and 90 volumes of methylene chloride R. Allow the plate to dry in air. Spray with diethylamine R and heat at 125 °C for 10 min. Allow to cool and spray with silver nitrate solution R2. Expose to ultraviolet light at 365 nm until brown to black spots appear. Any spot corresponding to carmustine impurity A in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) (1 per cent). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. Water (2.5.12). Not more than 1.0 per cent, determined on 0.50 g by the semi-micro determination of water.

CHARACTERS Appearance : pale yellow or yellow powder, flakes or hard masses. Solubility : practically insoluble in water, soluble on heating in ethyl acetate and in xylene, practically insoluble in alcohol. Relative density : about 0.97.

IDENTIFICATION Thin-layer chromatography (2.2.27). Test solution. Dissolve 0.10 g of the substance to be examined with heating in 5 ml of chloroform R. Use the warm solution. Reference solution. Dissolve 5 mg of menthol R, 5 µl of menthyl acetate R and 5 mg of thymol R in 10 ml of toluene R. Plate : TLC silica gel plate R. Mobile phase : ethyl acetate R, chloroform R (2:98 V/V). Application : 30 µl of the test solution and 10 µl of the reference solution as bands 20 mm by 3 mm. Development : over half of the plate. Drying : in air. Detection : spray with a freshly prepared 200 g/l solution of phosphomolybdic acid R in alcohol R (about 10 ml for a 20 cm plate). Heat at 100-105 °C for 10-15 min. Results : the chromatogram obtained with the reference solution shows in the lower part a dark blue zone (menthol), above this zone a reddish zone (thymol) and in the upper part a dark blue zone (menthyl acetate). The chromatogram obtained with the test solution shows a large blue zone (triacontanol = melissyl alcohol) at a level between the thymol and menthol zones in the chromatogram obtained with the reference solution. Further blue zones are visible in the upper part of the chromatogram obtained with the ASSAY test solution, at levels between those of the menthyl acetate Dissolve 0.100 g in 30 ml of ethanol R and dilute to 100.0 ml and thymol zones in the chromatogram obtained with the with water R. Dilute 3.0 ml of the solution to 100.0 ml with reference solution ; above these zones further zones are water R. Measure the absorbance (2.2.25) at the maximum visible in the chromatogram obtained with the test solution ; at 230 nm. the zone with the highest Rf value is very pronounced. A Calculate the content of C5H9Cl2N3O2 taking the specific number of faint zones are visible below the triacontanol zone and the starting point is coloured blue. absorbance to be 270. General Notices (1) apply to all monographs and other texts

1191

Carteolol hydrochloride


TESTS Appearance of solution. The solution is clear (2.2.1) and not more intensely coloured than a 50 mg/l solution of potassium dichromate R (2.2.2, Method II). Dissolve 0.10 g with heating in chloroform R and dilute to 10 ml with the same solvent. Melting point (2.2.15) : 80 °C to 88 °C. Melt the substance to be examined carefully on a water-bath before introduction into the capillary tubes. Allow the tubes to stand in the refrigerator for 24 h or at 0 °C for 2 h. Acid value : 2 to 7. To 2.000 g (m g) in a 250 ml conical flask fitted with a reflux condenser add 40 ml of xylene R and a few glass beads. Heat with stirring until the substance is completely dissolved. Add 20 ml of alcohol R and 1 ml of bromothymol blue solution R3 and titrate the hot solution with 0.5 M alcoholic potassium hydroxide until a green colour persisting for at least 10 s is obtained (n1 ml). Carry out a blank test (n2 ml). Calculate the acid value from the expression :

Saponification value : 78 to 95. To 2.000 g (m g) in a 250 ml conical flask fitted with a reflux condenser add 40 ml of xylene R and a few glass beads. Heat with stirring until the substance is completely dissolved. Add 20 ml of alcohol R and 20.0 ml of 0.5 M alcoholic potassium hydroxide. Boil under a reflux condenser for 3 h. Add 1 ml of phenolphthalein solution R1 and titrate the hot solution immediately with 0.5 M hydrochloric acid until the red colour disappears. Repeat the heating and titration until the colour no longer reappears on heating (n3 ml). Carry out a blank test (n4 ml). Calculate the saponification value from the expression :

Total ash (2.4.16) : maximum 0.25 per cent, determined on 2.0 g. STORAGE Protected from light. 01/2005:1972

CARTEOLOL HYDROCHLORIDE Carteololi hydrochloridum

C16H25N2O3Cl

Mr 328.8

DEFINITION 5-[(2RS)-3-[(1,1-Dimethylethyl)amino]-2-hydroxypropoxy]-3,4dihydroquinolin-2(1H)-one hydrochloride. Content : 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white crystals or crystalline powder. 1192

Solubility : soluble in water, sparingly soluble in methanol, slightly soluble in alcohol, practically insoluble in methylene chloride. IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of carteolol hydrochloride. B. It gives reaction (a) of chlorides (2.3.1). TESTS Appearance of solution. The solution is clear (2.2.1) and colourless (2.2.2, Method II). Dissolve 0.300 g in water R and dilute to 10 ml with the same solvent. pH (2.2.3) : 5.0 to 6.0. Dissolve 0.250 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 20.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 10.0 ml with the mobile phase. Reference solution (c). Dissolve 10 mg of carteolol for system suitability CRS in the mobile phase and dilute to 5 ml with the mobile phase. Reference solution (d). Dilute 5.0 ml of reference solution (b) to 10.0 ml with the mobile phase. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : mix 1 volume of methanol R2, 20 volumes of acetonitrile R and 79 volumes of a 2.82 g/l solution of sodium hexanesulphonate R. Flow rate : 1 ml/min. Detection : spectrophotometer at 252 nm. Injection : 20 µl. System suitability : — the chromatogram obtained with reference solution (c) is similar to the chromatogram provided with carteolol for system suitability CRS ; the peaks due to impurity H and carteolol show base-line separation, — signal-to-noise ratio : minimum 10 for the principal peak in the chromatogram obtained with reference solution (d), — number of theoretical plates: minimum 6000, calculated for the principal peak in the chromatogram obtained with reference solution (a). Limits : locate impurity H by comparison with the chromatogram provided with carteolol for system suitability CRS, — impurity H : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (0.2 per cent), — any other impurity : not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent), — total : not more than half the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent),


Carvedilol


— disregard limit : 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.02 per cent). Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 3 h. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined E. 5,5′-[(2-hydroxypropan-1,3-diyl)bis(oxy)]bis(3,4on 1.0 g. dihydroquinolin-2(1H)-one), ASSAY Dissolve 0.250 g in 60 ml of alcohol R. Add 5.0 ml of 0.01 M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the 2 points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 32.88 mg of C16H25N2O3Cl.

H. 5-[(2RS)-3-[(1,1-dimethylethyl)amino]-2hydroxypropoxy]quinolin-2(1H)-one.

STORAGE In an airtight container.

01/2005:1745

CARVEDILOL

IMPURITIES

Carvedilolum

A. 4,6,7,8-tetrahydroquinoline-2,5(1H,3H)-dione, C24H26N2O4

B. 5-hydroxy-3,4-dihydroquinolin-2(1H)-one,

C. 5-[[(2RS)-oxiran-2-yl]methoxy]-3,4-dihydroquinolin-2(1H)one,

D. F. G. I.

Mr 406.5

DEFINITION (2RS)-1-(9H-Carbazol-4-yloxy)-3-[[2-(2-methoxyphenoxy)ethyl]amino]propan-2-ol. Content : 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white or almost white, crystalline powder. Solubility : practically insoluble in water, slightly soluble in alcohol, practically insoluble in dilute acids. It shows polymorphism. IDENTIFICATION Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of carvedilol. If the spectrum obtained shows differences, dissolve the substance to be examined in 2-propanol R, evaporate to dryness and record a new spectrum using the residue.

TESTS Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the mobile phase. R = Cl, R′ = H : 5-[(2RS)-3-chloro-2-hydroxypropoxy]-3,4Reference solution (a). Dilute 1.0 ml of the test solution dihydroquinolin-2(1H)-one, to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. R = OCH3, R′ = H : 5-[(2RS)-2-hydroxy-3-methoxypropoxy]Reference solution (b). Dissolve 5.0 mg of carvedilol 3,4-dihydroquinolin-2(1H)-one, impurity C CRS in 5.0 ml of the test solution and dilute to 100.0 ml with the mobile phase. R = OH, R′ = H : 5-[(2RS)-2,3-dihydroxypropoxy]-3,4Reference solution (c). Dilute 1.0 ml of reference solution (b) dihydroquinolin-2(1H)-one, to 100.0 ml with the mobile phase. Dilute 2.0 ml of this solution to 10.0 ml with the mobile phase. R = NH-C(CH3)3, R′ = Br : 7-bromo-5-[(2RS)-3-[(1, Column : 1-(dimethylethyl)amino]-2-hydroxypropoxy]-3,4— size : l = 0.125 m, Ø = 4.6 mm, dihydroquinolin-2(1H)-one,


1193

Cascara


— stationary phase: octylsilyl silica gel for chromatography R (5 µm), — temperature : 55 °C. Mobile phase : dissolve 1.77 g of potassium dihydrogen phosphate R in water R and dilute to 650 ml with the same solvent ; adjust to pH 2.0 with phosphoric acid R and add 350 ml of acetonitrile R. Flow rate : 1.0 ml/min. Detection : spectrophotometer at 240 nm. Injection : 20 µl. Run time : 8 times the retention time of carvedilol. Relative retention with reference to carvedilol (retention time = about 4 min) : impurity A = about 0.6 ; impurity C = about 3.5 ; impurity B = about 6.7. System suitability : reference solution (b) : — resolution : minimum 17 between the peaks due to carvedilol and to impurity C. Limits : — correction factor : for the calculation of content, multiply the peak area of impurity A by 2, — impurity A : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent), — impurity C : not more than twice the area of the corresponding peak in the chromatogram obtained with reference solution (c) (0.02 per cent), — any other impurity : not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), — total: not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent), — disregard limit: the area of the principal peak in the chromatogram obtained with reference solution (c) (0.01 per cent). Heavy metals (2.4.8) : maximum 10 ppm. 2.0 g complies with limit test C. Prepare the standard using 2.0 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g.

B. 1,1′-[[2-(2-methoxyphenoxy)ethyl]nitrilo]bis[3-(9Hcarbazol-4-yloxy)propan-2-ol],

C. (2RS)-1-[benzyl[2-(2-methoxyphenoxy)ethyl]amino]-3-(9Hcarbazol-4-yloxy)propan-2-ol. 01/2005:0105

CASCARA Rhamni purshianae cortex DEFINITION Cascara consists of the dried, whole or fragmented bark of Rhamnus purshianus D.C. (Frangula purshiana (D.C.) A. Gray ex J. C. Cooper). It contains not less than 8.0 per cent of hydroxyanthracene glycosides of which not less than 60 per cent consists of cascarosides, both expressed as cascaroside A (C27H32O14 ; Mr 580.5) and calculated with reference to the dried drug. CHARACTERS It has the macroscopic and microscopic characters described under identification tests A and B.

IDENTIFICATION ASSAY Dissolve 0.350 g in 60 ml of anhydrous acetic acid R. Titrate A. The bark occurs in slightly channelled or nearly flat pieces, usually 1 mm to 5 mm in thickness, usually with 0.1 M perchloric acid, determining the end-point varying greatly in length and width. The outer surface is potentiometrically (2.2.20). grey or dark greyish-brown and shows occasional lenticels 1 ml of 0.1 M perchloric acid is equivalent to 40.65 mg of that are orientated transversally. It is usually more or less C24H26N2O4. completely covered by a whitish coat of lichens, epiphytic IMPURITIES moss and foliaceous liverwort. The inner surface is yellow to reddish-brown or almost black with fine longitudinal striations ; it turns red when treated with alkali. The yellow fracture is short and granular in the outer part and somewhat fibrous at the inner part. B. Reduce to a powder (355). The powder is yellowish-brown. Examine under a microscope using chloral hydrate solution R. The powder shows : bundles of partly lignified phloem fibres accompanied by crystal sheaths containing prisms of calcium oxalate ; groups of sclereids accompanied by crystal sheaths ; cluster crystals of calcium oxalate ; some parenchymatous cells contain a yellow substance which becomes deep red when treated A. 1-[[9-[2-hydroxy-3-[[2-(2-methoxyphenoxy)ethyl]amino]prowith alkali ; cork cells and, frequently, epiphytes ; the pyl]-9H-carbazol-4-yl]oxy]-3-[[2-(2-methoxyphenoxy)ethlatter may be liverworts, entire or in fragments, having a yl]amino]propan-2-ol, 1194



lamina one cell thick without a midrib and composed of isodiametric cells, or leaves of mosses, having a lamina one cell thick composed of elongated cells and possessing a midrib several cells thick. C. Examine the chromatograms obtained in the test for “Other species of Rhamnus ; anthrones” after spraying with a 50 g/l solution of potassium hydroxide R in alcohol (50 per cent V/V) R and heating. The chromatogram obtained with the test solution shows several reddish-brown zones with different intensities : there are four faint zones, three being situated at about the mid-point of the chromatogram and one in the lower third and there is a strong zone in the upper third of the chromatogram. Examine in ultraviolet light at 365 nm. The chromatogram obtained with the test solution shows several zones with the same fluorescence, situated above and particularly below (cascarosides) that corresponding to barbaloin in the chromatogram obtained with the reference solution. D. Heat 0.2 g of the powdered drug (180) with 50 ml of water R on a water-bath for 15 min. Allow to cool and filter. To 10 ml of the filtrate add 20 ml of hydrochloric acid R1 and heat on a water-bath for 15 min. Allow to cool, transfer to a separating funnel and shake with three quantities, each of 20 ml, of ether R. Reserve the aqueous layer (solution A). (a) Combine the three ether extracts and shake with 10 ml of dilute ammonia R2. The aqueous layer becomes reddish-violet. (b) Transfer solution A to a small flask, add 5 g of ferric chloride R and heat on a water-bath for 30 min. Allow to cool, transfer to a separating funnel and shake with 15 ml of ether R. Wash the ether layer with 10 ml of water R, discard the water and shake the ether layer with 5 ml of dilute ammonia R2. A red colour develops in the aqueous layer.

Cascara

solution of nitrotetrazolium blue R in methanol R. Examine the chromatogram immediately. No violet or greyish-blue zones appear. Foreign matter (2.8.2). Not more than 1 per cent. Loss on drying (2.2.32). Not more than 10.0 per cent, determined on 1.000 g of the powdered drug (180) by drying in an oven at 100 °C to 105 °C for 2 h. Total ash (2.4.16). Not more than 7.0 per cent. ASSAY Carry out the assay protected from bright light in one day. Stir 1.00 g of the powdered drug (180) into 100 ml of boiling water R and continue boiling and stirring for 5 min. Allow to cool, dilute to 100.0 ml with water R, shake, filter and discard the first 20 ml of filtrate. Transfer 10.0 ml of the filtrate to a separating funnel, add 0.1 ml of 1 M hydrochloric acid and shake with two quantities, each of 20 ml, of a mixture of 1 volume of ether R and 3 volumes of hexane R. Wash the combined organic extracts with 5 ml of water R, discard the organic layer and return the rinsings to the aqueous layer. Shake the combined aqueous layers with four quantities, each of 30 ml, of ethyl acetate R freshly saturated with water R, (to 150 ml of ethyl acetate R add 15 ml of water R, shake for 3 min and allow to stand) on each occasion allowing separation to take place until the organic layer is clear. Combine the ethyl acetate extracts. Use the aqueous layer for the assay for cascarosides and the organic layer for the assay for hydroxyanthracene glycosides other than cascarosides.

Hydroxyanthracene glycosides other than cascarosides. Transfer the organic layer to a suitable flask and remove the solvent by distillation, evaporating almost to dryness. Dissolve the residue in 0.3 ml to 0.5 ml of methanol R and transfer to a volumetric flask, rinsing the first flask with warm water R and adding the rinsings to the methanolic solution. Allow to cool and dilute to 50.0 ml with water R. Transfer 20.0 ml of the solution to a 100 ml round-bottomed TESTS flask with a ground-glass neck and containing 2 g of ferric chloride R and 12 ml of hydrochloric acid R. Attach a reflux Other species of Rhamnus ; anthrones. Examine by condenser and place the flask in a water-bath so that the thin-layer chromatography (2.2.27), using a suitable silica level of the water is above that of the liquid in the flask gel as the coating substance. and heat for 4 h. Allow to cool, transfer the solution to Test solution. To 0.5 g of the powdered drug (180) add 5 ml a separating funnel and rinse the flask successively with of alcohol (70 per cent V/V) R and heat to boiling. Cool and 3 ml to 4 ml of 1 M sodium hydroxide and 3 ml to 4 ml of centrifuge. Decant the supernatant solution immediately water R, adding the rinsings to the separating funnel. Shake and use within 30 min. the contents of the separating funnel with three quantities, Reference solution. Dissolve 20 mg of barbaloin R in each of 30 ml, of a mixture of 1 volume of ether R and alcohol (70 per cent V/V) R and dilute to 10 ml with the 3 volumes of hexane R. Wash the combined organic layers same solvent. with two quantities, each of 10 ml, of water R and discard Apply separately to the plate as bands 10 µl of each solution. the rinsings. Dilute the organic phase to 100.0 ml with Develop over a path of 10 cm using a mixture of 13 volumes the mixture of ether and hexane. Take 20.0 ml, evaporate carefully to dryness on a water-bath and dissolve the residue of water R, 17 volumes of methanol R and 100 volumes of ethyl acetate R. Allow the plate to dry for 5 min, spray with in 10.0 ml of a 5 g/l solution of magnesium acetate R in about 10 ml of a 50 g/l solution of potassium hydroxide R methanol R. Measure the absorbance (2.2.25) at 515 nm in alcohol (50 per cent V/V) R and heat at 100 °C to 105 °C using methanol R as the compensation liquid. Calculate the percentage content of hydroxyanthracene for 15 min. Examine the chromatograms immediately after glycosides, expressed as cascaroside A, from the expression : heating. The chromatogram obtained with the reference solution shows, in the central part, a reddish-brown zone corresponding to barbaloin. Examine in ultraviolet light at 365 nm. The zone corresponding to barbaloin shows intense yellowish-brown fluorescence. In the chromatogram i.e. taking the specific absorbance to be 180. obtained with the test solution, no zone with orange-brown A = absorbance at 515 nm, fluorescence is seen between the zone of barbaloin and the m = mass of the sample in grams. zones of cascarosides. Apply to another plate as a band 10 µl of the test solution Measure the absorbance of the test solution at 440 nm. If and develop as described above. Allow the plate to dry for the ratio of the absorbance at 515 nm to that at 440 nm is not longer than 5 min and spray immediately with a 5 g/l less than 2.4, the assay is not valid and must be repeated. General Notices (1) apply to all monographs and other texts

1195

Cassia oil


Cascarosides. Dilute the aqueous layer reserved for this assay to 50.0 ml with water R. Treat 20.0 ml of the solution as described above in the assay of hydroxyanthracene glycosides other than cascarosides. Measure the absorbance of the test solution at 440 nm. If the ratio of the absorbance at 515 nm to that at 440 nm is less than 2.4, the assay is not valid and must be repeated. Calculate the percentage content of cascarosides, expressed as cascaroside A from the expression :

with the reference solution and may show a very faint zone corresponding to eugenol. Other faint zones are present. B. Examine the chromatograms obtained in the test for chromatographic profile. The retention times of the principal peaks in the chromatogram obtained with the test solution are similar to those in the chromatogram obtained with the reference solution. Eugenol may be absent from the chromatogram obtained with the test solution.

TESTS Relative density (2.2.5) : 1.052 to 1.070. i.e. taking the specific absorbance to be 180. Refractive index (2.2.6) : 1.600 to 1.614. A = absorbance at 515 nm, Optical rotation (2.2.7) : − 1° to + 1°. m = mass of the sample in grams. Chromatographic profile. Examine by gas chromatography Measure the absorbance of the test solution at 440 nm. If (2.2.28). the ratio of the absorbance at 515 nm to that at 440 nm is Test solution. The substance to be examined. less than 2.7, the assay is not valid and must be repeated. Reference solution. Dissolve 100 µl of trans-cinnamic aldehyde R, 10 µl of cinnamyl acetate R, 10 µl STORAGE of eugenol R, 20 mg of coumarin R and 10 µl of Store protected from light. trans-2-methoxycinnamaldehyde R in 1 ml of acetone R. The chromatographic procedure may be carried out using : 01/2005:1496 — a fused-silica column 60 m long and about 0.25 mm in internal diameter coated with macrogol 20 000 R as the bonded phase, CASSIA OIL — helium for chromatography R as the carrier gas at a flow rate of 1.5 ml/min, Cinnamomi cassiae aetheroleum — a flame-ionisation detector, DEFINITION — a split ratio of 1:100, Cassia oil is obtained by steam distillation of the leaves with the following temperature programme : and young branches of Cinnamomum cassia Blume Time Temperature Rate Comment (C. aromaticum Nees). CHARACTERS A clear, mobile, yellow to reddish-brown liquid, with a characteristic odour reminiscent of cinnamic aldehyde.

Column

IDENTIFICATION First identification : B. Second identification : A. A. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R. Test solution. Dissolve 0.5 ml of the substance to be examined in acetone R and dilute to 10 ml with the same solvent. Reference solution. Dissolve 50 µl of trans-cinnamic aldehyde R, 10 µl of eugenol R and 50 mg of coumarin R in acetone R and dilute to 10 ml with the same solvent. Apply to the plate, as bands, 10 µl of each solution. Develop over a path of 15 cm using a mixture of 10 volumes of methanol R and 90 volumes of toluene R. Allow the plate to dry in air and examine in ultraviolet light at 365 nm. The zone of blue fluorescence in the chromatogram obtained with the test solution is similar in position and colour to the zone in the chromatogram obtained with the reference solution (coumarin). Spray the plate with anisaldehyde solution R. Examine in daylight while heating at 100-105 °C for 5-10 min. The chromatogram obtained with the reference solution shows in its upper part a violet zone (eugenol) and above this zone a greenish-blue zone (trans-cinnamic aldehyde). The chromatogram obtained with the test solution shows a zone similar in position and colour to the zone due to trans-cinnamic aldehyde in the chromatogram obtained 1196

(min)

(°C)

0 - 10

60

(°C/min) –

isothermal

10 - 75

60 → 190

2

linear gradient

75 - 160

190

Injection port

200

Detector

240

isothermal

Inject 0.2 µl of the reference solution. When the chromatogram is recorded in the prescribed conditions, the components elute in the order indicated in the composition of the reference solution. Depending on the operating conditions and the state of the column, coumarin may elute before or after trans-2-methoxycinnamaldehyde. Record the retention times of these substances. The test is not valid unless the resolution between the peaks corresponding to coumarin and trans-2-methoxycinnamaldehyde is at least 1.5. Inject 0.2 µl of the test solution. Using the retention times determined from the chromatogram obtained with the reference solution, locate the components of the reference solution in the chromatogram obtained with the test solution. Determine the percentage content of each of these components by the normalisation procedure. The percentages are within the following ranges : — trans-cinnamic aldehyde : 70 per cent to 90 per cent, — cinnamyl acetate : 1.0 per cent to 6.0 per cent, — eugenol : less than 0.5 per cent, — coumarin : 1.5 per cent to 4.0 per cent, — trans-2-methoxycinnamaldehyde : 3.0 per cent to 15 per cent.


Castor oil, virgin


STORAGE Store in a well-filled, airtight container, protected from light and heat. 01/2005:1497 corrected

CASTOR OIL, HYDROGENATED

Inject 1 µl of each solution. Calculate the fraction of each fatty acid using the following expression :

Ax,s,c =

corrected peak area of the fatty acid in the test solution :

Ricini oleum hydrogenatum Rc DEFINITION Hydrogenated castor oil is the oil obtained by hydrogenation of Virgin Castor oil (0051). It consists mainly of the triglyceride of 12-hydroxystearic acid. CHARACTERS A fine, almost white or pale yellow powder or almost white or Rc pale yellow masses or flakes, practically insoluble in water, freely soluble in methylene chloride, slightly soluble in light m 1, petroleum, very slightly soluble in ethanol.

=

relative correction factor for the peak corresponding to methyl 12-hydroxystearate :

=

1 for peaks corresponding to each of the other specified fatty acids or any unspecified fatty acid,

=

mass of methyl 12-hydroxystearate in the reference solution,

=

mass of methyl stearate in the reference solution,

r

IDENTIFICATION A. Melting point (2.2.14) : 83 °C to 88 °C. B. It complies with the test for hydroxyl value (see Tests). C. It complies with the test for composition of fatty acids (see Tests).

m2, A1,r

=

area of any peak corresponding to methyl 12-hydroxystearate in the chromatogram obtained with the reference solution,

TESTS Acid value (2.5.1). Not more than 4.0, determined on 10.0 g dissolved in 75 ml of hot alcohol R. Hydroxyl value (2.5.3, Method A) : 145 to 165, determined on a warm solution. Iodine value (2.5.4). Not more than 5.0. Alkaline impurities. Dissolve 1.0 g by gentle heating in a mixture of 1.5 ml of alcohol R and 3 ml of toluene R. Add 0.05 ml of a 0.4 g/l solution of bromophenol blue R in alcohol R. Not more than 0.2 ml of 0.01 M hydrochloric acid is required to change the colour of the indicator to yellow. Composition of fatty acids. Gas chromatography (2.4.22) with the following modifications. Use the mixture of calibrating substances in Table 2.4.22.-3. Test solution. Introduce 75 mg of the substance to be examined in a 10 ml centrifuge tube with a screw cap. Dissolve in 2 ml of 1,1-dimethylethyl methyl ether R1 by shaking and heat gently (50-60 °C). Add, when still warm, 1 ml of a 12 g/l solution of sodium R in anhydrous methanol R, prepared with the necessary precautions, and mix vigorously for at least 5 min. Add 5 ml of distilled water R and mix vigorously for about 30 s. Centrifuge for 15 min at 1500 g. Use the upper layer. Reference solution. Dissolve 50 mg of methyl 12-hydroxystearate CRS and 50 mg of methyl stearate CRS in 10.0 ml of 1,1-dimethylethyl methyl ether R1. The chromatographic procedure may be carried out using : — a fused-silica column 30 m long and 0.25 mm in internal diameter coated with macrogol 20 000 R (film thickness 0.25 µm), — helium for chromatography R as the carrier gas at a flow rate of 0.9 ml/min, — a flame-ionisation detector, — a split injector (1:100), maintaining the temperature of the column at 215 °C for 55 min and maintaining the temperature of the injection port and that of the detector at 250 °C.

A2,r

=

Ax,s

=

area of any peak corresponding to methyl stearate in the chromatogram obtained with the reference solution, area of the peaks corresponding to any specified or unspecified fatty acid methyl esters.


r

The fatty acid fraction of the oil has the following composition : — palmitic acid : not more than 2.0 per cent, — stearic acid : 7.0 per cent to 14.0 per cent, — arachidic acid : not more than 1.0 per cent, — 12-oxostearic acid (equivalent chain length on macrogol 20 000 : 22.7) : not more than 5.0 per cent, — 12-hydroxystearic acid (equivalent chain length on macrogol 20 000 : 23.9) : 78.0 per cent to 91.0 per cent, — any other fatty acid : not more than 3.0 per cent. Nickel (2.4.27). Not more than 1 ppm of Ni. STORAGE Store in a well-filled container. IMPURITIES

A. 12-oxostearic acid. 01/2005:0051 corrected

CASTOR OIL, VIRGIN Ricini oleum virginale DEFINITION Fatty oil obtained by cold expression from the seeds of Ricinus communis L. A suitable antioxidant may be added. 1197

Cefaclor


CHARACTERS Appearance : clear, almost colourless or slightly yellow, viscous, hygroscopic liquid. Solubility : slightly soluble in light petroleum, miscible with alcohol and with glacial acetic acid. Relative density : about 0.958. Refractive index : about 1.479. IDENTIFICATION First identification : D. Second identification : A, B, C. A. It complies with the test for optical rotation (see Tests). B. It complies with the test for hydroxyl value (see Tests). C. Iodine value (2.5.4) : 82 to 90. D. It complies with the test for composition of fatty acids (see Tests). TESTS Optical rotation (2.2.7) : + 3.5° to + 6.0°. Specific absorbance (2.2.25) : maximum 1.0, determined at the absorption maximum at 269 nm ± 1 nm. To 1.0 g add alcohol R and dilute to 100.0 ml with the same solvent. Acid value (2.5.1) : maximum 2.0. Dissolve 5.0 g in 25 ml of the prescribed mixture of solvents. Hydroxyl value (2.5.3, Method A) : minimum 150. Peroxide value (2.5.5) : maximum 10.0. Unsaponifiable matter (2.5.7) : maximum 0.8 per cent, determined on 5.0 g. Composition of fatty acids. Gas chromatography (2.4.22) with the following modifications. Use the mixture of calibrating substances in Table 2.4.22.-3. Test solution. Introduce 75 mg of the substance to be examined into a 10 ml centrifuge tube with a screw cap. Dissolve in 2 ml of 1,1-dimethylethyl methyl ether R1 with shaking and heat gently (50-60 °C). Add, when still warm, 1 ml of a 12 g/l solution of sodium R in anhydrous methanol R, prepared with the necessary precautions, and mix vigorously for at least 5 min. Add 5 ml of distilled water R and mix vigorously for about 30 s. Centrifuge for 15 min at 1500 g. Use the upper layer. Reference solution. Dissolve 50 mg of methyl ricinoleate CRS and 50 mg of methyl stearate CRS in 10.0 ml of 1,1-dimethylethyl methyl ether R1. Column : — material : fused silica, — size : l = 30 m, Ø = 0.25 mm, — stationary phase: macrogol 20 000 R (film thickness 0.25 µm). Carrier gas : helium for chromatography R. Flow rate : 0.9 ml/min. Split ratio : 1:100. Temperature :

Column

Time (min)

Temperature (°C)

0 - 55

215

Injection port

250

Detector

250

Detection : flame ionisation. Injection : 1 µl. 1198

Calculate the percentage content of each fatty acid by normalisation. Correct the area of the peak due to methyl ricinoleate, multiplying by a factor R calculated using the following expression :

m1

=

mass of methyl ricinoleate in the reference solution,

m2

=

mass of methyl stearate in the reference solution,

A1

=

area of the peak due to methyl ricinoleate in the chromatogram obtained with the reference solution,

A2

=

area of the peak due to methyl stearate in the chromatogram obtained with the reference solution.

Composition of the fatty-acid fraction of the oil : — palmitic acid : maximum 2.0 per cent, — stearic acid : maximum 2.5 per cent, — oleic acid and isomers (C18:1 equivalent chain length on macrogol 20 000 : 18.3) : 2.5 per cent to 6.0 per cent, — linoleic acid (C18:2 equivalent chain length on macrogol 20 000 : 18.8) : 2.5 per cent to 7.0 per cent, — linolenic acid (C18:3 equivalent chain length on macrogol 20 000 : 19.2) : maximum 1.0 per cent, — eicosenoic acid (C20:1 equivalent chain length on macrogol 20 000 : 20.2) : maximum 1.0 per cent, — ricinoleic acid (equivalent chain length on macrogol 20 000 : 23.9) : 85.0 per cent to 92.0 per cent, — any other fatty acid : maximum 1.0 per cent. Water (2.5.12) : maximum 0.3 per cent, determined on 5.0 g. STORAGE In an airtight, well-filled container, protected from light. LABELLING The label states the name and concentration of any added antioxidant. 01/2005:0986 corrected

CEFACLOR Cefaclorum

C15H14ClN3O4S,H2O

Mr 385.8

DEFINITION Cefaclor is the monohydrate of (6R,7R)-7-[[(2R)-2amino-2-phenylacetyl]amino]-3-chloro-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. It contains not See the information section on general monographs (cover pages)

Cefaclor


less than 96.0 per cent and not more than the equivalent of 102.0 per cent of C15H14ClN3O4S, calculated with reference to the anhydrous substance. CHARACTERS A white or slightly yellow powder, slightly soluble in water, practically insoluble in methanol and in methylene chloride. IDENTIFICATION Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cefaclor CRS. TESTS pH (2.2.3). Suspend 0.250 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. The pH of the suspension is 3.0 to 4.5. Specific optical rotation (2.2.7). Dissolve 0.250 g in a 10 g/l solution of hydrochloric acid R and dilute to 25.0 ml with the same solution. The specific optical rotation is + 101 to + 111, calculated with reference to the anhydrous substance. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 50.0 mg of the substance to be examined in 10.0 ml of a 2.7 g/l solution of sodium dihydrogen phosphate R adjusted to pH 2.5 with phosphoric acid R. Reference solution (a). Dissolve 2.5 mg of cefaclor CRS and 5.0 mg of delta-3-cefaclor CRS in 100.0 ml of a 2.7 g/l solution of sodium dihydrogen phosphate R adjusted to pH 2.5 with phosphoric acid R. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with a 2.7 g/l solution of sodium dihydrogen phosphate R adjusted to pH 2.5 with phosphoric acid R. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with end-capped octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.0 ml/min : Mobile phase A. Adjust a 7.8 g/l solution of sodium dihydrogen phosphate R to pH 4.0 with phosphoric acid R, Mobile phase B. Mix 450 ml of acetonitrile R with 550 ml of mobile phase A, — as detector a spectrophotometer set at 220 nm, — a 20 µl loop injector. Equilibrate the column with a mixture of 5 volumes of mobile phase B and 95 volumes of mobile phase A for at least 15 min between each analysis. Inject the solutions. Operate by gradient elution increasing the concentration of mobile phase B continuously and linearly by 0.67 per cent V/V per minute for 30 min (25 per cent V/V). Then increase the concentration of mobile phase B continuously and linearly by 5 per cent V/V per minute for 15 min (100 per cent V/V). Finally elute with mobile phase B for 10 min. Change the composition to a mixture of 5 volumes of mobile phase B and 95 volumes of mobile phase A to re-equilibrate the column. Inject reference solution (a). The test is not valid unless the resolution between the peaks due to cefaclor and delta-3-cefaclor is at least 2 and the symmetry factor of the cefaclor peak is at most 1.2. If necessary, adjust the acetonitrile content of the mobile phase. General Notices (1) apply to all monographs and other texts

Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak and any peaks due to the mobile phase, is not greater than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent) ; and the sum of all such peaks is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (2 per cent). Disregard any peak with an area less than 0.1 times that of the principal peak in the chromatogram obtained with reference solution (b). Heavy metals (2.4.8). 1.0 g complies with limit test C (30 ppm). Prepare the standard using 3 ml of lead standard solution (10 ppm Pb) R. Water (2.5.12). 3.0 per cent to 6.5 per cent, determined on 0.200 g by the semi-micro determination of water. ASSAY Examine by liquid chromatography (2.2.29). Test solution. Dissolve 15.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (a). Dissolve 15.0 mg of cefaclor CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (b). Dissolve 15.0 mg of cefaclor CRS and 15.0 mg of delta-3-cefaclor CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.5 ml/min a mixture prepared by adding 220 ml of methanol R to a mixture of 780 ml of water R, 10 ml of triethylamine R and 1 g of sodium pentanesulphonate R, adjusted to pH 2.5 with phosphoric acid R, — as detector a spectrophotometer set at 265 nm, — a 20 µl loop injector. Inject reference solution (b). The assay is not valid unless the resolution between the peaks due to cefaclor and delta-3-cefaclor is at least 2.5. Adjust the concentration of methanol in the mobile phase, if necessary. The assay is not valid unless the symmetry factor of the cefaclor peak is at most 1.5. Inject reference solution (a) six times. The assay is not valid unless the relative standard deviation of the peak area of cefaclor is at most 1.0 per cent. Inject alternately the test solution and reference solution (a). IMPURITIES

A. (2R)-2-amino-2-phenylacetic acid (phenylglycine),

B. (6R,7R)-7-amino-3-chloro-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, 1199

Cefadroxil monohydrate



CEFADROXIL MONOHYDRATE Cefadroxilum monohydricum C. (6R,7R)-7-[[(2S)-2-amino-2-phenylacetyl]amino]-3-chloro-8oxo-5-thia- 1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid,

C16H17N3O5S,H2O

Mr 381.4

DEFINITION (6R,7R)-7-[[(2R)-2-Amino-2-(4-hydroxyphenyl)acetyl]amino]-3methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid monohydrate. Content : 95.0 per cent to 102.0 per cent (anhydrous substance).

D. (2R,6R,7R)- and (2S,6R,7R)-7-[[(2R)-2-amino2-phenylacetyl]amino]-3-chloro-8-oxo-5-thia1-azabicyclo[4.2.0]oct-3-ene-2-carboxylic acid (delta-3-cefaclor),

CHARACTERS Appearance : white or almost white powder. Solubility : slightly soluble in water, very slightly soluble in alcohol. IDENTIFICATION E. 2-[[(2R)-2-amino-2-phenylacetyl]amino]-2-(5-chloro-4-oxo-3, Infrared absorption spectrophotometry (2.2.24). 4-dihydro-2H-1,3-thiazin-2-yl)acetic acid,

F. 3-phenylpyrazin-2-ol.

G. (2R,6R,7R)- and (2S,6R,7R)-7-[[(2R)-2-amino-2phenylacetyl]amino]-3-methylene-8-oxo-5-thia-1azabicyclo[4.2.0]octane-2-carboxylic acid (isocefalexine),

H. (6R,7R)-7-[[(2R)-2-[[(2R)-2-amino-2-phenylacetyl]amino]2-phenylacetyl]amino]-3-chloro-8-oxo-5-thia1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (N-phenylglycyl cefaclor). 1200

TESTS pH (2.2.3) : 4.0 to 6.0. Suspend 1.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Specific optical rotation (2.2.7) : + 165 to + 178 (anhydrous substance). Dissolve 0.500 g in water R and dilute to 50.0 ml with the same solvent. Absorbance (2.2.25). Dissolve 20.0 mg in phosphate buffer solution pH 6.0 R and dilute to 100.0 ml with the same solvent. The absorbance of the solution determined at 330 nm is not greater than 0.05. Dilute 10.0 ml of the solution to 100.0 ml with phosphate buffer solution pH 6.0 R. Examined between 235 nm and 340 nm, the diluted solution shows an absorption maximum at 264 nm. The specific absorbance at this maximum is 225 to 250 (anhydrous substance). Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 50.0 mg of the substance to be examined in mobile phase A and dilute to 50.0 ml with mobile phase A. Reference solution (a). Dissolve 10.0 mg of D-α-(4-hydroxyphenyl)glycine CRS (impurity A) in mobile phase A and dilute to 10.0 ml with mobile phase A. Reference solution (b). Dissolve 10.0 mg of 7-aminodesacetoxycephalosporanic acid CRS (impurity B) in phosphate buffer solution pH 7.0 R5 and dilute to 10.0 ml with the same buffer solution. Reference solution (c). Dilute 1.0 ml of reference solution (a) and 1.0 ml of reference solution (b) to 100.0 ml with mobile phase A.


Cefadroxil monohydrate


Reference solution (d). Dissolve 10 mg of dimethylformamide R and 10 mg of dimethylacetamide R in mobile phase A and dilute to 10.0 ml with mobile phase A. Dilute 1.0 ml to 100.0 ml with mobile phase A. Reference solution (e). Dilute 1.0 ml of reference solution (c) to 25.0 ml with mobile phase A. Column : — size : l = 0.10 m, Ø = 4.6 mm, — stationary phase: spherical octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : — mobile phase A : phosphate buffer solution pH 5.0 R, — mobile phase B : methanol R2, Time (min)



0-1

98

2

1 - 20

98 → 70

2 → 30

20 - 23

70 → 98

30 → 2

23 - 30

98

2

Flow rate : 1.5 ml/min. Detection : spectrophotometer at 220 nm. Injection : 20 µl ; inject the test solution and reference solutions (c), (d) and (e). Relative retention with reference to cefadroxil (retention time = about 6 min) : dimethylformamide = about 0.4 ; dimethylacetamide = about 0.75. System suitability : — resolution : minimum 5.0 between the peaks due to impurity A and to impurity B in the chromatogram obtained with reference solution (c), — signal-to-noise ratio : minimum 10 for the second peak in the chromatogram obtained with reference solution (e). Limits : — impurity A : not more than the area of the first peak in the chromatogram obtained with reference solution (c) (1.0 per cent), — any other impurity : not more than the area of the second peak in the chromatogram obtained with reference solution (c) (1.0 per cent), — total: not more than 3 times the area of the second peak in the chromatogram obtained with reference solution (c) (3.0 per cent), — disregard limit : 0.05 times the area of the second peak in the chromatogram obtained with reference solution (c) (0.05 per cent) ; disregard the peaks due to dimethylformamide and dimethylacetamide. N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm. Water (2.5.12) : 4.0 per cent to 6.0 per cent, determined on 0.200 g. Sulphated ash (2.4.14) : maximum 0.5 per cent, determined on 1.0 g. ASSAY Liquid chromatography (2.2.29). Test solution. Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. General Notices (1) apply to all monographs and other texts

Reference solution (a). Dissolve 50.0 mg of cefadroxil CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (b). Dissolve 5 mg of cefadroxil CRS and 50 mg of amoxicillin trihydrate CRS in the mobile phase and dilute to 100 ml with the mobile phase. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : acetonitrile R, a 2.72 g/l solution of potassium dihydrogen phosphate R (4:96 V/V). Flow rate : 1 ml/min. Detection : spectrophotometer at 254 nm. Injection : 20 µl. System suitability : reference solution (b) : — resolution : minimum 5.0 between the peaks due to cefadroxil and to amoxicillin. Calculate the percentage content of cefadroxil. STORAGE Protected from light. IMPURITIES

A. (2R)-2-amino-2-(4-hydroxyphenyl)acetic acid,

B. (6R,7R)-7-amino-3-methyl-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (7-ADCA),

C. (2R,5RS)-2-[(R)-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]carboxymethyl]-5-methyl-5,6-dihydro-2H-1,3-thiazine-4-carboxylic acid,

D. (6R,7R)-7-[[(2S)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (L-cefadroxil), 1201

Cefalexin monohydrate


IDENTIFICATION Infrared absorption spectrophotometry (2.2.24). Comparison : cefalexin monohydrate CRS. TESTS pH (2.2.3) : 4.0 to 5.5. E. (6RS)-3-(aminomethylene)-6-(4-hydroxyphenyl)piperazine- Dissolve 50 mg in carbon dioxide-free water R and dilute to 10 ml with the same solvent. 2,5-dione, Specific optical rotation (2.2.7) : + 149 to + 158 (anhydrous substance). Dissolve 0.125 g in phthalate buffer solution pH 4.4 R and dilute to 25.0 ml with the same solvent. Absorbance (2.2.25). Dissolve 50 mg in water R and dilute to 100.0 ml with the same solvent. The absorbance of the solution determined at 330 nm is not greater than 0.05. Dilute 2.0 ml of the solution to 50.0 ml with water R. Examined between 220 nm and 300 nm, the diluted solution F. (6R,7R)-7-[[(2R)-2-[[(2RS)-2-amino-2-(4-hydroxyshows an absorption maximum at 262 nm. The specific phenyl)acetyl]amino]-2-(4-hydroxyphenyl)acetyl]amiabsorbance at this maximum is 220 to 245, calculated with no]-3-methyl-8-oxo-5-thia-1-azabicycreference to the anhydrous substance. lo[4.2.0]oct-2-ene-2-carboxylic acid, Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 50.0 mg of the substance to be examined in mobile phase A and dilute to 50.0 ml with mobile phase A. Reference solution (a). Dissolve 10.0 mg of D-phenylglycine R in mobile phase A and dilute to G. 3-hydroxy-4-methylthiophen-2(5H)-one, 10.0 ml with mobile phase A. Reference solution (b). Dissolve 10.0 mg of 7-aminodesacetoxycephalosporanic acid CRS in phosphate buffer solution pH 7.0 R5 and dilute to 10.0 ml with mobile phase A. Reference solution (c). Dilute 1.0 ml of reference solution (a) and 1.0 ml of reference solution (b) to 100.0 ml with mobile phase A. H. (6R,7R)-7-[(2,2-dimethylpropanoyl)amino]-3-methyl-8Reference solution (d). Dissolve 10 mg of oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid dimethylformamide R and 10 mg of dimethylacetamide R (7-ADCA pivalamide). in mobile phase A and dilute to 10.0 ml with mobile phase A. Dilute 1.0 ml to 100.0 ml with mobile phase A. 01/2005:0708 Reference solution (e). Dilute 1.0 ml of reference solution (c) to 20.0 ml with mobile phase A. Reference solution (f). Dissolve 10 mg of cefotaxime CEFALEXIN MONOHYDRATE sodium CRS in mobile phase A and dilute to 10.0 ml with mobile phase A. To 1.0 ml of the solution add 1.0 ml of the Cefalexinum monohydricum test solution and dilute to 100 ml with mobile phase A. Column : — size : l = 0.10 m, Ø = 4.6 mm, — stationary phase : spherical octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : — mobile phase A : phosphate buffer solution pH 5.0 R, — mobile phase B : methanol R2, C16H17N3O4S,H2O Mr 365.4 DEFINITION (6R,7R)-7-[[(2R)-2-Amino-2-phenylacetyl]amino]-3-methyl8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid monohydrate. Content : 95.0 per cent to 102.0 per cent (anhydrous substance). CHARACTERS Appearance : white or almost white, crystalline powder. Solubility : sparingly soluble in water, practically insoluble in alcohol. 1202

Time (min)



0-1

98

2

1 - 20

98 → 70

2 → 30

20 - 23

70 → 98

30 → 2

23 - 30

98

2

Flow rate : 1.5 ml/min. Detection : spectrophotometer at 220 nm. Injection : 20 µl ; inject the test solution and reference solutions (c), (d), (e) and (f).


Cefalotin sodium


System suitability : — resolution : minimum of 2.0 between the peaks due to impurity A and to impurity B in the chromatogram obtained with reference solution (c) and minimum of 1.5 between the peaks due to cefalexin and to cefotaxime in the chromatogram obtained with reference solution (f). B. (6R,7R)-7-amino-3-methyl-8-oxo-5-thia-1Limits : azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid — impurity B : not more than the area of the second peak (7-aminodesacetoxycephalosporanic acid, 7-ADCA), in the chromatogram obtained with reference solution (c) (1.0 per cent), — any other impurity (disregard the peaks due to dimethylformamide and dimethylacetamide) : not more than the area of the first peak in the chromatogram obtained with reference solution (c) (1.0 per cent), — total: not more than 3 times the area of the first peak in the chromatogram obtained with reference solution (c) (3.0 per cent), — disregard limit: the area of the second peak in the C. (6R,7R)-7-[[(2R)-2-[[(2R)-2-amino-2-phenylacetyl]amino]chromatogram obtained with reference solution (e) 2-phenylacetyl]amino]-3-methyl-8-oxo-5-thia-1(0.05 per cent). azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm. Water (2.5.12) : 4.0 per cent to 8.0 per cent, determined on 0.300 g. Sulphated ash (2.4.14) : maximum 0.2 per cent, determined on 1.0 g. D. 3-hydroxy-4-methylthiophen-2(5H)-one, ASSAY Liquid chromatography (2.2.29). Test solution. Dissolve 50.0 mg of the substance to be examined in water R and dilute to 100.0 ml with the same solvent. Reference solution (a). Dissolve 50.0 mg of cefalexin monohydrate CRS in water R and dilute to 100.0 ml with the same solvent. E. (6R,7R)-7-[(2,2-dimethylpropanoyl)amino]-3-methyl-8Reference solution (b). Dissolve 10 mg of cefradine CRS oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid in 20 ml of reference solution (a) and dilute to 100 ml with (7-ADCA pivalamide), water R. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase: octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : methanol R, acetonitrile R, a 13.6 g/l solution of potassium dihydrogen phosphate R, water R (2:5:10:83 V/V/V/V). F. (2RS,6R,7R)-7-[[(2R)-2-amino-2-phenylacetyl]amino]Flow rate : 1.5 ml/min. 3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-3-ene-2carboxylic acid (delta-2-cefalexin). Detection : spectrophotometer at 254 nm. Injection : 20 µl. System suitability : reference solution (b) : 01/2005:0987 — resolution : minimum 4.0 between the peaks due to cefalexin and to cefradine. Calculate the percentage content of cefalexin monohydrate. CEFALOTIN SODIUM STORAGE Protected from light.

Cefalotinum natricum

IMPURITIES

A. (2R)-2-amino-2-phenylacetic acid (D-phenylglycine), General Notices (1) apply to all monographs and other texts

C16H15N2NaO6S2

Mr 418.4 1203

Cefamandole nafate


The chromatographic procedure may be carried out using : DEFINITION Cefalotin sodium contains not less than 96.0 per cent and not — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for more than the equivalent of 101.0 per cent of sodium (6R, chromatography R (5 µm), 7R)-3-[(acetyloxy)methyl]-8-oxo-7-[[2-(thiophen-2-yl)acetyl]amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate, — as mobile phase at a flow rate of 1.0 ml/min a solution calculated with reference to the anhydrous substance. prepared as follows : dissolve 17 g of sodium acetate R in 790 ml of water R, add 0.6 ml of glacial acetic acid R and CHARACTERS adjust if necessary to pH 5.8 to 6.0 with dilute sodium A white or almost white powder, freely soluble in water, hydroxide solution R or glacial acetic acid R, add 150 ml slightly soluble in ethanol. of acetonitrile R and 70 ml of ethanol R and mix, — as detector a spectrophotometer set at 254 nm, IDENTIFICATION — a 10 µl loop injector, A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with maintaining the temperature of the column at 40 °C. cefalotin sodium CRS. Inject reference solution (c). Adjust the sensitivity of the B. It gives reaction (a) of sodium (2.3.1). system to obtain peaks with a height corresponding to at least half the full scale of the recorder. The chromatogram TESTS obtained shows two principal peaks corresponding to cefalotin and desacetylcefalotin. The assay is not valid Solution S. Dissolve 2.50 g in carbon dioxide-free water R unless the resolution between these two peaks is at least 9.0. and dilute to 25.0 ml with the same solvent. Adjust the concentration of acetonitrile in the mobile phase Appearance of solution. Solution S is clear (2.2.1). The if necessary. The assay is not valid unless the symmetry absorbance (2.2.25) of solution S measured at 450 nm is factor of the cefalotin peak is at most 1.8. Inject reference not greater than 0.20. solution (a) six times. The assay is not valid unless the pH (2.2.3). The pH of solution S is 4.5 to 7.0. relative standard deviation of the peak area for cefalotin is Specific optical rotation (2.2.7). Dissolve 1.25 g in water R at most 1.0 per cent. Inject alternately the test solution and reference solution (a). and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 124 to + 134, calculated with reference Calculate the percentage content of cefalotin sodium. to the anhydrous substance. STORAGE Related substances. Examine by liquid chromatography Store in an airtight container, protected from light. If the (2.2.29) as described under Assay. Inject the test solution and reference solution (b). Continue the chromatography for substance is sterile, store in a sterile, airtight, tamper-proof at least four times the retention time of the principal peak. In container. the chromatogram obtained with the test solution : the area LABELLING of any peak, apart from the principal peak, is not greater The label states, where applicable, that the substance is free than the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent) and the sum from bacterial endotoxins. of the areas of any such peaks is not greater than three times IMPURITIES the area of the principal peak in the chromatogram obtained with reference solution (b) (3 per cent). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent). N,N-Dimethylaniline (2.4.26, Method B). Not more than 20 ppm. 2-Ethylhexanoic acid (2.4.28). Not more than 0.5 per A. (6R,7R)-3-methyl-8-oxo-7-[[2-(thiophen-2-yl)acetyl]amino]cent m/m. 5-thia-1-azabicyclo[4.2.0]oct-2-ene- 2-carboxylic acid Water (2.5.12). Not more than 1.5 per cent, determined on (desacetoxycefalotin). 0.500 g by the semi-micro determination of water. Bacterial endotoxins (2.6.14) : less than 0.13 IU/mg, if 01/2005:1402 intended for use in the manufacture of parenteral dosage corrected forms without a further appropriate procedure for the removal of bacterial endotoxins.

CEFAMANDOLE NAFATE ASSAY Examine by liquid chromatography (2.2.29). Cefamandoli nafas Test solution. Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the mobile phase. Reference solution (a). Dissolve 25.0 mg of cefalotin sodium CRS in the mobile phase and dilute to 25.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 100.0 ml with the mobile phase. Reference solution (c). Heat 5 ml of reference solution (a) in Mr 512.5 a water-bath at 90 °C for 10 min. Cool and inject immediately. C19H17N6NaO6S2 1204


Cefamandole nafate


DEFINITION Sodium (6R,7R)-7-[[(2R)-2-(formyloxy)-2-phenylacetyl]amino]3-[[(1-methyl-1H-tetrazol-5-yl)sulphanyl]methyl]-8-oxo-5thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate with sodium carbonate. Content : — cefamandole (C18H18N6O5S2) : 84.0 per cent to 93.0 per cent (anhydrous and sodium carbonate-free substance),

Time (min)



0-1

100

0

1 - 35

100 → 0

0 → 100

35 - 45

0

100

45 - 50

0 → 100

100 → 0

Flow rate : 1.5 ml/min. — sodium carbonate (Na2CO3) : 4.8 per cent to 6.4 per cent. Detection : spectrophotometer at 254 nm. Injection : 20 µl loop injector. System suitability : reference solution (a) : CHARACTERS — resolution : minimum 5.0 between the peaks due to Appearance : white or almost white powder. cefamandole and to cefamandole nafate. Solubility : freely soluble in water, sparingly soluble in Limits : methanol. — any impurity : not more than the area of the principal peak in the chromatogram obtained with reference IDENTIFICATION solution (b) (1 per cent), A. Infrared absorption spectrophotometry (2.2.24). — total : not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (b) Preparation : discs. (5 per cent), Comparison : cefamandole nafate CRS. — disregard limit : 0.1 times the area of the principal peak B. It gives reaction (a) of sodium (2.3.1). in the chromatogram obtained with reference solution (b) (0.1 per cent). TESTS Cefamandole free acid : maximum 9.5 per cent (anhydrous and sodium carbonate-free substance), determined by liquid Solution S. Dissolve 2.5 g in carbon dioxide-free water R chromatography as described under Assay. and dilute to 25 ml with the same solvent. 2-Ethylhexanoic acid (2.4.28) : maximum 0.3 per cent m/m. Appearance of solution. Solution S is clear (2.2.1) and its absorbance (2.2.25) at 475 nm has a maximum of 0.03. Heavy metals (2.4.8) : maximum 20 ppm. 1.0 g complies with limit test C. Prepare the standard using pH : 6.0 to 8.0 for solution S, measured after 30 min. 2 ml of lead standard solution (10 ppm Pb) R. Specific optical rotation (2.2.7) : − 25.0 to − 33.0 (anhydrous Water (2.5.12) : maximum 2.0 per cent, determined on and sodium carbonate-free substance). 0.500 g. Dissolve 1.00 g in acetate buffer solution pH 4.7 R and Bacterial endotoxins (2.6.14) : less than 0.15 IU/mg, if dilute to 10.0 ml with the same solvent. intended for use in the manufacture of parenteral dosage Related substances. Liquid chromatography (2.2.29). forms without a further appropriate procedure for the Prepare the solutions immediately before use. removal of bacterial endotoxins. Solvent mixture. Mix 18 volumes of acetonitrile R and 75 volumes of a 10 per cent V/V solution of triethylamine R ASSAY Cefamandole. Liquid chromatography (2.2.29). Prepare the previously adjusted to pH 2.5 with phosphoric acid R. solutions immediately before use. Test solution. Dissolve 0.100 g of the substance to be Test solution. Dissolve 50.0 mg of the substance to be examined in the solvent mixture and dilute to 10.0 ml with examined in the mobile phase and dilute to 100.0 ml with the solvent mixture. the mobile phase. Reference solution (a). Dilute 1 ml of the test solution to Reference solution (a). Dissolve 50.0 mg of cefamandole 10 ml with the solvent mixture, then heat the solution at nafate CRS in the mobile phase and dilute to 100.0 ml with 60 °C for 30 min. the mobile phase. Reference solution (b). Dilute 1.0 ml of the test solution to Reference solution (b). Dilute 1 ml of the test solution to 100.0 ml with the solvent mixture. 10 ml with the mobile phase, then heat the solution at 60 °C for 30 min. Column : Column : — size : l = 0.25 m, Ø = 4.6 mm, — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : octadecylsilyl silica gel for — stationary phase : octadecylsilyl silica gel for chromatography R (5 µm). chromatography R (5 µm). Mobile phase : Mobile phase : mix 25 volumes of acetonitrile R and 75 volumes of a 10 per cent V/V solution of triethylamine R — triethylamine phosphate solution : dissolve 2.0 g of previously adjusted to pH 2.5 with phosphoric acid R. sodium pentanesulphonate R in 350 ml of water R, add 40 ml of triethylamine R, adjust to pH 2.5 with Flow rate : 1.0 ml/min. phosphoric acid R and dilute to 700 ml with water R, Detection : spectrophotometer at 254 nm. — mobile phase A : mix 1 volume of triethylamine phosphate Injection : 20 µl loop injector. solution and 2 volumes of water R, System suitability : — resolution : minimum of 7.0 between the 2 principal peaks — mobile phase B : mix equal volumes of triethylamine in the chromatogram obtained with reference solution (b), phosphate solution, methanol R and acetonitrile R. General Notices (1) apply to all monographs and other texts

1205

Cefapirin sodium


— repeatability : maximum relative standard deviation of 0.8 per cent after a series of single injections of a minimum 3 freshly prepared reference solutions (a). Calculate the percentage content of cefamandole (C18H18N6O5S2) from the sum of the contents of cefamandole nafate and cefamandole free acid using the declared contents of cefamandole nafate CRS. 1 mg of cefamandole nafate is E. (6R,7R)-7-[[(2R)-2-(formyloxy)-2-phenylacetyl]amino]-3equivalent to 0.9025 mg of cefamandole. [(acetyloxy)methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2Sodium carbonate. Dissolve 0.500 g in 50 ml of water R. ene-2-carboxylic acid (formylmandeloyl-7-ACA). Titrate with 0.1 M hydrochloric acid, determining the end-point potentiometrically (2.2.20). 01/2005:1650 1 ml of 0.1 M hydrochloric acid is equivalent to 10.6 mg of Na2CO3. STORAGE In an airtight container, protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof container.

CEFAPIRIN SODIUM Cefapirinum natricum

LABELLING The label states : — that the substance contains sodium carbonate, — where applicable, that the substance is free from bacterial C17H16N3NaO6S2 Mr 445.5 endotoxins. DEFINITION IMPURITIES Sodium (6R,7R)-3-[(acetyloxy)methyl]-8-oxo-7-[[[(pyridin-4yl)sulphanyl]acetyl]amino]-5-thia-1-azabicyclo[4.2.0]oct-2ene-2-carboxylate. Content : 96.0 per cent to 102.0 per cent (anhydrous substance).

A. (6R,7R)-7-[[(2R)-2-(formyloxy)-2-phenylacetyl]amino]3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylic acid (formylmandeloyl-7-amino-desacetoxycephalosporanic acid),

B. R = H : (6R,7R)-7-[[(2R)-2-hydroxy-2-phenylacetyl]amino]3-[[(1-methyl-1H-tetrazol-5-yl)sulphanyl]methyl]-8-oxo5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (cefamandole), C. R = CO-H3C : (6R,7R)-7-[[(2R)-2-(acetyloxy)-2phenylacetyl]amino]-3-[[(1-methyl-1H-tetrazol-5yl)sulphanyl]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2ene-2-carboxylic acid (O-acetylcefamandole),

D. 1-methyl-1H-tetrazole-5-thiol, 1206

CHARACTERS Appearance : white or pale yellow powder. Solubility : soluble in water, practically insoluble in methylene chloride. IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Comparison : cefapirin sodium CRS. B. It gives reaction (a) of sodium (2.3.1). TESTS Appearance of solution. Dissolve 2.0 g in water R and dilute to 10.0 ml with the same solvent. The solution is clear (2.2.1). Dilute 5.0 ml to 10.0 ml with water R. The absorbance (2.2.25) of this solution at 450 nm is maximum 0.25. pH (2.2.3) : 6.5 to 8.5. Dissolve 0.100 g in carbon dioxide-free water R and dilute to 10.0 ml with the same solvent. Specific optical rotation (2.2.7) : + 150 to + 165 (anhydrous substance). Dissolve 0.500 g in water R and dilute to 25.0 ml with the same solvent. Related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately before use. Test solution. Dissolve 42 mg of the substance to be examined in the mobile phase and dilute to 200.0 ml with the mobile phase. Reference solution (a). Dissolve 42 mg of cefapirin sodium CRS in the mobile phase and dilute to 200.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase.


Cefatrizine propylene glycol


Reference solution (c). Dilute 1.0 ml of reference solution (b) to 20.0 ml with the mobile phase. Reference solution (d). Mix 1 ml of the test solution, 8 ml of the mobile phase and 1 ml of hydrochloric acid R1. Heat at 60 °C for 10 min. Column : — size : l = 0.30 m, Ø = 4 mm, — stationary phase: octadecylsilyl silica gel for chromatography R (10 µm). Mobile phase : mix 80 ml of dimethylformamide R, 4.0 ml of glacial acetic acid R and 20 ml of a 4.5 per cent (m/m) solution of potassium hydroxide R. Dilute to 2 litres with water R. Flow rate : 2.0 ml/min. Detection : spectrophotometer at 254 nm. Injection : 20 µl of the test solution and reference solutions (b), (c) and (d). Run time : twice the retention time of cefapirin. Relative retention with reference to cefapirin (retention time = about 13 min) : impurity B = about 0.3 ; impurity C = about 0.5 ; impurity A = about 0.75. System suitability : reference solution (d) : — resolution : minimum 2.0 between the peaks due to cefapirin and impurity A. Limits : — any impurity : for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent), and not more than 1 such peak has an area greater than 0.3 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.3 per cent), — total: not more than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (2.0 per cent), — disregard limit: area of the principal peak in the chromatogram obtained with reference solution (c) (0.05 per cent). N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm. 2-Ethylhexanoic acid (2.4.28) : maximum 0.5 per cent. Water (2.5.12) : maximum 2.0 per cent, determined on 0.300 g. Bacterial endotoxins (2.6.14) : less than 0.17 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modification. Injection : test solution and reference solution (a). Calculate the percentage content of C17H16N3NaO6S2. STORAGE Protected from light. If the substance is sterile, store in a sterile, tamper-proof container. LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins. IMPURITIES Specified impurities : A, B, C. General Notices (1) apply to all monographs and other texts

A. (5aR,6R)-6-[[[(pyridin-4-yl)sulphanyl]acetyl]amino]-5a, 6-dihydro-3H,7H-azeto[2,1-b]furo[3,4-d][1,3]thiazine-1, 7(4H)-dione (deacetylcefapirin lactone),

B. R = OH : (6R,7R)-3-(hydroxymethyl)-8-oxo-7-[[[(pyridin-4yl)sulphanyl]acetyl]amino]-5-thia-1-azabicyclo[4.2.0]oct-2ene-2-carboxylic acid (deacetylcefapirin), C. R = H : (6R,7R)-3-methyl-8-oxo-7-[[[(pyridin-4yl)sulphanyl]acetyl]amino]-5-thia-1-azabicyclo[4.2.0]oct-2ene-2-carboxylic acid (deacetoxycefapirin). 01/2005:1403

CEFATRIZINE PROPYLENE GLYCOL Cefatrizinum propylen glycolum

C18H18N6O5S2, (C3H8O2)n

Mr 462.5 (base)

DEFINITION Cefatrizine propylene glycol contains not less than 95.0 per cent and not more than the equivalent of 102.0 per cent of (6R,7R)-7-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]8-oxo-3-[[(1H-1,2,3-triazol-4-yl)sulphanyl]methyl]-5-thia1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, calculated with reference to the anhydrous and propylene glycol free substance. It consists of cefatrizine and propane-1,2-diol (molecular proportions about 1:1). It contains not less than 13.0 per cent and not more than 18.0 per cent of propylene glycol. CHARACTERS A white or almost white powder, slightly soluble in water, practically insoluble in alcohol and in methylene chloride. IDENTIFICATION A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cefatrizine propylene glycol CRS. B. Examine the chromatograms obtained in the test for propylene glycol. The retention time and size of the principal peak in the chromatogram obtained with the 1207

Cefatrizine propylene glycol


test solution are approximately the same as those of the principal peak in the chromatogram obtained with reference solution (b).

Water (2.5.12). Not more than 1.5 per cent, determined on 0.500 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

TESTS Specific optical rotation (2.2.7). Dissolve 0.400 g of the substance to be examined in 1 M hydrochloric acid and dilute to 20.0 ml with the same solvent. The specific optical rotation is + 63 to + 69, calculated with reference to the anhydrous, propylene glycol free substance.

ASSAY Examine by liquid chromatography (2.2.29).

Test solution. Dissolve 60.0 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with Propylene glycol. 13.0 per cent to 18.0 per cent, determined the mobile phase. by gas chromatography (2.2.28), using dimethylacetamide R Reference solution (a). Dissolve 60.0 mg of cefatrizine as the internal standard. propylene glycol CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Internal standard solution. Dissolve 1.0 g of dimethylacetamide R in a mixture of 20 volumes of Reference solution (b). Dissolve 30.0 mg of cefatrizine acetone R and 80 volumes of water R, and dilute to 50.0 ml impurity A CRS in buffer solution pH 7.0 R and dilute to with the same mixture of solvents. 100.0 ml with the same buffer solution. Test solution. Introduce 0.40 g of the substance to be Reference solution (c). Dilute 0.6 ml of reference solution (a) examined into a ground-glass-stoppered test tube. Add to 100.0 ml with the mobile phase. 3.0 ml of the internal standard solution, 1.0 ml of a mixture of 20 volumes of acetone R and 80 volumes of water R and Reference solution (d). Dilute 1.0 ml of reference solution (b) to 100.0 ml with buffer solution pH 7.0 R. 2.0 ml of hydrochloric acid R. Seal the test tube and stir the solution. Reference solution (e). To 1.0 ml of reference solution (a)

Reference solution (a). Dissolve 2.0 g of propylene glycol R add 1.0 ml of reference solution (b) and dilute to 10.0 ml in a mixture of 20 volumes of acetone R and 80 volumes of with the mobile phase. water R and dilute to 100.0 ml with the same solvent. The chromatographic procedure may be carried out using : Reference solution (b). Introduce into a ground-glass— a stainless steel column 0.25 m long and 4 mm in stoppered test tube 1.0 ml of reference solution (a) and internal diameter packed with octadecylsilyl silica gel for 1.0 ml of the internal standard solution. chromatography R (5 µm), The chromatographic procedure may be carried out using : — as mobile phase at a flow rate of 2 ml/min a mixture of 5 volumes of acetonitrile R and 95 volumes of a — a stainless steel column 2 m long and 2 mm in internal 2.72 g/l solution of potassium dihydrogen phosphate R diameter packed with ethylvinylbenzene-divinylbenzene in water R, copolymer R (150-180 µm), — as detector a spectrophotometer set at 272 nm. — nitrogen for chromatography R as the carrier gas at a flow rate of about 30 ml/min, Adjust the sensitivity of the system so that the height of the principal peak obtained with 20 µl of reference solution (a) — a flame-ionisation detector, is at least 50 per cent of the full scale of the recorder. Inject maintaining the temperature of the column at 200 °C and 20 µl of reference solution (a) six times. The test is not valid that of the injection port and of the detector at 250 °C. unless the relative standard deviation is at most 1.0 per cent. Inject reference solution (e). The test is not valid unless the Inject separately 1 µl of the test solution and 1 µl of reference resolution between the peaks corresponding to cefatrizine solution (b). and cefatrizine impurity A is at least 5.0. 7-ACA triazole and other related substances. Examine by Inject alternately 20 µl of the test solution and 20 µl of liquid chromatography (2.2.29) as described under Assay. Adjust the sensitivity of the system so that the height of the reference solution (a) and calculate the percentage content of cefatrizine using the chromatogram obtained with reference principal peak obtained with 20 µl of reference solution (c) is at least 50 per cent of the full scale of the recorder. Inject solution (a). 20 µl of the test solution and continue the chromatography for at least twice the retention time of the principal peak. IMPURITIES In the chromatogram obtained with the test solution : the area of any peak corresponding to cefatrizine impurity A is not greater than that of the peak due to cefatrizine impurity A in the chromatogram obtained with reference solution (d) (0.5 per cent) ; the area of any peak apart from the principal peak and any peak corresponding to cefatrizine impurity A is not greater than the area of the principal peak in the chromatogram obtained with reference solution (c) (0.6 per cent) ; the sum of the areas of any such peaks is not greater than 3.5 times the area of the principal peak in the chromatogram obtained with reference solution (c) (2.1 per cent). Disregard any peak with an area less than 0.05 times A. (6R,7R)-7-amino-8-oxo-3-[[(1H-1,2,3-triazol-4yl)sulphanyl]methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2that of the principal peak in the chromatogram obtained carboxylic acid (7-ACA triazole). with reference solution (c). 1208


Cefazolin sodium


CEFAZOLIN Cefazolinum

C14H13N8NaO4S3

01/2005:0988 Reference solution (b). Dissolve 20 mg of the substance to be examined in 10 ml of a 2 g/l solution of sodium hydroxide R. Allow to stand for 15-30 min. Dilute 1.0 ml of SODIUM the solution to 20 ml with mobile phase A. Column : natricum — size : l = 0.125 m, Ø = 4.0 mm, — stationary phase : octadecylsilyl silica gel for chromatography R (3 µm), — temperature : 45 °C. Mobile phase : — mobile phase A : solution containing 14.54 g/l of disodium hydrogen phosphate R and 3.53 g/l of potassium dihydrogen phosphate R, M 476.5 — mobile phase B : acetonitrile for chromatography R, r

DEFINITION Sodium (6R,7R)-3-[[(5-methyl-1,3,4-thiadiazol-2yl)sulphanyl]methyl]-8-oxo-7-[(1H-tetrazol-1-ylacetyl)amino]5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate. Content : 95.0 per cent to 102.0 per cent (anhydrous substance). CHARACTERS Appearance : white or almost white powder, very hygroscopic. Solubility : freely soluble in water, very slightly soluble in alcohol. It shows polymorphism. IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Preparation : dissolve 0.150 g in 5 ml of water R, add 0.5 ml of dilute acetic acid R, swirl and allow to stand for 10 min in iced water. Filter the precipitate and rinse with 1-2 ml of water R. Dissolve in a mixture of 1 volume of water R and 9 volumes of acetone R. Evaporate the solvent almost to dryness, then dry in an oven at 60 °C for 30 min. Comparison : cefazolin CRS. B. It gives reaction (a) of sodium (2.3.1). TESTS Solution S. Dissolve 2.50 g in carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and its absorbance (2.2.25) at 430 nm has a maximum of 0.15. pH (2.2.3) : 4.0 to 6.0 for solution S. Specific optical rotation (2.2.7) : − 15 to − 24 (anhydrous substance). Dissolve 1.25 g in water R and dilute to 25.0 ml with the same solvent. Absorbance (2.2.25). Dissolve 0.100 g in water R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the solution to 100.0 ml with sodium hydrogen carbonate solution R. Examined between 220 nm and 350 nm, the solution shows an absorption maximum at 272 nm. The specific absorbance at the maximum is 260 to 300 (anhydrous substance). Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 50.0 mg of the substance to be examined in mobile phase A and dilute to 20.0 ml with the same mobile phase. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A. General Notices (1) apply to all monographs and other texts

Time (min)



λ (nm)

0-1

98

2

210

1-2

98

2

254

2-4

98 → 85

2 → 15

254

4 - 10

85 → 60

15 → 40

254

10 - 11.5

60 → 35

40 → 65

254

11.5 - 12

35

65

254

12 - 15

35 → 98

65 → 2

254

15 - 16

98

2

254

16 - 21

98

2

210

Flow rate : 1.2 ml/min. Detection : spectrophotometer at 210 nm and at 254 nm (see table above). Injection : 5 µl. System suitability : reference solution (b) : — resolution : minimum 2.0 between the peaks due to cefazolin and to impurity I (see Figure 0988.-1). Limits : — any impurity (seen at 210 nm or 254 nm) : not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (1.0 per cent), — total : not more than 3.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (3.5 per cent), — disregard limit : 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm. Water (2.5.12) : maximum 6.0 per cent, determined on 0.300 g. Bacterial endotoxins (2.6.14) : less than 0.15 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. ASSAY Liquid chromatography (2.2.29). Test solution. Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (a). Dissolve 50.0 mg of cefazolin CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (b). Dissolve 5.0 mg of cefuroxime sodium CRS in 10.0 ml of reference solution (a) and dilute to 100.0 ml with the mobile phase. 1209

Cefazolin sodium


1. impurity F

3. impurity E

5. cefazolin

2. impurity J

4. unknown

6. impurity I

Figure 0988.-1. – Chromatogram of reference solution (b) (in situ degradation) for the test for related substances of cefazolin sodium

Column :

IMPURITIES

— size : l = 0.25 m, Ø = 4.6 mm, — stationary phase: octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : mix 10 volumes of acetonitrile R and 90 volumes of a solution containing 2.77 g/l of disodium hydrogen phosphate R and 1.86 g/l of citric acid R. Flow rate : 1.0 ml/min.

A. R = H : (6R,7R)-7-amino-3-[[(5-methyl-1,3,4-thiadiazol-2yl)sulphanyl]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2ene-2-carboxylic acid,

Detection : spectrophotometer at 270 nm. Injection : 20 µl.

B. R = CO-C(CH3)3 : (6R,7R)-7-[(2,2-dimethylpropanoyl)amino]-3-[[(5-methyl-1,3,4-thiadiazol-2-yl)sulphanyl]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid,

System suitability : reference solution (b) : — resolution : minimum 2.0 between the peaks due to cefazolin and cefuroxime. Calculate the percentage content of cefazolin sodium by multiplying the percentage content of cefazolin by 1.048. STORAGE In an airtight container, protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins. 1210

C. R = H : (6R,7R)-3-methyl-8-oxo-7-[(1H-tetrazol-1ylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylic acid, D. R = O-CO-CH3 : (6R,7R)-3-[(acetyloxy)methyl]-8-oxo-7-[(1Htetrazol-1-ylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2ene-2-carboxylic acid,


Cefixime



CEFIXIME Cefiximum

E. 5-methyl-1,3,4-thiadiazol-2-thiol (MMTD),

F. (1H-tetrazol-1-yl)acetic acid, C16H15N5O7S2,3H2O

G. (5aR,6R)-6-[(1H-tetrazol-1-ylacetyl)amino]-5a,6-dihydro3H,7H-azeto[2,1-b]furo[3,4-d][1,3]thiazine-1,7(4H)-dione,

Mr 507.5

DEFINITION Cefixime is (6R,7R)-7-[[(Z)-2-(2-aminothiazol-4-yl)-2[(carboxymethoxy)imino]acetyl]amino]-3-ethenyl-8-oxo-5-thia1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid trihydrate. It contains not less than 95.0 per cent and not more than the equivalent of 101.0 per cent of C16H15N5O7S2, calculated with reference to the anhydrous and ethanol-free substance. CHARACTERS A white or almost white powder, slightly hygroscopic, slightly soluble in water, soluble in methanol, sparingly soluble in ethanol, practically insoluble in ethyl acetate.

H. (6R,7R)-3-[(acetyloxy)methyl]-7-amino-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (7-ACA),

IDENTIFICATION Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cefixime CRS. If the spectra obtained show differences, dissolve the substance to be examined and the reference substance separately in methanol R, evaporate to dryness and record new spectra using the residues.

TESTS pH (2.2.3). Suspend 0.5 g in carbon dioxide-free water R I. 2-[carboxy[(1H-tetrazol-1-ylacetyl)amino]methyl]-5-[[(5methyl-1,3,4-thiadiazol-2-yl)sulphanyl]methyl]-5,6-dihydro- and dilute to 10 ml with the same solvent. The pH of the suspension is 2.6 to 4.1. 2H-1,3-thiazine-4-carboxylic acid (cefazoloic acid), Related substances. Examine by liquid chromatography (2.2.29) as described under Assay. Inject reference solution (b). Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is at least 50 per cent of the full scale of the recorder. Inject the test solution and continue the chromatography for 3 times the retention time of the principal peak. In the chromatogram obtained with the test solution : the area of J. 2-[carboxy[(1H-tetrazol-1-ylacetyl)amino]methyl]-5any peak, apart from the principal peak, is not greater than (hydroxymethyl)-5,6-dihydro-2H-1,3-thiazine-4-carboxylic half the area of the principal peak in the chromatogram acid (hydrolysed cefazoloic acid), obtained with reference solution (b) (0.5 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than 3 times the area of the principal peak in the chromatogram obtained with reference solution (b) (3 per cent). Disregard any peak with an area less than 0.1 times that of the principal peak in the chromatogram obtained with reference solution (b). Ethanol (2.4.24). Not more than 1.0 per cent m/m, determined by head-space gas chromatography (2.2.28), using the standard additions method. Sample solution. Dissolve 0.250 g of the substance to be K. (6R,7R)-3-[[(5-methyl-1,3,4-thiadiazol-2examined in a mixture of 1 volume of dimethylacetamide R yl)sulphanyl]methyl]-8-oxo-7-[(1H-tetrazol-1and 4 volumes of water R and dilute to 25.0 ml with the ylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2same mixture of solvents. carboxamide (cefazolinamide). General Notices (1) apply to all monographs and other texts

1211

Cefoperazone sodium


Water (2.5.12) : 9.0 per cent to 12.0 per cent, determined on 0.200 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.2 per cent, determined on 1.0 g. ASSAY Examine by liquid chromatography (2.2.29). Test solution. Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the mobile phase. Reference solution (a). Dissolve 25.0 mg of cefixime CRS in the mobile phase and dilute to 25.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 100.0 ml with the mobile phase. Reference solution (c). Dissolve 10 mg of cefixime CRS in 10 ml of water R. Heat on a water-bath for 45 min. Cool and inject immediately. The chromatographic procedure may be carried out using : — a column 0.125 m long and 4 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.0 ml/min a mixture of 250 volumes of acetonitrile R and 750 volumes of a tetrabutylammonium hydroxide solution prepared as follows : dissolve 8.2 g of tetrabutylammonium hydroxide R in water R and dilute to 800 ml with the same solvent ; adjust to pH 6.5 with dilute phosphoric acid R and dilute to 1000 ml with water R, — as detector a spectrophotometer set at 254 nm, — a 10 µl loop injector, maintaining the temperature of the column at 40 °C. Inject reference solution (c). Adjust the sensitivity of the system so that the heights of the principal peaks are at least 20 per cent of the full scale of the recorder. The test is not valid unless the resolution between the 2 principal peaks (cefixime and E-isomer) is at least 2.0. If necessary, adjust the concentration of acetonitrile in the mobile phase. Inject reference solution (a) 6 times. The test is not valid unless the relative standard deviation of the peak area for cefixime is at most 1.0 per cent. Inject alternately the test solution and reference solution (a).

C. (6R,7S)-7-[[(Z)-2-(2-aminothiazol-4-yl)-2[(carboxymethoxy)imino]acetyl]amino]-3-ethenyl8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (cefixime 7-epimer),

D. (6R,7R)-7-[[(E)-2-(2-aminothiazol-4-yl)-2[(carboxymethoxy)imino]acetyl]amino]-3-ethenyl8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (cefixime E-isomer),

E. R = H, R′ = CH3 : (6R,7R)-7-[[(Z)-2-(2-aminothiazol-4-yl)-2[(carboxymethoxy)imino]acetyl]amino]-3-methyl-8-oxo-5thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, F. R = C2H5, R′ = CH=CH2 : (6R,7R)-7-[[(Z)-2-(2-aminothiazol4-yl)-2-[(2-ethoxy-2-oxoethoxy)imino]acetyl]amino]3-ethenyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylic acid. 01/2005:1404

CEFOPERAZONE SODIUM Cefoperazonum natricum


C25H26N9NaO8S2

Mr 668

A. R = CO2H : 2-[[(Z)-2-(2-aminothiazol-4-yl)-2[(carboxymethoxy)imino]acetyl]amino]-2-[(2R)-5methyl-7-oxo-1,2,5,7-tetrahydro-4H-furo[3,4-d][1,3]thiazin2-yl]acetic acid,

DEFINITION Cefoperazone sodium contains not less than 95.0 per cent and not more than the equivalent of 102.0 per cent of sodium (6R,7R)-7-[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1yl)carbonyl]amino]-2-(4-hydroxyphenyl)acetyl]amino]-3-[[(1methyl-1H-tetrazol-5-yl)sulphanyl]methyl]-8-oxo-5-thia-1B. R = H : 2-[[[(Z)-1-(2-aminothiazol-4-yl)-2-[[[(2R,5RS)-5methyl-7-oxo-1,2,5,7-tetrahydro-4H-furo[3,4-d][1,3]thiazin- azabicyclo[4.2.0]oct-2-ene-2-carboxylate, calculated with 2-yl]methyl]amino]-2-oxoethylidene]amino]oxy]acetic acid, reference to the anhydrous and acetone-free substance.

1212


Cefoperazone sodium


— transfer-line temperature : 110 °C, maintaining the temperature of the column at 40 °C for 10 min. Heavy metals (2.4.8). 2.0 g complies with limit test C (5 ppm). Prepare the standard using 1 ml of lead standard IDENTIFICATION solution (10 ppm Pb) R. A. Dissolve the substance to be examined in methanol R and Water (2.5.12). Not more than 5.0 per cent, determined on evaporate to dryness. Examine by infrared absorption 0.200 g. spectrophotometry (2.2.24), comparing with the Ph. Eur. Bacterial endotoxins (2.6.14) : less than 0.20 IU/mg, if reference spectrum of cefoperazone sodium. intended for use in the manufacture of parenteral dosage B. Examine the chromatograms obtained in the assay. forms without a further appropriate procedure for the The retention time and size of the principal peak in removal of bacterial endotoxins. the chromatogram obtained with test solution (a) are approximately the same as those of the principal peak in ASSAY the chromatogram obtained with reference solution (a). Examine by liquid chromatography (2.2.29). Prepare the C. It gives reaction (a) of sodium (2.3.1). solutions immediately before use. Test solution (a). Dissolve 25.0 mg of the substance to be TESTS examined in the mobile phase and dilute to 250.0 ml with Appearance of solution. Dissolve 2.5 g in water R and the mobile phase. dilute to 25.0 ml with the same solvent. The solution is clear (2.2.1). The absorbance of the solution measured at 430 nm Test solution (b). Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the (2.2.25) is not greater than 0.15. mobile phase. pH (2.2.3). Dissolve 2.5 g in carbon dioxide-free water R Reference solution (a). Dissolve 25.0 mg of cefoperazone and dilute to 10 ml with the same solvent. The pH of the dihydrate CRS in the mobile phase and dilute to 250.0 ml solution is 4.5 to 6.5. with the mobile phase. Related substances. Examine by liquid chromatography (2.2.29) as prescribed under Assay. Inject 20 µl of reference Reference solution (b). Dilute 5.0 ml of reference solution (a) to 100.0 ml with the mobile phase. solution (b) and adjust the sensitivity of the system so that the height of the principal peak in the chromatogram The chromatographic procedure may be carried out using : obtained is at least 50 per cent of the full scale of the — a stainless steel column 0.15 m long and 4.6 mm in recorder. Inject 20 µl of test solution (b). Continue the internal diameter packed with end-capped octadecylsilyl chromatography for at least 2.5 times the retention time silica gel for chromatography R (5 µm), of the principal peak. In the chromatogram obtained with — as mobile phase at a flow rate of 1 ml/min a mixture of test solution (b) : the area of any peak, apart from the 884 volumes of water R ; 110 volumes of acetonitrile R ; principal peak, is not greater than 1.5 times the area of the 3.5 volumes of a 60 g/l solution of acetic acid R ; principal peak in the chromatogram obtained with reference 2.5 volumes of a triethylammonium acetate solution solution (b) (1.5 per cent) ; the sum of the areas of any prepared by diluting 14 ml of triethylamine R and 5.7 ml such peaks is not greater than 4.5 times the area of the of glacial acetic acid R to 100 ml with water R, principal peak in the chromatogram obtained with reference solution (b) (4.5 per cent). Disregard any peak with an area — as detector a spectrophotometer set at 254 nm. less than 0.1 times the area of the principal peak in the Inject 20 µl of reference solution (a). When the chromatogram obtained with reference solution (b). chromatogram is recorded in the prescribed conditions, the Acetone. Not more than 2.0 per cent, determined by retention time is about 15 min for cefoperazone. Adjust the head-space gas chromatography (2.2.28), using the method sensitivity of the system so that the height of the principal of standard additions. peak in the chromatogram obtained is at least 50 per cent of the full scale of the recorder. The test is not valid unless Sample solution. Dissolve 0.500 g of the substance to be in the chromatogram obtained with reference solution (a), examined in water R and dilute to 10.0 ml with the same the number of theoretical plates calculated for the principal solvent. peak is at least 5000 and its symmetry factor is at most 1.6. If Solvent solution. Dissolve 0.350 g of acetone R in water R necessary adjust the content of acetonitrile R in the mobile and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml phase. Inject reference solution (a) 6 times. The test is not of the solution to 100.0 ml with water R. valid unless the relative standard deviation of the peak area Prepare each of 4 injection vials as shown in the table below : is at most 1.0 per cent. Inject alternately test solution (a) and reference solution (a). Water R Vial No. Sample solution Solvent solution Calculate the percentage content of cefoperazone sodium by (ml) (ml) (ml) multiplying the percentage content of cefoperazone by 1.034. 0 4.0 1 1.0 CHARACTERS A white or slightly yellow powder, hygroscopic, freely soluble in water, soluble in methanol, slightly soluble in alcohol. If crystalline, it shows polymorphism.

2

1.0

1.0

3.0

3

1.0

2.0

2.0

4

1.0

3.0

1.0

The chromatographic procedure may be carried out using system B of the test for residual solvents (2.4.24) and the following static head-space injection conditions : — equilibration time : 15 min, General Notices (1) apply to all monographs and other texts

STORAGE In an airtight container, protected from light, at a temperature of 2 °C to 8 °C. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins. 1213

Cefotaxime sodium


IMPURITIES

A. (5aR,6R)-6-[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1yl)carbonyl]amino]-2-(4-hydroxyphenyl)acetyl]amino]-5a, 6-dihydro-3H,7H-azeto[2,1-b]furo[3,4-d][1,3]thiazine-1, 7(4H)-dione,

F. (6R,7S)-7-[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazine-1yl)carbonyl]amino]-2-(4-hydroxyphenyl)acetyl]amino]-3[[(1-methyl-1H-tetrazol-5-yl)sulphanyl]methyl]-8-oxo-5-thia1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid.


CEFOTAXIME SODIUM Cefotaximum natricum

B. (6R,7R)-7-[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1yl)carbonyl]amino]-2-(4-hydroxyphenyl)acetyl]amino]-3[(4-methyl-5-thioxo-4,5-dihydro-1H-tetrazol-1-yl)methyl]-8oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid,

C16H16N5NaO7S2

Mr 477.4

DEFINITION Cefotaxime sodium contains not less than 96.0 per cent and not more than the equivalent of 101.0 per cent of sodium (6R,7R)-3-[(acetyloxy)methyl]-7-[[(Z)-2-(2aminothiazol-4-yl)-2-(methoxyimino)acetyl]amino]-8-oxo-5thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate, calculated with reference to the dried substance. CHARACTERS A white or slightly yellow powder, hygroscopic, freely soluble in water, sparingly soluble in methanol.

C. 1-methyl-1H-tetrazole-5-thiol,

IDENTIFICATION A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cefotaxime sodium CRS. B. It gives reaction (a) of sodium (2.3.1). D. (6R,7R)-7-amino-8-oxo-3-[(1H-1,2,3-triazol-4ylsulphanyl)methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene2-carboxylic acid (7-TACA),

E. (6R,7R)-3-[(acetyloxy)methyl]-7-amino-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (7-ACA), 1214

TESTS Solution S. Dissolve 2.5 g in carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1). Add 1 ml of glacial acetic acid R to 10 ml of solution S. The solution, examined immediately, is clear. The absorbance (2.2.25) of solution S measured at 430 nm is not greater than 0.20. pH (2.2.3). The pH of solution S is 4.5 to 6.5. Specific optical rotation (2.2.7). Dissolve 0.100 g in water R and dilute to 10.0 ml with the same solvent. The specific optical rotation is + 58 to + 64, calculated with reference to the dried substance.


Cefotaxime sodium


Absorbance (2.2.25). Dissolve 20.0 mg in water R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of this solution to 100.0 ml with water R. The specific absorbance determined at the maximum at 235 nm is 360 to 390, calculated with reference to the dried substance. Related substances. Examine by liquid chromatography (2.2.29) as described under Assay. Inject the test solution and reference solution (b). Continue the chromatography for at least eight times the retention time of the main peak. In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent) ; the sum of the areas of such peaks is at most three times the area of the principal peak in the chromatogram obtained with reference solution (b) (3 per cent).

adjust the methanol content of the mobile phase. The test is not valid if the symmetry factor of the cefotaxime peak is greater than 2.0. Inject reference solution (a) six times. The test is not valid unless the relative standard deviation of the area of the cefotaxime peak is at most 1.0 per cent. Inject alternately the test solution and reference solution (a). STORAGE Store in an airtight container, protected from light at a temperature not exceeding 30 °C. If the substance is sterile, store in an sterile, airtight, tamper-proof container. LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins. IMPURITIES

N,N-Dimethylaniline (2.4.26, Method B). Not more than 20 ppm. 2-Ethylhexanoic acid (2.4.28). Not more than 0.5 per cent m/m. Loss on drying (2.2.32). Not more than 3.0 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Bacterial endotoxins (2.6.14) : less than 0.05 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for removal of bacterial endotoxins.

A. R = R′ = H : (6R,7R)-7-[[(Z)-2-(2-aminothiazol-4yl)-2-methoxyimino)acetyl]amino]-3-methyl-8-oxo-5thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (desacetoxycefotaxime),

ASSAY

B. R = OH, R′ = H : (6R,7R)-7-[[(Z)-2-(2-aminothiazol-4yl)-2-methoxyimino)acetyl]amino]-3-(hydroxymethyl)-8oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (desacetylcefotaxime),

Examine by liquid chromatography (2.2.29). Test solution. Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the same solvent. Reference solution (a). Dissolve 25.0 mg of cefotaxime sodium CRS in the mobile phase and dilute to 25.0 ml with the same solvent. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 100.0 ml with the mobile phase.

C. R = O-CO-CH3, R′ = CHO : (6R,7R)-3-[(acetyloxy)methyl]7-[[(Z)-2-[2-(formylamino)thiazol-4-yl]-2(methoxyimino)acetyl]amino]-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (N-formylcefotaxime),

Reference solution (c). Add 1.0 ml of dilute hydrochloric acid R to 4.0 ml of the test solution. Heat the solution at 40 °C for 2 h. Add 5.0 ml of buffer solution pH 6.6 R and 1.0 ml of dilute sodium hydroxide solution R. The chromatographic procedure may be carried out using : — a column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.0 ml/min a mixture prepared as follows : dissolve 3.5 g of potassium dihydrogen phosphate R and 11.6 g of disodium hydrogen phosphate R in 1000 ml of water R at pH 7.0 and add 180 ml of methanol R,

D. (6R,7R)-3-[(acetyloxy)methyl]-7-[[(E)-2-(2-aminothiazol4-yl)-2-(methoxyimino)acetyl]amino]-8-oxo-5-thia1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (E-cefotaxime),

— as detector a spectrophotometer set at 235 nm, — an injector with a fixed loop of 10 µl. Inject reference solution (a) and reference solution (c). Adjust the attenuation to obtain principal peaks from reference solution (c) with a height of at least 50 per cent of the full-scale deflection of the recorder. The test is not valid unless cefotaxime is eluted as the second of the principal peaks and the resolution between the two principal peaks is at least 3.5. If necessary, use another stationary phase or General Notices (1) apply to all monographs and other texts

E. (5aR,6R)-6-[[(Z)-2-(2-aminothiazol-4-yl)-2(methoxyimino)acetyl]amino]-5a,6-dihydro-3H,7Hazeto[2,1-b]furo[3,4-d][1,3]thiazine-1,7(4H)-dione (deacetylcefotaxime lactone). 1215

Cefoxitin sodium

CEFOXITIN Cefoxitinum

C16H16N3NaO7S2


01/2005:0990 Reference solution (b). To 1.0 ml of the test solution add 7.0 ml of water R and 2.0 ml of methanol R. Add 25 mg of sodium carbonate R, stir for 10 min at room temperature, SODIUM then heat in a water-bath at 70 °C for 30 min. Allow to cool. Add 3 drops of glacial acetic acid R and 1 ml of the test natricum solution and mix. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : phenylsilyl silica gel for chromatography R (5 µm) with a specific surface area of 300 m2/g and a pore size of 7 nm. Mobile phase : — mobile phase A : water R adjusted to pH 2.7 with Mr 449.4 anhydrous formic acid R, — mobile phase B : acetonitrile R,

DEFINITION Sodium (6R,7S)-3-[(carbamoyloxy)methyl]-7-methoxy-8-oxo-7[[(thiophen-2-yl)acetyl]amino]-5-thia-1-azabicyclo[4.2.0]oct-2ene-2-carboxylate. Content : 95.0 per cent to 102.0 per cent (anhydrous substance). CHARACTERS Appearance : white or almost white powder, very hygroscopic. Solubility : very soluble in water, sparingly soluble in alcohol. IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Comparison : cefoxitin sodium CRS. B. It gives reaction (a) of sodium (2.3.1).

TESTS Solution S. Dissolve 2.50 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than intensity 5 of the range of reference solutions of the most appropriate colour (2.2.2, Method II). pH (2.2.3) : 4.2 to 7.0. Dilute 2 ml of solution S to 20 ml with carbon dioxide-free water R. Specific optical rotation (2.2.7) : + 206 to + 214 (anhydrous substance). Dissolve 0.250 g in methanol R and dilute to 25.0 ml with the same solvent. Absorbance (2.2.25). Dissolve 0.100 g in water R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the solution to 100.0 ml with sodium hydrogen carbonate solution R. Examined between 220 nm and 350 nm, the solution shows an absorption maximum at 236 nm and a broad absorption maximum at about 262 nm. The specific absorbance at this broad maximum is 190 to 210 (anhydrous substance). Related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately before use. Solution A. Dilute 20 ml of a 34.8 g/l solution of dipotassium hydrogen phosphate R adjusted to pH 6.8 with phosphoric acid R to 1000 ml with water R. Test solution. Dissolve 50.0 mg of the substance to be examined in solution A and dilute to 10.0 ml with the same solution. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with solution A. 1216

Time (min)



0 - 12

90

10

12 - 37

90 → 80

10 → 20

37 - 50

80 → 60

20 → 40

50 - 55

60 → 20

40 → 80

55 - 60

20

80

60 - 62

20 → 90

80 → 10

62 - 70

90

10

Flow rate : 1 ml/min. Detection : spectrophotometer at 235 nm. Injection : 50 µl. Relative retentions with reference to cefoxitin (retention time = about 34 min) : impurity A = about 0.82 ; impurity B = about 1.16 ; impurity C = about 1.27 ; impurity D = about 1.31. System suitability : reference solution (b) : — resolution : minimum 5.0 between the 2 principal peaks. Limits : — any impurity : not more than half the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent), — total : not more than 4 times the area of the principal peak in the chromatogram obtained with reference solution (a) (4.0 per cent), — disregard limit : 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). Water (2.5.12) : maximum 1.0 per cent, determined on 0.500 g. Bacterial endotoxins (2.6.14) : less than 0.13 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. ASSAY Liquid chromatography (2.2.29). Test solution. Dissolve 25.0 mg of the substance to be examined in water R and dilute to 25.0 ml with the same solvent. Reference solution (a). Dissolve 25.0 mg of cefoxitin sodium CRS in water R and dilute to 25.0 ml with the same solvent. Reference solution (b). Dissolve 20.0 mg of 2-(2-thienyl)acetic acid R in water R and dilute to 25.0 ml with the same solvent.


Cefradine


Reference solution (c). Mix 1.0 ml of reference solution (a) and 5.0 ml of reference solution (b). Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase: octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : acetic acid R, acetonitrile R, water R (1:19:81 V/V/V). Flow rate : 1 ml/min. Detection : spectrophotometer at 254 nm. Injection : 20 µl ; inject the test solution and reference solutions (a) and (c). System suitability : reference solution (c) : — resolution : minimum 3.5 between the 2 principal peaks. Calculate the percentage content of cefoxitin sodium. STORAGE In an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins. IMPURITIES


CEFRADINE Cefradinum

C16H19N3O4S

Mr 349.4

DEFINITION Cefradine contains not less than 90.0 per cent of (6R,7R)7-[[(2R)-2-amino-2-(cyclohexa-1,4-dienyl)acetyl]amino]-3methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, calculated with reference to the anhydrous substance. The sum of the percentage contents of C16H19N3O4S and of cefalexin (C16H17N3O4S ; Mr 347.4) is not less than 95.0 per cent and not more than the equivalent of 102.0 per cent, calculated with reference to the anhydrous substance. CHARACTERS A white or slightly yellow, hygroscopic powder, sparingly soluble in water, practically insoluble in alcohol. IDENTIFICATION

A. (6R,7S)-3-(hydroxymethyl)-7-methoxy-8-oxo-7-[[(thiophen2-yl)acetyl]amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylic acid (decarbamoylcefoxitin),

B. (2RS,6R,7S)-3-[(carbamoyloxy)methyl]-7-methoxy8-oxo-7-[[(thiophen-2-yl)acetyl]amino]-5-thia1-azabicyclo[4.2.0]oct-3-ene-2-carboxylic acid (delta-3-cefoxitin),

C. R = H : (5aR,6R)-6-[[(thiophen-2-yl)acetyl]amino]-5a,6dihydro-3H,7H-azeto[2,1-b]furo[3,4-d][1,3]thiazine-1, 7(4H)-dione (cefalotin lactone), D. R = OCH3 : (5aR,6S)-6-methoxy-6-[[(thiophen-2yl)acetyl]amino]-5a,6-dihydro-3H,7H-azeto[2,1-b]furo[3,4d][1,3]thiazine-1,7(4H)-dione (cefoxitin lactone). General Notices (1) apply to all monographs and other texts

Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cefradine CRS. If the spectra obtained in the solid state show differences, dissolve 30 mg of the substance to be examined and of the reference substance separately in 10 ml of methanol R, evaporate to dryness at 40 °C at a pressure less than 2 kPa and record new spectra using the residues. TESTS Solution S. Dissolve 2.50 g in sodium carbonate solution R and dilute to 25.0 ml with the same solvent. Appearance of solution. Solution S is not more opalescent than reference suspension II (2.2.1). Allow solution S to stand for 5 min. The absorbance of solution S measured at 450 nm (2.2.25) is not greater than 0.60. pH (2.2.3). Dissolve 0.100 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. The pH of the solution is 3.5 to 6.0. Specific optical rotation (2.2.7). Dissolve 0.250 g in acetate buffer solution pH 4.6 R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 80 to + 90, calculated with reference to the anhydrous substance. Absorbance (2.2.25). Dissolve 50.0 mg in water R and dilute to 100.0 ml with the same solvent. The absorbance measured at 330 nm is not greater than 0.05. Dilute 2.0 ml of the solution to 50.0 ml with water R. Examined between 220 nm and 300 nm, the diluted solution shows an absorption maximum at 262 nm. The specific absorbance at this maximum is 215 to 240, calculated with reference to the anhydrous substance. Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Impregnate the plate by development with a 5 per cent V/V 1217

Ceftazidime


Reference solution (b). Dissolve 10.0 mg of cefradine CRS and 10.0 mg of cefalexin CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. The chromatographic procedure may be carried out using : Test solution. Dissolve 0.25 g of the substance to be examined in dilute hydrochloric acid R and dilute to 10 ml — a column 0.25 m long and 4.6 mm in internal with the same acid. diameter, packed with octadecylsilyl silica gel for chromatography R (5 µm or 10 µm), Reference solution (a). Dilute 1 ml of the test solution to — as mobile phase at a flow rate of 1.0 ml/min a mixture of 100 ml with dilute hydrochloric acid R. 1 volume of dilute acetic acid R, 17 volumes of a 36.2 g/l Reference solution (b). Dissolve 25 mg of solution of sodium acetate R, 200 volumes of methanol R 7-aminodesacetoxycephalosporanic acid CRS in and 782 volumes of water R, dilute hydrochloric acid R and dilute to 10 ml with the same — as detector a spectrophotometer set at 254 nm, acid (reference solution (b′)). Dilute 1 ml of this solution to — a 20 µl loop injector. 10 ml with dilute hydrochloric acid R. Inject reference solution (b). Adjust the sensitivity of the Reference solution (c). Dissolve 25 mg of detector so that the height of the peaks is at least half the cyclohexa-1,4-dienylglycine CRS in dilute hydrochloric full scale of the recorder. The test is not valid unless the acid R and dilute to 10 ml with the same acid (reference resolution between the peaks corresponding to cefalexin solution (c′)). Dilute 1 ml of this solution to 10 ml with and cefradine is at least 4. If necessary, adjust the methanol dilute hydrochloric acid R. content of the mobile phase. Inject reference solution (a) Reference solution (d). Dissolve 0.25 g of the substance to 6 times. The test is not valid unless the relative standard be examined in a mixture of 1 ml of reference solution (b′) deviation of the peak area for cefradine is at most 1.0 per and 1 ml of reference solution (c′) and dilute to 10 ml with cent. Inject separately the test solution and reference dilute hydrochloric acid R. solution (a). Apply to the plate 5 µl of each solution. Develop over a Calculate the percentage content of cefradine and cefalexin. path of 15 cm using a mixture of 3 volumes of acetone R, STORAGE 80 volumes of a 72 g/l solution of disodium hydrogen phosphate R and 120 volumes of a 21 g/l solution of citric Store in an airtight container, protected from light, at a acid R. Dry the plate by heating at 90 °C for 3 min. Spray the temperature of 2 °C to 8 °C. hot plate with a 1 g/l solution of ninhydrin R in the mobile phase. Heat the plate at 90 °C for 15 min and allow to cool. 01/2005:1405 In the chromatogram obtained with the test solution : any spot corresponding to 7-aminodesacetoxycephalosporanic acid is not more intense than the spot in the chromatogram CEFTAZIDIME obtained with reference solution (b) (1.0 per cent) ; any spot corresponding to cyclohexa-1,4-dienylglycine (the Ceftazidimum position of which is defined by comparison with the chromatogram obtained with reference solution (d)), is not more intense than the spot in the chromatogram obtained with reference solution (c) (1.0 per cent) ; any spot, apart from the principal spot and the spots corresponding to 7-aminodesacetoxycephalosporanic acid and to cyclohexa-1,4-dienylglycine, is not more intense than the principal spot in the chromatogram obtained with reference solution (a) (1.0 per cent). The test is not valid unless the chromatogram obtained with reference solution (d) shows 3 clearly separated spots. C22H22N6O7S2,5H2O Mr 637 Cefalexin. Not more than 5.0 per cent, calculated with DEFINITION reference to the anhydrous substance and determined by Ceftazidime is (6R,7R)-7-[[(Z)-2-(2-aminothiazol-4-yl)-2liquid chromatography (2.2.29), as prescribed under Assay. Inject separately the test solution and reference solution (b). [(1-carboxy-1-methylethoxy)imino]acetyl]amino]-8-oxo-3[(1-pyridinio)methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2N,N-Dimethylaniline (2.4.26, Method B). Not more than carboxylate pentahydrate. It contains not less than 95.0 per 20 ppm. cent and not more than 102.0 per cent of C22H22N6O7S2, calculated with reference to the anhydrous substance. Water (2.5.12). Not more than 6.0 per cent, determined on 0.300 g by the semi-micro determination of water. CHARACTERS Sulphated ash (2.4.14). Not more than 0.2 per cent, A white or almost white, crystalline powder, slightly soluble determined on 1.0 g. in water and in methanol, practically insoluble in acetone and in alcohol. It dissolves in acid and alkali solutions. ASSAY IDENTIFICATION Examine by liquid chromatography (2.2.29). Examine by infrared absorption spectrophotometry Test solution. Dissolve 50.0 mg of the substance to be (2.2.24), comparing with the spectrum obtained with examined in the mobile phase and dilute to 100.0 ml with ceftazidime CRS. the mobile phase. Reference solution (a). Dissolve 50.0 mg of cefradine CRS TESTS in the mobile phase and dilute to 100.0 ml with the mobile Solution S. Dissolve 0.25 g in carbon dioxide-free water R phase. and dilute to 50 ml with the same solvent. solution of tetradecane R in hexane R. Allow the solvent to evaporate and carry out the chromatography in the same direction as the impregnation.

1218



Ceftazidime

Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). pH (2.2.3). The pH of solution S is 3.0 to 4.0. Related substances A. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R. Test solution. Dissolve 0.100 g of the substance to be examined in a 36 g/l solution of disodium hydrogen phosphate R and dilute to 2.0 ml with the same solution. Reference solution. Dilute 1 ml of the test solution to 200 ml with a 36 g/l solution of disodium hydrogen phosphate R. Apply to the plate 2 µl of each solution. Develop over a path of 15 cm using a mixture of 6 volumes of butanol R, 26 volumes of sodium acetate buffer solution pH 4.5 R, 32 volumes of butyl acetate R and 32 volumes of glacial acetic acid R. Dry the plate in a current of warm air and examine in ultraviolet light at 254 nm. Any spot with an Rf value greater than that of the principal spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution (0.5 per cent). B. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 0.100 g of the substance to be examined in the mobile phase and dilute to 20.0 ml with the mobile phase. Dilute 5.0 ml of the solution to 20.0 ml with the mobile phase. Reference solution (a). Dissolve 5.0 mg of ceftazidime impurity A CRS in the mobile phase and dilute to 20.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 20.0 ml with the mobile phase. Reference solution (b). Dissolve 5 mg of ceftazidime impurity A CRS and 5 mg of ceftazidime CRS in the mobile phase and dilute to 20.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 20.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.3 ml/min a mixture of 7 volumes of acetonitrile R and 93 volumes of a 22.6 g/l solution of ammonium dihydrogen phosphate R, adjusted to pH 3.9 with a 10 per cent V/V solution of phosphoric acid R, — as detector a spectrophotometer set at 255 nm, maintaining the temperature of the column at 35 °C. Inject 20 µl of reference solution (b). Adjust the sensitivity of the system so that the heights of the 2 peaks in the chromatogram obtained are at least 50 per cent of the full scale of the recorder. The test is not valid unless in the chromatogram obtained, the resolution between the peaks corresponding to ceftazidime and impurity A is at least 5.9. Inject 20 µl of the test solution and 20 µl of reference solution (a). Continue the chromatography of the test solution for 3 times the retention time of ceftazidime. In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than half the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than twice the area of the principal peak in the chromatogram

obtained with reference solution (a) (2 per cent). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (a). Impurity F. Not more than 500 ppm, determined by liquid chromatography (2.2.29). Prepare the solutions immediately before use. Test solution. Dissolve 0.500 g of the substance to be examined in a 10 per cent V/V solution of phosphate buffer solution pH 7.0 R4 and dilute to 100.0 ml with the same solvent. Reference solution (a). Dissolve 1.00 g of pyridine R in water R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of the solution to 200.0 ml with water R. To 1.0 ml of the solution, add 10 ml of phosphate buffer solution pH 7.0 R4 and dilute to 100.0 ml with water R. Reference solution (b). Dilute 1.0 ml of the test solution to 200.0 ml with a 10 per cent V/V solution of phosphate buffer solution pH 7.0 R4. To 1.0 ml of the solution add 20 ml of reference solution (a) and dilute to 200 ml with a 10 per cent V/V solution of phosphate buffer solution pH 7.0 R4. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.0 ml/min a mixture of 8 volumes of a 28.8 g/l solution of ammonium dihydrogen phosphate R previously adjusted to pH 7.0 with ammonia R, 24 volumes of acetonitrile R and 68 volumes of water R, — as detector a spectrophotometer set at 255 nm, Inject 20 µl of reference solution (b). The test is not valid unless in the chromatogram obtained, the resolution between the peaks due to ceftazidime and to impurity F is at least 7.0. Inject 20 µl of reference solution (a). Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is at least 50 per cent of the full scale of the recorder. Inject reference solution (a) 6 times. The test is not valid unless the relative standard deviation of the area of the principal peak is at most 1.0 per cent. Inject alternately 20 µl of the test solution and 20 µl of reference solution (a). Water (2.5.12) : 13.0 per cent to 15.0 per cent, determined on 0.200 g by the semi-micro determination of water. Bacterial endotoxins (2.6.14) : less than 0.10 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins.


1219

ASSAY Examine by liquid chromatography (2.2.29). Test solution. Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the mobile phase. Reference solution (a). Dissolve 25.0 mg of ceftazidime CRS in the mobile phase and dilute to 25.0 ml with the mobile phase. Reference solution (b). Dissolve 5 mg of ceftazidime impurity A CRS in 5.0 ml of reference solution (a). The chromatographic procedure may be carried out using : — a column 0.15 m long and 4.6 mm in internal diameter packed with hexylsilyl silica gel for chromatography R (5 µm),

Ceftriaxone sodium


— as mobile phase at a flow rate of 2 ml/min a mixture prepared as follows : dissolve 4.26 g of disodium hydrogen phosphate R and 2.73 g of potassium dihydrogen phosphate R in 980 ml of water R, then add 20 ml of acetonitrile R, — as detector a spectrophotometer set at 245 nm. Inject 20 µl of reference solution (b). Adjust the sensitivity of the system so that the heights of the 2 principal peaks in the chromatogram obtained are at least 50 per cent of the full scale of the recorder. The test is not valid unless, in the chromatogram obtained, the resolution between the peaks D. (6R,7R)-7-[[(Z)-2-[[2-(1,1-dimethylethoxy)-1,1-dimethyl-2corresponding to ceftazidime and impurity A is at least 1.0. oxoethoxy]imino]-2-[2-[(triphenylmethyl)amino]thiazolInject alternately the test solution and reference solution (a). 4-yl]acetyl]amino]-8-oxo-3-(pyridiniomethyl)-5-thia-1Calculate the percentage content of ceftazidime. azabicyclo[4.2.0]oct-2-ene-2-carboxylate, STORAGE Store in an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins. IMPURITIES By liquid chromatography (related substances test) : A, B, C. E. (6R,7R)-7-[[(Z)-2-(2-ammoniothiazol-4-yl)By thin-layer chromatography (related substances test) : 2-[[2-(1,1-dimethylethoxy)-1,1-dimethyl-2D, E. oxoethoxy]imino]acetyl]amino]-8-oxo-3-(pyridiniomethyl)By liquid chromatography (impurity F test) : F. 5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate chloride,

F. pyridine. 01/2005:0991 A. (2RS,6R,7R)-7-[[(Z)-2-(2-aminothiazol-4-yl)-2-[(1-carboxy1-methylethoxy)imino]acetyl]amino]-8-oxo-3-[(1pyridinio)methyl]-5-thia-1-azabicyclo[4.2.0]oct-3-ene-2carboxylate (∆-2-ceftazidime),

CEFTRIAXONE SODIUM Ceftriaxonum natricum

C18H16N8Na2O7S3, 31/2H2O B. (6R,7R)-7-[[(E)-2-(2-aminothiazol-4-yl)-2-[(1-carboxy1-methylethoxy)imino]acetyl]amino]-8-oxo-3-[(1pyridinio)methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylate,

C. (6R,7R)-2-carboxy-8-oxo-3-(pyridiniomethyl)-5-thia-1azabicyclo[4.2.0]oct-2-en-7-aminium dichloride, 1220

Mr 662

DEFINITION Disodium (6R,7R)-7-[[(Z)-(2-aminothiazol-4yl)(methoxyimino)acetyl]amino]-3-[[(2-methyl-6-oxido5-oxo-2,5-dihydro-1,2,4-triazin-3-yl)sulphanyl]methyl]-8-oxo-5thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate. Content : 96.0 per cent to 102.0 per cent (anhydrous substance). CHARACTERS Appearance : almost white or yellowish, crystalline powder, slightly hygroscopic. Solubility : freely soluble in water, sparingly soluble in methanol, very slightly soluble in ethanol.


Ceftriaxone sodium


IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Comparison : ceftriaxone sodium CRS. B. It gives reaction (a) of sodium (2.3.1). TESTS Solution S. Dissolve 2.40 g in carbon dioxide-free water R and dilute to 20.0 ml with the same solvent. Appearance of solution. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y5 or BY5 (2.2.2). Dilute 2 ml of solution S to 20 ml with water R. pH (2.2.3) : 6.0 to 8.0 for solution S. Specific optical rotation (2.2.7) : − 155 to − 170 (anhydrous substance). Dissolve 0.250 g in water R and dilute to 25.0 ml with the same solvent. Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 30.0 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (a). Dissolve 30.0 mg of ceftriaxone sodium CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (b). Dissolve 5.0 mg of ceftriaxone sodium CRS and 5.0 mg of ceftriaxone impurity A CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (c). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase: octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : dissolve 2.0 g of tetradecylammonium bromide R and 2.0 g of tetraheptylammonium bromide R in a mixture of 440 ml of water R, 55 ml of 0.067 M phosphate buffer solution pH 7.0 R, 5.0 ml of citrate buffer solution pH 5.0 prepared by dissolving 20.17 g of citric acid R in 800 ml of water R, adjusting topH 5.0 with strong sodium hydroxide solution R and diluting to 1000.0 ml with water R, and 500 ml of acetonitrile R. Flow rate : 1.5 ml/min. Detection : spectrophotometer at 254 nm. Injection : 20 µl ; inject the test solution and reference solutions (b) and (c). Run time : twice the retention time of ceftriaxone. System suitability : reference solution (b) : — resolution : minimum of 3.0 between the peaks due to ceftriaxone and impurity A. Limits : — any impurity : not more than the area of the principal peak in the chromatogram obtained with reference solution (c) (1 per cent), — total: not more than 4 times the area of the principal peak in the chromatogram obtained with reference solution (c) (4 per cent), — disregard limit : 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.1 per cent). N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm. General Notices (1) apply to all monographs and other texts

2-Ethylhexanoic acid (2.4.28) : maximum 0.8 per cent m/m. Water (2.5.12) : 8.0 per cent to 11.0 per cent, determined on 0.100 g. Bacterial endotoxins (2.6.14) : less than 0.20 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances. Injection : test solution and reference solution (a). Calculate the percentage content of ceftriaxone sodium. STORAGE In an airtight container protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins. IMPURITIES

A. (6R,7R)-7-[[(E)-(2-aminothiazol-4-yl)(methoxyimino)acetyl]amino]-3-[[(2-methyl-5,6-dioxo-1,2,5,6-tetrahydro-1,2,4-triazin-3-yl)sulphanyl]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (E-isomer),

B. (5aR,6R)-6-[[(Z)-(2-aminothiazol-4-yl)(methoxyimino)acetyl]amino]-5a,6-dihydro-3H,7H-azeto[2,1-b]furo[3,4-d][1,3]thiazine-1,7(4H)-dione,

C. 2-methyl-3-sulphanyl-1,2-dihydro-1,2,4-triazin-5,6-dione,

D. S-benzothiazol-2-yl (Z)-(2-aminothiazol-4yl)(methoxyimino)thioacetate, 1221

Cefuroxime axetil


Related substances. Examine by liquid chromatography (2.2.29) as described under Assay. Calculate the percentage content of related substances from the areas of the peaks in the chromatogram obtained with the test solution by the normalisation procedure, disregarding any peak with an area less than 0.05 times that of the 2 principal peaks in the chromatogram obtained with reference solution (a). The percentage sum of the pair of peaks corresponding to the E-isomers located by comparison with the chromatogram E. (6R,7R)-7-amino-3-[[(2-methyl-5,6-dioxo-1,2,5,6-tetrahydro- obtained with reference solution (c) is not greater than 1,2,4-triazin-3-yl)sulphanyl]methyl]-8-oxo-5-thia-11.0 per cent, the percentage sum of the pair of peaks azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. corresponding to the ∆3-isomers located by comparison with the chromatogram obtained with reference solution (b) is not greater than 1.5 per cent and the area of any other secondary peak is not greater than 0.5 per cent. The sum of 01/2005:1300 related substances is not greater than 3.0 per cent. Acetone (2.4.24). Not more than 1.1 per cent. Water (2.5.12). Not more than 1.5 per cent, determined on CEFUROXIME AXETIL 0.400 g by the semi-micro determination of water.

Cefuroximum axetili

C20H22N4O10S

Mr 510.5

DEFINITION Cefuroxime axetil contains not less than 96.0 per cent and not more than the equivalent of 102.0 per cent of a mixture of the 2 diastereoisomers of (1RS)-1-(acetyloxy)ethyl (6R,7R)-3-[(carbamoyloxy)methyl]-7-[[(Z)-2-(furan2-yl)-2-(methoxyimino)acetyl]amino]-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylate, calculated with reference to the anhydrous and acetone-free substance. CHARACTERS A white or almost white powder, slightly soluble in water, soluble in acetone, in ethyl acetate and in methanol, slightly soluble in alcohol. IDENTIFICATION A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cefuroxime axetil CRS. B. Examine the chromatograms obtained in the Assay. The retention time and size of the principal peaks in the chromatogram obtained with the test solution are the same as those of the peaks due to diastereoisomers A and B of cefuroxime axetil in the chromatogram obtained with reference solution (d). TESTS Diastereoisomer ratio. Examine by liquid chromatography (2.2.29) as described under Assay. In the chromatogram obtained with the test solution, the ratio of the peak due to cefuroxime axetil diastereoisomer A to the sum of the peaks due to cefuroxime axetil diastereoisomers A and B is between 0.48 and 0.55 by the normalisation procedure. 1222

ASSAY Examine by liquid chromatography (2.2.29). Test solution. Prepare the solution immediately before use. Dissolve 10.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Reference solution (b). Heat 5 ml of the test solution at 60 °C for 1 h to generate the ∆3-isomers. Reference solution (c). Expose 5 ml of the test solution to ultraviolet light at 254 nm for 24 h to generate E-isomers. Reference solution (d). Prepare the solution immediately before use. Dissolve 10.0 mg of cefuroxime axetil CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a column 0.25 m long and 4.6 mm in internal diameter packed with trimethylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.0 ml/min a mixture of 38 volumes of methanol R and 62 volumes of a 23 g/l solution of ammonium dihydrogen phosphate R, — as detector a spectrophotometer set at 278 nm. Inject 20 µl each of reference solutions (a), (b), (c) and (d). When the chromatograms are recorded in the prescribed conditions the retention times relative to cefuroxime axetil diastereoisomer A (second peak) are approximately 0.9 for cefuroxime axetil diastereoisomer B, 1.2 for the cefuroxime axetil ∆3-isomers and 1.7 and 2.1 for the E-isomers. The test is not valid unless in the chromatogram obtained with reference solution (d), the resolution between the peaks corresponding to cefuroxime axetil diastereoisomers A and B is at least 1.5. In the chromatogram obtained with reference solution (b), the resolution between the peaks corresponding to cefuroxime axetil diastereoisomer A and cefuroxime axetil ∆3-isomer is at least 1.5. Inject reference solution (d) solution 6 times. The assay is not valid unless the relative standard deviation of the sum of the peaks corresponding to cefuroxime axetil diastereoisomers A and B is at most 2.0 per cent. Calculate the percentage content of C20H22N4O10S from the sum of areas of the two diastereoisomer peaks and the declared content of C20H22N4O10S in cefuroxime axetil CRS. STORAGE Store in an airtight container, protected from light.



IMPURITIES

Cefuroxime sodium

Solubility : freely soluble in water, very slightly soluble in alcohol. IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Comparison : cefuroxime sodium CRS. B. It gives reaction (a) of sodium (2.3.1).

TESTS Solution S. Dissolve 2.0 g in carbon dioxide-free water R and dilute to 20.0 ml with the same solvent. A. 1-(acetyloxy)ethyl (6R,7R)-3-[(carbamoyloxy)methyl]Appearance of solution. Solution S is not more opalescent 7-[[(Z)-2-(furan-2-yl)-2-(methoxyimino)acetyl]amino]-8than reference suspension II (2.2.1). The absorbance (2.2.25) oxo-5-thia-1-azabicyclo[4.2.0]oct-3-ene-2-carboxylate of solution S measured at 450 nm is not greater than 0.25. (∆3-isomers), pH (2.2.3) : 5.5 to 8.5. Dilute 2 ml of solution S to 20 ml with carbon dioxide-free water R. Specific optical rotation (2.2.7) : + 59 to + 66 (anhydrous substance). Dissolve 0.500 g in acetate buffer solution pH 4.6 R and dilute to 25.0 ml with the same buffer solution. Related substances. Liquid chromatography (2.2.29). Test solution (a). Dissolve 25.0 mg of the substance to be B. (1RS)-1-(acetyloxy)ethyl (6R,7R)-3-[(carbamoyloxy)meexamined in water R and dilute to 25.0 ml with the same thyl]-7-[[(E)-2-(furan-2-yl)-2-(methoxyimino)acetyl]amisolvent. no]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate Test solution (b). Dilute 5.0 ml of test solution (a) to 50.0 ml (E-isomers), with water R. Reference solution (a). Dissolve 25.0 mg of cefuroxime sodium CRS in water R and dilute to 25.0 ml with the same solvent. Dilute 5.0 ml to 50.0 ml with water R. Reference solution (b). Place 20 ml of reference solution (a) in a water-bath at 80 °C for 15 min. Cool and inject immediately. Reference solution (c). Dilute 1.0 ml of test solution (a) to C. R = CO-CCl3 : (6R,7R)-7-[[(Z)-2-(furan-2100.0 ml with water R. yl)-2-(methoxyimino)acetyl]amino]-8-oxo-3Column : [[[(trichloroacetyl)carbamoyl]oxy]methyl]-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, — size : l = 0.125 m, Ø = 4.6 mm, — stationary phase : hexylsilyl silica gel for D. R = H : cefuroxime. chromatography R (5 µm). Mobile phase : mix 1 volume of acetonitrile R and 01/2005:0992 99 volumes of an acetate buffer solution pH 3.4, prepared by dissolving 6.01 g of glacial acetic acid R and 0.68 g of CEFUROXIME SODIUM sodium acetate R in water R and diluting to 1000 ml with the same solvent. Cefuroximum natricum Flow rate : 1.5 ml/min. Detection : spectrophotometer at 273 nm. Injection : 20 µl loop injector ; inject test solution (a) and reference solutions (b) and (c). Run time : 4 times the retention time of cefuroxime. System suitability : reference solution (b) : — resolution : minimum of 2.0 between the peaks due to C16H15N4NaO8S Mr 446.4 cefuroxime and to impurity A. Limits : DEFINITION — impurity A : not more than the area of the principal peak Sodium (6R,7R)-3-[(carbamoyloxy)methyl]-7-[[(Z)in the chromatogram obtained with reference solution (c) (furan-2-yl)(methoxyimino)acetyl]amino]-8-oxo-5-thia-1(1.0 per cent), azabicyclo[4.2.0]oct-2-ene-2-carboxylate. — any other impurity : not more than the area of the Content : 96.0 per cent to 102.0 per cent (anhydrous principal peak in the chromatogram obtained with substance). reference solution (c) (1.0 per cent), CHARACTERS — total : not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (c) Appearance : white or almost white powder, slightly (3.0 per cent), hygroscopic. General Notices (1) apply to all monographs and other texts

1223

Celiprolol hydrochloride


— disregard limit : 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.05 per cent). N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm. 2-Ethylhexanoic acid (2.4.28) : maximum 0.5 per cent m/m. Water (2.5.12) : maximum 3.5 per cent, determined on 0.400 g. H. (5aR,6R)-6-[[(Z)-(furan-2-yl)(methoxyimino)acetyl]amino]Bacterial endotoxins (2.6.14) : less than 0.10 IU/mg, if 5a,6-dihydro-3H,7H-azeto[2,1-b]furo[3,4-d][1,3]thiazine-1, intended for use in the manufacture of parenteral dosage 7(4H)-dione, forms without a further appropriate procedure for the removal of bacterial endotoxins. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modification. Injection : test solution (b) and reference solution (a). Calculate the percentage content of cefuroxime sodium. STORAGE In an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins.

I. (Z)-(furan-2-yl)(methoxyimino)acetic acid. 01/2005:1632

CELIPROLOL HYDROCHLORIDE Celiprololi hydrochloridum

IMPURITIES

A. R = OH : (6R,7R)-7-[[(Z)-(furan-2-yl)(methoxyimino)acetyl]amino]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (descarbamoylcefuroxime), B. R = O-CO-CH3 : (6R,7R)-3-[(acetyloxy)methyl]-7-[[(Z)(furan-2-yl)(methoxyimino)acetyl]amino]-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, C. R = H : (6R,7R)-7-[[(Z)-(furan-2-yl)(methoxyimino)acetyl]amino]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, D. R = O-CO-NH-CO-CCl3 : (6R,7R)-7-[[(Z)-(furan2-yl)(methoxyimino)acetyl]amino]-8-oxo-3[[[(trichloroacetyl)carbamoyl]oxy]methyl]-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid,

Mr 416.0

DEFINITION 3-[3-Acetyl-4-[(2RS)-3-[(1,1-dimethylethyl)amino]-2hydroxypropoxy]phenyl]-1,1-diethylurea hydrochloride. Content : 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white or very slightly yellow, crystalline powder. Solubility : freely soluble in water and in methanol, soluble in alcohol, very slightly soluble in methylene chloride. It shows polymorphism. IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Comparison : celiprolol hydrochloride CRS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in methanol R, evaporate to dryness and record new spectra using the residues. B. It gives reaction (a) of chlorides (2.3.1).

E. R = O-CO-NH2 : (6R,7R)-3-[(carbamoyloxy)methyl]-7[[(E)-(furan-2-yl)(methoxyimino)acetyl]amino]-8-oxo5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (trans-cefuroxime), F. R = OH : (6R,7R)-7-[[(E)-(furan-2-yl)(methoxyimino)acetyl]amino]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, G. R = O-CO-CH3 : (6R,7R)-3-[(acetyloxy)methyl]-7-[[(E)(furan-2-yl)(methoxyimino)acetyl]amino]-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, 1224

C20H34ClN3O4

TESTS Optical rotation (2.2.7) : − 0.10° to + 0.10°. Dissolve 1.0 g in water R and dilute to 10.0 ml with the same solvent. Related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately before use. Test solution. Dissolve 0.100 g of the substance to be examined in mobile phase A and dilute to 20.0 ml with mobile phase A.


Celiprolol hydrochloride


Reference solution (a). Dissolve 2 mg of the substance to be examined and 2 mg of acebutolol hydrochloride R in mobile phase A and dilute to 50.0 ml with mobile phase A. Reference solution (b). Dissolve 10 mg of the substance to be examined in 2 ml of mobile phase A and allow to stand for 24 h (for identification of impurity A). Reference solution (c). Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A. Dilute 1.0 ml of this solution to 10.0 ml with mobile phase A. Reference solution (d). Dissolve 10 mg of celiprolol for peak identification CRS in mobile phase A and dilute to 2 ml with mobile phase A. Reference solution (e). This solution is only prepared if required (see below) and is used to determine the identity of impurity I which co-elutes with impurity H (the 2 impurities originate from different synthesis routes). Dissolve 2 mg of celiprolol impurity I CRS in mobile phase A and dilute to 20 ml with mobile phase A. Dilute 1.0 ml of the solution to 10.0 ml with mobile phase A. Column : — size : l = 0.15 m, Ø = 4.6 mm, — stationary phase: octylsilyl silica gel for chromatography R (5 µm), — temperature : 30 °C. Mobile phase : — mobile phase A : mix 91 ml of tetrahydrofuran R, 63 ml of acetonitrile R1, 0.6 ml of pentafluoropropanoic acid R and 0.2 ml of trifluoroacetic acid R ; dilute to 1000 ml with water R ; — mobile phase B : acetonitrile R1 ; Time (min)



0 - 50

100 → 80

0 → 20

50 - 51

80 → 100

20 → 0

51 - 65

100

0

Flow rate : 1.4 ml/min. Detection : spectrophotometer at 232 nm. Injection : 10 µl. Relative retention with reference to celiprolol (retention time = about 10 min) : impurity A = about 0.3 ; impurity D = about 0.7 ; impurity G = about 1.2 ; impurity B = about 1.4 ; impurity F = about 1.6 ; impurity C = about 2.2 ; impurity H or I = about 2.5 ; impurity E = about 3.9. System suitability : reference solution (a) : — resolution : minimum 4.0 between the peaks due to celiprolol and acebutolol. Limits : use the chromatogram supplied with celiprolol for peak identification CRS and the chromatogram obtained with reference solution (d) to identify the peaks due to impurities B, E and F : — correction factors : for the calculation of contents, multiply the peak areas of the following impurities by the corresponding correction factor : impurity A = 4.0 ; impurity B = 1.5 ; impurity E = 2.3 ; impurity F = 0.5 ; impurity I = 1.7 ; — any impurity : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (c) (0.2 per cent), and not more than General Notices (1) apply to all monographs and other texts

1 such peak has an area greater than the area of the principal peak in the chromatogram obtained with reference solution (c) (0.1 per cent) ; — total : not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.5 per cent) ; — if any of the above limits are exceeded and if a peak occurs with a relative retention of about 2.5 (impurity H or I), the identity of this peak has to be clarified by use of a UV spectrum recorded with a diode array detector ; if this spectrum is different from the one obtained with reference solution (e), no correction factor is applied ; — disregard limit : 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.05 per cent). Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 3 h. ASSAY Dissolve 0.350 g under an atmosphere of nitrogen in 50 ml of alcohol R and add 1.0 ml of 0.1 M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the 2 points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 41.60 mg of C20H34ClN3O4. STORAGE Protected from light. IMPURITIES Specified impurities : A, B, C, D, E, F, G, H, I.

A. R1 = H, R2 = NH-C(CH3)3 : 1-[5-amino2-[(2RS)-3-[(1,1-dimethylethyl)amino]-2hydroxypropoxy]phenyl]ethanone, C. R1 = CO-NH-C(CH3)3, R2 = NH-C(CH3)3 : 1-[3-acetyl-4-[(2RS)-3-[(1,1-dimethylethyl)amino]-2hydroxypropoxy]phenyl]-3-(1,1-dimethylethyl)urea, D. R1 = CO-N(C2H5)2, R2 = N(C2H5)2 : 3-[3-acetyl-4-[(2RS)-3(diethylamino)-2-hydroxypropoxy]phenyl]-1,1-diethylurea, H. R1 = CO-N(C2H5)2, R2 = Br : 3-[3-acetyl-4-[(2RS)-3-bromo2-hydroxypropoxy]phenyl]-1,1-diethylurea (bromhydrin compound),

B. 1,3-bis[3-acetyl-4-[3-[(1,1-dimethylethyl)amino]-2hydroxypropoxy]phenyl]urea, 1225

Cellulose acetate


gently and allow to stand for 3 h. Filter, wash the flask and the filter with carbon dioxide-free water R. Combine the filtrate and washings. Add 0.1 ml of phenolphthalein solution R1. Titrate with 0.01 M sodium hydroxide until a faint pink colour is obtained. 1 ml of 0.01 M sodium hydroxide is equivalent to 0.6005 mg of free acid, calculated as acetic acid. Heavy metals (2.4.8) : maximum 10 ppm. 2.0 g complies with limit test D. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. E. 1,1′-[[(1,1-dimethylethyl)imino]bis[(2-hydroxypropane-1,3diyl)oxy(3-acetyl-1,4-phenylene)]]bis(3,3-diethylurea),

Loss on drying (2.2.32) : maximum 5.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 3 h. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. Microbial contamination. Total viable aerobic count (2.6.12) not more than 103 micro-organisms per gram of which not more than 102 fungi per gram determined by plate count. It complies with the tests for Escherichia coli and Salmonella (2.6.13).

F. R1 = R3 = H, R2 = CO-CH3 : 3-(3-acetyl-4-hydroxyphenyl)1,1-diethylurea,

ASSAY

I. R1 = CO-CH3, R2 = H, R3 = C2H5 : 1-acetyl-1-(4ethoxyphenyl)-3,3-diethylurea,

Cellulose acetate containing not more than 42.0 per cent of acetyl groups To 2.000 g in a 500 ml conical flask add 100 ml of acetone R and 10 ml of water R. Close the flask and stir with a magnetic stirrer until dissolution is complete. Add 30.0 ml of 1 M sodium hydroxide with constant stirring. Close the flask and stir with a magnetic stirrer for 30 min. Add 100 ml of water R at 80 °C, washing down the sides of the flask, stir for 2 min and cool to room temperature. Titrate with 0.5 M sulphuric acid, using 0.1 ml of phenolphthalein solution R as indicator. Carry out a blank titration.

G. 3-[3-acetyl-4-[[(RS)-oxiranyl]methoxy]phenyl]-1,1diethylurea. 01/2005:0887

Calculate the percentage content of acetyl groups from the expression :

CELLULOSE ACETATE Cellulosi acetas

d m

DEFINITION n1 Partly or completely O-acetylated cellulose. Content : 29.0 per cent to 44.8 per cent of acetyl groups (C2H3O) (dried substance) and 90.0 per cent to 110.0 per cent n2 of the acetyl content stated on the label (dried substance). CHARACTERS Appearance : white, yellowish-white or greyish-white powder or granules, hygroscopic. Solubility : practically insoluble in water, soluble in acetone, in formic acid and in a mixture of equal volumes of methanol and methylene chloride, practically insoluble in alcohol.

=


=


=

number of millilitres of 0.5 M sulphuric acid used in the test,

=

number of millilitres of 0.5 M sulphuric acid used in the blank titration.

Cellulose acetate containing more than 42.0 per cent of acetyl groups

To 2.000 g in a 500 ml conical flask add 30 ml of dimethyl sulphoxide R and 100 ml of acetone R. Close the flask and stir with a magnetic stirrer for 16 h. Add 30.0 ml of 1 M sodium hydroxide with constant stirring. Close the flask and stir with a magnetic stirrer for 6 min. Allow to IDENTIFICATION stand without stirring for 60 min. Add 100 ml of water R at Infrared absorption spectrophotometry (2.2.24). 80 °C, washing down the sides of the flask, stir for 2 min and Comparison : Ph. Eur. reference spectrum of cellulose cool to room temperature. Titrate with 0.5 M hydrochloric acetate. acid, using 0.1 ml of phenolphthalein solution R. Add 0.5 ml of 0.5 M hydrochloric acid in excess, stir for 5 min and allow TESTS to stand for 30 min. Titrate with 0.5 M sodium hydroxide, Free acid : maximum 0.1 per cent, calculated as acetic acid until a persistent pink colour is obtained, stirring with a (dried substance). magnetic stirrer. Calculate the net number of millimoles To 5.00 g in a 250 ml conical flask, add 150 ml of carbon of 0.5 M sodium hydroxide consumed, taking the mean of dioxide-free water R, insert the stopper, swirl the suspension 2 blank titrations into consideration. 1226



Cellulose acetate phthalate

ASSAY Examine by liquid chromatography (2.2.29). Test solution. To 1.000 g in a 500 ml conical flask, add 100 ml of acetone R and 10 ml of water R. Close the flask and stir with a magnetic stirrer until dissolution is complete. Add 30.0 ml of 1 M sodium hydroxide with constant stirring. d = loss on drying as a percentage, Close the flask and stir with a magnetic stirrer for 30 min. m = mass of the substance to be examined, in grams, Add 100 ml of hot water R at 80 °C, washing down the sides of the flask and stir for 2 min. Cool, centrifuge or filter the n = net number of millimoles of 0.5 M sodium suspension and wash the residue with water R. Combine the hydroxide. filtrate and washings, adjust the pH to 3 (2.2.3) using dilute phosphoric acid R and dilute to 500.0 ml with water R. STORAGE Reference solution. Dissolve 0.200 g of glacial acetic acid R In an airtight container. and 0.400 g of butyric acid R in water R, adjust the pH to 3 (2.2.3) using dilute phosphoric acid R and dilute to 500.0 ml LABELLING with water R. The label states the nominal percentage content of acetyl The chromatographic procedure may be carried out using : groups. — a column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), 01/2005:1406 — as mobile phase at a flow rate of 1.2 ml/min a mixture of 5 volumes of methanol R and 95 volumes of phosphate CELLULOSE ACETATE BUTYRATE buffer solution pH 3.0 R1, changing after 30 min, by linear gradient elution over 5 min, to a mixture of Cellulosi acetas butyras 20 volumes of methanol R and 80 volumes of phosphate buffer solution pH 3.0 R1 and eluting for a further DEFINITION 25 min. Restore the initial solvent mixture for 1 min, Cellulose acetate butyrate is partly or completely O-acetylated — as detector a spectrophotometer set at 210 nm. and O-butyrated cellulose. It contains not less than 2.0 per Inject successively 20 µl of the reference solution and 20 µl cent and not more than 30.0 per cent of acetyl groups of the test solution. (C2H3O) and not less than 16.0 per cent and not more than 53.0 per cent of butyryl groups (C4H7O) calculated with Calculate the percentage contents of acetic acid and butyric reference to the dried substance. Both the acetyl content acid in the substance to be examined from the peak areas in and the butyryl content are not less than 90.0 per cent and the chromatograms obtained with the two solutions. not more than 110.0 per cent of those stated on the label, To calculate the percentage contents of acetyl (C2H3O) calculated with reference to the dried substance. and of butyryl (C4H7O) groups, multiply the percentage contents of acetic acid and of butyric acid by 0.717 and CHARACTERS A white, yellowish-white or greyish-white powder or granules, 0.807, respectively. slightly hygroscopic, practically insoluble in water and in STORAGE alcohol, soluble in acetone, in formic acid and in a mixture Store in an airtight container. of equal volumes of methanol and methylene chloride. LABELLING IDENTIFICATION The label states the nominal percentage content of acetyl A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum and butyryl groups. Calculate the percentage content of acetyl groups from the expression :

of cellulose acetate butyrate. The intensities of the bands may vary according to the degree of substitution. B. It complies with the limits of the assay. TESTS Acidity. To 5.00 g in a 250 ml conical flask, add 150 ml of carbon dioxide-free water R, insert the stopper, swirl the suspension gently and allow to stand for 3 h. Filter, wash the flask and the filter with carbon dioxide-free water R. Combine the filtrate and washings. Add 0.1 ml of phenolphthalein solution R1. Not more than 3.0 ml of 0.01 M sodium hydroxide is required to change the colour of the indicator. Heavy metals (2.4.8). 1.0 g complies with limit test F for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 2.0 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C for 3 h. Total ash (2.4.16). Not more than 0.1 per cent, determined on 1.0 g.


01/2005:0314

CELLULOSE ACETATE PHTHALATE Cellulosi acetas phthalas DEFINITION Partly O-acetylated and O-phthalylated cellulose. Content : — phthaloyl groups (C8H5O3, relative mass of the group 149.1) : 30.0 per cent to 36.0 per cent (anhydrous and acid-free substance). — acetyl groups (C2H3O, relative mass of the group 43.05) : 21.5 per cent to 26.0 per cent (anhydrous and acid-free substance). CHARACTERS Appearance : white, free-flowing powder or colourless flakes, hygroscopic. 1227

Cellulose, microcrystalline


Solubility : practically insoluble in water, freely soluble in acetone, soluble in diethylene glycol, practically insoluble in ethanol and in methylene chloride. It dissolves in dilute solutions of alkalis. IDENTIFICATION

Acetyl groups. To 0.100 g add 25.0 ml of 0.1 M sodium hydroxide and heat on a water-bath under a reflux condenser for 30 min. Cool, add 0.1 ml of phenolphthalein solution R and titrate with 0.1 M hydrochloric acid until the colour is discharged. Carry out a blank titration. Calculate the percentage content of acetyl groups from the expression :

Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of cellulose acetate phthalate. a TESTS Viscosity (2.2.9) : 45 mPa·s to 90 mPa·s for the apparent viscosity determined at 25 °C. Dissolve 15 g, calculated with reference to the anhydrous substance, in 85 g of a mixture of 1 part of water R and 249 parts of acetone R. Free acid : maximum 3.0 per cent, calculated as phthalic acid (anhydrous substance).

=

percentage content of water,

m

=


n1

=

number of millilitres of 0.1 M hydrochloric acid used in the test,

n2

=

number of millilitres of 0.1 M hydrochloric acid used in the blank titration,

P

=

percentage content of phthaloyl groups,

S

=

percentage content of free acid (see Tests).

STORAGE Shake 3.0 g for 2 h with 100 ml of a 50 per cent V/V solution In an airtight container. of methanol R and filter. Wash the flask and the filter with 2 quantities, each of 10 ml, of a mixture of a 50 per cent V/V 01/2005:0316 solution of methanol R. Combine the filtrate and washings, add phenolphthalein solution R and titrate with 0.1 M CELLULOSE, MICROCRYSTALLINE sodium hydroxide until a faint pink colour is obtained. Carry out a blank titration.

Cellulosum microcristallinum

1 ml of 0.1 M sodium hydroxide is equivalent to 8.3 mg of free acid, calculated as phthalic acid. Heavy metals (2.4.8) : maximum 10 ppm. 2.0 g complies with limit test C. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Water (2.5.12) : maximum 5.0 per cent, determined on 0.500 g. Carry out the test using a mixture of 2 volumes of methylene chloride R and 3 volumes of ethanol R. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY

C6nH10n+2O5n+1 DEFINITION Purified, partly depolymerised cellulose prepared by treating alpha-cellulose, obtained as a pulp from fibrous plant material, with mineral acids.

Phthaloyl groups. Dissolve 1.000 g in 50 ml of a mixture of 2 volumes of acetone R and 3 volumes of alcohol R. Add 0.1 ml of phenolphthalein solution R and titrate with 0.1 M sodium hydroxide until a faint pink colour is obtained. Carry out a blank titration.

CHARACTERS Appearance : white or almost white, fine or granular powder. Solubility : practically insoluble in water, in acetone, in ethanol, in toluene, in dilute acids and in a 50 g/l solution of sodium hydroxide.

Calculate the percentage content of phthaloyl groups (P) from the expression :

IDENTIFICATION A. Place about 10 mg on a watch-glass and disperse in 2 ml of iodinated zinc chloride solution R. The substance becomes violet-blue. B. The degree of polymerisation is not more than 350. Transfer 1.300 g to a 125 ml conical flask. Add 25.0 ml of water R and 25.0 ml of cupriethylenediamine hydroxide solution R. Immediately purge the solution with nitrogen R, insert the stopper and shake until completely dissolved. Transfer 7.0 ml of the solution to a suitable capillary viscometer (2.2.9). Equilibrate the solution at 25 ± 0.1 °C for at least 5 min. Record the

a

=

percentage content of water,

m

=


n

=

number of millilitres of 0.1 M sodium hydroxide used,

S

=

percentage content of free acid (see Tests).

1228




flow time t1 in seconds, between the 2 marks on the viscometer. Calculate the kinematic viscosity ν1 of the solution using the formula :

Determine the intrinsic viscosity [η]c by interpolation, using the intrinsic viscosity table (Table 0316.-1). Calculate the degree of polymerisation P using the formula :

where k1 is the viscometer constant. Dilute a suitable volume of cupriethylenediamine hydroxide solution R with an equal volume of water R and measure the flow time t2 using a suitable capillary viscometer. Calculate the kinematic viscosity ν2 of the solvent using the formula :

where m is the mass in grams of the substance to be examined and b is the loss on drying as a percentage. TESTS Solubility. Dissolve 50 mg in 10 ml of ammoniacal solution of copper tetrammine R. It dissolves completely, leaving no residue.

where k2 is the viscometer constant. Determine the relative viscosity ηrel of the substance to be examined using the formula :

pH (2.2.3) : 5.0 to 7.5 for the supernatant liquid. Shake 5 g with 40 ml of carbon dioxide-free water R for 20 min and centrifuge.

Table 0316.-1. — Intrinsic viscosity table Intrinsic viscosity [η]c at different values of relative viscosity ηrel [ η ]c ηrel

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

1.1

0.098

0.106

0.115

0.125

0.134

0.143

0.152

0.161

0.170

0.180

1.2

0.189

0.198

0.207

0.216

0.225

0.233

0.242

0.250

0.259

0.268

1.3

0.276

0.285

0.293

0.302

0.310

0.318

0.326

0.334

0.342

0.350

1.4

0.358

0.367

0.375

0.383

0.391

0.399

0.407

0.414

0.422

0.430

1.5

0.437

0.445

0.453

0.460

0.468

0.476

0.484

0.491

0.499

0.507

1.6

0.515

0.522

0.529

0.536

0.544

0.551

0.558

0.566

0.573

0.580

1.7

0.587

0.595

0.602

0.608

0.615

0.622

0.629

0.636

0.642

0.649

1.8

0.656

0.663

0.670

0.677

0.683

0.690

0.697

0.704

0.710

0.717

1.9

0.723

0.730

0.736

0.743

0.749

0.756

0.762

0.769

0.775

0.782

2.0

0.788

0.795

0.802

0.809

0.815

0.821

0.827

0.833

0.840

0.846

2.1

0.852

0.858

0.864

0.870

0.876

0.882

0.888

0.894

0.900

0.906

2.2

0.912

0.918

0.924

0.929

0.935

0.941

0.948

0.953

0.959

0.965

2.3

0.971

0.976

0.983

0.988

0.994

1.000

1.006

1.011

1.017

1.022

2.4

1.028

1.033

1.039

1.044

1.050

1.056

1.061

1.067

1.072

1.078

2.5

1.083

1.089

1.094

1.100

1.105

1.111

1.116

1.121

1.126

1.131

2.6

1.137

1.142

1.147

1.153

1.158

1.163

1.169

1.174

1.179

1.184

2.7

1.190

1.195

1.200

1.205

1.210

1.215

1.220

1.225

1.230

1.235

2.8

1.240

1.245

1.250

1.255

1.260

1.265

1.270

1.275

1.280

1.285

2.9

1.290

1.295

1.300

1.305

1.310

1.314

1.319

1.324

1.329

1.333

3.0

1.338

1.343

1.348

1.352

1.357

1.362

1.367

1.371

1.376

1.381

3.1

1.386

1.390

1.395

1.400

1.405

1.409

1.414

1.418

1.423

1.427

3.2

1.432

1.436

1.441

1.446

1.450

1.455

1.459

1.464

1.468

1.473

3.3

1.477

1.482

1.486

1.491

1.496

1.500

1.504

1.508

1.513

1.517

3.4

1.521

1.525

1.529

1.533

1.537

1.542

1.546

1.550

1.554

1.558

3.5

1.562

1.566

1.570

1.575

1.579

1.583

1.587

1.591

1.595

1.600

3.6

1.604

1.608

1.612

1.617

1.621

1.625

1.629

1.633

1.637

1.642

3.7

1.646

1.650

1.654

1.658

1.662

1.666

1.671

1.675

1.679

1.683


1229



Intrinsic viscosity [η]c at different values of relative viscosity ηrel [ η ]c ηrel

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

3.8

1.687

1.691

1.695

1.700

1.704

1.708

1.712

1.715

1.719

1.723

3.9

1.727

1.731

1.735

1.739

1.742

1.746

1.750

1.754

1.758

1.762

4.0

1.765

1.769

1.773

1.777

1.781

1.785

1.789

1.792

1.796

1.800

4.1

1.804

1.808

1.811

1.815

1.819

1.822

1.826

1.830

1.833

1.837

4.2

1.841

1.845

1.848

1.852

1.856

1.859

1.863

1.867

1.870

1.874

4.3

1.878

1.882

1.885

1.889

1.893

1.896

1.900

1.904

1.907

1.911

4.4

1.914

1.918

1.921

1.925

1.929

1.932

1.936

1.939

1.943

1.946

4.5

1.950

1.954

1.957

1.961

1.964

1.968

1.971

1.975

1.979

1.982

4.6

1.986

1.989

1.993

1.996

2.000

2.003

2.007

2.010

2.013

2.017

4.7

2.020

2.023

2.027

2.030

2.033

2.037

2.040

2.043

2.047

2.050

4.8

2.053

2.057

2.060

2.063

2.067

2.070

2.073

2.077

2.080

2.083

4.9

2.087

2.090

2.093

2.097

2.100

2.103

2.107

2.110

2.113

2.116

5.0

2.119

2.122

2.125

2.129

2.132

2.135

2.139

2.142

2.145

2.148

5.1

2.151

2.154

2.158

2.160

2.164

2.167

2.170

2.173

2.176

2.180

5.2

2.183

2.186

2.190

2.192

2.195

2.197

2.200

2.203

2.206

2.209

5.3

2.212

2.215

2.218

2.221

2.224

2.227

2.230

2.233

2.236

2.240

5.4

2.243

2.246

2.249

2.252

2.255

2.258

2.261

2.264

2.267

2.270

5.5

2.273

2.276

2.279

2.282

2.285

2.288

2.291

2.294

2.297

2.300

5.6

2.303

2.306

2.309

2.312

2.315

2.318

2.320

2.324

2.326

2.329

5.7

2.332

2.335

2.338

2.341

2.344

2.347

2.350

2.353

2.355

2.358

5.8

2.361

2.364

2.367

2.370

2.373

2.376

2.379

2.382

2.384

2.387

5.9

2.390

2.393

2.396

2.400

2.403

2.405

2.408

2.411

2.414

2.417

6.0

2.419

2.422

2.425

2.428

2.431

2.433

2.436

2.439

2.442

2.444

6.1

2.447

2.450

2.453

2.456

2.458

2.461

2.464

2.467

2.470

2.472

6.2

2.475

2.478

2.481

2.483

2.486

2.489

2.492

2.494

2.497

2.500

6.3

2.503

2.505

2.508

2.511

2.513

2.516

2.518

2.521

2.524

2.526

6.4

2.529

2.532

2.534

2.537

2.540

2.542

2.545

2.547

2.550

2.553

6.5

2.555

2.558

2.561

2.563

2.566

2.568

2.571

2.574

2.576

2.579

6.6

2.581

2.584

2.587

2.590

2.592

2.595

2.597

2.600

2.603

2.605

6.7

2.608

2.610

2.613

2.615

2.618

2.620

2.623

2.625

2.627

2.630

6.8

2.633

2.635

2.637

2.640

2.643

2.645

2.648

2.650

2.653

2.655

6.9

2.658

2.660

2.663

2.665

2.668

2.670

2.673

2.675

2.678

2.680

7.0

2.683

2.685

2.687

2.690

2.693

2.695

2.698

2.700

2.702

2.705

7.1

2.707

2.710

2.712

2.714

2.717

2.719

2.721

2.724

2.726

2.729

7.2

2.731

2.733

2.736

2.738

2.740

2.743

2.745

2.748

2.750

2.752

7.3

2.755

2.757

2.760

2.762

2.764

2.767

2.769

2.771

2.774

2.776

7.4

2.779

2.781

2.783

2.786

2.788

2.790

2.793

2.795

2.798

2.800

7.5

2.802

2.805

2.807

2.809

2.812

2.814

2.816

2.819

2.821

2.823

7.6

2.826

2.828

2.830

2.833

2.835

2.837

2.840

2.842

2.844

2.847

7.7

2.849

2.851

2.854

2.856

2.858

2.860

2.863

2.865

2.868

2.870

7.8

2.873

2.875

2.877

2.879

2.881

2.884

2.887

2.889

2.891

2.893

1230





0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

7.9

2.895

2.898

2.900

2.902

2.905

2.907

2.909

2.911

2.913

2.915

8.0

2.918

2.920

2.922

2.924

2.926

2.928

2.931

2.933

2.935

2.937

8.1

2.939

2.942

2.944

2.946

2.948

2.950

2.952

2.955

2.957

2.959

8.2

2.961

2.963

2.966

2.968

2.970

2.972

2.974

2.976

2.979

2.981

8.3

2.983

2.985

2.987

2.990

2.992

2.994

2.996

2.998

3.000

3.002

8.4

3.004

3.006

3.008

3.010

3.012

3.015

3.017

3.019

3.021

3.023

8.5

3.025

3.027

3.029

3.031

3.033

3.035

3.037

3.040

3.042

3.044

8.6

3.046

3.048

3.050

3.052

3.054

3.056

3.058

3.060

3.062

3.064

8.7

3.067

3.069

3.071

3.073

3.075

3.077

3.079

3.081

3.083

3.085

8.8

3.087

3.089

3.092

3.094

3.096

3.098

3.100

3.102

3.104

3.106

8.9

3.108

3.110

3.112

3.114

3.116

3.118

3.120

3.122

3.124

3.126

9.0

3.128

3.130

3.132

3.134

3.136

3.138

3.140

3.142

3.144

3.146

9.1

3.148

3.150

3.152

3.154

3.156

3.158

3.160

3.162

3.164

3.166

9.2

3.168

3.170

3.172

3.174

3.176

3.178

3.180

3.182

3.184

3.186

9.3

3.188

3.190

3.192

3.194

3.196

3.198

3.200

3.202

3.204

3.206

9.4

3.208

3.210

3.212

3.214

3.215

3.217

3.219

3.221

3.223

3.225

9.5

3.227

3.229

3.231

3.233

3.235

3.237

3.239

3.241

3.242

3.244

9.6

3.246

3.248

3.250

3.252

3.254

3.256

3.258

3.260

3.262

3.264

9.7

3.266

3.268

3.269

3.271

3.273

3.275

3.277

3.279

3.281

3.283

9.8

3.285

3.287

3.289

3.291

3.293

3.295

3.297

3.298

3.300

3.302

9.9

3.304

3.305

3.307

3.309

3.311

3.313

3.316

3.318

3.320

3.321


0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

10

3.32

3.34

3.36

3.37

3.39

3.41

3.43

3.45

3.46

3.48

11

3.50

3.52

3.53

3.55

3.56

3.58

3.60

3.61

3.63

3.64

12

3.66

3.68

3.69

3.71

3.72

3.74

3.76

3.77

3.79

3.80

13

3.80

3.83

3.85

3.86

3.88

3.89

3.90

3.92

3.93

3.95

14

3.96

3.97

3.99

4.00

4.02

4.03

4.04

4.06

4.07

4.09

15

4.10

4.11

4.13

4.14

4.15

4.17

4.18

4.19

4.20

4.22

16

4.23

4.24

4.25

4.27

4.28

4.29

4.30

4.31

4.33

4.34

17

4.35

4.36

4.37

4.38

4.39

4.41

4.42

4.43

4.44

4.45

18

4.46

4.47

4.48

4.49

4.50

4.52

4.53

4.54

4.55

4.56

19

4.57

4.58

4.59

4.60

4.61

4.62

4.63

4.64

4.65

4.66

Conductivity (2.2.38). The conductivity of the test solution does not exceed the conductivity of the water by more than 75 µS·cm− 1. Use as test solution the supernatant liquid obtained in the test for pH. Measure the conductivity of the supernatant liquid after a stable reading has been obtained and measure the conductivity of the water used to prepare the test solution.


Ether-soluble substances : maximum 0.05 per cent (5 mg) for the difference between the weight of the residue and the weight obtained from a blank determination. Place 10.0 g in a column about 20 mm in internal diameter and pass 50 ml of peroxide-free ether R through the column. Evaporate the eluate to dryness and weigh. Carry out a blank determination under the same conditions. Water-soluble substances : maximum 0.25 per cent (12.5 mg) for the difference between the weight of the residue and the weight obtained from a blank determination. Shake 5.0 g with 80 ml of water R for 10 min. Filter with the aid of vacuum into a tared flask. Evaporate to dryness on a 1231

Cellulose, powdered


Solubility : practically insoluble in water, slightly soluble in a 50 g/l solution of sodium hydroxide, practically insoluble in acetone, in ethanol, in toluene, in dilute acids and in most organic solvents.

water-bath. Dry at 100-105 °C for 1 h and weigh. Carry out a blank determination under the same conditions. Heavy metals (2.4.8) : maximum 10 ppm. 2.0 g complies with limit test C. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32) : maximum 7.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 3 h. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. Microbial contamination. Total viable aerobic count (2.6.12) not more than 103 micro-organisms per gram and with a limit for fungi of 102 per gram, determined by plate count. It complies with the tests for Escherichia coli, for Pseudomonas aeruginosa, for Staphylococcus aureus and for Salmonella (2.6.13).

IDENTIFICATION A. Place about 10 mg on a watch-glass and disperse in 2 ml of iodinated zinc chloride solution R. The substance becomes violet-blue. B. The degree of polymerisation is 440 to 2250. Transfer 0.250 g to a 125 ml conical flask. Add 25.0 ml of water R and 25.0 ml of cupriethylenediamine hydroxide solution R. Immediately purge the solution with nitrogen R, insert the stopper and shake until completely dissolved. Transfer 7.0 ml of the solution to a suitable capillary viscometer (2.2.9). Equilibrate the solution at 25 ± 0.1 °C for at least 5 min. Record the flow time t1 in seconds, between the 2 marks on the viscometer. Calculate the kinematic viscosity ν1 of the solution using the formula : 01/2005:0315

CELLULOSE, POWDERED

where k1 is the viscometer constant. Dilute a suitable volume of cupriethylenediamine hydroxide solution R with an equal volume of water R and measure the flow time t2 using a suitable capillary viscometer. Calculate the kinematic viscosity ν2 of the solvent using the formula :

Cellulosi pulvis

where k2 is the viscometer constant. Determine the relative viscosity ηrel of the substance to be examined using the formula :

Determine the intrinsic viscosity [η]c by interpolation, using the intrinsic viscosity table (Table 0315.-1). Calculate the degree of polymerisation, P, using the formula :

C6nH10n+2O5n+1 DEFINITION Purified, mechanically disintegrated cellulose prepared by processing alpha-cellulose obtained as a pulp from fibrous plant material. CHARACTERS Appearance : white or almost white, fine or granular powder.

where m is the mass in grams of the substance to be examined and b is the loss on drying as a percentage.

Table 0315.-1. – Intrinsic viscosity table Intrinsic viscosity [η]c at different values of relative viscosity ηrel [η]c ηrel

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

1.1

0.098

0.106

0.115

0.125

0.134

0.143

0.152

0.161

0.170

0.180

1.2

0.189

0.198

0.207

0.216

0.225

0.233

0.242

0.250

0.259

0.268

1.3

0.276

0.285

0.293

0.302

0.310

0.318

0.326

0.334

0.342

0.350

1.4

0.358

0.367

0.375

0.383

0.391

0.399

0.407

0.414

0.422

0.430

1.5

0.437

0.445

0.453

0.460

0.468

0.476

0.484

0.491

0.499

0.507

1.6

0.515

0.522

0.529

0.536

0.544

0.551

0.558

0.566

0.573

0.580

1.7

0.587

0.595

0.602

0.608

0.615

0.622

0.629

0.636

0.642

0.649

1.8

0.656

0.663

0.670

0.677

0.683

0.690

0.697

0.704

0.710

0.717

1.9

0.723

0.730

0.736

0.743

0.749

0.756

0.762

0.769

0.775

0.782

1232


Cellulose, powdered


Intrinsic viscosity [η]c at different values of relative viscosity ηrel [η]c ηrel

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

2.0

0.788

0.795

0.802

0.809

0.815

0.821

0.827

0.833

0.840

0.846

2.1

0.852

0.858

0.864

0.870

0.876

0.882

0.888

0.894

0.900

0.906

2.2

0.912

0.918

0.924

0.929

0.935

0.941

0.948

0.953

0.959

0.965

2.3

0.971

0.976

0.983

0.988

0.994

1.000

1.006

1.011

1.017

1.022

2.4

1.028

1.033

1.039

1.044

1.050

1.056

1.061

1.067

1.072

1.078

2.5

1.083

1.089

1.094

1.100

1.105

1.111

1.116

1.121

1.126

1.131

2.6

1.137

1.142

1.147

1.153

1.158

1.163

1.169

1.174

1.179

1.184

2.7

1.190

1.195

1.200

1.205

1.210

1.215

1.220

1.225

1.230

1.235

2.8

1.240

1.245

1.250

1.255

1.260

1.265

1.270

1.275

1.280

1.285

2.9

1.290

1.295

1.300

1.305

1.310

1.314

1.319

1.324

1.329

1.333

3.0

1.338

1.343

1.348

1.352

1.357

1.362

1.367

1.371

1.376

1.381

3.1

1.386

1.390

1.395

1.400

1.405

1.409

1.414

1.418

1.423

1.427

3.2

1.432

1.436

1.441

1.446

1.450

1.455

1.459

1.464

1.468

1.473

3.3

1.477

1.482

1.486

1.491

1.496

1.500

1.504

1.508

1.513

1.517

3.4

1.521

1.525

1.529

1.533

1.537

1.542

1.546

1.550

1.554

1.558

3.5

1.562

1.566

1.570

1.575

1.579

1.583

1.587

1.591

1.595

1.600

3.6

1.604

1.608

1.612

1.617

1.621

1.625

1.629

1.633

1.637

1.642

3.7

1.646

1.650

1.654

1.658

1.662

1.666

1.671

1.675

1.679

1.683

3.8

1.687

1.691

1.695

1.700

1.704

1.708

1.712

1.715

1.719

1.723

3.9

1.727

1.731

1.735

1.739

1.742

1.746

1.750

1.754

1.758

1.762

4.0

1.765

1.769

1.773

1.777

1.781

1.785

1.789

1.792

1.796

1.800

4.1

1.804

1.808

1.811

1.815

1.819

1.822

1.826

1.830

1.833

1.837

4.2

1.841

1.845

1.848

1.852

1.856

1.859

1.863

1.867

1.870

1.874

4.3

1.878

1.882

1.885

1.889

1.893

1.896

1.900

1.904

1.907

1.911

4.4

1.914

1.918

1.921

1.925

1.929

1.932

1.936

1.939

1.943

1.946

4.5

1.950

1.954

1.957

1.961

1.964

1.968

1.971

1.975

1.979

1.982

4.6

1.986

1.989

1.993

1.996

2.000

2.003

2.007

2.010

2.013

2.017

4.7

2.020

2.023

2.027

2.030

2.033

2.037

2.040

2.043

2.047

2.050

4.8

2.053

2.057

2.060

2.063

2.067

2.070

2.073

2.077

2.080

2.083

4.9

2.087

2.090

2.093

2.097

2.100

2.103

2.107

2.110

2.113

2.116

5.0

2.119

2.122

2.125

2.129

2.132

2.135

2.139

2.142

2.145

2.148

5.1

2.151

2.154

2.158

2.160

2.164

2.167

2.170

2.173

2.176

2.180

5.2

2.183

2.186

2.190

2.192

2.195

2.197

2.200

2.203

2.206

2.209

5.3

2.212

2.215

2.218

2.221

2.224

2.227

2.230

2.233

2.236

2.240

5.4

2.243

2.246

2.249

2.252

2.255

2.258

2.261

2.264

2.267

2.270

5.5

2.273

2.276

2.279

2.282

2.285

2.288

2.291

2.294

2.297

2.300

5.6

2.303

2.306

2.309

2.312

2.315

2.318

2.320

2.324

2.326

2.329

5.7

2.332

2.335

2.338

2.341

2.344

2.347

2.350

2.353

2.355

2.358

5.8

2.361

2.364

2.367

2.370

2.373

2.376

2.379

2.382

2.384

2.387

5.9

2.390

2.393

2.396

2.400

2.403

2.405

2.408

2.411

2.414

2.417

6.0

2.419

2.422

2.425

2.428

2.431

2.433

2.436

2.439

2.442

2.444


1233

Cellulose, powdered



0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

6.1

2.447

2.450

2.453

2.456

2.458

2.461

2.464

2.467

2.470

2.472

6.2

2.475

2.478

2.481

2.483

2.486

2.489

2.492

2.494

2.497

2.500

6.3

2.503

2.505

2.508

2.511

2.513

2.516

2.518

2.521

2.524

2.526

6.4

2.529

2.532

2.534

2.537

2.540

2.542

2.545

2.547

2.550

2.553

6.5

2.555

2.558

2.561

2.563

2.566

2.568

2.571

2.574

2.576

2.579

6.6

2.581

2.584

2.587

2.590

2.592

2.595

2.597

2.600

2.603

2.605

6.7

2.608

2.610

2.613

2.615

2.618

2.620

2.623

2.625

2.627

2.630

6.8

2.633

2.635

2.637

2.640

2.643

2.645

2.648

2.650

2.653

2.655

6.9

2.658

2.660

2.663

2.665

2.668

2.670

2.673

2.675

2.678

2.680

7.0

2.683

2.685

2.687

2.690

2.693

2.695

2.698

2.700

2.702

2.705

7.1

2.707

2.710

2.712

2.714

2.717

2.719

2.721

2.724

2.726

2.729

7.2

2.731

2.733

2.736

2.738

2.740

2.743

2.745

2.748

2.750

2.752

7.3

2.755

2.757

2.760

2.762

2.764

2.767

2.769

2.771

2.774

2.776

7.4

2.779

2.781

2.783

2.786

2.788

2.790

2.793

2.795

2.798

2.800

7.5

2.802

2.805

2.807

2.809

2.812

2.814

2.816

2.819

2.821

2.823

7.6

2.826

2.828

2.830

2.833

2.835

2.837

2.840

2.842

2.844

2.847

7.7

2.849

2.851

2.854

2.856

2.858

2.860

2.863

2.865

2.868

2.870

7.8

2.873

2.875

2.877

2.879

2.881

2.884

2.887

2.889

2.891

2.893

7.9

2.895

2.898

2.900

2.902

2.905

2.907

2.909

2.911

2.913

2.915

8.0

2.918

2.920

2.922

2.924

2.926

2.928

2.931

2.933

2.935

2.937

8.1

2.939

2.942

2.944

2.946

2.948

2.950

2.952

2.955

2.957

2.959

8.2

2.961

2.963

2.966

2.968

2.970

2.972

2.974

2.976

2.979

2.981

8.3

2.983

2.985

2.987

2.990

2.992

2.994

2.996

2.998

3.000

3.002

8.4

3.004

3.006

3.008

3.010

3.012

3.015

3.017

3.019

3.021

3.023

8.5

3.025

3.027

3.029

3.031

3.033

3.035

3.037

3.040

3.042

3.044

8.6

3.046

3.048

3.050

3.052

3.054

3.056

3.058

3.060

3.062

3.064

8.7

3.067

3.069

3.071

3.073

3.075

3.077

3.079

3.081

3.083

3.085

8.8

3.087

3.089

3.092

3.094

3.096

3.098

3.100

3.102

3.104

3.106

8.9

3.108

3.110

3.112

3.114

3.116

3.118

3.120

3.122

3.124

3.126

9.0

3.128

3.130

3.132

3.134

3.136

3.138

3.140

3.142

3.144

3.146

9.1

3.148

3.150

3.152

3.154

3.156

3.158

3.160

3.162

3.164

3.166

9.2

3.168

3.170

3.172

3.174

3.176

3.178

3.180

3.182

3.184

3.186

9.3

3.188

3.190

3.192

3.194

3.196

3.198

3.200

3.202

3.204

3.206

9.4

3.208

3.210

3.212

3.214

3.215

3.217

3.219

3.221

3.223

3.225

9.5

3.227

3.229

3.231

3.233

3.235

3.237

3.239

3.241

3.242

3.244

9.6

3.246

3.248

3.250

3.252

3.254

3.256

3.258

3.260

3.262

3.264

9.7

3.266

3.268

3.269

3.271

3.273

3.275

3.277

3.279

3.281

3.283

9.8

3.285

3.287

3.289

3.291

3.293

3.295

3.297

3.298

3.300

3.302

9.9

3.304

3.305

3.307

3.309

3.311

3.313

3.316

3.318

3.320

3.321

1234


Centaury



0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

10

3.32

3.34

3.36

3.37

3.39

3.41

3.43

3.45

3.46

3.48

11

3.50

3.52

3.53

3.55

3.56

3.58

3.60

3.61

3.63

3.64

12

3.66

3.68

3.69

3.71

3.72

3.74

3.76

3.77

3.79

3.80

13

3.80

3.83

3.85

3.86

3.88

3.89

3.90

3.92

3.93

3.95

14

3.96

3.97

3.99

4.00

4.02

4.03

4.04

4.06

4.07

4.09

15

4.10

4.11

4.13

4.14

4.15

4.17

4.18

4.19

4.20

4.22

16

4.23

4.24

4.25

4.27

4.28

4.29

4.30

4.31

4.33

4.34

17

4.35

4.36

4.37

4.38

4.39

4.41

4.42

4.43

4.44

4.45

18

4.46

4.47

4.48

4.49

4.50

4.52

4.53

4.54

4.55

4.56

19

4.57

4.58

4.59

4.60

4.61

4.62

4.63

4.64

4.65

4.66

TESTS Solubility. Dissolve 50 mg in 10 ml of ammoniacal solution of copper tetrammine R. It dissolves completely, leaving no residue. pH (2.2.3) : 5.0 to 7.5 for the supernatant liquid. Mix 10 g with 90 ml of carbon dioxide-free water R and allow to stand with occasional stirring for 1 h. Ether-soluble substances : maximum 0.15 per cent (15 mg) for the difference between the weight of the residue and the weight obtained from a blank determination. Place 10.0 g in a column about 20 mm in internal diameter and pass 50 ml of peroxide-free ether R through the column. Evaporate the eluate to dryness and weigh. Carry out a blank determination under the same conditions. Water-soluble substances : maximum 1.5 per cent (75.0 mg) for the difference between the weight of the residue and the weight obtained from a blank determination. Shake 5.0 g with 80 ml of water R for 10 min. Filter with the aid of vacuum into a tared flask. Evaporate to dryness on a water-bath. Dry at 100-105 °C for 1 h and weigh. Carry out a blank determination under the same conditions. Heavy metals (2.4.8) : maximum 10 ppm. 2.0 g complies with limit test C. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32) : maximum 6.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 3 h. Sulphated ash (2.4.14) : maximum 0.3 per cent, determined on 1.0 g. Microbial contamination. Total viable aerobic count (2.6.12) not more than 103 micro-organisms per gram and with a limit for fungi of 102 per gram, determined by plate count. It complies with the tests for Escherichia coli, for Pseudomonas aeruginosa, for Staphylococcus aureus and for Salmonella (2.6.13).

CHARACTERS Centaury has a very bitter taste. It has the macroscopic and microscopic characters described under identification tests A and B.


1235

IDENTIFICATION A. The hollow cylindrical, light green to dark brown stem has longitudinal ridges, and is branched only in its upper part. The sessile leaves are entire, decussately arranged, and have an ovate to lanceolate lamina, up to about 3 cm long. Both surfaces are glabrous and green to brownish-green. The inflorescence is diaxially branched. The tubular calyx is green and has five lanceolate, acuminate teeth. The corolla consists of a whitish tube divided in five elongated lanceolate pink lobes, about 5 mm to 8 mm long. Five stamens are present attached to the top of the corolla tube. The ovary is superior and has a short style, a broad bifid stigma and numerous ovules. Cylindrical capsules, about 7 mm to 10 mm long, with small brown markedly rough seeds are frequently present. B. Reduce to a powder (355). The powder is greenish-yellow to brownish. Examine under a microscope, using chloral hydrate solution R. The powder shows numerous fragments of stem containing sclerenchymatous fibres and narrow vessels with bordered pits or spiral of reticulate thickening, as well as rectangular pitted cells of the pith and medullary rays ; fragments of leaves with sinuous epidermal cells and striated cuticle, especially over the margins and surrounding the stomata, anisocytic stomata (2.8.3) and cells of the mesophyll with crystals of calcium oxalate of various types ; fragments of calyx and corolla, those of the calyx with straight-walled epidermal cells, epidermis of the corolla with obtuse papillae and radially striated cuticle ; parts of the endothecium with reticulate or ridge-shaped wall thickenings ; triangularly rounded to elliptical, yellow pollen grains, about 30 µm in diameter, with a finely grained exine and three germinal pores ; fragments of the wall of the fruit capsule composed of crossed layers of fusiform cells ; small yellowish-brown 01/2005:1301 seeds with a raised reticulate, dark brown structure formed by the coarse lateral walls of their epidermis. CENTAURY C. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R. Centaurii herba Test solution. To 1.0 g of the powdered drug (355) add 20 ml of methanol R and heat under a reflux condenser DEFINITION for 10 min. Cool and filter. Centaury consists of the whole or cut dried flowering aerial Reference solution. Dissolve 10 mg of rutin R in 10 ml parts of Centaurium erythraea Rafn (C. minus Moench, of methanol R. C. umbellatum Gilib., Erythraea centaurium (L.) Pers.).

Centella


Apply to the plate as bands 30 µl of the test solution and 10 µl of the reference solution. Develop over a path of 12 cm using a mixture of 16 volumes of anhydrous acetic acid R, 16 volumes of water R and 68 volumes of ethyl acetate R. Allow the plate to dry in a current of cold air and develop again in the same manner. Allow the plate to dry in a current of cold air. Examine the plate in ultraviolet light at 254 nm. The chromatogram obtained with the test solution shows as the main quenching zone a strong zone (swertiamarin) similar in position to the quenching zone of rutin in the chromatogram obtained with the reference solution ; further weaker quenching zones may also be present. Spray the plate with anisaldehyde solution R. Examine in daylight whilst heating at 100 °C to 105 °C for 5 min to 10 min. The chromatogram obtained with the reference solution shows a yellowish-brown zone (rutin). The chromatogram obtained with the test solution shows : a violet-brown zone (swertiamarin) ; a little above this zone, a faint yellowish-brown zone ; and above this a few, very faint, mostly grey zones leading to a reddish-violet zone at the solvent front. Examine the plate in ultraviolet light at 365 nm. The chromatogram obtained with the test solution shows : a strong brown to brownish-yellow fluorescent zone (swertiamarin) ; a little above this zone, a blue to yellowish-green fluorescent zone ; and above this a few faint blue to yellow fluorescent zones, leading to a faint reddish-violet fluorescent zone at the solvent front. Below the zone due to swertiamarin occur zones of intense light green to yellowish-green fluorescence as well as a number of faint brown fluorescent zones. TESTS Foreign matter (2.8.2). Not more than 3 per cent. Bitterness value. Not less than 2000. The bitterness value is determined by comparison with quinine hydrochloride, the bitterness value of which is set at 200 000 ; the bitterness value is the reciprocal of the dilution that still has a bitter taste. Quinine hydrochloride stock solution. Dissolve 0.100 g of quinine hydrochloride R in water R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with water R. Centaury extract. To 1.0 g of powdered drug (355) add 1000 ml of boiling water R. Heat on a water-bath for 30 min, stirring continuously. Allow to cool and dilute to 1000 ml with water R. Shake vigorously and filter, discarding the first 20 ml of filtrate. Prepare a series of dilutions by placing in the first tube 4.2 ml of quinine hydrochloride stock solution and increasing the volume by 0.2 ml in each subsequent tube to a total of 5.8 ml ; dilute the contents of each tube to 10.0 ml with water R. Determine as follows the dilution with the lowest concentration that still has a bitter taste. Take 10.0 ml of the weakest solution into the mouth and pass it from side to side over the back of the tongue for 30 s. If the solution is not found to be bitter, spit it out and wait for 1 min. Rinse the mouth with water R. After 10 min, use the next dilution in order of increasing concentration. Calculate the correction factor k from the expression :

n

1236

=

number of millilitres of quinine hydrochloride stock solution in the dilution of lowest concentration that is bitter.

Dilute 10/k ml of the centaury extract to 20.0 ml with water R. 10.0 ml of this dilution has a bitter taste. Loss on drying (2.2.32). Not more than 10.0 per cent, determined on 1.000 g of powdered drug (355) by drying in an oven at 100 °C to 105 °C for 2 h. Total ash (2.4.16). Not more than 6.0 per cent. STORAGE Store protected from light. 01/2005:1498

CENTELLA Centellae asiaticae herba DEFINITION Centella consists of the dried, fragmented aerial parts of Centella asiatica (L.) Urban. It contains not less than 6.0 per cent of total triterpenoid derivatives, expressed as asiaticoside (C48H78O19 ; Mr 959.15), calculated with reference to the dried drug. CHARACTERS It has the macroscopic and microscopic characters described under identification tests A and B. The leaves are very variable in size ; the petiole is usually 5 to 10, sometimes 15, times longer than the lamina, which is 10 mm to 40 mm long and 20 mm to 40 mm, sometimes up to 70 mm, wide. IDENTIFICATION A. The leaves are alternate, sometimes grouped together at the nodes, reniform or orbicular or oblong-elliptic and have palmate nervation, usually with seven veins, and a crenate margin. Young leaves show a few trichomes on the lower surface while adult leaves are glabrous. The inflorescence, if present, is a single umbel which usually consists of three flowers, rarely two or four ; the flowers are very small (about 2 mm) pentamerous and have an inferior ovary ; the fruit, a brownish-grey, orbicular cremocarp, up to 5 mm long, is very flattened laterally and has seven to nine prominent curved ridges. B. Reduce the drug to a powder (355). The powder is greenish-grey. Examine under a microscope using chloral hydrate solution R. The powder shows numerous fragments of leaf epidermis with polygonal cells having an irregularly striated cuticle, and paracytic stomata (2.8.3) that are more numerous in the lower epidermis ; fragments of petiole epidermis with elongated cells ; uniseriate, long, flexuous unicellular covering trichomes, occasionally multicellular ; young leaves ; spiral vessels ; resiniferous canals ; calcium oxalate prisms and macles up to 40 µm in diameter ; bundles of narrow septate fibres from the stem ; fragments of the fruit : layers of wide cells in a parquetry arrangement, annular vessels, parenchyma cells containing simple or compound starch granules. C. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R. Test solution. To 5.0 g of the powdered drug (355) add 50 ml of alcohol (30 per cent V/V) R ; heat to boiling under a reflux condenser and centrifuge. Reference solution. Dissolve 5 mg of asiaticoside R in methanol R and dilute to 10 ml with the same solvent. Apply to the plate as bands 10 µl of each solution. Develop over a path of 15 cm using a mixture of 11 volumes of acetic acid R, 11 volumes of formic acid R, 27 volumes


Cetirizine dihydrochloride


of water R and 100 volumes of ethyl acetate R. Allow the plate to dry in air. Spray with anisaldehyde solution R and heat at 100 °C to 105 °C. Examine in daylight. The chromatograms obtained with the reference and the test solutions show in the lower third a greenish-blue zone (asiaticoside). The chromatogram obtained with the test solution shows also below this zone a violet zone (madecassoside) ; near the solvent front it shows a light blue zone (asiatic acid) and just below a pinkish-violet zone (madecassic acid) ; in the lower half it shows brown, grey and brownish-green zones between the starting point and the zone due to madecassoside, and other brownish-yellow or light yellow zones above the zone due to asiaticoside.

m1 HPLCP

= mass of asiaticoside in the reference solution, in milligrams, = purity determined for asiaticoside.

Calculate the mean response factor for asiaticoside from the expression :

=

sum of response factors of asiaticoside for the chromatograms obtained with the reference solution,

N = number of injections of reference solution TESTS (N = 4, at least). Foreign matter (2.8.2). Not more than 7 per cent of which Calculate the percentage content of total triterpenoid not more than 5 per cent of underground organs and not derivatives, expressed as asiaticoside from the expression : more than 2 per cent of other foreign matter. Loss on drying (2.2.32). Not more than 10.0 per cent, determined on 1.000 g of the powdered drug by drying in an oven at 100 °C to 105 °C for 2 h. Total ash (2.4.16). Not more than 12.0 per cent. V = volume of the test solution, in millilitres, ASSAY m = mass of the substance to be examined in the test Examine by liquid chromatography (2.2.29). solution, in milligrams, Test solution. Place 5.0 g of the powdered drug (355) in a A = area of the peak corresponding to asiaticoside in cellulose fingerstall in a continuous extraction apparatus the chromatogram with the test solution, (Soxhlet type). Add 100 ml of methanol R and heat for 8 h. Cool and dilute the extract to 100.0 ml with methanol R. B = area of the peak corresponding to madecassoside Filter through a 0.45 µm filter. Dilute 2.0 ml of the filtrate in the chromatogram with the test solution, and dilute to 20.0 ml with methanol R. C = area of the peak corresponding to madecassic Reference solution. Dissolve 20.0 mg of asiaticoside R in acid in the chromatogram with the test solution, methanol R, if necessary using sonication, and dilute to D = area of the peak corresponding to asiatic acid in 20.0 ml with the same solvent. Dilute 2.0 ml of the solution the chromatogram with the test solution, to 100.0 ml with methanol R. = mean response factor of asiaticoside. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4 mm in Relative retention with reference to the solvent : internal diameter packed with octadecylsilyl silica gel for madecassoside = about 5.8 ; asiaticoside = about 8.1 ; chromatography R (5 µm), madecassic acid = about 17.6 ; asiatic acid = about 21.7. — as mobile phase at a flow rate of 1.0 ml/min : STORAGE Mobile phase A. Acetonitrile for chromatography R, Store protected from light. Mobile phase B. Dilute 3 ml of phosphoric acid R to 1000 ml with water R, 01/2005:1084 Time (min)



CETIRIZINE DIHYDROCHLORIDE

0 - 65

22

78

65 - 66

55

45

Cetirizini dihydrochloridum

66 - 76

95

5

76 - 85

22

78

— as detector a spectrophotometer set at 200 nm. Inject 20 µl of each solution. Calculate the response factor of asiaticoside from the expression :

A1

= area of the peak corresponding to asiaticoside in the chromatogram obtained with the reference solution,

V1

= volume of the reference solution, in millilitres,


C21H27Cl3N2O3

Mr 461.8

DEFINITION (RS)-2-[2-[4-[(4-Chlorophenyl)phenylmethyl]piperazin-1yl]ethoxy]acetic acid dihydrochloride. Content : 99.0 per cent to 100.5 per cent (dried substance). CHARACTERS Appearance : white or almost white powder. 1237

Cetirizine dihydrochloride


Solubility : freely soluble in water, practically insoluble in acetone and in methylene chloride. IDENTIFICATION First identification : B, D. Second identification : A, C, D. A. Dissolve 20.0 mg in 50 ml of a 10.3 g/l solution of hydrochloric acid R and dilute to 100.0 ml with the same acid. Dilute 10.0 ml of the solution to 100.0 ml with a 10.3 g/l solution of hydrochloric acid R. Examined between 210 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 231 nm. The specific absorbance at the maximum is 359 to 381. B. Infrared absorption spectrophotometry (2.2.24).

Reference solution (a). Dissolve 5.0 mg of cetirizine dihydrochloride CRS and 5.0 mg of cetirizine impurity A CRS in the mobile phase and dilute to 25.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 100.0 ml with the mobile phase. Reference solution (b). Dilute 2.0 ml of the test solution to 50.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : silica gel for chromatography R (5 µm). Mobile phase : dilute sulphuric acid R, water R, acetonitrile R (0.4:6.6:93 V/V/V).

Preparation : discs.

Flow rate : 1 ml/min.

Comparison : cetirizine dihydrochloride CRS.


C. Thin-layer chromatography (2.2.27). Test solution. Dissolve 10 mg of the substance to be examined in water R and dilute to 5 ml with the same solvent. Reference solution (a). Dissolve 10 mg of cetirizine dihydrochloride CRS in water R and dilute to 5 ml with the same solvent. Reference solution (b). Dissolve 10 mg of chlorphenamine maleate CRS in water R and dilute to 5 ml with the same solvent. To 1 ml of the solution add 1 ml of reference solution (a). Plate : TLC silica gel GF254 plate R.

Run time : 3 times the retention time of cetirizine. System suitability : reference solution (a) : — resolution : minimum 3 between the peaks due to cetirizine and impurity A, — symmetry factors : maximum 2.0. Limits : — impurities A, B, C, D, E, F : for each impurity, not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent), — any other impurity : for each impurity, not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent),

Mobile phase : ammonia R, methanol R, methylene chloride R (1:10:90 V/V/V). Application : 5 µl.

— total : not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.3 per cent),

Development : over 2/3 of the plate. Drying : in a current of cold air. Detection : examine in ultraviolet light at 254 nm. System suitability : reference solution (b) :

— disregard limit : 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.02 per cent).

— the chromatogram obtained shows 2 clearly separated spots.

Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C.

Results : the principal spot in the chromatogram obtained Sulphated ash (2.4.14) : maximum 0.2 per cent, determined with the test solution is similar in position and size to on 1.0 g. the principal spot in the chromatogram obtained with reference solution (a). ASSAY D. It gives reaction (a) of chlorides (2.3.1). Dissolve 0.100 g in 70 ml of a mixture of 30 volumes TESTS Solution S. Dissolve 1.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution BY7 (2.2.2, Method II). pH (2.2.3) : 1.2 to 1.8 for solution S.

of water R and 70 volumes of acetone R. Titrate with 0.1 M sodium hydroxide to the second point of inflexion. Determine the end-point potentiometrically (2.2.20). Carry out a blank titration. 1 ml of 0.1 M sodium hydroxide is equivalent to 15.39 mg of C21H27Cl3N2O3. STORAGE

Related substances. Liquid chromatography (2.2.29).

Protected from light.

Test solution. Dissolve 20.0 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase.

IMPURITIES

1238

Specified impurities : A, B, C, D, E, F.



Cetostearyl alcohol (type A), emulsifying

Acid value (2.5.1). Not more than 1.0. Hydroxyl value (2.5.3, Method A) : 208 to 228. Iodine value (2.5.4). Not more than 2.0, determined on 2.00 g, dissolved in 25 ml of chloroform R. Saponification value (2.5.6). Not more than 2.0. ASSAY Examine by gas chromatography (2.2.28). Test solution. Dissolve 0.100 g of the substance to be B. R1 = CH2-CO2H, R2 = H, R3 = Cl : (RS)-2-[4-[(4examined in ethanol R and dilute to 10.0 ml with the same chlorophenyl)phenylmethyl]piperazin-1-yl]acetic acid, solvent. Reference solution (a). Dissolve 60.0 mg of cetyl C. R1 = CH2-CH2-O-CH2-CO2H, R2 = Cl, R3 = H : alcohol CRS in ethanol R and dilute to 10.0 ml with the (RS)-2-[2-[4-[(2-chlorophenyl)phenylmethyl]piperazin-1same solvent. yl]ethoxy]acetic acid, Reference solution (b) Dissolve 40.0 mg of stearyl E. R1 = CH2-[CH2-O-CH2]2-CO2H, R2 = H, R3 = Cl : (RS)-2-[2-[2-[4-[(4-chlorophenyl)phenylmethyl]piperazin-1- alcohol CRS in ethanol R and dilute to 10.0 ml with the same solvent. yl]ethoxy]ethoxy]acetic acid (ethoxycetirizine), Reference solution (c). Mix 1 ml of reference solution (a) F. R1 = CH2-CH2-O-CH2-CO2H, R2 = R3 = H : and 1 ml of reference solution (b) and dilute to 10.0 ml with [2-[4-(diphenylmethyl)piperazin-1-yl]ethoxy]acetic ethanol R. acid, The chromatographic procedure may be carried out using : — a stainless steel column 3 m long and 4 mm in internal diameter packed with diatomaceous earth for gas chromatography R impregnated with 10 per cent m/m of poly(dimethyl)siloxane R, — nitrogen for chromatography R as the carrier gas at a flow rate of 30 ml/min, — a flame-ionisation detector, maintaining the temperature of the column at 200 °C and that of the injection port and the detector at 250 °C. Inject separately 2 µl of each solution. Adjust the flow rate so that the resolution between the 2 principal peaks in the D. 1,4-bis[(4-chlorophenyl)phenylmethyl]piperazine. chromatogram obtained with the test solution is not less than 1.25. The test is not valid unless the chromatogram 01/2005:0702 obtained with reference solution (c) shows 2 principal peaks with a signal-to-noise ratio of at least 5. Determine the content of cetyl alcohol and of stearyl alcohol from CETOSTEARYL ALCOHOL the chromatogram obtained with the test solution, using the normalisation procedure and identifying the peaks by Alcohol cetylicus et stearylicus comparison with the chromatograms obtained with reference solution (a) and reference solution (b), respectively. DEFINITION Cetostearyl alcohol is a mixture of solid aliphatic alcohols. 01/2005:0801 It contains not less than 40.0 per cent of stearyl alcohol (C18H38O ; Mr 270.5) and the sum of the contents of cetyl alcohol (C16H34O ; Mr 242.4) and of stearyl alcohol is not less CETOSTEARYL ALCOHOL (TYPE A), than 90.0 per cent. A. R1 = R2 = H, R3 = Cl : (RS)-1-[(4-chlorophenyl)phenylmethyl]piperazine,

CHARACTERS White or pale yellow, wax-like mass, plates, flakes or granules, practically insoluble in water, soluble in alcohol (90 per cent V/V) and in light petroleum ; when melted, it is miscible with fatty oils, with liquid paraffin and with melted wool fat.

EMULSIFYING

Alcohol cetylicus et stearylicus emulsificans A

DEFINITION Emulsifying cetostearyl alcohol (type A) is a mixture which contains not less than 80.0 per cent of cetostearyl alcohol IDENTIFICATION and not less than 7.0 per cent of sodium cetostearyl sulphate, Examine the chromatograms obtained in the assay. The both calculated with reference to the anhydrous substance. 2 principal peaks in the chromatogram obtained with the test A suitable buffer may be added. solution have similar retention times to the principal peaks in the chromatograms obtained with reference solution (a) CHARACTERS and reference solution (b), respectively. White or pale yellow, wax-like mass, plates, flakes or granules, soluble in hot water giving an opalescent solution, TESTS practically insoluble in cold water, slightly soluble in alcohol. Appearance of solution. Dissolve 0.50 g in 20 ml of boiling alcohol R. The solution is clear (2.2.1) and not more intensely IDENTIFICATION coloured than reference solution B6 (2.2.2, Method II). First identification : B, C, D. Melting point (2.2.14) : 49 °C to 56 °C. Second identification : A, C. General Notices (1) apply to all monographs and other texts

1239

Cetostearyl alcohol (type A), emulsifying


A. Examine by thin-layer chromatography (2.2.27), using a TLC silanised silica gel plate R. Test solution (a). Dissolve 0.1 g of the substance to be examined in 10 ml of trimethylpentane R, heating on a water-bath. Shake with 2 ml of alcohol (70 per cent V/V) R and allow to separate. Use the lower layer as test solution (b). Dilute 1 ml of the upper layer to 8 ml with trimethylpentane R. Test solution (b). Use the lower layer obtained in the preparation of test solution (a). Reference solution (a). Dissolve 40 mg of cetostearyl alcohol R in 10 ml of trimethylpentane R. Reference solution (b). Dissolve 20 mg of sodium cetostearyl sulphate R in 10 ml of alcohol (70 per cent V/V) R, heating on a water-bath.

Test solution (b). Dissolve 0.300 g of the substance to be examined in 50 ml of ethanol R, add 50 ml of water R and shake with 4 quantities, each of 25 ml, of pentane R, adding sodium chloride R, if necessary, to facilitate the separation of the layers. Combine the organic layers. Wash with 2 quantities, each of 30 ml, of water R, dry over anhydrous sodium sulphate R and filter. Reference solution. Dissolve 50 mg of cetyl alcohol CRS and 50 mg of stearyl alcohol CRS in ethanol R and dilute to 10 ml with the same solvent. The chromatographic procedure may be carried out using : — a fused-silica column 25 m long and 0.25 mm in internal diameter coated with poly(dimethyl)siloxane R, — nitrogen for chromatography R as the carrier gas at a flow rate of 1 ml/min, — a flame-ionisation detector, — a split ratio of 1:100, with the following temperature programme :

Apply to the plate 2 µl of each solution. Develop over a path of 12 cm using a mixture of 20 volumes of water R, 40 volumes of acetone R and 40 volumes of methanol R. Allow the plate to dry in air and spray with a 50 g/l Time Temperature Rate Comment solution of phosphomolybdic acid R in alcohol R. Heat at 120 °C until spots appear (about 3 h). The 2 principal (min) (°C) (°C/min) spots in the chromatogram obtained with test solution (a) Column 0 - 20 150 → 250 5 linear gradient are similar in position and colour to the principal spots in Injection port 250 the chromatogram obtained with reference solution (a). 2 of the spots in the chromatogram obtained with test Detector 250 solution (b) are similar in position and colour to the The substances are eluted in the following order : cetyl principal spots in the chromatogram obtained with alcohol, heptadecanol (internal standard) and stearyl alcohol. reference solution (b). Inject 1 µl of test solution (a) and 1 µl of test solution (b). If B. Examine the chromatograms obtained in the assay. the chromatogram obtained with test solution (b) shows a The retention times of the 2 principal peaks in the chromatogram obtained with test solution (b) are similar peak with the same retention time as the peak corresponding to the internal standard in the chromatogram obtained with to those of the 2 principal peaks in the chromatogram test solution (a), calculate the ratio : obtained with the reference solution. C. It gives a yellow colour to a non-luminous flame. D. To 0.3 g add 20 ml of ethanol R and heat on a water-bath with shaking. Filter the mixture immediately, evaporate Sci to dryness and take up the residue in 7 ml of water R. To 1 ml of the solution add 0.1 ml of a 1 g/l solution of methylene blue R, 2 ml of dilute sulphuric acid R and Si 2 ml of methylene chloride R and shake. A blue colour develops in the lower layer. TESTS Acid value (2.5.1). Not more than 2.0. Iodine value (2.5.4). Not more than 3.0, determined on 2.00 g dissolved in 25 ml of methylene chloride R. Saponification value (2.5.6). Not more than 2.0. Water (2.5.12). Not more than 3.0 per cent, determined on 2.50 g by the semi-micro determination of water. ASSAY Cetostearyl alcohol. Examine by gas chromatography (2.2.28).

= =

area of the peak corresponding to cetyl alcohol in the chromatogram obtained with test solution (b), area of the peak with the same retention time as the peak corresponding to the internal standard in the chromatogram obtained with test solution (a).

If r is less than 300, calculate the corrected area SHa(corr) of the peak corresponding to the internal standard in the chromatogram obtained with test solution (a) :

S′Ha =

area of the peak corresponding to the internal standard in the chromatogram obtained with test solution (a),

Sc

area of the peak corresponding to cetyl alcohol in the chromatogram obtained with test solution (a).

=

Inject, under the same conditions, equal volumes of the reference solution and of test solution (a). Identify the peaks in the chromatogram obtained with test solution (a) by comparing their retention times with those of the peaks in the chromatogram obtained with the reference solution and Test solution (a). Dissolve 0.300 g of the substance to be determine the area of each peak. examined in 50 ml of the internal standard solution, add 50 ml of water R and shake with 4 quantities, each of 25 ml, Calculate the percentage content of cetyl alcohol using the of pentane R, adding sodium chloride R, if necessary, to expression : facilitate the separation of the layers. Combine the organic layers. Wash with 2 quantities, each of 30 ml, of water R, dry over anhydrous sodium sulphate R and filter.

Internal standard solution. Dissolve 0.60 g of heptadecanol CRS in ethanol R and dilute to 150 ml with the same solvent.

1240


Cetostearyl alcohol (type B), emulsifying


SA

= area of the peak corresponding to cetyl alcohol in the chromatogram obtained with test solution (a),

mH

= mass of the internal standard in test solution (a), in milligrams, = corrected area of the peak corresponding to the internal standard in the chromatogram obtained with test solution (a),

SHa(corr) m

= mass of the substance to be examined in test solution (a), in milligrams.

Calculate the percentage content of stearyl alcohol using the expression :

SB

=

area of the peak corresponding to stearyl alcohol in the chromatogram obtained with test solution (a).

The percentage content of cetostearyl alcohol corresponds to the sum of the percentage content of cetyl alcohol and of stearyl alcohol. Sodium cetostearyl sulphate. Disperse 0.300 g in 25 ml of methylene chloride R. Add 50 ml of water R and 10 ml of dimidium bromide-sulphan blue mixed solution R. Titrate with 0.004 M benzethonium chloride, using sonication and heating and allowing the layers to separate before each addition, until the colour of the lower layer changes from pink to grey. 1 ml of 0.004 M benzethonium chloride is equivalent to 1.434 mg of sodium cetostearyl sulphate. LABELLING The label states, where applicable, the name and concentration of any added buffer. 01/2005:0802

CETOSTEARYL ALCOHOL (TYPE B), EMULSIFYING Alcohol cetylicus et stearylicus emulsificans B

cent V/V) R and allow to separate. Use the lower layer as test solution (b). Dilute 1 ml of the upper layer to 8 ml with trimethylpentane R. Test solution (b). Use the lower layer obtained in the preparation of test solution (a). Reference solution (a). Dissolve 40 mg of cetostearyl alcohol R in 10 ml of trimethylpentane R. Reference solution (b). Dissolve 20 mg of sodium laurilsulfate R in 10 ml of alcohol (70 per cent V/V) R, heating on a water-bath. Apply to the plate 2 µl of each solution. Develop over a path of 12 cm using a mixture of 20 volumes of water R, 40 volumes of acetone R and 40 volumes of methanol R. Allow the plate to dry in air and spray with a 50 g/l solution of phosphomolybdic acid R in alcohol R. Heat at 120 °C until spots appear (about 3 h). The 2 principal spots in the chromatogram obtained with test solution (a) are similar in position and colour to the principal spots in the chromatogram obtained with reference solution (a). 1 of the spots in the chromatogram obtained with test solution (b) is similar in position and colour to the principal spot in the chromatogram obtained with reference solution (b). B. Examine the chromatograms obtained in the assay. The retention times of the 2 principal peaks in the chromatogram obtained with test solution (b) are similar to those of the 2 principal peaks in the chromatogram obtained with the reference solution. C. It gives a yellow colour to a non-luminous flame. D. To 0.3 g add 20 ml of ethanol R and heat to boiling on a water-bath with shaking. Filter the mixture immediately, evaporate to dryness and take up the residue in 7 ml of water R. To 1 ml of the solution add 0.1 ml of a 1 g/l solution of methylene blue R, 2 ml of dilute sulphuric acid R and 2 ml of methylene chloride R and shake. A blue colour develops in the lower layer. TESTS Acid value (2.5.1). Not more than 2.0. Iodine value (2.5.4). Not more than 3.0, determined on 2.00 g dissolved in 25 ml of methylene chloride R. Saponification value (2.5.6). Not more than 2.0. Water (2.5.12). Not more than 3.0 per cent, determined on 2.50 g by the semi-micro determination of water.

ASSAY Cetostearyl alcohol. Examine by gas chromatography (2.2.28). Internal standard solution. Dissolve 0.60 g of heptadecanol CRS in ethanol R and dilute to 150 ml with the same solvent. Test solution (a). Dissolve 0.300 g of the substance to be CHARACTERS examined in 50 ml of the internal standard solution, add White or pale yellow, wax-like mass, plates, flakes or 50 ml of water R and shake with 4 quantities, each of 25 ml, granules, soluble in hot water giving an opalescent solution, of pentane R, adding sodium chloride R, if necessary, to practically insoluble in cold water, slightly soluble in alcohol. facilitate the separation of the layers. Combine the organic layers. Wash with 2 quantities, each of 30 ml, of water R, dry IDENTIFICATION over anhydrous sodium sulphate R and filter. First identification : B, C, D. Test solution (b). Dissolve 0.300 g of the substance to be Second identification : A, C. examined in 50 ml of ethanol R, add 50 ml of water R and A. Examine by thin-layer chromatography (2.2.27), using a shake with 4 quantities, each of 25 ml, of pentane R, adding TLC silanised silica gel plate R. sodium chloride R, if necessary, to facilitate the separation Test solution (a). Dissolve 0.1 g of the substance to of the layers. Combine the organic layers. Wash with be examined in 10 ml of trimethylpentane R, heating 2 quantities, each of 30 ml, of water R, dry over anhydrous on a water-bath. Shake with 2 ml of alcohol (70 per sodium sulphate R and filter. DEFINITION Emulsifying cetostearyl alcohol (type B) is a mixture which contains not less than 80.0 per cent of cetostearyl alcohol and not less than 7.0 per cent of sodium laurilsulfate, both calculated with reference to the anhydrous substance. A suitable buffer may be added.


1241

Cetostearyl isononanoate


Reference solution. Dissolve 50 mg of cetyl alcohol CRS and 50 mg of stearyl alcohol CRS in ethanol R and dilute to 10 ml with the same solvent. The chromatographic procedure may be carried out using : — a fused-silica column 25 m long and 0.25 mm in internal diameter coated with poly(dimethyl)siloxane R, — nitrogen for chromatography R as the carrier gas at a flow rate of 1 ml/min, — a flame-ionisation detector, — a split ratio of 1:100, with the following temperature programme : Time (min) 0 - 20

Column

Temperature Rate (°C) (°C/min) 150 → 250

Injection port

250

Detector

250

5

Comment linear gradient

SHa(corr) = corrected area of the peak corresponding to the internal standard in the chromatogram obtained with test solution (a), m = mass of the substance to be examined in test solution (a), in milligrams. Calculate the percentage content of stearyl alcohol using the expression :

SB

=

area of the peak corresponding to stearyl alcohol in the chromatogram obtained with test solution (a).

The percentage content of cetostearyl alcohol corresponds to the sum of the percentage content of cetyl alcohol and of stearyl alcohol.

Sodium laurilsulfate. Disperse 0.300 g in 25 ml of methylene chloride R. Add 50 ml of water R and 10 ml of The substances are eluted in the following order : cetyl alcohol, heptadecanol (internal standard) and stearyl alcohol. dimidium bromide-sulphan blue mixed solution R. Titrate with 0.004 M benzethonium chloride, using sonication and Inject 1 µl of test solution (a) and 1 µl of test solution (b). If heating, and allowing the layers to separate before each the chromatogram obtained with test solution (b) shows a addition, until the colour of the lower layer changes from peak with the same retention time as the peak corresponding pink to grey. to the internal standard in the chromatogram obtained with 1 ml of 0.004 M benzethonium chloride is equivalent to test solution (a), calculate the ratio : 1.154 mg of sodium laurilsulfate.

Sci

=

Si

=

area of the peak corresponding to cetyl alcohol in the chromatogram obtained with test solution (b), area of the peak with the same retention time as the peak corresponding to the internal standard in the chromatogram obtained with test solution (a).

If r is less than 300, calculate the corrected area SHa(corr) of the peak corresponding to the internal standard in the chromatogram obtained with test solution (a) :

S′Ha =

area of the peak corresponding to the internal standard in the chromatogram obtained with test solution (a),

Sc

area of the peak corresponding to cetyl alcohol in the chromatogram obtained with test solution (a).

=

Inject, under the same conditions, equal volumes of the reference solution and of test solution (a). Identify the peaks in the chromatogram obtained with test solution (a) by comparing their retention times with those of the peaks in the chromatogram obtained with the reference solution and determine the area of each peak. Calculate the percentage content of cetyl alcohol using the expression :

SA

= area of the peak corresponding to cetyl alcohol in the chromatogram obtained with test solution (a),

mH

= mass of the internal standard in test solution (a), in milligrams,

1242

LABELLING The label states, where applicable, the name and concentration of any added buffer. 01/2005:1085

CETOSTEARYL ISONONANOATE Cetostearylis isononanoas DEFINITION Cetostearyl isononanoate is a mixture of esters of cetostearyl alcohol with isononanoic acid, mainly 3,5,5-trimethylhexanoic acid. CHARACTERS A clear, colourless or slightly yellowish liquid, practically insoluble in water, soluble in alcohol and in light petroleum, miscible with fatty oils and with liquid paraffins. It has a viscosity of 15 mPa·s to 30 mPa·s, a relative density of 0.85 to 0.86 and a refractive index of 1.44 to 1.45. IDENTIFICATION A. On cooling, turbidity occurs below 15 °C. B. It complies with the test for saponification value (see Tests). C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of cetostearyl isononanoate. TESTS Appearance. The substance to be examined is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method I). Acid value (2.5.1). Not more than 1.0, determined on 5.0 g. Hydroxyl value (2.5.3, Method A). Not more than 5.0. Iodine value (2.5.4). Not more than 1.0.



Cetyl alcohol

Saponification value (2.5.6) : 135 to 148, determined on 1.0 g. Heavy metals (2.4.8). 2.0 g complies with limit test D for heavy metals (10 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Water (2.5.12). Not more than 0.2 per cent, determined on 10.0 g by the semi-micro determination of water. Total ash (2.4.16). Not more than 0.2 per cent, determined on 2.0 g.

TESTS Solution S. Dissolve 2.0 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Acidity or alkalinity. To 50 ml of solution S add 0.1 ml of bromocresol purple solution R. Not more than 0.1 ml of 0.1 M hydrochloric acid or 0.1 M sodium hydroxide is required to change the colour of the indicator. Amines and amine salts. Dissolve 5.0 g in 30 ml of a mixture 01/2005:0378 of 1 volume of 1 M hydrochloric acid and 99 volumes of methanol R and add 100 ml of 2-propanol R. Pass a stream of nitrogen R slowly through the solution. Gradually add CETRIMIDE 15.0 ml of 0.1 M tetrabutylammonium hydroxide and record the potentiometric titration curve (2.2.20). If the curve Cetrimidum shows 2 points of inflexion, the volume of titrant added between the 2 points is not greater than 2.0 ml. Loss on drying (2.2.32) : maximum 2.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 2 h. Sulphated ash (2.4.14) : maximum 0.5 per cent, determined on 1.0 g. DEFINITION Cetrimide consists of trimethyltetradecylammonium ASSAY bromide and may contain smaller amounts of dodecyl- and Dissolve 2.000 g in water R and dilute to 100.0 ml with hexadecyl-trimethylammonium bromides. the same solvent. Transfer 25.0 ml of the solution to a Content : 96.0 per cent to 101.0 per cent of separating funnel, add 25 ml of chloroform R, 10 ml of 0.1 M alkyltrimethylammonium bromides, calculated as sodium hydroxide and 10.0 ml of a freshly prepared 50 g/l C17H38BrN (Mr 336.4) (dried substance). solution of potassium iodide R. Shake, allow to separate and discard the chloroform layer. Shake the aqueous layer CHARACTERS with 3 quantities, each of 10 ml, of chloroform R and discard Appearance : white or almost white, voluminous, free-flowing the chloroform layers. Add 40 ml of hydrochloric acid R, powder. allow to cool and titrate with 0.05 M potassium iodate until Solubility : freely soluble in water and in alcohol. the deep brown colour is almost discharged. Add 2 ml of chloroform R and continue the titration, shaking vigorously, IDENTIFICATION until the colour of the chloroform layer no longer changes. A. Dissolve 0.25 g in alcohol R and dilute to 25.0 ml with the Carry out a blank titration on a mixture of 10.0 ml of the same solvent. At wavelengths from 260 nm to 280 nm, freshly prepared 50 g/l solution of potassium iodide R, the absorbance (2.2.25) of the solution has a maximum 20 ml of water R and 40 ml of hydrochloric acid R. of 0.05. 1 ml of 0.05 M potassium iodate is equivalent to 33.64 mg B. Dissolve about 5 mg in 5 ml of buffer solution pH 8.0 R. of C17H38BrN. Add about 10 mg of potassium ferricyanide R. A yellow precipitate is formed. Prepare a blank in the same manner 01/2005:0540 but omitting the substance to be examined : a yellow solution is observed but no precipitate is formed. CETYL ALCOHOL C. Solution S (see Tests) froths copiously when shaken. D. Thin-layer chromatography (2.2.27). Alcohol cetylicus Test solution. Dissolve 0.10 g of the substance to be DEFINITION examined in water R and dilute to 5 ml with the same Cetyl alcohol is a mixture of solid alcohols consisting mainly solvent. of hexadecanol (CH3-(CH2)14-CH2OH ; Mr 242.4). Reference solution. Dissolve 0.10 g of trimethyltetradecylammonium bromide CRS in water R and dilute to 5 ml with the same solvent. Plate : TLC silica gel F254 silanised plate R. Mobile phase : acetone R, 270 g/l solution of sodium acetate R, methanol R (20:35:45 V/V/V). Application : 1 µl. Development : over a path of 12 cm. Drying : in a current of hot air. Detection : allow to cool ; expose the plate to iodine vapour and examine in daylight. Result : the principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. E. It gives reaction (a) of bromides (2.3.1).


CHARACTERS White, unctuous mass, powder, flakes or granules, practically insoluble in water, freely to sparingly soluble in alcohol ; when melted, it is miscible with vegetable and animal oils, with liquid paraffin and with melted wool fat. IDENTIFICATION A. Melting point (2.2.14) : 46 °C to 52 °C. B. Hydroxyl value (2.5.3, Method A) : 218 to 238. TESTS Appearance of solution. Dissolve 0.5 g in boiling alcohol R, allow to cool and dilute to 20 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution B6 (2.2.2, Method II). Acid value (2.5.1). Not more than 1.0. 1243

Cetyl palmitate


Iodine value (2.5.4). Not more than 2.0, determined on 2.00 g dissolved in 25 ml of chloroform R. Saponification value (2.5.6). Not more than 2.0.

Reference solution (a). Dissolve 20.0 mg of cetyl palmitate 95 CRS in hexane R and dilute to 20.0 ml with the same solvent. Reference solution (b). Dissolve 20.0 mg of cetyl palmitate 15 CRS in hexane R and dilute to 01/2005:1906 20.0 ml with the same solvent. Column : CETYL PALMITATE — material : stainless steel, — size : l = 10 m, Ø = 0.53 mm, Cetylis palmitas — stationary phase : poly(dimethyl)siloxane R (film thickness : 2.65 µm). DEFINITION Carrier gas : helium for chromatography R. Mixture of C14-C18 esters of lauric, myristic, palmitic and Flow rate : 6.5 ml/min. stearic acids (“Cetyl esters wax”). Split ratio : 1:10. Content (expressed as hexadecyl hexadecanoate) : 10.0 per Temperature : cent to 20.0 per cent for Cetyl palmitate 15, 60.0 per cent to 70.0 per cent for Cetyl palmitate 65 and not less than Time Temperature 90.0 per cent for Cetyl palmitate 95. (min) (°C) CHARACTERS Appearance : white, waxy plates, flakes or powder. Solubility : practically insoluble in water, soluble in boiling ethanol and in methylene chloride, slightly soluble in light petroleum, practically insoluble in ethanol. mp : about 45 °C for Cetyl palmitate 15 and Cetyl palmitate 65 and about 52 °C for Cetyl palmitate 95. IDENTIFICATION A. It complies with the limits of the assay and the chromatogram obtained with the test solution shows the typical main peak(s). B. It complies with the test for saponification value (see Tests). TESTS Appearance of solution. The solution is not more intensely coloured than reference solution Y6 (2.2.2, Method II). Dissolve 4.0 g in methylene chloride R and dilute to 20 ml with the same solvent. Acid value (2.5.1) : maximum 4.0. Dissolve 10.0 g in 50 ml of the solvent mixture described by heating under reflux on a water-bath for 5 min. Hydroxyl value (2.5.3, Method A) : maximum 20.0. Iodine value (2.5.4) : maximum 2.0. Saponification value (2.5.6) : 105 to 120. Heat under reflux for 2 h. Alkaline impurities. Dissolve with gentle heating 2.0 g in a mixture of 1.5 ml of alcohol R and 3 ml of toluene R. Add 0.05 ml of a 0.4 g/l solution of bromophenol blue R in alcohol R. Not more than 0.4 ml of 0.01 M hydrochloric acid is required to change the colour of the solution to yellow. Nickel (2.4.27) : maximum 1 ppm. Water (2.5.12) : maximum 0.3 per cent, determined on 1.0 g using a mixture of equal volumes of anhydrous methanol R and methylene chloride R as solvent. Total ash (2.4.16) : maximum 0.2 per cent, determined on 1.0 g. ASSAY Gas chromatography (2.2.28) : use the normalisation procedure. Test solution. Dissolve 20.0 mg of the substance to be examined in hexane R and dilute to 20.0 ml with the same solvent. 1244

Column

0 - 10

100 → 300

10 - 15

300

Injection port

350

Detector

350

Detection : flame ionisation. Injection : 1 µl. Relative retention with reference to cetyl palmitate (retention time = about 9 min) : cetyl alcohol = about 0.3 ; palmitic acid = about 0.4 ; lauric ester = about 0.8 ; myristic ester = about 0.9 ; stearic ester = about 1.1. System suitability : reference solution (b) : — resolution : minimum of 1.5 between the peaks due to cetyl palmitate and cetyl stearate. STORAGE At a temperature not exceeding 25 °C. LABELLING The label states the type of cetyl palmitate. 01/2005:0379

CETYLPYRIDINIUM CHLORIDE Cetylpyridinii chloridum

C21H38ClN,H2O

Mr 358.0

DEFINITION Cetylpyridinium chloride contains not less than 96.0 per cent and not more than the equivalent of 101.0 per cent of 1-hexadecylpyridinium chloride, calculated with reference to the anhydrous substance. CHARACTERS A white powder, slightly soapy to the touch, soluble in water and in alcohol. An aqueous solution froths copiously when shaken. IDENTIFICATION First identification : B, D. Second identification : A, C, D.



A. Dissolve 0.10 g in water R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of this solution to 100.0 ml with water R. Examined between 240 nm and 300 nm (2.2.25), the solution shows an absorption maximum at 259 nm and 2 shoulders at about 254 nm and at about 265 nm. The specific absorbance at the maximum is 126 to 134, calculated with reference to the anhydrous substance. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cetylpyridinium chloride CRS. Examine the substances in the solid state. C. To 5 ml of dilute sodium hydroxide solution R add 0.1 ml of bromophenol blue solution R1 and 5 ml of chloroform R and shake. The chloroform layer is colourless. Add 0.1 ml of solution S (see Tests) and shake. The chloroform layer becomes blue. D. Solution S gives reaction (a) of chlorides (2.3.1).

Chamomile flower, Roman

1 ml of 0.05 M potassium iodate is equivalent to 34.0 mg of C21H38ClN. 01/2005:0380

CHAMOMILE FLOWER, ROMAN Chamomillae romanae flos DEFINITION Roman chamomile flower consists of the dried flower-heads of the cultivated double variety of Chamaemelum nobile (L.) All. (Anthemis nobilis L.). It contains not less than 7 ml/kg of essential oil. CHARACTERS It consists of flower-heads with a white to yellowish-grey colour, being composed of solitary hemispherical capitula, made up of a solid conical receptacle bearing the florets, each subtended by a transparent small palea. It has a strong and characteristic odour. It has the macroscopic and microscopic characters described under identification tests A and B.

TESTS Solution S. Dissolve 1.0 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Appearance of solution. Solution S is not more opalescent than reference suspension II (2.2.1) and is colourless (2.2.2, IDENTIFICATION A. The capitula have a diameter of 8 mm to 20 mm ; Method II). the receptacle is solid ; the base of the receptacle is Acidity. To 50 ml of solution S add 0.1 ml of phenolphthalein surrounded by an involucre consisting of 2 or 3 rows of solution R. Not more than 2.5 ml of 0.02 M sodium hydroxide compact and imbricated bracts with scarious margins. is required to change the colour of the indicator. Most florets are ligulate, but a few pale yellow tubular Amines and amine salts. Dissolve 5.0 g with heating in florets occur in the central region. Ligulate florets are 20 ml of a mixture of 3 volumes of 1 M hydrochloric acid and white, dull, lanceolate and reflexed with a dark brown, 97 volumes of methanol R and add 100 ml of 2-propanol R. inferior ovary, a filiform style and a bifid stigma ; tubular Pass a stream of nitrogen R slowly through the solution. florets have a five-toothed corolla tube, 5 syngenesious, Gradually add 12.0 ml of 0.1 M tetrabutylammonium epipetalous stamens and a gynoecium similar to that of hydroxide and record the potentiometric titration curve the ligulate florets. (2.2.20). If the curve shows 2 points of inflexion, the volume B. Separate the capitulum into its different parts. Examine of titrant added between the two points is not greater under a microscope using chloral hydrate solution R. All than 5.0 ml. If the curve shows no point of inflexion, the parts of the flower-heads are covered with numerous small substance to be examined does not comply with the test. If yellow glistening glandular trichomes. The involucral the curve shows one point of inflexion, repeat the test but bracts and paleae have epidermal cells in longitudal rows, add 3.0 ml of a 25.0 g/l solution of dimethyldecylamine R sclerified at the base and they are covered with conical in 2-propanol R before the titration. If the titration curve trichomes, about 500 µm long, each composed of 3 or after the addition of 12.0 ml of the titrant shows only one 4 very short base cells and a long, bent, terminal cell about point of inflexion, the substance to be examined does not 20 µm wide. The corolla of the ligulate flowers consists comply with the test. of papillary cells with cuticular striations. The ovaries of both kinds of florets have at their base a sclerous ring Water (2.5.12) : 4.5 per cent to 5.5 per cent, determined on consisting of a single row of cells. The receptacle and the 0.300 g by the semi-micro determination of water. ovaries contain small clusters of calcium oxalate. The Sulphated ash (2.4.14). Not more than 0.2 per cent, pollen grains have a diameter of about 35 µm and are determined on 1.0 g. rounded and triangular with 3 germinal pores and a spiny exine. ASSAY C. Examine by thin-layer chromatography (2.2.27), using a Dissolve 2.00 g in water R and dilute to 100.0 ml with the suitable silica gel as the coating substance. same solvent. Transfer 25.0 ml of the solution to a separating Test solution. To 0.5 g of the powdered drug (710) funnel, add 25 ml of chloroform R, 10 ml of 0.1 M sodium add 10 ml of methanol R and heat with shaking in a hydroxide and 10.0 ml of a freshly prepared 50 g/l solution water-bath at 60 °C for 5 min. Allow to cool and filter. of potassium iodide R. Shake well, allow to separate and Reference solution. Dissolve 2.5 mg of apigenin R and discard the chloroform layer. Shake the aqueous layer with 2.5 mg of apigenin-7-glucoside R in 10 ml of methanol R. three quantities, each of 10 ml, of chloroform R and discard the chloroform layers. To the aqueous layer add 40 ml of Apply to the plate as bands 10 µl of each solution. Develop hydrochloric acid R, allow to cool and titrate with 0.05 M over a path of 10 cm using a mixture of 17 volumes potassium iodate until the deep-brown colour is almost of glacial acetic acid R, 17 volumes of water R and discharged. Add 2 ml of chloroform R and continue the 66 volumes of butanol R. Dry the plate at 100-105 °C for titration, shaking vigorously, until the chloroform layer no 5 min and spray the warm plate with a 10 g/l solution of longer changes colour. Carry out a blank titration on a diphenylboric acid aminoethyl ester R in methanol R, mixture of 10.0 ml of the freshly prepared 50 g/l solution using about 10 ml for a plate 200 mm square. Spray of potassium iodide R, 20 ml of water R and 40 ml of the plate with the same volume of a 50 g/l solution of hydrochloric acid R. macrogol 400 R in methanol R. Allow to stand for about General Notices (1) apply to all monographs and other texts

1245

Charcoal, activated


30 min and examine in ultraviolet light at 365 nm. The chromatogram obtained with the reference solution shows in the upper third a zone of yellowish-green fluorescence (apigenin) and in the middle third a zone of yellowish fluorescence (apigenin-7-glucoside). The chromatogram obtained with the test solution shows a zone of yellowish-green fluorescence corresponding in position and fluorescence to the zone due to apigenin and a zone of yellowish fluorescence corresponding in position and fluorescence to the zone due to apigenin-7-glucoside in the chromatogram obtained with the reference solution ; above the apigenin-7-glucoside zone there is a zone of brownish fluorescence (luteolin) ; immediately below the apigenin-7-glucoside zone there is a zone of light brownish fluorescence (apiin) ; immediately below the apiin zone there is a zone of bright blue fluorescence and below this zone a zone of bright blue fluorescence ; other faint zones may be present. TESTS Diameter of the flower-heads. Not more than 3 per cent of flower-heads have a diameter smaller than 8 mm. Deteriorated flower-heads. Brown or darkened flower-heads are absent. Loss on drying (2.2.32) : maximum 11.0 per cent, determined on 1.000 g of the powdered drug (355) by drying in an oven at 100-105 °C for 2 h. Total ash (2.4.16). Not more than 8.0 per cent. ASSAY Carry out the determination of essential oils in vegetable drugs (2.8.12). Use 20.0 g of whole drug, a 500 ml round-bottomed flask, 250 ml of water R as the distillation liquid and 0.50 ml of xylene R in the graduated tube. Distil at a rate of 3-3.5 ml/min for 3 h. STORAGE Store protected from light. 01/2005:0313

CHARCOAL, ACTIVATED Carbo activatus DEFINITION Activated charcoal is obtained from vegetable matter by suitable carbonisation processes intended to confer a high adsorption power. CHARACTERS A black, light powder free from grittiness, practically insoluble in all usual solvents. IDENTIFICATION A. When heated to redness it burns slowly without a flame. B. It complies with the test for adsorption power (see Tests). TESTS Solution S. To 2.0 g in a conical flask with a ground-glass neck add 50 ml of dilute hydrochloric acid R. Boil gently under a reflux condenser for 1 h, filter and wash the filter with dilute hydrochloric acid R. Evaporate the combined filtrate and washings to dryness on a water-bath, dissolve the residue in 0.1 M hydrochloric acid and dilute to 50.0 ml with the same acid. 1246

Acidity or alkalinity. To 2.0 g add 40 ml of water R and boil for 5 min. Cool, restore to the original mass with carbon dioxide-free water R and filter. Reject the first 20 ml of the filtrate. To 10 ml of the filtrate add 0.25 ml of bromothymol blue solution R1 and 0.25 ml of 0.02 M sodium hydroxide. The solution is blue. Not more than 0.75 ml of 0.02 M hydrochloric acid is required to change the colour of the indicator to yellow. Acid-soluble substances. To 1.0 g add 25 ml of dilute nitric acid R and boil for 5 min. Filter whilst hot through a sintered-glass filter (10) and wash with 10 ml of hot water R. Evaporate the combined filtrate and washings to dryness on a water-bath, add to the residue 1 ml of hydrochloric acid R, evaporate to dryness again and dry the residue to constant mass at 100 °C to 105 °C. The residue weighs not more than 30 mg (3 per cent). Alkali-soluble coloured substances. To 0.25 g add 10 ml of dilute sodium hydroxide solution R and boil for 1 min. Cool, filter and dilute the filtrate to 10 ml with water R. The solution is not more intensely coloured than reference solution GY4 (2.2.2, Method II). Alcohol-soluble substances. To 2.0 g add 50 ml of alcohol R and boil under a reflux condenser for 10 min. Filter immediately, cool, and dilute to 50 ml with alcohol R. The filtrate is not more intensely coloured than reference solution Y6 or BY6 (2.2.2, Method II). Evaporate 40 ml of the filtrate to dryness and dry to constant mass at 100 °C to 105 °C. The residue weighs not more than 8 mg (0.5 per cent). Fluorescent substances. In an intermittent-extraction apparatus, treat 10.0 g with 100 ml of cyclohexane R1 for 2 h. Collect the liquid and dilute to 100 ml with cyclohexane R1. Examine in ultraviolet light at 365 nm. The fluorescence of the solution is not more intense than that of a solution of 83 µg of quinine R in 1000 ml of 0.005 M sulphuric acid examined in the same manner. Sulphides. To 1.0 g in a conical flask add 5 ml of hydrochloric acid R1 and 20 ml of water R. Heat to boiling. The fumes released do not turn lead acetate paper R brown. Copper. Not more than 25 ppm of Cu, determined by atomic absorption spectrometry (2.2.23, Method I). Test solution. Use solution S. Reference solutions. Prepare the reference solutions using copper standard solution (0.1 per cent Cu) R and diluting with 0.1 M hydrochloric acid. Measure the absorbance at 325.0 nm using a copper hollow-cathode lamp as source of radiation and an air-acetylene flame. Lead. Not more than 10 ppm of Pb, determined by atomic absorption spectrometry (2.2.23, Method I). Test solution. Use solution S. Reference solutions. Prepare the reference solutions using lead standard solution (100 ppm Pb) R and diluting with 0.1 M hydrochloric acid. Measure the absorbance at 283.3 nm using a lead hollow-cathode lamp as source of radiation and an air-acetylene flame. Depending on the apparatus the line at 217.0 nm may be used. Zinc. Not more than 25 ppm of Zn, determined by atomic absorption spectrometry (2.2.23, Method I). Test solution. Use solution S. Reference solutions. Prepare the reference solutions using zinc standard solution (100 ppm Zn) R and diluting with 0.1 M hydrochloric acid.



Chenodeoxycholic acid

Measure the absorbance at 214.0 nm using a zinc hollow-cathode lamp as source of radiation and an air-acetylene flame. Loss on drying (2.2.32). Not more than 15 per cent, determined on 1.00 g by drying in an oven at 120 °C for 4 h. Sulphated ash (2.4.14). Not more than 5.0 per cent, determined on 1.0 g. Adsorption power. To 0.300 g in a 100 ml ground-glass-stoppered conical flask add 25.0 ml of a freshly prepared solution of 0.5 g of phenazone R in 50 ml of water R. Shake thoroughly for 15 min. Filter and reject the first 5 ml of filtrate. To 10.0 ml of the filtrate add 1.0 g of potassium bromide R and 20 ml of dilute hydrochloric acid R. Using 0.1 ml of methyl red solution R as indicator, titrate with 0.0167 M potassium bromate until the red colour is discharged. Titrate slowly (1 drop every 15 s) towards the end of the titration. Carry out a blank titration using 10.0 ml of the phenazone solution. Calculate the quantity of phenazone adsorbed per 100 g of activated charcoal from the expression :

IDENTIFICATION First identification : A. Second identification : B, C. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with chenodeoxycholic acid CRS. Examine the substances prepared as discs using potassium bromide R. B. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). C. Dissolve about 10 mg in 1 ml of sulphuric acid R. Add 0.1 ml of formaldehyde solution R and allow to stand for 5 min. Add 5 ml of water R. The suspension obtained is greenish-blue.


1247

TESTS Specific optical rotation (2.2.7). Dissolve 0.500 g in methanol R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 11.0 to + 13.0, calculated with reference to the dried substance. Related substances. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating substance. a = number of millilitres of 0.0167 M potassium Test solution (a). Dissolve 0.40 g of the substance to be bromate used for the blank, examined in a mixture of 1 volume of water R and 9 volumes b = number of millilitres of 0.0167 M potassium of acetone R and dilute to 10 ml with the same mixture of bromate used for the test, solvents. m = mass in grams of the substance to be examined. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of 1 volume of water R and 9 volumes of Not less than 40 g of phenazone is adsorbed per 100 g of acetone R. activated charcoal, calculated with reference to the dried Reference solution (a). Dissolve 40 mg of chenodeoxycholic substance. acid CRS in a mixture of 1 volume of water R and 9 volumes Microbial contamination. Total viable aerobic count (2.6.12) of acetone R and dilute to 10 ml with the same mixture of 3 not more than 10 micro-organisms per gram, determined solvents. by plate count. Reference solution (b). Dissolve 20 mg of lithocholic acid CRS in a mixture of 1 volume of water R and 9 volumes STORAGE of acetone R and dilute to 10 ml with the same mixture of Store in an airtight container. solvents. Dilute 2 ml of the solution to 100 ml with a mixture of 1 volume of water R and 9 volumes of acetone R. 01/2005:1189 Reference solution (c). Dissolve 20 mg of ursodeoxycholic acid CRS in a mixture of 1 volume of water R and 9 volumes CHENODEOXYCHOLIC ACID of acetone R and dilute to 50 ml with the same mixture of solvents. Acidum chenodeoxycholicum Reference solution (d). Dissolve 20 mg of cholic acid CRS in a mixture of 1 volume of water R and 9 volumes of acetone R and dilute to 100 ml with the same mixture of solvents. Reference solution (e). Dilute 0.5 ml of test solution (a) to 20 ml with a mixture of 1 volume of water R and 9 volumes of acetone R. Dilute 1 ml of the solution to 10 ml with a mixture of 1 volume of water R and 9 volumes of acetone R. Reference solution (f). Dissolve 10 mg of chenodeoxycholic acid CRS in reference solution (c) and dilute to 25 ml with the same solution. C24H40O4 Mr 392.6 Apply separately to the plate 5 µl of each solution. Develop in an unsaturated tank over a path of 15 cm using a mixture DEFINITION of 1 volume of glacial acetic acid R, 30 volumes of acetone R and 60 volumes of methylene chloride R. Dry the plate Chenodeoxycholic acid contains not less than 99.0 per at 120 °C for 10 min. Spray the plate immediately with a cent and not more than the equivalent of 101.0 per cent 47.6 g/l solution of phosphomolybdic acid R in a mixture of 3α,7α-dihydroxy-5β-cholan-24-oic acid, calculated with of 1 volume of sulphuric acid R and 20 volumes of glacial reference to the dried substance. acetic acid R and heat again at 120 °C until blue spots CHARACTERS appear on a lighter background. In the chromatogram A white or almost white powder, very slightly soluble in obtained with test solution (a) : any spot corresponding to water, freely soluble in alcohol, soluble in acetone, slightly lithocholic acid is not more intense than the principal spot soluble in methylene chloride. in the chromatogram obtained with reference solution (b)

Chitosan hydrochloride


(0.1 per cent) ; any spot corresponding to ursodeoxycholic acid is not more intense than the principal spot in the chromatogram obtained with reference solution (c) (1 per cent) ; any spot corresponding to cholic acid is not more intense than the principal spot in the chromatogram obtained with reference solution (d) (0.5 per cent) ; any spot apart from the principal spot and any spots corresponding to lithocholic acid, ursodeoxycholic acid and cholic acid, is not more intense than the principal spot in the chromatogram obtained with reference solution (e) (0.25 per cent). The test is not valid unless the chromatogram obtained with reference solution (f) shows two clearly separated principal spots. Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 1.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

partial deacetylation of chitin normally leading to a degree of deacetylation of 70.0 per cent to 95.0 per cent. Chitin is extracted from the shells of shrimp and crab. PRODUCTION The animals from which chitosan hydrochloride is derived must fulfil the requirements for the health of animals suitable for human consumption to the satisfaction of the competent authority. It must have been shown to what extent the method of production allows inactivation or removal of any contamination by viruses or other infectious agents. CHARACTERS Appearance : white or almost white fine powder. Solubility : sparingly soluble in water, practically insoluble in ethanol.

IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Preparation : discs. Comparison : chitosan hydrochloride CRS. ASSAY B. It gives reaction (a) of chlorides (2.3.1). Dissolve 0.350 g in 50 ml of alcohol R, previously neutralised C. Dilute 50 ml of solution S (see Tests) to 250 ml with a to 0.2 ml of phenolphthalein solution R. Add 50 ml of 25 per cent V/V solution of ammonia R. A voluminous water R and titrate with 0.1 M sodium hydroxide until a gelatinous mass is formed. pink colour is obtained. D. To 10 ml of solution S add 90 ml of acetone R. A 1 ml of 0.1 M sodium hydroxide is equivalent to 39.26 mg of voluminous gelatinous mass is formed. C24H40O4. TESTS IMPURITIES Solution S. Dissolve 1.0 g in 100 ml of water R and stir vigorously for 20 min with a mechanical stirrer. Appearance of solution. Solution S is not more opalescent than reference suspension II (2.2.1) and not more intensely coloured than reference solution BY5 (2.2.2, Method II). Matter insoluble in water : maximum 0.5 per cent. Add 2.00 g to 400.0 ml of water R while stirring until no further dissolution takes place. Transfer the solution to a 2 litre beaker, add 200 ml of water R. Boil the solution gently for 2 h, covering the beaker during the operation. Filter A. R = H, R1 = OH, R2 = H, R3 = H : ursodeoxycholic acid, through a sintered-glass filter (40) wash the residue with B. R = H, R1 = H, R2 = OH, R3 = OH : 3α,7α,12α-trihydroxy- water and dry to constant weight in an oven at 100-105 °C. 5β-cholan-24-oic acid (cholic acid), The residue weighs a maximum of 10 mg. C. R = H, R1 = H, R2 = H, R3 = H : 3α-hydroxy-5β-cholan-24-oic pH (2.2.3) : 4.0 to 6.0 for solution S. acid (lithocholic acid), Viscosity (2.2.10) : 80 per cent to 120 per cent of the value D. R = H, R1 = OH, R2 = H, R3 = OH : 3α,7β,12α-trihydroxy- stated on the label, determined on solution S. 5β-cholan-24-oic acid (ursocholic acid), Determine the viscosity using a rotating viscometer at 20 °C with a spindle rotating at 20 r/min, using a suitable spindle E. R = H, R1 = H, R2 = H, R3 = OH : 3α,12α-dihydroxy-5βfor the range of the expected viscosity. cholan-24-oic acid (deoxycholic acid), Degree of deacetylation F. R = H, R1+R2 = = O, R3 = H : 3α-hydroxy-7-oxo-5β-cholanTest solution. Dissolve 0.250 g in water R and dilute to 24-oic acid, 50.0 ml with the same solvent stirring vigorously. Dilute G. R = CH3, R1 = OH, R2 = H, R3 = H : methyl 1.0 ml of the solution to 100.0 ml with water R. Measure 3α,7β-dihydroxy-5β-cholan-24-oate. the absorbance (2.2.25) from 200 nm to 205 nm as the first derivative of the absorbance curve. Determine the pH of the solution. 01/2005:1774 Reference solutions. Prepare solutions of 1.0 µg/ml, 5.0 µg/ml, 15.0 µg/ml and 35.0 µg/ml CHITOSAN HYDROCHLORIDE of N-acetylglucosamine R in water R. Measure the absorbance (2.2.25) from 200 nm to 205 nm of each solution Chitosani hydrochloridum as first derivative of the absorption curve. Make a standard curve by plotting the first derivative at 202 nm as a function DEFINITION of the concentration of N-acetylglucosamine, and calculate Chitosan hydrochloride is the chloride salt of an unbranched the slope of the curve by least squares linear regression. Use the standard curve to determine the equivalent amount of binary heteropolysaccharide consisting of the two units N-acetylglucosamine for the substance to be examined. N-acetyl-D-glucosamine and D-glucosamine, obtained by 1248



Chloral hydrate

Calculate the degree of deacetylation (molar) using the expression :

01/2005:0265

CHLORAL HYDRATE Chlorali hydras

C1

=

concentration of chitosan hydrochloride in the test solution in micrograms per millilitre,

C2

=

concentration of N-acetylglucosamine in the test solution, as determined from the standard curve prepared using the reference solution in micrograms per millilitre,

M1

=

203 (relative molecular mass of N-acetylglucosamine unit (C8H13NO5) in polymer),

M3

=

relative molecular mass of chitosan hydrochloride.

M3 is calculated from the pH in solution, assuming a pKa value of 6.8, using the following expressions :

C2H3Cl3O2

Mr 165.4

DEFINITION Chloral hydrate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of 2,2,2-trichloroethane-1,1-diol. CHARACTERS Colourless, transparent crystals, very soluble in water, freely soluble in alcohol. IDENTIFICATION A. To 10 ml of solution S (see Tests) add 2 ml of dilute sodium hydroxide solution R. The mixture becomes cloudy and, when heated, gives off an odour of chloroform. B. To 1 ml of solution S add 2 ml of sodium sulphide solution R. A yellow colour develops which quickly becomes reddish-brown. On standing for a short time, a red precipitate may be formed.

TESTS Solution S. Dissolve 3.0 g in carbon dioxide-free water R and dilute to 30 ml with the same solvent. Chlorides : 10.0 per cent to 20.0 per cent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Introduce 0.200 g into a 250 ml borosilicate flask fitted with pH (2.2.3). The pH of solution S is 3.5 to 5.5. a reflux condenser. Add 40 ml of a mixture of 1 volume Chloral alcoholate. Warm 1.0 g with 10 ml of dilute sodium of nitric acid R and 2 volumes of water R. Boil gently hydroxide solution R, filter the supernatant solution and under a reflux condenser for 5 min. Cool and add 25 ml of water R through the condenser. Add 16.0 ml of 0.1 M silver add 0.05 M iodine dropwise until a yellow colour is obtained. nitrate, shake vigorously and titrate with 0.1 M ammonium Allow to stand for 1 h. No precipitate is formed. thiocyanate, using 1 ml of ferric ammonium sulphate Chlorides (2.4.4). 5 ml of solution S diluted to 15 ml with solution R2 as indicator, and shaking vigorously towards the water R complies with the limit test for chlorides (100 ppm). end-point. Carry out a blank titration. Heavy metals (2.4.8). 10 ml of solution S diluted to 20 ml with water R complies with limit test A for heavy metals 1 ml of 0.1 M silver nitrate is equivalent to 3.55 mg of Cl. (20 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. Heavy metals (2.4.8) : maximum 40 ppm. Non-volatile residue. Evaporate 2.000 g on a water-bath. 1.0 g complies with limit test F. Prepare the standard using The residue weighs not more than 2 mg (0.1 per cent). 4 ml of lead standard solution (10 ppm Pb) R. ASSAY Loss on drying (2.2.32) : maximum 10 per cent, determined Dissolve 4.000 g in 10 ml of water R and add 40.0 ml on 1.000 g by drying in an oven at 100-105 °C. of 1 M sodium hydroxide. Allow to stand for exactly Sulphated ash (2.4.14) : maximum 1.0 per cent, determined 2 min and titrate with 0.5 M sulphuric acid, using 0.1 ml on 1.0 g. of phenolphthalein solution R as indicator. Titrate the neutralised solution with 0.1 M silver nitrate, using 0.2 ml of potassium chromate solution R as indicator. Calculate the number of millilitres of 1 M sodium hydroxide used by deducting from the volume of 1 M sodium hydroxide, STORAGE added at the beginning of the titration, the volume of 0.5 M At a temperature of 2-8 °C, protected from moisture and sulphuric acid used in the first titration and two-fifteenths light. of the volume of 0.1 M silver nitrate used in the second titration. 1 ml of 1 M sodium hydroxide is equivalent to 0.1654 g of C 2H3Cl3O2. LABELLING The label states the nominal viscosity in millipascal seconds STORAGE for a 10 g/l solution in water R. Store in an airtight container.

M2

=

161 (relative molecular mass of deacetylated unit (glucosamine) (C6H11NO4) in polymer).


1249

Chlorambucil


CHLORAMBUCIL Chlorambucilum

C14H19Cl2NO2

01/2005:0137 at most 1 such spot is more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). Water (2.5.12). Not more than 0.5 per cent, determined on 1.000 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.200 g in 10 ml of acetone R and add 10 ml of water R. Titrate with 0.1 M sodium hydroxide, using 0.1 ml of phenolphthalein solution R as indicator. 1 ml of 0.1 M sodium hydroxide is equivalent to 30.42 mg Mr 304.2 of C14H19Cl2NO2.

DEFINITION Chlorambucil contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of 4-4-[di(2-chloroethyl)amino]phenylbutyric acid, calculated with reference to the anhydrous substance. CHARACTERS A white, crystalline powder, practically insoluble in water, freely soluble in acetone and in alcohol. IDENTIFICATION First identification : A, B. Second identification : A, C, D. A. Melting point (2.2.14) : 64 °C to 67 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with chlorambucil CRS. C. To 0.4 g add 10 ml of dilute hydrochloric acid R, mix and allow to stand for 30 min, shaking from time to time. Filter and wash the precipitate with 2 quantities, each of 10 ml, of water R. To 10 ml of the combined filtrate and washings add 0.5 ml of potassium tetraiodomercurate solution R. A pale-brown precipitate is formed. To another 10 ml of the combined filtrate and washings add 0.2 ml of potassium permanganate solution R. The colour of the latter is discharged immediately. D. Dissolve 50 mg in 5 ml of acetone R and dilute to 10 ml with water R. Add 0.05 ml of dilute nitric acid R and 0.2 ml of silver nitrate solution R2. No opalescence is produced immediately. Heat the solution on a water-bath ; an opalescence develops. TESTS Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Carry out all operations as rapidly as possible protected from light. Prepare the solutions immediately before use. Test solution. Dissolve 0.2 g of the substance to be examined in acetone R and dilute to 10 ml with the same solvent. Reference solution (a). Dilute 1 ml of the test solution to 50 ml with acetone R. Reference solution (b). Dilute 25 ml of reference solution (a) to 100 ml with acetone R. Apply separately to the plate 5 µl of each solution. Develop over a path of 10 cm using a mixture of 20 volumes of methyl ethyl ketone R, 20 volumes of heptane R, 25 volumes of methanol R and 40 volumes of toluene R. Examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (a) (2.0 per cent) and 1250


CHLORAMPHENICOL Chloramphenicolum

C11H12Cl2N2O5

Mr 323.1

DEFINITION Chloramphenicol is 2,2-dichloro-N-[(1R,2R)-2-hydroxy-1(hydroxymethyl)-2-(4-nitrophenyl)ethyl]acetamide, produced by the growth of certain strains of Streptomyces venezuelae in a suitable medium. It is normally prepared by synthesis. It contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of C11H12Cl2N2O5, calculated with reference to the dried substance. CHARACTERS A white, greyish-white or yellowish-white, fine, crystalline powder or fine crystals, needles or elongated plates, slightly soluble in water, freely soluble in alcohol and in propylene glycol. A solution in ethanol is dextrorotatory and a solution in ethyl acetate is laevorotatory. IDENTIFICATION First identification : A, B. Second identification : A, C, D, E. A. Melting point (2.2.14) : 149 °C to 153 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with chloramphenicol CRS. C. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with 1 µl of the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). D. Dissolve about 10 mg in 1 ml of alcohol (50 per cent V/V) R, add 3 ml of a 10 g/l solution of calcium chloride R and 50 mg of zinc powder R and heat on a water-bath for 10 min. Filter the hot solution and allow to cool. Add 0.1 ml of benzoyl chloride R and shake for


Chloramphenicol palmitate


1 min. Add 0.5 ml of ferric chloride solution R1 and 2 ml of chloroform R and shake. The aqueous layer is coloured light violet-red to purple. E. To 50 mg in a porcelain crucible add 0.5 g of anhydrous sodium carbonate R. Heat over an open flame for 10 min. Allow to cool. Take up the residue with 5 ml of dilute nitric acid R and filter. To 1 ml of the filtrate add 1 ml of water R. The solution gives reaction (a) of chlorides (2.3.1). TESTS Acidity or alkalinity. To 0.1 g add 20 ml of carbon dioxide-free water R, shake and add 0.1 ml of bromothymol blue solution R1. Not more than 0.1 ml of 0.02 M hydrochloric acid or 0.02 M sodium hydroxide is required to change the colour of the indicator. Specific optical rotation (2.2.7). Dissolve 1.50 g in ethanol R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 18.5 to + 20.5. Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 0.10 g of the substance to be examined in acetone R and dilute to 10 ml with the same solvent. Reference solution (a). Dissolve 0.10 g of chloramphenicol CRS in acetone R and dilute to 10 ml with the same solvent. Reference solution (b). Dilute 0.5 ml of reference solution (a) to 100 ml with acetone R. Apply separately to the plate 1 µl and 20 µl of the test solution, 1 µl of reference solution (a) and 20 µl of reference solution (b). Develop over a path of 15 cm using a mixture of 1 volume of water R, 10 volumes of methanol R and 90 volumes of chloroform R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with 20 µl of the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). Chlorides (2.4.4). To 1.00 g add 20 ml of water R and 10 ml of nitric acid R and shake for 5 min. Filter through a filter paper previously washed by filtering 5 ml portions of water R until 5 ml of filtrate no longer becomes opalescent on addition of 0.1 ml of nitric acid R and 0.1 ml of silver nitrate solution R1. 15 ml of the filtrate complies with the limit test for chlorides (100 ppm). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 2.0 g. Pyrogens (2.6.8). If intended for use in the manufacture of a parenteral dosage form without a further appropriate procedure for the removal of pyrogens, it complies with the test for pyrogens. Inject per kilogram of the rabbit’s mass 2.5 ml of a solution containing per millilitre 2 mg of the substance to be examined.

STORAGE Store protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states, where applicable, that the substance is apyrogenic. 01/2005:0473

CHLORAMPHENICOL PALMITATE Chloramphenicoli palmitas

C27H42Cl2N2O6

Mr 561.6

DEFINITION Chloramphenicol palmitate contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of (2R,3R)-2-[(dichloroacetyl)amino]-3-hydroxy-3-(4nitrophenyl)propyl hexadecanoate, calculated with reference to the dried substance. CHARACTERS A white or almost white, fine, unctuous powder, practically insoluble in water, freely soluble in acetone, sparingly soluble in alcohol, very slightly soluble in hexane. It melts at 87 °C to 95 °C. It shows polymorphism and the thermodynamically stable form has low bioavailability following oral administration.

IDENTIFICATION A. Examine by thin-layer chromatography (2.2.27), using silanised silica gel H R as the coating substance. Test solution. Dissolve 50 mg of the substance to be examined in a mixture of 1 ml of 1 M sodium hydroxide and 5 ml of acetone R and allow to stand for 30 min. Add 1.1 ml of 1 M hydrochloric acid and 3 ml of acetone R. Reference solution (a). Dissolve 10 mg of chloramphenicol CRS in acetone R and dilute to 5 ml with the same solvent. Reference solution (b). Dissolve 10 mg of palmitic acid R in acetone R and dilute to 5 ml with the same solvent. Reference solution (c). Dissolve 10 mg of the substance to be examined in acetone R and dilute to 5 ml with the same solvent. Apply to the plate 4 µl of each solution. Develop over a path of 15 cm using a mixture of 30 volumes of a 100 g/l solution of ammonium acetate R and 70 volumes of alcohol R. Allow the plate to dry in air and spray with a solution containing 0.2 g/l of dichlorofluorescein R and 0.1 g/l of rhodamine B R in alcohol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. ASSAY The chromatogram obtained with the test solution shows Dissolve 0.100 g in water R and dilute to 500.0 ml with the three spots corresponding in position to the principal same solvent. Dilute 10.0 ml of this solution to 100.0 ml with spots in the chromatograms obtained with reference water R. Measure the absorbance (2.2.25) at the maximum solutions (a), (b) and (c). at 278 nm. B. Dissolve 0.2 g in 2 ml of pyridine R, add 2 ml of a Calculate the content of C11H12Cl2N2O5 taking the specific 100 g/l solution of potassium hydroxide R and heat on a absorbance to be 297. water-bath. A red colour is produced. General Notices (1) apply to all monographs and other texts

1251

Chloramphenicol sodium succinate


C. Dissolve 10 mg in 5 ml of alcohol R and add 4.5 ml of dilute sulphuric acid R and 50 mg of zinc powder R. Allow to stand for 10 min and if necessary decant the supernatant liquid or filter. Cool the solution in iced water and add 0.5 ml of sodium nitrite solution R. Allow to stand for 2 min and add 1 g of urea R, 2 ml of strong sodium hydroxide solution R and 1 ml of β-naphthol solution R. A red colour develops. TESTS Acidity. Dissolve 1.0 g in 5 ml of a mixture of equal volumes of alcohol R and ether R, warming to 35 °C. Add 0.2 ml of phenolphthalein solution R. Not more than 0.4 ml of 0.1 M sodium hydroxide is required to produce a pink colour persisting for 30 s. Specific optical rotation (2.2.7). Dissolve 1.25 g in ethanol R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 22.5 to + 25.5. Free chloramphenicol. Not more than 450 ppm. Dissolve 1.0 g, with gentle heating, in 80 ml of xylene R. Cool and shake with three quantities, each of 15 ml, of water R. Dilute the combined aqueous extracts to 50 ml with water R and shake with 10 ml of toluene R. Allow to separate and discard the toluene layer. Centrifuge a portion of the aqueous layer and measure the absorbance (A) (2.2.25) at the maximum at 278 nm using as the compensation liquid a blank solution having an absorbance not greater than 0.05. Calculate the content of free chloramphenicol in parts per million from the expression :

Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 0.1 g of the substance to be examined in acetone R and dilute to 10 ml with the same solvent. Reference solution (a). Dissolve 20 mg of chloramphenicol palmitate isomer CRS in acetone R and dilute to 10 ml with the same solvent. Dilute 1 ml of this solution to 10 ml with acetone R. Reference solution (b). Dissolve 20 mg of chloramphenicol dipalmitate CRS in acetone R and dilute to 10 ml with the same solvent. Dilute 1 ml of this solution to 10 ml with acetone R. Reference solution (c). Dissolve 5 mg of chloramphenicol CRS in acetone R and dilute to 10 ml with the same solvent. Dilute 1 ml of this solution to 10 ml with acetone R. Apply to the plate 10 µl of each solution. Develop over a path of 15 cm using a mixture of 10 volumes of methanol R, 40 volumes of chloroform R and 50 volumes of cyclohexane R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. In the chromatogram obtained with the test solution, any spots corresponding to chloramphenicol palmitate isomer and chloramphenicol dipalmitate are not more intense than the corresponding spots in the chromatograms obtained with reference solutions (a) and (b) respectively (2.0 per cent) and any spot, apart from the principal spot and the spots corresponding to chloramphenicol palmitate isomer and chloramphenicol dipalmitate, is not more intense than the principal spot in the chromatogram obtained with reference solution (c) (0.5 per cent). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by heating at 80 °C over diphosphorus pentoxide R at a pressure not exceeding 0.1 kPa for 3 h. 1252

Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 90.0 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of this solution to 250.0 ml with alcohol R. Measure the absorbance (2.2.25) of the solution at the maximum at 271 nm. Calculate the content of C27H42Cl2N2O6taking the specific absorbance to be 178. STORAGE Store protected from light. IMPURITIES

A. (1R,2R)-2-[(dichloroacetyl)amino]-3-hydroxy-1-(4nitrophenyl)propyl hexadecanoate (chloramphenicol palmitate isomer),

B. (1R,2R)-2-[(dichloroacetyl)amino]-1-(4-nitrophenyl)propane-1,3-diyl bishexadecanoate (chloramphenicol dipalmitate). 01/2005:0709

CHLORAMPHENICOL SODIUM SUCCINATE Chloramphenicoli natrii succinas

C15H15Cl2N2NaO8

Mr 445.2

DEFINITION Chloramphenicol sodium succinate, a mixture in variable proportions of sodium (2R,3R)-2-[(dichloroacetyl)amino]-3hydroxy-3-(4-nitrophenyl)propyl butanedioate (3 isomer) and of sodium (1R,2R)-2-[(dichloroacetyl)amino]-3-hydroxy-1-(4nitrophenyl)propyl butanedioate (1 isomer), contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of C15H15Cl2N2NaO8, calculated with reference to the anhydrous substance.



Chlorcyclizine hydrochloride

CHARACTERS The chromatographic procedure may be carried out using : A white or yellowish-white powder, hygroscopic, very soluble — a column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for in water, freely soluble in alcohol. chromatography R (5 µm), IDENTIFICATION — as mobile phase at a flow rate of 1.0 ml/min a mixture A. Examine by thin-layer chromatography (2.2.27), using of 5 volumes of a 20 g/l solution of phosphoric acid R, silica gel GF254 R as the coating substance. 40 volumes of methanol R and 55 volumes of water R, Test solution. Dissolve 20 mg of the substance to be — as detector a spectrophotometer set at 275 nm, examined in 2 ml of acetone R. — a 20 µl fixed-loop injector. Reference solution (a). Dissolve 20 mg of Inject the test solution and each of the reference solutions. chloramphenicol sodium succinate CRS in 2 ml of The test is not valid unless, in the chromatogram obtained acetone R. with reference solution (c), the two peaks corresponding to those in the chromatograms obtained with reference Reference solution (b). Dissolve 20 mg of solutions (a) and (b) are clearly separated from the chloramphenicol CRS in 2 ml of acetone R. peaks corresponding to the two principal peaks in the Apply separately to the plate 2 µl of each solution. Develop over a path of 15 cm using a mixture of 1 volume chromatogram obtained with the test solution. If necessary, adjust the methanol content of the mobile phase. of dilute acetic acid R, 14 volumes of methanol R and In the chromatogram obtained with the test solution, the area 85 volumes of chloroform R. Allow the plate to dry in of any peak corresponding to chloramphenicol is not greater air and examine in ultraviolet light at 254 nm. The two than the area of the principal peak in the chromatogram principal spots in the chromatogram obtained with the test solution are similar in position and size to those in the obtained with reference solution (a) (2.0 per cent) ; the area chromatogram obtained with reference solution (a). Their of any peak corresponding to chloramphenicol disodium disuccinate is not greater than the area of the principal peak positions are different from that of the principal spot in in the chromatogram obtained with reference solution (b) the chromatogram obtained with reference solution (b). (2.0 per cent). B. Dissolve about 10 mg in 1 ml of alcohol (50 per Water (2.5.12). Not more than 2.0 per cent, determined on cent V/V) R, add 3 ml of a 10 g/l solution of calcium 0.500 g by the semi-micro determination of water. chloride R and 50 mg of zinc powder R and heat on a water-bath for 10 min. Filter the hot solution and allow Pyrogens (2.6.8). If intended for use in the manufacture to cool. Add 0.1 ml of benzoyl chloride R and shake for of a parenteral dosage form without a further appropriate 1 min. Add 0.5 ml of ferric chloride solution R1 and procedure for removal of pyrogens, it complies with the test 2 ml of chloroform R and shake. The upper layer is light for pyrogens. Inject per kilogram of the rabbit’s mass 2.5 ml violet-red to purple. of a solution in water for injections R containing 2 mg of C. Dissolve 50 mg in 1 ml of pyridine R. Add 0.5 ml of dilute the substance to be examined per millilitre. sodium hydroxide solution R and 1.5 ml of water R. Heat ASSAY in a water-bath for 3 min. A red colour develops. Add 2 ml of nitric acid R and cool under running water. Add Dissolve 0.200 g in water R and dilute to 500.0 ml with the 1 ml of 0.1 M silver nitrate. A white precipitate is formed same solvent. Dilute 5.0 ml of this solution to 100.0 ml with water R. Measure the absorbance (2.2.25) at the maximum slowly. at 276 nm. D. It gives reaction (a) of sodium (2.3.1). Calculate the content of C15H15Cl2N2NaO8, taking the specific TESTS absorbance to be 220. pH (2.2.3). Dissolve 2.50 g in carbon dioxide-free water R STORAGE and dilute to 10 ml with the same solvent. The pH of the Store in an airtight container, protected from light. If the solution is 6.4 to 7.0. substance is sterile, store in a sterile, airtight, tamper-proof Specific optical rotation (2.2.7). Dissolve 0.50 g in water R container, protected from light. and dilute to 10.0 ml with the same solvent. The specific optical rotation is + 5.0 to + 8.0, calculated with reference LABELLING to the anhydrous substance. The label states, where applicable, that the substance is apyrogenic. Chloramphenicol and chloramphenicol disodium disuccinate. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 25.0 mg of the substance to be 01/2005:1086 examined in the mobile phase and dilute to 100.0 ml with the mobile phase. CHLORCYCLIZINE HYDROCHLORIDE Reference solution (a). Dissolve 10.0 mg of chloramphenicol CRS in the mobile phase and Chlorcyclizini hydrochloridum dilute to 100.0 ml with the mobile phase (solution a′). Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (b). Dissolve 10.0 mg of chloramphenicol disodium disuccinate CRS in the mobile phase and dilute to 100.0 ml with the mobile phase (solution b′). Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (c). Dissolve 25 mg of the substance to be examined in the mobile phase, add 5 ml of solution (a′) and 5 ml of solution (b′) and dilute to 100 ml with the mobile phase. C18H22Cl2N2 Mr 337.3 General Notices (1) apply to all monographs and other texts

1253

Chlordiazepoxide


DEFINITION Chlorcyclizine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of (RS)-1-[(4-chlorophenyl)phenylmethyl]-4-methylpiperazine hydrochloride, calculated with reference to the dried substance. CHARACTERS A white, crystalline powder, freely soluble in water and in methylene chloride, soluble in alcohol. IDENTIFICATION First identification : B, D. Second identification : A, C, D. A. Dissolve 10.0 mg in a 5 g/l solution of sulphuric acid R and dilute to 100.0 ml with the same acid. Dilute 10.0 ml of the solution to 100.0 ml with a 5 g/l solution of sulphuric acid R. Examined between 215 nm and 300 nm (2.2.25), the solution shows an absorption maximum at 231 nm. The specific absorbance at the maximum is 475 to 525, calculated with reference to the dried substance. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with chlorcyclizine hydrochloride CRS. Examine the substances prepared as discs. C. Examine the chromatograms obtained in the test for related substances (see Tests). The principal spot in the chromatogram obtained with test solution (b) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). D. It gives reaction (a) of chlorides (2.3.1). TESTS Appearance of solution. Dissolve 0.5 g in water R and dilute to 10 ml with the same solvent. The solution is clear (2.2.1) and colourless (2.2.2, Method II). pH (2.2.3). Dissolve 0.10 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. The pH of the solution is 5.0 to 6.0. Related substances. Examine by thin-layer chromatography (2.2.27), using a plate coated with a suitable silica gel. Test solution (a). Dissolve 0.20 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Test solution (b). Dilute 5 ml of test solution (a) to 100 ml with methanol R. Reference solution (a). Dissolve 10 mg of chlorcyclizine hydrochloride CRS in methanol R and dilute to 10 ml with the same solvent. Reference solution (b). Dissolve 5 mg of methylpiperazine R in methanol R and dilute to 50 ml with the same solvent. Reference solution (c). Dilute 1 ml of test solution (b) to 25 ml with methanol R. Reference solution (d). Dissolve 10 mg of hydroxyzine hydrochloride CRS and 10 mg of chlorcyclizine hydrochloride CRS in methanol R and dilute to 10 ml with the same solvent. Apply separately to the plate 10 µl of each solution and develop over a path of 15 cm using a mixture of 2 volumes of concentrated ammonia R, 13 volumes of methanol R and 85 volumes of methylene chloride R. Allow the plate to dry in air and expose it to iodine vapour for 10 min. In the chromatogram obtained with test solution (a) : any spot corresponding to methylpiperazine is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent) ; any spot, apart from the principal 1254

spot and any spot corresponding to methylpiperazine, is not more intense than the spot in the chromatogram obtained with reference solution (c) (0.2 per cent). The test is not valid unless the chromatogram obtained with reference solution (d) shows two clearly separated spots. Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g by drying in an oven at 130 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.200 g in a mixture of 1 ml of 0.1 M hydrochloric acid and 50 ml of methanol R. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the two points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 33.73 mg of C18H22Cl2N2. STORAGE Store protected from light. IMPURITIES

A. N-methylpiperazine. 01/2005:0656

CHLORDIAZEPOXIDE Chlordiazepoxidum

C16H14ClN3O

Mr 299.8

DEFINITION 7-Chloro-N-methyl-5-phenyl-3H-1,4-benzodiazepin-2-amine 4-oxide. Content : 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : almost white or light yellow, crystalline powder. Solubility : practically insoluble in water, sparingly soluble in alcohol. IDENTIFICATION Infrared absorption spectrophotometry (2.2.24). Comparison : chlordiazepoxide CRS. TESTS Related substances. Liquid chromatography (2.2.29). Carry out the test protected from bright light and prepare the solutions immediately before use. Test solution. Dissolve 20.0 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase.


Chlordiazepoxide hydrochloride


Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 2.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (b). Dissolve 5 mg of nitrazepam R in the mobile phase, add 25.0 ml of the test solution and dilute to 100.0 ml with the mobile phase. Dilute 2.0 ml of this solution to 50.0 ml with the mobile phase. Reference solution (c). Dissolve 4.0 mg of aminochlorobenzophenone R in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 100.0 ml with the mobile phase. Column : — size : l = 0.15 m, Ø = 4.6 mm, — stationary phase: octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : acetonitrile R, water R (50:50 V/V). Flow rate : 1.0 ml/min. Detection : spectrophotometer at 254 nm. Injection : 10 µl. Run time : 6 times the retention time of chlordiazepoxide. Retention time : nitrazepam = about 3.1 min ; chlordiazepoxide = about 3.6 min. Relative retention with reference to chlordiazepoxide : impurity A = about 0.7 ; impurity B = about 2.3 ; impurity C = about 3.9. System suitability : reference solution (b) : — resolution : minimum 2.0 between the peaks due to nitrazepam and chlordiazepoxide. Limits : — impurities A, B : for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent), — impurity C : not more than the area of the principal peak in the chromatogram obtained with reference solution (c) (0.2 per cent), — any other impurity : not more than half the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), — total: not more than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent), — disregard limit : 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.250 g, with heating if necessary, in 80 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 29.98 mg of C16H14ClN3O. STORAGE Protected from light. IMPURITIES Specified impurities : A, B, C. General Notices (1) apply to all monographs and other texts

A. 7-chloro-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one 4-oxide,

B. 6-chloro-2-(chloromethyl)-4-phenylquinazoline 3-oxide,

C. (2-amino-5-chlorophenyl)phenylmethanone (aminochlorobenzophenone). 01/2005:0474

CHLORDIAZEPOXIDE HYDROCHLORIDE Chlordiazepoxidi hydrochloridum

C16H15Cl2N3O

Mr 336.2

DEFINITION 7-Chloro-N-methyl-5-phenyl-3H-1,4-benzodiazepin-2-amine 4-oxide hydrochloride. Content : 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white or slightly yellow, crystalline powder, slightly hygroscopic. Solubility : soluble in water, sparingly soluble in alcohol. IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Comparison : chlordiazepoxide hydrochloride CRS. B. Dissolve 50 mg in 5 ml of water R, add 1 ml of dilute ammonia R1, mix, allow to stand for 5 min and filter. Acidify the filtrate with dilute nitric acid R. The solution gives reaction (a) of chlorides (2.3.1). 1255

Chlorhexidine diacetate


TESTS Appearance of solution. The solution is clear (2.2.1) and not more intensely coloured than reference solution GY6 (2.2.2, Method II). Dissolve 2.5 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Related substances. Liquid chromatography (2.2.29). Carry out the following operations protected from bright light and prepare the solutions immediately before use. Test solution. Dissolve 20.0 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 2.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (b). Dissolve 5 mg of nitrazepam R in the mobile phase, add 25.0 ml of the test solution and dilute to 100.0 ml with the mobile phase. Dilute 2.0 ml of this solution to 50.0 ml with the mobile phase. Reference solution (c). Dissolve 4.0 mg of aminochlorobenzophenone R in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase. Column : — size : l = 0.15 m, Ø = 4.6 mm, — stationary phase: octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : acetonitrile R, water R (50:50 V/V). Flow rate : 1.0 ml/min. Detection : spectrophotometer at 254 nm. Injection : 10 µl. Run time : 6 times the retention time of chlordiazepoxide. Retention time : nitrazepam = about 3.1 min ; chlordiazepoxide = about 3.6 min. Relative retention with reference to chlordiazepoxide : impurity A = about 0.7 ; impurity B = about 2.3 ; impurity C = about 3.9. System suitability : reference solution (b) : — resolution : minimum 2.0 between the peaks due to nitrazepam and chlordiazepoxide. Limits : — impurities A, B : for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent), — impurity C : not more than the area of the principal peak in the chromatogram obtained with reference solution (c) (0.2 per cent), — any other impurity : not more than half the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), — total: not more than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent), — disregard limit : 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in vacuo at 60 °C for 4 h. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. 1256

ASSAY Dissolve 0.250 g in 80 ml of anhydrous acetic acid R, heating if necessary. Cool and add 10 ml of mercuric acetate solution R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 33.62 mg of C16H15Cl2N3O. STORAGE In an airtight container, protected from light. IMPURITIES Specified impurities : A, B, C.

A. 7-chloro-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one 4-oxide,

B. 6-chloro-2-(chloromethyl)-4-phenylquinazoline 3-oxide,

C. (2-amino-5-chlorophenyl)phenylmethanone (aminochlorobenzophenone). 01/2005:0657

CHLORHEXIDINE DIACETATE Chlorhexidini diacetas

C26H38Cl2N10O4

Mr 625.6

DEFINITION Chlorhexidine diacetate contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of 1,1′-(hexane-1,6-diyl)bis[5-(4-chlorophenyl)biguanide] diacetate, calculated with reference to the dried substance.



Chlorhexidine diacetate

— as detector a spectrophotometer set at 254 nm. Equilibrate the column with the mobile phase for at least 1 h. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 10 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder. IDENTIFICATION Inject 10 µl of reference solution (a). The test is not valid First identification : A. unless the resulting chromatogram is similar to the specimen Second identification : B, C, D. chromatogram provided with chlorhexidine for performance A. Examine by infrared absorption spectrophotometry test CRS in that the peaks due to impurity-A and impurity-B (2.2.24), comparing with the spectrum obtained with precede that due to chlorhexidine. If necessary, adjust the chlorhexidine diacetate CRS. concentration of acetic acid in the mobile phase (increasing B. Dissolve about 5 mg in 5 ml of a warm 10 g/l solution of the concentration decreases the retention times). cetrimide R and add 1 ml of strong sodium hydroxide Inject separately 10 µl of the test solution and 10 µl each of solution R and 1 ml of bromine water R. A deep red reference solutions (b) and (c). Record the chromatograms colour is produced. of reference solutions (b) and (c) until the peak due to chlorhexidine has been eluted and record the chromatogram C. Dissolve 0.3 g in 10 ml of a mixture of equal volumes of hydrochloric acid R and water R. Add 40 ml of water R, of the test solution for six times the retention time of the filter if necessary and cool in ice water. Make alkaline to peak due to chlorhexidine. In the chromatogram obtained titan yellow paper R by adding dropwise and with stirring with the test solution, the sum of the areas of all the peaks, apart from the principal peak is not greater than the area strong sodium hydroxide solution R and add 1 ml in of the principal peak in the chromatogram obtained with excess. Filter, wash the precipitate with water R until reference solution (b) (2.5 per cent). Disregard any peak with the washings are free from alkali and recrystallise from a with a relative retention time of 0.25 or less with respect to alcohol (70 per cent V/V) R. Dry at 100 °C to 105 °C. the principal peak and any peak whose area is less than that The residue melts (2.2.14) at 132 °C to 136 °C. of the principal peak in the chromatogram obtained with D. It gives reaction (a) of acetates (2.3.1). reference solution (c). Loss on drying (2.2.32). Not more than 3.5 per cent, TESTS determined on 1.000 g by drying in an oven at 100 °C to Chloroaniline. Dissolve 0.20 g of the substance to be 105 °C. examined in 25 ml of water R with shaking if necessary. Sulphated ash (2.4.14). Not more than 0.15 per cent, Add 1 ml of hydrochloric acid R and dilute to 30 ml with determined on 1.0 g. water R. Add rapidly and with thorough mixing after each addition : 2.5 ml of dilute hydrochloric acid R, 0.35 ml ASSAY of sodium nitrite solution R, 2 ml of a 50 g/l solution of ammonium sulphamate R, 5 ml of a 1.0 g/l solution of Dissolve 0.140 g in 100 ml of anhydrous acetic acid R and naphthylethylenediamine dihydrochloride R and 1 ml of titrate with 0.1 M perchloric acid. Determine the end-point alcohol R, dilute to 50.0 ml with water R and allow to stand potentiometrically (2.2.20). for 30 min. Any reddish-blue colour in the solution is not 1 ml of 0.1 M perchloric acid is equivalent to 15.64 mg of more intense than that in a standard prepared at the same C26H38Cl2N10O4. time in the same manner using a mixture of 10.0 ml of a 0.010 g/l solution of chloroaniline R in dilute hydrochloric IMPURITIES acid R and 20 ml of dilute hydrochloric acid R instead of the solution of the substance to be examined (500 ppm). Related substances. Examined by liquid chromatography (2.2.29). Test solution. Dissolve 0.200 g of the substance to be examined in the mobile phase and dilute to 100 ml with the mobile phase. Reference solution (a). Dissolve 15 mg of chlorhexidine for performance test CRS in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (b). Dilute 2.5 ml of the test solution to 100 ml with the mobile phase. Reference solution (c). Dilute 2.0 ml of reference solution (b) to 10 ml with the mobile phase. Dilute 1.0 ml of this solution to 10 ml with the mobile phase. A. 1-(4-chlorophenyl)-5-[6-(3-cyanoguanidino)hexyl]biguanide, The chromatographic procedure may be carried out using : CHARACTERS A white or almost white, microcrystalline powder, sparingly soluble in water, soluble in alcohol, slightly soluble in glycerol and in propylene glycol.

— a stainless steel column 0.2 m long and 4 mm in internal diameter, packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow-rate of 1.0 ml/min, a solution of 2.0 g of sodium octanesulphonate R in a mixture of 120 ml of glacial acetic acid R, 270 ml of water R and 730 ml of methanol R, General Notices (1) apply to all monographs and other texts

B. [[[6-[5-(4-chlorophenyl)guanidino]hexyl]amino]iminomethyl]urea, 1257

Chlorhexidine digluconate solution


Test solution. Dilute 10.0 ml of the solution to be examined to 50 ml with water R. Reference solution. Dissolve 25 mg of calcium gluconate CRS in 1 ml of water R. Apply separately to the plate 5 µl of each solution. Develop over a path of 10 cm using a mixture of 10 volumes of ethyl acetate R, 10 volumes of concentrated ammonia R, 30 volumes of water R and 50 volumes of alcohol R. Dry the plate at 100 °C for 20 min, allow to cool and spray with a 50 g/l solution of potassium dichromate R in C. 1,1′-[hexane-1,6-diylbis[imino(iminocarbonyl)]]bis[3-(4a 40 per cent m/m solution of sulphuric acid R. After chlorophenyl)urea], 5 min, the principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. C. To 1 ml add 40 ml of water R, cool in iced water, make alkaline to titan yellow paper R by adding dropwise and D. 1,1′-[[[[[(4-chlorophenyl)amino]iminomethyl]imino]mewith stirring strong sodium hydroxide solution R and add thylene]bis[imino(hexane-1,6-diyl)]]bis[5-(4-chlorophe1 ml in excess. Filter, wash the precipitate with water R nyl)biguanide]. until the washings are free from alkali and recrystallise from alcohol (70 per cent V/V) R. Dry at 100 °C to 105 °C. The residue melts (2.2.14) at 132 °C to 136 °C. 01/2005:0658 D. To 0.05 ml add 5 ml of a 10 g/l solution of cetrimide R, corrected 1 ml of strong sodium hydroxide solution R and 1 ml of bromine water R ; a deep red colour is produced.

CHLORHEXIDINE DIGLUCONATE SOLUTION

TESTS Relative density (2.2.5) : 1.06 to 1.07. pH (2.2.3). Dilute 5.0 ml to 100 ml with carbon dioxide-free Chlorhexidini digluconatis solutio water R. The pH of the solution is 5.5 to 7.0. Chloroaniline. Dilute 2.0 ml to 100 ml with water R. To 10 ml of the solution add 2.5 ml of dilute hydrochloric acid R and dilute to 20 ml with water R. Add rapidly and with thorough mixing after each addition : 0.35 ml of sodium nitrite solution R, 2 ml of a 50 g/l solution of ammonium sulphamate R, 5 ml of a 1 g/l solution of naphthyle thylenediamine dihydrochloride R, 1 ml of alcohol R, dilute to 50.0 ml with water R and allow to stand for 30 min. Any reddish-blue colour in the solution is not greater than that in a standard prepared at the same time in the same C34H54Cl2N10O14 Mr 898 manner using a mixture of 10.0 ml of a 0.010 g/l solution of chloroaniline R in dilute hydrochloric acid R and 10 ml of DEFINITION water R instead of the dilution of the solution to be examined (0.25 per cent with reference to chlorhexidine digluconate Chlorhexidine digluconate solution is an aqueous at a nominal concentration of 200 g/l). solution which contains not less than 190 g/l and not more than 210 g/l of 1,1′-(hexane-1,6-diyl)bis[5-(4Related substances. Examined by liquid chromatography chlorophenyl)biguanide] di-D-gluconate. (2.2.29). Test solution. Dilute 5.0 ml of the sample to be examined to CHARACTERS 50.0 ml with the mobile phase. Dilute 5.0 ml of this solution An almost colourless or pale-yellowish liquid, miscible with to 50.0 ml with the mobile phase. water, with not more than 3 parts of acetone and with not Reference solution (a). Dissolve 15 mg of chlorhexidine more than 5 parts of ethanol (96 per cent). for performance test CRS in the mobile phase and dilute to 10.0 ml with the mobile phase. IDENTIFICATION Reference solution (b). Dilute 3.0 ml of the test solution to First identification : A, B. 100 ml with the mobile phase. Second identification : B, C, D. Reference solution (c). Dilute 1.0 ml of reference solution (b) A. To 1 ml add 40 ml of water R, cool in iced water, make to 50 ml with the mobile phase. alkaline to titan yellow paper R by adding dropwise The chromatographic procedure may be carried out using : and with stirring strong sodium hydroxide solution R — a stainless steel column 0.2 m long and 4 mm in internal and add 1 ml in excess. Filter, wash the precipitate diameter, packed with octadecylsilyl silica gel for with water R until the washings are free from alkali chromatography R (5 µm), and recrystallise from alcohol (70 per cent V/V) R. Dry at 100 °C to 105 °C. Examine the residue by infrared — as mobile phase at a flow-rate of 1.0 ml/min, a solution absorption spectrophotometry (2.2.24), comparing with of 2.0 g of sodium octanesulphonate R in a mixture of the spectrum obtained with chlorhexidine CRS. 120 ml of glacial acetic acid R, 270 ml of water R and 730 ml of methanol R, B. Examine by thin-layer chromatography (2.2.27), using — as detector a spectrophotometer set at 254 nm. silica gel G R as the coating substance. 1258


Chlorhexidine dihydrochloride


Equilibrate the column with mobile phase for at least 1 hour. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 10 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder. Inject 10 µl of reference solution (a). The test is not valid unless the resulting chromatogram is similar to the specimen chromatogram provided with chlorhexidine for performance test CRS in that the peaks due to impurity A and impurity B C. 1,1′-[hexane-1,6-diylbis[imino(iminocarbonyl)]]bis[3-(4precede that due to chlorhexidine. If necessary, adjust the chlorophenyl)urea], concentration of acetic acid in the mobile phase (increasing the concentration decreases the retention times). Inject separately 10 µl of the test solution and 10 µl each of reference solutions (b) and (c). Record the chromatograms of reference solutions (b) and (c) until the peak due to chlorhexidine has been eluted and record the chromatogram D. 1,1′-[[[[[(4-chlorophenyl)amino]iminomethyl]imino]meof the test solution for six times the retention time of the thylene]bis[imino(hexane-1,6-diyl)]]bis[5-(4-chlorophepeak due to chlorhexidine. In the chromatogram obtained nyl)biguanide]. with the test solution, the sum of the areas of the peaks, apart from the principal peak is not greater than the area 01/2005:0659 of the principal peak in the chromatogram obtained with reference solution (b) (3.0 per cent). Disregard any peak CHLORHEXIDINE with a relative retention time of 0.25 or less with respect to the principal peak and any peak whose area is less than that DIHYDROCHLORIDE of the principal peak in the chromatogram obtained with reference solution (c).

Chlorhexidini dihydrochloridum

ASSAY Determine the density (2.2.5) of the solution to be examined. Transfer 1.00 g to a 250 ml beaker and add 50 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid. Determine the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 22.44 mg of C34H54Cl2N10O14. STORAGE Store protected from light. IMPURITIES

C22H32Cl4N10

Mr 578.4

DEFINITION Chlorhexidine dihydrochloride contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of 1,1’-(hexane-1,6-diyl)bis[5-(4-chlorophenyl)biguanide] dihydrochloride, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, sparingly soluble in water and in propylene glycol, very slightly soluble in alcohol.

A. 1-(4-chlorophenyl)-5-[6-(3-cyanoguanidino)hexyl]biguanide,

B. [[[6-[5-(4-chlorophenyl)guanidino]hexyl]amino]iminomethyl]urea, General Notices (1) apply to all monographs and other texts

IDENTIFICATION First identification : A, D. Second identification : B, C, D. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with chlorhexidine dihydrochloride CRS. B. Dissolve about 5 mg in 5 ml of a warm 10 g/l solution of cetrimide R and add 1 ml of strong sodium hydroxide solution R and 1 ml of bromine water R. A dark red colour is produced. C. Dissolve 0.3 g in 10 ml of a mixture of equal volumes of hydrochloric acid R and water R. Add 40 ml of water R, filter if necessary and cool in iced water. Make alkaline to titan yellow paper R by adding dropwise, and with stirring, strong sodium hydroxide solution R and add 1 ml in excess. Filter, wash the precipitate with water R 1259

Chlorhexidine dihydrochloride


until the washings are free from alkali and recrystallise from alcohol (70 per cent V/V) R. Dry at 100 °C to 105 °C. The residue melts (2.2.14) at 132 °C to 136 °C.

chromatogram obtained with reference solution (b) (2.5 per cent). Disregard any peak with a relative retention time of 0.25 or less, with respect to chlorhexidine, and any peak with an area less than that of the principal peak in the D. It gives reaction (a) of chlorides (2.3.1). chromatogram obtained with reference solution (c). Loss on drying (2.2.32). Not more than 1.0 per cent, TESTS determined on 1.000 g by drying in an oven at 100–105 °C. Chloroaniline. To 0.20 g of the substance to be examined add 1 ml of hydrochloric acid R, shake for about 30 s, dilute Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. to 30 ml with water R and shake until a clear solution is obtained. Add rapidly and with thorough mixing after each ASSAY addition : 2.5 ml of dilute hydrochloric acid R, 0.35 ml Dissolve 100.0 mg in 5 ml of anhydrous formic acid R of sodium nitrite solution R, 2 ml of a 50 g/l solution of and add 70 ml of acetic anhydride R. Titrate with 0.1 M ammonium sulphamate R, 5 ml of a 1.0 g/l solution of perchloric acid, determining the end-point potentiometrically naphthylethylenediamine dihydrochloride R and 1 ml of alcohol R ; dilute to 50.0 ml with water R and allow to stand (2.2.20). for 30 min. Any reddish-blue colour in the solution is not 1 ml of 0.1 M perchloric acid is equivalent to 14.46 mg of more intense than that in a standard prepared at the same C22H32Cl4N10. time and in the same manner using a mixture of 10.0 ml of a 0.010 g/l solution of chloroaniline R in dilute hydrochloric IMPURITIES acid R and 20 ml of dilute hydrochloric acid R instead of the solution of the substance to be examined (500 ppm). Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 0.200 g of the substance to be examined in the mobile phase and dilute to 100 ml with the mobile phase. Reference solution (a). Dissolve 15 mg of chlorhexidine for performance test CRS in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (b). Dilute 2.5 ml of the test solution to 100 ml with the mobile phase. Reference solution (c). Dilute 2.0 ml of reference solution (b) to 10 ml with the mobile phase. Dilute 1.0 ml of the solution A. 1-(4-chlorophenyl)-5-[6-(3-cyanoguanidino)hexyl]biguto 10 ml with the mobile phase. anide, The chromatographic procedure may be carried out using : — a stainless steel column 0.2 m long and 4 mm in internal diameter, packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.0 ml/min, a solution of 2.0 g of sodium octanesulphonate R in a mixture of 120 ml of glacial acetic acid R, 270 ml of water R and 730 ml of methanol R,

B. [[[6-[5-(4-chlorophenyl)guanidino]hexyl]amino]iminomethyl]urea,

— as detector a spectrophotometer set at 254 nm. Equilibrate the column with the mobile phase for at least 1 h. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 10 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder. Inject 10 µl of reference solution (a). The test is not valid unless the resulting chromatogram is similar to the specimen chromatogram provided with chlorhexidine for performance test CRS in that the peaks due to impurity A and impurity B C. 1,1′-[hexane-1,6-diylbis[imino(iminocarbonyl)]]bis[3-(4precede that due to chlorhexidine. If necessary, adjust the chlorophenyl)urea], concentration of acetic acid in the mobile phase (increasing the concentration decreases the retention times). Inject 10 µl of the test solution and 10 µl each of reference solutions (b) and (c). Record the chromatograms of reference solutions (b) and (c) until the peak due to chlorhexidine is eluted and record the chromatogram of the test solution for six times the retention time of the peak due to chlorhexidine. D. 1,1′-[[[[[(4-chlorophenyl)amino]iminomethyl]imino]meIn the chromatogram obtained with the test solution, the thylene]bis[imino(hexane-1,6-diyl)]]bis[5-(4-chlorophesum of the areas of the peaks, apart from the principal peak, nyl)biguanide]. is not greater than the area of the principal peak in the 1260


Chlorobutanol hemihydrate


01/2005:0382 STORAGE In an airtight container.

CHLOROBUTANOL, ANHYDROUS Chlorobutanolum anhydricum

01/2005:0383

CHLOROBUTANOL HEMIHYDRATE C4H7Cl3O

Mr 177.5

Chlorobutanolum hemihydricum

DEFINITION Anhydrous chlorobutanol contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of 1 Mr 186.5 1,1,1-trichloro-2-methylpropan-2-ol, calculated with reference C4H7Cl3O, /2H2O to the anhydrous substance. DEFINITION CHARACTERS Chlorobutanol hemihydrate contains not less than 98.0 per A white, crystalline powder or colourless crystals, sublimes cent and not more than the equivalent of 101.0 per cent of 1,1,1-trichloro-2-methylpropan-2-ol, calculated with reference readily, slightly soluble in water, very soluble in alcohol, to the anhydrous substance. soluble in glycerol (85 per cent). It melts at about 95 °C, determined without previous drying. CHARACTERS IDENTIFICATION A. Add about 20 mg to a mixture of 1 ml of pyridine R and 2 ml of strong sodium hydroxide solution R. Heat in a water-bath and shake. Allow to stand. The pyridine layer becomes red. B. Add about 20 mg to 5 ml of ammoniacal silver nitrate solution R and warm slightly. A black precipitate is formed. C. To about 20 mg add 3 ml of 1 M sodium hydroxide and shake to dissolve. Add 5 ml of water R and then, slowly, 2 ml of iodinated potassium iodide solution R. A yellowish precipitate is formed. D. It complies with the test for water (see Tests). TESTS Solution S. Dissolve 5 g in alcohol R and dilute to 10 ml with the same solvent. Appearance of solution. Solution S is not more opalescent than reference suspension II (2.2.1) and not more intensely coloured than reference solution BY5 (2.2.2, Method II). Acidity. To 4 ml of solution S add 15 ml of alcohol R and 0.1 ml of bromothymol blue solution R1. Not more than 1.0 ml of 0.01 M sodium hydroxide is required to change the colour of the indicator to blue. Chlorides (2.4.4). Dissolve 0.17 g in 5 ml of alcohol R and dilute to 15 ml with water R. The solution complies with the limit test for chlorides (300 ppm). When preparing the standard, replace the 5 ml of water R by 5 ml of alcohol R. Water (2.5.12). Not more than 1.0 per cent, determined on 2.00 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.100 g in 20 ml of alcohol R. Add 10 ml of dilute sodium hydroxide solution R, heat in a water-bath for 5 min and cool. Add 20 ml of dilute nitric acid R, 25.0 ml of 0.1 M silver nitrate and 2 ml of dibutyl phthalate R and shake vigorously. Add 2 ml of ferric ammonium sulphate solution R2 and titrate with 0.1 M ammonium thiocyanate until an orange colour is obtained. 1 ml of 0.1 M silver nitrate is equivalent to 5.92 mg of C4H7Cl3O. General Notices (1) apply to all monographs and other texts

A white, crystalline powder or colourless crystals, sublimes readily, slightly soluble in water, very soluble in alcohol, soluble in glycerol (85 per cent). It melts at about 78 °C, determined without previous drying. IDENTIFICATION A. Add about 20 mg to a mixture of 1 ml of pyridine R and 2 ml of strong sodium hydroxide solution R. Heat in a water-bath and shake. Allow to stand. The pyridine layer becomes red. B. Add about 20 mg to 5 ml of ammoniacal silver nitrate solution R and warm slightly. A black precipitate is formed. C. To about 20 mg add 3 ml of 1 M sodium hydroxide and shake to dissolve. Add 5 ml of water R and then, slowly, 2 ml of iodinated potassium iodide solution R. A yellowish precipitate is formed. D. It complies with the test for water (see Tests). TESTS Solution S. Dissolve 5 g in alcohol R and dilute to 10 ml with the same solvent. Appearance of solution. Solution S is not more opalescent than reference suspension II (2.2.1) and not more intensely coloured than reference solution BY5 (2.2.2, Method II). Acidity. To 4 ml of solution S add 15 ml of alcohol R and 0.1 ml of bromothymol blue solution R1. Not more than 1.0 ml of 0.01 M sodium hydroxide is required to change the colour of the indicator to blue. Chlorides (2.4.4). To 1 ml of solution S add 4 ml of alcohol R and dilute to 15 ml with water R. The solution complies with the limit test for chlorides (100 ppm). When preparing the standard, replace the 5 ml of water R by 5 ml of alcohol R. Water (2.5.12). 4.5 per cent to 5.5 per cent, determined on 0.300 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.100 g in 20 ml of alcohol R. Add 10 ml of dilute sodium hydroxide solution R, heat in a water-bath for 5 min and cool. Add 20 ml of dilute nitric acid R, 25.0 ml of 0.1 M silver nitrate and 2 ml of dibutyl phthalate R and 1261

Chlorocresol


shake vigorously. Add 2 ml of ferric ammonium sulphate solution R2 and titrate with 0.1 M ammonium thiocyanate until an orange colour is obtained. 1 ml of 0.1 M silver nitrate is equivalent to 5.92 mg of C4H7Cl3O. STORAGE In an airtight container.

CHLOROCRESOL Chlorocresolum

— nitrogen for chromatography R as the carrier gas at a flow rate of 30 ml/min, — a flame-ionisation detector, maintaining the temperature of the column at 125 °C, that of the injection port at 210 °C and that of the detector at 230 °C. Continue the chromatography for three times the period of time (about 8 min) required for the appearance of the peak corresponding to chlorocresol. In the chromatogram obtained, the sum of the areas of the peaks apart from that corresponding to chlorocresol 01/2005:0384 does not exceed 1 per cent of the total area of the peaks. Disregard the peak corresponding to the solvent. Non-volatile matter. Evaporate 2.0 g to dryness on a water-bath and dry the residue at 100 °C to 105 °C. The residue weighs not more than 2 mg (0.1 per cent).

ASSAY In a ground-glass-stoppered flask, dissolve 70.0 mg in 30 ml of glacial acetic acid R. Add 25.0 ml of 0.0167 M potassium bromate, 20 ml of a 150 g/l solution of potassium bromide R and 10 ml of hydrochloric acid R. Allow to stand protected from light for 15 min. Add 1 g of potassium iodide R and C7H7ClO Mr 142.6 100 ml of water R. Titrate with 0.1 M sodium thiosulphate, shaking vigorously and using 1 ml of starch solution R, DEFINITION added towards the end of the titration, as indicator. Carry Chlorocresol contains not less than 98.0 per cent and out a blank titration. not more than the equivalent of 101.0 per cent of 1 ml of 0.0167 M potassium bromate is equivalent to 4-chloro-3-methylphenol. 3.565 mg of C7H7ClO. CHARACTERS STORAGE A white or almost white, crystalline powder or compacted crystalline masses supplied as pellets or colourless or white Store protected from light. crystals, slightly soluble in water, very soluble in alcohol, freely soluble in fatty oils. It dissolves in solutions of alkali hydroxides. 01/2005:0544 IDENTIFICATION A. Melting point (2.2.14) : 64 °C to 67 °C. B. To 0.1 g add 0.2 ml of benzoyl chloride R and 0.5 ml of dilute sodium hydroxide solution R. Shake vigorously until a white, crystalline precipitate is formed. Add 5 ml of water R and filter. The precipitate, recrystallised from 5 ml of methanol R and dried at 70 °C, melts (2.2.14) at 85 °C to 88 °C. C. To 5 ml of solution S (see Tests) add 0.1 ml of ferric chloride solution R1. A bluish colour is produced. TESTS Solution S. To 3.0 g, finely powdered, add 60 ml of carbon dioxide-free water R, shake for 2 min and filter. Appearance of solution. Dissolve 1.25 g in alcohol R and dilute to 25 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY6 (2.2.2, Method II). Acidity. To 10 ml of solution S add 0.1 ml of methyl red solution R. The solution is orange or red. Not more than 0.2 ml of 0.01 M sodium hydroxide is required to produce a pure yellow colour. Related substances. Examine by gas chromatography (2.2.28). Test solution. Dissolve 1.0 g of the substance to be examined in acetone R and dilute to 100 ml with the same solvent. The chromatographic procedure may be carried out using : — a glass column 1.80 m long and 3 mm to 4 mm in internal diameter packed with silanised diatomaceous earth for gas chromatography R impregnated with 3 per cent m/m to 5 per cent m/m of polymethylphenylsiloxane R, 1262

CHLOROQUINE PHOSPHATE Chloroquini phosphas

C18H32ClN3O8P2

Mr 515.9

DEFINITION Chloroquine phosphate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of N4-(7-chloroquinolin-4-yl)-N1,N1-diethylpentane-1,4-diamine bis(dihydrogen phosphate), calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, hygroscopic, freely soluble in water, very slightly soluble in alcohol and in methanol. It exists in 2 forms, one of which melts at about 195 °C and the other at about 218 °C. IDENTIFICATION First identification : B, D. Second identification : A, C, D.


Chloroquine sulphate


A. Dissolve 0.100 g in water R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this solution to 100.0 ml with water R. Examined between 210 nm and 370 nm (2.2.25), the solution shows absorption maxima at 220 nm, 235 nm, 256 nm, 329 nm and 342 nm. The specific absorbances at the maxima are respectively 600 to 660, 350 to 390, 300 to 330, 325 to 355 and 360 to 390. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with the base isolated from chloroquine sulphate CRS. Record the spectra using solutions prepared as follows : dissolve separately 0.1 g of the substance to be examined and 80 mg of the reference substance in 10 ml of water R, add 2 ml of dilute sodium hydroxide solution R and shake with 2 quantities, each of 20 ml, of methylene chloride R ; combine the organic layers, wash with water R, dry over anhydrous sodium sulphate R, evaporate to dryness and dissolve the residues separately, each in 2 ml of methylene chloride R. C. Dissolve 25 mg in 20 ml of water R and add 8 ml of picric acid solution R1. The precipitate, washed with water R, with alcohol R and finally with methylene chloride R, melts (2.2.14) at 206-209 °C. D. Dissolve 0.1 g in 10 ml of water R, add 2 ml of dilute sodium hydroxide solution R and shake with 2 quantities, each of 20 ml, of methylene chloride R. The aqueous layer, acidified by the addition of nitric acid R, gives reaction (b) of phosphates (2.3.1).

ASSAY Dissolve 0.200 g in 50 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 25.79 mg of C18H32ClN3O8P2.

TESTS Solution S. Dissolve 2.5 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution BY5 or GY5 (2.2.2, Method II). pH (2.2.3). The pH of solution S is 3.8 to 4.3. Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 0.50 g of the substance to be examined in water R and dilute to 10 ml with the same solvent. Reference solution (a). Dilute 1 ml of the test solution to 100 ml with water R. Reference solution (b). Dilute 5 ml of reference solution (a) to 10 ml with water R. Apply to the plate 2 µl of each solution. Develop over a path of 12 cm using a mixture of 10 volumes of diethylamine R, 40 volumes of cyclohexane R and 50 volumes of chloroform R. Allow the plate to dry in air. Examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (a) (1.0 per cent) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). Heavy metals (2.4.8). Dissolve 2.0 gin 10 ml of water R. Add 5 ml of concentrated ammonia R and shake with 40 ml of methylene chloride R. Filter the aqueous layer and neutralise the filtrate with glacial acetic acid R. Heat on a water-bath to eliminate methylene chloride, allow to cool and dilute to 20.0 ml with water R. 12 ml of this solution complies with limit test A for heavy metals (20 ppm). Prepare the standard using lead standard solution (2 ppm Pb) R. Loss on drying (2.2.32) : maximum 2.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C.

DEFINITION Chloroquine sulphate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of N4-(7-chloroquinolin-4-yl)-N1,N1-diethylpentane-1,4-diamine sulphate, calculated with reference to the anhydrous substance.


STORAGE In an airtight container, protected from light.

01/2005:0545

CHLOROQUINE SULPHATE Chloroquini sulfas

C18H28ClN3O4S,H2O

Mr 436.0

CHARACTERS A white or almost white, crystalline powder, freely soluble in water and in methanol, very slightly soluble in alcohol. It melts at about 208 °C (instantaneous method). IDENTIFICATION First identification : B, D. Second identification : A, C, D. A. Dissolve 0.100 g in water R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this solution to 100.0 ml with water R. Examined between 210 nm and 370 nm (2.2.25), the solution shows absorption maxima at 220 nm, 235 nm, 256 nm, 329 nm and 342 nm. The specific absorbances at the maxima are respectively 730 to 810, 430 to 470, 370 to 410, 400 to 440 and 430 to 470. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with the base isolated from chloroquine sulphate CRS. Record the spectra using solutions prepared as follows : dissolve separately 0.1 g of the substance to be examined and of the reference substance in 10 ml of water R, add 2 ml of dilute sodium hydroxide solution R and shake with two quantities, each of 20 ml, of chloroform R ; combine the chloroform layers, wash with water R, dry over anhydrous sodium sulphate R, evaporate to dryness and dissolve the residues separately each in 2 ml of chloroform R. C. Dissolve 25 mg in 20 ml of water R and add 8 ml of picric acid solution R1. The precipitate, washed with water R, with alcohol R and finally with ether R, melts (2.2.14) at 206 °C to 209 °C. D. It gives reaction (a) of sulphates (2.3.1). 1263

Chlorothiazide


TESTS Solution S. Dissolve 2.0 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution BY5 or GY5 (2.2.2, Method II). pH (2.2.3). The pH of solution S is 4.0 to 5.0. Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 0.50 g of the substance to be examined in water R and dilute to 10 ml with the same solvent. Reference solution (a). Dilute 1 ml of the test solution to 100 ml with water R. Reference solution (b). Dilute 5 ml of reference solution (a) to 10 ml with water R. Apply separately to the plate 2 µl of each solution. Develop over a path of 12 cm using a mixture of 10 volumes of diethylamine R, 40 volumes of cyclohexane R and 50 volumes of chloroform R. Allow the plate to dry in air. Examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (a) (1.0 per cent) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). Heavy metals (2.4.8). Dissolve 2.0 g in 10 ml of water R. Add 5 ml of concentrated ammonia R and shake with 40 ml of ether R. Filter the aqueous layer and neutralise the filtrate with glacial acetic acid R. Heat on a water-bath to eliminate ether, allow to cool and dilute to 20.0 ml with water R. 12 ml of this solution complies with limit test A for heavy metals (20 ppm). Prepare the standard using lead standard solution (2 ppm Pb) R. Water (2.5.12) : 3.0 per cent to 5.0 per cent, determined on 0.500 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

DEFINITION Chlorothiazide contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of 6-chloro-2H-1,2,4-benzothiadiazine-7-sulphonamide 1,1-dioxide, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, very slightly soluble in water, sparingly soluble in acetone, slightly soluble in alcohol. It dissolves in dilute solutions of alkali hydroxides.

IDENTIFICATION First identification : B, C. Second identification : A, C, D. A. Dissolve 80.0 mg in 100 ml of 0.1 M sodium hydroxide and dilute to 1000.0 ml with water R. Dilute 10.0 ml of the solution to 100.0 ml with 0.01 M sodium hydroxide. Examined between 220 nm and 320 nm (2.2.25), the solution shows 2 absorption maxima, at 225 nm and 292 nm, and a shoulder at about 310 nm. The specific absorbances at the maxima are 725 to 800 and 425 to 455, respectively. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with chlorothiazide CRS. C. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 25 mg of the substance to be examined in acetone R and dilute to 5 ml with the same solvent. Reference solution. Dissolve 25 mg of chlorothiazide CRS in acetone R and dilute to 5 ml with the same solvent. Apply to the plate 2 µl of each solution. Develop over a path of 10 cm using ethyl acetate R. Dry the plate in a current of air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. D. To 0.1 g add a pellet of sodium hydroxide R and heat strongly. Gas is evolved which turns red litmus paper R ASSAY blue. After cooling, take up the residue with 10 ml of Dissolve 0.400 g in 50 ml of anhydrous acetic acid R. dilute hydrochloric acid R. Gas is evolved which turns Titrate with 0.1 M perchloric acid determining the end-point lead acetate paper R black. potentiometrically (2.2.20). TESTS 1 ml of 0.1 M perchloric acid is equivalent to 41.8 mg of Solution S. To 1.0 g of the powdered substance to be C18H28ClN3O4S. examined add 50 ml of water R, shake for 2 min and filter. STORAGE Acidity or alkalinity. To 10 ml of solution S add 0.2 ml Store in an airtight container, protected from light. of 0.01 M sodium hydroxide and 0.15 ml of methyl red solution R. The solution is yellow. Not more than 0.4 ml of 0.01 M hydrochloric acid is required to change the colour of the indicator to red. 01/2005:0385 Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. CHLOROTHIAZIDE Test solution. Dissolve 25 mg of the substance to be examined in acetone R and dilute to 5 ml with the same Chlorothiazidum solvent. Reference solution. Dilute 1 ml of the test solution to 100 ml with acetone R. Apply to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 15 volumes of 2-propanol R and 85 volumes of ethyl acetate R. Dry the plate in a current of air until the solvents have evaporated (about 10 min) and C7H6ClN3O4S2 Mr 295.7 spray with a mixture of equal volumes of alcoholic solution 1264



of sulphuric acid R and alcohol R ; use about 10 ml for a plate 200 mm square and spray in small portions, allowing the solvent to evaporate each time to avoid excessive wetting. Heat at 100-105 °C for 30 min and immediately place the plate above, but not in contact with, 10 ml of a saturated solution of sodium nitrite R in a glass tank. Carefully add 0.5 ml of sulphuric acid R to the sodium nitrite solution, close the tank, and allow to stand for 15 min. Remove the plate, heat in a ventilated oven at 40 °C for 15 min and spray with 3 quantities, each of 5 ml, of a freshly prepared 5 g/l solution of naphthylethylenediamine dihydrochloride R in alcohol R. Examine the plate by transmitted light. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution (1.0 per cent). Chlorides (2.4.4). 15 ml of solution S complies with the limit test for chlorides (160 ppm). Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.250 g in 50 ml of dimethylformamide R. Titrate with 0.1 M tetrabutylammonium hydroxide in 2-propanol determining the end-point potentiometrically (2.2.20) at the first point of inflexion. Carry out a blank titration. 1 ml of 0.1 M tetrabutylammonium hydroxide in 2-propanol is equivalent to 29.57 mg of C7H6ClN3O4S2.

Chlorphenamine maleate

B. Dissolve 30.0 mg in 0.1 M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute 10.0 ml of this solution to 100.0 ml with 0.1 M hydrochloric acid. Examined between 230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 265 nm. The specific absorbance at the maximum is 200 to 220. C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with chlorphenamine maleate CRS. Examine the substances prepared as discs. D. Dissolve 0.1 g in 10 ml of water R and add dropwise and with shaking 25 ml of picric acid solution R. Collect the precipitate on a sintered-glass filter. The precipitate, washed with 3 ml of alcohol R, recrystallised from a mixture of equal volumes of alcohol R and water R and dried at 100 °C to 105 °C, melts (2.2.14) at 196 °C to 200 °C. E. To 0.2 g add 3 ml of water R and 1 ml of strong sodium hydroxide solution R. Shake with three quantities, each of 5 ml, of ether R. To 0.1 ml of the aqueous layer add a solution of 10 mg of resorcinol R in 3 ml of sulphuric acid R. Heat on a water-bath for 15 min. No colour develops. To the remainder of the aqueous layer add 2 ml of bromine solution R. Heat in a water-bath for 15 min, heat to boiling and cool. To 0.2 ml of the solution add a solution of 10 mg of resorcinol R in 3 ml of sulphuric acid R and heat on a water-bath for 15 min. A blue colour develops.

TESTS Solution S. Dissolve 2.0 g in water R and dilute to 20 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution BY6 (2.2.2, Method II). 01/2005:0386 Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 0.5 g of the substance to be examined CHLORPHENAMINE MALEATE in chloroform R and dilute to 10 ml with the same solvent. Reference solution. Dilute 1 ml of the test solution to 50 ml Chlorphenamini maleas with chloroform R. Dilute 1 ml of this solution to 10 ml with chloroform R. Apply to the plate 10 µl of each solution. Develop over a path of 12 cm using a mixture of 10 volumes of diethylamine R, 40 volumes of chloroform R and 50 volumes of cyclohexane R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution (0.2 per cent). Disregard C20H23ClN2O4 Mr 390.9 any spots remaining on the starting-line. Heavy metals (2.4.8). 1.0 g complies with limit test C for DEFINITION heavy metals (20 ppm). Prepare the standard using 2 ml of Chlorphenamine maleate contains not less than 98.0 per lead standard solution (10 ppm Pb) R. cent and not more than the equivalent of 101.0 per cent of (3RS)-3-(4-chlorophenyl)-N,N-dimethyl-3-(pyridin-2-yl)propan- Loss on drying (2.2.32). Not more than 0.5 per cent, 1-amine hydrogen (Z)-butenedioate, calculated with reference determined on 1.00 g by drying in an oven at 100 °C to 105 °C for 4 h. to the dried substance. Sulphated ash (2.4.14). Not more than 0.1 per cent, CHARACTERS determined on 1.0 g. A white, crystalline powder, freely soluble in water, soluble ASSAY in alcohol. Dissolve 0.150 g in 25 ml of anhydrous acetic acid R. IDENTIFICATION Titrate with 0.1 M perchloric acid determining the end-point First identification : A, C. potentiometrically (2.2.20). Second identification : A, B, D, E. 1 ml of 0.1 M perchloric acid is equivalent to 19.54 mg of A. Melting point (2.2.14) : 132 °C to 135 °C. C20H23ClN2O4. General Notices (1) apply to all monographs and other texts

1265

Chlorpromazine hydrochloride



Reference solution. Dilute 1 ml of the test solution to 200 ml with a mixture of 5 volumes of diethylamine R and 95 volumes of methanol R. Apply to the plate 10 µl of each solution. Develop 01/2005:0475 over a path of 15 cm using a mixture of 10 volumes of acetone R, 10 volumes of diethylamine R and 80 volumes of cyclohexane R. Allow the plate to dry in air and examine in CHLORPROMAZINE ultraviolet light at 254 nm. Any spot in the chromatogram HYDROCHLORIDE obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram Chlorpromazini hydrochloridum obtained with the reference solution (0.5 per cent). Disregard any spot at the starting point. Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (10 ppm). Prepare the standard using 1 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, C17H20Cl2N2S Mr 355.3 determined on 1.0 g. DEFINITION Chlorpromazine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of 3-(2-chloro-10H-phenothiazin-10-yl)-N,Ndimethylpropan-1-amine hydrochloride, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, very soluble in water, freely soluble in alcohol. It decomposes on exposure to air and light. It melts at about 196 °C. IDENTIFICATION First identification : B, C, D. Second identification : A, C, D. A. Prepare the solutions protected from bright light and measure the absorbances immediately. Dissolve 50.0 mg in 0.1 M hydrochloric acid and dilute to 500.0 ml with the same acid. Dilute 5.0 ml of the solution to 100.0 ml with 0.1 M hydrochloric acid. Examined between 230 nm and 340 nm (2.2.25), the solution shows two absorption maxima, at 254 nm and 306 nm respectively. The specific absorbance at the maximum at 254 nm is 890 to 960. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with chlorpromazine hydrochloride CRS. Examine the substances as 60 g/l solutions in methylene chloride R using a 0.1 mm cell. C. It complies with the identification test for phenothiazines by thin-layer chromatography (2.3.3). D. It gives reaction (b) of chlorides (2.3.1). TESTS pH (2.2.3). Dissolve 1.0 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. The pH of the freshly prepared solution is 3.5 to 4.5. Related substances. Carry out the test protected from bright light. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 0.2 g of the substance to be examined in a mixture of 5 volumes of diethylamine R and 95 volumes of methanol R and dilute to 10 ml with the same mixture of solvents. Prepare immediately before use. 1266

ASSAY Dissolve 0.250 g in a mixture of 5.0 ml of 0.01 M hydrochloric acid and 50 ml of alcohol R. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume of 0.1 M sodium hydroxide added between the two points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 35.53 mg of C17H20Cl2N2S. STORAGE Store in an airtight container, protected from light. 01/2005:1087

CHLORPROPAMIDE Chlorpropamidum

C10H13ClN2O3S

Mr 276.7

DEFINITION Chlorpropamide contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of 1-[(4-chlorophenyl)sulphonyl]-3-propylurea, calculated with reference to the dried substance. CHARACTERS A white, crystalline powder, practically insoluble in water, freely soluble in acetone and in methylene chloride, soluble in alcohol. It dissolves in dilute solutions of alkali hydroxides. It shows polymorphism. IDENTIFICATION First identification : C, D. Second identification : A, B, D. A. Melting point (2.2.14) : 126 °C to 130 °C. B. Dissolve 0.10 g in methanol R and dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of the solution to 100.0 ml with 0.01 M hydrochloric acid. Dilute 10.0 ml of the solution to 100.0 ml with 0.01 M hydrochloric


Chlorpromazine hydrochloride



Reference solution. Dilute 1 ml of the test solution to 200 ml with a mixture of 5 volumes of diethylamine R and 95 volumes of methanol R. Apply to the plate 10 µl of each solution. Develop 01/2005:0475 over a path of 15 cm using a mixture of 10 volumes of acetone R, 10 volumes of diethylamine R and 80 volumes of cyclohexane R. Allow the plate to dry in air and examine in CHLORPROMAZINE ultraviolet light at 254 nm. Any spot in the chromatogram HYDROCHLORIDE obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram Chlorpromazini hydrochloridum obtained with the reference solution (0.5 per cent). Disregard any spot at the starting point. Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (10 ppm). Prepare the standard using 1 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, C17H20Cl2N2S Mr 355.3 determined on 1.0 g. DEFINITION Chlorpromazine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of 3-(2-chloro-10H-phenothiazin-10-yl)-N,Ndimethylpropan-1-amine hydrochloride, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, very soluble in water, freely soluble in alcohol. It decomposes on exposure to air and light. It melts at about 196 °C. IDENTIFICATION First identification : B, C, D. Second identification : A, C, D. A. Prepare the solutions protected from bright light and measure the absorbances immediately. Dissolve 50.0 mg in 0.1 M hydrochloric acid and dilute to 500.0 ml with the same acid. Dilute 5.0 ml of the solution to 100.0 ml with 0.1 M hydrochloric acid. Examined between 230 nm and 340 nm (2.2.25), the solution shows two absorption maxima, at 254 nm and 306 nm respectively. The specific absorbance at the maximum at 254 nm is 890 to 960. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with chlorpromazine hydrochloride CRS. Examine the substances as 60 g/l solutions in methylene chloride R using a 0.1 mm cell. C. It complies with the identification test for phenothiazines by thin-layer chromatography (2.3.3). D. It gives reaction (b) of chlorides (2.3.1). TESTS pH (2.2.3). Dissolve 1.0 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. The pH of the freshly prepared solution is 3.5 to 4.5. Related substances. Carry out the test protected from bright light. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 0.2 g of the substance to be examined in a mixture of 5 volumes of diethylamine R and 95 volumes of methanol R and dilute to 10 ml with the same mixture of solvents. Prepare immediately before use. 1266

ASSAY Dissolve 0.250 g in a mixture of 5.0 ml of 0.01 M hydrochloric acid and 50 ml of alcohol R. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume of 0.1 M sodium hydroxide added between the two points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 35.53 mg of C17H20Cl2N2S. STORAGE Store in an airtight container, protected from light. 01/2005:1087

CHLORPROPAMIDE Chlorpropamidum

C10H13ClN2O3S

Mr 276.7

DEFINITION Chlorpropamide contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of 1-[(4-chlorophenyl)sulphonyl]-3-propylurea, calculated with reference to the dried substance. CHARACTERS A white, crystalline powder, practically insoluble in water, freely soluble in acetone and in methylene chloride, soluble in alcohol. It dissolves in dilute solutions of alkali hydroxides. It shows polymorphism. IDENTIFICATION First identification : C, D. Second identification : A, B, D. A. Melting point (2.2.14) : 126 °C to 130 °C. B. Dissolve 0.10 g in methanol R and dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of the solution to 100.0 ml with 0.01 M hydrochloric acid. Dilute 10.0 ml of the solution to 100.0 ml with 0.01 M hydrochloric


Chlorprothixene hydrochloride


acid. Examined between 220 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 232 nm. The specific absorption at the maximum is 570 to 630. C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with chlorpropamide CRS. Examine the substances prepared as discs. If the spectra obtained show differences, dissolve the substance to be examined and the reference substance in methylene chloride R, evaporate to dryness and record the new spectra using the residues. D. Heat 0.1 g with 2 g of anhydrous sodium carbonate R until a dull red colour appears for 10 min. Allow to cool, extract the residue with about 5 ml of water R, dilute to 10 ml with water R and filter. The solution gives the reaction (a) of chloride (2.3.1). TESTS Related substances. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating substance. Test solution. Dissolve 0.50 g of the substance to be examined in acetone R and dilute to 10 ml with the same solvent. Reference solution (a). Dissolve 15 mg of 4-chlorobenzenesulphonamide R (chlorpropamide impurity A) in acetone R and dilute to 100 ml with the same solvent. Reference solution (b). Dissolve 15 mg of chlorpropamide impurity B CRS in acetone R and dilute to 100 ml with the same solvent. Reference solution (c). Dilute 0.3 ml of the test solution to 100 ml with acetone R. Reference solution (d). Dilute 5 ml of reference solution (c) to 15 ml with acetone R. Reference solution (e). Dissolve 0.10 g of the substance to be examined, 5 mg of 4-chlorobenzenesulphonamide R and 5 mg of chlorpropamide impurity B CRS in acetone R and dilute to 10 ml with the same solvent. Apply to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 11.5 volumes of concentrated ammonia R, 30 volumes of cyclohexane R, 50 volumes of methanol R and 100 volumes of methylene chloride R. Allow the plate to dry in a current of cold air, heat at 110 °C for 10 min. At the bottom of a chromatographic tank, place an evaporating dish containing a mixture of 1 volume of hydrochloric acid R, 1 volume of water R and 2 volumes of a 50 g/l solution of potassium permanganate R, close the tank and allow to stand for 15 min. Place the dried hot plate in the tank and close the tank. Leave the plate in contact with the chlorine vapour for 2 min. Withdraw the plate and place it in a current of cold air until the excess of chlorine is removed and an area of coating below the points of application does not give a blue colour with a drop of potassium iodide and starch solution R. Spray with potassium iodide and starch solution R. In the chromatogram obtained with the test solution : any spot corresponding to impurity A is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.3 per cent) ; any spot corresponding to impurity B is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.3 per cent) ; any spot, apart from the principal spot and any spot corresponding to impurity A and B, is not more intense than the spot in the chromatogram obtained with reference solution (c) (0.3 per cent) ; not more than two such spots are more intense than the spot in the chromatogram obtained with reference solution (d) (0.1 per cent). The test is not valid unless the chromatogram obtained with reference General Notices (1) apply to all monographs and other texts

solution (e) shows three clearly separated spots with approximate Rf values of 0.4, 0.6 and 0.9 corresponding to chlorpropamide, impurity A and impurity B respectively. Heavy metals (2.4.8). Dissolve 2.0 g in a mixture of 15 volumes of water R and 85 volumes of acetone R and dilute to 20 ml with the same mixture of solvents. 12 ml of solution complies with limit test B for heavy metals (20 ppm). Prepare the standard using lead standard solution (2 ppm Pb) prepared by diluting lead standard solution (100 ppm Pb) R with a mixture of 15 volumes of water R and 85 volumes of acetone R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.250 g in 50 ml of alcohol R previously neutralised using phenolphthalein solution R1 as indicator and add 25 ml of water R. Titrate with 0.1 M sodium hydroxide until a pink colour is obtained. 1 ml of 0.1 M sodium hydroxide is equivalent to 27.67 mg of C10H13ClN2O3S. STORAGE Store protected from light. IMPURITIES

A. R = H : 4-chlorobenzenesulphonamide, C. R = CO-NH2 : [(4-chlorophenyl)sulphonyl]urea.

B. 1,3-dipropylurea, 01/2005:0815

CHLORPROTHIXENE HYDROCHLORIDE Chlorprothixeni hydrochloridum

C18H19Cl2NS

Mr 352.3

DEFINITION (Z)-3-(2-Chloro-9H-thioxanthen-9-ylidene)-N,Ndimethylpropan-1-amine hydrochloride. Content : 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white or almost white, crystalline powder. 1267

Chlorprothixene hydrochloride


Flow rate : 1.5 ml/min. Detection : spectrophotometer at 254 nm. Equilibration : for about 30 min with the mobile phase. Injection : 20 µl. IDENTIFICATION Run time : twice the retention time of chlorprothixene. First identification : A, E. Relative retention with reference to chlorprothixene : Second identification : B, C, D, E. impurity E = about 1.55. A. Infrared absorption spectrophotometry (2.2.24). System suitability : reference solution (a) : Preparation : dissolve 0.25 g in 10 ml of water R. Add — retention time : chlorprothixene = about 10 min, 1 ml of dilute sodium hydroxide solution R. Shake with — relative retention with reference to chlorprothixene : 20 ml of methylene chloride R. Separate the organic E-isomer = about 1.35. layer and wash with 5 ml of water R. Evaporate the organic layer to dryness and dry the residue at 40-50 °C. Limits : Examine the residues prepared as discs. — E-isomer : not more than 2.0 per cent, calculated from the area of the corresponding peak in the chromatogram Comparison : chlorprothixene hydrochloride CRS. obtained with reference solution (a) and taking B. Dissolve 0.2 g in a mixture of 5 ml of dioxan R and 5 ml into account the assigned content of this isomer in of a 1.5 g/l solution of sodium nitrite R. Add 0.8 ml of chlorprothixene hydrochloride CRS, nitric acid R. After 10 min add the solution to 20 ml of water R. 1 h later filter the precipate formed. The filtrate — impurity E : not more than 3 times the area of the principal peak in the chromatogram obtained with is used immediately for identification test C. Dissolve reference solution (b) (0.3 per cent taking into account a the precipitate by warming in about 15 ml of alcohol R response factor of 3), and add the solution to 10 ml of water R. Filter and dry the precipitate at 100-105 °C for 2 h. The melting point — any other impurity : not more than the area of the (2.2.14) is 152 °C to 154 °C. principal peak in the chromatogram obtained with reference solution (b) (0.3 per cent), C. To 1 ml of the filtrate obtained in identification test B, add 0.2 ml of a suspension of 50 mg of fast red B salt R in — total of any other impurity : not more than 2.33 times the area of the principal peak in the chromatogram obtained 1 ml of alcohol R. Add 1 ml of 0.5 M alcoholic potassium with reference solution (b) (0.7 per cent), hydroxide. A dark red colour is produced. Carry out a blank test. — disregard limit : 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b) D. Dissolve about 20 mg in 2 ml of nitric acid R. A red (0.03 per cent). colour is produced. Add 5 ml of water R and examine in ultraviolet light at 365 nm. The solution shows green Heavy metals (2.4.8) : maximum 20 ppm. fluorescence. 1.0 g complies with limit test F. Prepare the standard using E. It gives reaction (a) of chlorides (2.3.1). 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32) : maximum 0.5 per cent, determined TESTS on 1.000 g by drying in vacuo at 60 °C for 3 h. Solution S. Dissolve 0.25 g in carbon dioxide-free water R Sulphated ash (2.4.14) : maximum 0.1 per cent, determined and dilute to 25 ml with the same solvent. on 1.0 g. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). ASSAY Dissolve 0.300 g in a mixture of 5.0 ml of 0.01 M hydrochloric pH (2.2.3) : 4.4 to 5.2 for solution S. acid and 50 ml of alcohol R. Carry out a potentiometric Related substances. Liquid chromatography (2.2.29). Carry titration (2.2.20), using 0.1 M sodium hydroxide. Read out the test protected from bright light. the volume added between the 2 points of inflexion. Test solution. Dissolve 20.0 mg of the substance to be 1 ml of 0.1 M sodium hydroxide is equivalent to 35.23 mg of examined in the mobile phase and dilute to 20.0 ml with the C H Cl NS. 18 19 2 mobile phase. Reference solution (a). Dissolve 20.0 mg of chlorprothixene STORAGE hydrochloride CRS (with a defined content of E-isomer) in Protected from light. the mobile phase and dilute to 20.0 ml with the mobile phase. IMPURITIES Reference solution (b). Dilute 2.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 3.0 ml of this Specified impurities : A, B, C, D, E, F. solution to 20.0 ml with the mobile phase. Column : — size : l = 0.12 m, Ø = 4.0 mm, — stationary phase: base-deactivated octadecylsilyl silica gel for chromatography R (3 µm or 5 µm). Mobile phase : solution containing 6.0 g/l of potassium dihydrogen phosphate R, 2.9 g/l of sodium laurilsulfate R and 9 g/l of tetrabutylammonium bromide R in a mixture of 50 volumes of methanol R, 400 volumes of acetonitrile R A. (RS)-2-chloro-9-[3-(dimethylamino)propyl]-9Hand 550 volumes of distilled water R. thioxanthen-9-ol, Solubility : soluble in water and in alcohol, slightly soluble in methylene chloride. mp : about 220 °C.

1268



Chlortalidone

B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with chlortalidone CRS. Examine the substances prepared as discs using potassium bromide R. C. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 10 mg of the substance to be examined in acetone R and dilute to 10 ml with the same B. R1 = H, R2 = CH-CH2-CH2-N(CH3)2, R3 = H : N,N-dimethylsolvent. 3-(9H-thioxanthen-9-ylidene)propan-1-amine, Reference solution (a). Dissolve 10 mg of chlortalidone CRS in acetone R and dilute to 10 ml with C. R1 = Cl, R2 = CH-CH2-CH2-NH-CH3, R3 = H : (Z)-3-(2-chlorothe same solvent. 9H-thioxanthen-9-ylidene)-N-methylpropan-1-amine, Reference solution (b). Dissolve 10 mg of D. R1 = H, R2 = CH-CH2-CH2-N(CH3)2, R3 = Cl : (Z)-3-(4-chlorochlortalidone CRS and 10 mg of hydrochlorothiazide CRS 9H-thioxanthen-9-ylidene)-N,N-dimethylpropan-1-amine, in acetone R and dilute to 10 ml with the same solvent. E. R1 = Cl, R2 = O, R3 = H : 2-chloro-9H-thioxanthen-9-one, Apply separately to the plate 5 µl of each solution. Develop over a path of 10 cm using a mixture of 1.5 volumes of water R and 98.5 volumes of ethyl acetate R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows 2 clearly separated spots. F. (E)-3-(2-chloro-9H-thioxanthen-9-ylidene)-N,ND. Dissolve about 10 mg in 1 ml of sulphuric acid R. An dimethylpropan-1-amine (E-isomer). intense yellow colour develops. E. It complies with the test for optical rotation (see Tests). 01/2005:0546 TESTS CHLORTALIDONE Appearance of solution. Dissolve 1.0 g in dilute sodium hydroxide solution R and dilute to 10 ml with the same solution. The solution is clear (2.2.1) and not more intensely Chlortalidonum coloured than intensity 6 of the range of reference solutions of the most appropriate colour (2.2.2, Method II). Acidity. Dissolve 1.0 g with heating in a mixture of 25 ml of acetone R and 25 ml of carbon dioxide-free water R. Cool. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20). Not more than 0.75 ml of 0.1 M sodium hydroxide is required. C14H11ClN2O4S Mr 338.8 Optical rotation (2.2.7). Dissolve 0.20 g in methanol R and dilute to 20 ml with the same solvent. The angle of optical DEFINITION rotation is − 0.15° to + 0.15°. Chlortalidone contains not less than 98.0 per cent Related substances. Examine by thin-layer chromatography and not more than the equivalent of 102.0 per cent of (2.2.27), using as the coating substance a suitable silica gel 2-chloro-5-[(1RS)-1-hydroxy-3-oxo-2,3-dihydro-1H-isoindol-1with a fluorescent indicator having an optimal intensity at yl]benzenesulphonamide, calculated with reference to the 254 nm. dried substance. Test solution. Dissolve 0.2 g of the substance to be examined CHARACTERS in a mixture of 1 volume of water R and 4 volumes of acetone R and dilute to 5 ml with the same mixture of A white or yellowish-white powder, practically insoluble in solvents. water, soluble in acetone and in methanol, slightly soluble in alcohol, practically insoluble in methylene chloride. It Reference solution (a). Dissolve 20 mg of chlortalidone dissolves in dilute solutions of the alkali hydroxides. impurity B CRS and 20 mg of chlortalidone CRS in a mixture of 1 volume of water R and 4 volumes of acetone R It melts at about 220 °C, with decomposition. and dilute to 50 ml with the same mixture of solvents. IDENTIFICATION Reference solution (b). Dilute 1 ml of the test solution to First identification : B. 200 ml with a mixture of 1 volume of water R and 4 volumes of acetone R. Second identification : A, C, D, E. Apply separately to the plate 5 µl of each solution. A. Dissolve 50.0 mg in alcohol R and dilute to 50.0 ml Develop over a path of 15 cm using a mixture of 5 volumes with the same solvent. Dilute 10.0 ml of this solution to 100.0 ml with alcohol R. Examined between 230 nm and of toluene R, 10 volumes of xylene R, 20 volumes of 340 nm (2.2.25), the solution shows 2 absorption maxima, concentrated ammonia R, 30 volumes of dioxan R and 30 volumes of 2-propanol R. Allow the plate to dry in at 275 nm and 284 nm. The ratio of the absorbance measured at the absorption maximum at 284 nm to that a current of warm air and examine in ultraviolet light measured at the absorption maximum at 275 nm is 0.73 at 254 nm. In the chromatogram obtained with the test solution : any spot corresponding to impurity B is not more to 0.88. General Notices (1) apply to all monographs and other texts

1269

Chlortetracycline hydrochloride


intense than the corresponding spot in the chromatogram obtained with reference solution (a) (1 per cent) ; and any spot, apart from the principal spot and the spot corresponding to impurity B, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). The test is not valid unless the chromatogram obtained with reference solution (a) shows 2 clearly separated spots. Chlorides (2.4.4). Triturate 0.3 g, add 30 ml of water R, shake for 5 min and filter. 15 ml of the filtrate complies with the limit test for chlorides (350 ppm). Prepare the standard using 10 ml of chloride standard solution (5 ppm Cl) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.200 g in 50 ml of acetone R. In an atmosphere of nitrogen, titrate with 0.1 M tetrabutylammonium hydroxide, determining the end-point potentiometrically (2.2.20). Carry out a blank titration. 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to 33.88 mg of C14H11ClN2O4S.

01/2005:0173

CHLORTETRACYCLINE HYDROCHLORIDE Chlortetracyclini hydrochloridum

Compound

R

Molecular formula


Cl

C22H24Cl2N2O8

515.3

Tetracycline hydrochloride

H

C22H25ClN2O8

480.9

Mr

DEFINITION Mixture of antibiotics, the main component being the hydrochloride of (4S,4aS,5aS,6S,12aS)-7-chloro-4(dimethylamino)-3,6,10,12,12a-pentahydroxy-6-methyl-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide IMPURITIES (chlortetracycline hydrochloride), a substance produced by Specified impurities : A, B, C, D, E, F, G, H, I. the growth of certain strains of Streptomyces aureofaciens or obtained by any other means. Content : — C22H24Cl2N2O8 : minimum 89.5 per cent (anhydrous substance), — C22H25ClN2O8 : maximum 8.0 per cent (anhydrous substance), A. R = H, R′ = OH : 2-(4-chloro-3-sulphobenzoyl)benzoic acid, — 94.5 per cent to 102.0 per cent for the sum of the contents of chlortetracycline hydrochloride and tetracycline B. R = H, R′ = NH2 : 2-(4-chloro-3-sulphamoylbenzoyl)benzoic hydrochloride (anhydrous substance). acid, C. R = C2H5, R′ = NH2 : ethyl 2-(4-chloro-3sulphamoylbenzoyl)benzoate, I. R = CH(CH3)2, R′ = NH2 : 1-methylethyl 2-(4-chloro-3-sulphamoylbenzoyl)benzoate,

D. E. G. H.

F.

CHARACTERS Appearance : yellow powder. Solubility : slightly soluble in water and in alcohol. It dissolves in solutions of alkali hydroxides and carbonates.

IDENTIFICATION A. Thin-layer chromatography (2.2.27). Test solution. Dissolve 5 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution (a). Dissolve 5 mg of chlortetracycline R = OC2H5, R′ = SO2-NH2 : 2-chloro-5-[(1RS)-1-ethoxy-3hydrochloride CRS in methanol R and dilute to 10 ml oxo-2,3-dihydro-1H-isoindol-1-yl]benzenesulphonamide, with the same solvent. R = H, R′ = SO2-NH2 : 2-chloro-5-[(1RS)-3-oxo-2,3-dihydroReference solution (b). Dissolve 5 mg of chlortetracycline 1H-isoindol-1-yl]benzenesulphonamide, hydrochloride CRS, 5 mg of doxycycline R and 5 mg R = OH, R′ = Cl : (3RS)-3-(3,4-dichlorophenyl)-3-hydroxy-2, of demeclocycline hydrochloride R in methanol R and 3-dihydro-1H-isoindol-1-one, dilute to 10 ml with the same solvent. Plate : TLC octadecylsilyl silica gel F254 plate R. R = OCH(CH3)2, R′ = SO2-NH2 : 2-chloro-5-[(1RS)Mobile phase : mix 20 volumes of acetonitrile R, 1-(1-methylethoxy)-3-oxo-2,3-dihydro-1H-isoindol-120 volumes of methanol R and 60 volumes of a 63 g/l yl]benzenesulphonamide, solution of oxalic acid R previously adjusted to pH 2 with concentrated ammonia R. Application : 1 µl. Development : over 3/4 of the plate. Drying : in air. Detection : examine in ultraviolet light at 254 nm. System suitability : the chromatogram obtained with bis[2-chloro-5-(1-hydroxy-3-oxo-2,3-dihydro-1H-isoindol-1reference solution (b) shows 3 clearly separated spots. yl)benzenesulphonyl]amine.

1270




Results : the principal spot in the chromatogram obtained System suitability : reference solution (d) : with the test solution is similar in position and size to — resolution : minimum 2.0 between the peaks due to the principal spot in the chromatogram obtained with impurity A and to chlortetracycline ; if necessary, adjust reference solution (a). the dimethyl sulphoxide content in the mobile phase, B. To about 2 mg add 5 ml of sulphuric acid R. A deep — symmetry factor : maximum 1.3 for the peak due to blue colour develops which becomes bluish-green. Add chlortetracycline. the solution to 2.5 ml of water R. The colour becomes Limits : brownish. — impurity A : not more than the area of the corresponding C. It gives reaction (a) of chlorides (2.3.1). peak in the chromatogram obtained with reference solution (e) (4.0 per cent), TESTS — total of other impurities eluting between the solvent peak and the peak corresponding to chlortetracycline: pH (2.2.3) : 2.3 to 3.3. not more than 0.25 times the area of the peak due to Dissolve 0.1 g in 10 ml of carbon dioxide-free water R, impurity A in the chromatogram obtained with reference heating slightly. solution (e) (1.0 per cent), Specific optical rotation (2.2.7) : − 235 to − 250 (anhydrous — disregard limit : area of the principal peak in the substance). chromatogram obtained with reference solution (f) (0.1 per cent). Dissolve 0.125 g in water R and dilute to 50.0 ml with the Heavy metals (2.4.8) : maximum 50 ppm. same solvent. 0.5 g complies with limit test C. Prepare the standard using Absorbance (2.2.25) : maximum 0.40 at 460 nm. 2.5 ml of lead standard solution (10 ppm Pb) R. Dissolve 0.125 g in water R and dilute to 25.0 ml with the Water (2.5.12) : maximum 2.0 per cent, determined on same solvent. 0.300 g. Related substances. Liquid chromatography (2.2.29). Sulphated ash (2.4.14) : maximum 0.5 per cent, determined Prepare the solutions immediately before use. on 1.0 g. Test solution. Dissolve 25.0 mg of the substance to be Bacterial endotoxins (2.6.14) : less than 1 IU/mg, if intended examined in 0.01 M hydrochloric acid and dilute to 25.0 ml for use in the manufacture of parenteral dosage forms with the same acid. without a further appropriate procedure for the removal of Reference solution (a). Dissolve 25.0 mg of chlortetracycline bacterial endotoxins. hydrochloride CRS in 0.01 M hydrochloric acid and dilute ASSAY to 25.0 ml with the same acid. Liquid chromatography (2.2.29) as described in the test for Reference solution (b). Dissolve 10.0 mg of related substances with the following modification. 4-epichlortetracycline hydrochloride CRS in 0.01 M hydrochloric acid and dilute to 25.0 ml with the same acid. Injection : test solution and reference solutions (a) and (e). Calculate the percentage content of C22H24Cl2N2O8 using Reference solution (c). Dissolve 20.0 mg of tetracycline hydrochloride CRS in 0.01 M hydrochloric acid and dilute the chromatogram obtained with reference solution (a). Calculate the percentage content of C22H25ClN2O8 using the to 25.0 ml with the same acid. chromatogram obtained with reference solution (e). Reference solution (d). Mix 5.0 ml of reference solution (a) and 10.0 ml of reference solution (b) and dilute to 25.0 ml STORAGE with 0.01 M hydrochloric acid. Protected from light. If the substance is sterile, store in a Reference solution (e). Mix 5.0 ml of reference solution (b) sterile, airtight, tamper-proof container. and 5.0 ml of reference solution (c) and dilute to 50.0 ml LABELLING with 0.01 M hydrochloric acid. Reference solution (f). Dilute 1.0 ml of reference solution (c) The label states, where applicable, that the substance is free from bacterial endotoxins. to 20.0 ml with 0.01 M hydrochloric acid. Dilute 5.0 ml of this solution to 200.0 ml with 0.01 M hydrochloric acid. IMPURITIES Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase: octylsilyl silica gel for chromatography R (5 µm), — temperature : 35 °C. Mobile phase : to 500 ml of water R, add 50 ml of perchloric acid solution R, shake and add 450 ml of dimethyl sulphoxide R, A. (4R,4aS,5aS,6S,12aS)-7-chloro-4-(dimethylamino)3,6,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-1,4, Flow rate : 1 ml/min. 4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Detection : spectrophotometer at 280 nm. (4-epichlortetracycline), Injection : 20 µl ; inject the test solution and reference solutions (d), (e) and (f). B. demeclocycline. General Notices (1) apply to all monographs and other texts

1271

Cholecalciferol


01/2005:0072 The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with a suitable silica gel (5 µm), CHOLECALCIFEROL — as mobile phase at a flow rate of 2 ml/min a mixture of 3 volumes of pentanol R and 997 volumes of hexane R, Cholecalciferolum — as detector a spectrophotometer set at 254 nm. An automatic injection device or a sample loop is recommended. Inject a suitable volume of reference solution (b). Adjust the sensitivity of the system so that the height of the principal peak is at least 50 per cent of the full scale of the recorder. Inject reference solution (b) 6 times. When the chromatograms are recorded in the prescribed conditions, the approximate relative retention times with reference to cholecalciferol are 0.4 for pre-cholecalciferol and 0.5 for trans-cholecalciferol. The relative standard deviation of the response for cholecalciferol is not greater than 1 per cent and the resolution between the peaks due to pre-cholecalciferol and trans-cholecalciferol is not less than 1.0. If necessary adjust the proportions of the constituents and the flow rate of the mobile phase to obtain C27H44O Mr 384.6 this resolution. Inject a suitable volume of reference solution (a). Adjust the DEFINITION sensitivity of the system so that the height of the principal Cholecalciferol contains not less than 97.0 per cent peak is at least 50 per cent of the full scale of the recorder. and not more than the equivalent of 103.0 per cent of Inject the same volume of the test solution and record the (5Z,7E)-9,10-secocholesta-5,7,10(19)-trien-3β-ol. chromatogram in the same manner. 1 milligram of cholecalciferol is equivalent to 40 000 IU of Calculate the percentage content of cholecalciferol from the antirachitic activity (vitamin D) in rats. expression : CHARACTERS White or almost white crystals, practically insoluble in water, freely soluble in alcohol, soluble in fatty oils. It is sensitive to air, heat and light. Solutions in volatile solvents are unstable m and are to be used immediately. A reversible isomerisation to pre-cholecalciferol takes place m′ in solution, depending on temperature and time. The activity is due to both compounds. SD IDENTIFICATION Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cholecalciferol CRS. Examine the substances prepared as discs. TESTS Specific optical rotation (2.2.7). Dissolve 0.200 g rapidly and without heating in aldehyde-free alcohol R and dilute to 25.0 ml with the same solvent. The specific optical rotation, determined within 30 min of preparing the solution, is + 105 to + 112.

S′ D

=

mass of the substance to be examined in the test solution, in milligrams,

=

mass of cholecalciferol CRS in reference solution (a), in milligrams,

=

area (or height) of the peak due to cholecalciferol in the chromatogram obtained with the test solution,

=

area (or height) of the peak due to cholecalciferol in the chromatogram obtained with reference solution (a).

STORAGE Store in an airtight container, under nitrogen, protected from light, at a temperature between 2 °C and 8 °C. The contents of an opened container are to be used immediately. IMPURITIES

ASSAY Carry out the operations as rapidly as possible, avoiding exposure to actinic light and air. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 10.0 mg of the substance to be examined without heating in 10.0 ml of toluene R and dilute to 100.0 ml with the mobile phase. Reference solution (a). Dissolve 10.0 mg of cholecalciferol CRS without heating in 10.0 ml of toluene R and dilute to 100.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of cholecalciferol for performance test CRS to 5.0 ml with the mobile phase. Heat in a water-bath at 90 °C under a reflux condenser for 45 min A. (5E,7E)-9,10-secocholesta-5,7,10(19)-trien-3β-ol (trans-cholecalciferol, trans-vitamin D3), and cool. 1272



Cholecalciferol concentrate (oily form)

The content of cholecalciferol stated on the label is not less than 500 000 IU/g and the concentrate contains not less than 90.0 per cent and not more than 110.0 per cent of the content stated on the label. The concentrate may contain suitable stabilisers such as antioxidants.

B. cholesta-5,7-dien-3β-ol (7,8-didehydrocholesterol, provitamin D3),

CHARACTERS A clear, yellow liquid, practically insoluble in water, slightly soluble in ethanol, miscible with solvents of fats. Partial solidification may occur, depending on the temperature.

IDENTIFICATION First identification : A, C. Second identification : A, B. A. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R. Test solution. Dissolve an amount of the substance to be examined corresponding to 400 000 IU in ethylene C. 9β,10α-cholesta-5,7-dien-3β-ol (lumisterol3), chloride R containing 10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R and dilute to 4 ml with the same solvent. Prepare immediately before use. Reference solution (a). Dissolve 10 mg of cholecalciferol CRS in ethylene chloride R containing 10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R and dilute to 4 ml with the same solvent. Prepare immediately before use. Reference solution (b). Dissolve 10 mg of ergocalciferol CRS in ethylene chloride R containing 10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R and dilute to 4 ml with the same solvent. Prepare immediately before use. Apply to the plate 20 µl of each solution. Develop immediately, protected from light, over a path of 15 cm D. (6E)-9,10-secocholesta-5(10),6,8(14)-trien-3β-ol using a mixture of equal volumes of cyclohexane R and (iso-tachysterol3), peroxide-free ether R, the mixture containing 0.1 g/l of butylhydroxytoluene R. Allow the plate to dry in air and spray with sulphuric acid R. Compare the principal spot in the chromatogram obtained with the test solution with the principal spot in each of the chromatograms obtained with reference solution (a) and reference solution (b), respectively. The chromatogram obtained with the test solution shows immediately a bright yellow principal spot which rapidly becomes orange-brown, then gradually greenish-grey and remains so for 10 min. This spot is similar in position, colour and size to the spot in the chromatogram obtained with reference solution (a). The chromatogram obtained with reference solution (b) shows immediately at the same level an orange principal spot which gradually becomes reddish-brown and remains so E. (6E)-9,10-secocholesta-5(10),6,8-trien-3β-ol (tachysterol3). for 10 min. B. Prepare a solution in cyclohexane R containing the equivalent of about 400 IU/ml. Examined between 01/2005:0575 250 nm and 300 nm (2.2.25), the solution shows an absorption maximum at 267 nm. CHOLECALCIFEROL CONCENTRATE C. Examine the chromatograms obtained in the assay. The principal peak in the chromatogram obtained with the test (OILY FORM) solution has a similar retention time to the principal peak in the chromatogram obtained with reference solution (a).

Cholecalciferolum densatum oleosum

DEFINITION Cholecalciferol concentrate (oily form) consists of a solution of Cholecalciferol (0072) in a suitable vegetable fatty oil, authorised by the competent authority. General Notices (1) apply to all monographs and other texts

TESTS Acid value (2.5.1). Not more than 2.0, determined on 5.0 g dissolved in 25 ml of the prescribed mixture of solvents. Peroxide value (2.5.5). Not more than 20. 1273

Cholecalciferol concentrate (oily form)


ASSAY Carry out the assay as rapidly as possible, avoiding exposure to actinic light and air. Examine by liquid chromatography (2.2.29). Test solution. Dissolve a quantity of the preparation to be examined, weighed with an accuracy of 0.1 per cent, equivalent to about 400 000 IU, in 10.0 ml of toluene R and dilute to 100.0 ml with the mobile phase. Reference solution (a). Dissolve 10.0 mg of cholecalciferol CRS without heating in 10.0 ml of toluene R and dilute to 100.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of cholecalciferol for performance test CRS to 5.0 ml with the mobile phase. Heat in a water-bath at 90 °C under a reflux condenser for 45 min and cool. Reference solution (c). Dissolve 0.10 g of cholecalciferol CRS without heating in toluene R and dilute to 100.0 ml with the same solvent. Reference solution (d). Dilute 5.0 ml of reference solution (c) to 50.0 ml with the mobile phase. Keep the solution in iced water. Reference solution (e). Place 5.0 ml of reference solution (c) in a volumetric flask, add about 10 mg of butylhydroxytoluene R and displace air from the flask with nitrogen R. Heat in a water-bath at 90 °C under a reflux condenser protected from light and under nitrogen R for 45 min. Cool and dilute to 50.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with a suitable silica gel (5 µm), — as mobile phase at a flow rate of 2 ml/min a mixture of 3 volumes of pentanol R and 997 volumes of hexane R, — as detector a spectrophotometer set at 254 nm. An automatic injection device or a sample loop is recommended. Inject a suitable volume of reference solution (b). Adjust the sensitivity of the system so that the height of the principal peak is at least 50 per cent of the full scale of the recorder. Inject reference solution (b) 6 times. When the chromatograms are recorded in the prescribed conditions, the approximate relative retention times with reference to cholecalciferol are 0.4 for pre-cholecalciferol and 0.5 for trans-cholecalciferol. The relative standard deviation of the response for cholecalciferol is not greater than 1 per cent and the resolution between the peaks due to pre-cholecalciferol and trans-cholecalciferol is not less than 1.0. If necessary adjust the proportions of the constituents and the flow rate of the mobile phase to obtain this resolution. Inject a suitable volume of reference solution (d) and of reference solution (e). Calculate the conversion factor (f) from the expression :

K

=

area (or height) of the peak due to cholecalciferol in the chromatogram obtained with reference solution (d),

L

=

area (or height) of the peak due to cholecalciferol in the chromatogram obtained with reference solution (e),

M

=

area (or height) of the peak due to pre-cholecalciferol in the chromatogram obtained with reference solution (e).

1274

The value of f determined in duplicate on different days may be used during the entire procedure. Inject a suitable volume of reference solution (a). Adjust the sensitivity of the system so that the height of the principal peak is at least 50 per cent of the full scale of the recorder. Inject the same volume of the test solution and record the chromatogram in the same manner. Calculate the content of cholecalciferol in International Units per gram from the expression :

m

=

mass of the preparation to be examined in the test solution, in milligrams,

m′

=


V

=

volume of the test solution (100 ml),

V′

=

volume of reference solution (a) (100 ml),

SD

=


S′D

=

area (or height) of the peak due to cholecalciferol in the chromatogram obtained with reference solution (a),

Sp

=

f

=

area (or height) of the peak due to pre-cholecalciferol in the chromatogram obtained with the test solution, conversion factor.

STORAGE Store in an airtight, well-filled container, protected from light. The contents of an opened container are to be used as soon as possible ; any unused part is to be protected by an atmosphere of nitrogen. LABELLING The label states : — the number of International Units per gram, — the method of restoring the solution if partial solidification occurs, — the name of any added stabilisers. IMPURITIES

A. (5E,7E)-9,10-secocholesta-5,7,10(19)-trien-3β-ol (trans-cholecalciferol, trans-vitamin D3), See the information section on general monographs (cover pages)

Cholecalciferol concentrate (powder form)


The declared content of cholecalciferol is not less than 100 000 IU/g and the concentrate contains not less than 90.0 per cent and not more than 110.0 per cent of the content stated on the label. The concentrate may contain suitable stabilisers such as antioxidants. CHARACTERS B. cholesta-5,7-dien-3β-ol (7,8-di dehydrocholesterol, provitamin D3),

White or yellowish-white, small particles which, depending on their formulation, may be practically insoluble in water or may swell or form a dispersion. IDENTIFICATION First identification : A, C. Second identification : A, B. A. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R.

C. (9β,10α)-cholesta-5,7-dien-3β-ol (lumisterol3),

Test solution. Place 10.0 ml of the test solution prepared for the assay in a suitable flask and evaporate to dryness under reduced pressure by swirling in a water-bath at 40 °C. Cool under running water and restore atmospheric pressure with nitrogen R. Dissolve the residue immediately in 0.4 ml of ethylene chloride R containing 10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R. Prepare immediately before use. Reference solution (a). Dissolve 10 mg of cholecalciferol CRS in ethylene chloride R containing 10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R and dilute to 4 ml with the same solvent. Prepare immediately before use.

D. (6E)-9,10-secocholesta-5(10),6,8(14)-trien-3β-ol (iso-tachysterol3),

E. (6E)-9,10-secocholesta-5(10),6,8-trien-3β-ol (tachysterol3). 01/2005:0574

CHOLECALCIFEROL CONCENTRATE (POWDER FORM) Cholecalciferoli pulvis DEFINITION Cholecalciferol concentrate (powder form) is obtained by dispersing an oily solution of Cholecalciferol (0072) in an appropriate matrix which is usually based on a combination of gelatin and carbohydrates of suitable quality, authorised by the competent authority. General Notices (1) apply to all monographs and other texts

Reference solution (b). Dissolve 10 mg of ergocalciferol CRS in ethylene chloride R containing 10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R and dilute to 4 ml with the same solvent. Prepare immediately before use. Apply to the plate 20 µl of each solution. Develop immediately, protected from light, over a path of 15 cm using a mixture of equal volumes of cyclohexane R and peroxide-free ether R, the mixture containing 0.1 g/l of butylhydroxytoluene R. Allow the plate to dry in air and spray with sulphuric acid R. Compare the principal spot in the chromatogram obtained with the test solution with the principal spot in each of the chromatograms obtained with reference solution (a) and reference solution (b), respectively. The chromatogram obtained with the test solution shows immediately a bright-yellow principal spot which rapidly becomes orange-brown, then gradually greenish-grey and remains so for 10 min. This spot is similar in position, colour and size to the spot in the chromatogram obtained with reference solution (a). The chromatogram obtained with reference solution (b) shows immediately at the same level an orange principal spot which gradually becomes reddish-brown and remains so for 10 min. B. Place 5.0 ml of the test solution prepared for the assay in a suitable flask and evaporate to dryness under reduced pressure by swirling in a water-bath at 40 °C. Cool under running water and restore atmospheric pressure with nitrogen R. Dissolve the residue immediately in 50.0 ml of cyclohexane R. Examined between 250 nm and 300 nm (2.2.25), the solution shows an absorption maximum at 265 nm. C. Examine the chromatograms obtained in the assay. The principal peak in the chromatogram obtained with the test solution has a similar retention time to the principal peak in the chromatogram obtained with reference solution (a). 1275

Cholecalciferol concentrate (powder form)


ASSAY Carry out the assay as rapidly as possible, avoiding exposure to actinic light and air. Examine by liquid chromatography (2.2.29). Test solution. Introduce into a saponification flask a quantity of the preparation to be examined, weighed with an accuracy of 0.1 per cent, equivalent to about 100 000 IU. Add 5 ml of water R, 20 ml of ethanol R, 1 ml of sodium ascorbate solution R and 3 ml of a freshly prepared 50 per cent m/m solution of potassium hydroxide R. Heat under a reflux condenser on a water-bath for 30 min. Cool rapidly under running water. Transfer the liquid to a separating funnel with the aid of two quantities, each of 15 ml, of water R, one quantity of 10 ml of alcohol R and two quantities, each of 50 ml, of pentane R. Shake vigorously for 30 s. Allow to stand until the two layers are clear. Transfer the lower aqueous-alcoholic layer to a second separating funnel and shake with a mixture of 10 ml of alcohol R and 50 ml of pentane R. After separation, transfer the aqueous-alcoholic layer to a third separating funnel and the pentane layer to the first separating funnel, washing the second separating funnel with two quantities, each of 10 ml, of pentane R and adding the washings to the first separating funnel. Shake the aqueous-alcoholic layer with 50 ml of pentane R and add the pentane layer to the first funnel. Wash the pentane layer with two quantities, each of 50 ml, of a freshly prepared 30 g/l solution of potassium hydroxide R in alcohol (10 per cent V/V) R, shaking vigorously, then wash with successive quantities, each of 50 ml, of water R until the washings are neutral to phenolphthalein. Transfer the washed pentane extract to a ground-glass-stoppered flask. Evaporate the contents of the flask to dryness under reduced pressure by swirling in a water-bath at 40 °C. Cool under running water and restore atmospheric pressure with nitrogen R. Dissolve the residue immediately in 5.0 ml of toluene R and add 20.0 ml of the mobile phase to obtain a solution containing about 4000 IU/ml. Reference solution (a). Dissolve 10.0 mg of cholecalciferol CRS without heating in 10.0 ml of toluene R and dilute to 100.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 g of cholecalciferol for performance test CRS to 5.0 ml with the mobile phase. Heat in a water-bath at 90 °C under a reflux condenser for 45 min and cool. Reference solution (c). Dissolve 0.10 g of cholecalciferol CRS without heating in toluene R and dilute to 100.0 ml with the same solvent. Reference solution (d). Dilute 5.0 ml of reference solution (c) to 50.0 ml with the mobile phase. Keep the solution in iced water. Reference solution (e). Place 5.0 ml of reference solution (c) in a volumetric flask, add about 10 mg of butylhydroxytoluene R and displace air from the flask with nitrogen R. Heat in a water-bath at 90 °C under a reflux condenser protected from light and under nitrogen R for 45 min. Cool and dilute to 50.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with a suitable silica gel (5 µm), — as mobile phase at a flow rate of 2 ml/min, a mixture of 3 volumes of pentanol R and 997 volumes of hexane R, — as detector a spectrophotometer set at 254 nm. An automatic injection device or a sample loop is recommended. Inject a suitable volume of reference solution (b). Adjust the sensitivity of the system so that the 1276

height of the principal peak is at least 50 per cent of the full scale of the recorder. Inject reference solution (b) 6 times. When the chromatograms are recorded in the prescribed conditions, the approximate relative retention times with reference to cholecalciferol are 0.4 for pre-cholecalciferol and 0.5 for trans-cholecalciferol. The relative standard deviation of the response for cholecalciferol is not greater than 1 per cent and the resolution between the peaks due to pre-cholecalciferol and trans-cholecalciferol is not less than 1.0. If necessary adjust the proportions of the constituents and the flow rate of the mobile phase to obtain this resolution. Inject a suitable volume of reference solution (d) and of reference solution (e). Calculate the conversion factor (f) from the expression :

K

=

area (or height) of the peak due to cholecalciferol in the chromatogram obtained with reference solution (d),

L

=

area (or height) of the peak due to cholecalciferol in the chromatogram obtained with reference solution (e),

M

=

area (or height) of the peak due to pre-cholecalciferol in the chromatogram obtained with reference solution (e).

The value of f determined in duplicate on different days may be used during the entire procedure. Inject a suitable volume of reference solution (a). Adjust the sensitivity of the system so that the height of the principal peak is at least 50 per cent of the full scale of the recorder. Inject the same volume of the test solution and record the chromatogram in the same manner. Calculate the content of cholecalciferol in International Units per gram from the expression :

m

= mass of the substance to be examined in the test solution, in milligrams,

m′

= mass of cholecalciferol CRS in reference solution (a), in milligrams,

V

= volume of the test solution (25 ml), = volume of reference solution (a) (100 ml),

V′ SD

= area (or height) of the peak due to cholecalciferol in the chromatogram obtained with the test solution,

S′D

= area (or height) of the peak due to cholecalciferol in the chromatogram obtained with reference solution (a),

Sp

= area (or height) of the peak due to pre-cholecalciferol in the chromatogram obtained with the test solution,

f

= conversion factor.

STORAGE Store in an airtight, well-filled container, protected from light. The contents of an opened container are to be used as soon as possible ; any unused part is to be protected by an atmosphere of nitrogen.



Cholecalciferol concentrate (water-dispersible form)

LABELLING The label states : — the number of International Units per gram, — the name of any added stabilisers. IMPURITIES


CHOLECALCIFEROL CONCENTRATE (WATER-DISPERSIBLE FORM) Cholecalciferolum in aqua dispergibile A. (5E,7E)-9,10-secocholesta-5,7,10(19)-trien-3β-ol (trans-cholecalciferol, trans-vitamin D3),

B. cholesta-5,7-dien-3β-ol (7,8-di dehydrocholesterol, provitamin D3),


D. (6E)-9,10-secocholesta-5(10),6,8(14)-trien-3β-ol (iso-tachysterol3), General Notices (1) apply to all monographs and other texts

DEFINITION Cholecalciferol concentrate (water-dispersible form) consists of a solution of Cholecalciferol (0072) in a suitable vegetable fatty oil, authorised by the competent authority, to which suitable solubilisers have been added. The declared content of cholecalciferol is not less than 100 000 IU/g and the concentrate contains not less than 90.0 per cent and not more than 115.0 per cent of the content stated on the label. The concentrate may contain suitable stabilisers such as antioxidants. CHARACTERS A slightly yellowish liquid of variable opalescence and viscosity. Highly concentrated solutions may become cloudy at low temperatures or form a gel at room temperature. IDENTIFICATION First identification : A, C, D. Second identification : A, B, D. A. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R. Test solution. Place 10.0 ml of the test solution prepared for the assay in a suitable flask and evaporate to dryness under reduced pressure by swirling in a water-bath at 40 °C. Cool under running water and restore atmospheric pressure with nitrogen R. Dissolve the residue immediately in 0.4 ml of ethylene chloride R containing 10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R. Prepare immediately before use. Reference solution (a). Dissolve 10 mg of cholecalciferol CRS in ethylene chloride R containing 10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R and dilute to 4 ml with the same solvent. Prepare immediately before use. Reference solution (b). Dissolve 10 mg of ergocalciferol CRS in ethylene chloride R containing 10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R and dilute to 4 ml with the same solvent. Prepare immediately before use. Apply to the plate 20 µl of each solution. Develop immediately, protected from light, over a path of 15 cm using a mixture of equal volumes of cyclohexane R and peroxide-free ether R, the mixture containing 0.1 g/l of butylhydroxytoluene R. Allow the plate to dry in air and spray with sulphuric acid R. Compare the principal spot 1277

Cholecalciferol concentrate (water-dispersible form)


in the chromatogram obtained with the test solution with the principal spot in each of the chromatograms obtained with reference solution (a) and reference solution (b). The chromatogram obtained with the test solution shows immediately a bright-yellow principal spot which rapidly becomes orange-brown, then gradually greenish-grey and remains so for 10 min. This spot is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). The chromatogram obtained with reference solution (b) shows immediately at the same level an orange principal spot which gradually becomes reddish-brown and remains so for 10 min. B. Place 5.0 ml of the test solution prepared for the assay in a suitable flask and evaporate to dryness under reduced pressure by swirling in a water-bath at 40 °C. Cool under running water and restore atmospheric pressure with nitrogen R. Dissolve the residue immediately in 50.0 ml of cyclohexane R. Examined between 250 nm and 300 nm (2.2.25), the solution shows an absorption maximum at 265 nm.

Reference solution (a). Dissolve 10.0 mg of cholecalciferol CRS without heating in 10.0 ml of toluene R and dilute to 100.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 g of cholecalciferol for performance test CRS to 5.0 ml with the mobile phase. Heat in a water-bath at 90 °C under a reflux condenser for 45 min and cool. Reference solution (c). Dissolve 0.10 g of cholecalciferol CRS without heating in toluene R and dilute to 100.0 ml with the same solvent. Reference solution (d). Dilute 5.0 ml of reference solution (c) to 50.0 ml with the mobile phase. Keep the solution in iced water. Reference solution (e). Place 5.0 ml of reference solution (c) in a volumetric flask, add about 10 mg of butylhydroxytoluene R and displace air from the flask with nitrogen R. Heat in a water-bath at 90 °C under a reflux condenser protected from light and under nitrogen R for 45 min. Cool and dilute to 50.0 ml with the mobile phase.

C. Examine the chromatograms obtained in the assay. The The chromatographic procedure may be carried out using : principal peak in the chromatogram obtained with the test — a stainless steel column 0.25 m long and 4.6 mm in solution has a similar retention time to the principal peak internal diameter packed with a suitable silica gel (5 µm), in the chromatogram obtained with reference solution (a). — as mobile phase at a flow rate of 2 ml/min a mixture of D. Mix about 1 g with 10 ml of water R previously warmed 3 volumes of pentanol R and 997 volumes of hexane R, to 50 °C and cool to 20 °C. Immediately after cooling, a — as detector a spectrophotometer set at 254 nm. uniform, slightly opalescent and slightly yellow dispersion is obtained. An automatic injection device or a sample loop is recommended. Inject a suitable volume of reference solution (b). Adjust the sensitivity of the system so that the ASSAY height of the principal peak is at least 50 per cent of the full Carry out the assay as rapidly as possible, avoiding scale of the recorder. Inject reference solution (b) 6 times. exposure to actinic light and air. When the chromatograms are recorded in the prescribed conditions, the approximate relative retention times with Examine by liquid chromatography (2.2.29). reference to cholecalciferol are 0.4 for pre-cholecalciferol Test solution. Introduce into a saponification flask a quantity and 0.5 for trans-cholecalciferol. The relative standard of the preparation to be examined, weighed with an accuracy deviation of the response for cholecalciferol is not greater of 0.1 per cent, equivalent to about 100 000 IU. Add 5 ml than 1 per cent and the resolution between the peaks of water R, 20 ml of ethanol R, 1 ml of sodium ascorbate due to pre-cholecalciferol and trans-cholecalciferol is not solution R and 3 ml of a freshly prepared 50 per cent m/m less than 1.0. If necessary adjust the proportions of the solution of potassium hydroxide R. Heat under a reflux constituents and the flow rate of the mobile phase to obtain condenser on a water-bath for 30 min. Cool rapidly under this resolution. running water. Transfer the liquid to a separating funnel Inject a suitable volume of reference solution (d) and of with the aid of two quantities, each of 15 ml, of water R, reference solution (e). one quantity of 10 ml of alcohol R and two quantities, each of 50 ml, of pentane R. Shake vigorously for 30 s. Calculate the conversion factor (f) from the expression : Allow to stand until the two layers are clear. Transfer the aqueous-alcoholic layer to a second separating funnel and shake with a mixture of 10 ml of alcohol R and 50 ml of pentane R. After separation, transfer the aqueous-alcoholic layer to a third separating funnel and the pentane layer to K = area (or height) of the peak due to cholecalciferol the first separating funnel, washing the second separating in the chromatogram obtained with reference funnel with two quantities, each of 10 ml, of pentane R solution (d), and adding the washings to the first separating funnel. L = area (or height) of the peak due to cholecalciferol Shake the aqueous-alcoholic layer with 50 ml of pentane R in the chromatogram obtained with reference and add the pentane layer to the first funnel. Wash the solution (e), pentane layer with two quantities, each of 50 ml, of a freshly M = area (or height) of the peak due to prepared 30 g/l solution of potassium hydroxide R in pre-cholecalciferol in the chromatogram alcohol (10 per cent V/V) R, shaking vigorously, and then obtained with reference solution (e). wash with successive quantities, each of 50 ml, of water R until the washings are neutral to phenolphthalein. Transfer The value of f determined in duplicate on different days may the washed pentane extract to a ground-glass-stoppered flask. Evaporate the contents of the flask to dryness under be used during the entire procedure. reduced pressure by swirling in a water-bath at 40 °C. Cool Inject a suitable volume of reference solution (a). Adjust the under running water and restore atmospheric pressure with sensitivity of the system so that the height of the principal nitrogen R. Dissolve the residue immediately in 5.0 ml of peak is at least 50 per cent of the full scale of the recorder. toluene R and add 20.0 ml of the mobile phase to obtain a Inject the same volume of the test solution and record the solution containing about 4000 IU/ml. chromatogram in the same manner. 1278


Cholesterol


Calculate the content of cholecalciferol in International Units per gram from the expression :

m

=

mass of the preparation to be examined in the test solution, in milligrams,

m′

=


V

=

volume of the test solution (25 ml),

V′

=

volume of reference solution (a) (100 ml),

SD

=


S′D

=

area (or height) of the peak due to cholecalciferol in the chromatogram obtained with reference solution (a),

Sp

=

f

=

area (or height) of the peak due to pre-cholecalciferol in the chromatogram obtained with the test solution, conversion factor.

STORAGE Store in an airtight, well-filled container, protected from light, at the temperature stated on the label. Once the container has been opened, its contents are to be used as soon as possible ; any part of the contents not used at once is to be protected by an atmosphere of inert gas. LABELLING The label states : — the number of International Units per gram, — the name of the principal solubiliser or solubilisers used and the name of any added stabilisers, — the storage temperature. IMPURITIES


D. (6E)-9,10-secocholesta-5(10),6,8(14)-trien-3β-ol (iso-tachysterol3),


CHOLESTEROL Cholesterolum

A. (5E,7E)-9,10-secocholesta-5,7,10(19)-trien-3β-ol (trans-cholecalciferol, trans-vitamin D3),

C27H46O

Mr 386.7

DEFINITION Cholesterol contains not less than 95.0 per cent of cholest-5-en-3β-ol and not less than 97.0 per cent and not more than 103.0 per cent of total sterols, calculated with reference to the dried substance.

B. cholesta-5,7-dien-3β-ol (7,8-di dehydrocholesterol, provitamin D3), General Notices (1) apply to all monographs and other texts

CHARACTERS A white or almost white, crystalline powder, practically insoluble in water, sparingly soluble in acetone and in alcohol. It is sensitive to light. 1279

Chymotrypsin


IDENTIFICATION A. Melting point (2.2.14) : 147 °C to 150 °C. B. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R. Prepare the solutions immediately before use. Test solution. Dissolve 10 mg of the substance to be examined in ethylene chloride R and dilute to 5 ml with the same solvent. Reference solution. Dissolve 10 mg of cholesterol CRS in ethylene chloride R and dilute to 5 ml with the same solvent. Apply to the plate 20 µl of each solution. Develop immediately, protected from light, over a path of 15 cm using a mixture of 33 volumes of ethyl acetate R and 66 volumes of toluene R. Allow the plate to dry in air and spray 3 times with antimony trichloride solution R. Examine the chromatograms 3-4 min after spraying. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. C. Dissolve about 5 mg in 2 ml of methylene chloride R. Add 1 ml of acetic anhydride R, 0.01 ml of sulphuric acid R and shake. A pink colour is produced which rapidly changes to red, then to blue and finally to brilliant green.

maintaining the temperature of the column at 275 °C, that of the injection port at 285 °C and that of the detector at 300 °C. Inject 1.0 µl of each solution. The assay is not valid unless the resolution between the peaks due to pregnenolone isobutyrate and cholest-5-en-3β-ol in the chromatogram obtained with the reference solution is at least 10.0. Calculate the percentage content of cholest-5-en-3β-ol, using the declared content of cholest-5-en-3β-ol in cholesterol CRS. Calculate the percentage content of total sterols by adding together the contents of cholest-5-en-3β-ol and other substances with a retention time less than or equal to 1.5 times the retention time of cholest-5-en-3β-ol. Disregard the peaks due to the internal standard and the solvent. STORAGE Store protected from light. LABELLING The label states the source material for the production of cholesterol (for example bovine brain and spinal cord, wool fat or chicken eggs). IMPURITIES

TESTS Solubility in alcohol. In a stoppered flask, dissolve 0.5 g in 50 ml of alcohol R at 50 °C. Allow to stand for 2 h. No deposit or turbidity is formed. Acidity. Dissolve 1.0 g in 10 ml of ether R, add 10.0 ml of 0.1 M sodium hydroxide and shake for about 1 min. Heat gently to eliminate ether and then boil for 5 min. Cool, add A. 5α-cholest-7-en-3β-ol (lathosterol), 10 ml of water R and 0.1 ml of phenolphthalein solution R as indicator and titrate with 0.1 M hydrochloric acid until the pink colour just disappears, stirring the solution vigorously throughout the titration. Carry out a blank titration. The difference between the volumes of 0.1 M hydrochloric acid required to change the colour of the indicator in the blank and in the test is not more than 0.3 ml. Loss on drying (2.2.32). Not more than 0.3 per cent, determined on 1.000 g by drying in vacuo at 60 °C for 4 h. B. cholesta-5,24-dien-3β-ol (desmosterol), Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Examine by gas chromatography (2.2.28), using pregnenolone isobutyrate CRS as the internal standard. Internal standard solution. Dissolve 0.100 g of pregnenolone isobutyrate CRS in heptane R and dilute to 100.0 ml with the same solvent. Test solution. Dissolve 25.0 mg of the substance to be examined in the internal standard solution and dilute to 25.0 ml with the same solution. Reference solution. Dissolve 25.0 mg of cholesterol CRS in the internal standard solution and dilute to 25.0 ml with the same solution. The chromatographic procedure may be carried out using : — a fused-silica column 30 m long and 0.25 mm in internal diameter coated with poly(dimethyl)siloxane R (film thickness 0.25 µm), — helium for chromatography R as the carrier gas with a split ratio of 1:25 at a flow rate of 2 ml/min, — a flame-ionisation detector, 1280

C. 5α-cholesta-7,24-dien-3β-ol. 01/2005:0476

CHYMOTRYPSIN Chymotrypsinum DEFINITION Chymotrypsin is a proteolytic enzyme obtained by the activation of chrymotrypsinogen extracted from the pancreas of beef (Bos taurus L.). It has an activity not less than


Chymotrypsin


5.0 microkatals per milligram. In solution it has maximal enzymic activity at about pH 8 ; the activity is reversibly inhibited at pH 3, at which pH it is most stable. PRODUCTION The animals from which chymotrypsin is derived must fulfil the requirements for the health of animals suitable for human consumption to the satisfaction of the competent authority ; furthermore the tissues used shall not include any specified risk material as defined by any relevant international or, where appropriate, national legislation. It must have been shown to what extent the method of production allows inactivation or removal of any contamination by viruses or other infectious agents. The method of manufacture is validated to demonstrate that the product, if tested, would comply with the following test : Histamine (2.6.10). Not more than 1 µg (calculated as histamine base) per 5 microkatals of chymotrypsin activity. Before carrying out the test, heat the solution of the substance to be examined on a water-bath for 30 min. CHARACTERS A white, crystalline or amorphous powder, sparingly soluble in water ; the amorphous form is hygroscopic. IDENTIFICATION A. Dilute 1 ml of solution S (see Tests) to 10 ml with water R. In a depression in a white spot plate, mix 0.05 ml of this solution with 0.2 ml of substrate solution. A purple colour develops. Substrate solution. To 24.0 mg of acetyltyrosine ethyl ester R add 0.2 ml of alcohol R, and swirl until solution is effected. Add 2.0 ml of 0.067 M phosphate buffer solution pH 7.0 R and 1 ml of methyl red mixed solution R and dilute to 10.0 ml with water R. B. Dilute 0.5 ml of solution S to 5 ml with water R. Add 0.10 ml of a 20 g/l solution of tosylphenylalanylchloromethane R in alcohol R. Adjust to pH 7.0 and shake for 2 h. In a depression in a white spot plate, mix 0.05 ml of this solution with 0.2 ml of the substrate solution (see Identification test A). No colour develops within 3 min of mixing. TESTS Solution S. Dissolve 0.10 g in carbon dioxide-free water R and dilute to 10.0 ml with the same solvent. Appearance of solution. Solution S is not more opalescent than reference suspension II (2.2.1). pH (2.2.3). The pH of solution S is 3.0 to 5.0. Absorbance (2.2.25). Dissolve 30.0 mg in 0.001 M hydrochloric acid and dilute to 100.0 ml with the same acid. The solution shows an absorption maximum at 281 nm and a minimum at 250 nm. The specific absorbance at the maximum is 18.5 to 22.5 and that at the minimum is not greater than 8. Trypsin. Transfer to a depression in a white spot plate 0.05 ml of tris(hydroxymethyl)aminomethane buffer solution pH 8.1 R and 0.1 ml of solution S. Add 0.2 ml of substrate solution (test solution). At the same time and in the same manner, prepare a reference solution using the substance to be examined to which not more than 1 per cent m/m of trypsin BRP has been added. Start a timer. No colour appears in the test solution within 3 min to 5 min after the addition of the substrate solution. A purple colour is produced in the control solution. Substrate solution. To 98.5 mg of tosylarginine methyl ester hydrochloride R, suitable for assaying trypsin, add General Notices (1) apply to all monographs and other texts

5 ml of tris(hydroxymethyl)aminomethane buffer solution pH 8.1 R and swirl to dissolve. Add 2.5 ml of methyl red mixed solution R and dilute to 25.0 ml with water R. Loss on drying (2.2.32). Not more than 5.0 per cent, determined on 0.100 g by drying at 60 °C at a pressure not exceeding 0.7 kPa for 2 h. ASSAY The activity of chymotrypsin is determined by comparing the rate at which it hydrolyses acetyltyrosine ethyl ester R with the rate at which chymotrypsin BRP hydrolyses the same substrate under the same conditions. Apparatus. Use a reaction vessel of about 30 ml capacity provided with : — a device that will maintain a temperature of 25.0 ± 0.1 °C, — a stirring device, for example a magnetic stirrer, — a lid with holes for the insertion of electrodes, the tip of a burette, a tube for the admission of nitrogen and the introduction of reagents. An automatic or manual titration apparatus may be used. For the latter the burette is graduated in 0.005 ml and the pH meter is provided with a wide scale and glass-calomel electrodes. Test solution. Dissolve 25.0 mg of the substance to be examined in 0.001 M hydrochloric acid and dilute to 250.0 ml with the same acid. Reference solution. Dissolve 25.0 mg of chymotrypsin BRP in 0.001 M hydrochloric acid and dilute to 250.0 ml with the same acid. Store the solutions at 0 °C to 5 °C. Warm 1 ml of each solution to about 25 °C over 15 min and use 50 µl of each solution (corresponding to about 25 nanokatals) for each titration. Carry out the titration in an atmosphere of nitrogen. Transfer 10.0 ml of 0.01 M calcium chloride solution R to the reaction vessel and, while stirring, add 0.35 ml of 0.2 M acetyltyrosine ethyl ester solution R. When the temperature is steady at 25.0 ± 0.1 °C (after about 5 min) adjust the pH to exactly 8.0 with 0.02 M sodium hydroxide. Add 50 µl of the test solution (equivalent to about 5 µg of the substance to be examined) and start a timer. Maintain the pH at 8.0 by the addition of 0.02 M sodium hydroxide, noting the volume added every 30 s. Calculate the volume of 0.02 M sodium hydroxide used per second between 30 s and 210 s. Carry out a titration in the same manner using the reference solution and calculate the volume of 0.02 M sodium hydroxide used per second. Calculate the activity in microkatals per milligram using the expression :

m

=

mass of the substance to be examined, in milligrams,

m′

=

mass of chymotrypsin BRP, in milligrams,

V

=

volume of 0.02 M sodium hydroxide used per second by the test solution,

V′

=

volume of 0.02 M sodium hydroxide used per second by the reference solution,

A

=

activity of chymotrypsin BRP, in microkatals per milligram.

STORAGE Store in an airtight container at 2 °C to 8 °C, protected from light. 1281

Ciclopirox


LABELLING The label states : — the quantity of chymotrypsin and the total activity in microkatals per container, — for the amorphous substance, that it is hygroscopic. 01/2005:1407

CICLOPIROX Ciclopiroxum

C12H17NO2

Mr 207.3

DEFINITION Ciclopirox contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of 6-cyclohexyl-1-hydroxy-4-methylpyridin-2(1H)-one, calculated with reference to the dried substance. CHARACTERS A white or yellowish-white, crystalline powder, slightly soluble in water, freely soluble in ethanol and in methylene chloride. IDENTIFICATION First identification : B. Second identification : A, C. A. Melting point (2.2.14) : 140 °C to 145 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with ciclopirox CRS. C. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R. Test solution. Dissolve 20 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution. Dissolve 20 mg of ciclopirox CRS in methanol R and dilute to 10 ml with the same solvent. Before use predevelop the plate with a mixture of 10 volumes of concentrated ammonia R, 15 volumes of water R and 75 volumes of alcohol R until the solvent front has migrated to the top of the plate. Allow the plate to dry in air for 5 min. Apply to the plate 10 µl of each solution. Develop over a path of 15 cm using a mixture of 10 volumes of concentrated ammonia R, 15 volumes of water R and 75 volumes of alcohol R. Allow the plate to dry in air for 10 min and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. Spray the plate with a 20 g/l solution of ferric chloride R in ethanol R. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. 1282

TESTS Appearance of solution. Dissolve 2.0 g in methanol R and dilute to 10 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y5 (2.2.2, Method II). Related substances. Examine by liquid chromatography (2.2.29). Carry out the operations avoiding exposure to actinic light. All materials which are in direct contact with the substance to be examined like column materials, reagents, solvents and others should contain only very low amounts of extractable metal cations. Solvent mixture. Mix 1 volume of acetonitrile R and 9 volumes of the mobile phase. Test solution. Dissolve 30.0 mg of the substance to be examined in 15 ml of the solvent mixture. If necessary, use an ultrasonic bath. Dilute to 20.0 ml with the solvent mixture. Reference solution (a). Dissolve 15.0 mg of ciclopirox impurity A CRS and 15.0 mg of ciclopirox impurity B CRS in the solvent mixture and dilute to 10.0 ml with the same solvent mixture. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 200.0 ml with the solvent mixture. Reference solution (c). Dilute 2.0 ml of reference solution (b) to 10.0 ml with the solvent mixture. Reference solution (d). Mix 5 ml of reference solution (a) with 5 ml of the test solution. The chromatographic procedure may be carried out using : — a stainless steel column 80 mm long and 4 mm in internal diameter packed with nitrile silica gel for chromatography R2 (5 µm), — as rinsing solution a mixture of 1 volume of glacial acetic acid R, 1 volume of acetylacetone R, 500 volumes of acetonitrile R and 500 volumes of water R, — as mobile phase at a flow rate of 0.7 ml/min a mixture of 0.1 ml of glacial acetic acid R, 230 ml of acetonitrile R and 770 ml of a 0.96 g/l solution of sodium edetate R. If the retention time of the principal peak in the chromatogram obtained with the test solution is not between 8 min and 11 min adjust the ratio of the 0.96 g/l solution of sodium edetate : acetonitrile accordingly, — as detector a spectrophotometer set at 220 nm and 298 nm. In order to ensure desorption of disruptive metal ions, every new column is to be rinsed with the rinsing solution over a period of not less than 15 h and then with the mobile phase for not less than 5 h at a flow rate of 0.2 ml/min. Inject 10 µl each of the solvent mixture, the test solution and the reference solutions (b), (c) and (d) and record the chromatograms at 220 nm and 298 nm. Continue the chromatography of the test solution for 2.5 times the retention time of the principal peak. The relative retention times are : — impurity A : 0.5 — impurity C : 0.9 — ciclopirox : 1 — impurity B : 1.3 The test is not valid unless : in the chromatogram obtained with reference solution (d), the resolution between the peaks corresponding to impurity B and ciclopirox is not less than 2.0 ; the chromatogram obtained with reference solution (c) shows at 298 nm a peak corresponding to impurity B with a signal-to-noise ratio of not less than three ; in the chromatogram obtained with the test solution the symmetry factor of the principal peak is between 0.8 and 2.0.


Ciclopirox olamine


In the chromatogram obtained with the test solution at 220 nm, the area of the peak due to impurity A is not greater than the area of the corresponding peak in the chromatogram obtained with reference solution (b) at the same wavelength (0.5 per cent). In the chromatogram obtained with the test solution at 298 nm, the area of any peak apart from the principal peak is not greater than the area of the peak due to impurity B in the chromatogram obtained with reference solution (b) at the same wavelength (0.5 per cent) ; the sum of all peak areas at 298 nm, apart from the principal peak and any peak due to impurity B, is not greater than the area of the peak due to impurity B in the chromatogram obtained with reference solution (b) (0.5 per cent). At 298 nm disregard any peak due to the solvent and any peak with an area less than half the area of the peak due to impurity B in the chromatogram obtained with reference solution (c) at the same wavelength. Heavy metals (2.4.8). 2.0 g complies with limit test C for heavy metals (10 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 1.5 per cent, determined on 1.000 g by drying in vacuo at 60 °C over diphosphorus pentoxide R. Sulphated ash (2.4.14). Not more than 0.1 per cent determined on 1.0 g.

01/2005:1302

CICLOPIROX OLAMINE Ciclopirox olaminum

C14H24N2O3

Mr 268.4

DEFINITION 6-Cyclohexyl-1-hydroxy-4-methylpyridin-2(1H)-one and 2-aminoethanol. Content : — ciclopirox (C12H17NO2 ; Mr 207.3) : 76.0 per cent to 78.5 per cent (dried substance), — 2-aminoethanol (C2H7NO ; Mr 61.1) : 22.2 per cent to 23.3 per cent (dried substance).

CHARACTERS Appearance : white or pale yellow, crystalline powder. ASSAY Solubility : sparingly soluble in water, very soluble in alcohol and in methylene chloride, slightly soluble in ethyl acetate, Dissolve 0.150 g in 20 ml of methanol R. Add 20 ml practically insoluble in cyclohexane. of water R and titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20). Carry It shows polymorphism. out a blank titration. IDENTIFICATION 1 ml of 0.1 M sodium hydroxide is equivalent to 20.73 mg First identification : A. of C12H17NO2. Second identification : B. A. Infrared absorption spectrophotometry (2.2.24). STORAGE Comparison : ciclopirox olamine CRS. Store protected from light. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference IMPURITIES substance separately in the minimum volume of ethyl acetate R, evaporate to dryness on a water-bath and record new spectra using the residues. B. Thin-layer chromatography (2.2.27). Test solution. Dissolve 25 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution. Dissolve 25 mg of ciclopirox olamine CRS in methanol R and dilute to 10 ml with the same solvent. Plate : TLC silica gel F254 plate R. A. (RS)-2-(3-cyclohexyl-5-methyl-4,5-dihydroisoxazol-5Before use wash 2 plates by allowing a mixture of yl)acetic acid, 10 volumes of concentrated ammonia R, 15 volumes of water R and 75 volumes of ethanol R to migrate until the solvent front has reached the top of the plate. Allow the plates to dry in air for 5 min. Mobile phase : concentrated ammonia R, water R, ethanol R (10:15:75 V/V/V). Application : 10 µl. Development : over a path of 15 cm. Drying : in air for 10 min. Detection A : examine in ultraviolet light at 254 nm. B. X = O : 6-cyclohexyl-4-methyl-2H-pyran-2-one, Results A : the principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained C. X = NH : 6-cyclohexyl-4-methylpyridin-2(1H)-one. with the reference solution. General Notices (1) apply to all monographs and other texts

1283

Ciclopirox olamine


Detection B : spray one plate with ferric chloride solution R3. Results B : the principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. Detection C : spray the second plate with ninhydrin solution R. Heat at 110 °C until the spots appear. Results C : the principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. TESTS Appearance of solution. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY7 (2.2.2, Method II). Dissolve 2.0 g in methanol R and dilute to 20 ml with the same solvent. pH (2.2.3) : 8.0 to 9.0. Dissolve 1.0 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Related substances. Liquid chromatography (2.2.29). Carry out the operations avoiding exposure to actinic light. All materials which are in direct contact with the substance to be examined, such as column materials, reagents, solvents, etc. should contain only small amounts of extractable metal cations. Test solution. Dissolve 40.0 mg of the substance to be examined (corresponding to about 30 mg of ciclopirox) in a mixture of 20 µl of anhydrous acetic acid R, 2 ml of acetonitrile R, and 15 ml of the mobile phase. If necessary, use an ultrasonic bath. Dilute to 20.0 ml with the mobile phase. Reference solution (a). Dissolve 15.0 mg of ciclopirox impurity A CRS and 15.0 mg of ciclopirox impurity B CRS in a mixture of 1 ml of acetonitrile R and 7 ml of the mobile phase. Dilute to 10.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 200.0 ml with a mixture of 1 volume of acetonitrile R and 9 volumes of the mobile phase. Reference solution (c). Dilute 2.0 ml of reference solution (b) to 10.0 ml with a mixture of 1 volume of acetonitrile R and 9 volumes of the mobile phase.

Detection : variable wavelength spectrophotometer capable of operating at 220 nm and 298 nm. In order to ensure desorption of interfering metal ions, a new column is to be rinsed with the rinsing solution over a period of at least 15 h and then with the mobile phase for at least 5 h at a flow rate of 0.2 ml/min. Injection : 10 µl ; inject the test solution and reference solutions (b), (c) and (d). Run time : 2.5 times the retention time of ciclopirox. Relative retention with reference to ciclopirox : impurity A = about 0.5 ; impurity C = about 0.9 ; impurity B = about 1.3. System suitability : — resolution : minimum of 2.0 between the peaks corresponding to impurity B and ciclopirox in the chromatogram obtained with reference solution (d), — signal-to-noise ratio : minimum of 10 for the peak corresponding to impurity B in the chromatogram obtained with reference solution (c) at 298 nm, — symmetry factor : 0.8 to 2.0 for the principal peak in the chromatogram obtained with the test solution. Limits : — impurity A at 220 nm : not more than the area of the corresponding peak in the chromatogram obtained with reference solution (b) at the same wavelength (0.5 per cent), — any impurity at 298 nm : not more than the area of the peak due to impurity B in the chromatogram obtained with reference solution (b) at the same wavelength (0.5 per cent), — total at 298 nm apart from impurity B : not more than the area of the peak due to impurity B in the chromatogram obtained with reference solution (b) (0.5 per cent), — disregard limit at 298 nm : area of the peak due to impurity B in the chromatogram obtained with reference solution (c) at the same wavelength (0.1 per cent). Heavy metals (2.4.8) : maximum 20 ppm. 1.0 g complies with limit test C. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32) : maximum 1.5 per cent, determined on 1.000 g by drying under high vacuum. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g.

ASSAY 2-Aminoethanol. Dissolve 0.250 g in 25 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining Column : the end-point potentiometrically (2.2.20). — size : l = 80 mm, Ø = 4 mm, 1 ml of 0.1 M perchloric acid is equivalent to 6.108 mg of — stationary phase : nitrile silica gel for chromatography R C2H7NO. (5 µm), Ciclopirox. Dissolve 0.200 g in 2 ml of methanol R. — rinsing solution : a mixture of 1 volume of anhydrous Add 38 ml of water R, swirl and titrate immediately with acetic acid R, 1 volume of acetylacetone R, 500 volumes 0.1 M sodium hydroxide, determining the end-point of acetonitrile R and 500 volumes of water R. potentiometrically (2.2.20). Carry out a blank titration. Mobile phase : a mixture of 0.1 volumes of anhydrous acetic Use 0.1 M sodium hydroxide, the titre of which has been determined under the conditions prescribed above using acid R, 230 volumes of acetonitrile R and 770 volumes of 0.100 g of benzoic acid RV. a 0.96 g/l solution of sodium edetate R. If the retention time of the principal peak in the chromatogram obtained 1 ml of 0.1 M sodium hydroxide is equivalent to 20.73 mg with the test solution is not between 8 min and 11 min of C12H17NO2. adjust the ratio of the 0.96 g/l solution of sodium edetate to acetonitrile accordingly. STORAGE Flow rate : 0.7 ml/min. Protected from light. Reference solution (d). Mix 5 ml of reference solution (a) with 5 ml of the test solution.

1284


Ciclosporin


TESTS Appearance of solution. Dissolve 1.5 g in ethanol R and dilute to 15 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y5, BY5 or R 7 (2.2.2, Method II). Specific optical rotation (2.2.7). Dissolve 0.125 g in methanol R and dilute to 25.0 ml with the same solvent. The A. (RS)-2-(3-cyclohexyl-5-methyl-4,5-dihydroisoxazol-5specific optical rotation is − 185 to − 193, calculated with yl)acetic acid, reference to the dried substance. Related substances. Examine by liquid chromatography (2.2.29) as prescribed under Assay. Inject separately the test solution and reference solution (b). Continue the chromatography for 1.7 times the retention time of the principal peak. In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than 0.7 times the area of the principal peak in the chromatogram obtained with reference solution (b) B. 6-cyclohexyl-4-methyl-2H-pyran-2-one, (0.7 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (1.5 per cent). Disregard any peak due to the solvent and any peak with an area less than 0.05 times that of the principal peak in the chromatogram with reference solution (b). Heavy metals (2.4.8). The residue obtained in the test for loss on drying complies with limit test C for heavy metals C. 6-cyclohexyl-4-methylpyridin-2(1H)-one. (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. 01/2005:0994 corrected Loss on drying (2.2.32). Not more than 2.0 per cent, determined on 1.000 g at 60 °C at a pressure not exceeding 15 Pa for 3 h. CICLOSPORIN Bacterial endotoxins (2.6.14) : less than 0.84 IU/mg, if intended for use in the manufacture of parenteral dosage Ciclosporinum forms without a further appropriate procedure for the removal of bacterial endotoxins. Dissolve 50 mg of the substance to be examined in a mixture of 280 mg of alcohol R and 650 mg of polyoxyethylated castor oil R and dilute to the required concentration using water LAL. IMPURITIES

IDENTIFICATION A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with ciclosporin CRS. B. Examine the chromatograms obtained in the assay. The principal peak in the chromatogram obtained with the test solution is similar in retention time to the principal peak in the chromatogram obtained with reference solution (a).

ASSAY Examine by liquid chromatography (2.2.29). Test solution. Dissolve 30.0 mg of the substance to be examined in a mixture of equal volumes of acetonitrile R and water R and dilute to 25.0 ml with the same mixture of solvents. Reference solution (a). Dissolve 30.0 mg of ciclosporin CRS in a mixture of equal volumes of acetonitrile R and water R and dilute to 25.0 ml with the same mixture of solvents. Reference solution (b). Dilute 2.0 ml of reference solution (a) to 200.0 ml with a mixture of equal volumes of acetonitrile R and water R. Reference solution (c). Dissolve the contents of a vial of ciclosporin for system suitability CRS in 5.0 ml of the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (3 µm to 5 µm) ; the column is connected to the injection port by a steel capillary tube about 1 m long and having an internal diameter of 0.25 mm, — as mobile phase at a flow rate of about 1.5 ml per minute a mixture of 1 volume of phosphoric acid R, 50 volumes of 1,1-dimethylethyl methyl ether R, 430 volumes of acetonitrile R and 520 volumes of water R, — as detector a spectrophotometer set at 210 nm,


1285

C62H111N11O12

Mr 1203

DEFINITION Ciclosporin contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent of cyclo[[(2S,3R,4R,6E)-3-hydroxy-4-methyl-2-(methylamino)oct6-enoyl]-L-2-aminobutanoyl-N-methylglycyl-N-methyl-L-leucylL-valyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-N-methyl-L-leucyl-Nmethyl-L-leucyl-N-methyl-L-valyl], calculated with reference to the dried substance. Ciclosporin is a substance produced by Beauveria nivea (Tolypocladium inflatum Gams) or obtained by any other means. CHARACTERS A white or almost white powder, practically insoluble in water, freely soluble in ethanol and in methylene chloride.

Cilastatin sodium


— a 20 µl loop injector, maintaining the temperature of the column and of the steel capillary tube at 80 °C. Inject reference solution (c). The test is not valid unless the peak-to-valley ratio is minimum 1.4 where Hp = height above the baseline of the peak due to ciclosporin U and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to ciclosporin. Adjust the ratio of 1,1 dimethylethylmethyl ether to acetonitrile, if necessary. The test is not valid unless the retention time of the principal peak is 25 min to 30 min. Adjust the ratio of acetonitrile to water, if necessary. Inject reference solution (a) 6 times. The test is not valid unless the relative standard deviation of the area of the principal peak is at most 1.0 per cent. Inject alternately the test solution and reference solution (a). Calculate the percentage content of ciclosporin. STORAGE Store in an airtight container, protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins. IMPURITIES

A. different ciclosporins [difference with ciclosporin (R = CH3 : ciclosporin A)] : ciclosporin B [7-L-Ala] ; ciclosporin C [7-L-Thr] ; ciclosporin D [7-L-Val] ; ciclosporin E [5-L-Val] ; ciclosporin G [7-(L-2-aminopentanoyl)] ; ciclosporin H [5-D-MeVal] ; ciclosporin L [R = H] ; ciclosporin T [4-L-Leu] ; ciclosporin U [11-L-Leu] ; ciclosporin V [1-L-Abu],

B. [6-[(2S,3R,4R)-3-hydroxy-4-methyl-2-(methylamino)octanoic acid]]ciclosporin A, C. isociclosporin A. 01/2005:1408

CILASTATIN SODIUM Cilastatinum natricum

C16H25N2NaO5S 1286

Mr 380.4

DEFINITION Cilastatin sodium contains not less than 98.0 per cent and not more than the equivalent of 101.5 per cent of sodium (Z)-7-[[(R)-2-amino-2-carboxyethyl]sulphanyl]-2-[[[(1S)2,2-dimethylcyclopropyl]carbonyl]amino]hept-2-enoate, calculated with reference to the anhydrous and solvent-free substance. CHARACTERS A white or light yellow amorphous powder, hygroscopic, very soluble in water and in methanol, soluble in dimethyl sulphoxide, slightly soluble in ethanol, practically insoluble in acetone and in methylene chloride. IDENTIFICATION A. It complies with the test for specific optical rotation (see Tests). B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cilastatin sodium CRS. C. It gives reaction (a) of sodium (2.3.1). TESTS Solution S. Dissolve 1.0 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). pH (2.2.3). The pH of solution S is 6.5 to 7.5. Specific optical rotation (2.2.7). Dissolve 0.250 g in a mixture of 1 volume of hydrochloric acid R and 120 volumes of methanol R and dilute to 25.0 ml with the same mixture of solvents. The specific optical rotation is + 41.5 to + 44.5, calculated with reference to the anhydrous and solvent-free substance. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 32.0 mg of the substance to be examined in water R and dilute to 20.0 ml with the same solvent. Reference solution (a). Dilute 2.0 ml of the test solution to 100.0 ml with water R. Dilute 5.0 ml of this solution to 100.0 ml with water R. Reference solution (b). Dilute 5.0 ml the test solution to 100.0 ml with water R. Dilute 2.0 ml of this solution to 20.0 ml with water R. Reference solution (c). Dissolve 16 mg of the substance to be examined in dilute hydrogen peroxide solution R and dilute to 10.0 ml with the same solvent. Allow to stand for 30 min. Dilute 1 ml of this solution to 100 ml with water R. Reference solution (d). Dissolve 32 mg of mesityl oxide R in 100 ml of water R. Dilute 1 ml of the solution to 50 ml with water R. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 2.0 ml/min, the following mixtures : Mobile phase A. Prepare a mixture of 300 volumes of acetonitrile R and 700 volumes of a 0.1 per cent V/V solution of phosphoric acid R in water R, Mobile phase B. A 0.1 per cent V/V solution of phosphoric acid R in water R,


Cilastatin sodium


Time (min)



15

85

0 - 30

15 → 100

85 → 0

30 - 46

100

0

46 - 56

100 → 15

0 → 85

— as detector a spectrophotometer set at 210 nm, — a 20 µl loop injector, maintaining the temperature of the column at 50 °C.

with the following temperature programme : Time (min) Column

0 - 2.5

Temperature Rate (°C) (°C/min) – 50

2.5 - 5

50 → 70

5 - 5.5

70

Injection port

160

Detector

220

8

Comment isothermal linear gradient isothermal

Inject 1 µl of the reference solution and then 1 µl of the test solution. Calculate the percentage contents of acetone, methanol and mesityl oxide using the following expression :

Equilibrate the column with a mixture of 15 per cent V/V of mobile phase A and 85 per cent V/V of mobile phase B. Inject separately each solution. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with reference solution (b) is at least in which C is the concentration in µg/ml of the solvent 15 per cent of the full scale of the recorder. in the reference solution, W is the quantity in milligrams The test is not valid unless, the chromatogram obtained with of cilastatin sodium in the test solution and Ru and Rs are reference solution (c) shows three principal peaks : the first the ratios of the corresponding solvent peak areas to the two peaks (cilastatin impurity A) may elute without being propanol peak area in the test solution and the reference completely resolved and the mass distribution ratio of the solution, respectively. third peak (cilastatin) is at least ten ; in the chromatogram obtained with reference solution (a), the principal peak has a Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2.0 ml of signal-to-noise ratio of at least 5.0. lead standard solution (10 ppm Pb) R. In the chromatogram obtained with the test solution, the Water (2.5.12). Not more than 2.0 per cent, determined on area of any peak, apart from the principal peak, is not greater 0.50 g by the semi-micro determination of water. than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent) ; the sum Bacterial endotoxins (2.6.14) : less than 0.17 IU/mg, if intended for use in the manufacture of parenteral dosage of the areas of all peaks, apart from the principal peak, is forms without a further appropriate procedure for the not greater than twice the area of the principal peak in removal of bacterial endotoxins. the chromatogram obtained with reference solution (b) (1 per cent). Disregard any peak due to the solvent, any peak with an area less than that of the principal peak in ASSAY the chromatogram obtained with reference solution (a) Dissolve 0.300 g in 30 ml of methanol R and add 5 ml of and any peak corresponding to the principal peak in the water R. Add 0.1 M hydrochloric acid to a pH of about 3.0. chromatogram obtained with reference solution (d). Carry out a potentiometric titration (2.2.20), using 0.1 M Mesityl oxide, acetone and methanol. Not more than sodium hydroxide. Three jumps of potential are observed. 1.0 per cent m/m of acetone, 0.5 per cent m/m of methanol Titrate to the third equivalence point. and 0.4 per cent m/m of mesityl oxide. Examine by gas 1 ml of 0.1 M sodium hydroxide is equivalent to 19.02 mg of chromatography (2.2.28) using propanol R as the internal C16H25N2NaO5S. standard. Internal standard solution. Dissolve 0.5 ml of propanol R in water R and dilute to 1000 ml with the same solvent. Test solution. Dissolve 0.200 g of the substance to be examined in water R, add 2.0 ml of the internal standard solution and dilute to 10.0 ml with water R. Reference solution. Dissolve 2.0 ml of acetone R, 0.5 ml of methanol R and 0.5 ml of mesityl oxide R in water R and dilute to 1000 ml with the same solvent. To 2.0 ml of this solution add 2.0 ml of the internal standard solution and dilute to 10.0 ml with water R. This solution contains 316 µg of acetone, 79 µg of methanol and 86 µg of mesityl oxide per millilitre.

STORAGE Store in an airtight container, at a temperature not exceeding 8 °C. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins. IMPURITIES

The chromatographic procedure may be carried out using : — a fused-silica column 30 m long and 0.53 mm in internal diameter coated with macrogol 20 000 R (film thickness 1.0 µm), — helium for chromatography R as the carrier gas at a flow A. (Z)-7-[(RS)-[(R)-2-amino-2-carboxyethyl]sulphinyl]-2-[[[(1S)rate of 9 ml/min, 2,2-dimethylcyclopropyl]carbonyl]amino]hept-2-enoic — a flame-ionisation detector, acid, General Notices (1) apply to all monographs and other texts

1287

Cilazapril


B. R = H : (Z)-7-[[(R)-2-[[(1RS)-1-methyl-3-oxobutyl]amino]-2-carboxyethyl]sulphanyl]-2-[[[(1S)-2,2-dimethylcyclopropyl]carbonyl]amino]hept-2-enoic acid, C. R = CH3 : (Z)-7-[[(R)-2-[(1,1-dimethyl-3-oxobutyl)amino]-2-carboxyethyl]sulphanyl]-2-[[[(1S)-2,2-dimethylcyclopropyl]carbonyl]amino]hept-2-enoic acid,

D. 4-methylpent-3-en-2-one (mesityl oxide). 01/2005:1499 corrected

CILAZAPRIL Cilazaprilum

C22H31N3O5,H2O

Mr 435.5

DEFINITION Cilazapril contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent of (1S,9S)-9-[[(1S)-1(ethoxycarbonyl)-3-phenylpropyl]amino]-10-oxooctahydro6H-pyridazino[1,2-a][1,2]diazepine-1-carboxylic acid, calculated with reference to the anhydrous substance. CHARACTERS A white or almost white, crystalline powder, slightly soluble in water, freely soluble in methanol and in methylene chloride. IDENTIFICATION A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cilazapril CRS. B. It complies with the test for specific optical rotation (see Tests). TESTS Specific optical rotation (2.2.7). Dissolve 0.200 g in 0.067 M phosphate buffer solution pH 7.0 R, with the aid of ultrasound if necessary and dilute to 50.0 ml with the same buffer solution. The specific optical rotation is − 383 to − 399, determined at 365 nm and calculated with reference to the anhydrous substance. Impurity A. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R. Test solution. Dissolve 0.20 g of the substance to be examined in methanol R and dilute to 5.0 ml with the same solvent. 1288

Reference solution (a). Dissolve 2 mg of cilazapril impurity A CRS in methanol R and dilute to 50.0 ml with the same solvent. Reference solution (b). Dissolve 5 mg of cilazapril impurity A CRS and 5 mg of the substance to be examined in methanol R and dilute to 10.0 ml with the same solvent. Apply to the plate 5 µl of each solution. Develop over a path of 10 cm using a mixture of 5 volumes of glacial acetic acid R, 5 volumes of water R, 15 volumes of hexane R, 15 volumes of methanol R and 60 volumes of ethyl acetate R. Dry the plate in a current of cold air for 10 min. Spray the plate with a freshly prepared mixture of 1 volume of potassium iodobismuthate solution R and 10 volumes of dilute acetic acid R and then with dilute hydrogen peroxide solution R. Any spot corresponding to impurity A in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.1 per cent). The test is not valid unless the chromatogram obtained with reference solution (b) shows 2 clearly separated spots. Other related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (a). Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 20.0 ml with the mobile phase. Reference solution (b). Dissolve 5.0 mg of cilazapril impurity D CRS in the test solution and dilute to 10.0 ml with the same solution. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.0 ml/min a mixture prepared as follows : mix 10 volumes of triethylamine R and 750 volumes of water R, adjust to pH 2.30 with phosphoric acid R, and add 200 volumes of tetrahydrofuran R, — as detector a spectrophotometer set at 214 nm. Inject 20 µl of reference solution (a) and 20 µl of reference solution (b). Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with reference solution (a) is at least 50 per cent of the full scale of the recorder. The test is not valid unless the resolution between the peaks corresponding to cilazapril and impurity D in the chromatogram obtained with reference solution (b) is at least 2.5 and the symmetry factor of the peak due to cilazapril is not more than 3.0. Inject 20 µl of the test solution and 20 µl of reference solution (a). Continue the chromatography for twice the retention time of the principal peak. When impurity A is present (relative retention time of 4 to 5), it may be necessary to continue the chromatography until it is eluted. When the chromatograms are recorded in the prescribed conditions, the retention times relative to cilazapril are : impurity B about 0.6 ; impurity D about 0.9 ; impurity C about 1.6. In the chromatogram obtained with the test solution : the area of any peak corresponding to impurity D is not greater than 0.4 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent) ; the area of any peak corresponding to impurity B is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent) ; the area of any peak corresponding to impurity C is not greater than 0.2 times the area of the principal peak in the chromatogram


Cimetidine


01/2005:0756

obtained with reference solution (a) (0.1 per cent) ; the area of any peak, apart from the principal peak and the peaks corresponding to impurities B, C and D, is not greater than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (1 per cent). Disregard any peak with an area less than 0.1 times that of the principal peak in the chromatogram obtained with reference solution (a) and any peak due to impurity A. Water (2.5.12) : 3.5 per cent to 5.0 per cent, determined on 0.300 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.300 g in 10 ml of ethanol R and add 50 ml of water R. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20). Carry out a blank titration. 1 ml of 0.1 M sodium hydroxide is equivalent to 41.75 mg of C22H31N3O5.

CIMETIDINE Cimetidinum

C10H16N6S

Mr 252.3

DEFINITION Cimetidine contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent of 2-cyano-1-methyl-3-[2-[[(5-methyl-1H-imidazol-4yl)methyl]sulphanyl]ethyl]guanidine, calculated with reference to the dried substance. CHARACTERS A white or almost white powder, slightly soluble in water, soluble in alcohol, practically insoluble in methylene chloride. It dissolves in dilute mineral acids. It shows polymorphism.

STORAGE

IDENTIFICATION First identification : B. Second identification : A, C, D. IMPURITIES A. Melting point (2.2.14) : 139 °C to 144 °C. If necessary, dissolve the substance to be examined in 2-propanol R, Specified impurities : A, B, C, D. evaporate to dryness and determine the melting point again. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cimetidine CRS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in 2-propanol R, evaporate to dryness and record the spectra again. C. Examine the chromatograms obtained in the test for related substances. The principal spot in the A. R = C(CH3)3, R′ = C2H5 : 1,1-dimethylethyl (1S,9S)-9-[[(S)-1chromatogram obtained with test solution (b) is similar (ethoxycarbonyl)-3-phenylpropyl]amino]-10-oxooctahydroin position, colour and size to the principal spot in the 6H-pyridazino[1,2-a][1,2]diazepine-1-carboxylate, chromatogram obtained with reference solution (d). D. Dissolve about 1 mg in a mixture of 1 ml of ethanol R and 5 ml of a freshly prepared 20 g/l solution of citric acid R B. R = R′ = H : (1S,9S)-9-[[(S)-1-carboxy-3in acetic anhydride R. Heat in a water-bath for 10 min to phenylpropyl]amino]-10-oxooctahydro-6H-pyridazino[1,215 min. A reddish-violet colour develops. a][1,2]diazepine-1-carboxylic acid, TESTS Appearance of solution. Dissolve 3.0 g in 12 ml of 1 M hydrochloric acid and dilute to 20 ml with water R. The C. R = R′ = C2H5 : ethyl (1S,9S)-9-[[(S)-1-(ethoxycarbonyl)-3phenylpropyl]amino]-10-oxooctahydro-6H-pyridazino[1,2- solution is clear (2.2.1) and not more intensely coloured than reference solution Y5 (2.2.2, Method II). a][1,2]diazepine-1-carboxylate, Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution (a). Dissolve 0.50 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with methanol R. Reference solution (a). Dilute 1 ml of test solution (a) to 100 ml with methanol R. Dilute 20 ml of this solution to D. (1S,9S)-9-[[(R)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]- 100 ml with methanol R. Reference solution (b). Dilute 5 ml of reference solution (a) 10-oxooctahydro-6H-pyridazino-[1,2-a][1,2]diazepine-1to 10 ml with methanol R. carboxylic acid. Protected from light.


1289

Cimetidine hydrochloride


Reference solution (c). Dilute 5 ml of reference solution (b) to 10 ml with methanol R. Reference solution (d). Dissolve 10 mg of cimetidine CRS in 2 ml of methanol R. A. Apply separately to the plate 4 µl of each solution. Allow the plate to stand for 15 min in the chromatographic tank saturated with vapour from the mobile phase which consists of a mixture of 15 volumes of concentrated ammonia R, 20 volumes of methanol R and 65 volumes of ethyl acetate R and develop immediately over a path of 15 cm using the same mixture of solvents. Dry the plate in a stream of cold air, expose to iodine vapour until maximum contrast of the spots has been obtained and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the principal spot in the chromatogram obtained with reference solution (a) (0.2 per cent) and not more than two such spots are more intense than the principal spot in the chromatogram obtained with reference solution (b) (0.1 per cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows a clearly visible spot. B. Apply separately to the plate 4 µl of each solution. Develop over a path of 15 cm using a mixture of 8 volumes of concentrated ammonia R, 8 volumes of methanol R and 84 volumes of ethyl acetate R. Dry the plate in a stream of cold air, expose to iodine vapour until maximum contrast of the spots has been obtained and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution (a), apart from the principal spot, is not more intense than the principal spot in the chromatogram obtained with reference solution (a) (0.2 per cent) and not more than two such spots are more intense than the principal spot in the chromatogram obtained with reference solution (b) (0.1 per cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows a clearly visible spot. Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.2 per cent, determined on 1.0 g.

01/2005:1500

CIMETIDINE HYDROCHLORIDE Cimetidini hydrochloridum

C10H17ClN6S

Mr 288.8

DEFINITION Cimetidine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent of 2-cyano-1-methyl-3-[2-[[(5-methyl-1H-imidazol4-yl)methyl]sulphanyl]ethyl]guanidine hydrochloride, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, freely soluble in water, sparingly soluble in ethanol. IDENTIFICATION First identification : B, E. Second identification : A, C, D, E. A. Dissolve 70 mg in 0.2 M sulphuric acid and dilute to 100.0 ml with the same acid. Dilute 2.0 ml of the solution to 100.0 ml with 0.2 M sulphuric acid. Measure the absorbance (2.2.25) at the absorption maximum at 218 nm. The specific absorbance at the maximum is 650 to 705. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cimetidine hydrochloride CRS. C. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (d). D. Dissolve about 1 mg in a mixture of 1 ml of ethanol R and 5 ml of a freshly prepared 20 g/l solution of citric acid R in acetic anhydride R. Heat on a water-bath for 10 min to 15 min. A reddish-violet colour develops. E. It gives reaction (a) of chlorides (2.3.1).

TESTS Appearance of solution. Dissolve 3.0 g in 12 ml of 1 M hydrochloric acid and dilute to 20 ml with water R. The solution is clear (2.2.1) and not more intensely coloured ASSAY than reference solution Y5 (2.2.2, Method II). pH (2.2.3). Dissolve 100 mg in carbon dioxide-free water R Dissolve 0.200 g in 60 ml of anhydrous acetic acid R. and dilute to 10.0 ml with the same solvent. The pH of the Titrate with 0.1 M perchloric acid determining the end-point solution is 4.0 to 5.0. potentiometrically (2.2.20). Related substances. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel GF254 plate R. 1 ml of 0.1 M perchloric acid is equivalent to 25.23 mg of Test solution (a). Dissolve 0.50 g of the substance to be C10H16N6S. examined in methanol R and dilute to 10 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with methanol R. STORAGE Reference solution (a). Dilute 2 ml of test solution (b) to 100 ml with methanol R. Store in an airtight container, protected from light.

1290


Cinchocaine hydrochloride


Reference solution (b). Dilute 5 ml of reference solution (a) to 10 ml with methanol R.

IMPURITIES

Reference solution (c). Dilute 5 ml of reference solution (b) to 10 ml with methanol R. Reference solution (d). Dissolve 10 mg of cimetidine hydrochloride CRS in 2 ml of methanol R. A. Apply to the plate 4 µl of each solution. Allow the plate to stand for 15 min in a chromatographic tank saturated with vapour from the mobile phase, which consists of a mixture of 15 volumes of concentrated ammonia R, 20 volumes of methanol R and 65 volumes of ethyl acetate R, and develop immediately over a path of 15 cm using the same mixture of solvents. Dry the plate in a stream of cold air, expose to iodine vapour until maximum contrast of the spots has been obtained and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the principal spot in the chromatogram obtained with reference solution (a) (0.2 per cent) and at most two such spots are more intense than the principal spot in the chromatogram obtained with reference solution (b) (0.1 per cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows a clearly visible spot.

A. R1 = CN, R2 = SCH3 : 3-cyano-2-methyl-1-[2-[[(5-methyl1H-imidazol-4-yl)methyl]sulphanyl]ethyl]isothiourea, B. R1 = CN, R2 = OCH3 : 3-cyano-2-methyl-1-[2-[[(5-methyl1H-imidazol-4-yl)methyl]sulphanyl]ethyl]isourea, C. R1 = CONH2, R2 = NHCH3 : 1-[(methylamino)[[2-[[(5-methyl-1H-imidazol-4-yl)methyl]sulphanyl]ethyl]amino]methylene]urea, D. R1 = H, R2 = NHCH3 : 1-methyl-3-[2-[[(5-methyl-1Himidazol-4-yl)methyl]sulphanyl]ethyl]guanidine,

E. 2-cyano-1-methyl-3-[2-[[(5-methyl-1H-imidazol-4yl)methyl]sulphinyl]ethyl]guanidine,

B. Apply to the plate 4 µl of each solution. Develop over a path of 15 cm using a mixture of 8 volumes of concentrated ammonia R, 8 volumes of methanol R and 84 volumes of ethyl acetate R. Dry the plate in a stream of cold air, expose to iodine vapour until maximum contrast of the spots has been obtained and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the principal spot in the chromatogram F. 2-cyano-1,3-bis[2-[[(5-methyl-1H-imidazol-4-yl)methyl]sulphanyl]ethyl]guanidine. obtained with reference solution (a) (0.2 per cent) and at most two such spots are more intense than the principal 01/2005:1088 spot in the chromatogram obtained with reference solution (b) (0.1 per cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows CINCHOCAINE HYDROCHLORIDE a clearly visible spot. Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R.

Cinchocaini hydrochloridum

Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.2 per cent, determined on 1.0 g. C20H30ClN3O2 ASSAY Dissolve 0.200 g of the substance to be examined in a mixture of 5 ml of 0.01 M hydrochloric acid and 50 ml of alcohol R. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the two points of inflexion.

Mr 379.9

DEFINITION Cinchocaine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of 2-butoxy-N-[2-(diethylamino)ethyl]quinoline-4-carboxamide hydrochloride, calculated with reference to the dried substance.

CHARACTERS 1 ml of 0.1 M sodium hydroxide is equivalent to 28.88 mg of A white or almost white, crystalline powder or colourless C10H17ClN6S. crystals, hygroscopic, very soluble in water, freely soluble in acetone, in alcohol and in methylene chloride. It agglomerates very easily. STORAGE IDENTIFICATION Store in an airtight container, protected from light. First identification : B, E. General Notices (1) apply to all monographs and other texts

1291

Cinchona bark


Second identification : A, C, D, E. A. Dissolve 60.0 mg in 1 M hydrochloric acid and dilute to 100 ml with the same acid. Dilute 2 ml of the solution to 100 ml with 1 M hydrochloric acid. Examined between 220 nm and 350 nm (2.2.25), the solution shows two absorption maxima, at 246 nm and 319 nm. The ratio of the absorbance measured at 246 nm to that measured at 319 nm is 2.7 to 3.0. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cinchocaine hydrochloride CRS. Examine the substances prepared as discs using potassium chloride R. C. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). D. Dissolve 0.5 g in 5 ml of water R. Add 1 ml of dilute ammonia R2. A white precipitate is formed. Filter, wash the precipitate with five quantities, each of 10 ml, of water R and dry in a desiccator. It melts at 64 °C to 66 °C (2.2.14). E. It gives reaction (a) of chlorides (2.3.1). TESTS Solution S. Dissolve 5.0 g in carbon dioxide-free water R prepared from distilled water R, and dilute to 50 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). pH (2.2.3). Dilute 10 ml of solution S to 50 ml with carbon dioxide-free water R. The pH of the solution is 5.0 to 6.0. Related substances. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm. Test solution (a). Dissolve 0.20 g of the substance to be examined in methanol R and dilute to 5 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with methanol R. Reference solution (a). Dissolve 20 mg of cinchocaine hydrochloride CRS in methanol R and dilute to 5 ml with the same solvent. Reference solution (b). Dilute 1 ml of test solution (b) to 20 ml with methanol R. Reference solution (c). Dilute 1 ml of test solution (b) to 50 ml with methanol R. Reference solution (d). Dissolve 20 mg of benzocaine CRS in methanol R and dilute to 5 ml with the same solvent. Dilute 1 ml of the solution and 1 ml of reference solution (a) to 20 ml with methanol R. Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 1 volume of ammonia R, 5 volumes of methanol R, 30 volumes of acetone R and 50 volumes of toluene R. Dry the plate in a current of warm air for 15 min. Examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the principal spot in the chromatogram obtained with reference solution (b) (0.5 per cent) and at most one such spot is more intense than the spot in the chromatogram obtained with reference solution (c) (0.2 per cent). The test is not valid unless the chromatogram obtained with reference solution (d) shows two clearly separated spots. 1292

Heavy metals (2.4.8). 12 ml of solution S complies with limit test A for heavy metals (20 ppm). Prepare the standard using lead standard solution (2 ppm Pb) R. Loss on drying (2.2.32). Not more than 2.0 per cent, determined on 0.500 g by drying in vacuo at 60 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.300 g in a mixture of 15.0 ml of 0.01 M hydrochloric acid and 50 ml of alcohol R. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the two points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 37.99 mg of C20H30ClN3O2. STORAGE Store in an airtight container, protected from light. IMPURITIES

A. R1 = Cl, R2 = NH-[CH2]2-N(C2H5)2 : 2-chloro-N-[2(diethylamino)ethyl]quinoline-4-carboxamide, B. R1 = R2 = OH : 2-hydroxyquinoline-4-carboxylic acid, C. R1 = OH, R2 = NH-[CH2]2-N(C2H5)2 : N-[2(diethylamino)ethyl]-2-hydroxyquinoline-4-carboxamide, D. R1 = O-[CH2]3-CH3, R2 = OH : 2-butoxyquinoline-4carboxylic acid. 01/2005:0174

CINCHONA BARK Cinchonae cortex DEFINITION Whole or cut, dried bark of Cinchona pubescens Vahl (Cinchona succirubra Pavon), of C. calisaya (Weddell), of C. ledgeriana (Moens ex Trimen) or of its varieties or hybrids. Content : minimum 6.5 per cent of total alkaloids, of which 30 per cent to 60 per cent consists of quinine-type alkaloids (dried drug). CHARACTERS Intense bitter, somewhat astringent taste. Macroscopic and microscopic characters described under identification tests A and B. IDENTIFICATION A. The stem and branch bark is supplied in quilled or curved pieces 2 mm to 6 mm thick. The outer surface is dull brownish-grey or grey and frequently bears lichens ; it is usually rough, marked with transverse fissures and longitudinally furrowed or wrinkled ; exfoliation of the outer surface occurs in some varieties. The inner surface is striated and deep reddish-brown ; the fracture is short in the outer part and fibrous in the inner part.


Cinchona bark


B. Reduce to a powder (355). The powder is reddish-brown. Examine under a microscope using chloral hydrate solution R. The powder shows the following diagnostic characters : thin-walled cork cells filled with reddish-brown contents ; yellow, spindle-shaped striated phloem fibres up to 90 µm in diameter and up to 1300 µm in length, very thick-walled with an uneven lumen and with conspicuous, funnel-shaped pits ; parenchymatous idioblasts filled with microprisms of calcium oxalate. Examine under a microscope using a 50 per cent V/V solution of glycerol R. The powder shows a few starch granules 6 µm to 10 µm in diameter ; mostly simple but occasionally with 2 or 3 components. C. Thin-layer chromatography (2.2.27). Test solution. To 0.10 g of the powdered drug (180) in a test-tube add 0.1 ml of concentrated ammonia R and 5 ml of methylene chloride R. Shake vigorously occasionally during 30 min and filter. Evaporate the filtrate to dryness on a water-bath and dissolve the residue in 1 ml of ethanol R. Reference solution. Dissolve 17.5 mg of quinine R, 2.5 mg of quinidine R, 10 mg of cinchonine R and 10 mg of cinchonidine R in 5 ml of ethanol R. Plate : TLC silica gel plate R. Mobile phase : diethylamine R, ethyl acetate R, toluene R (10:20:70 V/V/V). Application : 10 µl, as bands. Development : twice over a path of 15 cm. Drying : at 100-105 °C then allow to cool. Detection A : spray with anhydrous formic acid R and allow to dry in air. Examine in ultraviolet light at 365 nm. Results A : see below the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. Furthermore, other fluorescent zones are present in the chromatogram obtained with the test solution.

TESTS Foreign matter (2.8.2) : maximum 2 per cent m/m. Total ash (2.4.16) : maximum 6.0 per cent. Loss on drying (2.2.32) : maximum 10 per cent, determined on 1.000 g of the powdered drug (355) by drying in an oven at 100-105 °C for 2 h. ASSAY Test solution. In a 250 ml conical flask mix 1.000 g of the powdered drug (180) with 10 ml of water R and 7 ml of dilute hydrochloric acid R. Heat in a water-bath for 30 min, allow to cool and add 25 ml of methylene chloride R, 50 ml of ether R and 5 ml of a 200 g/l solution of sodium hydroxide R. Shake the mixture repeatedly for 30 min, add 3 g of powdered tragacanth R and shake until the mixture becomes clear. Filter through a plug of absorbent cotton and rinse the flask and the cotton with 5 quantities, each of 20 ml, of a mixture of 1 volume of methylene chloride R and 2 volumes of ether R. Combine the filtrate and washings, evaporate to dryness and dissolve the residue in 10.0 ml of ethanol R. Evaporate 5.0 ml of the solution to dryness, dissolve the residue in 0.1 M hydrochloric acid and dilute to 1000.0 ml with the same acid. Reference solutions. Dissolve separately 30.0 mg of quinine R and 30.0 mg of cinchonine R in 0.1 M hydrochloric acid and dilute each solution to 1000.0 ml with the same acid. Measure the absorbances (2.2.25) of the 3 solutions at 316 nm and 348 nm using 0.1 M hydrochloric acid as the compensation liquid. Calculate the percentage content of alkaloids from the following equations :


_______

Quinidine : a distinct blue fluorescent zone _______

A distinct blue fluorescent zone (quinidine) _______

m

=

mass of the drug used, in grams,

x

=

percentage content of quinine-type alkaloids,

Quinine : a distinct blue fluorescent zone

A distinct blue fluorescent zone (quinine)

y

=

percentage content of cinchonine-type alkaloids,

A316 = A348 = Detection B : spray with iodoplatinate reagent R. Results B : see below the sequence of the zones present in A316c = the chromatograms obtained with the reference solution and the test solution. Furthermore, other zones are A316q = present in the chromatogram obtained with the test solution. Reference solution

Test solution


_______

Cinchonine : a violet zone which becomes violet-grey Quinidine : a violet zone which becomes violet-grey Cinchonidine : an intense dark blue zone _______

A violet zone, which becomes violet-grey (cinchonine) A violet zone, which becomes violet-grey (quinidine) An intense dark blue zone (cinchonidine) _______

Quinine : a violet zone which becomes violet-grey

A violet zone, which becomes violet-grey (quinine)

Reference solution

absorbance of the test solution at 316 nm, absorbance of the test solution at 348 nm, absorbance of the reference solution containing cinchonine at 316 nm, corrected to a concentration of 1 mg/1000 ml, absorbance of the reference solution containing quinine at 316 nm, corrected to a concentration of 1 mg/1000 ml,

A348c =

absorbance of the reference solution containing cinchonine at 348 nm, corrected to a concentration of 1 mg/1000 ml,

A348q =

absorbance of the reference solution containing quinine at 348 nm, corrected to a concentration of 1 mg/1000 ml.

Calculate the content of total alkaloids, (x + y), and the relative content of quinine-type alkaloids, from the following expression :

Test solution


1293

Cineole


CINEOLE Cineolum

C10H18O DEFINITION 1,3,3-Trimethyl-2-oxabicyclo[2.2.2]octane.

01/2005:1973 Reference solution (a). To 2.0 ml of test solution (a) add 20.0 ml of the internal standard solution and dilute to 100.0 ml with heptane R. Reference solution (b). Dissolve 50 mg of 1,4-cineole R and 50 mg of the substance to be examined in heptane R and dilute to 50.0 ml with the same solvent. Column : — size : l = 30 m, Ø = 0.25 mm, — stationary phase : macrogol 20 000 R (film thickness 0.25 µm). Carrier gas : helium for chromatography R. Mr 154.3 Linear velocity : 45 cm/s. Split-ratio : 1:70. Temperature :

CHARACTERS Appearance : clear colourless liquid. Solubility : practically insoluble in water, miscible with alcohol and with methylene chloride. It solidifies at about 0.5 °C.

Column

IDENTIFICATION A. It complies with the test for refractive index (see Tests). B. Thin-layer chromatography (2.2.27). Test solution. Dilute 1 ml of solution S (see Tests) to 25 ml with alcohol R. Reference solution. Mix 80 mg of cineole CRS with alcohol R and dilute to 10 ml with the same solvent. Plate : TLC silica gel plate R. Mobile phase : ethyl acetate R, toluene R (10:90 V/V). Application : 2 µl. Development : over 2/3 of the plate. Drying : in a current of cold air. Detection : spray with anisaldehyde solution R, heat at 100-105 °C for 5 min. Results : the principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. C. To 0.1 ml add 4 ml of sulphuric acid R. An orange-red colour develops. Add 0.2 ml of formaldehyde solution R. The colour changes to deep brown. TESTS Solution S. Dilute 2.00 g to 10.0 ml with alcohol R. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method I). Chiral impurities. The optical rotation (2.2.7) of solution S is − 0.10° to + 0.10°. Refractive index (2.2.6) : 1.456 to 1.460. Related substances. Gas chromatography (2.2.28). Internal standard solution. Dissolve 1.0 g of camphor R in heptane R and dilute to 200 ml with the same solvent. Test solution (a). Dissolve 2.5 g of the substance to be examined in heptane R and dilute to 25.0 ml with the same solvent. Test solution (b). Dissolve 2.5 g of the substance to be examined in heptane R, add 5.0 ml of the internal standard solution and dilute to 25.0 ml with heptane R. 1294

Time (min)

Temperature (°C)

0 - 10 10 - 35 35 - 45 45 - 55

50 50 → 100 100 → 200 200

Injection port

220

Detector

250

Detection : flame ionisation. Injection : 1 µl. System suitability : reference solution (b) : — resolution : minimum 10 between the peaks due to impurity A and to cineole. Limits : — total : calculate the ratio (R) of the area of the peak due to cineole to the area of the peak due to the internal standard from the chromatogram obtained with reference solution (a) ; from the chromatogram obtained with test solution (b), calculate the ratio of the sum of the areas of any peaks, apart from the principal peak and the peak due to the internal standard, to the area of the peak due to internal standard : this ratio is not greater than R (2 per cent), — disregard limit : 0.025 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). Residue on evaporation : maximum 0.1 per cent. To 2.0 g add 5 ml of water R, evaporate to dryness on a water-bath and dry at 100-105 °C for 1 h. The residue weighs a maximum of 2 mg. STORAGE In an airtight container, protected from light. IMPURITIES

A. 1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptane (1,4-cineole).


Cinnamon bark oil, Ceylon


CINNAMON Cinnamomi cortex

01/2005:0387 ASSAY Carry out the determination of essential oils in vegetable drugs (2.8.12). Use a 500 ml flask, 200 ml of 0.1 M hydrochloric acid as the distillation liquid and 0.50 ml of xylene R in the graduated tube. Powder the drug (710) and proceed immediately with the determination, using 20.0 g. Distil at a rate of 2.5-3.5 ml/min for 3 h.

DEFINITION STORAGE Cinnamon consists of the dried bark, freed from the outer cork and the underlying parenchyma, of the shoots grown Store protected from light. on cut stock of Cinnamomum zeylanicum Nees. It contains not less than 12 ml/kg of essential oil. CHARACTERS Cinnamon has a characteristic, aromatic odour. It has the macroscopic and microscopic characters described under identification tests A and B. IDENTIFICATION A. The bark is about 0.2 mm to 0.8 mm thick and occurs in closely-packed compound quills made up of single or double quills. The outer surface is smooth, yellowish-brown with faint scars marking the position of leaves and axillary buds and has fine, whitish and wavy longitudinal striations. The inner surface is slightly darker and longitudinally striated. The fracture is short and fibrous. B. Reduce to a powder (355). The powder is yellowish to reddish-brown. Examine under a microscope using chloral hydrate solution R. The powder shows the following diagnostic characters : groups of rounded sclereids with pitted, channelled and moderately thickened walls ; numerous colourless single fibres, often whole with narrow lumen and thickened, lignified walls and few pits ; small acicular crystals of calcium oxalate. Examine under a microscope using a 50 per cent V/V solution of glycerol R ; the powder shows abundant starch granules. Cork fragments are absent or very rare. C. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Shake 0.1 g of powdered drug (500) with 2 ml of methylene chloride R for 15 min. Filter and evaporate the filtrate carefully almost to dryness on a water-bath. Dissolve the residue in 0.4 ml of toluene R. Reference solution. Dissolve 50 µl of cinnamic aldehyde R and 10 µl of eugenol R in toluene R and dilute to 10 ml with the same solvent. Apply separately to the plate as bands 20 mm by 3 mm, 10 µl of each solution. Develop over a path of 10 cm using methylene chloride R. Allow the plate to dry in air, examine in ultraviolet light at 254 nm and mark the quenching zones. Examine the plate in ultraviolet light at 365 nm and mark the fluorescent zones. In ultraviolet light at 254 nm, the chromatograms obtained with the test and reference solutions show a quenching zone (cinnamaldehyde) in the median part and just above it a weaker quenching zone (eugenol). In ultraviolet light at 365 nm, the chromatogram obtained with the test solution shows a zone of light-blue fluorescence (o-methoxy-cinnamaldehyde) just below the zone corresponding to cinnamaldehyde. Spray with phloroglucinol solution R. The zone corresponding to cinnamaldehyde is yellowish-brown and the zone corresponding to o-methoxycinn-amaldehyde is violet. TESTS Total ash (2.4.16). Not more than 6.0 per cent. General Notices (1) apply to all monographs and other texts

01/2005:1501

CINNAMON BARK OIL, CEYLON Cinnamomi zeylanicii corticis aetheroleum DEFINITION Ceylon cinnamon bark oil is obtained by steam distillation of the bark of the shoots of Cinnamomum zeylanicum Nees (C. Verum J.S. Presl.). CHARACTERS A clear, mobile, light yellow liquid becoming reddish over time, with a characteristic odour reminiscent of cinnamic aldehyde. IDENTIFICATION First identification : B. Second identification : A. A. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R. Test solution. Dissolve 1 ml of the substance to be examined in acetone R and dilute to 10 ml with the same solvent. Reference solution. Dissolve 50 µl of trans-cinnamic aldehyde R, 10 µl of eugenol R, 10 µl of linalol R and 10 µl of β-caryophyllene R in alcohol R and dilute to 10 ml with the same solvent. Apply to the plate, as bands, 10 µl of each solution. Develop over a path of 15 cm using a mixture of 10 volumes of methanol R and 90 volumes of toluene R. Allow the plate to dry in air and spray with anisaldehyde solution R. Examine in daylight while heating at 100-105 °C for 5-10 min. The chromatogram obtained with the test solution shows zones similar in position and colour to those in the chromatogram obtained with the reference solution. B. Examine the chromatograms obtained in the test for chromatographic profile. The retention times of the principal peaks in the chromatogram obtained with the test solution are similar to those in the chromatogram obtained with the reference solution. Safrole, coumarin and cineole may be absent from the chromatogram obtained with the test solution. TESTS Relative density (2.2.5) : 1.000 to 1.030. Refractive index (2.2.6) : 1.572 to 1.591. Optical rotation (2.2.7) : − 2° to + 1°. Chromatographic profile. Examine by gas chromatography (2.2.28). Test solution. The substance to be examined. 1295

Cinnamon leaf oil, Ceylon


Reference solution. Dissolve 10 µl of cineole R, 10 µl of linalol R, 10 µl of β-caryophyllene R, 10 µl of safrole R, 100 µl of trans-cinnamic aldehyde R, 10 µl of eugenol R, 20 mg of coumarin R, 10 µl of trans-2-methoxycinnamaldehyde R and 10 µl of benzyl benzoate R in 1 ml of acetone R. The chromatographic procedure may be carried out using : — a fused-silica column 60 m long and about 0.25 mm in internal diameter coated with macrogol 20 000 R as the bonded phase, — helium for chromatography R as the carrier gas at a flow rate of 1.5 ml/min, — a flame-ionisation detector, — a split ratio of 1:100, with the following temperature programme : Time (min) Column

0 - 10

Temperature Rate (°C) (°C/min) – 60

10 - 75

60 → 190

75 - 200

190

2

Comment isothermal linear gradient isothermal

CHARACTERS Clear, mobile, reddish-brown to dark brown liquid, with a characteristic odour reminiscent of eugenol. IDENTIFICATION First identification : B. Second identification : A. A. Thin-layer chromatography (2.2.27). Test solution. Dilute 1 ml of the substance to be examined in acetone R and dilute to 10 ml with the same solvent. Reference solution. Dilute about 50 µl of trans-cinnamic aldehyde R, 10 µl of eugenol R, 10 µl of linalol R and 10 µl of β-caryophyllene R in alcohol R and dilute to 10 ml with the same solvent. Plate : TLC silica gel plate R. Mobile phase : methanol R, toluene R (10:90 V/V). Application : 10 µl, as bands.

Injection port

200

Development : over a path of 15 cm.

Detector

240

Drying : in air.

Inject 0.2 µl of the reference solution. When the chromatogram is recorded in the prescribed conditions, the components elute in the order indicated in the composition of the reference solution. Depending on the operating conditions and the state of the column, coumarin may elute before or after trans-2-methoxycinnamaldehyde. Record the retention times of these substances. The test is not valid unless the resolution between the peaks corresponding to linalol and β-caryophyllene is at least 1.5. Inject 0.2 µl of the test solution. Using the retention times determined from the chromatogram obtained with the reference solution, locate the components of the reference solution in the chromatogram obtained with the test solution. Determine the percentage content of each of these components by the normalisation procedure. The percentages are within the following ranges : — cineole : less than 3.0 per cent, — linalol : 1.0 per cent to 6.0 per cent, — β-caryophyllene : 1.0 per cent to 4.0 per cent, — safrole : less than 0.5 per cent, — trans-cinnamic aldehyde : 55 per cent to 75 per cent, — eugenol : less than 7.5 per cent, — coumarin : less than 0.5 per cent, — trans-2-methoxycinnamaldehyde : 0.1 per cent to 1.0 per cent, — benzyl benzoate : less than 1.0 per cent. STORAGE Store in a well-filled, airtight container, protected from light and heat.

Detection : spray with anisaldehyde solution R. Examine in day light while heating at 100-105 °C for 5-10 min. Results : the zones in the chromatogram obtained with the test solution are similar in position and colour to those in the chromatogram obtained with the reference solution. The zone due to trans-cinnamic aldehyde may be very faint or absent. B. Examine the chromatogram obtained in the test for chromatographic profile. Results : the characteristic peaks in the chromatogram obtained with the test solution are similar in retention time to those in the chromatogram obtained with the reference solution. The peaks corresponding to cineole, safrole, trans-cinnamic aldehyde, cinnamyl acetate and coumarin may be absent in the chromatogram obtained with the test solution. TESTS Relative density (2.2.5) : 1.030 to 1.059. Refractive index (2.2.6) : 1.527 to 1.540. Optical rotation (2.2.7) : − 2.5° to + 2.0°. Chromatographic profile. Gas chromatography (2.2.28) : use the normalisation procedure. Test solution. The substance to be examined. Reference solution. Dissolve 10 µl of cineole R, 10 µl of linalol R, 10 µl of β-caryophyllene R, 10 µl of safrole R, 10 µl of trans-cinnamic aldehyde R, 10 µl of cinnamyl acetate R, 100 µl of eugenol R and 10 mg of coumarin R in 1 ml of acetone R.

01/2005:1608 Column : — material : fused silica,

CINNAMON LEAF OIL, CEYLON

— size : l = 60 m, Ø = 0.25 mm,

Cinnamomi zeylanici folii aetheroleum DEFINITION Oil obtained by steam distillation of the leaves of Cinnamomum verum J.S. Presl. 1296

— stationary phase : macrogol 20 000 R. Carrier gas : helium for chromatography R. Flow rate : 1.5 ml/min. Split ratio : 1/100.


Cinnarizine


Temperature :

Column

Time (min)

Temperature (°C)

0 - 10 10 - 78 78 - 88

45 45 → 180 180

Injection port

200

Detector

240

Detection : flame ionisation. Injection : 0.2 µl. Elution order : the order indicated in the composition of the reference solution. Record the retention times of these substances. System suitability : reference solution : — resolution : minimum of 1.5 between the peaks due to linalol and β-caryophyllene. Using the retention times determined from the chromatogram obtained with the reference solution, locate the components of the reference solution in the chromatogram obtained with the test solution. Determine the percentage content of these components. The percentages are within the following ranges : — cineole : less than 1.0 per cent, — linalol : 1.5 per cent to 3.5 per cent, — β-caryophyllene : 1.5 per cent to 7.0 per cent, — safrole : less than 3.0 per cent, — trans-cinnamic aldehyde : less than 3.0 per cent, — cinnamyl acetate : less than 2.0 per cent, — eugenol : 70 per cent to 85 per cent, — coumarin : less than 1.0 per cent. STORAGE In a well-filled, airtight container, protected from light and heat.

Mobile phase : methylene chloride R. Application : 20 µl, as bands. Development : over a path of 10 cm. Drying : in air. Detection A : examine in ultraviolet light at 365 nm. Results A : see below the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. Top of the plate _______

_______

Trans-2-methoxycinnamaldehyde : a light blue fluorescent zone _______

A greenish fluorescent zone (above the starting line) Reference solution

Test solution

Detection B : spray with a 200 g/l solution of phosphomolybdic acid R in ethanol R. Examine in daylight while heating at 100-105 °C for 5-10 min. Results B : see below the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. Furthermore, other zones may be present in the chromatogram obtained with the test solution. Top of the plate 1 blue zone (terpenhydrocarbons) _______

_______

Eugenol: a blue zone

A blue zone (eugenol)

Trans-cinnamic aldehyde : a blue zone

A blue zone (trans-cinnamic aldehyde)

Trans-2-methoxycinnamaldehyde : an orange-brown zone (the colour fades away) _______

A weak orange-brown zone (trans-2-methoxycinnamaldehyde)

01/2005:1819

CINNAMON TINCTURE

A light blue fluorescent zone (trans-2- methoxycinnamaldehyde) _______

_______ 2 or 3 blue zones above the starting line

Reference solution

Test solution

TESTS Ethanol (2.9.10) : 64 per cent V/V to 70 per cent V/V. DEFINITION Methanol and 2-propanol (2.9.11) : maximum 0.05 per Tincture produced from Cinnamon (0387). cent V/V of methanol and maximum 0.05 per cent V/V of 2-propanol. PRODUCTION The tincture is produced from 1 part of the drug and 5 parts Dry residue (2.8.16) : minimum 1.5 per cent m/m, determined on 5.0 g. of ethanol (70 per cent V/V) by an appropriate procedure.

Cinnamomi corticis tinctura

CHARACTERS Appearance : clear, brownish-red liquid, with a characteristic odour. IDENTIFICATION Thin-layer chromatography (2.2.27). Test solution. Place 10 ml of the tincture to be examined, 10 ml of saturated sodium chloride solution R and 5 ml of toluene R in a ground glass-stoppered tube. Shake for 2 min and centrifuge for 10 min. Use the organic layer. Reference solution. Dissolve 5 µl of eugenol R, 25 µl of trans-cinnamic aldehyde R and 5 µl of trans-2-methoxycinnamaldehyde R in toluene R and dilute to 10 ml with the same solvent. Plate : TLC silica gel G plate R. C26H28N2 General Notices (1) apply to all monographs and other texts

01/2005:0816

CINNARIZINE Cinnarizinum

Mr 368.5 1297

Cinnarizine


DEFINITION Cinnarizine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of (E)-1-(diphenylmethyl)-4-(3-phenylprop-2-enyl)piperazine, calculated with reference to the dried substance. CHARACTERS A white or almost white powder, practically insoluble in water, freely soluble in methylene chloride, soluble in acetone, slightly soluble in alcohol and in methanol. IDENTIFICATION First identification : A, B. Second identification : A, C, D. A. Melting point (2.2.14) : 118 °C to 122 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cinnarizine CRS. Examine the substances prepared as discs. C. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable octadecylsilyl silica gel with a fluorescent indicator having an optimal intensity at 254 nm. Test solution. Dissolve 10 mg of the substance to be examined in methanol R and dilute to 20 ml with the same solvent. Reference solution (a). Dissolve 10 mg of cinnarizine CRS in methanol R and dilute to 20 ml with the same solvent. Reference solution (b). Dissolve 10 mg of cinnarizine CRS and 10 mg of flunarizine dihydrochloride CRS in methanol R and dilute to 20 ml with the same solvent. Apply to the plate 5 µl of each solution. Develop in an unsaturated tank over a path of 15 cm using a mixture of 20 volumes of 1 M sodium chloride, 30 volumes of methanol R and 50 volumes of acetone R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. D. Dissolve 0.2 g of anhydrous citric acid R in 10 ml of acetic anhydride R in a water-bath at 80 °C and maintain the temperature of the water-bath at 80 °C for 10 min. Add about 20 mg of the substance to be examined. A purple colour develops.

Reference solution (a). Dissolve 12.5 mg of cinnarizine CRS and 15.0 mg of flunarizine dihydrochloride CRS in methanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this solution to 20.0 ml with methanol R. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with methanol R. Dilute 5.0 ml of this solution to 20.0 ml with methanol R. The chromatographic procedure may be carried out using : — a stainless steel column 0.1 m long and 4.0 mm in internal diameter packed with base-deactivated octadecylsilyl silica gel for chromatography R (3 µm), — as mobile phase at a flow rate of 1.5 ml/min a gradient programme using the following conditions : Mobile phase A. A 10 g/l solution of ammonium acetate R, Mobile phase B. A 0.2 per cent V/V solution of glacial acetic acid R in acetonitrile R, Time (min)



Comment

0 – 20

75 → 10

25 → 90

linear gradient

20 - 25

10

90

isocratic elution

25 - 30

75

25


30 = 0

75

25

restart gradient

— as detector a spectrophotometer set at 230 nm. Equilibrate the column for a least 30 min at the initial eluent composition. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 10 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder. If necessary, adjust the concentration of glacial acetic acid in mobile phase B to obtain a horizontal base-line in the chromatogram. Inject 10 µl of reference solution (a). When the chromatogram is recorded in the prescribed conditions, the retention times are : cinnarizine about 11 min and flunarizine about 11.5 min. The test is not valid unless the resolution between the peaks corresponding to cinnarizine and flunarizine is at least 5.0. If necessary, adjust the time programme for the gradient elution. Inject 10 µl of methanol R as a blank, 10 µl of the test solution and 10 µl of reference solution (b). In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.25 per cent) ; the sum of the areas of the peaks, apart from the principal peak, is TESTS not greater than twice the area of the principal peak in the Appearance of solution. Dissolve 0.5 g in methylene chromatogram obtained with reference solution (b) (0.5 per chloride R and dilute to 20 ml with the same solvent. The cent). Disregard any peak due to the blank and any peak solution is clear (2.2.1) and not more intensely coloured with an area less than 0.2 times the area of the principal peak than reference solution BY7 (2.2.2, Method II). in the chromatogram obtained with reference solution (b). Acidity or alkalinity. Suspend 0.5 g in 15 ml of water R. Heavy metals (2.4.8). Dissolve 1.0 g in a mixture of Boil for 2 min. Cool and filter. Dilute the filtrate to 20 ml 15 volumes of water R and 85 volumes of acetone R. Add with carbon dioxide-free water R. To 10 ml add 0.1 ml of dilute hydrochloric acid R until dissolution is complete. phenolphthalein solution R and 0.25 ml of 0.01 M sodium Dilute to 20 ml with the same mixture of water R and hydroxide. The solution is pink. To 10 ml add 0.1 ml of acetone R. 12 ml of the solution complies with limit test B methyl red solution R and 0.25 ml of 0.01 M hydrochloric for heavy metals (20 ppm). Prepare the standard using 10 ml acid. The solution is red. of lead standard solution (1 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with the mixture of Related substances. Examine by liquid chromatography water R and acetone R. (2.2.29). Loss on drying (2.2.32). Not more than 0.5 per cent, Test solution. Dissolve 25.0 mg of the substance to be examined in methanol R and dilute to 10.0 ml with the same determined on 1.000 g by drying in an oven in vacuo at 60 °C for 4 h. solvent.

1298


Ciprofibrate


01/2005:2013


CIPROFIBRATE

ASSAY

Ciprofibratum

Dissolve 0.150 g in 50 ml of a mixture of 1 volume of anhydrous acetic acid R and 7 volumes of ethyl methyl ketone R. Titrate with 0.1 M perchloric acid, using 0.2 ml of naphtholbenzein solution R as indicator. 1 ml of 0.1 M perchloric acid is equivalent to 18.43 mg of C26H28N2. STORAGE

C13H14Cl2O3

Store protected from light.

DEFINITION 2-[4-[(1RS)-2,2-Dichlorocyclopropyl]phenoxy]-2methylpropanoic acid. Content : 99.0 per cent to 101.0 per cent (anhydrous substance).

IMPURITIES

A. 1-(diphenylmethyl)piperazine,

Mr 289.2

CHARACTERS Appearance : white or slightly yellow, crystalline powder. Solubility : practically insoluble in water, freely soluble in anhydrous ethanol, soluble in toluene. mp : about 115 °C. IDENTIFICATION Infrared absorption spectrophotometry (2.2.24). Comparison : ciprofibrate CRS.

B. (Z)-1-(diphenylmethyl)-4-(3-phenylprop-2-enyl)piperazine,

C. (4-(diphenylmethyl)-1,1-bis[(E)-3-phenylprop-2enyl]piperazinium chloride,

D. 1-(diphenylmethyl)-4-[(1RS,3E)-4-phenyl-1-[(E)-2phenylethenyl]but-3-enyl]piperazine,

E. 1,4-bis(diphenylmethyl)piperazine. General Notices (1) apply to all monographs and other texts

TESTS Appearance of solution. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY4 (2.2.2, Method II). Dissolve 1.0 g in anhydrous ethanol R and dilute to 10.0 ml with the same solvent. Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 0.125 g of the substance to be examined in a mixture of equal volumes of acetonitrile R and water R and dilute to 50 ml with the same mixture of solvents. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with a mixture of equal volumes of acetonitrile R and water R. Dilute 1.0 ml of this solution to 10.0 ml with a mixture of equal volumes of acetonitrile R and water R. Reference solution (b). Dissolve the contents of a vial of ciprofibrate for system suitability CRS in 2.0 ml of a mixture of equal volumes of acetonitrile R and water R. Column : — size : l = 0.15 m, Ø = 4.6 mm, — stationary phase : octylsilyl silica gel for chromatography R (5 µm). Mobile phase : — mobile phase A : 1.36 g/l solution of potassium dihydrogen phosphate R adjusted to pH 2.2 with phosphoric acid R, — mobile phase B : acetonitrile R, Time (min)



0 - 30

75 → 30

25 → 70

30 - 40

30

70

40 - 42

30 → 75

70 → 25

Flow rate : 1.5 ml/min. Detection : spectrophotometer at 230 nm. 1299

Ciprofloxacin


Injection : 10 µl. Identification of impurities : use the chromatogram supplied with ciprofibrate for system suitability CRS to identify the peaks due to impurities A, B, C, D and E. Relative retention with reference to ciprofibrate (retention time = about 18 min) : impurity A = about 0.7 ; impurity B = about 0.8 ; impurity C = about 0.95 ; impurity D = about 1.3 ; impurity E = about 1.5. System suitability : reference solution (b) : — resolution : baseline separation between the peaks due to impurity C and ciprofibrate. Limits : — correction factor : for the calculation of content, multiply the peak area of impurity A by 2.3, — impurities A, C, D : for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), — impurity B : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent), — impurity E : not more than 8 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.8 per cent), — any other impurity : for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), — total of other impurities : not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent), — disregard limit : 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). Chlorides (2.4.4) : maximum 350 ppm. To 0.190 g add 20 ml of water R and treat in an ultrasonic bath for 8 min. Filter. 15 ml of the filtrate complies with the test. Water (2.5.12) : maximum 0.5 per cent, determined on 1.000 g. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.250 g in a mixture of 20 ml of water R and 40 ml of anhydrous ethanol R. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M sodium hydroxide is equivalent to 28.92 mg of C13H14Cl2O3. STORAGE In an airtight container, protected from light. IMPURITIES Specified impurities : A, B, C, D, E.

A. 2-(4-ethenylphenoxy)-2-methylpropanoic acid,

B. 4-[(1RS)-2,2-dichlorocyclopropyl]phenol, 1300

C. R = CH2OH : 2-[4-[(1RS)-2,2-dichlorocyclopropyl]phenoxy]2-methylpropan-1-ol, D. R = CO-OCH3 : methyl 2-[4-[(1RS)-2,2-dichlorocyclopropyl]phenoxy]-2-methylpropanoate, E. R = CO-OC2H5 : ethyl 2-[4-[(1RS)-2,2-dichlorocyclopropyl]phenoxy]-2-methylpropanoate. 01/2005:1089 corrected

CIPROFLOXACIN Ciprofloxacinum

C17H18FN3O3

Mr 331.4

DEFINITION 1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4dihydroquinoline-3-carboxylic acid. Content : 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : almost white or pale yellow, crystalline powder, slightly hygroscopic. Solubility : practically insoluble in water, very slightly soluble in ethanol and in methylene chloride. IDENTIFICATION Infrared absorption spectrophotometry (2.2.24). Comparison : ciprofloxacin CRS. TESTS Appearance of solution. The solution is clear (2.2.1) and not more intensely coloured than reference solution GY5 (2.2.2, Method II). Dissolve 0.25 g in 0.1 M hydrochloric acid and dilute to 20 ml with the same solvent. Impurity A. Thin-layer chromatography (2.2.27). Test solution. Dissolve 50 mg of the substance to be examined in dilute ammonia R1 and dilute to 5 ml with the same solvent. Reference solution. Dissolve 10 mg of ciprofloxacin impurity A CRS in a mixture of 0.1 ml of dilute ammonia R1 and 90 ml of water R and dilute to 100 ml with water R. Dilute 2 ml of the solution to 10 ml with water R. Plate : TLC silica gel F254 plate R. Application : 5 µl. At the bottom of a chromatographic tank, place an evaporating dish containing 50 ml of concentrated ammonia R. Expose the plate to the ammonia vapour for 15 min in the closed tank. Withdraw the plate, transfer to a second chromatographic tank and proceed with development.


Ciprofloxacin


Mobile phase : acetonitrile R, concentrated ammonia R, methanol R, methylene chloride R (10:20:40:40 V/V/V/V). Development : over 3/4 of the plate. Drying : in air. Detection : examine in ultraviolet light at 254 nm. Limit : — impurity A : any spot corresponding to impurity A is not more intense than the principal spot in the chromatogram obtained with the reference solution (0.2 per cent). Related substances. Liquid chromatography (2.2.29). Test solution. To 25.0 mg of the substance to be examined add 0.2 ml of dilute phosphoric acid R and dilute to 50.0 ml with the mobile phase and treat in an ultrasonic bath until a clear solution is obtained. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 5.0 ml with the mobile phase. Reference solution (b). Dissolve 5 mg of ciprofloxacin hydrochloride for peak identification CRS in the mobile phase and dilute to 10.0 ml with the mobile phase. Column : — size : l = 0.25 m, Ø = 4.6 mm ; — stationary phase: base-deactivated octadecylsilyl silica gel for chromatography R (5 µm) ; — temperature : 40 °C. Mobile phase : mix 13 volumes of acetonitrile R and 87 volumes of a 2.45 g/l solution of phosphoric acid R, previously adjusted to pH 3.0 with triethylamine R. Flow rate : 1.5 ml/min. Detection : spectrophotometer at 278 nm. Injection : 50 µl. Run time : twice the retention time of ciprofloxacin. Relative retention with reference to ciprofloxacin (retention time = about 9 min) : impurity E = about 0.4 ; impurity F = about 0.5 ; impurity B = about 0.6 ; impurity C = about 0.7 ; impurity D = about 1.2. System suitability : reference solution (b) : — resolution : minimum 1.3 between the peaks due to impurity B and impurity C. Limits : — correction factors : for the calculation of contents, multiply the peak areas of the following impurities by the corresponding correction factor : impurity B = 0.7 ; impurity C = 0.6 ; impurity D = 1.4 ; impurity E = 6.7 ; use the chromatogram obtained with reference solution (b) and the type chromatogram supplied with the CRS to identify the corresponding peaks ; — impurities B, C, D, E : for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent) ; — any other impurity : not more than half the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent) ; — total: not more than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent) ; — disregard limit : 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). Heavy metals (2.4.8) : maximum 20 ppm. Dissolve 0.5 g in dilute acetic acid R and dilute to 30 ml with the same solvent. Add 2 ml of water R instead of 2 ml General Notices (1) apply to all monographs and other texts

of buffer solution pH 3.5 R. The filtrate complies with limit test E. Prepare the standard using 10 ml of lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32) : maximum 1.0 per cent, determined on 1.000 g by drying under vacuum at 120 °C. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g in a platinum crucible. ASSAY Dissolve 0.300 g in 80 ml of glacial acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 33.14 mg of C17H18FN3O3. STORAGE In an airtight container, protected from light. IMPURITIES Specified impurities : A, B, C, D, E. Other detectable impurities : F.

A. R = Cl : 7-chloro-1-cyclopropyl-6-fluoro-4-oxo-1,4dihydroquinoline-3-carboxylic acid (fluoroquinolonic acid), C. R = NH-[CH2]2-NH2 : 7-[(2-aminoethyl)amino]-1cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3carboxylic acid (ethylenediamine compound),

B. R = CO2H, R′ = H : 1-cyclopropyl-4-oxo-7-(piperazin-1yl)-1,4-dihydroquinoline-3-carboxylic acid (desfluoro compound), E. R = H, R′ = F : 1-cyclopropyl-6-fluoro-7-(piperazin-1yl)quinolin-4(1H)-one (decarboxylated compound), F. R = CO2H, R′ = OH : 1-cyclopropyl-6-hydroxy-4-oxo-7(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid,

D. 7-chloro-1-cyclopropyl-4-oxo-6-(piperazin-1-yl)-1,4dihydroquinoline-3-carboxylic acid. 1301

Ciprofloxacin hydrochloride


01/2005:0888 Limit : — impurity A : any spot corresponding to impurity A is not more intense than the principal spot in the chromatogram CIPROFLOXACIN HYDROCHLORIDE obtained with the reference solution (0.2 per cent). Related substances. Liquid chromatography (2.2.29). Ciprofloxacini hydrochloridum Test solution. Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (a). Dissolve 25.0 mg of ciprofloxacin hydrochloride CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (b). Dissolve 5 mg of ciprofloxacin hydrochloride for peak identification CRS in the mobile phase and dilute to 10.0 ml with the mobile phase. C17H19ClFN3O3 Mr 367.8 Reference solution (c). Dilute 1.0 ml of the test solution to DEFINITION 50.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. 1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4Column : dihydroquinoline-3-carboxylic acid hydrochloride. — size : l = 0.25 m, Ø = 4.6 mm ; Content : 98.0 per cent to 102.0 per cent (anhydrous substance). — stationary phase : base-deactivated octadecylsilyl silica gel for chromatography R (5 µm) ; CHARACTERS — temperature : 40 °C. Appearance : pale yellow, crystalline powder, slightly Mobile phase : mix 13 volumes of acetonitrile R and hygroscopic. 87 volumes of a 2.45 g/l solution of phosphoric acid R, Solubility : soluble in water, slightly soluble in methanol, previously adjusted to pH 3.0 with triethylamine R. very slightly soluble in ethanol, practically insoluble in Flow rate : 1.5 ml/min. acetone, in ethyl acetate and in methylene chloride. Detection : spectrophotometer at 278 nm. IDENTIFICATION Injection : 50 µl. A. Infrared absorption spectrophotometry (2.2.24). Run time : twice the retention time of ciprofloxacin. Preparation : discs. Relative retention with reference to ciprofloxacin (retention time = about 9 min) : impurity E = about 0.4 ; Comparison : ciprofloxacin hydrochloride CRS. impurity F = about 0.5 ; impurity B = about 0.6 ; B. 0.1 g gives reaction (b) of chlorides (2.3.1). impurity C = about 0.7 ; impurity D = about 1.2. TESTS System suitability : reference solution (b) : Solution S. Dissolve 0.5 g in carbon dioxide-free water R — resolution : minimum 1.3 between the peaks due to and dilute to 20 ml with the same solvent. impurity B and impurity C. Appearance of solution. The solution is clear (2.2.1) and Limits : not more intensely coloured than reference solution GY5 — correction factors : for the calculation of contents, (2.2.2, Method II). multiply the peak areas of the following impurities by the corresponding correction factor : impurity B = 0.7 ; Dilute 10 ml of solution S to 20 ml with carbon dioxide-free impurity C = 0.6 ; impurity D = 1.4 ; impurity E = 6.7; use water R. the chromatogram obtained with reference solution (b) pH (2.2.3) : 3.5 to 4.5 for solution S. and the type chromatogram supplied with the CRS to Impurity A. Thin-layer chromatography (2.2.27). identify the corresponding peaks; Test solution. Dissolve 50 mg of the substance to be — impurities B, C, D, E : for each impurity, not more than examined in water R and dilute to 5 ml with the same solvent. the area of the principal peak in the chromatogram Reference solution. Dissolve 10 mg of ciprofloxacin obtained with reference solution (c) (0.2 per cent); impurity A CRS in a mixture of 0.1 ml of dilute ammonia R1 — any other impurity : not more than half the area of and 90 ml of water R and dilute to 100 ml with water R. the principal peak in the chromatogram obtained with Dilute 2 ml of the solution to 10 ml with water R. reference solution (c) (0.1 per cent); Plate : TLC silica gel F254 plate R. — total : not more than 2.5 times the area of the principal Application : 5 µl. peak in the chromatogram obtained with reference solution (c) (0.5 per cent); At the bottom of a chromatographic tank, place an evaporating dish containing 50 ml of concentrated — disregard limit : 0.25 times the area of the principal peak ammonia R. Expose the plate to the ammonia vapour for in the chromatogram obtained with reference solution (c) 15 min in the closed tank. Withdraw the plate, transfer (0.05 per cent). to a second chromatographic tank and proceed with Heavy metals (2.4.8) : maximum 20 ppm. development. Dissolve 0.25 g in water R and dilute to 30 ml with the same Mobile phase : acetonitrile R, concentrated ammonia R, solvent. Carry out the prefiltration. The filtrate complies methanol R, methylene chloride R (10:20:40:40 V/V/V/V). with limit test E. Prepare the standard using 5 ml of lead Development : over 3/4 of the plate. standard solution (1 ppm Pb) R. Drying : in air. Water (2.5.12) : maximum 6.7 per cent, determined on 0.200 g. Detection : examine in ultraviolet light at 254 nm. 1302


Cisapride monohydrate


Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g in a platinum crucible. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modifications. Injection : 10 µl ; inject the test solution and reference solution (a). Calculate the percentage content of C17H19ClFN3O3.

01/2005:0995

CISAPRIDE MONOHYDRATE Cisapridum monohydricum

STORAGE In an airtight container, protected from light. IMPURITIES Specified impurities : A, B, C, D, E. Other detectable impurities : F.

C23H29ClFN3O4,H2O

Mr 484.0

DEFINITION Cisapride monohydrate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of 4-amino-5-chloro-N-[(3RS,4SR)-1-[3-(4-fluorophenoxy)propyl]3-methoxypiperidin-4-yl]-2-methoxybenzamide, calculated with reference to the anhydrous substance. CHARACTERS A white or almost white powder, practically insoluble in water, freely soluble in dimethylformamide, soluble in methylene chloride, sparingly soluble in methanol. It shows polymorphism.

A. R = Cl : 7-chloro-1-cyclopropyl-6-fluoro-4-oxo-1,4dihydroquinoline-3-carboxylic acid (fluoroquinolonic acid), C. R = NH-[CH2]2-NH2 : 7-[(2-aminoethyl)amino]-1cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3carboxylic acid (ethylenediamine compound),

B. R = CO2H, R′ = H : 1-cyclopropyl-4-oxo-7-(piperazin-1yl)-1,4-dihydroquinoline-3-carboxylic acid (desfluoro compound), E. R = H, R′ = F : 1-cyclopropyl-6-fluoro-7-(piperazin-1yl)quinolin-4(1H)-one (decarboxylated compound), F. R = CO2H, R′ = OH : 1-cyclopropyl-6-hydroxy-4-oxo-7(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid,

D. 7-chloro-1-cyclopropyl-4-oxo-6-(piperazin-1-yl)-1,4dihydroquinoline-3-carboxylic acid. General Notices (1) apply to all monographs and other texts

IDENTIFICATION Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cisapride monohydrate CRS. Examine the substances prepared as discs. If the spectra obtained show differences, dissolve the substance to be examined and the reference substance separately in the minimum volume of methanol R, evaporate to dryness in a current of air and record new spectra using the residues. TESTS Solution S. Dissolve 0.20 g in methylene chloride R and dilute to 20.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution BY6 (2.2.2, Method II). Optical rotation (2.2.7). The angle of optical rotation, determined on solution S, is − 0.1° to + 0.1°. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 0.100 g of the substance to be examined in methanol R and dilute to 10.0 ml with the same solvent. Reference solution (a). Dissolve 5.0 mg of cisapride monohydrate CRS and 40.0 mg of haloperidol CRS in methanol R and dilute to 100.0 ml with the same solvent. Reference solution (b). Dilute 5.0 ml of the test solution to 100.0 ml with methanol R. Dilute 1.0 ml of this solution to 10.0 ml with methanol R. The chromatographic procedure may be carried out using : — a stainless steel column 0.1 m long and 4.0 mm in internal diameter packed with base-deactivated octadecylsilyl silica gel for chromatography R (3 µm), — as mobile phase at a flow rate of 1.2 ml/min : Mobile phase A. A 20 g/l solution of tetrabutylammonium hydrogen sulphate R, Mobile phase B. Methanol R, 1303

Cisapride tartrate


Time (min)



Comment

0 - 20

80 → 55

20 → 45

linear gradient

20 - 21

55 → 5

45 → 95

switch to next step

21 - 25

5

95

isocratic

25 - 26

5 → 80

95 → 20


26 - 30

80

20

re-equilibration

30 = 0

80

20

restart gradient

— as detector a spectrophotometer set at 275 nm. Equilibrate the column with the initial mobile phase composition for at least 5 min. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 10 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder.

A. R1 = R3 = H, R2 = OCH3 : 4-amino-5-chloro-2-methoxyN-[(3RS,4SR)-3-methoxy-1-(3-phenoxypropyl)piperidin4-yl]benzamide, B. R1 = F, R2 = R3 = H : 4-amino-5-chloro-N-[1[3-(4-fluorophenoxy)propyl]piperidin-4-yl]-2methoxybenzamide, C. R1 = F, R2 = H, R3 = OCH3 : 4-amino-5-chloro-N-[(3RS, 4RS)-1-[3-(4-fluorophenoxy)propyl]-3-methoxypiperidin4-yl-2-methoxybenzamide, E. R1 = F, R2 = OH, R3 = H : 4-amino-5-chloro-N-[(3RS,4SR)1-[3-(4-fluorophenoxy)propyl]-3-hydroxypiperidin-4-yl]-2methoxybenzamide,

Inject 10 µl of reference solution (a). When the chromatogram is recorded in the prescribed conditions, the retention times are : cisapride about 15 min and haloperidol about 16 min. The test is not valid unless the resolution between the peaks due to cisapride and haloperidol is at least 2.5. If necessary, adjust the concentration of methanol in the mobile phase or adjust the time programme for the linear gradient. Inject separately 10 µl of methanol R as a blank, 10 µl of the test solution and 10 µl of reference solution (b). In the chromatogram obtained with the test solution : the area D. 4-[(4-amino-5-chloro-2-methoxybenzoyl)amino]-5of any peak, apart from the principal peak, is not greater chloro-N-[(3RS,4SR)-1-[3-(4-fluorophenoxy)propyl]-3than the area of the principal peak in the chromatogram methoxypiperidin-4-yl]-2-methoxybenzamide. obtained with reference solution (b) (0.5 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is 01/2005:1503 not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per CISAPRIDE TARTRATE cent). Disregard any peak due to the solvent (blank) and any peak with an area less than 0.1 times the area of the Cisapridi tartras principal peak in the chromatogram obtained with reference solution (b). Water (2.5.12) : 3.4 per cent to 4.0 per cent, determined on 0.500 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g in a platinum crucible. ASSAY

C27H35ClFN3O10

Dissolve 0.350 g in 70 ml of a mixture of 1 volume of anhydrous acetic acid R and 7 volumes of methyl ethyl ketone R and titrate with 0.1 M perchloric acid. Determine the end-point potentiometrically (2.2.20).

DEFINITION Cisapride tartrate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of 4-amino-5-chloro-N-[(3RS,4SR)-1-[3-(4-fluorophenoxy)propyl]3-methoxypiperidin-4-yl]-2-methoxybenzamide (2R,3R)-2,3-dihydroxybutanedioate, calculated with reference to the dried substance.

1 ml of 0.1 M perchloric acid is equivalent to 46.60 mg of C23H29ClFN3O4. STORAGE Store protected from light. IMPURITIES Specified impurities : A, B, C, E. Other detectable impurities : D. 1304

Mr 616.0

CHARACTERS A white or almost white powder, slightly soluble in water, freely soluble in dimethylformamide, slightly soluble in methanol, very slightly soluble in alcohol. It shows polymorphism. IDENTIFICATION A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cisapride tartrate CRS. Examine the substances prepared See the information section on general monographs (cover pages)

Cisapride tartrate


chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.25 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent). Disregard any peak due to the solvent (blank) and any peak with an area less than 0.2 times the area of the TESTS principal peak in the chromatogram obtained with reference Specific optical rotation (2.2.7). Dissolve 0.100 g in solution (b). methanol R and dilute to 25.0 ml with the same solvent. The Loss on drying (2.2.32). Not more than 0.5 per cent, specific optical rotation is + 5.0 to + 10.0, calculated with determined on 1.000 g by drying in an oven at 100 °C to reference to the dried substance. 105 °C. Related substances. Examine by liquid chromatography Sulphated ash (2.4.14). Not more than 0.1 per cent, (2.2.29). determined on 1.0 g in a platinum crucible. Test solution. Dissolve 0.100 g of the substance to be examined in a mixture of 10 volumes of water R and ASSAY 90 volumes of methanol R, with gentle warming if necessary, Dissolve 0.500 g in 70 ml of anhydrous acetic acid R and and dilute to 10.0 ml with the same mixture of solvents. titrate with 0.1 M perchloric acid. Determine the end-point Reference solution (a). Dissolve 5.0 mg of cisapride potentiometrically (2.2.20). tartrate CRS and 30.0 mg of haloperidol CRS in a mixture 1 ml of 0.1 M perchloric acid is equivalent to 61.60 mg of of 10 volumes of water R and 90 volumes of methanol R and C H ClFN O . 27 35 3 10 dilute to 100.0 ml with the same mixture of solvents. STORAGE Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with a mixture of 10 volumes of water R and Store protected from light. 90 volumes of methanol R. Dilute 5.0 ml of the solution to 20.0 ml with the same mixture of solvents. IMPURITIES Specified impurities : A, C. The chromatographic procedure may be carried out using : — a stainless steel column 0.1 m long and 4.0 mm in internal Other detectable impurities : B, D, E. diameter packed with base-deactivated octadecylsilyl silica gel for chromatography R (3 µm), — as mobile phase at a flow rate of 1.2 ml/min : Mobile phase A. A 20 g/l solution of tetrabutylammonium hydrogen sulphate R, Mobile phase B. Methanol R, as discs. If the spectra obtained show differences, dissolve the substance to be examined and the reference substance separately in the minimum volume of alcohol R, evaporate to dryness in a current of air and record new spectra using the residues. B. It complies with the test for specific optical rotation (see Tests).

Time (min)



Comment

0 - 20

80 → 55

20 → 45

linear gradient

20 - 21

55 → 5

45 → 95

switch to next step

21 - 25

5

95

isocratic

25 - 26

5 → 80

95 → 20


26 - 30

80

20

re-equilibration

30 = 0

80

20

restart gradient

A. R1 = R3 = H, R2 = OCH3 : 4-amino-5-chloro-2-methoxyN-[(3RS,4SR)-3-methoxy-1-(3-phenoxypropyl)piperidin4-yl]benzamide, B. R1 = F, R2 = R3 = H : 4-amino-5-chloro-N-[1[3-(4-fluorophenoxy)propyl]piperidin-4-yl]-2methoxybenzamide, C. R1 = F, R2 = H, R3 = OCH3 : 4-amino-5-chloro-N-[(3RS, 4RS)-1-[3-(4-fluorophenoxy)propyl]-3-methoxypiperidin4-yl]-2-methoxybenzamide,

— as detector a spectrophotometer set at 275 nm. E. R1 = F, R2 = OH, R3 = H : 4-amino-5-chloro-N-[(3RS,4SR)Equilibrate the column with the initial mobile phase 1-[3-(4-fluorophenoxy)propyl]-3-hydroxypiperidin-4-yl]-2composition for at least 5 min. methoxybenzamide. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 10 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder. Inject 10 µl of reference solution (a). When the chromatogram is recorded in the prescribed conditions, the retention times are : cisapride about 15 min and haloperidol about 16 min. The test is not valid unless the resolution between the peaks due to cisapride and haloperidol is at least 2.5. If necessary, adjust the concentration of methanol in the mobile phase or adjust the time programme for the linear gradient. Inject separately 10 µl of a mixture of 10 volumes of water R D. 4-[(4-amino-5-chloro-2-methoxybenzoyl)amino]-5and 90 volumes of methanol R as a blank, 10 µl of the chloro-N-[(3RS,4SR)-1-[3-(4-fluorophenoxy)propyl]-3test solution and 10 µl of reference solution (b). In the methoxypiperidin-4-yl]-2-methoxybenzamide, General Notices (1) apply to all monographs and other texts

1305

Cisplatin


01/2005:0599 Reference solution (b). Dilute 1 ml of solution S2 to 50 ml with dimethylformamide R. CISPLATIN Apply separately to the plate 2.5 µl of test solution (a), 2.5 µl of reference solution (a), 5 µl of test solution (b) and 5 µl of reference solution (b). Develop over a path of 15 cm using Cisplatinum a mixture of 10 volumes of acetone R and 90 volumes of dimethylformamide R. Allow the plate to dry in air and spray with a 50 g/l solution of stannous chloride R in a mixture of equal volumes of dilute hydrochloric acid R and water R. After 1 h, the chromatogram obtained with test [PtCl2(NH3)2] Mr 300.0 solution (b) shows no spot with an Rf value less than that of the principal spot and any spot with an Rf value greater than DEFINITION that of the principal spot is not more intense than the spot in the chromatogram obtained with reference solution (b). Cisplatin contains not less than 97.0 per cent and not more than the equivalent of 102.0 per cent of Silver. Not more than 250 ppm of Ag, determined by atomic cis-diamminedi-chloroplatinum (II). absorption spectrometry (2.2.23, Method I). Test solution. Dissolve 0.100 g of the substance to be CHARACTERS A yellow powder or yellow or orange-yellow crystals, slightly examined in 15 ml of nitric acid R, heating to 80 °C. Cool and dilute to 25.0 ml with water R. soluble in water, sparingly soluble in dimethylformamide, Reference solutions. To suitable volumes (10 ml to 30 ml) of practically insoluble in alcohol. silver standard solution (5 ppm Ag) R add 50 ml of nitric It decomposes with blackening at about 270 °C. acid R and dilute to 100.0 ml with water R. Carry out identification test B, the tests (except that for Measure the absorbance at 328 nm using a silver silver) and the assay protected from light. hollow-cathode lamp as source of radiation, a fuel-lean IDENTIFICATION air-acetylene flame and, preferably, a spectral slit width of 0.5 nm. Carry out a blank determination. First identification : A, B. Second identification : B, C. ASSAY A. Examine by infrared absorption spectrophotometry Examine by liquid chromatography (2.2.29). (2.2.24), comparing with the spectrum obtained with cisplatin CRS. Examine the substances prepared as discs Test solution. Dissolve 50.0 mg of the substance to be examined in a 9 g/l solution of sodium chloride R and dilute in potassium bromide R. to 100.0 ml with the same solvent. B. Examine the chromatograms obtained in the test Reference solution. Dissolve 50.0 mg of cisplatin CRS in a for related substances. The principal spot in the 9 g/l solution of sodium chloride R and dilute to 100.0 ml chromatogram obtained with test solution (a) is similar with the same solvent. in position, colour and size to the principal spot in the The chromatographic procedure may be carried out using : chromatogram obtained with reference solution (a). C. Add 50 mg to 2 ml of dilute sodium hydroxide solution R — a column 0.25 m long and 4.6 mm in internal diameter packed with strong-anion-exchange silica gel for in a glass dish. Evaporate to dryness. Dissolve the residue chromatography R (10 µm), in a mixture of 0.5 ml of nitric acid R and 1.5 ml of hydrochloric acid R. Evaporate to dryness. The residue — as mobile phase at a flow rate of 1.2 ml/min a mixture of is orange. Dissolve the residue in 0.5 ml of water R and 10 volumes of a 9 g/l solution of sodium chloride R and add 0.5 ml of ammonium chloride solution R. A yellow, 90 volumes of methanol R, crystalline precipitate is formed. — as detector a spectrophotometer set at 220 nm. Use a sample loop. Inject separately 20 µl of the test solution TESTS and 20 µl of the reference solution. Solution S1. Dissolve 25 mg in a 9 g/l solution of sodium chloride R prepared with carbon dioxide-free water R and STORAGE dilute to 25 ml with the same solvent. Store in an airtight container, protected from light. Solution S2. Dissolve 0.20 g in dimethylformamide R and dilute to 10 ml with the same solvent. 01/2005:0455 Appearance of solution S1. Solution S1 is clear (2.2.1) and not more intensely coloured than reference solution GY5 (2.2.2, Method II). CITRIC ACID, ANHYDROUS Appearance of solution S2. Solution S2 is clear (2.2.1). Acidum citricum anhydricum pH (2.2.3). The pH of solution S1, measured immediately after preparation, is 4.5 to 6.0. Related substances. Examine by thin-layer chromatography (2.2.27), using cellulose for chromatography R1 as the coating substance. Activate the plate by heating at 150 °C C6 H 8 O 7 Mr 192.1 for 1 h. Test solution (a). Dilute 1 ml of solution S2 to 10 ml with DEFINITION dimethylformamide R. Anhydrous citric acid contains not less than 99.5 per cent Test solution (b). Use solution S2. and not more than the equivalent of 100.5 per cent of 2-hydroxypropane-1,2,3-tricarboxylic acid, calculated with Reference solution (a). Dissolve 10 mg of cisplatin CRS in reference to the anhydrous substance. 5 ml of dimethylformamide R. 1306


Citric acid monohydrate


CHARACTERS A white, crystalline powder, colourless crystals or granules, very soluble in water, freely soluble in alcohol. It melts at about 153 °C with decomposition. IDENTIFICATION First identification : B, E. Second identification : A, C, D, E. A. Dissolve 1 g in 10 ml of water R. The solution is strongly acidic (2.2.4). B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with anhydrous citric acid CRS after drying both the substance to be examined and the reference substance at 100-105 °C for 2 h. C. Add about 5 mg to a mixture of 1 ml of acetic anhydride R and 3 ml of pyridine R. A red colour develops. D. Dissolve 0.5 g in 5 ml of water R, neutralise using 1 M sodium hydroxide (about 7 ml), add 10 ml of calcium chloride solution R and heat to boiling. A white precipitate is formed. E. It complies with the test for water (see Tests). TESTS Appearance of solution. Dissolve 2.0 g in water R and dilute to 10 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y7, BY7 or GY7 (2.2.2, Method II). Readily carbonisable substances. To 1.0 g in a cleaned test tube add 10 ml of sulphuric acid R and immediately heat the mixture in a water-bath at 90 ± 1 °C for 60 min. Immediately cool rapidly. The solution is not more intensely coloured than a mixture of 1 ml of red primary solution and 9 ml of yellow primary solution (2.2.2, Method I). Oxalic acid. Dissolve 0.80 g in 4 ml of water R. Add 3 ml of hydrochloric acid R and 1 g of zinc R in granules. Boil for 1 min. Allow to stand for 2 min. Transfer the supernatant liquid to a test-tube containing 0.25 ml of a 10 g/l solution of phenylhydrazine hydrochloride R and heat to boiling. Cool rapidly, transfer to a graduated cylinder and add an equal volume of hydrochloric acid R and 0.25 ml of a 50 g/l solution of potassium ferricyanide R. Shake and allow to stand for 30 min. Any pink colour in the solution is not more intense than that in a standard prepared at the same time in the same manner using 4 ml of a 0.1 g/l solution of oxalic acid R (360 ppm, calculated as anhydrous oxalic acid). Sulphates (2.4.13). Dissolve 2.0 g in distilled water R and dilute to 30 ml with the same solvent. The solution complies with the limit test for sulphates (150 ppm). Aluminium (2.4.17). If intended for use in the manufacture of dialysis solutions, it complies with the test for aluminium. Dissolve 20 g in 100 ml of water R and add 10 ml of acetate buffer solution pH 6.0 R. The solution complies with the limit test for aluminium (0.2 ppm). Use as the reference solution a mixture of 2 ml of aluminium standard solution (2 ppm Al) R, 10 ml of acetate buffer solution pH 6.0 R and 98 ml of water R. To prepare the blank use a mixture of 10 ml of acetate buffer solution pH 6.0 R and 100 ml of water R. Heavy metals (2.4.8). Dissolve 5.0 g in several portions in 39 ml of dilute sodium hydroxide solution R and dilute to 50 ml with distilled water R. 12 ml complies with limit test A for heavy metals (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. Water (2.5.12). Not more than 1.0 per cent, determined on 2.000 g by the semi-micro determination of water. General Notices (1) apply to all monographs and other texts

Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. Bacterial endotoxins (2.6.14) : less than 0.5 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. ASSAY Dissolve 0.550 g in 50 ml of water R. Titrate with 1 M sodium hydroxide, using 0.5 ml of phenolphthalein solution R as indicator. 1 ml of 1 M sodium hydroxide is equivalent to 64.03 mg of C6H8O7. LABELLING The label states : — where applicable, that the substance is free from bacterial endotoxins, — where applicable, that the substance is intended for use in the manufacture of dialysis solutions. 01/2005:0456

CITRIC ACID MONOHYDRATE Acidum citricum monohydricum

C6H8O7,H2O

Mr 210.1

DEFINITION Citric acid monohydrate contains not less than 99.5 per cent and not more than the equivalent of 100.5 per cent of 2-hydroxypropane-1,2,3-tricarboxylic acid, calculated with reference to the anhydrous substance. CHARACTERS A white, crystalline powder, colourless crystals or granules, efflorescent, very soluble in water, freely soluble in alcohol. IDENTIFICATION First identification : B, E. Second identification : A, C, D, E. A. Dissolve 1 g in 10 ml of water R. The solution is strongly acidic (2.2.4). B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with citric acid monohydrate CRS after drying both the substance to be examined and the reference substance at 100-105 °C for 2 h. C. Add about 5 mg to a mixture of 1 ml of acetic anhydride R and 3 ml of pyridine R. A red colour develops. D. Dissolve 0.5 g in 5 ml of water R, neutralise using 1 M sodium hydroxide (about 7 ml), add 10 ml of calcium chloride solution R and heat to boiling. A white precipitate is formed. E. It complies with the test for water (see Tests). TESTS Appearance of solution. Dissolve 2.0 g in water R and dilute to 10 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y7, BY7 or GY7 (2.2.2, Method II). 1307

Citronella oil


Readily carbonisable substances. To 1.0 g in a cleaned test tube add 10 ml of sulphuric acid R and immediately heat the mixture in a water-bath at 90 ± 1 °C for 60 min. Immediately cool rapidly. The solution is not more intensely coloured than a mixture of 1 ml of red primary solution and 9 ml of yellow primary solution (2.2.2, Method I). Oxalic acid. Dissolve 0.80 g in 4 ml of water R. Add 3 ml of hydrochloric acid R and 1 g of zinc R in granules. Boil for 1 min. Allow to stand for 2 min. Transfer the supernatant liquid to a test-tube containing 0.25 ml of a 10 g/l solution of phenylhydrazine hydrochloride R and heat to boiling. Cool rapidly, transfer to a graduated cylinder and add an equal volume of hydrochloric acid R and 0.25 ml of a 50 g/l solution of potassium ferricyanide R. Shake and allow to stand for 30 min. Any pink colour in the solution is not more intense than that in a standard prepared at the same time in the same manner using 4 ml of a 0.1 g/l solution of oxalic acid R (360 ppm, calculated as anhydrous oxalic acid).

01/2005:1609

CITRONELLA OIL Citronellae aetheroleum DEFINITION Oil obtained by steam distillation from the fresh or partially dried aerial parts of Cymbopogon winterianus Jowitt. CHARACTERS Pale yellow to brown-yellow liquid, with a very strong odour of citronellal.

IDENTIFICATION First identification : B. Second identification : A. A. Thin-layer chromatography (2.2.27). Test solution. Dilute 0.1 g of citronella oil in 10.0 ml of Sulphates (2.4.13). Dissolve 2.0 g in distilled water R and alcohol R. dilute to 30 ml with the same solvent. The solution complies with the limit test for sulphates (150 ppm). Reference solution. Dilute 20 µl of citronellal R in 10.0 ml of alcohol R. Aluminium (2.4.17). If intended for use in the manufacture Plate : TLC silica gel plate R. of dialysis solutions, it complies with the test for aluminium. Dissolve 20 g in 100 ml of water R and add 10 ml of acetate Mobile phase : ethyl acetate R, toluene R (10:90 V/V). buffer solution pH 6.0 R. The solution complies with the Application : 5 µl, as bands. limit test for aluminium (0.2 ppm). Use as the reference Development : over a path of 15 cm. solution a mixture of 2 ml of aluminium standard solution Drying : in air. (2 ppm Al) R, 10 ml of acetate buffer solution pH 6.0 R and Detection : spray with anisaldehyde solution R and heat 98 ml of water R. To prepare the blank use a mixture of 10 ml at 100-105 °C for 10 min. Examine in ultraviolet light at of acetate buffer solution pH 6.0 R and 100 ml of water R. 365 nm. Heavy metals (2.4.8). Dissolve 5.0 g in several portions in Result : see below the sequence of the zones present in 39 ml of dilute sodium hydroxide solution R and dilute to the chromatograms obtained with the reference and test 50 ml with distilled water R. 12 ml complies with limit test A solutions. Furthermore, other zones are present in the for heavy metals (10 ppm). Prepare the standard using lead chromatogram obtained with the test solution. standard solution (1 ppm Pb) R. Water (2.5.12) : 7.5 per cent to 9.0 per cent, determined on 0.500 g by the semi-micro determination of water.

Top of the plate Citronellal : a violet zone

Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. Bacterial endotoxins (2.6.14) : less than 0.5 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins.

A zone similar in colour to the citronellal zone An orange zone (citronellolgeraniol)

Reference solution

Test solution

B. Examine the chromatograms obtained in the test for chromatographic profile. Results : the characteristic peaks in the chromatogram obtained with the test solution are similar in retention ASSAY time to those in the chromatogram obtained with the reference solution. Neral and geranial may be absent in Dissolve 0.550 g in 50 ml of water R. Titrate with 1 M sodium the chromatogram obtained with the test solution. hydroxide, using 0.5 ml of phenolphthalein solution R as indicator. TESTS Relative density (2.2.5) : 0.881 to 0.895. 1 ml of 1 M sodium hydroxide is equivalent to 64.03 mg of C6H8O7. Refractive index (2.2.6) : 1.463 to 1.475. Optical rotation (2.2.7) : − 4° to + 1.5°. STORAGE Chromatographic profile. Gas chromatography (2.2.28) : use the normalisation procedure. In an airtight container. Test solution. The substance to be examined. Reference solution. Dilute 25 µl of limonene R, 100 µl of LABELLING citronellal R, 25 µl of citronellyl acetate R, 25 µl of citral R, 25 µl of geranyl acetate R, 25 µl of citronellol R and 100 µl The label states : of geraniol R in 5 ml of hexane R. — where applicable, that the substance is free from bacterial Column : endotoxins, — material : fused silica, — size : l = 60 m, Ø = 0.25 mm, — where applicable, that the substance is intended for use — stationary phase : macrogol 20 000 R (0.2 µm). in the manufacture of dialysis solutions. 1308


Clarithromycin


DEFINITION (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-4-[(2,6-Dideoxy3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]14-ethyl-12,13-dihydroxy-7-methoxy-3,5,7,9,11,13hexamethyl-6-[[3,4,6-trideoxy-3-(dimethylamino)-β-Dxylo-hexopyranosyl]oxy]oxacyclotetradecane-2,10-dione (6-O-methylerythromycin A). Content : 96.0 per cent to 102.0 per cent (anhydrous substance).

Carrier gas : helium for chromatography R. Flow rate : 1.0 ml/min. Split ratio : 1:100. Temperature : Time (min) Column

Temperature (°C)

0-2

80

2 - 26

80 → 150

26 - 42

150 → 185

42 - 49

185 → 250

Injection port

260

Detector

260

CHARACTERS Appearance : white or almost white, crystalline powder. Solubility : practically insoluble in water, soluble in acetone and in methylene chloride, slightly soluble in methanol.

Detection : flame ionisation. Injection : 1 µl of the reference solution, 0.2 µl of the test solution. Elution order : the order indicated in the composition of the reference solution. Record the retention times of these substances. System suitability : reference solution : — resolution : minimum of 1.2 between the peaks due to geranyl acetate and citronellol. Using the retention times determined from the chromatogram obtained with the reference solution, locate the components of the reference solution in the chromatogram obtained with the test solution. Determine the percentage content of each of these components. The percentages are within the following values : — limonene : 1.0 per cent to 5.0 per cent, — citronellal : 30.0 per cent to 45.0 per cent, — citronellyl acetate : 2.0 per cent to 4.0 per cent, — neral: less than 2.0 per cent, — geranial : less than 2.0 per cent, — geranyl acetate : 3.0 per cent to 8.0 per cent, — citronellol : 9.0 per cent to 15.0 per cent, — geraniol : 20.0 per cent to 25.0 per cent.

IDENTIFICATION Infrared absorption spectrophotometry (2.2.24). Comparison : clarithromycin CRS.

TESTS Solution S. Dissolve 0.500 g in methylene chloride R and dilute to 50.0 ml with the same solvent. Appearance of solution. Solution S is clear or not more opalescent than reference suspension II (2.2.1) and not more intensely coloured than reference solution Y7 (2.2.2, Method II). Specific optical rotation (2.2.7) : − 94 to − 102 (anhydrous substance), determined on solution S. Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 75.0 mg of the substance to be examined in 25 ml of acetonitrile R1 and dilute to 50.0 ml with water R. Reference solution (a). Dissolve 75.0 mg of clarithromycin CRS in 25 ml of acetonitrile R1 and dilute to 50.0 ml with water R. Reference solution (b). Dilute 5.0 ml of reference solution (a) to 100.0 ml with a mixture of equal volumes of acetonitrile R1 and water R. Reference solution (c). Dilute 1.0 ml of reference solution (b) to 10.0 ml with a mixture of equal volumes of acetonitrile R1 and water R. Reference solution (d). Dissolve 15.0 mg of clarithromycin STORAGE for peak identification CRS in 5.0 ml of acetonitrile R1 and In a well-filled container, protected from light. dilute to 10.0 ml with water R. Blank solution. Dilute 25.0 ml of acetonitrile R1 to 50.0 ml 01/2005:1651 with water R and mix. corrected Column : — size : l = 0.10 m, Ø = 4.6 mm, CLARITHROMYCIN — stationary phase : octadecylsilyl silica gel for chromatography R (3.5 µm), Clarithromycinum — temperature : 40 °C. Mobile phase : — mobile phase A : a 4.76 g/l solution of potassium dihydrogen phosphate R adjusted to pH 4.4 with dilute phosphoric acid R or a 45 g/l solution of potassium hydroxide R, filtered through a C18 filtration kit, — mobile phase B : acetonitrile R1,

C38H69NO13

Mr 748


Time (min)



0 - 32

75 → 40

25 → 60

32 - 34

40

60

34 - 36

40 → 75

60 → 25

36 - 42

75

25

1309

Clarithromycin


Flow rate : 1.1 ml/min. Detection : spectrophotometer at 205 nm. Injection : 10 µl ; inject the blank solution, the test solution and reference solutions (b), (c) and (d). Relative retention with reference to clarithromycin (retention time = about 11 min) : impurity I = about 0.38 ; impurity A = about 0.42 ; impurity J = about 0.63 ; impurity L = about 0.74 ; impurity B = about 0.79 ; impurity M = about 0.81 ; impurity C = about 0.89 ; impurity D = about 0.96 ; impurity N = about 1.15 ; impurity E = about 1.27 ; impurity O = about 1.31 ; impurity F = about 1.33 ; impurity P = about 1.35 ; impurity K = about 1.59 ; impurity G = about 1.72 ; impurity H = about 1.82. System suitability : — symmetry factor : maximum 1.7 for the peak due to clarithromycin in the chromatogram obtained with reference solution (b), — peak-to-valley ratio : minimum 3.0, where Hp = height above the baseline of the peak due to impurity D and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to clarithromycin in the chromatogram obtained with reference solution (d). Limits : — correction factors : for the calculation of contents, multiply the peak areas of the following impurities by the corresponding correction factor : impurity G = 0.27 ; impurity H = 0.15 ; use the chromatogram supplied with clarithromycin for peak identification CRS to identify the peaks ; — any impurity : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (c) (1.0 per cent), and not more than 4 such peaks have an area greater than 0.8 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.4 per cent) ; — total: not more than 7 times the area of the principal peak in the chromatogram obtained with reference solution (c) (3.5 per cent) ; — disregard limit : 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.1 per cent) ; disregard the peaks eluting before impurity I and after impurity H. Heavy metals (2.4.8) : maximum 20 ppm. Dissolve 1.0 g in a mixture of 15 volumes of water R and 85 volumes of dioxan R and dilute to 20 ml with the same mixture of solvents. 12 ml of the solution complies with limit test B. Prepare the standard using lead standard solution (1 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a mixture of 15 volumes of water R and 85 volumes of dioxan R. Water (2.5.12) : maximum 2.0 per cent, determined on 0.500 g. Sulphated ash (2.4.14) : maximum 0.2 per cent, determined on 0.5 g. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modifications. Injection : test solution and reference solution (a). Calculate the percentage content of C38H69NO13. 1310

IMPURITIES Specified impurities : A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P.

A. R1 = CH3, R2 = OH, R3 = H : 2-demethyl-2-(hydroxymethyl)6-O-methylerythromycin A (clarithromycin F), B. R1 = R2 = R3 = H : 6-O-methyl-15-norerythromycin A, P. R1 = R3 = CH3, R2 = H : 4′,6-di-O-methylerythromycin A,

C. R1 = R2 = CH3, R3 = H : 6-O-methylerythromycin A (E)-9-oxime, G. R1 = R2 = R3 = CH3 : 6-O-methylerythromycin A (E)-9-(O-methyloxime), J. R1 = CH3, R2 = R3 = H : erythromycin A (E)-9-oxime, M. R1 = R3 = H, R2 = CH3 : 3″-N-demethyl-6-Omethylerythromycin A (E)-9-oxime,

D. R1 = R2 = R3 = H : 3″-N-demethyl-6-O-methylerythromycin A, E. R1 = R2 = CH3, R3 = H : 6,11-di-O-methylerythromycin A, F. R1 = R3 = CH3, R2 = H : 6,12-di-O-methylerythromycin A, H. R1 = CHO, R2 = R3 = H : 3″-N-demethyl-3′-N-formyl-6-Omethylerythromycin A,


Clary sage oil


01/2005:1850

CLARY SAGE OIL Salviae sclareae aetheroleum DEFINITION Essential oil obtained by steam distillation from the fresh or dried flowering stems of Salvia sclarea L. I. 3-O-decladinosyl-6-O-methylerythromycin A,

K. (1S,2R,5R,6S,7S,8R,9R,11Z)-2-ethyl-6-hydroxy-9methoxy-1,5,7,9,11,13-hexamethyl-8-[[3,4,6-trideoxy3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]-3, 15-dioxabicyclo[10.2.1]pentadeca-11,13-dien-4-one (3-O-decladinosyl-8,9:10,11-dianhydro-6-Omethylerythromycin A-9,12-hemiketal,

CHARACTERS Appearance : colourless to brownish-yellow liquid, usually pale yellow, with a characteristic odour. IDENTIFICATION First identification : B. Second identification : A. A. Thin-layer chromatography (2.2.27). Test solution. Dissolve 1 ml of the substance to be examined in toluene R and dilute to 10 ml with the same solvent. Reference solution. Dissolve 60 µl of linalol R, 200 µl of linalyl acetate R and 60 µl of α-terpineol R in toluene R and dilute to 10 ml with the same solvent. Plate : TLC silica gel plate R. Mobile phase : ethyl acetate R, toluene R (5:95 V/V). Application : 5 µl of the test solution and 10 µl of the reference solution, as bands. Development : over a path of 15 cm. Drying : in air. Detection : spray with vanillin reagent R and heat at 100-105 °C for 5-10 min ; examine in daylight within 5 min. Results : see below the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. Furthermore, other faint zones are present in the chromatogram obtained with the test solution. Top of the plate α-Terpineol : a dark violet zone

A dark violet zone

_______

L. R = H : 6-O-methylerythromycin A (Z)-9-oxime, O. R = CH3 : 6-O-methylerythromycin A (Z)-9-(O-methyloxime),

N. (10E)-10,11-didehydro-11-deoxy-6-O-methylerythromycin A. General Notices (1) apply to all monographs and other texts

_______

Linalyl acetate : a dark violet zone _______

A dark violet zone

Linalol: a dark violet zone

A dark violet zone

Reference solution

_______

Test solution

B. Examine the chromatograms obtained in the test for chromatographic profile. Results : the chromatogram obtained with the test solution shows 5 peaks similar in position to the 5 peaks in the chromatogram obtained with the reference solution. The 2 peaks corresponding to α- and β-thujone may be absent. TESTS Relative density (2.2.5) : 0.890 to 0.908. Refractive index (2.2.6) : 1.456 to 1.466. Optical rotation (2.2.7) : − 26° to − 10°. Acid value (2.5.1) : maximum 1.0. Chromatographic profile. Gas chromatography (2.2.28) : use the normalisation procedure. Test solution. The substance to be examined. 1311

Clazuril for veterinary use


Reference solution. To 1 g of hexane R, add 5 µl of thujone R, 5 µl of linalol R, 100 µl of linalyl acetate R, 10 µl of α-terpineol R and 25 mg (± 20 per cent) of sclareol R. Mix thoroughly by stirring. Column :

01/2005:1714

CLAZURIL FOR VETERINARY USE Clazurilum ad usum veterinarium

— material : fused silica, — size : l = 30 m (a film thickness of 1 µm may be used) to 60 m (a film thickness of 0.2 µm may be used), Ø = 0.25-0.53 mm, — stationary phase: macrogol 20 000 R. Carrier gas : helium for chromatography R. Split ratio : 1:100.

C17H10Cl2N4O2

Temperature : Time (min) Column

DEFINITION (2RS)-[2-Chloro-4-(3,5-dioxo-4,5-dihydro-1,2,4-triazin-2(3H)yl)phenyl](4-chlorophenyl)acetonitrile. Content : 99.0 per cent to 101.0 per cent (dried substance).

Temperature (°C)

0 - 10

60

10 - 75

60 → 190

75 - 120

190

Injection port

220

Detector

240

Mr 373.2

CHARACTERS Appearance : white or light yellow powder. Solubility : practically insoluble in water, freely soluble in dimethylformamide, slightly soluble in alcohol and in methylene chloride.

Detection : flame ionisation. Injection : 0.2 µl. Elution order : order indicated in the composition of the reference solution. Record the retention times of these substances.

IDENTIFICATION A. Melting point (2.2.14) : 199 °C to 203 °C. B. Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of clazuril.

System suitability : reference solution :

TESTS Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 20.0 mg of the substance to be Using the retention times determined from the chromatogram examined in a mixture of equal volumes of tetrahydrofuran R obtained with the reference solution, locate the components and water R and dilute to 20.0 ml with the same mixture of the reference solution in the chromatogram obtained of solvents. with the test solution (disregard any peak due to hexane). Thujone R is a mixture of α- and β-thujone. α-Thujone elutes Reference solution (a). Dissolve 5 mg of clazuril for system suitability CRS in a mixture of equal volumes of before β-thujone under the described conditions. tetrahydrofuran R and water R and dilute to 5.0 ml with Determine the percentage content of each of these the same mixture of solvents. components. Reference solution (b). Dilute 1.0 ml of the test Also determine the percentage content of germacrene-D. The solution to 100.0 ml with a mixture of equal volumes of tetrahydrofuran R and water R. Dilute 2.0 ml of this germacrene-D peak can be identified in the chromatogram solution to 10.0 ml with a mixture of equal volumes of obtained with the test solution by its relative retention of 1.23 with reference to linalol under the described operating tetrahydrofuran R and water R. conditions. Column : — size : l = 0.10 m, Ø = 4.6 mm, The percentages are within the following ranges : — stationary phase : octadecylsilyl silica gel for — α- and β-thujone : less than 0.2 per cent, chromatography R (3 µm), — linalol : 6.5 per cent to 24 per cent, — temperature : 35 °C. — linalyl acetate : 56 per cent to 78 per cent, Mobile phase : — α-terpineol: less than 5.0 per cent, — mobile phase A : mix 100 volumes of a 7.7 g/l solution of ammonium acetate R adjusted to pH 6.2 with a — germacrene-D : 1.0 per cent to 12 per cent, 10 per cent V/V solution of anhydrous formic acid R, 150 volumes of acetonitrile R and 750 volumes of — sclareol : 0.4 per cent to 2.6 per cent. water R, — mobile phase B : mix 100 volumes of a 7.7 g/l solution STORAGE of ammonium acetate R adjusted to pH 6.2 with a In an airtight container, protected from light, at a 10 per cent V/V solution of anhydrous formic acid R, temperature not exceeding 25 °C. 850 volumes of acetonitrile R and 50 volumes of water R, — resolution : minimum 1.5 between the peaks due to linalol and linalyl acetate,

1312


Clazuril for veterinary use


Time (min)



0 - 20

100 → 0

0 → 100

20 - 25

0

100

25 - 30

0 → 100

100 → 0

30 - 40

100

0

Flow rate : 1.0 ml/min. Detection : spectrophotometer at 230 nm. Injection : 5 µl. System suitability : reference solution (a) : — peak-to-valley ratio : minimum 1.5, where Hp = height above the baseline of the peak due to impurity G and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to clazuril, — the chromatogram obtained is concordant with the chromatogram supplied with clazuril for system suitability CRS. Limits : — correction factors : for the calculation of contents, multiply the peak areas of the following impurities by the corresponding correction factor : impurity G = 1.4 ; impurity H = 0.8 ; — any impurity : not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.2 per cent) ; — total: not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.6 per cent) ; — disregard limit : 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.05 per cent) ; disregard the peaks due to the solvents. Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 4 h. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g.

C. R = NH2 : (2RS)-2-[2-chloro-4-(3,5-dioxo-4,5-dihydro-1,2,4triazin-2(3H)-yl)phenyl]-2-(4-chlorophenyl)acetamide,

B. R = NH2 : 2-[3-chloro-4-[(RS)-(4-chlorophenyl)cyanomethyl]phenyl]-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide, D. R = N(CH3)2 : 2-[3-chloro-4-[(RS)-(4-chlorophenyl)cyanomethyl]phenyl]-N,N-dimethyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide, E. R = OCH3 : methyl 2-[3-chloro-4-[(RS)-(4chlorophenyl)cyanomethyl]phenyl]-3,5-dioxo-2,3, 4,5-tetrahydro-1,2,4-triazine-6-carboxylate, F. R = OC2H5 : ethyl 2-[3-chloro-4-[(RS)-(4chlorophenyl)cyanomethyl]phenyl]-3,5-dioxo-2,3, 4,5-tetrahydro-1,2,4-triazine-6-carboxylate,

G. 2-[3-chloro-4-(4-chlorobenzoyl)phenyl]-1,2,4-triazine-3, 5(2H,4H)-dione,

ASSAY Dissolve about 0.260 g in 35 ml of tetrahydrofuran R and add 35 ml of water R. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20). Carry out a blank titration. 1 ml of 0.1 M sodium hydroxide is equivalent to 37.32 mg of C17H10Cl2N4O2. STORAGE Protected from light. IMPURITIES

A. R = OH : (2RS)-[2-chloro-4-(3,5-dioxo-4,5-dihydro-1,2,4triazin-2(3H)-yl)phenyl](4-chlorophenyl)acetic acid, General Notices (1) apply to all monographs and other texts

H. [2-chloro-4-(3,5-dioxo-4,5-dihydro-1,2,4-triazin-2(3H)yl)phenyl][4-[[2-chloro-4-(3,5-dioxo-4,5-dihydro-1,2, 4-triazin-2(3H)-yl)phenyl]cyanomethyl]phenyl](4chlorophenyl)acetonitrile,

I. (Z)-2-[[3-chloro-4-[(RS)-(4-chlorophenyl)cyanomethyl]phenyl]diazanylidene]acetamide. 1313

Clebopride malate


01/2005:1303

CLEBOPRIDE MALATE Clebopridi malas

C24H30ClN3O7

Mr 508.0

DEFINITION Clebopride malate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of 4-aminoN-(1-benzylpiperidin-4-yl)-5-chloro-2-methoxybenzamide acid (RS)-2-hydroxybutanedioate, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, sparingly soluble in water and in methanol, slightly soluble in ethanol, practically insoluble in methylene chloride. It melts at about 164 °C, with decomposition. IDENTIFICATION First identification : B, C. Second identification : A, C, D. A. Dissolve 20.0 mg in water R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of the solution to 100.0 ml with water R. Examined between 230 nm and 350 nm (2.2.25), the solution shows two absorption maxima, at 270 nm and 307 nm. The specific absorbances at the maxima are 252 to 278 and 204 to 226, respectively. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with clebopride malate CRS. Examine the substances prepared as discs. C. Dissolve 20 mg in 1 ml of sulphuric acid R, add 1 ml of β-naphthol solution R1 and mix. The solution examined in daylight has a yellow colour with blue fluorescence. D. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm. Test solution. Dissolve 5 mg of the substance to be examined in ethanol R and dilute to 10 ml with the same solvent. Reference solution (a). Dissolve 5 mg of clebopride malate CRS in ethanol R and dilute to 10 ml with the same solvent. Reference solution (b). Dissolve 5 mg of clebopride malate CRS and 5 mg of metoclopramide hydrochloride CRS in ethanol R and dilute to 10 ml with the same solvent. Apply to the plate as bands 10 mm by 3 mm, 5 µl of each solution. Develop over a path of 15 cm using a mixture of 2 volumes of concentrated ammonia R, 14 volumes of acetone R, 14 volumes of methanol R and 70 volumes of toluene R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal band in the chromatogram obtained with the test solution is similar in position and size to the principal band in the 1314

chromatogram obtained with the reference solution (a). The identification is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated bands. TESTS Solution S. Dissolve 1.0 g in carbon dioxide-free water R and dilute to 100.0 ml with the same solvent. Appearance of solution. Examined immediately after preparation, solution S is clear (2.2.1) and colourless (2.2.2, Method I). pH (2.2.3). The pH of solution S is 3.8 to 4.2. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 0.10 g of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (b). Dissolve 10.0 mg of clebopride malate CRS and 10.0 mg of metoclopramide hydrochloride CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.12 m long and 4.0 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1 ml/min a mixture of 20 volumes of acetonitrile R and 80 volumes of a 1 g/l solution of heptane sulphonate sodium R adjusted to pH 2.5 with phosphoric acid R, — as detector a spectrophotometer set at 215 nm. Equilibrate the column with the mobile phase for 30 min. Inject 20 µl of reference solution (b). Adjust the sensitivity of the system so that the heights of the peaks in the chromatogram obtained are at least 30 per cent of the full scale of the recorder. The test is not valid unless the retention time of the second peak (clebopride) is about 15 min and the relative retention time of the first peak is about 0.45. Inject 20 µl of the test solution and 20 µl of reference solution (a). Continue the chromatography of the test solution for twice the retention time of the principal peak. In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak and the two peaks eluting within 2 min, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent) ; the sum of the areas of all peaks, apart from the principal peak and the two peaks eluting within 2 min, is not greater than three times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.3 per cent). Disregard any peak with an area less than 0.25 times that of the principal peak in the chromatogram obtained with reference solution (a). Chlorides. Prepare the solutions at the same time. Test solution. Dissolve 0.530 g of the substance to be examined in 20.0 ml of anhydrous acetic acid R, add 6 ml of dilute nitric acid R and dilute to 50.0 ml with water R. Reference solution. To 1.5 ml of 0.001 M hydrochloric acid, add 20.0 ml of anhydrous acetic acid R and 6 ml of dilute nitric acid R and dilute to 50.0 ml with water R. Transfer separately both solutions recently prepared to test tubes. Add to each tube 1 ml of silver nitrate solution R2. Allow to stand for 5 min protected from light. Examine the


Clemastine fumarate


tubes laterally against a black background. Any opalescence in the test solution is not more intense than that in the reference solution (100 ppm). Sulphates. Prepare the solutions at the same time.

01/2005:1190

CLEMASTINE FUMARATE Clemastini fumaras

Test solution. Dissolve 3.00 g of the substance to be examined in 20.0 ml of glacial acetic acid R, heating gently if necessary. Allow to cool and dilute to 50.0 ml with water R. Reference solution. To 9 ml of sulphate standard solution (10 ppm SO4) R1, add 6 ml of glacial acetic acid R. Into two test tubes introduce 1.5 ml of sulphate standard solution (10 ppm SO4) R1 and add 1 ml of a 250 g/l solution of barium chloride R. Shake and allow to stand for 1 min. To one of the tubes add 15 ml of the test solution and to the C25H30ClNO5 other one add 15 ml of the reference solution.

Mr 460.0

After 5 min, any opalescence in the tube containing the test solution is not more intense than that containing the reference solution (100 ppm). Heavy metals (2.4.8). 1.0 g complies with limit test D for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R.

DEFINITION Clemastine fumarate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of (2R)-2-[2-[(R)-1-(4-chlorophenyl)-1-phenylethoxy]ethyl]1-methylpyrrolidine (E)-butenedioate, calculated with reference to the dried substance.


CHARACTERS A white or almost white, crystalline powder, very slightly soluble in water, sparingly soluble in alcohol (70 per cent V/V), slightly soluble in alcohol (50 per cent V/V) and in methanol.


IDENTIFICATION First identification : A, B. Dissolve 0.400 g in 50 ml of anhydrous acetic acid R. Titrate Second identification : A, C, D. with 0.1 M perchloric acid, determining the end-point A. It complies with the test for specific optical rotation (see potentiometrically (2.2.20). Tests). 1 ml of 0.1 M perchloric acid is equivalent to 50.80 mg of B. Examine by infrared absorption spectrophotometry C24H30ClN3O7. (2.2.24), comparing with the spectrum obtained with clemastine fumarate CRS. STORAGE C. Examine the chromatograms obtained in the test for Store protected from light. related substances (see Tests). The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the IMPURITIES chromatogram obtained with reference solution (a). D. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. Dissolve 40 mg of the substance to be examined in methanol R and dilute to 2 ml with the same solvent. Reference solution. Dissolve 50 mg of fumaric acid CRS A. 4-amino-5-chloro-2-methoxybenzoic acid, in alcohol R and dilute to 10 ml with the same solvent. Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 5 volumes of water R, 25 volumes of anhydrous formic acid R and 70 volumes of di-isopropyl ether R. Dry the plate at 100 °C to 105 °C for 30 min, allow to cool and spray with a 16 g/l solution of potassium permanganate R. in daylight. In the chromatogram obtained Examine B. 1-benzylpiperidin-4-amine, with test solution the spot with the highest Rf value is similar in position, colour and size to the spot in the chromatogram obtained with the reference solution. ASSAY

C. 4-amino-N-(1-benzylpiperidin-4-yl)-2-methoxybenzamide. General Notices (1) apply to all monographs and other texts

TESTS Solution S. Dissolve 0.500 g of substance to be examined in methanol R and dilute to 50.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution BY7 (2.2.2, Method II). 1315

Clemastine fumarate


pH (2.2.3). Suspend 1.0 g of the substance to be examined in 10 ml of carbon dioxide-free water R. The pH of the suspension is 3.2 to 4.2. Specific optical rotation (2.2.7). The specific optical rotation is + 15.0 to + 18.0, determined on solution S and calculated with reference to the dried substance. Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution (a). Dissolve 0.100 g of the substance to be examined in methanol R and dilute to 5.0 ml with the same solvent. Test solution (b). Dilute 1.0 ml of test solution (a) to 10.0 ml with methanol R. Reference solution (a). Dissolve 20.0 mg of clemastine fumarate CRS in methanol R and dilute to 10.0 ml with the same solvent. Reference solution (b). Dilute 1.5 ml of test solution (b) to 50.0 ml with methanol R. Reference solution (c). Dilute 0.5 ml of test solution (b) to 50.0 ml with methanol R. Reference solution (d). Dissolve 10.0 mg of diphenhydramine hydrochloride CRS in 5.0 ml of reference solution (a). Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 1 volume of concentrated ammonia R, 20 volumes of methanol R and 80 volumes of tetrahydrofuran R. Dry the plate in a current of cold air for 5 min, spray with a freshly prepared mixture of 1 volume of potassium iodobismuthate solution R and 10 volumes of dilute acetic acid R and then with dilute hydrogen peroxide solution R. Cover the plate immediately with a glass plate of the same size and examine the chromatograms after 2 min. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.3 per cent) and at most four such spots are more intense than the spot in the chromatogram obtained with reference solution (c) (0.1 per cent). Disregard any spot remaining at the starting point (fumaric acid). The test is not valid unless the chromatogram obtained with reference solution (d) shows two clearly separated spots. 1-(4-Chlorophenyl)-1-phenylethanol. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 20 mg of the substance to be examined in a mixture of 25 volumes of acetonitrile R and 75 volumes of a 10 g/l solution of ammonium dihydrogen phosphate R and dilute to 100 ml with the same mixture of solvents. Reference solution (a). Dissolve 6 mg of 1-(4-chlorophenyl)-1-phenylethanol CRS in a mixture of 25 volumes of acetonitrile R and 75 volumes of a 10 g/l solution of ammonium dihydrogen phosphate R and dilute to 100 ml with the same mixture of solvents. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with a mixture of 25 volumes of acetonitrile R and 75 volumes of a 10 g/l solution of ammonium dihydrogen phosphate R. Reference solution (c). Dissolve 10 mg of the substance to be examined in a mixture of 25 volumes of acetonitrile R and 75 volumes of a 10 g/l solution of ammonium dihydrogen phosphate R and dilute to 100 ml with the same mixture of solvents. To 1 ml of the solution add 1 ml of reference solution (a) and dilute to 100 ml with a mixture of 25 volumes of acetonitrile R and 75 volumes of a 10 g/l solution of ammonium dihydrogen phosphate R. 1316

The chromatographic procedure may be carried out using : — a stainless steel column 0.1 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1 ml/min a mixture of 0.1 volumes of phosphoric acid R, 45 volumes of acetonitrile R and 55 volumes of a 10 g/l solution of ammonium dihydrogen phosphate R, — as detector a spectrophotometer set at 220 nm. Inject 100 µl of each solution. The test is not valid unless the resolution between the peaks due to clemastine and 1-(4-chlorophenyl)-1-phenylethanol in the chromatogram obtained with reference solution (c) is greater than 2.2. In the chromatogram obtained with the test solution : the area of any peak corresponding to 1-(4-chlorophenyl)-1-phenylethanol is not greater than the area of the peak in the chromatogram obtained with reference solution (b) (0.3 per cent). Loss on drying (2.2.32). Not more than 0.5 per cent. determined on 1.000 g by drying in an oven at 100 °C to 105 °C for 6 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.350 g of the substance to be examined in 60 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 46.00 mg of C25H30ClNO5. IMPURITIES Specified impurities : A, B, C. Other detectable impurities : D.

A. (1RS,2R)-2-[2-[(R)-1-(4-chlorophenyl)-1-phenylethoxy]ethyl]-1-methylpyrrolidine 1-oxide,

B. 4-[1-(4-chlorophenyl)-1-phenylethoxy]-1-methylazepane,

C. (RS)-1-(4-chlorophenyl)-1-phenylethanol,


Clenbuterol hydrochloride


D. 2-[(2RS)-1-methylpyrrolidin-2-yl]ethanol.

01/2005:1409

CLENBUTEROL HYDROCHLORIDE Clenbuteroli hydrochloridum

C12H19Cl3N2O

Mr 313.7

DEFINITION Clenbuterol hydrochloride contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of (RS)-1-(4-amino-3,5-dichlorophenyl)-2-[(1,1dimethylethyl)amino]ethanol hydrochloride, calculated with reference to the anhydrous substance. CHARACTERS A white or almost white, crystalline powder, soluble in water and in alcohol, slightly soluble in acetone. It melts at about 173 °C, with decomposition. IDENTIFICATION First identification : A, C. Second identification : B, C. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with clenbuterol hydrochloride CRS. B. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R. Test solution. Dissolve 10 mg of the substance to be examined in 10 ml of methanol R. Reference solution. Dissolve 10 mg of clenbuterol hydrochloride CRS in 10 ml of methanol R. Apply to the plate 10 µl of each solution. Develop over a path of 10 cm using a mixture of 0.15 volumes of ammonia R, 10 volumes of ethanol R and 15 volumes of toluene R. Allow the plate to dry in air. Spray with a 10 g/l solution of sodium nitrite R in 1 M hydrochloric acid and dip after 10 min in a 4 g/l solution of naphthylethylenediamine dihydrochloride R in methanol R. Allow the plate to dry in air. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. C. It gives reaction (a) of chlorides (2.3.1). TESTS Solution S. Dissolve 0.5 g in 10 ml of carbon dioxide-free water R. General Notices (1) apply to all monographs and other texts

Appearance of solution. Solution S is not more opalescent than reference suspension II (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). pH (2.2.3). The pH of solution S is 5.0 to 7.0. Optical rotation (2.2.7). Dissolve 0.30 g in water R and dilute to 10.0 ml with the same solvent. If necessary filter. The angle of optical rotation is − 0.10° to + 0.10°. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Disperse 100.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (a). Dilute 0.1 ml of the test solution to 100.0 ml with water R. Reference solution (b). Dissolve 10 mg of clenbuterol impurity B CRS in 20 ml of the mobile phase, add 5 ml of the test solution and dilute to 50.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.125 m long and 4 mm in internal diameter, packed with end-capped octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 0.5 ml/min a mixture of 200 volumes of acetonitrile R, 200 volumes of methanol R and 600 volumes of a solution prepared as follows : dissolve 3.0 g of sodium decanesulphonate R and 5.0 g of potassium dihydrogen phosphate R in 900 ml ofwater R, adjust to pH 3.0 with dilute phosphoric acid R and dilute to 1000 ml with water R, — as detector a spectrophotometer set at 215 nm, maintaining the temperature of the column at 40 °C. Inject 5 µl of reference solution (a) and 5 µl of reference solution (b). Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with reference solution (a) is at least 50 per cent of the full scale of the recorder. When the chromatogram are recorded in the prescribed conditions, the retention time of clenbuterol is about 11 min. The test is not valid until in the chromatogram obtained with reference solution (b) the resolution between the peaks corresponding to impurity B and clenbuterol is at least 2.5. Inject 5 µl of the test solution and continue the chromatography for 1.5 times the retention time of the principal peak. In the chromatogram obtained with the test solution : the area of any peak apart from the principal peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent) and the sum of the areas of all the peaks apart of the principal peak is not greater than 2 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (a). Water (2.5.12). Not more than 1.0 per cent, determined on 0.500 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.250 g in 50 ml of alcohol R and add 5.0 ml of 0.01 M hydrochloric acid. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20). Read the volume added between the two points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 31.37 mg of C12H19Cl3N2O. 1317

Clindamycin hydrochloride


Plate : TLC silica gel G plate R. Mobile phase : mix 19 volumes of 2-propanol R, 38 volumes of a 150 g/l solution of ammonium acetate R adjusted to pH 9.6 with ammonia R, and 43 volumes of ethyl acetate R. Application : 5 µl. Development : over a path of 15 cm using the upper layer of the mobile phase. R1 = H, R2 = Cl : 4-amino-3,5-dichlorobenzaldehyde, Drying : in air. Detection : spray with a 1 g/l solution of potassium R1 = CH2-NH-C(CH3)3, R2 = Cl : 1-(4-amino-3,5permanganate R. dichlorophenyl)-2-[(1,1-dimethylethyl)amino]ethanone (clenbuterol-ketone), System suitability : the chromatogram obtained with reference solution (b) shows 2 clearly separated spots. R1 = CH3, R2 = Cl : 1-(4-amino-3,5-dichlorophenyl)ethaResults : the principal spot in the chromatogram obtained none, with the test solution is similar in position, colour and R1 = CH3, R2 = H : 1-(4-aminophenyl)ethanone, size to the principal spot in the chromatogram obtained with reference solution (a). R1 = CH2Br, R2 = Cl : 1-(4-amino-3,5-dichlorophenyl)-2bromoethanone. C. Dissolve about 10 mg in 2 ml of dilute hydrochloric acid R and heat on a water-bath for 3 min. Add 3 ml of sodium carbonate solution R and 1 ml of a 20 g/l 01/2005:0582 solution of sodium nitroprusside R. A violet-red colour develops. CLINDAMYCIN HYDROCHLORIDE D. Dissolve 0.1 g in water R and dilute to 10 ml with the same solvent. The solution gives reaction (a) of chlorides (2.3.1). Clindamycini hydrochloridum

IMPURITIES

A. B.

C. D. E.

TESTS pH (2.2.3) : 3.0 to 5.0. Dissolve 1.0 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. Specific optical rotation (2.2.7) : + 135 to + 150 (anhydrous substance). Dissolve 1.000 g in water R and dilute to 25.0 ml with the same solvent. C18H34Cl2N2O5S Mr 461.5 Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 50.0 mg of the substance to be DEFINITION examined in the mobile phase and dilute to 50.0 ml with the Methyl 7-chloro-6,7,8-trideoxy-6-[[[(2S,4R)-1-methyl-4mobile phase. propylpyrrolidin-2-yl]carbonyl]amino]-1-thio-L-threo-α-DReference solution (a). Dissolve 50.0 mg of clindamycin galacto-octopyranoside hydrochloride. It contains a variable hydrochloride CRS in the mobile phase and dilute to 50.0 ml quantity of water. with the mobile phase. Content : 84.0 per cent to 93.0 per cent of clindamycin Reference solution (b). Dilute 2.0 ml of the test solution to (C18H33ClN2O5S) (anhydrous substance). 100.0 ml with the mobile phase. Column : CHARACTERS — size : l = 0.25 m, Ø = 4.6 mm, Appearance : white or almost white, crystalline powder. Solubility : very soluble in water, slightly soluble in alcohol. — stationary phase : octadecylsilyl silica gel for chromatography R (5 µm). IDENTIFICATION Mobile phase : mix 45 volumes of acetonitrile R and 55 volumes of a 6.8 g/l solution of potassium dihydrogen First identification : A, D. phosphate R adjusted to pH 7.5 with a 250 g/l solution of Second identification : B, C, D. potassium hydroxide R. A. Infrared absorption spectrophotometry (2.2.24). Flow rate : 1 ml/min. Comparison : clindamycin hydrochloride CRS. Detection : spectrophotometer at 210 nm. B. Thin-layer chromatography (2.2.27). Injection : 20 µl. Test solution. Dissolve 10 mg of the substance to be Run time : twice the retention time of clindamycin. examined in methanol R and dilute to 10 ml with the System suitability : reference solution (a) : same solvent. — relative retentions with reference to clindamycin Reference solution (a). Dissolve 10 mg of clindamycin (retention time = about 10 min) : impurity A = about 0.4 ; hydrochloride CRS in methanol R and dilute to 10 ml impurity B = about 0.65 ; impurity C = about 0.8. with the same solvent. Limits : Reference solution (b). Dissolve 10 mg of clindamycin hydrochloride CRS and 10 mg of lincomycin — impurity B : not more than the area of the principal peak hydrochloride CRS in methanol R and dilute to 10 ml in the chromatogram obtained with reference solution (b) with the same solvent. (2.0 per cent), 1318


Clindamycin phosphate


— impurity C : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (4.0 per cent), — any other impurity : not more than half the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent),

01/2005:0996

CLINDAMYCIN PHOSPHATE Clindamycini phosphas

— total: not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (b) (6.0 per cent), — disregard limit: 0.025 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.05 per cent). Water (2.5.12) : 3.0 per cent to 6.0 per cent, determined on 0.500 g. Sulphated ash (2.4.14) : maximum 0.5 per cent, determined on 1.0 g. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modifications. Injection : 20 µl ; inject the test solution and reference solution (a). System suitability : reference solution (a) : — repeatability : maximum relative standard deviation of 0.85 per cent for 6 replicate injections. STORAGE In an airtight container. IMPURITIES

A. R1 = CH2-CH2-CH3, R2 = R4 = H, R3 = OH : methyl 6,8-dideoxy-6-[[[(2S,4R)-1-methyl-4-propylpyrrolidin2-yl]carbonyl]amino]-1-thio-D-erythro-α-D-galactooctopyranoside (lincomycin),

B. R1 = R3 = H, R2 = C2H5, R4 = Cl : methyl 7-chloro-6,7,8-trideoxy-6-[[[(2S,4S)-4-ethyl-1methylpyrrolidin-2-yl]carbonyl]amino]-1-thio-L-threo-α-Dgalacto-octopyranoside (clindamycin B),

C. R1 = CH2-CH2-CH3, R2 = R4 = H, R3 = Cl : methyl 7-chloro-6,7,8-trideoxy-6-[[[(2S,4R)-1-methyl-4propylpyrrolidin-2-yl]carbonyl]amino]-1-thio-D-erythro-αD-galacto-octopyranoside (7-epiclindamycin). General Notices (1) apply to all monographs and other texts

C18H34ClN2O8PS

Mr 505.0

DEFINITION Clindamycin phosphate contains not less than 95.0 per cent and not more than the equivalent of 100.5 per cent of methyl 7-chloro-6,7,8-trideoxy-6-[[[(2S,4R)-1-methyl-4propylpyrrolidin-2-yl]carbonyl]amino]-1-thio-L-threo-α-Dgalacto-octopyranoside 2-(dihydrogen phosphate), calculated with reference to the anhydrous substance. CHARACTERS A white or almost white powder, slightly hygroscopic, freely soluble in water, very slightly soluble in alcohol, practically insoluble in methylene chloride. It shows polymorphism. IDENTIFICATION First identification : A, D. Second identification : B, C, D. A. Examine by infrared spectrophotometry (2.2.24), comparing with the spectrum obtained with clindamycin phosphate CRS. In two separate tubes place 50 mg of the substance to be examined and 50 mg of clindamycin phosphate CRS. Add 0.2 ml of water R and heat until completely dissolved. Evaporate to dryness under reduced pressure and dry at 100 °C to 105 °C for 2 h. Examine the substances prepared as discs using potassium bromide R. B. Examine by thin-layer chromatography (2.2.27), using silica gel H R as the coating substance. Test solution. Dissolve 20 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution (a). Dissolve 20 mg of clindamycin phosphate CRS in methanol R and dilute to 10 ml with the same solvent. Reference solution (b). Dissolve 10 mg of lincomycin hydrochloride CRS in 5 ml of reference solution (a). Apply separately to the plate 5 µl of each solution. Develop over a path of 12 cm using a mixture of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes of butanol R. Dry the plate at 100 °C to 105 °C for 30 min and spray with a 1 g/l solution of potassium permanganate R. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two principal spots. 1319

Clindamycin phosphate


Reference solution (b). Dissolve 5.0 mg of lincomycin hydrochloride CRS and 15.0 mg of clindamycin hydrochloride CRS in 5.0 ml of reference solution (a) and dilute to 100.0 ml with the mobile phase. Reference solution (c). Dilute 1.0 ml of reference solution (a) to 100.0 ml with the mobile phase. The chromatographic procedure may be carried out using : D. Boil 0.1 g under a reflux condenser with a mixture of — a stainless steel column 0.25 m long and 4.6 mm in 5 ml of strong sodium hydroxide solution R and 5 ml of internal diameter packed with octylsilyl silica gel for water R for 90 min. Cool and add 5 ml of nitric acid R. chromatography R (5 µm to 10 µm), Extract with three quantities, each of 15 ml, of methylene chloride R and discard the extracts. Filter the upper layer — as mobile phase at a flow rate of 1.0 ml/min a mixture of 200 ml of acetonitrile R and 800 ml of a 13.6 g/l solution through a paper filter. The filtrate gives reaction (b) of of potassium dihydrogen phosphate R, previously phosphates (2.3.1). adjusted to pH 2.5 with phosphoric acid R, — as detector a spectrophotometer set at 210 nm, — a 20 µl loop injector. TESTS Solution S. Dissolve 1.00 g in carbon dioxide-free water R. Inject reference solution (b). Adjust the sensitivity of the detector so that the height of the peaks is not less than 50 per Heat gently if necessary. Cool and dilute to 25.0 ml with cent of the full scale of the recorder. The assay is not valid carbon dioxide-free water R. unless the first peak (lincomycin) is clearly separated from Appearance of the solution. Solution S is clear (2.2.1) and the solvent peak and the resolution between the second peak colourless (2.2.2, Method II). (clindamycin phosphate) and the third peak (clindamycin) pH (2.2.3). Dilute 5.0 ml of solution S to 20 ml with carbon is at least 6.0. Adjust the concentration of acetonitrile in the mobile phase, if necessary. The assay is not valid unless dioxide-free water R. The pH of the solution is 3.5 to 4.5. the symmetry factor of the clindamycin phosphate peak Specific optical rotation (2.2.7). Dissolve 0.250 g in water R is at most 1.5. Inject reference solution (a) six times. The and dilute to 25.0 ml with the same solvent. The specific test is not valid unless the relative standard deviation of optical rotation is + 115 to + 130, calculated with reference the peak area for clindamycin phosphate is at most 1.0 per to the anhydrous substance. cent. If necessary adjust the integrator parameters. Inject alternately the test solution and reference solution (a). Related substances. Examine by liquid chromatography (2.2.29), as described under Assay. Inject reference Calculate the percentage content of clindamycin phosphate. solution (c). Adjust the sensitivity of the detector so that the height of the principal peak is not less than 50 per cent STORAGE of the full scale of the recorder. Inject the test solution Store in an airtight container, at a temperature not exceeding and continue the chromatography for the retention time of clindamycin : the area of any peak, apart from the principal 30 °C. If the substance is sterile, store in a sterile, airtight, tamper-proof container. peak and any peak due to the solvent, is not greater than 2.5 times the area of the peak corresponding to clindamycin LABELLING phosphate in the chromatogram obtained with reference The label states, where applicable, that the substance is free solution (c) (2.5 per cent) and the sum of the areas of all from bacterial endotoxins. the peaks, apart from the principal peak and any peak due to the solvent, is not greater than four times the area of IMPURITIES the peak corresponding to clindamycin phosphate in the chromatogram obtained with reference solution (c) (4.0 per cent). Disregard any peak with an area less than 10.0 per A. lincomycin, cent of that of the principal peak in the chromatogram obtained with reference solution (c).

C. Dissolve about 10 mg in 2 ml of dilute hydrochloric acid R and heat in a water-bath for 3 min. Add 4 ml of sodium carbonate solution R and 1 ml of a 20 g/l solution of sodium nitroprusside R. Prepare a standard in the same manner using clindamycin phosphate CRS. The colour in the test solution corresponds to that in the standard.

Water (2.5.12). Not more than 6.0 per cent, determined on 0.250 g by the semi-micro determination of water. Bacterial endotoxins (2.6.14) : less than 0.6 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for removal of bacterial endotoxins. B. R1 = PO3H2, R2 = R3 = H, R4 = C2H5 : clindamycin B 2-(dihydrogen phosphate),

ASSAY Examine by liquid chromatography (2.2.29). Test solution. Dissolve 75.0 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the mobile phase. Reference solution (a). Dissolve 75.0 mg of clindamycin phosphate CRS in the mobile phase and dilute to 25.0 ml with the mobile phase. 1320

C. R1 = R3 = H, R2 = PO3H2, R4 = C3H7 : clindamycin 3-(dihydrogen phosphate), D. R1 = R2 = H, R3 = PO3H2, R4 = C3H7 : clindamycin 4-(dihydrogen phosphate), E. R1 = R2 = R3 = H, R4 = C3H7 : clindamycin.



Clioquinol

01/2005:2111 Reference solution (c). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 20.0 ml with the mobile phase. CLIOQUINOL Column : — size : l = 0.15 m, Ø = 3.9 mm, Clioquinolum — stationary phase : octylsilyl silica gel for chromatography R (5 µm). Mobile phase : dissolve 0.50 g of sodium edetate R in 350 ml of water R, add 4.0 ml of hexylamine R and mix. Adjust to pH 3.0 with phosphoric acid R. Add 600 ml of methanol R and dilute to 1000 ml with water R. Flow rate : 1.3 ml/min. C9H5ClINO Mr 305.5 Detection : spectrophotometer at 254 nm. Injection : 20 µl. DEFINITION Run time : 4 times the retention time of clioquinol. 5-Chloro-7-iodoquinolin-8-ol. Relative retention with reference to clioquinol (retention Content : 98.0 per cent to 102.0 per cent (dried substance). time = about 10 min) : impurity A = about 0.4 ; impurity B = about 0.7 ; impurity C = about 1.3. CHARACTERS Appearance : almost white, light yellow, brownish-yellow or System suitability : reference solution (a) : yellowish-grey powder. — resolution : minimum 3.0 between the peaks due to clioquinol and impurity C. Solubility : practically insoluble in water, sparingly soluble in methylene chloride, very slightly soluble or slightly soluble Limits : in ethanol (96 per cent). — impurity A : not more than the area of the corresponding peak in the chromatogram obtained with reference IDENTIFICATION solution (b) (2.0 per cent), First identification : B. — impurity B : not more than the area of the corresponding Second identification : A, C, D. peak in the chromatogram obtained with reference A. Dissolve 40.0 mg in methanol R and dilute to 100.0 ml solution (b) (1.0 per cent), with the same solvent. Dilute 10.0 ml to 100.0 ml with — impurity C : not more than the area of the corresponding methanol R (solution A). Examined between 280 nm peak in the chromatogram obtained with reference and 350 nm (2.2.25), solution A shows an absorption solution (b) (1.0 per cent), maximum at 321 nm. Dilute 10.0 ml of solution A to — any other impurity : for each impurity, not more than 100.0 ml with methanol R (solution B). Examined twice the area of the principal peak in the chromatogram between 230 nm and 280 nm, solution B shows an obtained with reference solution (c) (0.1 per cent), absorption maximum at 255 nm. The specific absorbance at this absorption maximum is 1530 to 1660. — total of the nominal contents of impurities A, B, C and any other impurities : maximum 3.0 per cent, B. Infrared absorption spectrophotometry (2.2.24). — disregard limit : the area of the principal peak in the Preparation : discs of potassium bromide R. chromatogram obtained with reference solution (c) Comparison : clioquinol CRS. (0.05 per cent). C. When heated, violet fumes are produced. Halides : maximum 140 ppm, expressed as chlorides. D. Dissolve about 1 mg in 5 ml of ethanol (96 per cent) R. Add 0.05 ml of ferric chloride solution R1. A dark green Shake 0.5 g with 25 ml of water R for 1 min and filter. To the filtrate add 0.5 ml of dilute nitric acid R and 0.5 ml of colour develops. silver nitrate solution R2. Allow to stand for 5 min. Any TESTS opalescence is not more intense than that in a standard prepared at the same time by adding 0.5 ml of silver nitrate Acidity or alkalinity. Shake 0.5 g with 10 ml of carbon dioxide-free water R and filter. To the filtrate add 0.2 ml of solution R2 to 25 ml of water R containing 0.2 ml of 0.01 M phenolphthalein solution R. The solution is colourless. Not hydrochloric acid and 0.5 ml of dilute nitric acid R. more than 0.5 ml of 0.01 M sodium hydroxide is required to Loss on drying (2.2.32) : maximum 0.5 per cent, determined change the colour of the indicator to pink. on 1.000 g by drying over diphosphorus pentoxide R at a pressure not exceeding 0.7 kPa for 24 h. Related substances. Liquid chromatography (2.2.29). Sulphated ash (2.4.14) : maximum 0.1 per cent, determined Test solution. Dissolve 50.0 mg of the substance to be examined in methanol R and dilute to 50.0 ml with the same on 1.0 g. solvent, heating gently if necessary. Dilute 10.0 ml of the ASSAY solution to 25.0 ml with the mobile phase. Reference solution (a). Dissolve 20.0 mg of 5-chloroquinolin- Dissolve 0.200 g in 20 ml of acetic anhydride R and add 30 ml of glacial acetic acid R. Titrate with 0.1 M perchloric 8-ol R, 10.0 mg of 5,7-dichloroquinolin-8-ol R, 5 mg of acid, determining the end-point potentiometrically (2.2.20). the substance to be examined and 10.0 mg of 5,7-diiodoquinolin-8-ol R in methanol R, heating gently if 1 ml of 0.1 M perchloric acid is equivalent to 30.55 mg of necessary and dilute to 20.0 ml with the same solvent. Dilute total quinolines, calculated as clioquinol. 4.0 ml of the solution to 50.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of reference solution (a) STORAGE Protected from light. to 10.0 ml with the mobile phase. General Notices (1) apply to all monographs and other texts

1321

Clobazam



A. R1 = Cl, R2 = H : 5-chloroquinolin-8-ol, B. R1 = R2 = Cl : 5,7-dichloroquinolin-8-ol, C. R1 = R2 = I : 5,7-diiodoquinolin-8-ol.

CLOBAZAM Clobazamum

C16H13ClN2O2

Reference solution (c). Dilute 1.0 ml of the test solution to 200.0 ml with the mobile phase. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : acetonitrile R, water R (40:60 V/V). Flow rate : 1 ml/min. Detection : spectrophotometer at 230 nm. Injection : 20 µl. Run time : 5 times the retention time of clobazam. Retention time : clobazam = about 15 min. System suitability : reference solution (b) : — resolution : minimum 1.3 between the peaks due to chlordiazepoxide and clonazepam. 01/2005:1974 Limits : corrected — impurity A : not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent), — any other impurity : not more than 0.4 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.2 per cent), — total of other impurities: not more than twice the area of the principal peak in the chromatogram obtained with reference solution (c) (1.0 per cent), — disregard limit : 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.05 per cent). Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Mr 300.7 Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on the residue obtained in the test for loss on drying.

DEFINITION 7-Chloro-1-methyl-5-phenyl-1,5-dihydro-3H-1,5benzodiazepine-2,4-dione. Content : 97.0 per cent to 103.0 per cent (dried substance). CHARACTERS Appearance : white or almost white, crystalline powder. Solubility : slightly soluble in water, freely soluble in methylene chloride, sparingly soluble in alcohol.

ASSAY Dissolve 50.0 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the solution to 250.0 ml with alcohol R. Measure the absorbance (2.2.25) at the maximum at 232 nm. Calculate the content of C16H13ClN2O2 taking the specific absorbance to be 1380. IMPURITIES

IDENTIFICATION Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of clobazam. TESTS Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 10.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (a). Dissolve 5.0 mg of clobazam impurity A CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 100.0 ml with the mobile phase. Reference solution (b). Dissolve 5 mg of chlordiazepoxide CRS and 5 mg of clonazepam CRS in the mobile phase and dilute to 50 ml with the mobile phase. Dilute 1 ml of the solution to 100 ml with the mobile phase. 1322

A. R1 = R3 = R4 = H, R2 = Cl : 7-chloro-5-phenyl-1,5-dihydro3H-1,5-benzodiazepine-2,4-dione, B. R1 = CH3, R2 = R3 = R4 = H : 1-methyl-5-phenyl-1,5dihydro-3H-1,5-benzodiazepine-2,4-dione, C. R1 = R3 = CH3, R2 = Cl, R4 = H : (3RS)-7-chloro-1,3dimethyl-5-phenyl-1,5-dihydro-3H-1,5-benzodiazepine-2,4dione, D. R1 = R3 = R4 = CH3, R2 = Cl : 7-chloro-1,3,3-trimethyl-5phenyl-1,5-dihydro-3H-1,5-benzodiazepine-2,4-dione,



Clobetasone butyrate

Reference solution (a). Dissolve 10 mg of clobetasone butyrate CRS in a mixture of equal volumes of methanol R and methylene chloride R and dilute to 10 ml with the same mixture of solvents. Reference solution (b). Dissolve 10 mg of clobetasol propionate R in a mixture of equal volumes of methanol R and methylene chloride R and dilute to 10 ml with the same mixture of solvents. Dilute 5 ml of this solution to 10 ml with reference solution (a). Apply separately to the plate 5 µl of each solution. E. N-[4-chloro-2-(phenylamino)phenyl]-N-methylacetamide, Develop over a path of 15 cm using a mixture of equal volumes of cyclohexane R and methyl acetate R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. C. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is F. methyl 3-[[4-chloro-2-(phenylamino)phenyl]methylamino]obtained (usually less than 5 min). Allow to cool, add 1 ml 3-oxopropanoate. of water R, 0.05 ml of phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the 01/2005:1090 solution colourless. Filter. To a freshly prepared mixture corrected of 0.1 ml alizarin S solution R and 0.1 ml of zirconyl nitrate solution R, add 1.0 ml of the filtrate. Mix, allow to stand for 5 min and compare the colour of the solution CLOBETASONE BUTYRATE with that of a blank prepared in the same manner. The colour of the test solution is yellow and that of the blank Clobetasoni butyras is red. TESTS Specific optical rotation (2.2.7). Dissolve 0.250 g in dioxan R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 127 to + 133, calculated with reference to the dried substance. Related substances. Prepare the solutions immediately before use. Examine by liquid chromatography (2.2.29). C26H32ClFO5 Mr 479.0 Test solution. Dissolve 50.0 mg of the substance to be examined in 5.0 ml of ethanol R and dilute to 50.0 ml with DEFINITION the mobile phase. Clobetasone butyrate contains not less than 97.0 per Reference solution (a). Dissolve 2 mg of clobetasone cent and not more than the equivalent of 102.0 per cent butyrate CRS and 1.5 mg of clobetasol propionate R in 5 ml of 21-chloro-9-fluoro-16β-methyl-3,11,20-trioxopregna-1,4of ethanol R and dilute to 100 ml with the mobile phase. dien-17-yl butanoate, calculated with reference to the dried substance. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. CHARACTERS The chromatographic procedure may be carried out using : A white or almost white powder, practically insoluble in — a stainless steel column 0.20 m long and 4.6 mm in water, freely soluble in acetone and in methylene chloride, internal diameter, packed with octadecylsilyl silica gel slightly soluble in alcohol. for chromatography R (5 µm), It melts at about 178 °C. — as mobile phase at a flow rate of 1 ml/min a mixture of 45 volumes of ethanol R and 55 volumes of water R, IDENTIFICATION — as detector a spectrophotometer set at 241 nm, A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with maintaining the temperature of the column at 60 °C. Inject clobetasone butyrate CRS. Examine the substances 20 µl of reference solution (a). When using a recorder, adjust prepared as discs. the sensitivity of the system so that the height of the two principal peaks are at least 50 per cent of the full scale of the B. Examine by thin-layer chromatography (2.2.27), using recorder. The test is not valid unless the resolution between as the coating substance a suitable silica gel with a the first peak (clobetasol propionate) and the second peak fluorescent indicator having an optimal intensity at (clobetasone butyrate) is at least five. 254 nm. Test solution. Dissolve 10 mg of the substance to be Inject 20 µl of the test solution and 20 µl of reference examined in a mixture of equal volumes of methanol R solution (b). Continue the chromatography for 2.5 times the and methylene chloride R and dilute to 10 ml with the retention time of the principal peak. In the chromatogram same mixture of solvents. obtained with the test solution : the area of any peak, apart General Notices (1) apply to all monographs and other texts

1323

Clofazimine


from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent) and not more than one such peak has an area greater than half the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (1.5 per cent). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (b). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. ASSAY Dissolve 20.0 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of the solution to 50.0 ml with alcohol R. Measure the absorbance (2.2.25) at the maximum at 235 nm. Calculate the content of C26H32ClFO5, taking the specific absorbance to be 327. STORAGE Store protected from light. IMPURITIES

A. clobetasone,

B. (17R)-4′-chloro-9-fluoro-16β-methyl-5′-propylspiro[androsta-1,4-diene-17,2′(3′H)furan]-3,3′,11-trione. 01/2005:2054

CLOFAZIMINE Clofaziminum

C27H22Cl2N4 1324

DEFINITION N,5-Bis(4-chlorophenyl)-3-[(1-methylethyl)imino]-3,5dihydrophenazin-2-amine. Content : 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : reddish-brown, fine powder. Solubility : practically insoluble in water, soluble in methylene chloride, very slightly soluble in ethanol (96 per cent). It shows polymorphism. IDENTIFICATION First identification : A. Second identification : B, C. A. Infrared absorption spectrophotometry (2.2.24). Comparison : clofazimine CRS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in methylene chloride R, evaporate to dryness and record new spectra using the residues. B. Thin-layer chromatography (2.2.27). Test solution. Dissolve 10 mg of the substance to be examined in methylene chloride R and dilute to 10 ml with the same solvent. Reference solution. Dissolve 10 mg of clofazimine CRS in methylene chloride R and dilute to 10 ml with the same solvent. Plate : TLC silica gel GF254 plate R. Mobile phase : propanol R, methylene chloride R (6:85 V/V). Application : 5 µl. First development: over 2/3 of the plate. Drying : horizontally in air for 5 min. Second development : over 2/3 of the plate. Drying : in air for 5 min. Detection : examine in ultraviolet light at 254 nm. Results : the principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. C. Dissolve 2 mg in 3 ml of acetone R and add 0.1 ml of hydrochloric acid R. An intense violet colour is produced. Add 0.5 ml of a 200 g/l solution of sodium hydroxide R ; the colour changes to orange-red.

TESTS Related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately before use. Test solution. Dissolve 50 mg of the substance to be examined in the mobile phase and dilute to 100 ml with the mobile phase. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (b). Dissolve 5.0 mg of clofazimine for system suitability CRS in the mobile phase and dilute to 10.0 ml with the mobile phase. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : octylsilyl silica gel for Mr 473.4 chromatography R (5 µm). See the information section on general monographs (cover pages)

Clofibrate


Mobile phase : dissolve 2.25 g of sodium laurilsulfate R, 0.85 g of tetrabutylammonium hydrogen sulphate R and 0.885 g of disodium hydrogen phosphate R in water R. Adjust to pH 3.0 with dilute phosphoric acid R and dilute to 500 ml with water R. Mix 35 volumes of this solution and 65 volumes of acetonitrile R. Flow rate : 1 ml/min. Detection : spectrophotometer at 280 nm. Injection : 20 µl. Run time : 3 times the retention time of clofazimine. Identification of impurities : use the chromatogram supplied with clofazimine for system suitability CRS to identify the peak due to impurity B.

A. R1 = Cl, R2 = H : N,5-bis(4-chlorophenyl)-3-imino-3,5dihydrophenazin-2-amine, B. R1 = H, R2 = CH(CH3)2 : 5-(4-chlorophenyl)-3-[(1methylethyl)imino]-N-phenyl-3,5-dihydrophenazin-2amine. 01/2005:0318

Relative retention with reference to clofazimine (retention time = about 15 min) : impurity A = about 0.7 ; impurity B = about 0.8.

CLOFIBRATE

System suitability : reference solution (b) :

Clofibratum

— resolution : baseline separation between the peaks due to impurity B and clofazimine. Limits : — impurity A : not more than the area of the principal peak in the chromatogram obtained with reference solution (a) C12H15ClO3 (0.1 per cent), — impurity B : not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.3 per cent),

Mr 242.7

DEFINITION Clofibrate is ethyl 2-(4-chlorophenoxy)-2-methylpropionate.

CHARACTERS — any other impurity : for each impurity, not more than the A clear, almost colourless liquid, very slightly soluble in area of the principal peak in the chromatogram obtained water, miscible with alcohol. with reference solution (a) (0.1 per cent), IDENTIFICATION — total: not more than 5 times the area of the principal peak A. Examine by infrared absorption spectrophotometry in the chromatogram obtained with reference solution (a) (2.2.24), comparing with the spectrum obtained with (0.5 per cent), clofibrate CRS. B. Dissolve 0.10 g in methanol R and dilute to 100.0 ml — disregard limit : 0.5 times the area of the principal peak with the same solvent. Dilute 10.0 ml of the solution in the chromatogram obtained with reference solution (a) to 100.0 ml with methanol R (solution a). Examined (0.05 per cent). between 250 nm and 350 nm (2.2.25), solution (a) shows Heavy metals (2.4.8) : maximum 10 ppm. two absorption maxima, at 280 nm and at 288 nm. The specific absorbances at the maxima are about 44 and 31, 2.0 g complies with limit test C. Prepare the reference respectively. Dilute 10.0 ml of solution (a) to 100.0 ml solution using 2 ml of lead standard solution (10 ppm Pb) R. with methanol R. Examined between 220 nm and 250 nm, Loss on drying (2.2.32) : maximum 0.5 per cent, determined the solution shows an absorption maximum at 226 nm. on 1.000 g by drying in an oven at 100-105 °C. The specific absorbance at the maximum is about 460. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined TESTS on 1.0 g. Refractive index (2.2.6) : 1.500 to 1.505. Relative density (2.2.5) : 1.138 to 1.147. ASSAY Acidity. To 1.0 g add 10 ml of ethanol R and 0.1 ml of phenol red solution R. Not more than 1.0 ml of 0.01 M sodium Dissolve 0.400 g in 5 ml of methylene chloride R and add hydroxide is required to change the colour of the indicator. 20 ml of acetone R and 5 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point Volatile related substances. Examine by gas chromatography (2.2.28). potentiometrically (2.2.20). Test solution. To 10.0 g of the substance to be examined add 1 ml of 0.1 M perchloric acid is equivalent to 47.34 mg a mixture of 10 ml of dilute sodium hydroxide solution R of C27H22Cl2N4. and 10 ml of water R. Shake, separate the lower (organic) layer, wash with 5 ml of water R and add the washings to the aqueous layer. Dry the organic layer with anhydrous IMPURITIES sodium sulphate R and use as the test solution. Reserve the aqueous layer for the test for 4-chlorophenol. Specified impurities : A, B. General Notices (1) apply to all monographs and other texts

1325

Clomifene citrate


Reference solution (a). Dissolve 0.12 g of the substance to be examined in chloroform R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 10.0 ml with chloroform R. Reference solution (b). Dissolve 0.12 g of methyl 2-(4-chlorophenoxy)-2-methylpropionate CRS in the substance to be examined and dilute to 10.0 ml with the same solvent. Dilute 1.0 ml of the solution to 10.0 ml with the substance to be examined. Dilute 1.0 ml of this solution to 10.0 ml with the substance to be examined. The chromatographic procedure may be carried out using : — a column 1.5 m long and 4 mm in internal diameter packed with either : silanised diatomaceous earth for gas chromatography R (250 µm to 420 µm) impregnated with 30 per cent m/m of poly(dimethyl)siloxane R ; or silanised diatomaceous earth for gas chromatography R (150 µm to 180 µm) impregnated with 10 per cent m/m of poly(dimethyl)siloxane R, — nitrogen for chromatography R as the carrier gas, — a flame-ionisation detector. Maintain the temperature of the column at 185 °C. Inject 2 µl of each solution. In the chromatogram obtained with the test solution, the sum of the areas of the peaks, except that corresponding to clofibrate, is not greater than ten times the area of the peak corresponding to clofibrate in the chromatogram obtained with reference solution (a) (0.1 per cent). In the chromatogram obtained with reference solution (b), measure from the baseline the height (A) of the peak corresponding to methyl 2-(4-chlorophenoxy)-2-methylpropionate and the height (B) of the lowest point of the curve separating this peak from the peak corresponding to clofibrate (see Figure 0318-1). The test is not valid unless A is equal to at least 30 per cent of the recorder-chart scale and A − B is greater than 75 per cent of A.

of 5 ml, of chloroform R and discard the organic layers. Acidify the aqueous layer by the dropwise addition of hydrochloric acid R. Shake with three quantities, each of 3 ml, of chloroform R. Combine the organic layers and dilute to 10.0 ml with chloroform R. Reference solution. Dissolve 0.25 g of chlorophenol R in chloroform R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with chloroform R. The chromatographic procedure may be carried out as described in the test for volatile related substances. Inject 2 µl of each solution. The area of any peak corresponding to 4-chlorophenol in the chromatogram obtained with the test solution is not greater than the area of the peak corresponding to 4-chlorophenol in the chromatogram obtained with the reference solution (25 ppm). 01/2005:0997

CLOMIFENE CITRATE Clomifeni citras

C32H36ClNO8

Mr 598.1

DEFINITION Clomifene citrate contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of a mixture of the E- and Z-isomers of 2-[4-(2-chloro1,2-diphenylethenyl)phenoxy]-N,N-diethylethanamine dihydrogen citrate, calculated with reference to the anhydrous substance. CHARACTERS A white or pale yellow, crystalline powder, slightly soluble in water, sparingly soluble in alcohol. IDENTIFICATION A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with clomifene citrate CRS. Examine the substances as discs prepared using potassium bromide R. B. Dissolve about 5 mg in 5 ml of a mixture of 1 volume of acetic anhydride R and 5 volumes of pyridine R and heat in a water-bath. A deep red colour is produced.

Figure 0318.-1. – Typical chromatogram for the test for volatile related substances 4-Chlorophenol. Examine by gas chromatography (2.2.28). Test solution. Shake the aqueous layer reserved in the test for volatile related substances with two quantities, each 1326

TESTS Prepare the solutions protected from light in brown-glass vessels. Ensure minimum exposure of the solutions to daylight until they are required for chromatography. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 12.5 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (a). Dissolve 12.5 mg of clomifene citrate for performance test CRS in the mobile phase and dilute to 10.0 ml with the mobile phase.



Clomifene citrate

The chromatographic procedure may be carried out using : — a stainless steel column 0.3 m long and 4 mm in internal diameter packed with silica gel for chromatography R The chromatographic procedure may be carried out using : (10 µm), — a stainless steel column 0.25 m long and 4.6 mm — as mobile phase at a flow rate of 2 ml/min a mixture of internal diameter packed with butylsilyl silica gel for 1 volume of triethylamine R, 200 volumes of ethanol-free chromatography R (5 µm), chloroform R and 800 volumes of hexane R, — as mobile phase at a flow rate of 1.2 ml/min a mixture — as detector a spectrophotometer set at 302 nm. prepared as follows : mix 400 ml of acetonitrile R with Equilibrate the column with the mobile phase for about 2 h. 600 ml of water R and add 8.0 ml of diethylamine R ; adjust the the mixture to pH 6.2 by the addition of about Inject 50 µl of the reference solution. The chromatogram 1 ml to 2 ml of phosphoric acid R taking care to reduce obtained shows a peak due to the E-isomer just before a progressively the volume of each addition as the required peak due to the Z-isomer. The test is not valid unless the resolution between the peaks corresponding to the E- and pH is approached, Z-isomers is at least 1.0. If necessary, adjust the relative — as detector a spectrophotometer set at 233 nm. proportions of ethanol-free chloroform and hexane in the Equilibrate the column with the mobile phase at a flow rate mobile phase. Measure the area of the peak due to the Z-isomer in the chromatograms obtained with the test of 1.2 ml/min for about 1 h. solution and the reference solution. Calculate the content Adjust the sensitivity of the system so that the height of the of the Z-isomer as a percentage of the total clomifene citrate principal peak in the chromatogram obtained with 10 µl of present using the declared content of clomifene citrate CRS. reference solution (b) is not less than 50 per cent of the full Water (2.5.12). Not more than 1.0 per cent, determined on scale of the recorder. 1.000 g by the semi-micro determination of water. Inject 10 µl of reference solution (a). Continue the chromatography for twice the retention time of the principal ASSAY peak. Measure the height (A) above the base-line of the peak Dissolve 0.500 g in 50 ml of anhydrous acetic acid R. due to impurity A and the height (B) above the base-line Titrate with 0.1 M perchloric acid determining the end-point of the lowest point of the curve separating this peak from potentiometrically (2.2.20). the peak due to clomifene. The test is not valid unless A is 1 ml of 0.1 M perchloric acid is equivalent to 59.81 mg of greater than fifteen times B and the chromatogram obtained C H ClNO . 32 36 8 resembles the reference chromatogram. If necessary, adjust the concentration of acetonitrile in the mobile phase. STORAGE Store protected from light. Inject separately 10 µl of the test solution and 10 µl of reference solution (b). Continue the chromatography for IMPURITIES four times the retention time of the principal peak. In the chromatogram obtained with the test solution : the area of any peak due to impurity A is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (2.0 per cent) ; the area of any other peak, apart from the principal peak, is not greater than half the area of the principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than 1.25 times the area of the principal peak in the chromatogram obtained with reference solution (b) (2.5 per cent). Disregard any peak with a retention time relative to A. 2-[4-(1,2-diphenylethenyl)phenoxy]-N,Nthe clomifene peak of 0.2 or less and any peak with an area diethylethanamine, less than 0.025 times the area of the principal peak in the chromatogram obtained with reference solution (b). Reference solution (b). Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase.

Z-isomer : 30.0 per cent to 50.0 per cent, determined by liquid chromatography (2.2.29). B. [4-[2-(diethylamino)ethoxy]phenyl]phenylmethanone, Test solution. Dissolve 25 mg of the substance to be examined in 25 ml of 0.1 M hydrochloric acid, add 5 ml of 1 M sodium hydroxide and shake with three quantities, each of 25 ml, of ethanol-free chloroform R. Wash the combined extracts with 10 ml of water R, dry over anhydrous sodium sulphate R and dilute to 100 ml with ethanol-free chloroform R. To 20 ml of the solution add 0.1 ml of C. (2RS)-2-[4-[2-(diethylamino)ethoxy]phenyl]-1,2triethylamine R and dilute to 100 ml with hexane R. diphenylethanone, Reference solution. Dissolve 25 mg of clomifene citrate CRS in 25 ml of 0.1 M hydrochloric acid, add 5 ml of 1 M sodium hydroxide and shake with three quantities, each of 25 ml, of ethanol-free chloroform R. Wash the combined extracts with 10 ml of water R, dry over anhydrous sodium sulphate R and dilute to 100 ml with ethanol-free chloroform R. To 20 ml of the solution add 0.1 ml of triethylamine R and D. 2,2-bis[4-[2-(diethylamino)ethoxy]phenyl]-1,2dilute to 100 ml with hexane R. diphenylethanone, General Notices (1) apply to all monographs and other texts

1327

Clomipramine hydrochloride


Comparison : clomipramine hydrochloride CRS. C. Thin-layer chromatography (2.2.27). Prepare the solutions immediately before use and protected from light. Test solution. Dissolve 20 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution. Dissolve 20 mg of clomipramine E. 2-[4-[1,2-bis(4-chlorophenyl)ethenyl]phenoxy]-N,Nhydrochloride CRS in methanol R and dilute to 10 ml diethylethanamine, with the same solvent. Plate : TLC silica gel G plate R. Mobile phase : concentrated ammonia R, acetone R, ethyl acetate R (5:25:75 V/V/V). Application : 5 µl. Development : over a path of 15 cm. F. 2-[4-[2-chloro-2-(4-chlorophenyl)-1-phenylethenyl]pheDrying : in air. noxy]-N,N-diethylethanamine, Detection : spray with a 5 g/l solution of potassium dichromate R in a 20 per cent V/V solution of sulphuric acid R. Examine immediately. Results : the principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. GH. 2-[2-chloro-4-(2-chloro-1,2-diphenylethenyl)phenoxy]-N, N-diethylethanamine (G. higher-melting point isomer ; H. D. Dissolve about 5 mg in 2 ml of nitric acid R. An intense blue colour develops. lower-melting point isomer). E. Dissolve about 50 mg in 5 ml of water R and add 1 ml of dilute ammonia R1. Mix, allow to stand for 5 min and filter. Acidify the filtrate with dilute nitric acid R. The 01/2005:0889 solution gives reaction (a) of chlorides (2.3.1).

CLOMIPRAMINE HYDROCHLORIDE

TESTS Solution S. Dissolve 2.0 g in carbon dioxide-free water R Clomipramini hydrochloridum and dilute to 20 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y5 (2.2.2, Method I). pH (2.2.3) : 3.5 to 5.0 for solution S. Related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately before use and protected from light. Test solution. Dissolve 20.0 mg of the substance to be examined in a mixture of 25 volumes of mobile phase B and C19H24Cl2N2 Mr 351.3 75 volumes of mobile phase A and dilute to 10.0 ml with the same mixture of mobile phases. DEFINITION Reference solution (a). Dissolve 22.6 mg of imipramine hydrochloride CRS, 4.0 mg of clomipramine 3-(3-Chloro-10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)-N,Nimpurity C CRS, 4.0 mg of clomipramine impurity D CRS dimethylpropan-1-amine hydrochloride. and 2.0 mg of clomipramine impurity F CRS in a mixture Content : 99.0 per cent to 101.0 per cent (dried substance). of 25 volumes of mobile phase B and 75 volumes of mobile CHARACTERS phase A and dilute to 100.0 ml with the same mixture of mobile phases. Dilute 1.0 ml of this solution to 10.0 ml with Appearance : white or slightly yellow, crystalline powder, a mixture of 25 volumes of mobile phase B and 75 volumes slightly hygroscopic. of mobile phase A. Solubility : freely soluble in water and in methylene chloride, Reference solution (b). Dilute 1.0 ml of the test solution to soluble in alcohol. 100.0 ml with a mixture of 25 volumes of mobile phase B It shows polymorphism. and 75 volumes of mobile phase A. Reference solution (c). Dissolve 10.0 mg of clomipramine IDENTIFICATION hydrochloride CRS and 3.0 mg of clomipramine First identification : B, E. impurity C CRS in a mixture of 25 volumes of mobile Second identification : A, C, D, E. phase B and 75 volumes of mobile phase A and dilute to A. Melting point (2.2.14) : 191 °C to 195 °C. 20.0 ml with the same mixture of mobile phases. Dilute 1.0 ml of this solution to 10.0 ml with a mixture of 25 volumes B. Infrared absorption spectrophotometry (2.2.24). of mobile phase B and 75 volumes of mobile phase A. Preparation : discs of potassium bromide R. The −1 transmittance at about 2000 cm (5 µm) is at least 65 per Column : cent without compensation. — size : l = 0.25 m, Ø = 4.6 mm, 1328


Clomipramine hydrochloride


— stationary phase : cyanopropylsilyl silica gel for chromatography R (5 µm),

Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g.

— temperature : 30 °C.

ASSAY Dissolve 0.250 g in 50 ml of alcohol R and add 5.0 ml of 0.01 M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the 2 points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 35.13 mg of C19H24Cl2N2.

Mobile phase : — mobile phase A : dissolve 1.2 g of sodium dihydrogen phosphate R in water R, add 1.1 ml of nonylamine R, adjust to pH 3.0 with phosphoric acid R and dilute to 1000 ml with water R, — mobile phase B : acetonitrile R. Time (min)



0 - 10

75

25

10 - 20

75 → 65

25 → 35

20 - 32

65

35

32 - 34

65 → 75

35 → 25

34 - 44

75

25

STORAGE In an airtight container, protected from light. IMPURITIES

Flow rate : 1.5 ml/min. Detection : spectrophotometer at 254 nm. Injection : 20 µl. Relative retentions with reference to clomipramine (retention time = about 8 min) : impurity A = about 0.5 ; impurity B = about 0.7 ; impurity C = about 0.9 ; impurity D = about 1.7 ; impurity E = about 2.5 ; impurity F = about 3.4 ; impurity G = about 4.3.

A. N-[3-(3-chloro-10,11-dihydro-5H-dibenzo[b,f]azepin-5yl)propyl]-N,N′,N′-trimethylpropane-1,3-diamine, B. imipramine,

System suitability : reference solution (c) : — resolution : minimum 3.0 between the peaks due to clomipramine and to impurity C. Limits : — impurity B : not more than the area of the corresponding peak in the chromatogram obtained with reference solution (a) (1.0 per cent), — impurity C, D : for each impurity, not more than the area of the corresponding peak in the chromatogram obtained C. 3-(3-chloro-5H-dibenzo[b,f]azepin-5-yl)-N,Ndimethylpropan-1-amine, with reference solution (a) (0.2 per cent), — impurity F : not more than the area of the corresponding peak in the chromatogram obtained with reference solution (a) (0.1 per cent), — any other impurity : not more than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent), — total of other impurities: not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.2 per cent), — total: not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent),

D. R1 = R3 = Cl, R2 = CH2-CH2-CH2-N(CH3)2 : 3-(3,7-dichloro-10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)N,N-dimethylpropan-1-amine,

— disregard limit : 0.01 times the area of the principal peak in the chromatogram obtained with reference solution (b) E. R1 = R2 = R3 = H : 10,11-dihydro-5H-dibenzo[b,f]azepine (iminodibenzyl), (0.01 per cent). Heavy metals (2.4.8) : maximum 20 ppm. 2.0 g complies with limit test C. Prepare the standard using 4 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. General Notices (1) apply to all monographs and other texts

F. R1 = Cl, R2 = R3 = H : 3-chloro-10,11-dihydro-5Hdibenzo[b,f]azepine, G. R1 = Cl, R2 = CH2-CH=CH2, R3 = H : 3-chloro-5-(prop-2enyl)-10,11-dihydro-5H-dibenzo[b,f]azepine. 1329

Clonazepam


01/2005:0890 Detection : spectrophotometer at 254 nm. Injection : 10 µl. CLONAZEPAM Run time : 3 times the retention time of clonazepam. Relative retention with reference to clonazepam (retention time = about 7 min) : impurity B = about 2.1 ; Clonazepamum impurity A = about 2.4. System suitability : reference solution (b) : — resolution : minimum 1.8 between the peaks due to flunitrazepam and to clonazepam. Limits : — impurity A : not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), — impurity B : not more than the area of the principal peak in the chromatogram obtained with reference solution (c) C15H10ClN3O3 Mr 315.7 (0.1 per cent) DEFINITION — any other impurity : for each impurity, not more than the area of the principal peak in the chromatogram obtained 5-(2-Chlorophenyl)-7-nitro-1,3-dihydro-2H-1,4-benzodiazepinwith reference solution (a) (0.1 per cent), 2-one. — total : not more than twice the area of the principal peak Content : 99.0 per cent to 101.0 per cent (dried substance). in the chromatogram obtained with reference solution (a) (0.2 per cent), CHARACTERS — disregard limit : 0.5 times the area of the principal peak Appearance : slightly yellowish, crystalline powder. in the chromatogram obtained with reference solution (a) Solubility : practically insoluble in water, slightly soluble (0.05 per cent). in alcohol and in methanol. Loss on drying (2.2.32) : maximum 0.5 per cent, determined mp : about 239 °C. on 1.000 g by drying in an oven at 100-105 °C for 4 h. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined IDENTIFICATION on 1.0 g. Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of clonazepam. ASSAY Dissolve 0.275 g in 50 ml of acetic anhydride R. Titrate TESTS with 0.1 M perchloric acid, determining the end-point Related substances. Liquid chromatography (2.2.29). Carry potentiometrically (2.2.20). out the test protected from light and prepare the solutions 1 ml of 0.1 M perchloric acid is equivalent to 31.57 mg immediately before use. of C15H10ClN3O3. Solvent mixture : tetrahydrofuran R, methanol R, water R STORAGE (10:42:48 V/V/V). Protected from light. Test solution. Dissolve 0.100 g of the substance to be examined in methanol R and dilute to 20.0 ml with the same IMPURITIES solvent. Dilute 1.0 ml to 10.0 ml with the solvent mixture. Specified impurities : A, B. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of the solution to 10.0 ml with the solvent mixture. Reference solution (b). Dissolve 5 mg of the substance to be examined and 5 mg of flunitrazepam R in the solvent mixture and dilute to 100.0 ml with the solvent mixture. Reference solution (c). Dissolve 1.0 mg of clonazepam impurity B CRS in the solvent mixture and dilute to 20.0 ml with the solvent mixture. Dilute 1.0 ml of the solution to 100.0 ml with the solvent mixture. A. (2-amino-5-nitrophenyl)(2-chlorophenyl)methanone, Column : — size : l = 0.15 m, Ø = 4.6 mm, — stationary phase: end-capped octylsilyl silica gel for chromatography R (5 µm). Mobile phase : mix 10 volumes of tetrahydrofuran R, 42 volumes of methanol R and 48 volumes of a 6.6 g/l solution of ammonium phosphate R previously adjusted to pH 8.0 with a 40 g/l solution of sodium hydroxide R or dilute phosphoric acid R. Flow rate : 1.0 ml/min. B. 3-amino-4-(2-chlorophenyl)-6-nitroquinolin-2(1H)-one. 1330



Closantel sodium dihydrate for veterinary use

01/2005:0477 over a path of 15 cm. Allow the plate to dry in air and spray with potassium iodobismuthate solution R2. Allow the plate to dry in air for 1 h, spray again with potassium CLONIDINE HYDROCHLORIDE iodobismuthate solution R2 and then immediately spray with a 50 g/l solution of sodium nitrite R. Any spot in the Clonidini hydrochloridum chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. C9H10Cl3N3 Mr 266.6 Sulphated ash (2.4.14). Not more than 0.1 per cent, DEFINITION determined on 1.0 g. Clonidine hydrochloride contains not less than 98.5 per ASSAY cent and not more than the equivalent of 101.0 per cent of 2,6-dichloro-N-(imidazolidin-2-ylidene)aniline, calculated with Dissolve 0.200 g in 70 ml of alcohol R. Titrate with 0.1 M ethanolic sodium hydroxide determining the end-point reference to the dried substance. potentiometrically (2.2.20). CHARACTERS 1 ml of 0.1 M sodium hydroxide is equivalent to 26.66 mg A white or almost white, crystalline powder, soluble in water of C9H10Cl3N3. and in ethanol. IDENTIFICATION 01/2005:1716 First identification : B, D. Second identification : A, C, D. CLOSANTEL SODIUM DIHYDRATE A. Dissolve 30.0 mg in 0.01 M hydrochloric acid and dilute FOR VETERINARY USE to 100.0 ml with the same acid. Examined between 245 nm and 350 nm (2.2.25), the solution shows two Closantelum natricum dihydricum absorption maxima, at 272 nm and at 279 nm, and a point of inflexion at 265 nm. The specific absorbances at the ad usum veterinarium maxima are about 18 and about 16, respectively. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with clonidine hydrochloride CRS. C. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). D. It gives reaction (a) of chlorides (2.3.1). C22H13Cl2I2N2NaO2,2H2O Mr 721 TESTS DEFINITION Solution S. Dissolve 1.25 g in carbon dioxide-free water R N-[5-Chloro-4-[(RS)-(4-chlorophenyl)cyanomethyl]-2and dilute to 25 ml with the same solvent. methylphenyl]-2-hydroxy-3,5-diiodobenzamide sodium salt Appearance of solution. Solution S is clear (2.2.1) and not dihydrate. more intensely coloured than reference solution Y7 (2.2.2, Content : 98.5 per cent to 101.5 per cent (anhydrous Method II). substance). pH (2.2.3). The pH of solution S is 4.0 to 5.0. CHARACTERS Related substances. Examine by thin-layer chromatography Appearance : yellow powder, slightly hygroscopic. (2.2.27), using silica gel G R as the coating substance. Solubility : very slightly soluble in water, freely soluble in Test solution (a). Dissolve 0.1 g of the substance to be examined in methanol R and dilute to 10 ml with the same ethanol (96 per cent), soluble in methanol. It shows polymorphism. solvent. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml IDENTIFICATION with methanol R. Infrared absorption spectrophotometry (2.2.24). Reference solution (a). Dissolve 10 mg of clonidine hydrochloride CRS in methanol R and dilute to 10 ml with Preparation : discs without recrystallisation. the same solvent. Comparison : closantel sodium dihydrate CRS. Reference solution (b). Dilute 1 ml of test solution (a) to 10 ml with methanol R. Dilute 5 ml of this solution to 100 ml TESTS Appearance of solution. The solution is clear (2.2.1) and with methanol R. Apply to the plate 10 µl of each solution. Shake a mixture of not more intensely coloured than reference solution GY4 10 volumes of glacial acetic acid R, 40 volumes of butanol R (2.2.2, Method II). Dissolve 0.50 g in ethanol (96 per cent) R and dilute to and 50 volumes of water R. Allow to separate. Filter the upper layer and use the filtrate as the mobile phase. Develop 50 ml with the same solvent. General Notices (1) apply to all monographs and other texts

1331

Closantel sodium dihydrate for veterinary use


Related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately before use and protect from light.

Limits :

— size : l = 0.10 m, Ø = 4.6 mm,

— any other impurity : for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.2 per cent) ;

— correction factors : for the calculation of contents, multiply the peak areas of the following impurities by the corresponding correction factor : impurity A = 1.5 ; Test solution. Dissolve 0.100 g of the substance to be impurity B = 1.3 ; examined in methanol R and dilute to 10.0 ml with the same solvent. — impurity G : not more than 2.5 times the area of the principal peak in the chromatogram obtained with Reference solution (a). Dissolve 10 mg of closantel for reference solution (b) (0.5 per cent) ; system suitability CRS (containing impurities A to J) in — impurities F, H, I : for each impurity, not more methanol R and dilute to 1.0 ml with the same solvent. than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) Reference solution (b). Dilute 1.0 ml of the test solution to (0.3 per cent) ; 100.0 ml with methanol R. Dilute 5.0 ml of this solution to 25.0 ml with methanol R. — impurities A, B, C, D, E, J : for each impurity, not more than the area of the principal peak in the chromatogram Column : obtained with reference solution (b) (0.2 per cent) ;

— stationary phase: base-deactivated octadecylsilyl silica gel for chromatography R (3 µm), — temperature : 35 °C. Mobile phase : — mobile phase A : to 100 ml of a 7.7 g/l solution of ammonium acetate R previously adjusted to pH 4.3 with acetic acid R, add 50 ml of acetonitrile R and 850 ml of water R, — mobile phase B : to 100 ml of a 7.7 g/l solution of ammonium acetate R previously adjusted to pH 4.3 with acetic acid R, add 50 ml of water R and 850 ml of acetonitrile R, Time (min)



0-2

50

50

2 - 22

50 → 20

50 → 80

22 - 27

20

80

27 - 28

20 → 50

80 → 50

28 - 32

50

50

— total : not more than 7.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (1.5 per cent) ; — disregard limit : 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.05 per cent). Water (2.5.12) : 4.8 per cent to 5.8 per cent, determined on 0.250 g. Use a mixture of 1 volume of dimethylformamide R and 4 volumes of methanol R as the solvent. ASSAY Dissolve 0.500 g in 50 ml of a mixture of 1 volume of anhydrous acetic acid R and 7 volumes of methyl ethyl ketone R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 68.5 mg of C22H13Cl2I2N2NaO2. STORAGE In an airtight container, protected from light. IMPURITIES

Flow rate : 1.5 ml/min.

Specified impurities : A, B, C, D, E, F, G, H, I, J.

Detection : spectrophotometer at 240 nm. Injection : 10 µl. Relative retention with reference to closantel (retention time = about 16 min) : impurity A = about 0.07 ; impurity B = about 0.48 ; impurity C = about 0.62 ; impurity D = about 0.65 ; impurity E = about 0.82 ; impurity F = about 0.89 ; impurity G = about 0.93 ; impurity H = about 1.13 ; impurity I = about 1.16 ; impurity J = about 1.55.

A. 2-hydroxy-3,5-diiodobenzoic acid,

System suitability : reference solution (a) : — resolution : baseline separation between the peaks due to impurity G and closantel, — the chromatogram obtained is similar to the chromatogram supplied with closantel for system suitability CRS. 1332

B. (2RS)-(4-amino-2-chloro-5-methylphenyl)(4chlorophenyl)ethanenitrile, See the information section on general monographs (cover pages)


Clotrimazole

DEFINITION Clotrimazole contains not less than 98.5 per cent and not more than the equivalent of 100.5 per cent of 1-[(2-chlorophenyl)diphenylmethyl]-1H-imidazole, calculated with reference to the dried substance.

C. R1 = H, R2 = CO2H, R3 = I : (2RS)-[2-chloro-4-[(2hydroxy-3,5-diiodobenzoyl)amino]-5-methylphenyl](4chlorophenyl)acetic acid, D. R1 = H, R2 = CONH2, R3 = I : N-[4-[(1RS)-2-amino-1-(4chlorophenyl)-2-oxoethyl]-5-chloro-2-methylphenyl]-2hydroxy-3,5-diiodobenzamide, E. R1 = H, R2 = CN, R3 = Cl : 3-chloro-N-[5-chloro-4-[(RS)-(4chlorophenyl)cyanomethyl]-2-methylphenyl]-2-hydroxy-5iodobenzamide, F. R1 + R2 = O, R3 = I : N-[5-chloro-4-(4-chlorobenzoyl)-2methylphenyl]-2-hydroxy-3,5-diiodobenzamide, G. R1 = H, R2 = C(=NH)OCH3, R3 = I : methyl (2RS)-2-[2-chloro-4-[(2-hydroxy-3,5-diiodobenzoyl)amino]5-methylphenyl]-2-(4-chlorophenyl)acetimidate, H. R1 = H, R2 = CO-OCH3, R3 = I : methyl (2RS)-[2-chloro-4-[(2-hydroxy-3,5-diiodobenzoyl)amino]-5methylphenyl](4-chlorophenyl)acetate, I. R1 = R3 = H, R2 = CN : N-[5-chloro-4-[(RS)-(4chlorophenyl)cyanomethyl]-2-methylphenyl]-2-hydroxy-5iodobenzamide,

CHARACTERS A white or pale yellow, crystalline powder, practically insoluble in water, soluble in alcohol and in methylene chloride. IDENTIFICATION First identification : B. Second identification : A, C, D. A. Melting point (2.2.14) : 141 °C to 145 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with clotrimazole CRS. C. Examine before spraying in ultraviolet light at 254 nm, the chromatograms obtained in the test for (2-chlorophenyl)diphenylmethanol. The principal spot in the chromatogram obtained with test solution (b) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). D. Dissolve about 10 mg in 3 ml of sulphuric acid R. The solution is pale yellow. Add 10 mg of mercuric oxide R and 20 mg of sodium nitrite R. Allow to stand with occasional shaking. An orange colour develops, becoming orange-brown.

TESTS Appearance of solution. Dissolve 1.25 g in alcohol R and dilute to 25 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY6 (2.2.2, Method II). (2-Chlorophenyl)diphenylmethanol. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution (a). Dissolve 0.50 g of the substance to be examined in alcohol R and dilute to 5 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with alcohol R. J. N-[5-chloro-4-[[4-[[2-chloro-4-[(2-hydroxy-3,5-diiodobenReference solution (a). Dissolve 50 mg of clotrimazole CRS zoyl)amino]-5-methylphenyl]cyanomethyl]phenyl](4-chlo- in alcohol R and dilute to 5 ml with the same solvent. rophenyl)cyanomethyl]-2-methylphenyl]-2-hydroxy-3,5-di- Reference solution (b). Dissolve 10 mg of iodobenzamide. (2-chlorophenyl)diphenylmethanol CRS in alcohol R and dilute to 5 ml with the same solvent. Dilute 1 ml of the solution to 10 ml with alcohol R. 01/2005:0757 Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm using a mixture of 0.5 volumes of CLOTRIMAZOLE concentrated ammonia R1, 10 volumes of propanol R and 90 volumes of toluene R. Allow the plate to dry in air. Spray with a 10 per cent V/V solution of sulphuric acid R Clotrimazolum in alcohol R and heat at 100 °C to 105 °C for 30 min. Any spot corresponding to (2-chlorophenyl)diphenylmethanol in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.2 per cent). Imidazole. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. Dissolve 0.50 g of the substance to be examined in alcohol R and dilute to 10 ml with the same C22H17ClN2 Mr 344.8 solvent. General Notices (1) apply to all monographs and other texts

1333

Clove


upper part of the hypanthium. The head is globular and Reference solution. Dissolve 10 mg of imidazole R in dome-shaped, composed of four imbricated petals that alcohol R and dilute to 10 ml with the same solvent. Dilute enclose numerous incurved stamens and a short, erect 1 ml of the solution to 10 ml with alcohol R. style with a nectary disc at the base. The hypanthium Apply separately to the plate 10 µl of each solution. Develop exudes essential oil when indented with the finger-nail. over a path of 15 cm using a mixture of 0.5 volumes of concentrated ammonia R1, 10 volumes of propanol R and B. Reduce to a powder (355). The powder is dark brown and has the odour and taste of the unground drug. 90 volumes of toluene R. Allow the plate to dry in air. At the Examine under a microscope using chloral hydrate bottom of a chromatography tank, place an evaporating dish solution R. The powder shows the following diagnostic containing a mixture of 1 volume of hydrochloric acid R1, characters : fragments of the hypanthium showing the 1 volume of water R and 2 volumes of a 15 g/l solution epidermis and underlying parenchyma containing large of potassium permanganate R, close the tank and allow oil glands ; short fibres occurring singly or in small to stand for 15 min. Place the dried plate in the tank and groups, with thickened, lignified walls and few pits ; close the tank. Leave the plate in contact with the chlorine abundant fragments of parenchyma containing cluster vapour for 5 min. Withdraw the plate and place it in a crystals of calcium oxalate ; numerous triangular pollen current of cold air until the excess of chlorine is removed grains about 15 µm in diameter with three pores in the and an area of coating below the points of application does angles. Starch granules are absent. not give a blue colour with a drop of potassium iodide and starch solution R. Spray with potassium iodide and starch C. Examine by thin-layer chromatography (2.2.27), using solution R. Any spot corresponding to imidazole in the silica gel GF254 R as the coating substance. chromatogram obtained with the test solution is not more Test solution. Shake 0.1 g of the powdered drug (500) intense than the spot in the chromatogram obtained with with 2 ml of methylene chloride R for 15 min. Filter and the reference solution (0.2 per cent). carefully evaporate the filtrate to dryness on a water-bath. Loss on drying (2.2.32). Not more than 0.5 per cent, Dissolve the residue in 2 ml of toluene R. determined on 1.000 g by drying in an oven at 100 °C to Reference solution. Dissolve 20 µl of eugenol R in 2 ml 105 °C. of toluene R. Sulphated ash (2.4.14). Not more than 0.1 per cent, Apply to the plate as bands 20 mm by 3 mm, 10 µl of determined on 1.0 g. the reference solution and 20 µl of the test solution. Develop in an unsaturated tank over a path of 10 cm ASSAY using toluene R. Allow the plate to stand for 5 min and Dissolve 0.300 g in 80 ml of anhydrous acetic acid R. Titrate develop again in the same manner. Allow the plate to dry with 0.1 M perchloric acid using 0.3 ml of naphtholbenzein in air, examine in ultraviolet light at 254 nm and mark the solution R as indicator until the colour changes from quenching zones. In the chromatogram obtained with the brownish-yellow to green. test solution there is in the median part a quenching zone 1 ml of 0.1 M perchloric acid is equivalent to 34.48 mg of (eugenol) similar in position to the quenching zone in C22H17ClN2. the chromatogram obtained with the reference solution and there may be a weak quenching zone (acetyleugenol) STORAGE just below the zone corresponding to eugenol. Spray the plate with anisaldehyde solution R using 10 ml for Store protected from light. a plate 200 mm square and examine in daylight while heating at 100 °C to 105 °C for 5 min to 10 min. The zones corresponding to eugenol in the chromatograms obtained with the test and reference solutions have a 01/2005:0376 strong brownish-violet colour and the zone corresponding to acetyleugenol in the chromatogram obtained with the CLOVE test solution is faint violet-blue. In the chromatogram obtained with the test solution there are other coloured zones, particularly a faint red zone in the lower part and Caryophylli flos a reddish-violet zone (caryophyllene) in the upper part. DEFINITION TESTS Clove consists of the whole flower buds of Syzygium aromaticum (L.) Merill et L. M. Perry (Eugenia caryophyllus Foreign matter (2.8.2). Not more than 6 per cent of peduncles, petioles and fruits, not more than 2 per cent of (C. Spreng.) Bull. et Harr.) dried until they become deteriorated cloves and not more than 0.5 per cent of other reddish-brown. It contains not less than 150 ml/kg of foreign matter. essential oil. Total ash (2.4.16). Not more than 7.0 per cent. CHARACTERS ASSAY Clove has a characteristic, aromatic odour. It has the macroscopic and microscopic characters described Carry out the determination of essential oils in vegetable drugs (2.8.12). Use a 250 ml flask, 100 ml of water R as the under identification tests A and B. distillation liquid and 0.50 ml of xylene R in the graduated IDENTIFICATION tube. Grind 5.0 g of the drug with 5.0 g of diatomaceous earth R to form a fine, homogeneous powder and proceed A. The flower bud is reddish-brown and consists of a quadrangular stalked portion, the hypanthium, 10 mm to immediately with the determination using 4.0 g of the 12 mm long and 2 mm to 3 mm in diameter, surmounted mixture. Distil at a rate of 2.5 ml/min to 3.5 ml/min for 2 h. by four divergent lobes of sepals which surround a STORAGE globular head 4 mm to 6 mm in diameter. A bilocular Store protected from light. ovary containing numerous ovules is situated in the 1334


Cloxacillin sodium


01/2005:1091 Chromatographic profile. Examine by gas chromatography (2.2.28). Test solution. Dissolve 0.2 g of the substance to be examined CLOVE OIL in 10 g of hexane R. Reference solution. Dissolve 7 mg of β-caryophyllene R, Caryophylli floris aetheroleum 80 mg of eugenol R and 4 mg of acetyleugenol R in 10 g of hexane R. DEFINITION The chromatographic procedure may be carried out using : Clove oil is obtained by steam distillation from the dried — a fused silica capillary column 60 m long and about flower buds of Syzygium aromaticum (L.) Merill et 0.25 mm in internal diameter, coated with macrogol L. M. Perry (Eugenia caryophyllus C. Spreng. Bull. et Harr.). 20 000 R, — helium for chromatography R as the carrier gas at a flow CHARACTERS rate of 1.5 ml per min, A clear, yellow liquid which becomes brown when exposed to — a flame-ionisation detector, air, miscible with methylene chloride, toluene and fatty oils. — a split ratio of 1/100, maintaining the temperature of the column at 60 °C for IDENTIFICATION 8 min, then raising the temperature at a rate of 3 °C First identification : B, per min to 180 °C and maintaining at 180 °C for 5 min and Second identification : A. maintaining the temperature of the injection port and that of the detector at 270 °C. A. Examine by thin-layer chromatography (2.2.27) using a Inject about 1.0 µl of the reference solution. Identify suitable silica gel with a fluorescent indicator having an the eluted components following the order indicated in optimal intensity at 254 nm as the coating substance. the composition of the reference solution. Record the Test solution. Dissolve 20 µl of the substance to be retention times of these substances. examined in 2.0 ml of toluene R. The test is not valid unless : the number of theoretical plates Reference solution. Dissolve 15 µl of eugenol R and 15 µl is at least 30 000, calculated from the β-caryophyllene peak at of acetyleugenol R in 2.0 ml of toluene R. 110 °C and the resolution between the peak corresponding Apply separately to the plate, as bands, 20 µl of the test to eugenol and that corresponding to acetyleugenol is at solution and 15 µl of the reference solution. Develop in an least 1.5. unsaturated tank over a path of 10 cm using toluene R. Inject about 1.0 µl of the substance to be examined. Using Allow the plate to stand for 5 min and develop again in the retention times determined from the chromatogram the same manner. Allow the plate to dry in air, examine in obtained with the reference solution, locate the components ultraviolet light at 254 nm and mark the quenching zones. of the reference solution on the chromatogram obtained The chromatogram obtained with the test solution shows with the test solution. Disregard the peak due to the solvent. in the medium part a quenching zone (eugenol) which Determine the percentage content of each of the three corresponds in position to the quenching zone in the chromatogram obtained with the reference solution ; there components by the normalisation procedure. The contents is a weak quenching zone (acetyleugenol) just below the are within the following ranges : quenching zone of eugenol which corresponds in position — β-caryophyllene : 5.0 per cent to 14.0 per cent, to the zone of acetyleugenol in the chromatogram — eugenol : 75.0 per cent to 88.0 per cent, obtained with the reference solution. Spray the plate — acetyleugenol : 4.0 per cent to 15.0 per cent. with anisaldehyde solution R and examine in daylight STORAGE while heating at 100 °C to 105 °C for 5 to 10 min. The zones corresponding to eugenol in the chromatogram Store in a well-filled, airtight container, protected from light obtained with the test and reference solutions have a and heat. strong brownish-violet colour and the zone corresponding to acetyleugenol in the chromatogram obtained with the 01/2005:0661 test solution is faint violet-blue. In the chromatogram obtained with the test solution there are other coloured zones particularly a faint red zone in the lower part and a CLOXACILLIN SODIUM reddish-violet zone (β-caryophyllene) in the upper part. B. Examine the chromatograms obtained in the test for Cloxacillinum natricum chromatographic profile. The chromatogram obtained with the test solution shows three main peaks similar in retention time to the three peaks obtained with the reference solution. TESTS Relative density (2.2.5) : 1.030 to 1.063. Refractive index (2.2.6) : 1.528 to 1.537. Angle of optical rotation (2.2.7) : 0° to − 2°. Fatty oils and resinified essential oils (2.8.7). It complies with the test for fatty oils and resinified oils. Solubility in alcohol (2.8.10). 1.0 ml is soluble in 2.0 ml or more of alcohol (70 per cent V/V) R. General Notices (1) apply to all monographs and other texts

C19H17ClN3NaO5S,H2O

Mr 475.9

DEFINITION Cloxacillin sodium contains not less than 95.0 per cent and not more than the equivalent of 101.0 per cent of sodium (2S,5R,6R)-6-[[[3-(2-chlorophenyl)-51335

Cloxacillin sodium


methylisoxazol-4-yl]carbonyl]amino]-3,3-dimethyl-7-oxo-4thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, calculated with reference to the anhydrous substance. CHARACTERS A white or almost white, crystalline powder, hygroscopic, freely soluble in water and in methanol, soluble in alcohol. IDENTIFICATION First identification : A, D. Second identification : B, C, D. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cloxacillin sodium CRS. Examine the substances prepared as discs. B. Examine by thin-layer chromatography (2.2.27), using silanised silica gel H R as the coating substance. Test solution. Dissolve 25 mg of the substance to be examined in 5 ml of water R. Reference solution (a). Dissolve 25 mg of cloxacillin sodium CRS in 5 ml of water R. Reference solution (b). Dissolve 25 mg of each of cloxacillin sodium CRS, dicloxacillin sodium CRS and flucloxacillin sodium CRS in 5 ml of water R. Apply separately to the plate 1 µl of each solution. Develop over a path of 15 cm using a mixture of 30 volumes of acetone R and 70 volumes of a 154 g/l solution of ammonium acetate R, adjusted to pH 5.0 with glacial acetic acid R. Allow the plate to dry in air and expose it to iodine vapour until the spots appear. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly separated spots. C. Place about 2 mg in a test-tube about 150 mm long and 15 mm in diameter. Moisten with 0.05 ml of water R and add 2 ml of sulphuric acid-formaldehyde reagent R. Mix the contents of the tube by swirling ; the colour of the solution is slightly greenish-yellow. Place the test-tube in a water-bath for 1 min ; the solution becomes yellow. D. It gives reaction (a) of sodium (2.3.1).

the areas of all the peaks, apart from the principal peak, is not greater than five times the area of the principal peak in the chromatogram obtained with reference solution (b) (5 per cent). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (b). N,N-Dimethylaniline (2.4.26, Method B). Not more than 20 ppm. 2-Ethylhexanoic acid (2.4.28). Not more than 0.8 per cent m/m. Water (2.5.12) : 3.0 per cent to 4.5 per cent, determined on 0.300 g by the semi-micro determination of water. Bacterial endotoxins (2.6.14) : less than 0.40 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins.

ASSAY Examine by liquid chromatography (2.2.29). Test solution (a). Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Test solution (b). Dilute 5.0 ml of test solution (a) to 50.0 ml with the mobile phase. Reference solution (a). Dissolve 50.0 mg of cloxacillin sodium CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 5.0 ml of the solution to 50.0 ml with the mobile phase. Reference solution (b). Dilute 5.0 ml of test solution (b) to 50.0 ml with the mobile phase. Reference solution (c). Dissolve 5 mg of flucloxacillin sodium CRS and 5 mg of cloxacillin sodium CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.0 ml/min a mixture of 25 volumes of acetonitrile R and 75 volumes of a 2.7 g/l solution of potassium dihydrogen phosphate R adjusted to pH 5.0 with dilute sodium hydroxide solution R, — as detector a spectrophotometer set at 225 nm, — a 20 µl loop injector. TESTS Inject reference solution (c). Adjust the sensitivity of the Solution S. Dissolve 2.50 g in carbon dioxide-free water R system so that the heights of the principal peaks in the and dilute to 25.0 ml with the same solvent. chromatogram obtained are at least 50 per cent of the Appearance of solution. Solution S is clear (2.2.1). The full scale of the recorder. The test is not valid unless the absorbance (2.2.25) of solution S measured at 430 nm is resolution between the first peak (cloxacillin) and the not greater than 0.04. second peak (flucloxacillin) is at least 2.5. Inject reference solution (a) six times. The test is not valid unless the relative pH (2.2.3). The pH of solution S is 5.0 to 7.0. standard deviation of the peak area for cloxacillin is at most Specific optical rotation (2.2.7). Dissolve 0.250 g in water R 1.0 per cent. Inject alternately test solution (b) and reference and dilute to 25.0 ml with the same solvent. The specific solution (a). optical rotation is + 160 to + 169, calculated with reference to the anhydrous substance. STORAGE Related substances. Examine by liquid chromatography Store in an airtight container, at a temperature not exceeding (2.2.29) as prescribed under Assay. Inject test solution (a) 25 °C. If the substance is sterile, store in a sterile, airtight, and continue the chromatography for five times the retention tamper-proof container. time of the principal peak. Inject reference solution (b). In the chromatogram obtained with test solution (a) : the area LABELLING of any peak, apart from the principal peak, is not greater The label states, where applicable, that the substance is free than the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent) ; the sum of from bacterial endotoxins.

1336



IMPURITIES

Clozapine

DEFINITION Clozapine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of 8-chloro11-(4-methylpiperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine, calculated with reference to the dried substance. CHARACTERS A yellow, crystalline powder, practically insoluble in water, freely soluble in methylene chloride, soluble in alcohol. It dissolves in dilute acetic acid.

A. R = CO2H : (4S)-2-[carboxy[[[3-(2-chlorophenyl)-5methylisoxazol-4-yl]carbonyl]amino]methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penicilloic acids of IDENTIFICATION A. Melting point (2.2.14) : 182 °C to 186 °C. cloxacillin), B. R = H : (2RS,4S)-2-[[[[3-(2-chlorophenyl)-5-methylisoxazol- B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with 4-yl]carbonyl]amino]methyl]-5,5-dimethylthiazolidine-4clozapine CRS. Examine the substances prepared as carboxylic acid (penilloic acids of cloxacillin), discs.

TESTS Related substances. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm. Before use, develop the plate with a mixture of C. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia25 volumes of methanol R and 75 volumes of methylene 1-azabicyclo[3.2.0]heptane-2-carboxylic acid chloride R. Allow the plate to dry in air for 15 min. (6-aminopenicillanic acid), Test solution (a). Dissolve 0.20 g of the substance to be examined in methylene chloride R and dilute to 5 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with methylene chloride R. Reference solution (a). Dissolve 20 mg of clozapine CRS in methylene chloride R and dilute to 5 ml with the same solvent. Reference solution (b). Dilute 1.5 ml of test solution (b) to D. 3-(2-chlorophenyl)-5-methylisoxazole-4-carboxylic acid, 50 ml with methylene chloride R. Reference solution (c). Dilute 1 ml of test solution (b) to 50 ml with methylene chloride R. Reference solution (d). Dilute 5 ml of reference solution (c) to 10 ml with methylene chloride R. Reference solution (e). Dissolve 10 mg of clozapine CRS and 10 mg of oxazepam CRS in methylene chloride R and dilute to 5 ml with the same solvent. Apply separately to the plate 5 µl of each solution. Develop over a path of 10 cm using a mixture of 25 volumes of methanol R and 75 volumes of methylene chloride R. Allow E. (2S,5R,6R)-6-[[(2S,5R,6R)-6-[[[3-(2-chlorophenyl)-5the plate to dry in air and examine in ultraviolet light at methylisoxazol-4-yl]carbonyl]amino]-3,3-dimethyl-7-oxo254 nm. Any spot in the chromatogram obtained with test 4-thia-1-azabicyclo[3.2.0]heptane-2-carbonyl]amino]-3, solution (a), apart from the principal spot, is not more intense 3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2than the spot in the chromatogram obtained with reference carboxylic acid (6-APA cloxacillin amide). solution (b) (0.3 per cent) ; at most one such spot is more intense than the spot in the chromatogram obtained with 01/2005:1191 reference solution (c) (0.2 per cent) ; and at most two such spots are more intense than the spot in the chromatogram CLOZAPINE obtained with reference solution (d) (0.1 per cent). The test is not valid unless the chromatogram obtained with reference solution (e) shows two clearly separated spots Clozapinum and the chromatogram obtained with reference solution (d) shows a clearly visible spot. Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, C18H19ClN4 Mr 326.8 determined on 1.0 g. General Notices (1) apply to all monographs and other texts

1337

Cocaine hydrochloride


ASSAY Dissolve 0.100 g in 50 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 16.34 mg of C18H19ClN4. IMPURITIES

A. 8-chloro-5,10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11one,

IDENTIFICATION First identification : B, D. Second identification : A, C, D, E. A. Dissolve 20.0 mg in 0.01 M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute 5.0 ml of the solution to 50.0 ml with 0.01 M hydrochloric acid. Examined between 220 nm and 350 nm (2.2.25), the solution shows 2 absorption maxima, at 233 nm and 273 nm. The specific absorbance at 233 nm is 378 to 402. B. Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of cocaine hydrochloride. C. Dissolve 0.1 g in 5 ml of water R and add 1 ml of dilute ammonia R2. A white precipitate is formed. Initiate crystallisation by scratching the wall of the tube with a glass rod. The crystals, washed with water R and dried in vacuo, melt (2.2.14) at 96 °C to 99 °C. D. It gives reaction (a) of chlorides (2.3.1). E. It gives the reaction of alkaloids (2.3.1).

TESTS Solution S. Dissolve 0.5 g in water R and dilute to 25 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Acidity. To 10 ml of solution S add 0.05 ml of methyl red solution R. Not more than 0.2 ml of 0.02 M sodium hydroxide is required to change the colour of the indicator. B. 11,11′-(piperazin-1,4-diyl)bis(8-chloro-5H-dibenzo[b,e][1, Specific optical rotation (2.2.7) : − 70 to − 73 (dried 4]diazepine), substance). Dissolve 0.50 g in water R and dilute to 20.0 ml with the same solvent. Readily carbonisable substances. To 0.2 g add 2 ml of sulphuric acid R. After 15 min, the solution is not more intensely coloured than reference solution BY5 (2.2.2, Method I). Related substances. Examine by liquid chromatography C. 8-chloro-11-(piperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine. (2.2.29). Test solution. Dissolve 25.0 mg of the substance to be 01/2005:0073 examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (a). Dilute 1.0 ml of the test solution to COCAINE HYDROCHLORIDE 50.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Cocaini hydrochloridum Reference solution (b). Dissolve 25 mg of the substance to be examined in 0.01 M sodium hydroxide and dilute to 10.0 ml with the same solvent. Dilute 1.0 ml of the solution to 10.0 ml with 0.01 M sodium hydroxide. Allow the solution to stand for 15 min. Column : — size : l = 0.15 m, Ø = 4.6 mm, — stationary phase : end-capped octadecylsilyl silica gel C17H22ClNO4 Mr 339.8 for chromatography R (5 µm) with a specific surface area of 335 m2/g, a pore size of 10 nm and a carbon loading DEFINITION of 19.1 per cent, Methyl (1R,2R,3S,5S)-3-(benzoyloxy)-8-methyl-8— temperature : 35 °C. azabicyclo[3.2.1]octane-2-carboxylate hydrochloride. Mobile phase : triethylamine R, tetrahydrofuran R, Content : 98.5 per cent to 101.0 per cent (dried substance). acetonitrile R, water R (0.5:100:430:479.5 V/V/V/V). Flow rate : 1 ml/min. CHARACTERS Appearance : white, crystalline powder or colourless crystals. Detection : spectrophotometer at 216 nm. Injection : 20 µl. Solubility : very soluble in water, freely soluble in alcohol, slightly soluble in methylene chloride. Relative retention with reference to cocaine (retention mp : about 197 °C, with decomposition. time = about 7.4 min) : degradation product = about 0.7. 1338


Coconut oil, refined


System suitability : reference solution (b) : — resolution : minimum of 5 between the peaks due to cocaine and to the degradation product. Limits : — any impurity eluting after the principal peak : not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), — total: not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent), — disregard limit : 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on the residue from the test for loss on drying.

01/2005:1410

COCONUT OIL, REFINED Cocois oleum raffinatum DEFINITION Refined coconut oil is the fatty oil obtained from the dried, solid part of the endosperm of Cocos nucifera L., then refined. CHARACTERS A white or almost white, unctuous mass, practically insoluble in water, freely soluble in methylene chloride and in light petroleum (bp : 65 °C to 70 °C), very slightly soluble in alcohol. The refractive index is about 1.449, determined at 40 °C.

IDENTIFICATION A. It complies with the test for melting point (see Tests). ASSAY B. It complies with the test for composition of fatty acids Dissolve 0.250 g in a mixture of 5.0 ml of 0.01 M hydrochloric (see Tests). acid and 50 ml of alcohol R. Carry out a potentiometric TESTS titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the 2 points of inflexion. Melting point (2.2.14) : 23 °C to 26 °C. 1 ml of 0.1 M sodium hydroxide is equivalent to 33.98 mg of Acid value (2.5.1). Not more than 0.5, determined on 20.0 g. C17H22ClNO4. Peroxide value (2.5.5). Not more than 5.0. STORAGE Unsaponifiable matter (2.5.7). Not more than 1.0 per cent, determined on 5.0 g. Protected from light. Alkaline impurities in fatty oils (2.4.19). It complies with IMPURITIES the test for alkaline impurities in fatty oils. Composition of fatty acids (2.4.22, Method B). Coconut oil is melted under gentle heating to a homogeneous liquid prior to sampling. Reference solution. Dissolve 15.0 mg of tricaproin CRS, 80.0 mg of tristearin CRS, 0.150 g of tricaprin CRS, 0.200 g of tricaprylin CRS, 0.450 g of trimyristin CRS and 1.25 g of trilaurin CRS in a mixture of 2 volumes of methylene chloride R and 8 volumes of heptane R and dilute to 50 ml with the same mixture of solvents heating at 45 °C to 50 °C. Transfer 2 ml to a 10 ml centrifuge tube with a screw cap and evaporate the solvent in a current of nitrogen R. Dissolve A. methyl (1R,2R,3S,5S)-8-methyl-3-[[(E)-3with 1 ml of heptane R and 1 ml of dimethyl carbonate R phenylpropenoyl]oxy]-8-azabicyclo[3.2.1]octane-2and mix vigorously under gentle heating (50 °C to 60 °C). carboxylate (cinnamoylcocaine), Add, while still warm, 1 ml of a 12 g/l solution of sodium R in anhydrous methanol R, prepared with the necessary precautions and mix vigorously for about 5 min. Add 3 ml of distilled water R and mix vigorously for about 30 s. Centrifuge for 15 min at 1500 g. Inject 1 µl of the organic phase. Calculate the percentage content of each fatty acid using the following expression : B. bis[(1R,2R,3S,5S)-2-(methoxycarbonyl)-8-methyl8-azabicyclo[3.2.1]oct-3-yl] (1r,2c,3t,4t)-2,4diphenylcyclobutane-1,3-dicarboxylate (α-truxilline), Ax,s,c is the corrected peak area of each fatty acid in the test solution :

Rc is the relative correction factor : C. bis[(1R,2R,3S,5S)-2-(methoxycarbonyl)-8-methyl8-azabicyclo[3.2.1]oct-3-yl] (1r,2c,3t,4t)-3,4diphenylcyclobutane-1,2-dicarboxylate (β-truxilline). General Notices (1) apply to all monographs and other texts

for the peaks corresponding to caproic, caprylic, capric, lauric and myristic acid methyl esters. 1339

Cocoyl caprylocaprate


mx,r

=

mass of tricaproin, tricaprylin, tricaprin, trilaurin or trimyristin in the reference solution, in milligrams,

m1,r

=

Ax,r

=

mass of tristearin in the reference solution, in milligrams, area of the peaks corresponding to caproic, caprylic, capric, lauric and myristic acid methyl esters in the reference solution,

A1,r

=

Ax,s

=

Rc

=

area of the peak corresponding to stearic acid methyl ester in the reference solution, area of peaks corresponding to any specified or unspecified fatty acid methyl esters, 1 for peaks corresponding to each of the remaining specified fatty acid methyl esters or any unspecified fatty acid methyl ester.

C. It complies with the test for composition of fatty acids and fatty alcohols (see Tests). TESTS Appearance. The substance to be examined is not more intensely coloured than reference solution Y5 (2.2.2, Method I). Acid value (2.5.1). Not more than 0.5, determined on 5.0 g. Hydroxyl value (2.5.3, Method A). Not more than 5.0. Iodine value (2.5.4). Not more than 1.0. Saponification value (2.5.6) : 160 to 173. Composition of fatty acids and fatty alcohols (2.4.22, Method C). Use the chromatogram obtained with the following reference solution for identification of the peaks corresponding to the fatty alcohols.

The fatty acid fraction of the oil has the following Reference solution. Dissolve the following amounts of the composition : substances listed in Table 1411.-1 in 10 ml of heptane R. — caproic acid (RRt 0.11) : not more than 1.5 per cent, Table 1411.-1 — caprylic acid (RRt 0.23) : 5.0 per cent to 11.0 per cent, Substance Amount (mg) — capric acid (RRt 0.56) : 4.0 per cent to 9.0 per cent, 10 Methyl caproate R — lauric acid (RRt 0.75) : 40.0 per cent to 50.0 per cent, 90 Methyl caprylate R — myristic acid (RRt 0.85) : 15.0 per cent to 20.0 per cent, 50 Methyl caprate R — palmitic acid (RRt 0.93) : 7.0 per cent to 12.0 per cent, 20 — stearic acid (RRt 1.00) : 1.5 per cent to 5.0 per cent, Methyl laurate R — oleic acid and isomers (RRt 1.01) : 4.0 per cent to 10.0 per 10 Methyl myristate R cent, 10 Methyl palmitate R — linoleic acid (RRt 1.03) : 1.0 per cent to 3.0 per cent, 10 Methyl stearate R — linolenic acid (RRt 1.06) : not more than 0.2 per cent, 10 Capric alcohol R — arachidic acid (RRt 1.10) : not more than 0.2 per cent, 100 Lauryl alcohol R — eicosenoic acid (RRt 1.11) : not more than 0.2 per cent. 40 Myristyl alcohol R STORAGE 30 Cetyl alcohol CRS Store in a well-filled container, protected from light. 20 Stearyl alcohol CRS Consider the sum of the areas of the peaks due to the fatty acids listed below to be equal to 100 and the sum of the 01/2005:1411 areas of the peaks due to the fatty alcohols listed below to be equal to 100.

COCOYL CAPRYLOCAPRATE

The fatty-acid fraction of the substance has the following composition : — caproic acid : not more than 2.0 per cent,

Cocoylis caprylocapras DEFINITION Cocoyl caprylocaprate is a mixture of esters of saturated C12 to C18 alcohols with caprylic (octanoic) and capric (decanoic) acids obtained by the reaction of these acids with vegetable saturated fatty alcohols. CHARACTERS A slightly yellowish liquid, practically insoluble in water, miscible with alcohol and with liquid paraffin. It has a relative density of about 0.86, a refractive index of about 1.445 and a viscosity of about 11 mPa·s. IDENTIFICATION A. Freezing point (2.2.18) : not more than 15 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cocoyl caprylocaprate CRS. 1340

— caprylic acid : 50.0 per cent to 80.0 per cent, — capric acid : 20.0 per cent to 50.0 per cent, — lauric acid : not more than 3.0 per cent, — myristic acid : not more than 1.0 per cent. The fatty-alcohol fraction of the substance has the following composition : — capric alcohol : not more than 3.0 per cent, — lauryl alcohol : 48.0 per cent to 59.0 per cent, — myristyl alcohol : 18.0 per cent to 25.0 per cent, — cetyl alcohol : 6.0 per cent to 12.0 per cent, — stearyl alcohol : 9.0 per cent to 16.0 per cent. Water (2.5.12). Not more than 0.1 per cent, determined on 5.00 g by the semi-micro determination of water. Total ash (2.4.16). Not more than 0.1 per cent, determined on 1.0 g.



Codeine

01/2005:0076 Reference solution (b). Dilute 1.0 ml of reference solution (a) to 20.0 ml with the mobile phase. Reference solution (c). Dilute 1.0 ml of the test solution to CODEINE 50.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Codeinum Reference solution (d). Dilute 0.5 ml of the test solution to 5.0 ml with reference solution (a). Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : end-capped octylsilyl silica gel for chromatography R (5 µm). Mobile phase : dissolve 1.08 g of sodium octanesulphonate R in a mixture of 20 ml of glacial acetic acid R and 250 ml of acetonitrile R and dilute to 1000 ml with water R. C18H21NO3,H2O Mr 317.4 Flow rate : 2 ml/min. Detection : spectrophotometer at 245 nm. DEFINITION Injection : 10 µl. 7,8-Didehydro-4,5α-epoxy-3-methoxy-17-methylmorphinan6α-ol. Run time : 10 times the retention time of codeine. Content : 99.0 per cent to 101.0 per cent (dried substance). Relative retention with reference to codeine (retention time = about 6 min) : impurity B = about 0.6 ; CHARACTERS impurity E = about 0.7 ; impurity A = about 2.0 ; Appearance : white or almost white, crystalline powder or impurity C = about 2.3 ; impurity D = about 3.6. colourless crystals. System suitability : reference solution (d) : Solubility : soluble in boiling water, freely soluble in alcohol. — resolution : minimum 3 between the peaks due to codeine and impurity A. IDENTIFICATION Limits : First identification : A, C. — correction factor : for the calculation of content, multiply Second identification : A, B, D, E. the peak area of impurity C by 0.25, A. Melting point (2.2.14) : 155 °C to 159 °C. — impurity A : not more than twice the area of the principal B. To 2.0 ml of solution S (see Tests) add 50 ml of water R peak in the chromatogram obtained with reference then 10 ml of 1 M sodium hydroxide and dilute to solution (b) (1.0 per cent), 100.0 ml with water R. Examined between 250 nm and — impurities B, C, D, E : for each impurity, not more than 350 nm (2.2.25), the solution shows only 1 absorption twice the area of the principal peak in the chromatogram maximum at 284 nm. The specific absorbance at the obtained with reference solution (c) (0.2 per cent), absorption maximum is about 50 (dried substance). — any other impurity : for each impurity, not more than the C. Infrared absorption spectrophotometry (2.2.24). area of the principal peak in the chromatogram obtained Preparation : dried substance prepared as a disc of with reference solution (c) (0.1 per cent), potassium bromide R. — total of impurities other than A: not more than 10 times Comparison : Ph. Eur. reference spectrum of codeine. the area of the principal peak in the chromatogram D. To about 10 mg add 1 ml of sulphuric acid R and 0.05 ml obtained with reference solution (c) (1.0 per cent), of ferric chloride solution R2 and heat on a water-bath. A blue colour develops. Add 0.05 ml of nitric acid R. The — disregard limit : 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (c) colour changes to red. (0.05 per cent). E. It gives the reaction of alkaloids (2.3.1). Loss on drying (2.2.32) : 4.0 per cent to 6.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. TESTS Sulphated ash (2.4.14) : maximum 0.1 per cent, determined Solution S. Dissolve 50 mg in carbon dioxide-free water R on 1.0 g. and dilute to 10.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and ASSAY colourless (2.2.2, Method II). Dissolve 0.250 g in 10 ml of anhydrous acetic acid R. Add Specific optical rotation (2.2.7) : − 142 to − 146 (dried 20 ml of dioxan R. Titrate with 0.1 M perchloric acid using substance). 0.05 ml of crystal violet solution R as indicator. Dissolve 0.50 g in alcohol R and dilute to 25.0 ml with the 1 ml of 0.1 M perchloric acid is equivalent to 29.94 mg same solvent. of C18H21NO3. Related substances. Liquid chromatography (2.2.29). STORAGE Test solution. Dissolve 0.100 g of the substance to be examined and 0.100 g of sodium octanesulphonate R in the Protected from light. mobile phase and dilute to 10.0 ml with the mobile phase. IMPURITIES Reference solution (a). Dissolve 5.0 mg of codeine Specified impurities : A, B, C, D, E. impurity A CRS in the mobile phase and dilute to 5.0 ml with the mobile phase. Other detectable impurities : F, G. General Notices (1) apply to all monographs and other texts

1341

Codeine hydrochloride dihydrate


01/2005:1412

CODEINE HYDROCHLORIDE DIHYDRATE Codeini hydrochloridum dihydricum A. R1 = OCH3, R2 = R3 = H : 7,8-didehydro-4,5α-epoxy-3,6αdimethoxy-17-methylmorphinan (methylcodeine),

E. R1 = R2 = OH, R3 = H : 7,8-didehydro-4,5α-epoxy-3methoxy-17-methylmorphinan-6α,10-diol, C18H22ClNO3,2H2O

F. R1 = R3 = OH, R2 = H : 7,8-didehydro-4,5α-epoxy-3methoxy-17-methylmorphinan-6α,14-diol,

Mr 371.9

DEFINITION 7,8-Didehydro-4,5α-epoxy-3-methoxy-17-methylmorphinan6α-ol hydrochloride dihydrate. Content : 99.0 per cent to 101.0 per cent (anhydrous substance).

B. morphine,

CHARACTERS Appearance : white or almost white, crystalline powder or small, colourless crystals. Solubility : soluble in water, slightly soluble in ethanol, practically insoluble in cyclohexane.

C. 7,7′,8,8′-tetradehydro-4,5α:4′,5′α-diepoxy-3,3′-dimethoxy17,17′-dimethyl-2,2′-bimorphinanyl-6α,6′α-diol (codeine dimer),

D. 7,8-didehydro-2-[(7,8-didehydro-4,5α-epoxy-6α-hydroxy17-methylmorphinan-3-yl)oxy]-4,5α-epoxy-3-methoxy-17methylmorphinan-6α-ol (3-O-(codein-2-yl)morphine),

G. 6,7,8,14-tetradehydro-4,5α-epoxy-3,6-dimethoxy-17methylmorphinan (thebaine). 1342

IDENTIFICATION First identification : A, D. Second identification : B, C, D, E. A. Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of codeine hydrochloride dihydrate. B. To 5 ml of solution S (see Tests) add 1 ml of a mixture of equal volumes of strong sodium hydroxide solution R and water R and initiate crystallisation, if necessary, by scratching the wall of the tube with a glass rod and cooling in iced water. Wash the precipitate with water R and dry at 100-105 °C. It melts (2.2.15) at 155 °C to 159 °C. C. To about 10 mg add 1 ml of sulphuric acid R and 0.05 ml of ferric chloride solution R2 and heat on a water-bath. A blue colour develops. Add 0.05 ml of nitric acid R. The colour changes to red. D. Solution S gives reaction (a) of chlorides (2.3.1). E. It gives the reaction of alkaloids (2.3.1). TESTS Solution S. Dissolve 2.00 g in carbon dioxide-free water R prepared from distilled water R and dilute to 50.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). Acidity or alkalinity. To 5 ml of solution S add 5 ml of carbon dioxide-free water R. Add 0.05 ml of methyl red solution R and 0.2 ml of 0.02 M hydrochloric acid ; the solution is red. Add 0.4 ml of 0.02 M sodium hydroxide ; the solution becomes yellow. Specific optical rotation (2.2.7) : − 117 to − 121 (anhydrous substance). Dilute 5.0 ml of solution S to 10.0 ml with water R.


Codeine hydrochloride dihydrate


Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 0.100 g of the substance to be examined and 0.100 g of sodium octanesulphonate R in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (a). Dissolve 5.0 mg of codeine impurity A CRS in the mobile phase and dilute to 5.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 20.0 ml with the mobile phase. Reference solution (c). Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (d). Dilute 0.5 ml of the test solution to 5.0 ml with reference solution (a). Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase: end-capped octylsilyl silica gel for chromatography R (5 µm). Mobile phase : dissolve 1.08 g of sodium octanesulphonate R in a mixture of 20 ml of glacial acetic acid R and 250 ml of acetonitrile R and dilute to 1000 ml with water R. Flow rate : 2 ml/min. Detection : spectrophotometer at 245 nm. Injection : 10 µl. Run time : 10 times the retention time of codeine. Relative retention with reference to codeine (retention time = about 6 min) : impurity B = about 0.6 ; impurity E = about 0.7 ; impurity A = about 2.0 ; impurity C = about 2.3 ; impurity D = about 3.6. System suitability : reference solution (d) : — resolution : minimum 3 between the peaks due to codeine and impurity A. Limits : — correction factor : for the calculation of content, multiply the peak area of impurity C by 0.25, — impurity A : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent), — impurities B, C, D, E : for each impurity, not more than twice the area of the principal peak in the chromatogram obtained with reference solution (c) (0.2 per cent), — any other impurity : for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (c) (0.1 per cent), — total of impurities other than A : not more than 10 times the area of the principal peak in the chromatogram obtained with reference solution (c) (1.0 per cent), — disregard limit : 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.05 per cent). Sulphates (2.4.13) : maximum 0.1 per cent. Dilute 5 ml of solution S to 20 ml with distilled water R. 15 ml of the solution complies with the limit test for sulphates. Water (2.5.12) : 8.0 per cent to 10.5 per cent, determined on 0.250 g.

1 ml of 0.1 M sodium hydroxide is equivalent to 33.59 mg of C18H22ClNO3. STORAGE Protected from light. IMPURITIES Specified impurities : A, B, C, D, E. Other detectable impurities : F, G.

A. R1 = OCH3, R2 = R3 = H : 7,8-didehydro-4,5α-epoxy-3,6αdimethoxy-17-methylmorphinan (methylcodeine), E. R1 = R2 = OH, R3 = H : 7,8-didehydro-4,5α-epoxy-3methoxy-17-methylmorphinan-6α,10-diol, F. R1 = R3 = OH, R2 = H : 7,8-didehydro-4,5α-epoxy-3methoxy-17-methylmorphinan-6α,14-diol, B. morphine,



ASSAY Dissolve 0.300 g in a mixture of 5 ml of 0.01 M hydrochloric acid R and 30 ml of alcohol R. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read G. 6,7,8,14-tetradehydro-4,5α-epoxy-3,6-dimethoxy-17the volume added between the 2 points of inflexion. methylmorphinan (thebaine). General Notices (1) apply to all monographs and other texts

1343

Codeine phosphate hemihydrate


01/2005:0074 pH (2.2.3) : 4.0 to 5.0 for solution S. Specific optical rotation (2.2.7) : − 98 to − 102 (dried CODEINE PHOSPHATE substance). HEMIHYDRATE Dilute 5.0 ml of solution S to 10.0 ml with water R. Related substances. Liquid chromatography (2.2.29). Codeini phosphas hemihydricus Test solution. Dissolve 0.100 g of the substance to be examined and 0.100 g of sodium octanesulphonate R in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (a). Dissolve 5.0 mg of codeine impurity A CRS in the mobile phase and dilute to 5.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 20.0 ml with the mobile phase. Reference solution (c). Dilute 1.0 ml of the test solution to C18H24NO7P,1/2H2O Mr 406.4 50.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. DEFINITION Reference solution (d). Dilute 0.5 ml of the test solution to 7,8-Didehydro-4,5α-epoxy-3-methoxy-17-methylmorphinan5.0 ml with reference solution (a). 6α-ol phosphate hemihydrate. Column : Content : 98.5 per cent to 101.0 per cent (dried substance). — size : l = 0.25 m, Ø = 4.6 mm, CHARACTERS — stationary phase : end-capped octylsilyl silica gel for Appearance : white or almost white, crystalline powder or chromatography R (5 µm). small, colourless crystals. Mobile phase : dissolve 1.08 g of sodium octanesulphonate R Solubility : freely soluble in water, slightly soluble or very in a mixture of 20 ml of glacial acetic acid R and 250 ml of slightly soluble in alcohol. acetonitrile R and dilute to 1000 ml with water R. IDENTIFICATION Flow rate : 2 ml/min. First identification : B, E, F. Detection : spectrophotometer at 245 nm. Second identification : A, C, D, E, F, G. Injection : 10 µl. A. Dilute 1.0 ml of solution S (see Tests) to 100.0 ml with water R. To 25.0 ml of this solution add 25 ml of water R Run time : 10 times the retention time of codeine. then 10 ml of 1 M sodium hydroxide and dilute to Relative retention with reference to codeine (retention 100.0 ml with water R. Examined between 250 nm and time = about 6 min) : impurity B = about 0.6 ; 350 nm (2.2.25), the solution shows only 1 absorption impurity E = about 0.7 ; impurity A = about 2.0 ; maximum, at 284 nm. The specific absorbance at the impurity C = about 2.3 ; impurity D = about 3.6. absorption maximum is about 38 (dried substance). System suitability : reference solution (d) : B. Infrared absorption spectrophotometry (2.2.24). Preparation : dissolve 0.20 g in 4 ml of water R. Add 1 ml — resolution : minimum 3 between the peaks due to codeine and impurity A. of a mixture of equal volumes of strong sodium hydroxide Limits : solution R and water R and initiate crystallisation, if necessary, by scratching the wall of the tube with a glass — correction factor : for the calculation of content, multiply rod and cooling in iced water. Wash the precipitate the peak area of impurity C by 0.25, with water R and dry at 100-105 °C. Examine the dried precipitate prepared as discs using potassium bromide R. — impurity A : not more than twice the area of the principal peak in the chromatogram obtained with reference Comparison : Ph. Eur. reference spectrum of codeine. solution (b) (1.0 per cent), C. Dissolve 0.20 g in 4 ml of water R. Add 1 ml of a mixture of equal volumes of strong sodium hydroxide solution R — impurities B, C, D, E : for each impurity, not more than twice the area of the principal peak in the chromatogram and water R and initiate crystallisation, if necessary, obtained with reference solution (c) (0.2 per cent), by scratching the wall of the tube with a glass rod and — any other impurity : for each impurity, not more than the cooling in iced water. The precipitate, washed with area of the principal peak in the chromatogram obtained water R and dried at 100-105 °C, melts (2.2.14) at 155 °C with reference solution (c) (0.1 per cent), to 159 °C. D. To about 10 mg add 1 ml of sulphuric acid R and 0.05 ml — total of impurities other than A: not more than 10 times of ferric chloride solution R2 and heat on a water-bath. the area of the principal peak in the chromatogram A blue colour develops. Add 0.05 ml of nitric acid R. The obtained with reference solution (c) (1.0 per cent), colour changes to red. — disregard limit : 0.5 times the area of the principal peak E. It complies with the test for loss on drying (see Tests). in the chromatogram obtained with reference solution (c) F. Solution S gives reaction (a) of phosphates (2.3.1). (0.05 per cent). G. It gives the reaction of alkaloids (2.3.1). Sulphates (2.4.13) : maximum 0.1 per cent. Dilute 5 ml of solution S to 20 ml with distilled water R. 15 ml TESTS of the solution complies with the limit test for sulphates. Solution S. Dissolve 1.00 g in carbon dioxide-free water R prepared from distilled water R and dilute to 25.0 ml with Loss on drying (2.2.32) : 1.5 per cent to 3.0 per cent, the same solvent. determined on 1.000 g by drying in an oven at 100-105 °C. 1344


Codeine phosphate sesquihydrate


ASSAY Dissolve 0.350 g in a mixture of 10 ml of anhydrous acetic acid R and 20 ml of dioxan R. Titrate with 0.1 M perchloric acid using 0.05 ml of crystal violet solution R as indicator. 1 ml of 0.1 M perchloric acid is equivalent to 39.74 mg of C18H24NO7P.

01/2005:0075

CODEINE PHOSPHATE SESQUIHYDRATE Codeini phosphas sesquihydricus

STORAGE Protected from light. IMPURITIES Specified impurities : A, B, C, D, E. Other detectable impurities : F, G.

C18H24NO7P,11/2H2O

Mr 424.4

DEFINITION 7,8-Didehydro-4,5α-epoxy-3-methoxy-17-methylmorphinan6α-ol phosphate sesquihydrate. Content : 98.5 per cent to 101.0 per cent (dried substance). A. R1 = OCH3, R2 = R3 = H : 7,8-didehydro-4,5α-epoxy-3,6αdimethoxy-17-methylmorphinan (methylcodeine), E. R1 = R2 = OH, R3 = H : 7,8-didehydro-4,5α-epoxy-3methoxy-17-methylmorphinan-6α,10-diol, F. R1 = R3 = OH, R2 = H : 7,8-didehydro-4,5α-epoxy-3methoxy-17-methylmorphinan-6α,14-diol, B. morphine,



G. 6,7,8,14-tetradehydro-4,5α-epoxy-3,6-dimethoxy-17methylmorphinan (thebaine). General Notices (1) apply to all monographs and other texts

CHARACTERS Appearance : white or almost white, crystalline powder or small, colourless crystals. Solubility : freely soluble in water, slightly soluble in alcohol. IDENTIFICATION First identification : B, E, F. Second identification : A, C, D, E, F, G. A. Dilute 1.0 ml of solution S (see Tests) to 100.0 ml with water R. To 25.0 ml of this solution add 25 ml of water R then 10 ml of 1 M sodium hydroxide and dilute to 100.0 ml with water R. Examined between 250 nm and 350 nm (2.2.25), the solution shows only 1 absorption maximum, at 284 nm. The specific absorbance at the absorption maximum is about 38 (dried substance). B. Infrared absorption spectrophotometry (2.2.24). Preparation : dissolve 0.20 g in 4 ml of water R. Add 1 ml of a mixture of equal volumes of strong sodium hydroxide solution R and water R and initiate crystallisation, if necessary, by scratching the wall of the tube with a glass rod and cooling in iced water. Wash the precipitate with water R and dry at 100-105 °C. Examine the dried precipitate prepared as discs using potassium bromide R. Comparison : Ph. Eur. reference spectrum of codeine. C. Dissolve 0.20 g in 4 ml of water R. Add 1 ml of a mixture of equal volumes of strong sodium hydroxide solution R and water R and initiate crystallisation, if necessary, by scratching the wall of the tube with a glass rod and cooling in iced water. The precipitate, washed with water R and dried at 100-105 °C, melts (2.2.14) at 155 °C to 159 °C. D. To about 10 mg add 1 ml of sulphuric acid R and 0.05 ml of ferric chloride solution R2 and heat on a water-bath. A blue colour develops. Add 0.05 ml of nitric acid R. The colour changes to red. E. It complies with the test for loss on drying (see Tests). F. Solution S gives reaction (a) of phosphates (2.3.1). G. It gives the reaction of alkaloids (2.3.1). TESTS Solution S. Dissolve 1.00 g in carbon dioxide-free water R prepared from distilled water R and dilute to 25.0 ml with the same solvent. pH (2.2.3) : 4.0 to 5.0 for solution S. 1345

Codeine phosphate sesquihydrate


Specific optical rotation (2.2.7) : − 98 to − 102 (dried substance). Dilute 5.0 ml of solution S to 10.0 ml with water R. Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 0.100 g of the substance to be examined and 0.100 g of sodium octanesulphonate R in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (a). Dissolve 5.0 mg of codeine impurity A CRS in the mobile phase and dilute to 5.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 20.0 ml with the mobile phase. Reference solution (c). Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (d). Dilute 0.5 ml of the test solution to 5.0 ml with reference solution (a). Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase: end-capped octylsilyl silica gel for chromatography R (5 µm). Mobile phase : dissolve 1.08 g of sodium octanesulphonate R in a mixture of 20 ml of glacial acetic acid R and 250 ml of acetonitrile R and dilute to 1000 ml with water R. Flow rate : 2 ml/min. Detection : spectrophotometer at 245 nm. Injection : 10 µl. Run time : 10 times the retention time of codeine. Relative retention with reference to codeine (retention time = about 6 min) : impurity B = about 0.6 ; impurity E = about 0.7 ; impurity A = about 2.0 ; impurity C = about 2.3 ; impurity D = about 3.6. System suitability : reference solution (d) : — resolution : minimum 3 between the peaks due to codeine and impurity A. Limits : — correction factor : for the calculation of content, multiply the peak area of impurity C by 0.25, — impurity A : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent), — impurities B, C, D, E : for each impurity, not more than twice the area of the principal peak in the chromatogram obtained with reference solution (c) (0.2 per cent), — any other impurity : for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (c) (0.1 per cent), — total of impurities other than A : not more than 10 times the area of the principal peak in the chromatogram obtained with reference solution (c) (1.0 per cent), — disregard limit : 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.05 per cent). Sulphates (2.4.13) : maximum 0.1 per cent. Dilute 5 ml of solution S to 20 ml with distilled water R. 15 ml of the solution complies with the limit test for sulphates. Loss on drying (2.2.32) : 5.0 per cent to 7.5 per cent, determined on 0.500 g by drying in an oven at 100-105 °C. ASSAY Dissolve 0.350 g in a mixture of 10 ml of anhydrous acetic acid R and 20 ml of dioxan R. Titrate with 0.1 M perchloric acid using 0.05 ml of crystal violet solution R as indicator. 1346

1 ml of 0.1 M perchloric acid is equivalent to 39.74 mg of C18H24NO7P. STORAGE Protected from light. IMPURITIES Specified impurities : A, B, C, D, E. Other detectable impurities : F, G.

A. R1 = OCH3, R2 = R3 = H : 7,8-didehydro-4,5α-epoxy-3,6αdimethoxy-17-methylmorphinan (methylcodeine), E. R1 = R2 = OH, R3 = H : 7,8-didehydro-4,5α-epoxy-3methoxy-17-methylmorphinan-6α,10-diol, F. R1 = R3 = OH, R2 = H : 7,8-didehydro-4,5α-epoxy-3methoxy-17-methylmorphinan-6α,14-diol, B. morphine,



G. 6,7,8,14-tetradehydro-4,5α-epoxy-3,6-dimethoxy-17methylmorphinan (thebaine).


Codergocrine mesilate


01/2005:2060 lower alcohols. Where necessary, the production method is validated to demonstrate that alkyl mesilates are not detectable in the final product.

CODERGOCRINE MESILATE Codergocrini mesilas

DEFINITION A mixture of : — (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2,5bis(1-methylethyl)-3,6-dioxooctahydro-8H-oxazolo[3,2a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10, 10a-octahydroindolo[4,3-fg]quinoline-9-carboxamide methanesulphonate (dihydroergocornine mesilate) ; — (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-10bhydroxy-2-(1-methylethyl)-3,6-dioxooctahydro-8Hoxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4, 6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9carboxamide methanesulphonate (dihydroergocristine mesilate) ; — (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2-(1methylethyl)-5-(2-methylpropyl)-3,6-dioxooctahydro8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9carboxamide methanesulphonate (α-dihydroergocryptine mesilate) ; — (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2-(1methylethyl)-5-[(1RS)-1-methylpropyl]-3,6-dioxooctahydro8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9carboxamide methanesulphonate (β-dihydroergocryptine mesilate or epicriptine mesilate). Content : 98.0 per cent to 102.0 per cent (dried substance). PRODUCTION The production method must be evaluated to determine the potential for formation of alkyl mesilates, which is particularly likely to occur if the reaction medium contains General Notices (1) apply to all monographs and other texts

CHARACTERS Appearance : white or yellowish powder. Solubility : sparingly soluble in water, sparingly soluble to soluble in ethanol (96 per cent), slightly soluble in methylene chloride. IDENTIFICATION A. Thin-layer chromatography (2.2.27). Test solution. Dissolve 0.20 g of the substance to be examined in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 5 ml with the same mixture of solvents. Reference solution. Dissolve 0.20 g of methanesulphonic acid R in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 5 ml with the same mixture of solvents. Plate : TLC silica gel plate R. Mobile phase : water R, concentrated ammonia R, butanol R, acetone R (5:10:20:65 V/V/V/V). Application : 10 µl. Development : over 2/3 of the plate. Drying : in a current of cold air for not more than 1 min. Detection : spray with a 1 g/l solution of bromocresol purple R in methanol R, adjusted to a violet-red colour with 0.05 ml of dilute ammonia R1. Drying : in a current of hot air at 100 °C. Results : the principal spot in the chromatogram obtained with the test solution is similar in position and colour to the principal spot in the chromatogram obtained with the reference solution. B. Examine the chromatograms obtained in the test for composition. Results : the 4 principal peaks in the chromatogram obtained with the test solution are similar in retention time and size to the 4 principal peaks in the chromatogram obtained with the reference solution. TESTS pH (2.2.3) : 4.2 to 5.2. Dissolve 0.10 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Composition. Liquid chromatography (2.2.29) : use the normalisation procedure. Test solution. Dissolve 20 mg of the substance to be examined in a mixture of 1 volume of anhydrous ethanol R and 2 volumes of a 10 g/l solution of tartaric acid R and dilute to 10 ml with the same mixture of solvents. Reference solution. Dissolve 20 mg of codergocrine mesilate CRS in a mixture of 1 volume of anhydrous ethanol R and 2 volumes of a 10 g/l solution of tartaric acid R and dilute to 10 ml with the same mixture of solvents. Column : — size : l = 0.15 m, Ø = 4.6 mm, — stationary phase : octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : triethylamine R, acetonitrile R, water R (2.5:25:75 V/V/V). Flow rate : 1.5 ml/min. Detection : spectrophotometer at 280 nm. Injection : 20 µl. 1347

Cod-liver oil (type A)


Run time : 20 min. Elution order : dihydroergocornine, α-dihydroergocryptine, dihydroergocristine, β-dihydroergocryptine. System suitability : test solution : — resolution : minimum 3 between any 2 consecutive principal peaks. Composition : — dihydroergocornine : 30.0 per cent to 35.0 per cent, — α-dihydroergocryptine : 20.0 per cent to 25.0 per cent, — dihydroergocristine : 30.0 per cent to 35.0 per cent, — β-dihydroergocryptine : 10.0 per cent to 13.0 per cent, — disregard limit : 1.0 per cent. Related substances. Thin-layer chromatography (2.2.27). Perform the test as rapidly as possible and protected from direct light. Prepare the test solution last and immediately before application on the plate. Test solution. Dissolve 0.40 g of the substance to be examined in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 5.0 ml with the same mixture of solvents. Reference solution (a). Dissolve 40 mg of dihydroergocristine mesilate CRS in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 10.0 ml with the same mixture of solvents. Dilute 3.0 ml of the solution to 50.0 ml with a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R. Reference solution (b). To 2.0 ml of reference solution (a), add 1.0 ml of a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R. Reference solution (c). To 1.0 ml of reference solution (a), add 2.0 ml of a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R. Reference solution (d). To 1.0 ml of reference solution (a), add 5.0 ml of a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R. Plate : TLC silica gel plate R. Mobile phase : concentrated ammonia R, methanol R, ethyl acetate R, methylene chloride R (1:3:50:50 V/V/V/V). Application : 10 µl. Drying : in the dark for 2 min after the application of the last solution. First development : in an unsaturated tank, over 2/3 of the plate. Drying : in a current of cold air for not more than 1 min. Second development : in an unsaturated tank, over 2/3 of the plate ; use freshly prepared mobile phase. Drying : in a current of cold air for not more than 1 min. Detection : spray thoroughly with dimethylaminobenzaldehyde solution R7 and dry in a current of hot air until the spot in the chromatogram obtained with reference solution (d) is clearly visible. System suitability : test solution : — the chromatogram shows at least 3 separated secondary spots. Limits : — any impurity : any spots, apart from the principal spot, are not more intense than the spot in the chromatogram obtained with reference solution (a) (0.3 per cent) ; not more than 4 such spots are more intense than the spot in the chromatogram obtained with reference solution (c) (0.1 per cent) and 2 of these may be more intense than the spot in the chromatogram obtained with reference solution (b) (0.2 per cent). 1348

Loss on drying (2.2.32) : maximum 5.0 per cent, determined on 0.500 g by drying at 120 °C under high vacuum. ASSAY Dissolve 0.500 g in 60 ml of pyridine R. Pass a stream of nitrogen R over the surface of the solution and titrate with 0.1 M tetrabutylammonium hydroxide, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to 68.04 mg of codergocrine mesilate (average Mr = 680). STORAGE Protected from light. 01/2005:1192

COD-LIVER OIL (TYPE A) Iecoris aselli oleum A DEFINITION Purified fatty oil obtained from the fresh livers of Gadus morhua L. and other species of Gadidae, solid substances being removed by cooling and filtering. A suitable antioxidant may be added. Content : 600 IU (180 µg) to 2500 IU (750 µg) of vitamin A per gram and 60 IU (1.5 µg) to 250 IU (6.25 µg) of vitamin D3 per gram. CHARACTERS Appearance : clear, yellowish, viscous liquid. Solubility : practically insoluble in water, slightly soluble in alcohol, miscible with light petroleum. IDENTIFICATION First identification : A, B, C. Second identification : C, D. A. In the assay for vitamin A using method A, the test solution shows an absorption maximum (2.2.25) at 325 ± 2 nm. In the assay for vitamin A using method B, the chromatogram obtained with the test solution shows a peak corresponding to the peak of all-trans-retinol in the chromatogram obtained with the reference solution. B. In the assay for vitamin D3, the chromatogram obtained with test solution (a) shows a peak corresponding to the peak of cholecalciferol in the chromatogram obtained with reference solution (b). C. It complies with the test for composition of fatty acids (see Tests). D. To 0.1 g add 0.5 ml of methylene chloride R and 1 ml of antimony trichloride solution R. Mix. A deep blue colour develops in about 10 s. TESTS Colour : not more intensely coloured than a reference solution prepared as follows : to 3.0 ml of red primary solution add 25.0 ml of yellow primary solution and dilute to 50.0 ml with a 10 g/l solution of hydrochloric acid R (2.2.2, Method II). Relative density (2.2.5) : 0.917 to 0.930. Refractive index (2.2.6) : 1.477 to 1.484. Acid value (2.5.1) : maximum 2.0. Anisidine value (2.5.36) : maximum 30.0. Iodine value (2.5.4, Method B) : 150 to 180. Use starch solution R2.




Peroxide value (2.5.5, Method B) : maximum 10.0. Unsaponifiable matter (2.5.7) : maximum 1.5 per cent, determined on 2.0 g, and extracting with 3 quantities, each of 50 ml, of peroxide-free ether R. Stearin. 10 ml remains clear after cooling in iced water for 3 h. Composition of fatty acids. Gas chromatography (2.2.28). Trivial name of fatty acid

Nomenclature

Lower limit area (per cent)

Upper limit area (per cent)

14:0 16:0 18:0

2.0 7.0 1.0

6.0 14.0 4.0

16:1 n-7 18:1 n-7 18:1 n-9 20:1 n-11 20:1 n-9 22:1 n-9

4.5 2.0 12.0 1.0 5.0 0

11.5 7.0 21.0 5.5 17.0 1.5

22:1 n-11+13

5.0

12.0

Saturated fatty acids : Myristic acid Palmitic acid Stearic acid

Mono-unsaturated fatty acids : Palmitoleic acid cis-Vaccenic acid Oleic acid Gadoleic acid Gondoic acid Erucic acid Cetoleic acid (22:1 n-11)

Poly-unsaturated fatty acids : Linoleic acid α-Linolenic acid Moroctic acid

18:2 n-6 18:3 n-3 18:4 n-3

0.5 0 0.5

3.0 2.0 4.5

Timnodonic (eicosapentaenoic) acid (EPA)

20:5 n-3

7.0

16.0

Cervonic (docosahexaenoic) acid (DHA)

22:6 n-3

6.0

18.0

Temperature :

Column

Time (min)

Temperature (°C)

0 - 55

170 → 225

55 - 75

225

Injection port

250

Detection

280

Detection : flame ionisation. Injection : 1 µl, twice. System suitability : — the 15 fatty acids to be tested are satisfactorily identified from the chromatogram shown in Figure 1192.-1, — injection of a mixture of equal amounts of methyl palmitate R, methyl stearate R, methyl arachidate R and methyl behenate R give area percentages of 24.4, 24.8, 25.2 and 25.6 (± 0.5 per cent), respectively, — resolution : minimum of 1.3 between the peaks due to methyl oleate and methyl cis-vaccenate ; the resolution between the pair due to methyl gadoleate and methyl gondoate is sufficient for purposes of identification and area measurement. Calculate the area per cent for each fatty acid methyl ester from the expression :

Ax

=

peak area of fatty acid x,

At

=

sum of the peak areas (up to C22:6 n-3).

The calculation is not valid unless : — the total area is based only on peaks due to solely fatty acids methyl esters, Test solution. Introduce about 0.45 g of the substance to be examined into a 10 ml volumetric flask, dissolve in — the number of fatty acid methyl ester peaks exceeding hexane R containing 50 mg of butylhydroxytoluene R per 0.05 per cent of the total area is at least 24, litre and dilute to 10.0 ml with the same solvent. Transfer — the 24 largest peaks of the methyl esters account for more 2.0 ml of the solution into a quartz tube and evaporate the than 90 per cent of the total area. (These correspond solvent with a gentle current of nitrogen R. Add 1.5 ml of a to, in common elution order : 14:0, 15:0, 16:0, 16:1 n-7, 20 g/l solution of sodium hydroxide R in methanol R, cover 16:4 n-1, 18:0, 18:1 n-9, 18:1 n-7, 18:2 n-6, 18:3 n-3, with nitrogen R, cap tightly with a polytetrafluoroethylene 18:4 n-3, 20:1 n-11, 20:1 n-9, 20:1 n-7, 20:2 n-6, 20:4 n-6, lined cap, mix and heat in a water-bath for 7 min. Cool, add 20:3 n-3, 20:4 n-3, 20:5 n-3, 22:1 n-11, 22:1 n-9, 21:5 n-3, 2 ml of boron trichloride-methanol solution R, cover with 22:5 n-3, 22:6 n-3). nitrogen R, cap tightly, mix and heat in a water-bath for 30 min. Cool to 40-50 °C, add 1 ml of trimethylpentane R, ASSAY cap and vortex or shake vigorously for at least 30 s. Vitamin A. Carry out the test as rapidly as possible, Immediately add 5 ml of saturated sodium chloride avoiding exposure to actinic light and air, oxidising agents, solution R, cover with nitrogen R, cap and vortex or shake oxidation catalysts (for example, copper and iron) and thoroughly for at least 15 s. Allow the upper layer to become acids. clear and transfer to a separate tube. Shake the methanol Use method A. If method A is found not to be valid, use layer once more with 1 ml of trimethylpentane R and combine the trimethylpentane extracts. Wash the combined method B. extracts with 2 quantities, each of 1 ml, of water R and dry METHOD A over anhydrous sodium sulphate R. Prepare 2 solutions for Ultraviolet absorption spectrophotometry (2.2.25). each sample. Test solution. To 1.00 g in a round-bottomed flask, add Column : 3 ml of a freshly prepared 50 per cent m/m solution of potassium hydroxide R and 30 ml of ethanol R. Boil under — material : fused silica, reflux in a current of nitrogen R for 30 min. Cool rapidly — size : l = 30 m, Ø = 0.25 mm, and add 30 ml of water R. Extract with 50 ml of ether R. Repeat the extraction 3 times and discard the lower layer — stationary phase: macrogol 20 000 R (film thickness after complete separation. Wash the combined upper 0.25 µm). layers with 4 quantities, each of 50 ml, of water R and Carrier gas : hydrogen for chromatography R or helium for evaporate to dryness under a gentle current of nitrogen R chromatography R, where oxygen scrubber is applied. at a temperature not exceeding 30 °C or in a rotary evaporator at a temperature not exceeding 30 °C under Split ratio : 1:200. General Notices (1) apply to all monographs and other texts

1349



Figure 1192.-1. – Chromatogram for the test for composition of fatty acids of cod-liver oil (type A)

reduced pressure (water ejector). Dissolve the residue in sufficient 2-propanol R1 to give an expected concentration of vitamin A equivalent to 10-15 IU/ml. Measure the absorbances of the solution at 300 nm, 310 nm, 325 nm and 334 nm and at the wavelength of maximum absorption with a suitable spectrophotometer in 1 cm specially matched cells, using 2-propanol R1 as the compensation liquid. Calculate the content of vitamin A, as all-trans-retinol, in International Units per gram from the expression :

METHOD B Liquid chromatography (2.2.29). Test solution. To 2.00 g in a round-bottomed flask, add 5 ml of a freshly prepared 100 g/l solution of ascorbic acid R and 10 ml of a freshly prepared 800 g/l solution of potassium hydroxide R and 100 ml of ethanol R. Boil under a reflux condenser on a water-bath for 15 min. Add 100 ml of a 10 g/l solution of sodium chloride R and cool. Transfer the solution to a 500 ml separating funnel rinsing the round-bottomed flask with about 75 ml of a 10 g/l solution of sodium chloride R and then with 150 ml of a mixture of equal volumes of light petroleum R1 and ether R. Shake for 1 min. When the layers have separated completely, discard the lower layer and wash the upper layer, first with 50 ml A325 = absorbance at 325 nm, of a 30 g/l solution of potassium hydroxide R in a 10 per cent V/V solution of ethanol R and then with 3 quantities, m = mass of the substance to be examined, in grams, each of 50 ml, of a 10 g/l solution of sodium chloride R. V = total volume of solution containing 10-15 IU of Filter the upper layer through 5 g of anhydrous sodium vitamin A per millilitre, sulphate R on a fast filter paper into a 250 ml flask suitable 1830 = conversion factor for the specific absorbance of for a rotary evaporator. Wash the funnel with 10 ml of fresh all-trans-retinol, in International Units. extraction mixture, filter and combine the upper layers. The above expression can be used only if A325 has a value of Distil them at a temperature not exceeding 30 °C under not greater than A325,corr/0.970 where A325,corr is the corrected reduced pressure (water ejector) and fill with nitrogen R when evaporation is completed. Alternatively evaporate the absorbance at 325 nm and is given by the equation : solvent under a gentle current of nitrogen R at a temperature not exceeding 30 °C. Dissolve the residue in 2-propanol R, transfer to a 25 ml volumetric flask and dilute to 25 ml with 2-propanol R. Gentle heating in an ultrasonic bath may be required. (A large fraction of the white residue is cholesterol, A designates the absorbance at the wavelength indicated constituting approximately 50 per cent of the unsaponifiable by the subscript. matter of cod-liver oil). If A325 has a value greater than A325,corr/0.970, calculate the content of vitamin A from the following expression : Reference solution (a). Prepare a solution of retinol acetate CRS in 2-propanol R1 so that 1 ml contains about 1000 IU of all-trans-retinol. The assay is not valid unless : — the wavelength of maximum absorption lies between 323 nm and 327 nm, — the absorbance at 300 nm relative to that at 325 nm is at most 0.73. 1350

The exact concentration of reference solution (a) is assessed by ultraviolet absorption spectrophotometry (2.2.25). Dilute reference solution (a) with 2-propanol R1 to a presumed concentration of 10-15 IU/ml and measure the absorbance at 326 nm in matched 1 cm cells using 2-propanol R1 as the compensation liquid.




Calculate the content of vitamin A in International Units per millilitre of reference solution (a) from the following expression, taking into account the assigned content of retinol acetate CRS :

A326 = V1 =


V2

volume of the diluted solution,

=

1900 =

volume of reference solution (a) used,

A1

=

A2

=

C

=

V m

= =

area of the peak due to all-trans-retinol in the chromatogram obtained with the test solution, area of the peak due to all-trans-retinol in the chromatogram obtained with reference solution (b), concentration of retinol acetate CRS in reference solution (a) as assessed prior to the saponification, in International Units per millilitre (= 1000 IU/ml), volume of reference solution (a) treated (2.00 ml), mass of the substance to be examined in the test solution (2.00 g).

conversion factor for the specific absorbance of retinol acetate CRS, in International Units.

Vitamin D3. Liquid chromatography (2.2.29). Carry out the assay as rapidly as possible, avoiding exposure to actinic light and air. Reference solution (b). Proceed as described for the test solution but using 2.00 ml of reference solution (a) in place Internal standard solution. Dissolve 0.50 mg of of the substance to be examined. ergocalciferol CRS in 100 ml of ethanol R. The exact concentration of reference solution (b) is assessed Test solution (a). To 4.00 g in a round-bottomed flask, add by ultraviolet absorption spectrophotometry (2.2.25). Dilute 5 ml of a freshly prepared 100 g/l solution of ascorbic acid R, 10 ml of a freshly prepared 800 g/l solution of reference solution (b) with 2-propanol R1 to a presumed concentration of 10-15 IU/ml of all-trans-retinol and measure potassium hydroxide R and 100 ml of ethanol R. Boil under a reflux condenser on a water-bath for 30 min. Add 100 ml the absorbance at 325 nm in matched 1 cm cells using of a 10 g/l solution of sodium chloride R and cool the 2-propanol R1 as the compensation liquid. solution to room temperature. Transfer the solution to a Calculate the content of all-trans-retinol in International 500 ml separating funnel rinsing the round-bottomed flask Units per millilitre of reference solution (b) from the with about 75 ml of a 10 g/l solution of sodium chloride R expression : and then with 150 ml of a mixture of equal volumes of light petroleum R1 and ether R. Shake for 1 min. When the layers have separated completely, discard the lower layer and wash the upper layer, first with 50 ml of a 30 g/l solution of potassium hydroxide R in a 10 per cent V/V solution A325 = absorbance at 325 nm, of ethanol R, and then with 3 quantities, each of 50 ml, of a 10 g/l solution of sodium chloride R. Filter the upper V3 = volume of the diluted solution, layer through 5 g of anhydrous sodium sulphate R on a V4 = volume of reference solution (b) used, fast filter paper into a 250 ml flask suitable for a rotary 1830 = conversion factor for the specific absorbance of evaporator. Wash the funnel with 10 ml of fresh extraction all-trans-retinol, in International Units. mixture, filter and combine the upper layers. Distil them at a temperature not exceeding 30 °C under reduced pressure Column : (water ejector) and fill with nitrogen R when evaporation is completed. Alternatively evaporate the solvent under a — size : l = 0.25 m, Ø = 4.6 mm, gentle current of nitrogen R at a temperature not exceeding — stationary phase: octadecylsilyl silica gel for 30 °C. Dissolve the residue in 1.5 ml of the mobile phase chromatography R (film thickness 5-10 µm). described under Purification. Gentle heating in an ultrasonic bath may be required. (A large fraction of the white residue Mobile phase : water R, methanol R (3:97 V/V). is cholesterol, constituting approximately 50 per cent m/m Flow rate : 1 ml/min. of the unsaponifiable matter of cod-liver oil). Detection : spectrophotometer at 325 nm. Test solution (b). To 4.00 g add 2.0 ml of the internal standard solution and proceed as described for test Injection : 10 µl ; inject in triplicate the test solution and solution (a). reference solution (b). Reference solution (a). Dissolve 0.50 mg of Retention time : all-trans-retinol = 5 ± 1 min. cholecalciferol CRS in 100.0 ml of ethanol R. Reference solution (b). In a round-bottomed flask, add 2.0 ml System suitability : of reference solution (a) and 2.0 ml of the internal standard — the chromatogram obtained with the test solution shows solution and proceed as described for test solution (a). a peak due to that of all-trans-retinol in the chromatogram PURIFICATION obtained with reference solution (b), Column : — when using the method of standard additions to the test solution there is greater than 95 per cent recovery of the — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : nitrile silica gel for chromatography R added retinol acetate CRS, (film thickness 10 µm). — the recovery of all-trans-retinol in reference solution (b) Mobile phase : isoamyl alcohol R, hexane R (1.6:98.4 V/V). as assessed by direct absorption spectrophotometry is Flow rate : 1.1 ml/min. greater than 95 per cent. Detection : spectrophotometer at 265 nm. Calculate the content of vitamin A using the following Inject 350 µl of reference solution (b). Collect the eluate expression : from 2 min before until 2 min after the retention time of cholecalciferol, in a ground-glass-stoppered tube containing 1 ml of a 1 g/l solution of butylhydroxytoluene R in General Notices (1) apply to all monographs and other texts

1351

Cod-liver oil (type B)


hexane R. Repeat the procedure with test solutions (a) and (b). Evaporate the eluates obtained from reference solution (b) and from test solutions (a) and (b), separately, to dryness at a temperature not exceeding 30 °C under a gentle current of nitrogen R. Dissolve each residue in 1.5 ml of acetonitrile R. DETERMINATION Column : — size : l = 0.15 m, Ø = 4.6 mm, — stationary phase: octadecylsilyl silica gel for chromatography R (film thickness 5 µm). Mobile phase : phosphoric acid R, 96 per cent V/V solution of acetonitrile R (0.2:99.8 V/V). Flow rate : 1.0 ml/min. Detection : spectrophotometer at 265 nm. Injection : 2 quantities not exceeding 200 µl of each of the 3 solutions obtained under Purification. System suitability : — resolution : minimum 1.4 between the peaks corresponding to ergocalciferol and cholecalciferol in the chromatogram obtained with reference solution (b), — when using the method of standard additions to test solution (a) there is greater than 95 per cent recovery of the added cholecalciferol CRS when due consideration has been given to correction by the internal standard. Calculate the content of vitamin D3 in International Units per gram using the following expression, taking into account the assigned content of cholecalciferol CRS :

m1

=

mass of the sample in test solution (b) in grams,

m2

=

A1

=

A2

=

A3

=

A4

=

A5

=

A6

=

V1

=

total mass of cholecalciferol CRS used for the preparation of reference solution (a) in micrograms (500 µg), area (or height) of the peak due to cholecalciferol in the chromatogram obtained with test solution (a), area (or height) of the peak due to cholecalciferol in the chromatogram obtained with test solution (b), area (or height) of the peak due to ergocalciferol in the chromatogram obtained with reference solution (b), area (or height) of the peak due to ergocalciferol in the chromatogram obtained with test solution (b), area (or height) of a possible peak in the chromatogram obtained with test solution (a) with the same retention time as the peak co-eluting with ergocalciferol in test solution (b), area (or height) of the peak due to cholecalciferol in the chromatogram obtained with reference solution (b), total volume of reference solution (a) (100 ml),

V2

=

volume of reference solution (a) used for preparing reference solution (b) (2.0 ml).

STORAGE In an airtight and well-filled container, protected from light. If no antioxidant is added, store under an inert gas. 1352

Once the container has been opened, its contents are used as soon as possible and any part of the contents not used at once is protected by an atmosphere of inert gas. LABELLING The label states : — the number of International Units of vitamin A, — the number of International Units of vitamin D3, — the name and concentration of any added antioxidant. 01/2005:1193

COD-LIVER OIL (TYPE B) Iecoris aselli oleum B DEFINITION Purified fatty oil obtained from the fresh livers of Gadus morhua L. and other species of Gadidae, solid substances being removed by cooling and filtering. A suitable antioxidant may be added. Content : 600 IU (180 µg) to 2500 IU (750 µg) of vitamin A per gram and 60 IU (1.5 µg) to 250 IU (6.25 µg) of vitamin D3 per gram. CHARACTERS Appearance : clear, yellowish, viscous liquid. Solubility : practically insoluble in water, slightly soluble in alcohol, miscible with light petroleum. IDENTIFICATION First identification : A, B, C. Second identification : C, D. A. In the assay for vitamin A using method A, the test solution shows an absorption maximum (2.2.25) at 325 ± 2 nm. In the assay for vitamin A using method B, the chromatogram obtained with the test solution shows a peak corresponding to the peak of all-trans-retinol in the chromatogram obtained with the reference solution. B. In the assay for vitamin D3, the chromatogram obtained with test solution (a) shows a peak corresponding to the peak of cholecalciferol in the chromatogram obtained with reference solution (b). C. It complies with the test for composition of fatty acids (see Tests). D. To 0.1 g add 0.5 ml of methylene chloride R and 1 ml of antimony trichloride solution R. Mix. A deep blue colour develops in about 10 s. TESTS Colour : not more intensely coloured than a reference solution prepared as follows : to 3.0 ml of red primary solution add 25.0 ml of yellow primary solution and dilute to 50.0 ml with a 10 g/l solution of hydrochloric acid R (2.2.2, Method II). Relative density (2.2.5) : 0.917 to 0.930. Refractive index (2.2.6) : 1.477 to 1.484. Acid value (2.5.1) : maximum 2.0. Iodine value (2.5.4, Method B) : 150 to 180. Use starch solution R2. Peroxide value (2.5.5, Method B) : maximum 10.0. Unsaponifiable matter (2.5.7) : maximum 1.5 per cent, determined on 2.0 g and extracting with 3 quantities, each of 50 ml, of peroxide-free ether R.




Stearin. 10 ml remains clear after cooling in iced water for 3 h.

Temperature :

Composition of fatty acids. Gas chromatography (2.2.28). Column Trivial name of fatty acid

Nomenclature

Lower limit area (per cent)

Upper limit area (per cent)

2.0 7.0 1.0

6.0 14.0 4.0

16:1 n-7 18:1 n-7 18:1 n-9 20:1 n-11 20:1 n-9 22:1 n-9

4.5 2.0 12.0 1.0 5.0 0

11.5 7.0 21.0 5.5 17.0 1.5

22:1 n-11+13

5.0

12.0

Saturated fatty acids : Myristic acid Palmitic acid Stearic acid

14:0 16:0 18:0

Mono-unsaturated fatty acids : Palmitoleic acid cis-Vaccenic acid Oleic acid Gadoleic acid Gondoic acid Erucic acid Cetoleic acid (22:1 n-11)

Poly-unsaturated fatty acids : Linoleic acid α-Linolenic acid Moroctic acid

18:2 n-6 18:3 n-3 18:4 n-3

0.5 0 0.5

3.0 2.0 4.5

Timnodonic (eicosapentaenoic) acid (EPA)

20:5 n-3

7.0

16.0

Cervonic (docosahexaenoic) acid (DHA)

22:6 n-3

6.0

18.0

Test solution. Introduce about 0.45 g of the substance to be examined into a 10 ml volumetric flask, dissolve in hexane R containing 50 mg of butylhydroxytoluene R per litre and dilute to 10.0 ml with the same solvent. Transfer 2.0 ml of the solution into a quartz tube and evaporate the solvent with a gentle current of nitrogen R. Add 1.5 ml of a 20 g/l solution of sodium hydroxide R in methanol R, cover with nitrogen R, cap tightly with a polytetrafluoroethylene lined cap, mix and heat in a water-bath for 7 min. Cool, add 2 ml of boron trichloride-methanol solution R, cover with nitrogen R, cap tightly, mix and heat in a water-bath for 30 min. Cool to 40-50 °C, add 1 ml of trimethylpentane R, cap and vortex or shake vigorously for at least 30 s. Immediately add 5 ml of saturated sodium chloride solution R, cover with nitrogen R, cap and vortex or shake thoroughly for at least 15 s. Allow the upper layer to become clear and transfer to a separate tube. Shake the methanol layer once more with 1 ml of trimethylpentane R and combine the trimethylpentane extracts. Wash the combined extracts with 2 quantities, each of 1 ml, of water R and dry over anhydrous sodium sulphate R. Prepare 2 solutions for each sample.

Time (min)

Temperature (°C)

0 - 55

170 → 225

55 - 75

225

Injection port

250

Detector

280

Detection : flame ionisation. Injection : 1 µl, twice. System suitability : — the 15 fatty acids to be tested are satisfactorily identified from the chromatogram shown in Figure 1193.-1. — injection of a mixture of equal amounts of methyl palmitate R, methyl stearate R, methyl arachidate R, and methyl behenate R give area percentages of 24.4, 24.8, 25.2 and 25.6 (± 0.5 per cent), respectively, — resolution : minimum of 1.3 between the peaks due to methyl oleate and methyl cis-vaccenate ; the resolution between the pair due to methyl gadoleate and methyl gondoate is sufficient for purposes of identification and area measurement. Calculate the area per cent for each fatty acid methyl ester from the expression :

Ax

=

peak area of fatty acid x,

At

=

sum of the peak areas (up to C22:6 n-3).

The calculation is not valid unless : — the total area is based only on peaks due to solely fatty acids methyl esters, — the number of fatty acid methyl ester peaks exceeding 0.05 per cent of the total area is at least 24, — the 24 largest peaks of the methyl esters account for more than 90 per cent of the total area. (These correspond to, in common elution order : 14:0, 15:0, 16:0, 16:1 n-7, 16:4 n-1, 18:0, 18:1 n-9, 18:1 n-7, 18:2 n-6, 18:3 n-3, 18:4 n-3, 20:1 n-11, 20:1 n-9, 20:1 n-7, 20:2 n-6, 20:4 n-6, 20:3 n-3, 20:4 n-3, 20:5 n-3, 22:1 n-11, 22:1 n-9, 21:5 n-3, 22:5 n-3, 22:6 n-3).

ASSAY Vitamin A. Carry out the test as rapidly as possible, avoiding exposure to actinic light and air, oxidising agents, oxidation catalysts (for example, copper and iron) and acids. Use method A. If method A is found not to be valid, use method B. METHOD A Column : Ultraviolet absorption spectrophotometry (2.2.25). — material : fused silica, Test solution. To 1.00 g in a round-bottomed flask, add 3 ml of a freshly prepared 50 per cent m/m solution of — size : l = 30 m, Ø = 0.25 mm, potassium hydroxide R and 30 ml of ethanol R. Boil under reflux in a current of nitrogen R for 30 min. Cool rapidly — stationary phase: macrogol 20 000 R (film thickness and add 30 ml of water R. Extract with 50 ml of ether R. 0.25 µm). Repeat the extraction 3 times and discard the lower layer after complete separation. Wash the combined upper Carrier gas : hydrogen for chromatography R or helium for layers with 4 quantities, each of 50 ml, of water R and chromatography R, where oxygen scrubber is applied. evaporate to dryness under a gentle current of nitrogen R at a temperature not exceeding 30 °C or in a rotary Split ratio : 1:200. evaporator at a temperature not exceeding 30 °C under General Notices (1) apply to all monographs and other texts

1353



Figure 1193.-1. – Chromatogram for the test for composition of fatty acids of cod-liver oil (type B) hydroxide R and 100 ml of ethanol R. Boil under a reflux condenser on a water-bath for 15 min. Add 100 ml of a 10 g/l solution of sodium chloride R and cool. Transfer the solution to a 500 ml separating funnel rinsing the round-bottomed flask with about 75 ml of a 10 g/l solution of sodium chloride R and then with 150 ml of a mixture of equal volumes of light petroleum R1 and ether R. Shake for 1 min. When the layers have separated completely, discard the lower layer and wash the upper layer, first with 50 ml of a 30 g/l solution of potassium hydroxide R in a 10 per cent V/V solution of ethanol R and then with 3 quantities, each of 50 ml, of a 10 g/l solution of sodium chloride R. Filter the upper layer through 5 g of anhydrous sodium sulphate R on a fast filter paper into a 250 ml flask suitable A325 = absorbance at 325 nm, for a rotary evaporator. Wash the funnel with 10 ml of fresh extraction mixture, filter and combine the upper layers. m = mass of the substance to be examined, in grams, Distil them at a temperature not exceeding 30 °C under V = total volume of solution containing 10-15 IU of reduced pressure (water ejector) and fill with nitrogen R vitamin A per millilitre, when evaporation is completed. Alternatively evaporate the 1830 = conversion factor for the specific absorbance of solvent under a gentle current of nitrogen R at a temperature all-trans-retinol, in International Units. not exceeding 30 °C. Dissolve the residue in 2-propanol R, transfer to a 25 ml volumetric flask and dilute to 25 ml with The above expression can be used only if A325 has a value of not greater than A325,corr/0.970 where A325,corr is the corrected 2-propanol R. Gentle heating in an ultrasonic bath may be required. (A large fraction of the white residue is cholesterol, absorbance at 325 nm and is given by the equation : constituting approximately 50 per cent of the unsaponifiable matter of cod-liver oil). Reference solution (a). Prepare a solution of retinol acetate CRS in 2-propanol R1 so that 1 ml contains about A designates the absorbance at the wavelength indicated 1000 IU of all-trans-retinol. by the subscript. The exact concentration of reference solution (a) is assessed If A325 has a value greater than A325,corr/0.970, calculate the by ultraviolet absorption spectrophotometry (2.2.25). Dilute content of vitamin A from the expression : reference solution (a) with 2-propanol R1 to a presumed concentration of 10-15 IU/ml and measure the absorbance at 326 nm in matched 1 cm cells using 2-propanol R1 as the compensation liquid. The assay is not valid unless : Calculate the content of vitamin A in International Units — the wavelength of maximum absorption lies between per millilitre of reference solution (a) using the following 323 nm and 327 nm, expression, taking into account the assigned content of — the absorbance at 300 nm relative to that at 325 nm is retinol acetate CRS : at most 0.73. METHOD B Liquid chromatography (2.2.29). Test solution. To 2.00 g in a round-bottomed flask, add 5 ml A326 = absorbance at 326 nm, of a freshly prepared 100 g/l solution of ascorbic acid R and V = volume of reference solution (a) used, 1 10 ml of a freshly prepared 800 g/l solution of potassium reduced pressure (water ejector). Dissolve the residue in sufficient 2-propanol R1 to give an expected concentration of vitamin A equivalent to 10-15 IU/ml. Measure the absorbances of the solution at 300 nm, 310 nm, 325 nm and 334 nm and at the wavelength of maximum absorption with a suitable spectrophotometer in 1 cm specially matched cells, using 2-propanol R1 as the compensation liquid. Calculate the content of vitamin A, as all-trans-retinol, in International Units per gram from the expression :

1354



V2


Vitamin D3. Liquid chromatography (2.2.29). Carry out the assay as rapidly as possible, avoiding exposure to actinic light and air. Internal standard solution. Dissolve 0.50 mg of Reference solution (b). Proceed as described for the test ergocalciferol CRS in 100 ml of ethanol R. solution but using 2.00 ml of reference solution (a) in place Test solution (a). To 4.00 g in a round-bottomed flask, add of the substance to be examined. 5 ml of a freshly prepared 100 g/l solution of ascorbic The exact concentration of reference solution (b) is assessed acid R, 10 ml of a freshly prepared 800 g/l solution of by ultraviolet absorption spectrophotometry (2.2.25). Dilute potassium hydroxide R and 100 ml of ethanol R. Boil under reference solution (b) with 2-propanol R1 to a presumed a reflux condenser on a water-bath for 30 min. Add 100 ml concentration of 10-15 IU/ml of all-trans-retinol and measure of a 10 g/l solution of sodium chloride R and cool the the absorbance at 325 nm in matched 1 cm cells using solution to room temperature. Transfer the solution to a 2-propanol R1 as the compensation liquid. 500 ml separating funnel rinsing the round-bottomed flask with about 75 ml of a 10 g/l solution of sodium chloride R Calculate the content of all-trans-retinol in International and then with 150 ml of a mixture of equal volumes of light Units per millilitre of reference solution (b) from the petroleum R1 and ether R. Shake for 1 min. When the expression : layers have separated completely, discard the lower layer and wash the upper layer, first with 50 ml of a 30 g/l solution of potassium hydroxide R in a 10 per cent V/V solution of ethanol R, and then with 3 quantities, each of 50 ml, of A325 = absorbance at 325 nm, a 10 g/l solution of sodium chloride R. Filter the upper layer through 5 g of anhydrous sodium sulphate R on a V3 = volume of the diluted solution, fast filter paper into a 250 ml flask suitable for a rotary V4 = volume of reference solution (b) used, evaporator. Wash the funnel with 10 ml of fresh extraction mixture, filter and combine the upper layers. Distil them at 1830 = conversion factor for the specific absorbance of a temperature not exceeding 30 °C under reduced pressure all-trans-retinol, in International Units. (water ejector) and fill with nitrogen R when evaporation Column : is completed. Alternatively evaporate the solvent under a gentle current of nitrogen R at a temperature not exceeding — size : l = 0.25 m, Ø = 4.6 mm, 30 °C. Dissolve the residue in 1.5 ml of the mobile phase — stationary phase: octadecylsilyl silica gel for described under Purification. Gentle heating in an ultrasonic chromatography R (film thickness 5-10 µm). bath may be required. (A large fraction of the white residue Mobile phase : water R, methanol R (3:97 V/V). is cholesterol, constituting approximately 50 per cent m/m of the unsaponifiable matter of cod-liver oil). Flow rate : 1 ml/min. Test solution (b). To 4.00 g add 2.0 ml of the internal Detection : spectrophotometer at 325 nm. standard solution and proceed as described for test Injection : 10 µl ; inject in triplicate the test solution and solution (a). reference solution (b). Reference solution (a). Dissolve 0.50 mg of Retention time : all-trans-retinol = 5 ± 1 min. cholecalciferol CRS in 100.0 ml of ethanol R. System suitability : Reference solution (b). In a round-bottomed flask, add 2.0 ml — the chromatogram obtained with the test solution shows of reference solution (a) and 2.0 ml of the internal standard a peak due to that of all-trans-retinol in the chromatogram solution and proceed as described for test solution (a). obtained with reference solution (b), PURIFICATION — when using the method of standard additions to the test Column : solution there is greater than 95 per cent recovery of the — size : l = 0.25 m, Ø = 4.6 mm, added retinol acetate CRS, — stationary phase : nitrile silica gel for chromatography R — the recovery of all-trans-retinol in reference solution (b) (film thickness 10 µm). as assessed by direct absorption spectrophotometry is Mobile phase : isoamyl alcohol R, hexane R (1.6:98.4 V/V). greater than 95 per cent. Flow rate : 1.1 ml/min. Calculate the content of vitamin A using the following Detection : spectrophotometer at 265 nm. expression : Inject 350 µl of reference solution (b). Collect the eluate from 2 min before until 2 min after the retention time of cholecalciferol, in a ground-glass-stoppered tube containing 1 ml of a 1 g/l solution of butylhydroxytoluene R in A1 = area of the peak due to all-trans-retinol in the hexane R. Repeat the procedure with test solutions (a) chromatogram obtained with the test solution, and (b). Evaporate the eluates obtained from reference A2 = area of the peak due to all-trans-retinol in solution (b) and from test solutions (a) and (b), separately, the chromatogram obtained with reference to dryness at a temperature not exceeding 30 °C under a solution (b), gentle current of nitrogen R. Dissolve each residue in 1.5 ml C = concentration of retinol acetate CRS in reference of acetonitrile R. solution (a) as assessed prior to the saponification, DETERMINATION in International Units per millilitre (= 1000 IU/ml), Column : V = volume of reference solution (a) treated (2.00 ml), — size : l = 0.15 m, Ø = 4.6 mm, m = mass of the substance to be examined in the test — stationary phase : octadecylsilyl silica gel for solution (2.00 g). chromatography R (film thickness 5 µm). = 1900 =

volume of the diluted solution,

conversion factor for the specific absorbance of retinol acetate CRS, in International Units.


1355

Cola


Mobile phase : phosphoric acid R, a 96 per cent V/V solution of acetonitrile R (0.2:99.8 V/V). Flow rate : 1.0 ml/min. Detection : spectrophotometer at 265 nm. Injection : 2 quantities not exceeding 200 µl of each of the 3 solutions obtained under Purification. System suitability : — resolution : minimum 1.4 between the peaks due to ergocalciferol and cholecalciferol in the chromatogram obtained with reference solution (b), — when using the method of standard additions to test solution (a) there is greater than 95 per cent recovery of the added cholecalciferol CRS when due consideration has been given to correction by the internal standard.

01/2005:1504

COLA Colae semen DEFINITION Cola consists of the whole or fragmented dried seeds, freed from the testa, of Cola nitida (Vent.) Schott et Endl. (C. vera K. Schum.) and its varieties, as well as of Cola acuminata (P. Beauv.) Schott et Endl. (Sterculia acuminata P. Beauv.). It contains not less than 1.5 per cent of caffeine (Mr 194.2), calculated with reference to the dried drug. CHARACTERS It has the macroscopic and microscopic characters described under identification tests A and B.

Calculate the content of vitamin D3 in International Units per gram using the following expression, taking into account IDENTIFICATION the assigned content of cholecalciferol CRS : A. The kernels have an oblong, somewhat obtuse, sub-tetragonal shape, with deformations resulting from mutual pressure inside the fruit ; they vary in size and mass, ranging from 5 g to 15 g ; the outside is hard, smooth and very dark brown, the inside is more reddish-brown. In C. nitida and its varieties, the m1 = mass of the sample in test solution (b) in grams, kernels are divided in two parts, almost plano-convex, corresponding to the cotyledons and usually occurring m2 = total mass of cholecalciferol CRS used for separated in the commercial drug ; the cotyledons are the preparation of reference solution (a) in 3 cm to 4 cm long, 2 cm to 2.5 cm wide and 1 cm to 2 cm micrograms (500 µg), thick. In C. acuminata, the cotyledons are smaller and A1 = area (or height) of the peak due to cholecalciferol divided into four to six irregular parts. in the chromatogram obtained with test B. Reduce to a powder (355). The powder is reddish-brown. solution (a), Examine under a microscope using a 50 per cent V/V A2 = area (or height) of the peak due to cholecalciferol solution of glycerol R. The powder shows numerous in the chromatogram obtained with test ovoid or reniform starch granules, 5 µm to 25 µm in size, solution (b), with concentric striations and a stellate, slightly eccentric A3 = area (or height) of the peak due to ergocalciferol hilum ; fragments of cotyledon tissue showing large, in the chromatogram obtained with reference thick-walled, reddish polygonal cells filled with starch solution (b), granules ; occasional fragments of the external epidermis A4 = area (or height) of the peak due to ergocalciferol in of the cotyledons. the chromatogram obtained with test solution (b), C. Examine by thin-layer chromatography (2.2.27), using a A5 = area (or height) of a possible peak in the TLC silica gel F254 plate R. chromatogram obtained with test solution (a) with Test solution. To 1.0 g of the powdered drug (355), add the same retention time as the peak co-eluting 5 ml of alcohol (60 per cent V/V) R. Shake mechanically with ergocalciferol in test solution (b), at 40 °C for 30 min and filter. A6 = area (or height) of the peak due to cholecalciferol Reference solution (a). Dissolve 25 mg of caffeine R in in the chromatogram obtained with reference 10 ml of alcohol (60 per cent V/V) R. solution (b), Reference solution (b). Dissolve 50 mg of theobromine R V1 = total volume of reference solution (a) (100 ml), in 10 ml of a mixture of 10 volumes of water R, 13 volumes V2 = volume of reference solution (a) used for preparing of methanol R and 77 volumes of ethyl acetate R. Filter. reference solution (b) (2.0 ml). Apply to the plate as bands 20 µl of each solution. Develop over a path of 10 cm using a mixture of STORAGE 10 volumes of water R, 13 volumes of methanol R and 77 volumes of ethyl acetate R. Allow the plate to dry in In an airtight and well-filled container, protected from light. air for 5 min. Examine in ultraviolet light at 254 nm. The If no antioxidant is added, store under an inert gas. chromatogram obtained with the test solution shows Once the container has been opened, its contents are used two principal quenching zones which are similar in as soon as possible and any part of the contents not used at position to the zones in the chromatograms obtained with once is protected by an atmosphere of inert gas. reference solutions (a) and (b), respectively. Spray the plates with a mixture of equal volumes of alcohol R and hydrochloric acid R and then with a solution prepared LABELLING immediately before use by dissolving 1 g of iodine R and The label states : 1 g of potassium iodide R in 100 ml of alcohol R. The chromatogram obtained with the test solution shows — the number of International Units of vitamin A, a reddish-brown principal zone similar in position and — the number of International Units of vitamin D3, colour to the zone in the chromatogram obtained with — the name and concentration of any added antioxidant. reference solution (a). 1356


Colchicine


01/2005:0758

TESTS Foreign matter (2.8.2). It complies with the test for foreign matter.

COLCHICINE

Loss on drying (2.2.32). Not more than 12.0 per cent, determined on 2.00 g of the powdered drug (355) by drying in an oven at 100-105 °C for 2 h.

Colchicinum

Total ash (2.4.16). Not more than 9.0 per cent. ASSAY Examine by liquid chromatography (2.2.29). Test solution. To 1.00 g (m1) of the powdered drug (355), add 50 ml of methanol R. Heat the mixture under a reflux condenser on a water-bath for 30 min. Allow to cool and filter. Rinse the filter with 10 ml of methanol R. Take up the residue with 50 ml of methanol R. Proceed as before. Combine the filtrates and the washings in a 200.0 ml volumetric flask and dilute to 200.0 ml with methanol R. Transfer 20.0 ml of this solution into a round-bottomed flask and evaporate to dryness under reduced pressure. Take up the residue with the mobile phase, transfer to a 50.0 ml volumetric flask and dilute to 50.0 ml with the mobile phase. Reference solution. In a 100.0 ml volumetric flask, dissolve 30.0 mg (m2) of caffeine R and 15.0 mg of theobromine R in the mobile phase and dilute to 100.0 ml with the mobile phase. Transfer 10.0 ml of this solution to a 100.0 ml volumetric flask and dilute to 100.0 ml with the mobile phase.

C22H25NO6

Mr 399.4

DEFINITION (-)-N-[(7S,12aS)-1,2,3,10-Tetramethoxy-9-oxo-5,6,7,9tetrahydrobenzo[a]heptalen-7-yl]acetamide. Content : 97.0 per cent to 102.0 per cent (anhydrous and ethyl acetate-free substance). CHARACTERS Appearance : yellowish-white, amorphous or crystalline powder. Solubility : very soluble in water, rapidly recrystallising from concentrated solutions as the sesquihydrate, freely soluble in alcohol, practically insoluble in cyclohexane.

The chromatographic procedure may be carried out using :

IDENTIFICATION — a stainless steel column 0.25 m long and 4.6 mm in First identification : B. internal diameter packed with octadecylsilyl silica gel for Second identification : A, C, D. chromatography R (5 µm), A. Dissolve 5 mg in alcohol R and dilute to 100.0 ml with — as mobile phase at a flow rate of 1 ml/min a mixture of the same solvent. Dilute 5.0 ml of the solution to 25.0 ml 25 volumes of methanol R and 75 volumes of water R, with alcohol R. Examined between 230 nm and 400 nm (2.2.25), the solution shows 2 absorption maxima, — as detector a spectrophotometer set at 272 nm, at 243 nm and 350 nm. The ratio of the absorbance measured at 243 nm to that measured at 350 nm is 1.7 — a loop injector. to 1.9. Inject appropriate volumes of each solution. The test B. Infrared absorption spectrophotometry (2.2.24). is not valid unless : in the chromatogram obtained with Preparation : discs of potassium bromide R. the reference solution, the resolution between the peaks corresponding respectively to cafeine and theobromine is at Comparison : colchicine CRS. least 2.5. If necessary, adjust the volume of water R in the C. To 0.5 ml of solution S (see Tests) add 0.5 ml of dilute mobile phase. hydrochloric acid R and 0.15 ml of ferric chloride solution R1. The solution is yellow and becomes Calculate the caffeine content using the expression : dark green on boiling for 30 s. Cool, add 2 ml of methylene chloride R and shake. The organic layer is greenish-yellow. D. Dissolve about 30 mg in 1 ml of alcohol R and add 0.15 ml of ferric chloride solution R1. A brownish-red A1 = area of the peak due to caffeine in the colour develops. chromatogram obtained with the test solution, A2 = area of the peak due to caffeine in the TESTS chromatogram obtained with the reference Solution S. Dissolve 0.10 g in water R and dilute to 20 ml solution, with the same solvent. m1 = mass of the drug to be examined in the test Appearance of solution. Solution S is clear (2.2.1) and not solution, in grams, more intensely coloured than reference solution GY3 (2.2.2, m2 = mass of caffeine R in the reference solution, in Method II). grams. Acidity or alkalinity. To 10 ml of solution S add 0.1 ml of bromothymol blue solution R1. Either the solution does not change colour or it becomes green. Not more than 0.1 ml of STORAGE 0.01 M sodium hydroxide is required to change the colour of the indicator to blue. Store protected from light. General Notices (1) apply to all monographs and other texts

1357

Colchicine


Specific optical rotation (2.2.7) : − 235 to − 250 (anhydrous and ethyl acetate-free substance). Dissolve 50.0 mg in alcohol R and dilute to 10.0 ml with the same solvent. Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 20.0 mg of the substance to be examined in a mixture of equal volumes of methanol R and water R and dilute to 20.0 ml with the same mixture of solvents. Reference solution (a). Dissolve 20.0 mg of colchicine for system suitability CRS in a mixture of equal volumes of methanol R and water R and dilute to 20.0 ml with the same mixture of solvents. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with a mixture of equal volumes of methanol R and water R. Reference solution (c). Dilute 1 ml of reference solution (b) to 20.0 ml with a mixture of equal volumes of methanol R and water R. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase: octylsilyl silica gel for chromatography R1 (5 µm). Mobile phase : mix 450 volumes of a 6.8 g/l solution of potassium dihydrogen phosphate R and 530 volumes of methanol R. After cooling to room temperature, adjust the volume to 1000 ml with methanol R. Adjust the apparent pH to 5.5 with dilute phosphoric acid R. Flow rate : 1 ml/min. Detection : spectrophotometer at 254 nm. Injection : 20 µl. Run time : 3 times the retention time of colchicine. Relative retention with reference to colchicine (retention time = about 7 min) : impurity D = about 0.4 ; impurity E = about 0.7 ; impurity B = about 0.8 ; impurity A = about 0.94 ; impurity C = about 1.2. System suitability : reference solution (a) : Peak-to-valley ratio : minimum 2, where HP = height above the baseline of the peak due to impurity A and HV = height above the baseline of the lowest point of the curve separating this peak from the peak due to colchicine. Limits : — impurity A : not more than 3.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (3.5 per cent), — any other impurity : not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent), — total: not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (5 per cent), — disregard limit: area of the principal peak in the chromatogram obtained with reference solution (c) (0.05 per cent). Colchiceine : maximum 0.2 per cent. Dissolve 50 mg in water R and dilute to 5 ml with the same solvent. Add 0.1 ml of ferric chloride solution R1. The solution is not more intensely coloured than a mixture of 1 ml of red primary solution, 2 ml of yellow primary solution and 2 ml of blue primary solution (2.2.2, Method II). 1358

Chloroform (2.4.24) : maximum 500 ppm. Ethyl acetate (2.4.24) : maximum 6.0 per cent m/m. Water (2.5.12) : maximum 2.0 per cent, determined on 0.500 g. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 0.5 g. ASSAY Dissolve 0.250 g with gentle heating in a mixture of 10 ml of acetic anhydride R and 20 ml of toluene R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 39.94 mg of C22H25NO6. STORAGE Protected from light. IMPURITIES

A. R1 = R3 = CH3, R2 = H : N-[(7S,12aS)-1,2,3,10tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7yl]formamide (N-deacetyl-N-formylcolchicine), E. R1 = H, R2 = R3 = CH3 : N-[(7S,12aS)-3-hydroxy-1,2,10trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7yl]acetamide (3-O-demethylcolchicine), F. R1 = R2 = CH3, R3 = H : N-[(7S,12aS)-10-hydroxy-1,2,3trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7yl]acetamide (colchiceine),

B. (-)-N-[(7S,12aR)-1,2,3,10-tetramethoxy-9-oxo-5,6, 7,9-tetrahydrobenzo[a]heptalen-7-yl]acetamide (conformationnal isomer),

C. N-[(7S,7bR,10aS)-1,2,3,9-tetramethoxy-8-oxo-5, 6,7,7b,8,10a-hexahydrobenzo[a]cyclopenta[3,4]cyclobuta[1,2-c]cyclohepten-7-yl]acetamide (β-lumicolchicine),



Colestyramine

the solution at the absorption maximum at 420 nm, using as compensation liquid a solution prepared in the same manner but using 5.0 ml of water R instead of the test solution. Repeat the operation using 5.0 ml of the reference solution. The absorbance obtained with the test solution is not greater than that obtained with the reference solution. Impurity A. Liquid chromatography (2.2.29). Test solution. Shake 5.0 g with 10 ml of acetone R for 30 min. Centrifuge and use the supernatant liquid. Reference solution (a). Dissolve 5 mg of styrene R in D. N-[(7S,12aS)-3-(β-D-glucopyranosyloxy)-1,2,10-trimethoxy- acetone R and dilute to 100.0 ml with the same solvent. 9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]acetamide Dilute 1.0 ml to 100.0 ml with acetone R. (colchicoside). Reference solution (b). Dissolve 0.35 ml of styrene R in acetone R and dilute to 100.0 ml with the same solvent. 01/2005:1775 Dilute 1.0 ml to 100.0 ml with acetone R. Reference solution (c). Dissolve 0.35 ml of toluene R in COLESTYRAMINE acetone R and dilute to 100.0 ml with the same solvent. Reference solution (d). Mix 1.0 ml of reference solution (b) Colestyraminum and 1.0 ml of reference solution (c) with acetone R and dilute to 100.0 ml with the same solvent. DEFINITION Column : Strongly basic anion-exchange resin in chloride form, — size : l = 0.30 m, Ø = 3.9 mm, consisting of styrene-divinylbenzene copolymer with — stationary phase : octadecylsilyl silica gel for quaternary ammonium groups. chromatography R (10 µm) with a specific surface area of Nominal exchange capacity : 1.8 g to 2.2 g of sodium 330 m2/g and a pore size of 12.5 nm. glycocholate per gram (dried substance). Mobile phase : acetonitrile R, water R (50:50 V/V). CHARACTERS Flow rate : 2.0 ml/min. Appearance : white or almost white, fine powder, Detection : spectrophotometer at 254 nm. hygroscopic. Injection : 20 µl of test solution, reference solutions (a) Solubility : insoluble in water, in methylene chloride and in and (d). ethanol (96 per cent). System suitability : reference solution (d) : IDENTIFICATION — resolution : minimum 1.5 between the peaks due to impurity A and toluene. A. Infrared absorption spectrophotometry (2.2.24). Limit : Comparison : colestyramine CRS. — impurity A : not more than the area of the principal peak B. It complies with the test for chlorides (see Tests). in the chromatogram obtained with reference solution (a) TESTS (1 ppm). pH (2.2.3) : 4.0 to 6.0. Chloride : 13.0 per cent to 17.0 per cent (dried substance). Suspend 0.100 g in 10 ml of water R and allow to stand for To 0.2 g add 100 ml of water R and 50 mg of potassium 10 min. nitrate R. Add, with stirring, 2 ml of nitric acid R and titrate with 0.1 M silver nitrate, determining the end-point Dialysable quaternary amines : maximum 500 ppm, potentiometrically (2.2.20). expressed as benzyltrimethylammonium chloride. 1 ml of 0.1 M silver nitrate is equivalent to 3.55 mg of Cl. Test solution. Place a 25 cm piece of cellulose dialysis tubing having a molecular weight cut-off of 12 000-14 000 Heavy metals (2.4.8) : maximum 20 ppm. and an inflated diameter of 3-6 cm (flat width of 5-9 cm) 1.0 g complies with limit test F. Prepare the reference in water R to hydrate until pliable, appropriately sealing solution using 2 ml of lead standard solution (10 ppm Pb) R. one end. Introduce 2.0 g of the substance to be examined Loss on drying (2.2.32) : maximum 12 per cent, determined into the tube and add 10 ml of water R. Seal the tube and on 1.000 g by drying in an oven at 70 °C over diphosphorus completely immerse it in 100 ml of water R in a suitable pentoxide R at a pressure not exceeding 7 kPa for 16 h. vessel and stir the liquid for 16 h to effect dialysis. Use the Sulphated ash (2.4.14) : maximum 0.1 per cent, determined dialysate as test solution. on 1.0 g. Reference solution. Prepare the reference solution in a similar manner but using 10 ml of a freshly prepared 0.1 g/l ASSAY solution of benzyltrimethylammonium chloride R instead Exchange capacity. Liquid chromatography (2.2.29). of the substance to be examined. Transfer 5.0 ml of the test solution to a separating funnel and Solution A. Dissolve 1.500 g of sodium glycocholate R add 5 ml of a 3.8 g/l solution of disodium tetraborate R, 1 ml in a solution containing 4 g/l of potassium dihydrogen of a solution containing 1.5 g/l of bromothymol blue R and phosphate R and 12 g/l of dipotassium hydrogen 4.05 g/l of sodium carbonate R and 10 ml of chloroform R. phosphate R and dilute to 100.0 ml with the same solution. Shake the mixture vigourously for 1 min, allow the phases Test solution. Add 20.0 ml of solution A to a quantity of the to separate and transfer the clear organic layer to a 25 ml substance to be examined equivalent to about 0.100 g of the volumetric flask. Repeat the extraction with a further 10 ml dried substance. Shake mechanically for 2 h and centrifuge of chloroform R, combine the organic layers and dilute to for 15 min. Dilute 5.0 ml of the supernatant liquid to 50.0 ml 25 ml with chloroform R. Measure the absorbance (2.2.25) of with water R. General Notices (1) apply to all monographs and other texts

1359

Colistimethate sodium


Reference solution (a). Dilute 4.0 ml of solution A to IDENTIFICATION 100.0 ml with water R. A. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Reference solution (b). Dissolve 60 mg of sodium glycocholate R and 30 mg of sodium taurodeoxycholate R Test solution. Dissolve 5 mg of the substance to be in water R and dilute to 100 ml with the same solvent. Dilute examined in 1 ml of a mixture of equal volumes of 1 ml of the solution to 10 ml with water R. hydrochloric acid R and water R. Heat at 135 °C in a sealed tube for 5 h. Evaporate to dryness on a water-bath Column : and continue the heating until the hydrochloric acid has — size : l = 0.25 m, Ø = 4.6 mm, evaporated. Dissolve the residue in 0.5 ml of water R. — stationary phase: octadecylsilyl silica gel for Reference solution (a). Dissolve 20 mg of leucine R in chromatography R (5 µm). water R and dilute to 10 ml with the same solvent. Mobile phase : mix 35 volumes of acetonitrile R and Reference solution (b). Dissolve 20 mg of threonine R in 65 volumes of a 10.9 g/l solution of potassium dihydrogen water R and dilute to 10 ml with the same solvent. phosphate R adjusted to pH 3.0 with phosphoric acid R. Reference solution (c). Dissolve 20 mg of phenylalanine R Flow rate : 1.5 ml/min. in water R and dilute to 10 ml with the same solvent. Detection : spectrophotometer at 214 nm. Reference solution (d). Dissolve 20 mg of serine R in Injection : 50 µl. water R and dilute to 10 ml with the same solvent. Run time : twice the retention time of glycocholate. Carry out the following procedures protected from light. System suitability : reference solution (b) : Apply to the plate as 10 mm bands 5 µl of each solution. — resolution : minimum 1.5 between the peaks due to Place the plate in the chromatographic tank so that it glycocholate and taurodeoxycholate. is not in contact with the mobile phase consisting of a mixture of 25 volumes of water R and 75 volumes of Calculate the nominal exchange capacity using the following phenol R. Leave the plate to become impregnated with expression : the vapour of the solvent for at least 12 h. Develop over a path of 12 cm using the same mobile phase. Dry the plate at 100-105 °C and spray with ninhydrin solution R1. Heat at 110 °C for 5 min. The chromatogram obtained with the test solution shows zones corresponding to A1 = area of the peak due to glycocholate in those in the chromatograms obtained with reference the chromatogram obtained with reference solutions (a) and (b), but shows no zones corresponding solution (a), to those in the chromatograms obtained with reference A2 = area of the peak due to glycocholate in the solutions (c) and (d). The chromatogram obtained with chromatogram obtained with the test solution, the test solution also shows a zone with a very low Rf m1 = mass, in milligrams, of sodium glycocholate R value (2,4-diaminobutyric acid). used in the preparation of solution A, B. Dissolve about 5 mg in 3 ml of water R. Add 3 ml of dilute sodium hydroxide solution R. Shake and add 0.5 ml of m2 = mass, in milligrams, of the dried substance to a 10 g/l solution of copper sulphate R. A violet colour be examined used in the preparation of the test is produced. solution, C. Dissolve about 50 mg in 1 ml of 1 M hydrochloric 0.73 = correction factor to convert the true exchange acid and add 0.5 ml of 0.01 M iodine. The solution is capacity to the conventionally used nominal decolourised and gives reaction (a) of sulphates (2.3.1). exchange capacity. D. It gives reaction (b) of sodium (2.3.1). STORAGE TESTS In an airtight container. Appearance of solution. Dissolve 0.16 g in 10 ml of water R. The solution is clear (2.2.1). pH (2.2.3). Dissolve 0.1 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. The pH of the A. styrene. solution, measured after 30 min, is 6.5 to 8.5. Specific optical rotation (2.2.7). Dissolve 1.25 g in water R 01/2005:0319 and dilute to 25.0 ml with the same solvent. The specific optical rotation is − 46 to − 51, calculated with reference to the dried substance. COLISTIMETHATE SODIUM Free colistin. Dissolve 80 mg in 3 ml of water R. Add 0.1 ml of a 100 g/l solution of silicotungstic acid R ; 10 s to Colistimethatum natricum 20 s after addition of the reagent, the solution is not more DEFINITION opalescent than reference suspension II (2.2.1). Colistimethate sodium is prepared from colistin by the action Total sulphite. Work in a fume cupboard. Dissolve 0.100 g of formaldehyde and sodium hydrogen sulphite. The potency in 50 ml of water R and add 5 ml of a 100 g/l solution of is not less than 11 500 IU/mg, calculated with reference to sodium hydroxide R and 0.3 g of potassium cyanide R. the dried substance. Boil gently for 3 min and then cool. Neutralise with 0.5 M sulphuric acid using 0.2 ml of methyl orange solution R as CHARACTERS indicator. Add an excess of 0.5 ml of the acid and 0.2 g of potassium iodide R. Titrate with 0.05 M iodine using 1 ml of A white or almost white powder, hygroscopic, very soluble starch solution R as indicator. The volume of 0.05 M iodine in water, slightly soluble in alcohol, practically insoluble in used in the titration is 5.5 ml to 7.0 ml. acetone. IMPURITIES Specified impurities : A.

1360



Colistin sulphate

Loss on drying (2.2.32). Not more than 5.0 per cent, Second identification : A, C, D, E. determined on 1.000 g by drying at 60 °C over diphosphorus A. Thin-layer chromatography (2.2.27). pentoxide R at a pressure not exceeding 670 Pa for 3 h. Test solution. Dissolve 5 mg of the substance to be Sulphated ash (2.4.14). 16 per cent to 21 per cent, examined in 1 ml of a mixture of equal volumes of determined on 0.50 g. hydrochloric acid R and water R. Heat at 135 °C in a sealed tube for 5 h. Evaporate to dryness on a water-bath Pyrogens (2.6.8). If intended for use in the manufacture and continue the heating until moistened blue litmus of parenteral dosage forms without a further appropriate paper R does not turn red. Dissolve the residue in 0.5 ml procedure for removal of pyrogens, it complies with the test of water R. for pyrogens. Inject per kilogram of the rabbit’s mass 1 ml of Reference solution (a). Dissolve 20 mg of leucine R in a solution in water for injections R containing 2.5 mg of the water R and dilute to 10 ml with the same solvent. substance to be examined per millilitre. Reference solution (b). Dissolve 20 mg of threonine R in ASSAY water R and dilute to 10 ml with the same solvent. Carry out the microbiological assay of antibiotics (2.7.2). Reference solution (c). Dissolve 20 mg of phenylalanine R in water R and dilute to 10 ml with the same solvent. STORAGE Reference solution (d). Dissolve 20 mg of serine R in Store in an airtight container, protected from light. If the water R and dilute to 10 ml with the same solvent. substance is sterile, store in a sterile, airtight, tamper-proof Plate : TLC silica gel G plate R. container. Mobile phase : water R, phenol R (25:75 V/V). LABELLING Carry out the following procedures protected from light. Application : 5 µl as 10 mm bands. The label states, where applicable, that the substance is free from pyrogens. Preconditioning : place the plate in the chromatographic tank, so that it is not in contact with the mobile phase, and allow to become impregnated with the vapour of the mobile phase for at least 12 h. 01/2005:0320 Development : over a path of 12 cm. Drying : at 100-105 °C. COLISTIN SULPHATE Detection : spray with ninhydrin solution R1 and heat at 110 °C for 5 min. Colistini sulfas Results : the chromatogram obtained with the test solution shows zones corresponding to those in the chromatograms obtained with reference solutions (a) and (b), but shows no zones corresponding to those in the chromatograms obtained with reference solutions (c) and (d). The chromatogram obtained with the test solution also shows a zone with a very low Rf value (2,4-diaminobutyric acid). B. Examine the chromatograms obtained in the assay. Results : the peaks due to colistin E1 and colistin E2 in the chromatogram obtained with the test solution are similar in retention time to the corresponding peaks in the chromatogram obtained with reference solution (a). C. Dissolve about 5 mg in 3 ml of water R. Add 3 ml of dilute sodium hydroxide solution R. Shake and add 0.5 ml of a 10 g/l solution of copper sulphate R. A violet colour DEFINITION is produced. A mixture of the sulphates of polypeptides produced by D. Dissolve about 50 mg in 1 ml of 1 M hydrochloric acid certain strains of Bacillus polymyxa var. colistinus or and add 0.5 ml of 0.01 M iodine. The solution remains obtained by any other means. coloured. Content : E. It gives reaction (a) of sulphates (2.3.1). — sum of colistins E1, E2, E3, E1-I and E1-7MOA : minimum TESTS 77.0 per cent (dried substance), pH (2.2.3) : 4.0 to 6.0. — colistin E1-I : maximum 10.0 per cent (dried substance), Dissolve 0.1 g in carbon dioxide-free water R and dilute to — colistin E1-7MOA : maximum 10.0 per cent (dried 10 ml with the same solvent. substance), Specific optical rotation (2.2.7) : − 63 to − 73 (dried — colistin E3 : maximum 10.0 per cent (dried substance). substance). Dissolve 1.25 g in water R and dilute to 25.0 ml with the CHARACTERS same solvent. Appearance : white or almost white powder, hygroscopic. Solubility : freely soluble in water, slightly soluble in alcohol, Related substances. Liquid chromatography (2.2.29) : use the normalisation procedure. practically insoluble in acetone. Test solution. Dissolve 25.0 mg of the substance to be IDENTIFICATION examined in 40 ml of water R and dilute to 50.0 ml with acetonitrile R. First identification : B, E. General Notices (1) apply to all monographs and other texts

1361

Colophony


Reference solution (a). Dissolve 25.0 mg of colistin sulphate CRS in 40 ml of water R and dilute to 50.0 ml with acetonitrile R. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 100.0 ml with a mixture of 20 volumes of acetonitrile R and 80 volumes of water R. Column : — size : l = 0.15 m, Ø = 4.6 mm, — stationary phase: end-capped octadecylsilyl silica gel for chromatography R (3.5 µm), — temperature : 30 °C. Mobile phase : mix 22 volumes of acetonitrile R and 78 volumes of a solution prepared as follows : dissolve 4.46 g of anhydrous sodium sulphate R in 900 ml of water R, add 2.5 ml of phosphoric acid R and dilute to 1000 ml with water R (pH 2.3 to 2.5). Flow rate : 1.0 ml/min. Detection : spectrophotometer at 215 nm. Injection : 20 µl. Run time : 1.5 times the retention time of colistin E1. Relative retention with reference to colistin E1 (retention time = about 16 min) : colistin E2 = about 0.45 ; colistin E3 = about 0.5 ; colistin E1-I = about 0.8 ; colistin E1-7MOA = about 1.1. System suitability : reference solution (a) : — resolution : minimum 8.0 between the peaks due to colistin E2 and colistin E1, minimum 6.0 between the peaks due to colistin E2 and colistin E1-I, minimum 2.5 between the peaks due to colistin E1-I and colistin E1, minimum 1.5 between the peaks due to colistin E1 and colistin E1-7MOA, — the chromatogram obtained is concordant with the chromatogram supplied with colistin sulphate CRS. Limits : — any impurity : maximum 4.0 per cent, — total: maximum 23.0 per cent, — disregard limit : the area of the peak due to colistin E1 in the chromatogram obtained with reference solution (b) ; disregard the peaks due to colistin E2, E3, E1-I, E1 and E1-7MOA. Sulphate : 16.0 per cent to 18.0 per cent (dried substance). Dissolve 0.250 g in 100 ml of water R and adjust to pH 11 using concentrated ammonia R. Add 10.0 ml of 0.1 M barium chloride and about 0.5 mg of phthalein purple R. Titrate with 0.1 M sodium edetate, adding 50 ml of alcohol R when the colour of the solution begins to change and continuing the titration until the violet-blue colour disappears. 1 ml of 0.1 M barium chloride is equivalent to 9.606 mg of SO4. Loss on drying (2.2.32) : maximum 3.5 per cent, determined on 1.000 g by drying at 60 °C over diphosphorus pentoxide R at a pressure not exceeding 670 Pa for 3 h. Sulphated ash (2.4.14) : maximum 1.0 per cent, determined on 1.0 g. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modification. Injection : test solution and reference solution (a). Calculate the percentage content of the sum of colistins E2, E3, E1-I, E1 and E1-7MOA, the percentage content of colistin E3, the percentage content of colistin E1-I and 1362

the percentage content of colistin E1-7MOA using the chromatogram obtained with reference solution (a) and the declared contents of colistin sulphate CRS. STORAGE In an airtight container, protected from light. 01/2005:1862

COLOPHONY Colophonium DEFINITION Residue remaining after distillation of the volatile oil from the oleoresin obtained from various species of Pinus. CHARACTERS Macroscopic characters described under identification A. IDENTIFICATION A. Translucent, pale yellow to brownish-yellow, angular, irregularly-shaped, brittle, glassy pieces of different sizes the surfaces of which bear conchoidal markings. B. Thin-layer chromatography (2.2.27). Test solution. Dissolve 1 g in 10 ml of methanol R by gently warming. Reference solution. Dissolve 10 mg of thymol R and 10 mg of linalol R in 10 ml of methanol R. Plate : TLC silica gel plate R. Mobile phase : methylene chloride R. Application : 10 µl, as bands. Development : over a path of 15 cm. Drying : in air. Detection : spray with anisaldehyde solution R and heat at 100-105 °C for 10 min ; examine in daylight. Results : see below the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. Furthermore, other coloured zones are present in the chromatogram obtained with the test solution. Top of the plate A purple band A purple band _______

_______ 2 purple bands

Thymol : an orange band _______ Linalol: a purple band

_______ Sequence of narrow purple bands Purple extended baseline band

Reference solution

Test solution

TESTS Acid value (2.5.1) : 145 to 180, determined on 1.0 g. Foreign matter (2.8.2) : maximum 2 per cent. Total ash (2.4.16) : maximum 0.2 per cent. STORAGE Do not reduce to a powder.



Copovidone

01/2005:0891 Reference solution. Dissolve 0.140 g of acetaldehyde ammonia trimer trihydrate R in water R and dilute to 200.0 ml with the same solvent. Dilute 1.0 ml of this solution COPOVIDONE to 100.0 ml with phosphate buffer solution pH 9.0 R. Into 3 identical spectrophotometric cells with a path length Copovidonum of 1 cm, introduce separately 0.5 ml of the test solution, 0.5 ml of the reference solution and 0.5 ml of water R (blank). To each cell, add 2.5 ml of phosphate buffer solution pH 9.0 R and 0.2 ml of nicotinamide-adenine dinucleotide solution R. Mix and stopper tightly. Allow to stand at 22 ± 2 °C for 2-3 min and measure the absorbance (2.2.25) of each solution at 340 nm, using water R as the compensation liquid. To each cell, add 0.05 ml of aldehyde dehydrogenase solution R, mix and stopper tightly. Allow to stand at 22 ± 2 °C for 5 min. Measure the absorbance of each solution at 340 nm using water R as compensation liquid. (C6H9NO)n, (C4H6O2)m Mr (111.1)n + (86.1)m Determine the content of aldehydes using the expression : DEFINITION Copovidone is a copolymer of 1-ethenylpyrrolidin-2-one and ethenyl acetate in the mass proportion 3:2. Content : At1 = absorbance of the test solution before the addition of aldehyde dehydrogenase, — nitrogen (N ; Ar 14.01) : 7.0 per cent to 8.0 per cent (dried substance), At2 = absorbance of the test solution after the addition of aldehyde dehydrogenase, — ethenyl acetate C4H6O2 ; Mr 86.10) : 35.3 per cent to 42.0 per cent (dried substance). As1 = absorbance of the reference solution before the K-value : 90.0 per cent to 110.0 per cent of the value stated addition of aldehyde dehydrogenase, on the label. As2 = absorbance of the reference solution after the addition of aldehyde dehydrogenase, CHARACTERS Ab1 = absorbance of the blank before the addition of Aspect : white or yellowish-white powder or flakes, aldehyde dehydrogenase, hygroscopic. A absorbance of the blank after the addition of = Solubility : freely soluble in water, in alcohol and in b2 aldehyde dehydrogenase, methylene chloride. m = mass of povidone, in grams, calculated with IDENTIFICATION reference to the dried substance, First identification : A. C = concentration (mg/ml), of acetaldehyde in the Second identification : B, C. reference solution, calculated from the weight of the acetaldehyde ammonia trimer trihydrate with A. Infrared absorption spectrophotometry (2.2.24). the factor 0.72. Comparison : Ph. Eur. reference spectrum of copovidone. Peroxides : maximum 400 ppm, expressed as H2O2. B. To 1 ml of solution S (see Tests) add 5 ml of water R and Dilute 10 ml of solution S to 25 ml with water R. Add 2 ml of 0.2 ml of 0.05 M iodine. A red colour appears. titanium trichloride-sulphuric acid reagent R and allow to stand for 30 min. The absorbance (2.2.25) of the solution, C. Dissolve 0.7 g of hydroxylamine hydrochloride R in measured at 405 nm using a mixture of 25 ml of a 40 g/l 10 ml of methanol R, add 20 ml of a 40 g/l solution of sodium hydroxide R and filter if necessary. To 5 ml of the solution of the substance to be examined and 2 ml of a 13 per cent V/V solution of sulphuric acid R as the compensation solution add 0.1 g of the substance to be examined and liquid, is not greater than 0.35. boil for 2 min. Transfer 50 µl to a filter paper and add 0.1 ml of a mixture of equal volumes of ferric chloride Hydrazine. Thin-layer chromatography (2.2.27). Use freshly solution R1 and hydrochloric acid R. A violet colour prepared solutions. appears. Test solution. To 25 ml of solution S add 0.5 ml of a 50 g/l solution of salicylaldehyde R in methanol R, mix and heat TESTS in a water-bath at 60 °C for 15 min. Allow to cool, add 2.0 ml Solution S. Dissolve 10 g in water R and dilute to 100 ml of xylene R, shake for 2 min and centrifuge. Use the clear with the same solvent. Add the substance to be examined to supernatant layer. the water R in small portions with constant stirring. Reference solution. Dissolve 9 mg of salicylaldehyde Appearance of solution. Solution S is not more opalescent azine R in xylene R and dilute to 100 ml with the same than reference suspension III (2.2.1) and not more intensely solvent. Dilute 1 ml of this solution to 10 ml with xylene R. coloured than reference solution B5, R5 or BY5 (2.2.2, Plate : TLC silanised silica gel plate R. Method II). Mobile phase : water R, methanol R (20:80 V/V). Aldehydes: maximum 500 ppm, expressed as acetaldehyde. Application : 10 µl. Test solution. Dissolve 1.0 g of the substance to be examined Development : over a path of 15 cm. in phosphate buffer solution pH 9.0 R and dilute to 100.0 ml with the same solvent. Stopper the flask and heat at 60 °C Drying : in air. Detection : examine in ultraviolet light at 365 nm. for 1 h. Allow to cool. General Notices (1) apply to all monographs and other texts

1363

Copper sulphate, anhydrous


Viscosity, expressed as K-value. Dilute 5.0 ml of solution S to 50.0 ml with water R. Allow to stand for 1 h and determine — hydrazine : any spot corresponding to salicylaldehyde the viscosity (2.2.9) of the solution at 25 ± 0.1 °C using azine in the chromatogram obtained with the test solution viscometer No. 1 with a minimum flow time of 100 s. is not more intense than the spot in the chromatogram Calculate the K-value from the expression : obtained with the reference solution (1 ppm). Limit :

Monomers : maximum 0.1 per cent. Dissolve 10.0 g in 30 ml of methanol R and add slowly 20.0 ml of iodine bromide solution R. Allow to stand for 30 min protected from light with repeated shaking. Add c = percentage concentration (g/100 ml) of the 10 ml of a 100 g/l solution of potassium iodide R and titrate substance to be examined, calculated with with 0.1 M sodium thiosulphate until a yellow colour is reference to the dried substance, obtained. Continue titration dropwise until the solution = viscosity of the solution relative to that of water. becomes colourless. Carry out a blank titration. Not more than 1.8 ml of 0.1 M sodium thiosulphate is used. ASSAY Impurity A. Liquid chromatography (2.2.29). Ethenyl acetate. Determine the saponification value (2.5.6) on 2.00 g of the substance to be examined. Multiply the Test solution. Dissolve 100 mg of the substance to be result obtained by 0.1534 to obtain the percentage content examined in water R and dilute to 50.0 ml with the same of the ethenyl acetate component. solvent. Nitrogen. Carry out the determination of nitrogen (2.5.9) Reference solution. Dissolve 100 mg of 2-pyrrolidone R in using 30.0 mg of the substance to be examined and 1 g of water R and dilute to 100 ml with the same solvent. Dilute a mixture of 3 parts of copper sulphate R and 997 parts of 1.0 ml to 100.0 ml with water R. dipotassium sulphate R, heating until a clear, light green solution is obtained and then for a further 45 min. Precolumn : STORAGE — size : l = 0.025 m, Ø = 4 mm, In an airtight container. — stationary phase: end-capped octadecylsilyl silica gel for chromatography R (5 µm).

LABELLING The label states the K-value.

Column :

IMPURITIES

— size : l = 0.25 m, Ø = 4 mm, — stationary phase: spherical aminohexadecylsilyl silica gel for chromatography R (5 µm),

A. pyrrolidin-2-one (2-pyrrolidone).

— temperature : 30 °C. Mobile phase : water R, adjusted to pH 2.4 with phosphoric acid R. Flow rate : 1 ml/min.

01/2005:0893

COPPER SULPHATE, ANHYDROUS Cupri sulfas anhydricus

Detection : spectrophotometer at 205 nm. A detector is placed between the precolumn and the analytical column. A second detector is placed after the analytical column.

CuSO4

Injection : 10 µl. When impurity A has left the precolumn (after about 1.2 min) switch the flow directly from the pump to the analytical column. Before the next chromatogram is run, wash the precolumn by reversed flow.

DEFINITION Anhydrous copper sulphate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of CuSO4, calculated with reference to the dried substance.

Limit :

CHARACTERS A greenish-grey powder, very hygroscopic, freely soluble in water, slightly soluble in methanol, practically insoluble in alcohol.

— impurity A : not more than the area of the principal peak obtained with the reference solution (0.5 per cent). Heavy metals (2.4.8) : maximum 20 ppm. 12 ml of solution S complies with limit test A. Prepare the standard using lead standard solution (2 ppm Pb) R. Loss on drying (2.2.32) : maximum 5.0 per cent, determined on 0.500 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. 1364

Mr 159.6

IDENTIFICATION A. Add several drops of dilute ammonia R2 to 1 ml of solution S (see Tests). A blue precipitate is formed on further addition of dilute ammonia R2, the precipitate dissolves and a dark blue colour is produced. B. It complies with the test for loss on drying (see Tests). C. Dilute 1 ml of solution S to 5 ml with water R. The solution gives reaction (a) of sulphates (2.3.1).


Coriander


TESTS Solution S. Dissolve 1.6 g in water R and dilute to 50 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1). Chlorides (2.4.4). Dilute 10 ml of solution S to 15 ml with water R. The solution complies with the limit test for chlor-ides (150 ppm). Examine the tubes laterally against a black background. Iron. Not more than 150 ppm of Fe, determined by atomic absorption spectrometry (2.2.23, Method I). Test solution. Dissolve 0.32 g in 10 ml of water R, add 2.5 ml of lead-free nitric acid R and dilute to 25.0 ml with water R. Reference solutions. Prepare the reference solutions using iron standard solution (20 ppm Fe) R, adding 2.5 ml of lead-free nitric acid R and diluting to 25.0 ml with water R. Measure the absorbance at 248.3 nm using an iron hollow-cathode lamp as a source of radiation and an air-butane flame. Lead. Not more than 80 ppm of Pb, determined by atomic absorption spectrometry (2.2.23, Method I). Test solution. Dissolve 1.6 g in 10 ml of water R, add 2.5 ml of lead-free nitric acid R and dilute to 25.0 ml with water R. Reference solutions. Prepare the reference solutions using lead standard solution (100 ppm Pb) R, adding 2.5 ml of lead-free nitric acid R and diluting to 25.0 ml with water R. Measure the absorbance at 217.0 nm using a lead hollow-cathode lamp as a source of radiation and an air-butane flame. Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 0.500 g by drying in an oven at 250 °C.

B. It complies with the test for loss on drying (see Tests). C. Dilute 1 ml of solution S to 5 ml with water R. The solution gives reaction (a) of sulphates (2.3.1). TESTS Solution S. Dissolve 5 g in water R and dilute to 100 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1). Chlorides (2.4.4). Dilute 10 ml of solution S to 15 ml with water R. The solution complies with the limit test for chlorides (100 ppm). Examine the tubes laterally against a black background. Iron. Not more than 100 ppm of Fe, determined by atomic absorption spectrometry (2.2.23, Method I). Test solution. Dissolve 0.5 g in 10 ml of water R, add 2.5 ml of lead-free nitric acid R and dilute to 25.0 ml with water R. Reference solutions. Prepare the reference solutions using iron standard solution (20 ppm Fe) R, adding 2.5 ml of lead-free nitric acid R and diluting to 25.0 ml with water R. Measure the absorbance at 248.3 nm using an iron hollow-cathode lamp as a source of radiation and an air-butane flame. Lead. Not more than 50 ppm of Pb, determined by atomic absorption spectrometry (2.2.23, Method I). Test solution. Dissolve 2.5 g in 10 ml of water R, add 2.5 ml of lead-free nitric acid R and dilute to 25.0 ml with water R. Reference solutions. Prepare the reference solutions using lead standard solution (100 ppm Pb) R, adding 2.5 ml of lead-free nitric acid R and diluting to 25.0 ml with water R. Measure the absorbance at 217.0 nm using a lead hollow-cathode lamp as a source of radiation and an air-butane flame. Loss on drying (2.2.32) : 35.0 per cent to 36.5 per cent, determined on 0.500 g by drying in an oven at 250 °C.

ASSAY Dissolve 0.125 g in 50 ml of water R. Add 2 ml of sulphuric acid R and 3 g of potassium iodide R. Titrate with 0.1 M sodium thiosulphate, using 1 ml of starch solution R, added ASSAY towards the end of the titration, as indicator. 1 ml of 0.1 M sodium thiosulphate is equivalent to 15.96 mg Dissolve 0.200 g in 50 ml of water R. Add 2 ml of sulphuric acid R and 3 g of potassium iodide R. Titrate with 0.1 M of CuSO4. sodium thiosulphate, adding 1 ml of starch solution R towards the end of the titration. STORAGE 1 ml 0.1 M sodium thiosulphate is equivalent to 24.97 mg Store in an airtight container. of CuSO4,5H2O. 01/2005:0894

COPPER SULPHATE PENTAHYDRATE Cupri sulfas pentahydricus CuSO4,5H2O

01/2005:1304

CORIANDER Coriandri fructus

Mr 249.7

DEFINITION Copper sulphate pentahydrate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of CuSO4,5H2O. CHARACTERS A blue, crystalline powder or transparent, blue crystals, freely soluble in water, soluble in methanol, practically insoluble in alcohol. IDENTIFICATION A. Add several drops of dilute ammonia R2 to 1 ml of solution S (see Tests). A blue precipitate is formed on further addition of dilute ammonia R2, the precipate dissolves and a dark blue colour is produced. General Notices (1) apply to all monographs and other texts

DEFINITION Coriander consists of the dried cremocarp of Coriandrum sativum L. It contains not less than 3 ml/kg of essential oil, calculated with reference to the dried drug. CHARACTERS The cremocarp is brown or light brown and is more or less spherical, about 1.5 mm to 5 mm in diameter, or oval form 2 mm to 6 mm long. It has the macroscopic and microscopic characters described under identification tests A and B. IDENTIFICATION A. The mericarps are usually tightly connected. The cremocarp is glabrous and has ten wavy, slightly raised primary ridges and eight straight, more prominent 1365

Coriander oil


secondary ridges. The stylopod crowns the apex. The mericarps are concave on the internal surface. A small fragment of the pedicel may be present. B. Reduce to a powder (355). The powder is brown. Examine under a microscope using chloral hydrate solution R. The powder shows numerous oil droplets ; fragments of endosperm with small thick-walled regular cells containing microcrystals and microrosettes of calcium oxalate and oil droplets ; fragments of endocarp with very narrow cells having a parquetry arrangement and usually associated with a layer of thin-walled rectangular sclereids of the mesocarp ; fragments from the sclerenchymatous layer of the mesocarp with short, strongly thickened, pitted, fusiform cells occurring in layers with the cells of adjacent layers approximately at right angles to one another ; fragments of parenchyma with small, thick-walled cells ; occasional fragments of vascular bundles. C. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating substance. Test solution. Shake 0.50 g of the freshly powdered drug (355) with 5.0 ml of hexane R for 2 min to 3 min and filter over 2 g of anhydrous sodium sulphate R. Reference solution. Dissolve 15 µl of linalol R and 25 µl of olive oil R in 5.0 ml of hexane R immediately before use. Apply to the plate as bands 20 µl of the test solution and 10 µl of the reference solution. Develop over a path of 10 cm using a mixture of 5 volumes of ethyl acetate R and 95 volumes of toluene R. Allow the plate to dry in air and develop again in the same conditions. Allow the plate to dry in air. Spray the plate with anisaldehyde solution R and examine in daylight while heating at 100 °C to 105 °C for 5 min to 10 min. The chromatogram obtained with the reference solution shows in the lower half a violet to greyish-violet zone (linalol) and in the upper half a bluish-violet zone (triglycerides). The chromatogram obtained with the test solution shows zones similar in position and colour to the zones in the chromatogram obtained with the reference solution. Several violet-grey to brownish zones, including the zone corresponding to geraniol, are between the starting point and the zone due to linalol in the chromatogram obtained with the reference solution. It may also show several faint violet-grey zones between the zone due to triglycerides and that due to linalol in the chromatogram obtained with the reference solution.

01/2005:1820

CORIANDER OIL Coriandri aetheroleum DEFINITION Essential oil obtained by steam distillation from the fruits of Coriandrum sativum L. CHARACTERS Appearance : clear, colourless or pale yellow liquid. It has a characteristic spicy odour. IDENTIFICATION First identification : B. Second identification : A. A. Examine by thin-layer chromatography (2.2.27). Test solution. Dissolve 10 µl of the substance to be examined in 1.0 ml of toluene R. Reference solution. Dissolve 10 µl linalol R and 2 µl of geranyl acetate R in 1.0 ml of toluene R. Plate : TLC silica gel plate R. Mobile phase : ethyl acetate R, toluene R (5:95 V/V). Application : 10 µl as bands. Development : over a path of 10 cm. Drying : in air. Detection : spray with anisaldehyde solution R and heat at 100-105 °C for 10-15 min. Examine immediately in daylight. Results : see below the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. Top of the plate _______

_______

Geranyl acetate : a violet-blue zone _______

A violet-blue zone (geranyl acetate) _______

Linalol: an intense violet zone

An intense violet zone (linalol) A violet-blue zone (geraniol)

Test solution TESTS Foreign matter (2.8.2). It complies with the test for foreign B. Examine the chromatograms obtained in the test for matter. None of the cremocarps show perforations due to chromatographic profile. animals. Results : the characteristic peaks in the chromatogram Loss on drying (2.2.32). Not more than 10.0 per cent, obtained with the test solution are similar in retention determined on 1.000 g of the powdered drug (355) by drying time to those in the chromatogram obtained with the in an oven at 100 °C to 105 °C for 2 h. reference solution. Total ash (2.4.16). Not more than 8.0 per cent. TESTS ASSAY Relative density (2.2.5) : 0.860 to 0.880. Carry out the determination of essential oils in vegetable Refractive index (2.2.6) : 1.462 to 1.470. drugs (2.8.12). Use a 500 ml round-bottomed flask, 200 ml Optical rotation (2.2.7) : + 7° to + 13°. of water R as the distillation liquid and 0.5 ml of xylene R in the graduated tube. Reduce the drug to a coarse powder Acid value (2.5.1) : maximum 3.0, determined on 5.00 g of and immediately use 30.0 g for the determination. Distil at a the substance to be examined. rate of 2 ml/min to 3 ml/min for 2 h. Chromatographic profile. Gas chromatography (2.2.28) : use the normalisation procedure. STORAGE Store protected from light. Test solution. The substance to be examined. Reference solution

1366


Cortisone acetate


Reference solution (a). Dissolve 10 µl of α-pinene R, 10 µl of limonene R, 10 µl of γ-terpinene R, 10 µl of p-cymene R, 10 mg of camphor R, 20 µl of linalol R, 10 µl of α-terpineol R, 10 µl of geranyl acetate R and 10 µl of geraniol R in 1 ml of hexane R. Reference solution (b). Dissolve 5 µl of geraniol R in hexane R and dilute to 10 ml with the same solvent. Column : — material : fused silica, — size : l = 60 m, Ø = 0.25 mm, — stationary phase: macrogol 20 000 R (film thickness 0.25 µm). Carrier gas : helium for chromatography R. Flow rate : 1 ml/min. Split ratio : 1:65. Temperature :

Column

Time (min)

Temperature (°C)

0 - 10 10 - 75 75 - 120

60 60 → 190 190

Injection port

220

Detector

240

Detection : flame ionisation. Injection : 0.2 µl. Elution order : order indicated in the composition of reference solution (a). Record the retention times of these substances. System suitability : reference solution (a) : — resolution : minimum 1.5 between the peaks due to linalol and camphor. Using the retention times determined from the chromatogram obtained with reference solution (a), locate the components of reference solution (a) in the chromatogram obtained with the test solution. Determine the percentage content of each of these components. The percentages are within the following ranges : — α-pinene : 3.0 per cent to 7.0 per cent, — limonene : 1.5 per cent to 5.0 per cent, — γ-terpinene : 1.5 per cent to 8.0 per cent, — p-cymene : 0.5 per cent to 4.0 per cent, — camphor : 3.0 per cent to 6.0 per cent, — linalol : 65.0 per cent to 78.0 per cent, — α-terpineol: 0.1 per cent to 1.5 per cent, — geranyl acetate : 0.5 per cent to 4.0 per cent, — geraniol : 0.5 per cent to 3.0 per cent, — disregard limit: area of the peak in the chromatogram obtained with reference solution (b) (0.05 per cent). Chiral purity. Gas chromatography (2.2.28). Test solution. Dissolve 0.02 g of the substance to be examined in pentane R and dilute to 10 ml with the same solvent. Reference solution. Dissolve 10 µl of linalol R and 5 mg of borneol R in pentane R and dilute to 10 ml with the same solvent. Column : — material : fused silica, — size : l = 25 m, Ø = 0.25 mm, — stationary phase: modified β-cyclodextrin for chiral chromatography R (film thickness 0.25 µm). General Notices (1) apply to all monographs and other texts

Carrier gas : helium for chromatography R. Flow rate : 1.3 ml/min. Split ratio : 1:30. Temperature :

Column

Time (min)

Temperature (°C)

0 - 65

50 → 180

Injection port

230

Detector

230

Detection : flame ionisation. Injection : 1 µl. System suitability : reference solution : — resolution : minimum 5.5 between the peaks due to (R)-linalol (1st peak) and (S)-linalol (2nd peak) and minimum 2.9 between the peaks due to (S)-linalol and borneol (3rd peak). Limit : calculate the percentage content of (R)-linalol from the expression :

AS

=

area of the peak due to (S)-linalol,

AR

=

area of the peak due to (R)-linalol.

— (R)-linalol : maximum 14 per cent. STORAGE In a well-filled, airtight container, protected from light and at a temperature not exceeding 25 °C. 01/2005:0321

CORTISONE ACETATE Cortisoni acetas

C23H30O6

Mr 402.5

DEFINITION Cortisone acetate contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent of 17-hydroxy-3,11,20-trioxopregn-4-en-21-yl acetate, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, practically insoluble in water, freely soluble in methylene chloride, soluble in dioxan, sparingly soluble in acetone, slightly soluble in alcohol and in methanol. It shows polymorphism. IDENTIFICATION First identification : A, B. Second identification : C, D, E. 1367

Cortisone acetate


through the solution for 5 min. Stopper the tube. Heat in a water-bath at 45 °C protected from light for 2.5 h. Allow to cool. Apply to the plate 5 µl of each solution. Prepare the mobile phase by adding a mixture of 1.2 volumes of water R and 8 volumes of methanol R to a mixture of 15 volumes of ether R and 77 volumes of methylene chloride R. Develop over a path of 15 cm. Allow the plate B. Examine by thin-layer chromatography (2.2.27), using a to dry in air and examine in ultraviolet light at 254 nm. TLC silica gel F254 plate R. The principal spot in each of the chromatograms obtained Test solution. Dissolve 10 mg of the substance to be with the test solutions is similar in position and size to examined in a mixture of 1 volume of methanol R and the principal spot in the chromatogram obtained with the 9 volumes of methylene chloride R and dilute to 10 ml corresponding reference solution. Spray with alcoholic with the same mixture of solvents. solution of sulphuric acid R and heat at 120 °C for 10 min or until the spots appear. Allow to cool. Examine Reference solution (a). Dissolve 20 mg of cortisone the plate in daylight and in ultraviolet light at 365 nm. acetate CRS in a mixture of 1 volume of methanol R and The principal spot in each of the chromatograms obtained 9 volumes of methylene chloride R and dilute to 20 ml with the test solutions is similar in position, colour in with the same mixture of solvents. daylight, fluorescence in ultraviolet light at 365 nm Reference solution (b). Dissolve 10 mg of hydrocortisone and size to the principal spot in the chromatogram acetate R in reference solution (a) and dilute to 10 ml obtained with the corresponding reference solution. with reference solution (a). The principal spots in the chromatograms obtained with test solution (b) and reference solution (b) have an Rƒ Apply to the plate 5 µl of each solution. Prepare the value distinctly lower than that of the principal spots in mobile phase by adding a mixture of 1.2 volumes of the chromatograms obtained with test solution (a) and water R and 8 volumes of methanol R to a mixture of reference solution (a). 15 volumes of ether R and 77 volumes of methylene chloride R. Develop over a path of 15 cm. Allow the plate D. Add about 2 mg to 2 ml of sulphuric acid R and shake to dry in air and examine in ultraviolet light at 254 nm. to dissolve. Within 5 min, a faint yellow colour develops. The principal spot in the chromatogram obtained with the Add the solution to 10 ml of water R and mix. The colour test solution is similar in position and size to the principal is discharged and a clear solution remains. spot in the chromatogram obtained with reference E. About 10 mg gives the reaction of acetyl (2.3.1). solution (a). Spray with alcoholic solution of sulphuric acid R. Heat at 120 °C for 10 min or until the spots TESTS appear. Allow to cool. Examine the plate in daylight and Specific optical rotation (2.2.7). Dissolve 0.250 g in in ultraviolet light at 365 nm. The principal spot in the dioxan R and dilute to 25.0 ml with the same solvent. The chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet specific optical rotation is + 211 to + 220, calculated with reference to the dried substance. light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (a). The Related substances. Examine by liquid chromatography (2.2.29). Prepare the solutions immediately before use. test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. Test solution. Dissolve 25.0 mg of the substance to be examined in acetonitrile R and dilute to 10.0 ml with the C. Examine by thin-layer chromatography (2.2.27), using a same solvent. TLC silica gel F254 plate R. Reference solution (a). Dissolve 2 mg of cortisone Test solution (a). Dissolve 25 mg of the substance to be acetate CRS and 2 mg of hydrocortisone acetate CRS in examined in methanol R with gentle heating and dilute acetonitrile R and dilute to 100.0 ml with the same solvent. to 5 ml with the same solvent. This solution is also used to prepare test solution (b). Dilute 2 ml of the solution to Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with acetonitrile R. 10 ml with methylene chloride R. The chromatographic procedure may be carried out using : Test solution (b). Transfer 2 ml of the solution obtained during preparation of test solution (a) to a 15 ml glass tube — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for with a ground-glass stopper or a polytetrafluoroethylene chromatography R (5 µm), cap. Add 10 ml of saturated methanolic potassium — as mobile phase at a flow rate of 1 ml/min a mixture hydrogen carbonate solution R and immediately pass prepared as follows : in a 1000 ml volumetric flask mix a stream of nitrogen R briskly through the solution for 400 ml of acetonitrile R with 550 ml of water R and allow 5 min. Stopper the tube. Heat in a water-bath at 45 °C to equilibrate ; adjust the volume to 1000 ml with water R protected from light for 2.5 h. Allow to cool. and mix again, Reference solution (a). Dissolve 25 mg of cortisone — as detector a spectrophotometer set at 254 nm. acetate CRS in methanol R with gentle heating and dilute to 5 ml with the same solvent. This solution is also Equilibrate the column with the mobile phase at a flow rate used to prepare reference solution (b). Dilute 2 ml of the of 1 ml/min for about 30 min. solution to 10 ml with methylene chloride R. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 20 µl of Reference solution (b). Transfer 2 ml of the solution reference solution (b) is at least 50 per cent of the full scale obtained during preparation of reference solution (a) of the recorder. to a 15 ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10 ml of saturated Inject 20 µl of reference solution (a). When the methanolic potassium hydrogen carbonate solution R chromatograms are recorded in the prescribed conditions and immediately pass a stream of nitrogen R briskly the retention times are : hydrocortisone acetate about A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cortisone acetate CRS. If the spectra obtained in the solid state show differences, record new spectra using 50 g/l solutions of the substance to be examined and of the reference substance in methylene chloride R in a 0.2 mm cell.

1368



Cotton, absorbent

10 min and cortisone acetate about 12 min. The test is not valid unless the resolution between the peaks due to hydrocortisone acetate and cortisone acetate is at least 4.2 ; if necessary, adjust the concentration of acetonitrile in the mobile phase. Inject 20 µl of the test solution, 20 µl of reference solution (b) and 20 µl of acetonitrile as a blank. Continue the chromatography for twice the retention time of the principal peak. In the chromatogram obtained with the test solution : the area of any peak apart from the principal peak, is not greater than half the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent) ; the sum of the areas of all the peaks apart from the principal peak, is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (1.5 per cent). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (b). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 0.500 g by drying in an oven at 100-105 °C.

TESTS Solution S. Place 15.0 g in a suitable vessel, add 150 ml of water R, close the vessel and allow to macerate for 2 h. Decant the solution, squeeze the residual liquid carefully from the sample with a glass rod and mix. Reserve 10 ml of the solution for the test for surface-active substances and filter the remainder. Acidity or alkalinity. To 25 ml of solution S add 0.1 ml of phenolphthalein solution R and to another 25 ml add 0.05 ml of methyl orange solution R. Neither solution is pink. Foreign fibres. Examined under a microscope, it is seen to consist exclusively of typical cotton fibres, except that occasionally a few isolated foreign fibres may be present. Fluorescence. Examine a layer about 5 mm in thickness under ultraviolet light at 365 nm. It displays only a slight brownish-violet fluorescence and a few yellow particles. It shows no intense blue fluorescence, apart from that which may be shown by a few isolated fibres. Neps. Spread about 1 g evenly between 2 colourless ASSAY transparent plates each 10 cm square. Examine for neps by Dissolve 0.100 g in alcohol R and dilute to 100.0 ml with the transmitted light and compare with Absorbent cotton RM. same solvent. Dilute 2.0 ml of the solution to 100.0 ml with The product to be examined is not more neppy than the alcohol R. Measure the absorbance (2.2.25) at the maximum standard. of 237 nm. Absorbency Calculate the content of C23H30O6 taking the specific Apparatus. A dry cylindrical copper wire basket 8.0 cm high absorbance to be 395. and 5.0 cm in diameter. The wire of which the basket is STORAGE constructed is about 0.4 mm in diameter, the mesh is 1.5 cm to 2.0 cm wide and the mass of the basket is 2.7 ± 0.3 g. Store protected from light. Sinking time. Not more than 10 s. Weigh the basket to the nearest centigram (m1). Take a total of 5.00 g in approximately equal quantities from 5 different places in the product to be examined, place loosely in the basket and weigh the filled basket to the nearest centigram (m2). Fill 01/2005:0036 a beaker 11 cm to 12 cm in diameter to a depth of 10 cm with water at about 20 °C. Hold the basket horizontally and drop it from a height of about 10 mm into the water. COTTON, ABSORBENT Measure with a stopwatch the time taken for the basket to sink below the surface of the water. Calculate the result as Lanugo gossypii absorbens the average of 3 tests. DEFINITION Water-holding capacity. Not less than 23.0 g of water per gram. After the sinking time has been measured, remove Absorbent cotton consists of new fibres or good quality the basket from the water, allow it to drain for exactly 30 s combers obtained from the seed-coat of various species of suspended in a horizontal position over the beaker, transfer the genus Gossypium L., cleaned, purified, bleached and it to a tared beaker (m3) and weigh to the nearest centigram carefully carded. It may not contain any compensatory (m4). Calculate the water-holding capacity per gram of colouring matter. absorbent cotton using the following expression : CHARACTERS It is white and is composed of fibres of average length not less than 10 mm, determined by a suitable method, and contains not more than traces of leaf residue, pericarp, seed-coat or other impurities. It offers appreciable resistance Calculate the result as the average of 3 tests. when pulled. It does not shed any appreciable quantity of Ether-soluble substances. Not more than 0.50 per cent. In dust when gently shaken. an extraction apparatus, extract 5.00 g with ether R for 4 h at a rate of at least 4 extractions per hour. Evaporate the IDENTIFICATION ether extract and dry the residue to constant mass at 100 °C A. Examined under a microscope, each fibre is seen to to 105 °C. consist of a single cell, up to about 4 cm long and up to Extractable colouring matter. In a narrow percolator, 40 µm wide, in the form of a flattened tube with thick and slowly extract 10.0 g with alcohol R until 50 ml of extract is rounded walls and often twisted. obtained. The liquid obtained is not more intensely coloured B. When treated with iodinated zinc chloride solution R, (2.2.2, Method II) than reference solution Y5, GY6 or a the fibres become violet. reference solution prepared as follows : to 3.0 ml of blue C. To 0.1 g add 10 ml of zinc chloride-formic acid solution R. primary solution add 7.0 ml of hydrochloric acid (10 g/l HCl). Heat to 40 °C and allow to stand for 2 h 30 min, shaking Dilute 0.5 ml of this solution to 10.0 ml with hydrochloric acid (10 g/l HCl). occasionally. It does not dissolve.

IMPURITIES A. hydrocortisone acetate.


1369

Cottonseed oil, hydrogenated


Peroxide value (2.5.5). Not more than 5.0. Unsaponifiable matter (2.5.7). Not more than 1.0 per cent, determined on 5.0 g. Alkaline impurities. Dissolve by gentle heating 2.0 g of the substance to be examined in a mixture of 1.5 ml of alcohol R and 3 ml of toluene R. Add 0.05 ml of a 0.4 g/l solution of bromophenol blue R in alcohol R. Not more than 0.4 ml of 0.01 M hydrochloric acid is required to change the colour to yellow. Composition of fatty acids (2.4.22, Method A). The chromatographic procedure may be carried out using : — a fused-silica capillary column 25 m long and 0.25 mm in internal diameter coated on the inner wall with poly(cyanopropyl)siloxane R (film thickness 0.2 µm), — helium for chromatography R as the carrier gas at a flow rate of 0.65 ml/min, — a flame-ionisation detector, — a split injector (1:100), maintaining the temperature of the column at 180 °C for 35 min and that of the injection port and the detector at 250 °C. The fatty acid fraction of the oil has the following composition : — saturated fatty acids of chain length less than C14 : not more than 0.2 per cent, — myristic acid : not more than 1.0 per cent, — palmitic acid : 19.0 per cent to 26.0 per cent, STORAGE — stearic acid : 68.0 per cent to 80.0 per cent, Store in a dust-proof package in a dry place. — oleic acid and isomers (C18:1 equivalent chain length on poly(cyanopropyl)siloxane 18.5 to 18.8): not more than 4.0 per cent, 01/2005:1305 — linoleic acid and isomers (C equivalent chain length 18:2 on poly(cyanopropyl)siloxane 19.4 to 19.8) : not more than 1.0 per cent, COTTONSEED OIL, HYDROGENATED — arachidic acid : not more than 1.0 per cent, Gossypii oleum hydrogenatum — behenic acid : not more than 1.0 per cent, — lignoceric acid : not more than 0.5 per cent. DEFINITION Nickel. Not more than 1 ppm of Ni, determined by atomic Hydrogenated cottonseed oil is the product obtained by absorption spectrometry (2.2.23, Method II). refining and hydrogenation of oil obtained from seeds of cultivated plants of various varieties of Gossypium Test solution. Introduce 5.0 g of the substance to be hirsutum L. or of other species of Gossypium. The product examined into a platinum or silica crucible tared after consists mainly of triglycerides of palmitic and stearic acids. ignition. Cautiously heat and introduce into the substance a wick formed from twisted ashless filter paper. Ignite the CHARACTERS wick. When the substance ignites, stop heating. After A white mass or powder which melts to a clear, pale yellow combustion, ignite in a muffle furnace at about 600 °C. liquid when heated, practically insoluble in water, freely Continue the incineration until white ash is obtained. After soluble in methylene chloride and in toluene, very slightly cooling, take up the residue with two quantities, each of soluble in alcohol. 2 ml, of dilute hydrochloric acid R and transfer into a 25 ml graduated flask. Add 0.3 ml of nitric acid R and dilute to IDENTIFICATION 25.0 ml with distilled water R. A. It complies with the test for melting point (see Tests). Reference solutions. Prepare three reference solutions by B. It complies with the test for foreign fatty oils (see Tests). adding 1.0 ml, 2.0 ml and 4.0 ml of nickel standard solution (0.2 ppm Ni) R to 2.0 ml portions of the test solution, TESTS diluting to 10.0 ml with distilled water R. Melting point (2.2.14) : 57 °C to 70 °C. Measure the absorbance at 232 nm using a nickel Acid value (2.5.1). Not more than 0.5, determined on 10.0 g. hollow-cathode lamp as a source of radiation, a graphite furnace as an atomic generator and argon R as the carrier Dissolve the substance to be examined in 50 ml of a hot gas. mixture of equal volumes of alcohol R and toluene R, previously neutralised with 0.1 M potassium hydroxide STORAGE using 0.5 ml of phenolphthalein solution R1 as indicator. Titrate the solution immediately while still hot. Store protected from light. Surface-active substances. Introduce the 10 ml portion of solution S reserved before filtration into a 25 ml graduated ground-glass-stoppered cylinder with an external diameter of 20 mm and a wall thickness of not greater than 1.5 mm, previously rinsed 3 times with sulphuric acid R and then with water R. Shake vigorously 30 times in 10 s, allow to stand for 1 min and repeat the shaking. After 5 min, any foam present must not cover the entire surface of the liquid. Water-soluble substances. Not more than 0.50 per cent. Boil 5.000 g in 500 ml of water R for 30 min, stirring frequently. Replace the water lost by evaporation. Decant the liquid, squeeze the residual liquid carefully from the sample with a glass rod and mix. Filter the liquid whilst hot. Evaporate 400 ml of the filtrate (corresponding to 4/5 of the mass of the sample taken) and dry the residue to constant mass at 100 °C to 105 °C. Loss on drying (2.2.32). Not more than 8.0 per cent, determined on 5.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.40 per cent. Introduce 5.00 g into a previously heated and cooled, tared crucible. Heat cautiously over a naked flame and then carefully to dull redness at 600 °C. Allow to cool, add a few drops of dilute sulphuric acid R, then heat and incinerate until all the black particles have disappeared. Allow to cool. Add a few drops of ammonium carbonate solution R. Evaporate and incinerate carefully, allow to cool and weigh again. Repeat the incineration for periods of 5 min to constant mass.

1370


Croscarmellose sodium


01/2005:1306 DEFINITION Mixture of 2-, 3- and 4-methylphenol.

COUCH GRASS RHIZOME Graminis rhizoma

DEFINITION Whole or cut, washed and dried rhizome of Agropyron repens (L.) Beauv. (Elymus repens (L.) Gould) ; the adventitious roots are removed. CHARACTERS Ma croscopic and microscopic characters described under identification tests A and B. IDENTIFICATION A. The shiny yellowish, light brown or yellowish-brown pieces of the rhizome are 2 mm to 3 mm thick and longitudinally furrowed. At the nodes are the remains of very thin, more or less branched roots and whitish or brownish scale-like leaves ; the internodes, up to 6 cm long, are furrowed and hollow inside. The transverse section of the nodes shows a yellowish medulla. B. Reduce to a powder (355). The powder is whitish-yellow. Examine under a microscope, using chloral hydrate solution R. The powder shows the following diagnostic characters : fragments of the epidermis covered with a thick cuticle and composed of rectangular and elongated thick-walled cells with pitted slightly wavy walls which alternate with small rounded to almost square cells ; U-shaped thickened endodermic cells ; numerous fragments of moderately thickened fibres and groups of vessels with slit-shaped pits or with spiral and annular thickening. TESTS Cynodon dactilon, Imperata cylindrica. Examine under a microscope using iodine solution R1. No blue starch grains are visible. Foreign matter (2.8.2) : maximum 15 per cent of greyish-black pieces of rhizome in the cut drug. Water-soluble extractive : minimum 25 per cent. To 5.0 g of the powdered drug (355) add 200 ml of boiling water R. Allow to stand for 10 min, shaking occasionally. Allow to cool, dilute to 200.0 ml with water R and filter. Evaporate 20.0 ml of the filtrate to dryness on a water-bath. Dry the residue in an oven at 100-105 °C. The residue weighs a minimum of 0.125 g. Loss on drying (2.2.32) : maximum 12.0 per cent, determined on 1.000 g of the powdered drug (355) by drying in an oven at 100-105 °C for 2 h. Total ash (2.4.16) : maximum 5.0 per cent. Ash insoluble in hydrochloric acid (2.8.1) : maximum 1.5 per cent.

CRESOL, CRUDE Cresolum crudum

C7H 8O

CHARACTERS Appearance : colourless or pale brown liquid. Solubility : sparingly soluble in water, miscible with alcohol and with methylene chloride. IDENTIFICATION A. To 0.5 ml add 300 ml of water R, mix and filter. To 10 ml of the filtrate add 1 ml of ferric chloride solution R1. A blue colour is produced. B. To 10 ml of the filtrate obtained in identification test A, add 1 ml of bromine water R. A pale yellow flocculent precipitate is produced. C. It complies with the test for relative density (see Tests). TESTS Solution S. To 2.5 g of the substance to be examined add 50 ml of water R, shake for 1 min and filter through a moistened filter. Acidity or alkalinity. To 10 ml of solution S add 0.1 ml of methyl red solution R and 0.2 ml of 0.01 M sodium hydroxide. The solution is yellow. Add 0.3 ml of 0.01 M hydrochloric acid. The solution is red. Relative density (2.2.5) : 1.029 to 1.044. Distillation range (2.2.11) : a maximum of 2.0 per cent V/V distils below 188 °C and a minimum of 80 per cent V/V distils between 195 °C and 205 °C. Sulphur compounds. Place 20 ml in a small conical flask. Over the mouth of the flask fix a piece of filter paper moistened with lead acetate solution R. Heat on a water-bath for 5 min. Not more than a light yellow colour is produced on the filter paper. Residue on evaporation : maximum 0.1 per cent. Evaporate 2.0 g to dryness on a water-bath and dry at 100-105 °C for 1 h. The residue weighs not more than 2 mg. STORAGE Protected from light. 01/2005:0985

CROSCARMELLOSE SODIUM Carmellosum natricum conexum DEFINITION Croscarmellose sodium (cross-linked sodium carboxymethylcellulose) is the sodium salt of a cross-linked, partly O-carboxymethylated cellulose.

CHARACTERS 01/2005:1628 A white or greyish-white powder, practically insoluble in acetone, in ethanol and in toluene. IDENTIFICATION A. Shake 1 g with 100 ml of a solution containing 4 ppm of methylene blue R and allow to settle. The substance to be examined absorbs the methylene blue and settles as a blue, fibrous mass. B. Shake 1 g with 50 ml of water R. Transfer 1 ml of the mixture to a test-tube, add 1 ml of water R and 0.05 ml of a freshly prepared 40 g/l solution of α-naphthol R in Mr 108.1


1371

Croscarmellose sodium


methanol R. Incline the test-tube and add carefully 2 ml of sulphuric acid R down the side so that it forms a lower layer. A reddish-violet colour develops at the interface. C. It gives reaction (a) of sodium (2.3.1). TESTS pH (2.2.3). Shake 1 g with 100 ml of carbon dioxide-free water R for 5 min. The pH of the suspension is 5.0 to 7.0. Degree of substitution. Place 1.000 g in a 500 ml conical flask, add 300 ml of a 100 g/l solution of sodium chloride R, 25.0 ml of 0.1 M sodium hydroxide, stopper the flask and allow to stand for 5 min, shaking occasionally. Add 0.05 ml of m-cresol purple solution R and about 15 ml of 0.1 M hydrochloric acid from a burette. Insert the stopper and shake. If the solution is violet, add 0.1 M hydrochloric acid in 1 ml portions until the solution becomes yellow, shaking after each addition. Titrate with 0.1 M sodium hydroxide until the colour turns to violet. Calculate the number of milliequivalents (M) of base required for the neutralisation equivalent to 1 g of dried substance. Calculate the degree of acid carboxymethyl substitution (A) from the expression :

C

=

Transfer 2.0 ml of the test solution and 2.0 ml of each of the reference solutions to separate 25 ml volumetric flasks. Heat the uncovered flasks for 20 min on a water-bath to eliminate acetone. Allow to cool and add 5.0 ml of 2,7-dihydroxynaphthalene solution R to each flask. Mix, add a further 15.0 ml of 2,7-dihydroxynaphthalene solution R and mix again. Close the flasks with aluminium foil and heat on a water-bath for 20 min. Cool and dilute to 25.0 ml with sulphuric acid R. Measure the absorbance (2.2.25) of each solution at 540 nm. Prepare a blank using 2.0 ml of a solution containing 5 per cent V/V each of glacial acetic acid R and water R in acetone R. Prepare a standard curve using the absorbances obtained with the reference solutions. From the standard curve and the absorbance of the test solution, determine the mass a, in milligrams, of glycollic acid in the substance to be examined, and calculate the content of sodium glycollate from the expression :

sulphated ash as a percentage.

Calculate the degree of sodium carboxymethyl substitution (S) from the expression :

The degree of substitution is the sum of A + S and it is between 0.60 and 0.85, calculated with reference to the dried substance. Sodium chloride and sodium glycollate. The sum of the percentage contents of sodium chloride and sodium glycollate is not more than 0.5 per cent, calculated with reference to the dried substance. Sodium chloride. Place 5.00 g in a 250 ml conical flask, add 50 ml of water R and 5 ml of strong hydrogen peroxide solution R and heat on a water-bath for 20 min, stirring occasionally to ensure total hydration. Cool, add 100 ml of water R and 10 ml of nitric acid R. Titrate with 0.05 M silver nitrate determining the end-point potentiometrically (2.2.20) using a silver indicator electrode and a double-junction reference electrode containing a 100 g/l solution of potassium nitrate R in the outer jacket and a standard filling solution in the inner jacket, and stirring constantly. 1 ml of 0.05 M silver nitrate is equivalent to 2.922 mg of NaCl. Sodium glycollate. Place a quantity of the substance to be examined equivalent to 0.500 g of the dried substance in a 100 ml beaker. Add 5 ml of glacial acetic acid R and 5 ml of water R and stir to ensure total hydration (about 15 min). Add 50 ml of acetone R and 1 g of sodium chloride R. Stir for several minutes to ensure complete precipitation of the carboxymethylcellulose. Filter through a fast filter paper impregnated with acetone R into a volumetric flask, rinse the beaker and filter with 30 ml of acetone R and dilute the filtrate to 100.0 ml with the same solvent. Allow to stand for 24 h without shaking. Use the clear supernatant to prepare the test solution. Prepare the reference solutions as follows : in a 100 ml volumetric flask, dissolve 0.100 g of glycollic acid R, previously dried in vacuo over diphosphorus pentoxide R, 1372

in water R and dilute to 100.0 ml with the same solvent. Use the solution within 30 days. Transfer 1.0 ml, 2.0 ml, 3.0 ml and 4.0 ml of the solution to separate volumetric flasks ; dilute the contents of each flask to 5.0 ml with water R, add 5 ml of glacial acetic acid R, dilute to 100.0 ml with acetone R and mix.

1.29 =

the factor converting glycollic acid to sodium glycollate,

b m

=


=

mass of the substance to be examined, in grams.

Water-soluble substances. Not more than 10.0 per cent. Disperse 10.00 g in 800.0 ml of water R and stir for 1 min every 10 min during the first 30 min. Allow to stand for 1 h and centrifuge, if necessary. Decant 200.0 ml of the supernatant liquid onto a fast filter paper in a vacuum filtration funnel, apply vacuum and collect 150.0 ml of the filtrate. Evaporate to dryness and dry the residue at 100 °C to 105 °C for 4 h. Heavy metals (2.4.8). To the residue obtained in the determination of the sulphated ash add 1 ml of hydrochloric acid R and evaporate on a water-bath. Take up the residue in 20 ml of water R. 12 ml of the solution complies with limit test A for heavy metals (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32). Not more than 10.0 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C for 6 h. Sulphated ash (2.4.14) : 14.0 per cent to 28.0 per cent, determined on 2.000 g, using a mixture of equal volumes of sulphuric acid R and water R, and calculated with reference to the dried substance. Settling volume. Place 75 ml of water R in a 100 ml graduated cylinder and add 1.5 g of the substance to be examined in 0.5 g portions, shaking vigorously after each addition. Dilute to 100.0 ml with water R and shake again until the substance is homogeneously distributed. Allow to stand for 4 h. The settling volume is between 10.0 ml and 30.0 ml. Microbial contamination. Total viable aerobic count (2.6.12) not more than 103 bacteria and 102 fungi per gram, determined by plate count. It complies with the test for Escherichia coli (2.6.13).



Crospovidone

01/2005:0892 of a mechanical stirrer, taking care not to damage the filter. Transfer 50.0 ml of the clear filtrate to a tared 100 ml beaker, evaporate to dryness and dry at 105-110 °C for 3 h. The CROSPOVIDONE residue weighs a maximum of 50 mg. Impurity A. Liquid chromatography (2.2.29). Crospovidonum Test solution. Suspend 1.250 g in 50.0 ml of methanol R and shake for 60 min. Leave the bulk to settle and filter through a 0.2 µm filter. Reference solution (a). Dissolve 50 mg of 1-vinylpyrrolidin-2-one R in methanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with methanol R. Dilute 5.0 ml of this (C6H9NO)n Mr (111.1)n solution to 100.0 ml with the mobile phase. Reference solution (b). Dissolve 10 mg of DEFINITION 1-vinylpyrrolidin-2-one R and 0.50 g of vinyl acetate R in Crospovidone is a cross-linked homopolymer of methanol R and dilute to 100 ml with the same solvent. 1-ethenylpyrrolidin-2-one. It is available in different degrees Dilute 1.0 ml of the solution to 100.0 ml with the mobile of powder fineness (type A and type B). phase. Content : 11.0 per cent to 12.8 per cent of nitrogen (N ; Precolumn : Ar 14.01) (dried substance). — size : l = 0.025 m, Ø = 4 mm, CHARACTERS — stationary phase : octadecylsilyl silica gel for chromatography R (5 µm). Appearance : white or yellowish-white powder or flakes, hygroscopic. Column : Solubility : practically insoluble in water, in alcohol and in — size : l = 0.25 m, Ø = 4 mm, methylene chloride. — stationary phase : octadecylsilyl silica gel for chromatography R (5 µm), IDENTIFICATION — temperature : 40 °C. A. Infrared absorption spectrophotometry (2.2.24). Mobile phase : acetonitrile R, water R (10:90 V/V). Comparison : Ph. Eur. reference spectrum of Flow rate : adjusted so that the retention time of the peak crospovidone. corresponding to impurity A is about 10 min. B. Suspend 1 g in 10 ml of water R, add 0.1 ml of 0.05 M iodine and shake for 30 s. Add 1 ml of starch solution R Detection : spectrophotometer at 235 nm. and shake. No blue colour develops within 30 s. Injection : 50 µl. After each injection of the test solution, C. To 10 ml of water R, add 0.1 g and shake. A suspension is wash the precolumn by passing the mobile phase backward, at the same flow rate as applied in the test, for 30 min. formed and no clear solution is obtained within 15 min. D. Weigh a suitable quantity of the substance to be examined System suitability : (for example 10 mg to 100 mg) and suspend it in 10.0 ml — resolution : minimum of 2.0 between the peaks of water R, adding a wetting agent. Observe under a corresponding to impurity A and to vinyl acetate in the microscope at a suitable magnification using a calibrated chromatogram obtained with reference solution (b), ocular micrometer. If the majority of particles are in — repeatability : maximum relative standard deviation of the range 50 µm to 300 µm, the product is classified as 2.0 per cent after 5 injections of reference solution (a). type A. If almost all the particles are below 50 µm, the Limits : product is classified as type B. — impurity A : not more than the area of the principal peak TESTS in the chromatogram obtained with reference solution (a) (10 ppm). Peroxides. Type A : maximum 400 ppm expressed as H2O2 ; type B : maximum 1000 ppm expressed as H2O2. Heavy metals (2.4.8) : maximum 10 ppm. Suspend 2.0 g in 50 ml of water R. To 25 ml of this 2.0 g complies with limit test D. Prepare the standard using suspension add 2 ml of titanium trichloride-sulphuric 2 ml of lead standard solution (10 ppm Pb) R. acid reagent R. Allow to stand for 30 min and filter. The Loss on drying (2.2.32) : maximum 5.0 per cent, determined absorbance (2.2.25) of the filtrate, measured at 405 nm on 0.500 g by drying in an oven at 100-105 °C. using a mixture of 25 ml of a filtered 40 g/l suspension of the substance to be examined and 2 ml of a 13 per cent V/V Sulphated ash (2.4.14) : maximum 0.1 per cent, determined solution of sulphuric acid R as the compensation liquid has on 1.0 g. a maximum of 0.35. ASSAY For type B use 10 ml of the suspension diluted to 25 ml with Place 100.0 mg of the substance to be examined (m mg) water R for the test. in a combustion flask, add 5 g of a mixture of 1 g of Water-soluble substances: maximum 1.0 per cent. copper sulphate R, 1 g of titanium dioxide R and 33 g Transfer 25.0 g to a 400 ml beaker, add 200 ml of water R and of dipotassium sulphate R, and 3 glass beads. Wash any stir for 1 h using a magnetic stirrer. Transfer the suspension adhering particles from the neck into the flask with a small quantity of water R. Add 7 ml of sulphuric acid R, to a 250.0 ml volumetric flask, rinsing with water R, and allowing it to run down the sides of the flask, and mix the dilute to volume with the same solvent. Allow the bulk of contents by rotation. Close the mouth of the flask loosely, the solids to settle. Filter about 100 ml of the almost clear for example by means of a glass bulb with a short stem, to supernatant liquid through a 0.45 µm membrane filter, protected by superimposing a 3 µm membrane filter. While avoid excessive loss of sulphuric acid. Heat gradually at filtering, stir the liquid above the filter manually or by means first, then increase the temperature until there is vigorous General Notices (1) apply to all monographs and other texts

1373

Crotamiton


boiling with condensation of sulphuric acid in the neck of the flask ; precautions are to be taken to prevent the upper part of the flask from becoming overheated. Continue the heating for 45 min. Cool, dissolve the solid material by cautiously adding to the mixture 20 ml of water R, cool again and place in a steam-distillation apparatus. Add 30 ml of strong sodium hydroxide solution R through the funnel, rinse the funnel cautiously with 10 ml of water R and distil immediately by passing steam through the mixture. Collect 80-100 ml of distillate in a mixture of 30 ml of a 40 g/l solution of boric acid R and 0.05 ml of bromocresol green-methyl red solution R and enough water R to cover the tip of the condenser. Towards the end of the distillation lower the receiver so that the tip of the condenser is above the surface of the acid solution and rinse the end part of the condenser with a small quantity of water R. Titrate the distillate with 0.025 M sulphuric acid until the colour of the solution changes from green through pale greyish-blue to pale greyish-red-purple (n1 ml of 0.025 M sulphuric acid). Repeat the test using about 100 mg of glucose R in place of the substance to be examined (n2 ml of 0.025 M sulphuric acid).

STORAGE In an airtight container. LABELLING The label states the type (type A or type B). IMPURITIES

Second identification : A, C, D. A. Dissolve 25.0 mg in cyclohexane R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 10.0 ml with cyclohexane R. Examined between 220 nm and 300 nm (2.2.25), the solution shows an absorption maximum at 242 nm. The specific absorbance at the maximum is 300 to 330. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with crotamiton CRS. C. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm. Test solution. Dissolve 25 mg of the substance to be examined in ethanol R and dilute to 10 ml with the same solvent. Reference solution. Dissolve 25 mg of crotamiton CRS in ethanol R and dilute to 10 ml with the same solvent. Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm using the mixture prepared as follows : shake 98 volumes of methylene chloride R with 2 volumes of concentrated ammonia R, dry over anhydrous sodium sulphate R, filter and mix 97 volumes of the filtrate with 3 volumes of 2-propanol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. D. To 10 ml of a saturated solution add a few drops of a 3 g/l solution of potassium permanganate R. A brown colour is produced and a brown precipitate is formed on standing.

TESTS Relative density (2.2.5) : 1.006 to 1.011. Refractive index (2.2.6) : 1.540 to 1.542. A. 1-ethenylpyrrolidin-2-one (1-vinylpyrrolidin-2-one). Free amines. Dissolve 5.00 g in 16 ml of methylene 01/2005:1194 chloride R and add 4.0 ml of glacial acetic acid R. Add 0.1 ml of metanil yellow solution R and 1.0 ml of 0.02 M perchloric acid. The solution is red-violet (500 ppm as CROTAMITON ethylaminotoluene). Chlorides. Boil 5.0 g under a reflux condenser for 1 h with Crotamitonum 25 ml of alcohol R and 5 ml of a 200 g/l solution of sodium hydroxide R. Cool, add 5 ml of water R and shake with 25 ml of ether R. Dilute the lower layer to 20 ml with water R ; add 5 ml of nitric acid R, dilute to 50 ml with water R and add 1 ml of a freshly prepared 50 g/l solution of silver nitrate R. Any opalescence in the solution is not more intense than that in a mixture of 1 ml of a freshly prepared 50 g/l solution C13H17NO Mr 203.3 of silver nitrate R and a solution prepared by diluting 5 ml of a 200 g/l solution of sodium hydroxide R to 20 ml with DEFINITION water R and adding 1.5 ml of 0.01 M hydrochloric acid, 5 ml Crotamiton contains not less than 96.0 per cent and of nitric acid R and sufficient water R to produce 50 ml not more than the equivalent of 102.0 per cent of (100 ppm). N-ethyl-N-(2-methylphenyl)but-2-enamide, calculated as the Related substances. Examine by liquid chromatography sum of the E- and Z-isomers, and not more than 15.0 per (2.2.29) as described under Assay. cent of the Z-isomer. Inject 20 µl each of test solution (a), reference solution (b) CHARACTERS and reference solution (c). Continue the chromatography for 2.5 times the retention time of the principal peak. In A colourless or pale yellow, oily liquid, slightly soluble in the chromatogram obtained with test solution (a) : the area water, miscible with alcohol. At low temperatures it may of any peak corresponding to crotamiton impurity A is not partly or completely solidify. greater than the area of the corresponding peak in the IDENTIFICATION chromatogram obtained with reference solution (b) (3 per cent) ; the sum of the areas of any peaks, apart from the First identification : B. 1374


Cyanocobalamin


principal peak, the peak corresponding to the Z-isomer and IMPURITIES any peak corresponding to crotamiton impurity A, is not greater than the sum of the areas of the peaks corresponding to the Z- and E-isomers in the chromatogram obtained with reference solution (c) (1 per cent). Disregard any peak with an area less than 0.02 times the area of the principal peak in the chromatogram obtained with reference solution (c). Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

A. N-ethyl-N-(2-methylphenyl)but-3-enamide. 01/2005:0547

ASSAY Examine by liquid chromatography (2.2.29). Test solution (a). Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase.

CYANOCOBALAMIN Cyanocobalaminum

Test solution (b). Dilute 1.0 ml of test solution (a) to 20.0 ml with the mobile phase. Reference solution (a). Dissolve 50.0 mg of crotamiton CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 20.0 ml with the mobile phase. Reference solution (b). Dissolve 15.0 mg of crotamiton impurity A CRS in the mobile phase and dilute to 20.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 50.0 ml with the mobile phase. Reference solution (c). Dilute 1.0 ml of test solution (a) to 100.0 ml with the mobile phase. Reference solution (d). Dissolve 15 mg of crotamiton impurity A CRS in the mobile phase and dilute to 100 ml with the mobile phase. Dilute 1 ml of the solution to 10 ml with test solution (a). The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4 mm in internal diameter packed with silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.0 ml/min a mixture of 8 volumes of tetrahydrofuran R and 92 volumes of cyclohexane R, — as detector a spectrophotometer set at 242 nm. Inject 20 µl of reference solution (b) and 20 µl of reference solution (d). When the chromatograms are recorded in the prescribed conditions, the retention times relative to the principal peak (E-isomer) are : Z-isomer about 0.5 and crotamiton impurity A about 0.8. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with reference solution (b) is at least 70 per cent of the full scale of the recorder. The test is not valid unless, in the chromatogram obtained with reference solution (d), the resolution between the peaks corresponding to impurity A and the E-isomer is at least 4.5. Inject alternately test solution (b) and reference solution (a). Calculate the percentage content of C13H17NO from the sum of the areas of the peaks due to the Z- and E-isomers in the chromatograms obtained. Calculate the content of the Z-isomer, as a percentage of the total content of the Eand Z-isomers, from the chromatogram obtained with test solution (b). STORAGE Protected from light. General Notices (1) apply to all monographs and other texts

C63H88CoN14O14P

Mr 1355

DEFINITION Cyanocobalamin contains not less than 96.0 per cent and not more than the equivalent of 102.0 per cent of α-(5,6-dimethylbenzimidazol-1-yl)cobamide cyanide, calculated with reference to the dried substance. CHARACTERS A dark-red, crystalline powder or dark-red crystals, sparingly soluble in water and in alcohol, practically insoluble in acetone. The anhydrous substance is very hygroscopic. IDENTIFICATION A. Dissolve 2.5 mg in water R and dilute to 100.0 ml with the same solvent. Examined between 260 nm and 610 nm (2.2.25), the solution shows 3 absorption maxima, at 278 nm, 361 nm and at 547 nm to 559 nm. The ratio of the absorbance at the maximum at 361 nm to that at the maximum at 547 nm to 559 nm is 3.15 to 3.45. The ratio of the absorbance at the maximum at 361 nm to that at the maximum at 278 nm is 1.70 to 1.90. B. Carry out the test protected from light. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. Dissolve 2 mg of the substance to be examined in 1 ml of a mixture of equal volumes of alcohol R and water R. 1375

Cyclizine hydrochloride


Reference solution. Dissolve 2 mg of cyanocobalamin CRS in 1 ml of a mixture of equal volumes of alcohol R and water R. Apply to the plate 10 µl of each solution. Develop in a non-saturated tank over a path of 12 cm using a mixture of 9 volumes of dilute ammonia R1, 30 volumes of methanol R and 45 volumes of methylene chloride R. Allow the plate to dry in air. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. TESTS Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 10.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the same mixture of solvents. Use within 1 h. Reference solution (a). Dilute 3.0 ml of the test solution to 100.0 ml with the mobile phase. Use within 1 h. Reference solution (b). Dilute 5.0 ml of the test solution to 50.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase. Use within 1 h. Reference solution (c). Dissolve 25 mg of the substance to be examined in 10 ml of water R, warming if necessary. Allow to cool and add 5 ml of a 1.0 g/l solution of chloramine R and 0.5 ml of 0.05 M hydrochloric acid. Dilute to 25 ml with water R. Shake and allow to stand for 5 min. Dilute 1 ml of this solution to 10 ml with the mobile phase and inject immediately. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4 mm in internal diameter packed with octylsilyl silica gel for chromatography R (5 µm), — as the mobile phase at a flow rate of 0.8 ml/min a mixture prepared as follows : mix 26.5 volumes of methanol R and 73.5 volumes of a 10 g/l solution of disodium hydrogen phosphate R adjusted to pH 3.5 using phosphoric acid R and use within 2 days, — a spectrophotometer set at 361 nm as detector, — a loop injector. Inject separately 20 µl of each solution and continue the chromatography for 3 times the retention time of cyanocobalamin. In the chromatogram obtained with the test solution, the sum of the areas of any peaks apart from the principal peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (3 per cent). Disregard any peak whose area is less than that of the principal peak in the chromatogram obtained with reference solution (b). The test is not valid unless the chromatogram obtained with reference solution (c) shows 2 principal peaks, the resolution between these peaks is not less than 2.5 and the chromatogram obtained with reference solution (b) shows one principal peak with a signal-to-noise ratio of not less than 5. Loss on drying (2.2.32). Not more than 12.0 per cent, determined on 20.00 mg by drying in vacuo at 100-105 °C for 2 h. ASSAY Dissolve 25.00 mg in water R and dilute to 1000.0 ml with the same solvent. Measure the absorbance (2.2.25) of the solution at the maximum at 361 nm. Calculate the content of C63H88CoN14O14P, taking the specific absorbance to be 207. 1376

STORAGE Store in an airtight container, protected from light. 01/2005:1092

CYCLIZINE HYDROCHLORIDE Cyclizini hydrochloridum

C18H23ClN2

Mr 302.8

DEFINITION Cyclizine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of 1-(diphenylmethyl)-4-methylpiperazine hydrochloride, calculated with reference to the dried substance. CHARACTERS A white, crystalline powder, slightly soluble in water and in alcohol. IDENTIFICATION First identification : B, E. Second identification : A, C, D, E. A. Dissolve 20.0 mg in a 5 g/l solution of sulphuric acid R and dilute to 100.0 ml with the same acid solution (solution A). Examined between 240 nm and 350 nm (2.2.25), solution A shows two absorption maxima, at 258 nm and 262 nm. The ratio of the absorbance measured at the maximum at 262 nm to that measured at the maximum at 258 nm is 1.0 to 1.1. Dilute 10.0 ml of solution A to 100.0 ml with a 5 g/l solution of sulphuric acid R (solution B). Examined between 210 nm and 240 nm, solution B shows an absorption maximum at 225 nm. The specific absorbance at the maximum is 370 to 410. Verify the resolution of the apparatus (2.2.25) ; the test is not valid unless the ratio of the absorbances is at least 1.7. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cyclizine hydrochloride CRS. Examine the substances prepared as discs using potassium chloride R. C. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). D. Dissolve 0.5 g in 10 ml of alcohol (60 per cent V/V) R using heat, if necessary. Cool in iced water. Add 1 ml of dilute sodium hydroxide solution R and 10 ml of water R. Filter, wash the precipitate with water R and dry it at 60 °C at a pressure not exceeding 0.7 kPa for 2 h. The melting point (2.2.14) is 105 °C to 108 °C. E. It gives reaction (a) of chlorides (2.3.1). TESTS pH (2.2.3). Dissolve 0.5 g in a mixture of 40 volumes of alcohol R and 60 volumes of carbon dioxide-free water R and dilute to 25 ml with the same mixture of solvents. The pH of the solution is 4.5 to 5.5.


Cyclopentolate hydrochloride


01/2005:1093

Related substances. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R. Prepare the solutions immediately before use. Test solution (a). Dissolve 0.20 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent.

CYCLOPENTOLATE HYDROCHLORIDE Cyclopentolati hydrochloridum

Test solution (b). Dilute 5 ml of test solution (a) to 100 ml with methanol R. Reference solution (a). Dissolve 10 mg of cyclizine hydrochloride CRS in methanol R and dilute to 10 ml with the same solvent. Reference solution (b). Dissolve 5 mg of methylpiperazine R in methanol R and dilute to 50 ml with the same solvent. C17H26ClNO3 Reference solution (c). Dilute 1 ml of test solution (b) to 10 ml with methanol R. Reference solution (d). Dissolve 10 mg of cyclizine hydrochloride CRS and 10 mg of hydroxyzine hydrochloride CRS in methanol R and dilute to 10 ml with the same solvent. Apply to the plate 20 µl of each solution. Develop over a path of 15 cm using a mixture of 2 volumes of concentrated ammonia R, 13 volumes of methanol R and 85 volumes of methylene chloride R. Allow the plate to dry in air for 30 min. Expose the plate to iodine vapour for 10 min. In the chromatogram obtained with test solution (a) : any spot corresponding to 1-methylpiperazine is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent) ; any spot, apart from the principal spot and any spot corresponding to 1-methylpiperazine, is not more intense than the principal spot in the chromatogram obtained with reference solution (c) (0.5 per cent). The test is not valid unless the chromatogram obtained with reference solution (d) shows two clearly separated spots. Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g by drying in an oven at 130 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY

Mr 327.9

DEFINITION Cyclopentolate hydrochloride contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent of 2-(dimethylamino)ethyl (RS)-2-(1-hydroxycyclopentyl)-2-phenylacetate hydrochloride, calculated with reference to the dried substance. CHARACTERS A white, crystalline powder, very soluble in water, freely soluble in alcohol. IDENTIFICATION First identification : B, D. Second identification : A, C, D. A. Melting point (2.2.14) : 135 °C to 141 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cyclopentolate hydrochloride CRS. Examine the substances as discs prepared using potassium chloride R. If the spectra obtained show differences, dissolve the substance to be examined and the reference substance separ-ately in alcohol R, evaporate to dryness and record new spectra using the residues. C. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position, fluorescence and size to the principal spot in the chromatogram obtained with reference solution (b). D. It gives reaction (a) of chlorides (2.3.1).

Dissolve 0.200 g in 15 ml of anhydrous formic acid R and add 40 ml of acetic anhydride R. Titrate with 0.1 M perchloric TESTS acid, determining the end-point potentiometrically (2.2.20). pH (2.2.3). Dissolve 0.2 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. The pH of the 1 ml of 0.1 M perchloric acid is equivalent to 15.14 mg of solution is 4.5 to 5.5. C18H23ClN2. Related substances. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating substance. STORAGE Test solution (a). Dissolve 0.20 g of the substance to be examined in alcohol R and dilute to 5 ml with the same Store protected from light. solvent. Test solution (b). Dilute 1 ml of test solution (a) to 20 ml IMPURITIES with alcohol R. Reference solution (a). Dilute 1 ml of test solution (a) to 200 ml with alcohol R. Reference solution (b). Dissolve 10 mg of cyclopentolate hydrochloride CRS in alcohol R and dilute to 5 ml with the same solvent. Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm using a mixture of 5 volumes of concentrated ammonia R, 15 volumes of water R, 30 volumes of butyl acetate R and 50 volumes of 2-propanol R. Allow A. 1-methylpiperazine. General Notices (1) apply to all monographs and other texts

1377

Cyclophosphamide


the plate to dry in air. Spray with analcoholic solution of sulphuric acid R and heat the plate at 120 °C for 30 min. Examine in ultraviolet light at 365 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.5 per cent). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C for 4 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.250 g in a mixture of 1.0 ml of 0.1 M hydrochloric acid and 50 ml of alcohol R. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the two points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 32.79 mg of C17H26ClNO3.

01/2005:0711

CYCLOPHOSPHAMIDE Cyclophosphamidum

C7H15Cl2N2O2P,H2O

Mr 279.1

DEFINITION Cyclophosphamide contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of (2RS)-N,N-bis(2-chloroethyl)tetrahydro-2H-1,3,2oxazaphosphorin-2-amine 2-oxide, calculated with reference to the anhydrous substance. CHARACTERS A white or almost white, crystalline powder, soluble in water, freely soluble in alcohol. IDENTIFICATION First identification : B. Second identification : A, C, D. A. Determine the melting point (2.2.14) of the substance to be examined. Mix equal parts of the substance to be examined and cyclophosphamide CRS and determine the melting point of the mixture. The difference between the melting points (which are about 51 °C) is not greater than 2 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cyclophosphamide CRS. C. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). 1378

D. Dissolve 0.1 g in 10 ml of water R and add 5 ml of silver nitrate solution R1 ; the solution remains clear. Boil, a white precipitate is formed which dissolves in concentrated ammonia R and is reprecipitated on the addition of dilute nitric acid R. TESTS Solution S. Dissolve 0.50 g in carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). pH (2.2.3). The pH of solution S is 4.0 to 6.0, determined immediately after preparation of the solution. Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution (a). Dissolve 0.10 g of the substance to be examined in alcohol R and dilute to 5 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with alcohol R. Reference solution (a). Dissolve 10 mg of cyclophosphamide CRS in alcohol R and dilute to 5 ml with the same solvent. Reference solution (b). Dilute 0.1 ml of test solution (a) to 10 ml with alcohol R. Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm using a mixture of 2 volumes of anhydrous formic acid R, 4 volumes of acetone R, 12 volumes of water R and 80 volumes of methyl ethyl ketone R. Dry the plate in a current of warm air and heat at 110 °C for 10 min. At the bottom of a chromatography tank, place an evaporating dish containing a 50 g/l solution of potassium permanganate R and add an equal volume of hydrochloric acid R. Place the plate whilst still hot in the tank and close the tank. Leave the plate in contact with the chlorine gas for 2 min. Withdraw the plate and place it in a current of cold air until the excess of chlorine is removed and an area of coating below the points of application gives at most a very faint blue colour with a drop of potassium iodide and starch solution R. Avoid prolonged exposure to cold air. Spray with potassium iodide and starch solution R and allow to stand for 5 min. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (1.0 per cent). Disregard any spot remaining at the starting-point. Chlorides (2.4.4). Dissolve 0.15 g in water R and dilute to 15 ml with the same solvent. The freshly prepared solution complies with the limit test for chlorides (330 ppm). Phosphates (2.4.11). Dissolve 0.10 g in water R and dilute to 100 ml with the same solvent. The solution complies with the limit test for phosphates (100 ppm). Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Water (2.5.12) : 6.0 per cent to 7.0 per cent, determined on 0.300 g by the semi-micro determination of water. ASSAY Dissolve 0.100 g in 50 ml of a 1 g/l solution of sodium hydroxide R in ethylene glycol R and boil under a reflux condenser for 30 min. Allow to cool and rinse the condenser with 25 ml of water R. Add 75 ml of 2-propanol R, 15 ml


Cyproheptadine hydrochloride


of dilute nitric acid R, 10.0 ml of 0.1 M silver nitrate and 2.0 ml of ferric ammonium sulfate solution R2 and titrate with 0.1 M ammonium thiocyanate. 1 ml of 0.1 M silver nitrate is equivalent to 13.05 mg of C7H15Cl2N2O2P.

01/2005:0817

CYPROHEPTADINE HYDROCHLORIDE Cyproheptadini hydrochloridum

Apply to the plate 2 µl of each solution. Develop over a path of 15 cm using a mixture of 5 volumes of diethylamine R, 20 volumes of ether R and 75 volumes of cyclohexane R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows 2 clearly separated principal spots. D. A saturated solution gives reaction (b) of chlorides (2.3.1). TESTS Acidity. Dissolve 0.10 g in water R and dilute to 25 ml with the same solvent. Add 0.1 ml of methyl red solution R. Not more than 0.15 ml of 0.01 M sodium hydroxide is required to change the colour of the indicator. Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. Dissolve 50 mg of the substance to be examined in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 5 ml with the same mixture of solvents.

C21H22ClN,11/2 H2O

Mr 350.9 Reference solution (a). Dissolve 10 mg of dibenzocycloheptene CRS (5H-dibenzo[a,d]cycloheptene) DEFINITION in a mixture of 1 volume of methanol R and 9 volumes of Cyproheptadine hydrochloride contains not less than 98.5 per methylene chloride R and dilute to 50 ml with the same mixture of solvents. Dilute 1 ml to 10 ml with a mixture cent and not more than the equivalent of 101.0 per cent of 4-(5H-dibenzo[a,d]cyclohepten-5-ylidene)-1-methylpiperidine of 1 volume of methanol R and 9 volumes of methylene chloride R. hydrochloride, calculated with reference to the anhydrous substance. Reference solution (b). Dilute 1 ml of the test solution to 100 ml with a mixture of 1 volume of methanol R and CHARACTERS 9 volumes of methylene chloride R. Dilute 1 ml to 10 ml A white or slightly yellow, crystalline powder, slightly soluble with a mixture of 1 volume of methanol R and 9 volumes of in water, freely soluble in methanol, sparingly soluble in methylene chloride R. alcohol. Apply to the plate 10 µl of each solution. Develop over a path of 15 cm using a mixture of 10 volumes of methanol R IDENTIFICATION and 90 volumes of methylene chloride R. Allow the First identification : B, D. plate to dry in air and spray with alcoholic solution of sulphuric acid R. Heat at 110 °C for 30 min and examine in Second identification : A, C, D. ultraviolet light at 365 nm while hot. In the chromatogram A. Dissolve 50.0 mg in alcohol R and dilute to 50.0 ml with obtained with the test solution : any spot corresponding to the same solvent. Dilute 2.0 ml of the solution to 100.0 ml dibenzocycloheptene is not more intense than the spot in the with alcohol R. Examined between 230 nm and 320 nm chromatogram obtained with reference solution (a) (0.2 per (2.2.25), the solution shows an absorption maximum cent) ; any spot apart from the principal spot and any spot at 286 nm. The specific absorbance at the maximum is corresponding to dibenzocycloheptene is not more intense 335 to 365, calculated with reference to the anhydrous than the spot in the chromatogram obtained with reference substance. solution (b) (0.1 per cent). B. Examine by infrared absorption spectrophotometry Water (2.5.12) : 7.0 per cent to 9.0 per cent, determined on (2.2.24), comparing with the spectrum obtained with 0.200 g. cyproheptadine hydrochloride CRS. Examine the Sulphated ash (2.4.14). Not more than 0.1 per cent, substances as mulls in liquid paraffin R. determined on 1.0 g. C. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. ASSAY Test solution. Dissolve 25 mg of the substance to be Dissolve 0.250 g in a mixture of 5.0 ml of 0.01 M hydrochloric examined in methanol R and dilute to 25 ml with the acid and 50 ml of alcohol R. Carry out a potentiometric same solvent. titration (2.2.20), using 0.1 M sodium hydroxide. Read Reference solution (a). Dissolve 10 mg of cyproheptadine the volume added between the 2 points of inflexion. hydrochloride CRS in methanol R and dilute to 10 ml 1 ml of 0.1 M sodium hydroxide is equivalent to 32.39 mg with the same solvent. of C21H22ClN. Reference solution (b). Dissolve 10 mg of imipramine hydrochloride CRS in methanol R and dilute to 10 ml with the same solvent. Dilute 1 ml to 2 ml with reference STORAGE solution (a). Store protected from light.


1379

Cyproterone acetate

IMPURITIES

A. R = H2 : dibenzo[a,d]cycloheptene,


C. To about 1 mg add 2 ml of sulphuric acid R and heat on a water-bath for 2 min. A red colour develops. Cool. Add the solution cautiously to 4 ml of water R and shake. The solution becomes violet. D. Incinerate about 30 mg with 0.3 g of anhydrous sodium carbonate R over a naked flame for about 10 min. Cool and dissolve the residue in 5 ml of dilute nitric acid R. Filter. To 1 ml of the filtrate add 1 ml of water R. The solution gives reaction (a) of chlorides (2.3.1). E. It gives the reaction of acetyl (2.3.1).

B. R = O : 5H-dibenzo[a,d]cyclohepten-5-one (dibenzosuberone).

TESTS Specific optical rotation (2.2.7). Dissolve 0.25 g in acetone R and dilute to 25.0 ml with the same solvent. The 01/2005:1094 specific optical rotation is + 152 to + 157, calculated with reference to the dried substance. CYPROTERONE ACETATE Related substances. Examine by liquid chromatography (2.2.29). Cyproteroni acetas Test solution. Dissolve 10.0 mg of the substance to be examined in acetonitrile R and dilute to 10.0 ml with the same solvent. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with acetonitrile R. Reference solution (b). Dissolve 5 mg of medroxyprogesterone acetate CRS in acetonitrile R and dilute to 50.0 ml with the same solvent. Dilute 1.0 ml of the solution to 10.0 ml with reference solution (a). The chromatographic procedure may be carried out using : C24H29ClO4 Mr 416.9 — a stainless steel column 0.125 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for DEFINITION chromatography R (3 µm), Cyproterone acetate contains not less than 97.0 per — as mobile phase at a flow rate of 1.5 ml/min a mixture of cent and not more than the equivalent of 103.0 per cent 40 volumes of acetonitrile R and 60 volumes of water R, of 6-chloro-3,20-dioxo-1β,2β-dihydro-3′H-cyclopropa[1, — as detector a spectrophotometer set at 254 nm. 2]pregna-1,4,6-trien-17-yl acetate, calculated with reference Inject 20 µl of reference solution (a) and 20 µl of reference to the dried substance. solution (b). Adjust the sensitivity of the system so that the CHARACTERS height of the principal peak in the chromatogram obtained A white or almost white, crystalline powder, practically with reference solution (a) is at least 50 per cent of the full insoluble in water, very soluble in methylene chloride, freely scale of the recorder. The test is not valid unless, in the soluble in acetone, soluble in methanol, sparingly soluble chromatogram obtained with reference solution (b), the in ethanol. resolution between the peak corresponding to cyproterone acetate and the peak corresponding to medroxyprogesterone It melts at about 210 °C. acetate is at least 3.0. IDENTIFICATION Inject 20 µl of the test solution. Continue the chromatography First identification : A. for twice the retention time of cyproterone acetate. In the Second identification : B, C, D, E. chromatogram obtained with the test solution, the sum of the areas of all the peaks, apart from the principal peak, is A. Examine by infrared absorption spectrophotometry not greater than 0.5 times the area of the principal peak (2.2.24), comparing with the spectrum obtained with in the chromatogram obtained with reference solution (a) cyproterone acetate CRS. (0.5 per cent). Disregard any peak with an area less than B. Examine by thin-layer chromatography (2.2.27), using a 0.05 times that of the principal peak in the chromatogram TLC silica gel F254 plate R. obtained with reference solution (a). Test solution. Dissolve 20 mg of the substance to be Loss on drying (2.2.32). Not more than 0.5 per cent, examined in methylene chloride R and dilute to 10 ml determined on 1.000 g by drying at 80 °C at a pressure not with the same solvent. exceeding 0.7 kPa. Reference solution. Dissolve 10 mg of cyproterone acetate CRS in methylene chloride R and dilute to 5 ml Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. with the same solvent. Apply to the plate 5 µl of each solution. Develop over ASSAY a path of 15 cm using a mixture of equal volumes of cyclohexane R and ethyl acetate R. Allow the plate to dry Dissolve 50.0 mg in methanol R and dilute to 50.0 ml with in air. Repeat the development. Allow the plate to dry in the same solvent. Dilute 1.0 ml of the solution to 100.0 ml air. Examine in ultraviolet light at 254 nm. The principal with methanol R. Measure the absorbance (2.2.25) at the spot in the chromatogram obtained with the test solution maximum at 282 nm. is similar in position and size to the principal spot in the Calculate the content of C24H29ClO4 taking the specific chromatogram obtained with the reference solution. absorbance to be 414. 1380



Cysteine hydrochloride monohydrate


TESTS Solution S. Dissolve 2.5 g in distilled water R and dilute to 50 ml with the same solvent. IMPURITIES Appearance of solution. Dilute 10 ml of solution S to 20 ml with water R. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY6 (2.2.2, Method II). Specific optical rotation (2.2.7). Dissolve 2.00 g in hydrochloric acid R1 and dilute to 25.0 ml with the same acid. The specific optical rotation is + 5.5 to + 7.0, calculated with reference to the dried substance. Ninhydrin-positive substances. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R. Test solution (a). Dissolve 0.20 g of the substance to be A. R = H : 3,20-dioxo-1β,2β-dihydro-3′H-cyclopropa[1, examined in water R and dilute to 10 ml with the same 2]pregna-1,4,6-trien-17-yl acetate, solvent. To 5 ml of the solution add 5 ml of a 40 g/l solution of N-ethylmaleimide R in alcohol R. Allow to stand for 5 min. B. R = OCH3 : 6-methoxy-3,20-dioxo-1β,2β-dihydro-3′Hcyclopropa[1,2]pregna-1,4,6-trien-17-yl acetate. Test solution (b). Dilute 1 ml of test solution (a) to 50 ml with water R. Reference solution (a). Dissolve 20 mg of cysteine hydrochloride monohydrate CRS in water R and dilute to 01/2005:0895 10 ml with the same solvent. Add 10 ml of a 40 g/l solution of N-ethylmaleimide R in alcohol R. Allow to stand for 5 min. CYSTEINE HYDROCHLORIDE Reference solution (b). Dilute 2 ml of reference solution (a) to 10 ml with water R. MONOHYDRATE Reference solution (c). Dilute 5 ml of test solution (b) to Cysteini hydrochloridum monohydricum 20 ml with water R. Reference solution (d). Dissolve 10 mg of tyrosine CRS in 10 ml of reference solution (a) and dilute to 25 ml with water R. Apply separately to the plate 5 µl of each test solution and reference solutions (b), (c), and (d). Develop over a path C3H8ClNO2S,H2O Mr 175.6 of 15 cm using a mixture of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes of butanol R. DEFINITION Dry the plate at 80 °C for 30 min. Spray with ninhydrin Cysteine hydrochloride monohydrate contains not less than solution R and heat at 100 °C to 105 °C for 15 min. Any 98.5 per cent and not more than the equivalent of 101.0 per spot in the chromatogram obtained with test solution (a), cent of (2R)-2-amino-3-sulfanylpropanoic acid hydrochloride, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference calculated with reference to the dried substance. solution (c) (0.5 per cent). The test is not valid unless the CHARACTERS chromatogram obtained with reference solution (d) shows 2 clearly separated principal spots. A white, crystalline powder or colourless crystals, freely soluble in water, slightly soluble in alcohol. Sulphates (2.4.13). Dilute 10 ml of solution S to 15 ml with distilled water R. The solution complies with the limit test IDENTIFICATION for sulphates (300 ppm). First identification : A, B, E. Ammonium (2.4.1). 50 mg complies with limit test B for Second identification : A, C, D, E. ammonium (200 ppm). Prepare the standard using 0.1 ml of A. It complies with the test for specific optical rotation (see ammonium standard solution (100 ppm NH4) R. Tests). Iron (2.4.9). In a separating funnel, dissolve 0.50 g in 10 ml of dilute hydrochloric acid R. Shake with 3 quantities, each B. Examine by infrared absorption spectrophotometry of 10 ml, of methyl isobutyl ketone R1, shaking for 3 min (2.2.24), comparing with the spectrum obtained with cysteine hydrochloride monohydrate CRS. Examine the each time. To the combined organic layers add 10 ml of water R and shake for 3 min. The aqueous layer complies substances prepared as discs. with the limit test for iron (20 ppm). C. Examine the chromatograms obtained in the test for Heavy metals (2.4.8). Dissolve 2.0 g in water R. Adjust to ninhydrin-positive substances. The principal spot in the pH 3 to 4 with concentrated ammonia R and dilute to 20 ml chromatogram obtained with test solution (b) is similar with water R. 12 ml of the solution complies with limit test A in position, colour, and size to the principal spot in the for heavy metals (10 ppm). Prepare the standard using lead chromatogram obtained with reference solution (b). D. Dissolve about 5 mg in 1 ml of dilute sodium hydroxide standard solution (1 ppm Pb) R. Loss on drying (2.2.32) : 8.0 per cent to 12.0 per cent, solution R. Add 1 ml of a 30 g/l solution of sodium nitroprusside R. An intense violet colour develops which determined on 1.000 g by drying at a pressure not exceeding 0.7 kPa for 24 h. becomes brownish-red and then orange. Add 1 ml of hydrochloric acid R. The solution becomes green. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. E. It gives reaction (a) of chlorides (2.3.1). General Notices (1) apply to all monographs and other texts

1381

Cystine


Ninhydrin-positive substances. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R. Test solution (a). Dissolve 0.10 g of the substance to be examined in 1 M hydrochloric acid and dilute to 10 ml with the same acid. Test solution (b). Dilute 1 ml of test solution (a) to 50 ml with water R. Reference solution (a). Dissolve 10 mg of cystine CRS in 1 ml of 1 M hydrochloric acid and dilute to 50 ml with water R. Reference solution (b). Dilute 2 ml of test solution (b) to STORAGE 20 ml with water R. Store protected from light. Reference solution (c). Dissolve 10 mg of cystine CRS and 10 mg of arginine hydrochloride CRS in 1 ml of 1 M 01/2005:0998 hydrochloric acid and dilute to 25 ml with water R. Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 30 volumes of CYSTINE concentrated ammonia R and 70 volumes of 2-propanol R. Allow the plate to dry in air. Spray with ninhydrin solution R Cystinum and heat at 100 °C to 105 °C for 15 min. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.2 per cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows two clearly separated spots. C6H12N2O4S2 Mr 240.3 Chlorides (2.4.4). Dissolve 0.25 g in 5 ml of dilute nitric acid R and dilute to 15 ml with water R. The solution, DEFINITION without further addition of nitric acid, complies with the Cystine contains not less than 98.5 per cent and limit test for chlorides (200 ppm). not more than the equivalent of 101.0 per cent of 3,3′-disulfanediylbis[(2R)-2-aminopropanoic acid], calculated Sulphates (2.4.13). Dissolve 0.5 g in 5 ml of dilute hydrochloric acid R and dilute to 15 ml with distilled with reference to the dried substance. water R. The solution complies with the limit test for CHARACTERS sulphates (300 ppm). A white, crystalline powder, practically insoluble in water Ammonium (2.4.1). 0.10 g complies with limit test B for and in alcohol. It dissolves in dilute solutions of alkali ammonium (200 ppm). Prepare the standard using 0.2 ml of hydroxides. ammonium standard solution (100 ppm NH4) R. Iron (2.4.9). In a separating funnel, dissolve 1.0 g in 10 ml IDENTIFICATION of dilute hydrochloric acid R. Shake with three quantities, First identification : A, B. each of 10 ml, of methyl isobutyl ketone R1, shaking for Second identification : A, C, D. 3 min each time. To the combined organic layers add 10 ml A. It complies with the test for specific optical rotation (see of water R and shake for 3 min. The aqueous layer complies Tests). with the limit test for iron (10 ppm). B. Examine by infrared absorption spectrophotometry Heavy metals (2.4.8). 2.0 g complies with limit test D for (2.2.24), comparing with the spectrum obtained with heavy metals (10 ppm). Prepare the standard using 2 ml of cystine CRS. Examine the substances prepared as discs. lead standard solution (10 ppm Pb) R. C. Examine the chromatograms obtained in the test for Loss on drying (2.2.32). Not more than 0.5 per cent, ninhydrin-positive substances. The principal spot in the determined on 1.000 g by drying in an oven at 100 °C to chromatogram obtained with test solution (b) is similar 105 °C. in position, colour and size to the principal spot in the Sulphated ash (2.4.14). Not more than 0.1 per cent, chromatogram obtained with reference solution (a). determined on 1.0 g. D. To 0.1 g carefully add 1 ml of strong hydrogen peroxide solution R and 0.1 ml of ferric chloride solution R1. Allow to cool. Add 1 ml of dilute hydrochloric acid R and ASSAY 5 ml of water R. Add 1 ml of barium chloride solution R1. In a flask with a ground-glass stopper, dissolve 0.100 g in Turbidity or a white precipitate develops within 3 min. a mixture of 2 ml of dilute sodium hydroxide solution R and 10 ml of water R. Add 10 ml of a 200 g/l solution of TESTS potassium bromide R, 50.0 ml of 0.0167 M potassium bromate and 15 ml of dilute hydrochloric acid R. Stopper Appearance of solution. Dissolve 1.0 g in dilute hydrochloric acid R and dilute to 10 ml with the same acid. the flask and cool in iced water. Allow to stand in the dark The solution is clear (2.2.1) and not more intensely coloured for 10 min. Add 1.5 g of potassium iodide R. After 1 min, titrate with 0.1 M sodium thiosulphate, using 2 ml of starch than reference solution Y7 (2.2.2, Method II). solution R, added towards the end-point, as indicator. Carry Specific optical rotation (2.2.7). Dissolve 0.50 g in 1 M hydrochloric acid and dilute to 25.0 ml with the same acid. out a blank titration. 1 ml of 0.0167 M potassium bromate is equivalent to The specific optical rotation is − 218 to − 224, calculated 2.403 mg of C6H12N2O4S2. with reference to the dried substance. ASSAY In a ground-glass stoppered flask dissolve 0.300 g of the substance to be examined and 4 g of potassium iodide R in 20 ml of water R. Cool the solution in iced water and add 3 ml of hydrochloric acid R1 and 25.0 ml of 0.05 M iodine. Stopper the flask and allow to stand in the dark for 20 min. Titrate with 0.1 M sodium thiosulphate using 3 ml of starch solution R, added towards the end of the titration, as indicator. Carry out a blank titration. 1 ml of 0.05 M iodine is equivalent to 15.76 mg of C3H8ClNO2S.

1382



Cytarabine

Specific optical rotation (2.2.7). Dissolve 0.250 g in water R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 154 to + 160, calculated with reference to the dried substance. Related substances. Examine by thin-layer chromatography 01/2005:0760 (2.2.27), using silica gel GF254 R as the coating substance. Test solution (a). Dissolve 0.25 g of the substance to be examined in water R and dilute to 5 ml with the same solvent. CYTARABINE Test solution (b). Dilute 2 ml of test solution (a) to 50 ml with water R. Cytarabinum Reference solution (a). Dissolve 10 mg of cytarabine CRS in water R and dilute to 5 ml with the same solvent. Reference solution (b). Dilute 0.5 ml of test solution (a) to 100 ml with water R. Reference solution (c). Dissolve 20 mg of uridine R and 20 mg of uracil arabinoside CRS in methanol R and dilute to 10 ml with the same solvent. Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 15 volumes of water R, 20 volumes of acetone R and 65 volumes of methyl ethyl ketone R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram C9H13N3O5 Mr 243.2 obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram DEFINITION obtained with reference solution (b) (0.5 per cent). The test is not valid unless the chromatogram obtained with reference Cytarabine contains not less than 99.0 per cent and solution (c) shows two clearly separated spots. not more than the equivalent of 100.5 per cent of 4-amino-1-β-D-arabinofuranosylpyrimidin-2(1H)-one, Loss on drying (2.2.32). Not more than 1.0 per cent, calculated with reference to the dried substance. determined on 0.250 g by drying over diphosphorus pentoxide R at 60 °C at a pressure of 0.2 kPa to 0.7 kPa CHARACTERS for 3 h. A white or almost white, crystalline powder, freely soluble Sulphated ash (2.4.14). Not more than 0.5 per cent, in water, very slightly soluble in alcohol and in methylene determined on 1.0 g. chloride. ASSAY It melts at about 215 °C. Dissolve 0.200 g in 60 ml of anhydrous acetic acid R, IDENTIFICATION warming if necessary. Titrate with 0.1 M perchloric acid A. Dissolve 20.0 mg in 0.1 M hydrochloric acid and dilute to determining the end-point potentiometrically (2.2.20). 100.0 ml with the same acid. Dilute 5.0 ml of the solution 1 ml of 0.1 M perchloric acid is equivalent to 24.32 mg of to 100.0 ml with 0.1 M hydrochloric acid. Examined C9H13N3O5. between 230 nm and 350 nm (2.2.25), the solution STORAGE shows an absorption maximum at 281 nm. The specific Store in an airtight container, protected from light. absorbance at the maximum is 540 to 570. B. Examine by infrared absorption spectrophotometry IMPURITIES (2.2.24), comparing with the spectrum obtained with cytarabine CRS. Examine the substances prepared as discs. C. Examine the chromatograms obtained in the test for related substances in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with test solution (b) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). STORAGE Store protected from light.

TESTS Appearance of solution. Dissolve 1.0 g in water R and dilute A. R = OH, R′ = H : 1-β-D-arabinofuranosylpyrimidine-2,4(1H, 3H)-dione (uracil arabonoside), to 10 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y5 B. R = H, R′ = OH : 1-β-D-ribofuranosylpyrimidine-2,4(1H, 3H)-dione (uridine). (2.2.2, Method II).


1383


Dalteparin sodium

01/2005:1195 Nitrite. Not more than 5 ppm. Examine by liquid chromatography (2.2.29). Rinse all volumetric flasks at least three times with water R before the preparation of the DALTEPARIN SODIUM solutions. Test solution. Dissolve 80.0 mg of the substance to be Dalteparinum natricum examined in water R and dilute to 10.0 ml with the same solvent. Allow to stand for at least 30 min. Reference solution (a). Dissolve 60.0 mg of sodium nitrite R in water R and dilute to 1000.0 ml with the same solvent. For the preparation of reference solution (b), use a pipette previously rinsed with reference solution (a). Reference solution (b). Dilute 1.00 ml of reference solution (a) to 50.0 ml with water R. Before preparing reference solutions (c), (d) and (e), rinse all pipettes with reference solution (b). Reference solution (c). Dilute 1.00 ml of reference solution (b) to 100.0 ml with water R (1 ppm nitrite). Reference solution (d). Dilute 3.00 ml of reference solution (b) to 100.0 ml with water R (3 ppm nitrite). Reference solution (e). Dilute 5.00 ml of reference DEFINITION solution (b) to 100.0 ml with water R (5 ppm nitrite). Dalteparin sodium is the sodium salt of a low-molecular-mass The chromatographic procedure may be carried out using : heparin that is obtained by nitrous acid depolymerisation of — a column 0.125 m long and 4.3 mm in internal diameter heparin from porcine intestinal mucosa. The majority of the packed with a strong anion-exchange resin, components have a 2-O-sulpho-α-L-idopyranosuronic — as mobile phase at a flow rate of 1.0 ml/min a solution acid structure at the non-reducing end and a consisting of 13.61 g of sodium acetate R dissolved in 6-O-sulpho-2,5-anhydro-D-mannitol structure at the water R, adjusted to pH 4.3 with phosphoric acid R and reducing end of their chain. diluted to 1000 ml with water R, Dalteparin sodium complies with the monograph — as detector an appropriate electrochemical device with on Low-molecular-mass heparins (0828) with the the following characteristics and settings : a suitable modifications and additional requirements below : working electrode, a detector potential of + 1.00 V versus The mass-average molecular mass ranges between 5600 and Ag/AgCl reference electrode and a detector sensitivity of 6400, with a characteristic value of about 6000. 0.1 µA full scale. The degree of sulphatation is 2.0 to 2.5 per disaccharide unit. Inject 100 µl of reference solution (d). When the The potency is not less than 110 IU and not more than chromatograms are recorded in the prescribed conditions, 210 IU of anti-factor Xa activity per milligram, calculated the retention time for nitrite is 3.3 to 4.0 min. The test is with reference to the dried substance. The anti-factor IIa activity is not less than 35 IU and not more than 100 IU/mg, not valid unless : — the number of theoretical plates calculated for the nitrite calculated with reference to the dried substance. The ratio peak is at least 7000 per metre per column (dalteparin of anti-factor Xa activity to anti-factor IIa activity is between sodium will block the binding sites of the stationary 1.9 and 3.2. phase, which will cause shorter retention times and PRODUCTION lower separation efficiency for the analyte ; the initial Dalteparin sodium is produced by a validated manufacturing performance of the column may be partially restored and purification procedure under conditions designed to using a 58 g/l solution of sodium chloride R at a flow minimise the presence of N-NO groups. rate of 1.0 ml/min for 1 h ; after regeneration the column is rinsed with 200 ml to 400 ml of water R), The manufacturing procedure must have been shown to reduce any contamination by N-NO groups to approved limits — the symmetry factor for the nitrite peak is less than three, using an appropriate, validated quantification method. — the relative standard deviation of the peak area for nitrite obtained from six injections is less than 3.0 per cent. IDENTIFICATION Carry out identification test C as described in the monograph Inject 100 µl each of reference solutions (c) and (e). The test is not valid unless : on Low-molecular-mass heparins (0828). The following requirements apply : — the correlation factor for a linear relationship between The mass-average molecular mass ranges between 5600 and concentration and response for reference solutions (c), 6400. The mass percentage of chains lower than 3000 is not (d) and (e) is at least 0.995, more than 13.0 per cent. The mass percentage of chains — the signal-to-noise ratio for reference solution (c) is higher than 8000 ranges between 15.0 per cent and 25.0 per not less than 5 (if the noise level is too high, electrode cent. recalibration is recommended), TESTS — a blank injection of water R does not give rise to spurious peaks. Appearance of solution. Dissolve 1 g in 10 ml of water R. The solution is clear (2.2.1) and not more intensely coloured Inject 100 µl of the test solution. Calculate the content of than intensity 5 of the range of reference solutions of the nitrite from the peak areas in the chromatogram obtained most appropriate colour (2.2.2, Method II). with reference solutions (c), (d) and (e). General Notices (1) apply to all monographs and other texts

1387

Dapsone


Boron. Not more than 1 ppm, determined by inductively coupled plasma atomic emission spectroscopy.

IDENTIFICATION

A. Melting point (2.2.14) : 175 °C to 181 °C. Boron is determined by measurement of the emission from an inductively coupled plasma (ICP) at a wavelength specific B. Dissolve 50.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this solution to to boron. The emission line at 249.733 nm is used. Use an 100.0 ml with methanol R. Examined between 230 nm appropriate apparatus, whose settings have been optimised and 350 nm (2.2.25), the solution shows 2 absorption as directed by the manufacturer. maxima, at 260 nm and 295 nm. The specific absorbances Test solution. Dissolve 0.2500 g of the substance to be at these maxima are 700 to 760 and 1150 to 1250, examined in about 2 ml of water for chromatography R, respectively. add 100 µl of nitric acid R and dilute to 10.00 ml with the C. Examine the chromatograms obtained in the test same solvent. for related substances. The principal spot in the Reference solution (a). Prepare a 1 per cent V/V solution of chromatogram obtained with test solution (b) is similar nitric acid R in water for chromatography R (blank). in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). Reference solution (b). Prepare a 11.4 µg/ml solution of boric acid R in a 1 per cent V/V solution of nitric acid R in water for chromatography R (STDcal). TESTS

Reference solution (c). Dissolve 0.2500 g of a reference Related substances. Examine by thin-layer chromatography dalteparin sodium with no detectable boron in about 2 ml of (2.2.27), using silica gel G R as the coating substance. water for chromatography R, add 100 µl of nitric acid R and Test solution (a). Dissolve 0.10 g of the substance to be dilute to 10.00 ml with the same solvent (STD0). examined in methanol R and dilute to 10 ml with the same Reference solution (d). Dissolve 0.2500 g of a reference solvent. dalteparin sodium with no boron detected in about 2 ml of a 1 per cent V/V solution of nitric acid R in water for chromatography R, add 10 µl of a 5.7 mg/ml solution of boric acid R and dilute to 10.00 ml with the same solvent (STD1). This solution contains 1 µg/ml of boron. Calculate the content of boron in the substance to be examined, using the following correction factor :

Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with methanol R. Reference solution (a). Dissolve 10 mg of dapsone CRS in methanol R and dilute to 10 ml with the same solvent. Reference solution (b). Dilute 1 ml of test solution (b) to 10 ml with methanol R. Reference solution (c). Dilute 2 ml of reference solution (b) to 10 ml with methanol R.

Loss on drying (2.2.32). Not more than 5.0 per cent, determined on 1.000 g by drying in an oven at 60 °C over diphosphorus pentoxide R at a pressure not exceeding 670 Pa for 3 h.

DAPSONE Dapsonum

Apply separately to the plate 1 µl of test solution (b), 1 µl of reference solution (a), 10 µl of test solution (a), 10 µl of reference solution (b) and 10 µl of reference solution (c). Develop in an unsaturated tank over a path of 15 cm using a mixture of 1 volume of concentrated ammonia R, 6 volumes of methanol R, 20 volumes of ethyl acetate R and 20 volumes of heptane R. Allow the plate to dry in air. Spray the plate with a 1 g/l solution of 4-dimethylaminocinnamaldehyde R 01/2005:0077 in a mixture of 1 volume of hydrochloric acid R and 99 volumes of alcohol R. Examine in daylight. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (1.0 per cent) and not more than 2 such spots are more intense than the spot in the chromatogram obtained with reference solution (c) (0.2 per cent). Loss on drying (2.2.32). Not more than 1.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C.

C12H12N2O2S

Mr 248.3


DEFINITION

ASSAY

Dapsone contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of 4,4′-sulphonyldianiline, calculated with reference to the dried substance.

Dissolve 0.100 g in 50 ml of dilute hydrochloric acid R. Carry out the determination of primary aromatic amino-nitrogen (2.5.8). 1 ml of 0.1 M sodium nitrite is equivalent to 12.42 mg of C12H12N2O2S.

CHARACTERS A white or slightly yellowish-white, crystalline powder, very slightly soluble in water, freely soluble in acetone, sparingly soluble in alcohol. It dissolves freely in dilute mineral acids. 1388




Daunorubicin hydrochloride

01/2005:0662 Reference solution (c). Dissolve 5.0 mg of daunorubicinone CRS and 5.0 mg of doxorubicin CRS in the mobile phase and dilute to DAUNORUBICIN HYDROCHLORIDE hydrochloride 100.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 10.0 ml with the mobile phase. Daunorubicini hydrochloridum Reference solution (d). Dilute 1.0 ml of reference solution (a) to 200.0 ml with the mobile phase. Column : — size : l = 0.25 m, Ø = 4.0 mm, — stationary phase : end-capped octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : mixture of equal volumes of acetonitrile R and a solution containing 2.88 g/l of sodium laurilsulfate R and 2.25 g/l of phosphoric acid R. Flow rate : 1 ml/min. Detection : spectrophotometer at 254 nm. Injection : 5 µl ; inject the test solution and reference C27H30ClNO10 Mr 564.0 solutions (b), (c) and (d). Run time : twice the retention time of daunorubicin. DEFINITION Relative retention with reference to daunorubicin (8S,10S)-8-Acetyl-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo(retention time = about 15 min) : impurity A = about hexopyranosyl)oxy]-6,8,11-trihydroxy-1-methoxy-7,8,9,100.4 ; impurity D = about 0.5 ; epirubicin = about 0.6 ; tetrahydrotetracene-5,12-dione hydrochloride. impurity B = about 0.7. Substance produced by certain strains of Streptomyces System suitability : reference solution (b) : coeruleorubidus or of Streptomyces peucetius or obtained — resolution : minimum of 2.0 between the peaks due to by any other means. impurity D and epirubicin. Content : 95.0 per cent to 102.0 per cent (anhydrous and Limits : solvent-free substance). — impurity A : not more than the area of the corresponding peak in the chromatogram obtained with reference PRODUCTION solution (c) (0.5 per cent), It is produced by methods of manufacture designed to — impurity B : not more than 3 times the area of the eliminate or minimise the presence of histamine. principal peak in the chromatogram obtained with reference solution (d) (1.5 per cent), CHARACTERS — impurity D : not more than the area of the corresponding Appearance : crystalline, orange-red powder, hygroscopic. peak in the chromatogram obtained with reference Solubility : freely soluble in water and in methanol, slightly solution (c) (0.5 per cent), soluble in alcohol, practically insoluble in acetone. — any other impurity : not more than the area of the principal peak in the chromatogram obtained with IDENTIFICATION reference solution (d) (0.5 per cent), A. Infrared absorption spectrophotometry (2.2.24). — total of other impurities : not more than 5 times the area Comparison : daunorubicin hydrochloride CRS. of the principal peak in the chromatogram obtained with reference solution (d) (2.5 per cent), B. Dissolve about 10 mg in 0.5 ml of nitric acid R, add 0.5 ml of water R and heat over a flame for 2 min. Allow — disregard limit : 0.1 times the area of the principal peak to cool and add 0.5 ml of silver nitrate solution R1. A in the chromatogram obtained with reference solution (d) white precipitate is formed. (0.05 per cent). Butanol (2.4.24, System B) : maximum 1.0 per cent. TESTS Water (2.5.12) : maximum 3.0 per cent, determined on pH (2.2.3) : 4.5 to 6.5. 0.100 g. Dissolve 50 mg in carbon dioxide-free water R and dilute to Bacterial endotoxins (2.6.14) : less than 4.3 IU/mg, if 10 ml with the same solvent. intended for use in the manufacture of parenteral dosage Related substances. Liquid chromatography (2.2.29). forms without a further appropriate procedure for the Prepare the solutions immediately before use. removal of bacterial endotoxins. Test solution. Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the ASSAY mobile phase. Liquid chromatography (2.2.29) as described in the test for related substances. Reference solution (a). Dissolve 50.0 mg of daunorubicin hydrochloride CRS in the mobile phase and dilute to 50.0 ml Injection : test solution and reference solution (a). with the mobile phase. Calculate the percentage content of C27H30ClNO10. Reference solution (b). Dissolve 10 mg of doxorubicin STORAGE hydrochloride CRS and 10 mg of epirubicin hydrochloride CRS in the mobile phase and dilute to In an airtight container, protected from light. If the 100.0 ml with the mobile phase. Dilute 1.0 ml of the solution substance is sterile, store in a sterile, airtight, tamper-proof to 10.0 ml with the mobile phase. container. General Notices (1) apply to all monographs and other texts

1389

Decyl oleate


LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins. IMPURITIES

A. R = CO-CH3 : (8S,10S)-8-acetyl-6,8,10,11-tetrahydroxy1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione (daunorubicin aglycone, daunorubicinone), E. R = CHOH-CH3 : (8S,10S)-6,8,10,11-tetrahydroxy-8-[(1RS)1-hydroxyethyl]-1-methoxy-7,8,9,10-tetrahydrotetracene-5, 12-dione (13-dihydrodaunorubicinone),

TESTS Relative density (2.2.5) : 0.860 to 0.870. Acid value (2.5.1). Not more than 1.0, determined on 10.0 g. Iodine value (2.5.4) : 55 to 70. Peroxide value (2.5.5). Not more than 10.0. Saponification value (2.5.6) : 130 to 140, determined on 2.0 g. Content of oleic acid (2.4.22, Method A). The fatty acid fraction of the substance contains at least 60.0 per cent of oleic acid. Water (2.5.12). Not more than 1.0 per cent, determined on 1.00 g by the semi-micro determination of water. Total ash (2.4.16). Not more than 0.1 per cent, determined on 2.0 g. STORAGE Store protected from light. LABELLING The label states, where applicable, the name and concentration of any added antioxidant. 01/2005:0896

DEFEROXAMINE MESILATE Deferoxamini mesilas B. R = CHOH-CH3 : (8S,10S)-10-[(3-amino-2,3,6-trideoxy-αL-lyxo-hexopyranosyl)oxy]-6,8,11-trihydroxy-8-[(1RS)-1hydroxyethyl]-1-methoxy-7,8,9,10-tetrahydrotetracene-5, 12-dione (daunorubicinol), C. R = CH2-CO-CH3 : (8S,10S)-10-[(3-amino-2,3,6-trideoxy-αL-lyxo-hexopyranosyl)oxy]-6,8,11-trihydroxy-1-methoxy8-(2-oxopropyl)-7,8,9,10-tetrahydrotetracene-5,12-dione (feudomycin B), D. R = CO-CH2-OH : doxorubicin,

C26H52N6O11S

Mr 657

F. R = CO-CH2-CH3 : (8S,10S)-10-[(3-amino-2,3,6-trideoxy-αL-lyxo-hexopyranosyl)oxy]-6,8,11-trihydroxy-1-methoxy8-propanoyl-7,8,9,10-tetrahydrotetracene-5,12-dione (8-ethyldaunorubicin).

DEFINITION Deferoxamine mesilate contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent expressed as N′-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]4-oxobutanoyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-Nhydroxybutanediamide methanesulfonate, calculated with 01/2005:1307 reference to the anhydrous substance.

DECYL OLEATE

PRODUCTION The production method must be evaluated to determine Decylis oleas the potential for formation of alkyl mesilates, which is particularly likely to occur if the reaction medium contains DEFINITION lower alcohols. Where necessary, the production method Decyl oleate is a mixture consisting of decyl esters of fatty is validated to demonstrate that alkyl mesilates are not acids, mainly oleic acid. A suitable antioxidant may be added. detectable in the final product. CHARACTERS CHARACTERS A clear, pale yellow or colourless liquid, practically insoluble A white or almost white powder, freely soluble in water, in water, miscible with alcohol, with methylene chloride and slightly soluble in methanol, very slightly soluble in alcohol. with light petroleum (40 °C to 60 °C). IDENTIFICATION IDENTIFICATION First identification : A, D. A. It complies with the test for relative density (see Tests). Second identification : B, C, D. B. It complies with the test for saponification value (see A. Examine by infrared absorption spectrophotometry Tests). (2.2.24), comparing with the spectrum obtained with deferoxamine mesilate CRS. Examine the substances C. It complies with the test for content of oleic acid (see prepared as discs. If the spectra obtained show Tests). 1390


Deferoxamine mesilate


differences, dissolve the substance to be examined and the obtained with the test solution : the area of any peak, reference substance separately in alcohol R, evaporate to apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with dryness and record new spectra using the residues. reference solution (b) (4.0 per cent) ; the sum of the area B. Dissolve about 5 mg in 5 ml of water R. Add of any such peaks is not greater than 1.75 times the area 2 ml of a 5 g/l solution of trisodium phosphate of the principal peak in the chromatogram obtained with dodecahydrate R and 0.5 ml of a 25 g/l solution of reference solution (b) (7.0 per cent). Disregard any peak with sodium naphthoquinonesulphonate R. A brownish-black an area less than 0.02 times that of the principal peak in the colour develops. chromatogram obtained with reference solution (b). C. The titrated solution obtained in the Assay (solution (a)) Chlorides (2.4.4). Dilute 2 ml of solution S to 20 ml with is brownish-red. To 10 ml of solution (a) add 3 ml of water R. 15 ml of the solution complies with the limit test for ether R. Shake. The organic layer is colourless. To 10 ml chlorides (330 ppm). of solution (a) add 3 ml of benzyl alcohol R. Shake. The Sulphates (2.4.13). Dilute 5 ml of solution S to 20 ml with organic layer is brownish-red. distilled water R. 15 ml of the solution complies with the D. Dissolve 0.1 g in 5 ml of dilute hydrochloric acid R. limit test for sulphates (400 ppm). Add 1 ml of barium chloride solution R2. The solution Heavy metals (2.4.8). 2.0 g complies with limit test C for is clear. In a porcelain crucible, mix 0.1 g with 1 g of heavy metals (10 ppm). Prepare the standard using 2 ml of anhydrous sodium carbonate R, heat and ignite over a naked flame. Allow to cool. Dissolve the residue in 10 ml lead standard solution (10 ppm Pb) R. of water R, heating if necessary, and filter. The filtrate Water (2.5.12). Not more than 2.0 per cent, determined on gives reaction (a) of sulphates (2.3.1). 1.000 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.1 per cent, TESTS determined on 1.0 g. Bacterial endotoxins (2.6.14) : less than 0.025 IU/mg, if Solution S. Dissolve 2.5 g in carbon dioxide-free water R prepared from distilled water R and dilute to 25 ml with the intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not removal of bacterial endotoxins.

more intensely coloured than reference solution Y5 (2.2.2, Method II). pH (2.2.3). The pH of freshly prepared solution S is 3.7 to 5.5. Related substances. Examine by liquid chromatography (2.2.29). Prepare the solutions immediately before use, protected from light.


ASSAY Dissolve 0.500 g in 25 ml of water R. Add 4 ml of 0.05 M sulphuric acid. Titrate with 0.1 M ferric ammonium sulphate. Towards the end of the titration, titrate uniformly and at a rate of about 0.2 ml/min. Determine the end-point potentiometrically (2.2.20) using a platinum indicator electrode and a calomel reference electrode. Retain the titrated solution for identification test C. 1 ml of 0.1 M ferric ammonium sulphate is equivalent to 65.68 mg of C26H52N6O11S.

Reference solution (a). Dissolve 10.0 mg of deferoxamine mesilate CRS in the mobile phase and dilute to 10.0 ml with STORAGE the mobile phase. Store protected from light, at a temperature of 2 °C to Reference solution (b). Dilute 1.0 ml of the test solution to 8 °C. If the substance is sterile, store in a sterile, airtight, tamper-proof container. 25.0 ml with the mobile phase. The chromatographic procedure may be carried out using :

LABELLING

— a stainless steel column 0.25 m long and 4.6 mm in The label states, where applicable, that the substance is free internal diameter packed with octadecylsilyl silica gel for from bacterial endotoxins. chromatography R (10 µm), IMPURITIES — as mobile phase at a flow rate of 2 ml/min a solution prepared as follows : dissolve 1.32 g of ammonium phosphate R and 0.37 g of sodium edetate R in 950 ml of water R ; adjust to pH 2.8 with phosphoric acid R (about 3-4 ml) and add 55 ml of tetrahydrofuran R, — as detector a spectrophotometer set at 220 nm. Inject 20 µl of reference solution (a). If a recorder is used, adjust the sensitivity of the detector so that the height of the peak with a relative retention time of about 0.8 is not less than 15 per cent of the full scale of the recorder. The test is not valid unless the resolution between the peak with the A. N′-[5-[[4-[[4-(acetylhydroxyamino)butyl]amino]-4relative retention time of about 0.8 and the principal peak oxobutanoyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-Nis at least 1.0. hydroxybutanediamide(desferrioxamine A1), Inject 20 µl of the test solution and 20 µl of reference solution (b). Continue the chromatography for 3 times the retention time of deferoxamine. In the chromatogram B. other desferrioxamines. General Notices (1) apply to all monographs and other texts

1391

Demeclocycline hydrochloride


01/2005:0176 TESTS pH (2.2.3) : 2.0 to 3.0. DEMECLOCYCLINE Dissolve 0.1 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. HYDROCHLORIDE Specific optical rotation (2.2.7) : − 248 to − 263 (anhydrous substance). Demeclocyclini hydrochloridum Dissolve 0.250 g in 0.1 M hydrochloric acid and dilute to 25.0 ml with the same acid. Specific absorbance (2.2.25) : 340 to 370 determined at the maximum at 385 nm (anhydrous substance). Dissolve 10.0 mg in 0.01 M hydrochloric acid and dilute to 100.0 ml with the same acid. To 10.0 ml of the solution add 12 ml of dilute sodium hydroxide solution R and dilute to 100.0 ml with water R. C21H22Cl2N2O8 Mr 501.3 Related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately before use. DEFINITION Test solution. Dissolve 25.0 mg of the substance to be examined in 0.01 M hydrochloric acid and dilute to 25.0 ml (4S,4aS,5aS,6S,12aS)-7-Chloro-4-(dimethylamino)-3,6, with the same acid. 10,12,12a-pentahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12aoctahydrotetracene-2-carboxamide hydrochloride. Reference solution (a). Dissolve 25.0 mg of demeclocycline hydrochloride CRS in 0.01 M hydrochloric acid and dilute Substance produced by certain strains of Streptomyces to 25.0 ml with the same acid. aureofaciens or obtained by any other means. Reference solution (b). Dissolve 5.0 mg of Content : 89.5 per cent to 102.0 per cent (anhydrous 4-epidemeclocycline hydrochloride CRS in 0.01 M substance). hydrochloric acid and dilute to 25.0 ml with the same acid. CHARACTERS Reference solution (c). Mix 1.0 ml of reference solution (a) Appearance : yellow powder. and 5.0 ml of reference solution (b) and dilute to 25.0 ml with 0.01 M hydrochloric acid. Solubility : soluble or sparingly soluble in water, slightly soluble in alcohol, very slightly soluble in acetone. It Reference solution (d). Dilute 5.0 ml of reference solution (a) dissolves in solutions of alkali hydroxides and carbonates. to 100.0 ml with 0.01 M hydrochloric acid. Column : IDENTIFICATION — size : l = 0.25 m, Ø = 4.6 mm, A. Thin-layer chromatography (2.2.27). — stationary phase : styrene-divinylbenzene copolymer R Test solution. Dissolve 5 mg of the substance to be (8 µm), examined in methanol R and dilute to 10 ml with the — temperature : 60 °C, same solvent. Mobile phase : weigh 80.0 g of 2-methyl-2-propanol R Reference solution (a). Dissolve 5 mg of demeclocycline and transfer to a 1000 ml volumetric flask with the aid hydrochloride CRS in methanol R and dilute to 10 ml of 200 ml of water R ; add 100 ml of a 35 g/l solution of with the same solvent. Reference solution (b). Dissolve 5 mg of demeclocycline dipotassium hydrogen phosphate R adjusted to pH 9.0 with dilute phosphoric acid R, 150 ml of a 10 g/l solution hydrochloride CRS, 5 mg of chlortetracycline of tetrabutylammonium hydrogen sulphate R adjusted to hydrochloride R and 5 mg of tetracycline hydrochloride R in methanol R and dilute to 10 ml with pH 9.0 with dilute sodium hydroxide solution R and 10 ml of a 40 g/l solution of sodium edetate R adjusted to pH 9.0 the same solvent. with dilute sodium hydroxide solution R ; dilute to 1000 ml Plate : TLC octadecylsilyl silica gel F254 plate R. with water R. Mobile phase : mix 20 volumes of acetonitrile R, Flow rate : 1 ml/min. 20 volumes of methanol R and 60 volumes of a 63 g/l solution of oxalic acid R previously adjusted to pH 2 with Detection : spectrophotometer at 254 nm. Injection : 20 µl ; inject the test solution and reference concentrated ammonia R. solutions (c) and (d). Application : 1 µl. System suitability : reference solution (c) : Development : over 3/4 of the plate. — resolution : minimum of 2.8 between the peaks due to Drying : in air. impurity B (1st peak) and demeclocycline (2nd peak) ; if Detection : examine in ultraviolet light at 254 nm. necessary, adjust the 2-methyl-2-propanol content of the System suitability : the chromatogram obtained with mobile phase or lower the pH of the mobile phase, reference solution (b) shows 3 clearly separated spots. — symmetry factor : maximum 1.25 for the peak due to Results : the principal spot in the chromatogram obtained demeclocycline. with the test solution is similar in position and size to Limits : the principal spot in the chromatogram obtained with — any impurity : not more than the area of the principal reference solution (a). peak in the chromatogram obtained with reference B. To about 2 mg add 5 ml of sulphuric acid R. A violet solution (d) (5.0 per cent), and not more than 1 such colour develops. Add the solution to 2.5 ml of water R. peak has an area greater than 0.8 times the area of The colour becomes yellow. the principal peak in the chromatogram obtained with C. It gives reaction (a) of chlorides (2.3.1). reference solution (d) (4.0 per cent), 1392



— total: not more than twice the area of the principal peak in the chromatogram obtained with reference solution (d) (10.0 per cent), — disregard limit : 0.02 times the area of the principal peak in the chromatogram obtained with reference solution (d) (0.1 per cent). Heavy metals (2.4.8) : maximum 50 ppm. 0.5 g complies with limit test C. Prepare the standard using 2.5 ml of lead standard solution (10 ppm Pb) R. Water (2.5.12) : maximum 3.0 per cent, determined on 1.000 g. Sulphated ash (2.4.14) : maximum 0.5 per cent, determined on 1.0 g. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modification. Injection : test solution and reference solution (a). Calculate the percentage content of C21H22Cl2N2O8. STORAGE Protected from light. IMPURITIES

Deptropine citrate

DEFINITION Deptropine citrate contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of (1R,3r,5S)-3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5yloxy)-8-methyl-8-azabicyclo[3.2.1]octane dihydrogen citrate, calculated with reference to the dried substance. CHARACTERS A white or almost white, microcrystalline powder, very slightly soluble in water and in ethanol, practically insoluble in methylene chloride. It melts at about 170 °C, with decomposition. IDENTIFICATION First identification : A. Second identification : B, C, D, E. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with deptropine citrate CRS. B. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (b). C. To about 1 mg add 0.5 ml of sulphuric acid R. A stable red-orange colour develops. D. Dissolve about 1 mg in 0.25 ml of perchloric acid R and warm gently until the solution becomes turbid. Add 5 ml of glacial acetic acid R ; a pink colour with an intense green fluorescence appears. E. To about 5 mg add 1 ml of acetic anhydride R and 5 ml of pyridine R. A purple colour develops.

TESTS A. (4S,4aS,5aS,6S,12aS)-4-(dimethylamino)-3,6,10,12, pH (2.2.3). Suspend 0.25 g in carbon dioxide-free water R, 12a-pentahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12adilute to 25 ml with the same solvent and filter. The pH of octahydrotetracene-2-carboxamide (demethyltetracycline), the solution is 3.7 to 4.5. Related substances. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm. Test solution (a). Dissolve 0.10 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml B. (4R,4aS,5aS,6S,12aS)-7-chloro-4-(dimethylamino)-3,6, with methanol R. 10,12,12a-pentahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12aReference solution (a). Dilute 1.0 ml of test solution (a) to octahydrotetracene-2-carboxamide (4-epidemeclocycline). 100.0 ml with methanol R. Reference solution (b). Dissolve 20 mg of deptropine citrate CRS in methanol R and dilute to 2 ml with the same 01/2005:1308 solvent. Dilute 1 ml of the solution to 10 ml with methanol R. Reference solution (c). Dissolve 5 mg of tropine CRS in methanol R and dilute to 100.0 ml with the same solvent. DEPTROPINE CITRATE Reference solution (d). Dissolve 10 mg of deptropine citrate CRS and 10 mg of tropine CRS in methanol R and Deptropini citras dilute to 25 ml with the same solvent. Apply to the plate 40 µl of each solution. Develop over a path of 10 cm using a mixture of 8 volumes of concentrated ammonia R and 92 volumes of butanol R. Dry the plate at 100 °C to 105 °C until the ammonia has completely evaporated. Examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (a) (1 per cent). Spray with dilute potassium C29H35NO8 Mr 525.6 iodobismuthate solution R and then with a 10 g/l solution General Notices (1) apply to all monographs and other texts

1393

Dequalinium chloride


of sodium nitrite R. Expose the plate to iodine vapours. Examine in daylight and in ultraviolet light at 254 nm. In the chromatogram obtained with test solution (a) : any spot corresponding to tropine is not more intense than the spot in the chromatogram obtained with reference solution (c) (0.5 per cent) ; any spot, apart from the principal spot and any spot corresponding to tropine, is not more intense than the spot in the chromatogram obtained with reference solution (a) (1 per cent). The test is not valid unless the chromatogram obtained with reference solution (d) shows two clearly separated spots. Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 2.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 4 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

D. (1R,3r,5S)-3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5yloxy)-8-azabicyclo[3.2.1]octane (demethyldeptropine). 01/2005:1413

DEQUALINIUM CHLORIDE Dequalinii chloridum

ASSAY Dissolve 0.400 g in 50 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 52.56 mg of C29H35NO8. C30H40Cl2N4


Mr 527.6

DEFINITION Dequalinium chloride contains not less than 95.0 per cent and not more than the equivalent of 101.0 per cent of 1,1′-(decane-1,10-diyl)bis(4-amino-2-methylquinolinium) dichloride, calculated with reference to the dried substance.

IMPURITIES

CHARACTERS A white or yellowish-white powder, hygroscopic, slightly soluble in water and in alcohol.

A. (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-ol (tropine),

B. (1R,3s,5S)-3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen5-yloxy)-8-methyl-8-azabicyclo[3.2.1]octane (pseudodeptropine),

C. 10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ol (dibenzocycloheptadienol), 1394

IDENTIFICATION First identification : B, E. Second identification : A, C, D, E. A. Dissolve about 10 mg in water R and dilute to 100 ml with the same solvent. Dilute 10 ml of the solution to 100 ml with water R. Examined between 230 nm and 350 nm (2.2.25), the solution shows 2 absorption maxima, at 240 nm and 326 nm and a shoulder at 336 nm. The ratio of the absorbance measured at the maximum at 240 nm to that measured at the maximum at 326 nm is 1.56 to 1.80 and the ratio of the absorbance measured at the maximum at 326 nm to that measured at the shoulder at 336 nm is 1.12 to 1.30. B. Examine by infrared absorption spectrophotometry between 600 cm− 1 and 2000 cm− 1 (2.2.24), comparing with the spectrum obtained with dequalinium chloride CRS. C. To 5 ml of solution S (see Tests) add 5 ml of potassium ferricyanide solution R. A yellow precipitate is formed. D. To 10 ml of solution S add 1 ml of dilute nitric acid R. A white precipitate is formed. Filter and reserve the filtrate for identification test E. E. The filtrate from identification test D gives reaction (a) of chlorides (2.3.1). TESTS Solution S. Dissolve 0.2 g in 90 ml of carbon dioxide-free water R, heating if necessary, and dilute to 100 ml with the same solvent.


Desipramine hydrochloride


Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Acidity or alkalinity. To 5 ml of solution S add 0.1 ml of bromothymol blue solution R1. Not more than 0.2 ml of 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is required to change the colour of the indicator. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 10.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase.

Dissolve 0.200 g in 5 ml of anhydrous formic acid R and add 50 ml of acetic anhydride R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 26.38 mg of C30H40Cl2N4. STORAGE In an airtight container. IMPURITIES

Reference solution (a). Dissolve 10.0 mg of dequalinium chloride for performance test CRS in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (b). Dissolve 10.0 mg of dequalinium chloride CRS in the mobile phase and dilute to 10.0 ml with A. 2-methylquinolin-4-amine, the mobile phase. Dilute 1.0 ml to 50.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with an end-capped octadecylsilyl silica gel for chromatography R, — as mobile phase at a flow rate of 1.5 ml/min the following solution : dissolve 2 g of sodium hexanesulphonate R in B. 4-amino-1-[10-[(2-methylquinolin-4-yl)amino]decyl]-2methylquinolinium chloride, 300 ml of water R. Adjust to pH 4.0 with acetic acid R and add 700 ml of methanol R, — as detector a spectrophotometer set at 240 nm. Adjust the sensitivity of the system so that the height of the peak due to impurity B in the chromatogram obtained with 10 µl of reference solution (a) is at least 25 per cent of the full scale of the recorder. Measure the height (A) above the baseline of the peak due to impurity B and the height (B) above the baseline at the lowest point of the curve separating this peak from the peak due to dequalinium chloride. The test is not valid unless A is greater than twice B. If necessary, C. 1-[10-(4-amino-2-methylquinolinio)decyl]-4-[[10-(4-amino2-methylquinolinio)decyl]amino]-2-methylquinolinium adjust the concentration of methanol in the mobile phase. trichloride. Inject 10 µl of the test solution and 10 µl of reference solution (b). Continue the chromatography of the test 01/2005:0481 solution for 5 times the retention time of the peak due to dequalinium chloride. In the chromatogram obtained with DESIPRAMINE HYDROCHLORIDE the test solution : the area of any peak due to impurity A is not greater than half the area of the principal peak in the Desipramini hydrochloridum chromatogram obtained with reference solution (b) (1 per cent) ; and the sum of the areas of all the peaks, apart from the principal peak, is not greater than 5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (10 per cent). Disregard any peak with an area less than 0.025 times the area of the principal peak in the chromatogram obtained with reference solution (b). Readily carbonisable substances. Dissolve 20 mg in 2 ml of sulphuric acid R. After 5 min the solution is not more intensely coloured than reference solution BY4 (2.2.2, C18H23ClN2 Mr 302.8 Method I). DEFINITION Loss on drying (2.2.32). Not more than 7.0 per cent, determined on 1.000 g by drying at 100-105 °C at a pressure Desipramine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of not exceeding 0.7 kPa. 3-(10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)-N-methylpropanSulphated ash (2.4.14). Not more than 0.1 per cent, 1-amine hydrochloride, calculated with reference to the dried determined on 1.0 g. substance. ASSAY In order to avoid overheating in the reaction medium, mix thoroughly throughout and stop the titration immediately after the end-point has been reached. General Notices (1) apply to all monographs and other texts

CHARACTERS A white or almost white, crystalline powder, soluble in water and in alcohol. It melts at about 214 °C. 1395

Deslanoside


IDENTIFICATION First identification : B, E. Second identification : A, C, D, E. A. Dissolve 40.0 mg in 0.01 M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute 5.0 ml of the solution to 100.0 ml with 0.01 M hydrochloric acid. Examined between 230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 251 nm and a shoulder at 270 nm. The specific absorbance at the maximum is 255 to 285. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with desipramine hydrochloride CRS. C. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). D. Dissolve about 50 mg in 3 ml of water R and add 0.05 ml of a 25 g/l solution of quinhydrone R in methanol R. An intense pink colour develops within about 15 min. E. To 0.5 ml of solution S (see Tests) add 1.5 ml of water R. The solution gives reaction (a) of chlorides (2.3.1). TESTS Solution S. Dissolve 1.25 g in carbon dioxide-free water R, warming to not more than 30 °C if necessary, and dilute to 25 ml with the same solvent. Appearance of solution. Solution S, examined immediately after preparation, is not more intensely coloured than reference solution BY6 (2.2.2, Method II). Acidity or alkalinity. To 10 ml of solution S add 0.1 ml of methyl red solution R and 0.3 ml of 0.01 M sodium hydroxide. The solution is yellow. Not more than 0.5 ml of 0.01 M hydrochloric acid is required to change the colour of the indicator to red. Related substances. Carry out the test protected from bright light. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R. Test solution (a). Dissolve 0.10 g of the substance to be examined in a mixture of equal volumes of ethanol R and methylene chloride R and dilute to 10 ml with the same mixture of solvents. Prepare immediately before use. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of equal volumes of ethanol R and methylene chloride R. Reference solution (a). Dissolve 25 mg of desipramine hydrochloride CRS in a mixture of equal volumes of ethanol R and methylene chloride R and dilute to 25 ml with the same mixture of solvents. Prepare immediately before use. Reference solution (b). Dilute 1 ml of reference solution (a) to 50 ml with a mixture of equal volumes of ethanol R and methylene chloride R. Apply to the plate 5 µl of each solution. Develop over a path of 7 cm using a mixture of 1 volume of water R, 10 volumes of anhydrous acetic acid R and 10 volumes of toluene R. Dry the plate in a current of air for 10 min, spray with a 5 g/l solution of potassium dichromate R in a mixture of 1 volume of sulphuric acid R and 4 volumes of water R and examine immediately. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.2 per cent). 1396

Heavy metals (2.4.8). 2.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 4 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.2500 g in a mixture of 5 ml of 0.01 M hydrochloric acid and 50 ml of alcohol R. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the two points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 30.28 mg of C18H23ClN2. STORAGE Store protected from light. 01/2005:0482

DESLANOSIDE Deslanosidum

C47H74O19

Mr 943

DEFINITION Deslanoside contains not less than 95.0 per cent and not more than the equivalent of 105.0 per cent of 3β-[(O-β-D-glucopyranosyl-(1→4)-O-2,6-dideoxy-β-D-ribohexopyranosyl-(1→4)-O-2,6-dideoxy-β-D-ribo-hexopyranosyl(1→4)-2,6-dideoxy-β-D-ribo-hexopyranosyl)oxy]-12β,14dihydroxy-5β,14β-card-20(22)-enolide, calculated with reference to the dried substance. CHARACTERS A white, crystalline or finely crystalline powder, hygroscopic, practically insoluble in water, very slightly soluble in alcohol. In an atmosphere of low relative humidity, it loses water. IDENTIFICATION First identification : A. Second identification : B, C, D.


Desmopressin


A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with deslanoside CRS. When comparing the spectra, special attention is given to the absence of a distinct absorption maximum at about 1260 cm–1 and to the intensity of the absorption maximum at about 1740 cm–1. Examine the substances in discs prepared by dissolving 1 mg of the substance to be examined or 1 mg of the reference substance in 0.3 ml of methanol R and triturating with about 0.4 g of dry, finely powdered potassium bromide R until the mixture is uniform and completely dry. B. Examine the chromatograms obtained in the test for related substances. The principal band in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal band in the chromatogram obtained with reference solution (a). C. Suspend about 0.5 mg in 0.2 ml of alcohol (60 per cent V/V) R. Add 0.1 ml of dinitrobenzoic acid solution R and 0.1 ml of dilute sodium hydroxide solution R. A violet colour develops. D. Dissolve about 5 mg in 5 ml of glacial acetic acid R and add 0.05 ml of ferric chloride solution R1. Cautiously add 2 ml of sulphuric acid R, avoiding mixing the two liquids. Allow to stand ; a brown but not reddish ring develops at the interface and a greenish-yellow, then bluish-green colour diffuses from it to the upper layer. TESTS Solution S. Dissolve 0.20 g in a mixture of equal volumes of chloroform R and methanol R and dilute to 10 ml with the same mixture of solvents. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Specific optical rotation (2.2.7). Dissolve 0.200 g in anhydrous pyridine R and dilute to 10.0 ml with the same solvent. The specific optical rotation is + 6.5 to + 8.5, calculated with reference to the dried substance. Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution (a). Use solution S. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of equal volumes of chloroform R and methanol R. Reference solution (a). Dissolve 20 mg of deslanoside CRS in a mixture of equal volumes of chloroform R and methanol R and dilute to 10 ml with the same mixture of solvents. Reference solution (b). Dilute 2.5 ml of reference solution (a) to 10 ml with a mixture of equal volumes of chloroform R and methanol R. Reference solution (c). Dilute 1 ml of reference solution (a) to 10 ml with a mixture of equal volumes of chloroform R and methanol R. Apply separately to the plate as 10 mm bands 5 µl of each solution. Develop immediately over a path of 15 cm using a mixture of 3 volumes of water R, 36 volumes of methanol R and 130 volumes of methylene chloride R. Dry the plate in a current of warm air, spray with a mixture of 5 volumes of sulphuric acid R and 95 volumes of alcohol R and heat at 140 °C for 15 min. Examine in daylight. In the chromatogram obtained with test solution (a), any band, apart from the principal band, is not more intense than the band in the chromatogram obtained with reference solution (b) (2.5 per cent) and at most two such bands are more intense than the band in the chromatogram obtained with reference solution (c) (1.0 per cent). General Notices (1) apply to all monographs and other texts

Loss on drying (2.2.32). Not more than 5.0 per cent, determined on 0.500 g by drying in vacuo at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on the residue obtained in the test for loss on drying. ASSAY Dissolve 50.0 mg in alcohol R and dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of this solution to 100.0 ml with alcohol R. Prepare a reference solution in the same manner, using 50.0 mg of deslanoside CRS (undried). To 5.0 ml of each solution add 3.0 ml of alkaline sodium picrate solution R and allow to stand protected from bright light in a water-bath at 20 ± 1 °C for 40 min. Measure the absorbance (2.2.25) of each solution at the maximum at 484 nm, using as the compensation liquid a mixture of 3.0 ml of alkaline sodium picrate solution R and 5.0 ml of alcohol R prepared at the same time. Calculate the content of C47H74O19 from the absorbances measured and the concentrations of the solutions. STORAGE Store in an airtight, glass container, protected from light, at a temperature below 10 °C.

01/2005:0712

DESMOPRESSIN Desmopressinum

C46H64N14O12S2

Mr 1069

DEFINITION Desmopressin is a synthetic cyclic nonapeptide having an antidiuretic action. It contains not less than 95.0 per cent and not more than the equivalent of 105.0 per cent of the peptide C46H64N14O12S2, calculated with reference to the anhydrous, acetic acid-free substance. It is available as an acetate. CHARACTERS A white, fluffy powder, soluble in water, in alcohol and in glacial acetic acid. IDENTIFICATION Examine the chromatograms obtained in the assay. The retention time and size of the principal peak in the chromatogram obtained with the test solution are approximately the same as those of the principal peak in the chromatogram obtained with the reference solution. TESTS Specific optical rotation (2.2.7). Dissolve 10.0 mg in a 1 per cent V/V solution of glacial acetic acid R and dilute to 5.0 ml with the same acid. The specific optical rotation is − 72 to − 82, calculated with reference to the anhydrous, acetic acid-free substance. 1397

Desmopressin


Amino acids. Examine by means of an amino-acid analyser. Standardise the apparatus with a mixture containing equimolar amounts of ammonia, glycine and the L-form of the following amino acids :

area of the peaks. Disregard any peak due to the solvent and any peak with an area less than 0.05 per cent of that of the principal peak. Acetic acid (2.5.34) : 3.0 per cent to 8.0 per cent. threonine lysine alanine leucine Test solution. Dissolve 20.0 mg of the substance to be serine tyrosine histidine valine examined in a mixture of 5 volumes of mobile phase B and 95 volumes of mobile phase A and dilute to 10.0 ml with the arginine methionine glutamic acid phenylalanine same mixture of solvents. aspartic acid proline isoleucine Water (2.5.32). Not more than 6.0 per cent, determined by the micro determination of water. together with half the equimolar amount of L-cystine. For the Bacterial endotoxins (2.6.14) : less than 500 IU/mg, if validation of the method, an appropriate internal standard, intended for use in the manufacture of parenteral dosage such as DL-norleucine R, is used. forms without a further appropriate procedure for the Test solution. Place 1.0 mg of the substance to be examined removal of bacterial endotoxins. in a rigorously cleaned hard-glass tube 100 mm long and 6 mm in internal diameter. Add a suitable amount of a ASSAY 50 per cent V/V solution of hydrochloric acid R. Immerse the tube in a freezing mixture at − 5 °C, reduce the pressure Examine by liquid chromatography (2.2.29). to below 133 Pa and seal. Heat at 110 °C to 115 °C for 16 h. Test solution. Dissolve 1.0 mg of the substance to be Cool, open the tube, transfer the contents to a 10 ml flask examined in 2.0 ml of water R. with the aid of five quantities, each of 0.2 ml, of water R and Reference solution. Dissolve the contents of a vial of evaporate to dryness over potassium hydroxide R under desmopressin CRS in water R to obtain a concentration of reduced pressure. Take up the residue in water R and 0.5 mg/ml. evaporate to dryness over potassium hydroxide R under Resolution solution. Dissolve the contents of a vial of reduced pressure ; repeat these operations once. Take up oxytocin/desmopressin validation mixture CRS in 500 µl the residue in a buffer solution suitable for the amino-acid analyser used and dilute to a suitable volume with the same of water R. buffer solution. Apply a suitable volume to the amino-acid The chromatographic procedure may be carried out using : analyser. — a stainless steel column 0.12 m long and 4.0 mm in Express the content of each amino acid in moles. Calculate internal diameter packed with octadecylsilyl silica gel for the relative proportions of the amino acids, taking one-sixth chromatography R (5 µm), of the sum of the number of moles of aspartic acid, glutamic — as mobile phase at a flow rate of 2.0 ml/min a mixture of acid, proline, glycine, arginine and phenylalanine as equal to 60 volumes of mobile phase A and 40 volumes of mobile one. The values fall within the following limits : aspartic acid phase B : 0.95 to 1.05 ; glutamic acid 0.95 to 1.05 ; proline 0.95 to 1.05 ; Mobile phase A. 0.067 M phosphate buffer solution glycine 0.95 to 1.05 ; arginine 0.95 to 1.05 ; phenylalanine 0.95 to 1.05 ; tyrosine 0.70 to 1.05 ; half-cystine 0.30 to 1.05. pH 7.0 R ; filter and degas, Lysine, isoleucine and leucine are absent ; not more than Mobile phase B. Mix equal volumes of mobile phase A and of traces of other amino acids are present. acetonitrile for chromatography R ; filter and degas, Related peptides. Examine by liquid chromatography — as detector a spectrophotometer set at 220 nm. (2.2.29) as described under Assay, following the elution Inject 50 µl of the resolution solution and identify the peaks conditions shown in the table below, at a flow rate of due to desmopressin and oxytocin (first and second peaks, 1.5 ml/min : respectively). If necessary, adjust the concentration of acetonitrile in the mobile phase to obtain a retention time of Time Comment Mobile phase A Mobile phase B about 5 min for the desmopressin peak. The test is not valid (min) (per cent V/V) (per cent V/V) unless the resolution between the peaks corresponding to 0-4 76 24 isocratic desmopression and oxytocin is at least 1.5. 24 → 42 4 - 18 76 → 58 linear gradient Inject 50 µl of the test solution and 50 µl of the reference 18 - 35 58 → 48 42 → 52 linear gradient solution. 48 → 76 35 - 40 52 → 24 Calculate the content of desmopressin (C46H64N14O12S2) from switch to initial eluent composition the peak areas in the chromatograms obtained with the test 76 24 40 - 50 re-equilibration solution and the reference solution and the declared content of C46H64N14O12S2 in desmopressin CRS. Inject 50 µl of the resolution solution and identify the peaks STORAGE due to desmopressin and oxytocin (first and second peaks, respectively). If necessary, adjust the concentration of Store in an airtight container, protected from light, at a acetonitrile in the mobile phase to obtain a retention time temperature of 2 °C to 8 °C. If the substance is sterile, store of about 16 min for the desmopressin peak. The test is not in a sterile, airtight, tamper-proof container. valid unless the resolution between the peaks corresponding to desmopressin and oxytocin is at least 1.5. LABELLING Inject 50 µl of the test solution. In the chromatogram obtained the area of any peak, apart from the principal peak, is not greater than 0.5 per cent of the total area of the peaks ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than 1.5 per cent of the total 1398

The label states : — the mass of peptide per container, — where applicable, that the substance is free from bacterial endotoxins.


Desoxycortone acetate


01/2005:0322

DESOXYCORTONE ACETATE Desoxycortoni acetas

in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. D. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a yellow colour develops. Add the solution to 2 ml of water R and shake. The resulting solution is dichroic showing an intense blue colour by transparency, and red fluorescence which is particularly intense in ultraviolet light at 365 nm. E. About 10 mg gives the reaction of acetyl (2.3.1).

TESTS Specific optical rotation (2.2.7). Dissolve 0.250 g in dioxan R and dilute to 25.0 ml with the same solvent. The C23H32O4 Mr 372.5 specific optical rotation is + 171 to + 179, calculated with reference to the dried substance. DEFINITION Desoxycortone acetate contains not less than 97.0 per cent Related substances. Examine by liquid chromatography (2.2.29). and not more than the equivalent of 103.0 per cent of 3,20-dioxopregn-4-en-21-yl acetate, calculated with reference Test solution. Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the to the dried substance. mobile phase. CHARACTERS Reference solution (a). Dissolve 2 mg of desoxycortone A white or almost white, crystalline powder or colourless acetate CRS and 2 mg of betamethasone 17-valerate CRS crystals, practically insoluble in water, freely soluble in in the mobile phase and dilute to 200.0 ml with the mobile methylene chloride, soluble in acetone, sparingly soluble in phase. alcohol, slightly soluble in propylene glycol and in fatty oils. Reference solution (b). Dilute 1.0 ml of the test solution to 200.0 ml with the mobile phase. IDENTIFICATION The chromatographic procedure may be carried out using : First identification : B, C. — a stainless steel column 0.25 m long and 4.6 mm in Second identification : A, C, D, E. internal diameter packed with octadecylsilyl silica gel for A. Melting point (2.2.14) : 157 °C to 161 °C. chromatography R (5 µm), B. Examine by infrared absorption spectrophotometry — as mobile phase at a flow rate of 1 ml/min a mixture (2.2.24), comparing with the spectrum obtained with prepared as follows : in a 1000 ml volumetric flask mix desoxycortone acetate CRS. 350 ml of water R with 600 ml of acetonitrile R and allow C. Examine by thin-layer chromatography (2.2.27), using to equilibrate ; adjust the volume to 1000 ml with water R as the coating substance a suitable silica gel with a and mix again, fluorescent indicator having an optimal intensity at — as detector a spectrophotometer set at 254 nm. 254 nm. Equilibrate the column with the mobile phase at a flow rate Test solution. Dissolve 10 mg of the substance to be of 1 ml/min for about 30 min. examined in a mixture of 1 volume of methanol R and Adjust the sensitivity of the system so that the height of the 9 volumes of methylene chloride R and dilute to 10 ml principal peak in the chromatogram obtained with 20 µl of with the same mixture of solvents. Reference solution (a). Dissolve 20 mg of desoxycortone reference solution (b) is not less than 50 per cent of the full acetate CRS in a mixture of 1 volume of methanol R and scale of the recorder. Inject 20 µl of reference solution (a). When the 9 volumes of methylene chloride R and dilute to 20 ml chromatograms are recorded in the prescribed conditions, with the same mixture of solvents. the retention times are : betamethasone 17-valerate, about Reference solution (b). Dissolve 10 mg of cortisone 7.5 min and desoxycortone acetate, about 9.5 min. The test acetate R in reference solution (a) and dilute to 10 ml is not valid unless the resolution between the peaks due with reference solution (a). to betamethasone 17-valerate and desoxycortone acetate Apply separately to the plate 5 µl of each solution. is at least 4.5 ; if necessary, adjust the concentration of Prepare the mobile phase by adding a mixture of acetonitrile in the mobile phase. 1.2 volumes of water R and 8 volumes of methanol R to Inject separately 20 µl of the test solution and 20 µl of a mixture of 15 volumes of ether R and 77 volumes of reference solution (b). Continue the chromatography for methylene chloride R. Develop over a path of 15 cm. three times the retention time of the principal peak. In the Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram chromatogram obtained with the test solution, the sum of obtained with the test solution is similar in position and the areas of all the peaks, apart from the principal peak, is not greater than the area of the principal peak in the size to the principal spot in the chromatogram obtained with reference solution (a). Spray with alcoholic solution chromatogram obtained with reference solution (b) (0.5 per cent). Disregard any peak with an area less than 0.1 times of sulphuric acid R. Heat at 120 °C for 10 min or until the area of the principal peak in the chromatogram obtained the spots appear. Allow to cool. Examine the plate in daylight and in ultraviolet light at 365 nm. The principal with reference solution (b). spot in the chromatogram obtained with the test solution Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 0.500 g by drying in an oven at 100 °C to is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot 105 °C. General Notices (1) apply to all monographs and other texts

1399

Detomidine hydrochloride for veterinary use


Reference solution (b). Dissolve 1 mg of detomidine impurity B CRS in the mobile phase and dilute to 100 ml with the mobile phase. Dilute 1 ml of the solution to 10 ml with reference solution (a). The chromatographic procedure may be carried out using : — a stainless steel column 0.15 m long and 4.6 mm in internal diameter packed with octylsilyl silica gel for STORAGE chromatography R (5 µm), Store protected from light. — as mobile phase at a flow rate of 1 ml/min a mixture of 35 volumes of acetonitrile R and 65 volumes of a 2.64 g/l solution of ammonium phosphate R, — as detector a spectrophotometer set at 220 nm. 01/2005:1414 corrected When the chromatograms are recorded in the prescribed conditions, the retention time of detomidine is about 7 min and the relative retention times of impurities A, B and C with DETOMIDINE HYDROCHLORIDE FOR respect to detomidine are about 0.4, 2.0 and 3.0, respectively. Inject 20 µl of reference solution (a). Adjust the sensitivity VETERINARY USE of the system so that the height of the principal peak in the chromatogram obtained is at least 50 per cent of the full Detomidini hydrochloridum ad usum scale of the recorder. Inject 20 µl of reference solution (b). The test is not valid unless : the resolution between the peaks veterinarium corresponding to detomidine and to impurity B is at least 5. Inject 20 µl of the test solution. Continue the chromatography for four times the retention time of the principal peak. Multiply the area of any peak (corresponding to the impurity C and its diastereoisomer) eluting with a relative retention time of about 3, by the correction factor 2.7. The sum of the areas of such peaks is not greater than C12H15ClN2 Mr 222.7 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent) ; the area DEFINITION of any other peak apart from the principal peak and the Detomidine hydrochloride for veterinary use contains not peak corresponding to the impurity C is not greater than the less than 98.5 per cent and not more than the equivalent area of the principal peak in the chromatogram obtained of 101.5 per cent of 4-(2,3-dimethylbenzyl)-1H-imidazole with reference solution (a) (0.2 per cent) ; the sum of the hydrochloride, calculated with reference to the dried areas of all the peaks apart from the principal peak is not substance. greater than five times the area of the principal peak in the chromatogram obtained with reference solution (a) (1 per CHARACTERS cent). Disregard any peak with an area less than 0.25 times the area of the principal peak in the chromatogram obtained A white or almost white, hygroscopic, crystalline powder, with reference solution (a). soluble in water, freely soluble in alcohol, very slightly soluble in methylene chloride, practically insoluble in Loss on drying (2.2.32). Not more than 0.5 per cent, acetone. determined on 1.000 g by drying in oven at 100 °C to 105 °C. It melts at about 160 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. IDENTIFICATION

ASSAY Dissolve 0.100 g in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml to 100.0 ml with alcohol R. Measure the absorbance (2.2.25) at the maximum at 240 nm. Calculate the content of C23H32O4 taking the specific absorbance to be 450.

A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with detomidine hydrochloride CRS. Examine the substances prepared as discs. If the spectra obtained show differences, dry the substance to be examined and the reference substance in an oven at 100 °C to 105 °C and record new spectra. B. It gives reaction (a) of chlorides (2.3.1).

ASSAY Dissolve 0.170 g in 50 ml of alcohol R. Add 5.0 ml of 0.01 M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the two points of inflection. 1 ml of 0.1 M sodium hydroxide corresponds to 22.27 mg of C12H15ClN2.

TESTS Appearance of solution. Dissolve 0.25 g in water R and dilute to 25 ml with the same solvent. The solution is clear (2.2.1) and colourless (2.2.2, Method II). Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 25.0 mg of the substance to be examined in 20 ml of the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (a). Dilute 0.20 ml of the test solution to 100.0 ml with the mobile phase.

STORAGE Store in an airtight container.

1400

IMPURITIES

A. (RS)-(2,3-dimethylphenyl)(1H-imidazol-4-yl)methanol,


Devil’s claw root


B. (RS)-(1-benzyl-1H-imidazol-5-yl)(2,3-dimethylphenyl)methanol,

C. 4-[(2,3-dimethylcyclohexyl)methyl]-1H-imidazole. 01/2005:1095

Apply to the plate as bands 20 µl of each solution. Develop over a path of 10 cm using a mixture of 8 volumes of water R, 15 volumes of methanol R and 77 volumes of ethyl acetate R. Dry the plate in a current of warm air. Examine in ultraviolet light at 254 nm. The chromatograms obtained with the test solution and the reference solution both show in the middle a quenching zone corresponding to harpagoside. The chromatogram obtained with the test solution shows other distinct bands, mainly above the zone corresponding to harpagoside. Spray with a 10 g/l solution of phloroglucinol R in alcohol R and then with hydrochloric acid R. Dry the plate at 80 °C for 5-10 min. In the chromatograms obtained with the reference solution and the test solution the zone corresponding to harpagoside is green. The chromatogram obtained with the test solution also shows several yellow to brown zones below and above the zone corresponding to harpagoside.

TESTS Starch. Examine the powdered drug (355) under a microscope using water R. Add iodine solution R1. No blue Harpagophyti radix colour develops. DEFINITION Foreign matter (2.8.2). It complies with the test for foreign matter. Devil’s claw root consists of the cut and dried tuberous, secondary roots of Harpagophytum procumbens D.C. Loss on drying (2.2.32). Not more than 12.0 per cent, and/or H. zeyheri L. Decne. It contains not less than 1.2 per determined on 1.000 g of the powdered drug (355) by drying cent of harpagoside (C24H30O11 ; Mr 494.5), calculated with in an oven at 100-105 °C. reference to the dried drug. Total ash (2.4.16). Not more than 10.0 per cent. CHARACTERS ASSAY Devil’s claw root is greyish-brown to dark brown and it has Examine by liquid chromatography (2.2.29) using methyl a bitter taste. It has the macroscopic and microscopic characters described cinnamate R as the internal standard. Internal standard solution. Dissolve 0.130 g of methyl in identification tests A and B. cinnamate R in 50 ml of methanol R and dilute to 100.0 ml IDENTIFICATION with the same solvent. A. It consists of thick, fan-shaped or rounded slices or Test solution. To 0.500 g of the powdered drug (355) add of roughly crushed discs. The darker outer surface is 50 ml of methanol R. Shake for 1 h and filter. Transfer traversed by tortuous longitudinal wrinkles. The paler the filter with the residue to a 100 ml flask, add 50 ml of cut surface shows a dark cambial zone and xylem bundles methanol R and heat under a reflux condenser for 1 h. Cool distinctly aligned in radial rows. The central cylinder and filter. Rinse the flask and the filter with 2 quantities, shows fine concentric striations. Seen under a lens, the each of 5 ml, of methanol R. Combine the filtrate and the cut surface presents yellow to brownish-red granules. rinsing solution and evaporate to dryness under reduced B. Reduce to a powder (355). The powder is brownish-yellow. pressure at a temperature not exceeding 40 °C. Take up Examine under a microscope using chloral hydrate the residue with 3 quantities, each of 5 ml, of methanol R solution R. The powder shows the following diagnostic and filter the extracts into a 25 ml volumetric flask. Whilst characters : fragments of cork layer consisting of washing the filter, dilute to 25.0 ml with methanol R. To yellowish-brown, thin-walled cells ; fragments of 10.0 ml of this solution add 1.0 ml of the internal standard cortical parenchyma consisting of large, thin-walled solution and dilute to 25.0 ml with methanol R. cells, sometimes containing reddish-brown granular Reference solution. Dilute 0.5 ml of the reference solution inclusions and isolated yellow droplets ; fragments of described in Identification test C to 2.0 ml with methanol R. reticulately thickened vessels and tracheidal vessels The chromatographic procedure may be carried out using : with associated lignified parenchyma from the central cylinder ; small needles and crystals of calcium oxalate — a stainless steel column 0.10 m long and 4 mm in are present in the parenchyma. The powder may show internal diameter packed with octadecylsilyl silica gel for rectangular or polygonal pitted sclereids with dark chromatography R (5 µm), reddish-brown contents. With a solution of phloroglucinol — as mobile phase at a flow rate of 1.5 ml/min a mixture of in hydrochloric acid, the parenchyma turns green. equal volumes of methanol R and water R, C. Examine by thin-layer chromatography (2.2.27), using a — as detector a spectrophotometer set at 278 nm, suitable silica gel as the coating substance. — a 10 µl loop injector. Test solution. Heat on a water-bath at 60 °C for Inject the test solution. Adjust the sensitivity of the detector 10 min 1.0 g of the powdered drug (355) with 10 ml of so that the height of the peak due to methyl cinnamate is methanol R. Filter and reduce the filtrate to about 2 ml about 50 per cent of the full scale of the recorder. under reduced pressure at a temperature not exceeding 40 °C. Determine the retention time of harpagoside using 10 µl of the reference solution examined under the same conditions Reference solution. Dissolve 1 mg of harpagoside R in as the test solution. 1 ml of methanol R.

DEVIL’S CLAW ROOT


1401

Dexamethasone


Test solution. Dissolve 10 mg of the substance to be examined in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 10 ml with the same mixture of solvents. Reference solution (a). Dissolve 20 mg of dexamethasone CRS in a mixture of 1 volume of m1 = mass of the drug, in grams, methanol R and 9 volumes of methylene chloride R and dilute to 20 ml with the same mixture of solvents. m2 = mass of methyl cinnamate R, in grams in the Reference solution (b). Dissolve 10 mg of internal standard solution, betamethasone CRS in reference solution (a) and dilute F1 = area of the peak corresponding to harpagoside in to 10 ml with the same solution. the chromatogram obtained with the test solution, Apply to the plate 5 µl of each solution. Develop over a F2 = area of the peak corresponding to methyl path of 15 cm, using a mixture of 5 volumes of butanol R cinnamate in the chromatogram obtained with saturated with water R, 10 volumes of toluene R and the test solution. 85 volumes of ether R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot STORAGE in the chromatogram obtained with the test solution is similar in position and size to the principal spot in Store protected from light. the chromatogram obtained with reference solution (a). Spray with alcoholic solution of sulphuric acid R. Heat at 120 °C for 10 min or until the spots appear. Allow to cool. Examine the chromatograms in daylight and in 01/2005:0388 ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar DEXAMETHASONE in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (a). The Dexamethasonum test is not valid unless the chromatogram obtained with reference solution (b) shows 2 spots which may, however, not be completely separated. D. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a faint reddish-brown colour develops. Add the solution to 10 ml of water R and mix. The colour is discharged. E. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is obtained (usually less than 5 min). Allow to cool, add 1 ml C22H29FO5 Mr 392.5 of water R, 0.05 ml of phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the DEFINITION solution colourless. Filter. To a freshly prepared mixture Dexamethasone contains not less than 97.0 per cent and of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl not more than the equivalent of 103.0 per cent of 9-fluoronitrate solution R, add 1.0 ml of the filtrate. Mix, allow to 11β,17,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione, stand for 5 min and compare the colour of the solution calculated with reference to the dried substance. with that of a blank prepared in the same manner. The test solution is yellow and the blank is red. CHARACTERS TESTS A white or almost white, crystalline powder, practically insoluble in water, sparingly soluble in ethanol, slightly Specific optical rotation (2.2.7). Dissolve 0.250 g in soluble in methylene chloride. dioxan R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 75 to + 80, calculated with IDENTIFICATION reference to the dried substance. First identification : B, C. Related substances. Examine by liquid chromatography (2.2.29). Second identification : A, C, D, E. Test solution. Place 25.0 mg of the substance to be examined A. Dissolve 10.0 mg in ethanol R and dilute to in a 10.0 ml volumetric flask, add 1.5 ml of acetonitrile R 100.0 ml with the same solvent. Place 2.0 ml of the and then 5 ml of mobile phase A. Mix with the aid of an solution in a stoppered test tube, add 10.0 ml of phenylhydrazine-sulphuric acid solution R, mix and heat ultrasonic bath until complete dissolution and dilute to 10.0 ml with mobile phase A. in a water-bath at 60 °C for 20 min. Cool immediately. The absorbance (2.2.25) of the solution at the maximum Reference solution (a). Dissolve 2 mg of dexamethasone CRS at 419 nm is not less than 0.4. and 2 mg of methylprednisolone CRS in mobile phase A and dilute to 100.0 ml with the same mobile phase. B. Examine by infrared absorption spectrophotometry Reference solution (b). Dilute 1.0 ml of the test solution to (2.2.24), comparing with the spectrum obtained with 100.0 ml with mobile phase A. dexamethasone CRS. The chromatographic procedure may be carried out using : C. Examine by thin-layer chromatography (2.2.27), using — a stainless steel column 0.25 m long and 4.6 mm in as the coating substance a suitable silica gel with a internal diameter packed with octadecylsilyl silica gel for fluorescent indicator having an optimal intensity at chromatography R (5 µm), 254 nm. Calculate the percentage content of harpagoside from the expression :

1402


Dexamethasone acetate


— as mobile phase at a flow rate of 2.5 ml/min a linear gradient programme using the following conditions : Mobile phase A. In a 1000 ml volumetric flask, mix 250 ml of acetonitrile R with 700 ml of water R and allow to equilibrate ; adjust the volume to 1000 ml with water R and mix again,

01/2005:0548

DEXAMETHASONE ACETATE Dexamethasoni acetas

Mobile phase B. Acetonitrile R, Time (min)



Comment

0

100

0

isocratic

15

100 → 0

0 → 100

begin linear gradient

40

0

100

end chromatogram, return to 100 A

41

100

0

begin equilibration with A

46 = 0

100

0

end equilibration, begin next chromatogram

— as detector a spectrophotometer set at 254 nm, maintaining the temperature of the column at 45 °C. Equilibrate the column for at least 30 min with mobile phase B at a flow rate of 2.5 ml/min and then with mobile phase A for 5 min. For subsequent chromatograms, use the conditions described from 40.0 min to 46.0 min. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 20 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder. Inject 20 µl of reference solution (a). When the chromatograms are recorded in the prescribed conditions, the retention times are : methylprednisolone about 11.5 min and dexamethasone about 13 min. The test is not valid unless the resolution between the peaks corresponding to methylprednisolone and dexamethasone is at least 2.8 ; if necessary, adjust the concentration of acetonitrile in mobile phase A. Inject 20 µl of mobile phase A as a blank, 20 µl of the test solution and 20 µl of reference solution (b). Record the chromatogram of the test solution for twice the retention time of the principal peak. In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent). Disregard any peak due to the blank and any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (b). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 0.500 g by drying in an oven at 100-105 °C. ASSAY Dissolve 0.100 g in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the solution to 100.0 ml with alcohol R. Measure the absorbance (2.2.25) at the maximum at 238.5 nm. Calculate the content of C22H29FO5 taking the specific absorbance to be 394. STORAGE Protected from light. General Notices (1) apply to all monographs and other texts

C24H31FO6

Mr 434.5

DEFINITION Dexamethasone acetate contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent of 9-fluoro-11β,17-dihydroxy-16α-methyl-3,20-dioxopregna-1, 4-dien-21-yl acetate, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, practically insoluble in water, freely soluble in acetone and in alcohol, slightly soluble in methylene chloride. Dexamethasone acetate shows polymorphism. IDENTIFICATION First identification : B, C. Second identification : A, C, D, E, F. A. Dissolve 10.0 mg in ethanol R and dilute to 100.0 ml with the same solvent. Place 2.0 ml of this solution in a ground-glass-stoppered tube, add 10.0 ml of phenylhydrazine-sulphuric acid solution R, mix and heat in a water-bath at 60 °C for 20 min. Cool immediately. The absorbance (2.2.25) of the solution at the maximum at 419 nm is not less than 0.35. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with dexamethasone acetate CRS. If the spectra obtained in the solid state with the substance to be examined and the reference substance show differences, record further spectra using saturated solutions (about 30 g/l) of the substance to be examined and of the reference substance in chloroform R in a 0.2 mm cell. C. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm. Test solution. Dissolve 10 mg of the substance to be examined in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 10 ml with the same mixture of solvents. Reference solution (a). Dissolve 20 mg of dexamethasone acetate CRS in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 20 ml with the same mixture of solvents. Reference solution (b). Dissolve 10 mg of cortisone acetate R in reference solution (a) and dilute to 10 ml with the same solution. Apply separately to the plate 5 µl of each solution. Prepare the mobile phase by adding a mixture of 1.2 volumes of water R and 8 volumes of methanol R to a mixture of 15 volumes of ether R and 77 volumes of methylene chloride R. Develop over a path of 15 cm. 1403

Dexamethasone sodium phosphate


Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). Spray with alcoholic solution of sulphuric acid R. Heat at 120 °C for 10 min or until the spots appear. Allow to cool. Examine the plate in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. D. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a faint reddish-brown colour develops. Add the solution to 10 ml of water R and mix. The colour is discharged and a clear solution remains. E. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is obtained (usually less than 5 min). Allow to cool, add 1 ml of water R, 0.05 ml of phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the solution colourless. Filter. To a freshly prepared mixture of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R, add 1.0 ml of the filtrate. Mix, allow to stand for 5 min and compare the colour of the solution with that of a blank prepared in the same manner. The test solution is yellow and the blank is red. F. About 10 mg gives the reaction of acetyl (2.3.1). TESTS Specific optical rotation (2.2.7). Dissolve 0.250 g in dioxan R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 84 to + 90, calculated with reference to the dried substance. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 25.0 mg of the substance to be examined in about 4 ml of acetonitrile R and dilute to 10.0 ml with water R. Reference solution (a). Dissolve 2 mg of dexamethasone acetate CRS and 2 mg of betamethasone acetate CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter, packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows : in a 1000 ml volumetric flask mix 380 ml of acetonitrile R with 550 ml of water R and allow to equilibrate ; dilute to 1000 ml with water R and mix again, — as detector a spectrophotometer set at 254 nm. Equilibrate the column with the mobile phase at a flow rate of 1 ml/min for about 30 min. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 20 µl of reference solution (b) is not less than 50 per cent of the full scale of the recorder. Inject 20 µl of reference solution (a). When the chromatograms are recorded in the prescribed conditions, the retention times are : betamethasone acetate, about 1404

19 min and dexamethasone acetate, about 22 min. The test is not valid unless the resolution between the peaks due to betamethasone acetate and dexamethasone acetate is at least 3.3 ; if necessary, adjust the concentration of acetonitrile in the mobile phase. Inject separately 20 µl of the test solution and 20 µl of reference solution (b). Continue the chromatography for 1.5 times the retention time of the principal peak. In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than half the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent) ; the sum of the areas of all such peaks is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (b). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 0.500 g by drying in vacuo in an oven at 100 °C to 105 °C. ASSAY Dissolve 0.100 g in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml to 100.0 ml with alcohol R. Measure the absorbance (2.2.25) at the maximum at 238.5 nm. Calculate the content of C24H31FO6 taking the specific absorbance to be 357. STORAGE Store protected from light. 01/2005:0549

DEXAMETHASONE SODIUM PHOSPHATE Dexamethasoni natrii phosphas

C22H28FNa2O8P

Mr 516.4

DEFINITION Dexamethasone sodium phosphate contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent of 9-fluoro-11β,17-dihydroxy-16α-methyl-3,20dioxopregna-1,4-dien-21-yl disodium phosphate, calculated with reference to the anhydrous, ethanol-free substance. CHARACTERS A white or almost white powder, very hygroscopic, freely soluble in water, slightly soluble in alcohol, practically insoluble in methylene chloride. It shows polymorphism. IDENTIFICATION First identification : B, C. Second identification : A, C, D, E, F.



Dexamethasone sodium phosphate

water R and neutralise to red litmus paper R with dilute ammonia R1. The solution gives reaction (a) of sodium (2.3.1) and reaction (b) of phosphates (2.3.1).

A. Dissolve 10.0 mg in 5 ml of water R and dilute to 100.0 ml with ethanol R. Place 2.0 ml of this solution in a ground-glass-stoppered tube, add 10.0 ml of phenylhydrazine-sulphuric acid solution R, mix and heat in a water-bath at 60 °C for 20 min. Cool immediately. The absorbance (2.2.25) of the solution measured at the maximum at 419 nm is at least 0.20. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with dexamethasone sodium phosphate CRS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in the minimum volume of alcohol R, evaporate to dryness on a water-bath and record new spectra using the residues. C. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm. Test solution. Dissolve 10 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution (a). Dissolve 20 mg of dexamethasone sodium phosphate CRS in methanol R and dilute to 20 ml with the same solvent. Reference solution (b). Dissolve 10 mg of prednisolone sodium phosphate CRS in reference solution (a) and dilute to 10 ml with the same solution. Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm, using a mixture of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes of butanol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). Spray with alcoholic solution of sulphuric acid R. Heat at 120 °C for 10 min or until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two spots which may, however, not be completely separated. D. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a faint yellowish-brown colour develops. Add the solution to 10 ml of water R and mix. The colour fades and a clear solution remains. E. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is obtained (usually less than 5 min). Allow to cool, add 1 ml of water R, 0.05 ml of phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the solution colourless. Filter. To a freshly prepared mixture of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R, add 1.0 ml of the filtrate. Mix, allow to stand for 5 min and compare the colour of the solution with that of a blank prepared in the same manner. The test solution is yellow and the blank is red. F. To 40 mg add 2 ml of sulphuric acid R and heat gently until white fumes are evolved, add nitric acid R dropwise, continue the heating until the solution is almost colourless and cool. Add 2 ml of water R, heat until white fumes are again evolved, cool, add 10 ml of

TESTS Solution S. Dissolve 1.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution B7 (2.2.2, Method II). pH (2.2.3). Dilute 1 ml of solution S to 5 ml with carbon dioxide-free water R. The pH of the solution is 7.5 to 9.5. Specific optical rotation (2.2.7). Dissolve 0.250 g in water R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 75 to + 83, calculated with reference to the anhydrous, ethanol-free substance. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (a). Dissolve 2 mg of dexamethasone sodium phosphate CRS and 2 mg of betamethasone sodium phosphate CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows : in a 250 ml conical flask, weigh 1.360 g of potassium dihydrogen phosphate R and 0.600 g of hexylamine R, mix and allow to stand for 10 min and then dissolve in 182.5 ml of water R ; add 67.5 ml of acetonitrile R, mix and filter (0.45 µm), — as detector a spectrophotometer set at 254 nm. Equilibrate the column with the mobile phase at a flow rate of 1 ml/min for about 45 min. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 20 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder. Inject 20 µl of reference solution (a). When the chromatograms are recorded in the prescribed conditions, the retention times are : betamethasone sodium phosphate, about 12.5 min and dexamethasone sodium phosphate about 14 min. The test is not valid unless the resolution between the peaks corresponding to betamethasone sodium phosphate and dexamethasone sodium phosphate is at least 2.2. If necessary, adjust slightly the concentration of acetonitrile or increase the concentration of water in the mobile phase. Inject separately 20 µl of the test solution and 20 µl of reference solution (b). Continue the chromatography for twice the retention time of the principal peak. In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than half the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent) ; the sum of the areas of all such peaks is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent). Disregard any peak with an area less than 0.05 times that of the principal peak in the chromatogram obtained with reference solution (b).


1405

Dexchlorpheniramine maleate


Inorganic phosphates. Dissolve 50 mg in water R and dilute to 100 ml with the same solvent. To 10 ml of this solution add 5 ml of molybdovanadic reagent R, mix and allow to stand for 5 min. Any yellow colour in the solution is not more intense than that in a standard prepared at the same time in the same manner using 10 ml of phosphate standard solution (5 ppm PO4) R (1 per cent). Ethanol. Not more than 3.0 per cent m/m, determined by gas chromatography (2.2.28), using propanol R as the internal standard. Internal standard solution. Dilute 1.0 ml of propanol R to 100.0 ml with water R. Test solution. Dissolve 0.50 g of the substance to be examined in 5.0 ml of the internal standard solution and dilute to 10.0 ml with water R. Reference solution. Dilute 1.0 g of ethanol R to 100.0 ml with water R. To 2.0 ml of the solution add 5.0 ml of the internal standard solution and dilute to 10.0 ml with water R. The chromatographic procedure may be carried out using : — a column 1 m long and 3.2 mm in internal diameter packed with ethylvinylbenzene-divinylbenzene copolymer R1 (150 µm to 180 µm), — nitrogen for chromatography R as the carrier gas at a flow rate of 30 ml/min, — a flame-ionisation detector, maintaining the temperature of the column at 150 °C, that of the injection port at 250 °C and that of the detector at 280 °C. Inject 2 µl of each solution. Ethanol and water. Determine the water content using 0.200 g by the semi-micro determination of water (2.5.12). Add the percentage content of water and the percentage content of ethanol obtained in the test for ethanol. The sum of the percentage contents of water and ethanol is not greater than 13.0 per cent m/m.

DEFINITION Dexchlorpheniramine maleate contains not less than 98.0 per cent and not more than the equivalent of 100.5 per cent of (3S)-3-(4-chlorophenyl)-N,N-dimethyl-3-(pyridin-2-yl)propan1-amine (Z)-butanedioate, calculated with reference to the dried substance. CHARACTERS A white crystalline powder, very soluble in water, freely soluble in alcohol, in methanol and in methylene chloride.

IDENTIFICATION First identification : A, C, E. Second identification : A, B, D, E. A. It complies with the test for specific optical rotation (see Tests). B. Melting point (2.2.14) : 110 °C to 115 °C. C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with dexchlorpheniramine maleate CRS. Examine the substances prepared as discs using potassium bromide R. D. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R. Test solution. Dissolve 0.10 g of the substance to be examined in methanol R and dilute to 5.0 ml with the same solvent. Reference solution. Dissolve 56 mg of maleic acid R in methanol R and dilute to 10 ml with the same solvent. Apply to the plate 5 µl of each solution. Develop over a path of 12 cm using a mixture of 3 volumes of water R, 7 volumes of anhydrous formic acid R, 20 volumes of methanol R and 70 volumes of di-isopropyl ether R. Allow the plate to dry in a current of air for a few minutes and examine in ultraviolet light at 254 nm. The chromatogram obtained with the test solution shows ASSAY two clearly separated spots. The upper spot is similar Dissolve 0.100 g in water R and dilute to 100.0 ml with the in position and size to the spot in the chromatogram same solvent. Dilute 10.0 ml of the solution to 500.0 ml with obtained with the reference solution. water R. Measure the absorbance (2.2.25) at the maximum E. To 0.15 g in a porcelain crucible add 0.5 g of anhydrous at 241.5 nm. sodium carbonate R. Heat over an open flame for 10 min. Calculate the content of C22H28FNa2O8P taking the specific Allow to cool. Take up the residue with 10 ml of dilute absorbance to be 303. nitric acid R and filter. To 1 ml of the filtrate add 1 ml STORAGE of water R. The solution gives reaction (a) of chlorides (2.3.1). Store in an airtight container, protected from light.

TESTS Solution S. Dissolve 2.0 g in water R and dilute to 20.0 ml A. dexamethasone, with the same solvent. B. betamethasone sodium phosphate. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution BY6 (2.2.2, 01/2005:1196 Method II). (2.2.3). Dissolve 0.20 g in 20 ml of water R. The pH of DEXCHLORPHENIRAMINE MALEATE pH the solution is 4.5 to 5.5. Specific optical rotation (2.2.7) : + 22 to + 23, determined Dexchlorpheniramini maleas on solution S and calculated with reference to the dried substance. Related substances. Examine by gas chromatography (2.2.28). Test solution. Dissolve 10.0 mg in 1.0 ml of methylene chloride R. Reference solution. Dissolve 5.0 mg of brompheniramine maleate CRS in 0.5 ml of methylene chloride R and add 0.5 ml of the test solution. Dilute 0.5 ml of this solution to C20H23ClN2O4 Mr 390.9 50.0 ml with methylene chloride R. IMPURITIES

1406


Dexpanthenol


The chromatographic procedure may be carried out using : — a glass column 2.3 m long and 2 mm in internal diameter packed with acid- and base- washed silanised diatomaceous earth for gas chromatography R (135 µm to 175 µm) impregnated with 3 per cent m/m of a mixture of 50 per cent of poly(dimethyl)siloxane and 50 per cent of poly(diphenyl)siloxane, — nitrogen for chromatography R as the carrier gas at a flow rate of 20 ml/min, — a flame-ionisation detector, maintaining the temperature of the column at 205 °C and that of the injection port and of the detector at 250 °C. Inject 1 µl of the reference solution. The test is not valid unless, in the chromatogram obtained with the reference solution, the resolution between the two peaks corresponding to dexchlorpheniramine and brompheniramine is at least 1.5. Inject 1 µl of the test solution. Continue the chromatography for at least 2.5 times the retention time of the principal peak. In the chromatogram obtained with the test solution : none of the peaks, apart from the principal peak, has an area greater than 0.8 times the area of the peak corresponding to dexchlorpheniramine in the chromatogram obtained with the reference solution (0.4 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than twice the area of the peak corresponding to dexchlorpheniramine in the chromatogram obtained with the reference solution (1 per cent). Enantiomeric purity. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 10.0 mg in 3 ml of water R. Add a few drops of concentrated ammonia R until an alkaline reaction is produced. Shake with 5 ml of methylene chloride R. Separate the layers. Evaporate the lower, methylene chloride layer to an oily residue on a water-bath. Dissolve the oily residue in 2-propanol R and dilute to 10.0 ml with the same solvent. Reference solution (a). Dissolve 10.0 mg of dexchlorpheniramine maleate CRS in 3 ml of water R. Add a few drops of concentrated ammonia R until an alkaline reaction is produced. Shake with 5 ml of methylene chloride R. Separate the layers. Evaporate the lower, methylene chloride layer to an oily residue on a water-bath. Dissolve the oily residue in 2-propanol R and dilute to 10.0 ml with the same solvent. Reference solution (b). Dissolve 10.0 mg of chlorphenamine maleate CRS in 3 ml of water R. Add a few drops of concentrated ammonia R until an alkaline reaction is produced. Shake with 5 ml of methylene chloride R. Separate the layers. Evaporate the lower, methylene chloride layer to an oily residue on a water-bath. Dissolve the oily residue in 2-propanol R and dilute to 10.0 ml with the same solvent. Reference solution (c). Dilute 1.0 ml of the test solution to 50 ml with 2-propanol R. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with amylose derivative of silica gel for chromatography R, — as mobile phase at a flow rate of 1 ml/min a mixture of 3 volumes of diethylamine R, 20 volumes of 2-propanol R and 980 volumes of hexane R, — as detector a spectrophotometer set at 254 nm. Under these conditions the peak of the (S)-isomer appears first.

Inject 10 µl of each solution. The test is not valid unless : in the chromatogram obtained with reference solution (b), the resolution between the peaks corresponding to the (R)-enantiomer and to the (S)-enantiomer is at least 1.5 ; the retention times of the principal peaks in the chromatograms obtained with the test solution and reference solution (a) are identical ((S)-enantiomer). In the chromatogram obtained with the test solution, the area of the peak corresponding to the (R)-enantiomer is not greater than the area of the principal peak in the chromatogram obtained with reference solution (c) (2 per cent) and the area of any peak, apart from the principal peak and any peak corresponding to the (R)-enantiomer, is not greater than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.5 per cent). Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 65 °C for 4 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.


1407

ASSAY Dissolve 0.150 g in 25 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 19.54 mg of C20H23ClN2O4. STORAGE Store protected from light. IMPURITIES

A. (3RS)-N,N-dimethyl-3-phenyl-3-(pyridin-2-yl)propan-1amine,

B. (3R)-3-(4-chlorophenyl)-N,N-dimethyl-3-(pyridin-2yl)propan-1-amine. 01/2005:0761

DEXPANTHENOL Dexpanthenolum

C9H19NO4

Mr 205.3

Dextran 1 for injection


DEFINITION Dexpanthenol contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of (2R)-2, 4-dihydroxy-N-(3-hydroxypropyl)-3,3-dimethylbutanamide, calculated with reference to the anhydrous substance.

in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). Heavy metals (2.4.8). 12 ml of solution S complies with limit test A for heavy metals (20 ppm). Prepare the reference solution using lead standard solution (1 ppm Pb) R. CHARACTERS Water (2.5.12). Not more than 1.0 per cent, determined on A colourless or slightly yellowish, viscous hygroscopic liquid, 1.000 g. or a white or almost white, crystalline powder, very soluble Sulphated ash (2.4.14). Not more than 0.1 per cent, in water, freely soluble in ethanol (96 per cent). determined on 1.0 g. IDENTIFICATION ASSAY First identification : A, B. To 0.400 g add 50.0 ml of 0.1 M perchloric acid. Boil under Second identification : A, C, D. a reflux condenser for 5 h protected from humidity. Allow A. It complies with the test for specific optical rotation (see to cool. Add 50 ml of dioxan R by rinsing the condenser, Tests). protected from humidity. Add 0.2 ml of naphtholbenzein solution R and titrate with 0.1 M potassium hydrogen B. Examine by infrared absorption spectrophotometry phthalate until the colour changes from green to yellow. (2.2.24), comparing with the spectrum obtained with Carry out a blank titration. dexpanthenol CRS. Examine the substances using discs prepared as follows : dissolve the substance to be 1 ml of 0.1 M perchloric acid is equivalent to 20.53 mg examined and the reference substance separately in of C9H19NO4. 1.0 ml of anhydrous ethanol R to obtain a concentration STORAGE of 5 mg/ml. Place dropwise 0.5 ml of this solution on a disc of potassium bromide R. Dry the disc at 100-105 °C In an airtight container. for 15 min. C. Examine the chromatograms obtained in the test for 01/2005:1506 3-aminopropanol. The principal spot in the chromatogram obtained with test solution (b) is similar in position, DEXTRAN 1 FOR INJECTION colour and size to the principal spot in the chromatogram obtained with reference solution (a). Dextranum 1 ad iniectabile D. To 1 ml of solution S (see Tests) add 1 ml of dilute sodium hydroxide solution R and 0.1 ml of copper sulphate DEFINITION solution R. A blue colour develops. Dextran 1 for injection is a low molecular weight fraction of dextran, consisting of a mixture of isomaltooligosaccharides. TESTS Solution S. Dissolve 2.500 g in carbon dioxide-free water R The average relative molecular mass is about 1000. and dilute to 50.0 ml with the same solvent. PRODUCTION Appearance of solution. Solution S is clear (2.2.1) and not It is obtained by hydrolysis and fractionation of dextrans more intensely coloured than reference solution B6 (2.2.2, produced by fermentation of sucrose using Leuconostoc Method II). mesenteroides strain NRRL B-512 = CIP 78.59 or substrains thereof (for example L. mesenteroides B-512 pH (2.2.3). The pH of solution S is not greater than 10.5. F = NCTC 10817). Specific optical rotation (2.2.7). The specific optical rotation is + 29.0 to + 32.0, determined on solution S and calculated It is prepared in conditions designed to minimise the risk of microbial contamination. with reference to the anhydrous substance. CHARACTERS 3-Aminopropanol. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. A white or almost white powder, hygroscopic, very soluble in water, very slightly soluble in alcohol. Test solution (a). Dissolve 0.25 g of the substance to be examined in anhydrous ethanol R and dilute to 5 ml with IDENTIFICATION the same solvent. A. Dissolve 3.000 g in water R, heat on a water-bath and Test solution (b). Dilute 1 ml of test solution (a) to 10 ml dilute to 100.0 ml with the same solvent. The specific with anhydrous ethanol R. optical rotation (2.2.7) is + 148 to + 164, calculated with Reference solution (a). Dissolve the contents of a vial of reference to the dried substance. Dry an aliquot of the dexpanthenol CRS in 1.0 ml of anhydrous ethanol R to solution first on a water-bath and then to constant weight obtain a concentration of 5 mg/ml. in vacuo at 70 °C. Calculate the dextran content after Reference solution (b). Dissolve 25 mg of 3-aminopropanol R correction for the content of sodium chloride. in anhydrous ethanol R and dilute to 100 ml with the same B. Examine by infrared absorption spectrophotometry solvent. (2.2.24), comparing with the spectrum obtained with Apply separately to the plate 10 µl of each solution. Develop dextran 1 CRS. Prepare the discs as follows : to 1-2 mg over a path of 15 cm using a mixture of 20 volumes of add one or a few drops of water R ; grind in an agate concentrated ammonia R, 25 volumes of methanol R and mortar for 1-2 min ; add about 300 mg of potassium 55 volumes of butanol R. Allow the plate to dry in air, bromide R and mix to a slurry (do not grind) ; dry in vacuo spray with a 100 g/l solution of trichloroacetic acid R in at 40 °C for 15 min, crush the residue (if it is not dry, methanol R and heat at 150 °C for 10 min. Spray with a 1 g/l dry for another 15 min). Prepare a disc using potassium solution of ninhydrin R in methanol R and heat at 120 °C bromide R. Run the infrared spectrum with a blank disc until a colour appears. Any spot due to 3-aminopropanol using potassium bromide R in the reference beam. 1408




C. It complies with the test for molecular-mass distribution (see Tests). TESTS Solution S. Dissolve 7.5 g in carbon dioxide-free water R, heat on a water-bath and dilute to 50 ml with the same solvent. Absorbance (2.2.25). Measure the absorbance of solution S at 375 nm. The absorbance is not more than 0.12. Acidity or alkalinity. To 10 ml of solution S add 0.1 ml of phenolphthalein solution R. The solution is colourless. Add 0.2 ml of 0.01 M sodium hydroxide. The solution is pink. Add 0.4 ml of 0.01 M hydrochloric acid. The solution is colourless. Add 0.1 ml of methyl red solution R. The solution is red or orange.

Calculate the average relative molecular mass Mw and the amount of the fraction with less than 3 and more than 9 glucose units, of dextran 1 CRS and of the substance to be examined. The test is not valid unless the values obtained for dextran 1 CRS are within the values stated on the label.

Mw mi

=

average molecular mass of the dextran,

=

molecular mass of oligosaccharide i,

wi

=

weight proportion of oligosaccharide i.

Use the following molecular mass values for the calculation : Oligosaccharide i

mi

glucose

180

Nitrogen-containing substances. Carry out the determination of nitrogen by sulphuric acid digestion (2.5.9), using 0.200 g and heating for 2 h. Collect the distillate in a mixture of 0.5 ml of bromocresol green solution R, 0.5 ml of methyl red solution R and 20 ml of water R. Titrate with 0.01 M hydrochloric acid. Not more than 0.15 ml of 0.01 M hydrochloric acid is required to change the colour of the indicator (110 ppm N).

isomaltose

342

isomaltotriose

504

isomaltotetraose

666

isomaltopentaose

828

isomaltohexaose

990

Sodium chloride. Not more than 1.5 per cent. Accurately weigh 3-5 g and dissolve in 100 ml of water R. Add 0.3 ml of potassium chromate solution R and titrate with 0.1 M silver nitrate until the yellowish-white colour changes to reddish-brown.

isomaltoheptaose

1152

isomaltooctaose

1314

1 ml of 0.1 M silver nitrate is equivalent to 5.844 mg of NaCl. Molecular-mass distribution. The average molecular mass (Mw) is 850 to 1150. The fraction with less than 3 units of glucose is less than 15 per cent, the fraction with more than 9 units of glucose is less than 20 per cent.

isomaltononaose

1476

isomaltodecaose

1638

isomaltoundecaose

1800

isomaltododecaose

1962

isomaltotridecaose

2124

isomaltotetradecaose

2286

isomaltopentadecaose

2448

Examine by size-exclusion chromatography (2.2.30).

isomaltohexadecaose

2610

Test solution. Dissolve 6.0-6.5 mg of the substance to be examined in 1.0 ml of the mobile phase.

isomaltoheptadecaose

2772

2934 Reference solution (a). Dissolve 6.0-6.5 mg of dextran 1 CRS 3096 isomaltononadecaose in 1.0 ml of the mobile phase. Reference solution (b). Dissolve the content of an ampoule Heavy metals (2.4.8). Dilute 20 ml of solution S to 30 ml with water R. 12 ml of this solution complies with limit of isomaltooligosaccharide CRS in 1 ml of the mobile test A (10 ppm). Prepare the reference solution using lead phase, and mix. This corresponds to approximately 45 µg standard solution (1 ppm Pb) R. of isomaltotriose (3 glucose units), approximately 45 µg of isomaltononaose (9 glucose units), and approximately 60 µg Loss on drying (2.2.32). Not more than 5.0 per cent, determined on 5.000 g by drying in an oven at 100-105 °C of sodium chloride per 100 µl. for 5 h. The chromatographic procedure may be carried out using : Bacterial endotoxins (2.6.14) : less than 25 IU/g. — 2 columns, 30 cm long and 10 mm in internal diameter, Microbial contamination. Total viable aerobic count (2.6.12) in series, prepacked with a packing material of dextran not more than 102 micro-organisms per gram, determined covalently bound to highly cross-linked porous agarose by plate-count. It complies with the test for Escherichia beads, allowing resolution of oligosaccharides in coli (2.6.13). the molecular mass range of 180 to 3000, kept at a temperature of 20-25 °C, — as mobile phase at a flow rate of 0.07-0.08 ml/min maintained constant to ± 1 per cent, a 2.92 g/l solution of sodium chloride R, — as detector a differential refractometer. Inject 100 µl of reference solution (b) and record the chromatogram for definition of the positions of isomaltotriose, isomaltononaose and sodium chloride. Inject 100 µl of the test solution and 100 µl of reference solution (a) and record the chromatograms. Determine the peak areas. Disregard any peak due to sodium chloride. General Notices (1) apply to all monographs and other texts

isomaltooctadecaose

01/2005:0999

DEXTRAN 40 FOR INJECTION Dextranum 40 ad iniectabile DEFINITION Dextran 40 for injection is a mixture of polysaccharides, principally of the α-1,6-glucan type. The average relative molecular mass is about 40 000. 1409



PRODUCTION It is obtained by hydrolysis and fractionation of dextrans produced by fermentation of sucrose using Leuconostoc mesenteroides strain NRRL B-512 = CIP 78.59 or substrains thereof (for example L. mesenteroides B-512F = NCTC 10817). It is prepared in conditions designed to minimise the risk of microbial contamination. CHARACTERS A white or almost white powder, very soluble in water, very slightly soluble in alcohol. IDENTIFICATION A. Dissolve 1.0 g in water R, heating on a water-bath, and dilute to 50.0 ml with the same solvent. The specific optical rotation (2.2.7) of the solution is + 195 to + 201, calculated with reference to the dried substance. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with dextran CRS. C. It complies with the test for molecular-mass distribution (see Tests). TESTS Solution S. Dissolve 5.0 g in distilled water R, heating on a water-bath, and dilute to 50 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Acidity or alkalinity. To 10 ml of solution S add 0.1 ml of phenolphthalein solution R. The solution remains colourless. Add 0.2 ml of 0.01 M sodium hydroxide. The solution is red. Add 0.4 ml of 0.01 M hydrochloric acid. The solution is colourless. Add 0.1 ml of methyl red solution R. The solution is red or orange. Nitrogen-containing substances. Carry out the determination of nitrogen by sulphuric acid digestion (2.5.9), using 0.200 g and heating for 2 h. Collect the distillate in a mixture of 0.5 ml of bromocresol green solution R, 0.5 ml of methyl red solution R and 20 ml of water R. Titrate with 0.01 M hydrochloric acid. Not more than 0.15 ml of 0.01 M hydrochloric acid is required to change the colour of the indicator (110 ppm N). Residual solvents. Examine by gas chromatography (2.2.28), using propanol R as internal standard. Test solution. Dissolve 5 g of the substance to be examined in 100 ml of water R and distil. Collect the first 45 ml of the distillate, add 1 ml of a 25 g/l solution of propanol R and dilute to 50 ml with water R. Reference solution. Mix 0.5 ml of a 25 g/l solution of ethanol R, 0.5 ml of a 25 g/l solution of propanol R and 0.5 ml of a 2.5 g/l solution of methanol R and dilute to 25.0 ml with water R. The chromatographic procedure may be carried out using : — a stainless steel column 1.8 m long and 2 mm in internal diameter packed with ethylvinylbenzene-divinylbenzene copolymer R (125-150 µm), — nitrogen for chromatography R as the carrier gas at a flow rate of 25 ml/min, — a flame-ionisation detector, maintaining the temperature of the column at 190 °C, that of the injection port at 240 °C and that of the detector at 210 °C. Inject the chosen volume of each solution. In the chromatogram obtained with the test solution, the area of any peak corresponding to ethanol or methanol is not greater than the area of the corresponding peak in the 1410

chromatogram obtained with the reference solution (0.5 per cent of ethanol and 0.05 per cent of methanol) and the sum of the areas of any peaks, apart from the peaks corresponding to ethanol, methanol and the internal standard, is not greater than the area of the peak corresponding to the internal standard (0.5 per cent calculated as propanol). Molecular-mass distribution (2.2.39). The average molecular mass (Mw) is 35 000 to 45 000. The average molecular mass of the 10 per cent high fraction is not more than 110 000. The average molecular mass of the 10 per cent low fraction is not less than 7000. Heavy metals (2.4.8). 12 ml of solution S complies with limit test A (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32). Not more than 7.0 per cent, determined on 0.200 g by heating in an oven at 105 ± 2 °C for 5 h. Sulphated ash (2.4.14). Not more than 0.3 per cent, determined on 0.50 g. Bacterial endotoxins (2.6.14) : less than 10 IU/g. Microbial contamination. Total viable aerobic count (2.6.12) not more than 102 micro-organisms per gram, determined by plate-count. It complies with the test for Escherichia coli (2.6.13). 01/2005:1000

DEXTRAN 60 FOR INJECTION Dextranum 60 ad iniectabile DEFINITION Dextran 60 for injection is a mixture of polysaccharides, principally of the α-1,6-glucan type. The average relative molecular mass is about 60 000. PRODUCTION It is obtained by hydrolysis and fractionation of dextrans produced by fermentation of sucrose using Leuconostoc mesenteroides strain NRRL B-512 = CIP 78.59 or substrains thereof (for example L. mesenteroides B-512F = NCTC 10817). It is prepared in conditions designed to minimise the risk of microbial contamination. CHARACTERS A white or almost white powder, very soluble in water, very slightly soluble in alcohol. IDENTIFICATION A. Dissolve 1.0 g in water R, heating on a water-bath, and dilute to 50.0 ml with the same solvent. The specific optical rotation (2.2.7) of the solution is + 195 to + 201, calculated with reference to the dried substance. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with dextran CRS. C. It complies with the test for molecular-mass distribution (see Tests). TESTS Solution S. Dissolve 5.0 g in distilled water R, heating on a water-bath, and dilute to 50 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II).



Acidity or alkalinity. To 10 ml of solution S add 0.1 ml of phenolphthalein solution R. The solution remains colourless. Add 0.2 ml of 0.01 M sodium hydroxide. The solution is red. Add 0.4 ml of 0.01 M hydrochloric acid. The solution is colourless. Add 0.1 ml of methyl red solution R. The solution is red or orange. Nitrogen-containing substances. Carry out the determination of nitrogen by sulphuric acid digestion (2.5.9), using 0.200 g and heating for 2 h. Collect the distillate in a mixture of 0.5 ml of bromocresol green solution R, 0.5 ml of methyl red solution R and 20 ml of water R. Titrate with 0.01 M hydrochloric acid. Not more than 0.15 ml of 0.01 M hydrochloric acid is required to change the colour of the indicator (110 ppm N).


01/2005:1001

DEXTRAN 70 FOR INJECTION Dextranum 70 ad iniectabile DEFINITION Dextran 70 for injection is a mixture of polysaccharides, principally of the α-1,6-glucan type. The average relative molecular mass is about 70 000.

PRODUCTION It is obtained by hydrolysis and fractionation of dextrans Residual solvents. Examine by gas chromatography (2.2.28), produced by fermentation of sucrose using Leuconostoc mesenteroides strain NRRL B-512 = CIP 78.59 or substrains using propanol R as internal standard. thereof (for example L. mesenteroides B-512F = NCTC Test solution. Dissolve 5 g of the substance to be examined 10817). in 100 ml of water R and distil. Collect the first 45 ml of the It is prepared in conditions designed to minimise the risk distillate, add 1 ml of a 25 g/l solution of propanol R and of microbial contamination. dilute to 50 ml with water R. CHARACTERS Reference solution. Mix 0.5 ml of a 25 g/l solution of A white or almost white powder, very soluble in water, very ethanol R, 0.5 ml of a 25 g/l solution of propanol R and slightly soluble in alcohol. 0.5 ml of a 2.5 g/l solution of methanol R and dilute to 25.0 ml with water R. IDENTIFICATION The chromatographic procedure may be carried out using : A. Dissolve 1.0 g in water R, heating on a water-bath, and dilute to 50.0 ml with the same solvent. The specific — a stainless steel column 1.8 m long and 2 mm in internal optical rotation (2.2.7) of the solution is + 195 to + 201, diameter packed with ethylvinylbenzene-divinylbenzene calculated with reference to the dried substance. copolymer R (125-150 µm), B. Examine by infrared absorption spectrophotometry — nitrogen for chromatography R as the carrier gas at a (2.2.24), comparing with the spectrum obtained with flow rate of 25 ml/min, dextran CRS. C. It complies with the test for molecular-mass distribution — a flame-ionisation detector, (see Tests). maintaining the temperature of the column at 190 °C, that of the injection port at 240 °C and that of the detector at TESTS 210 °C. Inject the chosen volume of each solution. In the Solution S. Dissolve 5.0 g in distilled water R, heating on a chromatogram obtained with the test solution, the area water-bath, and dilute to 50 ml with the same solvent. of any peak corresponding to ethanol or methanol is not Appearance of solution. Solution S is clear (2.2.1) and greater than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.5 per colourless (2.2.2, Method II). cent of ethanol and 0.05 per cent of methanol) and the sum Acidity or alkalinity. To 10 ml of solution S add 0.1 ml of the areas of any peaks, apart from the peaks corresponding of phenolphthalein solution R. The solution remains to ethanol, methanol and the internal standard, is not greater colourless. Add 0.2 ml of 0.01 M sodium hydroxide. The than the area of the peak corresponding to the internal solution is red. Add 0.4 ml of 0.01 M hydrochloric acid. The standard (0.5 per cent calculated as propanol). solution is colourless. Add 0.1 ml of methyl red solution R. Molecular-mass distribution (2.2.39). The average molecular The solution is red or orange. mass (Mw) is 54 000 to 66 000. The average molecular mass Nitrogen-containing substances. Carry out the of the 10 per cent high fraction is not more than 180 000. determination of nitrogen by sulphuric acid digestion (2.5.9), The average molecular mass of the 10 per cent low fraction using 0.200 g and heating for 2 h. Collect the distillate in a is not less than 14 000. mixture of 0.5 ml of bromocresol green solution R, 0.5 ml Heavy metals (2.4.8). 12 ml of solution S complies with limit of methyl red solution R and 20 ml of water R. Titrate with 0.01 M hydrochloric acid. Not more than 0.15 ml of 0.01 M test A (10 ppm). Prepare the standard using lead standard hydrochloric acid is required to change the colour of the solution (1 ppm Pb) R. indicator (110 ppm N). Loss on drying (2.2.32). Not more than 7.0 per cent, determined on 0.200 g by heating in an oven at 105 ± 2 °C Residual solvents. Examine by gas chromatography (2.2.28), using propanol R as internal standard. for 5 h. Test solution. Dissolve 5 g of the substance to be examined Sulphated ash (2.4.14). Not more than 0.3 per cent, in 100 ml of water R and distil. Collect the first 45 ml of the determined on 0.50 g. distillate, add 1 ml of a 25 g/l solution of propanol R and Bacterial endotoxins (2.6.14) : less than 16 IU/g. dilute to 50 ml with water R. Reference solution. Mix 0.5 ml of a 25 g/l solution of Microbial contamination. Total viable count (2.6.12) not ethanol R, 0.5 ml of a 25 g/l solution of propanol R and more than 102 micro-organisms per gram, determined by 0.5 ml of a 2.5 g/l solution of methanol R and dilute to plate-count. It complies with the test for Escherichia coli 25.0 ml with water R. (2.6.13). General Notices (1) apply to all monographs and other texts

1411

Dextrin


The chromatographic procedure may be carried out using : — a stainless steel column 1.8 m long and 2 mm in internal diameter packed with ethylvinylbenzene-divinylbenzene copolymer R (125-150 µm), — nitrogen for chromatography R as the carrier gas at a flow rate of 25 ml/min, — a flame-ionisation detector, maintaining the temperature of the column at 190 °C, that of the injection port at 240 °C and that of the detector at 210 °C. Inject the chosen volume of each solution. In the chromatogram obtained with the test solution, the area of any peak corresponding to ethanol or methanol is not greater than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.5 per cent of ethanol and 0.05 per cent of methanol) and the sum of the areas of any peaks, apart from the peaks corresponding to ethanol, methanol and the internal standard, is not greater than the area of the peak corresponding to the internal standard (0.5 per cent calculated as propanol). Molecular-mass distribution (2.2.39). The average molecular mass (Mw) is 64 000 to 76 000. The average molecular mass of the 10 per cent high fraction is not more than 185 000. The average molecular mass of the 10 per cent low fraction is not less than 15 000. Heavy metals (2.4.8). 12 ml of solution S complies with limit test A (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32). Not more than 7.0 per cent, determined on 0.200 g by heating in an oven at 105 ± 2 °C for 5 h. Sulphated ash (2.4.14). Not more than 0.3 per cent, determined on 0.50 g. Bacterial endotoxins (2.6.14) : less than 16 IU/g. Microbial contamination. Total viable count (2.6.12) not more than 102 micro-organisms per gram, determined by plate-count. It complies with the test for Escherichia coli (2.6.13).

TESTS pH (2.2.3). Disperse 5.0 g in 100 ml of carbon dioxide-free water R. The pH is 2.0 to 8.0. Chlorides. Dissolve 2.5 g in 50 ml of boiling water R, dilute to 100 ml with water R and filter. Dilute 1 ml of the filtrate to 15 ml, add 1 ml of dilute nitric acid R, pour the mixture as a single addition into 1 ml of silver nitrate solution R2 and allow to stand for 5 min protected from light. When viewed transversely against a black background any opalescence produced is not more intense than that obtained by treating a mixture of 10 ml of chloride standard solution (5 ppm Cl) R and 5 ml of water R, prepared in the same manner (0.2 per cent). Reducing sugars. To a quantity of dextrin equivalent to 2.0 g (dried substance) add 100 ml of water R, shake for 30 min, dilute with water R to 200.0 ml and filter. To 10.0 ml of alkaline cupri-tartaric solution R add 20.0 ml of the filtrate, mix, and heat on a hot plate adjusted to bring the solution to boil within 3 min. Boil for 2 min, and cool immediately. Add 5 ml of a 300 g/l solution of potassium iodide R and 10 ml of 1 M sulphuric acid, mix, and titrate immediately with 0.1 M sodium thiosulphate, using starch solution R, added towards the end of the titration, as indicator. Repeat the procedure beginning with “To 10.0 ml of...”, using, in place of the filtrate, 20.0 ml of a 1 g/l solution of glucose R, accurately prepared. Perform a blank titration. (VB − VU) is not greater than (VB − VS), in which VB, VU and VS are the number of millilitres of 0.1 M sodium thiosulphate consumed in the titrations of the blank, the dextrin and the glucose, respectively (10 per cent, calculated as glucose C6H12O6). Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 13.0 per cent, determined on 1.000 g by drying at 130 °C to 135 °C for 90 min. Sulphated ash (2.4.14). Not more than 0.5 per cent, determined on 1.0 g.

01/2005:1507 01/2005:0020

DEXTRIN Dextrinum DEFINITION Dextrin is maize, potato or cassava starch partly hydrolysed and modified by heating with or without the presence of acids, alkalis or pH control agents.

DEXTROMETHORPHAN HYDROBROMIDE Dextromethorphani hydrobromidum

CHARACTERS White or almost white, free-flowing powder, very soluble in boiling water forming a mucilaginous solution, slowly soluble in cold water, practically insoluble in alcohol. IDENTIFICATION A. Suspend 1 g in 50 ml of water R, boil for 1 min and cool. To 1 ml of the solution add 0.05 ml of iodine solution R1. A dark blue to reddish-brown colour is produced which disappears on heating. B. Centrifuge 5 ml of the mucilage obtained in identification test A. To the upper layer add 2 ml of dilute sodium hydroxide solution R and, dropwise with shaking, 0.5 ml of copper sulphate solution R and boil. A red precipitate is produced. C. It is very soluble in boiling water R, forming a mucilaginous solution. 1412

C18H26BrNO,H2O

Mr 370.3

DEFINITION ent-3-Methoxy-17-methylmorphinan hydrobromide monohydrate. Content : 99.0 per cent to 101.0 per cent (anhydrous substance). CHARACTERS Appearance : almost white, crystalline powder.


Dextromethorphan hydrobromide


Solubility : sparingly soluble in water, freely soluble in alcohol.

Reference solution (b). Dilute 1.0 ml of the test solution to 200.0 ml with the mobile phase.

mp : about 125 °C, with decomposition.

Column :

IDENTIFICATION First identification : A, B, D.

— size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : octadecylsilyl silica gel for chromatography R (5 µm).

A. It complies with the test for specific optical rotation (see Tests).

Mobile phase : dissolve 3.11 g of docusate sodium R in a mixture of 400 ml of water R and 600 ml of acetonitrile R. Add 0.56 g of ammonium nitrate R. Adjust to apparent pH 2.0 with glacial acetic acid R.

B. Infrared absorption spectrophotometry (2.2.24).


Second identification : A, C, D.

Preparation : discs.

Detection : spectrophotometer at 280 nm.

Comparison : dextromethorphan hydrobromide CRS.

Injection : 20 µl.

C. Thin-layer chromatography (2.2.27).

Run time : twice the retention time of dextromethorphan.

Test solution. Dissolve 25 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent.

Relative retention with reference to dextromethorphan (retention time = about 21.9 min) : impurity B = about 0.44 ; impurity C = about 0.85 ; impurity D = about 0.90 ; Reference solution. Dissolve 25 mg of dextromethorphan impurity A = about 1.13. hydrobromide CRS in methanol R and dilute to 10 ml System suitability : reference solution (a) : with the same solvent. — resolution : minimum 1.5 between the peaks due to Plate : TLC silica gel G plate R.

impurity A and dextromethorphan.

Mobile phase : concentrated ammonia R, methylene chloride R, methanol R, ethyl acetate R, toluene R (2:10:13:20:55 V/V/V/V/V).

Limits :

Application : 5 µl.

— any impurity : not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent), and not more than 1 such peak has an area greater than half the area of the principal peak in the chromatogram obtained with reference solution (b) (0.25 per cent),

Development : over 2/3 of the plate. Drying : in air. Detection : spray with potassium iodobismuthate solution R2.

— correction factor : for the calculation of content, multiply the peak area of impurity C by 0.2,

Results : the principal spot in the chromatogram obtained — total : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (b) with the test solution is similar in position and size to (1 per cent), the principal spot in the chromatogram obtained with the reference solution. — disregard limit : 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b) D. It gives reaction (a) of bromides (2.3.1). (0.05 per cent). N,N-Dimethylaniline : maximum 10 ppm. TESTS Dissolve 0.5 g with heating in 20 ml of water R. Allow to cool, add 2 ml of dilute acetic acid R and 1 ml of a 10 g/l solution of sodium nitrite R and dilute to 25 ml with water R. The Appearance of solution. Solution S is clear (2.2.1) and solution is not more intensely coloured than a reference colourless (2.2.2, Method II). solution prepared at the same time in the same manner using 20 ml of a 0.25 mg/l solution of dimethylaniline R. Acidity or alkalinity. Dissolve 0.4 g in carbon dioxide-free water R with gentle heating, cool and dilute to 20 ml with Water (2.5.12) : 4.0 per cent to 5.5 per cent, determined on the same solvent. Add 0.1 ml of methyl red solution R and 0.200 g. 0.2 ml of 0.01 M sodium hydroxide. The solution is yellow. Not more than 0.4 ml of 0.01 M hydrochloric acid is required Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. to change the colour of the indicator to red. Solution S. Dissolve 1.0 g in alcohol R and dilute to 20 ml with the same solvent.

Specific optical rotation (2.2.7). + 28 to + 30 ( anhydrous substance). Dissolve 0.200 g in 0.1 M hydrochloric acid and dilute to 10.0 ml with the same acid. Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 10.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (a). Dissolve 2 mg of dextromethorphan impurity A CRS in 2 ml of the test solution and dilute to 25.0 ml with the mobile phase. General Notices (1) apply to all monographs and other texts

ASSAY Dissolve 0.300 g in a mixture of 5.0 ml of 0.01 M hydrochloric acid and 20 ml of alcohol R. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20). Read the volume added between the 2 points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 35.23 mg of C18H26BrNO. STORAGE Protected from light. 1413

Dextromoramide tartrate


IMPURITIES

A. B. C.

D.

C. It gives reaction (b) of tartrates (2.3.1).

TESTS pH (2.2.3). Dissolve 0.2 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. The pH of the solution is 3.0 to 4.0. Specific optical rotation (2.2.7). Dissolve 0.50 g in 0.1 M hydrochloric acid and dilute to 10.0 ml with the same acid. The specific optical rotation is + 21 to + 23. Related substances. Examine by thin-layer chromatography R1 = CH3, R2 = H, X = H2 : ent-3-methoxymorphinan, (2.2.27), using silica gel G R as the coating substance. R1 = H, R2 = CH3, X = H2 : ent-17-methylmorphinan-3-ol, Test solution. Dissolve 0.2 g of the substance to be examined R1 = R2 = CH3, X = O : ent-3-methoxy-17-methylmorphinan- in methanol R and dilute to 10 ml with the same solvent. 10-one, Reference solution. Dilute 1 ml of the test solution to 100 ml with methanol R. Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm using methanol R. Allow the plate to dry in air and spray with dilute potassium iodobismuthate solution R. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution (1.0 per cent). Loss on drying (2.2.32). Not more than 0.5 per cent, ent-(14S)-3-methoxy-17-methylmorphinan. determined on 1.00 g by drying in an oven at 100 °C to 105 °C. 01/2005:0021 Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

DEXTROMORAMIDE TARTRATE Dextromoramidi tartras

ASSAY Dissolve 0.250 g in 30 ml of anhydrous acetic acid R. Titrate with 0.05 M perchloric acid using 0.15 ml of naphtholbenzein solution R as indicator. 1 ml of 0.05 M perchloric acid is equivalent to 27.13 mg of C29H38N2O8. 01/2005:0713

DEXTROPROPOXYPHENE HYDROCHLORIDE C29H38N2O8

Mr 542.6

Dextropropoxypheni hydrochloridum

DEFINITION Dextromoramide tartrate contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of 1-[(3S)3-methyl-4-(morpholin-4-yl)-2,2-diphenylbutanoyl]pyrrolidine hydrogen (2R,3R)-2,3-dihydroxybutanedioate, calculated with reference to the dried substance. CHARACTERS A white, amorphous or crystalline powder, soluble in water, sparingly soluble in alcohol. It melts at about 190 °C, with slight decomposition. IDENTIFICATION A. Dissolve 75 mg in 1 M hydrochloric acid and dilute to 100.0 ml with the same acid. Examined between 230 nm and 350 nm (2.2.25), the solution shows 3 absorption maxima, at 254 nm, 259 nm and 264 nm. The specific absorbances at the maxima are about 6.9, 7.7 and 6.5, respectively. B. Dissolve about 50 mg in water R and dilute to 10 ml with the same solvent. To 2 ml of the solution add 3 ml of ammoniacal silver nitrate solution R and heat on a water-bath. A grey or black precipitate is formed. 1414

C22H30ClNO2

Mr 375.9

DEFINITION Dextropropoxyphene hydrochloride contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of (1S,2R)-1-benzyl-3-(dimethylamino)-2-methyl-1phenylpropyl propanoate hydrochloride, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, very soluble in water, freely soluble in alcohol. It melts at about 165 °C.


Diazepam


IDENTIFICATION First identification : A, C, D. Second identification : A, B, D. A. It complies with the test for specific optical rotation (see Tests). B. Dissolve 50.0 mg in 0.01 M hydrochloric acid and dilute to 100.0 ml with the same acid. Examined between 220 nm and 360 nm (2.2.25), the solution shows 3 absorption maxima, at 252 nm, 257 nm and 263 nm and 2 shoulders, at 240 nm and 246 nm. The ratio of the absorbance at the maximum at 257 nm to that at the maximum at 252 nm is 1.22 to 1.28. The ratio of the absorbance at the maximum at 257 nm to that at the maximum at 263 nm is 1.29 to 1.35. The test is not valid unless, in the test for resolution (2.2.25), the ratio of the absorbances is at least 1.5. C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of dextropropoxyphene hydrochloride. D. Solution S (see Tests) gives reaction (a) of chlorides (2.3.1). TESTS Solution S. Dissolve 1.5 g in carbon dioxide-free water R and dilute to 30 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Acidity or alkalinity. Dilute 10 ml of solution S to 25 ml with carbon dioxide-free water R. To 10 ml of this solution, add 0.1 ml of methyl red solution R and 0.2 ml of 0.01 M sodium hydroxide. The solution is yellow. Add 0.4 ml of 0.01 M hydrochloric acid. The solution is red. Specific optical rotation (2.2.7). Dissolve 0.100 g in water R and dilute to 10.0 ml with the same solvent. The specific optical rotation is + 52 to + 57. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (a). Dilute 0.50 ml of the test solution to 100.0 ml with the mobile phase. Reference solution (b). Dissolve 50.0 mg of the substance to be examined in 2.5 ml of 2 M alcoholic potassium hydroxide. Add 2.5 ml of water R and boil under a reflux condenser for 30 min. Add 2.5 ml of dilute hydrochloric acid R and dilute to 50 ml with the mobile phase. The chromatographic procedure may be carried out using : — a column 0.125 m long and 4.6 mm in internal diameter packed with silica gel for chromatography R (5 µm), — a guard column packed with a suitable silica gel, equilibrated with the mobile phase and placed between the column and the injection device, — as mobile phase at a flow rate of 1.0 ml/min a mixture of 50 volumes of 0.2 M phosphate buffer solution pH 7.5 R, 84 volumes of tetrahydrofuran R, 350 volumes of methanol R and 516 volumes of water R, containing 0.9 g/l of cetyltrimethylammonium bromide R, — as detector a spectrophotometer set at 220 nm, — a loop injector. Equilibrate the chromatographic system by passage of the mobile phase for 16 h (the mobile phase may be recirculated after 6 h). General Notices (1) apply to all monographs and other texts

Inject 20 µl of each solution and record the chromatograms for twice the retention time of the principal peak. The test is not valid unless : — the chromatogram obtained with reference solution (a) shows a peak with a signal-to-noise ratio of at least 5, — the chromatogram obtained with reference solution (b) shows 2 peaks with a resolution of not less than 2.0. In the chromatogram obtained with the test solution, the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent). Heavy metals (2.4.8). 12 ml of solution S complies with limit test A (20 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.270 g in 60 ml of acetic anhydride R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 37.59 mg of C22H30ClNO2. STORAGE Protected from light. IMPURITIES

A. R = H : (2S,3R)-4-(dimethylamino)-1,2-diphenyl-3-methylbutan-2-ol, B. R = CO-CH3 : (1S,2R)-1-benzyl-3-(dimethylamino)-2-methyl1-phenylpropyl acetate.

01/2005:0022

DIAZEPAM Diazepamum

C16H13ClN2O

Mr 284.7 1415

Diazoxide


DEFINITION Diazepam contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of 7-chloro-1-methyl-5phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one, calculated with reference to the dried substance.

1 ml of 0.1 M perchloric acid is equivalent to 28.47 mg of C16H13ClN2O. STORAGE Store protected from light. 01/2005:0550

CHARACTERS A white or almost white, crystalline powder, very slightly soluble in water, soluble in alcohol. IDENTIFICATION A. Melting point (2.2.14) : 131 °C to 135 °C. B. Protect the solutions from bright light and measure the absorbances immediately. Dissolve 25 mg in a 5 g/l solution of sulphuric acid R in methanol R and dilute to 250.0 ml with the same acid solution (solution A). Dilute 5.0 ml of solution A to 100.0 ml with a 5 g/l solution of sulphuric acid R in methanol R. Examined between 230 nm and 330 nm (2.2.25), the solution shows 2 absorption maxima, at 242 nm and 285 nm. The specific absorbance at the maximum at 242 nm is about 1020. Dilute 25.0 ml of solution A to 100.0 ml with a 5 g/l solution of sulphuric acid R in methanol R. Examined between 325 nm and 400 nm, the solution shows a single absorption maximum at 366 nm. The specific absorbance at the maximum is 140 to 155. C. Dissolve about 10 mg in 3 ml of sulphuric acid R. The solution shows greenish-yellow fluorescence in ultraviolet light at 365 nm. D. To 80 mg in a porcelain crucible add 0.3 g of anhydrous sodium carbonate R. Heat over an open flame for 10 min. Allow to cool. Take up the residue with 5 ml of dilute nitric acid R and filter. To 1 ml of the filtrate add 1 ml of water R. The solution gives reaction (a) of chlorides (2.3.1).

DIAZOXIDE Diazoxidum

C8H7ClN2O2S

Mr 230.7

DEFINITION Diazoxide contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of 7-chloro-3-methyl-2H-1,2,4-benzothiadiazine 1,1-dioxide, calculated with reference to the dried substance. CHARACTERS A white or almost white, fine or crystalline powder, practically insoluble in water, freely soluble in dimethylformamide, slightly soluble in alcohol. It is very soluble in dilute solutions of the alkali hydroxides.

IDENTIFICATION First identification : B. Second identification : A, C, D. A. Dissolve 50.0 mg in 5 ml of 1 M sodium hydroxide and dilute to 50.0 ml with water R. Dilute 1.0 ml of this TESTS solution to 100.0 ml with 0.1 M sodium hydroxide. Examined between 230 nm and 350 nm (2.2.25), the Related substances and decomposition products. Carry out solution shows an absorption maximum at 280 nm and the test protected from bright light. Examine by thin-layer a shoulder at 304 nm. The specific absorbance at the chromatography (2.2.27), using silica gel GF254 R as the maximum is 570 to 610. coating substance. B. Examine by infrared absorption spectrophotometry Test solution. Dissolve 1.0 g of the substance to be examined (2.2.24), comparing with the spectrum obtained with in acetone R and dilute to 10 ml with the same solvent. diazoxide CRS. Examine the substances prepared as Prepare immediately before use. discs using potassium bromide R. Reference solution. Dilute 1 ml of the test solution to 100 ml C. Examine the chromatograms obtained in the test for with acetone R. Dilute 1 ml of this solution to 10 ml with related substances in ultraviolet light at 254 nm. The acetone R. principal spot in the chromatogram obtained with test Apply separately to the plate 5 µl of each solution. Develop solution (b) is similar in position and size to the principal over a path of 12 cm using a mixture of equal volumes of spot in the chromatogram obtained with reference ethyl acetate R and hexane R. Allow the plate to dry and solution (b). examine in ultraviolet light at 254 nm. Any spot in the D. Dissolve about 20 mg in a mixture of 5 ml of hydrochloric chromatogram obtained with the test solution, apart from acid R and 10 ml of water R. Add 0.1 g of zinc powder R. the principal spot, is not more intense than the spot in the Boil for 5 min, cool and filter. To the filtrate add 2 ml chromatogram obtained with the reference solution (0.1 per of a 1 g/l solution of sodium nitrite R and mix. Allow cent). to stand for 1 min and add 1 ml of a 5 g/l solution of Heavy metals (2.4.8). 2.0 g complies with limit test C for naphthylethylenediamine dihydrochloride R. A red or heavy metals (20 ppm). Prepare the standard using 4 ml of violet-red colour develops. lead standard solution (10 ppm Pb) R. TESTS Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying at 60 °C in vacuo for 4 h. Appearance of solution. Dissolve 0.4 g in 2 ml of 1 M sodium hydroxide and dilute to 20 ml with water R. The Sulphated ash (2.4.14). Not more than 0.1 per cent, solution is clear (2.2.1) and not more intensely coloured determined on 1.0 g. than reference solution Y7 (2.2.2, Method II). ASSAY Acidity or alkalinity. To 0.5 g of the powdered substance Dissolve 0.500 g in 50 ml of acetic anhydride R. Using 0.3 ml to be examined add 30 ml of carbon dioxide-free water R, of Nile blue A solution R as indicator, titrate with 0.1 M shake for 2 min and filter. To 10 ml of the filtrate add 0.2 ml perchloric acid until a yellowish-green colour is obtained. of 0.01 M sodium hydroxide and 0.15 ml of methyl red 1416


Dibutyl phthalate


solution R. The solution is yellow. Not more than 0.4 ml of 0.01 M hydrochloric acid is required to change the colour of the indicator to red. Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution (a). Dissolve 0.1 g of the substance to be examined in a mixture of 0.5 ml of 1 M sodium hydroxide and 1 ml of methanol R and dilute to 5 ml with methanol R. Test solution (b). Dilute 1 ml of test solution (a) to 5 ml with a mixture of 1 volume of 1 M sodium hydroxide and 9 volumes of methanol R. Reference solution (a). Dilute 0.5 ml of test solution (a) to 100 ml with a mixture of 1 volume of 1 M sodium hydroxide and 9 volumes of methanol R. Reference solution (b). Dissolve 20 mg of diazoxide CRS in a mixture of 0.5 ml of 1 M sodium hydroxide and 1 ml of methanol R and dilute to 5 ml with methanol R. Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 7 volumes of concentrated ammonia R, 25 volumes of methanol R and 68 volumes of chloroform R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.5 per cent). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C for 2 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.200 g with gentle heating in 50 ml of a mixture of 1 volume of water R and 2 volumes of dimethylformamide R. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20). Carry out a blank titration. 1 ml of 0.1 M sodium hydroxide is equivalent to 23.07 mg of C8H7ClN2O2S. 01/2005:0762

DIBUTYL PHTHALATE Dibutylis phthalas

C16H22O4

Mr 278.3

DEFINITION Dibutyl phthalate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent m/m of dibutyl benzene-1,2-dicarboxylate. CHARACTERS A clear, oily liquid, colourless or very slightly yellow, practically insoluble in water, miscible with alcohol. General Notices (1) apply to all monographs and other texts

IDENTIFICATION First identification : B, C. Second identification : A, D, E. A. Relative density (2.2.5) : 1.043 to 1.048. B. Refractive index (2.2.6) : 1.490 to 1.495. C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with dibutyl phthalate CRS. D. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 50 mg of the substance to be examined in ether R and dilute to 10 ml with the same solvent. Reference solution. Dissolve 50 mg of dibutyl phthalate CRS in ether R and dilute to 10 ml with the same solvent. Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm using a mixture of 30 volumes of heptane R and 70 volumes of ether R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. E. To about 0.1 ml add 0.25 ml of sulphuric acid R and 50 mg of resorcinol R. Heat in a water-bath for 5 min. Allow to cool. Add 10 ml of water R and 1 ml of strong sodium hydroxide solution R. The solution becomes yellow or brownish-yellow and shows a green fluorescence. TESTS Appearance. The substance to be examined is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). Acidity. Dissolve 20.0 g in 50 ml of alcohol R previously neutralised to phenolphthalein solution R1. Add 0.2 ml of phenolphthalein solution R1. Not more than 0.50 ml of 0.1 M sodium hydroxide is required to change the colour of the indicator. Related substances. Examine by gas chromatography (2.2.28), using bibenzyl R as internal standard. Internal standard solution. Dissolve 60 mg of bibenzyl R in methylene chloride R and dilute to 20 ml with the same solvent. Test solution (a). Dissolve 1.0 g of the substance to be examined in methylene chloride R and dilute to 20.0 ml with the same solvent. Test solution (b). Dissolve 1.0 g of the substance to be examined in methylene chloride R, add 2.0 ml of the internal standard solution and dilute to 20.0 ml with methylene chloride R. Reference solution. To 1.0 ml of test solution (a) add 10.0 ml of the internal standard solution and dilute to 100.0 ml with methylene chloride R. The chromatographic procedure may be carried out using : — a glass column 1.5 m long and 4 mm in internal diameter, packed with silanised diatomaceous earth for gas chromatography R (150 µm to 180 µm) impregnated with 3 per cent m/m of polymethylphenylsiloxane R, — nitrogen for chromatography R as the carrier gas at a flow rate of 30 ml per minute, — a flame-ionisation detector, maintaining the temperature of the column at 190 °C and that of the injection port and of the detector at 225 °C. 1417

Diclazuril for veterinary use


Inject 1 µl of the reference solution. The substances elute in the following order : bibenzyl and dibutyl phthalate. Adjust the sensitivity of the detector so that the height of the bibenzyl peak is not less than 70 per cent of the full scale of the recorder. The test is not valid unless the resolution between the peaks corresponding to bibenzyl and dibutyl phthalate is at least 12. Inject 1 µl of test solution (a). In the chromatogram obtained, verify that there is no peak with the same retention time as the internal standard. Inject separately 1 µl of test solution (b) and the reference solution. Continue the chromatography for 3 times the retention time of dibutyl phthalate, which is about 12 min. From the chromatogram obtained with the reference solution, calculate the ratio (R) of the area of the peak due to dibutyl phthalate to the area of the peak due to the internal standard. From the chromatogram obtained with test solution (b), calculate the ratio of the sum of the areas of any peaks, apart from the principal peak, the peak due to the internal standard and the peak due to the solvent, to the area of the peak due to the internal standard : this ratio is not greater than R (1.0 per cent). Water (2.5.12). Not more than 0.2 per cent, determined on 10.00 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

IDENTIFICATION Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of diclazuril.

TESTS Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 20.0 mg of the substance to be examined in dimethylformamide R and dilute to 20.0 ml with the same solvent. Reference solution (a). Dissolve 5 mg of diclazuril for system suitability CRS in dimethylformamide R and dilute to 5.0 ml with the same solvent. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with dimethylformamide R. Dilute 5.0 ml of the solution to 20.0 ml with dimethylformamide R. Column : — size : l = 0.10 m, Ø = 4.6 mm, — stationary phase : base-deactivated octadecylsilyl silica gel for chromatography R (3 µm), — temperature : 35 °C. Mobile phase : — mobile phase A : mix 10 volumes of a 6.3 g/l solution of ammonium formate R adjusted to pH 4.0 with anhydrous formic acid R, 15 volumes of acetonitrile R and 75 volumes of water R, — mobile phase B : mix 10 volumes of a 6.3 g/l solution ASSAY of ammonium formate R adjusted to pH 4.0 with Introduce 0.750 g into a 250 ml borosilicate glass flask. Add anhydrous formic acid R, 85 volumes of acetonitrile R 25.0 ml of 0.5 M alcoholic potassium hydroxide and a few and 5 volumes of water R, glass beads. Heat in a water-bath under a reflux condenser Time Mobile phase A Mobile phase B for 1 h. Add 1 ml of phenolphthalein solution R1 and titrate (min) (per cent V/V) (per cent V/V) immediately with 0.5 M hydrochloric acid until the colour changes from red to colourless. Carry out a blank titration. 0 - 20 100 → 0 0 → 100 Calculate the volume of potassium hydroxide used in the 0 20 - 25 100 saponification. 25 - 26 0 → 100 100 → 0 1 ml of 0.5 M alcoholic potassium hydroxide is equivalent to 69.59 mg of C16H22O4. 26 - 36 0 100


Flow rate : 1.0 ml/min. Detection : spectrophotometer at 230 nm. Injection : 5 µl. 01/2005:1718 System suitability : reference solution (a) : — peak-to-valley ratio : minimum of 1.5, where Hp = height DICLAZURIL FOR VETERINARY USE above the baseline of the peak due to impurity D and Hv = height above the baseline of the lowest point of the Diclazurilum ad usum veterinarium curve separating this peak from the peak due to diclazuril. Limits : — correction factors : for the calculation of contents, multiply the peak areas of the following impurities by the corresponding correction factor : impurity D = 1.9 ; impurity H = 1.4, — impurity D : not more than 0.4 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent), C17H9Cl3N4O2 Mr 407.6 — any other impurity : not more than the area of the principal peak in the chromatogram obtained with DEFINITION reference solution (b) (0.25 per cent), (RS)-(4-Chlorophenyl)[2,6-dichloro-4-(3,5-dioxo-4,5-dihydro-1, — total : not more than 4 times the area of the principal peak 2,4-triazin-2(3H)-yl)phenyl]acetonitrile. in the chromatogram obtained with reference solution (b) Content : 99.0 per cent to 101.0 per cent (dried substance). (1.0 per cent), CHARACTERS — disregard limit : 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (b) Appearance : white or light yellow powder. (0.05 per cent). Solubility : practically insoluble in water, sparingly soluble Loss on drying (2.2.32) : maximum 0.5 per cent, determined in dimethylformamide, practically insoluble in alcohol and on 1.000 g by drying in an oven at 100-105 °C for 4 h. methylene chloride. 1418


Diclofenac potassium


Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.150 g in 75 ml of dimethylformamide R. Carry out a potentiometric titration (2.2.20), using 0.1 M tetrabutylammonium hydroxide. Read the volume added at the second inflexion point. Carry out a blank titration. 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to 20.38 mg of C17H9Cl3N4O2. STORAGE Protected from light. IMPURITIES Specified impurities : A, B, C, D, E, F, G, H, I.

I. N,2-bis[3,5-dichloro-4-[(4-chlorophenyl)cyanomethyl]phenyl]-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide. 01/2005:1508

DICLOFENAC POTASSIUM Diclofenacum kalicum

A. R = Cl, R′ = CO2H : 2-[3,5-dichloro-4-[(RS)-(4chlorophenyl)cyanomethyl]phenyl]-3,5-dioxo-2,3,4,5tetrahydro-1,2,4-triazine-6-carboxylic acid, B. R = OH, R′ = H : (RS)-[2,6-dichloro-4-(3,5-dioxo4,5-dihydro-1,2,4-triazin-2(3H)-yl)phenyl](4hydroxyphenyl)acetonitrile, C. R = Cl, R′ = CONH2 : 2-[3,5-dichloro-4-[(RS)-(4chlorophenyl)cyanomethyl]phenyl]-3,5-dioxo-2,3,4,5tetrahydro-1,2,4-triazine-6-carboxamide, G. R = Cl, R′ = CO-O-[CH2]3-CH3 : butyl 2-[3,5-dichloro-4[(RS)-(4-chlorophenyl)cyanomethyl]phenyl]-3,5-dioxo-2,3, 4,5-tetrahydro-1,2,4-triazine-6-carboxylate,

D. X = O : 2-[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]-1,2,4triazine-3,5(2H,4H)-dione, F. X = H2 : 2-[3,5-dichloro-4-(4-chlorobenzyl)phenyl]-1,2,4triazine-3,5(2H,4H)-dione,

E. R = NH2 : (RS)-(4-amino-2,6-dichlorophenyl)(4chlorophenyl)acetonitrile, H. R = H : (RS)-(4-chlorophenyl)(2,6-dichlorophenyl)acetonitrile, General Notices (1) apply to all monographs and other texts

C14H10Cl2KNO2

Mr 334.2

DEFINITION Potassium diclofenac contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of potassium [2-[(2,6-dichlorophenyl)amino]phenyl]acetate, calculated with reference to the dried substance. CHARACTERS A white or slightly yellowish, crystalline powder, slightly hygroscopic, sparingly soluble in water, freely soluble in methanol, soluble in alcohol, slightly soluble in acetone. IDENTIFICATION First identification : A, D. Second identification : B, C, D. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with diclofenac potassium CRS. Examine the substances prepared as discs. B. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel GF254 plate R. Test solution. Dissolve 25 mg of the substance to be examined in methanol R and dilute to 5 ml with the same solvent. Reference solution (a). Dissolve 25 mg of diclofenac potassium CRS in methanol R and dilute to 5 ml with the same solvent. Reference solution (b). Dissolve 10 mg of indometacin R in reference solution (a) and dilute to 2 ml with the same solution. Apply to the plate 5 µl of each solution. Develop over a path of 10 cm using a mixture of 10 volumes of concentrated ammonia R, 10 volumes of methanol R and 80 volumes of ethyl acetate R. Allow the plate to dry in air. Examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the 1419

Diclofenac sodium


chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows 2 clearly separated spots. C. Dissolve about 10 mg in 10 ml of alcohol R. To 1 ml of the solution add 0.2 ml of a mixture, prepared immediately before use, of equal volumes of a 6 g/l solution of potassium ferricyanide R and a 9 g/l solution of ferric chloride R. Allow to stand protected from light for 5 min. Add 3 ml of a 10 g/l solution of hydrochloric acid R. Allow to stand protected from light for 15 min. A blue colour develops and a precipitate is formed. D. Suspend 0.5 g of the substance to be examined in 10 ml of water R. Stir, add water R until the substance is dissolved. Add 2 ml of hydrochloric acid R1, stir for 1 h and filter with the aid of vacuum. Neutralise the solution with sodium hydroxide solution R. The solution gives reaction (b) of potassium (2.3.1). TESTS Appearance of solution. Dissolve 1.25 g in methanol R and dilute to 25.0 ml with the same solvent. The solution is clear (2.2.1). The absorbance (2.2.25) measured at 440 nm is not greater than 0.05. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 50.0 mg of the substance to be examined in methanol R and dilute to 50.0 ml with the same solvent. Reference solution (a). Dilute 2.0 ml of the test solution to 100.0 ml with methanol R. Dilute 1.0 ml of the solution to 10.0 ml with methanol R. Reference solution (b). Dissolve 1.0 mg of diclofenac impurity A CRS in methanol R, add 1.0 ml of the test solution and dilute to 200.0 ml with methanol R. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with end-capped octylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1 ml/min a mixture of 34 volumes of a solution containing 0.5 g/l of phosphoric acid R and 0.8 g/l of sodium dihydrogen phosphate R adjusted to pH 2.5 with phosphoric acid R, and 66 volumes of methanol R, — as detector a spectrophotometer set at 254 nm. Inject 20 µl of reference solution (b). When the chromatograms are recorded in the prescribed conditions, the retention times are : diclofenac, about 25 min and impurity A, about 12 min. Adjust the sensitivity of the system so that the height of the peaks in the chromatogram obtained is at least 50 per cent of the full scale of the recorder. The test is not valid unless in the chromatogram obtained the resolution between the peaks corresponding to diclofenac and impurity A is not less than 6.5. Inject 20 µl of the test solution and 20 µl of reference solution (a). Continue the chromatography for 1.5 times the retention time of the principal peak in the chromatogram obtained with the test solution. In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than 2.5 times that of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent). Disregard any peak with an area less than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (a). 1420

Heavy metals (2.4.8). 2.0 g complies with limit test C (10 ppm). Use a quartz crucible. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 3 h. ASSAY Dissolve 0.250 g in 30 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 33.42 mg of C14H10Cl2KNO2. STORAGE In an airtight container, protected from light. IMPURITIES

A. 1-(2,6-dichlorophenyl)-1,3-dihydro-2H-indol-2-one,

B. R1 = CHO, R2 = Cl : 2-[(2,6-dichlorophenyl)amino]benzaldehyde, C. R1 = CH2OH, R2 = Cl : [2-[(2,6-dichlorophenyl)amino]phenyl]methanol, D. R1 = CH2-CO2H, R2 = Br : 2-[2-[(2-bromo-6chlorophenyl)amino]phenyl]acetic acid,

E. 1,3-dihydro-2H-indol-2-one. 01/2005:1002

DICLOFENAC SODIUM Diclofenacum natricum

C14H10Cl2NNaO2

Mr 318.1



DEFINITION Diclofenac sodium contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of sodium 2-[(2,6-dichlorophenyl)amino]phenyl]acetate, calculated with reference to the dried substance. CHARACTERS A white or slightly yellowish, crystalline powder, slightly hygroscopic, sparingly soluble in water, freely soluble in methanol, soluble in alcohol, slightly soluble in acetone. It melts at about 280 °C, with decomposition. IDENTIFICATION First identification : A, D. Second identification : B, C, D. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with diclofenac sodium CRS. Examine the substances prepared as discs. B. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel GF254 plate R. Test solution. Dissolve 25 mg of the substance to be examined in methanol R and dilute to 5 ml with the same solvent. Reference solution (a). Dissolve 25 mg of diclofenac sodium CRS in methanol R and dilute to 5 ml with the same solvent. Reference solution (b). Dissolve 10 mg of indometacin R in reference solution (a) and dilute to 2 ml with the same solution. Apply to the plate 5 µl of each solution. Develop over a path of 10 cm using a mixture of 10 volumes of concentrated ammonia R, 10 volumes of methanol R and 80 volumes of ethyl acetate R. Allow the plate to dry in air. Examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows 2 clearly separated spots. C. Dissolve about 10 mg in 10 ml of alcohol R. To 1 ml of the solution add 0.2 ml of a mixture, prepared immediately before use, of equal volumes of a 6 g/l solution of potassium ferricyanide R and a 9 g/l solution of ferric chloride R. Allow to stand protected from light for 5 min. Add 3 ml of a 10 g/l solution of hydrochloric acid R. Allow to stand, protected from light, for 15 min. A blue colour develops and a precipitate is formed. D. Dissolve 60 mg in 0.5 ml of methanol R and add 0.5 ml of water R. The solution gives reaction (b) of sodium (2.3.1).

Diclofenac sodium

Reference solution (b). Dissolve 1.0 mg of diclofenac impurity A CRS in methanol R, add 1.0 ml of the test solution and dilute to 200.0 ml with methanol R. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with end-capped octylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1 ml/min a mixture of 34 volumes of a solution containing 0.5 g/l of phosphoric acid R and 0.8 g/l of sodium dihydrogen phosphate R adjusted to pH 2.5 with phosphoric acid R, and 66 volumes of methanol R, — as detector a spectrophotometer set at 254 nm. Inject 20 µl of reference solution (b). When the chromatograms are recorded in the prescribed conditions, the retention times are about 25 min for diclofenac and about 12 min for impurity A. Adjust the sensitivity of the system so that the height of the peaks in the chromatogram obtained with reference solution (b) is not less than 50 per cent of the full scale of the recorder. Continue the chromatography for 1.5 times the retention time of diclofenac. The test is not valid unless in the chromatogram obtained the resolution between the peaks corresponding to diclofenac and impurity A is at least 6.5. Inject 20 µl of the test solution and 20 µl of reference solution (a). In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent) ; the sum of the areas of all the peaks apart from the principal peak is not greater than 2.5 times that of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent). Disregard any peak with an area less than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (a). Heavy metals (2.4.8). 2.0 g complies with limit test C (10 ppm). Use a quartz crucible. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 3 h. ASSAY Dissolve 0.250 g in 30 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 31.81 mg of C14H10Cl2NNaO2. STORAGE In an airtight container, protected from light.

TESTS Appearance of solution. Dissolve 1.25 g in methanol R and dilute to 25.0 ml with the same solvent. The solution is clear IMPURITIES (2.2.1). The absorbance (2.2.25) measured at 440 nm is not greater than 0.05. Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 50.0 mg of the substance to be examined in methanol R and dilute to 50.0 ml with the same solvent. Reference solution (a). Dilute 2.0 ml of the test solution to 100.0 ml with methanol R. Dilute 1.0 ml of the solution to A. 1-(2,6-dichlorophenyl)-1,3-dihydro-2H-indol-2-one, 10.0 ml with methanol R. General Notices (1) apply to all monographs and other texts

1421

Dicloxacillin sodium


Apply to the plate 1 µl of each solution. Develop over a path of 15 cm using a mixture of 30 volumes of acetone R and 70 volumes of a 154 g/l solution of ammonium acetate R the pH of which has been adjusted to 5.0 with glacial acetic acid R. Allow the plate to dry in air and expose it to iodine vapour until the spots appear. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not B. R1 = CHO, R2 = Cl : 2-[(2,6-dichlorophenyl)amino]benvalid unless the chromatogram obtained with reference zaldehyde, solution (b) shows three clearly separated spots. C. R1 = CH2OH, R2 = Cl : [2-[(2,6-dichlorophenyl)amino]pheC. Place about 2 mg in a test-tube about 150 mm long and nyl]methanol, 15 mm in diameter. Moisten with 0.05 ml of water R and D. R1 = CH2-CO2H, R2 = Br : 2-[2-[(2-bromo-6add 2 ml of sulphuric acid-formaldehyde reagent R. Mix chlorophenyl)amino]phenyl]acetic acid, the contents of the tube by swirling ; the colour of the solution is slightly greenish-yellow. Place the test-tube in a water-bath for 1 min ; a yellow colour develops. D. It gives reaction (a) of sodium (2.3.1). TESTS Solution S. Dissolve 2.50 g in carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. 01/2005:0663 Appearance of solution. Solution S is clear (2.2.1). The absorbance (2.2.25) of solution S measured at 430 nm is not greater than 0.04. DICLOXACILLIN SODIUM pH (2.2.3). The pH of solution S is 5.0 to 7.0. Dicloxacillinum natricum Specific optical rotation (2.2.7). Dissolve 0.250 g in water R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 128 to + 143, calculated with reference to the anhydrous substance. Related substances. Examine by liquid chromatography (2.2.29) as prescribed under Assay. Inject reference solution (b). Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is at least 50 per cent of the full scale of the recorder. Inject test solution (a) and continue the chromatography C19H16Cl2N3NaO5S,H2O Mr 510.3 for five times the retention time of the principal peak. In the chromatogram obtained with test solution (a) : the area DEFINITION of any peak, apart from the principal peak, is not greater Dicloxacillin sodium contains not less than 95.0 per than the area of the principal peak in the chromatogram cent and not more than the equivalent of 101.0 per obtained with reference solution (b) (1 per cent) ; the sum of cent of sodium (2S,5R,6R)-6-[[[3-(2,6-dichlorophenyl)-5the areas of all peaks, apart from the principal peak, is not methylisoxazol-4-yl]carbonyl]amino]-3,3-dimethyl-7-oxo-4greater than five times the area of the principal peak in the thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, calculated chromatogram obtained with reference solution (b) (5 per with reference to the anhydrous substance. cent). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained CHARACTERS with reference solution (b). A white or almost white, crystalline powder, hygroscopic, N,N-Dimethylaniline (2.4.26, Method B). Not more than freely soluble in water, soluble in alcohol and in methanol. 20 ppm. IDENTIFICATION 2-Ethylhexanoic acid (2.4.28). Not more than 0.8 per First identification : A, D. cent m/m. Second identification : B, C, D. Water (2.5.12) : 3.0 per cent to 4.5 per cent, determined on A. Examine by infrared absorption spectrophotometry 0.300 g by the semi-micro determination of water. (2.2.24), comparing with the spectrum obtained with Pyrogens (2.6.8). If intended for use in the manufacture dicloxacillin sodium CRS. Examine the substances of parenteral dosage forms without a further appropriate prepared as discs. procedure for the removal of pyrogens, it complies with the B. Examine by thin-layer chromatography (2.2.27), using test for pyrogens. Inject per kilogram of the rabbit’s mass silanised silica gel H R as the coating substance. 1 ml of a solution in water for injections R containing 20 mg Test solution. Dissolve 25 mg of the substance to be of the substance to be examined per millilitre. examined in 5 ml of water R. ASSAY Reference solution (a). Dissolve 25 mg of dicloxacillin Examine by liquid chromatography (2.2.29). sodium CRS in 5 ml of water R. Test solution (a). Dissolve 50.0 mg of the substance to be Reference solution (b). Dissolve 25 mg each of examined in the mobile phase and dilute to 50.0 ml with the cloxacillin sodium CRS, dicloxacillin sodium CRS and mobile phase. flucloxacillin sodium CRS in 5 ml of water R. E. 1,3-dihydro-2H-indol-2-one.

1422



Dicycloverine hydrochloride

Test solution (b). Dilute 5.0 ml of test solution (a) to 50.0 ml with the mobile phase. Reference solution (a). Dissolve 50.0 mg of dicloxacillin sodium CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 5.0 ml of the solution to 50.0 ml with the mobile phase. Reference solution (b). Dilute 5.0 ml of test solution (b) to 50.0 ml with the mobile phase.

C. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylic acid (6-aminopenicillanic acid),

Reference solution (c). Dissolve 5 mg of flucloxacillin sodium CRS and 5 mg of dicloxacillin sodium CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.0 ml/min a mixture of 75 volumes of a 2.7 g/l solution of potassium dihydrogen D. 3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carboxylic acid. phosphate R adjusted to pH 5.0 with dilute sodium hydroxide solution R and 25 volumes of acetonitrile R, 01/2005:1197 — as detector a spectrophotometer set at 225 nm, — a 20 µl loop injector.

DICYCLOVERINE HYDROCHLORIDE

Inject reference solution (c). When the chromatograms are Dicycloverini hydrochloridum recorded in the prescribed conditions, the retention time of dicloxacillin is about 10 min. Adjust the sensitivity of the system so that the heights of the principal peaks in the chromatogram obtained are at least 50 per cent of the full scale of the recorder. The test is not valid unless in the chromatogram obtained, the resolution between the first peak (flucloxacillin) and the second peak (dicloxacillin) is at least 2.5. Inject reference solution (a) six times. The test is not valid unless the relative standard deviation of the peak Mr 346.0 area for dicloxacillin is at most 1.0 per cent. Inject alternately C19H36ClNO2 test solution (b) and reference solution (a). DEFINITION Dicycloverine hydrochloride contains not less than 99.0 per STORAGE cent and not more than the equivalent of 101.0 per Store in an airtight container, at a temperature not exceeding cent of 2-(diethylamino)ethyl bicyclohexyl-1-carboxylate 25 °C. If the substance is sterile, store in a sterile, airtight, hydrochloride, calculated with reference to the dried tamper-proof container. substance. LABELLING The label states, where applicable, that the substance is apyrogenic. IMPURITIES

A. R = CO2H : (4S)-2-[carboxy[[[3-(2,6-dichlorophenyl)5-methylisoxazol-4-yl]carbonyl]amino]methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penicilloic acids of dicloxacillin), B. R = H : (2RS,4S)-2-[[[[3-(2,6-dichlorophenyl)-5methylisoxazol-4-yl]carbonyl]amino]methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penilloic acids of dicloxacillin), General Notices (1) apply to all monographs and other texts

CHARACTERS A white or almost white, crystalline powder, soluble in water, freely soluble in alcohol and in methylene chloride. It shows polymorphism. IDENTIFICATION First identification : A, D. Second identification : B, C, D. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with dicycloverine hydrochloride CRS. Examine the substances prepared as discs using potassium chloride R. If the spectra obtained show differences, dissolve the substance to be examined and the reference substance separately in acetone R, evaporate to dryness and record new spectra using the residues. B. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (b). C. To 3 ml of a 1.0 g/l solution of sodium laurilsulfate R add 5 ml of methylene chloride R and 0.05 ml of a 2.5 g/l solution of methylene blue R, mix gently and allow to stand ; the lower layer is blue. Add 2 ml of a 20 g/l 1423

Dienestrol


01/2005:0483

solution of the substance to be examined, mix gently and allow to stand ; the upper layer is blue and the lower layer is colourless.

DIENESTROL

D. It gives reaction (a) of chlorides (2.3.1).

Dienestrolum

TESTS pH (2.2.3). Dissolve 0.5 g in water R and dilute to 50 ml with the same solvent. The pH of the solution is 5.0 to 5.5. Related substances. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating substance. Test solution (a). Dissolve 0.25 g to the substance to be examined in methanol R and dilute to 5 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 50 ml with methanol R. Reference solution (a). Dilute 1 ml of test solution (b) to 10 ml with methanol R. Reference solution (b). Dissolve 10 mg of dicycloverine hydrochloride CRS in methanol R and dilute to 10 ml with the same solvent. Reference solution (c). Dissolve 5 mg of tropicamide CRS in reference solution (b) and dilute to 5 ml with the same solution. Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm using a mixture of 5 volumes of concentrated ammonia R, 10 volumes of ethyl acetate R, 10 volumes of water R and 75 volumes of propanol R. Dry the plate in a current of warm air. Spray with dilute potassium iodobismuthate solution R. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.2 per cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows two clearly separated spots. Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY

C18H18O2

Mr 266.3

DEFINITION Dienestrol contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent of (E,E)-4,4′-(1,2-diethylidene-ethylene)diphenol, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, practically insoluble in water, freely soluble in acetone and in alcohol. It dissolves in dilute solutions of the alkali hydroxides. IDENTIFICATION First identification : A, D. Second identification : B, C, D. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with dienestrol CRS. Examine the substances prepared as discs. B. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the spot in the chromatogram obtained with reference solution (a). C. Dissolve about 1 mg in 5 ml of glacial acetic acid R, add 1 ml of a 1 per cent V/V solution of bromine R in glacial acetic acid R and heat in a water-bath for 2 min. Place 0.5 ml of this solution in a dry test-tube, add 0.5 ml of ethanol R, mix and add 10 ml of water R. A reddish-violet colour is produced. Add 5 ml of chloroform R, shake vigorously and allow to separate. The chloroform layer is red and the aqueous layer is almost colourless. D. Dissolve about 0.5 mg in 0.2 ml of glacial acetic acid R, add 1 ml of phosphoric acid R and heat on a water-bath for 3 min. A reddish-violet colour is produced.

Dissolve 0.300 g in a mixture of 5.0 ml of 0.01 M hydrochloric acid and 50 ml of alcohol R. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read TESTS the volume added between the two points of inflexion. Melting range. Determined by the capillary method (2.2.14), 1 ml of 0.1 M sodium hydroxide is equivalent to 34.60 mg of the melting point is 227 °C to 234 °C. The temperature interval between the formation of a definite meniscus in C19H36ClNO2. the melt and the disappearance of the last particle does not exceed 3 °C. IMPURITIES Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution (a). Dissolve 0.2 g of the substance to be examined in 2 ml of alcohol R. Test solution (b). Dilute 1 ml of test solution (a) to 20 ml with alcohol R. Reference solution (a). Dissolve 25 mg of dienestrol CRS in alcohol R and dilute to 5 ml with the same solvent. Reference solution (b). Dilute 1 ml of reference solution (a) to 10 ml with alcohol R. A. bicyclohexyl-1-carboxylic acid. 1424



Diethyl phthalate

Reference solution (c). Dissolve 10 mg of Test solution. Dissolve 50 mg of the substance to be diethylstilbestrol CRS in 2 ml of alcohol R. To 1 ml examined in ether R and dilute to 10 ml with the same of this solution add 1 ml of reference solution (a). solvent. Apply separately to the plate 1 µl of each solution. Develop Reference solution. Dissolve 50 mg of diethyl over a path of 15 cm using a mixture of 10 volumes of phthalate CRS in ether R and dilute to 10 ml with the diethylamine R and 90 volumes of toluene R. Allow the same solvent. plate to dry in air, spray with alcoholic solution of sulphuric Apply separately to the plate 10 µl of each solution. acid R and heat at 120 °C for 10 min. Any spot in the Develop over a path of 15 cm using a mixture of chromatogram obtained with test solution (a), apart from 30 volumes of heptane R and 70 volumes of ether R. the principal spot, is not more intense than the spot in Allow the plate to dry in air and examine in ultraviolet the chromatogram obtained with reference solution (b) light at 254 nm. The principal spot in the chromatogram (0.5 per cent). The test is not valid unless the chromatogram obtained with the test solution is similar in position and obtained with reference solution (c) shows at least two clearly size to the principal spot in the chromatogram obtained separated spots having approximately the same intensity. with the reference solution. Loss on drying (2.2.32). Not more than 0.5 per cent, E. To about 0.1 ml add 0.25 ml of sulphuric acid R and determined on 1.000 g by drying in an oven at 100 °C to 50 mg of resorcinol R. Heat on a water-bath for 5 min. 105 °C. Allow to cool. Add 10 ml of water R and 1 ml of strong Sulphated ash (2.4.14). Not more than 0.1 per cent, sodium hydroxide solution R. The solution becomes determined on 1.0 g. yellow or brownish-yellow and shows green fluorescence. ASSAY Dissolve 25.0 mg in ethanol R and dilute to 100.0 ml with the same solvent. To 5.0 ml of this solution add 10 ml of ethanol R and dilute to 250.0 ml with 0.1 M sodium hydroxide. Prepare a reference solution in the same manner using 25.0 mg of dienestrol CRS. Measure the absorbance (2.2.25) of the solutions at the maximum at 245 nm. Calculate the content of C18H18O2 from the measured absorbances and the concentrations of the solutions.

TESTS Appearance. The substance to be examined is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). Acidity. Dissolve 20.0 g in 50 ml of alcohol R previously neutralised to phenolphthalein solution R1. Add 0.2 ml of phenolphthalein solution R1. Not more than 0.1 ml of 0.1 M sodium hydroxide is required to change the colour of the indicator to pink. STORAGE Related substances. Examine by gas chromatography Store protected from light. (2.2.28), using naphthalene R as the internal standard. Internal standard solution. Dissolve 60 mg of naphthalene R 01/2005:0897 in methylene chloride R and dilute to 20 ml with the same solvent. DIETHYL PHTHALATE Test solution (a). Dissolve 1.0 g of the substance to be examined in methylene chloride R and dilute to 20.0 ml with the same solvent. Diethylis phthalas Test solution (b). Dissolve 1.0 g of the substance to be examined in methylene chloride R, add 2.0 ml of the internal standard solution and dilute to 20.0 ml with methylene chloride R. Reference solution. To 1.0 ml of test solution (a) add 10.0 ml of the internal standard solution and dilute to 100.0 ml with methylene chloride R. C12H14O4 Mr 222.2 The chromatographic procedure may be carried out using : — a glass column 2 m long and 2 mm in internal diameter DEFINITION packed with silanised diatomaceous earth for gas Diethyl phthalate contains not less than 99.0 per cent and chromatography R (150 µm to 180 µm) impregnated with not more than the equivalent of 101.0 per cent m/m of 3 per cent m/m of polymethylphenylsiloxane R, diethyl benzene-1,2-dicarboxylate. — nitrogen for chromatography R as the carrier gas at a flow rate of 30 ml/min, CHARACTERS — a flame-ionisation detector, A clear, oily liquid, colourless or very slightly yellow, practically insoluble in water, miscible with alcohol. maintaining the temperature of the column at 150 °C and that of the injection port and of the detector at 225 °C. IDENTIFICATION Inject 1 µl of the reference solution. The substances are First identification : B, C. eluted in the following order : naphthalene and diethyl Second identification : A, D, E. phthalate. Adjust the sensitivity of the detector so that the A. Relative density (2.2.5) : 1.117 to 1.121. height of the peak due to naphthalene is not less than 50 per B. Refractive index (2.2.6) : 1.500 to 1.505. cent of the full scale of the recorder. The test is not valid unless the resolution between the peaks corresponding to C. Examine by infrared absorption spectrophotometry naphthalene and diethyl phthalate is at least 10. (2.2.24), comparing with the spectrum obtained with diethyl phthalate CRS. Examine as thin films. Inject 1 µl of test solution (a). In the chromatogram obtained, D. Examine by thin-layer chromatography (2.2.27), using verify that there is no peak with the same retention time as silica gel GF254 R as the coating substance. the internal standard. General Notices (1) apply to all monographs and other texts

1425

Diethylcarbamazine citrate


Inject separately 1 µl of test solution (b) and 1 µl of the reference solution. Continue the chromatography for three times the retention time of diethyl phthalate. From the chromatogram obtained with the reference solution, calculate the ratio (R) of the area of the peak due to diethyl phthalate to the area of the peak due to the internal standard. From the chromatogram obtained with test solution (b), calculate the ratio of the sum of the areas of any peaks, apart from the principal peak and the peaks due to the internal standard and the solvent, to the area of the peak due to the internal standard ; this ratio is not greater than R (1.0 per cent). Water (2.5.12). Not more than 0.2 per cent, determined on 5.0 g by the semi-micro determination of water. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Introduce 0.750 g into a 250 ml borosilicate glass flask. Add 25.0 ml of 0.5 M alcoholic potassium hydroxide and a few glass beads. Boil on a water-bath under a reflux condenser for 1 h. Add 1 ml of phenolphthalein solution R1 and titrate immediately with 0.5 M hydrochloric acid. Carry out a blank titration. Calculate the volume of 0.5 M alcoholic potassium hydroxide used in the saponification. 1 ml of 0.5 M alcoholic potassium hydroxide is equivalent to 55.56 mg of C12H14O4. STORAGE Store in an airtight container. 01/2005:0271

DIETHYLCARBAMAZINE CITRATE Diethylcarbamazini citras

C16H29N3O8

Mr 391.4

DEFINITION Diethylcarbamazine citrate contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of N,N-diethyl-4-methylpiperazine-1-carboxamide dihydrogen 2-hydroxypropane-1,2,3-tricarboxylate, calculated with reference to the dried substance. CHARACTERS A white, crystalline powder, slightly hygroscopic, very soluble in water, soluble in alcohol, practically insoluble in acetone. It melts at about 138 °C, with decomposition.

corresponds in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). C. Dissolve 0.1 g in 5 ml of water R. The solution gives the reaction of citrates (2.3.1). TESTS Solution S. Shake 2.5 g with water R until dissolved and dilute to 25 ml with the same solvent. Appearance of solution. Solution S is not more opalescent than reference suspension II (2.2.1) and is not more intensely coloured than reference solution BY6 (2.2.2, Method II). Dimethylpiperazine and methylpiperazine. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. Dissolve 0.5 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution (a). Dissolve 0.1 g of diethylcarbamazine citrate CRS in methanol R and dilute to 2.0 ml with the same solvent. Reference solution (b). Dissolve 10 mg of methylpiperazine R in methanol R and dilute to 100 ml with the same solvent. Reference solution (c). Dissolve 10 mg of dimethylpiperazine R in methanol R and dilute to 100 ml with the same solvent. Apply to the plate 10 µl of each solution. Develop over a path of 12 cm using a mixture of 5 volumes of concentrated ammonia R, 30 volumes of methyl ethyl ketone R and 65 volumes of methanol R. Dry the plate at 100-105 °C and expose to iodine vapour for 30 min. In the chromatogram obtained with the test solution, any spots corresponding to methylpiperazine and dimethylpiperazine are not more intense than the spots in the chromatograms obtained with reference solutions (b) and (c) respectively (0.2 per cent). Heavy metals (2.4.8). 12 ml of solution S complies with limit test A for heavy metals (20 ppm). Prepare the standard using 10 ml of lead standard solution (2 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.0 g by drying in vacuo at 60 °C for 4 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.350 g in 25 ml of anhydrous acetic acid R and add 25 ml of acetic anhydride R. Using 0.2 ml of crystal violet solution R as indicator, titrate with 0.1 M perchloric acid until a greenish-blue colour is obtained. 1 ml of 0.1 M perchloric acid is equivalent to 39.14 mg of C16H29N3O8. STORAGE Store in an airtight container.

IDENTIFICATION DIETHYLENE First identification : A, C. Second identification : B, C. A. Examine by infrared absorption spectrophotometry Diethylenglycoli (2.2.24), comparing with the spectrum obtained with diethylcarbamazine citrate CRS. B. Examine the chromatograms obtained in the test for dimethylpiperazine and methylpiperazine. The principal spot in the chromatogram obtained with the test solution C6H14O3 1426

01/2005:1198

GLYCOL MONOETHYL ETHER monoethylicum aetherum Mr 134.2


Diethylene glycol monoethyl ether


Injection : 0.5 µl. Relative retentions with reference to diethylene glycol monoethyl ether (retention time = about 4 min) : ethylene glycol monomethyl ether = about 0.4 ; ethylene glycol CHARACTERS monoethyl ether = about 0.5 ; ethylene glycol = about 0.55 ; Appearance : clear, colourless, hygroscopic liquid. diethylene glycol = about 1.1. Solubility : miscible with water, with acetone and with System suitability : alcohol, miscible in certain proportions with vegetable oils, — resolution : minimum 3.0 between the peaks due to not miscible with mineral oils. ethylene glycol monoethyl ether and to ethylene glycol in Relative density : about 0.991. the chromatogram obtained with reference solution (b), — signal-to-noise ratio : minimum 3.0 for the peak due to IDENTIFICATION ethylene glycol monomethyl ether in the chromatogram A. Refractive index (2.2.6) : 1.426 to 1.428. obtained with reference solution (a), B. Infrared absorption spectrophotometry (2.2.24). Limits (take into account the impurity/internal standard Comparison : Ph. Eur. reference spectrum of diethylene peak area ratio) : glycol monoethyl ether. — ethylene glycol monomethyl ether : not more than the area of the corresponding peak in the chromatogram TESTS obtained with reference solution (a) (50 ppm), Acid value (2.5.1) : maximum 0.1. — ethylene glycol monoethyl ether : not more than the area Mix 30.0 ml with 30 ml of alcohol R previously neutralised of the corresponding peak in the chromatogram obtained with 0.1 M potassium hydroxide using phenolphthalein with reference solution (a) (160 ppm), solution R as indicator. Titrate with 0.01 M alcoholic — ethylene glycol: not more than the area of the potassium hydroxide. corresponding peak in the chromatogram obtained with Peroxide value (2.5.5) : maximum 8.0, determined on 2.00 g. reference solution (a) (620 ppm), Related substances. Gas chromatography (2.2.28). — diethylene glycol : not more than the area of the Internal standard solution. Dilute 1.00 g of decane R to corresponding peak in the chromatogram obtained with 100.0 ml with methanol R. reference solution (a) (250 ppm), Test solution. To 5.00 g of the substance to be examined, — total : not more than the area of the principal peak in add 0.1 ml of the internal standard solution and dilute to the chromatogram obtained with reference solution (c) 10.0 ml with methanol R. (0.2 per cent). Reference solution (a). Dilute 25.0 mg of ethylene glycol Ethylene oxide. Head-space gas chromatography (2.2.28). monomethyl ether R, 80.0 mg of ethylene glycol monoethyl Test solution. To 1.00 g of the substance to be examined in ether R, 0.310 g of ethylene glycol R and 0.125 g of a vial, add 50 µl of water R. diethylene glycol R to 100.0 ml with methanol R. To 1.0 ml of this solution add 0.1 ml of the internal standard solution Reference solution. To 1.00 g of the substance to be examined in a vial, add 50 µl of ethylene oxide solution R4 and dilute to 10.0 ml with methanol R. and close tightly. Reference solution (b). Dilute 25.0 mg of ethylene glycol Column : monoethyl ether R and 25.0 mg of ethylene glycol R to 100.0 ml with methanol R. Dilute 1.0 ml of this solution to — material : fused silica, 5.0 ml with methanol R. — size : l = 30 m, Ø = 0.32 mm, Reference solution (c). Dilute 1.00 g of the substance to be — stationary phase : poly(cyanopropyl)(7)(phenyl)(7)methexamined to 100.0 ml with methanol R. To 1.0 ml of this yl(86)siloxane R (film thickness 1 µm). solution add 0.1 ml of the internal standard solution and Carrier gas : helium for chromatography R. dilute to 10.0 ml with methanol R. Flow rate : 1.1 ml/min. Column : Static head-space conditions which may be used : — material : fused silica, — equilibration temperature : 80 °C, — size : l = 30 m, Ø = 0.32 mm, — stationary phase: poly(cyanopropyl)(7)(phenyl)(7)meth- — equilibration time : 45 min, — transfer line temperature : 110 °C, yl(86)siloxane R (film thickness 1 µm). Carrier gas : nitrogen for chromatography R or helium for — pressurisation time : 2 min, chromatography R. — injection time : 12 s. Flow rate : 2.0 ml/min. Temperature : Split ratio : 1:80. Time Temperature Temperature : (min) (°C) DEFINITION 2-(2-Ethoxyethoxy)ethanol, produced by condensation of ethylene oxide and alcohol, followed by distillation.

Column

Time (min)

Temperature (°C)

0-1

120

1 - 10

120 → 225

10 - 12

225

Injection port

275

Detector

250

Detection : flame ionisation. General Notices (1) apply to all monographs and other texts

Column

0-5

40

5 - 18

40 → 200

Injection port

150

Detector

250

Detection : flame ionisation. Injection : 1.0 ml. The peak due to ethylene oxide is identified by injecting solutions of ethylene oxide of increasing concentration. 1427

Diethylene glycol monopalmitostearate


Determine the content of ethylene oxide (ppm) in the substance to be examined using the following expression :

Composition of fatty acids (2.4.22, Method A). The fatty acid fraction has the following composition : — stearic acid : 40.0 per cent to 60.0 per cent,

ST

=

area of the peak corresponding to ethylene oxide in the chromatogram obtained with the test solution,

SS

=

area of the peak corresponding to ethylene oxide in the chromatogram obtained with the reference solution,

— sum of contents of palmitic acid and stearic acid : not less than 90.0 per cent. Free diethylene glycol. Not more than 8.0 per cent, determined as prescribed under Assay. Total ash (2.4.16). Not more than 0.1 per cent, determined on 1.0 g. ASSAY

MT

=

mass of the substance to be examined in the test solution, in grams,

Determine the free diethylene glycol content and monoesters content by size-exclusion chromatography (2.2.30).

MS

=

mass of the substance to be examined in the reference solution, in grams,

C

=

mass of added ethylene oxide in the reference solution, in micrograms.

Test solution. Into a 15 ml flask, weigh about 0.2 g (m), to the nearest 0.1 mg. Add 5.0 ml of tetrahydrofuran R and shake to dissolve. Heat gently, if necessary. Reweigh the flask and calculate the total mass of solvent and substance (M).

Limit : — ethylene oxide : maximum 1 ppm. Water (2.5.12) : maximum 0.1 per cent, determined on 10.0 g. STORAGE Under an inert gas, in an airtight container.

Reference solutions. Into four 15 ml flasks, respectively weigh, to the nearest 0.1 mg, about 2.5 mg, 5.0 mg, 10.0 mg and 20.0 mg of diethylene glycol R. Add 5.0 ml of tetrahydrofuran R and shake to dissolve. Weigh the flasks again and calculate the concentration of diethylene glycol in milligrams per gram for each reference solution. The chromatographic procedure may be carried out using :

LABELLING The label states that the substance is stored under an inert gas.

— a gel-permeation column 0.6 m long and 7 mm in internal diameter packed with styrene-divinylbenzene copolymer R (particle diameter 5 µm and pore size 10 nm), — as mobile phase at a flow rate of 1 ml/min tetrahydrofuran R,

01/2005:1415 — a differential refractive index detector. Inject 40 µl of each solution. When the chromatograms are recorded in the prescribed conditions, the retention times DIETHYLENE GLYCOL relative to diethylene glycol are about 0.84 for the MONOPALMITOSTEARATE monoesters and about 0.78 for the diesters. From the calibration curve obtained with the reference solutions determine the concentration (C) in milligrams per gram of Diethylenglycoli monopalmitostearas diethylene glycol in the test solution. DEFINITION Calculate the percentage content of free diethylene glycol in the substance to be examined using the following expression : Diethylene glycol monopalmitostearate is a mixture of diethylene glycol mono- and diesters of stearic and palmitic acids. It contains not less than 45.0 per cent of monoesters produced from the condensation of diethylene glycol and stearic acid 50 of vegetable or animal origin. From the peak area of the monoesters (A) and the diesters (B), calculate the percentage content of monoesters using CHARACTERS the following expression : A white or almost white, waxy solid, practically insoluble in water, soluble in acetone and in hot alcohol. IDENTIFICATION A. It complies with the test for melting point (see Tests). B. It complies with the test for composition of fatty acids (see Tests). C. It complies with the assay (monoesters content).

D

=

the percentage content of free diethylene glycol and the percentage content of free fatty acids.

Calculate the percentage content of free fatty acids using the expression :

TESTS Melting point (2.2.15) : 43 °C to 50 °C. Acid value (2.5.1). Not more than 4.0, determined on 10.0 g. IA = acid value. Iodine value (2.5.4). Not more than 3.0. STORAGE Saponification value (2.5.6) : 150 to 170, determined on 2.0 g. Store protected from light. 1428


Diflunisal


01/2005:0484 Reference solution (d). Dissolve 10 mg of dienestrol CRS in 2 ml of alcohol R. To 1 ml of this solution add 1 ml of reference solution (a). DIETHYLSTILBESTROL Apply to the plate 1 µl of each solution. Develop over a path of 15 cm using a mixture of 10 volumes of diethylamine R Diethylstilbestrolum and 90 volumes of toluene R. Allow the plate to dry in air, spray with alcoholic solution of sulphuric acid R and heat at 120 °C for 10 min. In the chromatogram obtained with test solution (a), any spots corresponding to diethylstilbestrol monomethyl ether and diethylstilbestrol dimethyl ether are not more intense than the spots in the chromatograms obtained with reference solutions (b) and (c) respectively (0.5 per cent). Diethylstilbestrol gives one or sometimes C18H20O2 Mr 268.4 two spots. The test is not valid unless the chromatogram obtained with reference solution (d) shows at least two clearly DEFINITION separated spots having approximately the same intensity. Diethylstilbestrol contains not less than 97.0 per cent Loss on drying (2.2.32). Not more than 0.5 per cent, and not more than the equivalent of 101.0 per cent of determined on 1.000 g by drying in an oven at 100 °C to (E)-4,4′-(1,2-diethylethene-1,2-diyl)diphenol, calculated with 105 °C. reference to the dried substance. Sulphated ash (2.4.14). Not more than 0.1 per cent, CHARACTERS determined on 1.0 g. A white or almost white, crystalline powder, practically ASSAY insoluble in water, freely soluble in alcohol. It dissolves in solutions of the alkali hydroxides. Dissolve 20.0 mg in ethanol R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of the solution to 100.0 ml with It melts at about 172 °C. ethanol R. To 25.0 ml of the resulting solution add 25.0 ml IDENTIFICATION of a solution of 1 g of dipotassium hydrogen phosphate R in First identification : B, D. 55 ml of water R. Prepare in the same manner a reference solution using 20.0 mg of diethylstilbestrol CRS. Transfer Second identification : A, C, D. an equal volume of each solution to separate 1 cm quartz A. Examined between 230 nm and 450 nm (2.2.25), the cells and close the cells ; place the two cells about 5 cm from irradiated solution of the substance to be examined prepared as prescribed in the assay shows two absorption a low-pressure, short-wave 2 W to 20 W mercury lamp and irradiate for about 5 min. Measure the absorbance (2.2.25) maxima, at 292 nm and 418 nm. of the irradiated solutions at the maximum at 418 nm, using B. Examine by infrared absorption spectrophotometry water R as the compensation liquid. Continue the irradiation (2.2.24), comparing with the spectrum obtained with for successive periods of 3 min to 15 min, depending on diethylstilbestrol CRS. Examine the substances prepared the power of the lamp, and repeat the measurement of the as discs. absorbances at 418 nm until the maximum absorbance C. Examine the chromatograms obtained in the test for (about 0.7) is obtained. If necessary, adjust the geometry of mono-and dimethyl ethers. The principal spot in the the irradiation apparatus to obtain a maximum, reproducible chromatogram obtained with test solution (b) is similar absorbance at 418 nm. in position, colour and size to the principal spot in the Calculate the content of C18H20O2 from the measured chromatogram obtained with reference solution (a). absorbances and the concentrations of the solutions. D. Dissolve about 0.5 mg in 0.2 ml of glacial acetic acid R, add 1 ml of phosphoric acid R and heat on a water-bath STORAGE for 3 min. A deep-yellow colour develops. Store protected from light. TESTS 4,4′-Dihydroxystilbene and related ethers. Dissolve 0.100 g in ethanol R and dilute to 10.0 ml with the same solvent. The absorbance (2.2.25) of the solution measured at 325 nm is not greater than 0.50. Mono- and dimethyl ethers. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution (a). Dissolve 0.2 g of the substance to be examined in 2 ml of alcohol R. Test solution (b). Dilute 1 ml of test solution (a) to 20 ml with alcohol R. Reference solution (a). Dissolve 10 mg of diethylstilbestrol CRS in 2 ml of alcohol R. Reference solution (b). Dissolve 5 mg of diethylstilbestrol monomethyl ether CRS in alcohol R and dilute to 10 ml with the same solvent. Reference solution (c). Dissolve 5 mg of diethylstilbestrol dimethyl ether CRS in alcohol R and dilute to 10 ml with the same solvent.

DEFINITION Diflunisal contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of 2′,4′-difluoro-4-hydroxybiphenyl-3-carboxylic acid, calculated with reference to the dried substance.


1429

01/2005:0818

DIFLUNISAL Diflunisalum

C13H8F2O3

Mr 250.2

Diflunisal


CHARACTERS A white or almost white, crystalline powder, practically insoluble in water, soluble in alcohol. It dissolves in dilute solutions of alkali hydroxides. IDENTIFICATION

spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.15 per cent). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated principal spots. B. Examine by liquid chromatography (2.2.29).

First identification : B. Second identification : A, C, D. A. Dissolve 10 mg in a 0.3 per cent V/V solution of hydrochloric acid R in methanol R and dilute to 100.0 ml with the same acid solution. Dilute 2.0 ml of the solution to 10.0 ml with a 0.3 per cent V/V solution of hydrochloric acid R in methanol R. Examined between 230 nm and 350 nm (2.2.25), the solution shows two absorption maxima, at 251 nm and 315 nm. The ratio of the absorbance measured at the maximum at 251 nm to that measured at the maximum at 315 nm is 4.2 to 4.6. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with diflunisal CRS. Examine the substances prepared as discs. If the spectra obtained show differences, dissolve the substance to be examined and the reference substance separately in alcohol R, evaporate to dryness and record new spectra using the residues. C. Dissolve about 2 mg in 10 ml of alcohol R and add 0.1 ml of ferric chloride solution R1. A violet-red colour is produced. D. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is obtained (usually less than 5 min). Allow to cool, add 1 ml of water R, 0.05 ml of phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the solution colourless. Filter. Add 1.0 ml of the filtrate to a freshly prepared mixture of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R. Mix, allow to stand for 5 min and compare the colour of the solution with that of a blank prepared in the same manner. The test solution is yellow and the blank is red. TESTS Appearance of solution. Dissolve 0.5 g in alcohol R and dilute to 50 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y7 (2.2.2, Method II). Related substances A. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 0.20 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent.

Test solution. Dissolve 50.0 mg of the substance to be examined in the reference solution and dilute to 10.0 ml with the same solution. Reference solution. Dissolve 55.0 mg of fluoranthene R in a mixture of 1 volume of water R and 4 volumes of acetonitrile R and dilute to 100.0 ml with the same mixture of solvents. Dilute 1.0 ml to 100.0 ml with a mixture of 1 volume of water R and 4 volumes of acetonitrile R. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (10 µm), — as mobile phase at a flow rate of 2 ml/min a mixture of 2 volumes of glacial acetic acid R, 25 volumes of methanol R, 55 volumes of water R and 70 volumes of acetonitrile R, — as detector a spectrophotometer set at 254 nm. Inject 20 µl of the reference solution and adjust the sensitivity of the detector so that the height of the principal peak in the chromatogram obtained is not less than 10 per cent of the full scale of the recorder. Inject separately 20 µl of the test solution and 20 µl of the reference solution. Continue the chromatography for three times the retention time of fluoranthene. In the chromatogram obtained with the test solution, the sum of the areas of any peaks, apart from the principal peak, with a retention time greater than that of fluoranthene is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (0.1 per cent). Disregard any peak with an area less than 0.05 times that of the principal peak in the chromatogram obtained with the reference solution. Heavy metals (2.4.8). 2.0 g complies with limit test C for heavy metals (10 ppm). Use a platinum crucible. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.3 per cent, determined on 1.000 g by drying at 60 °C at a pressure not exceeding 700 Pa for 2 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g in a platinum crucible.

Reference solution (a). Dissolve 30 mg of biphenyl-4-ol R ASSAY in methanol R and dilute to 100 ml with the same solvent. Dissolve 0.200 g in 40 ml of methanol R. Add 5 ml of water R Dilute 1 ml of the solution to 10 ml with methanol R. and 0.2 ml of phenol red solution R. Titrate with 0.1 M Reference solution (b). Dissolve 20 mg of biphenyl-4-ol R sodium hydroxide until the colour changes from yellow to reddish-violet. in methanol R, add 1 ml of the test solution and dilute to 10 ml with methanol R. 1 ml of 0.1 M sodium hydroxide is equivalent to 25.02 mg Apply to the plate 10 µl of each solution. Develop over a of C13H8F2O3. path of 15 cm using a mixture of 10 volumes of glacial acetic acid R, 20 volumes of acetone R and 70 volumes STORAGE of methylene chloride R. Dry the plate in a current of warm air and examine in ultraviolet light at 254 nm. Any Store protected from light. 1430


Digitalis leaf


IMPURITIES

A. R1 = R2 = R3 = H : biphenyl-4-ol, B. R1 = H, R2 = R3 = F : 2′,4′-difluorobiphenyl-4-ol, C. R1 = CO-CH3, R2 = R3 = F : 2′,4′-difluorobiphenyl-4-yl acetate, D. condensation products. 01/2005:0117

DIGITALIS LEAF Digitalis purpureae folium DEFINITION Digitalis leaf consists of the dried leaf of Digitalis purpurea L. It contains not less than 0.3 per cent of cardenolic glycosides, expressed as digitoxin (Mr 765), and calculated with reference to the drug dried at 100 °C to 105 °C. CHARACTERS Digitalis leaf has a faint but characteristic odour. The whole leaf is about 10 cm to 40 cm long and 4 cm to 15 cm wide. The lamina is ovate lanceolate to broadly ovate. The winged petiole is from one quarter as long as to equal in length to the lamina. It has the macroscopic and microscopic characters described in identication tests A and B. IDENTIFICATION A. The leaf is brittle and often occurs broken. The upper surface is green and the lower surface is greyish-green. The apex is subacute and the margin is irregularly crenate, dentate or serrate. The base is decurrent. The venation is pinnate, the lateral veins being prominent especially on the lower surface, leaving the midrib at about 45° and anastomosing near the margin ; a veinlet terminates in each tooth of the margin and the lower veins run down the winged petiole. The upper surface is rugose and pubescent ; the lower surface shows a network of raised veinlets and is densely pubescent. B. Reduce to a powder (355). Examine under a microscope using chloral hydrate solution R. The powder shows the following diagnostic characters : epidermal cells with anticlinal walls which are straight or slightly sinuous on the upper surface and markedly sinuous on the lower surface ; the cuticle is smooth. Trichomes are of two types : uniseriate, bluntly pointed non-glandular, usually of three to five cells, often with one or more collapsed cells, walls mostly finely warty or faintly striated ; glandular trichomes usually with a unicellular, sometimes a multicellular uniseriate stalk and a unicellular or bicellular or exceptionally tetracellular head. Anomocytic stomata (2.8.3) are absent or very rare on the upper surface, numerous on the lower surface. Calcium oxalate crystals and sclerenchyma are absent. C. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. To 1.0 g of the powdered drug (180) add a mixture of 20 ml of alcohol (50 per cent V/V) R and 10 ml of lead acetate solution R. Boil for 2 min, allow to cool and centrifuge. Shake the supernatant solution with General Notices (1) apply to all monographs and other texts

two quantities, each of 15 ml, of chloroform R ; separate the two layers by centrifugation if necessary. Dry the chloroform layers over anhydrous sodium sulphate R and filter. Evaporate 10 ml of the solution to dryness on a water-bath and dissolve the residue in 1 ml of a mixture of equal volumes of chloroform R and methanol R. Reference solution. Dissolve 5 mg of purpureaglycoside A CRS, 2 mg of purpureaglycoside B CRS, 5 mg of digitoxin R and 2 mg of gitoxin R in a mixture of equal volumes of chloroform R and methanol R and dilute to 10 ml with the same mixture of solvents. Apply separately to the plate as bands 2 cm by 0.3 cm 20 µl of each solution. Develop over a path of 10 cm using a mixture of 7.5 volumes of water R, 10 volumes of methanol R and 75 volumes of ethyl acetate R. Allow the solvents to evaporate. Spray with a mixture of 2 volumes of a 10 g/l solution of chloramine R and 8 volumes of a 250 g/l solution of trichloroacetic acid R in alcohol R. Heat at 100 °C to 105 °C for 10 min. Examine in ultraviolet light at 365 nm. The chromatogram obtained with the reference solution shows a zone of light-blue fluorescence in the lower part of the chromatogram, corresponding to purpureaglycoside B, and just above it a zone of brownish-yellow fluorescence, corresponding to purpureaglycoside A. A zone of light-blue fluorescence, corresponding to gitoxin, appears in the middle of the chromatogram and above it a zone of brownish-yellow fluorescence, corresponding to digitoxin. The zones in the chromatogram obtained with the test solution are similar in position, colour and size to the zones in the chromatogram obtained with the reference solution. Other zones of fluorescence may also appear in the chromatogram obtained with the test solution. D. Evaporate 5 ml of the chloroformic solution obtained in identification test C to dryness on a water-bath. To the residue add 2 ml of dinitrobenzoic acid solution R and 1 ml of 1 M sodium hydroxide. A reddish-violet colour develops within 5 min. E. Evaporate 5 ml of the chloroformic solution obtained in identification test C to dryness on a water-bath. To the residue add 3 ml of xanthydrol solution R and heat on a water-bath for 3 min. A red colour develops. TESTS Foreign matter (2.8.2). There are no leaves with few or no trichomes and epidermal cells showing, in surface view, beaded anticlinal walls (Digitalis lanata). Loss on drying (2.2.32). Not more than 6.0 per cent, determined on 1.000 g of the powdered drug (355) by drying in an oven at 100 °C to 105 °C. Total ash (2.4.16). Not more than 12.0 per cent. Ash insoluble in hydrochloric acid (2.8.1). Not more than 5.0 per cent. ASSAY Shake 0.250 g of the powdered drug (180) with 50.0 ml of water R for 1 h. Add 5.0 ml of a 150 g/l solution of lead acetate R, shake and after a few minutes add 7.5 ml of a 40 g/l solution of disodium hydrogen phosphate R. Filter through a pleated filter paper. Heat 50.0 ml of the filtrate with 5 ml of hydrochloric acid (150 g/l HCl) under a reflux condenser on a water-bath for 1 h. Transfer to a separating funnel, rinse the flask with two quantities, each of 5 ml, of water R and shake with three quantities, each of 25 ml, of chloroform R. Dry the combined chloroform layers over anhydrous sodium sulphate R and dilute to 100.0 ml with chloroform R. Evaporate 40.0 ml of the chloroformic 1431

Digitoxin


solution to dryness, dissolve the residue in 7 ml of alcohol (50 per cent V/V) R and add 2 ml of dinitrobenzoic acid solution R and 1 ml of 1 M sodium hydroxide. At the same time prepare a reference solution as follows. Dissolve 50.0 mg of digitoxin CRS in alcohol R and dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of the solution to 50.0 ml with alcohol R. To 5.0 ml of the resulting solution add 25 ml of water R and 3 ml of hydrochloric acid (150 g/l HCl). Heat the solution under a reflux condenser on a water-bath for 1 h and complete the preparation as described above. Measure the absorbance (2.2.25) of the two solutions at 540 nm several times during the first 12 min until the maximum is reached, using as the compensation liquid a mixture of 7 ml of alcohol (50 per cent V/V) R, 2 ml of dinitrobenzoic acid solution R and 1 ml of 1 M sodium hydroxide. From the absorbances measured and the concentrations of the solutions, calculate the content of cardenolic glycosides, expressed as digitoxin.

A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with digitoxin CRS. B. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). C. Suspend about 0.5 mg in 0.2 ml of alcohol (60 per cent V/V) R. Add 0.1 ml of dinitrobenzoic acid solution R and 0.1 ml of dilute sodium hydroxide solution R. A violet colour develops. D. Dissolve about 0.5 mg in 1 ml of glacial acetic acid R, heating gently, allow to cool and add 0.05 ml of ferric chloride solution R1. Cautiously add 1 ml of sulphuric acid R, avoiding mixing the two liquids. A brown ring develops at the interface and on standing a green, then blue colour passes to the upper layer.

STORAGE Store protected from light and moisture.

TESTS Appearance of solution. Dissolve 50 mg in a mixture of equal volumes of methanol R and methylene chloride R and dilute to 10 ml with the same mixture of solvents. The 01/2005:0078 solution is clear (2.2.1) and colourless (2.2.2, Method I). Specific optical rotation (2.2.7). Dissolve 0.25 g in DIGITOXIN chloroform R and dilute to 10.0 ml with the same solvent. The specific optical rotation is + 16.0 to + 18.5. Digitoxinum Related substances. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R. Test solution. Dissolve 20 mg of the substance to be examined in a mixture of equal volumes of methanol R and methylene chloride R and dilute to 2 ml with the same mixture of solvents. Reference solution (a). Dissolve 20 mg of digitoxin CRS in a mixture of equal volumes of methanol R and methylene chloride R and dilute to 2 ml with the same mixture of solvents. Reference solution (b). Dilute 0.5 ml of reference solution (a) to 50 ml with a mixture of equal volumes of methanol R and methylene chloride R. Reference solution (c). Dissolve 10 mg of gitoxin CRS with stirring in a mixture of equal volumes of methanol R and methylene chloride R and dilute to 50 ml with the same mixture of solvents. Reference solution (d). Dilute 1 ml of reference solution (b) to 2 ml with a mixture of equal volumes of methanol R and methylene chloride R. C41H64O13 Mr 765 Reference solution (e). Mix 1 ml of reference solution (a) and 1 ml of reference solution (c). DEFINITION Apply to the plate 5 µl of each solution. Develop immediately over a path of 15 cm using a mixture of 15 volumes of Digitoxin contains not less than 95.0 per cent and methanol R, 40 volumes of cyclohexane R and 90 volumes not more than the equivalent of 103.0 per cent of of methylene chloride R. Dry the plate in a stream of cold 3β-[(O-2,6-dideoxy-β-D-ribo-hexopyranosyl-(1→4)-O-2,6air for 5 min. Repeat the development and dry the plate dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-D-riboin a stream of cold air for 5 min. Spray with a mixture of hexopyranosyl)oxy]-14-hydroxy-5β,14β-card-20(22)-enolide, 1 volume of sulphuric acid R and 9 volumes of alcohol R calculated with reference to the dried substance. and heat at 130 °C for 15 min. Examine in daylight. CHARACTERS Gitoxin. Any spot corresponding to gitoxin in the A white or almost white powder, practically insoluble in chromatogram obtained with the test solution is not more water, freely soluble in a mixture of equal volumes of intense than the spot in the chromatogram obtained with methanol and methylene chloride, slightly soluble in alcohol reference solution (c) (2.0 per cent). and in methanol. Other glycosides. Any spot in the chromatogram obtained with the test solution, apart from the principal spot and IDENTIFICATION the spot corresponding to gitoxin, is not more intense than First identification : A. the spot in the chromatogram obtained with reference solution (b) (1.0 per cent). Second identification : B, C, D. 1432



Digoxin

IDENTIFICATION First identification : A. Second identification : B, C, D. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with digoxin CRS. B. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). ASSAY C. Suspend about 0.5 mg in 0.2 ml of alcohol (60 per Dissolve 40.0 mg in alcohol R and dilute to 50.0 ml with the cent V/V) R. Add 0.1 ml of dinitrobenzoic acid solution R same solvent. Dilute 5.0 ml of the solution to 100.0 ml with and 0.1 ml of dilute sodium hydroxide solution R. A alcohol R. Prepare a reference solution in the same manner, violet colour develops. using 40.0 mg of digitoxin CRS. To 5.0 ml of each solution D. Dissolve about 0.5 mg in 1 ml of glacial acetic acid R, add 3.0 ml of alkaline sodium picrate solution R, allow to heating gently, allow to cool and add 0.05 ml of ferric stand protected from bright light for 30 min and measure chloride solution R1. Cautiously add 1 ml of sulphuric the absorbance (2.2.25) of each solution at the maximum acid R avoiding mixing the two liquids. A brown ring at 495 nm, using as the compensation liquid a mixture of develops at the interface and on standing a green, then 5.0 ml of alcohol R and 3.0 ml of alkaline sodium picrate blue colour passes to the upper layer. solution R prepared at the same time. The test is not valid unless the chromatogram obtained with reference solution (e) shows clearly separated spots corresponding to digitoxin, gitoxin and other glycosides and the spot in the chromatogram obtained with reference solution (d) is clearly visible. Loss on drying (2.2.32). Not more than 1.5 per cent, determined on 0.500 g by drying in an oven at 100 °C to 105 °C for 2 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on the residue from the test for loss on drying.

Calculate the content of C41H64O13 from the absorbances measured and the concentrations of the solutions.

TESTS Appearance of solution. Dissolve 50 mg in a mixture of equal volumes of methanol R and methylene chloride R STORAGE and dilute to 10 ml with the same mixture of solvents. The Store protected from light. solution is clear (2.2.1) and colourless (2.2.2, Method I). 01/2005:0079 Specific optical rotation (2.2.7). Dissolve 0.20 g in anhydrous pyridine R and dilute to 10.0 ml with the same solvent. The specific optical rotation is + 10.0 to + 13.0, DIGOXIN calculated with reference to the dried substance. Related substances. Examine by thin-layer chromatography Digoxinum (2.2.27), using kieselguhr G R as the coating substance. Impregnate the plate by placing it in a closed chromatographic tank containing the necessary quantity of a mixture of 10 volumes of formamide R and 90 volumes of acetone R so that the plate dips about 5 mm into the liquid. When the impregnation mixture has risen at least 15 cm from the lower edge of the plate, remove the plate and allow the solvent to evaporate for 30 min. Use the plate immediately. Test solution. Dissolve 50 mg of the substance to be examined in a mixture of equal volumes of methanol R and methylene chloride R and dilute to 5 ml with the same mixture of solvents. Reference solution (a). Dissolve 20 mg of digoxin CRS in a mixture of equal volumes of methanol R and methylene chloride R and dilute to 2 ml with the same mixture of solvents. Reference solution (b). Dilute 1 ml of reference solution (a) to 50 ml with a mixture of equal volumes of methanol R and methylene chloride R. Reference solution (c). Dilute 2 ml of reference solution (b) C41H64O14 Mr 781 to 4 ml with a mixture of equal volumes of methanol R and methylene chloride R. DEFINITION Reference solution (d). Dissolve 5 mg of digitoxin CRS in Digoxin contains not less than 95.0 per cent and a mixture of equal volumes of methanol R and methylene not more than the equivalent of 103.0 per cent of chloride R and dilute to 50 ml with the same mixture of 3β-[(O-2,6-dideoxy-β-D-ribo-hexopyranosyl-(1→4)-O-2,6solvents. dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-D-ribohexopyranosyl)oxy]-12β,14-dihydroxy-5β-card-20(22)-enolide, Reference solution (e). Dissolve 5 mg of gitoxin CRS in a mixture of equal volumes of methanol R and methylene calculated with reference to the dried substance. chloride R and dilute to 25 ml with the same mixture of solvents. CHARACTERS Apply separately to the plate 2 µl of each solution. Develop A white or almost white powder or colourless crystals, over a path of 12 cm using a mixture of 4 volumes of practically insoluble in water, freely soluble in a mixture of equal volumes of methanol and methylene chloride, slightly formamide R, 50 volumes of methyl ethyl ketone R and 50 volumes of xylene R. Dry the plate in a current of cold soluble in alcohol. General Notices (1) apply to all monographs and other texts

1433

Dihydralazine sulphate, hydrated


air until only the lower edge is still visibly moist. Repeat the development and dry the plate at 115 °C for 20 min. Allow to cool, spray with a mixture of 1 volume of a freshly prepared 30 g/l solution of chloramine R and 15 volumes of a 250 g/l solution of trichloroacetic acid R in alcohol R and heat at 115 °C for 5 min. Examine in ultraviolet light at 365 nm. Any spot corresponding to digitoxin in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (d) (1.0 per cent). Any spot corresponding to gitoxin in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (e) (2.0 per cent). Any spot in the chromatogram obtained with the test solution, apart from the principal spot and spots corresponding to digitoxin and gitoxin, is not more intense than the spot in the chromatogram obtained with reference solution (b) (2.0 per cent) and at most one such spot is more intense than the spot in the chromatogram obtained with reference solution (c) (1.0 per cent). Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 0.500 g by drying in vacuo. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on the residue from the test for loss on drying. ASSAY Dissolve 40.0 mg in alcohol R, heating if necessary, and dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of the solution to 100.0 ml with alcohol R. Prepare a reference solution in the same manner, using 40.0 mg of digoxin CRS. To 5.0 ml of each solution add 3.0 ml of alkaline sodium picrate solution R, allow to stand protected from bright light for 30 min and measure the absorbance (2.2.25) of each solution at the maximum at 495 nm, using as the compensation liquid a mixture of 5.0 ml of alcohol R and 3.0 ml of alkaline sodium picrate solution R prepared at the same time. Calculate the content of C41H64O14 from the absorbances measured and the concentrations of the solutions. STORAGE Store protected from light. IMPURITIES A. digitoxin,

B. 3β-[(O-2,6-dideoxy-β-D-ribo-hexopyranosyl-(1→4)-O-2,6dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-Dribo-hexopyranosyl)oxy]-14,16β-dihydroxy-5β-card-20(22)enolide (gitoxin). 1434


DIHYDRALAZINE SULPHATE, HYDRATED Dihydralazini sulfas hydricus

C8H12N6O4S,21/2H2O

Mr 333.3

DEFINITION Hydrated dihydralazine sulphate contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of (phthalazine-1,4(2H,3H)-diylidene)dihydrazine sulphate, calculated with reference to the dried substance. CHARACTERS A white or slightly yellow, crystalline powder, slightly soluble in water, practically insoluble in ethanol. It dissolves in dilute mineral acids. IDENTIFICATION A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of dihydralazine sulphate hydrated. B. Dissolve about 50 mg in 5 ml of dilute hydrochloric acid R. The solution gives reaction (a) of sulphates (2.3.1). TESTS Appearance of solution. Dissolve 0.20 g in dilute nitric acid R and dilute to 10 ml with the same acid. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY6 (2.2.2, Method II). Related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately before use. Test solution. Dissolve 50.0 mg of the substance to be examined in a 6 g/l solution of glacial acetic acid R and dilute to 50.0 ml with the same solvent. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase containing 0.5 g/l of sodium edetate R. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase containing 0.5 g/l of sodium edetate R. Reference solution (b). Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase containing 0.5 g/l of sodium edetate R. Reference solution (c). Dissolve 5 mg of dihydralazine for system suitability CRS in a 6 g/l solution of glacial acetic acid R and dilute to 5.0 ml with the same solvent. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : nitrile silica gel for chromatography R (5 µm). Mobile phase : to 22 volumes of acetonitrile R add 78 volumes of a solution containing 1.44 g/l of sodium laurilsulfate R and 0.75 g/l of tetrabutylammonium bromide R and adjust to pH 3.0 with 0.05 M sulphuric acid. Flow rate : 1.5 ml/min. Detection : spectrophotometer at 230 nm. Injection : 20 µl.


Dihydrocodeine hydrogen tartrate


Run time : twice the retention time of dihydralazine. Relative retention with reference to dihydralazine : impurity A = about 0.8. System suitability : reference solution (c) : — the peaks due to impurity A and dihydralazine are baseline separated as in the chromatogram supplied with dihydralazine for system suitability CRS. Limits : — impurity A : not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (2 per cent), — impurity C : not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), — any other impurity : for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), — total of impurities other than A : not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent), — disregard limit : 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (a). Hydrazine. Examine by liquid chromatography (2.2.29). Prepare the solutions immediately before use. Test solution. Dissolve 40.0 mg of hydrazine sulphate R in water R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 25.0 ml with water R. To 0.50 ml of the solution, add 0.200 g of the substance to be examined and dissolve in 6 ml of dilute hydrochloric acid R, then dilute to 10.0 ml with water R. In a centrifuge tube with a ground-glass stopper, place immediately 0.50 ml of the solution and 2.0 ml of a 60 g/l solution of benzaldehyde R in a mixture of equal volumes of methanol R and water R. Shake for 90 s. Add 1.0 ml of water R and 5.0 ml of heptane R. Shake for 1 min and centrifuge. Use the upper layer. Reference solution. Dissolve 40.0 mg of hydrazine sulphate R in water R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 25.0 ml with water R. To 0.50 ml of the solution, add 6 ml of dilute hydrochloric acid R and dilute to 10.0 ml with water R. In a centrifuge tube with a ground-glass stopper, place 0.50 ml of the solution and 2.0 ml of a 60 g/l solution of benzaldehyde R in a mixture of equal volumes of methanol R and water R. Shake for 90 s. Add 1.0 ml of water R and 5.0 ml of heptane R. Shake for 1 min and centrifuge. Use the upper layer. Blank solution. Prepare in the same manner as for the reference solution but replacing the 0.50 ml of hydrazine sulphate solution by 0.50 ml of water R. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1 ml/min a mixture of 30 volumes of a 0.3 g/l solution of sodium edetate R and 70 volumes of acetonitrile R, — as detector a spectrophotometer set at 305 nm. Inject 20 µl each of the test solution, the reference solution and the blank solution. Comparing the chromatograms obtained with the reference solution and the blank solution, identify the peak of benzaldehyde azine (benzalazine) corresponding to hydrazine having a retention time relative to the principal peak (benzaldehyde) of about 1.8. In the chromatogram obtained with the test solution, the area of the peak corresponding to benzaldehyde azine is not General Notices (1) apply to all monographs and other texts

greater than twice that of the corresponding peak in the chromatogram obtained with the reference solution (10 ppm of hydrazine). Iron (2.4.9). To the residue obtained in the test for sulphated ash add 0.2 ml of sulphuric acid R and heat carefully until the acid is almost completely eliminated. Allow to cool and dissolve the residue with heating in 5.5 ml of hydrochloric acid R1. Filter the hot solution through a filter previously washed 3 times with dilute hydrochloric acid R. Wash the crucible and the filter with 5 ml of water R. Combine the filtrate and the washings and neutralise with about 3.5 ml of strong sodium hydroxide solution R. Adjust the pH to between 3 and 4 with acetic acid R and dilute to 20 ml with water R. The solution complies with the limit test for iron (20 ppm). Prepare the standard with 5 ml of iron standard solution (2 ppm Fe) R and 5 ml of water R. Loss on drying (2.2.32) : 13.0 per cent to 15.0 per cent, determined on 1.000 g by drying in an oven at 50 °C at a pressure not exceeding 0.7 kPa for 5 h. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 60.0 mg in 25 ml of water R. Add 35 ml of hydrochloric acid R and titrate slowly with 0.05 M potassium iodate, determining the end-point potentiometrically (2.2.20), using a calomel reference electrode and a platinum indicator electrode. 1 ml of 0.05 M potassium iodate is equivalent to 7.208 mg of C8H12N6O4S. IMPURITIES Specified impurities : A, B, C.

A. R = NH2 : 4-hydrazinophthalazin-1-amine, C. R = H :(phthalazin-1-yl)hydrazine (hydralazine), B. H2N-NH2 : hydrazine. 01/2005:1776

DIHYDROCODEINE HYDROGEN TARTRATE Dihydrocodeini hydrogenotartras

C22H29NO9

Mr 451.5

DEFINITION 4,5α-Epoxy-3-methoxy-17-methylmorphinan-6α-ol hydrogen (2R,3R)-2,3-dihydroxybutanedioate. Content : 98.5 per cent to 101.0 per cent (anhydrous substance). 1435

Dihydrocodeine hydrogen tartrate


CHARACTERS

System suitability : reference solution (a) :

Appearance : white or almost white, crystalline powder.

— resolution : minimum of 2 between the peaks due to dihydrocodeine and to impurity A.

Solubility : freely soluble in water, sparingly soluble in alcohol, practically insoluble in cyclohexane.

Limits : — impurity A : not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent),

IDENTIFICATION First identification : A.

— any other peak : not more than 0.6 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.3 per cent),

Second identification : B, C, D. A. Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of dihydrocodeine hydrogen tartrate. B. To about 0.1 g add 1 ml of sulphuric acid R and 0.05 ml of ferric chloride solution R1 and heat on a water-bath. A brownish-yellow colour develops. Add 0.05 ml of dilute nitric acid R. The colour does not become red. C. To 1 ml of solution S (see Tests) add 5 ml of picric acid solution R. Heat on a water-bath until a clear solution is obtained. Allow to cool. A precipitate is formed. Filter, wash with 5 ml of water R and dry at 100-105 °C. The crystals melt (2.2.14) at 220 °C to 223 °C. D. It gives reaction (b) of tartrates (2.3.1). TESTS Solution S. Dissolve 2.50 g in carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution BY5 (2.2.2, Method II). pH (2.2.3) : 3.2 to 4.2 for solution S.

— total : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent); disregard any peak due to tartaric acid (relative retention with reference to dihydrocodeine = about 0.25), — disregard limit : 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.05 per cent). Water (2.5.12) : maximum 0.7 per cent, determined on 1.00 g. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.350 g in 60 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 45.15 mg of C22H29NO9. STORAGE Protected from light.

Specific optical rotation (2.2.7) : − 70.5 to − 73.5 (anhydrous IMPURITIES substance). Dilute 10.0 ml of solution S to 20.0 ml with water R. Related substances. Liquid chromatography (2.2.29).

A. codeine,


B. morphine,

Reference solution (a). Dissolve 2.0 mg of codeine phosphate R in 2.0 ml of the test solution and dilute to 25.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of the test solution to 200 ml with the mobile phase. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase: octylsilyl silica gel for chromatography R (5 µm).

C. 4,5α-epoxy-3-methoxy-17-methylmorphinan-6-one (hydrocodone),

Mobile phase : to 1.0 g of sodium heptanesulphonate R, add 10.0 ml of glacial acetic acid R and 4.0 ml of a solution of 5.0 ml of triethylamine R diluted to 25.0 ml with a mixture of equal volumes of water R and acetonitrile R. Add 170 ml of acetonitrile R and dilute to 1000 ml with water R. Flow rate : 1 ml/min. Detection : spectrophotometer at 284 nm. Injection : 20 µl. Run time : 5 times the retention time of dihydrocodeine. Retention time : dihydrocodeine = about 14 min. 1436

D. 4,5α-epoxy-3,6α-dimethoxy-17-methylmorphinan (tetrahydrothebaine).



Dihydroergocristine mesilate

01/2005:1416 C. Thin-layer chromatography (2.2.27). Test solution. Dissolve 0.20 g of the substance to be examined in a mixture of 1 volume of methanol R and DIHYDROERGOCRISTINE MESILATE 9 volumes of methylene chloride R and dilute to 5 ml with the same mixture of solvents. Dihydroergocristini mesilas Reference solution. Dissolve 0.20 g of methanesulphonic acid R in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 5 ml with the same mixture of solvents. Dilute 1 ml of the solution to 10 ml with a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R. Plate : TLC silica gel F254 plate R. Mobile phase : water R, concentrated ammonia R, butanol R, acetone R (5:10:20:65 V/V/V/V). Application : 10 µl. Development : over a path of 10 cm protected from light. C36H45N5O8S Mr 708 Drying : in a current of cold air for not more than 1 min. DEFINITION Detection : spray with a 1 g/l solution of bromocresol (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-Benzyl-10b-hydroxypurple R in methanol R, adjusting the colour to violet-red 2-(1-methylethyl)-3,6-dioxo-octahydro-8H-oxazolo[3,2with one drop of dilute ammonia R1 and dry the plate in a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10, a current of hot air at 100 °C. 10a-octahydroindolo[4,3-fg]quinoline-9-carboxamide Results : the principal spot in the chromatogram obtained methanesulphonate. with the test solution is similar in position, colour and Content : 98.0 per cent to 102.0 per cent (dried substance). size to the principal spot in the chromatogram obtained with the reference solution. PRODUCTION The production method must be evaluated to determine TESTS the potential for formation of alkyl mesilates, which is Appearance of solution. The solution is clear (2.2.1) and not particularly likely to occur if the reaction medium contains more intensely coloured than reference solution B7 (2.2.2, lower alcohols. Where necessary, the production method Method II). is validated to demonstrate that alkyl mesilates are not Dissolve 0.50 g in methanol R and dilute to 25.0 ml with detectable in the final product. the same solvent. CHARACTERS pH (2.2.3) : 4.0 to 5.0. Appearance : white or almost white, fine crystalline powder. Dissolve 0.10 g in carbon dioxide-free water R and dilute to Solubility : slightly soluble in water, soluble in methanol. 20 ml with the same solvent. Specific optical rotation (2.2.7) : − 37 to − 43 (dried IDENTIFICATION substance). A. Infrared absorption spectrophotometry (2.2.24). Dissolve 0.250 g in anhydrous pyridine R and dilute to Preparation : discs. 25.0 ml with the same solvent. Comparison : dihydroergocristine mesilate CRS. Related substances. Liquid chromatography (2.2.29). Carry B. Thin-layer chromatography (2.2.27). out the test and preparation of the solutions protected from Test solution. Dissolve 0.10 g of the substance to be bright light. examined in a mixture of 1 volume of methanol R and Test solution. Dissolve 75.0 mg of the substance to be 9 volumes of methylene chloride R and dilute to 5 ml examined in 10 ml of acetonitrile R. Add 10 ml of a 1.0 g/l with the same mixture of solvents. solution of phosphoric acid R and dilute to 50.0 ml with Reference solution. Dissolve 0.10 g of water R. dihydroergocristine mesilate CRS in a mixture Reference solution. Dissolve 20.0 mg of codergocrine of 1 volume of methanol R and 9 volumes of methylene mesilate CRS in 10 ml of acetonitrile R. Add 10 ml of a chloride R and dilute to 5 ml with the same mixture of 1.0 g/l solution of phosphoric acid R and dilute to 50.0 ml solvents. with water R. Dilute 6.0 ml of the solution to 50.0 ml with Plate : TLC silica gel F254 plate R. a mixture of 20 volumes of acetonitrile R, 20 volumes of a 1.0 g/l solution of phosphoric acid R and 60 volumes of Mobile phase : concentrated ammonia R, water R. dimethylformamide R, ether R (2:15:85 V/V/V). Column : Application : 5 µl. Development : over 2/3 of the plate protected from light. — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : octadecylsilyl silica gel for Drying : in a current of cold air for 5 min. chromatography R (5 µm) with a pore size of 10 nm and Detection : spray with dimethylaminobenzaldehyde a carbon loading of 19 per cent. solution R7 and dry in a current of hot air for 2 min. Results : the principal spot in the chromatogram obtained Mobile phase : with the test solution is similar in position, colour and — mobile phase A : mix 100 volumes of acetonitrile R size to the principal spot in the chromatogram obtained with 900 volumes of water R and add 10 volumes of with the reference solution. triethylamine R, General Notices (1) apply to all monographs and other texts

1437

Dihydroergocristine mesilate


— mobile phase B : mix 100 volumes of water R with 900 volumes of acetonitrile R and add 10 volumes of triethylamine R. Time (min)



0-5

75

25

5 - 20

75 → 25

25 → 75

20 - 22

25 → 75

75 → 25

22 - 30

75

25

B. (6aR,9S,10aS)-7-methyl-4,6,6a,7,8,9,10,10aoctahydroindolo[4,3-fg]quinoline-9-carboxamide (6-methylisoergoline-8α-carboxamide),

Flow rate : 1.2 ml/min. Detection : spectrophotometer at 280 nm. Injection : 10 µl. Relative retention with reference to dihydroergocristine (retention time = about 13.7 min) : impurity F = about 0.8 ; impurity H = about 0.9 ; impurity I = about 1.02. System suitability : reference solution : — the chromatogram shows 4 peaks, — resolution : minimum 1 between the peaks corresponding C. (6aR,9R,10aR)-N-[(2S,5S,10aS,10bS)-5-benzyl-10bhydroxy-2-(1-methylethyl)-3,6-dioxooctahydro-8Hto dihydroergocristine and impurity I. oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4, Limits : 6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9carboxamide (2′-epidihydroergocristine), — any impurity : not more than the area of the peak corresponding to dihydroergocristine in the chromatogram obtained with the reference solution (1 per cent), — total: not more than twice the area of the peak corresponding to dihydroergocristine in the chromatogram obtained with the reference solution (2 per cent), — disregard limit : 0.1 times the area of the peak corresponding to dihydroergocristine in the D. R1 = CH(CH3)2, R2 = CH3 : (6aR,9R,10aR)-N-[(2R,5S, chromatogram obtained with the reference solution 10aS,10bS)-10b-hydroxy-2-methyl-5-(1-methylethyl)-3,6(0.1 per cent). dioxooctahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazinLoss on drying (2.2.32) : maximum 3.0 per cent, determined 2-yl]-7-methyl-4,6,6a,7,8,9,10,10a-octahydroindolo[4,3on 0.500 g by drying under high vacuum at 80 °C. fg]quinoline-9-carboxamide (dihydroergosine), ASSAY Dissolve 0.300 g in 60 ml of pyridine R. Pass a stream of nitrogen R over the surface of the solution and titrate with 0.1 M tetrabutylammonium hydroxide, determining the end-point potentiometrically (2.2.20). Note the volume used at the second point of inflexion. 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to 35.39 mg of C36H45N5O8S. STORAGE Store protected from light. IMPURITIES

A. (6aR,9R,10aR)-7-methyl-4,6,6a,7,8,9,10,10aoctahydroindolo[4,3-fg]quinoline-9-carboxamide (6-methylergoline-8β-carboxamide), 1438

E. R1 = CH2-C6H5, R2 = CH3 : (6aR,9R,10aR)-N-[(2R,5S,10aS, 10bS)-5-benzyl-10b-hydroxy-2-methyl-3,6-dioxooctahydro8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline9-carboxamide (dihydroergotamine), F. R1 = R2 = CH(CH3)2 : (6aR,9R,10aR)-N-[(2R,5S, 10aS,10bS)10b-hydroxy-2,5-bis(1-methylethyl)-3,6dioxooctahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin2-yl]-7-methyl-4,6,6a,7,8,9,10,10a-octahydroindolo[4,3fg]quinoline-9-carboxamide (dihydroergocornine), G. R1 = CH2-C6H5, R2 = CH2-CH3 : (6aR,9R,10aR)-N[(2R,5S,10aS,10bS)-5-benzyl-2-ethyl-10b-hydroxy-3,6dioxooctahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin2-yl]-7-methyl-4,6,6a,7,8,9,10,10a-octahydroindolo[4,3fg]quinoline-9-carboxamide (dihydroergostine), H. R1 = CH2-CH(CH3)2, R2 = CH(CH3)2 : (6aR,9R,10aR)N-[(2R,5S,10aS,10bS)-10b-hydroxy-2-(1-methylethyl)5-(2-methylpropyl)-3,6-dioxooctahydro-8H-oxazolo[3, 2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10, 10a-octahydroindolo[4,3-fg]quinoline-9-carboxamide (α-dihydroergocryptine),



Dihydroergotamine mesilate

Content : 98.0 per cent to 101.0 per cent (dried substance). I. R1 = C*H(CH3)-CH2-CH3, R2 = CH(CH3)2 : (6aR,9R,10aR)N-[(2R,5S,10aS,10bS)-10b-hydroxy-2-(1-methylethyl)-5[(1RS-1-methylpropyl]-3,6-dioxooctahydro-8H-oxazolo[3, 2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10, 10a-octahydroindolo[4,3-fg]quinoline-9-carboxamide (β-dihydroergocryptine or epicriptine), J. R1 = CH2-C6H5, R2 = C*H(CH3)-CH2-CH3 : (6aR,9R,10aR)N-[(2R,5S,10aS,10bS)-5-benzyl-10b-hydroxy-2-[(1RS)1-methylpropyl]-3,6-dioxooctahydro-8H-oxazolo[3,2a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10, 10a-octahydroindolo[4,3-fg]quinoline-9-carboxamide (dihydroergosedmine),

K. (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-10bhydroxy-2-(1-methylethyl)-3,6-dioxooctahydro-8Hoxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a, 7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (ergocristine),

L. (6aR,7RS,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl10b-hydroxy-2-(1-methylethyl)-3,6-dioxooctahydro-8Hoxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4, 6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9carboxamide 7-oxide (dihydroergocristine 6-oxide).

PRODUCTION The production method must be evaluated to determine the potential for formation of alkyl mesilates, which is particularly likely to occur if the reaction medium contains lower alcohols. Where necessary, the production method is validated to demonstrate that alkyl mesilates are not detectable in the final product. CHARACTERS Appearance : white or almost white, crystalline powder or colourless crystals. Solubility : slightly soluble in water, sparingly soluble in methanol, slightly soluble in alcohol. IDENTIFICATION First identification : B, C. Second identification : A, C, D. A. Dissolve 5.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Examined between 250 nm and 350 nm (2.2.25), the solution shows 2 absorption maxima, at 281 nm and 291 nm, and a shoulder at 275 nm. Above 320 nm the absorbance is negligible. The specific absorbance at the absorption maximum at 281 nm is 95 to 105 (dried substance). B. Infrared absorption spectrophotometry (2.2.24). Preparation : discs. Comparison : dihydroergotamine mesilate CRS. C. Examine the chromatograms obtained in the test for related substances. Results : the principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). D. To 0.1 g add 5 ml of dilute hydrochloric acid R and shake for about 5 min. Filter and add 1 ml of barium chloride solution R1. The filtrate remains clear. Mix 0.1 g with 0.4 g of powdered sodium hydroxide R, heat to fusion and continue to heat for 1 min. Cool, add 5 ml of water R, boil and filter. Acidify the filtrate by the addition of hydrochloric acid R1 and filter again. The filtrate gives reaction (a) of sulphates (2.3.1).

01/2005:0551 TESTS Appearance of solution. The solution is clear (2.2.1) and not DIHYDROERGOTAMINE MESILATE more intensely coloured than reference solution Y7 or BY7 (2.2.2, Method II). Dissolve 0.10 g in a mixture of 0.1 ml of a 70 g/l solution of Dihydroergotamini mesilas methanesulphonic acid R and 50 ml of water R. pH (2.2.3) : 4.4 to 5.4. Dissolve 0.10 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Specific optical rotation (2.2.7) : − 42 to − 47 (dried substance). Dissolve 0.250 g in anhydrous pyridine R and dilute to 25.0 ml with the same solvent. C34H41N5O8S Mr 680 Related substances. Thin-layer chromatography (2.2.27). Prepare the reference solutions and the test solutions DEFINITION immediately before use and in the order indicated below. Reference solution (a). Dissolve 10.0 mg of (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-Benzyl-10bdihydroergotamine mesilate CRS in a mixture of hydroxy-2-methyl-3,6-dioxooctahydro-8H-oxazolo[3,21 volume of methanol R and 9 volumes of methylene a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10, chloride R and dilute to 5 ml with the same mixture of 10a-octahydroindolo[4,3-fg]quinoline-9-carboxamide solvents. methanesulphonate. General Notices (1) apply to all monographs and other texts

1439

Dihydroergotamine tartrate


Reference solution (b). Dilute 2.5 ml of reference solution (a) to 50 ml with a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R. Reference solution (c). Dilute 2 ml of reference solution (b) to 5 ml with a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R.

01/2005:0600

DIHYDROERGOTAMINE TARTRATE Dihydroergotamini tartras

Test solution (a). Dissolve 0.10 g of the substance to be examined in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 5 ml with the same mixture of solvents. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R. Plate : TLC silica gel G plate R. Mobile phase : concentrated ammonia R, methanol R, ethyl C H N O Mr 1317 70 80 10 16 acetate R, methylene chloride R (1:6:50:50 V/V/V/V). DEFINITION Application : 5 µl. Bis[(6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-10bhydroxy-2-methyl-3,6-dioxooctahydro-8H-oxazolo[3,2Development : protected from light over a path of 15 cm. Dry a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10, 10a-octahydroindolo[4,3-fg]quinoline-9-carboxamide] the plate in a current of cold air for not longer than 1 min. Repeat the development protected from light over a path of (2R,3R)-2,3-dihydroxybutanedioate. 15 cm using a freshly prepared amount of the mobile phase. Content : 98.0 per cent to 101.0 per cent (dried substance). Drying : in a current of cold air. Detection : spray abundantly with dimethylaminobenzaldehyde solution R7 and dry in a current of hot air for about 2 min. Limits : test solution (a) : — any impurity : any spot, apart from the principal spot, is not more intense than the principal spot in the chromatogram obtained with reference solution (b) (0.5 per cent) and not more than 2 such spots are more intense than the principal spot in the chromatogram obtained with reference solution (c) (0.2 per cent). Loss on drying (2.2.32) : maximum 4.0 per cent, determined on 0.500 g by drying at 100-105 °C at a pressure not exceeding 0.1 kPa for 5 h.

ASSAY Dissolve 0.500 g in a mixture of 10 ml of anhydrous acetic acid R and 70 ml of acetic anhydride R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 68.00 mg of C34H41N5O8S.

STORAGE Protected from light. 1440

CHARACTERS Appearance : white or almost white, crystalline powder or colourless crystals. Solubility : very slightly soluble in water, sparingly soluble in alcohol. IDENTIFICATION First identification : B, C. Second identification : A, C, D. A. Dissolve 5.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Examined between 250 nm and 350 nm (2.2.25), the solution shows 2 absorption maxima, at 281 nm and 291 nm, and a shoulder at 275 nm. Above 320 nm the absorbance is negligible. The specific absorbance at the maximum at 281 nm is 95 to 115 (dried substance). B. Infrared absorption spectrophotometry (2.2.24). Preparation : discs. Comparison : dihydroergotamine tartrate CRS. C. Examine the chromatograms obtained in the test for related substances. Results : the principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). D. Suspend about 15 mg in 1 ml of water R. 0.1 ml of the suspension gives reaction (b) of tartrates (2.3.1). TESTS Appearance of solution. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y7 or BY7 (2.2.2, Method II). Dissolve 0.1 g in alcohol (85 per cent V/V) R warming carefully in a water-bath at 40 °C and dilute to 50 ml with the same solvent.



Dihydrostreptomycin sulphate for veterinary use

pH (2.2.3) : 4.0 to 5.5 for the clear supernatant. Suspend 50 mg in 50 ml of carbon dioxide-free water R and shake for 10 min. Allow to stand. Specific optical rotation (2.2.7) : − 52 to − 57 (dried substance). Dissolve 0.250 g in anhydrous pyridine R and dilute to 25.0 ml with the same solvent. Related substances. Thin-layer chromatography (2.2.27). Prepare the reference solutions and the test solutions immediately before use and in the order indicated.

01/2005:0485

DIHYDROSTREPTOMYCIN SULPHATE FOR VETERINARY USE Dihydrostreptomycini sulfas ad usum veterinarium

Reference solution (a). Dissolve 20 mg of dihydroergotamine tartrate CRS in a mixture of 1 volume of methanol R and 9 volumes of chloroform R and dilute to 10 ml with the same mixture of solvents. Reference solution (b). Dilute 2.5 ml of reference solution (a) to 50 ml with a mixture of 1 volume of methanol R and 9 volumes of chloroform R. Reference solution (c). Dilute 2 ml of reference solution (b) to 5 ml with a mixture of 1 volume of methanol R and 9 volumes of chloroform R. Test solution (a). Dissolve 0.10 g of the substance to be examined in a mixture of 1 volume of methanol R and 9 volumes of chloroform R and dilute to 5 ml with the same mixture of solvents. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of 1 volume of methanol R and 9 volumes of chloroform R.

C42H88N14O36S3

Mr 1461

DEFINITION Dihydrostreptomycin sulphate for veterinary use is Plate : TLC silica gel G plate R. bis[N,N′-bis(aminoiminomethyl)-4-O-[5-deoxy-2-O-[2-deoxy-2Mobile phase : concentrated ammonia R, methanol R, ethyl (methylamino)-α-L-glucopyranosyl]-3-C-(hydroxymethyl)-αL-lyxofuranosyl]-D-streptamine] tris(sulphate), the sulphate acetate R, methylene chloride R (1:6:50:50 V/V/V/V). of a substance obtained by catalytic hydrogenation of Application : 5 µl. streptomycin or by any other means. Stabilisers may be Development : protected from light over a path of 15 cm. Dry added. The potency is not less than 730 IU/mg, calculated with reference to the dried substance. the plate in a current of cold air for not longer than 1 min. Repeat the development protected from light over a path of 15 cm using a freshly prepared amount of the mobile phase. PRODUCTION The method of manufacture is validated to demonstrate that Drying : in a current of cold air. the product if tested would comply with the following test : Detection : spray the plate abundantly with Abnormal toxicity (2.6.9). Inject into each mouse 1 mg dimethylaminobenzaldehyde solution R7 and dry dissolved in 0.5 ml of water for injections R. in a current of hot air for about 2 min. Limits : in the chromatogram obtained with test solution (a) : CHARACTERS A white or almost white powder, freely soluble in water, — any impurity : any spot, apart from the principal spot, practically insoluble in acetone, in alcohol and in methanol. is not more intense than the principal spot in the It may be hygroscopic. chromatogram obtained with reference solution (b) (0.5 per cent) and not more than 2 such spots are more IDENTIFICATION intense than the principal spot in the chromatogram A. Examine by thin-layer chromatography (2.2.27) using obtained with reference solution (c) (0.2 per cent). a plate coated with a 0.75 mm layer of the following Loss on drying (2.2.32) : maximum 5.0 per cent, determined mixture : mix 0.3 g of carbomer R with 240 ml of water R on 0.200 g by drying in an oven at 100-105 °C. and allow to stand, with moderate shaking, for 1 h ; adjust to pH 7 by the gradual addition, with continuous shaking, of dilute sodium hydroxide solution R and add 30 g of ASSAY silica gel H R. Dissolve 0.250 g in 50 ml of anhydrous acetic acid R. Titrate Heat the plate at 110 °C for 1 h, allow to cool and use with 0.05 M perchloric acid, determining the end-point immediately. potentiometrically (2.2.20). Test solution. Dissolve 10 mg of the substance to be 1 ml of 0.05 M perchloric acid is equivalent to 32.93 mg examined in water R and dilute to 10 ml with the same of C70H80N10O16. solvent. Reference solution (a). Dissolve 10 mg of STORAGE dihydrostreptomycin sulphate CRS in water R and dilute to 10 ml with the same solvent. Protected from light. General Notices (1) apply to all monographs and other texts

1441

Dihydrostreptomycin sulphate for veterinary use


Reference solution (b). Dissolve 10 mg of dihydrostreptomycin sulphate CRS, 10 mg of kanamycin monosulphate CRS and 10 mg of neomycin sulphate CRS in water R and dilute to 10 ml with the same solvent. Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm using a 70 g/l solution of potassium dihydrogen phosphate R. Dry the plate in a current of warm air and spray with a mixture of equal volumes of a 2 g/l solution of 1,3-dihydroxynaphthalene R in alcohol R and a 460 g/l solution of sulphuric acid R. Heat at 150 °C for 5-10 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows 3 clearly separated spots. B. Dissolve 0.1 g in 2 ml of water R, add 1 ml of α-naphthol solution R and 2 ml of a mixture of equal volumes of strong sodium hypochlorite solution R and water R. A red colour develops. C. Dissolve 10 mg in 5 ml of water R and add 1 ml of 1 M hydrochloric acid. Heat in a water-bath for 2 min. Add 2 ml of a 5 g/l solution of α-naphthol R in 1 M sodium hydroxide and heat in a water-bath for 1 min. A violet-pink colour is produced. D. It gives reaction (a) of sulphates (2.3.1).

Maintain the column at a constant temperature between 120 °C and 140 °C and the injection port and the detector at a temperature at least 50 °C higher than that of the column. The area of the peak corresponding to methanol in the chromatogram obtained with the test solution is not greater than the area of the peak in the chromatogram obtained with the reference solution. Streptomycin. Dissolve 0.100 g in water R and dilute to 5.0 ml with the same solvent. Add 5.0 ml of 0.2 M sodium hydroxide and heat in a water-bath for exactly 10 min. Cool in ice for exactly 5 min, add 3 ml of a 15 g/l solution of ferric ammonium sulphate R in 0.25 M sulphuric acid, dilute to 25.0 ml with water R and mix (test solution). Prepare at the same time and in the same manner a reference solution using 10 mg of streptomycin sulphate CRS in 50 ml of water R. Using 5.0 ml of this solution, operate as for the test solution. Exactly 20 min after addition of the ferric ammonium sulphate solution, measure the absorbance (2.2.25) of each solution at the maximum at 525 nm in a 2 cm cell, using as the compensation liquid a solution prepared at the same time and in the same manner as the test solution but omitting the substance to be examined. The absorbance of the test solution is not greater than that of the reference solution (1 per cent). Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 5.0 per cent, determined on 1.000 g by drying at 60 °C over diphosphorus pentoxide R at a pressure not exceeding 0.1 kPa for 4 h. Sulphated ash (2.4.14). Not more than 1.0 per cent, determined on 1.0 g.

TESTS Solution S. Dissolve 2.5 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. Appearance of solution. Solution S is not more intensely coloured than intensity 5 of the range of reference solutions of the most appropriate colour (2.2.2, Method II). Allow to stand protected from light at a temperature of about 20 °C for 24 h ; solution S is not more opalescent than reference suspension II (2.2.1).

Sulphate. 18.0 per cent to 21.5 per cent of sulphate (SO4), calculated with reference to the dried substance. Dissolve 0.250 g in 100 ml of water R and adjust the solution to pH 11 using concentrated ammonia R. Add 10.0 ml of 0.1 M barium chloride and about 0.5 mg of phthalein purple R. Titrate with 0.1 M sodium edetate, adding 50 ml of alcohol R when the colour of the solution begins to change and continue the titration until the violet-blue colour disappears. 1 ml of 0.1 M barium chloride is equivalent to 9.606 mg of sulphate (SO4).

pH (2.2.3). The pH of solution S is 5.0 to 7.0.

Specific optical rotation (2.2.7). Dissolve 0.200 g in water R Bacterial endotoxins (2.6.14) : less than 0.50 IU/mg, if and dilute to 10.0 ml with the same solvent. The specific intended for use in the manufacture of parenteral dosage optical rotation is − 83 to − 91, calculated with reference to forms without a further appropriate procedure for removal the dried substance. of bacterial endotoxins. Methanol. Not more than 0.2 per cent, determined by gas chromatography (2.2.28). ASSAY Test solution. Dissolve 1.00 g of the substance to be Carry out the microbiological assay of antibiotics (2.7.2). examined in water R and dilute to 25.0 ml with the same solvent. STORAGE Reference solution. Dilute 8.0 mg of methanol R to 100 ml In an airtight container, protected from light. If the with water R. substance is sterile, store in a sterile, airtight, tamper-proof The chromatographic procedure may be carried out using : container. — a column 1.5 m to 2.0 m long and 2.0 mm to 4.0 mm in internal diameter packed with ethylvinylbenzene-divinylbenzene copolymer R (150-180 µm),

LABELLING The label states :

— nitrogen for chromatography R as the carrier gas at a constant flow rate of 30-40 ml/min, — a flame-ionisation detector. 1442

— where applicable, the name and quantity of any added stabiliser, — where applicable, that the substance is free from bacterial endotoxins.



Diltiazem hydrochloride

01/2005:1004 Specific optical rotation (2.2.7). Dilute 5.0 ml of solution S to 25.0 ml with water R. The specific optical rotation is + 115 to + 120, calculated with reference to the dried substance. DILTIAZEM HYDROCHLORIDE Related substances. Examine by liquid chromatography (2.2.29). Diltiazemi hydrochloridum Test solution. Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 200.0 ml with the mobile phase. Reference solution(a). Dissolve 50.0 mg of diltiazem hydrochloride CRS in the mobile phase and dilute to 200.0 ml with the mobile phase. Reference solution (b). Dissolve 3 mg of diltiazem impurity A CRS in the mobile phase and dilute to 10 ml with the mobile phase. To 1 ml of this solution, add 1.2 ml of C22H27ClN2O4S Mr 451.0 reference solution (a) and dilute to 100.0 ml with the mobile phase. DEFINITION Reference solution(c). Dilute 0.3 ml of the test solution to Diltiazem hydrochloride contains not less than 98.5 per 100.0 ml with the mobile phase. cent and not more than the equivalent of 101.0 per cent The chromatographic procedure may be carried out using : of (2S,3S)-5-[2-(dimethylamino)ethyl]-2-(4-methoxyphenyl)— a stainless steel column 0.10 m long and 4.6 mm in 4-oxo-2,3,4,5-tetrahydro-1,5-benzothiazepin-3-yl acetate internal diameter packed with octadecylsilyl silica gel for hydrochloride, calculated with reference to the dried chromatography R (3 µm), substance. — as mobile phase at a flow rate of 1.5 ml/min a mixture CHARACTERS of 5 volumes of ethanol R, 25 volumes of acetonitrile R A white, crystalline powder, freely soluble in water, in and 70 volumes of a solution containing, in 1 litre, 6.8 g methanol and in methylene chloride, slightly soluble in of potassium dihydrogen phosphate R and 0.1 ml of ethanol. It melts at about 213 °C with decomposition. N,N-dimethyloctylamine R, adjusted to pH 4.5 using dilute phosphoric acid R, IDENTIFICATION — as detector a spectrophotometer set at 240 nm, First identification : A, D. — a loop injector. Second identification : B, C, D. Inject separately 20 µl of each solution. Adjust the sensitivity A. Examine by infrared absorption spectrophotometry of the detector so that the height of the principal peak in (2.2.24), comparing with the spectrum obtained with the chromatogram obtained with reference solution (c) diltiazem hydrochloride CRS. Examine the substances is at least 10 per cent of the full scale of the recorder. prepared as discs. Continue the chromatography for five times the retention B. Examine by thin-layer chromatography (2.2.27), using time of the principal peak. The test is not valid unless in as the coating substance a suitable silica gel with a the chromatogram obtained with reference solution (b), the fluorescent indicator having an optimal intensity at resolution between the peaks due to diltiazem impurity A 254 nm. and diltiazem is at least 4.0 and the symmetry factors are Test solution. Dissolve 0.10 g of the substance to be not more than 2.0. If necessary, adjust the concentration examined in methylene chloride R and dilute to 10 ml of N,N-dimethyloctylamine in the mobile phase. In the with the same solvent. chromatogram obtained with the test solution, the sum Reference solution. Dissolve 0.10 g of diltiazem of the areas of any peaks, apart from the principal peak, hydrochloride CRS in methylene chloride R and dilute is not greater than the area of the principal peak in the to 10 ml with the same solvent. chromatogram obtained with reference solution (c) (0.3 per cent). Disregard any peak with an area less than 0.025 times Apply to the plate 10 µl of each solution. Develop over that of the peak in the chromatogram obtained with a path of 10 cm using a mixture of 1 volume of acetic reference solution (c). acid R, 3 volumes of water R, 10 volumes of methylene chloride R and 12 volumes of ethanol R. Allow the plate Heavy metals (2.4.8). Dissolve 2.0 g in water R and dilute to dry in air and examine in ultraviolet light at 254 nm. to 20.0 ml with the same solvent. 12 ml of the solution The principal spot in the chromatogram obtained with complies with limit test A for heavy metals (10 ppm). Prepare the test solution is similar in position and size to the the standard using lead standard solution (1 ppm Pb) R. principal spot in the chromatogram obtained with the Loss on drying (2.2.32). Not more than 0.5 per cent, reference solution. determined on 1.000 g by drying in an oven at 100-105 °C C. Dissolve 50 mg in 5 ml of water R. Add 1 ml of ammonium for 2 h. reineckate solution R. A pink precipitate is produced. Sulphated ash (2.4.14). Not more than 0.1 per cent, D. It gives reaction (a) of chlorides (2.3.1). determined on 1.0 g. TESTS ASSAY Solution S. Dissolve 1.00 g in carbon-dioxide free water R Dissolve 0.400 g in a mixture of 2 ml of anhydrous and dilute to 20.0 ml with the same solvent. formic acid R and 60 ml of acetic anhydride R and titrate Appearance of solution. Solution S is clear (2.2.1) and with 0.1 M perchloric acid, determining the end-point colourless (2.2.2, Method II). potentiometrically (2.2.20). pH (2.2.3). Dilute 2.0 ml of solution S to 10.0 ml with carbon 1 ml of 0.1 M perchloric acid is equivalent to 45.1 mg of C22H27ClN2O4S. dioxide-free water R. The pH of the solution is 4.3 to 5.3. General Notices (1) apply to all monographs and other texts

1443

Dimenhydrinate



A. (2R,3S)-5-[2-(dimethylamino)ethyl]-2-(4-methoxyphenyl)-4oxo-2,3,4,5-tetrahydro-1,5-benzothiazepin-3-yl acetate,

B. R1 = CO-CH3, R2 = H, R3 = OCH3 : (2S,3S)-2(4-methoxyphenyl)-4-oxo-2,3,4,5-tetrahydro-1,5benzothiazepin-3-yl acetate, C. R1 = CO-CH3, R2 = CH2-CH2-N(CH3)2, R3 = OH : (2S,3S)-5-[2-(dimethylamino)ethyl]-2-(4-hydroxyphenyl)-4oxo-2,3,4,5-tetrahydro-1,5-benzothiazepin-3-yl acetate, D. R1 = CO-CH3, R2 = CH2-CH2-NH-CH3, R3 = OCH3 : (2S,3S)-2-(4-methoxyphenyl)-5-[2-(methylamino)ethyl]-4oxo-2,3,4,5-tetrahydro-1,5-benzothiazepin-3-yl acetate, E. R1 = R2 = H, R3 = OCH3 : (2S,3S)-3-hydroxy-2-(4methoxyphenyl)-2,3-dihydro-1,5-benzothiazepin-4(5H)one, F. R1 = H, R2 = CH2-CH2-N(CH3)2, R3 = OCH3 : (2S,3S)-5-[2(dimethylamino)ethyl]-3-hydroxy-2-(4-methoxyphenyl)-2,3dihydro-1,5-benzothiazepin-4(5H)-one.

IDENTIFICATION First identification : C. Second identification : A, B, D. A. Melting point (2.2.14) : 102 °C to 106 °C. B. Dissolve 0.1 g in a mixture of 3 ml of water R and 3 ml of alcohol R, add 6 ml of water R and 1 ml of dilute hydrochloric acid R and cool in iced water for 30 min, scratching the wall of the tube with a glass rod if necessary to initiate crystallisation. Dissolve about 10 mg of the precipitate obtained in 1 ml of hydrochloric acid R, add 0.1 g of potassium chlorate R and evaporate to dryness in a porcelain dish. A reddish residue is obtained which becomes violet-red when exposed to ammonia vapour. C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with dimenhydrinate CRS. D. Dissolve 0.2 g in 10 ml of alcohol R. Add 10 ml of picric acid solution R and initiate crystallisation by scratching the wall of the tube with a glass rod. The precipitate, washed with water R and dried at 100 °C to 105 °C, melts (2.2.14) at 130 °C to 134 °C.

TESTS Appearance of solution. Dissolve 1.0 g in alcohol R and dilute to 20 ml with the same solvent. The solution is clear (2.2.1) and colourless (2.2.2, Method II). pH (2.2.3). To 0.4 g add 20 ml of carbon dioxide-free water R, shake for 2 min and filter. The pH of the filtrate is 7.1 to 7.6. Theophylline and substances related to diphenhydramine. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution. Dissolve 0.40 g of the substance to be examined in methylene chloride R and dilute to 10 ml with the same solvent. Reference solution (a). Dissolve 20 mg of theophylline R in methylene chloride R and dilute to 100 ml with the same 01/2005:0601 solvent. Reference solution (b). Dilute 5 ml of the test solution to DIMENHYDRINATE 100 ml with methylene chloride R. Dilute 10 ml of this solution to 100 ml with methylene chloride R. Dimenhydrinatum Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 1 volume of concentrated ammonia R, 9 volumes of methanol R and 90 volumes of methylene chloride R. Dry the plate in a current of cold air and examine in ultraviolet light at 254 nm. Any spot corresponding to theophylline in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.5 per cent). Spray with potassium iodobismuthate solution R. Allow the plate to dry in air and C24H28ClN5O3 Mr 470.0 spray with dilute hydrogen peroxide solution R. Any spot in the chromatogram obtained with the test solution, apart DEFINITION from the principal spot, is not more intense than the spot Dimenhydrinate contains not less than 53.0 per cent in the chromatogram obtained with reference solution (b) and not more than 55.5 per cent of diphenhydramine (0.5 per cent). Disregard any spot extending from the [2-(diphenylmethoxy)-N,N-dimethylethylamine, C17H21NO, starting point to an Rf of about 0.1. Mr 255.4] and not less than 44.0 per cent and not Heavy metals (2.4.8). Dissolve 2.5 g in a mixture of more than 46.5 per cent of 8-chlorotheophylline 15 volumes of water R and 85 volumes of acetone R (8-chloro-3,7-dihydro-1,3-dimethyl-1H-purine-2,6-dione, C7H7ClN4O2, Mr 214.6), both calculated with reference to the and dilute to 25 ml with the same mixture of solvents. The solution complies with limit test B for heavy metals dried substance. (20 ppm). Prepare the standard using lead standard solution CHARACTERS (2 ppm Pb) prepared by diluting lead standard solution (100 ppm Pb) R with a mixture of 15 volumes of water R A white, crystalline powder or colourless crystals, slightly and 85 volumes of acetone R. soluble in water, freely soluble in alcohol. 1444


Dimethyl sulfoxide


Acidity or alkalinity. Dissolve 0.2 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. Add 0.25 ml of bromocresol green solution R and 0.3 ml of 0.01 M hydrochloric acid. The solution is yellow. Not more than 0.5 ml of 0.01 M sodium hydroxide is required to change the colour of the indicator to blue. ASSAY Diphenhydramine. Dissolve 0.200 g in 60 ml of anhydrous Refractive index (2.2.6) : 1.568 to 1.574. acetic acid R. Titrate with 0.1 M perchloric acid, determining Halides. To 2.0 g add 25 ml of alcoholic potassium the end-point potentiometrically (2.2.20). hydroxide solution R and boil under a reflux condenser for 2 h. Eliminate the alcohol by evaporation in a stream of hot 1 ml of 0.1 M perchloric acid is equivalent to 25.54 mg of air, add 20 ml of water R and cool. Add 40 ml of water R and C17H21NO. 8-Chlorotheophylline. To 0.800 g add 50 ml of water R, 3 ml 10 ml of strong hydrogen peroxide solution R, boil gently for 10 min, cool and filter rapidly. Add 10 ml of dilute nitric of dilute ammonia R1 and 0.6 g of ammonium nitrate R acid R and 5.0 ml of 0.1 M silver nitrate. Using 2 ml of ferric and heat on a water-bath for 5 min. Add 25.0 ml of 0.1 M ammonium sulphate solution R2 as indicator, titrate with silver nitrate and continue heating on a water-bath for 0.1 M ammonium thiocyanate until a reddish-yellow colour 15 min with frequent swirling. Cool, add 25 ml of dilute is obtained. Carry out a blank titration. The difference nitric acid R and dilute to 250.0 ml with water R. Filter between the titration volumes is not greater than 1.0 ml. and discard the first 25 ml of the filtrate. Using 5 ml of ferric ammonium sulphate solution R2 as indicator, titrate ASSAY 100.0 ml of the filtrate with 0.1 M ammonium thiocyanate Dissolve 0.100 g in 40 ml of methanol R. Add 20 ml of 0.1 M until a yellowish-brown colour is obtained. hydrochloric acid and 50.0 ml of 0.05 M iodine. Allow to 1 ml of 0.1 M silver nitrate is equivalent to 21.46 mg of stand for 10 min and titrate with 0.1 M sodium thiosulphate. C7H7ClN4O2. Carry out a blank titration. 1 ml of 0.05 M iodine is equivalent to 6.21 mg of C3H8OS2. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in vacuo. Sulphated ash (2.4.14). Not more than 0.2 per cent, determined on 1.0 g.

DIMERCAPROL

01/2005:0389 STORAGE Store in a well-filled, airtight container, protected from light, at a temperature of 2 °C to 8 °C. 01/2005:0763

Dimercaprolum

DIMETHYL SULFOXIDE Dimethylis sulfoxidum

C3H8OS2

Mr 124.2

DEFINITION Dimercaprol contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent of (2RS)-2,3-disulfanylpropan-1-ol. CHARACTERS A clear, colourless or slightly yellow liquid, soluble in water and in arachis oil, miscible with alcohol and with benzyl benzoate. IDENTIFICATION A. Dissolve 0.05 ml in 2 ml of water R. Add 1 ml of 0.05 M iodine. The colour of the iodine is discharged immediately. B. Dissolve 0.1 ml in 5 ml of water R and add 2 ml of copper sulphate solution R. A bluish-black precipitate is formed which quickly becomes dark grey. C. In a ground-glass-stoppered tube, suspend 0.6 g of sodium bismuthate R, previously heated to 200 °C for 2 h, in a mixture of 2.8 ml of dilute phosphoric acid R and 6 ml of water R. Add 0.2 ml of the substance to be examined, mix and allow to stand for 10 min with frequent shaking. To 1 ml of the supernatant liquid add 5 ml of a 4 g/l solution of chromotropic acid, sodium salt R in sulphuric acid R and mix. Heat for 15 min in a water-bath. A violet-red colour develops.

C2H6OS

Mr 78.1

DEFINITION Dimethyl sulfoxide is sulphinylbismethane. CHARACTERS A colourless liquid or colourless crystals, hygroscopic, miscible with water and with alcohol. IDENTIFICATION First identification : C. Second identification : A, B, D. A. It complies with the test for relative density (see Tests). B. It complies with the test for refractive index (see Tests). C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with dimethyl sulfoxide CRS. D. Dissolve 50 mg of nickel chloride R in 5 ml of the substance to be examined. The solution is greenish-yellow. Heat in a water-bath at 50 °C. The colour changes to green or bluish-green. Cool. The colour changes to greenish-yellow.

TESTS Acidity. Dissolve 50.0 g in 100 ml of carbon dioxide-free TESTS water R. Add 0.1 ml of phenolphthalein solution R1. Not Appearance. It is clear (2.2.1) and not more intensely more than 5.0 ml of 0.01 M sodium hydroxide is required to coloured than reference solution B6 or BY6 (2.2.2, Method II). produce a pink colour.


1445

Dimethylacetamide


01/2005:1667

Relative density (2.2.5) : 1.100 to 1.104. Refractive index (2.2.6) : 1.478 to 1.479. Freezing point (2.2.18). Not less than 18.3 °C. Absorbance (2.2.25). Purge with nitrogen R for 15 min. The absorbance, measured using water R as the compensation liquid, is not more than 0.30 at 275 nm and not more than 0.20 at both 285 nm and 295 nm. Examined between 270 nm and 350 nm, the substance to be examined shows no absorption maximum. Related substances. Examine by gas chromatography (2.2.28), using bibenzyl R as the internal standard. Internal standard solution. Dissolve 0.125 g of bibenzyl R in acetone R and dilute to 50 ml with the same solvent. Test solution (a). Dissolve 5.0 g of the substance to be examined in acetone R and dilute to 10.0 ml with the same solvent. Test solution (b). Dissolve 5.0 g of the substance to be examined in acetone R, add 1.0 ml of the internal standard solution and dilute to 10.0 ml with acetone R.

DIMETHYLACETAMIDE Dimethylacetamidum

C4H9NO

Mr 87.1

DEFINITION N,N-Dimethylacetamide. CHARACTERS Appearance : clear, colourless, slightly hygroscopic liquid. Solubility : miscible with water, with alcohol, and with most common organic solvents. bp : about 165 °C.

Reference solution. Dissolve 50.0 mg of the substance to be examined and 50 mg of dimethyl sulphone R in acetone R, IDENTIFICATION add 10.0 ml of the internal standard solution and dilute to First identification : C. 100.0 ml with acetone R. Second identification : A, B, D. The chromatographic procedure may be carried out using : A. Relative density (2.2.5) : 0.941 to 0.944. — a glass column 1.5 m long and 4 mm in internal B. Refractive index (2.2.6) : 1.435 to 1.439. diameter, packed with diatomaceous earth for gas C. Infrared absorption spectrophotometry (2.2.24). chromatography R (125 µm to 180 µm) impregnated with Preparation : films. 10 per cent m/m of polyethyleneglycol adipate R, Comparison : dimethylacetamide CRS. — nitrogen for chromatography R as the carrier gas at a D. Dilute 50 mg with 1 ml of methanol R. Add 1 ml of a flow rate of 30 ml/min, 15 g/l solution of hydroxylamine hydrochloride R and — a flame-ionisation detector, mix. Add 1 ml of dilute sodium hydroxide solution R, mix and allow to stand for 30 min. Add 1 ml of dilute maintaining the temperature of the column at 165 °C and hydrochloric acid R and add 1 ml of a 100 g/l solution that of the injection port and of the detector at 190 °C. of ferric chloride R in 0.1 M hydrochloric acid. A Inject 1 µl of the reference solution and adjust the sensitivity reddish-brown colour develops, reaching a maximum of the system so that the heights of the three peaks, apart intensity after about 5 min. from the solvent peak, are not less than 70 per cent of the full scale of the recorder. The substances elute in the following TESTS order : dimethyl sulfoxide, dimethyl sulphone, bibenzyl. The Appearance. The substance to be examined is clear (2.2.1) test is not valid unless, in the chromatogram obtained with and not more intensely coloured than reference solution Y7 the reference solution, the resolution between the peaks due (2.2.2, Method II). to dimethyl sulfoxide and dimethyl sulphone is at least 3. Acidity. Dilute 50 ml with 50 ml of water R previously Inject 1 µl of test solution (a). In the chromatogram obtained, adjusted with 0.02 M potassium hydroxide or 0.02 M verify that there is no peak with the same retention time as hydrochloric acid to a bluish-green colour, using 0.5 ml of the internal standard. bromothymol blue solution R1 as indicator. Not more than 5.0 ml of 0.02 M potassium hydroxide is required to restore Inject 1 µl of test solution (b) and 1 µl of the reference the initial (bluish-green) colour. solution. Continue the chromatography for four times the retention time of dimethyl sulfoxide, which is about Alkalinity. To 50 ml add 50 ml of water R previously adjusted 5 min. From the chromatogram obtained with the reference with 0.02 M potassium hydroxide or 0.02 M hydrochloric solution, calculate the ratio (R) of the area of the peak due acid to a yellow colour, using 0.5 ml of bromothymol blue to dimethyl sulfoxide to the area of the peak due to the solution R1 as indicator. Titrate with 0.02 M hydrochloric internal standard. From the chromatogram obtained with acid. Not more than 0.5 ml of 0.02 M hydrochloric acid is test solution (b), calculate the ratio of the sum of the areas required to restore the initial (yellow) colour. of any peaks, apart from the principal peak, the peak due to Related substances. Gas chromatography (2.2.28) : use the the internal standard and the peak due to the solvent, to the normalisation procedure. area of the peak due to the internal standard ; this ratio is Test solution. The substance to be examined. not greater than R (0.1 per cent). Reference solution (a). Dilute a mixture of 1 ml of the Water (2.5.12). Not more than 0.2 per cent, determined on substance to be examined and 1 ml of dimethylformamide R 10.0 g by the semi-micro determination of water. to 20 ml with methylene chloride R. Reference solution (b). Dilute 1 ml of the substance to be STORAGE examined to 20.0 ml with methylene chloride R. Dilute Store in an airtight, glass container, protected from light. 0.1 ml of the solution to 10.0 ml with methylene chloride R. 1446


Dimeticone


Column : — material : fused silica, — size : l = 30 m, Ø = 0.32 mm, — stationary phase: macrogol 20 000 R (film thickness 1 µm). Carrier gas : nitrogen for chromatography R. Linear velocity : 30 cm/s. Split ratio : 1:20. Temperature :

Column

Time (min)

Temperature (°C)

0 - 15

80 → 200

Injection port

250

Detector

250

Detection : flame ionisation. Injection : 0.5 µl. System suitability : — resolution : minimum 5.0 between the peaks due to dimethylacetamide and dimethylformamide in the chromatogram obtained with reference solution (a), — signal-to-noise ratio : minimum 10 for the principal peak in the chromatogram obtained with reference solution (b). Limits : — any impurity : maximum 0.1 per cent, — total: maximum 0.3 per cent, — disregard limit: area of the peak in the chromatogram obtained with reference solution (b) (0.05 per cent). Heavy metals (2.4.8) : maximum 10 ppm. Dilute 4.0 g to 20.0 ml with water R. 12 ml of the solution complies with limit test A. Prepare the standard using lead standard solution (2 ppm Pb) R. Non-volatile matter : maximum 20 ppm. Evaporate 50 g to dryness using a rotary evaporator at a pressure not exceeding 1 kPa and on a water-bath. Dry the residue in an oven at 170-175 °C. The residue weighs not more than 1 mg. Water (2.5.32) : maximum 0.1 per cent, determined on 0.100 g. STORAGE In an airtight container, protected from light. IMPURITIES A. acetic acid,

B. R = H : N,N-dimethylformamide, C. R = C2H5 : N,N-dimethylpropanamide, D. R = CH2-CH2-CH3 : N,N-dimethylbutanamide. General Notices (1) apply to all monographs and other texts

01/2005:0138

DIMETICONE Dimeticonum

DEFINITION Dimeticone is a poly(dimethylsiloxane) obtained by hydrolysis and polycondensation of dichlorodimethylsilane and chlorotrimethylsilane. Different grades exist which are distinguished by a number indicating the nominal viscosity placed after the name. Their degree of polymerisation (n = 20 to 400) is such that their kinematic viscosities are nominally between 20 mm2·s− 1 and 1300 mm2·s− 1. Dimeticones with a nominal viscosity of 50 mm2·s− 1 or lower are intended for external use only. CHARACTERS Clear, colourless liquids of various viscosities, practically insoluble in water, very slightly soluble to practically insoluble in ethanol, miscible with ethyl acetate, with methyl ethyl ketone and with toluene. IDENTIFICATION A. It is identified by its kinematic viscosity at 25 °C (see Tests). B. Examine by infrared absorption spectrophotometry (2.2.24) comparing with the spectrum obtained with dimeticone CRS. The region of the spectrum from 850 cm− 1 to 750 cm− 1 is not taken into account. C. Heat 0.5 g in a test-tube over a small flame until white fumes begin to appear. Invert the tube over a 2nd tube containing 1 ml of a 1 g/l solution of chromotropic acid, sodium salt R in sulphuric acid R so that the fumes reach the solution. Shake the 2nd tube for about 10 s and heat on a water-bath for 5 min. The solution is violet. D. In a platinum crucible, prepare the sulphated ash (2.4.14) using 50 mg. The residue is a white powder that gives the reaction of silicates (2.3.1). TESTS Acidity. To 2.0 g add 25 ml of a mixture of equal volumes of ethanol R and ether R, previously neutralised to 0.2 ml of bromothymol blue solution R1 and shake. Not more than 0.15 ml of 0.01 M sodium hydroxide is required to change the colour of the solution to blue. Viscosity (2.2.9). Determine the kinematic viscosity at 25 °C. For dimeticones, the kinematic viscosity is not less than 90 per cent and not more than 110 per cent of the nominal viscosity stated on the label. Mineral oils. Place 2 g in a test-tube and examine in ultraviolet light at 365 nm. The fluorescence is not more intense than that of a solution containing 0.1 ppm of quinine sulphate R in 0.005 M sulphuric acid examined in the same conditions. Phenylated compounds. Dissolve 5.0 g with shaking in 10 ml of cyclohexane R. At wavelengths from 250 nm to 270 nm, the absorbance (2.2.25) of the solution is not greater than 0.2. 1447

Dimetindene maleate


Optical rotation (2.2.7). Determined on solution S, the angle of optical rotation is − 0.10° to + 0.10°. Related substances. Examine by gas chromatography (2.2.28). Test solution. Dissolve 50.0 mg of the substance to be examined in a mixture of equal volumes of acetone R and methylene chloride R and dilute to 5.0 ml with the same mixture of solvents. Reference solution (a). Dilute 1 ml of the test solution to 100.0 ml with a mixture of equal volumes of acetone R and methylene chloride R. Reference solution (b). Dissolve 5.0 mg of 2-ethylpyridine R in a mixture of equal volumes of acetone R and methylene chloride R and dilute to 50.0 ml with the same mixture of solvents. Dilute 10.0 ml of this solution to 100.0 ml with the same mixture of solvents. The chromatographic procedure may be carried out using : — a fused-silica column 30 m long and 0.32 mm in internal diameter coated with polymethylphenylsiloxane R (film thickness 0.25 µm), LABELLING — helium for chromatography R as the carrier gas at a The label states : linear velocity of about 30 cm/s, — the nominal viscosity by a number placed after the name — a flame-ionisation detector, of the product, maintaining the temperature of the column at 60 °C for — where applicable, that the product is intended for external 1 min, then raising the temperature at a rate of 6 °C/min use. to 260 °C and maintaining at 260 °C for 12 min. Maintain the temperature of the injection port at 240 °C and that of the detector at 260 °C. 01/2005:1417 Inject via a split injector with a split flow of 30 ml/min for 1.3 times the retention time of the principal peak. Inject 2 µl of reference solution (a). The symmetry factor DIMETINDENE MALEATE of the principal peak in the chromatogram obtained is not greater than 1.3. Dimetindeni maleas Inject 2 µl of the test solution and 2 µl of reference solution (b). In the chromatogram obtained with the test solution the area of the peak corresponding to 2-ethylpyridine is not greater than the area of the peak in the chromatogram obtained with reference solution (b) (0.1 per cent). In the chromatogram obtained with the test solution the area of any peak, apart from the principal peak and any peak appearing during the first 8 min (2-ethylpyridine, the solvent C24H28N2O4 Mr 408.5 mixture and maleic acid), is not greater than 0.2 times the area of the peak in the chromatogram with reference solution (a) (0.2 per cent) and the sum of the areas of all DEFINITION peaks is not greater than 0.5 times the area of the peak in the Dimetindene maleate contains not less than 99.0 per cent chromatogram obtained with reference solution (a) (0.5 per and not more than the equivalent of 101.0 per cent of cent). Disregard any peak with an areas less than 0.05 times N,N-dimethyl-2-[3-[(RS)-1-(pyridin-2-yl)ethyl]-1H-inden-2the area of the principal peak in the chromatogram obtained yl]ethanamine (Z)-butenedioate, calculated with reference with the reference solution (a). to the dried substance. Loss on drying (2.2.32). Not more than 0.1 per cent, CHARACTERS determined on 1.000 g by drying in an oven at 100 °C to A white to almost white, crystalline powder, slightly soluble 105 °C for 2 h. in water, soluble in methanol. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. IDENTIFICATION Examine by infrared absorption spectrophotometry (2.2.24), ASSAY comparing with the spectrum obtained with dimetindene Dissolve 0.150 g in 80 ml of anhydrous acetic acid R. Titrate maleate CRS. Examine the substance prepared as discs. with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). TESTS 1 ml of 0.1 M perchloric acid is equivalent to 20.43 mg of Solution S. Dissolve 0.20 g in methanol R and dilute to C24H28N2O4. 20.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not STORAGE more intensely coloured than Y6 (2.2.2, Method II). Store protected from light. Heavy metals. Mix 1.0 g with methylene chloride R and dilute to 20 ml with the same solvent. Add 1.0 ml of a freshly prepared 0.02 g/l solution of dithizone R in methylene chloride R, 0.5 ml of water R and 0.5 ml of a mixture of 1 volume of dilute ammonia R2 and 9 volumes of a 2 g/l solution of hydroxylamine hydrochloride R. At the same time, prepare a standard as follows : to 20 ml of methylene chloride R add 1.0 ml of a freshly prepared 0.02 g/l solution of dithizone R in methylene chloride R, 0.5 ml of lead standard solution (10 ppm Pb) R and 0.5 ml of a mixture of 1 volume of dilute ammonia R2 and 9 volumes of a 2 g/l solution of hydroxylamine hydrochloride R. Immediately shake each solution vigorously for 1 min. Any red colour in the test solution is not more intense than that in the standard (5 ppm). Volatile matter. For dimeticones with a nominal viscosity greater than 50 mm2·s− 1, not more than 0.3 per cent, determined on 1.00 g by heating in an oven at 150 °C for 2 h. Carry out the test using a dish 60 mm in diameter and 10 mm deep.

1448


Dinoprost trometamol


01/2005:1312

IMPURITIES

DINOPROST TROMETAMOL Dinoprostum trometamolum A. 2-ethylpyridine,

C24H45NO8 B. 2-(1H-inden-2-yl)-N,N-dimethylethanamine,

Mr 475.6

DEFINITION Dinoprost trometamol contains not less than 96.0 per cent and not more than the equivalent of 102.0 per cent of trometamol (Z)-7-[(1R,2R,3R,5S)-3,5-dihydroxy-2-[(E)(3S)-3-hydroxyoct-1-enyl]cyclopentyl]hept-5-enoate (PGF2α), calculated with reference to the anhydrous substance. CHARACTERS A white or almost white powder, very soluble in water, freely soluble in alcohol, practically insoluble in acetonitrile.

C. R = C2H5 : ethyl (2RS)-2-benzyl-4-(dimethylamino)butanoate, D. R = H : (2RS)-2-benzyl-4-(dimethylamino)butanoic acid,

E. (2RS)-2-[2-(dimethylamino)ethyl]indan-1-one,

F. R = [CH2]3-CH3 : 2-(3-butyl-1H-inden-2-yl)-N,Ndimethylethanamine, G. R = C6H5 : N,N-dimethyl-2-(3-phenyl-1H-inden-2yl)ethanamine,

H. R = CH = CH2 : 2-[(1RS)-1-(2-ethenyl-1H-inden-3yl)ethyl]pyridine, I. R = CH2-CH2-NH-CH3 : N-methyl-2-[3-[(1RS)-1-(pyridin-2yl)ethyl]-1H-inden-2-yl]ethanamine. General Notices (1) apply to all monographs and other texts

IDENTIFICATION A. Dissolve 0.100 g in alcohol R and dilute to 10.0 ml with the same solvent. The specific optical rotation (2.2.7) is + 19 to + 26, calculated with reference to the anhydrous substance. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with dinoprost trometamol CRS. TESTS Related substances. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 10.0 mg of the substance to be examined in a mixture of 23 volumes of acetonitrile R and 77 volumes of water R and dilute to 10.0 ml with the same mixture of solvents. Reference solution (a). Degradation of dinoprost trometamol to impurity B. Dissolve 1 mg of the substance to be examined in 1 ml of the mobile phase and heat the solution on a water-bath at 85 °C for 5 min and cool. Reference solution (b). Dilute 2.0 ml of the test solution to 20.0 ml with a mixture of 23 volumes of acetonitrile R and 77 volumes of water R. Dilute 2.0 ml of the solution to 20.0 ml with a mixture of 23 volumes of acetonitrile R and 77 volumes of water R. The chromatographic procedure may be carried out using : — a stainless steel column 0.15 m long and 3.9 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R1 (5 µm) with a pore size of 10 nm and a carbon loading of 19 per cent, — as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows : dissolve 2.44 g of sodium dihydrogen phosphate R in water R and dilute to 1000 ml with water R ; adjust the pH to 2.5 with phosphoric acid R (about 0.6 ml) ; mix 770 ml of the buffer solution with 230 ml of acetonitrile R1, — as detector a spectrophotometer set at 200 nm. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 20 µl of reference solution (b) is 25 per cent to 50 per cent of the full scale of the recorder. 1449

Dinoprostone


Inject 20 µl of reference solution (a). When the chromatograms are recorded in the prescribed conditions, the retention times are : (15R)-PGF2α (impurity B) about 55 min ; 5,6-trans-PGF2α (impurity A) about 60 min and dinoprost about 66 min. Continue the chromatography for 2.5 times the retention time of the principal peak (to elute degradation products formed during heating). The test is not valid unless the resolution between the peaks corresponding to impurity A and impurity B is at least 1.5 and the resolution between the peaks corresponding to impurity A and dinoprost is at least 2.0 ; the symmetry factor for the peaks corresponding to impurity A and impurity B is less than 1.2. If necessary, adjust the composition of the mobile phase by increasing the concentration of acetonitrile to decrease the retention times. Inject separately 20 µl of the test solution and 20 µl of reference solution (b). Continue the chromatography of the test solution for 10 min after the elution of the principal peak. In the chromatogram obtained with the test solution the area of any peak corresponding to impurity A is not greater than twice the area of the principal peak obtained with reference solution (b) (2 per cent) ; the area of any other peak, apart from the principal peak and any peak corresponding to impurity A, is not greater than 1.5 times the area of the principal peak obtained with reference solution (b) (1.5 per cent) and not more than one such peak has an area greater than half the area of the principal peak obtained with reference solution (b) (0.5 per cent). The sum of the areas of all the peaks, apart from the principal peak and the peak corresponding to impurity A, is not greater than twice the area of the principal peak obtained with reference solution (b) (2 per cent). Disregard any peak due to the mobile phase and any peak due to trometamol (retention time about 1.5 min) and also any peak with an area of less than 0.05 times the area of the principal peak obtained with reference solution (b). Water (2.5.12). Not more than 1.0 per cent, determined on 0.500 g by the semi-micro determination of water. ASSAY Examine by liquid chromatography (2.2.29). Test solution. Dissolve 10.0 mg of the substance to be examined in a mixture of 23 volumes of acetonitrile R and 77 volumes of water R and dilute to 10.0 ml with the same mixture. Reference solution. Dissolve 10.0 mg of dinoprost trometamol CRS in a mixture of 23 volumes of acetonitrile R and 77 volumes of water R and dilute to 10.0 ml with the same mixture. The chromatographic procedure may be carried out using : — a stainless steel column 0.15 m long and 3.9 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R1 (5 µm) with a pore size of 10 nm and a carbon loading of 19 per cent, — as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows : dissolve 2.44 g of sodium dihydrogen phosphate R in water R and dilute to 1000 ml with water R ; adjust the pH to 2.5 with phosphoric acid R (about 0.6 ml) ; mix 730 ml of the buffer solution with 270 ml of acetonitrile R1, — as detector a spectrophotometer set at 200 nm. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 20 µl of the reference solution is 70 per cent to 90 per cent of the full scale of the recorder. When the chromatogram is recorded under the conditions described above, the retention time is about 23 min for dinoprost. 1450

Inject the reference solution six times. The test is not valid unless the relative standard deviation of the peak area for dinoprost is at most 2.0 per cent. Inject 20 µl of the test solution. Calculate the percentage of dinoprost trometamol. IMPURITIES

A. (E)-7-[(1R,2R,3R,5S)-3,5-dihydroxy-2-[(E)-(3S)-3hydroxyoct-1-enyl]cyclopentyl]hept-5-enoic acid ((5E)-PGF2α ; 5,6-trans-PGF2α),

B. (Z)-7-[(1R,2R,3R,5S)-3,5-dihydroxy-2-[(E)-(3R)-3hydroxyoct-1-enyl]cyclopentyl]hept-5-enoic acid ((15R)-PGF2α ;15-epiPGF2α),

C. (Z)-7-[(1S,2R,3R,5S)-3,5-dihydroxy-2-[(E)-(3S)-3hydroxyoct-1-enyl]cyclopentyl]hept-5-enoic acid ((8S)-PGF2α ; 8-epiPGF2α),

D. (Z)-7-[(1R,2R,3S,5S)-3,5-dihydroxy-2-[(E)-(3S)-3hydroxyoct-1-enyl]cyclopentyl]hept-5-enoic acid (11β-PGF2α ; 11-epiPGF2α). 01/2005:1311

DINOPROSTONE Dinoprostonum

C20H32O5

Mr 352.5

DEFINITION (Z)-7-[(1R,2R,3R)-3-hydroxy-2-[(E)-(3S)-3-hydroxyoct-1-enyl]-5oxocyclopentyl]hept-5-enoic acid (PGE2). Content : 95.0 per cent to 102.0 per cent (anhydrous substance).



Dinoprostone

CHARACTERS Appearance : white or almost white, crystalline powder or colourless crystals. Solubility : practically insoluble in water, very soluble in methanol, freely soluble in alcohol. The substance degrades at room temperature.

to increase the retention time for dinoprostone and impurity C and increase the concentration of methanol to decrease the retention time for both compounds). Limits : — correction factors : for the calculation of contents, multiply the peak areas of the following impurities by the corresponding correction factor : impurity D = 0.2 ; impurity E = 0.7, IDENTIFICATION A. Specific optical rotation (2.2.7) : − 82 to − 90 (anhydrous — impurity C : not more than 3 times the area of the principal peak in the chromatogram obtained with substance). reference solution (b) (1.5 per cent), Immediately before use, dissolve 50.0 mg in alcohol R — impurity D : not more than twice the area of the principal and dilute to 10.0 ml with the same solvent. peak in the chromatogram obtained with reference B. Infrared absorption spectrophotometry (2.2.24). solution (b) (1 per cent), Comparison : dinoprostone CRS. — impurity E : not more than the area of the principal peak in the chromatogram obtained with reference solution (b) TESTS (0.5 per cent), Prepare the solutions immediately before use. — any other impurity : not more than the area of the Related substances. Liquid chromatography (2.2.29). principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent), Test solution (a). Dissolve 10.0 mg of the substance to be examined in a 58 per cent V/V solution of methanol R2 and — total of other impurities: not more than twice the area dilute to 2.0 ml with the same solvent. of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent), Test solution (b). Dissolve 20.0 mg of the substance to be examined in a 58 per cent V/V solution of methanol R2 and — disregard limit : 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b) dilute to 20.0 ml with the same solvent. (0.05 per cent). Reference solution (a). Dissolve 1 mg of dinoprostone CRS If any peak with a relative retention to dinoprostone of and 1 mg of dinoprostone impurity C CRS in a 58 per cent V/V solution of methanol R2 and dilute to 10.0 ml with about 0.8 is greater than 0.5 per cent or if the total of other impurities is greater than 1.0 per cent, record the the same solvent. Dilute 4.0 ml of the solution to 10.0 ml chromatogram of test solution (a) with a detector set at with a 58 per cent V/V solution of methanol R2. 230 nm. If the area of the peak at 230 nm is twice the area Reference solution (b). Dilute 0.5 ml of test solution (a) to of the peak at 210 nm, multiply the area at 210 nm by 0.2 10.0 ml with a 58 per cent V/V solution of methanol R2. (correction factor for impurity F). Dilute 1.0 ml of the solution to 10.0 ml with a 58 per Water (2.5.12) : maximum 0.5 per cent, determined on 0.50 g. cent V/V solution of methanol R2. Reference solution (c). In order to prepare in situ the ASSAY degradation compounds (impurity D and impurity E), Prepare the solutions immediately before use. dissolve 1 mg of the substance to be examined in 100 µl of 1 M sodium hydroxide (the solution becomes brownish-red), Liquid chromatography (2.2.29) as described in the test for related substances. wait 4 min, add 150 µl of 1 M acetic acid (yellowish-white opalescent solution) and dilute to 5.0 ml with a 58 per Injection : test solution (b) and reference solution (d). cent V/V solution of methanol R2. Calculate the percentage content of C20H32O5. Reference solution (d). Dissolve 20 mg of dinoprostone CRS in a 58 per cent V/V solution of methanol R2 and dilute to STORAGE 20.0 ml with the same solvent. At a temperature not exceeding - 15 °C. Column : IMPURITIES — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase: end-capped octadecylsilyl silica gel for chromatography R, — temperature : 30 °C. Mobile phase : mix 42 volumes of a 0.2 per cent V/V solution of acetic acid R and 58 volumes of methanol R2. Flow rate : 1.0 ml/min. A. (Z)-7-[(1R,2R,3R)-3-hydroxy-2-[(E)-(3R)-3-hydroxyoct-1enyl]-5-oxocyclopentyl]hept-5-enoic acid (15-epiPGE2 ; Detection : spectrophotometer at 210 nm. (15R)-PGE2), Injection : 20 µl ; inject test solution (a) and reference solutions (a), (b) and (c). Relative retention with reference to dinoprostone (retention time = about 18 min) : impurity C = about 1.2 ; impurity D = about 1.8 ; impurity E = about 2.0. System suitability : reference solution (a) : — resolution : minimum of 3.8 between the peaks due to B. (Z)-7-[(1S,2R,3R)-3-hydroxy-2-[(E)-(3S)-3-hydroxyoct-1dinoprostone and to impurity C. If necessary adjust the enyl]-5-oxocyclopentyl]hept-5-enoic acid (8-epiPGE2 ; concentration of the acetic acid solution and/or methanol (8S)-PGE2), (increase the concentration of the acetic acid solution


1451

Diosmin


CHARACTERS Appearance : greyish-yellow or light yellow hygroscopic powder. Solubility : practically insoluble in water, soluble in dimethyl sulphoxide, practically insoluble in alcohol. It dissolves in dilute solutions of alkali hydroxides. C. (E)-7-[(1R,2R,3R)-3-hydroxy-2-[(E)-(3S)-3-hydroxyoct-1enyl]-5-oxocyclopentyl]hept-5-enoic acid (5-trans-PGE2 ; (5E)-PGE2),

IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Comparison : diosmin CRS. B. Examine the chromatograms obtained in the assay. Results : the principal peak in the chromatogram obtained with the test solution is similar in retention time and size to the principal peak in the chromatogram obtained with reference solution (a).

TESTS Iodine : maximum 0.1 per cent. Determine the total content of iodine by potentiometry, using an iodide-selective electrode (2.2.36), after oxygen combustion (2.5.10). Test solution. Wrap 0.100 g of the substance to be examined in a piece of filter paper and place it in a sample carrier. Introduce into the flask 50 ml of a 0.2 g/l solution of hydrazine R. Flush the flask with oxygen for 10 min. Ignite E. (Z)-7-[2-[(E)-(3S)-3-hydroxyoct-1-enyl]-5-oxocyclopent-1the filter paper. Stir the contents of the flask immediately enyl]hept-5-enoic acid (PGB2), after the end of the combustion to dissolve completely the combustion products. Continue stirring for 1 h. Reference solution. Dilute 2.0 ml of a 16.6 g/l solution of potassium iodide R to 100.0 ml with water R. Dilute 10.0 ml of the solution to 100.0 ml with water R. Introduce into a beaker 30 ml of a 200 g/l solution of potassium nitrate R in 0.1 M nitric acid. Immerse the electrodes and stir for 10 min. The potential of the solution F. (Z)-7-[(1R,2R,3R)-3-hydroxy-2-[(E)-3-oxo-oct-1-enyl](nT1) must remain stable. Add 1 ml of the test solution and 5-oxocyclopentyl]hept-5-enoic acid (15-oxo-PGE2 ; measure the potential (nT2). 15-keto-PGE2). Introduce into a beaker 30 ml of a 200 g/l solution of potassium nitrate R in 0.1 M nitric acid. Immerse the 01/2005:1611 electrodes and stir for 10 min. The potential of the solution must remain stable (nR1). Add 80 µl of the reference solution and measure the potential (nR2). DIOSMIN The absolute value |nT2 - nT1| is not higher than the absolute value |nR2 - nR1|. Diosminum Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 25.0 mg of the substance to be examined in dimethyl sulphoxide R and dilute to 25.0 ml with the same solvent. Reference solution (a). Dissolve 25.0 mg of diosmin CRS in dimethyl sulphoxide R and dilute to 25.0 ml with the same solvent. Reference solution (b). Dilute 5.0 ml of reference solution (a) to 100.0 ml with dimethyl sulphoxide R. C28H32O15 Mr 609 Reference solution (c). Dissolve 5.0 mg of diosmin for system suitability CRS in dimethyl sulphoxide R and dilute DEFINITION to 5.0 ml with the same solvent. 7-[[6-O-(6-Deoxy-α-L-mannopyranosyl)-β-D-glucopyranColumn : osyl]oxy]-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-ben- — size : l = 0.10 m, Ø = 4.6 mm, zopyran-4-one. — stationary phase : octadecylsilyl silica gel for Substance obtained through iodine-assisted oxidation chromatography R (3 µm), of (2S)-7-[[6-O-(6-deoxy-α-L-mannopyranosyl)-β— temperature : 40 °C. D-glucopyranosyl]oxy]-5-hydroxy-2-(3-hydroxy-4Mobile phase : acetonitrile R, glacial acetic acid R, methoxyphenyl)-2,3-dihydro-4H-1-benzopyran-4-one methanol R, water R (2:6:28:66 V/V/V/V). (hesperidin) of natural origin. Flow rate : 1.5 ml/min. Content : 90.0 per cent to 102.0 per cent (anhydrous Detection : spectrophotometer at 275 nm. substance). D. (Z)-7-[(1R,2S)-2-[(E)-(3S)-3-hydroxyoct-1-enyl]-5oxocyclopent-3-enyl]hept-5-enoic acid (PGA2),

1452


Diosmin


Injection : 10 µl loop injector ; inject the test solution and reference solutions (b) and (c). Run time : 6 times the retention time of diosmin. Relative retention with reference to diosmin (retention time = about 4.6 min) : impurity A = about 0.5, impurity B = about 0.6, impurity C = about 0.8, impurity D = about 2.2, impurity E = about 2.6, impurity F = about 4.5.

STORAGE In an airtight container. IMPURITIES

System suitability : reference solution (c) : — resolution : minimum of 2.5 between the peaks due to impurities B and C. Limits : — correction factors : for the calculation of contents, multiply the peak areas of the following impurities by the corresponding correction factor : impurity A = 0.38 ; impurity F = 0.61,

A. 1-(3-hydroxy-4-methoxyphenyl)ethanone (acetoisovanillone),

— impurity A : not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent), — impurity B : not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (5 per cent), — impurity C : not more than 0.6 times the area of the principal peak in the chromatogram obtained with reference solution (b) (3 per cent),

B. (2S)-7-[[6-O-(6-deoxy-α-L-mannopyranosyl)-β-Dglucopyranosyl]oxy]-5-hydroxy-2-(3-hydroxy-4methoxyphenyl)-2,3-dihydro-4H-1-benzopyran-4-one (hesperidin),

— impurity E : not more than 0.6 times the area of the principal peak in the chromatogram obtained with reference solution (b) (3 per cent), — impurity F : not more than 0.6 times the area of the principal peak in the chromatogram obtained with reference solution (b) (3 per cent), — any other impurity : not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent), — total of other impurities and impurity A : not more C. R1 = R3 = H, R2 = OH : 7-[[6-O-(6-deoxy-α-Lthan 0.2 times the area of the principal peak in the mannopyranosyl)-β-D-glucopyranosyl]oxy]-5-hydroxy-2-(4chromatogram obtained with reference solution (b) (1 per hydroxyphenyl)-4H-1-benzopyran-4-one (isorhoifin), cent), — total: not more than twice the area of the principal peak D. R1 = OH, R2 = OCH , R3 = I : 7-[[6-O-(6-deoxy-α-L3 in the chromatogram obtained with reference solution (b) mannopyranosyl)-β-D-glucopyranosyl]oxy]-5-hydroxy-2-(3(10 per cent), hydroxy-4-methoxyphenyl)-6-iodo-4H-1-benzopyran-4-one (6-iododiosmin), — disregard limit : 0.02 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent). E. R1 = R3 = H, R2 = OCH3 : 7-[[6-O-(6-deoxy-α-LHeavy metals (2.4.8) : maximum 20 ppm. mannopyranosyl)-β-D-glucopyranosyl]oxy]-5-hydroxy-2-(4methoxyphenyl)-4H-1-benzopyran-4-one (linarin), 2.0 g complies with limit test C. Prepare the standard using 4.0 ml of lead standard solution (10 ppm Pb) R. Water (2.5.12) : maximum 6.0 per cent, determined on 0.300 g. Sulphated ash (2.4.14) : maximum 0.2 per cent, determined on 1.0 g. ASSAY Liquid chromatography (2.2.29), as described in the test for related substances. Injection : test solution and reference solution (a). General Notices (1) apply to all monographs and other texts

F. 5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1benzopyran-4-one (diosmetin). 1453

Diphenhydramine hydrochloride


01/2005:0023 mobile phase. Dilute 2.0 ml of the solution to 10.0 ml with corrected the mobile phase. Reference solution (a). Dilute 1.0 ml of the test solution to 10.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 20.0 ml with the mobile phase.

DIPHENHYDRAMINE HYDROCHLORIDE

Reference solution (b). Dissolve 5 mg of diphenhydramine impurity A CRS and 5 mg of diphenylmethanol R in the mobile phase and dilute to 10.0 ml with the mobile phase. To 2.0 ml of this solution add 1.5 ml of the test solution and dilute to 10.0 ml with the mobile phase.

Diphenhydramini hydrochloridum

Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : base-deactivated octylsilyl silica gel for chromatography R (5 µm). C17H22ClNO

Mobile phase : mix 35 volumes of acetonitrile R and 65 volumes of a 5.4 g/l solution of potassium dihydrogen Mr 291.8 phosphate R adjusted to pH 3.0 using phosphoric acid R. Flow rate : 1.2 ml/min.

DEFINITION 2-(Diphenylmethoxy)-N,N-dimethylethanamine hydrochloride.


Content : 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white or almost white, crystalline powder. Solubility : very soluble in water, freely soluble in alcohol.

Run time : 7 times the retention time of diphenhydramine. Relative retention with reference to diphenhydramine (retention time = about 6 min) : impurity A = about 0.9 ; impurity B = about 1.5 ; impurity C = about 1.8 ; impurity D = about 2.6 ; impurity E = about 5.1. System suitability : reference solution (b) : — resolution : minimum 2.0 between the peaks due to diphenhydramine and to impurity A.

IDENTIFICATION First identification : C, D.

Limits :

Second identification : A, B, D.

— correction factor : for the calculation of content, multiply the peak area of impurity D by 0.7,

A. Melting point (2.2.14) : 168 °C to 172 °C.

B. Dissolve 50 mg in alcohol R and dilute to 100.0 ml with — impurity A : not more than the area of the principal peak the same solvent. Examined between 230 nm and 350 nm, in the chromatogram obtained with reference solution (a) the solution shows 3 absorption maxima (2.2.25), at (0.5 per cent), 253 nm, 258 nm and 264 nm. The ratio of the absorbance — any other impurity : not more than 0.6 times the area of measured at the maximum at 258 nm to that measured the principal peak in the chromatogram obtained with at the maximum at 253 nm is 1.1 to 1.3. The ratio of the reference solution (a) (0.3 per cent), absorbance measured at the maximum at 258 nm to that measured at the maximum at 264 nm is 1.2 to 1.4. — total : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (a) C. Infrared absorption spectrophotometry (2.2.24). (1.0 per cent), Preparation : discs. — disregard limit : 0.1 times the area of the principal peak Comparison : diphenhydramine hydrochloride CRS. in the chromatogram obtained with reference solution (a) (0.05 per cent). D. It gives the reactions of chlorides (2.3.1). Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. TESTS Sulphated ash (2.4.14) : maximum 0.1 per cent, determined Solution S. Dissolve 1.0 g in carbon dioxide-free water R on 1.0 g. and dilute to 20 ml with the same solvent. Appearance of solution. Solution S and a fivefold dilution of solution S are clear (2.2.1). Solution S is not more intensely coloured than reference solution BY6 (2.2.2, Method II). Acidity or alkalinity. To 10 ml of solution S add 0.15 ml of methyl red solution R and 0.25 ml of 0.01 M hydrochloric acid. The solution is pink. Not more than 0.5 ml of 0.01 M sodium hydroxide is required to change the colour of the indicator to yellow. Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 70 mg of the substance to be examined in the mobile phase and dilute to 20.0 ml with the 1454

ASSAY Dissolve 0.250 g in 50 ml of alcohol R and add 5.0 ml of 0.01 M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the 2 points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 29.18 mg of C17H22ClNO. STORAGE Protected from light.



Diphenoxylate hydrochloride

IMPURITIES Specified impurities : A, B, C, D, E.

B. Dissolve about 30 mg in 5 ml of methanol R. Add 0.25 ml of nitric acid R and 0.4 ml of silver nitrate solution R1. Shake and allow to stand. A curdled precipitate is formed. Centrifuge and rinse the precipitate with three quantities, each of 2 ml, of methanol R. Carry out this operation rapidly and protected from bright light. Suspend the precipitate in 2 ml of water R and add 1.5 ml of ammonia R. The precipitate dissolves easily.

A. R = R′ = H : 2-(diphenylmethoxy)-N-methylethanamine, B. R = R′ = CH3 : 2-[(RS)-(4-methylphenyl)phenylmethoxy]N,N-dimethylethanamine, C. R = Br, R′ = CH3 : 2-[(RS)-(4-bromophenyl)phenylmethoxy]N,N-dimethylethanamine,

D. R = OH, R′ = H : diphenylmethanol (benzhydrol), E. R + R′ = O : diphenylmethanone (benzophenone).

01/2005:0819

DIPHENOXYLATE HYDROCHLORIDE Diphenoxylati hydrochloridum

C30H33ClN2O2

Mr 489.1

DEFINITION Diphenoxylate hydrochloride contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of ethyl 1-(3-cyano-3,3-diphenylpropyl)-4-phenylpiperidine-4carboxylate hydrochloride, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, very slightly soluble in water, freely soluble in methylene chloride, sparingly soluble in alcohol. It melts at about 220 °C, with decomposition.

TESTS Appearance of solution. Dissolve 1.0 g in methylene chloride R and dilute to 10 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). Related substances. Examine by thin-layer chromatography (2.2.27), using a plate coated with a suitable octadecylsilyl silica gel (5 µm) with a fluorescent indicator having an optimal intensity at 254 nm. Test solution. Dissolve 1.0 g in a mixture of 1 volume of methanol R and 2 volumes of methylene chloride R and dilute to 20 ml with the same mixture of solvents. Reference solution (a). Dilute 0.5 ml of the test solution to 100 ml with a mixture of 1 volume of methanol R and 2 volumes of methylene chloride R. Reference solution (b). Dissolve 0.50 g of the substance to be examined in 25 ml of a 15 g/l solution of potassium hydroxide R in methanol R and add 1 ml of water R. Heat on a water-bath under a reflux condenser for 4 h. Cool and add 25 ml of 0.5 M hydrochloric acid. Shake with 100 ml of methylene chloride R. Evaporate the lower layer to dryness on a water-bath. Dissolve the residue in 10 ml of a mixture of 1 volume of methanol R and 2 volumes of methylene chloride R, add 10 ml of the test solution and dilute to 25 ml with a mixture of 1 volume of methanol R and 2 volumes of methylene chloride R. Apply separately to a plate (100 mm square) 1 µl of each solution. Develop in an unsaturated tank over a path of 7 cm using a mixture of 10 volumes of methanol R, 30 volumes of a 59 g/l solution of sodium chloride R and 60 volumes of dioxan R. Allow the plate to dry in an oven at 160 °C for 15 min and place the hot plate in a closed tank containing about 20 ml of fuming nitric acid R for 30 min. Remove the plate and heat it again at 160 °C for 15 min. Allow to cool and examine immediately in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.5 per cent). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated principal spots. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.400 g in 40 ml of alcohol R and add 5.0 ml of 0.01 M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1 M ethanolic sodium hydroxide. Read the volume added between the two points of inflexion. 1 ml of 0.1 M ethanolic sodium hydroxide is equivalent to 48.91 mg of C30H33ClN2O2.

IDENTIFICATION A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum STORAGE of diphenoxylate hydrochloride. Store protected from light. General Notices (1) apply to all monographs and other texts

1455

Dipivefrine hydrochloride


01/2005:1719 Injection : 10 µl. Retention times : impurity A = about 2.2 min ; impurity B = about 3.2 min. DIPIVEFRINE HYDROCHLORIDE System suitability : reference solution : Dipivefrini hydrochloridum — resolution : minimum 2.0 between the peaks due to impurity A and impurity B. Limits : — impurity A : not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.1 per cent), — impurity B : not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.1 per cent). Related substances. Liquid chromatography (2.2.29). C19H30ClNO5 Mr 387.9 Solvent mixture. Mix 40 volumes of methanol R2 and 60 volumes of acetonitrile R. Mix 55 volumes of this mixture DEFINITION and 45 volumes of 0.01 M hydrochloric acid. Hydrochloride of 4-[(1RS)-1-hydroxy-2-(methylamino)ethyl]-1, Test solution. Dissolve 50.0 mg of the substance to be 2-phenylene bis(2,2-dimethylpropanoate). examined in the solvent mixture and dilute to 5.0 ml with the Content : 97.5 per cent to 102.0 per cent (dried substance). solvent mixture. Reference solution (a). Dilute 1.0 ml of the test solution to CHARACTERS 100.0 ml with the solvent mixture. Appearance : white or almost white, crystalline powder. Reference solution (b). Dissolve 5 mg of dipivefrine for Solubility : freely soluble in water, very soluble in methanol, system suitability CRS (containing impurities C, D and E) freely soluble in ethanol (96 per cent) and in methylene in the solvent mixture and dilute to 2.0 ml with the solvent chloride. mixture. mp : about 160 °C. Reference solution (c). Dissolve 5.0 mg of dipivefrine hydrochloride CRS in the solvent mixture and dilute to IDENTIFICATION 2.0 ml with the solvent mixture. Dilute 1.0 ml of the solution A. Infrared absorption spectrophotometry (2.2.24). to 25.0 ml with the solvent mixture. Preparation : discs. Column : Comparison : dipivefrine hydrochloride CRS. — size : l = 0.15 m, Ø = 4.6 mm, B. It gives reaction (a) of chlorides (2.3.1). — stationary phase : end-capped polar-embedded octadecylsilyl amorphous organosilica polymer R TESTS (5 µm). Impurities A and B. Liquid chromatography (2.2.29). Mobile phase : mix 45 volumes of a 0.7 g/l solution of Test solution. Dissolve 0.100 g of the substance to be concentrated ammonia R adjusted to pH 10.0 with dilute examined in 0.01 M hydrochloric acid and dilute to 10.0 ml acetic acid R and 55 volumes of a mixture of 40 volumes of with the same acid. methanol R2 and 60 volumes of acetonitrile R. Reference solution. Dissolve 10.0 mg of adrenaline R Flow rate : 1 ml/min. and 10.0 mg of adrenalone hydrochloride R in 0.01 M Detection : spectrophotometer at 260 nm. hydrochloric acid and dilute to 100.0 ml with the same Injection : 10 µl. acid. Dilute 1.0 ml of this solution to 10.0 ml with 0.01 M hydrochloric acid. Protect this solution from light. Run time : 2.5 times the retention time of dipivefrine. Column : Relative retention with reference to dipivefrine (retention time = about 7 min) : impurities C and D = about 0.4 ; — size : l = 0.15 m, Ø = 4.6 mm, impurity E = about 1.3 ; impurity F = about 2.0. — stationary phase : end-capped polar-embedded System suitability : reference solution (b) : octadecylsilyl amorphous organosilica polymer R — resolution : minimum 3.0 between the peaks due to (5 µm). dipivefrine and impurity E. Mobile phase : — mobile phase A : 0.1 per cent V/V solution of anhydrous Limits : — correction factors : for the calculation of contents, formic acid R, multiply the peak areas of the following impurities — mobile phase B : methanol R2, acetonitrile R (40:60 V/V), by the corresponding correction factor : impurities C Time Mobile phase A Mobile phase B and D = 0.5 ; impurity E = 0.06 ; (min) (per cent V/V) (per cent V/V) — sum of impurities C and D : not more than 0.3 times the 0-3 0 100 area of the principal peak in the chromatogram obtained with reference solution (a) (0.3 per cent) ; 0 → 60 3-5 100 → 40 — impurities E, F : for each impurity, not more than 0.1 times 40 60 5 - 10 the area of the principal peak in the chromatogram 60 → 0 10 - 11 40 → 100 obtained with reference solution (a) (0.1 per cent) ; — any other impurity : for each impurity, not more 0 11 - 25 100 than 0.1 times the area of the principal peak in the Flow rate : 1 ml/min. chromatogram obtained with reference solution (a) (0.1 per cent) ; Detection : spectrophotometer at 260 nm. 1456



— total: not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent) ; — disregard limit : 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent), disregard any peak with a mass distribution ratio of less than 0.5. Loss on drying (2.2.32) : maximum 1.0 per cent, determined on 1.000 g by drying in vacuo at 60 °C for 6 h. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modification. Injection : 20 µl of reference solutions (a) and (c). Calculate the percentage content of C19H30ClNO5 using the chromatograms obtained with reference solutions (a) and (c) and the declared content of dipivefrine hydrochloride CRS. IMPURITIES Specified impurities : A, B, C, D, E, F.

A. R1 = R2 = R3 = H : 4-[(1RS)-1-hydroxy-2(methylamino)ethyl]benzene-1,2-diol ((±)-adrenaline), C. R1 = R3 = H, R2 = CO-C(CH3)3 : 2-hydroxy-5[(1RS)-1-hydroxy-2-(methylamino)ethyl]phenyl 2,2-dimethylpropanoate, D. R1 = CO-C(CH3)3, R2 = R3 = H : 2-hydroxy-4[(1RS)-1-hydroxy-2-(methylamino)ethyl]phenyl 2,2-dimethylpropanoate, F. R1 = R2 = CO-C(CH3)3, R3 = C2H5 : 4-[(1RS)-2(ethylmethylamino)-1-hydroxyethyl]-1,2-phenylene bis(2,2-dimethylpropanoate),

Dipotassium clorazepate

DEFINITION Potassium (3RS)-7-chloro-2-oxo-5-phenyl-2,3-dihydro-1H-1, 4-benzodiazepine-3-carboxylate compound with potassium hydroxide (1:1). Content : 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white or light yellow, crystalline powder. Solubility : freely soluble to very soluble in water, very slightly soluble in alcohol, practically insoluble in methylene chloride. Solutions in water and in alcohol are unstable and are to be used immediately. IDENTIFICATION First identification : B, E. Second identification : A, C, D, E. A. Dissolve 10.0 mg in a 0.3 g/l solution of potassium carbonate R and dilute to 100.0 ml with the same solution (solution A). Dilute 10.0 ml of solution A to 100.0 ml with a 0.3 g/l solution of potassium carbonate R (solution B). Examined between 280 nm and 350 nm (2.2.25), solution A shows a broad absorption maximum at about 315 nm. The specific absorbance at the absorption maximum at 315 nm is 49 to 56. Examined between 220 nm and 280 nm (2.2.25), solution B shows an absorption maximum at 230 nm. The specific absorbance at the absorption maximum at 230 nm is 800 to 870. B. Infrared absorption spectrophotometry (2.2.24). Preparation : discs. Comparison : Ph. Eur. reference spectrum of dipotassium clorazepate. C. Dissolve about 20 mg in 2 ml of sulphuric acid R. Observed in ultraviolet light at 365 nm, the solution shows yellow fluorescence. D. Dissolve 0.5 g in 5 ml of water R. Add 0.1 ml of thymol blue solution R. The solution is violet-blue. E. Place 1.0 g in a crucible and add 2 ml of dilute sulphuric acid R. Heat at first on a water-bath, then ignite until all black particles have disappeared. Allow to cool. Take up the residue with water R and dilute to 20 ml with the same solvent. The solution gives reaction (b) of potassium (2.3.1).

TESTS B. R = H : 1-(3,4-dihydroxyphenyl)-2-(methylamino)ethanone Appearance of solution. The solution is clear (2.2.1) and not more intensely coloured than reference solution GY5 (adrenalone), (2.2.2, Method II). E. R = CO-C(CH3)3 : 4-[(methylamino)acetyl]-1,2-phenylene bis(2,2-dimethylpropanoate) (adrenalone dipivalate ester). Rapidly dissolve 2.0 g with shaking in water R and dilute to 20.0 ml with the same solvent. Observe immediately. 01/2005:0898 Related substances. Thin-layer chromatography (2.2.27). Prepare the solutions immediately before use and carry out the test protected from light. DIPOTASSIUM CLORAZEPATE Test solution. Dissolve 0.20 g of the substance to be examined in water R and dilute to 5.0 ml with the same Dikalii clorazepas solvent. Shake immediately with 2 quantities, each of 5.0 ml, of methylene chloride R. Combine the organic layers and dilute to 10.0 ml with methylene chloride R. Reference solution (a). Dissolve 10 mg of aminochlorobenzophenone R in methylene chloride R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of the solution to 25.0 ml with methylene chloride R. Reference solution (b). Dissolve 5 mg of nordazepam CRS in methylene chloride R and dilute to 25.0 ml with the same solvent. Dilute 5.0 ml of the solution to 25.0 ml with C16H11ClK2N2O4 Mr 408.9 methylene chloride R. General Notices (1) apply to all monographs and other texts

1457

Dipotassium phosphate


Reference solution (c). Dilute 10.0 ml of reference solution (b) to 20.0 ml with methylene chloride R. Reference solution (d). Dissolve 5 mg of nordazepam CRS and 5 mg of nitrazepam CRS in methylene chloride R and dilute to 25 ml with the same solvent. Plate : TLC silica gel F254 plate R. Mobile phase : acetone R, methylene chloride R (15:85 V/V). Application : 5 µl. Development : over 2/3 of the plate.

B. 7-chloro-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (nordazepam).

Drying : in air. Detection A : examine in ultraviolet light at 254 nm. System suitability : the chromatogram obtained with reference solution (d) shows 2 clearly separated spots. Limits A :

01/2005:1003

DIPOTASSIUM PHOSPHATE

Dikalii phosphas — impurity B : any spot due to impurity B is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.2 per cent), K2HPO4 Mr 174.2 — any other impurity : any spot, apart from any spot due DEFINITION to impurity B, is not more intense than the spot in the Dipotassium phosphate contains not less than 98.0 per chromatogram obtained with reference solution (c) cent and not more than the equivalent of 101.0 per cent of (0.1 per cent). K2HPO4, calculated with reference to the dried substance. Detection B : spray with a freshly prepared 10 g/l solution of sodium nitrite R in dilute hydrochloric acid R. Dry in CHARACTERS a current of warm air and spray with a 4 g/l solution of A white powder or colourless crystals, very hygroscopic, very naphthylethylenediamine dihydrochloride R in alcohol R. soluble in water, very slightly soluble in alcohol. Limits B : IDENTIFICATION — impurity A : any spot due to impurity A is not more A. Solution S (see Tests) is slightly alkaline (2.2.4). intense than the spot in the chromatogram obtained with B. Solution S gives reaction (b) of phosphates (2.3.1). reference solution (a) (0.1 per cent). C. Solution S gives reaction (a) of potassium (2.3.1). Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in vacuo at 60 °C for 4 h. TESTS Solution S. Dissolve 5.0 g in distilled water R and dilute to ASSAY 50 ml with the same solvent. Dissolve 0.130 g in 10 ml of anhydrous acetic acid R. Add Appearance of solution. Solution S is clear (2.2.1) and 30 ml of methylene chloride R. Titrate with 0.1 M perchloric colourless (2.2.2, Method II). acid, determining the 2 points of inflexion by potentiometry Reducing substances. Heat on a water-bath for 5 min a (2.2.20). mixture of 5 ml of solution S, 5 ml of dilute sulphuric acid R At the 2nd point of inflexion, 1 ml of 0.1 M perchloric acid is and 0.25 ml of 0.02 M potassium permanganate. The solution remains faintly pink. equivalent to 13.63 mg of C16H11ClK2N2O4. Monopotassium phosphate. Calculated from the number of millilitres of 1 M hydrochloric acid (10.0 ml) and of 1 M STORAGE sodium hydroxide (n1 ml and n2 ml) used in the assay, the In an airtight container, protected from light. ratio : IMPURITIES Specified impurities : A, B.

A. (2-amino-5-chlorophenyl)phenylmethanone (aminochlorobenzophenone), 1458

does not exceed 0.025 (2.5 per cent). Chlorides (2.4.4). To 2.5 ml of solution S add 10 ml of dilute nitric acid R and dilute to 15 ml with water R. The solution complies with the limit test for chlorides (200 ppm). Sulphates (2.4.13). To 1.5 ml of solution S add 2 ml of dilute hydrochloric acid R and dilute to 15 ml with distilled water R. The solution complies with the limit test for sulphates (0.1 per cent). Arsenic (2.4.2). 5 ml of solution S complies with limit test A for arsenic (2 ppm). Iron (2.4.9). 10 ml of solution S complies with the limit test for iron (10 ppm). See the information section on general monographs (cover pages)


Heavy metals (2.4.8). Dissolve 2.0 g in 8 ml of water R. Acidify with about 6 ml of dilute hydrochloric acid R (pH 3 to 4) and dilute to 20 ml with water R. 12 ml of the solution complies with limit test A for heavy metals (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. Sodium. If intended for use in the manufacture of parenteral dosage forms, it contains not more than 0.1 per cent of Na, determined by atomic emission spectrometry (2.2.22, Method I). Test solution. Dissolve 1.00 g of the substance to be examined in water R and dilute to 100.0 ml with the same solvent. Reference solutions. Prepare the reference solutions using sodium standard solution (200 ppm Na) R, diluted as necessary with water R. Measure the emission intensity at 589 nm. Loss on drying (2.2.32). Not more than 2.0 per cent, determined on 1.000 g by drying in an oven at 125 °C to 130 °C. Bacterial endotoxins (2.6.14) : less than 1.1 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins.

Diprophylline

DEFINITION Diprophylline contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of 7-[(2RS)-2,3-dihydroxypropyl]-1,3-dimethyl-3,7-dihydro-1Hpurine-2,6-dione, calculated with reference to the dried substance. CHARACTERS A white, crystalline powder, freely soluble in water, slightly soluble in alcohol.

IDENTIFICATION First identification : B, C. Second identification : A, C, D. A. Melting point (2.2.14) : 160 °C to 165 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with diprophylline CRS. Examine the substances as discs prepared using 0.5 mg to 1 mg of the substance to be examined in 0.3 g of potassium bromide R. C. Dissolve 1 g in 5 ml of acetic anhydride R and boil under a reflux condenser for 15 min. Allow to cool and add 100 ml of a mixture of 20 volumes of ether R and 80 volumes of light petroleum R. Cool in iced water for at least 20 min, shaking from time to time. Filter, wash ASSAY the precipitate with a mixture of 20 volumes of ether R Dissolve 0.800 g (m) in 40 ml of carbon dioxide-free water R and 80 volumes of light petroleum R, recrystallise from and add 10.0 ml of 1 M hydrochloric acid. Using 1 M sodium alcohol R and dry in vacuo. The crystals melt (2.2.14) hydroxide, titrate potentiometrically (2.2.20) to the first at 142 °C to 148 °C. inflexion point of the pH curve (n1 ml). Continue the titration D. It gives the reaction of xanthines (2.3.1). to the second inflexion point of the curve (total volume of 1 M sodium hydroxide required, n2 ml). TESTS Calculate the percentage content of K2HPO4 from the Solution S. Dissolve 2.5 g in carbon dioxide-free water R expression : and dilute to 50 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Acidity or alkalinity. To 10 ml of solution S add 0.25 ml of d = percentage loss on drying. bromothymol blue solution R1. The solution is yellow or green. Not more than 0.4 ml of 0.01 M sodium hydroxide is STORAGE required to change the colour of the indicator to blue. Store in an airtight container. Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel HF254 R as the coating substance. LABELLING Test solution. Dissolve 0.3 g of the substance to be examined The label states : — where applicable, that the substance is suitable for use in in a mixture of 20 volumes of water R and 30 volumes of methanol R and dilute to 10 ml with the same mixture of the manufacture of parenteral dosage forms, solvents. Prepare immediately before use. — where applicable, that the substance is free from bacterial Reference solution (a). Dilute 1 ml of the test solution to endotoxins. 100 ml with methanol R. Reference solution (b). Dilute 0.2 ml of the test solution to 01/2005:0486 100 ml with methanol R. Reference solution (c). Dissolve 10 mg of theophylline R in methanol R, add 0.3 ml of the test solution and dilute to DIPROPHYLLINE 10 ml with methanol R. Apply to the plate 10 µl of each solution. Develop over a Diprophyllinum path of 15 cm using a mixture of 1 volume of concentrated ammonia R, 10 volumes of ethanol R and 90 volumes of chloroform R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (a) (1 per cent) and at most one such spot is more intense than the spot in the chromatogram obtained with reference solution (b) (0.2 per cent). The test is not valid unless the chromatogram obtained C10H14N4O4 Mr 254.2 with reference solution (c) shows two clearly separated spots. General Notices (1) apply to all monographs and other texts

1459

Dipyridamole


Chlorides (2.4.4). Dilute 2.5 ml of solution S to 15 ml (2.2.25), the solution shows two absorption maxima with water R. The solution complies with the limit test for at 232 nm and 284 nm. The ratio of the absorbance chlorides (400 ppm). measured at the maximum at 284 nm to that measured at the maximum at 232 nm is 1.25 to 1.45. Heavy metals (2.4.8). 12 ml of solution S complies with limit test A for heavy metals (20 ppm). Prepare the standard using C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with lead standard solution (1 ppm Pb) R. dipyridamole CRS. Examine the substances as discs Loss on drying (2.2.32). Not more than 0.5 per cent, prepared using potassium bromide R. determined on 1.000 g by drying in an oven at 100 °C to D. Dissolve about 5 mg in a mixture of 0.1 ml of nitric acid R 105 °C. and 2 ml of sulphuric acid R. An intense violet colour Sulphated ash (2.4.14). Not more than 0.1 per cent, is produced. determined on 1.0 g. TESTS ASSAY In order to avoid overheating in the reaction medium, mix Related substances. Examine by liquid chromatography thoroughly throughout and stop the titration immediately (2.2.29). Test solution. Dissolve 10.0 mg in the mobile phase and after the end-point has been reached. dilute to 20.0 ml with the mobile phase. Dissolve 0.200 g in 3.0 ml of anhydrous formic acid R Reference solution (a). Dilute 1.0 ml of the test solution to and add 50.0 ml of acetic anhydride R. Titrate with 20.0 ml with the mobile phase. Dilute 5.0 ml of this solution 0.1 M perchloric acid, determining the end-point to 50.0 ml with the mobile phase. potentiometrically (2.2.20). Reference solution (b). Dissolve 10.0 mg of diltiazem 1 ml of 0.1 M perchloric acid is equivalent to 25.42 mg of hydrochloride CRS in the mobile phase and dilute to 10.0 ml C10H14N4O4. with the mobile phase. Dilute 1.0 ml of this solution to STORAGE 20.0 ml with reference solution (a). Store protected from light. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in 01/2005:1199 internal diameter packed with octylsilyl silica gel for chromatography R (5 µm), DIPYRIDAMOLE — as mobile phase at a flow rate of 1.3 ml/min a mixture prepared as follows : dissolve 0.504 g of potassium Dipyridamolum dihydrogen phosphate R in 370 ml of water R and adjust to pH 3.0 with phosphoric acid R ; add 80 ml of acetonitrile R and 550 ml of methanol R, — as detector a spectrophotometer set at 290 nm, maintaining the temperature of the column at 30 °C. Inject 20 µl of each solution and continue the chromatography of the test solution for nine times the retention time of dipyridamole. The test is not valid unless, in the chromatogram obtained with reference solution (b), the resolution between the peaks corresponding respectively to diltiazem and dipyridamole is at least 2.0. In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak is not greater than C24H40N8O4 Mr 504.6 the area of the peak in the chromatogram obtained with reference solution (a) (0.5 per cent) ; and the sum of the DEFINITION areas of all the peaks, apart from the principal peak, is not Dipyridamole contains not less than 98.5 per cent greater than twice the area of the peak in the chromatogram and not more than the equivalent of 101.5 per cent of obtained with reference solution (a) (1 per cent). Disregard 2,2′,2″,2′′′-[[4,8-di(piperidin-1-yl)pyrimido[5,4-d]pyrimidine-2, any peak with an area less than 0.1 times that of the peak in 6-diyl]dinitrilo]tetraethanol, calculated with reference to the the chromatogram obtained with reference solution (a). dried substance. Chlorides (2.4.4). To 0.250 g add 10 ml of water R and strongly shake. Filter, rinse the filter with 5 ml of water R CHARACTERS and dilute to 15 ml with water R. The combined filtrates A bright yellow, crystalline powder, practically insoluble comply with the limit test for chlorides (200 ppm). in water, freely soluble in acetone, soluble in ethanol. It Loss on drying (2.2.32). Not more than 0.5 per cent, dissolves in dilute solutions of mineral acids. determined on 1.000 g by drying in an oven at 100 °C to IDENTIFICATION 105 °C. First identification : C. Sulphated ash (2.4.14). Not more than 0.1 per cent, Second identification : A, B, D. determined on 1.0 g. A. Melting point (2.2.14) : 162 °C to 168 °C. ASSAY B. Dissolve 10 mg in a mixture of 1 volume of 0.1 M Dissolve 0.400 g in 70 ml of methanol R. Titrate with hydrochloric acid and 9 volumes of methanol R and 0.1 M perchloric acid, determining the end-point dilute to 50.0 ml with the same mixture of solvents. potentiometrically (2.2.20). Dilute 5.0 ml of this solution to 100.0 ml with a mixture 1 ml of 0.1 M perchloric acid is equivalent to 50.46 mg of of 1 volume of 0.1 M hydrochloric acid and 9 volumes C24H40N8O4. of methanol R. Examined between 220 nm and 350 nm 1460




Dirithromycin

B. Examine the chromatograms obtained in the assay. The retention time and size of the principal peak in the chromatogram obtained with test solution (a) are similar to those of the principal peak in the chromatogram obtained with reference solution (a).

TESTS Related substances. Examine by liquid chromatography (2.2.29), as described under Assay. Inject 10 µl of reference solution (b). Adjust the sensitivity of the system so that the height of the principal peak is at least 50 per cent of the full scale of the recorder. Inject 10 µl of test solution (b) and continue the chromatography for three times the retention time of the principal peak. In the chromatogram obtained A. R = R′ = NC5H10 : 2,2′-[[4,6,8-tri(piperidin-1-yl)pyrimido[5, with test solution (b) : the area of any peak corresponding 4-d]pyrimidin-2-yl]nitrilo]diethanol, to impurity A is not greater than 0.75 times the area of the principal peak in the chromatogram obtained with reference B. R = R′ = N(CH2-CH2OH)2 : 2,2′,2″,2′′′,2′′′′,2′′′′′solution (b) (1.5 per cent) ; the area of any peak, apart from [[8-(piperidin-1-yl)pyrimido[5,4-d]pyrimidine-2,4,6the principal peak and any peak corresponding to impurity A triyl]trinitrilo]hexaethanol, and any peak corresponding to 15S-epimer, is not greater C. R = NC5H10, R′ = Cl : 2,2′-[[2-chloro-4,8-di(piperidin-1than half the area of the principal peak in the chromatogram yl)pyrimido[5,4-d]pyrimidin-6-yl]nitrilo]diethanol. obtained with reference solution (b) (1 per cent). 01/2005:1313 Dirithromycin 15S-epimer. Not more than 1.5 per cent, determined by liquid chromatography (2.2.29), as described under Assay. Inject 10 µl of reference solution (b). Adjust the DIRITHROMYCIN sensitivity of the system so that the height of the principal peak is at least 50 per cent of the full scale of the recorder. Dirithromycinum Inject reference solution (b) six times. The test is not valid unless the relative standard deviation of the peak area for dirithromycin is at most 5.0 per cent. Inject alternately test solution (b) and reference solution (b). Calculate the percentage content of dirithromycin 15S-epimer using the chromatogram obtained with reference solution (b). Acetonitrile (2.4.24, System A). Not more than 0.1 per cent. Prepare the solutions using dimethylformamide R instead of water R. Sample solution. Dissolve 0.200 g of the substance to be examined in dimethylformamide R and dilute to 20.0 ml with the same solvent. The following static head-space injection conditions may be used : — equilibration temperature : 120 °C, C42H78N2O14 Mr 835 — equilibration time : 60 min, — transfer-line temperature : 125 °C. DEFINITION Heavy metals (2.4.8). Dissolve 1.0 g in 20 ml of a mixture Dirithromycin is (1R,2S,3R,6R,7S,8S,9R,10R,12R, of equal volumes of methanol R and water R. 12 ml of the 13S,15R,17S)-9-[[3-(dimethylamino)-3,4,6-trideoxy-β-Dsolution complies with limit test B for heavy metals (20 ppm). xylo-hexopyranosyl]oxy]-3-ethyl-2,10-dihydroxy-15-[(2Prepare the standard using lead standard solution (1 ppm methoxyethoxy)methyl]-2,6,8,10,12,17-hexamethyl-7-[(3-CPb) obtained by diluting lead standard solution (100 ppm methyl-3-O-methyl-2,6-dideoxy-α-L-ribo-hexopyranosyl)oxy]Pb) R with a mixture of equal volumes of methanol R and 4,16-dioxa-14-azabicyclo[11.3.1]heptadecan-5-one (or water R. (9S)-9,11-[imino[(1R)-2-(2-methoxyethoxy)ethylidene]oxy]-9deoxo-11-deoxyerythromycin). The sum of the percentage Water (2.5.12). Not more than 1.0 per cent, determined on contents of C42H78N2O14 and dirithromycin 15S-epimer is not 1.00 g by the semi-micro determination of water. less than 96.0 per cent and not more than the equivalent of Sulphated ash (2.4.14). Not more than 0.1 per cent, 102.0 per cent, calculated with reference to the anhydrous determined on 1.0 g. substance. ASSAY CHARACTERS Examine by liquid chromatography (2.2.29). A white or almost white powder, very slightly soluble in Solvent mixture. Mix 30 volumes of methanol R and water, very soluble in methanol and in methylene chloride. 70 volumes of acetonitrile R. It shows polymorphism. Test solution (a). Dissolve 20.0 mg of the substance to be examined in the solvent mixture and dilute to 10.0 ml with IDENTIFICATION the solvent mixture. A. Examine by infrared absorption spectrophotometry Test solution (b). Dissolve 0.10 g of the substance to be (2.2.24), comparing with the spectrum obtained with dirithromycin CRS. Examine the substance prepared as examined in the solvent mixture and dilute to 10.0 ml with the solvent mixture. discs. General Notices (1) apply to all monographs and other texts

1461

Disodium edetate


Reference solution (a). Dissolve 20.0 mg of dirithromycin CRS in the solvent mixture and dilute to 10.0 ml with the solvent mixture. Reference solution (b). Dilute 5.0 ml of reference solution (a) to 50.0 ml with the solvent mixture. Reference solution (c). Dissolve 20 mg of dirithromycin CRS in the mobile phase and dilute to 10 ml with the mobile phase. Allow to stand for 24 h before use. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 2.0 ml/min a mixture of : 9 volumes of water R ; 19 volumes of methanol R ; 28 volumes of a solution containing 1.9 g/l of potassium dihydrogen phosphate R and 9.1 g/l of dipotassium hydrogen phosphate R adjusted to pH 7.5 if necessary with a 100 g/l solution of potassium hydroxide R ; 44 volumes of acetonitrile R, — as detector a spectrophotometer set at 205 nm,

C. R = CH2-O-CH2-CH2-O-CH3, R′ = H, R2 = H, R3 = CH3 : (9S)9,11-[imino[(1RS)-2-(2-methoxyethoxy)ethylidene]oxy]-9deoxo-11,12-dideoxyerythromycin (dirithromycin B), D. R = CH2-O-CH2-CH2-O-CH3, R′ = H, R2 = OH, R3 = H : (9S)9,11-[imino[(1RS)-2-(2-methoxyethoxy)ethylidene]oxy]3′-O-demethyl-9-deoxo-11-deoxyerythromycin (dirithromycin C),

E. R = CH3, R′ = CH3, R2 = OH, R3 = CH3 : 9,11-[imino(1methylethylidene)oxy]-9-deoxo-11-deoxyerythromycin. maintaining the temperature of the column at 40 °C. Inject 10 µl of reference solution (a). Adjust the sensitivity of the system so that the height of the principal peak in the 01/2005:0232 chromatogram obtained is at least 50 per cent of the full scale of the recorder. Inject 10 µl of reference solution (c). DISODIUM EDETATE When the chromatogram is recorded in the prescribed conditions, the retention times relative to dirithromycin Dinatrii edetas are : impurity A about 0.7 and 15S-epimer about 1.1. The test is not valid unless, in the chromatogram obtained with reference solution (c), the resolution between the peaks corresponding to dirithromycin and its 15S-epimer is at least 2.0 (if necessary adjust the concentration of the organic modifiers in the mobile phase). Inject reference solution (a) six times. The test is not valid unless the relative standard deviation of the peak area for dirithromycin is at most 1.0 per cent. Inject alternately test solution (a) and reference C10H14N2Na2O8,2H2O Mr 372.2 solution (a). DEFINITION Disodium dihydrogen (ethylenedinitrilo)tetraacetate IMPURITIES dihydrate. Content : 98.5 per cent to 101.0 per cent. CHARACTERS Appearance : white, crystalline powder. Solubility : soluble in water, practically insoluble in alcohol.

A. (9S)-9-amino-9-deoxoerythromycin,

B. R = H : (9S)-9-amino-3-de(2,6-dideoxy-3-C-methyl-3-Omethyl-α-L-ribo-hexopyranosyl)-9-deoxoerythromycin, 1462

IDENTIFICATION First identification : A, B, D. Second identification : B, C, D. A. Infrared absorption spectrophotometry (2.2.24). Preparation : discs. Comparison : disodium edetate CRS. B. Dissolve 2 g in 25 ml of water R, add 6 ml of lead nitrate solution R, shake and add 3 ml of potassium iodide solution R. No yellow precipitate is formed. Make alkaline to red litmus paper R by the addition of dilute ammonia R2. Add 3 ml of ammonium oxalate solution R. No precipitate is formed. C. Dissolve 0.5 g in 10 ml of water R and add 0.5 ml of calcium chloride solution R. Make alkaline to red litmus paper R by the addition of dilute ammonia R2 and add 3 ml of ammonium oxalate solution R. No precipitate is formed. D. It gives the reactions of sodium (2.3.1).


Disodium phosphate, anhydrous


TESTS Solution S. Dissolve 5.0 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). pH (2.2.3) : 4.0 to 5.5 for solution S. Impurity A. Liquid chromatography (2.2.29). Carry out the test protected from light. Solvent mixture. Dissolve 10.0 g of ferric sulphate pentahydrate R in 20 ml of 0.5 M sulphuric acid and add 780 ml of water R. Adjust to pH 2.0 with 1 M sodium hydroxide and dilute to 1000 ml with water R. Test solution. Dissolve 0.100 g of the substance to be examined in the solvent mixture and dilute to 25.0 ml with the solvent mixture. Reference solution. Dissolve 40.0 mg of nitrilotriacetic acid R in the solvent mixture and dilute to 100.0 ml with the solvent mixture. To 1.0 ml of the solution add 0.1 ml of the test solution and dilute to 100.0 ml with the solvent mixture. Column : — size : l = 0.10 m, Ø = 4.6 mm, — stationary phase: spherical graphitised carbon for chromatography R1 (5 µm) with a specific surface area of 120 m2/g and a pore size of 25 nm. Mobile phase : dissolve 50.0 mg of ferric sulphate pentahydrate R in 50 ml of 0.5 M sulphuric acid and add 750 ml of water R. Adjust to pH 1.5 with 0.5 M sulphuric acid or 1 M sodium hydroxide, add 20 ml of ethylene glycol R and dilute to 1000 ml with water R. Flow rate : 1 ml/min. Detection : spectrophotometer at 273 nm. Injection : 20 µl ; filter the solutions and inject immediately. Run time : 4 times the retention time of the iron complex of impurity A. Retention times : iron complex of impurity A = about 5 min ; iron complex of edetic acid = about 10 min. System suitability : reference solution : — resolution : minimum 7 between the peaks due to the iron complex of impurity A and the iron complex of edetic acid, — signal-to-noise ratio : minimum 50 for the peak due to impurity A. Limit : — impurity A : not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.1 per cent). Iron (2.4.9) : maximum 80 ppm. Dilute 2.5 ml of solution S to 10 ml with water R. Add 0.25 g of calcium chloride R to the test solution and the standard before the addition of the thioglycollic acid R. Heavy metals (2.4.8) : maximum 20 ppm. 1.0 g complies with limit test D. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. ASSAY Dissolve 0.300 g in water R and dilute to 300 ml with the same solvent. Add 2 g of hexamethylenetetramine R and 2 ml of dilute hydrochloric acid R. Titrate with 0.1 M lead nitrate, using about 50 mg of xylenol orange triturate R as indicator. 1 ml of 0.1 M lead nitrate is equivalent to 37.22 mg of C10H14N2Na2O8,2H2O. General Notices (1) apply to all monographs and other texts

STORAGE Protected from light. IMPURITIES Specified impurities : A.

A. nitrilotriacetic acid. 01/2005:1509 corrected

DISODIUM PHOSPHATE, ANHYDROUS Dinatrii phosphas anhydricus Na2HPO4

Mr 142.0

DEFINITION Anhydrous disodium phosphate contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of Na2HPO4, calculated with reference to the dried substance. CHARACTERS A white powder, hygroscopic, soluble in water, practically insoluble in alcohol. IDENTIFICATION A. Solution S (see Tests) is slightly alkaline (2.2.4). B. It complies with the test for loss on drying (see Tests). C. Solution S (see Tests) gives reaction (b) of phosphates (2.3.1). D. Solution S (see Tests) gives reaction (a) of sodium (2.3.1). TESTS Solution S. Dissolve 5.0 g in distilled water R and dilute to 100.0 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Reducing substances. To 10 ml of solution S add 5 ml of dilute sulphuric acid R and 0.25 ml of 0.02 M potassium permanganate and heat on a water-bath for 5 min. The solution retains a slight red colour. Monosodium phosphate. Calculated from the number of millilitres of 1 M hydrochloric acid (25 ml) and of 1 M sodium hydroxide (n1 ml and n2 ml) used in the assay, the ratio :

does not exceed 0.025. Chlorides (2.4.4). 5 ml of solution S diluted to 15 ml with dilute nitric acid R complies with the limit test for chlorides (200 ppm). Sulphates (2.4.13). To 6 ml of solution S add 2 ml of dilute hydrochloric acid R and dilute to 15 ml with distilled water R. The solution complies with the limit test for sulphates (500 ppm). Arsenic (2.4.2). 10 ml of solution S complies with limit test A for arsenic (2 ppm). 1463

Disodium phosphate dihydrate


Iron (2.4.9). 10 ml of solution S complies with the limit test for iron (20 ppm). Heavy metals (2.4.8). 12 ml of solution S complies with limit test A for heavy metals (10 ppm). Prepare the standard using 5 ml of lead standard solution (1 ppm Pb) R and 5 ml of water R. Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C for 4 h. ASSAY Dissolve 1.600 g (m) in 25.0 ml of carbon dioxide-free water R and add 25.0 ml of 1 M hydrochloric acid. Using 1 M sodium hydroxide, titrate potentiometrically (2.2.20) to the first inflexion point (n1 ml). Continue the titration to the second inflexion point (total volume of 1 M sodium hydroxide required, n2 ml). Calculate the percentage content of Na2HPO4 from the expression :

d

=

percentage loss on drying.


DISODIUM PHOSPHATE

Monosodium phosphate. Calculated from the number of millilitres of 1 M hydrochloric acid (25 ml) and of 1 M sodium hydroxide (n1 ml and n2 ml) used in the assay, the ratio :

does not exceed 0.025. Chlorides (2.4.4). To 2.5 ml of solution S add 10 ml of dilute nitric acid R and dilute to 15 ml with water R. The solution complies with the limit test for chlorides (400 ppm). Sulphates (2.4.13). To 3 ml of solution S add 2 ml of dilute hydrochloric acid R and dilute to 15 ml with distilled water R. The solution complies with the limit test for sulphates (0.1 per cent). Arsenic (2.4.2). 5 ml of solution S complies with limit test A for arsenic (4 ppm). Iron (2.4.9). 5 ml of solution S diluted to 10 ml with water R complies with the limit test for iron (40 ppm). Heavy metals (2.4.8). 12 ml of solution S complies with limit test A for heavy metals (20 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying (2.2.32) : 19.5 per cent to 21.0 per cent, determined on 1.000 g by drying in an oven at 130 °C.

ASSAY Dissolve 2.000 g (m g) in 50 ml of water R and add 25.0 ml of 1 M hydrochloric acid. Using 1 M sodium hydroxide, titrate potentiometrically (2.2.20) to the first inflexion point of the pH curve (n1 ml). Continue the titration to the second 01/2005:0602 inflexion point of the curve (total volume of 1 M sodium hydroxide required, n2 ml). DIHYDRATE Calculate the percentage content of Na2HPO4 from the expression :

Dinatrii phosphas dihydricus Na2HPO4,2H2O

Mr 178.0

DEFINITION Disodium phosphate dihydrate contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of Na2HPO4, calculated with reference to the dried substance. CHARACTERS A white or almost white powder or colourless crystals, soluble in water, practically insoluble in alcohol.

=

percentage loss on drying.

01/2005:0118

DISODIUM PHOSPHATE DODECAHYDRATE Dinatrii phosphas dodecahydricus

IDENTIFICATION A. Solution S (see Tests) is slightly alkaline (2.2.4). B. It complies with the test for loss on drying (see Tests). C. Solution S gives reaction (b) of phosphates (2.3.1). D. Solution S gives reaction (a) of sodium (2.3.1). TESTS Solution S. Dissolve 5.0 g in distilled water R and dilute to 100 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Reducing substances. To 5 ml of solution S add 5 ml of dilute sulphuric acid R and 0.25 ml of 0.02 M potassium permanganate and heat on a water-bath for 5 min. The solution retains a slight red colour. 1464

d

Na2HPO4,12H2O

Mr 358.1

DEFINITION Disodium phosphate dodecahydrate contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of Na2HPO4, calculated with reference to the anhydrous substance. CHARACTERS Colourless, transparent crystals, very efflorescent, very soluble in water, practically insoluble in alcohol. IDENTIFICATION A. Solution S (see Tests) gives the reactions of phosphates (2.3.1). B. Solution S gives the reactions of sodium (2.3.1).


Disopyramide


01/2005:1006

TESTS Solution S. Dissolve 5.0 g in distilled water R and dilute to 50 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Reducing substances. To 5 ml of solution S add 5 ml of dilute sulphuric acid R and 0.25 ml of 0.02 M potassium permanganate and heat on a water-bath for 5 min. The solution retains a slight red colour. Monosodium phosphate. Calculated from the number of millilitres of 1 M hydrochloric acid (25 ml) and of 1 M sodium hydroxide (n1 ml and n2 ml) used in the assay, the ratio :

does not exceed 0.025. Chlorides (2.4.4). To 2.5 ml of solution S add 10 ml of dilute nitric acid R and dilute to 15 ml with water R. The solution complies with the limit test for chlorides (200 ppm). Sulphates (2.4.13). To 3 ml of solution S add 2 ml of dilute hydrochloric acid R and dilute to 15 ml with distilled water R. The solution complies with the limit test for sulphates (500 ppm). Arsenic (2.4.2). 5 ml of solution S complies with limit test A for arsenic (2 ppm). Iron (2.4.9). 5 ml of solution S diluted to 10 ml with water R complies with the limit test for iron (20 ppm). Heavy metals (2.4.8). 12 ml of solution S complies with limit test A for heavy metals (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. Water (2.5.12). 57.0 per cent to 61.0 per cent, determined on 50.0 mg by the semi-micro determination of water. Use a mixture of 10 volumes of anhydrous methanol R and 40 volumes of formamide R as solvent.

DISOPYRAMIDE Disopyramidum

C21H29N3O

Mr 339.5

DEFINITION Disopyramide contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent of (2RS)-4-[bis(1methylethyl)amino]-2-phenyl-2-(pyridin-2-yl)butanamide, calculated with reference to the dried substance. CHARACTERS A white or almost white powder, slightly soluble in water, freely soluble in methylene chloride, soluble in alcohol.

IDENTIFICATION First identification : B. Second identification : A, C. A. Dissolve 40.0 mg in a 5 g/l solution of sulphuric acid R in methanol R and dilute to 100.0 ml with the same solution. Dilute 5.0 ml of this solution to 50.0 ml with a 5 g/l solution of sulphuric acid R in methanol R. Examined between 240 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 269 nm and a shoulder at 263 nm. The specific absorbance at the ASSAY maximum is 190 to 210. Dissolve 4.00 g (m g) in 25 ml of water R and add 25.0 ml B. Examine by infrared absorption spectrophotometry of 1 M hydrochloric acid. Using 1 M sodium hydroxide, (2.2.24), comparing with the spectrum obtained with titrate potentiometrically (2.2.20) to the first inflexion point disopyramide CRS. Examine the substances as discs of the pH curve (n1 ml). Continue the titration to the second prepared by placing 50 µl of a 50 g/l solution in inflexion point of the curve (total volume of 1 M sodium methylene chloride R on a disc of potassium bromide R. hydroxide required, n2 ml). Dry the discs at 60 °C for 1 h before use. Calculate the percentage content of Na2HPO4 from the C. Examine the chromatograms obtained in the test for expression : related substances in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with test solution (b) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). Spray with dilute potassium iodobismuthate solution R. Examine in daylight. The principal spot in the d = percentage water content. chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). TESTS Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution (a). Dissolve 0.20 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with methanol R. Reference solution (a). Dissolve 20 mg of disopyramide CRS in methanol R and dilute to 10 ml with the same solvent. General Notices (1) apply to all monographs and other texts

1465

Disopyramide phosphate


Reference solution (b). Dilute 0.5 ml of test solution (b) to 20 ml with methanol R.

01/2005:1005

Apply to the plate 10 µl of each solution. Develop over a DISOPYRAMIDE PHOSPHATE path of 15 cm using a mixture of 1 volume of concentrated ammonia R, 30 volumes of acetone R and 30 volumes of Disopyramidi phosphas cyclohexane R. Dry the plate in a current of warm air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.25 per cent). Heavy metals (2.4.8). 2.0 g complies with limit test C for heavy metals (10 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying at 80 °C over diphosphorus pentoxide R at a pressure not exceeding 0.7 kPa for 2 h. C21H32N3O5P Mr 437.5 Sulphated ash (2.4.14). Not more than 0.2 per cent, determined on 1.0 g. DEFINITION Disopyramide phosphate contains not less than 98.0 per ASSAY cent and not more than the equivalent of 102.0 per cent Dissolve 0.130 g in 30 ml of anhydrous acetic acid R. Add of (2RS)-4-[bis(1-methylethyl)amino]-2-phenyl-2-(pyridin0.2 ml of naphtholbenzein solution R. Titrate with 0.1 M 2-yl)butanamide dihydrogen phosphate, calculated with perchloric acid until the colour changes from yellow to reference to the dried substance. green. CHARACTERS 1 ml of 0.1 M perchloric acid is equivalent to 16.97 mg of A white or almost white powder, soluble in water, sparingly C21H29N3O. soluble in alcohol, practically insoluble in methylene chloride. STORAGE Store protected from light.

IDENTIFICATION First identification : B. IMPURITIES Second identification : A, C, D. A. Dissolve 50.0 mg in a 5 g/l solution of sulphuric acid R in methanol R and dilute to 100.0 ml with the same solution. Dilute 5.0 ml of this solution to 50.0 ml with a 5 g/l solution of sulphuric acid R in methanol R. Examined between 240 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 269 nm and a shoulder at 263 nm. The specific absorbance at the maximum is 147 to 163. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with disopyramide phosphate CRS. Examine the substances prepared as discs. A. R = CN, R′ = CH(CH3)2 : (2RS)-4-[bis(1-methylethyl)amino]C. Examine the chromatograms obtained in the test for 2-phenyl-2-(pyridin-2-yl)butanenitrile (di-isopyronitrile), related substances in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with test solution (b) is similar in position and size to the principal B. R = H, R′ = CH(CH3)2 : (3RS)-N,N-bis(1-methylethyl)-3spot in the chromatogram obtained with reference phenyl-3-(pyridin-2-yl)propan-1-amine, solution (a). Spray with dilute potassium iodobismuthate solution R. Examine in daylight. The principal spot in the chromatogram obtained with test solution (b) is similar C. R = CO-NH2, R′ = H : (2RS)-4-[(1-methylethyl)amino]-2in position, colour and size to the principal spot in the phenyl-2-(pyridin-2-yl)butanamide, chromatogram obtained with reference solution (a). D. Solution S (see Tests) gives reaction (a) of phosphates (2.3.1).

D. (RS)-phenyl(pyridin-2-yl)acetonitrile (pyronitrile). 1466

TESTS Solution S. Dissolve 1.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). pH (2.2.3). The pH of solution S is 4.0 to 5.0. See the information section on general monographs (cover pages)

Disulfiram


Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution (a). Dissolve 0.25 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with methanol R. Reference solution (a). Dissolve 25 mg of disopyramide phosphate CRS in methanol R and dilute to 10 ml with the same solvent. Reference solution (b). Dilute 1 ml of test solution (b) to 20 ml with methanol R. Apply to the plate 10 µl of each solution. Develop over a path of 15 cm using a mixture of 1 volume of concentrated ammonia R, 30 volumes of acetone R and 30 volumes of cyclohexane R. Dry the plate in a current of warm air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). Heavy metals (2.4.8). 2.0 g complies with limit test C for heavy metals (10 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. ASSAY Dissolve 0.180 g in 30 ml of anhydrous acetic acid R. Add 0.2 ml of naphtholbenzein solution R. Titrate with 0.1 M perchloric acid until the colour changes from yellow to green. 1 ml of 0.1 M perchloric acid is equivalent to 21.88 mg of C21H32N3O5P. STORAGE Store protected from light. IMPURITIES

A. B. C.

D.

01/2005:0603

DISULFIRAM Disulfiramum

C10H20N2S4

Mr 296.5

DEFINITION Disulfiram contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of tetraethyldisulfanedicarbothioamide, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, practically insoluble in water, freely soluble in methylene chloride, sparingly soluble in alcohol. IDENTIFICATION First identification : A, B. Second identification : A, C, D. A. Melting point (2.2.14) : 70 °C to 73 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with disulfiram CRS. Examine the substances prepared as discs. C. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). D. Dissolve about 10 mg in 10 ml of methanol R. Add 2 ml of a 0.5 g/l solution of cupric chloride R in methanol R. A yellow colour develops which becomes greenish-yellow.

TESTS Related substances. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm. Test solution (a). Dissolve 0.20 g of the substance to be examined in ethyl acetate R and dilute to 10 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml R = CN, R’ = CH(CH3)2 : (2RS)-4-[bis(1-methylethyl)amino]- with ethyl acetate R. 2-phenyl-2-(pyridin-2-yl)butanenitrile (di-isopyronitrile), Reference solution (a). Dissolve 10 mg of disulfiram CRS in ethyl acetate R and dilute to 5 ml with the same solvent. R = H, R’ = CH(CH3)2 : (3RS)-N,N-bis(1-methylethyl)-3Reference solution (b). Dilute 1 ml of test solution (b) to phenyl-3-(pyridin-2-yl)propan-1-amine, 20 ml with ethyl acetate R. R = CO-NH2, R′ = H : (2RS)-4-[(1-methylethyl)amino]-2Apply to the plate 10 µl of each solution. Develop over a path phenyl-2-(pyridin-2-yl)butanamide, of 15 cm using a mixture of 30 volumes of butyl acetate R and 70 volumes of hexane R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). Diethyldithiocarbamate. Dissolve 0.20 g in 10 ml of peroxide-free ether R, add 5 ml of buffer solution pH 8.0 R (RS)-phenyl(pyridin-2-yl)acetonitrile (pyronitrile). and shake vigorously. Discard the upper layer and wash the


1467

Dithranol


lower layer with 10 ml of peroxide-free ether R. Add to the lower layer 0.2 ml of a 4 g/l solution of copper sulphate R and 5 ml of cyclohexane R. Shake. Any yellow colour in the upper layer is not more intense than that of a standard prepared at the same time using 0.2 ml of a freshly prepared 0.15 g/l solution of sodium diethyldithiocarbamate R (150 ppm). Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in vacuo at 50 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.450 g in 80 ml of acetone R and add 20 ml of a 20 g/l solution of potassium nitrate R. Titrate with 0.1 M silver nitrate. Determine the end-point potentiometrically (2.2.20), using a silver electrode and a silver-silver chloride double-junction electrode saturated with potassium nitrate. 1 ml of 0.1 M silver nitrate is equivalent to 59.30 mg of C10H20N2S4. STORAGE Store protected from light. IMPURITIES

A. diethylthiocarbamic thioanhydride (sulfiram),

Carry out all tests protected from bright light and use freshly prepared solutions. IDENTIFICATION First identification : A, B. Second identification : A, C, D. A. It melts (2.2.14) at 178 °C to 182 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with dithranol CRS. C. Examine by thin-layer chromatography (2.2.27), using silica gel H R as the coating substance. Test solution. Dissolve 10 mg of the substance to be examined in methylene chloride R and dilute to 10 ml with the same solvent. Reference solution (a). Dissolve 10 mg of dithranol CRS in methylene chloride R and dilute to 10 ml with the same solvent. Reference solution (b). Dissolve about 5 mg of dantron R in 5 ml of reference solution (a). Apply to the plate 10 µl of each solution. Develop over a path of 12 cm using a mixture of equal volumes of hexane R and methylene chloride R. Allow the plate to dry in air. Place the plate in a tank saturated with ammonia vapour until the spots appear. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows 2 clearly separated spots. D. To 5 mg add 0.1 g of anhydrous sodium acetate R and 1 ml of acetic anhydride R. Boil for 30 s. Add 20 ml of alcohol R. Examined in ultraviolet light at 365 nm, the solution shows a blue fluorescence.

TESTS Related substances A. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 0.200 g of the substance to be examined in 20 ml of methylene chloride R, add 1.0 ml B. diethyldithiocarbamate. of glacial acetic acid R and dilute to 100.0 ml with hexane R. 01/2005:1007 Reference solution. Dissolve 5.0 mg each of anthrone R, dantron R, dithranol impurity C CRS and dithranol CRS DITHRANOL in methylene chloride R and dilute to 5.0 ml with the same solvent. To 1.0 ml of the solution, add 19.0 ml of Dithranolum methylene chloride R and 1.0 ml of glacial acetic acid R, and dilute to 50.0 ml with hexane R. The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with silica gel for chromatography R (5 µm), C14H10O3 Mr 226.2 — as mobile phase at a flow rate of 2 ml/min a mixture of 1 volume of glacial acetic acid R, 5 volumes of DEFINITION methylene chloride R and 82 volumes of hexane R, Dithranol contains not less than 98.5 per cent and — as detector a spectrophotometer set at 260 nm. not more than the equivalent of 101.0 per cent of Inject 20 µl of each solution. Continue the 1,8-dihydroxyanthracen-9(10H)-one, calculated with chromatography for 1.5 times the retention time reference to the dried substance. of dithranol impurity C. Adjust the sensitivity of CHARACTERS the system so that the height of the principal peak in the chromatogram obtained with the reference A yellow or brownish-yellow, crystalline powder, practically solution is about 70 per cent of the full scale of the insoluble in water, soluble in methylene chloride, sparingly recorder. When the chromatograms are recorded in soluble in acetone, slightly soluble in alcohol. It dissolves in the prescribed conditions, the substances are eluted in dilute solutions of alkali hydroxides. 1468


Dobutamine hydrochloride


the following sequence : dithranol, dantron, anthrone, dithranol impurity C. The test is not valid unless, in the chromatogram obtained with the reference solution, the resolution between the peaks due to dithranol and dantron is greater than 2.0. In the chromatogram obtained with the test solution : the area of any peak corresponding to anthrone, dantron or dithranol impurity C is not greater than that of the corresponding peak in the chromatogram obtained with the reference solution (1 per cent) ; the area of any peak apart from the principal peak and any peaks due to anthrone, dantron and dithranol impurity C is not greater than that of the peak due to dithranol in the chromatogram obtained with the reference solution (1 per cent). B. Examine by liquid chromatography (2.2.29). Test solution. Dissolve 25.0 mg of the substance to be examined in 5 ml of tetrahydrofuran R and dilute to 25.0 ml with the mobile phase. Reference solution. Dissolve 5.0 mg of dithranol impurity D CRS and 5.0 mg of dithranol CRS in 5 ml of tetrahydrofuran R and dilute to 10.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 20.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.20 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 0.9 ml/min a mixture of 2.5 volumes of glacial acetic acid R, 40 volumes of tetrahydrofuran R and 60 volumes of water R, — as detector a spectrophotometer set at 254 nm. Inject 20 µl of each solution. Continue the chromatography for 3 times the retention time of the peak due to dithranol. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with the reference solution is about 50 per cent of the full scale of the recorder. The test is not valid unless, in the chromatogram obtained with the reference solution, the resolution between the peaks due to dithranol impurity D and dithranol is greater than 2.5. In the chromatogram obtained with the test solution, the area of any peak corresponding to dithranol impurity Dis not greater than that of the corresponding peak in the chromatogram obtained with the reference solution (2.5 per cent). The total content of related substances as determined in tests A and B is not more than 3.0 per cent. Chlorides (2.4.4). Shake 1.0 g with 20 ml of water R for 1 min and filter. Dilute 10 ml of the filtrate to 15 ml with water R. The solution complies with the limit test for chlorides (100 ppm). Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.


A. R1 = R2 = H, X = H2 : anthracen-9(10H)-one (anthrone), B. R1 = R2 = OH, X = O : 1,8-dihydroxyanthracene-9,10-dione (danthron), D. R1 = OH, R2 = H, X = H2 : 1-hydroxyanthracen-9(10H)-one,

C. C. 4,4′,5,5′-tetrahydroxy-9,9′-bianthracenyl-10,10′(9H, 9′H)-dione. 01/2005:1200

DOBUTAMINE HYDROCHLORIDE Dobutamini hydrochloridum

C18H24ClNO3

Mr 337.9

DEFINITION Dobutamine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of (RS)-4-[2-[[3-(4-hydroxyphenyl)-1methylpropyl]amino]ethyl]benzene-1,2-diol hydrochloride, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, sparingly soluble in water, soluble in methanol, sparingly soluble in alcohol.

IDENTIFICATION First identification : C, E. ASSAY Second identification : A, B, D, E. Dissolve 0.200 g in 50 ml of anhydrous pyridine R. A. Melting point (2.2.14) : 189 °C to 192 °C. Titrate with 0.1 M tetrabutylammonium hydroxide under B. Dissolve 20.0 mg in methanol R and dilute to 100.0 ml nitrogen R. Determine the end-point potentiometrically with the same solvent. Dilute 10.0 ml of the solution to (2.2.20), using a glass indicator electrode and a calomel 100.0 ml with methanol R. Examined between 220 nm reference electrode containing, as the electrolyte, a saturated and 300 nm (2.2.25), the solution shows two absorption solution of potassium chloride R in methanol R. maxima, at 223 nm and 281 nm. The ratio of the 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent absorbance measured at the maximum at 281 nm to that to 22.62 mg of C14H10O3. measured at the maximum at 223 nm is 0.34 to 0.36.


1469

Dobutamine hydrochloride


C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with dobutamine hydrochloride CRS. Examine the substances prepared as discs. D. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. Dissolve 10 mg in a mixture of equal volumes of glacial acetic acid R and methanol R and dilute to 10 ml with the same mixture of solvents. Reference solution (a). Dissolve 10.0 mg of dobutamine hydrochloride CRS in a mixture of equal volumes of glacial acetic acid R and methanol R and dilute to 10 ml with the same mixture of solvents. Reference solution (b). Dissolve 5.0 mg of dopamine hydrochloride CRS in 5 ml of the test solution. Apply to the plate 10 µl of each solution. Develop over a path of 15 cm using a mixture of 5 volumes of water R, 15 volumes of glacial acetic acid R, 30 volumes of ether R and 45 volumes of butanol R. Dry the plate and spray with a 1 g/l solution of potassium permanganate R. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. E. It gives reaction (a) of chlorides (2.3.1) using a mixture of equal volumes of methanol R and water R.

— as mobile phase at a flow rate of 1 ml/min : Mobile phase A. Dissolve 2.60 g of sodium octanesulphonate R in 1000 ml of water R and add 3 ml of triethylamine R ; adjust to pH 2.5 with phosphoric acid R, Mobile phase B. A mixture of 18 volumes of acetonitrile R and 82 volumes of methanol R, Time (min)



0-5

65

35

5 - 20

65 → 20

35 → 80

20 - 25

20

80

— as detector a spectrophotometer set at 280 nm. Inject 20 µl of reference solution (a). The test is not valid unless the resolution between the peaks corresponding to dobutamine hydrochloride and anisaldehyde is at least 4.0. Inject 20 µl of the test solution and 20 µl of reference solution (b). In the chromatogram obtained with the test solution : any peak apart from the principal peak is not greater than the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent) ; and the sum of the areas of all the peaks apart from the principal peak is not greater than twice the area of the principal peak in the chromatogram obtained with the reference solution (b) (1 per cent). Disregard any peak due to the solvent and any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b). TESTS Heavy metals (2.4.8). 2.0 g complies with limit test C for Acidity or alkalinity. Dissolve 0.1 g in water R with gentle heavy metals (10 ppm). Prepare the standard using 2 ml of heating and dilute to 10 ml with the same solvent. Add 0.1 ml of methyl red solution R and 0.2 ml of 0.01 M sodium lead standard solution (10 ppm Pb) R. hydroxide. The solution is yellow. Add 0.4 ml of 0.01 M Loss on drying (2.2.32). Not more than 0.5 per cent, hydrochloric acid. The solution is red. determined on 1.000 g by drying in an oven at 100 °C to Optical rotation (2.2.7). Dissolve 0.50 g in methanol R and 105 °C. dilute to 10.0 ml with the same solvent. The angle of optical Sulphated ash (2.4.14). Not more than 0.1 per cent, rotation is − 0.05° to + 0.05°. determined on 1.0 g. Absorbance (2.2.25). Dissolve 0.5 g in a mixture of ASSAY equal volumes of methanol R and of water R with heating, if necessary, at 30 °C to 35 °C and dilute to 25 ml with In order to avoid overheating in the reaction medium, mix the same mixture of solvents. Cool quickly. Measure the thoroughly throughout and stop the titration immediately absorbance immediately at 480 nm. The absorbance is not after the end-point has been reached. greater than 0.04. Dissolve 0.250 g in 10 ml of anhydrous formic acid R. Add Related substances. Examine by liquid chromatography 50 ml of acetic anhydride R. Titrate with 0.1 M perchloric (2.2.29). acid, determining the end-point potentiometrically (2.2.20). Test solution. Dissolve 0.10 g in a mixture of 35 volumes of 1 ml of 0.1 M perchloric acid is equivalent to 33.79 mg of mobile phase B and 65 volumes of mobile phase A and dilute C H ClNO . 18 24 3 to 20.0 ml with the same mixture of mobile phases. Reference solution (a). Dilute 4.0 ml of the test solution to STORAGE 100.0 ml with a 0.05 g/l solution of anisaldehyde R in a Store protected from light. mixture of 35 volumes of mobile phase B and 65 volumes of mobile phase A. Dilute 1.0 ml of the solution to 10.0 ml with a mixture of 35 volumes of mobile phase B and 65 volumes IMPURITIES of mobile phase A. Reference solution (b). Dilute 5.0 ml of the test solution to A. dopamine, 100.0 ml with a mixture of 35 volumes of mobile phase B and 65 volumes of mobile phase A. Dilute 1.0 ml of the solution to 10.0 ml with a mixture of 35 volumes of mobile phase B and 65 volumes of mobile phase A. The chromatographic procedure may be carried out using : — a stainless steel column 0.15 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), B. 4-(4-hydroxyphenyl)butan-2-one, 1470


Docusate sodium


C. (2RS)-N-[2-(3,4-dimethoxyphenyl)ethyl]-4-(4methoxyphenyl)butan-2-amine. 01/2005:1418

DOCUSATE SODIUM Natrii docusas

C20H37NaO7S

Mr 444.6

DEFINITION Sodium 1,4-bis[(2-ethylhexyl)oxy]-1,4-dioxobutane-2sulphonate. Content : 98.0 to 101.0 per cent (anhydrous substance). CHARACTERS Appearance : white or almost white, waxy masses or flakes, hygroscopic. Solubility : sparingly soluble in water, freely soluble in alcohol and in methylene chloride. IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Preparation : place about 3 mg of the substance to be examined on a sodium chloride plate, add 0.05 ml of acetone R and immediately cover with another sodium chloride plate. Rub the plates together to dissolve the substance to be examined, slide the plates apart and allow the acetone to evaporate. Comparison : Ph. Eur. reference spectrum of docusate sodium. B. In a crucible, ignite 0.75 g in the presence of dilute sulphuric acid R, until an almost white residue is obtained. Allow to cool and take up the residue with 5 ml of water R. Filter. 2 ml of the filtrate gives reaction (a) of sodium (2.3.1). TESTS Alkalinity. Dissolve 1.0 g in 100 ml of a mixture of equal volumes of methanol R and water R, previously neutralised to methyl red solution R. Add 0.1 ml of methyl red solution R. Not more than 0.2 ml of 0.1 M hydrochloric acid is required to change the colour of the indicator to red. Related non-ionic substances. Gas chromatography (2.2.28). Internal standard solution. Dissolve 10 mg of methyl behenate R in hexane R and dilute to 50 ml with the same solvent. Test solution (a). Dissolve 0.10 g of the substance to be examined in 2.0 ml of the internal standard solution and dilute to 5.0 ml with hexane R. Pass the solution, at a rate of about 1.5 ml/min, through a column 10 mm in internal diameter, packed with 5 g of basic aluminium oxide R and previously washed with 25 ml of hexane R. Elute with 5 ml of hexane R and discard the eluate. Elute with 20 ml General Notices (1) apply to all monographs and other texts

of a mixture of equal volumes of ether R and hexane R. Evaporate the eluate to dryness and dissolve the residue in 2.0 ml of hexane R. Test solution (b). Prepare as described for test solution (a) but dissolving 0.10 g of the substance to be examined in hexane R, diluting to 5.0 ml with the same solvent, and using a new column. Reference solution. Dilute 2.0 ml of the internal standard solution to 5.0 ml with hexane R. Column : — material : glass, — size : l = 2 m, Ø = 2 mm, — stationary phase : silanised diatomaceous earth for gas chromatography R impregnated with 3 per cent m/m of polymethylphenylsiloxane R. Carrier gas : nitrogen for chromatography R. Flow rate : 30 ml/min. Temperature : — column : 230 °C, — injection port and detector : 280 °C. Detection : flame ionisation. Injection : 1 µl. Run time : 2.5 times the retention time of the internal standard. System suitability : there is no peak with the same retention time as the internal standard in the chromatogram obtained with test solution (b). Limits : test solution (a) : — any impurity : for each impurity, not more than the area of the peak due to the internal standard (0.4 per cent). Chlorides : maximum 350 ppm. Dissolve 5.0 g in 50 ml of alcohol (50 per cent V/V) R and add 0.1 ml of potassium dichromate solution R. Not more than 0.5 ml of 0.1 M silver nitrate is required to change the colour of the indicator from yellow to orange. Sodium sulphate : maximum 2 per cent. Dissolve 0.25 g in 40 ml of a mixture of 20 volumes of water R and 80 volumes of 2-propanol R. Adjust to pH between 2.5 and 4.0 using perchloric acid solution R. Add 0.4 ml of naphtharson solution R and 0.1 ml of a 0.125 g/l solution of methylene blue R. Not more than 1.5 ml of 0.025 M barium perchlorate is required to change the colour of the indicator from yellowish-green to yellowish-pink. Heavy metals (2.4.8) : maximum 10 ppm. Dissolve 4.0 g in alcohol (80 per cent V/V) R and dilute to 20 ml with the same solvent. 12 ml of the solution complies with limit test B. Prepare the standard using lead standard solution (2 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with alcohol (80 per cent V/V) R. Water (2.5.12) : maximum 3.0 per cent, determined on 0.250 g. ASSAY To 1.000 g in a 250 ml conical flask fitted with a reflux condenser add 25.0 ml of 0.5 M alcoholic potassium hydroxide and heat on a water-bath under reflux for 45 min. Allow to cool. Add 0.25 ml of phenolphthalein solution R1 and titrate with 0.5 M hydrochloric acid until the red colour disappears. Carry out a blank titration. 1 ml of 0.5 M hydrochloric acid is equivalent to 0.1112 g of C20H37NaO7S. STORAGE In an airtight container. 1471

Dodecyl gallate


01/2005:2078 System suitability : reference solution (d) : — the chromatogram shows 2 clearly separated principal spots. DODECYL GALLATE Limit : test solution (a) : Dodecylis gallas — impurity A : any spot due to impurity A is not more intense than the spot in the chromatogram obtained with reference solution (c) (0.5 per cent). Chlorides (2.4.4) : maximum 100 ppm. To 1.65 g add 50 ml of water R. Shake for 5 min. Filter. 15 ml of the filtrate complies with the test. Heavy metals (2.4.8) : maximum 10 ppm. 2.0 g complies with limit test C. Prepare the reference C19H30O5 Mr 338.4 solution using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32) : maximum 0.5 per cent, determined DEFINITION on 1.000 g by drying in an oven at 70 °C. Dodecyl 3,4,5-trihydroxybenzoate. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined Content : 97.0 per cent to 103.0 per cent (dried substance). on 1.0 g. CHARACTERS ASSAY Appearance : white or almost white, crystalline powder. Dissolve 0.100 g in methanol R and dilute to 250.0 ml with Solubility : very slightly soluble or practically insoluble in the same solvent. Dilute 5.0 ml of the solution to 200.0 ml water, freely soluble in ethanol (96 per cent), slightly soluble with methanol R. Measure the absorbance (2.2.25) at the in methylene chloride. absorption maximum at 275 nm. IDENTIFICATION Calculate the content of C19H30O5 taking the specific absorbance to be 321. A. Melting point (2.2.14). Determine the melting point of the substance to be STORAGE examined. Mix equal parts of the substance to be In a non-metallic container, protected from light. examined and dodecyl gallate CRS and determine the melting point of the mixture. The difference between the IMPURITIES melting points (which are about 96 °C) is not greater Specified impurities : A. than 2 °C. B. Examine the chromatograms obtained in the test for impurity A. Results : the principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). A. 3,4,5-trihydroxybenzoic acid (gallic acid). TESTS Impurity A. Thin-layer chromatography (2.2.27). Test solution (a). Dissolve 0.20 g of the substance to be examined in acetone R and dilute to 10 ml with the same solvent. Test solution (b). Dilute 1.0 ml of test solution (a) to 20 ml with acetone R. Reference solution (a). Dissolve 10 mg of dodecyl gallate CRS in acetone R and dilute to 10 ml with the same solvent. Reference solution (b). Dissolve 20 mg of gallic acid R in acetone R and dilute to 20 ml with the same solvent. Reference solution (c). Dilute 1.0 ml of reference solution (b) to 10 ml with acetone R. Reference solution (d). Dilute 1.0 ml of reference solution (b) to 5 ml with test solution (a). Plate : TLC silica gel plate R. Mobile phase : anhydrous formic acid R, ethyl formate R, toluene R (10:40:50 V/V/V). Application : 5 µl of test solutions (a) and (b) and reference solutions (a), (c) and (d). Development : over 2/3 of the plate. Drying : in air for 10 min. Detection : spray with a mixture of 1 volume of ferric chloride solution R1 and 9 volumes of ethanol (96 per cent) R. 1472

01/2005:1510

DOG ROSE Rosae pseudo-fructus DEFINITION Dog rose consists of the rose hips made up by the receptacle and the remains of the dried sepals of Rosa canina L., R. pendulina L. and other Rosa species, with the achenes removed. It contains not less than 0.3 per cent of ascorbic acid (C6H8O6 ; Mr 176.1), calculated with reference to the dried drug. CHARACTERS It has the macroscopic and microscopic characters described under identification tests A and B. IDENTIFICATION A. It consists of fragments of the fleshy, hollow, urceolate receptacle, bearing the remains of the reduced sepals, light pink to orange-pink, the convex outer surface shiny and strongly wrinkled ; bearing on its lighter inner surface abundant bristle-like hairs. B. Reduce to a powder (355). The powder is orange-yellow. Examine under a microscope using chloral hydrate solution R. The powder shows numerous fragments of receptacle, the outer epidermis with orange-yellow


Domperidone


contents and a thick cuticle, the inner epidermis composed of thin-walled cells containing cluster crystals and occasional prisms of calcium oxalate ; scattered lignified cells, isodiametric, with thickened and pitted walls forming the trichome bases ; abundant long, unicellar trichomes, up to 2 mm long and 30 µm to 45 µm thick, tapering towards each end, walls heavily thickened and with a waxy cuticle which may show fissures in a spiral arrangement ; numerous oily orange-yellow globules. C. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R. Test solution. To 5 g of the powdered drug (355) add 25 ml of alcohol R. Shake for 30 min and filter. Reference solution. Dissolve 10 mg of ascorbic acid R in 5.0 ml of alcohol (60 per cent V/V) R. Apply to the plate 20 µl of the test solution and 2 µl of the reference solution. Develop over a path of 15 cm using a mixture of 5 volumes of acetic acid R, 5 volumes of acetone R, 20 volumes of methanol R and 70 volumes of toluene R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The chromatogram obtained with the test solution shows a quenching zone similar in position to the principal zone in the chromatogram obtained with the reference solution. Spray with a 0.2 g/l solution of dichlorophenolindophenol, sodium salt R in alcohol R. Examine in daylight. The chromatogram obtained with the test solution shows a white zone on a pink background similar in position and colour to the principal zone in the chromatogram obtained with the reference solution. The chromatogram also shows an intense orange-yellow zone near the solvent front and a yellow zone in the upper third (carotenoids). TESTS Foreign matter (2.8.2). Not more than 1 per cent. Loss on drying (2.2.32). Not more than 10.0 per cent, determined on 1.000 g of the powdered drug (355) by drying in an oven at 100-105 °C. Total ash (2.4.16). Not more than 7.0 per cent. ASSAY Test solution. In a round-bottomed flask, weigh 0.500 g of the freshly powdered drug (710). Add a solution of 1.0 g of oxalic acid R in 50.0 ml of methanol R. Boil under a reflux condenser for 10 min, and cool in iced water until the temperature reaches 15 °C to 20 °C. Filter. Transfer 2.0 ml of the filtrate to a 50 ml conical flask. Add successively, with gentle shaking after each addition, 2.0 ml of dichlorophenolindophenol standard solution R and then, exactly 60 s later, 0.5 ml of a 100 g/l solution of thiourea R in alcohol (50 per cent V/V) R and 0.7 ml of dinitrophenylhydrazine-sulphuric acid solution R. Heat under a reflux condenser at 50 °C for 75 min, and place immediately in iced water for 5 min. Add dropwise 5.0 ml of a mixture of 12 ml of water R and 50 ml of sulphuric acid R, taking care to carry out the addition over a period of not less than 90 s and not more than 120 s while maintaining vigorous stirring in iced water. Allow to stand for 30 min at room temperature and measure the absorbance (2.2.25) at 520 nm using solution A as compensation liquid. Solution A. Treat 2.0 ml of the filtrate obtained during the preparation of the test solution as described but adding the dinitrophenylhydrazine-sulphuric acid solution R just before the absorbance is measured. Reference solution. Dissolve 40.0 mg of ascorbic acid R in a freshly prepared 20 g/l solution of oxalic acid R in methanol R and dilute to 100.0 ml with the same General Notices (1) apply to all monographs and other texts

solvent. Dilute 5.0 ml of the solution to 100.0 ml with the methanolic solution of oxalic acid. Treat 2.0 ml of the solution as described above for the filtrate obtained during the preparation of the test solution. Measure the absorbance (2.2.25) at 520 nm using solution B as compensation liquid. Solution B. Treat 2.0 ml of the reference solution as described above for solution A. Calculate the percentage content of ascorbic acid from the expression :

A1

=

absorbance of the test solution,

A2 m1

=

absorbance of the reference solution,

=


m2

=

mass of ascorbic acid used, in grams. 01/2005:1009

DOMPERIDONE Domperidonum

C22H24ClN5O2

Mr 425.9

DEFINITION Domperidone contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of 5-chloro-1-[1-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-1yl)propyl]piperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one, calculated with reference to the dried substance. CHARACTERS A white or almost white powder, practically insoluble in water, soluble in dimethylformamide, slightly soluble in alcohol and in methanol. IDENTIFICATION First identification : A, B. Second identification : A, C, D. A. Melting point (2.2.14) : 244 °C to 248 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with domperidone CRS. Examine the substances prepared as discs. C. Examine by thin-layer chromatography (2.2.27), using a suitable octadecylsilyl silica gel as the coating substance. Test solution. Dissolve 20 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution (a). Dissolve 20 mg of domperidone CRS in methanol R and dilute to 10 ml with the same solvent. Reference solution (b). Dissolve 20 mg of domperidone CRS and 20 mg of droperidol CRS in methanol R and dilute to 10 ml with the same solvent. 1473

Domperidone


Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 20 volumes of ammonium acetate solution R, 40 volumes of dioxan R and 40 volumes of methanol R. Dry the plate in a current of warm air for 15 min and expose it to iodine vapour until the spots appear. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. D. It gives the reaction of non-nitrogen substituted barbiturates (2.3.1).

cent). Disregard any peak in the chromatogram obtained with the blank run and any peak with an area less than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (b). Heavy metals (2.4.8). 1.0 g complies with limit test D for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.

TESTS Appearance of solution. Dissolve 0.20 g in dimethylformamide R and dilute to 20.0 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). Related substances. Examine by liquid chromatography (2.2.29). Prepare the solutions immediately before use. Test solution. Dissolve 0.10 g of the substance to be examined in dimethylformamide R and dilute to 10.0 ml with the same solvent. Reference solution (a). Dissolve 10.0 mg of domperidone CRS and 15.0 mg of droperidol CRS in dimethylformamide R and dilute to 100.0 ml with the same solvent. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with dimethylformamide R. Dilute 5.0 ml of this solution to 20.0 ml with dimethylformamide R. The chromatographic procedure may be carried out using : — a stainless steel column 0.1 m long and 4.6 mm in internal diameter packed with base-deactivated octadecylsilyl silica gel for chromatography R (3 µm), — as mobile phase at a flow rate of 1.5 ml/min a mixture of 3 volumes methanol R and 7 volumes of a 5 g/l solution of ammonium acetate R, changing by linear gradient to methanol R over 10 min, followed by elution with methanol R for 2 min, — as detector a spectrophotometer set at 280 nm. Equilibrate the column for at least 30 min with methanol R and then equilibrate at the initial mobile phase composition for at least 5 min. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 10 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder. Inject 10 µl of reference solution (a). When the chromatogram is recorded in the prescribed conditions, the retention times are : domperidone about 6.5 min ; droperidol about 7 min. The test is not valid unless the resolution between the peaks due to domperidone and droperidol is at least 2.0. If necessary, adjust the concentration of methanol in the mobile phase or adjust the time programme for the linear gradient. Inject separately 10 µl of dimethylformamide R as a blank, 10 µl of the test solution and 10 µl of reference solution (b). In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.25 per cent) ; the sum of the areas of all peaks, apart from the principal peak, is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per

ASSAY Dissolve 0.300 g in 50 ml of a mixture of 1 volume of anhydrous acetic acid R and 7 volumes of methyl ethyl ketone R. Titrate with 0.1 M perchloric acid until the colour changes from orange-yellow to green using 0.2 ml of naphtholbenzein solution R as indicator. 1 ml of 0.1 M perchloric acid is equivalent to 42.59 mg of C22H24ClN5O2.

1474


A. 5-chloro-1-(piperidin-4-yl)-1,3-dihydro-2H-benzimidazol2-one,

B. 4-(5-chloro-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)-1formylpiperidine,

C. cis-4-(5-chloro-2-oxo-2,3-dihydro-1H-benzimidazol1-yl)-1-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-1yl)propyl]piperidine 1-oxide,

D. 5-chloro-3-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-1yl)propyl]-1-[1-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-1yl)propyl]piperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2one,



Domperidone maleate

B. Examine by thin-layer chromatography (2.2.27), using a suitable octadecylsilyl silica gel as the coating substance. Test solution. Dissolve 20 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution (a). Dissolve 20 mg of domperidone maleate CRS in methanol R and dilute to 10 ml with the E. 1-[3-[4-(5-chloro-2-oxo-2,3-dihydro-1H-benzimidazolsame solvent. 1-yl)piperidin-1-yl]propyl]-3-[3-(2-oxo-2,3-dihydro-1HReference solution (b). Dissolve 20 mg of domperidone benzimidazol-1-yl)propyl]-1,3-dihydro-2H-benzimidazolmaleate CRS and 20 mg of droperidol CRS in methanol R 2-one, and dilute to 10 ml with the same solvent. Apply to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 20 volumes of ammonium acetate solution R, 40 volumes of dioxan R and 40 volumes of methanol R. Dry the plate in a current of warm air for 15 min and expose it to iodine vapour until the spots appear. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the F. 1,3-bis[3-[4-(5-chloro-2-oxo-2,3-dihydro-1H-benzimidazolchromatogram obtained with reference solution (a). The 1-yl)piperidin-1-yl]propyl]-1,3-dihydro-2H-benzimidazoltest is not valid unless the chromatogram obtained with 2-one. reference solution (b) shows two clearly separated spots. C. Triturate 0.1 g with a mixture of 1 ml of strong sodium hydroxide solution R and 3 ml of water R. Shake with 01/2005:1008 three quantities, each of 5 ml, of ether R. To 0.1 ml of the corrected aqueous layer add a solution of 10 mg of resorcinol R in 3 ml of sulphuric acid R. Heat on a water-bath for 15 min. No colour develops. To the remainder of the DOMPERIDONE MALEATE aqueous layer add 2 ml of bromine solution R. Heat on a water-bath for 15 min and then heat to boiling. Cool. Domperidoni maleas To 0.1 ml of the solution add a solution of 10 mg of resorcinol R in in 3 ml of sulphuric acid R. Heat on a water-bath for 15 min. A violet colour develops. TESTS Appearance of solution. Dissolve 0.20 g in dimethylformamide R and dilute to 20.0 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). C26H28ClN5O6 Mr 542.0 Related substances. Examine by liquid chromatography (2.2.29). Prepare the solutions immediately before use. DEFINITION Test solution. Dissolve 0.10 g of the substance to be Domperidone maleate contains not less than 99.0 per examined in dimethylformamide R and dilute to 10.0 ml cent and not more than the equivalent of 101.0 per cent with the same solvent. of 5-chloro-1-[1-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-1yl)propyl]piperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one Reference solution (a). Dissolve 10.0 mg of domperidone hydrogen (Z)-butenedioate, calculated with reference to the maleate CRS and 15.0 mg of droperidol CRS in dried substance. dimethylformamide R and dilute to 100.0 ml with the same solvent. CHARACTERS Reference solution (b). Dilute 1.0 ml of the test solution to A white or almost white powder, very slightly soluble in 100.0 ml with dimethylformamide R. Dilute 5.0 ml of this water, sparingly soluble in dimethylformamide, slightly solution to 20.0 ml with dimethylformamide R. soluble in methanol, very slightly soluble in alcohol. The chromatographic procedure may be carried out using : It shows polymorphism. — a stainless steel column 0.1 m long and 4.6 mm in internal diameter packed with base-deactivated octadecylsilyl IDENTIFICATION silica gel for chromatography R (3 µm), First identification : A. — as mobile phase at a flow rate of 1.5 ml per minute of a Second identification : B, C. mixture of 3 volumes of methanol R and 7 volumes of A. Examine by infrared absorption spectrophotometry a 5 g/l solution of ammonium acetate R, changing by (2.2.24), comparing with the spectrum obtained with linear gradient to methanol R over 10 min, followed by domperidone maleate CRS. Examine the substances elution with methanol R for 2 min, prepared as discs. If the spectra obtained show differences, dissolve the substance to be examined and the — as detector a spectrophotometer set at 280 nm. reference substance separately in the minimum volume of Equilibrate the column for at least 30 min with methanol R 2-propanol R, evaporate to dryness on a water-bath and and then equilibrate at the initial mobile phase composition record new spectra using the residues. for at least 5 min. General Notices (1) apply to all monographs and other texts

1475

Dopamine hydrochloride


Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 10 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder. Inject 10 µl of reference solution (a). When the chromatogram is recorded in the prescribed conditions, the C. cis-4-(5-chloro-2-oxo-2,3-dihydro-1H-benzimidazolretention times are : domperidone maleate, about 6.5 min 1-yl)-1-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-1and droperidol, about 7 min. The test is not valid unless yl)propyl]piperidine 1-oxide, the resolution between the peaks due to domperidone maleate and droperidol is at least 2.0. If necessary, adjust the concentration of methanol in the mobile phase or adjust the time programme for the linear gradient. Inject 10 µl of dimethylformamide R as a blank, 10 µl of the test solution and 10 µl of reference solution (b). In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram D. 5-chloro-3-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-1obtained with reference solution (b) (0.25 per cent) ; the yl)propyl]-1-[1-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-1sum of the areas of all peaks, apart from the principal peak, yl)propyl]piperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2is not greater than twice the area of the principal peak in one, the chromatogram obtained with reference solution (b) (0.5 per cent). Disregard any peak in the chromatogram obtained with the blank run, any peak due to maleic acid at the beginning of the chromatogram and any peak with an area less than 0.2 times that of the principal peak in the chromatogram obtained with reference solution (b). Heavy metals (2.4.8). 1.0 g complies with limit test D for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. E. 1-[3-[4-(5-chloro-2-oxo-2,3-dihydro-1H-benzimidazol1-yl)piperidin-1-yl]propyl]-3-[3-(2-oxo-2,3-dihydro-1HLoss on drying (2.2.32). Not more than 0.5 per cent, benzimidazol-1-yl)propyl]-1,3-dihydro-2H-benzimidazoldetermined on 1.000 g by drying in an oven at 100 °C to 2-one, 105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.400 g in 50 ml of anhydrous acetic acid R. Using 0.2 ml of naphtholbenzein solution R as indicator, titrate with 0.1 M perchloric acid until the colour changes from orange-yellow to green. 1 ml of 0.1 M perchloric acid is equivalent to 54.20 mg of C26H28ClN5O6.

F. 1,3-bis[3-[4-(5-chloro-2-oxo-2,3-dihydro-1H-benzimidazol1-yl)piperidin-1-yl]propyl]-1,3-dihydro-2H-benzimidazol2-one.


01/2005:0664

IMPURITIES

DOPAMINE HYDROCHLORIDE Dopamini hydrochloridum

C8H12ClNO2 A. 5-chloro-1-(piperidin-4-yl)-1,3-dihydro-2H-benzimidazol2-one,

B. 4-(5-chloro-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)-1formylpiperidine, 1476

Mr 189.6

DEFINITION Dopamine hydrochloride contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of 4-(2-aminoethyl)benzene-1,2-diol hydrochloride, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, freely soluble in water, soluble in alcohol, sparingly soluble in acetone and in methylene chloride.


Dosulepin hydrochloride


IDENTIFICATION First identification : B, E. Second identification : A, C, D, E. A. Dissolve 40.0 mg in 0.1 M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute 10.0 ml of the solution to 100.0 ml with 0.1 M hydrochloric acid. Examined between 230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 280 nm. The specific absorbance at the maximum is 136 to 150. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with dopamine hydrochloride CRS. Examine the substances as discs prepared using potassium chloride R. C. Dissolve about 5 mg in a mixture of 5 ml of 1 M hydrochloric acid and 5 ml of water R. Add 0.1 ml of sodium nitrite solution R containing 100 g/l of ammonium molybdate R. A yellow colour develops which becomes red on the addition of strong sodium hydroxide solution R. D. Dissolve about 2 mg in 2 ml of water R and add 0.2 ml of ferric chloride solution R2. A green colour develops which changes to bluish-violet on the addition of 0.1 g of hexamethylenetetramine R. E. It gives reaction (a) of chlorides (2.3.1). TESTS Appearance of solution. Dissolve 0.4 g in water R and dilute to 10 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution B6 or Y6 (2.2.2, Method II). Acidity or alkalinity. Dissolve 0.5 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. Add 0.1 ml of methyl red solution R and 0.75 ml of 0.01 M sodium hydroxide. The solution is yellow. Add 1.5 ml of 0.01 M hydrochloric acid. The solution is red. Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance. Test solution. Dissolve 0.15 g of the substance to be examined in methanol R and dilute to 5 ml with the same solvent. Reference solution (a). Dissolve 7.5 mg of 4-O-methyldopamine hydrochloride R in methanol R and dilute to 100 ml with the same solvent. Reference solution (b). Dissolve 7.5 mg each of 3-O-methyldopamine hydrochloride R and 4-O-methyldopamine hydrochloride R in methanol R and dilute to 100 ml with the same solvent. Apply to the plate 10 µl of each solution. Develop over a path of 15 cm using a mixture of 2 volumes of anhydrous formic acid R, 7 volumes of water R, 36 volumes of methanol R and 52 volumes of chloroform R. Allow the plate to dry in air for 15 min. Spray evenly and abundantly with a mixture of equal volumes of potassium ferricyanide solution R and ferric chloride solution R1, prepared immediately before use. Any spot in the chromatogram obtained with the test solution with an Rf value higher than that of the principal spot is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.25 per cent). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C for 2 h. General Notices (1) apply to all monographs and other texts

Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY In order to avoid overheating in the reaction medium, mix thoroughly throughout and stop the titration immediately after the end-point has been reached. Dissolve 0.1500 g in 10 ml of anhydrous formic acid R. Add 50 ml of acetic anhydride R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 18.96 mg of C8H12ClNO2. STORAGE Store in an airtight container, protected from light. IMPURITIES

A. 5-(2-aminoethyl)-2-methoxyphenol (4-O-methyldopamine). 01/2005:1314

DOSULEPIN HYDROCHLORIDE Dosulepini hydrochloridum

C19H22ClNS

Mr 331.9

DEFINITION (E)-3-(Dibenzo[b,e]thiepin-11(6H)-ylidene)-N,Ndimethylpropan-1-amine hydrochloride. Content : 98.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white or faintly yellow, crystalline powder. Solubility : freely soluble in water, in alcohol and in methylene chloride. IDENTIFICATION First identification : B, D. Second identification : A, C, D. A. Dissolve 25.0 mg in a 1 g/l solution of hydrochloric acid R in methanol R and dilute to 100.0 ml with the same solution. Dilute 2.0 ml to 50.0 ml with a 1 g/l solution of hydrochloric acid R in methanol R. Examined between 220 nm and 350 nm (2.2.25), the solution shows 2 absorption maxima at 231 nm and 306 nm and a shoulder at about 260 nm. The specific absorbance at the maximum at 231 nm is 660 to 730. B. Infrared absorption spectrophotometry (2.2.24). Preparation : discs. Comparison : dosulepin hydrochloride CRS. C. Dissolve about 1 mg in 5 ml of sulphuric acid R. A dark red colour is produced. D. It gives reaction (b) of chlorides (2.3.1). 1477

Dosulepin hydrochloride


TESTS

— disregard limit : 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (a) Appearance of solution. The solution is clear (2.2.1) and not (0.05 per cent). more intensely coloured than reference solution Y5 (2.2.2, Heavy metals (2.4.8) : maximum 20 ppm. Method II). 1.0 g complies with limit test C. Prepare the standard using Dissolve 1 g in water R and dilute to 20 ml with the same 2 ml of lead standard solution (10 ppm Pb) R. solvent. Loss on drying (2.2.32) : maximum 0.5 per cent, determined pH (2.2.3) : 4.2 to 5.2. on 1.000 g by drying in an oven at 100-105 °C. Dissolve 1 g in carbon dioxide-free water R and dilute to Sulphated ash (2.4.14) : maximum 0.1 per cent, determined 10 ml with the same solvent. on 1.0 g. Impurity E and related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately before use and ASSAY protect from light. Dissolve 0.250 g in a mixture of 5 ml of anhydrous Test solution. Dissolve 50.0 mg of the substance to be acetic acid R and 35 ml of acetic anhydride R. Titrate examined in 5 ml of methanol R and dilute to 100.0 ml with with 0.1 M perchloric acid, determining the end-point the mobile phase. potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 33.19 mg Reference solution (a). Dissolve 12.5 mg of dosulepin impurity A CRS in 5 ml of methanol R and dilute to 50.0 ml of C19H22ClNS. with the mobile phase. Dilute 0.5 ml to 100.0 ml with the mobile phase. STORAGE Protected from light. Reference solution (b). Dissolve 10.0 mg of dosulepin hydrochloride CRS in 5 ml of methanol R and dilute to IMPURITIES 20.0 ml with the mobile phase. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase: nitrile silica gel for chromatography R1 (5 µm), — temperature : 35 °C. Mobile phase : 0.83 per cent V/V solution of perchloric acid R, propanol R, methanol R, water R (1:10:30:60 V/V/V/V).

A. X = SO : (E)-3-(5-oxo-5λ4-dibenzo[b,e]thiepin-11(6H)ylidene)-N,N-dimethylpropan-1-amine,

Flow rate : 1 ml/min. Detection : spectrophotometer at 229 nm.

D. X = SO2 : (E)-3-(5,5-dioxo-5λ6-dibenzo[b,e]thiepin-11(6H)ylidene)-N,N-dimethylpropan-1-amine,

Injection : 5 µl. Run time : 2.5 times the retention time of dosulepin (E-isomer). Relative retention with reference to dosulepin (E-isomer ; retention time = about 25 min) : impurity E = about 0.9. System suitability : reference solution (b) : — peak-to-valley ratio : minimum 4, where Hp = height above the baseline of the peak due to impurity E and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to dosulepin (E-isomer).

B. R + R′ = O : dibenzo[b,e]thiepin-11(6H)-one,

Limits :

C. R = OH, R′ = [CH2]3-N(CH3)2 : (11RS)-11-[3(dimethylamino)propyl]-6,11-dihydrodibenzo[b,e]thiepin— impurity E : not more than 5 per cent of the sum of the 11-ol, areas of the peak due to impurity E and the principal peak in the chromatogram obtained with the test solution, — impurity A : not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.25 per cent), — any other impurity : not more than 0.4 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), — total of other impurities and impurity A : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent), 1478

E. (Z)-3-(dibenzo[b,e]thiepin-11(6H)-ylidene)-N,Ndimethylpropan-1-amine.



Doxapram hydrochloride

01/2005:1201 Optical rotation (2.2.7) : − 0.10° to + 0.10°, determined on solution S. DOXAPRAM HYDROCHLORIDE Related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately before use. Doxaprami hydrochloridum Test solution. Dissolve 10.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 5.0 ml with the mobile phase. Reference solution (c). Dissolve 5 mg of doxapram impurity B CRS in the mobile phase and dilute to 5.0 ml with the mobile phase. To 1.0 ml of the solution, add 1.0 ml of the C24H31ClN2O2,H2O Mr 433.0 test solution and dilute to 100.0 ml with the mobile phase. Column : DEFINITION — size : l = 0.25 m, Ø = 4.6 mm, (4RS)-1-Ethyl-4-[2-(morpholin-4-yl)ethyl]-3,3— stationary phase : spherical end-capped octadecylsilyl diphenylpyrrolidin-2-one hydrochloride. silica gel for chromatography R (5 µm) with a carbon Content : 98.0 per cent to 100.5 per cent (dried substance). loading of 14 per cent, a specific surface area of 350 m2/g and a pore size of 10 nm. CHARACTERS Mobile phase : mix 50 volumes of acetonitrile R and Appearance : white or almost white, crystalline powder. 50 volumes of a 0.82 g/l solution of sodium acetate R Solubility : soluble in water, in alcohol and in methylene adjusted to pH 4.5 with glacial acetic acid R. chloride. Flow rate : 1.5 ml/min. IDENTIFICATION Detection : spectrophotometer at 214 nm. First identification : A, C. Injection : 20 µl. Second identification : B, C. Run time : 4 times the retention time of doxapram. A. Infrared absorption spectrophotometry (2.2.24). Retention time : doxapram = about 6 min. Preparation : discs. System suitability : reference solution (c) : Comparison : doxapram hydrochloride CRS. — resolution : minimum of 3.0 between the peaks B. Thin-layer chromatography (2.2.27). corresponding to doxapram and to impurity B. Test solution. Dissolve 10 mg of the substance to be Limits : examined in methanol R and dilute to 10 ml with the — any impurity : not more than the area of the principal same solvent. peak in the chromatogram obtained with reference Reference solution. Dissolve 10 mg of doxapram solution (b) (0.2 per cent), hydrochloride CRS in methanol R and dilute to 10 ml — total : not more than the area of the principal peak in with the same solvent. the chromatogram obtained with reference solution (a) Plate : TLC silica gel plate R. (1.0 per cent), Mobile phase : solution of ammonia R containing — disregard limit : 0.05 times the area of the principal peak 17 g/l of NH3, 2-propanol R, 2-methylpropanol R in the chromatogram obtained with reference solution (a) (10:10:80 V/V/V). (0.05 per cent). Application : 10 µl. Heavy metals (2.4.8) : maximum 20 ppm. Development : over a path of 15 cm. Dissolve 2.0 g in a mixture of 15 volumes of water R and Drying : in air. 85 volumes of methanol R and dilute to 20 ml with the same Detection : spray with dilute potassium iodobismuthate mixture of solvents. 12 ml of the solution complies with limit solution R and examine immediately. test B. Prepare the standard using lead standard solution Results : the principal spot in the chromatogram obtained (2 ppm Pb) obtained by diluting lead standard solution with the test solution is similar in position, colour and (100 ppm Pb) R with a mixture of 15 volumes of water R and size to the principal spot in the chromatogram obtained 85 volumes of methanol R. with the reference solution. Loss on drying (2.2.32) : 3.0 per cent to 4.5 per cent, C. It gives reaction (a) of chlorides (2.3.1). determined on 1.000 g by drying in an oven at 100-105 °C. TESTS Sulphated ash (2.4.14) : maximum 0.1 per cent, determined Solution S. Dissolve 2.500 g in carbon dioxide-free water R on 1.0 g. and dilute to 50.0 ml with the same solvent. ASSAY Appearance of solution. The solution is clear (2.2.1) and Dissolve 0.300 g in a mixture of 10 ml of 0.01 M hydrochloric colourless (2.2.2, Method II). acid and 50 ml of alcohol R. Carry out a potentiometric Dilute 10 ml of solution S to 25 ml with water R. titration (2.2.20) using 0.1 M sodium hydroxide. Read the volume added between the 2 points of inflexion. pH (2.2.3) : 3.5 to 5.0. Dilute 5 ml of solution S to 25 ml with carbon dioxide-free 1 ml of 0.1 M sodium hydroxide is equivalent to 41.50 mg water R. of C24H31ClN2O2. General Notices (1) apply to all monographs and other texts

1479

Doxepin hydrochloride


IMPURITIES

A. R = Cl : (4RS)-4-(2-chloroethyl)-1-ethyl-3,3diphenylpyrrolidin-2-one, B. R = NH-CH2-CH2-OH : (4RS)-1-ethyl-4-[2-[(2hydroxyethyl)amino]ethyl]-3,3-diphenylpyrrolidin-2-one. 01/2005:1096 corrected

DOXEPIN HYDROCHLORIDE Doxepini hydrochloridum

C19H22ClNO

Mr 315.8

DEFINITION Doxepin hydrochloride is (E)-3-(dibenzo[b,e]oxepin-11(6H)ylidene)-N,N-dimethylpropan-1-amine hydrochloride. It contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of C19H22ClNO, calculated with reference to the dried substance. CHARACTERS A white or almost white, crystalline powder, freely soluble in water, in alcohol and in methylene chloride. IDENTIFICATION First identification : C, E. Second identification : A, B, D, E. A. Melting point (2.2.14) : 185 °C to 191 °C. B. Dissolve 50.0 mg in a 1 g/l solution of hydrochloric acid R in methanol R and dilute to 100.0 ml with the same acid solution. Dilute 5.0 ml to 50.0 ml with a 1 g/l solution of hydrochloric acid R in methanol R. Examined between 230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 297 nm. The specific absorbance at the maximum is 128 to 142. C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of doxepin hydrochloride. D. Dissolve about 5 mg in 2 ml of sulphuric acid R. A dark red colour is produced. E. Solution S (see Tests) gives reaction (a) of chlorides (2.3.1). TESTS Solution S. Dissolve 1.5 g in carbon dioxide-free water R and dilute to 30 ml with the same solvent. 1480

Appearance of solution. Dilute 10 ml of solution S to 25 ml with water R. The solution is clear (2.2.1) and colourless (2.2.2, Method II). Acidity. To 10 ml of solution S add 0.1 ml of methyl red solution R. Not more than 0.1 ml of 0.1 M sodium hydroxide is required to change the colour of the indicator to yellow. Related substances. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R (2-10 µm) with a concentration zone. Test solution. Dissolve 0.10 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution (a). Dissolve 10.0 mg of doxepin impurity A CRS in methanol R, add 1 ml of the test solution and dilute to 10 ml with methanol R. Dilute 1.0 ml of this solution to 50 ml with methanol R. Reference solution (b). Dissolve 10.0 mg of doxepin impurity B CRS in methanol R, add 1 ml of the test solution and dilute to 10 ml with methanol R. Dilute 1.0 ml of this solution to 50 ml with methanol R. Reference solution (c). Dissolve 10.0 mg of doxepin impurity B CRS in methanol R and dilute to 200 ml with the same solvent. Apply to one plate (plate A) 2 µl of the test solution and 2 µl of reference solution (a). Develop over a path of 5 cm using a mixture of 30 volumes of methyl ethyl ketone R and 60 volumes of heptane R. Doxepin remains on the starting line. Apply to another plate (plate B) 2 µl of the test solution and 2 µl of reference solutions (b) and (c). Develop over a path of 5 cm using a mixture of 10 volumes of methanol R and 90 volumes of methylene chloride R. Allow the plates to dry in a current of air. Spray with a solution prepared as follows : dissolve 20 g of zinc chloride R in 30 ml of glacial acetic acid R, add 3 ml of phosphoric acid R and 0.80 ml of strong hydrogen peroxide solution R and dilute to 60 ml with water R. Heat the plates at 120 °C for 15 min and examine immediately in ultraviolet light at 365 nm. Plate A. In the chromatogram obtained with the test solution : any spot corresponding to impurity A is not more intense than the corresponding spot in the chromatogram obtained with reference solution (a) (0.2 per cent) ; any spot, apart from the principal spot and the spot corresponding to impurity A, is not more intense than the spots in the chromatogram obtained with reference solution (a) (0.2 per cent). Plate B. In the chromatogram obtained with the test solution : any spot corresponding to impurity C (Rf about 0.12) is not more intense than the spot in the chromatogram obtained with reference solution (c) (0.5 per cent) ; any spot corresponding to impurity B is not more intense than the corresponding spot in the chromatogram obtained with reference solution (b) (0.2 per cent) ; any spot, apart from the principal spot, the spot corresponding to impurity B or the spot corresponding to impurity C, is not more intense than the spots in the chromatogram obtained with reference solution (b) (0.2 per cent). The test is not valid unless the chromatograms obtained with reference solutions (a) and (b) show clearly visible and separated principal spots. Z-Isomer. 13.0 per cent to 18.5 per cent of the Z-isomer, determined by liquid chromatography (2.2.29). Test solution. Dissolve 20.0 mg of the substance to be examined in the mobile phase and dilute to 20.0 ml with the


Doxorubicin hydrochloride


mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. The chromatographic procedure may be carried out using : — a stainless steel column 0.12 m long and 4 mm in internal diameter packed with spherical octylsilyl silica gel for chromatography R (5 µm) with a specific surface area of 220 m2/g and a pore size of 80 nm, C. (E)-3-(dibenzo[b,e]oxepin-11(6H)-ylidene)-N— as mobile phase at a flow rate of 1 ml/min a mixture of methylpropan-1-amine, 30 volumes of methanol R and 70 volumes of a 30 g/l solution of sodium dihydrogen phosphate R, previously adjusted to pH 2.5 with phosphoric acid R, — as detector a spectrophotometer set at 254 nm, maintaining the temperature of the column at 50 °C. Inject 20 µl of the test solution. Adjust the sensitivity of the system so that the height of the principal peak is at least 50 per cent of the full scale of the recorder. The test is not valid unless the resolution between the first peak (E-isomer) D. (Z)-3-(dibenzo[b,e]oxepin-11(6H)-ylidene)-N,Ndimethylpropan-1-amine. and the second peak (Z-isomer) is at least 1.5. The ratio of the area of the peak due to the E-isomer to the 01/2005:0714 area of the peak due to the Z-isomer is 4.4 to 6.7. Heavy metals (2.4.8). 1.0 g complies with limit test D DOXORUBICIN HYDROCHLORIDE (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Doxorubicini hydrochloridum Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.250 g in a mixture of 5 ml of anhydrous acetic acid R and 35 ml of acetic anhydride R. Titrate with 0.1 M perchloric acid until the colour changes from blue to green, using 0.2 ml of crystal violet solution R as indicator. 1 ml of 0.1 M perchloric acid is equivalent to 31.58 mg of C19H22ClNO. C27H30ClNO11 STORAGE Protected from light. IMPURITIES

A. dibenzo[b,e]oxepin-11(6H)-one,

Mr 580.0

DEFINITION (8S,10S)-10-[(3-Amino-2,3,6-trideoxy-α-L-lyxohexopyranosyl)oxy]-6,8,11-trihydroxy-8-(hydroxyacetyl)1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione hydrochloride. Substance produced by certain strains of Streptomyces coeruleorubidus or Streptomyces peucetiusor obtained by any other means. Content : 98.0 per cent to 102.0 per cent (anhydrous, solvent-free substance). CHARACTERS Appearance : orange-red, crystalline powder, hygroscopic. Solubility : soluble in water, slightly soluble in methanol. IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Comparison : doxorubicin hydrochloride CRS. B. Dissolve about 10 mg in 0.5 ml of nitric acid R, add 0.5 ml of water R and heat over a flame for 2 min. Allow to cool and add 0.5 ml of silver nitrate solution R1. A white precipitate is formed.

B. (11RS)-11-[3-(dimethylamino)propyl]-6,11dihydrodibenzo[b,e]oxepin-11-ol, General Notices (1) apply to all monographs and other texts

TESTS pH (2.2.3) : 4.0 to 5.5. Dissolve 50 mg in carbon dioxide-free water R and dilute to 10 ml with the same solvent. 1481

Doxycycline hyclate


Related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately before use. Test solution (a). Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Test solution (b). Dilute 10.0 ml of test solution (a) to 100.0 ml with the mobile phase. Reference solution (a). Dissolve 10.0 mg of doxorubicin hydrochloride CRS and 10 mg of epirubicin hydrochloride CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 10.0 ml of the solution to 100.0 ml with the mobile phase. Reference solution (b). Dilute 5.0 ml of reference solution (a) to 20.0 ml with the mobile phase. Reference solution (c). Dissolve 50.0 mg of doxorubicin hydrochloride CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 10.0 ml of the solution to 100.0 ml with the mobile phase. Column : — size : l = 0.25 m, Ø = 4.0 mm, — stationary phase: end-capped octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : mix equal volumes of acetonitrile R and a solution containing 2.88 g/l of sodium laurilsulfate R and 2.25 g/l of phosphoric acid R. Flow rate : 1 ml/min. Detection : spectrophotometer at 254 nm. Injection : 5 µl ; inject test solution (a) and reference solutions (a) and (b). Run time : 3.5 times the retention time of doxorubicin. Retention time : doxorubicin = about 8 min. System suitability : reference solution (a) : — resolution : minimum of 2.0 between the peaks due to doxorubicin and to epirubicin. Limits : — any impurity : not more than the area of the peak corresponding to doxorubicin in the chromatogram obtained with reference solution (b) (0.5 per cent), — disregard limit : 0.1 times the area of the peak corresponding to doxorubicin in the chromatogram obtained with reference solution (b) (0.05 per cent). Ethanol (2.4.24, System B) : maximum 1.0 per cent. Water (2.5.12) : maximum 4.0 per cent, determined on 0.100 g. Bacterial endotoxins (2.6.14) : less than 2.2 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances. Injection : test solution (b) and reference solution (c). Calculate the percentage content of C27H30ClNO11. STORAGE In an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins. 1482

IMPURITIES A. daunorubicin,

B. R = OCH3 : (8S,10S)-10[(3-amino-2,3,6-trideoxy-α-L-lyxohexopyranosyl)oxy]-8-(2-bromo-1,1-dimethoxyethyl)-6,8, 11-trihydroxy-1-methoxy-7,8,9,10-tetrahydrotetracene-5, 12-dione, C. R + R = O : (8S,10S)-10[(3-amino-2,3,6-trideoxy-α-L-lyxohexopyranosyl)oxy]-8-(bromoacetyl)-6,8,11-trihydroxy-1methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione,

D. (8S,10S)-6,8,10,11-tetrahydroxy-8-(hydroxyacetyl)1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione (doxorubicin aglycone, doxorubicinone). 01/2005:0272

DOXYCYCLINE HYCLATE Doxycyclini hyclas

(C22H25ClN2O8),1/2C2H6O,1/2H2O

Mr 512.9

DEFINITION Hydrochloride hemiethanol hemihydrate of (4S,4aR,5S,5aR,6R,12aS)-4-(dimethylamino)-3,5,10, 12,12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11, 12a-octahydrotetracene-2-carboxamide. Substance obtained from oxytetracycline or metacycline or by any other means. Content : 95.0 per cent to 102.0 per cent of C22H25ClN2O8 (anhydrous and ethanol-free substance). CHARACTERS Appearance : yellow, crystalline powder, hygroscopic. Solubility : freely soluble in water and in methanol, sparingly soluble in ethanol (96 per cent). It dissolves in solutions of alkali hydroxides and carbonates.



Doxycycline hyclate

TESTS pH (2.2.3) : 2.0 to 3.0. Dissolve 0.1 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. Specific optical rotation (2.2.7) : − 105 to − 120 (anhydrous and ethanol-free substance). Dissolve 0.250 g in a mixture of 1 volume of 1 M hydrochloric acid and 99 volumes of methanol R and dilute to 25.0 ml with the same mixture of solvents. Carry out the measurement within 5 min of preparing the solution. Specific absorbance (2.2.25) : 300 to 335, determined at the absorption maximum at 349 nm (anhydrous and ethanol-free substance). Dissolve 25.0 mg in a mixture of 1 volume of 1 M hydrochloric acid and 99 volumes of methanol R and dilute to 25.0 ml with the same mixture of solvents. Dilute 1.0 ml of the solution to 100.0 ml with a mixture of 1 volume of 1 M hydrochloric acid and 99 volumes of methanol R. Carry out the measurement within 1 h of preparing the solution. Light-absorbing impurities. The absorbance (2.2.25), determined at 490 nm is maximum 0.07 (anhydrous and ethanol-free substance). Dissolve 0.10 g in a mixture of 1 volume of 1 M hydrochloric acid and 99 volumes of methanol R and dilute to 10.0 ml with the same mixture of solvents. Carry out the measurement within 1 h of preparing the solution. Related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately before use. Test solution. Dissolve 20.0 mg of the substance to be examined in 0.01 M hydrochloric acid and dilute to 25.0 ml with the same acid. Reference solution (a). Dissolve 20.0 mg of doxycycline hyclate CRS in 0.01 M hydrochloric acid and dilute to 25.0 ml with the same acid. Reference solution (b). Dissolve 20.0 mg of 6-epidoxycycline hydrochloride CRS in 0.01 M hydrochloric acid and dilute to 25.0 ml with the same acid. Reference solution (c). Dissolve 20.0 mg of metacycline hydrochloride CRS in 0.01 M hydrochloric acid and dilute to 25.0 ml with the same acid. Reference solution (d). Mix 4.0 ml of reference solution (a), 1.5 ml of reference solution (b) and 1.0 ml of reference solution (c) and dilute to 25.0 ml with 0.01 M hydrochloric acid. Reference solution (e). Mix 2.0 ml of reference solution (b) and 2.0 ml of reference solution (c) and dilute to 100.0 ml with 0.01 M hydrochloric acid. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase: styrene-divinylbenzene copolymer R (8 µm), — temperature : 60 °C.

Mobile phase : weigh 60.0 g of 2-methyl-2-propanol R and transfer to a 1000 ml volumetric flask with the aid of 200 ml of water R ; add 400 ml of buffer solution pH 8.0 R, 50 ml of a 10 g/l solution of tetrabutylammonium hydrogen sulphate R adjusted to pH 8.0 with dilute sodium hydroxide solution R and 10 ml of a 40 g/l solution of sodium edetate R adjusted to pH 8.0 with dilute sodium hydroxide solution R ; dilute to 1000.0 ml with water R. Flow rate : 1.0 ml/min. Detection : spectrophotometer at 254 nm. Injection : 20 µl of the test solution and reference solutions (d) and (e). Relative retention with reference to doxycycline : impurity E = about 0.2 ; impurity D = about 0.3 ; impurity C = about 0.5 ; impurity F = about 1.2. System suitability : reference solution (d) : — resolution : minimum 1.25 between the peaks due to impurity B (1st peak) and impurity A (2nd peak) and minimum 2.0 between the peaks due to impurity A and doxycycline (3rd peak) ; if necessary, adjust the 2-methyl-2-propanol content in the mobile phase ; — symmetry factor : maximum 1.25 for the peak due to doxycycline. Limits : — impurity A : not more than the area of the corresponding peak in the chromatogram obtained with reference solution (e) (2.0 per cent), — impurity B : not more than the area of the corresponding peak in the chromatogram obtained with reference solution (e) (2.0 per cent), — impurities C, D, E, F : for each impurity, not more than 0.25 times the area of the peak due to impurity A in the chromatogram obtained with reference solution (e) (0.5 per cent), — any other impurity : for each impurity, not more than 0.25 times the area of the peak due to impurity A in the chromatogram obtained with reference solution (e) (0.5 per cent), — disregard limit : 0.05 times the area of the peak due to impurity A in the chromatogram obtained with reference solution (e) (0.1 per cent). Ethanol. Gas chromatography (2.2.28). Internal standard solution. Dilute 0.50 ml of propanol R to 1000.0 ml with water R. Test solution (a). Dissolve 0.10 g of the substance to be examined in water R and dilute to 10.0 ml with the same solvent. Test solution (b). Dissolve 0.10 g of the substance to be examined in the internal standard solution and dilute to 10.0 ml with the same solution. Reference solution. Dilute 0.50 ml of ethanol R to 100.0 ml with the internal standard solution. Dilute 1.0 ml of this solution to 10.0 ml with the internal standard solution. Column : — size : l = 1.5 m, Ø = 4.0 mm, — stationary phase : ethylvinylbenzene-divinylbenzene copolymer R (150-180 µm). Carrier gas : nitrogen for chromatography R. Temperature : — column : 135 °C, — injection port and detector : 150 °C. Detection : flame ionisation. Calculate the content of ethanol taking the density (2.2.5) at 20 °C to be 0.790 g/ml.


1483

IDENTIFICATION A. Examine the chromatograms obtained in the assay. Results : the principal peak in the chromatogram obtained with the test solution is similar in retention time and size to the principal peak in the chromatogram obtained with reference solution (a). B. To about 2 mg add 5 ml of sulphuric acid R. A yellow colour develops. C. It gives reaction (a) of chlorides (2.3.1).

Doxycycline monohydrate


Limit : — ethanol: 4.3 per cent to 6.0 per cent. Heavy metals (2.4.8) : maximum 50 ppm. 0.5 g complies with limit test C. Prepare the reference solution using 2.5 ml of lead standard solution (10 ppm Pb) R. Water (2.5.12) : 1.4 per cent to 2.8 per cent, determined on 1.20 g. Sulphated ash (2.4.14) : maximum 0.4 per cent, determined on 1.0 g. Bacterial endotoxins (2.6.14) : less than 1.14 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modification. Injection : test solution and reference solution (a). Calculate the percentage content of C22H25ClN2O8 (Mr = 480.9). STORAGE In an airtight container, protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof container. LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins. IMPURITIES Specified impurities : A, B, C, D, E, F.

01/2005:0820

DOXYCYCLINE MONOHYDRATE Doxycyclinum monohydricum

C22H24N2O8,H2O

Mr 462.5

DEFINITION (4S,4aR,5S,5aR,6R,12aS)-4-(Dimethylamino)-3,5,10,12, 12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12aoctahydrotetracene-2-carboxamide monohydrate. Substance obtained from oxytetracycline or metacycline or by any other means. Content : 95.0 per cent to 102.0 per cent (anhydrous substance). CHARACTERS Appearance : yellow, crystalline powder. Solubility : very slightly soluble in water and in alcohol. It dissolves in dilute solutions of mineral acids and in solutions of alkali hydroxides and carbonates. IDENTIFICATION A. Examine the chromatograms obtained in the assay. Results : the principal peak in the chromatogram obtained with the test solution is similar in retention time and size to the principal peak in the chromatogram obtained with reference solution (a). B. To about 2 mg add 5 ml of sulphuric acid R. A yellow colour develops. C. Dissolve 25 mg in a mixture of 0.2 ml of dilute nitric acid R and 1.8 ml of water R. The solution does not give reaction (a) of chlorides (2.3.1).

A. R1 = NH2, R2 = R5 = H, R3 = N(CH3)2, R4 = CH3 : (4S,4aR,5S,5aR,6S,12aS)-4-(dimethylamino)-3,5,10,12, TESTS 12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11, 12a-octahydrotetracene-2-carboxamide (6-epidoxycycline), pH (2.2.3) : 5.0 to 6.5. Suspend 0.1 g in carbon dioxide-free water R and dilute to B. R1 = NH2, R2 = H, R3 = N(CH3)2, R4 + R5 = CH2 : 10 ml with the same solvent. (4S,4aR,5S,5aR,12aS)-4-(dimethylamino)-3,5,10,12,12apentahydroxy-6-methylene-1,11-dioxo-1,4,4a,5,5a,6,11,12a- Specific optical rotation (2.2.7) : − 113 to − 130 (anhydrous octahydrotetracene-2-carboxamide (metacycline), substance). Dissolve 0.250 g in a mixture of 0.5 volumes of hydrochloric C. R1 = NH2, R2 = N(CH3)2, R3 = R4 = H, R5 = CH3 : acid R and 99.5 volumes of methanol R and dilute to (4R,4aR,5S,5aR,6R,12aS)-4-(dimethylamino)-3,5,10,12, 25.0 ml with the same mixture of solvents. Carry out the 12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11, 12a-octahydrotetracene-2-carboxamide (4-epidoxycycline), measurement within 5 min of preparing the solution. Specific absorbance (2.2.25) : 325 to 363 determined at the D. R1 = NH2, R2 = N(CH3)2, R3 = R5 = H, R4 = CH3 : maximum at 349 nm (anhydrous substance). (4R,4aR,5S,5aR,6S,12aS)-4-(dimethylamino)-3,5, Dissolve 25.0 mg in a mixture of 0.5 volumes of hydrochloric 10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a, acid R and 99.5 volumes of methanol R and dilute to 50.0 ml 5,5a,6,11,12a-octahydrotetracene-2-carboxamide with the same mixture of solvents. Dilute 2.0 ml of the (4-epi-6-epidoxycycline), solution to 100.0 ml with a mixture of 0.5 volumes of 1 M E. R1 = NH2, R2 = H, R3 = N(CH3)2, R4 = OH, R5 = CH3 : hydrochloric acid and 99.5 volumes of methanol R. Carry oxytetracycline, out the measurement within 1 h of preparing the solution. Light-absorbing impurities. The absorbance (2.2.25) F. R1 = CH3, R2 = R4 = H, R3 = N(CH3)2, R5 = CH3 : determined at 490 nm has a maximum of 0.07 (anhydrous (4S,4aR,5S,5aR,6R,12aS)-2-acetyl-4-(dimethylamino)substance). 3,5,10,12,12a-pentahydroxy-6-methyl-4a,5a, Dissolve 0.10 g in a mixture of 0.5 volumes of hydrochloric 6,12a-tetrahydrotetracene-1,11(4H,5H)-dione acid R and 99.5 volumes of methanol R and dilute to (2-acetyl-2-decarbamoyldoxycycline). 1484


Doxycycline monohydrate


10.0 ml with the same mixture of solvents. Carry out the measurement within 1 h of preparing the solution.

— any other impurity : not more than 0.25 times the area of the peak due to impurity A in the chromatogram obtained with reference solution (e) (0.5 per cent), Related substances. Liquid chromatography (2.2.29). — disregard limit : 0.05 times the area of the peak due to Prepare the solutions immediately before use. impurity A in the chromatogram obtained with reference Test solution. Dissolve 20.0 mg of the substance to be solution (e) (0.1 per cent). examined in 0.01 M hydrochloric acid and dilute to 25.0 ml Heavy metals (2.4.8) : maximum 50 ppm. with the same acid. 0.5 g complies with limit test C. Prepare the standard using Reference solution (a). Dissolve 20.0 mg of doxycycline 2.5 ml of lead standard solution (10 ppm Pb) R. hyclate CRS in 0.01 M hydrochloric acid and dilute to 25.0 ml with the same acid. Water (2.5.12) : 3.6 per cent to 4.6 per cent, determined on Reference solution (b). Dissolve 20.0 mg of 6-epidoxycycline 0.200 g. hydrochloride CRS in 0.01 M hydrochloric acid and dilute Sulphated ash (2.4.14) : maximum 0.4 per cent, determined to 25.0 ml with the same acid. on 1.0 g. Reference solution (c). Dissolve 20.0 mg of metacycline hydrochloride CRS in 0.01 M hydrochloric acid and dilute to 25.0 ml with the same acid. Reference solution (d). Mix 4.0 ml of reference solution (a), 1.5 ml of reference solution (b) and 1.0 ml of reference solution (c) and dilute to 25.0 ml with 0.01 M hydrochloric acid.

ASSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modification. Injection : test solution and reference solution (a). Calculate the percentage content of C22H24N2O8.

Reference solution (e). Mix 2.0 ml of reference solution (b) and 2.0 ml of reference solution (c) and dilute to 100.0 ml with 0.01 M hydrochloric acid.


Column :

IMPURITIES

— size : l = 0.25 m, Ø = 4.6 mm, — stationary phase: styrene-divinylbenzene copolymer R (8 µm), — temperature : 60 °C. Mobile phase : weigh 60.0 g of 2-methyl-2-propanol R and transfer into a 1000 ml volumetric flask with the aid of 200 ml of water R ; add 400 ml of buffer solution pH 8.0 R, 50 ml of a 10 g/l solution of tetrabutylammonium hydrogen A. R1 = NH2, R2 = R5 = H, R3 = N(CH3)2, R4 = CH3 : (4S,4aR,5S,5aR,6S,12aS)-4-(dimethylamino)-3,5,10,12, sulphate R adjusted to pH 8.0 with dilute sodium hydroxide 12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11, solution R and 10 ml of a 40 g/l solution of sodium edetate R 12a-octahydrotetracene-2-carboxamide (6-epidoxycycline), adjusted to pH 8.0 with dilute sodium hydroxide solution R ; dilute to 1000.0 ml with water R. Flow rate : 1.0 ml/min. Detection : spectrophotometer at 254 nm. Injection : 20 µl ; inject the test solution and reference solutions (d) and (e). Relative retention with reference to doxycycline : impurity E = about 0.2 ; impurity D = about 0.3 ; impurity C = about 0.5 ; impurity F = about 1.2. System suitability : reference solution (d) : — resolution : minimum 1.25 between the peaks due to impurity B (1st peak) and impurity A (2nd peak) and minimum 2.0 between the peaks due to impurity A and doxycycline (3rd peak) ; if necessary, adjust the 2-methyl-2-propanol content in the mobile phase, — symmetry factor : maximum 1.25 for the peak due to doxycycline. Limits :

B. R1 = NH2, R2 = H, R3 = N(CH3)2, R4 + R5 = CH2 : (4S,4aR,5S,5aR,12aS)-4-(dimethylamino)-3,5,10,12,12apentahydroxy-6-methylene-1,11-dioxo-1,4,4a,5,5a,6,11,12aoctahydrotetracene-2-carboxamide (metacycline), C. R1 = NH2, R2 = N(CH3)2, R3 = R4 = H, R5 = CH3 : (4R,4aR,5S,5aR,6R,12aS)-4-(dimethylamino)-3,5,10,12, 12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11, 12a-octahydrotetracene-2-carboxamide (4-epidoxycycline), D. R1 = NH2, R2 = N(CH3)2, R3 = R5 = H, R4 = CH3 : (4R,4aR,5S,5aR,6S,12aS)-4-(dimethylamino)-3,5, 10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a, 5,5a,6,11,12a-octahydrotetracene-2-carboxamide (4-epi-6-epidoxycycline), E. R1 = NH2, R2 = H, R3 = N(CH3)2, R4 = OH, R5 = CH3 : oxytetracycline,

— impurity A : not more than the area of the corresponding peak in the chromatogram obtained with reference solution (e) (2.0 per cent),

F. R1 = CH3, R2 = R4 = H, R3 = N(CH3)2, R5 = CH3 : (4S,4aR,5S,5aR,6R,12aS)-2-acetyl-4-(dimethylamino)3,5,10,12,12a-pentahydroxy-6-methyl-4a,5a, — impurity B : not more than the area of the corresponding 6,12a-tetrahydrotetracene-1,11(4H,5H)-dione peak in the chromatogram obtained with reference (2-acetyl-2-decarbamoyldoxycycline). solution (e) (2.0 per cent),


1485

Doxylamine hydrogen succinate


01/2005:1589 Reference solution (a). Dilute 1.0 ml of the test solution to 200.0 ml with ethanol R.

DOXYLAMINE HYDROGEN SUCCINATE

Reference solution (b). Dissolve 40 mg of doxylamine impurity A CRS and 40 mg of 2-benzoylpyridine R in ethanol R and dilute to 20 ml with the same solvent. Dilute 1 ml of this solution to 20 ml with ethanol R. Column :

Doxylamini hydrogenosuccinas

— material : fused silica, — size : l = 30 m, Ø = 0.53 mm, — stationary phase : poly(dimethyl)(diphenyl)siloxane R (film thickness 1.5 µm). Carrier gas : helium for chromatography R. Flow rate : 7 ml/min. Temperature : C21H28N2O5

Mr 388.5

DEFINITION N,N-dimethyl-2-[(1RS)-1-phenyl-1-(pyridin-2yl)ethoxy(ethanamine hydrogen butanedioate. Content : 99.0 per cent to 101.0 per cent (anhydrous substance).

Column

Time (min)

Temperature (°C)

0 - 12

160 → 220

12 - 27

220

Injection port

250

Detector

250

CHARACTERS Appearance : a white or almost white powder. Solubility : very soluble in water, freely soluble in alcohol.

Detection : flame ionisation.

IDENTIFICATION First identification : C. Second identification : A, B. A. Melting point (2.2.14) : 103 °C to 108 °C. B. Dissolve 0.200 g in 0.1 M hydrochloric acid and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with 0.1 M hydrochloric acid. Examined between 230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 262 nm. The specific absorbance at the maximum is 229 to 243 (anhydrous substance). C. Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of doxylamine hydrogen succinate.

— resolution : minimum 1.5 between the peaks due to impurity A and impurity D. Limits :

TESTS Appearance of solution. The solution is clear (2.2.1) and colourless (2.2.2, Method II). Dissolve 0.4 g of the substance to be examined in water R and dilute to 20 ml with the same solvent. Optical rotation (2.2.7) : - 0.10° to + 0.10°. Dissolve 2.50 g of the substance to be examined in water R and dilute to 25.0 ml with the same solvent. Related substances. Gas chromatography (2.2.28). Test solution. Dissolve 0.650 g of the substance to be examined in 20 ml of 0.1 M hydrochloric acid. Add 3 ml of a 100 g/l solution of sodium hydroxide R and extract 3 times with 25 ml of methylene chloride R. Combine the methylene chloride extracts and filter using hydrophobic phase-separation filter paper. Rinse the filter with 10 ml of methylene chloride R and combine the rinsings with the methylene chloride extracts. Evaporate the solvent under reduced pressure at a temperature not exceeding 40 °C. Dissolve the residue in 20.0 ml of ethanol R. 1486

Injection : 1 µl. System suitability : reference solution (b) :

— any impurity : not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent), — total : not more than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (1 per cent), — disregard limit : 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). Water (2.5.12) : maximum 0.5 per cent, determined on 2.00 g. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.150 g in 50 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to 19.43 mg of C21H28N2O5. IMPURITIES

A. N,N-dimethyl-2-[1(RS)-1-phenyl-1-(pyridin-4yl)ethoxy]ethanamine,



Droperidol

Reference solution (a). Dissolve 30 mg of droperidol CRS in a mixture of 1 volume of acetone R and 9 volumes of methanol R and dilute to 10 ml with the same mixture of solvents. Reference solution (b). Dissolve 30 mg of droperidol CRS and 30 mg of benperidol CRS in a mixture of 1 volume of acetone R and 9 volumes of methanol R and dilute to 10 ml with the same mixture of solvents. B. R1 = CH3, R2 = H : (1RS)-1-phenyl-1-(pyridin-2-yl)ethanol, Apply to the plate 10 µl of each solution. Develop over a C. R1 = H, R2 = CH2-CH2-N(CH3)2 : N,N-dimethyl-2-[(RS)-1path of 15 cm using a mixture of 1 volume of acetone R phenyl(pyridin-2-yl)methoxy]ethanamine, and 9 volumes of methanol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows 2 clearly separated spots. C. Dissolve about 10 mg in 5 ml of ethanol R. Add 0.5 ml of dinitrobenzene solution R and 0.5 ml of 2 M alcoholic D. phenyl(pyridin-2-yl)methanone (2-benzoylpyridine). potassium hydroxide R. A violet colour is produced and becomes brownish-red after 20 min. 01/2005:1010 D. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is DROPERIDOL obtained (usually less than 5 min). Allow to cool, add 1 ml of water R, 0.05 ml of phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the Droperidolum solution colourless. Filter. To a freshly prepared mixture of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R, add 1.0 ml of the filtrate. Mix, allow to stand for 5 min and compare the colour of the solution with that of a blank prepared in the same manner. The test solution is yellow and the blank is red. TESTS Appearance of solution. Dissolve 0.20 g in methylene C22H22FN3O2 Mr 379.4 chloride R and dilute to 20.0 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY5 (2.2.2, Method II). DEFINITION Droperidol contains not less than 99.0 per cent and Related substances. Examine by liquid chromatography not more than the equivalent of 101.0 per cent of (2.2.29). Prepare the solutions immediately before use. 1-[1-[4-(4-fluorophenyl)-4-oxobutyl]-1,2,3,6-tetrahydropyridin- Test solution. Dissolve 0.10 g of the substance to be 4-yl]-1,3-dihydro-2H-benzimidazol-2-one, calculated with examined in dimethylformamide R and dilute to 10.0 ml reference to the dried substance. with the same solvent. Reference solution (a). Dissolve 2.5 mg of droperidol CRS CHARACTERS and 2.5 mg of benperidol CRS in dimethylformamide R and A white or almost white powder, practically insoluble in water, freely soluble in dimethylformamide and in methylene dilute to 100.0 ml with the same solvent. chloride, sparingly soluble in alcohol. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with dimethylformamide R. Dilute 5.0 ml of this It shows polymorphism. solution to 20.0 ml with dimethylformamide R. IDENTIFICATION The chromatographic procedure may be carried out using : First identification : A. — a stainless steel column 0.10 m long and 4.6 mm Second identification : B, C, D. in internal diameter packed with base-deactivated A. Examine by infrared absorption spectrophotometry octadecylsilyl silica gel for chromatography R (3 µm), (2.2.24), comparing with the spectrum obtained with — as mobile phase at a flow rate of 1.5 ml/min : droperidol CRS. Examine the substances prepared as mobile phase A : acetonitrile R, discs. If the spectra obtained show differences, dissolve the substance to be examined and the reference substance mobile phase B : 10 g/l solution of tetrabutylammonium separately in the minimum volume of acetone R, hydrogen sulphate R1, evaporate to dryness on a water-bath and record new Time Mobile phase A Mobile phase B spectra using the residues. (min) (per cent V/V) (per cent V/V) B. Examine by thin-layer chromatography (2.2.27), using 0 → 40 0 - 15 100 → 60 silica gel GF254 R as the coating substance. 40 60 15 - 20 Test solution. Dissolve 30 mg of the substance to be examined in a mixture of 1 volume of acetone R and 40 → 0 20 - 25 60 → 100 9 volumes of methanol R and dilute to 10 ml with the same mixture of solvents. — as detector a spectrophotometer set at 275 nm. General Notices (1) apply to all monographs and other texts

1487

Droperidol


Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 10 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder. Inject 10 µl of reference solution (a). When the chromatogram A. 1-(1,2,3,6-tetrahydropyridin-4-yl)-1,3-dihydro-2His recorded in the prescribed conditions, the retention times benzimidazol-2-one, are : benperidol about 6.5 min and droperidol about 7 min. The test is not valid unless the resolution between the peaks due to droperidol and benperidol is at least 2.0. If necessary, adjust the final concentration of acetonitrile in the mobile phase or adjust the time programme for the linear gradient. Inject 10 µl of dimethylformamide R as a blank, 10 µl of the test solution and 10 µl of reference solution (b). In the chromatogram obtained with the test solution : the area B. 1-[1-[4-(2-fluorophenyl)-4-oxobutyl]-1,2,3,6of any peak, apart from the principal peak, is not greater tetrahydropyridin-4-yl]-1,3-dihydro-2H-benzimidazol-2than the area of the principal peak in the chromatogram one, obtained with reference solution (b) (0.25 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent). Disregard any peak obtained with the blank and any peak with an area less than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (b). Heavy metals (2.4.8). 1.0 g complies with limit test D for C. 1-[4-(4-fluorophenyl)-4-oxobutyl]-4-(2-oxo-2,3-dihydro-1Hheavy metals (20 ppm). Prepare the standard using 2 ml of benzimidazol-1-yl)pyridinium chloride, lead standard solution (10 ppm Pb) R. Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.300 g in 50 ml of a mixture of 1 volume of anhydrous acetic acid R and 7 volumes of methyl ethyl ketone R. Using 0.2 ml of naphtholbenzein solution R, titrate with 0.1 M perchloric acid until the colour changes from orange-yellow to green. 1 ml of 0.1 M perchloric acid is equivalent to 37.94 mg of C22H22FN3O2.

D. (1RS)-1-[4-(4-fluorophenyl)-4-oxobutyl]-4-(2-oxo-2,3dihydro-1H-benzimidazol-1-yl)-1,2,3,6-tetrahydropyridine 1-oxide,

STORAGE Store protected from light. IMPURITIES E. 1-[1-[4-[4-[4-(2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)-3, 6-dihydropyridin-1(2H)-yl]-1-oxobutyl]phenyl]-1,2,3,6tetrahydropyridin-4-yl]-1,3-dihydro-2H-benzimidazol-2one.

1488



Ebastine

01/2005:2015 Run time : 1.4 times the retention time of ebastine. corrected Relative retention with reference to ebastine : impurity A = about 0.04 ; impurity B = about 0.05 ; impurity D = about 0.20 ; impurity C = about 0.22 ; EBASTINE impurity F = about 0.42 ; impurity G = about 0.57 ; impurity E = about 1.14. Ebastinum System suitability : reference solution (a) : — resolution : minimum 2.0 between the peaks due to impurity D and impurity C. Limits : — impurities A, B, C, D, E, F, G : for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent), — any other impurity : for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent), C32H39NO2 Mr 469.7 — total : not more than 4 times the area of the principal peak in the chromatogram obtained with reference solution (b) DEFINITION (0.4 per cent), 1-[4-(1,1-Dimethylethyl)phenyl]-4-[4-(diphenylmethoxy)pipe— disregard limit : 0.5 times the area of the principal peak ridin-1-yl]butan-1-one. in the chromatogram obtained with reference solution (b) Content : 99.0 per cent to 101.0 per cent (anhydrous (0.05 per cent). substance). Sulphates (2.4.13) : maximum 100 ppm. CHARACTERS Suspend 2.5 g in 25 ml of dilute nitric acid R. Boil under a reflux condenser for 10 min. Cool and filter. 15 ml of the Appearance : white or almost white, crystalline powder. filtrate complies with the limit test for sulphates. Solubility : practically insoluble in water, very soluble in Water (2.5.12) : maximum 0.5 per cent, determined on methylene chloride, sparingly soluble in methanol. 0.500 g. mp : about 86 °C. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined IDENTIFICATION on 1.0 g. Infrared absorption spectrophotometry (2.2.24). ASSAY Comparison : Ph. Eur. reference spectrum of ebastine. Dissolve 0.350 g in 50 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point TESTS potentiometrically (2.2.20). Related substances. Liquid chromatography (2.2.29). Keep 1 ml of 0.1 M perchloric acid is equivalent to 46.97 mg the solutions protected from light. of C32H39NO2. Solution A. Mix 65 volumes of acetonitrile R and 35 volumes of a 1.1 g/l solution of phosphoric acid R adjusted to pH 5.0 STORAGE with a 40 g/l solution of sodium hydroxide R. Protected from light. Test solution. Dissolve 0.125 g of the substance to be examined in solution A and dilute to 50.0 ml with the same IMPURITIES solution. Reference solution (a). Dissolve 5.0 mg of ebastine impurity C CRS and 5.0 mg of ebastine impurity D CRS in solution A and dilute to 20.0 ml with the same solution. Dilute 1.0 ml of the solution to 100.0 ml with solution A. Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with solution A. Dilute 1.0 ml of this solution to 10.0 ml with solution A. A. R1–H : diphenylmethanol (benzhydrol), Column : B. R2–CH3 : 1-[4-(1,1-dimethylethyl)phenyl]ethanone, — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : nitrile silica gel for chromatography R (5 µm). Mobile phase : mix 35 volumes of acetonitrile R and 65 volumes of a 1.1 g/l solution of phosphoric acid R C. 4-(diphenylmethoxy)piperidine, adjusted to pH 5.0 with a 40 g/l solution of sodium hydroxide R. Adjust the percentage of acetonitrile to between 30 per cent V/V and 40 per cent V/V so that the retention time of ebastine is about 110 min. Flow rate : 1 ml/min. Detection : spectrophotometer at 210 nm. D. 1-[4-(1,1-dimethylethyl)phenyl]-4-(4-hydroxypiperidin-1yl)butan-1-one, Injection : 10 µl. General Notices (1) apply to all monographs and other texts

1491

Econazole


Reference solution (a). Dissolve 10 mg of econazole for system suitability CRS in methanol R and dilute to 1.0 ml with the same solvent. Reference solution (b). Dilute 1.0 ml of the test solution to 20.0 ml with methanol R. Dilute 1.0 ml of this solution to 25.0 ml with methanol R. Column : E. 1-[4-(1,1-dimethylpropyl)phenyl]-4-[4-(diphenylmethoxy)pi- — size : l = 0.10 m, Ø = 4.6 mm, peridin-1-yl]butan-1-one, — stationary phase : base-deactivated octadecylsilyl silica gel for chromatography R (3 µm), — temperature : 35 °C. Mobile phase : — mobile phase A : mix 20 volumes of methanol R and 80 volumes of a 0.77 g/l solution of ammonium F. 1-[4-(1,1-dimethylethyl)phenyl]-4-[cis-4-(diphenylmethoxy)acetate R, 1-oxidopiperidin-1-yl]butan-1-one, — mobile phase B : methanol R, acetonitrile R (40:60 V/V),

G. 1-[4-(1,1-dimethylethyl)phenyl]-4-[trans-4(diphenylmethoxy)-1-oxidopiperidin-1-yl]butan-1-one.

Time (min)



0 - 25

60 → 10

40 → 90

25 - 27

10

90

27 - 28

10 → 60

90 → 40

28 - 33

60

40

Flow rate : 1.5 ml/min. 01/2005:2049 Detection : spectrophotometer at 225 nm. corrected Injection : 10 µl. Relative retention with reference to econazole (retention ECONAZOLE time = about 15 min) : impurity A = about 0.2 ; impurity B = about 0.6 ; impurity C = about 1.1. Econazolum System suitability : reference solution (a) : — peak-to-valley ratio : minimum 1.5, where Hp = height above the baseline of the peak due to impurity C and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to econazole. Limits : — correction factor : for the calculation of content, multiply the peak area of impurity A by 1.4, — impurities A, B, C : for each impurity, not more than the area of the principal peak in the chromatogram obtained C18H15Cl3N2O Mr 381.7 with reference solution (b) (0.2 per cent), — total : not more than 1.5 times the area of the principal DEFINITION peak in the chromatogram obtained with reference 1-[(2RS)-2-[(4-Chlorobenzyl)oxy]-2-(2,4-dichlorophenyl)ethyl]solution (b) (0.3 per cent), 1H-imidazole. — disregard limit : 0.25 times the area of the principal peak Content : 99.0 per cent to 101.0 per cent (dried substance). in the chromatogram obtained with reference solution (b) (0.05 per cent). CHARACTERS Appearance : white or almost white powder. Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in vacuo at 60 °C for 4 h. Solubility : practically insoluble in water, very soluble in alcohol and in methylene chloride. Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g. IDENTIFICATION A. Melting point (2.2.14) : 88 °C to 92 °C. ASSAY B. Infrared absorption spectrophotometry (2.2.24). Dissolve 0.300 g in 75 ml of anhydrous acetic acid R. Titrate Comparison : Ph. Eur. reference spectrum of econazole. with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). Carry out a blank titration. TESTS 1 ml of 0.1 M perchloric acid is equivalent to 38.17 mg of Related substances. Liquid chromatography (2.2.29). C18H15Cl3N2O. Test solution. Dissolve 0.100 g of the substance to be examined in methanol R and dilute to 10.0 ml with the same STORAGE solvent. Protected from light. 1492


Econazole nitrate



Solubility : very slightly soluble in water, soluble in methanol, sparingly soluble in methylene chloride, slightly soluble in alcohol. mp : about 165 °C, with decomposition. IDENTIFICATION Infrared absorption spectrophotometry (2.2.24). Comparison : Ph. Eur. reference spectrum of econazole nitrate.

A. (1RS)-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanol,

B. (2RS)-2-[(4-chlorobenzyl)oxy]-2-(2,4-dichlorophenyl)ethanamine,

C. 1-(4-chlorobenzyl)-3-[(2RS)-2-[(4-chlorobenzyl)oxy]-2-(2,4dichlorophenyl)ethyl]imidazolium.

TESTS Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 0.100 g of the substance to be examined in methanol R and dilute to 10.0 ml with the same solvent. Reference solution (a). Dissolve 10 mg of econazole for system suitability CRS in methanol R and dilute to 1.0 ml with the same solvent. Reference solution (b). Dilute 1.0 ml of the test solution to 20.0 ml with methanol R. Dilute 1.0 ml of the solution to 25.0 ml with methanol R. Column : — size : l = 0.10 m, Ø = 4.6 mm, — stationary phase : base-deactivated octadecylsilyl silica gel for chromatography R (3 µm), — temperature : 35 °C. Mobile phase : — mobile phase A : mix 20 volumes of methanol R and 80 volumes of a 0.77 g/l solution of ammonium acetate R, — mobile phase B : methanol R, acetonitrile R (40:60 V/V), Time (min)



0 - 25

60 → 10

40 → 90

25 - 27

10

90

27 - 28

10 → 60

90 → 40

28 - 33

60

40

01/2005:0665 Flow rate : 1.5 ml/min. corrected Detection : spectrophotometer at 225 nm. Injection : 10 µl. ECONAZOLE NITRATE Relative retention with reference to econazole (retention time = about 15 min) : impurity A = about 0.2 ; impurity B = about 0.6 ; impurity C = about 1.1. Econazoli nitras System suitability : reference solution (a) : — peak-to-valley ratio : minimum of 1.5, where Hp = height above the baseline of the peak due to impurity C, and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to econazole. Limits : — correction factor : for the calculation of content, multiply the peak area of impurity A by 1.4, — impurities A, B, C : for each impurity, not more than the area of the principal peak in the chromatogram obtained C18H16Cl3N3O4 Mr 444.7 with reference solution (b) (0.2 per cent), DEFINITION — total : not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference 1-[(2RS)-2-[(4-Chlorobenzyl)oxy]-2-(2,4-dichlorophenyl)ethyl]solution (b) (0.3 per cent), 1H-imidazole nitrate. — disregard limit : 0.25 times the area of the principal peak Content : 99.0 per cent to 101.0 per cent (dried substance). in the chromatogram obtained with reference solution (b) CHARACTERS (0.05 per cent) ; disregard the peak due to the nitrate ion at the beginning of the chromatogram. Appearance : white or almost white crystalline powder. General Notices (1) apply to all monographs and other texts

1493

Edetic acid


Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for 4 h.

01/2005:1612

EDETIC ACID

Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g.

Acidum edeticum

ASSAY Dissolve 0.400 g in 50 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 44.47 mg of C18H16Cl3N3O4.

Mr 292.2

DEFINITION (Ethylenedinitrilo)tetraacetic acid. Content : 98.0 per cent to 101.0 per cent.


CHARACTERS Appearance : white, crystalline powder or colourless crystals. Solubility : practically insoluble in water and in alcohol. It dissolves in dilute solutions of alkali hydroxides.


A. (1RS)-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanol,

B. (2RS)-2-[(4-chlorobenzyl)oxy]-2-(2,4-dichlorophenyl)ethanamine,

C. 1-(4-chlorobenzyl)-3-[(2RS)-2-[(4-chlorobenzyl)oxy]-2-(2,4dichlorophenyl)ethyl]imidazolium. 1494

C10H16N2O8

IDENTIFICATION First identification : A. Second identification : B, C. A. Infrared absorption spectrophotometry (2.2.24). Preparation : discs, after drying the substance to be examined in an oven at 100-105 °C for 2 h. Comparison : sodium edetate R, treated as follows : dissolve 0.25 g of sodium edetate R in 5 ml of water R, add 1.0 ml of dilute hydrochloric acid R. Filter, wash the residue with 2 quantities, each of 5 ml, of water R and dry the residue in an oven at 100-105 °C for 2 h. B. To 5 ml of water R add 0.1 ml of ammonium thiocyanate solution R and 0.1 ml of ferric chloride solution R1 and mix. The solution is red. Add 0.5 ml of solution S (see Tests). The solution becomes yellowish. C. To 10 ml of solution S add 0.5 ml of calcium chloride solution R. Make alkaline to red litmus paper R by the addition of dilute ammonia R2 and add 3 ml of ammonium oxalate solution R. No precipitate is formed. TESTS Solution S. Dissolve 5.0 g in 20 ml of dilute sodium hydroxide solution R and dilute to 100 ml with water R. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Impurity A. Liquid chromatography (2.2.29). Carry out the test protected from light. Solvent mixture. Dissolve 10.0 g of ferric sulphate pentahydrate R in 20 ml of 0.5 M sulphuric acid and add 780 ml of water R. Adjust to pH 2.0 with 1 M sodium hydroxide and dilute to 1000 ml with water R. Test solution. Dissolve 0.100 g of the substance to be examined in 1.0 ml of 1 M sodium hydroxide and dilute to 25.0 ml with the solvent mixture. Reference solution. Dissolve 40.0 mg of nitrilotriacetic acid R in the solvent mixture and dilute to 100.0 ml with the solvent mixture. To 1.0 ml of the solution add 0.1 ml of the test solution and dilute to 100.0 ml with the solvent mixture. Column : — size : l = 0.10 m, Ø = 4.6 mm, — stationary phase : spherical graphitised carbon for chromatography R1 (5 µm) with a specific surface area of 120 m2/g and a pore size of 25 nm.


Edrophonium chloride


Mobile phase : dissolve 50.0 mg of ferric sulphate pentahydrate R in 50 ml of 0.5 M sulphuric acid and add 750 ml of water R. Adjust to pH 1.5 with 0.5 M sulphuric acid or 1 M sodium hydroxide, add 20 ml of ethylene glycol R and dilute to 1000 ml with water R. Flow rate : 1 ml/min. Detection : spectrophotometer at 273 nm. Injection : 20 µl ; filter the solutions and inject immediately. Run time : 4 times the retention time of the iron complex of impurity A. Retention time : iron complex of impurity A = about 5 min ; iron complex of edetic acid = about 10 min. System suitability : reference solution : — resolution : minimum 7 between the peaks due to the iron complex of impurity A and the iron complex of edetic acid, — signal-to-noise ratio : minimum 50 for the peak due to impurity A. Limit : — impurity A : not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.1 per cent). Chlorides (2.4.4) : maximum 200 ppm. To 10 ml of solution S add 8 ml of nitric acid R and stir for 10 min. A precipitate is formed. Filter and wash the filter with water R. Collect the filtrate and the washings and dilute to 20 ml with water R. Dilute 10 ml of this solution to 15 ml with water R. Iron (2.4.9) : maximum 80 ppm. Dilute 2.5 ml of solution S to 10 ml with water R and add 0.25 g of calcium chloride R before adding the thioglycollic acid R. Allow to stand for 5 min. Also add 0.25 g of calcium chloride R to the standard. Heavy metals (2.4.8) : maximum 20 ppm. 1.0 g complies with limit test D. Use a fused-silica crucible. If necessary moisten the residue on ignition with nitric acid R and ignite until a white residue is obtained. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Sulphated ash (2.4.14) : maximum 0.2 per cent, determined on 1.0 g. ASSAY Dissolve 0.250 g in 2.0 ml of dilute sodium hydroxide solution R and dilute to 300 ml with water R. Add 2 g of hexamethylenetetramine R and 2 ml of dilute hydrochloric acid R. Titrate with 0.1 M zinc sulphate using about 50 mg of xylenol orange triturate R as indicator. 1 ml of 0.1 M zinc sulphate corresponds to 29.22 mg of C10H16N2O8. STORAGE Protected from light. IMPURITIES Specified impurities : A.

A. nitrilotriacetic acid. General Notices (1) apply to all monographs and other texts

01/2005:2106

EDROPHONIUM CHLORIDE Edrophonii chloridum

C10H16ClNO

Mr 201.7

DEFINITION N-Ethyl-3-hydroxy-N,N-dimethylanilinium chloride. Content : 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS Appearance : white or almost white, crystalline powder. Solubility : very soluble in water, freely soluble in ethanol (96 per cent), practically insoluble in methylene chloride. IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24). Comparison : edrophonium chloride CRS. B. It gives reaction (a) of chlorides (2.3.1). TESTS Appearance of solution. The solution is clear (2.2.1) and colourless (2.2.2, Method II). Dissolve 0.5 g in water R and dilute to 25 ml with the same solvent. pH (2.2.3) : 4.0 to 5.0. Dissolve 1.0 g in carbon dioxide-free water R and dilute to 10.0 ml with the same solvent. Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 50.0 mg in water R and dilute to 50.0 ml with the same solvent. Reference solution (a). Dissolve 10.0 mg of 3-dimethylaminophenol R in acetonitrile R and dilute to 10.0 ml with the same solvent. Reference solution (b). Mix 1.0 ml of the test solution and 1.0 ml of reference solution (a) and dilute to 100.0 ml with water R. Dilute 10.0 ml of this solution to 100.0 ml with water R. Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase : styrene-divinylbenzene copolymer R (8-10 µm). Mobile phase : mix 10 volumes of acetonitrile R and 90 volumes of a 7.7 g/l solution of tetramethylammonium bromide R previously adjusted to pH 3.0 with phosphoric acid R. Flow rate : 1 ml/min. Detection : spectrophotometer at 281 nm. Injection : 20 µl. Run time : twice the retention time of edrophonium. Relative retention with reference to edrophonium (retention time = about 3.8 min) : impurity A = about 1.3. System suitability : reference solution (b) : — resolution : minimum 2.0 between the peaks due to edrophonium and impurity A. 1495

Elder flower


B. Reduce to a powder (355). The powder is greenish-yellow. Limits : Examine under a microscope using chloral hydrate — impurity A : not more than the area of the corresponding solution R. The powder shows numerous spherical, peak in the chromatogram obtained with reference sometimes ellipsoidal, pollen grains about 30 µm in solution (b) (0.1 per cent), diameter, with three germinal pores and very finely pitted — any other impurity : for each impurity, not more than the exine ; calyx epidermal cells with a striated cuticle and area of the peak due to edrophonium in the chromatogram occasional unicellular marginal teeth from the basal obtained with reference solution (b) (0.1 per cent), region ; corolla fragments with numerous small globules — total: not more than 5 times the area of the peak due of volatile oil, those of the upper epidermis with slightly to edrophonium in the chromatogram obtained with thickened and beaded walls and a striated cuticle ; reference solution (b) (0.5 per cent), mesophyll cells of petals and sepals with idioblasts — disregard limit: 0.5 times the area of the peak due containing numerous sandy crystals of calcium oxalate. to edrophonium in the chromatogram obtained with C. Examine in ultraviolet light at 365 nm the chromatograms reference solution (b) (0.05 per cent). obtained in the test for Sambucus ebulus. The Loss on drying (2.2.32) : maximum 0.5 per cent, determined chromatogram obtained with the test solution shows an on 1.000 g by drying in a desiccator over diphosphorus intense zone of light blue fluorescence due to chlorogenic pentoxide R at a pressure not exceeding 0.7 kPa for 24 h. acid, a zone of orange fluorescence due to rutin as well as a zone of orange fluorescence due to isoquercitrin, which Sulphated ash (2.4.14) : maximum 0.1 per cent, determined appears slightly above the zone due to hyperoside in on 1.0 g. the chromatogram obtained with the reference solution. Bacterial endotoxins (2.6.14) : less than 8.3 IU/mg. A zone of greenish-blue fluorescence appears in the chromatogram obtained with the test solution a little ASSAY below the caffeic acid zone in the chromatogram obtained Dissolve 0.150 g in 60 ml of a mixture of equal volumes of with the reference solution. Additional faint fluorescent acetic anhydride R and anhydrous acetic acid R. Titrate zones may be present. In daylight, only the orange with 0.1 M perchloric acid, determining the end-point fluorescent zones due to rutin and isoquercitroside in potentiometrically (2.2.20). the chromatogram obtained with the test solution are 1 ml of 0.1 M perchloric acid is equivalent to 20.17 mg clearly visible. of C10H16ClNO. STORAGE Protected from light. IMPURITIES Specified impurities : A.

A. 3-(dimethylamino)phenol.

ELDER FLOWER

TESTS Foreign matter (2.8.2). Determined on 10 g, not more than 8 per cent of fragments of coarse pedicels and other foreign matter ; not more than 15 per cent of discoloured, brown flowers. Sambucus ebulus L. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating substance. Test solution. To 0.5 g of the powdered drug (355) add 10 ml of methanol R and heat in a water bath at 65 °C for 5 min, shaking frequently. Allow to cool and filter. Dilute the filtrate to 10 ml with methanol R. Reference solution. Dissolve 1 mg of caffeic acid R, 1 mg of 01/2005:1217 chlorogenic acid R, 2.5 mg of hyperoside R and 2.5 mg of rutin R in 10 ml of methanol R.

Apply separately to the plate, as bands, 10 µl of each solution. Develop over a path of 15 cm using a mixture of 10 volumes of anhydrous formic acid R, 10 volumes of water R, 30 volumes Sambuci flos of methyl ethyl ketone R and 50 volumes of ethyl acetate R. DEFINITION Dry the plate at 100-105 °C and spray it while still warm Elder flower consists of the dried flowers of Sambucus with a 10 g/l solution of diphenylboric acid aminoethyl nigra L. It contains not less than 0.80 per cent of flavonoids, ester R in methanol R. Subsequently spray the plate with a calculated as isoquercitroside (C21H20O12 ; Mr 464.4) with 50 g/l solution of macrogol 400 R in methanol R. Allow the reference to the dried drug. plate to dry in air for 30 min and examine in ultraviolet light at 365 nm. The chromatogram obtained with the reference CHARACTERS solution shows in the lower half, with increasing Rf values, It has the macroscopic and microscopic characters described the zone of orange fluorescence due to rutin, the zone of under identification tests A and B. light blue fluorescence due to chlorogenic acid and the zone of orange-yellow to orange-brown fluorescence due to IDENTIFICATION hyperoside. The upper third presents a zone of greenish-blue A. The flower, about 5 mm in diameter, has three small fluorescence due to caffeic acid. The chromatogram obtained bracts (hand lens) and may have a peduncle. The with the test solution does not show a pink zone below the five-toothed calyx is small ; the corolla is light yellow, with zone due to rutin in the chromatogram obtained with the five broadly oval petals fused at their bases into a tube. reference solution. The filaments of the five yellow stamens alternate with Loss on drying (2.2.32). Not more than 10.0 per cent, the petals. The corolla is often isolated or attached to determined on 1.000 g of the powdered drug (355) by drying the stamens, to which it is fused at the base. The ovary in an oven at 100-105 °C for 2 h. is inferior with three locules and it bears a short style with three obtuse stigmata. Total ash (2.4.16). Not more than 10.0 per cent. 1496


Eleutherococcus


ASSAY fibres in the bark and is coarsely fibrous, especially in the internal part of the xylem. The lower surface bears Stock solution. In a 100 ml round-bottomed flask, introduce numerous cylindrical and knotty roots, 3.5 cm to 15 cm 0.600 g of the powdered drug (355), add 1 ml of a 5 g/l long and 0.3 cm to 1.5 cm in diameter ; with a smooth, solution of hexamethylenetetramine R, 20 ml of acetone R greyish-brown to blackish-brown surface ; the bark is and 2 ml of hydrochloric acid R1. Boil the mixture under a about 0.5 mm thick, closely adhering to the pale yellow reflux condenser for 30 min. Filter the liquid through a plug xylem ; the fracture is slightly fibrous ; in places where of absorbent cotton into a flask. Add the absorbent cotton to the outer layer has been removed, the outer surface is the residue in the round-bottomed flask and extract with two yellowish-brown. quantities, each of 20 ml, of acetone R, each time boiling under a reflux condenser for 10 min. Allow to cool, filter each B. Reduce to a powder (355). The powder is yellowish-brown. extract through the plug of absorbent cotton into the flask. Examine under a microscope, using chloral hydrate After cooling, filter the combined acetone extracts through a solution R. The powder shows numerous groups of paper-filter into a volumetric flask, dilute to 100.0 ml with thick-walled, lignified fibres ; fragments of reticulate and acetone R by rinsing the flask and the paper-filter. Introduce bordered pitted vessels with a wide lumen ; groups of 20.0 ml of the solution into a separating funnel, add 20 ml of secretory canals, up to 20 µm in diameter with brown water R and shake the mixture with one quantity of 15 ml contents ; parenchymatous cells containing cluster and then three quantities, each of 10 ml, of ethyl acetate R. crystals of calcium oxalate 10 µm to 50 µm in diameter. Combine the ethyl acetate extracts in a separating funnel, Examine under a microscope, using a 50 per cent V/V wash with two quantities, each of 50 ml, of water R, and filter solution of glycerol R. The powder shows small starch the extracts over 10 g of anhydrous sodium sulphate R into granules, rounded to slightly angular in outline, single a volumetric flask and dilute to 50.0 ml with ethyl acetate R. compounds or with 2 or 3 components. Test solution. To 10.0 ml of the stock solution add 1 ml C. Thin-layer chromatography (2.2.27). of aluminium chloride reagent R and dilute to 25.0 ml Test solution. To 1.0 g of the powdered drug (355) add with a 5 per cent V/V solution of glacial acetic acid R in 10 ml of alcohol (50 per cent V/V) R and boil under methanol R. reflux for 1 h. Cool and filter. Evaporate the filtrate to Compensation solution. Dilute 10.0 ml of the stock solution dryness on a water-bath. Dissolve the residue in 2.5 ml to 25.0 ml with a 5 per cent V/V solution of glacial acetic of a mixture of 5 volumes of water R and 20 volumes of acid R in methanol R. alcohol (50 per cent V/V) R and filter. Measure the absorbance (2.2.25) of the test solution after Reference solution. Dissolve 2.0 mg of esculin R and 30 min, by comparison with the compensation solution at 2.0 mg of catalpol R in 20 ml of a mixture of 2 volumes of 425 nm. Calculate the percentage content of flavonoids, water R and 8 volumes of alcohol (50 per cent V/V) R. calculated as isoquercitroside, from the expression : Plate : TLC silica gel plate R. Mobile phase : water R, methanol R, methylene chloride R (4:30:70 V/V/V). Application : 20 µl, as bands. i.e. taking the specific absorbance of isoquercitroside to Development : over a path of 10 cm. be 500. Drying : in air. A = absorbance at 425 nm, Detection A : examine in ultraviolet light at 365 nm. m = mass of the substance to be examined, in grams. Results A : the chromatogram obtained with the reference solution shows in the upper half a blue fluorescent zone STORAGE (esculin). Store protected from light. Detection B : spray with anisaldehyde solution R and examine in daylight while heating at 100-105 °C for 01/2005:1419 5-10 min. corrected Results B : see below the sequence of the zones present in the chromatograms obtained with the reference solution ELEUTHEROCOCCUS and the test solution. Furthermore, other faint zones are present in the chromatogram obtained with the test Eleutherococci radix solution. DEFINITION Top of the plate Dried, whole or cut underground organs of Eleutherococcus A brown zone (eleutheroside B) senticosus (Rupr. et Maxim.) Maxim. Esculin : a blue fluorescent zone Content : minimum 0.08 per cent for the sum of (marked at 365 nm) eleutheroside B (Mr 372.4) and eleutheroside E (Mr 742.7). CHARACTERS Macroscopic and microscopic characters described under identification tests A and B. IDENTIFICATION A. The rhizome is knotty, of irregular cylindrical shape, 1.5 cm to 4.0 cm in diameter ; the surface is rugged, longitudinally wrinkled and greyish-brown to blackish-brown ; the bark, about 2 mm thick, closely adheres to the xylem ; the heartwood is light brown and the sapwood is pale yellow ; the fracture shows short thin General Notices (1) apply to all monographs and other texts

A reddish-brown zone (eleutheroside E)

_______

_______

Catalpol: a violet-brown zone _______

_______ 2 brown zones

Reference solution

Test solution

TESTS Foreign matter (2.8.2) : maximum 3 per cent. 1497

Eleutherococcus


Loss on drying (2.2.32) : maximum 10.0 per cent, determined on 1.000 g of the powdered drug (355) by drying in an oven at 100-105 °C for 2 h. Total ash (2.4.16) : maximum 8.0 per cent. ASSAY Liquid chromatography (2.2.29).

Figure 1419.-1. – UV spectrum of eleutheroside B for the assay of eleutherococcus Test solution. To 0.500 g of the powdered drug (355) in a 100 ml round-bottomed flask, add 30 ml of a mixture of equal volumes of alcohol R and water R. Heat in a water-bath at 60 °C for 30 min. Allow to cool and filter through a sintered-glass filter. Collect the liquid in a 250 ml round-bottomed flask. Repeat this operation twice, using the residue obtained in the filtration step instead of the powdered drug. Add both fractions of supernatant liquid to the 250 ml round-bottomed flask. Evaporate under reduced pressure until about 10 ml of supernatant liquid is left in the flask. Transfer the supernatant liquid quantitatively to a 20.0 ml volumetric flask and dilute to 20.0 ml with a mixture of equal volumes of alcohol R and water R. Filter through a nylon filter (pore size 0.45 µm). 1498

Reference solution (a). Dissolve 10 mg of ferulic acid R in a mixture of equal volumes of methanol R and water R and dilute to 20.0 ml with the same mixture of solvents. Reference solution (b). Dissolve 10 mg of caffeic acid R in a mixture of equal volumes of methanol R and water R and dilute to 20.0 ml with the same mixture of solvents. Reference solution (c). Transfer 1 ml of reference solution (a) to a 25 ml volumetric flask and dilute to 25.0 ml with a mixture of equal volumes of methanol R and water R. Filter through a nylon filter (pore size 0.45 µm). Reference solution (d). Transfer 1 ml of reference solution (a) and 1 ml of reference solution (b) in a mixture of equal volumes of methanol R and water R and dilute to 25.0 ml with the same mixture of solvents. Filter through a nylon filter (pore size 0.45 µm).

Figure 1419.-2. – UV spectrum of eleutheroside E for the assay of eleutherococcus


Emetine hydrochloride heptahydrate


01/2005:0080

Precolumn : — size : l = 0.04 m, Ø = 4.6 mm,

EMETINE HYDROCHLORIDE HEPTAHYDRATE

— stationary phase: octadecylsilyl silica gel for chromatography R (5 µm).

Emetini hydrochloridum heptahydricum

Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase: octadecylsilyl silica gel for chromatography R (5 µm). Mobile phase : — mobile phase A : phosphoric acid R, water R (0.5:99.5 V/V),

C29H42Cl2N2O4,7H2O

— mobile phase B : acetonitrile for chromatography R, Time (min)



0-5

90

10

5 - 27

90 → 80

10 → 20

27 - 30

80 → 50

20 → 50

30 - 35

50

50

35 - 40

50 → 90

50 → 10

Mr 680

DEFINITION Emetine hydrochloride heptahydrate contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of (2S,3R,11bS)-2-[[(1R)-6,7-dimethoxy1,2,3,4-tetrahydroisoquinolin-1-yl]methyl]-3-ethyl-9,10dimethoxy-1,3,4,6,7,11b-hexahydro-2H-benzo[a]quinolizine dihydrochloride, calculated with reference to the dried substance. CHARACTERS A white or slightly yellowish, crystalline powder, freely soluble in water and in alcohol.


IDENTIFICATION First identification : A, E. Detection : spectrophotometer at 220 nm. Second identification : B, C, D, E. Injection : 20 µl of the test solution and reference A. Examine by infrared absorption spectrophotometry solutions (c) and (d). (2.2.24), comparing with the spectrum obtained with emetine hydrochloride CRS. Retention time : eleutheroside B = about 10 min ; B. Examine the chromatograms obtained in the test for eleutheroside E = about 22 min. related substances in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the Locate the peaks due to eleutheroside B and eleutheroside E test solution is similar in position, fluorescence and size using the UV spectra shown in Figures 1419.-1 and 1419.-2. to the spot in the chromatogram obtained with reference solution (a). System suitability : reference solution (d) : C. Dissolve about 10 mg in 2 ml of dilute hydrogen peroxide solution R, add 1 ml of hydrochloric acid R and heat. — resolution : minimum 15 between the peaks due to caffeic An orange colour develops. acid and ferulic acid. D. Sprinkle about 5 mg on the surface of 1 ml of Calculate the total percentage content of eleutheroside B sulphomolybdic reagent R2. A bright-green colour and eleutheroside E from the expression : develops. E. It gives reaction (a) of chlorides (2.3.1).

AB

=

area of the peak due to eleutheroside B in the chromatogram obtained with the test solution,

AE

=

area of the peak due to eleutheroside E in the chromatogram obtained with the test solution,

AR

=

area of the peak due to ferulic acid in the chromatogram obtained with reference solution (c),

C

=

concentration of ferulic acid in reference solution (c), in micrograms per millilitre,

m

=

mass of the drug to be examined, in milligrams.


TESTS Solution S. Dissolve 1.25 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y5 or BY5 (2.2.2, Method II). pH (2.2.3). Dilute 4 ml of solution S to 10 ml with carbon dioxide-free water R. The pH of the solution is 4.0 to 6.0. Specific optical rotation (2.2.7). Dissolve in water R a quantity of the substance to be examined corresponding to 1.250 g of dried substance and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 16 to + 19, calculated with reference to the dried substance. 1499

Emetine hydrochloride pentahydrate


Related substances. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R. Prepare the solutions immediately before use. Test solution. Dissolve 50 mg of the substance to be examined in methanol R containing 1 per cent V/V of dilute ammonia R2 and dilute to 100 ml with the same solvent. Reference solution (a). Dissolve 50 mg of emetine hydrochloride CRS in methanol R containing 1 per cent V/V of dilute ammonia R2 and dilute to 100 ml with the same solvent.

01/2005:0081

EMETINE HYDROCHLORIDE PENTAHYDRATE Emetini hydrochloridum pentahydricum

Reference solution (b). Dissolve 10 mg of isoemetine hydrobromide CRS in methanol R containing 1 per cent V/V of dilute ammonia R2 and dilute to 100 ml with the same solvent. Dilute 5 ml of this solution to 50 ml with methanol R containing 1 per cent V/V of dilute ammonia R2. Reference solution (c). Dissolve 10 mg of cephaëline C H Cl N O ,5H O Mr 644 hydrochloride CRS in methanol R containing 1 per cent V/V 29 42 2 2 4 2 of dilute ammonia R2 and dilute to 100 ml with the same DEFINITION solvent. Dilute 5 ml of this solution to 50 ml with methanol R Emetine hydrochloride pentahydrate contains not less containing 1 per cent V/V of dilute ammonia R2. than 98.0 per cent and not more than the equivalent of Reference solution (d). Dilute 1 ml of reference solution (a) 102.0 per cent of (2S,3R,11bS)-2-[[(1R)-6,7-dimethoxyto 100 ml with methanol R containing 1 per cent V/V of 1,2,3,4-tetrahydroisoquinolin-1-yl]methyl]-3-ethyl-9,10dilute ammonia R2. dimethoxy-1,3,4,6,7,11b-hexahydro-2H-benzo[a]quinolizine dihydrochloride, calculated with reference to the dried Reference solution (e). To 1 ml of reference solution (a) substance. add 1 ml of reference solution (b) and 1 ml of reference solution (c). CHARACTERS Apply to the plate 10 µl of the test solution and each of A white or slightly yellowish, crystalline powder, freely reference solutions (a), (b), (c) and (d) and 30 µl of reference soluble in water and in alcohol. solution (e). Develop over a path of 15 cm using a mixture IDENTIFICATION of 0.5 volumes of diethylamine R, 2 volumes of water R, 5 volumes of methanol R, 20 volumes of ethylene glycol First identification : A, E. monomethyl ether R and 100 volumes of chloroform R. Allow the plate to dry in air until the solvent has evaporated. Second identification : B, C, D, E. Spray in a well ventilated fume-cupboard with chloroformic A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with solution of iodine R and heat at 60 °C for 15 min. Examine emetine hydrochloride CRS. in ultraviolet light at 365 nm. In the chromatogram obtained with the test solution, any spots corresponding to isoemetine B. Examine the chromatograms obtained in the test for and cephaëline are not more intense than the spots in the related substances in ultraviolet light at 365 nm. The chromatograms obtained with reference solutions (b) and principal spot in the chromatogram obtained with the (c) respectively (2.0 per cent) ; any spot, apart from the test solution is similar in position, fluorescence and size principal spot and the spots corresponding to isoemetine to the spot in the chromatogram obtained with reference and cephaëline, is not more intense than the spot in the solution (a). chromatogram obtained with reference solution (d) (1.0 per cent). The test is not valid unless the chromatogram obtained C. Dissolve about 10 mg in 2 ml of dilute hydrogen peroxide solution R, add 1 ml of hydrochloric acid R and heat. with reference solution (e) shows three clearly separated An orange colour develops. spots. D. Sprinkle about 5 mg on the surface of 1 ml of Loss on drying (2.2.32). 15.0 per cent to 19.0 per cent, sulphomolybdic reagent R2. A bright-green colour determined on 1.00 g by drying in an oven at 100 °C to develops. 105 °C for 3 h. E. It gives reaction (a) of chlorides (2.3.1). Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. TESTS Solution S. Dissolve 1.25 g in carbon dioxide-free water R ASSAY and dilute to 25 ml with the same solvent. Dissolve 0.200 g in a mixture of 5.0 ml of 0.01 M hydrochloric Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y5 or BY5 acid and 50 ml of alcohol R. Carry out a potentiometric (2.2.2, Method II). titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the 2 points of inflexion. pH (2.2.3). Dilute 4 ml of solution S to 10 ml with carbon dioxide-free water R. The pH of the solution is 4.0 to 6.0. 1 ml of 0.1 M sodium hydroxide is equivalent to 27.68 mg of C29H42Cl2N2O4. Specific optical rotation (2.2.7). Dissolve in water R a quantity of the substance to be examined corresponding to 1.250 g of dried substance and dilute to 25.0 ml with the STORAGE same solvent. The specific optical rotation is + 16 to + 19, Store protected from light. calculated with reference to the dried substance. 1500



Related substances. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R. Prepare the solutions immediately before use. Test solution. Dissolve 50 mg of the substance to be examined in methanol R containing 1 per cent V/V of dilute ammonia R2 and dilute to 100 ml with the same solvent.

Enalapril maleate

01/2005:1420

ENALAPRIL MALEATE Enalaprili maleas

Reference solution (a). Dissolve 50 mg of emetine hydrochloride CRS in methanol R containing 1 per cent V/V of dilute ammonia R2 and dilute to 100 ml with the same solvent. Reference solution (b). Dissolve 10 mg of isoemetine hydrobromide CRS in methanol R containing 1 per cent V/V of dilute ammonia R2 and dilute to 100 ml with the same C24H32N2O9 Mr 492.5 solvent. Dilute 5 ml of this solution to 50 ml with methanol R containing 1 per cent V/V of dilute ammonia R2. DEFINITION Enalapril maleate contains not less than 98.5 per Reference solution (c). Dissolve 10 mg of cephaëline hydrochloride CRS in methanol R containing 1 per cent V/V cent and not more than the equivalent of 101.5 per cent of (2S)-1-[(2S)-2-[[(1S)-1-(ethoxycarbonyl)-3of dilute ammonia R2 and dilute to 100 ml with the same phenylpropyl]amino]propanoyl]pyrrolidine-2-carboxylic acid solvent. Dilute 5 ml of this solution to 50 ml with methanol R (Z)-butenedioate, calculated with reference to the dried containing 1 per cent V/V of dilute ammonia R2. substance. Reference solution (d). Dilute 1 ml of reference solution (a) CHARACTERS to 100 ml with methanol R containing 1 per cent V/V of dilute ammonia R2. A white or almost white crystalline powder, sparingly soluble in water, freely soluble in methanol, practically insoluble in Reference solution (e). To 1 ml of reference solution (a) methylene chloride. It dissolves in dilute solutions of alkali add 1 ml of reference solution (b) and 1 ml of reference hydroxides. solution (c). Apply to the plate 10 µl of the test solution and each of reference solutions (a), (b), (c) and (d) and 30 µl of reference solution (e). Develop over a path of 15 cm using a mixture of 0.5 volumes of diethylamine R, 2 volumes of water R, 5 volumes of methanol R, 20 volumes of ethylene glycol monomethyl ether R and 100 volumes of chloroform R. Allow the plate to dry in air until the solvent has evaporated. Spray in a well ventilated fume-cupboard with chloroformic solution of iodine R and heat at 60 °C for 15 min. Examine in ultraviolet light at 365 nm. In the chromatogram obtained with the test solution, any spots corresponding to isoemetine and cephaëline are not more intense than the spots in the chromatograms obtained with reference solutions (b) and (c) respectively (2.0 per cent) ; any spot, apart from the principal spot and the spots corresponding to isoemetine and cephaëline, is not more intense than the spot in the chromatogram obtained with reference solution (d) (1.0 per cent). The test is not valid unless the chromatogram obtained with reference solution (e) shows three clearly separated spots. Loss on drying (2.2.32). 11.0 per cent to 15.0 per cent, determined on 1.00 g by drying in an oven at 100 °C to 105 °C for 3 h.

IDENTIFICATION First identification : B. Second identification : A, C, D. A. Melting point (2.2.14) : 143 °C to 145 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with enalapril maleate CRS. C. Dissolve about 30 mg in 3 ml of water R. Add 1 ml of bromine water R and heat on a water bath until bromine has disappeared completely and cool. To 0.2 ml of this solution, add 3 ml of a 3 g/l solution of resorcinol R in sulphuric acid R and heat on a water bath for 15 min. A reddish-brown colour develops. D. To about 30 mg, add 0.5 ml of a 100 g/l solution of hydroxylamine hydrochloride R in methanol R and 1.0 ml of a 100 g/l solution of potassium hydroxide R in alcohol R. Heat to boiling, allow to cool and acidify with dilute hydrochloric acid R. Add 0.2 ml of ferric chloride solution R1 diluted 1 to 10 ; a reddish-brown colour appears.

TESTS Solution S. Dissolve 0.25 g in carbon dioxide-free water R Sulphated ash (2.4.14). Not more than 0.1 per cent, and dilute to 25.0 ml with the same solvent. determined on 1.0 g. Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II). ASSAY Dissolve 0.200 g in a mixture of 5.0 ml of 0.01 M hydrochloric pH (2.2.3). The pH of solution S is 2.4 to 2.9. Specific optical rotation (2.2.7) : − 48 to − 51, determined acid and 50 ml of alcohol R. Carry out a potentiometric on solution S and calculated with reference to the dried titration (2.2.20), using 0.1 M sodium hydroxide. Read substance. the volume added between the two points of inflexion. Related substances. Examine by liquid chromatography 1 ml of 0.1 M sodium hydroxide is equivalent to 27.68 mg (2.2.29). of C29H42Cl2N2O4. Buffer solution A. Dissolve 2.8 g of sodium dihydrogen phosphate monohydrate R in 950 ml of water R. Adjust to STORAGE pH 2.5 with phosphoric acid R and dilute to 1000 ml with water R. Store protected from light. General Notices (1) apply to all monographs and other texts

1501

Enalapril maleate


Buffer solution B. Dissolve 2.8 g of sodium dihydrogen phosphate monohydrate R in 950 ml of water R. Adjust to pH 6.8 with strong sodium hydroxide solution R and dilute to 1000 ml with water R. Dissolution mixture. Mix 50 ml of acetonitrile R1 and 950 ml of buffer solution A. Test solution. Dissolve 30.0 mg of the substance to be examined in the dissolution mixture and dilute to 100.0 ml with the same mixture. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the dissolution mixture. Reference solution (b). Dissolve 3.0 mg of enalapril for system suitability CRS in the dissolution mixture and dilute to 10.0 ml with the same mixture. The chromatographic procedure may be carried out using : — a stainless steel column 0.15 m long and 4.1 mm in internal diameter packed with styrene-divinylbenzene copolymer R (5 µm), — as mobile phase at a flow rate of 1.4 ml/min, Mobile phase A. Mix 50 ml of acetonitrile R1 and 950 ml of buffer solution B, Mobile phase B. Mix 340 ml of buffer solution B and 660 ml of acetonitrile R1, Time (min)



0 - 20

95 → 40

5 → 60

20 - 25

40

60

25 - 26

40 → 95

60 → 5

26 - 30

95

5

STORAGE Store protected from light. IMPURITIES Specified impurities : A, B, C, D, E, H. Other detectable impurities : F, G, I.

A. (2S)-1-[(2S)-2-[[(1R)-1-(ethoxycarbonyl)-3phenylpropyl]amino]propanoyl]pyrrolidine-2-carboxylic acid,

B.

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