Analytical profiles of drug substances and excipients

Analytical Profiles of Substances Volume 8 Edited by

Klaus Florey The Squibb Institute for Medical Research New Brunswick, New Jersey

Contributing Editors

Norman W. Atwater Lester Chafetz Rafik Bishara Boen T. Kho Glenn A. Brewer, Jr. Hans-Georg Leemann Bruce C. Rudy Compiled under the auspices of the Pharmaceutical Analysis and Control Section Academy of Pharmaceutical Sciences

Academic Press New York San Francisco London 1979 A Subsidiary of Harcourt Brace Jovanovich. Publishers

EDITORIAL BOARD Norman W. Atwater Rafik Bishara Jerome I. Bodin Glenn A. Brewer, Jr. Lester Chafetz Edward M. Cohen Klaus Florey Salvatore A. Fusari

Derek W. Houghton Erik H. Jensen Boen T. Kho Hans-Georg Leemann Arthur F. Michaelis Gerald J. Papariello Bruce C. Rudy Bernard Z. Senkowski

Academic Press Rapid Manuscript Reproduction

COPYRIGHT @ 1979, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR A N Y INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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79 80 81 82

9 8 7 6 5 4 3 2 1

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS H . Y. Aboul-Enein, Riyadh University, Riyadh, Saudi Arabia A . A . Al-Badr, Riyadh University, Riyadh, Saudi Arabia T. Alexander, Food and Drug Administration, Washington, D.C. N. W. Atwater, E. R. Squibb and Sons, Princeton, New Jersey S . A . Benezra, Burroughs Wellcome Company, Research Triangle Park, North Carolina R. Bishara, Eli Lilly and Company, Indianapolis, Indiana J. I . Bodin, Carter Wallace, Inc., Cranbury, New Jersey G . A . Brewer, The Squibb Institute for Medical Research, New Brunswick, New Jersey L. Chafetz, Warner-Lambert Research Institute, Morris Plains, New Jersey Z. L. Chang, Abbott Laboratories, North Chicago, Illinois A. P . K. Cheung, SRI International, Menlo Park, California E. M . Cohen, Merck Sharp & Dohme, West Point, Pennsylvania R . D . Daley, Ayerst Laboratories, Rouses Point, New York E. Debesis, Hoffmann-LaRoche, Inc., Nutley, New Jersey K . Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey K . Furnkranz, Food and Drug Administration, Washington, D.C. S. A . Fusari, Parke-Davis, Inc., Detroit, Michigan D. A . Giron-Forest, Sandoz Limited, Basel, Switzerland P . E. Crubb, Sterling- Winthrop Research Institute, Rensselaer, New York W. F. Heyes, The Squibb Institute for Medical Research, Moreton, Wirral, England D . W . Houghton, G . D. Searle and Company Ltd., High Wycombe, Bucks, England E. Jensen, The Upjohn Company, Kalamazoo, Michigan B. T. Kho, Ayerst Laboratories, Rouses Point, New York J . Kirschbaum, T h e Squibb Institute for Medical R e s e a r c h , N e w Brunswick, New Jersey vii

viii

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

W . L . Koch, Eli Lilly and Company, Indianapolis, Indiana H. G. Leemann, Sandoz Limited, Basel, Switzerland P . Lim, SRI International, Menlo Park, California H. B. Long, Eli Lilly and Company, Indianapolis, Indiana J. W . McRae, Burroughs Wellcome Company, Research Triangle Park, North Carolina A. F. Michaelis, KV Pharmaceutical Company, St. Louis, Missouri N. G. Nash, Ayerst Laboratories, Rouses Point, New York C. E. Orzech, Ayerst Laboratories, Rouses Point, New York G. Pupariello, Wyeth Laboratories, Philadelphia, Pennsylvania L . 0. Pont, SRI International, Menlo Park, California B. Rudy, Burroughs Wellcome Company, Greenville, North Carolina W . D . SchBnleber, Sandoz Limited, Basel, Switzerland G. Schwartzman, United States Pharmacopeia, Rockville, Maryland G. Selzer, Food and Drug Administration, Washington, D.C. B. Senkowski, Alcon Laboratories, Fort Worth, Texas E. R . Townley, Schering Plough Corporation, Bloomfield, New Jersey L . Wuyland, Food and Drug Administration, Washington, D.C. C-H. Yung, Burroughs Wellcome Company, Research Triangle Park, North Carolina

PREFACE Although the official compendia list tests and limits for drug substances related to identity, purity, and strength, they normally do not provide other physical or chemical data, nor do they list methods of synthesis or pathways of physical or biological degradation and metabolism. For drug substances important enough to be accorded monographs in the official compendia, such supplemental information should also be made readily available. To this end the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences, has undertaken a cooperative venture to compile and publish Analytical Profiles of Drug Substances in a series of volumes of which this is the seventh. The concept of analytical profiles is taking hold not only for compendia1 drugs but, increasingly, in the industrial research laboratories. Analytical profiles are being prepared and periodically updated to provide physicochemical and analytical information of new drug substances during the consecutive stages of research and development. Hopefully, then, in the not too distant future, the publication of an analytical profile will require a minimum of effort whenever a new drug substance is selected for compendial status. The cooperative spirit of our contributors has made this venture possible. All those who have found the profiles useful are requested to contribute a monograph of their own. The editors stand ready to receive such contributions. Thanks to the dedicated efforts of Dr. Morton E. Goldberg, a long cherished dream has come to fruition with the publication of Pharmacological and Biochemical Properties of Drug Substances, M. E. Goldberg, editor, published by APhA Academy of Pharmaceutical Sciences. This new series supplements the comprehensive description of the physical, chemical, and analytical characteristics of drug substances covered in Analytical Profiles of Drug Substances with the equally important description of pharmacological and biochemical properties. ix

X

PREFACE

Drug substances appearing in the new series will be cross-referenced in the cumulative index. The goal to cover all drug substances with comprehensive monographs is still a distant one. It is up to our perseverance to make it a reality. Klaus Florey

Analytical Profiles of Drug Substances, 8

ASPIRIN Klaus Florey I . Introduction 1 . 1 Foreword 1.2 History 2. Description 2. I Name, Formula, Molecular Weight 2.2 Appearance, Color, Odor 3. Synthesis 4. Physical Properties 4.1 Spectra 4.11 Infrared 4.12 Ultraviolet 4.13 Fluorescence-Phosphorescence 4.14 Raman 4.15 Nuclear Magnetic Resonance 4.16 Mass 4.2 Solid Properties 4.2 I Melting Range 4.22 Differential Thermal Analysis 4.23 Thermogravimetric Analysis 4.24 Crystal Properties 4.3 Solution Properties 4.3 I Solubility 4.32 Dissociation Constant (pKa) 4.33 Partition Coefficients 4.34 Dielectric Constant, Dipole Moment 4.35 Radiation Absorption 5. Methods of Analysis 5.1 Historical Synopsis 5.2 Identity and Color Tests 5.3 Quantitative Analysis 5.31 Elemental 5.32 Colorimetric 5.33 Ultraviolet 5.34 Infrared 5.35 Fluorescence-Phosphorescence 5.36 Titrimetric-Electrochemical 5.37 Miscellaneous (NMR) 5.4 Chromatographic Methods 5.41 Paper 5.42 Thin-Layer 5.43 Column I

Copyright @ 1979 by Academic Press, inc. All rights of reproduction in any form reserved. ISBN 0-12-260808-9

2

KLAUS FLOREY 5.44 High Pressure Liquid 5.45 Gas-Liquid 5.5 Electrophoretic Methods 5.6 Determination of Impurities 5.61 Salicylic Acid 5.62 Acetic Acid 5.63 Acetylsalicylic Anhydride and Acetylsalicylsalicylic Acid 6. Stability-Degradation 7. Pharmacokinetics-Drug Metabolism Products 8. Bioavailability-Dissolution 9. Determination in Biological Fluids and Tissues 10. Determination in Pharmaceutical Preparations 1 1 . Acknowledgments 12. References

ASPIRIN

3

Introduction 1.1 Foreword The writing of an analytical profile of aspirin, this drug of drugs, poses two major dilemmas. The first is in the name itself. There are many countries where Aspirin is still a tradename of the German firm Bayer AG, and Acetylsalicylic Acid is used as the generic. Yet, I decided to use the former because it is the U . S . P . and B.P., the better known worldwide and the more elegant name. Aspirin has now been available for close to 80 years, and its usefulness and popularity are undiminished. Consequently, the literature is voluminous and also undiminished. A complete coverage would be beyond the scope of an analytical profile. I have endeavored to cover the newer literature as comprehensibly as possible and have included only those older references which I found of historical interest. To all those who have labored in the vineyard of aspirin and who go unreferenced in this profile, I tender my sincere apologies. 1.

1.2

Bistor d u m e n t e d facts of the discovery of aspirin are quickly told. It was synthesized-by the German chemist Felix Hoffmann (1868-1946) in the laboratories of Farbenfabriken Bayer, Elberfeld, Germany in 1897 (Fig. 1). The compound was tested pharmacologically by H. Dreser' and clinically among others by Wohlgemuth and Witthauer3 who documented the antirheumatic, antipyretic and analgesic properties free of the undesirable side effects of salicylic acid. Apparently, there was some initial reluctance at Bayer to market the new compound since it was thought that the field was already crowded with new drugs. But opposition faded when the new drug got the support of Carl Duisberg, then the general manager of Bayer. Duisberg, of course, was the great chemist and industrialist who built Bayer into the chemical giant of world renown. After the inspired trade name Aspirin, a contraction of acetyl and "spirssure" (salicylic acid), was coined in the offices of Bayer - Euspirin was also considered and fortunately discarded4 -it was marketed in tablet form in 1899 and conquered the world.

KLAUS FLOREY

44

-. .. .

&...

. ,.,... . .

.

..

.

'

,

.

..I .

F i g u r e 1.

L a b o r a t o r y notebook e n t r y of F e l i x Hoffmann, d e s c r i b i n g h i s f i r s t p r e p a r a t i o n of a s p i r i n . The i n i t i a l s CD o n t h e p a g e are t h o s e of C a r l D u i s b e r g . ( C o u r t e s y of Bayer A . G . , L e v e r k u s e n , Germany)

ASPIRIN

5

What motivated Hoffmann to undertake this momentous synthesis? Legend has it that he wanted to help his father who was suffering from rheumatism and who was no longer able to tolerate sodium salicylate, then widely used in rheumatic and arthritic diseases. Salicylic acid occurs naturally in several plants. The analgesic and antipyretic properties of willow bark were already known in antiquity to Hippocrates and the blossoms of spiraea ulmaria (meadow sweet) were used in the middle ages. Salicylic acid was crystallized from willow bark extracts in the early years of the last century, and Kolbe, in 1859, was able to synthesize it from sodium phenolate and carbon dioxide. His student von Heyden worked out a commercially feasible process and started a factory to produce salicylic acid which made possible its widespread use in rheumatic diseases. However, its bad taste, stomach irritation and other side effects were a strong incentive to search for derivatives which retained its efficacy without its disadvantages. Acetylation of the hydroxyl group was one of the logical modifications. Acetylated salicylic acid had already been described three times in the literature (see Section 3 ) . Von Heyden and possibly also Merck, Darmstadt,are reputed to have experimented with aspirin without being able to produce the pure drug. At the time when Felix Hoffmann prepared pure aspirin successfully in the Bayer laboratories, one of his colleagues was Arthur Eichengrun, who had been hired by Carl Duisberg in 1896, while Hoffmann had been hired in 1894. Eichengrun,as an old man, had to undergo the horrors of the infamous Nazi concentration camp in Theresienstadt which he survived. In 1949, Eichengrun published his memoirs relating to the invention of aspirin5 which was then a half-century old. He claimed that it was he who told Hoffmann to prepare acetylsalicylic acid. Acetylation certainly was on Eichengrh's mind, since he had also experimented successfully with the acetylation of cellulose about the same time. He went cn to fame as the inventor and developer of rayon and safety film. Eichengrcn also claimed that anothercolleague of Bayer, the pharmacologist Dreser, opposed clinical trials. However, the memory of the 82 year-old Eichengrun must have been faulty when he

KLAUS FLOREY

6

wrote these rather bitter reminiscences concerning Hoffmann's, Dreser's and his own role in the discovery of aspirin because, in 1913, Eichengrun wrote a chapter on "The Pharmaceutical Research Laboratory" in the book History and Development of Farbenfabriken Bayer, V o r m . Friedr. Bayer & Co., Elber?eld by F. Fischer, 1913'+, where he laid no paternity claim to aspirin and described Dreser's role correctly. The pertinent passage (p. 412) translates as follows: "Acetylsalicylic acid, prepared by Felix rested unnoticed for 1+ years Hoffmann among thepreparations rejected by the pharmacological laboratory until in 1898,during unrelated investigations, Dreser's attention was again drawn to it. On account of the observation that the acetyl compound was increasing cardiac activity in contrast to salicylic acid itself, he recommended a clinical trial of the product . . . . I '

........

Felix Hoffmann did not publish his version of the discovery, nor did he obtain a German patent, since the synthesis had been previously described. Farbenfabriken Bayer did obtain a U.S. Patent6 in 1900 which named him as the inventor. Chemical Abstracts reveal no subsequent publications by him, nor is there any record that he was publiclyhonored for his contribution. However, in 1899, he was appointed director of the pharmaceutical research and marketing division of Bayer. He retired in 19287. In many ways, the story of the discovery of aspirin is typical for the way in which new drugs are invented and developed in pharmaceutical research laboratories, where many individuals have to make a contribution and where it is often difficult to fathom completely what thought processes, suggestions and interactions lead to a successful new drug. Description 2.1 Name, Formula, Molecular Weight Aspirin is acetylsalicylic acid, also salicylic acid acetate and 2-(acetyloxy)-benzoic acid (50-78-2). The last name is currently popular in Chemical Abstracts. 2.

ASPIRIN

‘gH8’4

M.W.

180.16

Appearance, Color, Odor Aspirin is a white, crystalline powder. It is odorless but might have a faint odor of acetic acid. 2.2

S nthesis h synthesis of aspirin is credited to Gerhardt* in 1853. Gerhardt was investigating

3.

mixed organic acid anhydrides and, among others, reacted acetylchloride with sodium salicylate. He obtained a solid product, undoubtedly impure acetylsalicylic acid,which immediately and without further characterization he hydrolyzed with aqueous sodium carbonate to salicylic and acetic acids. Next it was prepared by reaction of salicylic acid with acetylchloride by H. von Gilmg in 1859, who described a crystalline product. In 1869, K.Krautlo had a student, A . Prinzhorn, prepare acetylsalicylic acid by the methods of Gerhardt and von Gilm and obtained an identical product by both methods with a reported melting point of 118.5O. Kraut also correctly observed that the product is not an acid anhydride as assumed by Gerhardt but rather a phenolic ester. Felix Hoffmann6 used acetic anhydride for its preparation. CH 3,COC1

(gooH +

OH

or

CH CO 3 \ CH3CO” or CH2=C0

catalyst

KLAUS FLOREY

8

Essentially, all methods of synthesis are variations of the reaction of acetylchloride, acetic anhydride or ketenell with salicylic acid using a variety of catalysts such as pyridine12 or sulfuric acid13 and reaction conditions (c.f. 14). The preparation of aspirin labeled with a 14C-labeled acetyl group has also been reported.15 Efforts to improve the commercial processes continue to the present day. 4.

Physical Properties 4.1 Spectra 4.11 Infrared The assignment of the KBr infrared spectrum (Figure 2) of aspirin (U.S.P. reference standard #0675-F-4) is summarized in Table 1.16 It agrees essentially with a spectrum published previ~usly?~A reflection spectrum has also been presented.

*

TABLE 1 Infrared Spectrum Interpretation Wavelength (c m-') 2300-2500 1760 1690 1490 1220 1 1190 760

Assignment carboxyl OH vinyl ester C=O aromatic acid C=O aromatic C=C stretch =C-0 (acid and ester) ortho subst. phenyl C-H bending

4.12

Ultraviolet Aspirin in 0.1N sulfuric acidlg and in dilute1trichloroacetic acid20 exhibits maxima at 229 nm (El 484) and 276 nm (E 65.5) .1 In chloroform a maximum was found at 2 7 nm (El 68).21 Fluorescence - Phosphorescence The native fluorescence of aspirin, in contrast to salicylic acid, is a weak one and has been studied only recently.22 Excitation wavelength maximum is at 280 nm and emission maximum is at 335 nm. Maxima for salicylic acid are at 308 and 450 nm respectively. 4.13

id

rd

aE

KLAUS FLOREY

10

The phosphorescence emission maximum was found at 410 nm.23 4.14

Raman. Raman spectra are described and discussed in the following references: 24, 25 4.15

Nuclear Magnetic Resonance The 60 and 100 MHz proton macrnetic resonance spectra of aspirin have been published as part of a n a l y t i ~ a l ~and ~-~ biochemical ~ studie~.~O?~l The 100 MHz pmr spectrum of a deuter ochloroform solution containing tetramethylsilane as an internal reference was obtained on a Varian Associates XL-100-15 spectrometer equipped with a Nicolet pulsed Fourier accessory. 3 2 (Figure 3 ) The rms error for the experimental and calculated spectra shown in Figure 4 is 0.2. The proton assignment is shown below. 6 J = 8.05Hz H1 12.04 3?4 = 1.34Hz J H* 2.34(3H) 3?5 J3,6 = 0.3Hz H3 7.13 J = 7.80Hz H4 7.61 4?5 = 1.74Hz J H5 7.33 4?6 = 7.96Hz J H6 8.11 5?6

.

The differences in the pmr spectrum previously reported in aqueous media30 are attributed to the solvent used. The proton-proton couplings of the aryl protons are virtually identical, however. The fully decoupled I3C-NMR spectrum of aspirin in CD30D (200 mg/ml) is shown in Figure 5. The spectrum was obtained on a Varian XL-100-15 NMR spectrometer equipped with a Transform Technology TT-100 FT data system. The data represent the transformation of a 400 pulse FID obtained using 4096 data points with a 15 second delay time between accumulations.33 Peaks a-i (Figure 5) arise from the nine carbons of aspirin. The seven peak multiplet centered at 6=49.0 ppm

12.0

11.0

10.0

9.o

8 .O

7.0

6.0

5.0

4 .O

3.0

2.0

I.o

PPM

Figure 3.

100 MHz PMR Spectrum of Aspirin.

Instrument:

Varian XL-100-15

0.o

- -

c

=z

12

d

k

-4

a

-4

2 rCI

0 Id

)-I

a,

u

+J

a p:

v)

E

a

Id

a, 4J

I

rl

u

u

Id

rl

Id

c

a a

a,

Id

4J

2

rl

m

-4

a,

w

m

k 3

kl

.rl

13

6,

5

.-

KLAUSFLOREY

14

arises from the solvent. Assignment of each of the peaks can be made on the basis of their chemical shifts and the coupling information summarized in Peak a is the sole peak in the aliphatic Table 2 . region of the spectrum and is, therefore, assigned to methyl carbon (C-9). As expected, the fully coupled spectrum exhibits four peaks in this region - - - and f can with a 'JCH of 130 + 2 hz. Peaks b,d,e be assigned to the Four singly protonated aromatic carbons Each is strongly coupled to one proton ~ '65 ~ + 2 hz. Weak long range coupwith a i J of - - - and f can be ling can also be observed. Peak b,d,e assigned to carbons 3,5,6 and 4, respectivery by comparison of their chemical shifts to values predicted on the basis of substituent effects observed in model systems. Peaks c,g,h and i must arise from non-protonated carbons since they do not exhibit appreciable splitting in the fully coupled spectrum. Chemical shift predictions based on substituent effects enable assignment of peaks c and 2 to the C-1 and C-2 aromatic ring carbons, respectively. The remaining peaks h and i must arise from the carbonyl carbons. Comparrson 07 their respective chemical shifts to those of model compounds suggest their assignment to the carboxyl (C-7) and a-cetoxy carbonyls (C-81, respectively. This assignment is confirmed by the observation of a long range coupling of peak i to the three protons of the methyl group. TABLE 2 l3C-NMR Data for Aspirin Assignment Jcab Multiplicityb Peak G(ppm)a Carbon # 21.6 124.6 124.9 126.9 132.6 134.7 151.9 167.4 171.2

9 3 1 5 6 4 2 7 8

a) ppm from TMS external via the relationship 6 (CD30D)=49.0 ppm. b) obtained from the fully coupled spectrum.

ASPIRIN

c) none observed. m) weakly coupled multiplets due to long range effects. 4.16

Mass The low resolution mass

spectrum was obtained on an AEI MS-902 double-focussing mass spectrometer equipped with a frequency-modulated analog tape recorder at a source temperature of looo C. above ambient (approximately 1300 C.). 32 By adjusting the sample flow and the electron multiplier gain, the maximum sensitivity was obtained without clipping the most intense peak. The recorded analog spectrum was processed on a PDP-11.34 Except for differences in the intensity of the molecular ion (M’), the intensities shown in Figure 6 are virtually identical to the previously published spectrum.35 Differences could be ascribed to instrument design, source temperatures or even source design. The assignment of a number of fragment ions of the mass spectrum is shown in Figure 7. Figure 7.

120

+H 138

m* 104 3 m/z 138 -H20

92

‘HCO -m/z

m/z 120

m/z 64 63

The metastable ion at m/z 104.3 supports the loss of the elements of water from the m/z 138 rearrangement ion.

KLAUS FLOREY

16

5292 R S P I R I N 03-RUG-78

MF16761

100 30

90 >IH

cn

Z W IZ H

W

>

H

l-

a

-I W rY

80

25

70

W : Z

0 H

I-

a

kl H

60

20

Z

0 H

50

_I

15 a I-

40

cj

I-

30

10

I-

Z W

0

20

5

10

a: W

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0

I N T E N S I T Y SUM = 4 0 1 4 0

Figure 6.

BRSE PERK X = 3 3 . 0 7

Mass Spectrum of Aspirin. Instrument: AEI MS 902

ASPIRIN

17

The mass spectrum of aspirin has been used as an aid in the rapid identification of toxic materials isolated from urine, blood or gastric aspirates of drug abuse patients.36r37 4.2

Solid Properties 4.21 Melting Range The melting point of aspirin is nothing very definitive having variously been given between 118-144°38 and a good deal of work has been done to get the best methods for compendia1 Early commercial preparation melted around 135°.44 The European P h a r m a ~ o p e i agives ~~ a melting point of 141° to 144O as determined by the instantaneous method. For further discussion, see Polymorphism (Section 4.242)

.

4.22

Differential Thermal Analysis When aspirin (USP reference standard) was heated at a rate of 15O/min. in air, a single endotherm was observed with a T onset=1340 and T peak = 139°.46 DTA and TGA patterns of aspirin have also been previously studied4&nd used for forensic drug identification.48 4.23

Thermogravimetric Analysis When aspirin (USP reference standard) was heated at 20°/min. and a N2 flow of 20 cc/min.,no weight loss was observed at less than 1300. 4.24

Crystal Properties 4.241 Single Crystal X-Ray Diffraction The crystal structure of aspirin was determined by Wheatley.49 The monoclinic crystals have a space group of P21/C. The dimension of the unit cell are: a=11.446A; b=6.596A; c=11.388 A; ~ = 9 5 033'; n=4. 4.242

Powder X-Ray Diffraction The powder x-ray diffraction pattern of aspirin is presented in Table 3 and Figure 8. 5 0 4.243

Polymorphism Tn 1968, Tawashi5' claimed that aspirin exists in several polymorphic forms.

a,

0

aJ

8k

2

W

.

PI

4

4 0

W

aJ

k

ASPIRIN

19

TABLE 3

P o w d e r X-Ray D i f f r a c t i o n P a t t e r n o f A s p i r i n (U.S.P. Reference S t a n d a r d )

20 (Deg . I 7.80 14.09 15.63 16.78 18.19 20.63 20.95 21.53 22.56 23.20 25.00 27.05

D

(b)

11.3 6.25 5.68 5.28 4.88 4.30 4.22 4.12 3.93 3.83 3.55 3.30

R e l at i v e

Intensity 0.539 0.033 1,000 0.087 0.038 0.079 0.035 0.030 0.268 0.210 0.033 0.427

20 (Deg . I 27.56 28.85 29.62 30.26 31.54 32.57 33.85 34.50 36.04 36.55 37.45 39.37

R el ati v e . .

3.23 3.08 3.02 2.96 2.84 2.74 2.67 2.60 2.50 2.46 2.40 2.29

Intensity 0.054 0.068 0.040 0.039 0.120 0.110 0.051 0.037 0.049 0.048 0.034 0.038

This started a f l u r r y of a c t i v i t y . DeBis~chop~~ claimed t o have o b t a i n e d t h r e e d i f f e r e n t c r y s t a l forms b u t s t r e s s e d t h a t t h e o n l y s t a b l e one i s t h e monoclinic one m e l t i n g a t 142O C . However, t h e c l a i m f o r t r u e polymorphism of a s p i r i n w a s u e s t i o n e d o r ref u t e d i n several l a b ~ r a t o r i e s ~ ~and - ~ c%a n be b e s t summarized i n t h e words o f G . Schwartzman:57 " I t i s g e n e r a l l y a c c e p t e d t h a t t r u e polymorphism r e s u l t s i n d i s t i n c t o p t i c a l and s p e c t r a l p r o p e r t i e s . The data p r e s e n t e d a r e e n t i r e l y n e g a t i v e i n t h e s e r e s p e c t s . The e v i d e n c e accumulated a g r e e s w i t h t h e f i n d i n g s o f P f e i f f e r 5 5 and q u e s t i o n s t h e f o r m a t i o n of a s p i r i n polymorphs. W e believe t h a t the d i f f e r e n t crystal habits w e r e c a u s e d by t h e s o l v e n t s used f o r c r y s t a l l i z a t i o n . The d i s s i m i l a r m e l t i n g p o i n t s a r e probabl y due t o t h e poor t r a n s f e r of h e a t caused by the larger crystal s i z e o r t o possible crystal d e f e c t s . I'

4.244

Optical Constants A s p i r i n h a s been d e s c r i b 8=95O ed a s m o n o c l i n i c , a:b:c = 1.7322:1:1.7322, 8=1.6424 4.25'. I n d i c e s f o r 576 p p a r e : d=1.5042;

KLAUS FLOREY

20

and 2=1.6554; 2V=15' 46'. The optical plane is normal to (010) and lies in obtuse angle f3.58 Very similar constants are presented in reference 41. Polarized Crystal Absorption Spectrum The polarized absorption spectrum of a single crystal of aspirin was measured which indicated that aspirin may be spectroscopically treated as perturbed benzoic acid.59 4.245

Calorimetry The heat of combustion at constant volume was determined as 859.3 kcal/Mol.60 Aspirin tablets make good samples for use in oxygen bomb calorimetry.61 4.25

4.3

Solution Properties 4.31 S ~ l u b i l i t v ~ ~ ' ~ ~

g/ml Water at 25' 0.0033 Water at 37O 0.01 Water at 100' 0.03 0.2 - 0.4 Ethanol Chloroform 0.025 - 0.06 Carbon tetrachloride 0.0004 0.1 - 0.2 Ether Abs. ether sparingly soluble Benzene 0.0033 Petroleum ether insoluble The solubility in polyethylene glycol 400 and in aqueous solution of other polyethylene glycols has been described.65-66 The effect of selected surfactants above and below the critical micelle concentration (CMC) on aspirin solubility6 was studied. Dissociation Constant (pKa) In 1913, Springer and determined the dissociation in-aqueous solution at various temperatures. At 25O they determined the (pKa 3.55). The dissociation constant as 2.8 x Merck Index62 gives a value of 3.27 x (pKa 3.49) at 25O. When the apparent pKa was de4.32

ASPIRIN

21

termined in DMF, using quaternary butyl ammonium hydroxide as the titrant, the pKa observed depended on the solvent of crystallization. From ethanol (m.p. 140-142O) , a value of 8.99; from hexane (m.p. 121-124O), a value of 9.19 was obtained. The latter higher value was ascribed to internal hydrogen bonding of the carbonyl to the hydroxy group.69 This apparent pKa should not be confused with the true pKa of 3.5 (see above). Partition Coefficients When aspirin was partitioned between buffers pH 1-7 and octyl alcoho1,partition coefficients ranging from k=17.7 (pH 1) to k=0.025 (pH 7) were obtained.70 Earlier, coefficients of 0.32 in to1uene:water and 1.81 in ch1oroform:water were determined.71 4.33

4.34

Dielectric Constant, Dipole Moment

Dielectric constant: fb 2.35 5 to 7

Dipole Moment: 2.09

-

Ref. 72 73 74

5.65 calc.; 4.36 observed 0.93 (acc. to 75 Onsager by immersion)

Radiation Absorption The absorption coerricient of a collimated beam of 6oCo z-radiation was determined for aspirin. See reference 76 for details. 4.35

Methods of Analysis 5.1 Historical Synopsis As the following pages of this section will show, there is hardly a new method of analysis which is not immediately tried for the determination of aspirin as such, or in formulations and biological fluids. The analysis of aspirin is intricately interwoven with that of salicylic acid, its precursor and degradation product. From the very first,residual salicylic acid was determinedby the convenient reaction with ferric salts --typical for phenols -- which give a violet complex with salicylic acid. In spite of the plethora of methods, the 5.

22

KLAUS FLOREY

compendia1 approach to determine purity and strength of aspirin has been very conservative. A monograph for aspirin was introduced into U.S.P. not earlier than Volume X (1926) and has not been changed in its essentials for the last fifty years; Aside from such niceties as ash, carbonizable substances, chloride, sulfate and heavy metals, the mainstays then (U.S.P. X, 1926) and now (U.S.P. XIX, 1975) are: a) identification by a color test with ferric chloride after heating, saponification to salicylic acid, identified as a white precipitate and an odor test (ethyl acetate), b) residual salicylates as determined by a color matching test with ferric ammonium sulfate with a 0.1% limit and c) an assay involving saponification to salicylic and acetic acids and back titration of excess alkali with a purity specification of 99.5 to 100.5%. The European P h a r m a c ~ p e i auses ~ ~ essentially the same analytical methods as U.S.P. In contrast to aspirin itself, the U.S.P. monograph for aspirin tablets has undergone considerable changes. For some reason, U.S.P. does not use the ferric salt test for free salicylic acid, as does the British Pharmacopeia of 1973. Apparently, certain excipients such as citric and tartaric acid interfere with this reaction.77 Already in 1913, a double titration method was developed78 which was made an official method in 1926.79 This method was used as the assay method when the aspirin tablets monograph was introduced into U.S.P. XI1 in 1942. For identification, the same two tests as for aspirin itself were prescribed then (U.S.P. XII) and now (U.S.P. XIX) ; however, due to the pioneering work of Higuchi, Banes, Smith and Levine in the ' ~ O ' S ,a ~ ~ test for non-aspirin salicylates was introduced using a siliceous earth column for separation from excipients and aspirin, and spectrophotometric finish at 306 nm. A limit of 0.3% is specified. The same column method with different eluting solvents and a spectrophotometric finish at 280 nm is used for the assay with limits of 95 tb 105 percent. 5.2 Identity and Color Tests Aspirin can be identified by the following name tests:

ASPIRIN

23

Test Color Ref. Trinder's reagent Purple after hydrolysis 63 McNally's test Red 63 Mandelin's reagent Green with blue 81 (Ammonium vanadate ) tint; changes to red-violet Feigl Saponif. to salicylic 82 acid. Reaction with KOH at 130°: Violet fluorescence Kulberg Saponif. to salicylic 83 acid. Addition of FeC13 in HC1: red-violet coloration Vitali Morin Orange to red 84 Kofler Microscopic Identifi85 cation

For identification by infrared, see Section 4.11. Identification in combination products by mass spectrometry has been described.86 5.3

Quantitative Analysis 5.31 Elemental The percent of carbon, hydrogen and oxygen is as follows:62 % (theoretical) 60.00 c9 4.48 H8 35.53 O4 5.32

Colorimetric The use of basic organic dyes for ion pair extraction-photometric determination has been described.87 After ammonia treatment,,an orange-red color with CuSoq and H202 (Deniges) can be quantitated.88 A water insoluble violet complex ( X max. 620 nm) with 2-picoline-Cu(II) has also been reported.89 5.33

Ultraviolet The maximum at 277 nm has been used to determine aspirin in tablets after chromatography (see Section 5.43). It also has been used to determine aspirin in mixtures with other drugs (cf. 21). For simultaneous determination of

KLAUS FLOREY

24

aspirin and salicylic acid, see Section 5.61. 5.34

Infrared The infrared spectrum has been used to determine aspirin in combination products. Accuracy of 1-2% has been claimed (cf. 90,911. For aspirin, the absorption maximum at 1765 cm-1 has been used.92 Fluorescence - Phosphorescence Although fluorescence of aspirin, as contrasted to that of salicylic acid, is weak (see Section 4.13), it has been used for the determination in tablets.2 2 Phosphorimetry (see Section 4.13) has been described as useful in the determination of aspirin in blood serum and plasma.23 Since the phosphorescence of salicylic acid at the maximum of 410 nm is about 500 times weaker, it does not interfere. 5.35

Titrimetric - Electrochemical Although theoretically aspirin could be titrated directly with alkali, this tends to give inaccurate results due to its instability in alkali and, therefore, the compendia1 methods back titrate after saponification (cf. 7 9 ) . However, non-aqueous titration is possible and desirable, particularly for determination in combination products. Sodium methoxide in benzene-methanol is used as the titrant and methylisobutyl ketone as the solvent. The end point is determined potentiometrically.93 Alternately, tetrabutyl ammonium hydroxide and DMF as titration solvent have also been used.94,95 Titration in ethylene diamine has also been described.96 Potentiometric measurements of ion-pair association and selective acid stren th in ethylene diamine and water has been reported.7 7 Potentiometric titration in aqueous medium has also been described9* as has colorimetric.99 5.36

The direct current and alternating current polarographic response of aspirin in an a aprotic organic solvent system (acetonitrile - 0.1M tetrabut 1 ammonium perchlorate) has been studied.Y0 The following values were obtained: 1. dc half-wave potential: E%= -1.64 2. ac fundamental harmonic peak potential: Ep = -1.76

ASPIRIN

25

3. ac second harmonic minimum potential: E min = 1.87 n values calculated from the three modes are: 0.44; 0.45 and 0.40. Approximate detection limits (moles/liter) for the three modes are: 5 x 10-5; 1 x 10-4; 1 x 10-4. On a rotating disk electrode, aspirin was reduced to the aldehyde.lo1 5.37

Miscellaneous (NMR) Nuclear magnetic resonance spectrometry has been used to quantitate aspirin in a combination product with a coefficient of variation of 1.1.1°2 For quantitation, the shift at 2.3 ppm representing the ester methyl group was used. 5.4

Chromatographic Methods 5.41 Paper Paper chromatographic systems have been tabulated in Table 4 . TABLE 4 Solvent systems: Pet-ether, Methanol, Benzene and Water (25:20:20:0.05) Iso-propyl alcohol, Water, Ammonia (15:85: 10) Methanol, Water, Ammonia (10:90:10) Butanol, 25% Ammonia (4:1) 0.75% Nitric Acid *RfA

=

aspirin; RfS

=

RfA* RfS* -

Detection

Ref.

0.05

-

FeC13

103

-

-

FeC13

104

-

-

FeC13

104

0.45 0.8

0.6

U.V. 105 Fluorimetry 106

salicylic acid.

Cellulose anion-exchange paper chromatography with 5 % acetic acid-n-propanol (5:l) gave good separation,detected by U.V. light, of aspirin ( R f . 0.07) from salicylic acid ( R f . 0.51) . l o 7

KLAUS FLOREY

26

5.42

Thin Layer TLC has been used to identifv and quantify aspirin in pharmaceutical preparations and body fluids. Data have been summarized in Table 5. Readout by d e n s i t o m e t e r ~ ~and ~ ~ TLC , ~ ~separation ~ as student experimentsllO have also been described. 5.43

Column mentioned in the historical synopsis (Section 5.1) , Levine perfected the compendia1 partition column procedure in which aspirin in chloroform is first trapped in an immobile phase of sodium bicarbonate on a column of siliceous earth (celite) then eluted with a solution of acetic acid in chloroform and measured spectrophotometrically. This has been also used for separation in combination products.80 For the determination of salicylic acid in presence of aspirin by this method, see Section 5.61. Ion exchange columns filled with strongly or weakly basic anion exchange resin in the acetate or chloride cycle have also been used for se aration of aspirin ,724 This has also in combination products. 2 2 I been adapted for a student experiment.125 A Sephadex-G25 column has been used for the separation of aspirin from salicylic acid. As

5.44

High Pressure Liquid This newest of chromatographic techniques has already been used quite-extensively for the determination of aspirin in pharmaceutical products. Separation on columns filled with anionexchange resin127 or cation-exchange resin with and without counter ions128r129were investigated in detail. Silica surface columns have also been used130 as have been reverse phase (octadecyl) ones!31-134 U . V . detection was used throughout. 5.45

Gas-liquid Determination of aspirin by qaschromato raphy was first reported by Hoffkan and Mitchellq35 in 1963 who separated it from other tablet ingredients by direct chromatography on tetrafluoroethylene polymer coated with Dow-Corning silicone, using a flame ionization detector and an integrator. A glass-bead column, coated with carbowax and isophthalic acid has also been used.136 Most other investigators made the methyl- or trimethylsilyl-derivative prior to chromatography. A s

TABLE 5 Thin Layer Chromatography of Aspirin Support Silica Silica Gel G Silica Po1yamide Polyamide Polyamide Polyamide Silica Gel G Polyamide

Silica Gel G

Solvent System MeOH-HOAc-Et2O-CgH6 (1:18: 60/120) Et20-AcOEt (1:4) C6H6-HOAC-MeOH-CHC13Pet-ether CH3C13-C6H6-90% HC02H (5:l:O.l) iso-PrOH-H20-90% HC02H' (1:5: 6: 01) CH3C13-90% HC03H (20:0.1) Cyclohexane-CHC13-HOAc (4:5:1) Cyclohexane-CHC13-HOAc (50:40:10) CHC13-Cyclohexane-HOAcDioxane (40:60:1:10) CHC13-Cyclohexane-HOAc (40:60:1) EtOH-IsoProOH-XyleneCHC13 (12.5:12.5:25:50)

Rf.Asp.

Rf.Sa1.

QJo.9

-

0.26

'

-

Detection

W

Ref.

-

111 112

0.42

0.44

w

113

0.44

0.24

W

114

0.32

0.08

w

114

0.47

0.24

W

114

0.61

0.41

w

114

0.36

-

K4(Fe(CNa))3

115

0.48

0.14

W

116

0.37

0.12

w

116

Iodine

117

0.04

-

TABLE 5 (continued) Support

Solvent System

Rf.Asp.

Rf.Sa1.

Detection

Ref.

NH4 Vanadate NH4 Vanadate

118 118

CHC13-(CH3)2C0 (9:1) MeOH NH3(100:1.5)

0.075 0.75

-

Silica Gel GF

EtOAc-t-PrOH-NH3 (40:30:3)

0.25

S i l i c a Gel GF

E ~ O H - H O A C - H ~ O(60:30:10) CgHg

0.92 0.10

-

-

-

Silica

-

Silica Gel GF Silica Gel 6 0

C6Hg-Dioxane-HOAc ( 6 0 : 20: 2)

-

W W

w

119 119 119

Fluorimetry

120

ASPIRIN

29

meth lating agents, diazomethane in ether alcohollY7 or tetrahydrofurane, 3 8 methanol with boron t r i f l ~ o r i d e land ~ ~ methyl iodide with potassium carbonate140 were used. For trimethylsilylation hexamethyl disilazane,141-143N-0-bis(trimethy1silyl) acetamide and other trimethylsilyl BSTFA145-146and MSTFA147 have been tried. It was noted that derivatization with BSTFA or MSTFA resulted in slight hydrolysis of aspirin.147 Electrophoretic Methods Aspirin can be separated from salicylic Separation acid by ionophoresis at a pH of 4-5.14* of aspirin in combination products has been achieved with paper strip electrophoresis, ysinq4$uffers at pH 2-8 and a 200 V. applied potential. Aspirin was separated from metabolites by paper electrophoresis in a phthalate buffer of pH 3.2 and an ionic strength of 0.0125-0.0500.150 5.5

Determination of Impurities 5.61 Salicylic Acid As has alreadv been pointed out, the determination of salicylic acid is intricately‘ interwoven with that of aspirin itself. There is the convenient color reaction with ferric chloride which was already used by Dreserl to determine free salicylic acid in his own urine after the ingestion of aspirin. However, this reaction is not too specific and considerable work has gone in the development of interference free methods. 5.6

My task has been made easy since there is an excellent review by C.A. Kelly151 on the determination of salicylic acid in aspirin and aspirin products. A more recent review has been compiled by S.L. Ali.152

I , therefore, have restricted my tabulation of methods to those references which discuss determination of salicylic acid in biological specimens, which were not covered by Kelly, to important references which have been published since the Kelly review and references which are pertinent to other sections of this profile. References for the determination of salicylic acid: 1. Colorimetric (iron complex): 77,78,153,154 (Folin-Ciocalteu reagent): 218,219

KLAUS FLOREY

30

Nonaqueous titration: 104 Iodometric: 131 Spectrophotometric: 20, 156-158 Fluorometric: 22,159,160 Infrared: 92 Column: 57,137,138,103 PC (for Rf values and solvent systems see Section 5.41): 106,107 9. TLC (for Rf values and solvent systems see Section 5.42): 114,116,119 10. VPC: 106,107,140,141,144,145,146,147 11. HPLC: 152,164 12. Automated: 165-167 2. 3. 4. 5. 6. 7. 8.

5.62

Acetic Acid It is easily forgotten that aspirin degrades to acetic as well as salicylic acid. And, indeed, any smell aspirin might have is due to acetic acid. However, the volatility of acetic acid does not make the determination of acetic acid a reliable tool to measure stability or degradation. A.N. Smith in 192016* devised a method to determine acetic acid in aspirin by bubbling dry air through a thin layer of powdered aspirin, trapping the acetic acid in water and backtitrating it with alkali. Gas chr~matographyl~~ has also been used. 5.63

Acetylsalicylic Anhydride and Acetylsalicylsalicylic Acid In addition to salicylic and acetic acids, very small quantities of acetylsalicylanhydride (0.0012 to 0.024%) and acetylsalicylsalicylic acid (0.03 to 0.1%) have been found in aspirin preparations. The former has been determined by gas chromatography, TLC1 and spectro hotometry , the latter by gas chromatography. tT4 A controversy is still ongoing (cf. 152) whether the occasionally observed hypersensitivity against aspirinis caused by these two impurities and whether the basis of the adverse reaction is immunological. Stability - Deqradation That aspirin is sensitive to moisture is wellknown and most of us at one time or another have observed the growth of salicylic acid whiskers on aspirin tablets left f o r too long in a humid bath6.

ASPIRIN

31

room wall cabinet. The hydrolysis of aspirin to salicylic acid was already of concern to the pharmacologist, Dreser' (see Section 1.21, who in 1899 prepared what nowadays one would call a preformulation profile. He tested the hydrolysis of aspirin in acid and alkaline solutions and,by perusing textbooks of Ostwald and Nernst to devise the proper equations, determined hydrolysis constants of 3.3 x at "body temperature" in acid medium and 2.5 x at room temperature in alkaline medium. He also observed that aspirin was easily split in weakly alkaline medium which had consequences for the metabolism (see Section 7 ) . One could say that he established the first profile for the pH dependence of hydrolysis of aspirin. Of course, the definition of pH was unknown at the time. The next systematic study of the hydrolysis of aspirin in water, albeit at 1000 C., was undertaken by Rath172 who determined a hydrolysis constant of about 0.17 depending on experimental conditions. Much work has been done since, and it is quite evident that this seemingly simple hydrolysis to acetic and salicylic acids is both complex and controversial. I am fortunate that I can refer the reader to the excellent and detailed review by Clark A . KellylS1 already mentioned in Section 5.61. The most complete and thorough kinetic studies of the factors involved in the hydro1 sis of aspirin are undoubtedly those by Edwards 73, which were further elaborated by Garrett.174 Stability and decomposition kinetics of aspirin both as a solid and in solution continue to be studied. The topochemical decomposition pattern of aspirin tablets has been explored.175 The degradation of aspirin in the presence of sodium carbonate and hi h humidity was studied by x-ray diffraction. l q 6 The activation energy of decomposition by water vapor in the solid state was found to be 30 kcal/mo1.177 The effect of common tablet excipients on aspirin in aqueous suspension was also studied.178 An exhaustive study of the stability of aspirin in polyethylene glycols (substituted, unsubstituted and esterified), as well as other polyhydric alcohols, was undertaken by Whithworth and collaborator^!^^-^^^

32

KLAUS FLOREY

It was found that decomposition is in part due to transesterification and that substitution increases stability. The effect of gamma radiation on aspirin has been described.lE4 The pH stability profile of aspirin according to Edwards has been made the subject of a student experiment.lE5 Pharmacokinetics - Drug Metabolic Products When Dreser' investigated the pharmacological properties of aspirin in 1897 (see Section 1.21, he postulated that, based on the easy hydrolysis of aspirin in weakly alkaline medium, aspirin also should be converted to salicylic acid in vivo. And, indeed, he found that only 22 minutes Z t a n gestion of aspirin, his own urine gave a positive reaction for salicylic acid with ferric chloride. After 12 hours, he could no longer detect salicylic acid in his urine. He found no evidence for aspirin itself in urine, nor did he find a combination of aspirin with glycine analogous to the formation of hippuric acid already known at the time. He had evidence for formation of another nitrogen containing derivative. This early investigation should give food for thought to those who believe that pro-drugs and pharmacokinetics are recent discoveries.

7.

In 1911, NeuberglE6 detected small amounts of gentisic acid (2,5-dihydroxybenzoic acid) in the urine of dogs dosed with aspirin. The metabolism of aspirin is intertwined with that of salicylic acid, but I was unable to ascertain who first reported the metabolic formation of salicyluric acid, the major metabolite of both salicylic acid and aspirin, specifically after administration of aspirin. Reviews by PuetterlE7 and by Levy,188-190taken together present a comprehensive picture of the pharmacokinetics and metabolism of aspirin. Once absorbed, aspirin is rapidly converted to salicylic acid. After i.v. administration, the half life of aspirin in the human organism was found to be only 15 minuteslgl by Rowland and Riegelman who also estimated that only 2 0 % of the -in vivo hydrolysis takes place in blood.192 Apparently, all tissues investigated possess

ASPIRIN

33

esterases which can split aspirin; however, most of it seems to be hydrolyzed in the liver.187 While the major pathway of hydrolysis leads to free acetic acid, it is noteworthy that a significant portion of the acetic acid is not set free but used for transacetylation of certain factors which pla a role in the inhibition of platelet aggregation1J 3 only shown by aspirin but not by salicylic acid. The kinetics of this important pathway still need exploration.187 Aspirin serum esterase activity is species dependent.Ig4 In man it is sex linked since it was found to be higher in men than women.lg5 It also seems to be age dependent.lg6 Once aspirin is hydrolyzed to salicylic acid, it follows the metabolic pathway of the latter. The metabolic products of salicylic acid are presented in Figure 9 . Over the last decade, the pharmacokinetics of salicylates in the healthy and diseased organism have been explored in great detail by Levy.188 Salicylic acid (11) is eliminated by five parallel and competing pathways (Fig. 9 ) leading to renal excretion. These are: excretion unchanged (II), conjugation with glycine to form salicyluric acid (111) -- the major pathway -conjugation of the carboxyl or phenolic hydroxyl group to form glucuronides IV and V and hydroxylation to gentisic acid (VI) To a minor degree, salicyluric acid can be further conjugated with glucuronic and sulfuric acids.187 The major metabolic pathways (salicyluric and salicyl phenol glucuronide) are easily saturated in the usual dosage range for treatment of inflammation.lg7 For the clinical implications of these non-linear pharmacokinetic characteristics, I refer to the reviews by Levy.188'190

.

To properly treat bovine "sufferers of headaches" (inflammation) the pharmacokinetics of aspirin in cattle have also been explored.1g8 Bioavailability - Dissolution In the last two decades, the concept of bioavailability has gained prominence and-with it dissolution as a possible in vitro model for drug absorption. In 1960, S w i n t o s k y d BlytheIg9 8.

I I

0

W Ocn U

E o' u

/ I

8

8u o'

I

E

X

0

0

rcl

ASPIRIN

35

compared the relative availability of aspirin from entexic coated and compressed tablets by measuring the excretion of salicylate. About the same time, Levy200,201started to compare dissolution and absorption rates of different commercial aspirin tablets, found good correlation and proposed that the U.S.P. tablet disintegration test be replaced by a dissolution test; a suggestion which as of this writing has not been heeded. These studies were extended and further perfected by Gibaldi.163 Many other papers on this subject have appeared. Particularly,the influence of the crystal habitat (see Section 4.243) on absorption has been studied (cf. 202). Significant intraindividua1,but not i n t e r i n d i v i d u a 1 , v a r i a t i o n s in blood plasma aspirin levels were found in young male subjects after administration of aspirin as tablets or solutions. The plasma level curves obtained for tablets were more variable than those obtained for solutions.203 Levy204 found that using urinary excretion measurements for evaluation of aspirin dosage forms,with different absorption rates in man, requires that such measurements be made during the first hour after drug administration. The use of a pHstat for testing dissolution of various aspirin dosage forms has been described.205 Determination in Biological Fluids and Tissues All the advances in pharmacokinetics and drug metabolism described in Sections 7 and 8 would not have been possible without the availability of the proper analytical methods. The following is a tabulation of publications in this field, most of which have already been discussed in Section 5. It should be mentioned that a few publications talk about aspirin blood levels, but really mean salicylate levels. The following tabulation covers only those papers where aspirin was differentiated from other salicylates by chromatography or other means. It seems that the "workhorse" for serum salicylate levels is still the colorimetric (ferricnitrate) method of Brodie, Udenfriend and C ~ b u r n ' ~ ~ published in 1944, or modifications thereof. Simplified versions (cf. 206) may lead to erroneous results under certain conditions.207 The method is also applicable for urinary metabolites after proper hydrolysis (cf. 2 0 8 ) . For other methods restricted to salicylic acid, see Section 5.61. The first gas chromatographic separation of aspirin 9.

KLAUS FLOREY

36

and salicylic acid in plasma was described by Rowland and Riegelman, 4 1 who presented a brief review of earlier methods. Methods for aspirin: Colorimetric (with differential extraction):218,219 Spectrophotometric (differential): 20, 209 Fluorometric (as salicylic acid): 160, 210 Phosphorimetric: 23 VPC: 137,141,143,145,146 PC: 106,107,119 TLC: 118,211 Mass Spectrometric: 37 Radioisotopic: 212-214 Determination in Pharmaceutical Preparations The following tabulation of references highlights those methods (see Section 5 ) useful in pharmaceutical analysis. 10.

1.

Determination in tablets: Differential U.V.: 157,158 Automation: 166 22 Fluorometric : 121 Column : TLC : 120 GC : 136,139,144

2.

Determination in buffered tablets: Fluorometric: 159 161,162 Column :

3.

Determination in combination products: a. General Identity tests: 83,86,103,104,105, 112 b. Aspirin, caffeine, acetophenetidine (and more components) w: 21,215 IR: 91-93 NMR: 102 Electrophoresis: 149 110 ,111 TLC : Column: 80 Ion-exchange column: 123 HPLC : 127,130,131 135 ,142 VPC : Pot. titration: 216

ASPIRIN

11.

37

c.

Aspirin, acetaminophen, caffeine: Nonaqueous titration: 94,217 Potentiometric: 102

d.

Aspirin-barbiturate combinations: Nonaqueous titration: 93

e.

Cough-cold mixtures: HPLC: 132

Acknowledgments

I would like to express my gratitude to the Archives of Bayer AG, Leverkusen, Germany for historical information, to G. Levy, School of Pharmacy, S.U.N.Y., Buffalo for a critical review of the section on pharmacokinetics, to A . I. Cohen, M. Porubcan and B. Toeplitz for providing spectral information and interpretations, and to M. Bruno for her expert secretarial assistance.

KLAUS FLOREY

38

12.

References

H. Dreser, Pflug. Arch. Gen. Physiol., 76, 306 (1899). 2. J. Wohlgemuth, Ther. Mh., 13, 276 (1899). 3. K. Witthauer, Ther. Mh., 13,330 (1899). 4. Bayer A.G. Grchives 5. A. Eichengrun, Pharmazie, 4, 582 (1949). 6. F. Hoffmann, U.S. Patent c44077 of February 27, 1900. 7. Neue Deutsche Biographien, p. 415. 8. C. Gerhardt, Annalen der Chemie, 67, 149(1853). 9. H. von Gilm, Annalen, 112, 181 (1859). 1 5 0 3 (1869). 10. K. Kraut, Annalen, 11. D.A. Nightingale, U.S. Patent 1,604,472; C.A. 21, 157 (1927). 12. BayerT Co., DRP 386679 (1924); Beilstein 111, p . 41. 21, 23 13. Fernandez, Torres, An.Soc. espaz., (1923); Beilstein 111, p. 41. 14. Norwich Pharmacal.Co., Brit. Pat. 893,967 (1962); C.A. 57, 3572f (1962). J.P. Hummel, W.D. Paul and 15. J.W. Clappiso: J. I. Routh, J. Am. Pharm. ASSOC., 40, 532 (1951). 16. B. Toeplitz, The Squibb Institute for Medical Research, Personal Communication. 17. O.R. Sammul, W.L. Brannon and A.L. Hayden, J. Assoc. Offic. Agr. Chemists, 47, 918 (1964). - 91861p 18. S.G. Il'yasov, V.L. Dragun, C.A.82, (1975). 19. Isolation and Identification of Drugs, E.C.G. Clarke, Editor, The Pharmaceutical Press, London (1969). 6, 275 20. A. Garner, J.K. Sugden, Anal. Lett. (1973). 21. M. Jones and R.L. Thatcher, Anal. Chem., 23, 957 (1951). 22. C.I. Miles and G.H. Schenk, Anal. Chem., 42, 656 (1970). 23. J.D. Winefordner and H.W. Latz, Anal. Chem., 35, 1517 (1963). 74, 24. L. Kahovec and K.W.F. Kohlrausch, Monotsh. 333 (1943); C.A. 38, 51475 (1944). 25. G.V.L.N. Murty andT.R. Seshadri, Proc.Indian Acad. Sci., 19A, 17 (1944); C.A. 38, 6197-8 (1944). 1.

ASPIRIN

26. 27. 28. 29. 30. 31. 32. 33. 34.

35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.

39

E.G. Brame, Encycl. Ind. Chem. Anal., 2, 707 (1966). G.A. Olah, P.W. Westerman, J. Org. Chem., 2 1307 (1974). B.L. Shapiro, L.E. Mohrmann, J. Phys. Chem. Ref. Data, 6 919 (1977). K.N. Scott,-J. Magn. Resonance, 5, 55 (1972). B.D. Sykes, W.E. Hull, Ann. N.Y. Acad. Sci., 226, 60 (1973); C.A. 80, 74282s (1974). A.J. Marcus, S.E. Lasker, Haernostasis, 2, 92 (1973); C.A. 82, 8 0 3 6 0 ~(1975). A.I. Cohen, The Squibb Institute for Medical Research, Personal Communication. M. Porubcan, The Squibb Institute for Medical Research, Personal Communication. P.J. Black, A.I. Cohen, presented at the 20th Annual Conference on Mass Spectrometry and Allied Topics, Dallas, Texas, June 4-9, 1972. H.M. Fales, G.W.A. Milne, N.C. Law, Arch. Mass Spectral Data, 2, 628 (1971). N.C. Law, V. Aandahl; H.M. Fales, G.W.A. Milne, Clin. Chim. Acta, 32, 221 (1971). W. Boerner, S. Abbzt, J.C. Eidson, C.E. C.E. Becker, H.T. Horio, K. Loeffler, Clin. Chim. Acta, 49, 445 (1973). L. Kofler, Pharm. Zentralhalle, 89, 223 (1950); C.A. 45, 1301a (1951). G.D. =a1 and C.R. Szalkowski, J. Am. Pharm. ASSOC., 22, 36 (1933). T.S. Carswell, J. Am. Pharm. ASSOC., 16,306 (1927). J.L. Hayman, L.R. Wagener and E.F. Holden, J. Am. Pharm. ASSOC., 14, 388 (1925). H.W. Johnson and C.W. Ballard, Pharm. J., 157, 88 (1946). M. deBisschop, J. Pharm. Belg., 25, 330 (1970). Beilstein X, p. 67 (1927). Europeian Pharmacopeia I, 236 (1969). T. Prusik, The Squibb Institute for Medical Research, Personal Communication. W.W. Wendlandt, J. Hoiberg, Anal. Chim. Acta, 29, 539 (1963). K W . Wendlandt, L.W. Collins, Anal. Chim. Acta, 71, 411 (1974). P.J. Wheatley, J. Chem. S O C . Suppl., 1964,6039. Q. Ochs, The Squibb Institute for Medical Research, Personal Communication.

40

51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74.

KLAUSFLOREY

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358,

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w.

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J.V. Swintosky and R.H. Blythe, Drug Standards, 28, 5 (1960). 200. G. Levy, J.Pharm. Sci., 50, 3.88 (1961). 201. G . Levy, J.R. Leonards anhJ.A. Procknal, J. Pharm. Sci., 54, 1719 (1965). 202. A.G. Mitchell, B.L. Milaire, D.J. Saville, R.V. Griffiths, J. Pharm. Pharmacol., 23,. 534 (1971). 203. K. Frislid, E.M. Haram, R. Norberg, S. Oie, Pharm. Acta Helv., 48, 610 (1973). 204. G. Levy and A. Yacobi, J. Clin. Pharmacol., 15, 525 (1975). 205. S. Goto, J. Nakashima, M. Tsuruta, H. Sato, T. Nakayama, Yakuzaigaku, 34, 160 (1974); C.A. 83, 152268~(1975). 206. W.C. gller, Am. J. Clin. Path., 17, 22 (1947). 207. L.J. Fenton, M.E. Beresford, W.J. Keenan, Pediatrics, 59, 55 (1977). 208. G. Levy and T A . Procknal, J. Pharm. Sci., 57, 1330 (1968). 209. J.I. Routh, N.A. Shane, E. Arrendondo, W.D. Paul, Clin. Chem., 13, 734 (1967). 210. S.L. Kanter, W.R. Horbalz J. Pharm. Sci., 60, 1898 (1971). 211. Z.I. El-Darawy, Z;M. Mobarak, Forensic Sci., -4 , 171 (1974). 212. E.M. Burnham, W.D. Paul and J.I. Routh, Proc. Iowa Acad. Sci., 63, 403 (1956); C.A. 50, 21671.1 (1956). 213. W.R. Borst, J.E. Christian and T.S. Miya, J. Pharm. Sci., 45, 511 (1956). 214. H.G. Mandel, N.M.Cambosos, P.K. Smith, J. Pharmacol. Exptl. Therap., 112, 495 (1954). 215. A.W. Clayton and R.E. Thiers, J. Pharm. Sci., 55, 404 (1966). 216. S.L. Lin and M.I. Blake, Anal. Chem., 38, 549 (1966). 217. S.P. Agarwal, M.I. Blake, Can. J. Pharm. Sci., 7, 123 (1972). 218. E.J.H. Smith, J. Pharm. Pharmacol., 3, 409 (1951). 219. I.A. Muni, J.L. Leeling, R.J. Helms, N. Johnson, Jr., J.J. Bare and B.M. Phillips, J. Pharm. Sci., 67, 289 (1978). The literature has been covered systematically through Chemical Abstracts,Volume 88 (1978), with a few stray references beyond.

-

-

Analytical Profiles of Drug Substances, 8

BROMOCRIPTINE METHANESULPHONATE Danielle A . Giron-Forest and W . Dieter Schonleber

3. 4.

5.

6. 7.

8.

Introduction 1 . 1 History I .2 Name, Formula, Molecular Weight 1.3 Appearance, Color, Odor Ph ysicochemical Properties 2. I Elemental Analysis 2.2 Spectra 2.21 Infrared 2.22 Ultraviolet 2.23 Fluorescence 2.24 Proton Nuclear Magnetic Resonance 2.25 Carbon-13 Nuclear Magnetic Resonance 2.26 Mass 2.3 Crystal Properties 2.3 I Melting Characteristics 2.32 Polymorphism 2.33 X-Ray Diffraction 2.34 Differential Scanning Calorimetry 2.35 Thermogravimetry 2.4 Solubility 2.5 Dissociation Constant 2.6 Partition Coefficients 2.7 Optical Rotation Synthesis Stability and Degradation Processes 4.1 InBulk 4 . 2 In Solution 4.3 In the Dosage Form Biopharmaceutics 5. I Pharmacokinetics 5.2 Metabolism Toxicology Analytical Methods 7. I Application of General Tests 7.2 Titration 7.3 Spectroscopic Methods 7.31 Infrared 7.32 Ultraviolet 7.33 Colorimetry 7.34 Nuclear Magnetic Resonance 7 . 4 Chromatography 7.41 Paper 7.42 Thin-Layer 7.43 Gas-Liquid 7.44 High Performance Liquid 7.5 Differential Scanning Calorimetry 7.6 Phase Solubility 7.7 Analysis of the Dosage Form 7.8 Determination in Body Fluids and Tissues References All 41

Copyright @ 1979 by Academic Press, Inc. rights of reproduction in any form reserved. ISBN 0 12-260808-9

DANIELLE A. GIRON-FOREST AND W. DIETER SCHONLEBER

48

1.

Introduction 1.1 History

The most valuable pharmacological p r o p e r t i e s of the pept i d e type e r g o t a l c a l o i d s induced a v a r i e t y of attempts of chemical d e r i v a t i s a t i o n of t h e p a r e n t compounds ( 1 , 2 + lit. quoted t h e r e i n ) . I n one of these the bromination of a-ergoc r y p t i n e l e d t o a product of h i g h l y i n t e r e s t i n g pharmacology, namely bromocriptine. I t became known t o suppress p r o l a c t i n e s e c r e t i o n , and i t i s t h e r e f o r e a u s e f u l t o o l i n t h e treatment of p r o l a c t i n e dependent d i s o r d e r s , such a s g a l a c t o r r h e a a s s o c i a t e d with hyperprolactinemia and postpartum, a s w e l l as c e r t a i n kinds of s t e r i l i t y ( 3 - 9 ) . I n more e l e v a t e d doses, t h e drug i s a p o t e n t antiparkinsonicum. I n a d d i t i o n , t h e r e i s r e c e n t evidence of bromocriptine playing an important r o l e i n the trace heavy metals balance of t h e b r a i n ( 1 0 ) .

1.2

Name, Formula, Molecular Weight

Bromocriptine mesilate i s 2-bromo-a-ergocryptine methanesulphonate o r 2-bromo-12'-hydroxy-2'-(l-methylethyl)-5'-(2methylpropyl-5'a-ergotaman-3',6',18-trione methanesulphonate o r Bromocriptinum ( I N N ) . I t i s the a c t i v e i n g r e d i e n t i n ParlodelB dosage forms.

CH3

/..

x CH3S03H

BROMOCRIPTINE METHANESULPHONATE

49

Chemical A b s t r a c t s R e g i s t r y Numbers : 25614-03-3 (26409-15-4) ( 4783 0- 26- 2 )

Average Molecular Weights : base :

654.61

mesilate:

750.71

Average Formulas : base :

C

mesilate: C

H

32 4 0

BrN 0 5 5

H BrN 0 S 33 44 5 8

1 . 3 . Appearance, Colour, Odour Grey tinged white o r l i g h t yellow, f i n e l y c r y s t a l l i n e powder, odourless o r of weak, c h a r a c t e r i s t i c odour. 2.

Physicochemical P r o p e r t i e s 2.1

Elemental Analysis

The elemental a n a l y s i s of bromocriptine mesilate y i e l d e d t h e following r e s u l t s (11): element

calculated

%

52.8 5.9 10.6 9.3 17.0 4.3

C

H Br N 0 S

2.2

%

found 53.2 6.0 10.5 9.2 16.8 4.4

Spectra

2.21 I n f r a r e d

The i n f r a r e d spectrum of bromocriptine m e s i l a t e i n a KBr p e l l e t i s given i n f i g . 1. I t was recorded on a Perkin

Elmer 257 i n f r a r e d spectrophotometer. The main c h a r a c t e r i s t i c bands are t h e following

:

%Absorption

a,

m

cr ffl

rl .d

P

BROMOCRIPTINE METHANESULPHONATE wave number ( c m 3200 1728

-

2900

1642, 1672 1560

-1

)

51

assignment C-H-stretching v i b r a t i o n s C=O-stretching o f 5-membered r i n g lactam ( c y c l o l ) C=O-stretching o f 6-membered r i n g l a c t a m (amide I ) amide-11 band and C=C- st r e t c h i n g

The s p e c t r u m of t h e b a s e h a s been r e p o r t e d by P.A. S t a d l e r and co-workers (11). 2.22 U l t r a v i o l e t The u l t r a v i o l e t spectrum o f b r o m o c r i p t i n e mesilate was r e c o r d e d on a P h i l i p s 1700 uv s p e c t r o p h o t o m e t e r i n 0 . 1 M met h a n o l i c methanesulphonic a c i d s o l u t i o n . I t i s g i v e n i n f i g . 2. A maximum o c c u r s a t a b o u t 308 nm w i t h a l o g molar absorpt i v i t y o f 4.0. I n 1:l dichloromethane/methanol s o l u t i o n t h e a b s o r p t i o n maximum was r e p o r t e d as 306 nm l o g = 3.988 (11). 2.23 F l u o r e s c e n c e Bromocriptine m e s i l a t e e x h i b i t s fluorescence l i k e the o t h e r l y s e r g i c and i s o l y s e r g i c a c i d type a l c a l o i d s . I n 2 p e r c e n t e t h a n o l i c t a r t a r i c a c i d s o l u t i o n , t h e e m i s s i o n maximum a p p e a r s a t 402 m ( e x c i t a t i o n a t 325 nm) See f i g . 3 .

.

2 . 2 4 P r o t o n Nuclear Magnetic Resonance The PMR spectrum of b r o m o c r i p t i n e mesilate i n d e u t e r a t e d d i m e t h y l s u l p h o x i d e a s o b t a i n e d on a Bruker HX-90 NMR s p e c t r o meter i s p r e s e n t e d i n f i g . 4. TMS s e r v e d a s i n t e r n a l s t a n d a r d . The c h a r a c t e r i s t i c s of t h e spectrum a r e g i v e n i n the f o l l o w i n g t a b l e (see a l s o 1 2 , 1 3 and lit. q u o t e d t h e r e i n ) :

Chemical S h i f t downf i e l d (ppm)

PMR-spectrum and Assignment Intensity Multiplicity Assignment

11.85

1 H

Singlet

indole-Ng

10.45

1 H

Singlet

NH -

9.55

1 H

Singlet

CONH-

7.3

4H

I

7

-

and

(18-19)

OH

DANIELLE A. GIRON-FOREST AND

52

230

Figure 2.

270

310

350

w. DIETER SCHONLEBER

390

nm

Ultraviolet Spectrum of Bromocriptine Mesilate in 0.1 M Methanolic Methanesulphonic Acid. CA = 0.05 mg/ml, C = 0.012 m g / m l . B Instrument: Philips SP 1700

53

BRLOMI3CRIPTINE METHANESULPHONATE

nm

Figure 3.

Fluorescence Spectrum of Bromocriptine Mesilate in 2 % Ethanolic Tartaric Acid. C = 65,ug/ml. Instrument: Perkin Elmer MPF-3

Figure 4.

PMR Spectrum of Bromocriptine Mesilate in (CD ) SO, 3 2 Instrument: Bruker HX-90 E

55

BROMOCRIPTINE METHANESULPHONATE

1H

6.47 4.3

-

4.5 3.2

3

2.37

2.1

16 H

- 4.2

-

2*1 1.5

1

Singlet

{

3 H 8-9 H

{

Triplet singlet Multiplet

H-C

-

9

H-C

- 5' 6-CH ((2-17) -3 g-C C-!; ;H-C4; 8' 11' H-C5 ; F-C * H-C 7 ' 8 CH C0CK2CH3 ; H20 3 (recryst. solvent)

Singlet

CH -SO H

Singlet

CH COCH CH

Multiplet

-3

3

-3 2 3 5'-C€12 ; 5'-Cg ; 2 ' - C g

H-Cg,

0.75-1.15

12 H

Multiplet

2'CH

-3

;

; g-C 1 0 '

-

5'-CH -3

and

CH3COCH CH

2 -3

2.25 Carbon-13 Magnetic Resonance Spectrum The spectrum o f b r o m o c r i p t i n e m e s i l a t e h a s been r e c o r d e d i n d i m e t h y l s u l p h o x i d e u s i n g a Bruker HX-90 NMR s p e c t r o p h o t o meter ( f i g . 5 ) . The a s s i g n m e n t o f t h e i n d i v i d u a l s i g n a l s i s g i v e n i n f i g . 6. 2.26

Mass Spectrum

The low r e s o l u t i o n e l e c t r o n impact mass s p e c t r a l p a t t e r n of bromocriptine m e s i l a t e ( f i g . 7 ) corresponds q u i t e n i c e l y t o t h o s e o f t h e o t h e r non- and d i h y d r o g e n a t e d e r g o t a l c a l o i d s ( 1 2 , 1 3 and lit. quoted h e r e i n ) . The m o l e c u l a r peak M of t h e b a s e shows up a t 653 and 655, r e s p e c t i v e l y , r e f l e c t i n g t h e n a t u r a l abundance o f t h e bromine i s o t o p e s , and M - 1 8 a t 635/637 mass u n i t s . A s i n t h e p a r e n t compounds t h e n e x t s m a l l e r f r a g m e n t a p p e a r s a t M-228, i n d i c a t i c g a s u b s t a n t i a l loss of t h e p e p t i d e s e c t i o n c o n s t i t u e n t s w i t h t h e t e n t a t i v e fragment s t r u c t u r e F1. From t h i s s p e c i e s t h e i s o p r o p y l group s p l i t s o f f e a s i l y t o y i e l d t h e l i n e p a i r a t 382/384 m a s s u n i t s . L o s s o f t h e e n t i r e p e p t i d e moiety l e a d s t o t h e f o r m a t i o n o f 2-bromo-lysergamide F2 a t 345/347 m.u., t h e mass s p e c t r a l b e h a v i o u r of which c o r r e s p o n d s t o t h a t o f t h e bromine-free compound. Thus, i t g i v e s r i s e t o peak p a i r s a t 300/302 and 299/301, r e s p e c t i v e l y , presumably by l o s s of formamide o r by t h e r u p t u r e of t h e t e t r a - h y d r o - p y r i d i n e r i n g ,

6000

4003

2000

3000

2000

>m

'%a

rm

xa

I tm

I

-

4ca

I

t T

Figure 5 .

y1

T

lb

T

lbo

L

T

13-C-NMR Spectrum of Bromocriptine Mesilate in (CD ) SO, 3 2 Instrument: Bruker HX-90 E

I 0

57

100 90 80

I

1

I

I

'O

70

I__

60 50 YO

30

20 10

36

I

582

635 653

'25

f

1 " " " " ' 1 " " " ' " l ' " ' ' " " " " " ' ' ~

100

Figure 7 .

200

300

900

500

Low Resolution Electron Impact Mass Spectrum of Bromocriptine Mesilate Instrument: CEC 21-llOB; Energy 70 eV, Ion Source 0 Temperature 160 - 200 C

600

M/Z

700

BROMOCRIPTINE METHANESULPHONATE

59

a decay mode t h a t w a s proposed e a r l i e r (14,15) for l y s e r g i c a c i d d e r i v a t i v e s . Peak d o u b l e t s a t 285/287 and 274/276 m.u. i n d i c a t e fragments of i l l i c i t s t r u c t u r e s t i l l c o n t a i n i n g bromine.

425/427 m.u. The p e p t i d e moiety i t s e l f (308 rn.u.1 obviously does n o t appear i n t h e spectrum, whereas i t s fragmentation p a t t e r n with peaks a t m/e 195 (F3), 167 (F3-CO), 155 - 153 (F4), 125 (FS), 86 (F6), and base peak 70 (F7) mass u n i t s , i s c l e a r l y understood (14,15) The m e s i l a t e shows a s i g n a l a t m/e 96.

-

1

I 195 m.u.

F3

F4 1 5 4 m.u

DANIELLE A. GIRON-FOREST AND W. DIETER SCHONLEBER

60

D ' q0

H

F5

125 m.u.

2.3

F7 86 m.u.

70 m.u.

Crystal Properties

2.31 M e l t i n g C h a r a c t e r i s t i c s Bromocriptine m e s i l a t e decomposes between 180 and 200 O C , t h u s a m e l t i n g p o i n t o r m e l t i n g r a n g e c a n n o t be g i v e n . 2.32 Polymorphism I n v e s t i g a t i o n s f o r t h e o c c u r r e n c e o f polymorphism have been u n d e r t a k e n by i r s p e c t r o s c o p y , d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y and x-ray powder d i f f r a c t i o n ( G u i n i e r - d e W o l f f ) . N o polymorphism h a s been observed so f a r . An amorphous form may be p r e p a r e d a r t i f i c i a l l y by r a p i d e v a p o r a t i o n o f a methanolic s o l u t i o n of t h e drug substance. 2.33 X-Ray D i f f r a c t i o n X-ray s t r u c t u r a l a n a l y s i s o f b r o m o c r i p t i n e , c r y s t a l l i z e d a s t h e b a s e from dichloromethane/diethylether, h a s been carr i e d o u t on a CAD 4 - d i f f r a c t o m e t e r w i t h CuKa-radiation ( 1 6 ) . 3371 r e f l e x i o n s were w i t h i n s i n @/Alo00 mg/kg ( 2 ) . Thus, bromocriptine proved less t o x i c than t h e nonhydrogenated e r g o t a l c a l o i d s by one o r d e r of magnitude, resembling t h e behaviour of t h e dihydrogenated derivatives. Chronic t o x i c i t y s t u d i e s were c a r r i e d o u t with r a t s , dogs, and rhesus monkeys ( 2 ) . I n n e a r l y a l l c a s e s , the p r i n c i p a l e f f e c t produced w a s ischemia of some p a r t of t h e body. The well-known e m e t i c e f f e c t of e r g o t a l c a l o i d s i n dogs w a s p a r t i c u l a r l y pronounced with bromocriptine, even with o r a l doses of a s low a s 0 . 1 mg/kg.

BROMOCRIPTINE METHANESULPHONATE

7.

69

A n a l y t i c a l Methods 7.1

Application of General T e s t s

Compared with t h e u n s u b s t i t u t e d e r g o t a l c a l o i d s (1,181 t h e i n t r o d u c t i o n of bromine i n t o t h e 2-position of t h e indole nucleus diminishes t h e r e a c t i v i t y of t h e molecule i n r e s p e c t t o general a l c a l o i d t e s t s . Nevertheless, they s t i l l remain applicable. 1. KeLlgrLs-tgsL. Bromocriptine m e s i l a t e i s d i s s o l v e d i n g l a c i a l a c e t i c a c i d t o which have been added t r a c e s of f e r r i c c h l o r i d e . A f t e r c a r e f u l l y l a y e r i n g with concentrated s u l f u r i c a c i d , a green colour i s produced a t t h e i n t e r f a c e . U s u a l l y an i n t e n s e b l u e - v i o l e t colour occurs with 2-H-substituted compounds. 2 . I a n grk's gegcLion, ( 2 4 ) Bromocriptine m e s i l a t e i s dissolved i n methanol. After a d d i t i o n of van U r k ' s r e a g e n t and vigorous shaking a blue colour develops slowly, which i s s u b s t a n t i a l l y weaker than with 2-H-ergot a l c a l o i d s .

3. Meyeg's t e z t L Bromocriptine m e s i l a t e i s d i s s o l v e d i n methanol/water 3:lO. After a d d i t i o n of one drop of d i l u t e d hydrochloric a c i d and one drop of Meyer's reagent (mercuric potassium iodide) and s h a k b g , a white p r e c i p i t a t e i s produced. 7.2

Titration

Bromocriptine m e s i l a t e may be assayed i n g l a c i a l a c e t i c a c i d / a c e t i c anhydride 1 : 7 by t i t r a t i o n with 0 . 1 N p e r c h l o r i c a c i d . The endpoint may be determined p o t e n t i o m e t r i c a l l y using a glass/calomel e l e c t r o d e system. The methanesulphonic a c i d c o n t e n t of bromocriptine m e s i l a t e i s u s u a l l y determined by t i t r a t i o n with 0 . 1 N methanolic potassium hydroxide. The endpoint may be d e t e c t e d p o t e n t i o m e t r i c a l l y using a glass/calomel e l e c t r o d e system. Residual N-bromosuccinimide from t h e manufacturing process may be i d e n t i f i e d and/or q u a n t i f i e d by making use of i t s o x i d a t i o n p o t e n t i a l by t i t r a t i o n of l i b e r a t e d i o d i n e a f t e r a d d i t i o n of potassium iodide i n a c e t i c a c i d ( 2 5 ) .

70

DANIELLE A. GIRON-FOREST AND W. DIETER SCHONLEBER

7.3

Spectroscopic Methods

7.31 Infrared I n f r a r e d spectroscopy i s u t i l i z e d f o r i d e n t i f i c a t i o n purposes during t h e a n a l y s i s of t h e drug substance. (see 2 . 2 1 ) 7.32 U l t r a v i o l e t Spectrophotometric a n a l y s i s of bromocriptine m e s i l a t e i s c a r r i e d o u t d i r e c t l y using t h e uv maximum a t about 305 run i n methanolic methanesulphonic a c i d . However, t h e method i s n o t very s p e c i f i c . I t was p r e f e r r e d t o f i r s t s e p a r a t e t h e i m p u r i t i e s from bromocriptine by t h i n l a y e r chromatography and then t o i s o l a t e t h e substance by e l u t i o n from t h e s i l i c a g e l of t h e p l a t e with methanol. The i n t a c t a c t i v e i n g r e d i e n t i s measured i n 0.01 M methanolic methanesulphonic a c i d ( 2 6 ) .

7 . 3 3 Colorimetry I n moderately a c i d i c s o l u t i o n s bromocriptine m e s i l a t e r e a d i l y forms i o n p a i r s with a n i o n i c dyes such a s p i c r i c a c i d , bromothymol blue, methyl orange, which a r e e x t r a c t a b l e with an organic s o l v e n t . A procedure has been developed both f o r d i r e c t assay and f o r assay following chromatographic separat i o n from t h e i m p u r i t i e s . Therein bromocriptine m e s i l a t e i s allowed t o r e a c t with bromothymol blue a t pH 2.5. The r e s u l t i n g i o n p a i r i s then e x t r a c t e d with benzene and i t s concent r a t i o n determined a t 410 nm ( 2 5 ) . 7.34 Proton Magnetic Resonance PMR spectroscopy may be used f o r i d e n t i f i c a t i o n of t h e drug substance. ( s e e 2 . 2 4 )

7.4

Chromatography

7 . 4 1 Paper Paper chromatography was applied formerly t o t h e d e t e r mination of bromocriptine. conditions: system 1: s t a t i o n a r y phase: 25 % formamide mobile phase : carbon t e t r a c h l o r i d e / d i e t h y l e t h e r 1:l

BROMOCRIPTINE METHANESULPHONATE

71

system 2 : s t a t i o n a r y phase: 10 % dimethylphthalate mobile phase : dimethylformamide/ 0 . 5 N hydrochloric a c i d 15:85 The drug substance i s v i s u a l i z e d by r e a c t i o n with i o d i n e vapour

.

Rf values of bromocriptine and precursor system 1

system 2

0.88

0.09

0.70

0.31

Bromocriptine a- ergocr yp t i n e (precursor) 7 . 4 2 Thin Layer

The r e l a t i v e i n s t a b i l i t y of bromocriptine makes it very d i f f i c u l t t o avoid a r t i f a c t formation i n t e s t s o l u t i o n s o r during s p o t t i n g on t h e p l a t e . Therefore, bromocriptine mesil a t e , u s u a l l y d i s s o l v e d i n chloroform/methanol 1:1, has t o be s p o t t e d on t h e p l a t e very r a p i d l y and with t h e exclusion of l i g h t . The s e p a r a t i o n being terminated, t h e mobile phase i s removed by means of high vacuum f o r 30 minutes. A g r e a t number of t l c systems have been i n v e s t i g a t e d f o r t h e s e p a r a t i o n of by-products, degradation products, metabol i t e s , and e x c i p i e n t s . Also a v a r i e t y of spraying r e a g e n t s have been t e s t e d ( s e e t a b l e ) . The most advantageous one w a s Dragendorff's r e a g e n t with consecutive spraying by 30 % hydrogen peroxide. methods: s t a t i o n a r y phase: s i l i c a g e l 60 F254, Merck t l c p l a t e s , no.5715 mobile phase 1

:

(27)

dichloromethane/dioxane/96 p e r c e n t ethanol/conc.ammonia (v/v/v/v)

180:15:5:0.1

mobile phase 2

:

chloroform/methanol/formic a c i d 7 8 :20 : 2 (v/v/v)

mobile phase 3

:

chloroform/96 p e r c e n t ethanol/conc. ammonia 192:8:0.35 (v/v/v)

I n the procedure with mobile phase 1 and 2 , t h e visual i z a t i o n i s accomplished by screening under uv l i g h t (254 and 366 nm) and i n a d d i t i o n by spraying with Dragendorff's reagent, modified by Munier and Deboeuf, followed by 30 perc e n t hydrogen peroxide.

DANIELLE A. GIRON-FOREST AND W. DIETER SCHONLEBER

72

values st mobile phase 1 mobile phase 2 R

compound bromocr i p t i n e

2-bromo-a-ergocryptinine

1 . 0 (Rf 0.5) 1.5

a-ergocryptinine (isomer of precursor) a-ergocryptine (precursor) 2-bromo-lysergic a c i d 2-bromo-isolysergic a c i d 2-bromo-lysergamide 2-bromo-isolysergamide

1.4 0.6 0.0

0.0 0.5 0.5

1 . 0 (Rf 0.7) 1.1 1.0 0.5 0.25 0.35 0.15 0.35

Mobile phase 3 may be used f o r t h e d e t e c t i o n and s e m i q u a n t i t a t i v e determination of r e s i d u a l N-bromosuccinimide. Thereby, t h e p l a t e i s sprayed with water and then placed i n a c h l o r i n e atmosphere f o r 10 minutes. Excess c h l o r i n e i s removed by p l a c i n g t h e p l a t e i n a stream of w a r m a i r f o r another 1 0 minutes. A f t e r spraying with o - t o l u i d i n e reagent, the eval u a t i o n i s made a g a i n s t a d i l u t i o n of N-bromosuccinimide. i s 0.3 with r e s p e c t t o The d e t e c t i o n l i m i t i s 0 . 1 pg, t h e R st bromocriptine R f value. A high performance t l c system has been developped f o r t h e in-process-control (28) using Merck s i l i c a g e l HPTLC p l a t e s , no. 5628, a s t h e s t a t i o n a r y phase and tetrahydrofuran/ chloroform/n-heptane/methanol/conc. ammonia 20:20:57:7:1 p e r volume a s t h e mobile phase. The chromatography ( 6 cm ascending) i s c a r r i e d o u t without preceding chamber s a t u r a t i o n . The compounds separated a r e v i s u a l i z e d by uv a t 254 and 366 nm, r e s p e c t i v e l y , and by i o d i n e vapour. Rf of bromocriptine i s 0.27. The p o t e n t i a l by-products y i e l d s p o t s a t R 1.5 s (2-bromo-a-ergocryptinine) , and 0.55 (a-ergocryptiney 2-Bromo-lysergic a c i d remains a t the s t a r t i n g p o i n t .

.

Beside t h e modified Dragendorff's reagent/hydrogen peroxide a l r e a d y mentioned, a v a r i e t y of spraying reagents has been used f o r t h e v i s u a l i z a t i o n of bromocriptine. They a r e compiled i n t h e following l i s t .

BROMOCRIPTINE METHANESULPHONATE

reagent

spraying r e a g e n t s f o r bromocriptine detection l i m i t colour on s i l i c a g e l Merck no. 5715

Dragendorff (Munier,Deboeuf) + 30 p e r c e n t H 2 0 2 0.05 p e r c e n t aqueous permangana t e van U r k ' s r e a g e n t cinnamic aldehyde formaldehyde/hydrochloric acid formaldehyde/sulphuric a c i d chlorine/o-toluidine folin/ammonia ninhydrin/uv d e t . 360 iodine/potassium i o d i d e E h r l i c h ' s reagent

brown yellow grey yellow grey violet violet grey red brown orange

The i d e n t i t y of methanesulphonic a c i d may be determined by t l c on c e l l u l o s e p l a t e s , Merck no. 5728, with ethanol/ water/conc. ammonia 80:16:4 (v/v/v) a s a mobile phase. Detection i s achieved by spraying with acid-base i n d i c a t o r s , e.g. bromocresol green o r s i m i l a r s p e c i e s . Rf of methanesulphonic a c i d i s 0.5 ( t h a t of bromocriptine base = 0.9). 7.43 G a s Chromatography GC cannot be a p p l i e d t o t h e a n a l y s i s of bromocriptine m e s i l a t e due t o i t s low v o l a t i l i t y and i t s thermal i n s t a b i l i t y . A procedure according t o 29 o r 30, which claims e x c e l l e n t i d e n t i f i c a t i o n and q u a n t i t a t i o n on t h e b a s i s of well-defined p e p t i d e s e c t i o n p y r o l y s i s products, h a s n o t y e t been attempted. However, GC i s very u s e f u l determining t h e r e s i d u a l r e c r y s t a l l i z a t i o n s o l v e n t butanone-2. The c o n d i t i o n s a r e t h e following:

Column : Porapak Q 80/100 mesh, i n 1.8 m x 2mm g l a s s Temperatures: i n j e c t o r 240 OC, column 130 O C flame i o n i z a t i o n d e t e c t o r , 240 OC. 7.44 High Performance Liquid A s e r i e s of HPLC systems have been u t i l i z e d both f o r assay and p u r i t y of bromocriptine m e s i l a t e . S a t i s f a c t o r y procedures have been accomplished both on s t r a i g h t phase ( s i l i c a g e l ) and on reversed phase ( o c t a d e c y l s i l a n i s e d s i l i c a g e l ) columns.

74

DANIELLE A. GIRON-FOREST AND W. DIETER SCHONLEBER

HPLC-conditions s tr ai gh t-phase: -s t a t i o n a r y phase: s i l i c a g e l , 5 pm, i n s t a i n l e s s s t e e l 25 cm x 3 mm i.d. mobile phase: water-saturated dichloromethane/methanol 100:2 (v/v) uv d e t e c t i o n a t 254 nm. F i g . 11 shows a chromatogram of bromocriptine mesilate spiked w i t h dodecylbenzene, p o t e n t i a l by-products and t h e p r e c u r s o r . The flow was 1.7 ml/minute ( 3 1 ) . Key: 1 = dodecylbenzene, 2 = bromocriptinine, 3 = a-ergocrypt i n i n e , 4 = bromocriptine, 5 = a-ergocryptine ( p r e c u r s o r ) .

reversed - - - -phase, --

zyste?l_I:

s t a t i o n a r y phase: Merck F@-18, 1 0 pm i n s t a i n l e s s s t e e l 25 c m x 4.6 mm i . d . mobile phase: g r a d i e n t : 35 t o 60 p e r c e n t B w i t h i n 60 min. A = water, 0 . 2 p e r c e n t t r i e t h y l a m i n e added B = a c e t o n i t r i l e , 0.2 p e r c e n t t r i e t h y l a m i n e added. uv d e t e c t i o n a t 280 nm. This system was found optimal f o r t h e p u r i t y t e s t . Fig. 1 2 shows a chromatogram of t h e drug substance spiked with p o t e n t i a l by-products and the precursor. The flow was s e t a t 4.0 ml/minute. Key: I = 2-bromo-a-ergocryptinine, I1 = a-ergocryptinine, 111 = a-ergocryptine ( p r e c u r s o r ) , I V = bromocriptine, V = 2-bromo-lysergamide, V I = 2-bromo-lysergic a c i d , V I I = 2-bromo-isolysergamide.

reversed-p&azeL --ZyEtgm-II-

(26) :

s t a t i o n a r y phase: Merck RP-18, 10 pm i n s t a i n l e s s s t e e l , 25 cm x 4.6 mm mobile phase: 0.01 M ammonium carbonate o r 0.05 M ammonium hydrogen carbonate s o l u t i o n / a c e t o n i t r i l e 35:65 i s o c r a t i c , flow 1 . 5 ml/min.

BROMOCRIPTINE METHANESULPHONATE

75

I

1

3

I Figure 11.

5

High Performance Liquid Chromatogram of Bromocriptine Mesilate, spiked with Dodecylbenzene, Precursor and p o t e n t i a l By-products. Adsorption Mode, i s o c r a t i c , Uv-detection a t 254 nm

DANIELLE A. GIRON-FOREST AND W. DIETER SCHONLEBER

76

5

1 c

0

c 2

Figure 1 2 .

4

6

8

10

12

L 14

L 16

18

20 min

High Performance Liquid Chromatogram of Bromocriptine M e s i l a t e , spiked w i t h Precursor and p o t e n t i a l By-products. Reversed-phase Mode, Solvent Gradient, Uv-detection a t 280 nm.

BROMOCRIF'TINE METHANESULPHONATE

7.5

71

D i f f e r e n t i a l Scanning Calorimetry

D i f f e r e n t i a l scanning c a l o r i m e t r y cannot be a p p l i e d t o q u a n t i f y t h e p u r i t y of t h e drug substance f o r t h e reasons mentioned i n 2.31 and 2.34. However, i t may be v a l u a b l e i n q u a l i t a t i v e comparisons from sample t o sample o r batch t o batch on t h e b a s i s of t h e i r corresponding d i f f e r e n t i a l scanning c a l o r i m e t r y p a t t e r n s . 7.6

Phase S o l u b i l i t y Analysis

For phase s o l u b i l i t y a n a l y s i s , a c e t o n i t r i l e appears t o be t h e most s u i t a b l e s o l v e n t . A t y p i c a l p l o t i s given i n f i g u r e 13 along w i t h t h e experimental conditions. 7.7

Analysis of t h e Dosage Form

The i d e n t i f i c a t i o n o f bromocriptine m e s i l a t e i n t h e dosage form c a n be c a r r i e d o u t by t h i n l a y e r chromatography using Merck p l a t e s with dichloromethane/methanol/formic a c i d 78:20:2 (v/v/v) and subsequent u v - v i s u a l i z a t i o n a t 254 and 360 run. Using t h i s method, i t i s important t o only a i r - d r y t h e s p o t a f t e r a p p l i c a t i o n t o t h e p l a t e , s i n c e more vigorous evaporation of t h e s o l v e n t w i l l g i v e r i s e t o a r t i f a c t s ( 3 2 ) . Bromocriptine can a l s o be i d e n t i f i e d a s t h e base by i r spectroscopy a f t e r e x t r a c t i o n from t h e dosage form with ethanol and removal of t h e s o l v e n t , both i n s o l u t i o n and i n a KBr p e l l e t ( 3 3 ) . Bromocriptine m e s i l a t e i n P a r l o d e l a t a b l e t s may be assayed i n a non-specific way by d i r e c t uv-spectrophotometry following e x t r a c t i o n with methanol ( 3 2 )

.

Fluorimetry i n 0 . 1 N hydrochloric a c i d has been a p p l i e d during t h e measurement of d i s s o l u t i o n r a t e of t h e dosage forms with e x c i t a t i o n a t 335 and emission a t 425 nm, r e s p e c t i v e l y (26)

-

A s p e c i f i c a s s a y of bromocriptine m e s i l a t e i n t h e dosage form may be c a r r i e d o u t by t l c followed by uv-spectrophotometry ( 2 6 ) (The system can a l s o s e r v e f o r i d e n t i f i c a t i o n p u r p o s e s ) . The drug substance i s e x t r a c t e d with methanol i n t h e absence of l i g h t , t h e chromatographic c o n d i t i o n s a r e : Merck p l a t e s F 254, mobile phase: dichloromethane/dioxane/ ethanol abs./conc. ammonia 180:15:5:0.1 per volume. The s p o t corresponding t o bromocriptine i s e x t r a c t e d with methanol, and t h e c o n c e n t r a t i o n i s determined a t about 300 nm by spec tropho tometry.

78

DANIELLE A. GIRON-FOREST AND

SYSTEfl COMPOSITION: MG OF SAMPLE PER

Figure 13.

w. DIETER SCHONLEBER

G OF SOLVENl

Phase Solubility Analysis Plot of Bromocriptine Mesilate, dried in High Vacuum for 15 Hours. Solvent: dry Acetonitrile, Vibration for 24 hrs. in the Absence of Light. Slope 1.41~0.05 %.

BROMOCRIF'TINE METHANESULPHONATE

79

A f u r t h e r s p e c i f i c assay i s t h e HPLC-determination of bromocriptine m e s i l a t e following e x t r a c t i o n with methanol from t h e dosage form using RP-18 a s t h e s t a t i o n a r y and aceton i t r i l e / O . O l M ammonium carbonate s o l u t i o n 65:35 a s t h e mobile phase. Uv-detection wavelength i s s e t a t 300 nm ( 2 6 ) .

For t h e d e t e c t i o n and e s t i m a t i o n of degradation products

i n dosage forms, a s o l v e n t g r a d i e n t , c o n t a i n i n g t h e components mentioned above, i s u t i l i z e d with advantage. 7.8

Determination i n Body F l u i d s and T i s s u e s ( 2 3 )

Due t o i t s p o t e n t e f f i c a c y i n t h e treatment of hyperprolactinemia and acromegaly, bromocriptine i s administered i n low doses leading t o minute c o n c e n t r a t i o n s i n body f l u i d s and t i s s u e s . Therefore, none b u t t h e most s e n s i t i v e a n a l y t i c a l methods can be used t o measure i t s c o n c e n t r a t i o n i n b i o l o g i c a l specimens. The only method so f a r a p p l i c a b l e f o r pharmacok i n e t i c s t u d i e s with bromocriptine i s t h e u s e of t h e r a d i o a c t i v e l y l a b e l l e d drug, measurement of t o t a l r a d i o a c t i v i t y and i t s f r a c t i o n a t i o n by chromatographic s e p a r a t i o n techniques f o r t h e assay of p a r e n t drug and major metabolites. Recently, a radioimmunoassay k i t f o r t h e a n a l y s i s of picogram q u a n t i t i e s of unchanged bromocriptine i n body f l u i d s has become a v a i l a b l e . G a s chromatography, mass fragmentography and l i q u i d chromatography a l s o appear t o be s u i t a b l e f o r determining bromocriptine i n plasma from p a t i e n t s with P a r k i n s o n ' s d i s e a s e which a r e on treatment a t high dose l e v e l s . These c u r r e n t l y developped procedures permit q u a n t i t a t i v e determinations down t o c o n c e n t r a t i o n s of 0.5 (GC) , 1 . 0 (MF), and 1 0 ng/ml (LC), respectively ( 3 4 ) . The p a t t e r n of m e t a b o l i t e s i n b i l e (animals o n l y ) and i n u r i n e have been i n v e s t i g a t e d using column chromatography (Amberlite XAD 2 and Sephadex D E A E ) , t l c and reversed phase HPLC i n combination with r a d i o a c t i v i t y monitoring. The p r i n c i p a l metabolites have been i s o l a t e d from t h e b i l e of r a t s t r e a t e d with high doses of bromocriptine. The s t r u c t u r e of t h e i s o l a t e d m e t a b o l i t e s was e l u c i d a t e d by means of spectroscopic techniques.

DANIELLE A. GIRON-FOREST AND

80

w. DIETER SCHONLEBER

8.

References

1. 2.

F.Troxler and A.Hofmann, Helv.Chim.Acta 2, 2160 (1957) Handbook of Experimental Pharmacology, Vol. 49:"Ergot Alcaloids and Related Compounds", B.Berde and H.O.Schild, Ed., Springer-Verlag, Berlin, Heidelberg, New York, 1978 E.Fluckiger, F.Troxler, and A.Hofmann, German Offenlegungsschrift no. 1 926 045 (1969) E. Kovacs and E.Fluckiger, Experientia 30, 1172 (1974) J.K.Murdoch, Can.J.Hosp.Pharm. (1977) 149 H.Gjonnoes, Tidsskr.Nor.Laegeforen 97, 1405 (1977) A.Saarinen, V.Myllila, M.Reunanen, and E-Hokkanen, Duodecim 93, 1219 (1977) Triangle, Sandoz Journal of Medical Science, g(1)(1978) Symposium on Bromocriptine, Special Supplement, The Medical Journal of Australia, Vol 2, no. 3 (1978) W.J.Weiner, P.A.Nausieda, and H.I.Klawans, Neurology 28, 734 (1978) H.R.Schneider, P.A.Stadler, P.Stutz, F.Troxler,and J.Seres, Experientia 33, 1412 (1977) W.D.Schonleber , A.L. Jacobs, and G. A.Brewer, jr. :"Dihydroergotoxine mesilate" in Analytical Profiles of Drug Substances 1,81 B. Kreilgaard, "Ergotamine tartrate" in Analytical Profiles of Drug Substances 6, 113 T.Inone, Y.Nakahara, and T.Niwaguchi, Chem.Pharm.Bul1. 20, 409 (1972) D.Voigt, S.Johne, and D.Groger, Pharmazie 29, 697 (1974) H.P.Weber, Sandoz Ltd., personal communication H-Bethke, B.Delz, and K.Stich, J.Chromatog. 123, 193 (1976) A.Hofmann: Die Mutterkornalkaloide, F.Enke Verlag, Stuttgart, GFR, (1964) P. Gull and P.A.Stadler, Sandoz Ltd., personal communication Sandoz stability report for drug substance Sandoz stability reports for Parlodel capsules and tablets This part was kindly contributed by E.Schreier, Sandoz Ltd. A.Ehmann, J-Chromatog. 132, 267 (1977) V.Creutzburg-Biermanova, Sandoz Ltd., personal communication

3.

4. 5. 6. 7. 8. 9. 10. 11.

12.

13.

14.

L

15. 16. 17. 18.

19.

20. 22. 23. 24. 25.

BROMOCRIPTINE METHANESULPHONATE

26. 27. 28. 29. 30.

31. 32.

33. 34.

81

Control procedure for dosage form, Sandoz Ltd. Control procedure for drug substance, Sandoz Ltd. R.Stampfli, Sandoz Ltd., personal communication F.J.W.Van Mansvelt, J.E.Greving, and R.A.De Zeeuw, J.Chromatog. 151, 113 (1978) T.A.Plomp, J.G.Leferink, R.A.A.Maes, J.Chromatog. 151, 121 (1978) H.P.Keller, Sandoz Ltd., personal communication W-Ableidinger,Sandoz Ltd., personal communication M.Devaud, Sandoz Ltd., personal communication N.-E.Larsen, R.Oehman, M.Larsson, E.F.Hvidberg, to be published in Clin. Chem. Acta.

Acknowledgments The authors are indebted to many colleagues at SandozWander Ltd. for their most valuable help, in particular to Messrs. H.-R. Loosli, H.P. Weber and E. Schreier. The technical assistance for the proper presentation of the mass spectrum by Prof. W. Simon, at the Swiss Federal Institute of Technology in Zurich, is gratefully acknowledged. Furthermore, the authors wish to express special thanks to Miss Moret for her secretarial assistance in preparing this manuscript.

Analytical Profiles of Drug Substances, 8

CALCITROL Eileen Debesis I. 2.

3. 4. 5. 6.

7. 8.

Description I . I Name, Formula, Molecular Weight I .2 Appearance, Color, Odor Physical Properties 2.I Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Melting Range 2.6- Differential Scanning Calorimetry 2.7 Thermogravimetric Analysis 2.8 Hot Stage Microscopy 2.9 Solubility 2.10 X-Ray Powder Diffraction 2.1 I Optical Rotation Synthesis Stability and Degradation Drug Metabolic Products Methods of Analysis 6. I Elemental Analysis 6.2 Chromatographic Methods 6.2 I Thin-Layer Chromatography 6.22 High Performance Liquid Chromatography 6.23 Gas Chromatography-Mass Spectrometry 6.3 Biological Methods 6.3 I Radioimmunoassay 6.32 Protein Binding Assay 6.33 Bioassay 6.4 Polarography Acknowledgments References

83

Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.

ISBN 0-12-260808-9

EILEEN DEBESIS

84

1.

Description 1.1 Name, Formula, Molecular Weight Calcitriol is 1 a, 2 5 - d i h y d r o x y c h o l e c a l c i f e r o l .

MW = 416.65 27"44'3

1.2 Appearance, Color, Odor Calcitriol is an odorless white crystalline powder. 2.

Physical Properties 2.1 Infrared Spectrum The infrared spectrum of calcitriol is shown in Figure 1 (1). The spectrum was recorded on a Perkin-Elmer Model 283 Grating Infrared Spectrophotometer and was measured in a KBr pellet which contained 1 mg of calcitriol in 300 mg of KBr. Figure 1: a.

b. c. d.

The following absorptions have been assigned for OH stretching (bonded): 3391 cm-l Aliphatic CH stretching: 2943,12872 cm-l CH deformation: 1468, 1318 cmC-0 stretching: 1056 cm-

2.2 Nuclear Magnetic Resonance Spectrum (NMR) The NMR spectrum of calcitriol, recorded on a Varian XL-lOO/Nicolet TT-100 pulsed Fourier Transform NMR spectrometer, with internal deuterium lock, is shown in Figure 2 (2). The spectrum was recorded using a solution of 0.84 mg of sample dissolved in 50 microliters of CD30D (100%D) containing 1% v/v tetramethylsilane in a 1.7 mm capillary tube. The spectral assignments are given in Table I.

WAVELENGTH (MICRONS)

2.5

3.0

4.0

5.0

6.0

8.0

10

I

I

I

I

I

I

I

I

16 20 25 1

I

I

100 -

0

I

I

I

I

I

I

I

Figure 1 Infrared Spectrum oE Calcitriol

I

I

I

!

87

CALCITRIOL

H

Table I NMR Spectral Assignments for Calcitriol Proton

Chemical Shift (a)

Multiplicity

-CH3(C18)

0.57

Sing1et

-CH3(C21>

0.95

Doublet

-CH3(C26,27 \

\

X H 2 , ;CH HO. ;CH ~

1

-1.16

Singlet

-1.38-3

Complex

4.08

Comp 1ex

4.32

Triplet

4.90, 5.28

Complex

6.07, 6.32

AB Quartet JAB = 11 Hz

/ -

Ho> I CH -

- CH2

HDO + 3 x OH (exchangeable protons) 4.78

EILEEN DEBESIS

88

2.3 Ultraviolet Spectrum The ultraviolet spectrum of calcitriol (1 mg of Calcitrio1/100 ml of absolute ethanol) in the region of 220 to 400 nm exhibits one maximum at 264 nm (E = 1.9 x 10 ) and one minimum at 226 nm. The spectrum is shown in Figure 3 (3). Mass Spectrum The low resolution mass spectrum of calcitriol is shown in Figure 4 (4). The spectrum was obtained using a Varian MAT CH5 spectrometer, which was interfaced with a Varian data system 620 I. The data system accepts the output of the spectrometer, calculates the masses, compares the intensities to the base peak, and plots this information as a series of lines whose heights are proportional to the intensities, 2.4

'

The molecular ion was measured at m/e = 416. Other characteristic masses were observed at m/e = 398, 380 and 362, corresponding to the l o s s of one, two and three molecules of water, respectively, from the molecular ion; m/e = 383, corresponding to the loss of water and CH3 from the parent peak; and m/e = 365, corresponding to the l o s s of two water molecules and one CH from the molecular ion. The base peak is 3 observed at m/e = 134, and corresponds to the

2.5

+Hf$!

..

moiety H

Melting Range Calcitriol melts at 111-115"C (3).

2.6 Differential Scanning Calorimetry Melting was accompanied by decomposition (5). Thermogravimetric Analysis Calcitriol was subjected to thermogravimetric analysis on a Perkin-Elmer Model TGS-1 Thermogravimetric Analyzer. The sample exhibited two overlapping weight losses. The initial weight loss, of 0.4%, began at 55°C and ended at 105"C, and was due to surface moisture or solvent. The second weight l o s s , due to decomposition, amounted to 2.5% at 205"C, 7.5% at 255"C, 33% at 305"C, and 93% at 355°C (5). 2.7

2.8 Hot Stage Microscopy As observed on a Mettler Hot Stage FP 52 with FP5 controller, the sample appeared as birefringent needle-like crystals which melted from 115-117°C. The sample did not recrystallize from the melt (5).

CALCITRIOL

89

0.6

0.5

0.4 W

0

z a

3 0.3 8

m U

0.2

0.I

0 250

300 NANOMETERS

Figure 3 Ultraviolet Spectrum of Calcitriol

350

Q)

a Kn

91

CALCITRIOL

2.9 Solubility Extensive solubility data is not readily obtainable due to the scarcity of calcitriol. The material is slightly soluble in methanol, ethanol, ethyl acetate and tetrahydrofuran ( 3 ) . 2.10 X-Ray Powder Diffraction The X-ray powder diffraction data for calcitriol are presented in Table I11 ( 3 ) ; instrumental conditions are given below. The diffraction pattern is shown in Figure 5. Instrument and Operating Conditions Instrument

Guinier-De Wolff Camera

Generator

GE XRD-6 50 KV, 12.5 mA

Detector

Film

Sample

As is

Densitometer"

Gelman DCD-16

Optics

Tungsten Lamp 575 nm Visible mode

Detector

Silicon phototransistor

Optical density

0.25

Beam exit slit

0.53 mm

*The diffraction pattern was recorded on film; the density of the individual lines in the pattern was determined by densitometry.

o

m

u

id a

a c.

93

CALCITRIOL

TABLE I11

Calcitriol Powder Diffraction Data 20

I/Io**

26.52 13.33 22.41 20.89 18.01 28.97 27.33 23.70 33.55 30.54 31.77 17.19 24.95 21.54 28.04 32.66 19.46 20.35 16.07 11.37 8.03 34.18 12.07

4.993 9.863 5.892 6.316 7.314 4.578 4.847 5.577 3.968 4.348 4.183 7.662 5.302 6.129 4.727 4.072 6.776 6.480 8.192 11.559 16.343 3.896 10.889 A

100 90 88

82 70 66 49 44 36 34 33 33

26 26 15 12 12 12 2

2 2 2

1

(interplanar distance)

*d

--

**I/Io

= percent relative intensity (based on maximum

2 Sin 0

intensity of 1.00)

2.11 Optical Rotation The specific rotation of calcitriol in absolute methanol, measured at 589 nm and 2SoC, was +48' ( 7 ) .

EILEEN DEBESIS

94

Synthesis Several procedures for the synthesis of calcitriol have et a1.(8) and Norman been reported in the literature. Parren -et al. (9) described the preparation of calcitriol from 25-hydroxyvitamin D3. Uskokovic and others (10-18) have described lengthier syntheses utilizing more readily available starting materials. A typical reaction scheme, utilizing 1 a, 25-diacetoxy-7-dehydrocholesterol as the starting material, is shown in Figure 6. 3.

Stability and Degradation Calcitriol must be protected from air and light. The drug substance exhibits good stability when stored at -15'C to -25'C in an argon atmosphere. The material is stable at room temperature when dissolved in a vegetable oil derivative, containing antioxidants, such as is used in calcitriol soft gelatin capsules (19). 4.

5.

Drug Metabolic Products Calcitriol may be absorbed directly into the intestine or bone, or may be hydroxylated to form 1 a, 24, 25-trihydroxycholecalciferol prior to intestinal absorption (20-24). Methods of Analysis 6.1 Elemental Analysis A typical elemental analysis of a sample of calcitriol is presented in Table IV (7);

6.

TABLE IV Elemental Analysis of Calcitriol E 1ement

% Theory

C H

77.84 10.64 11.52

0

% Found

77.53 10.75 11.92 (by difference)

2 o 2 v

0 ,

yz

g

x3R

I 0

O--

0

r

0 I

I

8

m

rn

1

1

0

r

8

'.

L

.rl V

0

r(

c,

k

.rl *+-I

I .rl

EILEEN DEBESIS

96

6.2 Chromatographic Methods 6.21 Thin-Layer Chromatography (TLC) The following TLC procedure is useful for determining the purity of calcitriol. It separates the previtamin, 1 a, 25-dihydroxyprecholecalciferol. A silica gel GF plate is activated by heating for one hour at 105OC and is then cooled in a desiccator. A low actinic all glass chromatographic chamber is equilibrated with the developing solvent, and 0.8 mg of calcitriol is applied to the plate from ethyl acetate. The plate is developed in an ascending mode in ethyl acetate:spectroquality heptane:methanol (100:10:2) for 15 cm. After air drying, the plate is viewed under shortwave ultraviolet radiation, then sprayed with a 15% w/v solution of phosphomolybdic acid in ethanol, followed by heating at 105OC for 10 minutes to develop the colors. The approximate R f values are summarized in Table V ( 3 ) . TABLE V Summary of TLC Data Compound 1 a, 25-Dihydroxyprecholecalciferol Calcitriol

Approximate R 0.4 0.5

6.22 High Performance Liquid Chromatography High performance liquid chromatography is used to determine the purity of calcitriol, and to separate it from related compounds. Using a 10 micron silica column of 25 cm length, and a mobile phase of spectroquality heptane: ethyl acetate:methanol (50:SO:l) at a flow rate of 1.7 ml/ minute, separation and quantitation are achieved. p-Dimethylaminobenzaldehyde may be used as an internal standard to compensate for variations in injection technique and instrumental conditions. With a 254 nm ultraviolet absorbance detector, 0.01 ug of calcitriol may be detected ( 3 ) . This procedure, with a mobile phase of spectroquality heptane:ethyl acetate:methanol (70:25:5) is also useful for analyzing calcitriol in soft gelatin capsules. The capsule fill solution may be injected directly. The amount of calcitriol in the capsule is determined by comparison to a calcitriol reference standard, prepared in a medium similar to the capsule fill ( 3 ) .

97

CALCITRIOL

6.23 Gas Chromatography - Mass Spectrometry Halket and Lisboa (25) examined several Vitamin D derivatives by capillary gas chromatography coupled with mass spectrometry. This technique offered the advantages of great sensitivity and separating power. Retention times and fragmentation patterns for ergocalciferol, cholecalciferol and calcitriol were reported. 6.3 Biological Methods 6.31 Radioimmunoassav Several workers (26-32) have reported on the use of radioimmunoassay for measuring calcitriol at very low (pg) levels in serum. 6.32 Protein Binding Assays Various protein binding techniques are reported (32-42) for the determination of calcitriol in plasma. Generally, a preliminary purification step is required to avoid interference from other plasma components. 6.33 Bioassays Stern. et al. (43.44) reDorted a bioassav technique based on fetal rat bone absorption of calcitriol. Parkes and Reynolds (45) developed an in-vitro bioassay using duodenal tissue from chicken embryos. .

.


is linear with concentration in the 0.01 tol6203 m~ region (46). 7.

Acknowledgments The author wishes to thank the staffs of the Scientific Literature Department and Research Records Office of HoffmannLa Roche Inc., Nutley, NJ.

EILEEN DEBESIS

98

8.

References 1. 2. 3. 4. 5.

6.

7. 8. 9.

10.

11. 12.

13. 14. 15. 16. 17. 18. 19. 20.

Ng, S., Hoffmann-La Roche, Inc. Personal Communication. Grant, Anne, Hoffmann-La Roche, Inc. Personal Communication. Rubia, Linda, Hoffmann-La Roche, Inc. Personal Communication. Benz, W . , Hoffmann-La Roche, Inc. Personal Communication. Ramsland, Arnold, Hoffmann-La Roche, Inc. Personal Communication. Macri, Frank, Hoffmann-La Roche, Inc. Personal Communication. Scheidl, F . , Hoffmann-La Roche, Inc. Personal Communication. Paaren, H. E., Hamer, D. E., Schnoes, H. K., and DeLuca, H. F . , Proc. Nat2. Acad. S c i . USA, 75, 2080 (1978). Norman, A. W., Myrtle, J. F., Midgett, R. J., and Nowicki, H. G., U.S. 3 , 772, 150 (November 13, 1973); CA 80:105146g (1974). Uskokovic, M. Baggiolini, E., Mahgoub, A., Narwid, T., and Partridge, J. J. , Vitamin D and ProbZems Related t o Uremic Bone Disease, Walter de Gruyter, Berlin, New York, 279 (1975). Okamura, U. H., Am. Chem. Soc. Abstr. Pap. 275th ACS Natl. Meet., Anaheim, California, Mar. 13-17, 1978, MEDI #32 (1978). Matsunaga, I., Ochi, K., Nagano, H., Shindo, M., Ishikawa, M., Kaneko, C., and DeLuca, F . H., to Chugai Pharmaceutical Co., Ltd. Japan Kokai 76 100,056 (September 3, 1976); CA 8639014313 (1977). Suda, T. , Horiuchi, N., and O g a t c E. , Tampakushitsu Kakusan Koso, 21, 844 (1956). DeLuca, H. F., TetrahehTn Lee&, 1972, 4147. Cohen, Z., Keinan, E . , Mazur, Y., and Ulman, A., J . Org. Chern., 41, 2651 (1976). Mielczarek, I., Wiad. Chem., 28, 813 (1974). 33, Kaneko, C. , Yuki Gosei KagakuTyokai Shi, 75 (1975). Barton, D. H. R., Hesse, R. H., Pechet, M. M., and Rizzardo, E., J . Chem. Soc., Chem. Commun., 1974, 203. Johnson, J. B., Hoffmann-La Roche, Inc. Personal Communication. Napoli, J. L., Vitamin D and Its Metabolites, Annu. Rep. Med. Chem., 10, 295 (1975).

r,

CALCITRIOL

21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

31. 32. 33. 34.

35. 36.

99

Kumar, R., and Deluca, H. F., Biochm. Biophys. Res. Commun., 69, 197 (1976). Kumar, R., Harnden, D., and DeLuca, H. F., Biochemistry, 15, 2420 (1976). Harnden, D., Kzar, R., Holick, M. F., and DeLuca, H. F., Science, 193,493 (1976). Castillo, L., DeLuca, H. F., and Ikekawa, N., J . BioZ. Chem., 252, 1421 (1977). Halket, J. M., and Lisboa, B. P., Acta. Endocrinol., (Copenhagen), 87, (Suppl. 215), 120 (1978). Clemens, T. L., Hendy, G. N., Graham, R. F., Baggiolini, E. G., Uskokovic, M. R., and O'Riordan, J. L. H., Endocrinology, 102 (Suppl.), 490 (1978). Schaefer, P. C., LifschitrM. D., Fadem, S. Z. and Goldsmith, R. S., Endocrinology, 102, (Suppl.), 320, (1978). Clemens, T. L., Hendy, G. N., Graham, R. F., Baggiolini, E. G., Uskokovic, M. R., and O'Riordan, J. L. H., J . Endocrinol. 77, 49P (1978). Clemens, T. L., Hendy, G.N., Graham, R. F . , Baggiolini, E. G . , Uskokovic, M. R., and O'Riordan, J. L. H., Clin. Sci. Mol. Med. Med., 54, 329 (1978). Fairney, A . , Turner, C., Baggiolini, E . G., Uskokovic, M. R., in Vitamin D: BiochLmioal, Chemical, and Clinical Aspects Related t o Calciwn MetaboZism, A. W. Norman, K. Schaefer, J. W. Coburn, H. F. DeLuca, D. Fraser, H. G. Grigoleit, D. von Herrath [eds.], Walter de Gruy-ter, Berlin, New York, 459 (1977). Eisman, J. A . , Hanistra, A . J., Kream, B. E., DeLuca, H. F., Arch. Biochem. Biophys., 176, 235 1976). Etsuko, A . , Suda, T., Nakano, H., Igaku No A y w n i , 102, 509 (1977). Brumbaugh, P. F . , Haussler, D. H., Bressler, R., and Haussler, M. R., Science, 183, 1089 (1974). Caldas, A . E., Gray, R. W., Lemann, J., Jr., J . Lab. CZin. Med., 91, 840 (1978). Shimura, F . , and TamGa,.M., Vitamins, 52, 173 (1978). Horst, P. L., Merchant, C . , Shephard, R., Hamstra, A . Jorgensen, N. A . , DeLuca, H. F . , J . Dairy S c i . , 60, (Suppl. l), 123 (1977).

EILEEN DEBESIS

100

37.

38.

39. 40.

41. 42. 43. 44.

45.

46.

Kream, B. E., Eisman, J. A., DeLuca, H. F., in Vitamin D: Biochemical, Chemical and Clinical Aspects Related t o Calcium Metabolism, A. W. Norman, K. Schaefer, J . W. Coburn, H. F. DeLuca, D. Fraser, H. C. Grigoleit, and D. von Herrath, [eds.], Walter de Gruyter, Berlin, New York, 501 (1977). Haussler, M. R., Hughes, M. R., Pike, J. W., McCain, T. A,, in Vitamin D: Biochemical, Chemical and Clinical Aspects Related t o Calcium Metabolism, A. W. Norman, K. Schaefer, J . W. Coburn, H. F. DeLuca, D. Fraser, H. C. Grigoleit, and D. von Herrath, [eds.], Walter de Gruyter, Berlin, New York, 473 (1977). Norman, A. W., Henry, H., Bishop, J. E., Coburn, J. W., CZin. Res., 26, 423A (1978). Eisman, J. A., Hamstra, A. J., Kream, B. E., DeLuca, H. F., Science, 193, 1021 (1976). Hughes, M. R., Baylink, D. J., Jones, P. G., Haussler, M. .R., J. CZin. Inuest. , 58, 61 (1976). Brumbaugh, P. F., Haussler, D. H., Bursac, K. M., Haussler, M. R., Biochemistry, 13, 4091 (1974). Stern, P. H., Hamstra, A. J., Dxuca, H . F., Bell, N. H., J. Clin. Edocrinol. Metab., 46, 891 (1978). Stern, P. H., Phillips, T. E., Lucas, S. V., Hamstra, A. J., DeLuca, H. F., Bell, N. H., in Vitamin D: Biochemical, Chemical and Clinical Aspects Related t o Calciwn Metabolism, A. W. Norman, K. Schaefer, J. W. Coburn, H. F. DeLuca, D. Fraser, H. G. Grigoleit, D. von Herrath, [eds.], Walter de Gruyter, Berlin, New York, 459 (1977). Parkes, C. O., Reynolds, J . J . , Moz. Cell. Endocrinol., 7, 25 (1977). Rucki, R. J., Hoffmann-La Roche, Inc. Personal Communication.

Analytical Profiles of Drug Substances, 8

CHLORTETRACYCLINE HYDROCHLORIDE George Schwartzman I ;Lola Wayland, Thomas Alexander Kenneth Furnkranz, George Selzer, and the USASRG * I.

Description I . I Drug Properties I .2 Physical Description and Optical Crystallography 1.3 Chemical Properties 1.4 Structure I .5 The FDA Chlortetracycline Standard 2. Physical Properties 2. I Thermal Properties (DTA, TGA) 2.2 X-Ray Powder Diffraction 2.3 Solubility 2.4 Acid-Base Properties 2.5 Polymorphism3. Spectral Properties (Optical) 3.1 Ultraviolet 3.2-Infrared 3.3 Spectropolarimetry 3.4 Fluorescence 4. Spectral Properties (Other) 4. I Proton NMR 4.2 ' T - N M R 4.3 Mass Spectrometry 5 . Chromatography 5 . I Paper and Thin-Layer 5.2 Gas-Liquid 6. Manufacture 6. I Fermentation 6.2 Isolation 7. Stability 8. Analytical Methods 8. I Microbiological 8.2 Chemical 9. References *The US.Antibiotics Standards Research Group (USASRG) is an ad hor collaboration of antibiotics researchers at the U S . Food and Drug Administration. Contributors to this monograph from the Bureau of Drugs include: T. Alexander. R. Barron, V. Folen. K. Furnkranz. G . Mack (Baltimore District), M. Maientbal. G. Mazzola (Bureau of Foods). G . Schwartzman. G. Selzer. E. Sheinin. J. Taylor, L.Wayland. and J. Weber. The USASRG was formed at the request of P. Weiss. National Center for Antibiotics Analysis. Individual contributions are referenced where possible. 'Retired. Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. 101 ISBN 0-12-260808-9

GEORGE SCHWARTZMAN ET A L .

I02

1.

DESCRIPTION

1.1 Drug P r o p e r t i e s C h l o r t e t r a c y c l i n e h y d r o c h l o r i d e (CTC-HCL) i s the h y d r o c h l o r i d e s a l t of a n a n t i b i o t i c s u b s t a n c e produced by t h e growth of Streptomyces a u r e o f a c i e n s (Fam. Streptomycetaceae) I t was d i s c o v e r e d i n 1948 by Duggar (1).

.

CTC-HC1 is a broad-spectrum a n t i b i o t i c and a n t i protozoan a c t i v e a g a i n s t many Gram-positive b a c t e r i a , some Gram-negative b a c t e r i a , s p i r o c h e t e s , amebae, and c e r t a i n l a r g e v i r u s e s . S t a p h y l o c o c c i have become g e n e r a l l y r e s i s t a n t to t h e drug, and d r u g - r e s i s t a n t s t r a i n s may b e found among o t h e r b a c t e r i a l genera and s p e c i e s g e n e r a l l y s e n s i t i v e t o i t . C r o s s - r e s i s t a n c e t o o t h e r a n t i b i o t i c s of t h e t e t r a c y c l i n e family is a u t o m a t i c ( 2 ) . Organisms may be c o n s i d e r e d s u s c e p t i b l e i f t h e Minimum I n h i b i t o r y Concentration (MIC) is n o t more t h a n 4.0 pg/ml and i n t e r m e d i a t e i f t h e M I C is 4.0-12.5 pg/ml ( s e e Table 1). T e t r a c y c l i n e s are r e a d i l y absorbed and are bound t o plasma p r o t e i n s i n v a r y i n g d e g r e e s . They a r e c o n c e n t r a t e d by t h e l i v e r i n t h e b i l e and e x c r e t e d i n t h e u r i n e and f e c e s a t high c o n c e n t r a t i o n s and i n a b i o l o g i c a l l y a c t i v e form. The mode of a c t i o n a g a i n s t microorganisms i n v o l v e s t h e i n h i b i t i o n of phosphorylation p r o c e s s e s i n b a c t e r i a l c e l l s ( 3 ) . TABLE 1 (5) Antimicrobial Spectrum of Chlortetracycline Minimum Inhibitory Microorganism Concentration (Vg/ml)

E.

Micrococcus pyrogenes aureus 209P Micrococcus pyrogenes v z . aureus 1248A Streptococcus pyrogenes Streptococcus Q Streptococcus faecalis Micrococcus flavus Diplococcus pneumoniae Sarcina lutea ~Bacillus subtilis Escherichia Haemophilus influenzae Klebsiella pneumoniae Neisseria catarrhalis Aerobacter aerogenes Proteus vulgaris Pseudomonas aeruginosa Salmonella schottmuelleri Salmonella typhi Shigella dysenteriae Brucella bronchiseptica Mycobacterium tuberculosis Mycobacterium friedmanii Mycobacterium smegmatis Mycobacterium s p . Candida albicans Clostridium butyricum Pasteurella multocida Vibrio percolans

0.292 0.39 0.29 0.147 0.39 0.292 0.098 0.147 0.195 1.45 0.312 0.29 0.098 1.17 4.6 25 3.12 1.17 3.12 0.292 0.147 0.122 0.073 0.58 100 0.078 0.049 0.098

CHLORTETRACYCLINE HYDROCHLORIDE

103

1.2

Physical Description and Optical Crystallography CTC-HC1 is a yellow, odorless powder composed mainly of crystals in the shape of small hexagons, and has the following optical crystallographic characteristics (4):

a: 1.635; optic sign negative; 2V = 59" (calc.);

8:

1.706; orthorhombic; extinction parallel; and

y: 1.730; symmetrical It is stable in air, but is slowly affected by light (6). 1.3

Chemical Properties CTC-HC1 is the HC1 salt of amphoteric CTC; it is multifunctional with two chromophores. It is a parachlorophenol with an aYf3-unsaturatedketone in conjugation. The second chromophore involves another a,B-unsaturated ketone that is in conjugation with an anomalously behaving amide (7). The tertiary amine is responsible for the basic character and the phenolic group is acidic. CTC is fluorescent and can be assayed polarographically (8). A particularly intriguing aspect of the chemistry of the compounds of the tetracycline family is their ability to form metallic complexes. CTC shares in this intensively studied capability, which is very likely related to the therapeutic activity (9). This property is also used in the purification (10) and analysis (11) of CTC.

1.4

Structure C22H23ClN208.HCl Empirical Formula 515.35 Molecular Weight

7-Chloro-4-(dirnethylamino)-1,4,4a,5,5a,6,11,12aoctahydro-3,6,10,12,12a-pentahydroxy-6-methyl-l,ll-dioxo-2naphthacenecarboxamide monohydrochloride (CAS-64-72-2) (6,12-14) (Figure 1).

1.5

The FDA Chlortetracycline Standard The current official FDA Chlortetracycline Working Standard is chlortetracycline hydrochloride, Lot W501-632B95-1 (9/29/53), obtained from Lederle, which markets the antibiotic under the proprietary name Aureomycin. The current working standard has an assigned potency of 1000 pg/mg (the term pg applied to chlortetracycline means the chlortetracycline activity (potency) contained in l u g of the FDA Chlortetracycline Master Standard (Lot #990-107-141-1),which is also chlortetracycline hydrochloride).

I05

CHLORTETRACYCLINE HYDROCHLORIDE

The Working S t a n d a r d is s t o r e d i n l o t s of 250 mg a t -2O"C, p r o t e c t e d from l i g h t and m o i s t u r e , a t t h e h a t i o n a l C e n t e r f o r A n t i b i o t i c s A n a l y s i s , Washington, DC.

2.

PHYSICAL PROPERTIES 2.1 Thermal P r o p e r t i e s D i f f e r e n t i a l Thermal A n a l y s i s (DTA) and Thermal G r a v i m e t r i c A n a l y s i s (TGA).CTC-HC1 i s s t a b l e u n t i l i t b e g i n s t o decompose e x o t h e r m i c a l l y a t a p p r o x i m a t e l y 230°C ( F i g u r e s 2 and 3 ) ( 1 5 ) . The compound d o e s n o t l o s e any m a s s u n t i l t h e f i n a l d e c o m p o s i t i o n t a k e s p l a c e . No polymorphs have b e e n s e e n i n t h e samples examined. 2.2

X-Ray Powder D i f f r a c t i o n The X-ray d i f f r a c t i o n p a t t e r n of t h e FDA Working S t a n d a r d h a s been d e t e r m i n e d . It d e m o n s t r a t e s c r y s t a l l i n e s t r u c t u r e ; t h e d a t a are l i s t e d i n T a b l e 2 ( 1 6 ) . TABLE 2 X-Ray D i f f r a c t i o n Data

(1)

1/11

9.10 8.48 (7.75a D(7.43 6.65 6.38 (5.72 D(5.61 5.28 5.16 D(4.96 (4.88 4.68 4.54 4.42 4.29 4.24 (4.15 D(4.09 3.88 3.80 3.71 3.64 3.57 3.52 3.37

11 12 56 44 13 43 81 42 61 8 8 15 3 40 100 70 25 52 78 37 31 59 33 16 25 23

d

3

=

Doublet.

d (i) 3.34 3.27 3.23 (3.17 D(3.12 3.095 2.982 2.948 2.910 (2.878 D(2.854 2.788 2.710 D ( 2 . 653 (2.632 2.560 2.482 2.440 2.421 2.390 2.343 2.290 2.235 2.210 2.170 2.142 2.093

1/11

17 37 16 73 74 38 8 9 47 25 31 35 36 14 10 30 22 13 27 12 24 7 14 37 13 23 21

d 2.060B D(1.985 (1.962 1.897 1.880 1.868 1.856 1.827 1.805 1.784 ~(1.748 (1.737 1.705 1.682 1.638 1.631 1.608 1.595 1.583

1/11

13 11 13 12 10 13 13 8 9 6 8 11 9 6 5 4 5 4 4

GEORGE SCHWARTZMAN ET AL.

106

120'

140"

Figure 2.

120°

Figure 3.

160'

180" 200'

220'

240"

Differential thermogram of CTC-HC1.

160'

200"

240°

Thermal gravimetric analysis curve of CTC-HC1.

CHLORTETRACYCLINE HYDROCHLORIDE

I07

2.3

Solubility The s o l u b i l i t y of a n t i b i o t i c s , i n c l u d i n g CTC-HC1, w a s r e p o r t e d by Andrew and Weiss (17). CTC-HC1 i s a n amphoteric s u b s t a n c e and consequently i t is solu'ble i n aqueous a c i d and b a s e . However, i t can r a p i d l y degrade i n t h e s e s o l v e n t s . Its s o l u b i l i t y i n w a t e r is about 8 mg/ml and i n methanol about 1 7 mg/ml. I n h i g h e r m o l e c u l a r weight a l c o h o l s , t h e s o l u b i l i t y of CTC-HC1 i s c o n s i d e r a b l y le ss t h a n i n methanol. For p r a c t i c a l purposes, i t i s i n s o l u b l e i n many common s o l v e n t s such a s t h e a l i p h a t i c hydrocarbons, benzene, I t i s r e a d i l y s o l u b l e i n p y r i d i n e and e t h e r , and chloroform. t o t h e e x t e n t of about 5 mg/ml i n formamide. P y r i d i n e is an u n d e s i r a b l e s o l v e n t because of i t s b a s i c i t y , and formamide i s n o t d e s i r a b l e because of t h e d i f f i c u l t y i n o b t a i n i n g and maintaining i t a s a s t a b l e solvent.

2.4

Acid-Base P r o p e r t i e s

CTC e x h i b i t s t h r e e a c i d i c d i s s o c i a t i o n c o n s t a n t s when t i t r a t e d i n aqueous s o l u t i o n s ( 1 8 ) . Stephens e t a l . (19)

i d e n t i f i e d t h e t h r e e a c i d i c groups ( F i g u r e 4 ) , and r e p o r t e d thermodynamic pKa v a l u e s of 3.30, 7 . 4 4 , and 9.27. Leeson e t a l . (20) a s s i g n e d pKa v a l u e s t o t h e f o l l o w i n g a c i d i c groups:

pKa

Assignment

3.30

Tricarbonylmethane System (A)

7.44

P h e n o l i c Diketone System (B)

9.27

Dimethylamino System (C)

Kalnins and B e l e n ' s k i i (21) v e r i f i e d t h e assignments by i n f r a r e d (IR) spectroscopy. The pH o f a 10 mg/ml aqueous s o l u t i o n , as d e s c r i bed i n t h e Code of F e d e r a l R e g u l a t i o n s monograph f o r CTC-HC1 (22) should l i e between 2.3 and 3.3. 2.5

Polymorphism I n r e c e n t y e a r s , w i t h growing concern about t h e r e l a t i v e b i o a v a i l a b i l i t i e s of d i f f e r e n t samples of t h e same drug s u b s t a n c e , polymorphism h a s become of p r i m e i n t e r e s t . Miyazaki and co-workers (23) have r e p o r t e d t h e e x i s t e n c e of The X-ray powder d i f f r a c two c r y s t a l l i n e forms of CTC-HC1. t i o n p a t t e r n s , I R s p e c t r a , d i s s o l u t i o n b e h a v i o r s , and h y g r o s c o p i c i t i e s t h a t t h e y r e p o r t e d were d i s t i n c t l y d i f f e r e n t and t h e r e w e r e d i s c r e p a n c i e s i n t h e b i o a v a i l a b i l i t i e s . I n l a t e r work on two forms of CTC b a s e p r e p a r e d by c r y s t a l l i z i n g from water o r methanol, t h e X-ray p a t t e r n s and

CHLORTETRACYCLINE HYDROCHLORIDE

I09

water c o n t e n t s were d i f f e r e n t , b u t o t h e r p h y s i c a l p r o p e r t i e s were i n d i s t i n g u i s h a b l e . T h e r e a p p e a r e d t o be no d i f f e r e n c e i n b i o a v a i l a b i l i t y ( 2 4 ) . No polymorphs were found i n t h e FDA CTC s t a n d a r d . 3.

SPECTRAL PROPERTIES (OPTICAL) 3.1 Ultraviolet Medium

Maxima (nm)

0 . 1 N HC1 0 . 1 N NaOH Methanol

368, 340sh, 322sh, 265, 229 345, 283, 253, 222 372, 342sh, 322sh, 2 6 2 s h , 253, 233

See F i g u r e 5 ( 2 5 ) . 3.2

Infrared The I R a b s o r p t i o n s p e c t r a of CTC-HC1 a r e shown i n F i g u r e s 6 and 7 . S p e c t r a were r u n on a Perkin-Elmer Model 467 g r a t i n g s p e c t r o p h o t o m e t e r as a K C 1 p e l l e t ( 1 mg/200 mg K C I ) , The s p e c t r a are e s s e n and as a N u j o l m u l l ( 2 0 mg/3 d r o p s ) . t i a l l y i d e n t i c a l . The CTC-HC1 s p e c t r u m may a l s o b e f o u n d i n a c o m p i l a t i o n of I R s p e c t r a of Drug R e f e r e n c e S t a n d a r d s by Hayden e t a l . ( 2 6 ) . Major a b s o r p t i o n f r e q u e n c i e s h a v e b e e n compiled (27) and a s s i g n m e n t s h a v e b e e n made on inany of t h e bands(28-31). Some of t h e m a j o r f r e q u e n c i e s and band a s s i g n ments are:

&

x

3360

2.98

vNH of NH2 (asym.)

3315

3.02

vNH of NH2 (sym.)

3200-3450

2.90-3.12

vOH ( a l c o h o l i c , masks NH)

2800-2400

3.57-4.17

t e r t i a r y amine h a l i d e s a l t , g r o u p of b r o a d bands

1675

5.97

vc-0

1582

6.32

6NH2

1450

6.90

6CH ( b e n d i n g of CH3)

1368

7.31

VCN

1042

9.60

OH ( d e f o r m a t i o n , a l c o h o l s )

GEORGE SCHU ,RTZMAN ET AL.

110

a

b

m v)

0 XI 00

B

2 0

m

0

Figure 5 .

WAVELENGTH (nm) Ultraviolet absorption spectra of CTC-HC1 i n a , 0.1N HC1; b , 0.1N NaOH; and c , methanol.

a,

c, rl rl

a

-ai V

rl

v)

M

(d

W 0

t

B 2 a

8

I800 1600 1400 WAVELENGTH (CM")

Figure 7.

1200

Infrared absorption spectrum of CTC-HC1 a s Nujol mull.

100

CHLORTETRACYCLINE HYDROCHLORIDE

113

3.3

Spectropolarimetry Because c o n f i g u r a t i o n a l i n f o r m a t i o n can be d e r i v e d from o p t i c a l r o t a t o r y d i s p e r s i o n and c i r c u l a r d i c h r o i s m s c a n s , c o n s i d e r a b l e work h a s been conducted u s i n g t h e s e t e c h n i q u e s t o s t u d y t h e t e t r a c y c l i n e s ( 3 2 ) . The a b s o l u t e c o n f i g u r a t i o n of CTC w a s determined u s i n g o p t i c a l r o t a t o r y d i s p e r s i o n d a t a (33) S p e c t r a l c u r v e s are p r e s e n t e d i n F i g u r e s 8 and 9. The c i r c u l a r d i c h r o i s m spectrum is similar t o t h a t p r e s e n t e d by Mitscher e t a l . ( 3 4 ) , e x c e p t t h a t t h e v a l u e s d i f f e r by a f a c t o r of about 1.5. I n Table 3, d a t a o b t a i n e d by M i t s c h e r and i n FDA l a b o r a t o r i e s are compared.

3.4

Fluorescence The CTC-HC1 FDA Working Standard g i v e s a y e l l o w f l u o r e s c e n c e under 1or.pwave u l t r a v i o l e t (W) l i g h t (375 m) (35). The e x c i t a t i o n (324 and 357 nm) and emission (443 nm) s p e c t r a of t h i s s t a n d a r d d i s s o l v e d i n 0.05N NaOH (11.4 mg/50 ml) a r e p r e s e n t e d i n F i g u r e 10.

TABLE 3 Molecular E l l i p t i c i t i e s of CTC Wavelength (nm) M i t s c h e r ' s Valuea FDA Value (X 103) (X 103) 236 254 288 318 355 ( s h )

+10 -31 +4 8 -24

+2 3 -48 +88 -32 -12

-14 aThese v a l u e s w e r e o b t a i n e d from t h e g r a p h i n R e f e r e n c e

4.

Ratio

0.43 0.64 0.54 0.73 1.09 34

.

SPECTRAL PROPERTIES (OTHER) P r o t o n NMR The 60 MHz NMR s p e c t r a of CTC-HC1 i n DMSO-d6 and i n methanol-d4 are shown i n F i g u r e s 11 and 1 2 , r e s p e c t i v e l y . The s h a r p s i g n a l s due t o t h e methyl groups are r e a d i l y a s s i g n a b l e ; t h e i r chemical s h i f t s and i n t e n s i t i e s are c o n s i s t e n t w i t h t h e s t r u c t u r e . The 2 methylene p r o t o n s a t C-5 are n o t e q u i v a l e n t and g i v e two sets of m u l t i p l e t s . Only one m u l t i p l e t is a s s i g n a b l e i n t h e methanol spectrum: t h e s i g n a l c e n t e r e d a t 2.20 ppm. I n DMSO-db t h e s e p r o t o n s appear between 1 . 5 and 2 . 3 ppm. The o n l y methine p r o t o n a c t u a l l y a s s i g n e d i s t h e one a t C-4. The c l o s e p r o x i m i t y t o t h e p o s i t i v e l y charged N c a u s e s a downfield s h i f t away from t h e o t h e r s i g n a l s . When D20 i s added t o t h e DMSO-db s o l u t i o n , t h e H-4 s i g n a l remains c o n s t a n t However, t h e two amide p r o t o n s , o r i g i n a l l y a t 9.15 and 9.55 ppm, exchange w i t h t h e deuterium and a r e no l o n g e r observed. These s i g n a l s were n o t observed i n methanol-d4, a g a i n , due t o

4.1

~-

NOIWMSIO W O l V l O Y lWl1dO

I I4




I-

w 40 k

z -

30

20 10

0 200 FIGURE 7:

300

400 WAVELENGTH

500

Corrected Fluorescence and Emission Spectra of Griseofulvin: la, Excitation Spectrum with Emission at 420 nm; l b , Emission Spectrum with Excitation at 295 nm.

EDWARD R. TOWNLEY

234

2.11

D i f f e r e n t i a l Scanning C a l o r i m e t r y F i g u r e 8 shows t h e DSC thermogram of g r i s e o f u l v i n o b t a i n e d w i t h a DuPont Model 900 Thermal Analyzer. A single s h a r p m e l t i n g endokherm o c c u r s f o r t h i s s u b s t a n c e w i t h o n s e t t e m p e r a t u r e a t 216 C.

2.12

Thermogravimetry F i g u r e 9 shows t h e TG thermogram of g r i s e o f u l v i n o b t a i n e d w i t h a DuPont Model 950 Thermogravimetric Analyzer. The thermogram shows no weight l o s s from ambient t o about 2OO0C followed by weight loss due t o s u b l i m a t i o n . 2.13

Electrophoretic Properties Zeta p o t e n t i a l s of d i s p e r s e d g r i s e o f u l v i n have been s t u d i e d b o t h a l o n e , and i n t h e p r e s e n c e of s u r f a c e - a c t i v e a g e n t s , t h e l a t t e r a t a c o n t r o l l e d pH ( 8 ) . A M o b i l i t y / Z e t a P o t e n t i a l pH p l o t of = +25mV g r i s e o f u l v i n , shows a p o s i t i v e c h a r g e a t pH 1 . 5 which r a p i d l y d e c r e a s e s t o z e r o a t pH 2 . 4 . There i s t h e n r e v e r s a l of c h a r g e followed by a n i n c r e a s e o v e r t h e pH r a n g e 2 . 4 t o 7 . 0 . The z e t a p o t e n t i a l a t t h e l a t t e r pH i s -45 mV. The p o t e n t i a l t h e n s t a y s c o n s t a n t o v e r t h e pH range 7 t o 10.

2.14

Solubility The f o l l g w i n g d a t a are g i v e n f o r t h e s o l u b i l i t y of g r i s e o f u l v i n a t 25 C ; a c e t o n e 30 g/L, carbon t e t r a c h l o r i d e 2 g/L, d i c h l o r o e t h a n e 80 g/L, dimethylacetamide, 40 g/L, dioxane 30 g/L, e t h y l e t h e r 0 . 7 g/L, h e p t a n e 0.3 g/L, methanol 0 . 4 g/L; m i n e r a l o i l (0.1 g/L; propylene g l y c o l 2 g/L; Span 80 0.2 g/L, Tween 80, 7 g/L water 0 . 2 g/L ( 1 7 ) .

3.

P r o d u c t i o n and S y n t h e s i s Griseof u l v i n is b i o s y n t h e t i c a l l y manufactured by e l a b o r a t i o n w i t h P e n i c i l l i u m griseofulvum and r e l a t e d s t r a i n s of Penicillia. The b i o s y n t h e s i s h a s been t h e s u b j e c t of numerous chemical and b i o l o g i c a l s t u d i e s , t h e l a t e s t of which is g i v e n by Harris, e t . a l . ( 9 ) F i g u r e 10. Other proposed b i o s y n t h e t i c pathways a r e d i s c u s s e d . G r i s e o f u l v i n was f i r s t i s o l a t e d i n 1938 by Oxford ( 1 0 ) (1939); i t s t o t a l s y n t h e s i s was accomplished i n 1960 and f o l l o w i n g y e a r s i n s e v e r a l l a b o r a t o r i e s ( B r o s s i e t a l . , 1960 ( 1 1 ) Grove, 1963; ( 1 2 ) Mutant s t r a i n s of P. patulum are used f o r t h e commercial p r o d u c t i o n of t h e a n t i b i o t i c by f e r mentation (9).

et. al.,

4.

Impurities Some f e r m e n t e r b r o t h i m p u r i t i e s have been l i s t e d by Holbrook, B a i l e y and B a i l e y (13) and r e p e a t e d i n a d e s c r i p t i o n

FIGURE 8:

Differential Scanning Calorimetry Curve of Griseofulvin

20

0

\ 50

100 T. 'C

FIGURE 9 :

200 250 300 350 150 (CORRECTED FOR CHROMEL ALUMEL THERMOCOUPLES)

Thermogravimetry Curve of Griseofulvin

400

450

500

237

GRISEOFULVIN

acetate

-

0

0

0

0

-

CHJO

a

0

0

OH

-

FIGURE 10:

0

I CHJ

OH H,C

a

I

CHJ

A Biosynthetic Route f o r G r i s e o f ulvin.

EDWARD R. TOWNLEY

238

of e f f i c i e n t l i q u i d chromatographic s e p a r a t i o n systems by B a i l e y and B r i t t a i n ( 1 4 ) and i n a g a s chromatographic s e p a r a t i o n system by Margosis ( 1 5 ) . S t r u c t u r e s are i n F i g u r e 11. The common i m p u r i t y found i n commercial b a t c h e s of g r i s e o f u l v i n is d e c h l o r o g r i s e o f u l v i n ( 1 4 , 1 5 ) which a p p e a r s t o b e i n t h e range of 0.5 t o 3 . 5 % .

5.

Stability G r i s e o f u l v i n is a s t a b l e drug s u b s t a n c e . After 1 2 y e a r s s t o r a g e a t room t e m p e r a t u r e no decomposition was d e t e c t e d by d i f f e r e n t i a t i n g LC methods (16). There is no p h o t o d e g r a d a t i o n under r e a s o n a b l e c o n d i t i o n s of l i g h t exposure ( 7 ) . G r i s e o f u l v i n is c o n v e r t e d t o g r i s e o f u l v i c a c i d under a c i d i c c o n d i t i o n s . 6.

Drug Metabolic P r o d u c t s The major human m e t a b o l i t e of g r i s e o f u l v i n i s 6-demethylg r i s e o f u l v i n and i t s g l u c u r o n i d e ( 1 7 , l B ) which a c c o u n t f o r about 65% of t h e i n t r a v e n o u s d o s e ( 1 9 ) and 3 5 t o 65% of t h e o r a l dose ( 2 0 , 2 1 ) . The 6 - d e m e t h y l g r i s e o f u l v i n is a l s o t h e major m e t a b o l i t e i n dogs ( 2 2 ) and r a b b i t s ( 2 3 ) w h i l e b o t h 4d e m e t h y l g r i s e o f u l v i n and 6 - d e m e t h y l g r i s e o f u l v i n are major m e t a b o l i t e s i n r a t s ( 2 4 ) and mice ( 2 5 ) . These m e t a b o l i t e s can b e determined by g a s l i q u i d chromatography v i a i s o p r o poxyl d e r i v a t i v e s ( 1 8 ) o r t r i m e t h y l s i l y l e t h e r d e r i v a t i v e s ( 2 6 , 2 7 ) . The 6-demethylgriseofulvin h a s been measured i n u r i n e by h i g h performance l i q u i d chromatography ( 2 8 ) and u l t r a v i o l e t spectrophotometry ( 1 9 ) . Only t r a c e amounts of g r i s e o f u l v i n are found i n t h e u r i n e ( 2 8 ) .

7.

Methods of A n a l y s i s

7.01

Identification A wine r e d c o l o r is produced when about 5 mg of g r i s e o f u l v i n are d i s s o l v e d i n 1 m l of s u l f u r i c a c i d w i t h about 5 mg of powdered potassium dichromate ( 2 9 ) .

7.02

Elemental A n a l y s i s A n a l y s i s of g r i s e o f u l v i n , w a s determined f o r c a r b o n , hydrogen, and c h l o r i n e . The carbon, and hydrogen a n a l y s i s w a s performed on a P e r k i n . E l m e r Model 240 i n s t r u m e n t . A n a l y s i s f o r c h l o r i n e w a s performed by combustion of t h e sample and c o u l o m e t r i c t i t r a t i o n u s i n g a n American I n s t r u m e n t Co. Chloride T i t r a t o r . The r e s u l t s from t h e e l e m e n t a l a n a l y s i s are l i s t e d i n

Table I X .

239

GRISEOFULVIN

0 I1

C

0 0

CH03

0

CH 3

-- 0

Dechlorgr i seo fulvin

@i0 \ CHO

c1

0

-

cii 3 0

c1

=c

CII 3

Dihydrogr iseofulvin

CH 3

Dehydrogriseofulvin

C H 30

c1

CH 3

Tetrahydrogriseofulvin CH30

C1

CtI 3

OCR 3

Griseofulvic Acid

c1 Isogriseofulvin FIGURE 11:

Impurities Found in the Fermenter Broth

EDWARD R. TOWNLEY

240

Table I X Elemental A n a l y s i s of G r i s e o f u l v i n : Element C H

c1

Batch UGFP-1961

% Theory

% Found

57.88 4.86 10.05

57.97 4.84 9.92

7.03

Spectrophotometric Analysis Q u a n t i t a t i v e u l t r a v i o l e t a n a l y s i s of g r i s e o f u l v i n may b e performed by comparison t o a R e f e r e n c e S t a n d a r d . The u l t r a v i o l e t absorbance i s d e s c r i b e d i n S e c t i o n 2.04 and F i g u r e 5. 7.04

Spectrofluorometric Analysis Griseof u l v i n e x h i b i t s f l u o r e s c e n t p r o p e r t i e s which have been u t i l i z e d f o r h i g h l y s e n s i t i v e a n a l y s e s i n blood and serum (30-33) s k i n and sweat ( 3 4 ) . Riegelman (32) h a s combined TLC s e p a r a t i o n w i t h a f l u o r i m e t r i c d e n s i t o m e t e r r e a d o u t t o g i v e a h i g h l y s p e c i f i c and s e n s i t i v e g r i s e o f u l v i n d e t e r mination i n plasma. Other a n a l y s e s are commonly performed i n e i t h e r 1%aqueous e t h a n o l ( 3 0 ) , a c t i v a t i o n maxima 295 and 335 nm, f l u o r e s c e n c e maxima a t 450 nm o r anhydrous methanol (31) a c t i v a t i o n maxima unchanged a t 295 and 335 nm, f l u o r e s c e n c e maxima 420 nm. Values are u n c o r r e c t e d . Other a p p l i c a t i o n s t o t h e a n a l y s i s of b u l k d r u g s , dosage forms o r as a d e t e c t i o n method f o r high performance l i q u i d chromatography are f e a s i ble. 7.05

Colorimetric Analysis A c o l o r i m e t r i c a s s a y of g r i s e o f u l v i n , based on t h e yellow-orange c o l o r (Xmax=420 nm) which d e v e l o p s when g r i s e o f u l v i n is h e a t e d w i t h i s o n i c o t i n i c a c i d h y d r a z i d e i n a l k a l i n e medium h a s been d e s c r i b e d by Unterman (35) and t h e mechanism i n v e s t i g a t e d by Unterman and Duca (36). 7.06

Iodometric Analysis Iodometric a n a l y s i s has been a p p l i e d t o t h e d e t e r mination of g r i s e o f u l v i n i n s t a g e s of t h e manufacturing proc e s s (37). The mycelium i s e x t r a c t e d w i t h chloroform and t h e a n a l y s i s c a r r i e d out i n a l c o h o l i c solution. The 0.01N i o d i n e s o l u t i o n is s t a n d a r d i z e d w i t h g r i s e o f u l v i c a c i d . 7.07

Turbidimetric Analysis A t u r b i d i m e t r i c a s s a y f o r potency e v a l u a t i o n of g r i s e o f u l v i n h a s been r e p o r t e d (38). The drug i s d i s s o l v e d i n

GRISEOFULVIN

24 1

e t h y l e n e g l y c o l monomethyl e t h e r (niethyl c e l l o s o l v e ) . Polym e r i z a t i o n i s induced w i t h g l y c e r o l and guanosine-5'-triphosp h a t e (GTP). 7.08

Polarographic Analysis A s t u d y d i r e c t e d toward a comparison of t h e reduct i o n p o t e n t i a l s f o r g r i s e o f u l v i n homologs and a n a l o g s s u g g e s t s t h a t polarography i s a method of g r i s e o f u l v i n i d e n t i f i c a t i o n ( 3 9 ) . In e t h a n o l i c s o l u t i o n w i t h 0 . 2 M K C 1 s u p p o r t i n g elect r o l y t e , g r i s e o f u l v i n shows two p o l a r o g r a p h i c waves w i t h h a l f wave p o t e n t i a l s a t about -1.58 V and -1.84 V. T h i s system h a s been a p p l i e d w i t h good r e s u l t s t o t h e d e t e r m i n a t i o n of f i n i s h ed p r o d u c t s i n c l u d i n g t a b l e t s . The a c c u r a c y , p r e c i s i o n and s e l e c t i v i t y of t h e method w a s compared w i t h t h e i o d o m e t r i c method ( 4 0 ) . I s o g r i s e o f u l v i n and g r i s e o f u l v i c a c i d do n o t interfere. (41,42) 7.09

Chromatographic Analyses 7.091

P a r t i t i o n Column Chromatography G r i s e o f u l v i n maybe s e p a r a t e d from o b s e r v e d s t r u c t u r a l l y s i m i l a r i m p u r i t i e s i n t h e f e r m e n t e r b r o t h by means of p a r t i t i o n column chromatography ( 1 3 ) . A C e l i t e c o l umn packing and s o l v e n t system c o n s i s t i n g of methano1:water: hexane:chloroform ( 8 : 2 : 9 : 1 ) was used. Tetrahydrogriseofulvin, dihydrogriseofulvin, i s o g r i s e o f u l v i n , dechlorogriseofulvin are s e p a r a t e d from g r i s e o f u l v i n . The s t r u c t u r e s f o r t h e s e compounds have been g i v e n i n S e c t i o n 5. 7.092 T a b l e X.

P a p e r Chromatography A p a p e r chromatography system i s g i v e n i n

(43) Table X

S o l v e n t System Benzene :Cyclohexane; Methanol :Water (5:5:6:4) Glacial a c e t i c a c i d , 0.5%, w a s added t o t h e o r g a n i c phase of t h e s o l v e n t after equilibration. 7.093 i n T a b l e XI.

Paper

Detection

Reference

Whatman No. 1

uv

43

Thin Layer Chromatography Thin l a y e r chromatographic systems are g i v e n The d e t e c t i o n method w a s U.V.

EDWARD R. T O W N L E Y

242

Table XI Adsorbent

Tr

Reference

Methanol :n-bu tanol; 95% ethano1:conc. ammonium hydroxide (4:1:2:1, by ~01.)

Silica Gel

0.64

44

Ch1oroform:isopropanol (3:1, by vole)

Silica Gel

0.86

44

n-butano1:formic acid: Silica Gel water (77:10:13 by vol.)

0.86

44

n-Butanol:95% ethanol conc. ammonium hydroxide:water (4:1:2:1 by vole)

Silica Gel

0.64

44

Ch1oroform:acetone (93:7 by vole)

Silica Gel

0.65

45

Ch1oroform:methanol (lot1 by vole)

Silica Gel

0.82

46

-

47

Solvent System

Ch1oroform:acetic acid Silica Gel diethyl ether (17:1:3) Methanol :benzene (2:98 by vol.)

Silica Gel

0.50

48

Ethyl acetate

Silica Gel

0.50

17

7.094

Gas Chromatopraphy Successful griseofulvin analyses by gas chromatography are reported for simulated samples (49) fermentation extracts (45) and pharmaceutical bulk and dosage forms (50). Margosis (15) describes the application of gas chromatography to the purity determination of griseofulvin. Separation from the related compounds; dehydrogriseofulvin, isogriseofulvin and dechlorogriseofulvin was demonstrated. In a subsequent collaborative study (50) the accuracy and precision of the method was established. Although griseofulvin is thermally stable, the high GLC temperatures require precautions similar to those taken for steroids when preparing columns, column supports and associated equipment (45).

GRISEOFULVIN

243

The s p e c i f i c and s e n s i t i v e GLC d e t e r m i n a t i o n of g r i s e o f u l v i n i n body f l u i d s and t i s s u e s such as s k i n , sweat, u r i n e and plasma w i t h e l e c t r o n c a p t u r e d e t e c t i o n h a s been used by s e v e r a l i n v e s t i g a t o r s (34,51,52). Gas chromatographic c o n d i t i o n s are g i v e n in Table XII. Table XI1

Carrier Gas

Column

150 cm x 4 mm I.D.; Ushaped s t a i n l e s s s t e e l t u b i n g , 1.5% QF-1 on Anakrom ABS 3 f t x 4 mm I.D.; coiled g l a s s tubing, 3% OV-101 on Gas Chrom Q

N2

He2

Column Temp.

Internal Standard

230'

245'

5 f t x 4 mm I.D.; g l a s s 10% Methane column, 3% OV-17 on 90% Argon Chromosorb W. o r Gas or Chrom Q.

Reference

diphenylphthalate

tetraphenylcyclopentadienone

27OoC

diazepam

45,49

15

34,50, 51,52

N2

3 f t x 4 mm I.D.; glass coiled. % OV-17 on Gas Chrom. Q

He 2

225'

tetraphenyl15 cyc l o p e n t adienone

7.095

High Performance L i q u i d Chromatography G r i s e o f u l v i n can b e r e a d i l y chromatographed on e i t h e r normal o r r e v e r s e p h a s e columns. S e p a r a t i o n of dec h l o r o g r i s e o f u l v i n , t h e most common s y n t h e t i c i m p u r i t y i s accomplished on C / 1 8 , CN (17) o r ETH columns. (15) I s o g r i s e o f u l v i n , i s s e p a r a t e d on e i t h e r CN (17) o r ETH columns (15). L i q u i d chromatography c o n d i t i o n s are g i v e n i n T a b l e XIII.

EDWARD R. T O W N L E Y

244

Table XI11

Column PBondapak C/18 ( o c t y l d e c y l c h e m i c a l l y bonded to silica)

Reverse phase

mondapak C/18 ( o c t y l d e c y l c h e m i c a l l y bonded to silica)

II

Zorbax CN (cyanop r o p y l c h e m i c a l l y bonded to silica)

11

Mobile Phase

Internal Standard

Refer-

Methanol: water 3:2

n-butyl p-hydroxy benzoate

16

45% a c e t o - d i a z e p a n

53

ence

nitrile in 45mM KH PO4 pn = 3.6

Permaphase ETH Normal (C-7 e t h e r c h e m i c a l l y phase bonded t o p e l l i c u l a r 30-50 mesh g l a s s packing)

Methanol: water 3:2

5% c h l o r o form i n hexane

m-phenyl phenol

16

14

7.10

B i o l o g i c a l Methods o r A n a l y s i s M i c r o b i o l o g i c a l p r o c e d u r e s have been developed f o r a s s a y of g r i s e o f u l v i n and a p p l i e d t o t h e a n a l y s i s of b u l k d r u g s and dosage forms ( 1 ) . The c y l i n d e r p l a t e a g a r d i f f u s i o n method is t h e o f f i c i a l m i c r o b i o l o g i c a l method of d e t e r m i n a t i o n ( 5 5 ) . Microsporum gypseum (ATCC 14683) i s t h e t e s t organism. 8.

I d e n t i f i c a t i o n and D e t e r m i n a t i o n i n Body F l u i d s and T i s s u e G r i s e o f u l v i n h a s u s u a l l y been determined i n body f l u i d s and t i s s u e s by s p e c t r o f l u o r i m e t r i c (30-34) o r g a s chromatograp h i c methods (45,46,48). More r e c e n t l y g r i s e o f u l v i n h a s been determined i n plasma by h i g h performance l i q u i d chromatography (53,541. A n a l y s i s of Dosage forms Usual dosage forms of g r i s e o f u l v i n are c a p s u l e s , t a b l e t s and b o l u s e s . These may b e p r e p a r e d f o r a n a l y s i s by s i m p l e l i q u i d s o l i d e x t r a c t i o n of drug s u b s t a n c e . Margosis (15,50) used chloroform as an e x t r a c t i n g s o l v e n t w i t h g e n t l e h e a t . A compendia procedure d e s c r i b e s t h e e x t r a c t i o n of g r i s e o f u l v i n from t a b l e t s w i t h b o i l i n g a l c o h o l . The a n a l y s i s f o r e x t r a c t e d drug s u b s t a n c e has been performed by s e v e r a l methods. Most common is a simple u l t r a v i o l e t a n a l y s i s ( 2 , 3 , 5 5 ) . Polarography u t i l i z i n g t h e system g i v e n i n S e c t i o n 7.08 h a s been used (39). Thin-layer, g a s and l i q u i d chromagraphy may a l s o be 9.

G R I S EO F U LV I N

245

used u t i l i z i n g systems d e s c r i b e d i n S e c t i o n s 7.093, 7.094, and 7.095 r e s p e c t i v e l y . These l a t t e r methods are v a l u a b l e because of t h e i r s p e c i f i c i t y . I n t h e United S t a t e s , g r i s e o f u l v i n drug s u b s t a n c e and dosage forms must conform t o t h e r e g u l a t i o n s of t h e F e d e r a l Food and Drug A d m i n i s t r a t i o n concerning a n t i b i o t i c d r u g s (55, 5 6 ) . M i c r o b i o l o g i c a l a s s a y r e s u l t s o b t a i n e d by a n a l y t i c a l methods d e s c r i b e d i n t h e s e compendia a r e c o n c l u s i v e . 10.

Acknowledgements The a u t h o r wishes t o thank D r . M. D. Yudis and D r . H. Suprenant f o r t h e i r encouragement f o r t h i s work and t o acknowledge t h e v a l u a b l e a s s i s t a n c e of members of t h e S c h e r i n g C o r p o r a t i o n , P h y s i c a l Organic Research S e c t i o n : D r . R. B r a m b i l l a , M r . P. B a r t n e r , M r . C. Eckhart and M r . R. F o e s t e r f o r t h e a c q u i s i t i o n and i n t e r p r e t a t i o n of t h e p h y s i c a l d a t a , and t o t h e S c h e r i n g l i b r a r y s t a f f p a r t i c u l a r l y M s . J . Nocka, f o r t h e l i t e r a t u r e s e a r c h e s and S c h e r i n g General O f f i c e S t a f f f o r t h e c a r e f u l t y p i n g of t h i s monograph.

EDWARD R. T O W N L E Y

246

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A. Zlatkis, "Advances in Gas Cromatography 1967," Proceedings of the Fourth International Symposium held in New York, N.Y., April 3-6, 1967. Preston Technical Abstracts Co., Evanston, Ill. 1967, p. 129.

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28.

15,

E. Papp, K. Magyar and H. J. Schwartz, J. Pharm. Sci., 65, 441 (1976).

29

H. Auterhoff and M. Kliem, Arch der Pharmazie, 309, 326 (1976)

30

C. Bedford, K.J. Child and E.G. Tomich, Nature, 2,364 (1959)

31.

M. Kraml, J. Dubuc and D. Dvornik, J. Pharm. Sci., 5 4 , 655 (1965).

32.

L.J. Fisher and S . Riegelman, J. Chromatogr.,.u, 268 (1966)

EDWARD R. TOWNLEY

248

19, ( 6 )

33.

K. E i s e n b r a n d t , Pharmazie,

406 ( 1 9 6 4 ) .

34.

V. P Shah, S. Riegelman and W. L. E p s t e i n , J. Pharm. S c i . , 6 l , 634 ( 1 9 7 2 ) .

35.

H.W.

36

H.W. Unterman and A l . Duca, Rev. Roum Chim. (1971).

37.

H.W. Unterman and A l . Duca,. Chim. Anal. 196 ( 1 9 7 2 ) .

38

J. Hoebeke and G. Van V i j e n , L i f e S c i . , l7, 5 9 1 ( 1 9 7 5 ) .

39.

H.W. Unterman and A l . Duca, Isr. J. Chem., l2, ( 5 ) 985 (1974).

40.

J. Kadar-Pauncz, Acta Pharm Hung., 3 5 , 297 ( 1 9 6 3 ) .

41.

H.W. Unterman and A l . Duca, Rev. Chim. ( B u c h a r e s t ) ( 1 9 7 1 ) ; 2, 188 ( 1 9 7 2 ) .

42

H.W. Unterman, and A l . Duca, Chim. Anal. 1 ( 2 ) , 97 ( 1 9 7 1 ) .

43.

S. Symchowicz and K.K.

Unterman, Rev. Chim. ( B u c u r e s t i )

16,( 5 )

286 ( 1 9 6 5 ) .

16,1077

(Bucharest) 2 ( 3 )

1,97

(Bucharest)

Wong, Biochem. Pharmacol, l5,

1595 ( 1 9 6 6 ) . 44.

W.A. Creasey, K.G. Bensch and S.E. Pharmacol., 20, 1579 ( 1 9 7 1 ) .

45.

R.J. Cole, J . W . K i r k s e y and C.E. b i o l . 9 l9, 1 0 6 , ( 1 9 7 0 ) .

46.

C. L i n , R. Chang, C . Casmer, and S. Metab. Dispos., 1,6 1 1 ( 1 9 7 3 ) .

47.

2 . Durackova, V.

Malawista, Biochem.

Holaday, Appl. Micro-

Symchowicz, Drug

B e t i n a , P. Nemec, J. Chromatog.,

116,

141 ( 1 9 7 6 ) . 48

49.

I s s a q , E.W. Barr, T. Wei, C. Meyers, A. A s z a l o s , J. Chromatog., 133, 291 ( 1 9 7 7 ) .

H.J.

S. I g u c h i , M. Yamamoto and T. Goromaru, J. Chromatogr.,

24,

182 ( 1 9 6 6 ) .

GRISEOFULVIN

249

64,

50.

M. M a r g o s i s , J. Pharm. S c i . ,

51.

H . J . S c h w a r t z , B.A. Waldman, and V. Madrid, J. Pharm. S c i . , 65, 370 (1976).

52.

V.P.

Shah, S . Riegelman and W.L.

1020 ( 1 9 7 5 ) .

E p s t e i n , J. Pharm. S c i . ,

6 1 , 634 (1972).

155, 206

53.

L.P. Hackett and L . J . (1978).

54.

R.L. N a t i o n , G.W. Peng, V. Smith and W.L. Pharm. S c i . , 67, 805, 1978.

55.

US Code of F e d e r a l R e g u l a t i o n s (1976) Food and DruRs, T i t l e 21, p a r t 436.105, Washington, D.C., US Government

D u s i , J. Chromatog.,

Chiou, J.

printing office. 56.

E.M. Oden, G.H. Wagman, and M . J . W e i n s t e i n , A n a l y t i c a l M i c r o b i o l o g y , Vol. 11, Academic P r e s s , 1972, p 385.

Analytical Profiles of Drug Substances, 8

HALCINONIDE Joel Kirschbaum I.

2.

3.

4.

5. 6.

7.

History, Description, Precautions, Synthesis 1 . 1 History I . 2 Name, Formula, Molecular Weight I . 3 Appearance, Color. Odor I .4 Precautions I .5 Synthesis Physical Properties 2.01 Single Crystal X-RayDiffraction 2.02 Mass Spectrometry 2.03 Nuclear Magnetic Resonance Spectrometry (NMR) 2.031 'H-NMR 2.032 IT-NMR 2.04 Infrared Spectrometry 2.05 X-Ray Powder Diffraction 2.06 Ultraviolet Spectrometry 2.07 Optical Rotation 2.08 Fluorescence Spectrometry 2.09 Melting Range 2.10 Differential Thermal Analysis 2. I I Thermal Gravimetric Analysis 2.12 Microscopy and Crystal Size 2. I3 Particle Size 2.14 Surface Area 2. I5 Polymorphism 2.16 Hydration Solution Properties 3 . 1 Intrinsic Dissolution Rate 3 . 2 Solubilities in Aqueous and Nonaqueous Solvents 3.3 Partition Coefficients Methods of Analysis 4. I Elemental and Inorganic Analyses 4.2 Identification, Ultraviolet and Colorimetric Analyses 4.3 Chromatographic Analyses 4.3 I High Pressure Liquid Chromatography (HPLC) 4.32 Thin-Layer Chromatography (TLC) 4.33 Column Chromatography 4.34 Paper Chromatography 4.4 Polarographic Analysis 4.5 Halcinonide in Tissues and Body Fluids Stability Acknowledgments References 25 I

Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISBN 0-12-260808-9

JOEL KIRSCHBAUM

252

1 History, Description, Precautions and Synthesis

1.1 History Halcinonide is a topical antiinflammatory agent. 192 Introduction of a 9a-fluoro group3 to the hydrocortisone molecule resulted in an eight-fold increase in antiinflammatory activity. Such other groups as 16a-hydroxy, 16a-methyl, 168methyl, 16,17-acetonide and 6a-methyl moieties have been found to diminish or eliminate mineralocorticoid activity resulting from the 9a-fluoro substituent, while retaining he enhanced antiinflammatory activity of the halo derivative Halcinonide lipophilicity is increased by masking the 16,17-hydroxyl groups as the acetonide, which increases the availability of the steroid at the site of action in the skin. The 21-chloro group also increased antiinflammatory properties. Halcinonide has systemic3 as well as topical activity.

E.

1.2 Names, Formula and Molecular Weight Halcinonide is the United States adopted name5 (USAN). The preferred chemical names6 are 21-chloro-9-fluoro-11Bhydroxy-l6a, 17-[(l-methylethylidene)bis(oxy)]pregn-4-ene-3, 20-dione, and 21-chloro-9a-fluoro-ll~-l6a,l7-trihydroxypregn-4 -ene-3,20-dione cyclic 16,17-acetal with acetone. Other chemical names are 21-chloro-9-fluoro-ll~-hydroxy-l6~,l7a-isopropylidenedioxy-4-pregnene-3,2O-dione, 9a-fluoro-21-chloro-llf3, 16a, 17a-trihydroxypregn-4-ene-3, 20-dione 16,17 acetonide and 21chloro-9-fluoro-118,16~,l7-trihydroxypregn-4-ene-3,2O-dione cyclic 16,17-acetal with acetone. 21

12

in

I

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0 .O

2qH 32 5 454.97 Daltons

>tt(,, 24

14

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4

6

Halcinonide has also been called Halog and Squibb SQ 18,566. The Chemical Abstracts systematic number is CAS-3093-35-4.

HALCINONIDE

1.3

253

Appearance, Color and Odor

Halcinonide is a white or practically white, odorless powder consisting of free flowing crystals. 1.4

Precautions

Since halcinonide is a corticosteroid, large doses of unformulated steroid are needed systemically before the unwanted side effects appear. Halcinonide is usually formulated at concentrations of 0 . 1 % , or less. Normal handling precautions are adequate.

1.5

Synthesis

The of halcinonide is summarized in Figure 1, starting with 16a-hydroxy-9a-fluorohydrocortisone (A4-pregnene-9a-f luoro-11@, 16a,1 7 a , 21-tetrol-3,20-dione; dihydrotriamcinolone, I), which is available c ~ m m e r c i a l l y . l ~ - ~ ~ This tetrahydroxy steroid is slurried in acetone, and then 70% perchloric acid is added slowly. The acetonide, I1 (9a-fluoro11~,16a,l7,21-tetrahydroxypregn-4-ene-3,2O-dioney cyclic 16,17acetal with acetone; d i h y d r o t r i a m c i n o l o n e - a c e t o n i d e ) precipitates spontaneously from solution. Mesyl chloride is added to the acetonide in pyridine to give the 21-mesylate derivative (dihydrotriamcinolone acetonide-21-mesylate, 111). Compound I11 is dissolved in dimethylformamide, lithium chloride is added and the mixture is refluxed to produce halcinonide (IV), which is recrystallized from a solution of n-propanol in water. 2.

Physical Properties

2.01 Single Crystal X-Ray Diffraction

14

The three dimensional structure was obtained by means of single crystal X-ray diffraction. CuKa radiation, a graphite monochromator, and a photomultiplier tube were used to collect 1825 total reflections on an automated diffractometer. Of these, 1162 were used for the analysis. Figure 2 shows a computer generated drawing of halcinonide. The position of the chlorine atom was not clear from the Patterson map, but the direct method program "MLJLTAN" gave its position. Least squares refinement of coordinates together with anisotropic temperature factors, in the final stages, gave an R factor of 0.11.

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JOEL KIRSCHBAUM

256

2.02 Mass S p e c t r o m e t r y F i g u r e . 3 i s t h e p l o t t e d low r e s o l u t i o n mass s p e c t r u m of h a l c i n o n i d e found u s i n g a n A E I S c i e n t i f i c Apparat u s L t d , Model 902 mass s p e c t r o m e t e r . S p e c t r a were c o l l e c t ed on f r e q u e n c y modulated a n a l o g t a p e and p r o c e s s e d on a D i g i t a l Equipment C o r p . , P D P - l l . The t a b l e 1 5 below summarizes h i g h r e s o l u t i o n d a t a from m/z 39 t o t h e p r o m i n e n t m o l e c u l a r i o n a t 454.1922, which c o r r e s p o n d s t o C24H3205FC1. Each f r a g m e n t i o n c o n t a i n i n g c h l o r i n e a p p e a r s as a d o u b l e t p e a k b e c a u s e c h l o r i n e is a m i x t u r e of two i s o t o p e s ( 3 5 C l and 3 7 C l > . T h e s e p e a k s a r e two mass u n i t s a p a r t w i t h a n i n t e n s i t y r a t i o of 3 t o 1. Fragmentation proceeds a l o n g s e v e r a l pathways, a s shown below.

439 419 396?)m/z

361

377

m/z 2 6 4

m/z

278

These s t r u c t u r e s do n o t n e c e s s a r i l y r e p r e s e n t the a c t u a l i o n i c s t r u c t u r e b u t o n l y t h e o r i g i n of t h e f r a g m e n t .

C H

The weak i o n s a t m/z 121, 122 and 1 2 3 (CgHgO, 0) a r e c h a r a c t e r i s t i c of A4-3-one s t e r o i d s . 8 11

0 and C H

8 10

Figure 3 Low Resolution Mass Spectrum of Halcinonide. See text for details. 5433 S Q 1 8 , 5 6 6 BFI NN009NFl 175 DEG

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High-Resolution Massa Spectrum of H a l c i n o n i d e Found Mass

Calc. Mass

454.1925 439.1680 419.2252 396.1501 377.2147 361.1838 319.1665 278.1695 264.1524 246.1422 135.0808 123.0769 122.0729 121.0683 76.9790 43.0184

454.1922 439.1687 419.2233 396.1503 37 7.2128 361.1815 319.1709 278.1682 264.1525 246.1420 135.0810 123.0810 122.0732 121.0653 76.9794 43.0184

a

Unsat.

bO/EC

8. 0 8. 5 8.5 8. 0 7.5 8.5 7.5 6.0 6.0 7.0 4.5 3.5 4.0 4.5 1.5 1.5

24 23 24 21 22 21 19 17 16 16 9 8 8

32 29 32 26 30 26 24 23 21 19 11 11 10

8

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5 5 5 4 4 4 3 2 2 1 1 1 1 1 1 1

Only t h o s e peaks c o n s i d e r e d t o b e s i g n i f i c a n t t o t h e d i s c u s s i o n are l i s t e d . element map c a n b e o b t a i n e d on r e q u e s t .

bNumber of double bonds and r i n g s . C

0 E E 0 E E E 0 0 0 E E 0 E E E

c H

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1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0

c1 1 1 0

1 0 0 0 0 0 0 0 0 0 0

1 0

A complete

HALCINONIDE

259

2 . 0 3 Nuclear Magnetic Resonance Spectrometry

2.031

(NMR)

'H-NMR

Figure 4 is the 100 MHz NMR Spectrum of halcinonide in deuterochloroform containing tetramethylsilane as internal reference at 0 Hz. The instrument used is a Varian Associates, Inc., Model XL-100 NMR spectrometer. Below is an interpretation of the various resonances. 'H-NMR

Proton at Carbon

Assignments

Chemical Shift

Peak Appearance S = singlet D = doublet B = broad

c-4

5.78

S

c-11

4.58

B

C-16

5.05

D J15,16= 4 . 0 Hz

C-18

0.87

S

c-19

1.51

S

c-21

4.19 4.57

AB quartet

8-Acetonide, methyl

1.15

a-Acetonide, methyl

1.44

These data agree with the published results of the 21hydroxy-A' analog, triamcinolone acetonide.l 7

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26 1

13C-NMR

The 13C-NMR spectrum of halcinonide, Figure 5, was obtained18 using a Jeol FX-60Q NMR spectrometer operating in the Fourier transform mode at 15 MHz. Numbers on the vario u s peaks refer to the assignments in the table below. These assignments were made by comparison of chemical shifts and 3C-19F coupling constants to those assigned to such related steroids as 9a-fluorocortisol (9a-fluoro-llf3,17,21-trihydroxypregn-4-ene-3,20-dione). Four pairs of closely related peaks show chemical shift differences small enough to lead to possible reversals o f their tentative assignments. These pairs are the peaks assigned to the acetonide methyl groups (6 = 28.6 and 6 = 25.1 ppm), the two carbonyl peaks (6 = 202.1 and 199.1 ppm), the methylene carbons assigned to C-1 and C-7 (6 = 26.2 and 28.5 ppm), and the methylene carbons assigned to C-2 and C-15 (6 = 33.2 and 33.7 ppm). These assignments must be verified by additional experimentation. 'C-NMR Assignments Carbon

1 2 3

4

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Chemical Shift 6 (ppm>a 26.2 33.7 199.1 124.7 169.0 30.7 28.5 33.1 99.0 43.7 70.2 37.3

(28.5) (33.2) (202.1)

(26.2)

44.8

43.4 33.2 81.9 98.1 17.0 21.9 202.1 47.1 111.5 28.6 25.1

C-F Coupling Constant (Hz) 3

3 19 175 21

2 (33.7)

6 (199.1) (25.1) (28.6)

a Referenced from center peak of deuterochloroform = 77.0 pprn from tetramethyl silane. Numbers in parentheses refer to alternative assignments (see text for details).

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HALCINONIDE

2.04

I n f r a r e d Spectrometry

F i g u r e 6 shows t h e i n f r a r e d s p e c t r u m of h a l c i n o n i d e r u n as a p o t a s s i u m bromide p e l l e t , u s i n g a Perkin-Elmer Model 6 2 1 i n f r a r e d s p e c t r o m e t e r . Below a r e t h e i n t e r p r e t a t i o n s of v a r i o u s a b s o r b a n c e s . l 9

1

Frequency (cm )

2.05

Interpretation

3450

-OH S t r e t c h

2950

-OH S t r e t c h

17 3 0

C z 0 Keto

1660

C3

Keto

1620

A4

C=C

X-ray Powder D i f f r a c t i o n

F i g u r e 7 is t h e powder X-ray d i f f r a c t i o n p a t t e r n of h a l c i n o n i d e a s o b t a i n e d on a P h i l i p s gowder Using a d i f f r a c t i o n u n i t e m i t t i n g CuKa r a d i a t i o n a t 1.54A. s c i n t i l l a t i o n c o u n t e r d e t e c t o r , t h e sample w a s scanned and r e c o r d e d from a p p r o x i m a t e 1 2 t o 4 0 d e g r e e s ( 2 0 ) . The t a b l e below i s t h e s o r t e d d a t a . 21;

20 (Degrees)

' d ' (Angstrom) 1

14.80 12.53 18.49 17.94 19.82 29.31 18.10 11.67 32.14 39.59 25.86 21.47 26.80 25.24 15.67 40.37 22.33 33.08 25.47 34.02 'Interplanar

5.99 7.06 4.80 4.94 4.48 3.05 4.90 7.58 2.78 2.28 3.45 4.14 3.33 3.53 5.66 2.23 3.98 2.71 3.50 2.64

Relative Area

2

1.000 0.866 0.738 0.526 0.422 0.266 0.246 0.225 0.195 0.171 0.165 0.142 0.135 0.135 0.131 0.124 0.117 0.114 0.053 0.052

distance

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Figure 6 I n f r a r e d Spectrum of H a l c i n o n i d e , Potassium Bromide P e l l e t . See t e x t € o r d e t a i l s .

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JOEL KIRSCHBAUM

2.06

U l t r a v i o l e t Spectrometry

U l t r a v i o l e t s p e c t r a 2 ' of h a l c i n o n i d e w e r e r e c o r d e d on a Beckman Acta C I I I s p e c t r o p h o t o m e t e r . The s p e c t r a i n m e t h a n o l , 0.M m e t h a n o l i c h y d r o c h l o r i c a c i d and 0 . W m e t h a n o l i c sodium h y d r o x i d e show p e a k maxima a t 238 nm, and r e m a i n unchanged a f t e r 24 h o u r s . Absorbances are t a b u notation. l a t e d below u s i n g t r a d i t i o n a l E 1l % cm

1% Elcm So l v e n t

Initial

3 Hours

24 Hours

Me t h a n o 1

379

374

373

0.M Methanolic

370

372

370

380

382

380

H y d r o c h l o r i c Acid 0 . IM M e t h a n o l i c

Sodium Hydroxide The s p e c t r u m i n 95% e t h a n o l a l s o h a s a peak maximum a t 238 nm and a p p e a r s s t a b l e f o r a t l e a s t 3 h o u r s . I n a c e t o n i t r i l e and e t h y l e t h e r t h e maximum i s s h i f t e d t o 234 nm and 229 nm, r e s p e c t i v e l y . 2.07

Optical Rotation

were

The s p e c i f i c r o t a t i o n s i n d e t e r m i n e d 2 3 t o b e as f o l l o w s ,

Wavelength (nm)

2.08

Specific Rotation

589

+156

578

+164

546

+188

436

+348

305

+436

F l u o r e s c e n c e Spectrometry

Using a Perkin-Elme54Model 204 f l u o r e s c e n c e s p e c t r o p h o t o m e t e r , no f l u o r e s c e n c e w a s found a t e i t h e r 1 o r 1000 ug h a l c i n o n i d e p e r mL o f m e t h a n o l . No f l u o r e s c e n c e w a s induced i n 0 . U h y d r o c h l o r i c a c i d o r 0 . U sodium h y d r o x i d e .

267

HALCINONIDE

2.09

Melting Range

Following the USP procedure25 for class lA compounds, the melting range.of halcinonide is 270-272" (reference 21). This is in excellent agreement with the results of differential thermal analysis, below, as well as hot stage microscopy, section 2.12. 2.10

Differential Thermal Analysis

A duPont Model 900 Differential Thermal Analyzer shows halcinonide to have one endothermZ6 at 269". Decomposition on melting precludes differential scanning colorimetry studies for purity.

2.11

Thermal Gravimetric Analysis

Using a Perkin-Elmer Model TGS-2 Thermogravimetric Analyzer, halcinonide was found26 to lose 0.3% total volatile material at 70". No further l o s s was found up to 150". The heating rate was 20"/minute under a nitrogen atmosphere. 2.12 the method to 4, 5 to three lots (acicular)

Microscopy and Crystal Type

Microscopically, the crystal type depends on of preparation of halcinonide. Slabs, either 2 10, or 10 to 15 microns square, were found in of halcinonide, intermingled with needle-like crystals 5 to 10microns 10ng.27

14 Single crystal x-ray diffraction studies showed that the crystals of halcinonide recrystallized from n-propyl alcohol-water azeotrope (79:22) are orthorhombic and belong to the gpace group P2 2 2 , with unit sell constants 1 of a = 10.007 A , b = 11.875 an& c = 19.460 A. Density is 1.330 gm/cm3, as measured by flotation in a hexane-carbon tetrachloride gradient. The molecular weight calculated from the unit cell volume and density is 461 daltons (theoretical is 455 daltons).

A

Hot stage microscopy26 was performed using a Mettler FP52 temperature controller at a rate o f 3"/minute. Melting began at 268.2", and by 271.4" all crystals were melted. The yellow melt slowly turned orange-brown. Cooling showed no recrystallization at ambient temperature.

JOEL KIRSCHBAUM

268

2.13

Particle

Size

By l i g h t s c a t t e r i n g u s i n g a Royco i n s t r u m e n t , a l l p a r t i c l e s are below 20 microns.27 C o u l t e r Counter ana l y s i s of ground h a l c i n o n i d e suspended i n a sodium c h l o r i d e s o l u t i o n shows 100% t o be

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Assignment

Chemical S h i f t (ppm)

Multiplicity

J (Hz)

H 3 ' , 5'

6.64

d

8

H 2'c 6'

7.60

d

8

8.59

S

7.89

S

4.8

m (broad)

0 II

H-C-,

5

unknown H, 6

+

The above a s s i g n m e n t s a r e i n g e n e r a l a g r e e m e n t w i t h t h o s e of Pastore3 for folates. The C, p r o t o n i s o b s c u r e d by t h e l a r g e HOD p e a k , and i t s l o c a t i o n was c o n f i r m e d by s h i f t i n g t h e HOD a b s o r b a n c e a t 8 0 " C. The p e a k a t + 7.9 ppm d u e t o a n unknown s p e c i e s h a s b e e n p r e s e n t i n a l l c a l c i u m l e u c o v o r i n samples. The i d e n t i t y o f t h e s p e c i e s r e s p o n s i b l e f o r t h i s s i n g l e t r e m a i n s unknown, b u t a t t h e p r e s e n t t i m e t h e s e a u t h o r s h y p o t h e s i z e t h a t t h e dL d i a s t e r e o m e r ' s f o r m y l g r o u p c o n t r i b u t e s t h i s peak. Since chemically prepared calcium l e u c o v o r i n i s a s s a y e d a t t h i s l a b o r a t o r y , a n 'H nmr s p e c trum h a s n o t been o b t a i n e d on a b i o l o g i c a l l y o r enzymatic a l l y p r e p a r e d m a t e r i a l . E i t h e r s h o u l d b e f o l i n i c a c i d and t h e r e f o r e have t h e 1 L c o n f i g u r a t i o n . Investigations t o r e s o l v e t h i s problem a r e c o n t i n u i n g . 2.3

Carbon-13 Magnetic Resonance Spectrum

The 1 3 C nmr s p e c t r u m r e p r o d u c e d i n F i g u r e 3 w a s o b t a i n e d from a D,O/NaOD s o l u t i o n of c a l c i u m l e u c o v o r i n on a V a r i a n X L l O O S p e c t r o m e t e r . The a s s i g n m e n t s on t h e TMS s c a l e a n d r e f e r e n c e d t o d i o x a n e are l i s t e d below: Assignment

Chemical S h i f t (ppm)

c- 2 c- 4

93.7 102.0 0.0

C- 6

c- 7

-

23.3

0

I1

- C-H

c- 9

98.2

- 25.0

I

I

I

I

1

I

1

I

I

1

1

1

I

I

I

I

I

I

I

D IOXAN E

c PPm FIGURE 3

13C NMR OF CALCIUM LEUCOVORIN

LSJ-57

LESLIE 0 . PONT ET A L .

324

A s s i gnmen t

Chemical S h i f t (ppm)

C-4a o r C-8a

25.2

C-8a o r C-4a

86.5

c-1'

54.7

C-2

I ,

C-3',

C-6'

62.4

c-5'

45.7

c-4 c-7

84.8

'

C-Ci

C- B C-Y

102.8

-

10.8 37.9 32.3

a- coo-

112.6

y-coo-

115.6

The above a s s i g n m e n t s a r e i n r e a s o n a b l e agreement w i t h t h o s e previously reported f o r p t e r i d i n e s . 2.4

U l t r a v i o l e t Spectrum

F i g u r e 4 r e p r e s e n t s t h e uv s p e c t r u m of c a l c i u m l e u c o v o r i n i n pH 7.0 p h o s p h a t e b u f f e r (0.10 M ) , A,, = 286 nm, Amin = 243 nm, and i n 0.10N NaOH (pH 1 3 ) , A,, = 282 nm, A m i n = 242 nm. These s p e c t r a l f e a t u r e s a r e s i m i l a r t o t h o s e r e p o r t e d i n t h e l i t e r a t u r e . 5-12 The most commonly r e p o r t e d a b s o r p t i v i t i e s f o r l e u c o v o r i n o r f o l i n i c a c i d were o b t a i n e d from s o l u t i o n s prepa:ed i n 0.1N NaOH; t h e s e v a l u e s v a r y from 2.7 x lo4 M-' cm-' t o 3 . 2 6 x 10' M-' crn-l'l. Robinson" r e p o r t s E~~~ a p p r o x i m a t e l y 5% lower ( a t pH 8 . 4 ) t h a n t h o s e i n b a s i c s o l u t i o n . The s i n g l e a b s o r p t i o n i s due t o c o n t r i b u t i o n s from p-amino-benzoylglutamate and t h e u n s a t u r a t e d p o r t i o n of t h e p t e r i d i n e r i n g . Loss of a b s o r b a n c e a t s h o r t e r and l o n g e r w a v e l e n g t h s i n b a s i c s o l u t i o n (256 nm and 368 nm) i s c h a r a c t e r i s t i c o f r e d u c e d f o l a t e s . Spectral d a t a o b t a i n e d from s o l u t i o n s p r e p a r e d i n a c i d s do n o t r e f l e c t l e u c o v o r i n a b s o r b a n c e s b e c a u s e t h e compound i s n o t s t a b l e . Leucovorin d e h y d r a t e s under a c i d i c c o n d i t i o n s t o produce anhydroleucovorin, N5 ,N"-methenyltetrahydrofolic acid, IV. T h i s compound h a s been i s o l a t e d i n more t h a n o n e form, d e p e n d i n g on pH. l 3 Q

CALCIUM LEUCOVORIN

325

1.0

0.9 0.8

0.7 w

0

z a

2

0.6 0.5 0.4

0.3

0.2 0.1

0 200

250

nm

350

300

LSJ-58

FIGURE 4

ULTRAVIOLET SPECTRUM OF CALCIUM LEUCOVORIN 0.1N NaOH pH 7 Phosphate buffer -~

LESLIE 0. PONT ET A L .

326

2.5

Mass Spectrum-

An e l e c t r o n impact mass s p e c t r u m of c a l c i u m l e u c o v o r i n h a s n o t b e e n o b t a i n e d b e c a u s e t h e compound i s n o t s u f ficiently volatile. I t would b e d i f f i c u l t t o i s o l a t e t h e f r e e a c i d w i t h o u t f i r s t d e h y d r a t i n g t h e compound. Due t o i t s i o n i c n a t u r e , calcium leucovorin w i l l n o t d i s s o l v e i n common s i l y l a t i n g r e a g e n t s . F i e l d d e s o r p t i o n , a n o t h e r mass s p e c t r a l t e c h n i q u e , g e n e r a l l y l e n d s i t s e l f more t o compounds l i k e leucovorin. I n d e e d , t h i s t e c h n i q u e h a s been a p p l i e d s u c c e s s f u l l y t o m e t h o t r e x a t e and o t h e r f o l i c a c i d a n a l o g s . l 4

2.6

X-ray C r y s t a l l o g r a p h i c Data

C r y s t a l l o g r a p h i c d a t a have n o t been p u b l i s h e d f o r c a l c i u m l e u c o v o r i n , b u t l i t e r a t u r e v a l u e s do e x i s t f o r t h e D i f f r a c t i o n p a t t e r n s could n o t be obtained barium s a l t . ' on a l l s a m p l e s a l t h o u g h t h e y had b e e n r e c r y s t a l l i z e d s e v eral t i m e s . I n t e r p l a n a r s p a c i n g s a r e l i s t e d , b u t t h e s e numb e r s a p p e a r t o change w i t h t h e amount o f m o i s t u r e p r e s e n t i n t h e sample. 2.7

Optical Rotation 21

0

a]

= 5 8 9

21

a]

0.3

14.3 k 0.4"

(c1, N/10

NaOH)

1 7 . 9 k 0.4'

(c 1, N/10

NaOH)

0

546 21

+

=

0

=

+

14.9 ? 0.4"

(C 1, H20)

=

+

18.8 t 0.4"

(c1, H z O )

5 8 9

21

a] 5 4 6

The above v a l u e s were o b t a i n e d on a n a v e r a g e l o t o f c a l c i u m l e u c o v o r i n examined in t h i s l a b o r a t o r y . C o r r e c t i o n s were made f o r water c o n t e n t . D i f f e r e n c e s i n r o t a t i o n v a l u e s may p a r t l y r e f l e c t s o l u b i l i t y i n t h e two s o l v e n t s ; t h e compound w a s more d i f f i c u l t t o d i s s o l v e i n N / 1 0 NaOH. The H 2 0 r o t a tion is c l o s e t o t h e l i t e r a t u r e value: (rID = 1 4 . 2 6 " 3.42 H,O).'

+

(c,

CALCIUM LEUCOVORIN

2.8

327

C i r c u l a r Dichroism

The f o l l o w i n g d a t a were o b t a i n e d on an a v e r a g e sample of c a l c i u m l e u c o v o r i n : Solvent

&

(nm)

[ e l (deg

M-lcm-')

0.1N NaOH

282

3.89

H20

287

3.47 x lo3

103

The molar e l l i p t i c i t i e s a r e c a l c u l a t e d f o r l e u c o v o r i n f r e e a c i d and have been c o r r e c t e d f o r water c o n t e n t . These v a l u e s may b e q u i t e d i f f e r e n t from t h o s e of f o l i n i c a c i d , Although a s i n g l e v a l u e i s r e p o r t e d f o r each s o l v e n t , b o t h s p e c t r a c o n t a i n more t h a n one a b s o r p t i o n band. Whether t h e e x t r a n e o u s a b s o r p t i o n s a r e due t o i m p u r i t i e s o r a r e a c t u a l l y due t o l e u c o v o r i n ' s a b s o r p t i o n b e h a v i o r i s n o t known at this time.

2.9

Dissociation Constants

Three pKa v a l u e s have been r e p o r t e d f o r l e u c o v o r i n ( f r e e a c i d ) ; t h e y a r e 3 . 1 , 4 . 8 , and 1 0 . 4 , as d e t e r mined by e l e c t r o m e t r i c t i t r a t i o n . 6 The f i r s t two v a l u e s a r e a t t r i b u t e d t o t h e g l u t a m y l c a r b o x y l s , and 10.4 is a s s i g n e d t o t h e h y d r o x y l group a t t h e 4 p o s i t i o n , ' by comparison t o model compounds.

2.10 S o l u b i l i t y The i o n i c n a t u r e o f c a l c i u m l e u c o v o r i n s e v e r e l y r e s t r i c t s i t s s o l u b i l i t y i n common o r g a n i c s o l v e n t s ( s o l I n water, t h e s o l u b i l i t y is u b i l i t y i n DMSO 20 15.852 6.885 14.080 9.260 6.578

1.030 7.014 2.083 1.742

7.989 0.77* >20* 7.938

11.288 3.257 >20 >20

7.328 0.217 9.463 1.046

6.485 9.40* >20*

13.038 >20 >20 1.365 10.580

4.500 3.918 >20 0.2 1 7 3.862

>20

* -+

N e omy c i n

0.05mg.

0.188 0.0

WILLIAM F . HEYES

412

2.3 .Physical P r o p e r t i e s of S o l u t i o n s 2.31.

Density

K i g e l e t a l l 8 have r e p o r t e d t h e d e n s i t y o f 6 & 1 8 % a q u e o u s neomycin s u l p h a t g s o l u t i o n s o v e r t h e t e m p e r a t u r e r a n g e 20° t o 8 0 C . The p u b l i s h e d v a l u e s a r e g i v e n i n T a b l e 5 . T a ble 5 D e n s i t i e s o f Neomycin S u l p h a t e S o l u t i o n s N e omy c i n

Density

Concen t r a t i on %

ooc

4 O°C

6 O°C

8 O°C

-

L

6

1.025

1.021

1.014

1.007

18

1.086

1.081

1.076

1.068

2.32. V i s c o s i t y T a b l e 6 shows t h e r e p o r t e d 1 8 v i s c o s i t i e s f o r 6 and 1 8 % a q u e o u s n e o m y c i n o s u l p b a t e s o l u t i o n s o v e r t h e t e m p e r a t u r e r a n g e 2 0 C-80 C . Table 6

--V i s c o s i t y o f Neomycin S u l p h a t e S o l u t i o n s Neomycin Concentration

Viscosity (cps) 2 ooc

4OoC

6 O°C

8OoC

6

1.25

0.86

0.64

0.623

18

1.99

1.30

0.95

0.74

9:

2.33.

S u r f a c e Tensiofl

Kigel e t a l l 8 h a v e r e p o r t e d t h e s u r f a c e t e n s i o n v a l u e s of 6 & 1 8 % aqueous neomycin sulphate s o l u t i o n s (Table 7 ) .

NEOMYCIN

413

Table 7 S u r f a c e T e n s i o n V a l u e s f o r Neomycin _Sulphate Solutions N e omy c i n

S u r f ace T e n s i o n (dynes/cm)

Concentration

2 ooc

4OoC

6

69.81

18

72.01

-%

6 O°C

8O°C

66.00

63.30

60.30

68.12

65.45

62.36

I

2.34.

--

-

A d s o r p t i o n by C l a y s , S o i l s & M i n e r als

The a d s o r p t i o n of a n t i b i o t i c s from a q u e o u s s o l u t i o n by c l a y s and m i n e r a l s h a s r e p o r t e d by a number of a u t h o r s . P i n c h e t concluded t h a t , f o r s t r o n g l y b a s i c a n t i b i o t i c s s u c h a s neomycin, a d s o r p t i o n by c l a y s s u c h as montmorillonite d i s t o r t e d the c r y s t a l l a t t i c e o f t h e c l a y 4.42, c o r r e s p o n d i n g t o m o n o l a y e r a d s o r p t i o n . The same a u t h o r s also s u g g e s t e d t h e bonding i n t h e monolayers t o be s t r o n g l y e l e c t r o s t a t i c . A t t e m p t s t o r e l e a s e t h e a n t i b i o t i c by the addition of b u f f e r s has en demonstrated t o be o n l y p a r t i a l l y successful". Phosphate b u f f e r s r e l e a s e d a p p r o x . 50% o f t h e a d s o r b e d a n t i b i o t i c w h e r e a s c i t r a t e , g l y c i n e and u n i v e r s a l b u f f e r s were c o m p l e t e l y i f f e c t i v e . In a recent study h a v e shown d i v a l e n t magnesium McGinity a n d H i l l " i o n s t o be more e f f i c i e n t t h a n m o n o v a l e n t sodium i o n s i n d i s p l a c i n g neomycin from t h e n e g a t i v e l y c h a r g e d c l a y s a t t a p u l g i t e and m o n t m o r i l l o n i t e .

!??'

From t h e s e s t u d i e s t h e l i m i t i n g a d s o r p t i v e c a p c i t i e s f o r t h r e e c l a y s w e r e calc u l a t e d u s i n g t h e Langmuir e q u a t i o n . Wayman e t a126 s t u d i e d t h e b i n d i n g of neomycin t o t h e f i l t r a t i o n m a t e r i a l s c e l i t e , c e l l u l o s e powder and S e i t z f i l t e r s . Neomycin w a s f o u n d t o b e a d s o r b e d on a l l t h r e e materials. Acid-washing t h e c e l l u l o s e powder f a i l e d t o d e s o r b a l l o f t h e neomycin.

414

WILLIAM F. HEYES

2 . 3 5 . A d s o r p t i o n by I o n Exchange R e s i n s During a s t u d y o f t h e physicoc h e m i c a l p r o p e r t i s o f some a m i n o g l y c o s i d e a n t i b i o t i c s Kuzyaeval' d e t e r m i n e d t h e s t a t i c and dynamic e x c h a n g e c a p a c i t i e s o f neomycin on t h e c a t i o n e x c h a n g e r e s i n KB-4P-2 ( a p h e n o x y a c e t i c acid-formaldehyde r e s i n ) u s i n g t h e r e s i n i n t h e Na form. K i l l f i n e t a127 h a v e r e p o r t e d v a l u e s f o r t h e e x c h a n g e e n t h a l p y o f neomycin € o r t h r e e i o n - e x c h a n g e r e s i n s . The v a l u e s of A H w e r e c a l c u l a t e d by a p p l i c a t i o n o f t h e Gibbs-Helmholtz e q u a t i o n ; t h e p u b l i s h e d r e s u l t s are t a b u l a t e d below:-

T ab le 8 Some Exchange E n t h a l p i e s f o r Neomycin Ion exchange resin

+ form

Type

AH c a l e q u i-1 v

.

methacrylic acidd iv i n y 1ben z e n e

-40

KMDM6 ,Na+form

methacrylic acidhexame t h y l e n e d i m e t h y 1 a c r y 1a m i d e

+34 0

KB4P2 ,Na+form

phenoxyacetic acidformaldehyde

+5 7 0

KFU,Na

Ig8a f u r t h e r p u b l i c a t i o n reported investigations i n t o t h e v a r i a t i o n of the S e l e c t i v i t y Coefficient ( K ) w i t h t h e amount o f neomycin a d s o r b e d f o r f o u r t e e n d i f f e r e n t ion-exchange r e s i n s . In a l l cases t h e s e l e c t i v i t y c o e f f i c i e n t i n c r e a s e d w i t h t h e amount o f a d s o r b e d neomycin ( e x p r e s s e d as t h e % surface-coverage o f t h e i o n exchange r e s i n ) , t h o u g h d i f f e r e n t t y p e s of i o n e x c h a n g e r s r e s u l t e d i n d i f f e r e n t r e s p o n s e s . T h e d e g r e e o f crossl i n k i n g of t h e i o n e x c h a n g e r e s i n was a l s o r e p o r t e d t o a f f e c t e d t h e v a l u e of t h e s e l e c t i v i t y coefficient. The a u t h o r s c o n c l u d e d t h a t p o l y c o n d e n s a t i o n - t y p e i o n exchange r e s i n s a d s o r b K i l ' f i n and Samsonov

NEOMYCIN

415

neomycin more s e l e c t i v e l y t h a n t h e p o l y m e r i s a t i o n t y p e r e s i n s and t h a t v a r i a t i o n of t h e components of e i t h e r t y p e of r e s i n had l i t t l e e f f e c t on t h e a d s o r p t i o n of neomycin. Klyueva and G e l ’ p e r i n 2 ’ have compared t h e e q u i l i b r i u m d i s t r i b u t i o n c u r v e s f o r t h e a d s o r p t i o n o f neomycin on i o n - e x c h a n g e r e s i n s from b o t h p u r e s o l u t i o n s and t y p i c a l f e r m e n t a t i o n broths. 3.

S a l t s , Derivatives 3.1.

&

Complexes

Salts

Many a t t e m p t s t o a l t e r t h e p h y s i c a l p r o p e r t i e s o f neomycin by t h e f o r m a t i o n o f v a r i o u s s a l t s have b e e n d e s c r i b e d . Thus t h e neomycin s a l t s of t h e h i g h e r f a t t y a c i d s , s u c h a s t h e s t e a r a t e , p a l m i t a t e and m y r i s t a t e 3 0 r 31 were p r e p a r e d and b e i n g w a t e r - i n s o l u b l e w e r e f o r m u l a t e d i n ointment bases. Similarly, t h e undecylenate s a l t h a s been re a r e d and p r o c e s s e s f o r i t s p r o d u c t i o n p a t e n t e d 3 5 39, ~ 3 4 * The u n d e c y l e n a t e and c a p r y l a t e s a l t s h a v e been d e s c r i b e d as b e i n p a r t i c u l a r l y s u i t a b l e as a n t i m i t o t i c compounds 85

.

V a r i o u s c a r b o x y l i c a c i d s a l t s have a l s o gal lard^^^ p r o d u c e d t h e m a l e a t e been r e p o r t e d . s a l t o f neomycin w h i c h , i t was c l a i m e d , improved t h e a q u e o u s s t a b i l i t y of t h e a n t i b i o t i c . A p r a c t i c a l l y t a s t e l e s s compound t h e c i t r a t e s a l t , h a s b e e n d e s c r i b e d by S z y s ~ k a ~ Neomycin ~ . mandelate h a s been claimed t o be p a r t i c u l a r 1 u s e f u l i n t h e t r e a t m e n t of u r o g e n i t a l i n f e c t i o n s s g w h i l e t h e d i hydroxy-d i n a p h t h l m e t h a n e - d i c a r b o x y l a t e 40 and t h e pamoate s a l t s 4 1 , g 7 1 6 8 h a v e a l o w i n t e s t i n a l abs o r p t i o n and a r e t h u s e f f e c t i v e t r e a t m e n t s f o r intestinal infections. F i s h e r and H a l l r e p o r t e d t h e p r o p i o n a t e s a l t t o be u n s u i t a b l e f o r u s e i n o p h t h a l m i c preparation^^^. O t h e r s a l t s d e s c r i b e d i n t h e l i t e r a t u r e a r e neom c i n g l u c u r o n a t e 4 2 , s u c c i ~ t, h a l a t e ~ ~ a n d n a t e 4 3 I 4 4 , l a c t a t e 42 a ~ c o r b a t e ~ h t h e s u l p h o s ~ c c i n a t e4~8 ~which r has the property of improved s k i n p e n e t r a t i o n .

416

WILLIAM F. HEYES

Amongst t h e o r g a n i c s u l p h o a c i d s u s e d

t o p r e p a r e neomycin s a l t s , t h e p - t o l u e n e s u l p h o n a t e h a s been r e p o r t e d and i n c o r p o r a t e d i n an

a e r o s o l s p r a y f o r m u l a t i o n 4 9 . S a l t s o f neomycin w i t h h a l o g e n s u b s t i t u t e d 8 - h y d r o x y q u i n o l i n e 5s u l p h o n i c a c i d s h a v e been d e s c r i b e d a s p o s s e s s i n g l o w t o x i c i t y , a n t i s e p t i c and a n t i a m e b i c b e s i d e s a n t i m i c r o b i a l pro erties50f51. Stankovics e t a 1 5 2 and V e ~ i a n ap r~e ~c i p i t a t e d a complex s a l t of neomycin, by m i x i n g a q u e o u s s o l u t i o n s of neomycin s u l p h a t e and sodium l a u r y l s u l p h a t e , which had improved a c t i v i t y a g a i n s t Staphylocaccub a u k e u d . I n a more r e c e n t i n v e s t i g a t i o n o f t h i s r e a c t i o n , found t h a t t h e p r e c i p i t a t e d Leucuta e t complex d i s s o l v e d on a d d i t i o n o f e x c e s s sodium I.R. s t u d i e s of t h e complex lauryl sulphate. have i n d i c a t e d t h a t amide g r o u p s , c o u p l i n g rea c t i o n s and H-bonding a r e i n v o l v e d i n t h e format i o n o f t h e compound. M i c h e l e and V a l e t t e h a v e rep o r t e d an enhanced a n t i b i o t i c a c t i v i t y and a reduced t o x i c i t y f o r t h e e u c a l y p t 0 1 ~ u l p h o n a t e ~ ~ . P r e p a r a t i o n of t h e d o d e c l b e n z e n e - s u l p h o n a t e 5 5 and t h e cyclohexylsulphonatex6has a l s o been d e s c r i b e d . The p a n t o t h e n a t e s a l t of neomycin , f i r s t d e s c r i b e d by K e l l e r e t a 1 5 7 , a l s o e x h i b i t s t h e p r o p e r t y of a lower t o x i c i t y t h a n t h e p a r e n t a n t i b i o t i c . A f u r t h e r p a p e r by t h e same a u t h o r s 5 8 a t t r i b u t e d t h e lower t o x i c i t y o f t h e p a n t o t h e n a t e t o be due t o a r e d u c t i o n i n t h e c a l c i u m - b i n d i n g a b i l i t y of t h e a n t i b i o t i c when i n t h e form of t h e p a n t o t h e n a t e s a l t . A s i m i l a r e f f e c t h a s been n o t e d by W e i t n a u e r e t a159 w i t h a n e q u i m o l a r m i x t u r e of neomycin and c a l c i u m g l u c o n a t e . Numerous p r o cedures f o r the preparation o t e pantothenate ~ ~ s a l t have been p a t e n t e d 6 0 , 6 1 , ‘ 2 r k 3 . A b b ~ urep o r t e d t h e p r e p a r a t i o n of a complex s a l t of neomycin w i t h a m i x t u r e of g l u t a m i c and p a n t o t h e n i c a c i d s and d e s c r i b e d t h e compound a s p o s s e s s i n g b e t t e r o r g a n o l e p t i c p r o p e r t i e s t h a n o t h e r neomycin salts. S a l t s w i t h t h e amino a c i d s N-methyls t e a r o y l g l y c i n e 6 5 and l u t a m i c a c i d 6 6 , and t h e v i t a m i n s a s c o r b i c a c i d 2 3 and n i c o t i n i c a c i d 6 3 have a l s o been e v a l u a t e d .

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C h l o r o p h e n o l s a r e of a s u f f i c i e n t l y a c i d i c n a t u r e t o be a b l e t o form s a l t s w i t h neomycin and h a v e been r e p o r t e d t o p o s s e s s h t h a n t i b a c t e r i a l and m y c o c i d a l proper tie^^^ ,7 0 r 7p. Shaw70 h a s d e s c r i b e d t h e t o p i c a l u s e of s u c h a compound, neomycin h e x a k i s p e n t a c h l o r o p h e n a t e , i n t h e t r e a t m e n t o f d e n t a l r o o t i n f e c t i o n and p e r i o dontic cavities. A s a l t formed by r e a c t i n g neomycin w i t h p-amino b e n z e n e s u l h o a c e t a m i d e was d e s c r i b e d by t w o g r o u p s of w o r k e r s T 2 I 7 3 . The s a l t combined t h e c h e m o t h e r a p e u t i c p r o p e r t i e s of t h e components. A n o t h e r u n u s u a l s a l t of neomycin d e s c r i b e d i n t h e l i t e r a t u r e i s t h a t w i t h m- u l p h o n y l b e n z a l d e h y d e i s o n i c o t i n o y l h y d r a z o n e78’7’. The compound i s rep u t e d t o be l e s s t o x i c t h a n neomycin and t o be more a c t i v e a g a i n s t t u b e r c u l e b a c i l l i t h a n t h e individual constituents.

S a l t s of neomycin w i t h t h e i n o r g a n i c The s u l p h a t e s a l t a c i d s have a l s o b e e n d e s c r i b e d . i s t h e u s u a l c o m m e r c i a l form i n u s e t o d a y b u t neomycin b o r a t e h a s been u s e d i n o p h t h a l m i c p r e p a r a t ions46. Not a l l neomycin s a l t s , however, p o s s e s s a d v a n t a g e s s u c h as a r e d u c e d t o x i c i t y . I n f a c t , t h e orotic acid (1,2,3,6-tetrahydro-2 ,6d i o x o - 4 - p y r i m i d i n e c a r b o x y l i c a c i d ) s a l t w a s rep o r t e d t o h a v e a g r e a t e r t o x i c i t y t h a n neomycin i t ~ e l f ~Some ~ u ~ n c~e r ~ t a i .n t y of t h i s f a c t i s a p p a r e n t a s o t h e r a u t h o r s have r e p o r t e d t h e two compounds t o b e o f a s i m i l a r t o x i c i t y 7 6 . The s a l t of neomycin w i t h u s n i c a c i d ( a n a n t i b a c t e r i a l s u b s t a n c e f o u n d i n l i c h e n s ) h a s been r e p ~ r t e d ~ ~ t o h a v e l e s s a n t i b a c t e r i a l a c t i v i t y t h a n t h e neomycin used i n t h e p r e p a r a t i o n of t h e s a l t . 3.2. Derivatives The neomycin m o l e c u l e c o n t a i n s b o t h f r e e amino and f r e e .hydroxy g r o u p s . Many w o r k e r s have e x p l o i t e d t h e p o s s i b i l i t y o f c h e m i c a l d e rivatisation a t these positions in attempts t o r e d u c e t h e t o x i c i t y of t h e p a r e n t a n t i b i o t i c . S u b s t i t u t i o n of methane s u l p h o n a t e g r o u p s a t t h e amino n i t r o g e n h a s been r e p o r t e d by U m e z a w a e t a183184 and by B o i s s i e r e t a185. Both g r o u p s o f w o r k e r s d e s c r i b e d t h e d e r i v a t i v e t o b e less t o x i c

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than t h e p a r e n t a n t i b i o t i c . Boissier claimed t h a t t h e ' i n v i t r o ' and ' i n v i v o ' a n t i b a c t e r i a l a c t i v i t y o f t h e neomycin i s p r e s e r v e d f o l l o w i n g derivatisation. However, Umezawa, w o r k i n g w i t h f r a d i o m y c i n , d e s c r i b e d t h e N-methyl s u l p h o n a t e d e r i v a t i v e a s having h a l f t h e a c t i v i t y of f r a d i o mycin when r e a s u r e d a g a i n s t gram-pos i t i v e b a c t e r i a and o n l y one t e n t h t h e a c t i v i t y when measured again s t gram-negative b a c t e r i a . Russian workers89 have r e p o r t e d t h e r e d u c t i o n i n a n t i b a c t e r i a l a c t i v i t y t o b e p r o p o r t i o n a l t o t h e d e g r e e o f Ns u b s t i t u t i o n . Alternative procedures f o r t h e p r e p a r a t i o n o f N-methyl s u l p h o n y l neomycin h a v e been r e p o r t e d by a number o f a u t h o r s 8 6 1 8 7 1 8 8 and t h e pharmacology o f t h e compound h a s been e x t e n s i v e l y i n v e s t i g a t e d by D i Marco and B e r t a z z o i g o . T h e p r e p a r a t i o n of t h e f o l l o w i n g s u l p h o d e r i v a t i v e s h a s a l s o been d e s c r i b e d : N-me t hy l s u l p h i no n e omyc i n

I

N - p h e n y l s u l p h o n y l neomycin93 N- a c e t amidop hen y Is u l phony 1 neomyc i n

N-p-aminophenylsulphonyl neomycing3 N- t r i c h lorome t h y l t h i o neomycin9 N - e t h y l t h i o c a r b o n y l neomycing3 and neomycin N - s u l p h o n a t e g 4 Vanderhaeghe8O h a s s t u d i e d t h e a l k y l a t i o n of neomycin. A r e d u c t i o n i n t o x i c i t y was n o t e d f o l l o w i n g a l k y l a t i o n b u t t h i s w a s c o u p l e d w i t h a c o m p l e t e loss o f a n t i b a c t e r i a l properties. The r e s u l t i n g compound, however, w a s found t o p o s s e s s good h y p o c h o l e s t e r e m i c a c t i v i t y . The e f f e c t of N - a l k y l a t i o n , 0 - a l k y l a t i o n , Na c y l a t i o n and O - a c y l a t i o n h a s been i n v e s t i g a t e d by Magyar e t a181. These w o r k e r s r e p o r t e d t h e l o s s o f b i o l o g i c a l a c t i v i t y t o be p r o p o r t i o n a l t o t h e number o f g r o u p s s u b s t i t u t e d . F u r t h e r i n v e s t i g a t i o n s by t h e s e w o r k e r s d e m o n s t r a t e d t h a t conv e r s i o n o f amino g r o u p s i n t o q u a t e r n a r y ammonium g r o u p s r e s u l t e d i n t h e p r o d u c t i o n of compounds having a mild t u b e r c u l o s t a t i c e f f e c t . Penasse e t a182 have d e s c r i b e d an a l t e r n a t i v e p r o c e d u r e f o r t h e p r e p a r a t i o n of N-alkyl d e r i v a t i v e s .

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3 . 3 . Complexes

The f o r m a t i o n o f a zinc-neomycin complex h a s been r e p o r t e d by Chornock95 and by K e l l e r and C o s a r g 6 . E l e c t r o p h o r e s i s w a s u s e d t o d e m o n s t r a t e t h e f o r m a t i o n o f a complex which was d e s c r i b e d as p o s s e s s i n g a s i m i l a r b i o l o g i c a l a c t i v i t y t o neomycin b u t a d e c r e a s e d t o x i c i t y . More r e c e n t l y Agrawal, H a r m a l k e r and V i j a y a w a r g i y a g 7 have d e s c r i b e d t h e f o r m a t i o n o f a copper-neomycin complex. With a s t o i c h i o m e t r y o f 1:1, t h e b l u e c o l o u r e d complex h a s been made t h e b a s i s o f a s p e c t r o p h o t o m e t r i c a s s a y method f o r neomycin ( S e e S e c t i o n 6 . 2 6 ) . The c o m p l e x a t i o n o f neomycin w i t h a number of b i o c h e m i c a l l y i m p o r t a n t compounds h a s been r e p o r t e d . I n t e r a c t i o n of t h e a n t i b i o t i c with h e p a r i n w a s f i r s t i n v e s t i g a t e d by Higginbotham and D o u g h e r t y 9 8 . U s i n g a s p e c t r o p h o t o m e t r i c p r o c e d u r e , which measured t h e amount o f dye r e l e a s e d f r o m a t o l u i d i n e b l u e - h e p a r i n complex on a d d i t i o n of neomycin s o l u t i o n , t h e s e a u t h o r s s t u d i e d a number of a n t i b i o t i c s and t h e i r r e a c t i o r . w i t h heparin. G u b e r n i e v a and S i l a e v g 9 employed elect r o p h o r e s i s t o i n v e s t i g a t e t h e n a t u r e of t h e n e o m y c i n - h e p a r i n complex and s u g g e s t e d t h e compound may b e more t h a n a s i m p l e c a t i o n - a n i o n i n t e r a c t i o n . The p o s s i b i l i t y o f a d d i t i o n a l H-bonding f o l l o w i n g t h e i n i t i a l i o n i c i n t e r a c t i o n was p o s t u l a t e d . A more d e t a i l e d s t u d y o f t h e c o m p l e x a t i o n o f n e o mycin w i t h h e p a r i n , DNA,RNA and p o l y h l o r e t i n p h o s p h a t e h a s been d e s c r i b e d by HeinyOO. The c o m p l e x e s w e r e p r e p a r e d by m i x i n g a q u e o u s s o l u t i o n s of neomycin s u l p h a t e and t h e c o m p l e x i n g a g e n t u n d e r i n v e s t i g a t i o n . The p r e c i p i t a t e which formed r e d i s s o l v e d on a d d i t i o n o f sodium h y d r o x i d e s o l u t i o n o r aqueous s o l u t i o n s of s i l v e r n i t r a t e , manganese s u l p h a t e and c o b a l t c h l o r i d e . S u b j e c t i o n o f t h e p r e c i p i t a t e d complex t o an a g a r d i f f u s i o n e x p e r i m e n t d e m o n s t r a t e d t h e compound t o b e b i o logically active. U s i n g an a g a r o s e g e l s y s t e m Kunin and Tupasi.101 o b s e r v e d t h e f o r m a t i o n o f a p r e c i p i t a t i o n band between z o n e s of d e x t r a n s u l p h a t e and neomycin. The a u t h o r s a t t r i b u t e d t h e p r e c i p i t a t i o n t o be a r e s u l t o f complex f o r m a t i o n

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and n o t t h e r e s u l t of an a n t i g e n - a n t i b o d y i n t e r a c t i o n which t h e s y s t e m i s u s u a l l y employed t o detect. H a r r i s l o 2 h a s s t u d i e d t h e complexat i o n of neomycin w i t h p e c t i n and d e m o n s t r a t e d t h e i n h i b i t i o n of complex f o r m a t i o n i n t h e p r e s e n c e of an e l e c t r o l y t e . P o t e n t i o m e t r i c measurements i n d i c a t e t h e mechanism o f t h e r e a c t i o n t o be a cation-anion i n t e r a c t i o n . H-bonding between t h e hydroxy g r o u p s o f p e c t i n and s u g a r m o i e t i e s o f neomycin h a s been s u g g e s t e d and would f u r t h e r s t a b i l i s e t h e compound.

The c o m p l e x a t i o n of neomycin w i t h a n i o n i c d y e s s u c h a s a m a r a n t h ( F . D . & C . Red No.2) i s w e l l known100,102 and h a s been made t h e b a s i s of a q u a n t i t a t i v e a s s a y f o r n e o m y ~ i n ~ ~ These ~ 1 ~ 5 . complexes a r e a g a i n of t h e c a t i o n / a n i o n t y p e and t h e i r f o r m a t i o n i s d e p e n d a n t on t h e i o n i c s t r e n g t h of t h e s o l u t i o n . I n o r g a n i c c o n d e n s e d p h o s p h a t e s have been r e p o r t e d t o complex w i t h neomycin and i n a s t u d y of t h e s e compounds I S i n g h l o 3 d e m o n s t r a t e d t h e s t r e n g t h o f t h e complex t o be d e p e n d a n t on t h e d e g r e e of p o l y m e r i s a t i o n of t h e p h o s p h a t e . By i n c r e a s i n g t h e number of p h o s p h a t e u n i t s i n t h e polymer o v e r t h e r a n g e 1 t o 1 6 u n i t s an i n c r e a s i n g s t r e n g t h of c o m p l e x a t i o n w a s o b s e r v e d . Beyond t h i s u p p e r l i m i t , however, t h e s t r e n g t h of t h e complex remained c o n s t a n t . Relative complex-strengths w e r e a s s e s s e d by t i t r i m e t r y w i t h p o t a s s i u m c h l o r i d e solution. A number of w o r k e r s h a v e u t i l i s e d e l e c t r o p h o r e s i s t o e s t a b l i s h t h e f o r m a t i o n of a complex between neom c i n and t h e r o t e i n s i n b l o o d Serum and plasma104 ,Yo511 0 6 1 1 0 7 , ,881 1 0 9 , 1 1 0 , 1 1 1 . However, t h e n a t u r e of t h e complex i s s t i l l unknown. Geitman h a s e x t e n s i v e l y s t u d i e d t h e phenomena of p r o t e i n - b i n d i n g o f a n t i b i o t i c s l l 1 t 1 l 2 . I n a p a p e r p u b l i s h e d j o i n t l y w i t h Kivman1l2, Geitman r e p o r t e d t h a t neomycin d i d n o t b i n d t o any of t h e p r o t e i n f r a c t i o n s when added t o serum b u t bound t o t h e i s o l a t e d albumin and g l o b u l i n f r a c t i o n s . An e a r l i e r p a p e r l l l d e s c r i b e d t h e g l o b u l i n - n e o m y c i n complex t o b e o f g r e a t e r s t a b i l -

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i t y t h a n t h e albumin-neomycin complex and e s t a b l i s h e d lmg o f serum p r o t e i n t o b i n d 0 . 0 6 u n i t s o f neomycin

.

4.

S y n t h e s i s and P r o d u c t i o n 4.1.

Commercial B i o s y n t h e sis

Waksman e t a1286, i n 1 9 4 9 , f i r s t rep o r t e d t h e p r o d u c t i o n of neomycin by f e r m e n t a t i o n of a c u l t u r e o f S . d h a d i a ~ ( 3 5 3 5 ) .The same o r g a n i s m s u b s e q u e n t l y formed t h e b a s i s o f an i n d u s t r i a l f e r m e n t a t i o n rocess f o r t h e b i o s y n t h e s i s o f neomycin28712i8. I s o l a t i o n o f t h e a n t i b i o t i c from t h e f e r m e n t a t i o n media i s a c c o m p l i s h e d b y u s e of i o n - e x c h a n e r e s i n s , s u c h a s Amberlite I R C 50225 1 9 5 0 , 2 5 1 .

F o l l o w i n g t h e o r i g i n a l r e p o r t by Waksman, a number o f o t h e r a u t h o r s have d e s c r i b e d S . Q a a d i a e t o y i e l d a n t i b i o t i c s on f e r m e n t a t i o n . The r e s u l t i n g s u b s t a n c e s w e r e named s t r e p t o t h r i c i n B 1 2 8 9 , m y c e r i n 2 9 0 and c 0 l i m y c i n 2 9 1 ~b u t h a v e s i n c e b e e n shown t o be i d e n t i c a l t o t h e neomycin complex. 4.2.

S y n t h e s i s o f R a d i o - L a b e l l e d Neomycin

The b i o s y n t h e t i c p r e p a r a t i o n o f l 4 C l a b e l l e d neomycin w a s f i r s t d e s c r i b e d by Sebek267 who s t u d i e d a number o f s u b s t r a t e s f o r t h i s p u r p o s e b u t c o n c l u d e d 14C'labelled glucose was t h e most s a t i s f a c t o r y . A l t h o u g h 6 8 % o f t h e r a d i o n u c l i d e w a s l o s t as r e s p i r a t o r y I 4 C O , 1 9 % o f t h e t o t a l a c t i v i t y added p r i o r t o f e r m e n $ a t i o n w a s i n c o r p o r a t e d i n t o t h e neomycin s u l p h a t e . T h i s method o f p r e p a r i n g 1 4 C - l a b e l l e d neomycin h a s b e e n e x t e n s i v e l y u s e d t o s t u d y t h e mechanism o f neomycin b i o s y n t h e s i s . Stroshane13 has described t h e s y n t h e s i s o f 13C-neomycin and 15N-neomycin, a g a i n a s p a r t o f an i n v e s t i g a t i o n o f neomycin b i o s n t h e s i s . 13C-glucose s e r v e d a s p r e c u r s o r f o r y3C-neomycin and g l u c o s a m i n e l a b e l l e d w i t h I 5 N w a s u t i l i s e d f o r t h e p r e p a r a t i o n o f 15Nneomycin. A p r o c e d u r e f o r 1abe l l i n g neomycin w i t h t r i t i u m h a s been d e s c r i b e d by J a c k s o n e t

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a t 2 9 2 and i n v o l v e s p a s s i n g an e l e c t r i c d i s c h a r g e across a v e s s e l c o n t a i n i n g t r i t i u m and neomycin sulphate. L a b i l e t r i t i u m was removed by w a s h i n g w i t h a p o l a r s o l v e n t and t h e t r i t i u m - l a b e l l e d neomycin p u r i f i e d by m u l t i p l e r e c r y s t a l l i s a t i o n . 5.

S t a b i l i t y and D e g r a d a t i o n 5.1.

Hydrolytic Degradation

The d e g r a d a t i o n of neomycin h a s b e e n e G t e n s i v e l y s t u d i e d by R i n e h a r t l a s a means o f e s t a b l i s h i n g t h e s t r u c t u r e s o f t h e neomycin compon e n t s . F i g . 5 i l l u s t r a t e s t h e r o u t e by which comp l e t e d e g r a d a t i o n of t h e a n t i b i o t i c w a s a c h i e v e d . Extremely vigorous c o n d i t i o n s are n e c e s s a r y f o r t h e h y d r o l y s i s o f n e m i n e a s t h i s compound i s r e s i s t a n t t o acid-hydrolysis. However, u n d e r t h e s e c o n d i t i o n s t h e c h e m i c a l e n t i t i e s neosamine C ( f r o m n e a m i n e ) and r i b o s e are n o t o b s e r v e d b e c a u s e v i g o r o u s h y d r o l y s i s i m m e d i a t e l y decomposes t h e compounds f u r t h e r . By d e r i v a t i s i n g t h e amino g r o u p s o f t h e neamine and n e o b i o s a m i n i d e m o l e c u l e s R i n e h a r t w a s a b l e t o lower t h e r e s i s t a n c e of t h e s e compounds t o h y d r o l y s i s and t h u s u s e mild h y d r o l y t i c c o n d i t i o n s t o s u c c e s s f u l l y i s o l a t e neosamine C and r i b o s e : (CHJCO)

a ) neamine

CH30H

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t e t r a N - a c e t y l neamine

-

J

3N HC1

lOhr @ 9 5 O C

deoxystreptamine

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neosamine

PhCOCl

blmethyl neob i o s a m i n i d e

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ribose

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methyl N,N'-dibenzoyl neob i o s a m i n i d e 1.6N

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d4 CH OC H O(OH)3 3 5 6

Rus s i a n w o r k e r s attempted t o prep a r e t h e d e g r a d a t i o n p r o d u c t s i n a p u r e f o r m by c a r r y i n g o u t t h e h y d r o l y s i s i n t h e p r e s e n c e of an i o n e x c h a n g e r e s i n which a d s o r b e d t h e i n t e r m e d i a t e

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n eomy c i n I

F i g u r e 5.

H y d r o l y t i c d e g r a d a t i o n of neomycin C

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hydrolysis products,thereby preventing f u r t h e r dec o m p o s i t i o n . The d e g r a d a t i o n p r o d u c t s w e r e rel e a s e d from t h e r e s i n by g r a d i e n t - e l u t i o n . 5.2.

S t a b i l i t y o f Bulk M a t e r i g

J a p a n e s e w o r k e r s l5 h a v e d e s c r i b e d t h e s t a b i l i t y of b u l k f r a d i o m y c i n (neomycin B l s u l p h a t e which was shown t o h a v e r e t a i n e d 9 9 % o f i t s microb i o l o g i c a l p o t e n c y a f t e r s t o r a g e f o r 2 4 months. Neomycin s u l p h a t e powder h a s been r e o r t e d t o b e s t a b l e f o r a t l e a s t 3 y e a r s a t 2OoC 5 9 8 ,305. Neomycin s u l p h a t e may a l s o b e h e a t e d a t llO°C f o r 10 hours, during dry-heat sterilisat i o n , w i t h o u t l o s s o f p o t e n c y , t h o u g h some d e g r e e of yellowing i s apparent298. 5.3.

S t a b i l i t y i n Aqueous S o l u t i o n

Swart e t a 1 h a v e d e m o n s t r a t e d t h e s t a b i l i t y of neomycin h y d r o c h l o r i d e i n a q u e o u s Further ins o l u t i o n o v e r t h e pH r a n g e 2-9293. v e s t i g a t i o n s by Simone and P o p i n o 2 9 8 c o n f i r m e d t h e a q u e o u s s t a b i l i t y a t 23OC b u t a t 45OC p o t e n c y l o s s e s o f u p t o 9 4 % w e r e n o t e d o v e r a p e r i o d of 2 y e a r s w i t h s o l u t i o n s i n t h e pH r a n g e 4-8. Leach e t a 1 2 9 4 d e s c r i b e d a p u r i f i e d neomycin t o be s t a b l e t o t h e a c t i o n o f a l k a l i b u t Refluxing t h e a n t i b i o t i c w i t h barium not t o acids. h y d r o x i d e f o r a p e r i o d of e i g h t e e n h o u r s f a i l e d t o show a l o s s i n m i c r o b i o l o g i c a l p o t e n c y . Japanese w o r k e r s have d e s c r i b e d a stable a q u e o u s s o l u t i o n of neomycin w i t h t h e i n c o r p o r a t i o n o f a b o r a t e b u f f e r (pH 6 ) and E . D . T . A . 2 9 5 The p r e s e n c e of 1-10% o f g l y c e r o l , p r o p y l e n e g l y c o l o r m a n n i t o l h a s b e e n c l a i m e d t o improve t h e s o l u t i o n a p p e a r a n c e by p r e v e n t i n g d i s c o l ~ r a t i o n ~ The ~~. p r e s e n c e of p o l y o l s a l s o p r e v e n t e d a d e c r e a s e i n pH v a l u e o f t h e s o l u t i o n . D i s c o l o r a t i o n may a l s o b e p r e v e n t e d by a d d i t i o n of 0.1% sodium m e t a b i s u l p h i t e a t a s o l u t i o n pH of 6 . 6 t o 6 . 8 2 9 7 .

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S t a b i l i t y i n Pharmaceutical Formulations

Numerous r e p o r t s c o n c e r n i n g t h e s t a b i l i t y of neomycin i n v a r i o u s d o s a e forms have been p u b l i s h e d . Simone and Popino j 9 8 s t u d i e d t h e s t a b i l i t y o f neomycin i n l i q u i d dosage forms s u c h a s n a s a l d r o p s , mouth washes and t i n c t u r e s . The a n t i b i o t i c w a s s t a b l e i n a l l t h e formulations t e s t e d , e x c e p t D o b e l l s s o l u t i o n ( a mouth w a s h ) , f o r a t l e a s t 6 months a t 2 0 O C . Some f o r m u l a t i o n s were s t a b l e f o r considerably longer. Heyd2” h a s s t u d i e d t h e s t a b i l i t y o f neomycin i n aqueous g e l s and f o r m u l a t e d a s a t i s f a c t o r y p r o d u c t by a d s o r b i n g t h e a n t i b i o t i c on an i o n exchange r e s i n , Amberlite IRP-69M, p r i o r t o incorporation i n the gel. R e c o n s t i t u t e d aqueous s u s p e n s i o n s o f neomycin s u l p h a t e , c o n t a i n i n g t r a g a c a n t h and s u g a r have been shown t o b e s t a b l e f o r 10 d a y s when s t o r e d a t 4OC b u t some loss of p o t e n c y w a s o b s e r v e d when t h e s u s p e n s i o n was exposed t o d a y l i g h t 3 ” . Non-aqueous s u s p e n s i o n s c o n t a i n i n g p e a n u t o i l and l a n o l i n have been r e p o r t e d 2 9 8 which are s t a b l e f o r 1 year a t 200C. Simone and pop in^^'^ have c o n s i d e r e d t h e s t a b i l i t y of neomycin i n b o t h h y d r o p h o b i c and hydrophilic ointment bases. No l o s s of p o t e n c y o v e r a p e r i o d of 1 y e a r a t 2OoC w a s r e p o r t e d f o r formulations containing carboxymethylcellulose, polyethylene glycol(P.E.G.) o r white-soft paraffin. However, f o r m u l a t i o n s c o n t a i n i n g h y d r o u s A l l materlanolin w e r e reported t o be unstable. i a l s u s e d i n t h e f o r m u l a t i o n s were o b t a i n e d from U.S. sources. C o a t e s e t a1301 i n v e s t i g a t e d t h e u s e o f P . E . G . from B r i t i s h s o u r c e s and d e s c r i b e d neomycin a s b e i n g i n c o m p a t i b l e w i t h t h e m a t e r i a l s tested. S o l i d d o s a g e forms of neomycin as t a b l e t s and t r o c h e s have been p r e p a r e d 2 9 8 and a r e r e p o r t e d t o b e s t a b l e a t 2OoC and a t 56OC. I n t h e a r e a of animal h e a l t h n u t r i t i o n a feed-premix c o n t a i n i n g neomycin and oxyt e t r a c y c l i n e h a s been d e s c r i b e d . With neomycin

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a t a l e v e l of 20g/lb t h e p r o d u c t is s t a b l e f o r 1 y e a r a t 20°C, though an 8 % loss o f p o t e n c y was obs e r v e d on s t o r a g e f o r 6 months a t 380C305. A medi c a t e d d r i n k i n g - w a t e r c o n t a i n i n g 100 ppm o f neomycin and p r e p a r e d w i t h t a p - w a t e r , showed n o s i g n i f i c a n t l o s s of p o t e n c y a f t e r 48 h o u r s s t o r a g e a t 38OC i n e i t h e r g l a s s c o n t a i n e r s o r g a l v a n i s e d troughs305. 5 . 5 . C o m p a t i b i l i t y w i t h E x c i p i e n t Materials The r e a c t i o n of neomycin w i t h many compounds h a s been d e s c r i b e d i n S e c t i o n 3 , h e n c e numerous r e p o r t s o f neomycin i n c o m p a t i b i l i t y may b e e x p e c t e d . D a l e and Rundman304 h a v e e x t e n s i v e l y reviewed t h e c o m p a t i b i l i t y o f neomycin w i t h s u b s t a n c e s t h a t may b e e n c o u n t e r e d by t h e f o r m u l a t i o n pharmac i s t . K u d a l k e r e t a1303 have d e s c r i b e d t h e incomp a t i b i l i t y o f t h e a n t i b i o t i c w i t h r a n c i d o i l s , and the i n c o m p a t i b i l i t y with b e n t o n i t e , a montomorillo n i t e c l a y , h a s been r e p o r t e d by D a n t i and Guth306. The i n c o m p a t i b i l i t y w i t h l a c t o s e , c a u s i n g a d i s c o l o r a t i o n o f t h e m i x t u r e h a s b e e n s t u d i e d by Hammouda and Sa1akawy3O7. The amount of browning produced w a s shown t o be d e p e n d a n t on t h e i n i t i a l pH of t h e s o l u t i o n . The r a t e of d i s c o l o r a t i o n of t h e l a c t o s e / n e o m y c i n powder w a s d i r e c t l y r e l a t e d t o t h e t e m p e r a t u r e o f s t o r a g e and t h e r e l a t i v e h u m i d i t y of t h e a t m o s p h e r e . D i s c o l o r a t i o n w a s o v e r come by a d d i t i o n of sodium b i s u l p h i t e . F l o r e s t a n o e t a l 308 compared t h e rel e a s e of neomycin from P . E . G . d i e s t e r o i n t m e n t b a s e s and g r e a s e - t y p e b a s e s and c o n c l u d e d t h e former t o b e p r e f e r r e d though Coates e t a1301 h a v e d e s c r i b e d P . E . G . from B r i t i s h s o u r c e s t o b e incomp a t i b l e w i t h neomycin. 5.6.

Compatibility with Other Actives

Neomycin forms an i n s o l u b l e complex when added t o aqueous s o l u t i o n s of c o r t i c o s t e r o i d phosp h a t e s . T o overcome t h i s i n c o m p a t i b i l i t y , McGinty and Brown304 i n c o r p o r a t e d d i s o d i u m h y d r o g e n phosp h a t e i n t h e o p t h a l m i c f o r m u l a t i o n . The mechanism by which t h e c o m p l e x a t i o n i s p r e v e n t e d h a s n o t y e t been f u l l y d e t e r m i n e d .

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6 . Methods of A n a l y s i s 6.1.

I d e n t i f i c a t i on

T a b l e 9 l i s t s a number of c o l o r i m e t r i c t e s t s which h a v e b e e n u s e d a s i d e n t i t y t e s t s f o r neomycin. N o one t e s t , h o w e v e r , h a s been demons t r a t e d t o b e s p e c i f i c f o r neomycin and i t i s t h u s a l s o a d v i s a b l e t o e s t a b l i s h t h e a b s e n c e of o t h e r c h e m i c a l l y s i m i l a r a n t i b i o t i c s by s u i t a b l e means. Table 9 Colorimetric Identity T e s t s f o r N eomy c i n Test

G l u cos amine Molisch Ninhydrin Fur f u r a 1 Ph lorog l u c i n o1

Chemical g r o u p ing u t i l i s e d i n re a c t i o n

Colour obtained

Reference

g l y c o si d e pentose amino ribose ribose

cherry purple purple pink-red pink-red

114 115 115,116 116 117

Both t h i n - l a y e r and p a p e r chromatog r a p h y w i l l p r o v i d e s p e c i f i c i d e n t i f i c a t i o n of neomycin. Numerous s y s t e m s f o r t h i s p u r p o s e w i l l b e found l i s t e d i n s e c t i o n 6 . 3 4 . I n a d d i t i o n t o t h e above c h e m i c a l t e s t s a number o f m i c r o b i o l o g i c a l p r o c e d u r e s have b e e n have d e s c r i b e d a reported. Heinmann e t simple t e s t t o d i f f e r e n t i a t e neomycin-like m o l e c u l e s from t e t r a c y c l i n e s and c h l o r a m p h e n i c o l . When added t o a column of i n n o c u l a t e d a g a r o n l y t h e n e o m y c i n - l i k e m o l e c u l e s show a n a r e a of g r o w t h i n h i b i t i o n a t t h e t o p o f t h e column. A more s p e c i f i c m i c r o b i o l o g i c a l p r o c e d u r e h a s b e e n r e p o r t e d by F r e r e s and B u l l a n d 1 l g . By u s i n g f o u r t e s t OrganiSmS(8aCillUb c e k e u n , BacilCub b u b t i l i b , Sahcina C u t e a and M i c k u c o c c u b 6Cavub)patterns of i n h i b i t i o n w e r e determined f o r e a c h of t h e 1 2 a n t i b i o t i c s e x t r a c t e d i n t o t h r e e solvents. Comparing t h e i n h i b i t i o n p a t t e r n o f a n

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unknown a n t i b i o t i c w i t h t h e s t a n d a r d p a t t e r n s , t h e a u t h o r s were a b l e t o i d e n t i f y t h e p r e s e n c e o f i n d i v i d u a l s u b s t a n c e s i n animal t i s s u e . A s i m i l a r t e s t h a s a l s o b e e n d e s c r i b e d by T i e c c o e t 6.2.

Chemical P r o c e d u r e s 6.21.

T i t r i m e t r i c Assay

The d e t e r m i n a t i o n of neomycin by non-a u e o u s t i t r a t i o n h a s been d e s c r i b e d by Penau e t a l q 2 1 . Neomycin b a s e i s a l l o w e d t o r e a c t w i t h standardised perchloric acid; the excess acid i s t h e n b a c k - t i t r a t e d w i t h p o t a s s i u m hydrogen p h t h a l a t e u s i n g c r y s t a l v i o l e t a s i n d i c a t o r . To d e t e r mine t h e neomycin c o n t e n t of t h e s u l p h a t e s a l t t h e same a u t h o r s p r e c i p i t a t e d t h e s u l p h a t e w i t h b e n z i d i n e b e f o r e r e a c t i n g t h e neomycin w i t h p e r c h l o r i c a c i d . The amount o f b e n z i d i n e r e q u i r e d t o p r e c i p i t a t e t h e s u l p h a t e i s c a l c u l a t e d from t h e s u l p h a t e c o n t e n t which i s i t s e l f d e t e r m i n e d by t i t r a t i o n w i t h sodium h y d r o x i d e . An a l t e r n a t i v e method f o r d e t e r m i n i n g t h e s u l p h a t e c o n t e n t o f neomycin s u l h a t e h a s been r e p o r t e d by Roets and Vanderhaeghey22. The p r o c e d u r e i n v o l v e s a t i t r a t i o n w i t h b a r i u m c h l o r i d e and m u s t be c a r r i e d o u t i n a 30-40% s o l u t i o n of a l c o h o l i n o r d e r t o o b t a i n a s h a r p endA s the antibiotic is insoluble i n alcohol point. it i s n e c e s s a r y t o remove t h e neomycin p a r t o f t h e This i s accomplished molecule p r i o r t o t i t r a t i o n . u s i n g a c a t i o n e x c h a n g e r e s i n s u c h a s Dowex 5 0 - X 8 .

D u r i n g an i n v e s t i g a t i o n of t h e c o m p l e x a t i o n p r o p e r t i e s of neomycin, H a r r i s 1 O 2 r e p o r t e d t h e ti t r a t i o n o f neomycin conduc t o m e t r i c a l l y w i t h a m a r a n t h ( F . D . and C Red N o . 2 ) . The e n d - p o i n t o f t h e t i t r a t i o n i s s h a r p and h a s good reproducibility. 6.22.

Polarography

The u s e of d i f f e r e n t i a l p u l s e p o l a r o g r a p h y t o d e t e r m i n e neomycin s u l p h a t e i n aqueous s o l u t i o n s h a s been d e s c r i b e d by S i e g e r m a n et F o l l o w i n g a c i d h y d r o l y s i s of neomycin, t h e s o l u t i o n was a d j u s t e d t o pH 4 and 0 . 1 M a c e t a t e

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b u f f e r ( p H 4 ) added a s s u p p o r t i n g e l e c t r o l y t e . R e d u c t i o n p o t e n t i a l s of -0.19 and -0.36V w e r e obtained. The above a u t h o r s a l s o d e s c r i b e d t h e a p p l i c a t i o n of t h i s t e c h n i q u e t o t h e e x a m i n a t i o n o f s a m p l e s of s k i n p r e v i o u s l y t r e a t e d w i t h neomycin-containing formulations. 6.23. Polarimetry Neomycins B and C h a v e b e e n shown t o d i f f e r i n t h e i r s p e c i f i c r o t a t i o n v a l u e s , neomycin B h a v i n g a s p e c i f i c r o t a t i o n of + 80° and neomycin c a s e c i f i c r o t a t i o n o f + 1 2 0 0 1 2 4 , 1 2 7 . Brooks e t a 1 12g made t h i s f a c t t h e b a s i s f o r a number o f methods t o d e t e r m i n e t h e B & C c o n t e n t o f commercial neomycin. The s p e c i f i c r o t a t i o n o f t h e t e s t s o l u t i o n i s d e t e r m i n e d a t 25OC and t o t a l neomycin d e t e r m i n e d e i t h e r t i t r i m e t r i c a l l y o r s p e c t r o p h o t o m e t r i c a l l y . By s u b s t i t u t i o n of t h e s e v a l u e s i n a s u i t a b l e e q u a t i o n t h e c o n c e n t r a t i o n o f neoI n a s e c o n d method mycins B and C a r e c a l c u l a t e d . t h e same a u t h o r s d e t e r m i n e d t h e s p e c i f i c r o t a t i o n of t h e t e s t - s o l u t i o n a t t e m p e r a t u r e s o f 25O and 7 5 O C . The c h a n g e i n t h e v a l u e of s p e c i f i c r o t a t i o n on i n c r e a s i n g t h e t e m p e r a t u r e from 25Oto 75OC c a n t h e n b e u s e d t o c a l c u l a t e t h e amounts of neomycin B and C i n t h e s a m p l e . The u s e o f an a u t o m a t i c p o l a r i m e t e r w i t h a f l o w - c e l l h a s been r e p o r t e d by d e Rosri126 , t o m o n i t o r t h e e l u a t e from an i o n e x c h a n g e column (Bio-Rad A G l - X 2 ) t h r o u g h which a s o l u t i o n of neomycin w a s p a s s e d . The d e t e c t i o n o f an o p t i c a l l y a c t i v e s u b s t a n c e was r e c o r d e d e l e c t r o n i c a l l y w i t h a s u i t a b l e pen r e c o r d e r . By d e t e r m i n i n g t h e a r e a s of t h e p e a k s r e c o r d e d , t h e amounts of neomycir,s B and C and neamine i n a number of c o m m e r c i a l s a m p l e s have b e e n d e t e r m i n e d . 6.24.

R a d i o - c h e m i c a l Assay

To d e t e r m i n e t h e r e l a t i v e amounts o f neomycins B C and neamine by r a d i o chemical method, K a i s e r i 2 8 s e p a r a t e d t h e “C - l a b e l l e d Na c e t y l d e r i v a t i v e s by p a p e r c h r o m a t o g r a p h y and q u a n t i t a t e d t h e chromatograms by l i q u i d s c i n t i l l a t i o n c o u n t i n g . A c o e f f i c i e n t of v a r i a t i o n of 3 . 6 % w a s obtained.

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Seaman and Stewart1’’ have described a radio-chemical assay f o r determining neomycin on c o t t o n - f a b r i c . Neomycin i s r e a c t e d with carbon d i s u l p h i d e forming a d i t h i o c a r b o n a t e silver which i s t h e n decomposed w i t h [lloAg] n i t r a t e . The p r e c i p i t a t e d [ll0Ag] s i l v e r s u l p h i d e , which i s d i r e c t l y r e l a t e d t o t h e amount o f neomycin p r e s e n t , i s e s t i m a t e d by c o u n t i n g . Recently an i s o t o p e d i l u t i o n method h a s been r e p o r t e d 1 3 0 f o r a s s a y i n g neomycin s u l p h a t e . However, it i s f i r s t n e c e s s a r y t o p r e p a r e I 4 C - l a b e l l e d neomycin s u l p h a t e T h i s i s a c c o m p l i s h e d by a d d i n g 1 4 C - l a b e l l e d g l u c o s e t o a small-scale fer m entation of S . dtadiae.. 14C-labelled neomycin c a n t h e n be e x t r a c t e d by s o l v e n t - e x t r a c t i o n o r by ion-exchange c h r o m a t o g r a p h y .

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6.25.

Uorimetric Assay

The neomycin m o l e c u l e d o e s n o t e x h i b i t fluor escence b u t following s u i t a b l e der i v a t i s a t i o n two s p e c t r o f l u o r i m e t r i c d e t e r m i n a t i o n s reported the have been d e s c r i b e d . Maeda e t c o m p l e x a t i o n of s i m p l e h e x o s a m i n e s w i t h p y r i d o x a l and z i n c i o n s t o r e s u l t i n a f l u o r e s c e n t d e r i v a l y i n g t h i s p r o c e d u r e t o neomycin, tive. demonstrated a l i n e a r response over t h e Simpson c o n c e n t r a t i o n r a n g e 0-50 uq/ml o f neomycin s u l p h a t e .

33

The r e a c t i o n of neomycin w i t h f l u o r e s c a m i n e t o form a f l u o r e s c e n t complex h a s been r e p o r t e d by K u s n i r & Barna133. The f l u o r e s c e n c e i n t e n s i t y v a r i e s w i t h b o t h t h e pH of t h e s o l u t i o n (optimum pH r a n g e 7 . 5 - 9 . 5 ) and t h e amount of f l u o r e s c a m i n e added. T h i s p r o c e d u r e may b e u s e d t o d e t e r m i n e neomycin a t v e r y low l e v e l s , t h e minimum c o n c e n t r a t i o n d e t e r m i n a b l e b e i n g 45 ng/ml. 6.26.

S p e c t r o p h o t o m e t r i c Assay

Spectrophotometric a s s a y s of neomycin may be c o n v e n i e n t l y d i v i d e d i n t o t w o groups: -

(a) (b)

Direct methods, i n v o l v i n g t h e i n t a c t neomycin m o l e c u l e . I n d i r e c t methods , i n v o l v i n g h y d r o l y s i s of neomycin p r i o r t o a s s a y .

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( a )D i r e c t Methods 0 ' Keefe and R u ~ s o - A l e s i l ~ i r~sft r e p o r t e d t h e a p p l i c a t i o n of n i n h y d r i n t o t h e a s s a y o f n e o mycin, t h e p r o c e d u r e b e i n g an a d a p t a t i o n of t h a t d e s c r i b e d by Moore and S t e i n 1 3 5 f o r t h e a s s a y o f amino a c i d s and i n v o l v i n g measurement a t 570nm o f t h e p u r p l e - c o l o u r e d complex formed. Maehr and S h a f f n e r 1 3 6 have a p p l i e d t h i s p r o c e d u r e t o t h e d e t e r m i n a t i o n o f neomycin i n c h r o m a t o r a p h y column e l u a t e s . Gerosa and M e l a n d r i attempted t o a p p l y t h e Moore and S t e i n method t o t h e q u a n t i t a t i v e d e t e r m i n a t i o n of neomycin i n p h a r m a c e u t i c a l preparations b u t reported a l i n e a r response only o v e r t h e s h o r t c o n c e n t r a t i o n r a n g e o f 400-600pg/ml o f neomycin. B o r ~ w i e c k a land ~ ~ Thorburn-Burns e t a1139 however, r e p o r t e d no s u c h l i m i t a t i o n and s u c c e s s f u l l y a p p l i e d t h e method t o t h e a s s a y o f neomycin i n p h a r m a c e u t i c a l f o r m u l a t i o n s . P r e g n a l a t t o and S a b i n o 2 0 0 i n c o r p o r a t e d g l y c e r i n e i n t o t h e n i n h y d r i n s o l u t i o n and r e p o r t e d an i m proved s e n s i t i v i t y and r e p r o d u c i b i l i t y e n a b l i n g neomycin c o n c e n t r a t i o n s a s l o w a s 4 pg/ml t o be d e t e r m i n e d . An a u t o m a t e d n i n h y d r i n a s s a y h a s b e e n d e s c r i b e d by K a p t i o n a k , B i e r n a c k a and P a z d e r a l 4 0 b a s e d on a m o d i f i e d Moore and S t e i n m e t h o d l 4 1 . An a l t e r n a t i v e r e a g e n t which a l s o reacts w i t h t h e p r i m a r y amino g r o u p s of neomycin h a s r e c e n t l y been d e s c r i b e d l 7 2 . The r e a g e n t , d i c l o n e ( 2 , 3 - d i c h l o r o - l , 4 - n a p h t h o q u i n o n e ) , r e a c t s w i t h neomycin b a s e t o form an o r a n g e - c o l o u r e d p r o d u c t when t h e s o l u t i o n i s a d j u s t e d t o pH 4 .

C o m p l e x a t i o n of neomycin w i t h v a r i o u s d y e s was r e p o r t e d by H e i n l o o and t h e a q u e o u s i n s o l u b i l i t y o f t h e complex w i t h a m a r a n t h ( F . D . & C Red N o . 2 ) h a s been u t i l i s e d by H i l l 2 5 and by B u f t o n and S a d d l e r 1 4 2 t o a s s a y neomycin i n a q u e o u s s o l u t i o n . Amaranth may a l s o be u s e d t o d e t e r m i n e neomycin i n p r o d u c t i o n s a m p l e s from f e r m e n t a t i o n r e c o v e r 1 6 8 , The d y e , Orange 11, h a s b e e n s i m i l a r l y 1 4 3 d e s c r i b e d ( 1 max. = 484 nm) Complex f o r m a t i o n between neomycin and t h e dye i s v e r y d e p e n d a n t on t h e i o n i c s t r e n g t h o f t h e s o l u t i o n , thus n e c e s s i t a t i n g c a r e f u l c o n t r o l of reaction c o n d i t i o n s t o e n s u r e c o m p l e t e p r e c i p i t a t i o n of t h e complex d u r i n g t h e a s s a y p r o c e d u r e . R e a c t i o n of sodium l 1 2 - n a p h t h o - q u i n o n e - 4-su l p h o n a t e w i t h

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n e 0 m y c i n l 4 ~ p r o d u c e san o r a n g e / y e l l o w product,Xmax 460nm i n a c e t i c a c i d , which h a s been u t i l i s e d t o d e t e r m i n e t h e neomycin c o n t e n t o f o p h t h a l m i c s o l u t i o n s g i v i n g r e s u l t s i n good a g r e e m e n t w i t h microbiological assays. S t r o n g l y b a s i c a n t i b i o t i c s may be p r e c i p i t a t e d by f o r m a t i o n of t h e c o l o u r e d r e i n e c k a t e s a l t which may t h e n be d e t e r m i n e d s p e c t r o p h o t o m e t r i ~ a l l y l ~B~i c .k f o r d l 6 6 d i s s o l v e d t h e p r e c i p i t a t e d neomycin r e i n e c k a t e i n a c e t o n e and h a s s u c c e s s f u l l y u s e d t h i s p r o c e d u r e t o a s s a y neomycin e x t r a c t e d from t o p i c a l f o r m u l a t i o n s . Roushdi e t a1173 p r e f e r r e d t o oxidise the p r e c i p i t a t e with potassium p e r m a n g a n a t e and t h e n c o l o r i m e t r i c a l l y e s t i m a t e t h e chromate produced w i t h d i p h e n y l c a r b a z i d e . A r o m a t i c a l d e h y d e s r e a c t w i t h p r i m a r y amines f o r m i n g S c h i f f ' s b a s e s which a r e o f t e n c o l o u r e d . The c o l o u r may t h e n be u s e d t o q u a n t i t a t e t h e amine. Using t h i s p r i n c i p l e Kocy167 h a s shown neomycin t o form a y e l l o w - c o l o u r e d S c h i f f ' s b a s e w i t h s a l i c y l a l d e h y d e t h o u g h t h e q u a n t i t a t i v e a s p e c t of t h i s p r o c e d u r e i s , as y e t , i n c o m p l e t e . The c o m p l e x a t i o n o f neomycin w i t h c o p p e r r e s u l t s i n t h e f o r m a t i o n of a b l u e - c o l o u r e d compound which h a s a l s o been made t h e b a s i s o f a c o l o r i m e t r i c d e t e r m i n a t i o n o f neomycin i n pharmac e u t i c a l f o r m u l a t i o n s g 7 . Maximum c o l o u r i n t e n s i t y was o b s e r v e d a t a s o l u t i o n pH of 10. F u r t h e r i n v e s t i g a t i o n showed t h e s t o i c h i o m e t r y of t h e comp l e x t o be 1:l i n a l k a l i n e s o l u t i o n . ( b) I n d i r e c t Methods H y d r o l y s i s p r o d u c t s o f neomycin may b e an a m i n o - s u g a r , a p e n t o s e o r f u r f u r a l d e p e n d i n g on t h e r e a c t i o n c o n d i t i o n s c h o s e n . Each o f t h e s e e n t i t i e s h a s been u t i l i s e d f o r i n d i r e c t s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n o f neomycin. The p r e s e n c e o f a s u g a r m o i e t y i n neomycin was d e m o n s t r a t e d by Hamre e t a1145 who u s e d c a r b a z o l e and a n t h r o n e t o q u a n t i t a t e neomycins B and C i n commercial neomycin. The p r o c e d u r e s employed were t h o s e p r e v i o u s l y a p p l i e d t o manosido~ t r e p t o m y c i n,147. l ~ ~ Penau e t r e p o r t e d poor

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a g r e e m e n t b e t w e e n r e s u l t s o b t a i n e d by t h e a n t h r o n e a s s a y and m i c r o b i o l o g i c a l p r o c e d u r e s and s u g g e s t e d t h e p r e s e n c e of c a r b o h y d r a t e i m p u r i t i e s i n neomycin t o be t h e r e a s o n . O t h e r r e a g e n t s which f o r m c o l u d comp l e x e s w i t h s u g a r s s u c h a s o r c i n o l 1 4 % 1 5 ? 9 ,1 5 0 ( 3,5d i h y d r o x y t o l u e n e ) and p h l o r o g l ~ c i n o (1 l ~,3~,~ 5t r i h y d r o x y b e n z e n e ) have b e e n r e p o r t e d f o r t h e s p e c t r o p h o t o m e t r i c a s s a y o f neomycin. Hoodless c l a i m e d t h e p h l o r o g l u c i n o l p r o c e d u r e t o be less time-consuming t h a n o t h e r methods117 and a p p l i e d t h i s p r o c e d u r e t o neomycin s u l p h a t e raw m a t e r i a l . D ~ u l a k a s la d ~ o~p t e d a s i m i l a r p r o c e d u r e t o d e t e r mine t h e neomycin s u l p h a t e c o n t e n t o f o p h t h a l m i c ointments. I n t e r f e r i n g substances co-extracted from t h e o i n t m e n t were s e p a r a t e d from neomycin by TLC b e f o r e r e a c t i n g t h e a n t i b i o t i c w i t h p h l o r o glucinol. The o r c i n o l p r o c e d u r e h a s been u s e d t o a s s a y t h e neomycin c o n t e n t of f e r m e n t e r b r o t h s 1 4 8 I 1 4 9 I 150 though i t i s n e c e s s a r y t o f i r s t s e p a r a t e t h e a n t i b i o t i c by i o n - e x c h a n g e c h r o m a t o g r a p h y . Korchegin15O r e p o r t e d good a g r e e m e n t between t h e o r c i n o l a s s a y f production samand t h e m i c r o b i o l o g i c a l a s s a ples. Khromov-and S t a r u n o v ay7' and K o r c h a g i n e t compared t h e o r c i n o l and m i c r o b i o l o g i c a l a s s a y s f o r d e t e r m i n i n g t h e neomycin c o n t e n t of p o l y m e r - f i l m s a n d , a g a i n , good a g r e e m e n t b e t w e e n t h e two p r o c e d u r e s was n o t e d . A f t e r m i l d h y d r o l y s i s , neomycin h a s been shown t o g i v e a p o s i t i v e Elson-Morgan r e a c t i o n 1 5 1 , a r e a c t i o n c h a r a c t e r i s t i c o f amino-sugars152. A method i n v o l v i n g t h i s r e a c t i o n h a s been made t h e b a s i s of a q u a n t i t a t i v e a s s a y f o r n e ~ m y c i n l ~ ~ which h a s been u s e d t o d e t e r m i n e t h e neomycin cont e n t of f e r m e n t a t i o n b r o t h s 1 5 4 . V i g o r o u s h y d r o l y s i s o f neomycin r e s u l t s i n be q u a n t i t a t e d i n a number of d i f f e r e n t ways15xf156 . D u t c h e r e t a1157 u s e d a UV s p e c t r o p h o t o m e t r i c p r o c e d u r e , m e a s u r i n g t h e a b s o r b a n c e of t h e s o l u t i o n a t 280nm. A s i m i l a r p r o c e d u r e combined w i t h measurement of t h e o p t i c a l r o t a t i o n h a s b e e n u s e d by Brooks e t a l l 5 8 t o d e t e r m i n e t h e neomycin B and C c o n t e n t t h e f o r m a t i o n o f f ~ r f u r a l which l ~ ~ ma

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of t h e a n t i b i o t i c . Colorimetrically, the furfural o b t a i n e d from neomycin may be a s s a y e d w i t h an a n i l i n e by m e a s u r i n g t h e amount o f p i n k / r e d complex formed. Morgan e t a1159 u s e d 4 - b r o m o a n i l i n e i n t h i s c o n t e x t , t h e 4-bromo d e r i v a t i v e b e i n g more resist a n t t o t h e formation of i n t e r f e r i n g coloured prod u c t s t h a n a n i l i n e i t s e l f l 6 0 . T h i s method h a s been a p p l i e d t o v a r i o u s p h a r m a c e u t i c a l f o r m u l a t i o n s and r e s u l t s show good a g r e e m e n t w i t h m i c r o b i o l o g i c a l a s s a y s . A n i l i n e h a s been u s e d t o d e t e r m i n e t h e neomycin c o n t e n t o f f e r m e n t e r b r o t h s and o t h e r p r o d u c t i o n s a m p l e s l 6 1 , neomycin u n d e c y l e n a t e and neomycin c a p r y l a t e l 6 2 . Much work h a s been c a r r i e d o u t t o e s t a b l i s h optimum c o n d i t i o n s f o r a r e p r o d u c i b l e c o n v e r s i o n of neomycin t o f u r f u r a l , q u a n t i t a t i v e c o n v e r s i o n of t h e p e n t o s e m o i e t y t o f u r f u r a l b e i n g d i f f i c u l t and The a c i d s t r e n g t h t h e procedures tedious163t164. f o r optimum p r o d u c t i o n o f f u r f u r a l must b e s u c h t h a t i t i s s t r o n g enough t o h y d r o l y s e t h e s u g a r m o l e c u l e , y e t i n s u f f i c i e n t l y s t r o n g t o decompose t h e f u r f u r a l formed. A r e v i e w o f t h e l i t e r a t u r e by I v a s h k i v 1 7 0 showed 1 2 % h y d r o c h l o r i c a c i d t o b e a c c e p t e d a s optimum f o r c o n v e r s i o n o f p e n t o s e s t o f u r f u r a l . However, a s neomycin must f i r s t b e decomposed t o t h e pentose b e f o r e h y d r o l y s i s of t h e s u g a r can t a k e p l a c e , more v i o r o u s c o n d i t i o n s a r e r e q u i r e d . Dutcher e t a l l Z 4 used 4 0 % s u l p h u r i c a c i d t o produce an a c i d c o n c e n t r a t i o n o f 37% i n t h e s o l u t i o n f o r h y d r o l y s i s and o b t a i n e d maximum f o r m a t i o n o f f u r f u r a l a f t e r 1% h r s r e f l u x . Morgan e t a1159 however, u s e d 7 0 % s u l p h u r i c a c i d t o p r o d u c e a 25% a c i d conc e n t r a t i o n i n t h e h y d r o l y s i s s o l u t i o n and o b t a i n e d maximum f o r m a t i o n o f f u r f u r a l a f t e r one h o u r . I v a s h k i v 1 7 0 h a s e v a l u a t e d t h e u s e of b o t h hydroc h l o r i c and s u l p h u r i c a c i d s and found up t o f o u r t i m e s more f u r f u r a l t o be p r o d u c e d by s u l p h u r i c acid, the y i e l d being extremely s e n s i t i v e t o the c o n c e n t r a t i o n of a c i d employed. C o n t i n u i n g f u r t h e r work w i t h s u l p h u r i c a c i d , t h e f o r m a t i o n o f f u r f u r a l a s a f u n c t i o n o f neomycin c o n c e n t r a t i o n was e x amined and t h e c o n c l u s i o n r e a c h e d t h a t b e t t e r y i e l d s of f u r f u r a l a r e o b t a i n e d a t low c o n c e n t r a t i o n s of t h e a n t i b i o t i c . Heyes171 h a s shown a cons t a n t amount of f u r f u r a l t o be p r o d u c e d on hydrol y s i s w i t h 27-28% s u l p h u r i c a c i d , r e f l u x i n g f o r 1 h o u r . T h i s makes i t u n n e c e s s a r y t o a c c u r a t e l y

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p i p e t t e concentrated s o l u t i o n s of sulphuric acid. From t h e above d i s c u s s i o n i t would, t h e r e f o r e a p p e a r n e c e s s a r y t o e s t a b l i s h optimum c o n d i t i o n s f o r f u r f u r a l p r o d u c t i o n i n e a c h i n d i v i d u a l case. 6.3. Chromatographic Procedures 6.31. Counter-Current D i s t r i b u t i o n S w a r t , L e c h e v a l i e r and Waksman 1 7 6 have examined neomycin by t h e method o f c o u n t e r c u r r e n t d i s t r i b u t i o n u s i n g a p a r t i t i o n - s y s t e m of b o r a t e b u f f e r ( p H 7 . 6 ) / p e n t a s o l ( s y n t h e t i c amyl a l cohol)/stearic acid. T h r e e m a j o r components w i t h v a r y i n g amounts of neamine were found i n s a m p l e s of neomycin B from v a r i o u s s o u r c e s . D e t e r m i n a t i o n o f t h e amount of a n t i b i o t i c r e m a i n i n g i n e a c h t u b e f o l l o w i n g p a r t i t i o n was c a r r i e d o u t m i c r o b i o l o g i cally. I n an e x t e n s i o n o f t h i s s t u d y t o t h e whole g r o u p of n e o m y c i n - l i k e m o l e c u l e s S c h a f f n e r 1 7 7 however, h a s been u n a b l e t o s e p a r a t e neomycin B i n t o t h e s e p a r a t e components r e p o r t e d by S w a r t e t u s i n g e i t h e r o f f o u r p a r t i t i o n s y s t e m s . The s y s t e m s employed w e r e : Systems 1-3) 0 . 5 M b o r a t e b u f f e r a t pH 7 . 3 , 7.6 o r 7 . 8 / p e n t a s o l / s t e a r i c acid. 4 ) 0 . 5 % b i c a r b o n a t e buf f e r / p e n t a s o l / stearic acid.

Both c h e m i c a l and m i c r o b i o l o g i c a l assays w e r e u t i l i z e d t o determine the d i s t r i b u t i o n of a n t i b i o t i c i n t h e t u b e s . In a l l experiments t h e m i c r o b i o l o g i c a l a s s a y r e s u l t s w e r e more e r r a t i c t h a n t h e c o r r e s p o n d i n g c h e m i c a l a s s a y s and on t h i s b a s i s S c h a f f n e r e x p l a i n e d t h e d i f f e r e n c e between h i s r e s u l t s and t h o s e of S w a r t e t who u s e d o n l y m i c r o b i o l o g i c a l d e t e r m i n a t i o n s . I n t h i s same work S c h a f f n e r w a s a b l e t o d e m o n s t r a t e t h e same d i s t r i b u t i o n p a t t e r n f o r b o t h f r a m y c e t i n and neomycins B and C and t h u s t e n t a t i v e l y i d e n t i Similarly, but using f i e d f r a m y c e t i n a s neomycin. a p a r t i t i o n s y s t e m o f 3% p - t o l u e n e s u l p h o n i c a c i d i n 1N a c e t a t e b u f f e r (pH 3 . 8 ) / b u t a n o l B a i k i n a e t have i d e n t i f i e d m c e r i n and c o l i m y c i n a s neomycin a n d S a n n i k o v 1 7 5 h a s i d e n t i f i e d f r a m i c i n w i t h neomycin.

WILLIAM F. HEYES

436

From a p r e p a r a t i v e a s p e c t , Peck e t a l l 8 ' have employed t h e c o u n t e r - c u r r e n t d i s t r i b u t i o n t e c h n i q u e w i t h a p a r t i t i o n s y s t e m of water/ b u t a n o l / p - t o l u e n e s u l p h o n i c a c i d t o s e p a r a t e and p u r i f y t h e neamine (neomycin A ) p r e s e n t i n commerc i a l neomycin. 6.32 Electrophoresis

Neomycin i s a r e a d i l y i o n i s a b l e m o l e c u l e and s h o u l d t h u s b e s e p a r a b l e from o t h e r a n t i b i o t i c s by a p p l i c a t i o n of an e l e c t r i c f i e l d ( z o n e e l e c t r o p h o r e s i s ) . V a r i o u s w o r k e r s have s u c c e s s f u l l y a p p l i e d t h i s t e c h n i q u e t o neomycin and T a b l e 10 summarises t h e c o n d i t i o n s r e p o r t e d i n t h e l i t e r a t u r e . A number o f a u t h o r s d e s c r i b e d t h e q u a l i t a t i v e s e p a r a t i o n o f neomycin from o t h e r c h e m i c a l - t p e s of a n t i b i o t i c s u s i n g p a p e r - e l e c t r o p h o r e s i s l 8 y t 1 8 5 , 1 8 7 w h i l e Ochab189 d e s c r i b e d s y s r e m s s p e c i f i c a l l y d e s i g n e d t o s e p a r a t e compounds within the i n o g l y c o s i d e group of a n t i b i o t i c s . C a r r e t all' have r e p o r t e d t h e q u a n t i t a t i v e d e t e r m i n a t i o n o f neomycin i n t h e p r e s e n c e o f p o l y m i x i n and b a c i t r a c i n . Using p a p e r e l e c t r o p h o r e s i s q u a n t i t a t i o n of neomycin w a s a c c o m p l i s h e d c o l o r i metrically with ninhydrin. Lightbown and DeRossi 188 s u b s t i t u t e d an a g a r - g e l s u p p o r t f o r t h e more u s u a l c h r o m a t o g r a p h y - p a p e r and s u c c e s s f u l l y s e p a r a t e d neomycin from a v a r i e t y o f a n t i b i o t i c s b u t n o t from paromomycin. However, ' q u a n t i t a t i o n o f t h i s p r o c e d u r e w a s n o t p o s s i b l e d u e t o t h e e l o n g a t e d zone of i n h i b i t i o n produced by neomycin on i n c u b a t i o n o f the agar-gel. The same a u t h o r s a l s o r e p o r t e d some anomalous b e h a v i o u r of t h e neomycin i o n i n t h e p r e s e n c e of c e r t a i n t y p e s of a g a r . Neomycin i s a b a s i c m o l e c u l e and u n d e r n o r m a l c i r c u m s t a n c e s would be e x p e c t e d t o b e h a v e a s a c a t i o n and m i g r a t e t o t h e anode. However, w i t h c e r t a i n t y p e s of a g a r t h e neomycin w a s o b s e r v e d t o m i g r a t e t o w a r d s t h e c a t h o d e . T h i s phenomena w a s r e p e a t e d l y c o n f i r m e d though n o t e x p l a i n e d . A s y s t e m s u i t a b l e f o r t h e q u a n t i t a t i v e e s t i m a t i o n of neomycin u s i n an a g a r g e l s u p p o r t h a s been d e s c r i b e d by Grynney83. T o o b t a i n a good c l e a r zone f o r neomycin i t w a s n e c e s s a r y t o change t h e b u f f e r e d e l e c t r o l y t e / a g a r - g e l t o a p h o s p h a t e s y s t e m a t pH 6 . 5 . D e t e c t i o n of t h e

rl

rl

m

c

-d

k

h

a

c c c

-4

Eu

\

3 m

Ln rl

k a, a ru a

k

7

om w k

k a,

m W Q

W

u

2

E

.

Ln

3 0 \ 0s 3 0

oa,

m

..

W W

. .

k a,

w 3 Q

w 7

a

5

4

a m

0

rl

Eu k

7

om w k

om

k

0

k

Lnc N

3 0

7

lclk

7

W k

3 0

3

\

3 0 0

In

-

oa,

m

0s

m N

.d

e

m..c

Ln

m

\ - a ,

z a a -L!

m

. . N

L13

=I

ru a,

u c

0 PI

Table lO(Contd..

.)

E l e c t r o p h o r e s i s of Neomycin Electrolyte

P

5. Trishydroxymethylaminomethane/maleic a c i d / sodium h y d r o x i d e ( 0 . 9 0 7 % :0.870%:0 . 0 0 2 % ) pH 5 . 6

agar-gel buffered pH 5 . 6

6.

paper

m w

2 M f o r m i c acid/O.lM toluene sulphonate/ p r o p ano l / w a C e r ( 5 0 : 50:5 :40)pH 1 . 8

(40:5:5:50)

7.

Conditions

Medium ~-

I

2 ooov 2 0 0 mA

3 40v

D e t e c t i n g Agent (See a l s o S ectio n 6.34)

B .n u b L L L L A

B . A u bLLk5A

189

ATCC 6633

1 89 paper

340v

toluene sulphonate/ water ( 4 0 : 5 :55)pH 1 . 9

8 . 1 M ammonium h y d r o x i d e / 1 M sodium h y d r o x i d e / 0 . 1 M toluene sulphonate/ p r o p a n ol / w at e r ( 2 0 : O . 5 : 10: 1 0 : 6 9 . 5 ) pH 1 0 . 8

188

NCTC 8 2 4 1

pH 1 . 9

2 M f o r m i c acid/O.lM

Reference

73. Aub&.k%A

189

ATCC 6633

paper

340v

B . AubLL&iA ATCC 6633

189

T a b l e 10 (Contd.

.. )

E l e c t r o p h o r e s i s o f Neomycin

9.

Electrolyte

Medium -

1M ammonium h y d r o x i d e / 1M sodium h y d r o x i d e / w a t e r

paper

(20:0.25:79.75)pH

3 40v

Detect i n g Agent R e f e r en c e (See a l s o S e c t i o n 6.343

B.bubXilib

189

ATCC 6633

11.5

10. i M sodium h y d r o x i d e / w a t e r ( 5 : 9 5 ) pH 1 2 - 2

11. Acrylamide g e l

Conditions

pH 4 . 6

'd P

ic

1 2 . pH 6.5 p h o s p h a t e b u f f e r (KHPO4 22g/l; KH2P04,28g/l)diluted 1 : 4 0 with d i s t i l l e d water.

paper

3 40v

B.aubtiLib

189

ATCC 6633

a c r y 1amide ge 1 a g a r -ge 1 buffered a t pH 6 . 5

lOOV, 2mA

Naphthalene Black

190

310 t o 360v

Staphalococcub

183

(current 5 50mA)

c p i d etr mid i b

ATCC 1 2 2 2 8

WILLIAM F. HEYES

440

a n t i b i o t i c was a c h i e v e d b i o a u t o g r a p h i c a l l y . The o r g a n i s m S f a p h a l o c o c c u a e p i d e h m i d i a ATCC 12228 w a s found t o be t h e m o s t s e n s i t i v e f o r t h i s p u r p o s e . (The d e s c r i p t i o n o f a l t e r n a t i v e b i o a u t o g r a p h i c a l s y s t e m s w i l l be found i n s e c t i o n 6 . 3 4 . ) Using an a c r y l a m i d e - g e l as s u p p o r t , CoombelgO h a s d e m o n s t r a t e d t h e q u a n t i t a t i v e a s s a y of neomycin w i t h a r e c o v e r y o f 9 6 % i n t h e p r e s e n c e of b a c i t r a c i n and p o l y m i x i n . I n t h i s case q u a n t i t a t i o n w a s a c h i e v e d by d e n s i t o m e t r y a f t e r s t a i n i n g the g e l with naphthalene black. E l e c t r o p h o r e s i s h a s a l s o been employed t o s e p a r a t e neomycin f r o m a n a l y t i c a l l y i n t e r f e r i n g s u b s t a n c e s s u c h a s p r o t e i n s . Hence B r a m m e r and Hemson182 have d e t e r m i n e d t h e neomycin c o n t e n t of b l o o d serum. Neomycin was s e p a r a t e d from t h e serum p r o t e i n s by e l e c t r o p h o r e s i s on c e l l u l o s e a c e t a t e and a s s a y e d c o l o r i m e t r i c a l l y f o l l o w i n g e l u t i o n from t h e s u p p o r t . A modified technique c o n s i s t i n g of an e l e c t r i c f i e l d a p p l i e d p e r p e n d i c u l a r t o a f l o w i n g b u f f e r s o l u t i o n and u s i n g c h r o m a t o g r a p h y p a p e r a s support(cross-electrophoresis) h a s been e x p l o i t e d t o s t u d y t h e r e a c t i o n of neomycin w i t h h e ~ a r i n ~ ~ E t vl i~d e~n c. e f o r t h e f o r m a t i o n o f a neomycin-heparin complex was o b t a i n e d by t h i s means.

6.33.

Column Chromatoqraphy

The column c h r o m a t o g r a p h i c s e p a r a t i o n of neomycin B , neomycin C and neamine on an ion-exchan e column h a s been d e s c r i b e d by Maehr and S c h a f f n e r ?3 6 Dowex 1 X 2 ( O H f o r m ) was t h e i o n e x c h a n g e r e s i n and w a t e r t h e e l u t i n g s o l v e n t . The column e l u a t e was m o n i t o r e d by r e a c t i o n w i t h n i n h y d r i n . Using a slower e l u t i o n r a t e and a s m a l l e r p a r t i c l e s i z e of t h e same i o n - e x c h a n g e r e s i n Inouye and Ogawa19 r e p o r t e d improved r e s o l u t i o n of t h e p e a k s . The a p p l i c a t i o n o f t h i s p r o c e d u r e t o t h e q u a n t i t a t i v e d e t e r m i n a t i o n of neomycin C and n e a mine i n commercial s a m p l e s of raw m a t e r i a l h a s a l s o b e e n d e s c r i b e d 6 . When compared w i t h TLC res u l t s on t h e same s a m p l e s , t h e column chromatog r a p h i c method gave c o n s i s t e n t l y h i g h e r r e s u l t s f o r t h e neamine c o n t e n t . TLC e x a m i n a t i o n o f t h e

.

NEOMYCIN

44 I

f r a c t i o n of e l u a t e c o n t a i n i n g neamine , d e m o n s t r a t e d t h e p r e s e n c e o f t h r e e o t h e r u n i d e n t i f i e d compounds which gave a p o s i t i v e n i n h y d r i n r e a c t i o n t h u s exp l a i n i n g t h e o b s e r v e d d i f f e r e n c e between t h e t w o procedures. R o e t s and Vanderhaeghe l9 h a v e a l s o examined neomycin by c h r o m a t o g r a p h y on Dowex 1x2 b u t t h e s e a u t h o r s m o n i t o r e d t h e column e l u a t e c o n d u c t o m e t r i c a l l y . G i l l e t e t a l l g 4 appl i e d a s i m i l a r p r o c e d u r e t o t h e e x a m i n a t i o n of v a r i o u s s a m p l e s of neomycin t o a s c e r t a i n t h e r a t i o of neomycin B:C. A number of minor components p r e s e n t i n commercial neomycin h a v e been s e p a r a t e d by column c h r o m a t o g r a p h y on a c a r b o x y l i c c a t i o n exchange r e s i n I A m b e r l i t e ~ ~ - 5 The 0 ~components ~ ~ . were e l u t e d from t h e r e s i n w i t h ammonium h y d r o x i d e s o l u t i o n . T . L . C . o f t h e e l u e n t f r a c t i o n s showed t h e p r e s e n c e of two p r e v i o u s l y u n r e p o r t e d i m p u r i t i e s which were t h e n i s o l a t e d on Dowex 1 x 2 and t e n t a t i v e l y i d e n t i f i e d u s i n g NMR and mass s p e c t r o metry.

When i n v e s t i g a t i n g t h e a c i d h y d r o l y s i s o f neomycin I B r a z h n i k o v a and K u d i n o v a l g 8 added a s u l p h o c a t i on i c - t y p e i o n -exchange re s i n t o t h e a c i d s o l u t i o n of a n t i b i o t i c t o a d s o r b i n t e r m e d i a t e h y d r o l y s i s p r o d u c t s and t h u s p r e v e n t f u r t h e r decomposition d u r i n g h e a t i n g . Following e l u t i o n o f t h e r e s i n . i n a column t h e p r o d u c t s o f neomycin h y d r o l y s i s w e r e i d e n t i f i e d and shown t o be d e p e n d a n t on t h e t i m e o f h y d r o l y s i s . An i o n - e x c h a n g e c h r o m a t o g r a p h i c p r o c e d u r e u t i l i s i n g Dowex 1x2 h a s been made t h e b a s i s of an i n d u s t r i a l process f o r manufacturing neomycin B which i s f r e e from neomycin C l g 5 .

The i s o l a t i o n o f neamine on A m b e r l i t e I R C 5 0 ( N a f o r m ) f o l l o w i n g t h e a c i d hydrol y s i s of neomycin h a s been d e s c r i b e d l g 6 . Neamine i s e l u t e d from t h e i o n - e x c h a n g e column w i t h h d r o chloric acid. T h i s p r o c e s s had been p a t e n t e d 7 9 7 a s a means of m a n u f a c t u r i n g neamine.

WILLIAM F. HEYES

442

Many column c h r o m a t o g r a p h i c p r o c e d u r e s have been d e s c r i b e d f o r t h e commercial s e p a r a t i o n and p u r i f i c a t i o n of neomycin f o l l o w i n g fermentation. 6.34.

Paper and Thin-Layer Chromatograph

The e x a m i n a t i o n of neomycin by p a p e r and t h i n - l a y e r c h r o m a t o g r a p h y h a s b e e n f o r t w o main p u r p o s e s : a ) t o s e p a r a t e and i d e n t i f y neomycin i n t h e p r e s e n c e of o t h e r a n t i b i o t i c s , and b ) t o s e p a r a t e i n d i v i d u a l components and d e g r a d a t i o n p r o d u c t s o f neomycin. The s o l v e n t s y s t e m s employed f o r t h e s e p u r p o s e s a r e summarised i n T a b l e s 1 2 and 1 3 ( p a p e r c h r o m a t o g r a p h y ) and T a b l e s 1 4 and 1 5 (thin-layer procedures)

.

V i s u a l i s a t i o n of t h e chromatograms h a s been r e p o r t e d u s i n g b o t h c h e m i c a l and m i c r o b i o l o g i c a l means. Chemical s p r a y r e a g e n t s which h a v e been d e s c r i b e d a r e l i s t e d i n T a b l e 1 6 . The v a r i o u s o r g a n i s m s t h a t have b e e n employed f o r b i o a u t o g r a p h y a r e g i v e n i n T a b l e 11. T a b l e 11

V is u a l i s a t i o n o f Chromatograms Bioautographically Organism

Reference 201,202,204,228 234,235

.

B putniluh

22 4

€a c h e t r i c h i a c o l i

205,206

S t a p h a l o c o c c u h aukeuh

2 0 7 , 2 2 0 , 2 3 1 , 2 32

ATCC 6538 s t r a i n

Table 12 P a p e r C h r o m a t o g r a p h i c S y s t e m s S e p a r a t i n g Neomycin from o t h e r A n t i b i o t i c s Paper

Direction

S o l v e n t Sys t e m

Rf V a l u e

Use

Reference

Whatman N o . 1

A s c e n d i n9

Water - s a t u r a t e d butanol

0.84

Whatman No.1

Ascend i n g

n-Butano l/ace t i c a c i d / w a t e r ( 2 :1:1)

0.85

Whatman N o . 1

Ascending

n-Butanol/pyridine/ w a t e r (1:0 . 6 :1)

0.94

) Identifi-

202

Whatman N o . 1 A s c e n d i n g

3 % Aqueous ammonium chloride

0.00

202

Whatman No.1

Ascending

Benzene/acetic acid/ w a t e r ( 2 : 1:1), o r g a n i c 1a y e r

0.89

) c a t i o n of Ineomycin i n ) t h e presence )of o t h e r )antibiotics

Whatman No.1

A s cend i n g

n -Bu t a n 0 1- s a t u r a t e d

0.07

)

202

1 202

1

1 ) )

202

202

water

Toyo No. 5 0

Ascending

Methanol/3 % a q u e o u s sodium c h l o r i d e ( 5 :1)

Descending for 9 hrs.

n-Butanol/pyridine/ acetic acid/water ( 1 5 : 10: 3 :1 2 )

0.03

) S e p a r a t i o n of neomycin from )other I streptomyces )antibiotics

203

204

Table 1 2 (Contd.. 1 P a p e r C h r o m a t o g r a p h i c Systems S e p a r a t i n g Neomycin from o t h e r A n t i b i o t i c s D i r e c tion

S o l v e n t System

Toyo N o . 5 0

Descending for 9 hrs.

2 % p-toluene sulphoni c acid + 2% pyridine i n wet n - b u t a n o l

Toyo No. 5 0

Ascending

t-Butanol/acetic w a t e r ( 5 5 :6 :39)

Whatman No.1

Ascending

Whatman N o . 1

Ascending

Paper

Rf V a l u e

Use

Reference

0 . 4 8 - 0 . 5 4 ) S e p a r a t i o n of Ineomycin f r o m ) other 1s t r e p t o m y c e s )antibiotics.

204

0.06

1

20 4

Water

0.98

)

205

Water-saturated n -bu t a n 0 1

Oe0

!i d e n t i f i c a t i o n

acid/

P

Systematic

'of the antibiotics

2 05

Whatman N o . 1 Ascending

Water-saturated e t h y l acetate

Whatman N o . 1 Ascending

W a t e r - s a t u r a t e d benzene

0.0

Whatman No.1

Methanol/water ( 4 0 : 6 0 )

0.21 )

205,206

Whatman N o . 1 Ascending

n-Propanol/water ( 4 0 : 6 0 )

0.20 )

205,206

Whatman N o . 1

Ascending

Methanol/3% a q u e o u s ammonium c h l o r i d e ( 7 0 : 3 0 )

0.11 )1

205,206

Whatman No.1

Ascending

Methyl e t h y l k e t o n e / n b u t a n o l / w a t e r ( 3 0 : 5 :6 5 )

0.27

Ascending

2 05

1 )

2 05

1 )

1

205,206

Table 1 2 (Contd..

.)

P a p e r C h r o m a t o g r a p h i c Systems S e p a r a t i n g Neomycin from o t h e r A n t i b i o t i c s Paper Whatman No.1

Direction Ascending

S o l v e n t System 6 6 . 6 % aqueous propanol/benzene/ethylene glycol/acetic a c i d ( 5 :5 :1 . 5 :1)

0.03 )

0.27

Whatman No.1

Ascending

5 0 % aqueous p r o p a n o l / a c e t i c a c i d ( 2 5 : 1)

Whatman N o . 1

Ascending

Water-saturated butanol/acetic acid/potassium c y a n i d e ( 100: 1:0: 0 5 9 ) n-Butanol/water/acetic acid(30:13:8)

Whatman No.3

Ascending

Whatman No.3

Ascending

Whatman No. 3

Ascending

Rf V a l u e

n-Butanol/acetic acid/ pyridine/water/ethanol ( 6 0 : 15: 6 :5 :5) n-Butanol/water/acetic acid/pyridine/sodium c h l o r i d e ( 3 0 : 1 2 : 7 :2: 0.1)

__ Use

R e f e re n c e

207

)

1

1

O.O0

' S e p a r a t i o n of neomycin from ch l o r a m p h e n i c o l , tetracycline ) and p e n i c i l l i n

1

207

207

)

Oso2

0.04

' s e p a r a t i o n of neomycin f r o m 'polymixin B and b a c i t r a c i n

186,208

!

186,208

1

1 0.05 )

1

1

186,208

Table 13

P a p e r C h r o m a t o g r a p h i c S y s t e m s S e p a r a t i n g Neomycin Components and D e g r a d a t i o n P r o d u c t s Paper

Direction

S o l v e n t Sy s t e m

Rf Value Neomycin Neamine B

-

P m P

Whatman No.1

Descending

-

Toyo No.50

Descending for 9 hrs

Water /bu t a n o 1/ m e t h a n ol / m e t h y 1 orange . ( 2 : 4 : 1:1:5 ) Butanol/pyridine/ w a t e r ( 6 0 : 30: 4 0 )

Butanol/pyridine/ w a t e r ( 3 :2: 1)

n-Propanol/ pyridine/acetic acid/water (15:10:3:12)

Use

Reference

C

N o t available

0.29

0.19

N o t available

0.13

-

0.15

0.28) ) ) ) )

Stability testing fradiomycin

209

Separation o f B and C as a c e t y l derivatives

2 10

Separation of B and C as a c e t y l derivatives

211

Separation of neamine f r o m neomycins B and C

204

Table 13 (Contd.. ) P a p e r C h r o m a t o g r a p h i c Systems S e p a r a t i n g Neomycin Components and D e g r a d a t i o n P r o d u c t s Paper

Direction

S o l v e n t System

Rf Value Neomycin Neamine B

Toyo N o . 50 Toyo

Descending for 9 hrs Ascending

No. 5 0

2 % p-toluene sulphonic acid in w a t e r s a t u r a t e d n-butanol

0.11-

1 . 5 % sodium c h l o r i d e i n 80% m e t h a n o l

0.08-

Use

Reference

C

0.20

0.11

0.23

0.22

)Separation

)of neamine

20 4

) from neo)mycins B )and C

1

P P 4

Grade M

Ascending

Butanol/acetic acid/wat er (4:l: 5)

Whatman NO. 4

Descending f o r 24-3g h r s , 28 C

n-Butanol/water/ piperidine (84:16:2)

Not a v a i l a b l e

Separation of B and C as acetyl derivatives

0.70 0.35 1-00 Q u a n t i t a t i v e (values r e l a t i v e spectrophotot o neamine) m e t r i c determ i n a t i o n of components

212

213

Table 1 3 ( C o n t d . .

.)

P a p e r C h r o m a t o g r a p h i c Systems S e p a r a t i n g Neomycin Components and D e g r a d a t i o n P r o d u c t s Paper

Direction

S o l v e n t System

Rf V a l u e Neomycin B

Whatman No. 1

Descending f o r 2 4- 36 h r s , 2527OC

P

Methyl e t h y l ke t o n e / t -bu t ano 1/ methanol/6.5N ammonium h y d r o x i d e (16:3:1:6)

Use

Reference

Neamine

C

0.54 0.30 1.00 (values r e l a t i v e t o neamine)

Separation a s f r e e bases Q u a n ti t a t i on by s p e c t r o ph ot o m e t r y

m P

Whatman No. 1

-

Descending f o r 8-400 hrs , 24 C

Methyl e t h y l k e t o n e / 0 . 5 7 0.34 1.00 isopropanol/6.5N (values r e l a t i v e ammonium h y d r o x i d e t o neamine) (80:20:30)

Radial

But an o l / p y r i d i n e / a c e t i c acid/water (15: 10: 3 :1 2 )

Whatman D e s c e n d i n g No.limf o r 18 h r s pregnated with PH 8 buffer

N o t available

Butanol/water(l:2) 0.5 containing 4% t o l u e n e s u l p h o n ic a c i d and 2 % p i p e r i d i n e hydrochloride

0.6

0

214

2 15

Separation of degradation products

216

S e p a r a t i o n of neamine from neomycin B and C

228

Table 1 4 T h i n L a y e r C h r o m a t o g r a p h i c Systems S e p a r a t i n q Neomvcin from O t h e r A n t i b i o t i c s Ads o r b e n t

S o l v e n t Sys tern

Rf Value

Use

Reference

n-Propanol/ethylacetate/ w a t e r / 1 3 . 4 M ammonium h y d r o x i d e ( 5 0 : 10: 30: 10)

0.52

S e p a r a t i o n of neomycin from o t h e r aminoglycoside antibiotics

217

Cellulose MN

n-Propanol/pyridine/ a c e t i c acid/water ( 1 5 :10: 3 :1 2 1

0.10

S e p a r a t i o n of neomycin from other w a t e r soluble basic a n t i b i o t ic s

2 18

Kieselgel G

Wate r / m e t hano l / b u t an o l / butyl acetate/acetic a c i d ( 1 2 :2.5 :7 . 5 :4 0 :2 0 )

0.0

S i l i c a ge 1 G/

aluminium o x i d e (1:1)

P \c

Kieselgel G

Kieselgel G

Water/butanol/pyridine/ a c e t i c a c i d . ( 1 4 :30: 2 0 : 6 ) ( 1 6 :4 0 : 8: 1 6 ) Water/butanol/acetic a c i d (39 :55 :6 )

219

)

1 S e p a r a t i o n of 1 neomycin from 1 bacitracin, ) p o l y m i x i n and

0.12

)

tyrothricin

219

0.00 )

1 1 0.00

)

219

T a b l e 1 4 (Contd..

.)

Thin L a y e r C h r o m a t o g r a p h i c Systems S e p a r a t i n g Neomycin from O t h e r A n t i b i o t i c s Ad s or b e n t Kieselgel G

Se ph adex G-15 P VI 0

S o l v e n t System Methanol/l7% aqueous ammonia c h l o r o f o r m , a q u e o u s l a y e r (10:10:20)

pH6 p h o s p h a t e b u f f e r (0.025M) + 0.5M sodium chloride

Rf V a l u e

0.31

Use ) Sep aratio n of ) neomycin from )bacitracin, ) p o l y m i x i n and )tyrothricin

Reference

219

1 1 0.75

) Identification

220

)of antibiotics Silica gel G Kieselgel G

Kieselgel G

Ben z e n e / a c e t one/ ace t i c a c i d ( 4 :4 :2 ) But a n o l / a c e t i c a c i d / pyridine/water (30: 2 2 : 6 : 3 8 ) Butanol/water/acetic acid/ethanol/pyridine ( 6 0 : 10:15: 5: 6 )

0.35

1 1

221

? Separation 0.14

of ’? nebmycin from zinc bacitracin ’1 and polymixin

186 208

0.05

1

186 208

T a b l e 14 (Contd..

.)

Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin from Other A n t i b i o t i c s Ad so r ben t Kieselgel G Kieselgel G + Kieselguhr

S o l v e n t System Propanol/ethyl acetate/water/ 2 5 % ammonium hydroxide (100:20:60:20).

Use

Rf Value 0.08

)

Reference 222

1 0.14

)

222

1

G(1:l)

1

Kieselgel G + Kieselguhr G(1:2)

0.22

)

222

C e 1l u l o s e MN-300

0.32

) S e p a r a t i o n of 'glycosidic

222

antibiotics

1

Kieselgel G + aluminium oxide G type E ( 1 : l )

0.12

Kieselgel G impregnated w i t h sodium acetate

0.33

)

222

1

)

1

1

222

T a b l e 2 (Contd.. ) Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin from Other A n t i b i o t i c s Ads o r b e n t Kieselgel G Kieselgel G + Kieselguhr G ( 1 : 1) Kieselgel G + Kieselguhr G(1:2)

S o l v e n t System Prop a n o l / e t hy 1 ace t a t e / w a t e r / 2 5 % ammonium hydroxide/ p y r i d i n e / 3 . 8 5 % aqueous ammonium a c e t a t e ( 100:2 0 :6 0 :2 0 :10:2 00) II

Rf

Value 0.11

Kieselgel G impregnated w i t h 3.85% aqueous ammonium acetate Kieselgel G Kieselgel G + Kieselguhr G(1:l) Kieselgel G + Kieselguhr G(1:2)

1 1

0.16

222

1 1

222

1

1 0.08

S e p a r a t i o n of glycosidic antibiotics 222

1 0.11

(100:20:60:20:10:200) It

222 222

0.08

Ethanol/ethylacetate/water/ 25% ammonium hydroxide/ p y r i d i n e / 3 . 8 5 % aqueous ammonium a c e t a t e

Reference

0.13

VI P h,

Use

222

1 0.12

222

1 1

.

Table 1 4 (Contd. ) Thin Layer Chromatographic Systems S e p a r a t i n q Neomycin from Other A n t i b i o t i c s Adsorbent --

S o l v e n t System

Use

Rf Value

Reference

Kieselgel G + aluminium oxide G ( t y p e E ) (1:1)

Ethanol/ethylacetate/water/ 2 5 % ammonium hydroxide/ p y r i d i n e / 3 . 8 5 % aqueous ammonium a c e t a t e (100: 2 0 : 6 0 : 2 0 : 10: 2 0 0 )

0.08

Kieselgel G

Methanol/ethyl a c e t a t e / water/25 % ammonium hydroxi d e / p y r i d i n e / 3 . 8 5 % aqueous ammonium a c e t a t e (100:20:60:20:10:200)

0.06

)

222

0.17

)

222

222

)

1 1

1 VI P

Kieselgel G + Kieselguhr G (1:1) Kieselgel G + Kieselguhr

11

0.14

222

1 1

G(1:2)

Kieselgel G + aluminium oxide G , type E ( 1 : l )

’)

S e p a r a t i o n of glycosidic antibiotics

Methanol/ethyl a c e t a t e / water/25% ammonium hydroxi d e / p y r i d i n e / 3 . 8 5 % aqueous ammonium acetate ( 100 :2 0 :6 0 :2 0 : 10: 2 0 0 )

0.11

)

1 1

1

222

Table -1 4 (Contd. . I Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin from Other A n t i b i o t i c s

VI

Ads o r b e n t

So lven t System

Rf v a l u e

Kieselgel G impregnated w i t h 3.85% aqueous ammonium acetate

Methanol/ethyl acetate/ water/25% ammonium hydroxi d e / p y r i d i n e / 3 . 8 5 % aqueous ammonium a c e t a t e (100: 20: 60: 2 0 : 10: 2 0 0 )

0.08

Kieselgel G

Butanol/methanol/ethyl a c e t a t e / w a t e r / 2 5 % ammonium

0.06

fr

Kieselgel G + Kieselguhr G ( 1 : 1) Kieselgel G + Kieselguhr G(1:2)

hydroxide/pyridine/3.85% aqueous ammonium acetate

222

1 1 1 1 1 222

1

0.07

222

1

(100: 100: 20: 60: 20: 10: 2 0 0 )

0.13

) S e p a r a t i o n of ) glycosidic ) antibiotics

222

1 0.05

Kieselgel G + aluminium oxide G , type E ( 1 : l ) Kieselgel G + Kieselguhr G(1:2)

Reference

)

222

1 25 % ammonium hydroxide/ water/acetone (16: 144: 40)

0.36

1

222

T a b l e 1 4 (Contd.. 1 Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin from Other A n t i b i o t i c s Adsorbent Kieselgel G + Kieselguhr G(1:2)impregnated with 2 % sodium a c e t a t e

%

S o l v e n t System 2 5 % ammonium hydroxide/ w a t e r/ a c e t on e (16:144 :40)

Kieselgel G + aluminium oxide G , t y p e E (1:1)

If

Kieselgel G + Kieselguhr G ( 1 : 1)impregnated with 2 % sodium a c e t a t e

11

Kieselgel G

Use -

Rf Value 0.34

1 1

Reference 222

1 1

0.48

) S e p a r a t i o n of glycosidic antibiotics

222

1 0.43

222

)

1 1 1 1 Water/sodium c i t r a t e / c i t r i c acid (100:20:5)

0.95

Classification of a n t i b i o t i c s

223

Table 15 Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin Components and Degradation P r o d u c t s Adsorbent

Use

Rf Value

S o l v e n t System Neomycin B

Neomycin

Neamine

C

Acidified carbon

Water

0.10

0.10

0.60

Acidified carbon

0.5N s u l p h u r i c acid

0.21

0.43

0.61

Untreated carbon

Water

0.00

0.00

0.00

Untreated carbon

0.5N s u l p h u r i c acid

VI P

0.5N hydroCarbon chloric acid treated w i t h hydrochloric acid II

0.5N hydroc h l o r i c acid/ methanol ( 4 :1)

0.24 0.37-0.45

Reference

0.45 0.53-0.74

0.54 0.63-0.92

)Assay of ) neomycin )components ) i n commer) c i a 1 form)ulations

22 4

)

224

1 1

224

)

224

)

204

1 ) Separation ) of neomycins

)B and C and

0.44-0.72

0.58-0.81

0.64-0.91

) neamine )

1 1

20 4

T a bl e 15 (Contd.. -

.)

Thin L a y e r C h r o m a t o q r a p h i c Systems S e p a r a t i n g Neomycin Components and D e g r a d a t i o n P r o d u c t s Ad s or b e n t

S o l v e n t System Neomycin

B Carbon treated with w a t e r

Carbon/ gypsum treated with sulphuric acid

Carbon/ gypsum treated with water

0.5N hydrochloric acid

0.41

0.5N hydrochloric acid/ m e t h a n o l ( 4 : 1)

0.48

0.5N s u l p h u r i c acid

0.20

Rf V a l u e Neomycin

Use -

Reference

Neamine

C 0.57

20 4

0.62 )

0.58

0.68

1

204

)

1 0.59

0.80 ) Sep aratio n of neomycins ) B and C and ) neamine

20 4

1 1

1 0.5N s u l p h u r i c a c i d / m e t h a n o l ( 4 : 1) 0.5N hydrochloric acid

0.64

0.82

0.85 )

0.16

0.28

0.30

204

1 20 4 )

1

Table 15 (Contd.. ) Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin Components and Degradation P r o d u c t s Adsorbent

R f Value

S o l v e n t System Neomycin

Carbon/ gypsum treated with water P

0.5N s u l p h u r i c acid

B

Neomycin C

0.24

0.40

Use

R e f er e n c e

Neamine

--

20 4

1 1 )Separation ) o f neomycin )B and C and ) neamine

I1

0.5N hydrochloric acid/ methanol (1:1)

0.36

0.36

0.46

11

0.5N hydroc h l o r i c acid/ p r o p a n o l ( 1 : 1)

0.48

0.48

0.52

Kieselgel H

3.85% aqueous ammonium acetate

0.13

0.13

0.36

Assay of neamine

196,9

Kieselgel H

3 . 4 % ammonium hydroxide

0.37

0.44

0.47

Assay of neomycin C

196,9

v, W

1 1

20 4

20 4

Table 15 (Contd.. ) Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin Components and Degradation P r o d u c t s Ads orben t

C e 1l u l o s e MN

R f Value -

S o l v e n t System

Methyl e t h y l ketone/iso-

Neomycin

Neomycin

B

C

0.21

0.12

Use -

Neamine 0.31

propanol/6.5N ammonium hydroxide (80:20:30)

2 \o

Cellulose MN-300

Methyl e t h y l k e tone/me t h a n o 1/ isopropanol/7.9N ammonium hydro x i d e (10: 8.5 :3 :7 )

Cellulose MN-300

Propanol/pyridine/ a c e t i c acid/water ( 100 :6 6 :2 0 :80)

Silica gel G

11

R e fe r e n ce

Separation

of neomycin

2 15

components

0.52

0.24

single 0.25 double 0.33

development 0.20 0.47 development 0.25 0.60

0.57

0.66

0.61

236

)Assay of )neomycins B 226 land C and nea)mine i n pharm) a c e u t i c a l form0.73 ) u l a t i o n s 226

Table 15 (Contd.. ) Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin Components and Deqradation P r o d u c t s Ad s o r ben t

S o l v e n t System Neomycin

m

0

Use

Rf Value B

Neomycin C 0.46

Cellulose MN-300/ Kieselguhr G(1:l)

Propanol/pyridine/ acetic acid/water (100: 66 :20: 80)

0.53

Silica gel G

Prop an o l /e t hy 1 acetate/water/25% ammonium hydroxide (100:20:60:20)

0.23

Reference

Neamine 0.73 )

226

1 1 0.23

0.42

)

226

1 1

Cellulose MN-300/ Kieselguhr G(1:l)

Methanol/3% aqueous 0.36 sodium c h l o r i d e ( 2 :1)

0.17

Silica gel G/ Kieselguhr G(1:2)

3.85 % aqueous ammonium a c e t a t e

0.22

0.22

)Assay of )neomycins B 0.43 ) a n d C and 226 )nearnine i n )pharmaceutical ) formulations

1 '0.39 )

1 1 1

226

Table 1 5 (Contd.. ) Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin Components and Degradation P r o d u c t s Adsorbent

S o l v e n t System Neomycin B

Silica gel G

Kieselguhr

Rf Value Neomycin

Neamine

0.14

0.11

0.22

0.46

0.68

0.84

Propanol/e than0.18 ol/ethyl acetate/ water/25% ammonium hydroxide/pyridine/ 3.85%ammonium a c e t a t e (100: 100: 2 0 : 6 0 : 20: 10: 2 0 0 )

0.15

)Assay of )neomycins B ) a n d C and lneamine i n )pharmaceutical 0 - 38 f o r m u l a t i o n s

0.11

0.32 )

Methyl e t h y l ketone/ t b u t ano l / m e t han o1/ 6.5N ammonium hydroxide ( 1 6 0 : 30: 10: 6 0 ) II

purified

Silica g e l G/ Kieselguhr G(1:2)

Reference

C

G

Silica gel G

Use -

II

0.18

)

226

226

1 226

Table 1 5 (Contd.. ) Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin Components and Degradation P r o d u c t s Adsorbent

S o l v e n t System

Use -

R f Value

Neomycin B

Neomycin

Reference

Neamine

C

1.5M Sodium a c e t a t e , pH 8.5

0.15

0.15

0.28

227

25SA)

+ 58.49 sodium chloride/ t - b u t a n o l ( 1 0 : 1)

S e p a r a t i o n of neamine from neomycins B and C

Silica ge 1

3% Ammonium hydroxide / a c e t o n e ( 1 6 0 : 40)

0.33

0.33

-

Quantitative a n a l y s i s of n e omy c i n sulphate

229

Kieselgel G

3% Ammonium hydroxide / a c e t o n e ( 1 6 0 : 90)

S t a b i l i t y of neomycin a f t e r e t h y l e n e oxide st e r i l i s a t i o n

233

Dowex 50x8 sodium form(1onex

N o t available

463

N EOMY CIN

T a b l e 16

V i s u a l i s a t i o n of Chromatograms by C h e m i c a l Means Spray Reagent

1. E t h a n o l i c sodium h ypoch 1o r it e f o l l o w e d by s t a r c h / potassium iodide solution 2 . Ch l o r i n e /e t h a n o 1 f o l l o w e d by s t a r c h / potassium iodide solution 3. C h l o r i n e / c a r b o n tetrachloride f o l l o w e d by s t a r c h / p o t as s ium i o d i d e / pyridine 4 . t - b u t y l hypochlor i t e / d i ch 1oromethane/acetic acid f o l l o w e d by s t a r c h / potassium iodide solution 5. N i n h y d r i n

Ninhydrin/cadmium acetate 7. p - d i m e t h y l aminoben z a l d e h y d e

6.

Ninhydrin/pd i m e t h y l aminobenzaldehyde 9 . Copper s u l p h a t e / ammonium h y d r oxide

Colour of Neomycin zone

R e f e r e n ce

Dark b l u e

210,211

Dark b l u e

212

Dark b l u e

213

Dark b l u e

196

Purple

186,203,208 2 1 4 , 2 1 5 ,2 1 6 217,218,219 229,231

Purple/p ink

227

Pink o r purple

223 226

8.

Blue

221

464

WILLIAM F. HEYES

Q u a n t i t a t i v e a n a l y s i s o f neomycin a f t e r c h r o m a t o g r a p h y u t i l i s i n g some o f t h e v i s u a l i s a t i o n t e c h n i q u e s g i v e n i n T a b l e s 11 and 1 6 have been r e p o r t e d . Majumder a n d Majumder213 s e p a r a t e d neomycins B a n d C a s t h e a c e t y l d e r i v a t i v e s on p a p e r t h e n , f o l l o w i n g c o n v e r s i o n t o t h e c h l o r o d e r i v a t i v e s , t h e c o l o u r formed by r e a c t i o n w i t h t h e starch/iodine/hydrochloric a c i d r e a g e n t w a s measure d a t 570nm. A l a t e r p a p e r by t h e same a u t h o r s 2 1 4 d e s c r i b e d t h e s e p a r a t i o n o f neomycins B and C as t h e f r e e b a s e s . The r e s u l t i n g chromatograms w e r e ded e v e l o p e d w i t h n i n h y d r i n t h e n t h e c o l o u r e d complex e l u t e d i n t o m e t h a n o l and t h e a b s o r b a n c e o f t h e s o l u t i o n measured a t 570nm. Thin- l a y e r c h r o m a t o g r a p h y h a s been u s e d b y F o p p i a n o and Brown229 t o a s s a y t h e t o t a l neomycin B and C c o n t e n t of neomycin s u l p h a t e . The neomycin zone w a s s c r a p e d o f f t h e p l a t e and rea c t e d w i t h o r c i n o l / f e r r i c c h l o r i d e r e a g e n t , t h e abs o r b a n c e o f t h e r e s u l t i n g c o l o u r b e i n g measured a t 665nm. By t h i s p r o c e d u r e a p r e c i s i o n o f 2% was achieved. Ninhydrin h a s also been used t o quantitate neomycin c o l o r i m e t r i c a l l y f o l l o w i n g t h i n - l a e r c h r o m a t o g r a p h y . P r e g n o l a t t o and S a b i n 0 2 5 0 r e p o r t e d t h e a d d i t i o n of g l y c e r i n e t o t h e n i n h y d r i n s o l u t i o n t o improve t h e s e n s i t i v i t y and An a l t e r n a t i v e r e p r o d u c i b i l i t y of t h e p r o c e d u r e . c o l o r i m e t r i c p r o c e d u r e f o r t h e q u a n t i t a t i o n of neomycin a f t e r c h r o m a t o g r a p h y h a s been d e s c r i b e d by D o u l a k a s l 7 4 . Neomycin i s e l u t e d f r o m t h e s i l i c a g e l w i t h pH 1 2 . 5 p h o s p h a t e b u f f e r t h e n o x i d i s e d w i t h sodium h y p o b r o m i t e . The r e s u l t i n g a l d e h y d e i s complexed w i t h p h l o r o g l u c i n o l y i e l d i n g a p i n k c o l o u r e d p r o d u c t which i s measured s p e c t r o p h o t o metrically. The u s e o f b i o a u t o g r a p h y f o r t h e q u a n t i t a t i o n of chromatograms h a s b e e n d e s c r i b e d by many w o r k e r s . E m i l i a n o w i c z - C z e r s k a and Herman228 s e p a r a t e d neamine f r o m neomycins B and C t h e n q u a n t i t a t i v e l y d e t e r m i n e d t o t a l neomycin B and C by b i o a u t o g r a p h y w i t h B.hubtiCih. A l i n e a r res p o n s e f o r c o n c e n t r a t i o n s between 0 . 2 5 and 1 O p g o f neomycin w a s o b t a i n e d . With a s i m i l a r t e c h n i q u e b u t using a mathematical a n a l y s i s of the bioauto-

NEOMYCIN

465

g r a m s , Simon230 r e p o r t e d a q u a n t i t a t i v e d e t e r m i n a t i o n o f neomycin s u i t a b l e f o r u s e w i t h i r r e g u l a r Brodasky224 employed b i o a u t o g r a p h y shaped zones. w i t h BaciLLuh pumiluh f o r t h e a s s a y o f neomycin and neamine a f t e r s e p a r a t i o n by T.L.C. on a l a y e r of c a r b o n . The q u a n t i t a t i o n o f s m a l l amounts of n e o mycin C however, p r o v e d d i f f i c u l t , t h e d i f f i c u l t ies b e i n g a s s o c i a t e d w i t h t h e enhanced growth of t h e o r g a n i s m i n t h e p r e s e n c e o f c a r b o n ( f r o m t h e T.L.C. p l a t e ) and ammonium i o n s ( f r o m t h e c h r o m a t o g r a p h i c procedure). Similar d i f f i c u l t i e s w e r e experienced by S o k o l s k i and L ~ m m i s 2 3who ~ a t t r i b u t e d p o o r neomycin z o n e s t o be t h e r e s u l t o f a n t a g o n i s m by t h e sodium c h l o r i d e p r e s e n t i n t h e b i o a u t o g r a p h i c s y s tem. An e x t e n s i v e s t u d y by L a n g n e r 2 0 1 d e m o n s t r a t e d maximum s e n s i t i v i t y f o r t h e d e t e r m i n a t i o n of neomycin t o b e a p p a r e n t u s i n g t h e o r g a n i s m B.dubtiLih (ATCC 6633) t o g e t h e r w i t h pH 8 T r i s a g a r . The d e t e c t i o n l i m i t f o l l o w i n g chromatography w a s r e p o r t e d t o be 0.05pg. 6.35.

G a s - L i q u i d Chromatography

T s u j i and R o b e r t s o n l g 2 a c h i e v e d t h e s e p a r a t i o n of neomycin B , neomycin C and neamine as t h e t r i m e t h y l s i l y l e t h e r s on a 6 f t . column of 0 . 7 5 % OV-1 on G a s Chrom Q a t a t e m p e r a t u r e of 290OC. The same c o n d i t i o n s have a l s o b e e n shown t o s e p a r a t e n e o b i o s a m i n e B , neosamine and d e o x y s t r e p t a m i n e from neomycin and neamine. Hence t h e method c o u l d b e u s e d t o s t u d y t h e s t a b i l i t y o f neomycin o r t o m o n i t o r t h e b i o s y n t h e t i c p r o d u c t i o n U s e of t h e p r o c e d u r e t o assess t h e s t a b process. i l i t y o f neomycin i n p h a r m a c e u t i c a l f o r m u l a t i o n s h a s b e e n d e m o n s t r a t e d by Van G i e s s e n and T s u j i 2 3 7 w i t h t r i l a u r i n as i n t e r n a l s t a n d a r d . H o w e v e r , t h e s e a u t h o r s recommended a 2 f t . column packed w i t h 3 % OV-1 on G a s Chrom Q as l o n g e r columns r e q u i r e d a h i g h e r t e m p e r a t u r e t o c h r o m a t o g r a p h neomycin and c o n s e q u e n t l y h a d a r e d u c e d column l i f e . A concent r a t i o n o f 3 % OV-1 w a s p r e f e r r e d , a s 2 % o r less r e s u l t e d i n i n c r e a s e d column a d s o r p t i o n o f neomycin. A f t e r a f u r t h e r s t u d y of t h e p r o c e d u r e Margosis and T s u j i 2 3 8 recommended a number of improvements t o o p t i m i s e t h e a n a l y s i s o f neomycin by G . L . C . The improvements t o t h e a s s a y i n c l u d e d t h e modif icat i o n o f t h e i n j e c t i o n p o r t t o p r e v e n t s a m p l e dec o m p o s i t i o n by c o n t a c t o f i n j e c t e d m a t e r i a l w i t h

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m e t a l o r Teflon and t h e a d d i t i o n of an a c c u r a t e volume of s i l y l a t i n g r e a g e n t t o each sample and s t a n d a r d t o overcome i n c o n s i s t e n t d e r i v a t i s a t i o n . A comparison of t h e G.L.C. and microbiological(agar-diffusion) a s s a y s h a s been rep o r t e d by T s u j i e t a1239 who showed t h e neomycin c o n t e n t of neomycin s u l p h a t e powders determined by t h e G.L.C. method, t o c o r r e l a t e w e l l w i t h v a l u e s o b t a i n e d by t h e m i c r o b i o l o g i c a l a s s a y when it i s assumed neomycin C h a s 35% o f t h e b i o l o g i c a l act i v i t y of neomycin B.

Neomycin h a s been s e p a r a t e d from m i x t u r e s of o t h e r aminoglycoside a n t i b i o t i c s cont a i n i n g t h e 2-deoxystreptamine moiety a s b o t h t h e p e r t r i m e t h y l s i l y l d e r i v a t i v e and t h e N - t r i f l u o r o a c e t y l p e r t r i m e t h y l s i l y l d e r i v a t i v e u s i n g a column of 0.75% OV-1 on Gas Chrom Q 2 4 0 . The p r o c e d u r e may be used t o e s t i m a t e t h e number of s u g a r moieti e s bound i n t h e a n t i b i o t i c as a close r e l a t i o n s h i p e x i s t s between t h e number of r i n g s and t h e retention t i m e . Examination of neomycin B by a combined G.C.-M.S. procedure h a s been r e p o r t e d by Murata e t a l l 5 . G.C. w a s accomplished w i t h t h e t r i m e t h y l s i l y l d e r i v a t i v e on a l m column of 1%OV-1 on Chromosorb W , by which means neomycin was s e p a r a t ed from kanamycin. The r e s u l t i n g mass s p e c t r a of t h e neomycin d e r i v a t i v e e x h i b i t e d a minute molec u l a r i o n peak a t m / e 1550 i n d i c a t i n g t h a t a l l a c t i v e hydrogens of b o t h hydroxy and amine groups were completely s i l y l a t e d . 6.4.Microbiological Procedures 6 . 4 1 . T u r b i d i m e t r i c Assay Various organisms have 'been used t o a s s a y neomycin t u r b i d i m e t r i c a l l y . These are summarised i n T a b l e 1 7 w i t h t h e working concent r a t i o n f o r t h e a n t i b i o t i c as r e p o r t e d by t h e respective authors. O f t h e organisms t a b u l a t e d overl e a f K.pneumaniae i s g e n e r a l l y used f o r r o u t i n e assays.

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Table 1 7 C o n d i t i o n s of T u r b i d i m e t r i c Assay Organism

---

S.auheub NRRLB314 II II 209P 11

11

F1

s t h epzo cu cc U b

daecalib ATCC 1054 K l e bb i e l l a pneumaniae PC1602 Klebbiella pneumo n i a e

ATCC 10031

E.coli ATCC 10536 II

II

ATCC 11105

Lacto b a c i l l u b ahabino zua La c t o ba c i l l ub cab ei Lacto b a c i l l u a dehmenti L a c t u ba c i l l u n L eic hm a n n i Aeho b a c t e h aehogeneb I P L 22K5118

C o n c e n t r a t i o n Range of Assay ( T o t a l pg neomycin base required i n s o l u t i o n t o be assayed) 2.5 5.0

-

Reference

2 41

100-300

242

-

243

0.016

0.04

120-200

2 48

6-15

244

6-14

245 I 246 I 268

1- 3

247

1-20

253

3-7

2 48

20-60

248

2-6

248

4-6

248

0-6

249

H e i n 2 4 3 n o t e d an a p p a r e n t d e c r e a s e i n t h e p o t e n c y of neomycin when a s s a y e d t u r b i d i m e t r i c a l l y i f t h e i n n o c u l u m c o n t a i n e d e i t h e r sodium c h l o r i d e o r p o t a s s i u m phos h a t e . With S.auheub a s o r g a n i s m l S o k o l s k i e t a 125!? d e m o n s t r a t e d t h e p r e s e n c e of K C 1 t o a n t a g o n i s e t h e a c t i v i t y of neomycin C more t h a n t h a t of neomycin B.

WILLIAM F. HEYES

A c o m p a r a t i v e s t u d y o f t h e responce o f neomycin B and neomycin C i n t h e t u r b i d i metric a s s a y u s i n g K . pneumakziae (ATCC 10031) h a s been r e p o r t e d 2 3 9 . The r e s p o n s e - r a t i o neomycin B: neomycin C w a s 100:38.9. An a u t o m a t e d t u r b i d i m e t r i c p r o c e d u r e u s i n g a T e c h n i c o n A u t o a n a l y z e r h a s a l s o been d e s c r i b e d (See S e c t i o n 6.5)

.

6.42.Agar-Diffusion

Assay

T a b l e 18 l i s t s t h e o r g a n i s m s t h a t have been recommended f o r d e t e r m i n i n g t h e microb i o l o g i c a l p o t e n c y o f neomycin. T a ble 18 Recommended Organ i s m

Working Concentration (P g h l )

8. p u m i l i n

Reference

4-2 1

9

4-20

245

NCTC 8241

.

S aua eun ATCC 6538

.

S e p i d ekmidics ATCC 12228

0.64

-

1.56

245,268

I n a l l t h e above r e f e r e n c e s , t h e c y l i n d e r - p l a t e a s s a y p r o c e d u r e i s t h e one d e s c r i b e d though a l t e r n a t i v e p r o c e d u r e s have been r e p o r t e d i n t h e l i t e r a t u r e . Thus Davis and P a r k e 2 4 5 r e p o r t e d a l i n e a r - d i f f u s i o n s y s t e m i n which a s o l u t i o n o f t h e a n t i b i o t i c i s allowed t o d i f f u s e i n t o a g a r - f i l l e d The s t a n d a r d d e v i a t i o n s c a l glass capilliaries c u l a t e d f o r s e v e n e x p e r i m e n t s w e r e between 5 and 10&(100 r e s u l t s ) f o r t h e a s s a y o f neomycin. The p r o c e d u r e i s more economic i n t e r m s o f a g a r and n u t r i e n t s t h a n o t h e r d i f f u s i o n methods. K u z e l and Coffey255 m o d i f i e d t h e c o n v e n t i o n a l c y l i n d e r p l a t e p r o c e d u r e by u s i n g a c y l i n d e r s e a l e d a t one e n d , f o r m i n g a c u p . The c u p s are f i l l e d w i t h t h e a n t i b i o t i c s o l u t i o n s t o b e a s s a y e d , t h e n a p l a t e of i n o c u l a t e d a g a r i s i n v e r t e d and p l a c e d over t h e t o p of t h e c u p s . With t h i s method a s i g n i f i c a n t r e d u c t i o n i n t h e s t a n d a r d d e v i a t i o n f o r t h e a s s a y o f neomycin

.

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w a s c l a i m e d . The r e p l a c e m e n t o f c l i n d e r s by a e r 47Y d i s c s h a s a l s o been r e p o r t e d 2561275,278. ~~~~i claimed a standard d e v i a t i o n of 2% f o r the assay of neomycin by t h i s means and recommended t h e p a p e r d i s c p r o c e d u r e i n p r e f e r e n c e t o t h e c y l i n d e r method. The t y p e of p a p e r u s e d h a s been shown t o a f f e c t t h e ~~~ seed d i a m e t e r of t h e i n h i b i t i ~ n - z o n eWhatman t e s t p a p e r p r o d u c i n g t h e s m a l l e s t z o n e s . When d e t e r m i n i n g neomycin l e v e l s i n m i l k , K o s i k ~ w s k i ~ ~ ~ improved t h e s e n s i t i v i t y o f t h e p r o c e d u r e by s u b s t i t u t i n g d r i e d milk t a b l e t s f o r paper d i s c s . S e v e r a l s t u d i e s of t h e f a c t o r s a f f e c t i n g t h e i n h i b i t i o n - z o n e s i z e i n t h e neomycin a g a r d i f f u s i o n a s s a y have been r e p o r t e d . P i n z e l i k e t a1257 d e m o n s t r a t e d t h a t v a r i o u s d e l a y s d u r i n g t h e a n a l y t i c a l procedure a f f e c t e d t h e s i z e of t h e i n h i b i t i o n zone. Refrigeration of t h e agar-plates p r i o r t o i n c u b a t i o n c a u s e d an i n c r e a s e i n zones i z e when compared w i t h s i m i l a r p l a t e s h e l d a t 2OoC. T h i s p r i n c i p l e h a s been u t i l i z e d by S i d d i q u e e t a1264 t o improve s e n s i t i v i t y when d e t e r m i n i n g t h e neomycin c o n t e n t o f m i l k . The p r e s e n c e o f K C l , N a C l , C a C 1 2 h a s been shown t o m a r k e d l y i n c r e a s e neomycin d i f f u s i o n i n a g a r 2 5 8 , 2 6 5 194. C o n v e r s e l y t h e p r e f p h o s p h a t e s 2 5 8 and of g l u c o s e / s t a r c h w i t h NaCl d e c r e a s e s t h e s i z e of t h e i n h i b i t i o n z o n e . I n c o r p o r a t i o n of non- f at milk i n t h e a g a r s i g n i f i c a n t l y r e d u c e s t h e s i z e o f t h e i n h i b i t i o n z o n e bec a u s e o f t h e i n t e r a c t i o n o f neom c i n w i t h t h e f r e e c a r b o x 1 g r o u p of m i l k p r o t e i n 2 6 3 F e d o r k o and K a t z 2 6 3 d i l u t e d neomycin s o l u t i o n s w i t h b l o o d serum and r e p o r t e d an i n c r e a s e i n t h e s i z e o f t h e i n h i b i t i o n zone when compared w i t h s i m i l a r s o l u t i o n s d i l u t e d w i t h pH 8 phos h a t e b u f f e r . S o k o l s k i e t a1252 and Yousef e t a1559 d e m o n s t r a t e d t h e p h y s i c a l b i n d i n g o f neomycin t o a g a r , a p r o c e s s which may be r e v e r s e d by a d d i t i o n o f o t a s s i u m o r sodium c h l o r i d e . F u r t h e r s t u d i e s 2 6 0 t 5 6 1 , 2 5 2 have r e p o r t e d t h e b i n d i n g t o be a f u n c t i o n o f b o t h t h e p H o f t h e a g a r and t h e m a n u f a c t u r e r . I n c o r p o r a t i o n o f 0.1M t r i s b u f f e r w i t h t h e a g a r (pH 8 ) h a s b e e n recommende d 2 6 0 , 261 i n o r d e r t o a c h i e v e t h e h i g h e s t s e n s i tivity. T r i s buffer results in a greater sensitivi t y t h a n p h o s p h a t e b u f f e r 2 6 1 and d o e s n o t a f f e c t t h e s i z e o f t h e i n h i b i t i o n zone w h e r e a s t h e p r e sence o f p h o s p h a t e r e d u c e s zone s i z e 2 5 2 . G i l l e t e t , 1 1 9 4 c o n c l u d e d t h a t t h e q u a l i t y of t h e a g a r a f f e c -

.

WILLIAM F. HEYES

470

t e d t h e i n h i b i t i o n zone s i z e . A comparison of 3 a g a r s showed t h e b e s t response f o r a g i v e n concent r a t i o n of neomycin w a s o b t a i n e d w i t h agarose.Comb i n i n g a g a r o s e w i t h recommendations d e s c r i b e d by o t h e r a u t h o r s , such a s t h e a d d i t i o n of C a C l and t h e use of t r i s b u f f e r , f u r t h e r i n c r e a s e d tge s i z e of t h e i n h i b i t i o n zone. The l i t e r a t u r e d e s c r i b e d above r e f e r s t o t h e a s s a y of t h e t o t a l neomycin complex i . e . neomycins B and C and neamine. To a s s a y neomycin B i n t h e p r e s e n c e of neomycin C and neamine by a m i c r o b i a l method S o k o l s k i and Carpenter265 adopted t h e f o l l o w i n g p r o c e d u r e . These a u t h o r s e m ployed a neomycin C - r e s i s t a n t organism (8.b u b i i l i b UC 564) and added K C 1 t o t h e a g a r t o t o t a l l y dep r e s s d i f f u s i o n of neamine and i n c r e a s e d i f f u s i o n of neomycin B . Neomycin B and C d i f f e r i n t h e i r b i o l o g i c a l a c t i v i t i e s though t h i s d i f f e r e n c e may v a r y w i t h t h e c o n d i t i o n s of t h e a s s a y . T y p i c a l l y neomycin C h a s an a c t i v i t y of 30-50% of t h a t of The p r e s e n c e of potassium c h l o neomycin B 2 6 6 r 2 3 9 . r i d e o r phosphate r e d u c e s t h e d i f f u s i o n of neomycin C more t h a n t h a t o f neomycin B252. Furthermore, t h i s a n t a g o n i s t i c e f f e c t of t h e s a l t s h a s been shown t o be more pronounced when u s i n g S . U U t r e U b a s organism than when u s i n g B.bubiilib. By a l t e r i n g t h e i o n i c s t r e n g t h o f t h e medium a system h a s been developed i n which t h e r e s p o n s e s of neomycins B and C a r e i d e n t i c a l 2 6 6 . The m i c r o b i a l assay of neomycin i n t h e p r e s e n c e of o t h e r a n t i b i o t i c s can p r e s e n t d i f f i c u l t i e s i f b o t h a n t i b i o t i c s are a c t i v e a g a i n s t t h e same t e s t organisms. The a s s a y o f neomycin i n t h e p r e s e n c e of d i h y d r o s t r e p t o m y c i n , t o which t h e t e s t organism S . U U t r e U b i s a l s o s e n s i t i v e , h a s been Levine e t a 1 2 6 9 r e n d e r e d t h e d i h y d r o reported. s t r e p t o m y c i n i n a c t i v e by h y d r o l y s i s w i t h barium hydroxide p r i o r t o t e r m i n i n g neomycin w i t h S. UUfLeUb DeNuzio 2% however, chose t o overcome t h e same problem by c u l t i v a t i n g a s t r a i n of S.uutreub which was r e s i s t a n t t o d i h y d r o s t r e p t o mycin. When a s s a y i n g neomycin i n a m i x t u r e cont a i n i n g t e t r a c y c l i n e , e n i c i l l i n and d i h y d r o s t r e p t omycin, Tanguay e t a12T1 found i t n e c e s s a r y t o

.

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s e p a r a t e t e t r a c y c l i n e and p e n i c i l l i n by s o l v e n t ext r a c t i o n b e f o r e d e t e r m i n i n g neomycin by t h e method of DeNuzio. The d e t e r m i n a t i o n of neomycin i n animal f e e d s h a s r e c e i v e d much a t t e n t i o n . B a r b i e r s and Neff272 recommended t h e u s e of a t r i s b u f f e r a t pH 8 t o p r e p a r e neomycin s o l u t i o n s and t h e a d d i t i o n of magnesium o r calcium c h l o r i d e t o t h e a g a r f o r enhancement of t h e i n h i b i t i o n zones. E x t r a c t i o n of neomycin from t h e f e e d r e q u i r e d t h e u s e of sodium c h l o r i d e s o l u t i o n f o r 100%r e c o v e r y . A c o l l a b o r a t i v e s t u d y of t h i s procedure by e i g h t e e n l a b o r a t o r i e s r e s u l t e d i n an average r e c o v e r y of 100.3% w i t h a c o e f f i c i e n t of v a r i a t i o n of 15.2%279. Modif i c a t i o n of t h e p r e v i o u s method h a s been d e s c r i b e d by Williams and Wornick273 who i n c o r p o r a t e d t h e t r i s b u f f e r i n t h e a g a r and d e l e t e d t h e a d d i t i o n of calcium c h l o r i d e . A t h r e e - f o l d i n c r e a s e i n s e n s i t i v i t y was r e p o r t e d . However, a c o l l a b o r a t i v e s t u d y of t h e modified procedure r e s u l t e d i n an ave r a g e r e c o v e r of 1 1 5 % w i t h a c o e f f i c i e n t of v a r i a t i o n of 20.4%3 7 4

.

An i n v e s t i g a t i o n of t h e a g a r d i f f u s i o n a s s a y of neomycin a t v e r y low l e v e l s ( 5 0 n g 1000ng) i n meat p r o d u c t s h a s been r e p o r t e d 2 8 0 . The p o i n t s on t h e dose-response graph c o v e r i n g t h e above c o n c e n t r a t i o n range w e r e s c a t t e r e d about t h e a v e r age s t r a i g h t l i n e . However, by j o i n i n g a l l t h e p o i n t s t o g e t h e r a complex c u r v e r e s u l t e d , t h e equat i o n of which was d e r i v e d .

6.5.

Automated Procedures

An a t t e m p t t o automate t h e t u r b i d i metric method f o r t h e d e t e r m i n a t i o n of neomycin w i t h K.pneurnaniae has been r e p o r t e d by Gerke e t a1281 who o b t a i n e d s a t i s f a c t o r y a s s a y s w i t h s o l An u t i o n s c o n t a i n i n g 150-1200 pg/ml of neomycin. automated r e s p i r o m e t r i c method, which measured t h e amount of C 0 2 produced by i n t e r a c t i o n of a n t i b i o t i c and t h e b a c t e r i a E.cali w a s a l s o r e p o r t e d w i t h a similar sensitivity. I n b o t h methods Auto Analyzer systems were employed.

The m o d i f i c a t i o n of t h e above r e s p i r o m e t r i c procedure t o i n c l u d e c o n t i n u o u s c u l t u r e of

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t h e t e s t o r g a n i s m E . c o L i h a s been d e s c r i b e d 2 8 2 . With t h e o r g a n i s m grown i n t h i s manner t h e s e n s i t i v i t y o f t h e a s s a y i s improved. G r e e l y e t a1283 have a p p l i e d t h e automated r e s p i r o m e t r i c method t o t h e d e t e r m i n a t i o n o f neomycin i n p h a r m a c e u t i c a l p r o d u c t s and compared t h e s e a s s a y s w i t h t h e r e s u l t s o b t a i n e d by t h e c y l i n d e r - p l a t e p r o c e d u r e on t h e same s a m p l e s . Good c o r r e l a t i o n between t h e t w o p r o c e d u r e s was d e m o n s t r a t e d . A n automated c o l o r i m e t r i c a s s a y f o r t h e q u a n t i t a t i o n of t h e s e p a r a t e components o f neom c i n ( B , C and neamine) h a s a l s o been r e p o r t The method u t i l i z e s a s e p a r a t i o n o f t h e ed148. components on a column o f c a r b o n and K i e s l g u h r G (4:l). The column e l u a t e i s reacted w i t h n i n h y d r i n t o d e t e r m i n e t h e amounts o f neomycin B , C and neamine.

6.6.

U s e as a n A n a l y t i c a l Reagent

Turbidimetric procedures f o r d e t e r mining r i b o n u c lease2 and d e o x y r i b o n u c l e a s e 85 u s i n g neomycin as t h e p r e c i p i t a n t h a v e been d e s c r i bed. The t u r b i d i t y w a s shown t o b e d e p e n d a n t on t h e r e l a t i v e c o n c e n t r a t i o n s o f r e a c t a n t s , t h e molec u l a r w e i g h t o f t h e RNA o r DNA and t h e i o n i c s t r e n g t h of t h e s o l u t i o n t h u s n e c e s s i t a t i n g t h e c a r e f u l c o n t r o l of assay conditions. Extraneous p r o t e i n s do n o t i n t e r f e r e e n a b l i n g t h e p r o c e d u r e t o be a p p l i e d d i r e c t l y t o blood serum. 6.7.

D e t e r m i n a t i o n i n Body F l u i d s and Tissues

An a g a r d i f f u s i o n a s s a y , based upon t h e method d e s c r i b e d by Groves and Randa11246, h a s been u t i l i s e d by t h e m a j o r i t y of w o r k e r s f o r det e r m i n i n g neomycin l e v e l s i n c l i n i c a l samples. T o compensate f o r t h e p r o t e i n - b i n d i n g e f f e c t e x h i b i t ed by neomycin, Kivman and Geitman309 recommended t h e a d d i t i o n of serum and 3 % KC1 t o t h e neomycin s t a n d a r d s o l u t i o n . D a n i e l ~ v a ~however, ~ ~ , pref e r r e d t o release t h e bound a n t i b i o t i c by enzymat i c h y d r o l y s i s p r i o r t o m i c r o b i o l o g i c a l a s s a y . An a g a r d i f f u s i o n method r e q u i r i n g o n l y 2 0 ~ 1o f serum sample h a s been d e s c r i b e d by A x l i n e e t a1311.

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The a p p l i c a t i o n of a t u r b i d i m e t r i c method t o t h e e x a m i n a t i o n o f s e r u m s a m p l e s h a s been r e p o r t e d by Fe l s e n f e l d 3 l 2 . More r e c e n t l y a laser l i g h t s c a t t e r i n g b i o a s s a y f o r d e t e r m i n i n g t h e neomycin c o n t e n t of m i l k , s e r u m , u r i n e and b i l e h a s been r e p o r t e d 3 1 3 . The p r o c e d u r e u s e s a helium-neon l a s e r l i g h t s o u r c e and h a s a l i n e a r d o s e - r e s p o n s e g r a p h over t h e r a n g e 0.1 t o 1 0 u g / m l f o r serum. A n enzymic a s s a y i n v o l v i n g t h e r e a c t i o n of neomycin w i t h a m i n o g l y c o s i d e 4 ' a d e n y l t r a n s f e r a s e h a s b e e n d e s c r i b e d 3 1 4 . A l i n e a r r e s p o n s e f o r neomycin w a s o b s e r v e d over t h e r a n g e 2.5 t o 2Oug/ml serum.

The c o n c e n t r a t i o n of neomycin i n u r i n e h a s been d e t e r m i n e d w i t h a f l u o r i m e t r i c method315. I n t e r f e r i n g amines w e r e s e p a r a t e d by c h r o m a t o g r a p h y on SE Sephadex C-25 b e f o r e r e a c t i n g t h e i s o l a t e d neomycin w i t h f l u o r e s c a m i n e

.

B r a m m e r and Hemson182 employed a n e l e c t r o p h o r e t i c p r o c e d u r e t o s e p a r a t e neomycin from b l o o d - p r o t e i n s b e f o r e d e t e r m i n i n g t h e neomycin c o n c e n t r a t i o n c o l o r i m e t r i c a1l y

.

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Waksman S.A. a n d L e c h e v a l i e r H . A . , U.S. P a t e n t 2,799,620 (1957). Umezawa H . , T a k e u c h i T. a n d Yamagiwa S . , J a p a n . Med. J . , 3 , 25-30 ( 1 9 5 0 ) . A r a i T. a n d A i i s o K . , J a p a n e s e P a t e n t 5450 (1953). Game G.F., K o c h e t k o v a G . V . , P r o b r a z e n s k a y a T.P. a n d P e v z n e r N.S., A n t i b i o t i k i , 1 (51, 4-8 ( 1 9 5 6 ) . J a c k s o n F.L., K i t t i n g e r G.W. and K r a u s e F.P. , N u c l e o n i c s , 18 ( 8 ) ,102-5 ( 1 9 6 0 ) . S w a r t E.A. , H u t c h i n s o n D . a n d Waksman S.A. , A r c h . Biochem., 24, 92-103 ( 1 9 4 9 ) . L e a c h B.E., D e V r i e s W . H . , N e l s o n H.A., J a c k s o n W.G. a n d E v a n s J . S . , J.Am.Chem.Soc., 7 3 I 2797-2800 ( 1 9 5 0 ) Makino S. , H a y a s h i E . a n d Okamura K . , J a p a n . P a t e n t 6247 ( 1 9 6 0 ) . T a k e n a g a M. a n d Okada Am, G e r . P a t e n t 2,165,373 (1972). T a n a k a I . , Yoshizawa H., O t a n i S., N a k a y a s h i k i J. a n d Hara E . ,Y a k u z a i g a k u , 2 2 , 147-50 ( 1 9 6 2 ) . Simone R.M. a n d P o p i n o R.P., J.Am.Pharm. A S ~ O C .I 44, 275-80 ( 1 9 5 5 ) . Beyd A . , J . P h a r m . S c i . , 60 ( 9 ) ,1343-5 (1971). Gecgil S . , I s t a n b u l U n i v . E c z a c i l i k F a k . Mecmuasi , 2 (1),56-57 ( 1 9 6 8 ) Coates L . V T , P a s h l e y M.M. a n d T a t t e r s a l l K . , J.Pharm.Pharmaco1. , 13, 6 2 0 - 4 ( 1 9 6 1 ) . D a l e J . K . a n d Rundman S. J . , J.Am.Pharm. ASSOC., P r a c t . Pharm. E d . , 1 8 , 4 2 1 - 5 ( 1 9 5 7 ) . K u d a l k a r V.G., H a l l N . A . a n d R i s i n g L.W. , Am.J.Pharm., 1 3 1 , 166-73 ( 1 9 5 9 ) . M c G i n i t y J . W . a n d Brown R.L., J . P h a r m . S c i . , 64 ( 9 ) ,1528-30 ( 1 9 7 5 ) . E r n i c k R.C., P r o c . , Annu. P f i z e r R e s . C o n f . , 1 5 t h , 54-90 1 1 9 6 7 ) . Danti A . G . a n d G u t h E . P . , J.Am.Pharm.Assoc., S c i . E d . , 4 6 , 249-52 ( 1 9 5 7 ) . Hammouda Y T a n d S a l a k a w y S.A., P h a r m a z i e , 26 (10), 636-40 ( 1 9 7 1 ) . F l o r e s t a n o H . J . , B a h l e r M.E. a n d J e f f r i e s S . F . , J.Am.Pharm.Assoc. , 4 5 , 538-41 ( 1 9 5 6 ) . Kivman G. Y a . a n d G e i t m a n I. Y a . , A n t i b i o t i k i , 16 ( 4 ) ,331-5 ( 1 9 7 1 ) .

.

.

WILLIAM F. HEYES

488

310. 311. 312. 313. 314. 315.

D a n i e l o v a L . T . , A n t i b i o t i k i , 1 9 (111, 1032-8 ( 1 9 7 4 ) . Axline S.G., Yaffe S . J . a n d Simon H . J . , P e d i a t r i c s , 39 ( 1 ) , 9 7 - 1 0 7 ( 1 9 6 7 ) . F e l s e n f e l d O . , V o l i n i I . F . , K a d i s o n E.R., Zimmermann E . a n d I s h i h a r a S . J . , Am.J. Clin.Path., 3 , 670-1 (1950). Wyatt P . J . , P h i l l i p s D . T . and A l l e n E . H . , J . A g r i c . F o o d Chem., 24 ( 5 ) ,984-8 ( 1 9 7 6 ) . Santanam P . a n d K a y s e r F.H., A n t i m i c r o b . A g e n t s C h e m o t h e r . , lo ( 4 ) ,664-7 ( 1 9 7 6 ) . K u s n i r J . a n d B a r n a K . , Cesk. Farm., 24 ( 6 ) ,253-5 ( 1 9 7 6 )

This p r o f i l e attempts t o cover t h e published l i t e r a t u r e on neomycin u p t o a n d i n c l u d i n g C h e m i c a l Abstracts, Volume 8 5 .

Analytical Profiles of Drug Substances, 8

PSEUDOEPHEDRINE HYDROCHLORIDE Steven A . Benezra and John W . McRae 1.

2.

3. 4. 5. 6.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor Physical Properties 2. I Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Melting Point 2.6 Specific Rotation 2.7 Solubility 2.8 Partition Coefficient 2.9 Differential Scanning Calorimetry 2.10 Crystal Structure 2.1 I Dissociation Constant Synthesis Stability Metabolism and Pharmacokinetics Methods of Analysis 6.1 Elemental Analysis 6.2 Nonaqueous Titration 6.3 Ultraviolet Spectrophotometric Analysis 6.4 Colorimetric Analysis 6.5 Chromatography 6.51 High Performance Liquid Chromatography 6.52 Thin-Layer Chromatography 6.53 Paper Chromatography 6.54 Gas Chromatography

489

Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-260808-9

490

1.

STEVEN A . BENEZRA AND

JOHN W. MCRAE

Description

1.1 Name, Formula, Molecular Weight d-Pseudoephedrine hydrochloride is (+)-threo-a-(1(methy1amino)ethyl)benzyl alcohol hydrochloride. Throughout this analytical profile, d-pseudoephedrine will be referred to as pseudoephedrine.

*o

C10H15NO*HC1

H

0

HCI

I

CH3

201.72

1.2 Appearance, Color, Odor Pseudoephedrine hydrochloride occurs as fine white t o off-white crystals o r as a powder having a faint odor1

2.

Physical Properties

2.1 Infrared Spectrum The infrared spectrum of pseudoephedrine hydrochloride is shown in Figure 1. It was obtained as a 0.2% dispersion of pseudoephedrine hydrochloride in KBr with a Nicolet Model 7199 FT-IR spectrophotometer.2 Table I gives the infrared assignments consistent with the structure of pseudoephedrine hydrochloride. Table I Infrared Spectral Assignments for Pseudoephedrine Hydrochloride Frequency (cm-l) Assignment OH stretch 3270 3010 Asym. C-H stretch

1

0

0

1

1

8

1

I

3

I

I

0 d

1

0 (u

STEVEN A. BENEZRA AND

492

Sym. C-H stretch

2930

+NH

2700

stretch

C=C aromatic stretch OH bend, secondary alcohol C-H bend, monosubst. benzene

1587, 1490 1430 762, 702 2.2

JOHNW. MCRAE

Nuclear Magnetic Resonance (NMR) Spectrum The NMR spectrum o f pseudoephedrine hydrochloride

is shown in Figure 2.

The spectrum was obtained with a

Varian model CFT-20 80 MHz NMR spectrometer. Deuterated DMSO was used as the solvent with tetramethylsilane as an internal standard. Table I1 gives the NMR assignments consistent with the structure of pseudoephedrine hydrochloride.

G9

8 H H HN-

OH H

@@

Table I1

NMR Assignments for Pseudoephedrine Hydrochloride Proton a

No. of Protons 3

Shift (ppm) 0.96

Multiplicity doublet

b

3

2.55

singlet

C

1

3.25

quartet (partially obscured by H20)

d

1

e

1

f

5

8

2

4.54 6.32 7.34 8.90

doublet of doublets doublet singlet broad singlet

493

PSEUDOEPHEDRINE HYDROCHLORIDE

J

1

1

I

8

9

7

6

5

4

3

S

I

2

'

I

!

I

1

I

1

I'

0

PPm

Figure 2

-

NMR Spectrum of Pseudoephedrine Hydrochloride

I.o

ae

0.6 U

4 [L

v 0 )

a 0.4

0.2

0.9

Figure 3

-

nrn

Ultraviolet Spectrum of Pseudoephedrine Hydrochloride

STEVEN A. BENEZRA A N D

494

JOHN W. MCRAE

2.3 Ultraviolet Spectrum The ultraviolet spectrum of pseudoephedrine hydrochloride in ethanol was obtained with a Beckman ACTA CIII ultraviolet spectrophotometer and is shown in Figure 3. Pseudoephedrine hydrochloride exhibits absorption maxima at 208, 251, 257, and 264 nm with extinction coefficients of 8300, 161, 201, and 161, respectively. 2.4. Mass Spectrum The low resolution mass spectrum of pseudoephedrine hydrochloride is shown in Figure 4.5 It was obtained with a Varian MAT CH5-DF mass spectrometer. Direct probe at 80'C into the ion source was used to obtain the mass spectrum. The electron energy was 70 eV. The assignments of the major ions formed in the mass spectrometer are shown below.

The

molecular ion is not observed.

H +HC/NzCH3 'CHq3 m/e 58 (100%) 2.5

0 +

m/e 77 (9%)

HCI

m/e 36 (10%)

+

HNCH3

m/e 30 (12%)

Melting Point Pseudoephedrine hydrochloride melts between 182'

and 185'C.

2.5 Specific Rotation The specific rotation, of d-pseudoephedrine hydrochloride in water is between +61.0' and +62.5'.l

[cy]iO,

2.7

Solubility The solubility of pseudoephedrine hydrochloride in

495

PSEUDOEPHEDRINE HYDROCHLORIDE

1001

I50

I00

200

m /e

Figure 4

1 I

178

-

Mass Spectrum of Pseudoephedrine Hydrochloride

1

1

1

180

179

I

181

I

I

182

1 1

183

TEMPERATURE

Figure 5

-

1

i

184

I

1

185

1 1

186

1 1

187

OC

DSC Curve of Pseudoephedrine Hydrochloride

STEVEN A. BENEZRA AND JOHN W. MCRAE

496

various solvents at 25OC is given in Table 111.' Table I11 Solubility of Pseudoephedrine Hydrochloride at 25OC Solvent

Solubility (gm/ml)

Water

2.0

Chloroform

0.011

E thano1

0.278

Ether

1.4

2.8 Partition Coefficient The partition coefficients of pseudoephedrine hydrochloride at 25OC in n-octanol/aq. pH 1.2 and n-octanol/ aq. pH 6.0 are 0.010 and 0.049 respectively.6 2.9 Differential Scanning Calorimetry (DSC) The DSC curve of pseudoephedrine hydrochloride obtained with a Perkin Elmer DSC-1B differential scanning calorimeter is shown in Figure 5 . ?

The heating rate was

5OC/min. The heat of fusion is 6.4 Kcal/mol. The melting point (uncorrected) is 184OC. 2.10 Crystal Structure The crystal properties of pseudoephedrine hydrochloride were determined with a GE model XRD-6 x-ray diffractometer using Zr filtered MoKa radiation on a crystal grown from water.

Pseudoephedrine hydrochloride has an ortho-

rhombic crystal system belonging to the P2 2 2 space group. 0 l a 1 0 The cell dimensions are a=25.358 A , bz6.428 A , ~ ~ 6 . 9 0A1 with each cell containing four molecules. 2.11 Dissociation Constant The pKa of pseudoephedrine hydrochloride determined

PSEUDOEPHEDRINE HYDROCHLORIDE

491

titrimetrically in 80% aqueous methylcellosolve is 9.22. 3.

Synthesis Pseudoephedrine hydrochloride is prepared by a

Welsh rearrangementl o of R-ephedrine hydrochloride with acetic anhydride followed by deacetylation with hydrochloric acid. l 1

2-Ephedrine can be resolved from dR-ephedrine with

2-mandelic acid. l2

R-Ephedrine occurs naturally in certain

plants of the Ma Huang species. 4.

Stability Pseudoephedrine hydrochloride can be considered a

stable compound in bulk and in formulations. After 4-weeks under fluorescent lights (2400 ft. candles) and ultraviolet light (190 pw/cm2) no discoloration or chemical degradation was observed. The bulk drug was stable for at least 6 months at 37OC and 3 months at 5OoC. Tablet and syrup formulations stored at 15-30°C for 5 years showed no appreciable degradation. l 3

5.

Metabolism and Pharmacokinetics The major biotransformations of pseudoephedrine

hydrochloride are parahydroxylation, N-demethylation, and oxidative deamination. l4

The proposed pathways for the

metabolism of pseudoephedrine are shown in Figure 6 . In a study with human subjects, whose urine pH was controlled with sodium bicarbonate and ammonium chloride, it was found that 10-25% of the administered pseudoephedrine hydrochloride was metabolized to norpseudoephedrine and the elimination of pseudoephedrine and norpseudoephedrine was related to urine pH.

As the urine pH increased, the serum

half-life o f pseudoephedrine and norpseudoephedrine increased.15

In another similar study it was found that a decrease

H

CH3

p-&-H++-Q OH NH

\ I

I

CH3 Pseudoephedr ine

OH NH, Nor pseudoephedrine

o + ( / OH

I/

CH3

I-Hydroxy-l-phenyl-2-proponone

OW,H OH OH

I -Phenyl-l,Z-propanediol

OH Benzoic acid Figure 6

-

Metabolism of Pseudoephedrine Hydrochloride

PSEUDOEPHEDRINE HYDROCHLORIDE

499

in urine pH caused a decrease in plasma half-life of pseudoephedrine. l6

Plasma half-lives measured in normal human

subjects were 5 . 2 - 8 . 0 hours.l6 In a rat study using 14C-labelled dQ-ephedrine, 85% of the i.p.-administered drug was eliminated in the first 40 hours.

Two major metabolic pathways were postulated after

analysis of the metabolites. The major metabolic pathway was ring para-hydroxylation forming para-hydroxyephedrine and para-hydroxynorephedrine.

The minor metabolic pathway was

oxidative deamination, giving acidic metabolites such as hippuric and benzoic acids. l7 The relative tissue distribution in mice 15 minutes after i.v.-administered l4C2ephedrine was kidney > lung, adrenal, spleen, liver > intestines, stomach > brain, heart > plasma. l7 The LDs0 in mice o f pseudoephedrine administered i.p. is 1.0 mmole/kg.18 6.

Methods of Analysis Elemental Analysis

6.1

The results of the elemental analysis o f pseudoephedrine hydrochloride are given in Table IV.6 The analysis was performed on a NF Reference Standard. Table IV Elemental Analysis of Pseudoephedrine Hydrochloride Element

Theory (%)

Found ( X )

C

59.55

59.54

H

8.00

8.11

N

6.95

6.81

STEVEN A. BENEZRA AND

500

6.2

JOHN W.M C W E

Nonaqueous Titration Pseudoephedrine hydrochloride is dissolved in a

mixture of glacial acetic acid and mercuric acetate test solution. A standardized solution of 0 . 1 N perchloric acid is used to titrate the solution to a blue-green end point with crystal violet indicator. Each ml of 0.1N perchloric acid is equivalent to 0.1 mmole of pseudoephedrine hydrochloride. 6.3

Ultraviolet Spectrophotometric Analysis An ultraviolet spectrophotometric analysis is used

to assay pseudoephedrine hydrochloride in tablets. A portion of finely powdered tablets equivalent to approximately 30 mg of pseudoephedrine hydrochloride is placed in a distilling flask which is part of a micro-steam distillation apparatus. Sodium chloride, water, and concentrated sodium hydroxide are added. A minimum of 30 ml of distillate is collected in a volumetric flask containing dilute hydrochloric acid.

The

flask is made to volume with distilled water and the absorbance of the solution is determined at 257 nm in 1 cm cells and compared to a solution of known concentration of NF Pseudoephedrine Hydrochloride Reference Standard. An ultraviolet spectrophotometric method based on the absorbance of a periodate oxidation product o f pseudoephedrine hydrochloride will be the official method of analysis in the USP XX.19,20 A portion of tablets or syrup in water is placed in a separatory funne.1. Sodium bicarbonate and sodium metaperiodate are added. After standing for 15 minutes, 1 N HC1 is added. The solution is extracted with hexane. The hexane extract is filtered and its absorbance determined at 242 nm in 1 cm cells. The amount of the oxidation product of pseudoephedrine hydrochloride is determined by comparison of the sample absorbance against the absorbance of a Pseudoephedrine Hydrochloride Reference Standard treated in the same manner.

50 1

PSEUDOEPHEDRINE HYDROCHLORIDE

6.4 Colorimetric Analysis Pseudoephedrine hydrochloride in syrup formulations has been analyzed by colorimetry. Pseudoephedrine forms a stable blue-colored chelate with cupric sulfate at pH 12.5. The complex has a maximum absorbance at 500 nm. The complex is extracted from an aqueous layer with 1-pentanol. Interfering substances such as glycerine and sugars normally found in syrup formulations, which form complexes with cupric aulfate, are not extracted into l-pentanol.*l 6.5

Chromatography 6.51 High Performance Liquid Chromatography (HPLC) High performance liquid chromatography has been

used to analyze pseudoephedrine hydrochloride and dosage forms containing pseudoephedrine hydrochloride.

Table V

gives the HPLC conditions used for separations. Table V HPLC Conditions €or Pseudoephedrine Hydrochloride Column Corasil@/Phenyl

Mobile Phase Rention Time (min) 1.8 (phenyl) acetonitrile:

Corasil@/C18

0.1% ammonium

Reference 22

1.9 (C18)

carbonate (9:l) pH 8.9

Corasil@/Phenyl Corasil@/C18

acetonitrile: 1% ammonium

carbonate (6:4) pH 7.4

22

STEVEN A. BENEZRA AND JOHN W.M C M E

502

Zipax@/SCX

0.02 M dibasic ammonium phosphate:dioxane

7

23

6

24

8

25

(64:36) Nucleosil@/

methanol:0.5M

silica gel

sodium dihydrogen phosphate: phosphoric acid (195:50:2)

Spherisorb@/

ethanol:0.4%

silica gel

ammonium acetate (85: 15)

6.52 Thin Layer Chromatography (TLC) Table VI lists the various TLC systems used for pseudoephedrine hydrochloride. Table VI TLC Systems for Pseudoephedrine Hydrochloride

Rf

Reference

silica gel

0.25

26

silica gel

0.46

Mobile Phase

Adsorbent

ethyl acetate: cyclohexane: methano1:conc. M140H(70:15:10:5) n-butano1:ethanol: water:acetic acid (60:30:10:0.2)

27 0.18 (free base)

503

PSEUDOEPHEDRINE HYDROCHLORIDE

chlorof o rni:

silica gel

0.33

28

methanol : acetone (7:3:5) ethyl ether:

0.10 (free base) silica gel

benzene (1:l)

0.35 (as 4-chloro-

29

7-nitrobenzo-2,1,3oxadiazole derivative) chloroform :

alumina

0.70 (as

water:acetic

acetylated

acid (20:75:20:1)

product)

16

(lower phase) 6.53 Paper Chromatography Paper chromatography has been used to separate and detect pseudoephedrine hydrochloride from other pharmacologically active amines. Whatman No. 1 paper developed in n-butanol:water:95% acetic acid (4:5:1), n-butano1:toluene: water:95% acetic acid (10:10:5:5), ethyl acetate:water:95% acetic acid (3:3:1), o r chloroform:water:95% acetic acid (10:5:4) gave Rf values of 0.73, 0.35, 0.57, and 0.52 for pseudoephedrine hydrochloride respectively. Visualization of pseudoephedrine hydrochloride was done by spraying the chromatogram with 0.5% bromcresol green in methanol or 0.2% ninhydrin in acetic acid:butanol 5 :95.30 6.54 Gas Chromatography Pseudoephedrine hydrochloride has been separated from other arnines by gas chromatography. The oxazolidine

504

STEVEN A. BENEZRA AND JOHN

w.MCRAE

derivative has been prepared by the reaction of pseudoephedrine with anhydrous acetone. On a 1.15% SE-30, glass column, 2.4 m x 3 mm i.d. at 104OC the oxazolidine derivative has a retention time of 16.4 minutes.31

On a 15% PEG 6000, glass column 2 m x 4 mm i.d. at 175OC the oxazolidine derivative had a retention time of 10.1 minutes.32 The N-trifluroacetyl-L-prolylchloride derivative of pseudoephedrine has retention time of 105 minutes on a 3% SE-30, stainless steel column, 2 m x 3 mm i.d. at 170°C.33 An on-column acetic anhydride derivatization technique has been described for pseudoephedrine hydrochloride. Immediately after injection of a solution of pseudoephedrine onto a 20% SE-30, 1.8 m x 7 mm i.d. glass column at 125OC, an injection of acetic anhydride was made. The pseudoephedrine derivative formed on column has a retention time of 55.5 minutes as compared to a retention time of 8.7 minutes for underivatized pseudoephedrine.34 A variety of methods have been used to determine pseudoephedrine hydrochloride levels in plasma and urine by ~~ basefied gas chromatography. Bye and c o - w o r k e r ~extracted plasma or urine with diethyl ether. The ether extract concentrate was chromatographed on a 1.2 m x 2mm i.d. glass column packed with 2% Carbowax 20 M +5% KOH. The column was maintained at 187OC for plasma samples and 15OoC for urine samples. The heptafluorobutyric anhydride derivative of pseudoephedrine and electron capture detector have been used to enhance the sensitivity of the gas chromatographic method. Lin and c o - ~ o r k e r sand ~ ~ Cummins and Fourier37 extracted basefied urine or serum with benzene. Heptafluorobutyric anhydride is added to the benzene extract. The heptafluoroibutyric anhydride derivative extracted was chromatographed

505

PSEUDOEPHEDRINE HYDROCHLORIDE

on a 3% OV-17, 1.82 m x 2 mm i.d. glass column at 150°C36 or a 5% ethylene glycol succinate, 1.82 m x 2 mm i.d. stainless steel column at 140°C. 37 Pseudoephedrine in urine was analyzed by gas chromatography using a 2% polyethylene glycol 600 + 5% KOH,

2 m x 2 mm i.d. stainless steel column at 165OC. The urine was extracted with diethyl ether and then made basic with 5N NaOH. The pseudoephedrine was extracted with diethyl ether, concentrated, and injected directly into the gas chromatograph, or derivatized with acetone and then chromatographed.38 Pseudoephedrine was determined after acidification, precipitation, and acetic anhydride derivatization. The ester derivative was injected onto a 2.5% SE-30, 1.8 m x 4 mm i.d. column at 190°C.39 References 1.

N . F . XIV, Mack Printing Co., 1975

2. W. Martin, Burroughs Wellcome Co., personal communication 3 . R . Crouch, Burroughs Wellcome Co., personal communication 4.

J.W. McRae, unpublished data

5. D. Brent, Burroughs Wellcome Co., personal communication

6. B.S. Hurlbert, Burroughs Wellcome Co., personal communication 7. J. Ebron, Burroughs Wellcome, personal communication 8. M. Mathew, G.J. Palenik, Acta. Cryst., E, 1016 (1977)

9. V. Prelog, P. Haflinger, Helv. Chim. Acta., 33, 2021 (1950) 10. L.H. Welsh, J. h e r . Chem. SOC., 3500 (1949) 11. R. Seaman, Burroughs Wellcome Co., personal communication

c,

STEVEN A. BENEZRA AND JOHN W.MCRAE

506

12.

R.H. Manski, T.B. Johnson, J. h e r . Chem. SOC., 51,

1906

(1929) 13.

T. Morgan, Burroughs Wellcome Co., personal communication

14.

D.R. Feller, P. Basu, W. Mellon, J. Curott, L. Malspeis, Arch. Int. Pharmacodyn.,

15. 16.

17.

203,

187 (1973)

D.C. Brater, L.Z. Benet, E. Lin, R.C. Morris, Jr.,

K.L. Melmon, Clin. Res., 24, 252A (1976) R.G. Kuntzman, I. Tsai, L. Brand, L. Mark, Clin. Pharmacol. Ther., 12, 62 (1971) J. Bralet, Y. Cohen, G . Valette, Biochem. Pharm.,

11

2319 (1968) 18.

M.D. Fairchild, G.A. Alles, J. Pharmacol. Exp. Ther.

19. 20.

158, 135 (1967) J.E. Wallace, J. Pharm. S c i . , g ,1489 (1969) L. Chafetz, J. Pharm. Sci., 60, 291 (1971)

21.

J.A. McCrerie, Wellcome Foundation Ltd., personal communication

22.

I.L. Honigberg, J.T. Stewart, A.P. Smith, J. Pharm. Sci.,

3,766

(1974)

23.

T.L. Spriek, J. Pharm. Sci., 64, 5 9 1 (1974)

24.

G.T. Hill, Wellcome Foundation Ltd., personal communication

25.

M.L. Blackmon, Burroughs Wellcome Co., personal communication

26.

K.K. Kaistha, R. Tadrus, R. Janda, J. Chromatog.,

107,

359 (1975) 27. 28

29. 30.

Y. Hashimoto, Y. Ikeshiro, T. Higashiyama, T. Ando, M. Endo, Yakugaku Zasshi, 97, 1594 (1977) L.N. Mikhailova, M.N. Preobrazhenskaya, G.M. Kadatskii, S.D. Sokolov, Khim.-Farm. Zh., 2, 49 (1975) J.C. Hudson, W.P. Rice, J. Chromatog., 117, 449 (1976) A. WickstrZim, B. Salvesen, J. Pharm. Pharmacol., 4, 631 (1952)

PSEUDOEPHEDRINE HYDROCHLORIDE

31.

507

E.B.-Hanssen, A.B. Svendsen, J. Pharm. Sci.,

51,

938

(1962) 32.

K. Yamasaki, K. Fujia, M. Sakamoto, M. Okeda, M. Yoshida, 0. Tanaka, Chem. Pharm. Bull.,

33.

22,

2898 (1974)

A.H. Beckett, B. Testa, J. Pharm. Pharmacol., 9, 382 (1973)

34. 35.

M.W. Anders, G.J. Mannering, Anal. Chem., 34, 730 (1962) C. Bye, H.M. Hill, D.T.D. Hughes, A.W. Peck, Eur. J. Clin. Pharmacol., 8, 47 (1974)

36.

E.T. Lin, D.C. Brater, L.Z. Benet, J. Chromatog., 140, 273 (1977)

2,

37.

L.M. Cummins, M.J. Fourier, Anal. Lett.,

38.

A.H. Beckett, G.R. Wilkinson, J. Pharm. Pharmacol.,

39.

Suppl. 1 7 , 104s (1965) P. Leblish, B . S . Finkle, J . W . Chem.,

16,195

(1970)

403 (1969)

Brackett, Jr., Clin.

Analytical Profiles of Drug Substances, 8

TRIPROLIDINE HYDROCHLORIDE Steven A . Benezra and Chen-Hwa Yang I. 2.

3. 4. 5. 6.

Description I. I Name, Formula, Molecular Weight I .2 Appearance, Color, Odor Physical Properties 2. I Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Fluorescence Spectrum 2.6 Crystal and Molecular Structure 2:7 Melting Point 2.8 Solubility 2.9 Partition Coefficient 2.10 Dissociation Constant Synthesis Stability Drug Metabolic Products and Pharmacokinetics Methods of Analysis 6. I Elemental Analysis 6.2 Nonaqueous Titration 6.3 Ultraviolet Spectrophotometric Analysis 6.4 Quantitative Infrared Analysis 6.5 Chromatography 6.5 I High Performance Liquid Chromatography 6.52 Thin-Layer Chromatography 6.53 Quantitative Thin-Layer Chromatography 6.54 Gas Chromatography 6.6 Fluorimetric Analysis

509

Copy-t @ 1979 by Academic F’ress, Inc. AU rights of reproduction in any form reserved.

ISBN 0-12-2608089

5 10

1.

STEVEN A. BENEZRA AND CHEN-HWA YANG

Description 1.1

Name, Formula, Molecular Weight Triprolidine hydrochloride is E-2-[3-(l-pyrroli-

diny1)-1-p-tolylpropenyllpyridine,

monohydrochloride;

E-l-(2-pyridyl)-3-pyrrolidino-l-p-tolylprop-l-ene,

monohydro-

chloride. J

M.W. 314.85 1.2 Appearance, Color, Odor Triprolidine hydrochloride occurs as a white, crystalline powder, with no more than a slight unpleasant odor.

2.

Phvsical ProDerties 2.1

Infrared Spectrum (IR) The infrared spectrum of triprolidine hydrochloride

is shown in Figure 1.

The IR spectrum was recorded with a

Nicolet model 7199 FT-IR spectrophotometer as a 0.2% dispersion of triprolidine hydrochloride in KBr.2

Table I gives

the infrared assignments consistent with the structure of triprolidine hydrochloride.

511

TRIPROLIDINE HYDROCHLORIDE

Table I Infrared Spectral Assignments €or Triprolidine Hydrochloride Monohydrate Assignment

Band (em-')

3480 2958 2690 1630 1582 1562

OH stretch (hydrate) CH stretch (-CH3) NH' stretch C=C stretch (conj. diene) C=C stretch (aromatic) C=C stretch (pyridine)

1462 1386 1358 846

-CH a s p . bend 3 -CH3 sym. bend C-N stretch (tert. amine) =C-H rock

824 776

para subst. benzene 1-subst. pyridine

2.2 Nuclear Magnetic Resonance (NMR) Spectrum The NMR spectrum of triprolidine hydrochloride is shown in Figure 2. It was obtained with a Varian XL-100 100 MHz NMR spectrometer. Deuterated DMSO was used as the solvent with tetramethylsilane as an internal standard. The interpretation of the NMR spectrum is given in Table 11.

i 9

H3cQ

e

b

c I'

1

8

1

1

?=====-

c P

r-

{

1

8

I

8

I

33NVlllWSNVU

I

P

0

I

1

I

1

STEVEN A. BENEZRA AND CHEN-HWA YANG

5 I4

Table I1

NMR Assignments for Triprolidine Hydrochloride Chemical Shift (ppm) No. of Protons Multiplicity Assignments 1 1 11.55 j a 1 multiplet 8.63 b 6 1 7.74 multiplet C 1 7.35 d multiplet 7.00 1 4 4 benzene ring 7.12-7.38 e 1 3 6.95 f 2 2 3.78 h 1 (broad) 2 3.49 h 1 (broad) 2 2.94 1 i 2.37 3 mu1tiplet 1.88 4 lz =8, Jc,d=1.2, Jaf=7 J =4.7, Ja,d=0.9, Ja,b=2.0, J b,c

a,c

2.3 Ultraviolet (W)Spectrum The ultraviolet spectrum of triprolidine hydrochloride in 0.1N HC1 was taken with a Beckman ACTA CIII W spectrophotometer and is shown in Figure 3.4 Table I11 gives the W data for triprolidine hydrochloride in various solvents. Table I11 W Spectral Data for Triprolidine Hydrochloride Solvent h a x (NO)

EmaX

0.1N NaOH

0.1N HC1 Methanol

234 275 232 290 235 282

17000 8000 13000 9900 15000 7200

5 15

TRIPROLIDINE HYDROCHLORIDE

I-

0.6 W

0 Z

a m cr

0 cn

m

a

220 Figure 3

260

- Ultraviolet

300 340 nm

380

Spectrum of Triprolidine Hydrochloride

250 m /e

Figure 4

-

Mass Spectrum of Triprolidine Hydrochloride

300

STEVEN A. BENEZRA AND CHEN-HWA YANG

516

2.4 Mass Spectrum The mass spectrum of triprolidine hydrochloride is shown in Figure 4. It was obtained with a Varian MAT CH5-DF mass spectrometer. The electron energy was 70 eV and the sample was introduced via direct probe at 120°C.5 The characteristic fragments are in agreement with those found by Kuntzman and co-workers.6 The major fragments characteristic of triprolidine are shown in Figure 5. 2.5 Fluorescence Spectrum Triprolidine hydrochloride in 0.1M sulfuric acid has an excitation maximum at 305 nm and an emission maximum at 445 nm. The fluorescence is much diminished in hydrochloric acid and almost non-existent in distilled water. The addition of halide ions to 0.1M sulfuric acid solutions of triprolidine hydrochloride has a quenching effect on the fluorescent intensity. The degree of quenching is I- > BrZC1 > F-. A concentration of 10-3M C1- has little effect on the fluorescent intensity, while 0.1M C1- reduces the

s

intensity by a proximately 75% of the chloride-free sulfuric acid solution.

2.6 Crystal and Molecular Structure James and Williams have determined that triprolidine hydrochloride monohydrate crystals belong to the P2 /c 1 space group.8 There are 4 molecules per unit cell. The cell parameters are a = 14.777 b = 9.5785 c = 13.099 0 A, and f? = 90.48O. The 2-pyridyl ring and p-tolyl group make dihedral angles of 29.7O and 55.3O respectively with the double bond plane. The inter-aryl dihedral angle is 106.5O. The data was obtained on crystals grown from a solution of the compound in anisole. A Picker FACS-1 diffractometer with CuK01 radiation was used for the measurements.

i,

i,

R2,

r c=c

RI’

, c=c

R l?

R2 \

3

‘H

RI’

/-R3

R2,

+c=c

‘H

m/e 200 ( 10%)

c=c+

R,’ H ‘ m/e 194 ( 18 %)

R2=QCH

Figure 5

c=c R,’

‘H

m/e 209 (90%)

m/e 278 ( 33%)

R2,

+CH2 /

CH’;

- Major

‘H

m/e 208 (100%)

R2\+

/

CH

+CHz-R,

Rl m/e 182 (7%)

R3=-N

m/e 84 (15 %)

3

Ions in Mass Spectrum of Triprolidine Hydrochloride

STEVEN A. BENEZRA AND CHEN-HWA YANG

518

2.7 Melting Point Triprolidine hydrochloride melts at 115-12OoC in a sealed capillary tube.

2.8 Solubility The solubility of triprolidine hydrochloride, as the monohydrate, in various solvents at 25OC is given in Table IV.9 Table IV Solubility of Triprolidine Hydrochloride at 25OC Solvent

Solubility (mg/ml)

Water

316

0.1N HC1

319

95% Ethanol

374

n-Octanol

75

Chloroform

480 237

I

AllSN31NI

0

I

9

0

m

S4 I

0

0 0 (Y

0 0 d

0 0 9 0

2 0 0

P 0 0

2

8 5 0

B 0

0

z

0 0

8 0 N

0 0

aD

N

0

0 0

5

0

0

3

ZUI L. CHANG

542

-

FIGURE 9 ULTRAVIOLET SPECTRUM O F VALPROIC ACID

0.6

0.5

0.4

ez

2 SI

0.3

9

0.2

0.1

0 200

2 50

300 WAVELENGTH (nm)

350

543

SODIUM VALPROATE AND VALPROIC ACID

2.5

a.

3000-2800 cm-1

Complex of strong bands due to the overlapping C-H stretching vibrations of the various methyl and methylene groups.

b.

1660 cm-l

Weak band due to C=O stretching vibration of the carboxylic acid.

c.

1450 cm-l

Due to the superimposing C-H bending vibrations of the various methyl and methylene groups.

Ultraviolet Spectrum (UV) Sodium valproate in methanol solution has no W maximum between 400 and 205 nm. When the UV spectrum of 0.1% solution of valproic acid in methanol solution was scanned from 400,to205 nm, one maximum at 213 nm ( E = 86) was observed (Figure 9 ) . The spectrum was obtained with a Beckman Acta V Spectrophotometer.

2.6

Solubility Approximate solubility data have been determined for sodium valproate at room temperature. One gram of sodium valproate is soluble in 0 . 4 ml of water and also in 1.5 ml of ethanol. It is freely soluble in methanol (1 in 5 ) . It is practically insoluble in common organic solvents such as ether, chloroform, benzene, n-heptane, etc. The following solubility data have been determined for valproic acid at room temperature: n-Heptane Chloroform Ethyl Acetate Methanol 100% Ethanol Acetone Diethyl Ether Benzene 1 N Aqueous NaOH 0.1 N Aqueous HCl Water

Greater than Greater than Greater than Greater than Greater than Greater than Greater than Greater than Greater than 1.15 mg/ml 1 . 2 7 mg/ml

lo%, v/v lo%, v/v

v/v v/v v/v v/v lo%, v/v lo%, v/v lo%, v/v lo%, lo%, lo%, lo%,

ZUI L. CHANG

544

2.7

Crystal Properties The X-ray powder diffraction pattern of sodium valproate was determined by visual observation of a film obtained with a 143.2 mm Debye-Scherrer Powder Camera (Table IV). An Enraf-Nonius Difractis 6 0 1 Generator; 38 KV and 1 8 MA with nikel filtered copper radiation; A = 1.5418, was employed ( 4 ) . Table IV X-Ray Powder Diffraction Pattern d-Spacings and Intensities

2.8

dA -

111 1

dA

16.0 13.4 7.7 6.7 5.8 5.25 4.9 4.77 4.42 4.21 4.09 3.95 3.85 3.64 3.40 3.20 3.13 3.02 2.87 2.84

60 100 50 3 5 20 5 2 5 30 35 5 1 15 20 1 3 4 5 5

2.80 2.66 2.61 2.57 2.46 2.42 2.37 2.23 2.20 2.06 2.01 1.97 1.93 1.90 1.86 1.81 1.75 1.69 1.66

1/11 5 2

1 1

1 1 2 2 2 2

1 1 2

1 1 5 1 1 1

Dissociation Constants Sodium valproate exhibits basic properties. Titration of an aqueous solution of sodium valproate with aqueous hydrochloric acid gave a pKa value of 4.8 (proton gained). Titration of valproic acid with aqueous sodium hydroxide using acetone-water as the sample solvent and extrapolated to pure water gave a pKa value of 4.6 (proton lost).

SODIUM VALPROATE AND VALPROIC ACID

2.9

545

Hygroscopic Behavior Sodium valproate is hygroscopic. The rate of moisture absorption was tested in a humidity chamber using a Cahn Electro Balance and the results are shown in Table V (5). Table V Rate of Moisture Absorption of Sodium Valproate

Relative Time, min. 10 20 30 40 50 60 Overnight Humidity No gain

12, 22 33, 43% 53%

1.76

3.17 4.39

5.61

6.78 7.80

42.9%*

*Sample was completely liquified. Expressed as precent weight gain. 2.10

Sublimation Sodium valproate did not sublime when it was stored at 105°C for 10 days.

2.11 Melting Range Sodium valproate does not melt, decompose, or physically change form in the normal working range of the Thomas-Hoover Capillary Melting Point apparatus. 2.12

Boiling Point The following boiling points have been reported for valproic acid: bpi4 120-121°C (6), bp20 128-13OoC (6) and bp760 221-222"C (7).

2.13

Differential Thermal Analysis Differential thermal analysis of sodium valproate shows a large endotherm beginning at 100°C and ending at 118°C which is possible due to the loss of water. A sharp endothermic peak at 450°C is indicative of the melting point of sodium valproate. Differential thermal analysis of valproic acid shows. a sharp endothermic response at 225°C indicative of the boiling point of valproic acid.

FIGURE 10 I

I

- DIFFERENTIAL THERMAL ANALYSIS CURVE OF SODIUM VALPROATE I

I

I

I

I

I

I

0

I

I

I

I

50

100

150

200

T,

O C

I

250

I

I

I

300 350 400 (CORRECTED FOR CHROME1 ALUMEL THERMOCOUPLES)

I

450

500

FIGURE 11

- DIFFERENTIAL THERMAL ANALYSIS CURVE OF VALPROIC ACID

0

50

100

150

200

250

300

350

400

1, O C (CORRECTED FOR CHROME1 ALUMEL THERMOCOUPLES)

450

500

548

ZUI L. CHANG

2.14

Specific Gravity The specific gravity of valproic acid was determined It has a in a calibrated 25 ml pycnometer at 25'C. value of 0.904 g/ml (8).

2.15

Refractive Index Valproic acid has a refractive index of N D ~ ~ - ~ 1.425 (6).

3.

Synthesis The synthesis of valproic acid was first described in the literature by Oberreit (9) in 1896. The sodium valproate is preferably formed from valproic acid by the interaction of sodium hydroxide in an aqueous solution. The synthetic pathways are shown in Figure 12.

4. Stability-Degradation Sodium valproate was found to be extremely stable when refluxed in water, 1.0 N hydrochloric acid, or 1.0 sodium hydroxide for 3 Kours. Also, it was very stable when it was subjected to heat at llO°C for 10 days and to natural sunlight for 30 days in the dry state. Valproic acid is a very stable compound. No degradation has been observed by the action of heat, light, and strong aqueous alkali, or acid. 5. Drug Metabolism and Pharmacokinetics In 1971, Eymard et. al. (10) studied the distribution and the absorption of carbon-14 labeled sodium valproate in rats by oral administration. The metabolites and metabolic pathway of a new anticonvulsant drug, sodium valproate, in rats were investigated using carbon-14 labeled sodium valproate. Most of the metabolites in urine and bile were a glucuronide conjugate of ValprOic acid. Free sodium valproate was as little as one-seventh of the total metabolites. In feces, only free sodium valproate was detected, and the possibility of enterohepatic circulation of sodium valproate was presumed. A part of dosed sodium valproate was excreted in expired air in the form of C02. This degradative reaction took place in liter mitochondria and required CoA and oxygen. It was stimulated by ATP

FIGURE 12

- SYNTHETIC PATHWAYS OF VALPROIC ACID AND SODIUM VALPROATE

/ CH2

C02C2H5

\ C02C2H5

Diethyl Malonate

+

2CI-CH2CH

= CH2

c02r

CH2= CHCH2 * C ’‘

(or NaOCzHS) CHjOH

(or C,H,OH)

CH2= CHCH2

/ \ C02R

Esters of Diallyl Malonic Acid

Ally1 Chloride

R=CHs and/or C2H5

Pd/C H

CH3CH2CH2

\ / CH3CH2CH2/ c \

Co2R

CO2R

-

CH3CH2C H2

CH3CH2CH2

CHsCH2CH2

/ CH-C02H

2-Propylpentonoic Acid (Valproic Acid)

/ \ C02H

Dipropyl Malonic Acid

CH3CH2CH2

\

C02H

C ’‘

c

2) HCI

Esters of Dipropyl Malonic Acid

-c02

CH3CH2CH2

1) KOH, H 2 0

N aOH H2O

c

CH3CH2CH2

\

/ CH-c02No

Sodium 2-propylpentanoate (Sodium Valproate)

ZUI L. CHANG

550

and EDTA, and inhibited by various enzyme reaction inhibitors such as malonate, Antimycin-A, chloropromazine, p-chloromercuribenzoate (PCMB) and 2,4-dinitrophenol. Therefore, this degradation is not a one-step reaction, decarboxylation, but must be @-oxidation of a fatty acid. Thin-layer chromatography and gas-liquid chromatography were used for assay of metabolites (11). The pharmacokinetics of distribution and elimination of sodium valproate in mice and dogs has been reported by Schobben and van der Kleijn (12). The omega-oxidation of sodium valproate in rats has been reported by Kuhara, et. al. (13). The absorption, excretion, and biotransformation of valproic acid were studied by Kukino and Matsumoto (14). A preliminary pharmacokinetic profile of sodium valproate in monkey has been written by Levig, et. al. (15). The pharmacokinetics of sodium valproate have been studied in 7 patients by Schobben, et. al. (16). The plasma concentrations were determined by gas-liquid chromatography during and following subchronic treatment. Elimination of the drug appeared to follow a monophasic exponential course; biological half lives were 8 to 15 hours. The drug appeared to have a relatively restricted distribution: calculated relative distribution volumes ranged from 0.15 to 0.40 llkg. There were large interindividual differences in clearance rate. The therapeutic range was considered to be between 50 and 100 mg/l of plasma. In 1976, Matsumoto, et. al. (17, 18) discovered several new metabolites of sodium valproate in rat urine, which support the hypothesis that the drug is also metabolized by a B-oxidation mechanism. One of the metabolites, 2n-propyl 3-0x0-pentanoic acid, was recently found by Gompertz, et. al. (19) and Kochen, et. al. (20) to be a major constituent in urine of children who were receiving sodium valproate. The urinary 3-0x0 derivative of valproate was reported to account €or 20% of the administered dose. 6.

Methods of Analysis

6.1 Identification The presence of sodium cation may be identified by

55 1

SODIUM VALPROATE AND VALPROIC ACID

a flame test. A positive test for sodium is produced in a non-luminous flame imparting a yellow color. The presence of carboxylic anion may be ider!.tified by the following two tests:

6.2

A.

A 5% aqueous solution gives a violet precipitate with a 5% aqueous solution of cobalt nitrate.

B.

A 5% aqueous solution gives a violet precipitate with potassium iodobismuthite.

Elemental Analysis A typical elemental analysis of a sample of sodium valproate is present in Table VI (21).

Table VI Elemental Analysis of Sodium Valproate Element C

H 0

Na

% Theory 57.82 9.10 19.25 13.83

% Found

57.82 9.38

-----* -----*

*The oxygen value cannot be determined due to presence of sodium.

A typical elemental analysis of a sample of valproic acid is present in Table VII (22). Table VII Elemental Analysis of Valproic Acid Element C

H 0

6.3

% Theory

66.63 11.18 22.19

% Found

66.39 11.32 22.43

Chromatographic Analysis 6.31

Thin-Layer Chromatography A number of thin-layer chromatographic systems

552

ZUI L. CHANG

on silica gel have been found to induce the migration of the compound. However, no system has been found which gives resolution equivalent to the gas-liquid chromatographic system detailed in section 6.32. Thin-layer chromatography is not the preferred method for determining the impurities. The following systems have been studied : 1. Ch1oroform:Methanol:Glacial Acetic Acid (17:2 :1) 2.

Ethyl Acetate:Formic Acid:Water (5:l:l)

3.

Benzene:Methanol:Acetone:Ammonium Hydrox-

ide (2:2:5:1)

4. Ch1oroform:Methanol:Ammonium Hydroxide (20:15 :3)

5. n-Butanol Saturated with 10% Ammonium Hydroxide Solution (Aqueous) 6.32 Gas-Liquid Chromatography The author has found the following GLC procedure to be suitable to determine the purity of sodium valproate and valproic acid. Preparatory to chromatography, the sodium valproate was acidified with a strong aqueous hydrochloric acid solution, and the valproic acid which is practically insoluble in water was separated. The separated free acid was then analyzed. The following is the typical chromatographic condition for the GLC determination of valproic acid: Column: 10% DEGS-PS on Supelcoport 80/100 mesh, Two 6 ft x 114 in 0.d. stainless steel. Detection: Thermal Conductivity Detector. Temperature: Inj. Port Column Detector Flow Rate:

225OC 160°C 300°C -20 ml/min helium

SODIUM VALPROATE AND VALPROIC ACID

553

Attenuation: 2 x 4 Slope Sensitivity: 0.03 or adjust for proper sensitivity Sample Size:

2 1.11

Impurities as low as 0.01% could be detected using the above conditions. 6.4

Titrimetry Sodium valproate exhibits basic properties. It can be titrated with 0.1 N hydrochloric acid. In addition, sodium valproate can be potentiometrically titrated with standardized 0.1 perchloric acid using a modified glass-calomel electrode system, in which 0.1 N lithium perchlorate in acetic acid has been substituted for potassium chloride, and employing glacial acetic acid as the sample solvent

.

Valproic acid can be potentiometrically titrated with standardized 0.1 N tetra-n-butylammonium hydroxide in chlorobenzene using a modified glasscalomel electrode system, in which 1.0 aqueous tetra-n-butylammonium chloride has been substituted for potassium chloride, and employing acetone as the sample solvent. 7.

Determination of Valproic Acid and Its Metabolites in Biological Fluids Many gas-liquid chromatographic methods for determination of valproic acid in biological fluids have been reported. Early methods required derivatization of valproic acid ( 2 3 , 24, 25, 2 6 ) .

Although Meijer and Hessing-Brand in 1973 (27) developed a micro diffusion method without derivatization, it required special equipment. Recently, most of the methods which have been used for the analysis of valproic acid in plasma, serum, cerebral spinal fluid, saliva, breast milk, and urine involve acidification of the biological sample, extraction into an organic solvent, and direct injection onto a gasliquid chromatographic column (28, 29, 16, 30, 31, 3 2 ,

554

ZUI L. CHANG

33, 34, 35, 36, 37, 38, 39). Most of the methods employ an internal standard, and all are reported as accurate and reproducible. However, Schmidt, et. al. (40) recently reported great interlaboratory variation in the analysis of such biological samples. 8. References 1. W. Washburn, Abbott Laboratories, Personal Communication.

2. M. Cirovic and R. Egan, Abbott Laboratories, Personal Communication. 3. S. Mueller, Abbott Laboratories, Personal Communication. 4.

J. Quick, Abbott Laboratories, Personal Communication.

5. M. Yunker, Abbott Laboratories, Personal Communication. 6. The Merck Index, 9th Edition, 9574, Merck ti Co., Inc., Rahway, N.J., 1976. 7. Dictionary of Organic Compounds, Vol. 5, p. 2799, Oxford University Press, New York, (1965).

8. J. Fornnarino, Abbott Laboratories, Perwnal Communication. 9. E. Oberreit, Berichte der Deutschen Chemischen Gesellschaft, 3,1998 (1896).

10. P. Eymad, et. al., J. Pharmacol. (Paris), 2 (2), 251 (1971)

.

11. K. Kukino, K. Mineura, T. Deguchi, A. Ishii and H. Takahira. Yaku-Gaku Zasshi, 92 (71, 896 (1972). 12. F. Schobben and E. van der Kleijn, Pharm. Weekbl., 109 (2), 33 (1974). 13. T. Kuhara, et. al., Biomed. Mass Spectrom., 291 (1974).

1. ( 4 ) ,

SODIUM VALPROATE AND VALPROIC ACID

555

14. K. Kukino and I. Matsumoto, J. of Kurukome Med. ASSOC., 34 (41, 369 (1975). 15. R.H. Levig, et. al., Epilepsia 16 (l), 202 (1975). 16.

F. Schobben, E. van der Kleijn and F.J. M. Gabreels, Europ. J. Clin. Pharmacol., 8, 98 (1975).

17.

I. Matsumoto, T. Kuhara and M. Yoshino, Advances in Mass Spectrometry in Biochemistry and Medicine, 1, 17-26, (1976).

18. I. Matsumoto, T. Kuhara and M. Yoshino, Biomedical Mass Spectrometry, 3 (5), 235-240, (1976). 19. D. Gompertz, P. Tippett, K. Bartlett, and T. Baillie, Clinica Chimica Acta, 74, 153-160, (1977). 20. W. Kochen, H. Imbeck and C. Jakobs, ArzneimittelForschung, 27 (I) No. 5, 1090-1099, (1977). 21.

Galbraith Laboratories, Inc., Knoxville, TN.

22. J. Hood, Abbott Laboratories, Personal Communication. 23. J. Alary, D. Cantin, A. Coeur and G. Carraz, Trav. SOC. Pharm. Lyon., 16 (2), 53 (1972).

u.

24. B. Ferrands, P. Eymard and C. Gautier, Ann. Pharm. 5, 31 (4), 279 (1973). 25. J.M.H.G. Cremers and Ing. P.E. Verheesen, Pharm. Weekbl. , 109 (22), 522 (1974). 26. F.N. Ijdenberg, Pharm, Weekbl.,

110 (2),

21 (1975).

27. J.W.A. Meijer and L. Hessing-Brand, Clin. Chim. , .Acta 43 ( 2 ) , 215 (1973). 28.

I.C. Dijkhuis and E. Vervloet, Pharm. Weekbl., (2), 42 (1974).

109

29. F. Schobben, E. van der Kleijn and Neth. Nijmegen, Pharm. Weekbl, 109 (2), 30 (1974). 30. C.R. Chard, N.J. Legg, Ed., Clinical and Pharmacological Aspects of Sodium Valproate in the Treatment of Epilepsy, pp. 89-91 (1975).

ZUI L. CHANG

556

31. P.N. Patsalos, V.C. Goldberg and P.T. Lascelles, Proc. Analyt. Div. Chem. SOC., 12 (lo), 270 (1975). 32. M.C. Pfaff and G. Mahuzier, Ann. Pharm. Fr., (1975).

33, 355

33. T.B. Vree, E. van der Kleijn and H.J. Knop, J. Chromatography, 121 (l), 150 (1976). 34. N.H. Wood, D.C. Sampson and W.J. Hensley, Clinica Chimica Acta, 77, 343 (1977). 35. L.J. Dusci and L.P. Hackett, J. of Chromatography, 132, 145 (1977). 36. L.J. Dusci and L.P. Hackett, J. Chromatography, 132 (l), 145 (1977). 37. C.J. Jensen and R. Gugler, J. Chromatography, 137, 188 (1977). 38. W. Loescher, Epilepsia 18 (2), 225 (1977). 39. A. Sengupta and M.A. Peat, J. Chromatography, 137 206 (1977). 40.

D. Schmidt, B. Ferrandes, D. Grandjean, R. Gugler, C. Jakobs, S . Johannessen, U. Klotz, W. Kochen, H.J. Kupferberg, J.M.A. Meijer, A. Richens, H. Schaefer, H.U. Schulz and.A. Windorfer, Arzneim-ForschIDrug 27 (l), 1078 (1977).

w,

9. Acknowledgements The author wishes to thank Mr. V.E. Papendick and Mr. J. B. Martin for their review of the manuscript, and Miss Diane Penza for typing the manuscript.

CUMULATIVE INDEX Italic numerals refer to volume numbers Acetaminophen, 3, I Acetohexamide, I , 1 ;2,573 Allopurinal, 7, 1 Alpha-tocopheryl acetate, 3, 11 1 Amitriptyline hydrochloride, 3, 127 Amoxicillin, 7, 19 AmphotericinB,6, 1;7,502 Ampicillin,2, I ; # , 517 Aspirin, 8, 1 Bendroflumethiazide, 5 , I ;6,597 Betamethasone dipropionate, 6,43 Bromocriptine methanesulfonate, 8 , 4 7 Calcitriol, 8,83 Cefazolin*; 4, I Cephalexin, 4,21 Cephalothin sodium, I , 319 Cephradine*, 5 , 2 1 Chloral hydrate, 2,85 Chloramphenicol, 4,47,517 Chlordiazepoxide, I , I5 Chlordiazepoxide hydrochloride, I , 39; 4,517 Chloroquine phosphate, J , 6 1 Chlorpheniramine maleate, 7,43 Chloroprothixene, 2,63 Chlortetracycline hydrochloride, 8; 101 Clidinium bromide, 2, 145 Clonazepam, 6 , 6 1 Clorazepate dipotassium, 4,91 Cloxacillin sodium, 4,113 Cyclizine, 6,83; 7, 502 Cycloserine, I , 53 Cyclothiazide, I , 66 Dapsone, 5 , 8 7 Dexamethasone, 2, 163; 4,518 Diatrizoic acid, 4,137; 5,556 Diazepam,1,79;4,517

Digitoxin, 3, 149 Dihydroergotoxine methane sulfonate, 7,8 1 Dioctyl sodium sulfosuccinate, 2, 199 Diperodon, 6.99 Diphenhydramine hydrochloride, 3, 173 Diphenoxylate hydrochloride, 7, 149 Disulfram, 4,168 Dobutamine hydrochloride, 8 , 139 Droperidol, 7,171 Echothiophate iodide, 3,233 Epinephrine, 7, 193 Ergotamine tartrate, 6, 113 Erythromycin, 8 , 139 Erythromycin estolate, 1, 101; 2, 573 Estradiol valerate, 4,192 Ethambutol hydrochloride, 7,231 Ethynodiol diacetate, 3,253 Fenoprofencalcium*, 6, 161 Flucytosine, 5 , 115 Fludrocortisone acetate, 3,281 Fluorouracil, 2,221 Fluoxymesterone, 7,251 Fluphenazine enanthate, 2,245; 4,523 Fluphenazine hydrochloride, 2,263; 4,518 Gluthethimide,S, 139 Gramicidin, 8 , 179 Griseofulvin, 8,219 Halcinonide, 8,251 Halothane, I , 119;2,573 Hexetidine, 7,277 Hydralazine hydrochloride, 8,283 Hydroflumethiazide, 7,297 Hydroxyprogesterone caproate, 4,209 Hydroxyzine dihydrochloride, 7,319 Iodipamide, 3,333 Isocarboxazid, 2,295

*Monographs in “Pharmacological and Biochemical Properties of Drug Substances: M. E. Goldberg, D. Sc., Editor American Pharmaceutical Association.

558

Isoniazide, 6,183 Isopropamide, 2,315 Isosorbide dinitrate, 4,225;5,556 Kanamycin sulfate, 6,259 Ketamine, 6,297 Leucovonn Calcium, 8,3 I5 Levarterenol bitartrate, I , 49;2,573 Levallorphan tartrate, 2,339 Levodopa, 5,189 Levothyroxine sodium, 5,225 Meperidine hydrochloride, I, 175 Meprobamate, I, 209;4,519 &Mercaptopurine, 7,343 Methadone hydrochloride, 3,365;4,519 Methaqualone, 4,245,519 Methimazole, 8,351 Methotrexate, 5,283 Methyclothiazide, 5,307 Methyprylon, 2,363 Metronidazole, 5,327 Minocycline, 6,323 Nalidixic Acid, 8.371 Neomycin, 8,399 Nitrohrantoin, 5 , 345 Norethindrone, 4,268 Norgestrel, 4,294 Nortriptyline hydrochloride, I, 233;2,573 Nystatin, 6,341 Oxazepam, 3,441 Phenazopyridine hydrochloride, 3,465 Phenelzine sulfate, 2,383 Phenformin hydrochloride, 4,319;5,429 Phenobarbital, 7,359 Phenoxymethyl penicillin potassium, I, 249 Phenylephrine hydrochloride, 3,483 Piperazine estrone sulfate, 5,375 Primidone, 2,409 Procainamide hydrochloride, 4,333 Procarbazine hydrochloride, 5,403 Prornethazine hydrochloride, 5,429

CUMULATIVE INDEX

Proparacaine hydrochloride, 6,423 Propiomazine hydrochloride, 2,439 Propoxyphene hydrochloride, I, 301 ;4 , 5 19; 6,598 Propylthiouracil, 6,457 Pseudoephedrine hydrochloride, 8,489 Reserpine, 4,384;5,557 Rifampin, 5,467 Secobarbital sodium, I, 343 Spironolactone, 4,431 Sodium nitroprusside, 6,487 Sulphamerazine, 6,515 Sulfamethazine, 7,401 Sulfamethoxazole, 2,467;4,520 Sulfasalazine,5,515 Sulfisoxazole, 2,487 Testolactone,S, 533 Testosterone enanthate, 4,452 Theophylline, 4,466 Thiostrepton, 7,423 Tolbutamide,3,513;5,557 Triamcinolone, 1,367;2,571;4,520,523 Triamcinolone acetonide, I, 397,416;2,571; 4,520;7 Triamcinolone diacetate, I , 423 Triamcinolone hexacetonide, 6,579 Triclobisonium chloride, 2,507 Triflupromazine hydrochloride, 2,523;4,520; 5,557 Trimethaphan camsylate, 3,545 Trimethobenzamide hydrochloride, 2,551 Trimethoprim, 7,445 Triprolidine hydrochloride, 8,509 Tropicamide, 3,565 Tubocurarine chloride, 7,477 Tybamate, 4,494 Valproate Sodium and valproic acid*, 8, 529 Vinblastine sulfate, I, 443 Vincristine sulfate, I , 463