Profiles of Drug Substances, Excipients and Related Methodology vol 11, Volume 11 (Analytical Profiles of Drug Substances and Excipients)

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Analytical Profiles of

Drug Substances Volume 11 Edited by

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

Contributing Editors

Gerald S. Brenner Glenn A. Brewer, Jr. Lester Chafetz Nicholas DeAngelis

Lee T. Grady Hans-Georg Leemann Joseph Mollica Milton D. Yudis

Compiled under the auspices of the Pharmaceutical Analysis and Control Section Academy of Pharmaceutical Sciences

Paris

ACADEMIC PRESS 1982 A Subsidiary of Harcourt Brace Jovanovich,Publishers New York London San Diego San Francisco SPo Paulo Sydney Tokyo Toronto

EDITORIAL BOARD

Norman W. Atwater Rafik Bishara Gerald S . Brenner Glenn A. Brewer, Jr. Lester Chafetz Nicholas DeAngelis John E. Fairbrother Klaus Florey

Salvatore A. Fusari Lee T. Grady Boen T. Kho Hans-Georg Leemann Joseph A. Mollica Bruce C. Rudy Milton D. Yudis

Academic Press Rapid Manuscrlpt Reproduction

COI’YRIGHT @ 1982, BY ACADEMIC I’RESS, INC. ALL RIGHTS RESERVED. NO PART O F T H I S PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR hlECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS,INC.

I1 I Fifth Avenue. New York, New York 10003

Uiiifed Kirigdoni Edition pirblished b y ACADEMIC PKESS, INC. ( L O N D O N ) LTD. 24/28 Oval Road, London N W I

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ISBN 0-12-26081 1-9 PRINTED IN THE UNITED STATES OF AMERICA 82 83 84 85

Y 8 7 6 5 4 3 2 1

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

E . Abignente, University of Naples, Naples, Italy H . Y. Aboul-Enein, King Saud University, Riyadh, Saudi Arabia A. A . AZ-Badr, King Saud University, Riyadh, Saudi Arabia I . A . Al-Meshal, King Saud University, Riyadh, Saudi Arabia M . A . Al-Yahyu, King Saud University, Riyadh, Saudi Arabia S. L. Ali, Zentrallaboratorium Deutscher Apotheker e. V., Eschborn, Germany N. W. Atwater, E. R. Squibb and Sons, Princeton, New Jersey D. M. Baaske, American Critical Care, Chicago, Illinois R. Bishara, Eli Lilly and Company, Indianapolis, Indiana G. S. Brenner, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania G. A. Brewer, Jr., The Squibb Institute for Medical Research, New Brunswick, New Jersey P. de Caprariis, University of Naples, Naples, Italy J. E . Carter, Ortho Pharmaceuticals, S o m e d e , New Jersey L. Chajetz, Warner-Lambert Research Institute, Morris Plains, New Jersey N. DeAngelis, Wyeth Laboratories, Philadelphia, Pennsylvania W. DeWitte, Ciba-Geigy, Suffern, New York H . A . El-Obeid, King Saud University, Riyadh, Saudi Arabia A. A. Elazzouny, National Research Center, Dokki, Cairo, Egvpt J. Fairbroth, Stiefel Laboratories Ltd., Sligo, Ireland K. Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey S . A. Fwari, Parke-Davis, Inc., Detroit, Michigan L. T. Grudy, The United States Pharmacopeia, Rockville, Maryland M . M. A. Hassan, King Saud University, Riyadh, Saudi Arabia J. G. Hoogerhde, Schering-Plough Corporation, Bloomfield, New Jersey vii

viii

AFFILIATIONS OF EDITORS, CONTRIBUTORS, A N D REVIEWERS

J. H . Johnson,American Critical Care, Chicago, Illinois H . Kudin, The Squibb Institute for Medical Research, New Brunswick, New Jersey B. T. K b , Ayerst Laboratories, Rouses Point, New York F. Kreuzig, Biochemie GmbH, Kundl, Austria H. G. Leemunn, Sandoz, Basel, Switzerland E. A. Lo@, King Saud University, Riyadh, Saudi Arabia M. E. Mohamed, King Saud University, Riyadh, Saudi Arabia 1. MoZZica, Ciba-Geigy Corporation, Summit, New Jersey F. I. Muhtadi, King Saud University, Riyadh, Saudi Arabia V. D. Reij, Wyeth Laboratories, Philadelphia, Pennsylvania B. C. Rudy, Mary Kay Cosmetics, Dallas,Texas C. M . S h r m , Wyeth Laboratories, Philadelphia, Pennsylvania D. H . Sieh, The Squibb Institute for Medical Research, New Brunswick, New Jersey H. Stober, Ciba-Geigy, Suffern, New York K. D. Thkker, The United States Pharrnacopeia, Rockville, Maryland T. D. Wilson, Sterling Winthrop Research Institute, Rensselaer, New York B. E . Wyka, Schering- Plough Corporation, Bloomfield, New Jersey M. D. Yudis, Schering- Plough Corporation, Bloomfield, New Jersey

PREFACE

It is now well over a decade that I perceived the need to supplement the official compendial standards of drug substances with a comprehensive review of pertinent physical, chemical, and analytical data and methods. Ten years ago the first volume of Analytical Projiles of Drug Substanceswas published under the auspices of the Pharmaceutical Analysis and Control Section of the APhA Academy of Pharmaceutical Sciences. That we were able to publish one volume per year is a tribute to the diligence of the editors to solicit monographs and even more so to the enthusiastic response of our authors, an international group associated with pharmaceutical firms,academic institutions, and compendial authorities. I would like to express my sincere gratitude to them for making this venture possible. I am pleased to report that five years ago a companion series entitled Phurmacological and Biochemical Properties of Drug Substances was initiated by Morton E. Goldberg under the auspices of the section on Pharmacology and Toxicology, APhA Academy of Pharmaceutical Sciences. So far, three volumes have been published. Over the years, we have had queries concerning our publication policy. Our goal is to cover all drug substances of medical value and, therefore, we have welcomed any monographs of interest to an individual contributor. We also have endeavored to solicit profiles of the most useful and used medicines, but many in this category still need to be profiled. Starting with this, the eleventh volume, we shall also supplement previously published profiles with new data as we can find volunteers to write such s u p plements. In this volume, five of the original profiles in Volume 1have been updated. The goal to cover and update all drug substances of medical value with comprehensive monographsis still a distant one. I estimatethat only about a quarter of such compounds have been profiled so far. We would very much like to accelerate

ix

X

PREFACE

the rate of publication and hope that even more authors can be encouraged to write profiles. All those who have found these profiles useful are requested to contribute monographs of their own. We, the editors, stand ready to receive such contributions.

Klaus Florey

AMINOPHYLLINE Kailus D.Thakker and Lee T.Grady

1.

2.

3. 4.

5.

6.

I. 8. 9.

Description 1 . 1 Nomenclature 1.2 Formula, Molecular Weight, and Composition 1.3 Appearance, Color, and Odor Physical Properties 2.1 Spectral 2.2 Other Properties Methods of Preparation Stability-Degradation 4.1 Stability in Solution 4.2 Stability in Solid State Methods of Analysis 5.1 Identification Tests 5.2 Gravimetric Methods 5.3 Titrimetric Methods 5.4 Spectroscopic Methods 5.5 Chromatographic Methods 5.6 Immunoassays Metabolism Biopharmaceutics and Pharmacokinetics Toxicity References

Analytical Profilcs of Drug Substances Volume I I

1

2 2 2 2 3 3 3 9 9 9 9 10 10 11 11 13 13 26 31 31 33 34

Copyright 01982 by The American Pharmaceutical Association ISBN 0-12-260811-9

KAILAS D. THAKKER AND LEE T. GRADY

2

1.

Description 1.1

Nomenclature 1.11

Chemical Name

Aminophylline is chemically known as 1Hpurine-2,6-dione, 3,7-dih dro-l,3-dimethyl-, compound wTth 1,2-ethanediamine (2:l).

P

1.12

Adopted Names

Aminophylline was also known as theophylline ethylenediamine and euphylline. 1 1.13

Trade Names

Aminophylline is known as carena; inophylline; metaphylline; theophyldine; aminocardol; ammophylline; cardiocilina; cardophyllin; phylcardin; tefamin; cardiomin; grifomin; minaphil; peterphylline; stenovasan; the drox; diophylline; genophylline; phyllindon and theolamine.

P

1.2

Formula, Molecular Weight, and Composition:

11

Anhydrous:

C16H24N1004

420.44

Dihydrate:

C16H28N1006

456.44

Theophylline Ethylenediamine 1.3

85-87% 12-15%

Appearance, Color and Odor

Aminophylline is available in the anhydrous form or as the dihydrate. The dihydrate occurs as white or slightly yellowish granules or powd r. It has a faint ammoniacal odor and a bitter taste. f

AMINOPHYLLINE

2.

3

Physical Properties 2.1

Spectral 2.11

Infrared

The infrared spectrum of aminophylline in mineral oil mull, obtained on a B ckman 4250 spectrophotometer, is shown in Figure l.5 It is generally consistent wi h the reported infrared spectrum of theophylline.5,4 The stretches for -NH2 in ethylenediamine and -NH in theophylline appear as a broad band (combined with the mineral oil signal) in the region of 3.0-4.0 P M . Other signals are in the same vicinity as those for theophylline. The fingerprint region beyond 8.5 IJM is distinctive and can be used for identification. 2.12

Ultraviolet

Spectral characteristics of aminophylline solutions in the ultraviolet region were reported by Andrade an Inacio.' Absorption max ma occurred at 243-5 nm = 1701 and at 273-5 [E:im= 5001 in pH 9.5 borate buffer. LE1cm

''

Figure 2 shows the ultraviolet spectrum of aminophylline in water obtained on a Beckman 5260 recording spectrophotometer. 2.13

Nuclear Magnetic Resonance 2.13.1

Proton NMR

An 80 MHz proton magnetic resonance spectrum of aminoph lline in d6-dimethyl sulfoxide, obtained on a Varian FT-80A,' containing tetramethylsilane as an internal reference, is shown in Figure 3 . It is jimilar to the reported (60 MHz) proton NMR of theophylline. The assignments, based on assignments of theophylline protons, are shown in Table I. 2.13.2

Carbon-13 NMR

The 20 MHz proton-noise decoupled spectrum of aminophylline in d6-dimethyl sulfoxide, obtained on a Varian FT-80A is shown in Figure 4.6 The assignments are shown in Table 11. These are based on assignments of dimethyluracil and 1-methylhypoxanthine.7

t P

WAVELE W T H UM

Fig. 1.

IR Spectrum of Aminophylline

0.8

0.7

a.6

y Q!5

U

z

4

m

g

*1

v)

m

a

0. :

0 .i

.1

240

260

280

300

320

WAVELENGTH (nms)

Fig. 2.

W Spectrum of Aminophylline

5

340

36

l

'

l

~

l

'

l

~

~~ l l ' t \ l

l

10

9

a

I

'

l

6

PPm

Fig. 3 .

H'

NMR of Aminophylline

5

r

l 4

~ 3

l

~ 2

l 1

'

l 0

'

l

~

Solvent I

Fig. 4.

I 3 C NMR of Aminophylline

I

8


Table I Proton Assignment -CH2-N-CH3-

Proton Position (see structure) 10,ll 1 3 7

N-C%N-H C-H

8

Chemical Shift (ppm) 2.75 3.23 3.42 5.67 7.71

aIntensity of signal is proportional to the concentration. This proton may be delocalized in the ring. Table I1 Carbon Assignment

Carbon Position (see structure)

N-CH3 N-CH3

Chemical Shift 29.7 27.5 109.6 148.5 142.7 151.4 155.6

c=c c=c

N-C-N -c=o N-C=O-N

-CH2 on ethylenediamine is buried in the solvent signal as shown in Figure 4. 2.2

Other Properties 2.21

Differential Scanning Calorimetry and Melting Point

The thermogram of aminophylline2 shows two endothermic transitions, one at 120" and another at 272°C. The first transition reflects the melting point of aminophylline; the second transition reflects the melting point of theophylline. Theophylline is known to sublime on melting. 2 2.22

Solubility Aminophylline is soluble in water

AMINOPHYLLINE

(1 g/5 ml) .l

9

It is insoluble in dehydrated alcohol and in

ether.8 3.

Methods of Preparation:

Aminophylline was first prepared by Gruter' by dissolving theophylline in aqueous solutions of ethylenediamine in stoichiometric proportions and evaporating in vacuum over sodium hydroxide. Alternate methods include treating anhydrous or h drated crystals of theophylline with ethylenediamine vapor," and treating a 3 M solution of theophylline in a weak organic base (pyridine, quinoline or M aqueous solution of ethylenea-picoline) with a 2 diamine.11 4.

Stabilitv-Degradation 4.1

Stability in Solution

Solutions of aminophylline become turbid on standing due to absorption of carbon dioxide, with subsequent precipitation of theophylline.' S 8 During the preparation of aminophylline injection, excess ethylenediamine is necessary to keep aminophylline from decomposing.12 4.2

Stabilitv in Solid State

Aminophylline crystals, in the presence of moisture, can absorb carbon dioxide fr m air and decompose into theophylline and ethylenediamine.8 This accounts for its characteristic odor. Mixtures containing aminophylline and ephedrine hydrochloride were found to be discolored13 due to an exchange reaction between the two drugs. The ethylenediamine in aminophylline is presumed to liberate ephedrine base which decomposes rapidly. The color change was accelerated by temperature and humidity. 13

.

Numerous reports are found in the literature on the stability of aminophylline in suppository bases,14-21 especially those containing fatty acids. Dissolution of suppositories made with cocoa butter base was markedly lower than those ma e with macrogol base after storage at 22" for up to a yearaP5 Other physical properties of cocoa butter suppositories such as melting point (Tm) and melting time have been known to increase within weeks of storage at 22"

10


.

and 30" as a result of decomposition16 Increase in the in vitro melting point (above 37" in some casf$) was correlated with poor rate of release of theophylline. Decomposition of aminophylline suppositories is presumed to be due to the formation of insoluble amides ethylenediamine and fatty acids of the suppository b base. etwe'' our decomposition products that have been isolated" were identified as mixturf6 of amides of oleic, palmitic, lauric and myristic acids. Further characterization of the decomposition products by IR GLC, and TLC confirmed the presence of alkyl amides. 20"" Stabili rs such as hydroxylamine hydrochloride have been useful. %f

5.

Methods of Analysis

5.1

Identification Tests

Spectral identification tests include IR, W,Mass and N M R spectroscopy. The melting point of theophylline liberated from aminophylline is the basis of one of the compendia1 identification tests. 22 The following color reactions are also useful identification tests; many are used for dosage forms and for mixtures of pharmaceuticals. (i) Ethylenediamine present in aminophylline reacts with sodium rhodizon85e to form a water-soluble, violet-colored precipitate. Ethylenediamine must be released from aminophylline and converted to acetate or hydrochloride. 0

+2

a+

0

Ethylenediamine also foryz a yellow (ii) precipitate with 2,4-dinitrochlorobenzene. Primary aliphatic and aromatic amines interfere with the test.

AMINOPHYLLINE

11

(iii) Aminophylline develops an orange color with ferric chloride and a yellow color with Ehrlich’s reagent. Color de elopment is rapid. This test was designed by Cooper” €or identification of pharmaceuticals in tablets. (iv) Reaction between dimethylglyoxime and oxidized solutions of purines to form a colored product is the basis of the identification test developed by Kido. 26 (V) Reaction of theophylline with potassium chlorate in hydrochloric acid, followed by exposure to ammonia vapors, produces a purple residue. This reaction is the basis of on of the compendia1 identification tests for aminophylline.2 5

(vi) Bratton-Marshall: Aminophylline in acid solution, when treated with diazotized p-aminobenzene-sulfonic acid or p-aminonitrobenzene produces a yellow or a red color, respectively.27 (vii) Addition of 1-2 drops of a 5% solution of sodium nitroprusside to a solutig of aminophylline in acetone produces a violet color. (viii) Aminophylline powder, on mixing and heatin with copper sulfate powder, also produces a violet color.99 5.2

Gravimetric Methods

Earlier methods of analysis of aminophylline were based on extraction of theophylline with organic solvents such as chloroform and 2-propg~;4~ followed by gravimetric determination of the residue. 5.3

Titrimetric Methods 5.31

Alkalimet ric

The weakly acidic nature of theophylline lends itself to titrations with alkali. Titrations with alkali have be59 carried out t o Jgsay aminophylline using a potentiometric or colorimetric end-point (thymolphthalein as indicator) in mixtures of pha rmaceutica1s.


12

5.32

Acidimetric

Ethylenediamine's basic nature was used in many early methods of analysi strong acid were carried out. ethylenediamin by solvent extraction31 or ion-exchange ~ h r o m a t o g r a p h 3was ~ followed by titrations with hydrochloric acid using a colorimetric end-point (methyl orange as indicator). 5.33

Argentometric

When theophylline is treated with solutions of silver nitrate, it forms a silver salt that is insoluble in water and soluble in nitric acid solutions. Basej6gtlthis property, several assay methods have been develop including the compendia1 assay for aminophylline. general, silver theophyllate is precipitated, filtered, washed, re-dissolved in nitric acid and titrated with ammonium thio cy Aminophylline resent in mixtures of pharmaceuticals'8:5p;39 and xanthines" can be analyzed. Some presence of amm nia is necessary when precipitating silver theophyllate." It has been suggested that argentometric titrations give higher values due to adsorption of silver ions by the very voluminous silver theophyllate precipitate. 31

In an unusual application, silver (lloAg) nitrate was used and radi metric titration was carried out to analyze aminophylline. 41 5.34

Complexometric

Bosly developed a titrimetric method for aminophylline by Precipitating Hg-(the~phylline)~with mercuric acetate, itrating excess H g H ions with ammonium thiocyana:zd4' Similarly, the Cu salt of theophylline is also precipitated excess copper titrated with ethylenediaminetetraacetate.4y94 5.35

Non-Aqueous

Non-aqueous titrations of aminophylline using sodium methoxide as the titrant have been carried out with dirnethylf~rmamide~~ or ben~ene-methanol~~ as the solvent for aminophylline. A differential titrimetric method for aminophylline was dexgloped using acetous perchloric acid as the titrant. This method allows

13

AMINOPHY LLINE

determination of ethylenediamine and theophylline content in a single titration, and is useful for aminophylline tablets, injections and suppositories. 5.4

Spectroscopic Methods

Aminophylline solutions obey the Beer-Lambert Law for a zqncentration range of 0.5-1.2 mg%.5 Schack and Waxler developed a potentiometric assay for aminophylline in biological fluids using chloroform/2-propano1 extraction to isolate theophylline, followed by W-absorbance measurement at 275 nm. The method of Schack and Waxler was used most extensively for analysis of aminophylline in early pharmacokinetics research.a Several other spectrophotometric methods have been developed for aminophylline in mixtures of pharmaceuticals, each one using t organic solvent to isolate an extraction aminophylline. ''*kg In one case, graphical correction was applied to the W absorbances of mixtures of aminophylline and phen barbital, measured at two wavelengths of maxima.58 In another application, extraction of serum samples with a salt-solvent pair of ammonium sulfate and ch1oroform:hexane (7:3 v/v) was carried out followed b back extraction of theophyllinea into aqueous borate buffer ( PH 9 . 0 ) an measurement of UV absorbance at 275 nm. 51 PlavsicgZ used charcoal extraction to isolate theophyl ine from other interfering substances. Since theophylline was reversibly adsorbed on charcoal, elution with organic solvent was followed by measurement of W absorbance. Determination of aminophylline in the blood of patients was carried out after gfidation with potassium dichromate in an acidic medium, separation of the oxidation product by steam distillation and measurement of W absorbance at 257 nm. 5.5

Chromatographic Methods 5.51

Thin-Layer Chromatography

In addition to systems developed for theo~ h y l l i n e ,several ~ TLC systems have been developed for "Aminophylline and theophylline are indistinguishable at biological pH's. Therefore assays of theophylline in biological fluids are also included here due to their obvious applicability.

Table I11 Thin-Layer Chromatographic Systems for Aminophylline

No.

Solvent for Drug

Stationary Phase

Developing Solvent

Detection Methods

Application

1.

chloroform

Silica gel F254

ch1oroform:acetic acid (100 :20)

W,Dragendorff ' s reagent

Analysis of suppositories

54

2.

water

aluminum oxide

benzene:ethanol (9:l or 9:1.5)

W, iodine, modified Dragendorff ' s reagent

Mixture of pharmaceuticals

55

Silica gel F254

(i) acetone:chloroform:l-butanol:25% ammonium hydroxide (30:30:40:10) (ii) ch1oroform:ethyl ether (9O:lO) (iii) ch1oroform:ethanol (9O:lO)

ferric chloride, iodine

Analysis of mixtures of xanthines

56

Silica gel H

ch1oroform:acetone: methanol (8:l:l)

lJv

Analysis of suppositories

18

4.

-----a

Ref.

Table 111 (contd.) No.

Solvent for Drug

St at ionary Phase

Developing Solvent

Detect ion Methods

Application

5.

-----a

Silica g e l

methylene chloride: methano1:acetic acid (90:10:3)

uv

Analysis of tablets ampuls, suppositories

F254 anot mentioned.

Ref.

57


16

analysis of aminophylline and its dosage forms. lists the methods developed for aminophylline.

Table 111

Zorka et alS8 separated and identified several pharmaceuticals in a mixture including aminophylline using silica gel G plates and neutral, acidic, and alkaline mobile phases. Detection was done by UV. Riechert5' developed a "micro"-TLC method for analysis of theophylline in biological fluids. Kieselgel 60 F-254 DL-"Fertigplatten" thin-layer plates were used and developed with ch1oroform:methanol (9O:lO) for a mixture of caffeine and theophylline and with ethyl acetate:methanol:25% ammonia (80:ZO:lO) for a mixture of theobromine and theophylline present in saliva, plasma or urine. Sensitivity of detection claimed was 25 ng/lO ~ 1 . None of the dietary xanthines or other commonly coadministered drugs appear to interfere. 5.52

PaDer ElectroDhoresis60

Whatman Paper No. 1 was used with a potential gradient of 20 V/cm. Several xanthines were separated. Among these, theophylline and aminophylline were best chromatographed in Britton-Robinson buffer at pH 10. Detection agent was 1% solution of disodium-2-hydroxy-3,6naphthalenedisulfonate. 5.53

Pressurized Liquid Chromatography

Numerous liquid chromatographic methods for determinations of theophy 1 ne n biological fluids are listed in the literature.4,f1-g' The maj ri of the 'l-" few are methods use reverse-phase chromatog p listed that use i er normal-phase p b z ' or ion-exchange chromatography. In many cases, chromatographic conditions h e e n developed for a specific application,QY,8B~'9 since adaptation of reported methods may introduce some inttgference from co-adm nistered drugs such as acetazo&mide, trisulfapyrimidinebl or cephalosporins.

'',"

li9

Table IV lists some of the recent, most cited methods of analysis of theophylline in biological fluids by liquid chromatography.

Table IV No.

Stationary phase

Mobile Phase

Pre-treatment of sample

Comments

Reverse-phase C-18 (ion-pair)

methano1:sodium acetate (pH 4 . 2 ) containing 10 mM tet rabutylammonTum chloride (10:90)

Extraction of plasma with chloroform:2-propanol

Separation of theophylline from paraxanthine.

2.

Reverse-phase C-18 (ion-pair)

acetonitri1e:water containing 10 mM tet rabutylammonium chloride (2.5:97.5)

Urine:

3.

Reverse-phase C-18

ethano1:water (20:80)

4.

Reverse-phase C-18 (p-Bondapak C-18)

acetonitrile:O.Ol M - sodium acetate (pH 4 . 0 ) (1:9); 2 . 0 ml/min flow

1.

(50:50)

pH adjusted before chromatography Serum: ultrafiltration

Ref. No. 61

Analysis of theo62 phylline in urine and serum; separation from other xanthines and metabolites. Assay of theophylline 6 3 in plasma; separation of theophylline from sulfisoxazole and ampic i11in.

Molecular ultrafiltration to remove plasma proteins

Direct injection.

64

Table IV (contd.)

No.

Stationary phase

5. Reverse-phase C-18

Mobile Phase


Comments

acetonitri1e:water ( 6 : 9 4 ) ; 3.0 ml/min flow

De-proteinization with 2.5 volumes of acetonitrile

Micromethod--only 10-1.11sample is required.

Ref. No. 65

6.

Reverse-phase C-18 (p-Bondapak C-18)

acetonitri1e:acetate buffer (pH 4 . 0 ) ( 7 : 9 3 )

De-proteinization with aqueous acetonitriles olution

Microscale method-66 only 3 0 p 1 plasma needed for analysis; direct injection.

7.

Reverse-phase C-18 u-Bondapak C-18

methanol:l% propionic acid (20:80)

Extraction with chloroform evaporation; sample redissolved in methanol

50-1.1 1 sample needed.

8. Reverse-phase C-18 (p-Bondapak C-18)

methano1:tetrahydrofuran:water containing 10 d/liter sodium acetate

Same as Ref. 62

p-Hydroxyethyltheo-

Q)

(40:10:50)

67

68 phylline as internal standard; separation from other xanthines and commonly administered antibiotics.

Table IV (contd.) Stationary phase

Mobile Phase


Comments

9.

Reverse-phase

methano1:sodium acetate (15:85)

Deproteinization of serum samples with two volumes of methanol

B-Hydroxyethyltheophylline used as internal standard; no interference from antibiotics or metabolites.

69

10.

Reverse-phase

methano1:amonium phosphate

Extraction with organic solvent before analysis

100 u1 of serum sample can be analyzed; theophylline can be analyzed in presence of anticonvulsants.

70

11.

R verse-phase C-18

acet nitrile : acetate buffer (pH 4.0) (9O:lO)

Extraction with chloroform:2-propanol (95:5)

8-Hydroxyethyltheo71 phylline used as internal standard; theophylline recovery was found to be between 71 and 75%.

No.

Ref. NO

Table IV (contd.)

No.

Stationary phase

Mobile Phase


Comments

Ref.

NO


acetonitrile: acetate buffer (pH 4 . 0 ) (12:88)

Extraction of 0.5-0.2 ml of acidified plasma with dichloromethane


methanol:0.05 M phosphate buffer (pH 4 . 7 ) (12:88 v/v)

Molecular filtration of Method applicable serum to separate proteins to human serum, urine and saliva samples, also separation and quantitation of theophylline and its metabolites.

73

2 0 ~1 of blood are

74

h)

0

14.

Reverse-phase

Separation of xanthines; sensitivity of detection 0.1 mg/ liter

sufficient for analysis; detection by direct-curren t pulse and differential pulse amperometry.

72

Table IV (contd.) No.

Stationary phase

Mobile Phase


Comments

15.

Reverse-phase C-8

methano1:buffer (14:86 v/v)

Extraction with 2-propanol:chloroform (25 :75)

Completely automat75 ed analysis. Developed on Technicon "Fast-LC" 200-p 1 sample required. No interference was observed.

sodium acetate: 0.02 M methanol (2:l)-

Adjustment of pH to 5.5

Theophylline, sulfamethoxazole, ampicillin and caffeine well separated.

76

acetonitrile: phosphate buffer (pH 4.8) (1:l); 3.0 ml/min flow; 50"

Same as Ref. 85

Authors found that the retention time of internal standard varied from run t o run when using the conditions described in Ref. #5; column temp. was therefore maintained at 50°C.

77

16.

Reverse-phase RP-8


Ref. No.

.

Table IV (contd.)

No.

Stationary phase

Mobile Phase


Comments

18.

Reverse-phase C-18 Whatman Partisi1 10-ODS

acetonitrile: 10 mM phosphate buffer (10:90)

Protein denaturation by acetonitrile

No pre-column necessary; no interference observed.

78

19.

Reverse-phase C-18 p-Bondapak

methano1:pH 2.0 Precipitation of proteins by 50% v/v trichloroacehydrochloric acid solution (containing tic acid 0.02 M potassium chloride)

Theophylline analyzed in presence of methyl xanthines and caffeine.

79

20 *

Reverse-phase C-18

acetonitri1e:acetate buffer (8:92)

Theophylline well Plasma samples extracted with chloroform:2-propanol separated from paracetamol and (95:s); solvent removed and xanthines. and samples re-dissolved dissolved in mobile phase

21.

Reverse-phase C-18 5 p

Solvent A: water containing .01 M sodium acetate and 0.005 M tet rabu ty1ammonium hydrogen sulfate.

Ion-pair extraction using tetrabutylammonium sulfate and ethyl acetate: chloroform:2-propanol, (45:45:10 v/v) after adjustment of urine pH to 6.0-6.5

N

N

Simultaneous quantitation of theophylline and its major metabolites.

Ref. No.

80

81

Table IV (contd.)

No.

Stationary phase

21. contd.

g

22.

Reverse phase p-Bondapak C-18

Mobile Phase


Comments

Proteins precipitated by perchloric acid, supernatant neutralized and injected

50 111 of serum are

Ref. No.

Solvent B: ( 5 0 : 5 0 ) methano1:solvent A gradient elution with 9% solvent B at start, 46% solvent B at end of run (for program see Ref. 81). methanol:10 mM monobasic sodium phosphate (1:4); 0.8 ml/min flow

sufficient; theophylline is well separated from dietary xanthines, caffeine, theobromine and theophylline metabolites.

82

Table IV (contd.)

*

K 3 .

No.

Stationary phase

Mobile Phase


Comments

23.

Normal phase column (silica)

ch1orform:dioxane: formic acid (95.5:4.5:0.01 v/v)

Equal volume of saturated ammonium sulfate is added to plasma, and then extracted with chlorof orm:2-propanol (95 :5 v/v); solvent is evaporated and residue redissolved in mobile phase

100 u 1 of plasma 83 are sufficient; theophylline well separated from the metabolites.

24.

Normal phase Lichrosorb Si-60

(i) chloroform: Extraction from plasma 2-propanol: with chloroform 2glacial acetic acid propanol (95:5) (92:7:1) with 40% hexane (ii) Ethylene chloride: methanolic ammonium formate (98:2)

25.

Cation-exchange 0.66% acetic acid Partis i1 SCx column temp. at 5OoC

0.1-ml sample extracted with ethyl acetate

Ref.

NO

Mass spectrometry used for identification.

84

Applicable to plasma or saliva samples; no interference.

85

Table IV (contd.)

No.

26.

Stationary phase

Mobile Phase

Strongly basic anion exchange resin (35% crosslinkage)

acetate buffer; linear gradient from 0-6.0 M, flow 0.72 a / m i n


Comments

Ref.

NO Sample filtered to remove particulate matter

General method for W-absorbing compounds in urine; 1 30 compounds tested.

86


26

5.54

Gas Chromatography

Earlier attempts to analyze blood levels of theophylline resulted in evelopment of various gas chromatographic methods. g5h_f0' Most of these methods require extraction and derivatization before chromatography. Table V lists some of the more recent methods. 5.6

Immunoassays 5.61

Enzyme Immunoassay (EMIT)

The enzyme immunoassay (EMIT) developed by Syva Corporation (Palo Alto, California) is the most widely used method for the assay of the0 lline (in biological fluids) in clinical laboratories.

nSs

In principle, theophylline antibodies are preparedlo4 by injecting a solution of bovine gamma globulin linked to theophylline ( o r a chemically similar derivative) into sheep. Theophylline (or a derivative) is also linked to an enzyme, in this case, glucose-6-phosphate dehydrogenase. When a patient's serum containing free theophylline is mixed with a solution of antibodies and enzyme-labelled theophylline, the free drug and the enzymelabelled drug compete for the binding sites on antibodies. The reduction in enzyme activity when bound to antibodies can be monitored by using the proper substrate; in this case NADH. This assa s rapid, specific, and requires small s easily adaptable to ommercial sample size. kinetic analyzers, and can be modified"' to suit the application. Comparison of the EMIT assay with -_ chromat~~t;;~~#c methods shows good agreement between the two assays. Vinet et all2' developed another enzyme immunoassay. In this method, the sample is extracted with chloroform/2-propano1, and back-extracted into aqueous sodium hydroxide. Inhibition of beef liver phosphatase by theophylline is determined at 25" C, using p-nitrophenyl acetate as the substrate in a pH 9.4 2-amino-2-methyl-lpropanol buffer system.

In another application nephelometric, competitive immunoassay was developed. '21 A precipitate was obtained by combining theophylline-antibody complex with a macromolecule, and scattering of light by the precipitate

Table V No.

Stationary phase

1.

3% OV-17 on Gas Chrom Q

Conditions

Column at 230°C; nitrogen-phosphorus detector


Comments

Sample extracted with chloroform:2-propanol ( 5 0 : 5 0 ) , evaporation and redissolution of residue in 0.02 M tetrabutylammonium-h yd ro xid e

20 p 1 of plasma are

2.

F.I.D. Silicone stationary phase, 2% SP 2510 DA

Extraction of sample with salt-solvent pair of ammonium sulfate and methylene chloride: hexane:acetic acid (80:20:0.1)

3.

5% OV-225 on 80/100 Gas Chrom Q

Sample extracted with ethyl acetate, and then treated with pentafluorobenzyl bromide for derivatization

Electron-capture detector; column at 250°C

Ref.

NO

93

sufficient; detection sensitivity 100 pmol/liter; theophylline well separated from other xanthines and co-administered drugs. 94

100 p 1 of serum are

sufficient; detection sensitivity 0.1 ug/ml.

95

Table V (contd.) No.

Stationary phase

Conditions

4.

3% XE-60 on

80/100 Gas Chrom Q

5.

3% OV-17 on

100/120 Gas Chrom Q 6.

3% OV-17 on 100/120 Gas Chrom Q


Comments

Ref. No.

Electron-capture detector; injector and column at 220"C, detector at 225°C

Derivatization by pentafluorobenzyl bromide followed by column chromatography prior to injection

Sensitivity of detection 5 ng/ml.

Nitrogen detector; column at 240°C

Extraction with tetrahexyl- 25-pl sample is ammonium hydrogen sulfate sufficient. in aqueous sodium hydroxide

97

Nitrogen-phosphorus detector; column at 240°C

Off-column derivatization No interference as follows: Sample is ex- from drugs or tracted with dichlorometabolites. methane, dried and reacted with -N,N-dimethylacetamide, tetramethylammonium hydroxide and 1iodopentane. It is then transferred into cyclohexane: pentane mixture (95: S), the solvent evaporated, and it is redissolved in methanol.

98

96

Table V No.

Stationary phase

Conditions

7.

3% OV-17 on Gas Chrom Q

Flame-ionization detector; column at 190°C

Extraction of sample with ether:dichloromethane: 2-propanol ( 6 : 4 : 1 ) , reextraction of the organic layer with 1 N sodium hydroxide, acidification with phosphoric acid, re-extraction with organic solvent, evaporation and redissolution of residue in tetrapropylammonium hydroxide

99

8.

3% SP 2250 on 100/120 Supelcoport

Flame-ionization detector; column temperature programmed from 160°C to 240°C at 8" C/min

Sample extracted with dichloromethane, evaporated, and dissolved in toluene, butylating agent is then added.

100

h)

\o


Comments

Ref. No.

Table V (contd.) No.

Stationary phase

Conditions


Comments

Ref. No.

9.

3% OV-17 on 100/120 Chrom W HP column well conditioned

Column temperature programmed from 180°C to 280°C; detector at 280"C, mass spectral source at 150°C

Extraction with chloroform, butylation with tetramethylammonium hydroxide and -N,N-dimethylacetamide

Mass spectrometry using probabilitybased matching.

101

10.

3% SP2250 on 100/120 Supelcoport type 50-50 methylphenyl silicone

Column temperature programmed at 190°C to 3OO0C at 10"/min

Samples are introduced in a flash heater for ethylation

No interference.

102

AMINOPHYLLINE

31

was used to quantitate theophylline content. 5.62

Radioimmunoassay (RIA)

The principle of radioimmunoassay is similar to EMIT, except that in this case decrease in radioactivity is measured. Ra ioimmunoassays f r th ophylline have been developed using 'H-theophylline. lY2 ,123 8-Carboxytheophylline was used to prepare antibodies. There is no interference from endogenous purines or known metabolites of theophylline at the concentrations studied. 122 6.

Metabolism

UsinglJZ4C] aminophylline injection, Caldwell, Monks and Smith determined that the metabolites of aminophylline are the same as those of theophylline. 125,126 These are (i) 3-methylxanthine (ii) 1,3-dimethyluric acid and (iii) 1-methyluric acid. However, the rate and extent of conversion to 1,3-dimethyluric acid and 3-methylxanthine were higher or aminophylline than for theophylline.126 Therefore, "C recovery in urine (0-24 hours) was higher for aminophylline (87%) than for theophylline ( 7 6 % ) . The formation of 3-methylxanthine follows saturation kinetics; therefore, the presence of circulating methylxan ines from The foods affects the elimination of aminophylline. appearance of the other wo metabolites follows first order kinetics. Jenne et all2' determined that 1-demethylation of theophylline to 3-methylxanthine is the dominant reaction determining theophylline concentration in serum. Presence of ethylenediaminelyyst affect this conversion, but how it does is not known.

'"

7.

Biopharmaceutics and Pharmacokinetics

Pharmacokinetics of aminophylline has been studied extensively. Since aminophylline and theophylline are indistinguishable in biological fluids, pharmacokineticists d o not differentiate between the two. lthough the pharmacokinetics of theophylline was reported' earlier, the advent of newer analytical techniques has since led to an extensive amount of work. Aminophyl line is administered orally 129-133 as a 1329133,134,135 Or as a singlesustained-re1 dose capsulg- ordosage tablet, fo 15',130 intravenously (or by infusion) ntramuscularly, or rectally as suppositories or enema. 1389131 Among different aminophylline dosage

lwe

"'

32


forms, rect 1 positories give the widest variation.lf8,fYf Absorption of aminophylline from tablets or capsules is rapid, plasma levels reaching therapeutic range within 1-1 l/2 hours. Sustained-release preparations of aminophylline are used often to maintain theophylline levels within for about 12 hours in treatment of as theraPey51'15'~f39 thma The rectal route is used often with infants and children.

.

Plasma theophylline levels of 5-15 pg/ml (after administrati of aminophylline) are considered safe and toxic symptoms do O'' Although in most ca therapeutic. not appear at levels above 25 ug/ml,Bz8 the patient-tothat individualization of the patient variation is therapy is necessary. In some case intra-patient variation in doselblood levels is observedfh6 during milk levels continuous long-term therapy. Saliva and bre of theophylline are lower than plasma levels, but1 &75PZ$ correlation between plasma/saliva ratio is obtained.

fglhi@

ft5

.'

The pharmacokinetic behavior of aminophylline once it appears in the blood can be described by the same ode1 that The is used to describe theophylline pharmacokinetics volume of distri u i n at steady-state for all ages is 0.45 literlkg'158-E5P except in neonates where it is slightly larger.152 Elimination is rapid, with a half-life of about 6 hours in normal, healthy, non-smoking adults. Among the factors that affect the ation of theophylline fr sma are age,1f'i'P'9 physi lo and 163-18 diet ,165 co-administer disease stat 1'B-p89 time of day,fb6 and smoking habits.fg7di'8s'In a case when any of these factors is operating, the half-life of elimination varies from 5 to 30 hours. Very recently, Monks et have compared the disposition and elimination of theophylline and aminophylline. Elimination of aminophylline was faster than theophylline in the same subjects. Although qualitatively the disposition (and metabolism) of theophylline and aminophylline were similar, the authors claimed there were small but significant differences in rates of elimination and the extent of dose eliminated within 48 hours.

AMINOPHYLLINE

8.

Toxicitv

Toxicity of aminophylline results when bl d levels exceed the level of 20 pg/ml of theophylline.lY8 The severity of toxicity is directly related to plasma levels of theophylli In mild toxicity, symptoms are nausea and vomiting ,1981171 diarrhea, abdominal pain, nervousness, insomnia, tachycardia and headache. serious tachycardia, grand ma1 seizures, cardiac arrythmias may occur In some cases, death may result from acute toxicity. ij4

33


34

9.

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AMINOPHYLLINE

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40


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107.

J. Y. Chang and R. J. B a s t i a n i , C l i n i c a l Study No. 41, Summary r e p o r t , S p a C o r p o r a t i o n , P a l o A l t o , CA (1977).

108.

V. Henry, J. Deutsch, and G. Lum, C l i n . Chem.,

514 (1978).

24 ( 3 ) , --

109.

A. C a s t r o , J. I b a n e z , W. V o ig h t, T. Noto, and H. Malkus, C l i n . Chem., -24 ( 6 ) , 944 ( 1978) .

110.

D. N. D i e t z l e r , N. Weidner, V. L. T i e b e r , J. M. McDonald, C. H. Smith, J. H. Ladenson, and M. P. L a c k i e , C l i n . Chim. Acta, -1 0 1 (2 - 3) , 163 ( 1980) .

111.

N. Weidner, J. M. McDonald, V. L. T i e b e r , C. H. Smith, G. Kessler, J. H. Ladenson, and D. N. D i e t z l e r , C l i n . Chim. Acta, -97 ( l ) , 9 (1979).

112.

M. O e l l e r i c h , G. W.

113.

N. U r quh art, W. Godolphin, and D. J . Campbell, C l i n .

S y b r e c h t , and R. Haeckel, J. C l i n . 17 ( 5 ) , 299 (1979). Chem. C l i n . Biochem., --

Chem., -25 ( 5 ) , 785 (1979). 114.

H. M. He ick , A. Mohammad, and C. Golas, C l i n . Biochem., -12 ( 2 ) , 68 (1979).

AMINOPHYLLINE

115.

D. A. L a c h e r , J . A. S i n n , J. S a v o r y , and M. R. Wills, Ann. C l i n . Lab. S c i . , -10 (4), 305 (1980).

24 (3), 520 116. H. S t o n e and B. G i l l i l a n , C l i n . Chem., -(1978). 24 (2), 391 (1978). 117. J. P. Long, C l i n . Chem., -118. F. D. Lasky, J. A 1 R a z i , and A. Kramen, C l i n . Chem., 24 (8), 1381 (1978). -119. R. L. Boeckx, E. M. F r i t h , and F. E. Simons, "her. Drug Monit., _1 (l), 65 (1979). 120. B. V i n e t and L. Z i z i a n , C l i n . Chem., -25 (8), 1370 (1979). 121. T. Nishikawa, H. Kubo, and M. S a i t o , C l i n . Chim. Acta, 91 (l), 59 (1979). -122. A. L. Neese and L. F. Soyka, C l i n . Pharmacol. T h e r a p . , 21 (5), 633 (1977). 123. C. E . Cook, M. E. Twine, M. Myers, E. Amerson, J. A. K e p l e r , and G. F. T a y l o r , Res. Commun. Chem. P a t h . Pharm., - 13, 497 (1976).

124. J. C a l d w e l l , T. J. Monks, and R . L. S m i t h , B r . J . Pharmacol., 63, 369p (1978). 125. H. H. C o r n i s h and A. A . C h r i s t m a n , J. B i o l . Chem., 228, 315 (1957). 126. J. W. J e n n e , T. W. C h i c k , B. A. Miller, and R. D. S t r i c k l a n d , Am. J. Hosp. Pharm., 34, 408 (1977). 127. T. J . Monks, R. L. S m i t h , and J. C a l d w e l l , J. Pharm. Pharmacol., 33, 93 (1981). 128. J. W. J e n n e , H. T. Nagasawa, and R. D. Thompson, C l i n . Pharm. and Therap., -19 (3), 375 (1976). 129. L. H e n d e l e s , M. Weinberger, and L. B i g h l e y , Am. J. Hosp. Pharm., 34, 525 (1977). 130. M. L. S l o t f e l d t , C. E. Johnson, G. Grambau, and J. G. Weg, Am. J. Hosp. Pharm., -36 (l), 66 (1979).

41


42

131.

H. Lamont, E. Moermann, M. B o g a r t , M. Van Der S t r a e t e n , and R . Pauwels, Eur. J. C l i n . Pharmacol., 1 5 ( 6 ) , 401 (1979). --

132.

G. E. M a r l i n , M. A. B u t c h e r , J. A. Klumpp, and P. J. Thompson, B r . J. C l i n . Pharmacol., -10 (3), 265 (1980).

133.

P. W. Trembath and S. W. Boobis, C l i n . Pharmacol. Ther., 26 ( 5 ) , 654 (1979). - --

134.

S. McKenzie and E. B a i l l i e , J. I n t . Med. Res., Suppl. 1, 22 (1979).

135.

7 -

M. C. Meyer, A. B. S t r a u g h n , and P. Lieberman, C h e s t ,

78 ( 2 ) , 300 (1980). -136.

L. J. Lesko, A. T. Canada, G. Eastwood, D. Walker, and D. R. Brousseau, J. Pharm. S c i . , -6 8 ( l l ) , 1392 (1979).

137.

M. W. Weinberger, R. A. Matthay, E. J. Ginchansky, C. A. Chidsey, and T. L. P e t t y , J. Am. Med. Assoc., 235, 2110 (1978).

138.

E. B. T r u i t t , J r . , V. A. McKusick, and J. C. K r a n t z , J r . , J. Pharmacol. Exp. Therap., 1 6 0 , 309 (1950).

139.

J. Ahrens, Dtsch. Med. Wochenschr.,

(1977).

102 (13), 482 --

140.

M. W. Weinberger and E. A. Bronsky, J. P e d i a t r . , 421 (1974).

141.

E. Ginchansky and M. Weinberger, J . P e d i a t r . , -91 (4), 655 (1977).

142.

P. R a n g s i t h i e n c h a i and R. W. Newcomb, J. P e d i a t r . , 91 ( 2 ) , 325 (1977). --

143.

F. Nielsen-Kudsk, I. Magnussen, T. S . J e n s e n , and K. Naeser, Acta Pharmacol. T o x i c o l . , -46 ( 3 ) , 205 (1980).

144.

F. E . Simons, K. J. Simons, G. G. S h a p i r o , W. E . P i e r s o n , and C. W. Bierman, J. Med., 9 ( l ) , 81 (1976).

84,

43

AMINOPHYLLINE

145. M. H. J a c o b s , R. M. S e n i o r , and G. Kessler, J. Med. Assoc., 235, 1983 (1976).

Am.

J. 146. P. D. Watson, R. C. S t r u n k , and L. M. T a u s s i g , P e d i a t r . , -91 (2), 321 (1977). J. 147. N. N. Khanna, H. S. Bada, and S. M. Somani, Pediatr., 96, 494 (1980).

148. S. P. G a l a n t , S. A. Gillman, L. H. Cummins, P. P. Kozak, and J. J. O r c u t t , Am. J. D i s . C h i l d . , -131 (9), 970 (1977). 149. R. Koysooko, E . F. E l l i s , and G. Levy, C l i n . Pharmacol. Therap., -15 (5), 454 (1974). 150. P. A. Mitenko and R. I. O g i l v i e , C l i n . Pharmacol. Therap., 14, 509 (1973). 151. L. H e n d e l e s , M. Weinberger, and L. B i g h l e y , Am. Rev. R e s p i r . D i s . , 118, 97 (1978). 152. 3. V. Aranda, D. S. S i t a r , W. E. P a r s o n s , P. M. Loughnan, and A. H. N e i m s , N. Engl. J. Med., 295, 413 (1976). 153. G. G i a c o i a , W. J. J u s k o , J. Menke, and J. R. Koup, J. Pediatr., 89, 829 (1976). 154. J. P. Rosen, M. D a n i s h , M. C. Ragni. C. L. S a c c a r , S. -~ J. Y a f f e , and H. I. Lecks, P e d i a t r i c s , 64, 248 (1979). 155. F. E. R. Simons and K. J. Simons, J. C l i n . Pharmacol., 18, 472 (1978). 156. P. M. Loughnan, D. S. S i t a r , R. I. O g i l v i e , A. E i s e n , 2. Fox, and A. H. N e i m s , J. P e d i a t r . , 88, 874 (1976). 58, 157. E. F. E l l i s , R. Koysooko, and G. Levy, P e d i a t r i c s , 542 (1976). 158. K. M. P i a f s k y , D. S. S i t a r , R. E. Rangno, and R. I. O g i l v i e , N. Engl. J. Med., 296, 1495 (1977). 159. K. M. P i a f s k y , D. S. S i t a r , R. E. Rangno, and R. I. O g i l v i e , C l i n . Pharmacol. Therap., 21, 310 (1977).

44


160.

A. Mangione, T. E. I m h o f f , R. V. L e e , L. Y. Shum, and W. J u s k o , C h es t , 73, 616 (1978).

161.

N. Vicuna, J. L. McNay, T. M. Ludden, and H. S c h w e r t n e r , B r . J. C l i n . Pharamcol., 7, 33 (1979).

162.

K. C. Chang, T. D. B e l l , B. A. L a u e r , and H. C h a i , L n c e t , 1, 1132 (1978). -a -

163.

R. A. Landay, M. A. G o n z a l e z , and J. C. T a y l o r , J. A l l e r g y C l i n . Immunol., 62, 27 (1978).

164.

M. Weinberger, D. Hudgel, S. S p e c t o r , and C. C h i d s e y , J. A l l e r g y C l i n . Immunol., 59, 228 (1977).

165.

A. Kappas, K. E. Anderson, A. H. Conney, and A. P. Alvares, C l i n . Pharmacol. Ther., 23, 445 (1978).

166.

L. J. Lesko, D. B r o u s s e a u , A. T. Canada, and G . Eastwood, J. Pharm. S c i . , -69 (3), 358 (1980).

167.

J. J e n n e , M. Nagasawa, R. McMugh, F. McDonald, and E . Wyse, L i f e S c i . , 17, 195 (1975).

168.

J. R. P o w e l l , J. T h i e r c e l i n , S. Vozeh, L. Sansom, and S. Reigelman, Am. Rev. Resp. D i s . , 116, 17 (1977).

169.

W. J. J u s k o , J. J. S c h e n t a g , J. H. C l a r k , M. G a r d e n e r a n d A. M. Yurchak, C l i n . Pharmacol. T h e r a p . ,

406 (1978). 170.

24,

L. H e n d e l e s , L. B i g h l e y , R. H. R i c h a r d s o n , C. D. H e p l e r , a n d J. C a r m i c h a e l , Drug I n t e l l . C l i n . Pharm., 11, 12 (1977).

171.

T. R. Kordash, R. G. van D e l l e n , and J. T. M c C a l l , J. Am. Med. Assoc., 238, 139 (1977).

172.

L. W. Z w i l l i c h , F. D. S u t t o n , J r . , T. A. N e f f , W. M. Cohn, R. A. Matthay, and M. W. W e i n b e r g e r , Ann. I n t e r n . Med., 82, 784 (1975).

173.

M.

174.

C. L. Winek, J. D. B r i c k e r , W. D. Collom, and F. W. Fochtman, F o r e n s i c S c i . I n t . , 15 (3), 233 (1980).

S. Schwartz and D. F. S c o t t , E p i l e p s i a , 15, 501 (1974).

ASCORBIC ACID Ibrahim A . Al-Mesh1 and Mahmoud M . A. Hassan

1.

2.

3.

4. 5. 6. 7. 8. 9.

10.

Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight I .4 Elemental Composition 1.5 Appearance, Color, Odor, and Taste Physical Properties 2.1 Crystal Properties 2.2 Solubility 2.3 Specific Rotation 2.4 Dissociation Constant 2.5 Identification 2.6 Spectral Properties Preparation 3.1 Isolation 3.2 Synthesis Biosynthesis of Ascorbic Acid Metabolism Daily Requirement Mode of Action Vitamin Deficiency Methods of Analysis 9.1 Titrimetric Methods 9.2 Spectrophotometric Methods 9.3 Turbidimetric Method 9.4 Chromatographic Methods 9.5 Enzymatic Method 9.6 Polarographic Method 9.7 Chronometric Method References

Analytical Profiles of Drug Substances Volume II

46

46 46 47 47 47 47 47 48

48 49 49 51 61 61 63 63 66 66 66 67 67 67 70 73 73 75 15 75 76

Copyrighl 0 1982 by The Amenan

45

phrrm.ceuUcd Awduion ISBN 0-12-260811-9

IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

46

1,

Description 1.1. Nomenclature

1.1.1

Chemical Names

a) b) c) d) 1.1.2

L-Ascorbic a c i d L-Xyloascorbic a c i d 3-0x0-L-gluofuranolactone (enol form). L-3-Ketothreohexuronic a c i d l a c t o n e

Generic Names Vitamin C ; A s c o r b i c a c i d

1.1.3

Trade Names Adenex; A l l e r c o r b ; A n t i s c o r b u t i c Vitamin; Ascorbicap; Ascorbajen; A s c o r i l ; A sc o ri n ; A s c o r t e a l ; A s c o r v i t ; Cantan; C a n t a x i n ; Catav i n C ; Ce b i c u r e ; Cebid; Cebione; Cecon; Cegiol an ; Cell i n ; Cenetone; Cereon; Cergona; Ce s c o r b a t ; Cetamid; Cetan; Cetemican; Ceval i n ; Ce v a t i n e ; Cevex; Cevimin; Cevi-Bid; Ce-Vi-Sol; Cevitamin; C e v i t a n ; Cimin; C e v i t a mic Acid; C e v i t e x ; Ciamin; C i p c a ; C o l a s c o r ; Concemin; C-vimin; Davitamon C ; E r i v i t C ; Hybrin; L a r o s c o r b i n e ; Lemascorb; Megascorb; P l a n a v i t C ; Pr o s c o r b i n C ; Redoxon; Ribena; Sc o r b a c i d ; Scorbu-C; T e s t a s c o r b i c ; V i c e l a t ; V i t a c e ; V i t a c i mi n ; V i t a c i n ; V i t a s c o r b o l ; Vitix.

1.2

Formulae 1.2.1

Empirical ‘gH8’6

1.2.2

Structural

CtI*OtI

I

HR=o

HOCH

HO

OH

41

ASCORBIC ACID

1.2.3

CAS No.

(50-81-7) 1.2.4

(Wiswesser Line Notation TOSV EHJ CQ DQ EYQ IQL (1)

1.2.5

Stereochemistry The nature o f the ring system in ascorbic acid was determined by a study of the methylated derivatives of the acid. By this means complete confirmation was obtained of the accuracy o f the views advanced, concerning the stereochemical configuration of the molecule and the nature of the reactive enolic groups(2). The furanose structure for ascorbic acid shown above (1.2.2) was put forward by Lerbert el a1 ( 2 ) on the basis o f its chemical behaviour as well as its oxidation products.

1.3

Molecular Weight 176.12

1.4

Elemental Composition C, 40.91%; H, 4.58%; 0 , 54.51%.

1.5

Qpearance, Color, Odor and Taste White or slightly yellow crystals or powder. Odorless or almost odorless; pleasant sharp acidic taste(3).

2.


Crystal Properties Usually plates, sometimes needles, monoclinic system (4). 2.1.1

X-ray Diffraction The available data (2) of the X-ray, reveals that the total of 12 carbon and oxygen atoms

48


all but one can be accomodated in one plane without appreciable valency strain whilst the remaining carbon (C5) lies less than 1A" above the plane as shown in the following model :

2.1.2

Melting Range Ascorbic acid melts at 190-192" with decomposition ( 4 ) .

2.2

So lubi1ity

Ascorbic acid is soluble at 20", in 3.5 parts of water and 25 parts of alcohol (95 per cent); 50 parts of absolute alcohol, 100 ml of glycerol, 20 ml of propylene glycol. Solubility in hot water 40.0% at 40°, 80% at 100". Insoluble in ether, chloroform, benzene and light petroleum (boiling range 40-60"). 2.3

Specific Rotation [a] [a]

['I

I'[

is+ i3+

20.5" to 21.5" (C = 1, water) 48 (C = 1, methanol) (4).

l9 + 24 (water) (2). 5780 l8 + 116 (sodium salt in neutral solution) ( 4 ) 5780 l8 + 130 (N/20 NaoH) ( 2 ) . 5780 l8 5780 + 149 (N/l NaoH)

(2).

l8 + 155 (N/2 NaoH) 5780 l8 + 161 (2N NaoH) 5780

(2) (2)

49

ASCORBIC ACID

2.4

Dissociation Constant Ascorbic acid is a moderately strong organic acid, two ionization constants: pK1 4 . 1 7 and pK 11.57. pH = 3 (5mg/ml), pH = 2 2 (50 mg/ml) (4).

2.5

Identification i)

Solution of ascorbic acid decolorises, 2,6dicholorophenol-indophenol solution (5).

ii)

Solution and ascorbic acid reduces silver nitrate solution immediately in the cold producing a black precipitate (5).

iii)

Dissolve 0.1 g of ascorbic acid in sufficient

w a t e r to produce 100 ml and dilute 1 ml to 100 ml with 0.01M h y d r o c h l o r i c a c i d . The

light absorption of the resulting solution exhibits a maximum only at 244 nm; A (1 per cent, 1 cm) at 244 nm, about 560 (6).

iv)

To 2 ml of a 5 per cent w/v solution add 0.5 g of s o d i u m h y d r o g e n c a r b o n a t e ; carbon

dioxide is evolved ( 6 ) .

v)

To 1 ml of a 5 per cent w/v solution add about 0.2 ml of ZM n i t r i c a c i d and 0.2 ml of 0.1M silver n i t r a t e ; a grey precipitate is

produced (6).

vi)

To 5 ml of a 1 per cent w/v solution add 0.05 ml of a freshly-prepared 5 per cent w/v solution of s o d i u m n i t r o p r u s s i d e and 2 ml of 2M s o d i u m h y d r o x i d e followed by 0.6 to 0.7 ml of h y d r o c h l o r i c a c i d , added dropwise with stirring; the yellow color turns blue (6).

vii)

Specific optical rotation, in a 10 per cent w/v solution, +20.5' to +21.5O (6).

viii)

A

solution (1 in 50) reduces alkaline cupric tartrate TS slowly at room temperature but more readily upon heating ( 7 ) .


50

ix)

To 2 ml of a solution (1 in 50) add 4 drops of methylene blue TS, and warm to 40": the

deep blue color becomes appreciably lighter or is completely discharged within 3 minutes (7).

XI

Dissolve 15 mg in 15 ml of a solution of trichloroacetic acid (1 in 201, add about 200 mg of activated charcoal, shake the mixture vigorously for 1 minute, and filter through a small fluted filter, returning the filtrate, if necessary, until clear. To 5 ml of the filtrate add 1 drop of pyrrole, and agitate gently until dissolved, then heat in a bath at 50": a blue color develops ( 7 ) .

xi)

For the examination of vegetable infusions

for the presence of vitamin C, the ascending

- descending paper-chromatographic method of

Block was used on the dinitrosazones. Many combinations of solvent were used, but a mixture of xylene and nitrobenzene (95:5) were the most satisfactory. Of these, the latter solvent furnished compact spots of such definition that treatment with alcoholic potash to reveal them was unnecessary (8)

xii)

Amounts of vitamin C down to 3 pg can be detected as dark areas on the layer exposed to short-wave UV light; brief heating to 120°C renders it fluorescent in radiation of 365 nm ( 9 ) .

xiii)

The customary identification o f free ascorbic acid depends on its strong reducing properties and any of the reactions known from paper chromatography may be utilized. The limit of detection with indophenol reagent (blue) is around 0.1 pg dipyridly-iron (red) and molybdophosphoric acid (blue) are almost as sensitive; after brief heating, derivatives and decomposition products yield the colors also. Amounts of 3-5 pg can be visualized with iodoplatinate reagent (yellow) and with alkaline silver nitrate reagent (9).

51

ASCORBIC ACID

2.6

Spectral Properties 2.6.1

Ultraviolet Spectrum The UV spectrum of ascorbic acid (0.002%) in aqueous, acidic methanolic, ethanolic and alkaline solution was scanned from 200 to 400 nm using Varian Carry 119 Spectrophotometer (Fig. I ) . The UV maxima are as follows: 'max Aqueous solution Acidic solution Methanol Ethano 1

(nm)

263 243 244 245

Other reported data (2) are as foJlows: 'max Aqueous solution Acidic solution (pH3) Ethano 1 Methanol Sodium salt in aqueous solution 2.6.2

(nm>

260-265 245 245 263 265

Infrared Spectrum The IR spectrum of ascorb,icacid as KBr-disc was recorded on a Perkin-Elmer 580B FTspectrometer (Fig. 2 ) . The structural assignments have been correlated with the following band frequencies (Table 1).

Table

-

1: IR Characteristics of Ascorbic Acid,

Frequency Cm-I

Assignment

3510 3405 3306

OH

1755 1670

c=o

1110 1025

c-0-c

IBRAHIM A. AL-MESHAL AND MAHMOUD M. A . HASSAN

52

.

.

.

200 210 225 230 2 4 250 260 270 284 2YO

300 310 2 0 3%

3u)

350

-

3m 380 390

Fig. 1. UV Spectrum of Ascorbic Acid. Ascorbic Acid i n Water; --- Ascorbic Acid i n Ethanol; +scorbic Acid i n Ascorbic Acid i n Acid Solution. Methanol;

....

4w mnmlmbr(tlDm

Fig. 2.

am

fWe

mr

1

f4a

IR Spectrum of A s c o r b i c A c i d as KBr d i s c .

c

w

I

a

o

c

o

o

4

m

2

I


54

Other characteristic absorption bands are: 3208, 1500, 1390, 1372, 1320, 1275, 1250, 1222, 1200, 1140, 1075, 1068, 1045, 1025, 990, 870, 820, 755, 720, 680. 2.6.3

Nuclear Magnetic Resonance Spectra 2.6.3.1

Proton Spectra The PMR spectra of ascorbic acid in dueterium oxide,in pyridine and in pyridine D5 were recorded on a Varian-T-60-A, 60 MHz spectrometer using sodium-2,2-dimethyl-2-silapentane-5-sulphonate and tetramethylsilane as reference standard respectively (Fig.3, Fig. 4 and Fig. 5). The following structural assignments have been made (Table 2):

Table - 2:

PMR Characteristics o f Ascorbic Acid.

Chemical Shift (ppm) D20

Pyridine

Pyridine D5 Assignment

4.87(d) 4.10(m) 3.77(s) 3.68(d)

5.43(d) 4.68(m) 4.40(s) 4.30(s)

5.36 (d) 4.33 (m) 4.36 (s) 4.23 (d)

'

5-H 6-H 7-CH2

s=singlet, d=doublet, m=multiplet.

400

r

Fig.

3.

PMR Spectrum of A s c o r b i c A c i d in D20.

z

0

I m

0

0

0 m 0

1

;I 56

L I

L

m 4

Fig. 5.

PMR Spectrum of Ascorbic Acid i n Pyridine D5.

58


PMR data in D20 and in a mixture of DMSO D6 and CDC13 were also reported (10,11,12). 13

2.6.3.2

C-NMR

The 13C-NMR completely decoupled and off-resonance spectra are shown in Fig. 6 and Fig. 7 respectively. Both were recorded over 5000 Hz range in dimethylsulfoxide on Jeol FX-100, 100 MHz spectrometer. Using 10 mm sample tube and tetramethylsilane as reference standard, at ambient temperature. The carbon chemical shifts are assigned on the basis of the additivity principles and the protoncoupled spectrum (Table 3).

11

I HO

- c 6-

I Table

-

3:

Carbon Chemical Shifts of Ascorbic Acid.

Carbon No.

Chemical Shift ppm

c-1 c-2 c-3 c-4 c-5 C -6

170.31(s) 117.93 ( s ) 152.62 (s) 74.56 (d) 68.42 (d) 61.93(t)

s=singlet; d=doublet; t=triplet 13C-NMR data in Water have been also reported (1 3) ,

Fig. 6.

coupled).

13C-NMR Spectrum of Ascorbic Acid (Completely de-

~~

Fig. 7 .

13C-NMR Spectrum of Ascorbic Acid (off-Resonance)

.

61

ASCORBIC ACID

2.6.4

Mass Spectrum The mass spectrum o f ascorbic acid obtained by electron impact ionization, was recorded on a Ribermag R-10-10 mas spectrometer equipped with direct inlet probe. The spectrum (Fig. 8) shows a molecular ion peak M+ at m/e 176 with a relative intensity o f 5.9%. The most prominent fragments and their relative intensities are shown in Table 4:

Table M/e

-

4: Prominent Mass Fragments of Ascorbic Acid. Fragment

Relative Intensity %

177 176

M + l M +

1.0 5.9

1

116

100.0

85

36.0

71

24.7

70

23.1

HF

H

H o g

a' 61 3.

Preparation 3.1

29.7

HO-

C

PH - 0

I H

I H

'i'

C

Isolation Many methods were reported €or the isolation o f ascor bic acid from plants. However, the most popular is by using freshly prepared solution of 5-6% methaphosphoric acid (14). This solution is a good extractant as well as stabilizing agent f o r a limited period by complexing metal ions and minimizing the rate of oxidation. It has also been claimed that ascorbic acid can be stablized by diluted perchloric acid solution

ASCORBIC ACID

63

o r 2,3-dimercapto-l-propanol (15). An alternative method of extracting ascorbic acid from foods is by forming a slurry of the frozen material with absolute ethanol has been found to be as effective as extraction with metabphosphoric acid (16).Also a mixture of 8% acetic acid and 0.5% oxalic acid was used (17) 3.2

Synthesis L-ascorbic acid is conventionally synthesized (18,19) by hydrogenating D-glucose to D-sorbitol. The latter is made to yields L-sorbitol by oxidation with Acetobacter suboxydan, this followed by introducing carboxyl group at C 1 while the L-sorbose is in the form of its diacetone derivative. The resulting diacetone-2keto-L-gluconic acid is then heated with hydrochloric acid t o give ascorbic acid (Scheme 1 , p . 11). Alternative route from sorbose by oxidation with nitrogen peroxide. Another method for synthesizing L-ascorbic acid was reported ( 2 0 ) , involving a one-step oxidation of 1,20-isopropyhene-a-D-glucofuranose to 1,2-0-isopropylidene-a-D-xylo-hexofuranurono-6,3-Lactone-S-ulose and acid treatment of the later followed by reduction.

4. Biosynthesis of Ascorbic Acid In both plants and animals ascorbic acid is formed from Dglucuronic acid. UDP-glucuronic acid is first converted to D-glucuronic acid lactone via D-glucuronic acid-l-phosphate. This compound is then reduced at carbon atom 1 t o form L-gulonic acid. (Since in the numbering of the carbon atoms of carbohydrates the most highly oxidized carbon atom is given the lowest possible number, the original carbon atom 6 of glucuronic acid becomes carbon atom 1 of gulonic acid). After the conversion of the gulonic acid to the corresponding y-lactone, the hydroxyl group at carbon atom 2 is oxidized to a keto group. The 2-keto-Lgulonic acid lactone formed is subsequently converted t o L-ascrobic acid by enolization (21). Direct conversion of D-glucuronic acid to L-gulonic acid by isomerization at carbon atom 5 has not yet been conclusively established (scheme 2,~.12).


64

CH20H I HO - C I HO - C -

CH20H

I

H

IIO - C

H

I

I

H?

I

cat.

H -C-O€i

HO

-c - B

-

HO-

C-

B-

C-

HO -

I

CH20H I

H

co

OH

I C - I-;

Acetobactv suboxydans

I

I CHO D-glucose

I

HO-

H

C--B

I

H-

C-

I HO - c I

OH

H

Ct120H

CH20€i

G-sorbitol

L-sorbose COOH

I

co HO acetone H+

(1)*<Mn04

+

I

9:CMe2

I

ki

-

C

HO

-

C

~

( 2 ) H 2 0 (H+)

H

I

- cI

CH OII 2

2-keto-L-gulonic acid

(CH20H C -OH

II enolize

11

C

I

I

lactoniz

H - C-OH C-H I CH20H

bo

H

I

HO-

-E

I

2,3,4,6-diacetone sorbose

HO-

- OH

HO-C-H

OH

I

I

CH20H

L-ascorbic acid (Vitamin C) Scheme 1: Synthesis of ascorbic acid.

O€i

65

ASCORBIC ACID

6

-

0 II ..

Q"DpCOOH

COOH

Ho@o-p

HO

OH

OH

UDP-D-Glycuronic acid

C

,@"20H

D-Glucuronic acid1-P

CH20H

OH

D-Glucuronic acid lactone

CH20H

L-Gulonic acid

L-Gulonic acid lactone

4

I CH20H

2-Keto-L-gulonic acid lactone.

HO-C-H

I

CH20H

L-Ascorbic acid.

Scheme 2: The biosynthesis of L-ascorbic acid.


66

5.

Metabolism Ascorbic acid is readily absorbed and metabolised. However, after oral administration of large quantities, only small amounts are excreted in the urine while there is a steady rise in the level of ascorbic acid in the plasma. If the oral ingestion is continued for a sufficient period, the plasma concentration rises to a maximum, after which a rapid urinary excretion of a large part of the ingested ascorbic acid occurs (22). Ascorbic acid (ASA) and dehydroascorbic acid (DAsA) are metabolized by humans, and the levels of AsA in blood were maximum at the 4th hr after AsA administration but at 2nd hr after DAsA administration. The amount of AsA and DAsA excreted in urine in 6 hr was respectively. 60 and 70% of the amount excreted in 24 hr after administration, About 30 and 40% of administered dose were excreted by men and women respectively in 24 hr. Of the vitamin excreted after administration of either AsA or DAsA, about 90 and 10% were in the form of AsA and DAsA, respectively (23). The amino acids phenylalanine and tyrosine are not metabolized completely in vitamin C-deficient individuals, Under these conditions they are metabolized only partly and are excreted in the urine as homogentisic, p-hydroxyphenylpyruvic and p-hydroxyphenyllactic acids. It appears that vitamin C plays the role of a coenzyme in the metabolism of tyrosine through its deaminated product, because scorbutic liver slices cannot metabolize this amino acid in the absence of this vitamin. Vitamin C in adequate amounts delays the oxidation of epinephrine by the body (24 1.

6.

Daily Requirement! Ascorbic acid is needed in daily quantities of about 70 mg to sustain full stamina, and is an essential nutrient for human beings. In the case of insufficiency the symptoms of scurvy appears (21).

7.

Mode o f Action We have as yet no complete understanding o f the mode of action of ascorbic acid. It is, however, known that this substance is involved in certain hydroxylation reactions which are catalysed by mixed function oxygenases in the reduction o f folic acid to tetrahydrofolic acid as well as well in the regulation of the redox equilibrium between Fez+ and Fe3+ and Cu" and C U ~ +(21).

61

ASCORBIC ACID

8. Vitamin Defficiency

Patients placed on a diet deficient in vitamin C exhibited the following: 1) 10 days, plasma fell to a low level; 2) 30 days plasma level was zero; 3) 13 weeks, first clinical evidence of scurvy; 4) 132 days, hyperkeratotic papules developed; 5) 141 days, wounds failed to heal and 6) 162 days, perifollicular hemorrhages of scurvy developed; ascorbic acid value of white cell platelets fell to zero. Loss of weight occurred, accompanied by lowered blood pressure (24). 9. Methods of Analysis 9.1 Titrimetric Methods

British Pharmacopoeia 1973 The B.P. 1975 has described the assay as follows: Weigh and powder 20 tablets. Dissolve a quantity of the powder equivalent to 0.15 g of ascorbic acid as completely as possible in a mixture of 30 ml of water and 20 ml of d i l u t e sulphuric acid and titrate with 0.lN anunonium c e r i c sulphate, using f e r r o i n sulphate solution as indicator. Each ml of 0 . 1 1 m o niwn c e ri c sulphate is equivalent to 0.008806 g of C6H806. British Pharmacopoeia 1980 The B.P. 1980 has described the assay as follows : Dissolve 0.2 g in a mixture o f 80 ml of freshly boiled and cooled water and 10 ml of M sulfuric acid. Titrate with 0 . 0 5 M iodine VS using 1 ml of starch solution as indicator until a persistent blue color is obtained. Each ml of 0.05M iodine VS is equivalent to 0.00881 g of C H 0 6 8 6' United States Pharmacopoeia 1980 a) Ascorbic Acid: Dissolve about 400 mg of ascorbic acid, accurately weighed, in a

68


mixture of 100 ml of carbon dioxide-free water and 25 ml of 2N s u l f u r i c acid. Titrate the solution at once with 0.1 N iodine VS, adding 3 ml o f starch TS as the end-point is approached. Each ml of 0.1 N iodine is equivalent to 8.806 mg of C6H806. b)

Ascorbic acid injections: Transfer to a 100-ml volumetric flask an accurately measured volume of ascorbic acid injection, equivalent to about 50 mg of ascorbic acid and previously diluted with water if necessary. Add 20 ml of metaphosphoric-acetic acids TS, dilute with water to volume, and mix. Accurately measure a volume of the dilution, equivalent to about 2 mg of ascorbic acid, into a 50-ml conical flask, add 5 ml of metaphosphoric-acetic acids TS, and titrate with standard d i c h l o r o p h e n o l - i n d o p h e n o l solution until a rose-pink color persists f o r at least 5 seconds. Correct for the volume of the dichlorophenol-indophenol solution consumed by a mixture of 5.5 ml of metaphosphoricacetic acids TS and 15 ml of water. From the ascorbic acid equivalent o f the standard d i c h l o r o p h e n o l - i n d o p h e n o l solution calculate the ascorbic acid content in each ml o f the injection. Various other titrimetric methods of assay of vitamin C in vegetable tissues, whether as ascorbic acid or as total vitamin C (ascorbic plus dehydroascorbic acids), were studied and compared. For the extraction of the vitamin, an aqueous mixture of 8 per cent, acetic and 0.5 per cent oxalic acids was used instead of metaphosphoric acid. Hot and cold extractions gave practically the same results, except with hard, dry tissues, which required hot extraction. To effect removal of colloidal matter, which interferes with filtration and clarification of the extract, it is considered advisable to add 25 to 50 per cent o f ethanol. In the presence of ethanol causes an error o f at least 2 to 12 per cent. To establish (visually) the titration end-point in the indophenol method, it is recommended that a standard having the same composition as the sample under titration

69

ASCORBIC ACID

b u t p r e v i o u s l y o x i d i s e d w i t h i o d i n e and mercuric a c e t a t e be employed. T h i s g e n e r a l l y e n a b l e s 93 t o 100 p e r c e n t of v i t a m i n C e x p e r i m e n t a l l y added t o be determined. I n t h e method of d e t e r m i n a t i o n with methlene b l u e , t h e r a t i o o f methylene b l u e used t o v i t a m i n C c o n t e n t d e c r e a s e s as t h e l a t t e r v a l u e i n c r e a s e s . T h i s d e c r e a s e of t h e r a t i o i s small; t o o b t a i n s a t i s f a c t o r y r e s u l t s , t h e a l i q u o t t i t r a t e d should n o t c o n t a i n >0.04 mg o f v i t a m i n C . V i s u a l d e t e r m i n a t i o n o f t h e end-point w i t h t h e a i d o f a s t a n d a r d i s a g a i n recommended. The i o d i m e t r i c method, with potassium i o d a t e was a l s o s t u d i e d . The c o n c l u s i o n s drawn a r e : 1) t h a t t h e indophenol method i s s u f f i c i e n t l y a c c u r a t e , i s t h e most simple method and h a s t h e w i d e s t s p h e r e of a p p l i c a t i o n ; 2) t h a t t h e methylene b l u e method g i v e s r e s u l t s 3 t o 10 p e r c e n t lower t h a n t h e indophenol method and 3) t h a t t h e i o d i m e t r i c method g i v e s r ? s u l t s 20 t o 40 p e r cmt higher (17). Reduction i s e f f e c t e d by adding M sodium s u l p h i d e s o l u t i o n a c i d i f i e d with HC1, and removing t h e e x c e s s o f s u l p h i d e w i t h M e t h a n o l i c mercuric c h l o r i d e s o l u t i o n . Reduct i o n i s complete i n 10 t o 15 min. C l e a r f i l t r a t e s a r e r e a d i l y o b t a i n a b l e €or t i t r a t i o n w i t h dichlorophenol-indophenol ( 2 5 ) . Simple methods o f d e t e r m i n i n g a s c o r b i c a c i d , t h i a m i n e and n i c o t i n i c a c i d a r e applied t o mixtures containing t h e s e vitamins with various pharmaceutical p r e p a r a t i o n s . Ascorbic a c i d can b e determined i o d i m e t r i c a l l y i n t h e p r e s e n c e o f calcium l a c t a t e , p h y t i n g l u c o s e calcium g l y c e r o p h o s p h a t e , c a f f e i n e sodium b e n z o a t e , n i c o t i n i c a c i d , amidopyrine o r t h i a m i n e . For m i x t u r e c o n t a i n i n g a s c o r b i c a c i d and nicotinic acid, the ascorbic acid i s determined i o d i m e t r i c a l l y and t h e t o t a l a c i d i s

70


then titrated against 0.1 N solution of hydrochloric acid with phenol red as indicator. Thiamine i s determined in the presence of ascorbic acid by a modification of the U.S.S.R. Pharmacopoeia VIII argentimetric method ( 2 6 ) . The platinum - tungsten bimetallic electrode system is applicable to the quantitative iodimetric determination of ascorbic acid according to the B.P. 1953. The equivalence point is given by the very sharp break in the titration curve. The error, = f 0.0002 on samples of 0.05 to 0.15 g is much less than for the usual volumetric procedure (27). Other Sitrimetric method for the determination of ascorbic acid is by involving two stage oxidation by potasium iodate ( 2 8 ) . 9.2

Spectrophotometric Method 9.2.1

Colorimetric An assay method described for ascorbic acid involves the reaction with diazotised 4methoxy-2-nitroaniline in acid medium, and subsequent development of a blue color in alkaline solution. This color, with a maximum absorbancy at 570 nm, i s compared with standards in suitable photo-electric colorimeter. It can be carried out directly, e.g., in the presence of dehydro-ascorbic acid and all other vitamins. Its sensitivity permits the determination of quantities down to 0.5 mg with a low limit of 10 pg per ml, when a 50ml sample aliquot is used (29a).

9.2.2

Ultraviolet In aqueous solution ascorbic acid is characterised by a single very intense band with its head at 260-265.nm. The molecular extinction coefficient is approximately 7000 for solutions containing about 2 mg/100 cc. In stronger solutions (ca.50 mg per 100 c.c.) wide deviations from Beer's law are encoun-

71

ASCORBIC ACID

tered. But for concentrations ranging between 0.5 and 2.5 mg per 100 C.C. Beer's law holds with sufficient exactitude to permit of the use of spectrophotometric measurements for quantitative estimations of concentrations. The intensity of the band diminishes rapidly, falling to half value in a few hours (decomposition of ascorbic acid by oxidation (2). Other color reactions have been proposed as a basis for measuring ascorbic acid: reaction with diazotised p-aminobenzoic acid to produce a pink color and the interaction of ascorbic acid with dimethoxydiquinone to form a reddish -violet color which is measured spectrophotometrically at 510 nm (29b). Ascorbic acid, cystine, thioglycollic acid, a-mercaptopropionic acid and sodium mercaptobutane sulphonate are determined by the fluorescence produce by the reduction of sodium 1 : Z naphthaquinone-4-sulphonate (Folin's reagent) in U.V. The use of p-chloromercuribenzoic acid in the determination of ascorbic acid with 2,6-dichlorophenol-indophenol below pH3-5 is recommended because of the extent of spontaneous fading of indophenol that occurs. p Chloromercuribenzoic acid does not affect the reaction between indophenol and ascorbic acid, but it inhibits almost entirely the decolorisation of indophenol by various interfering substances (31). 9.2.3

Spectrofluorimetric The reaction of dehydroascorbic acid with ophenylenediamine to give a fluorescent quinoxaline was used as the basis of an assay to determine y quantities of ascorbic and dehydroascorbic acids, The development of the fluorescent derivative of the vitamin is prevented by forming a boric acid-dehydroascorbic acid complex prior to addition of the diamine solution. This provides a means of differentiating between the fluorescence from the vitamin and that from possible interfering substances. When applied to pharmaceutical preparations, beverages and special dietary foods,

72


the method shows a high degree of specificity.

No interfering substances were found (32).

A rapid colorimetric determination of ascorbic acid (10 to 200 mg per 100 g) with the sodium salt of 2,6-dichlorophenol-indophenol was studied. An amyl acetate extract of 2,6dichlorophenol-indophenol has max. absorption at 525 nm the extinction of which remains unchanged for 4 hrs. The sample is extracted with 2 per cent metaphosphoric acid and diluted to produce a 0 . 4 to 1.0 mg per cent solution of ascorbic acid. 2,6-dichlorophenolindophenol solution (5 ml in water) is added to the sample solution which is rapidly extracted with amyl acetate for the extinction to be measured. Another 5-ml portion of the 2,6-dichlorophenol-indophenol solution is added to metaphosphoric acid and similarly treated. The amount of ascorbic acid is determined from the difference in the extinction of the two extracts, which is propor-. tional to the concentration of ascorbic acid up to 4 mg per 100 ml ( 3 3 ) .

Other method depends on the reduction of ferric chloride by ascorbic acid and the colorimetric determination of the ferric chloride by means of reaction with aa-dipyridly; the reaction is carried out in the presence of phosphoric acid to eliminate interference by reduction. A solution (1 ml) containing N 0.02 mg of ascorbic acid is treted with 0.3 ml of 85 per cent. Phosphoric acid to give a pH of 1 to 2, 5 ml of 0.5 per cent aqueous aa-dipyridly solution and 1 ml of 1 per cent ferric chloride. The method has been applied to orange juice, honey and urine; it is sensitive to 5 lJg of ascorbic acid per ml ( 3 4 ) . A direct method for the determination of vitamin C without removal of the reagent, 2,4dinitrophenylhydrazine, is applied to blood and urine. The ascorbic acid content i s calculated from a chart (35).

Ascorbic acid can be semi-quantitatively estimated with the comparison method after color reaction with molybdophosphoric acid.

73

ASCORBIC ACID

The red zone of dehydroascorbic acid-DNP is scraped off, eluted with 85% sulphuric acid, filtered or, better, centrifuged, and the light absorption of the solution measured at 520 to 525 nm against water as blank. The analysis result is worked out with the help of a standard solution which is treated identically ( 9 ) . 9.3

Turbidimetric Method This method (36) is used €or the determination o f ascorbic acid in foods, by the reaction of selenious acid with ascorbic acid and stannous ions at low pH and room temperature.

9.4

Chromatographic Methods 9.4.1

Paper Chromatography Methods used €or the separation of ascorbic acid by paper chromatography are shown in Table 5 ( p . 21).

9.4.2

Gas-Liquid Chromatography Gerstl and Ranf€t (42) extracted food with metaphosphoric acid, separted the ascorbic acid on a column of cellulose and formed the trimethysilyl ether derivative by reaction with N.0-bis-(trimethylsily) acetamide before chromatographing on a column of Gas Q containing 3% SE-30.

9.4.3

High Pressure Liquid Chromatography Packla and Kissinger ( 4 3 ) , used a strong anion resin and elution with pH 4.75 buffer solution, has been applied to the determination of ascorbic acid in milk products, baby foods, fruit juice concentrates, whole fruits and fortified cereals. The procedure was not directly suitable for measuring dehydroascorbic acid. However, sood et a1 (44) used HPLC in

TABLE - -- -~

~

~~~

Chromatogram

-

5

~

~

Solvent System

Rf Value

Paper Chro- 1) n-butanol saturated matography. with water + oxalic 2) Phenol saturated with water + oxalic acid. Paper (Schleicher and Schull No. 602).

P

Methods used for separating ascorbic acid by paper chromatography

1) 50% Methanol.

0.7-0.8

2) Wat er-n-butano1-

0.6-0.7

glacial acetic acid (50:40:14:5)

Paper Chro- butanol-acetic acidmatography. water (4:1 :5) Paper Chro- n-butanol-acetic acidmatography. water (4:l:S)

0.36

Reagent 2,6-dichlorophenolindophenol with subsequent colorimetry.

Application Reference Plant and animal tissues.

37

1) 1% silver-nitrate Lemon juice 38 solution in 10% white,red and Aqueous ammonia. black-currents. 2 ) 0.005N iodine solu- tomatoes and tion in 0,04% starch green pappers. so 1ut ion. 3) 0.04% aqueous solution of 2,6-dichlorophenol-indophenol. 4) U.V. ammonium-molybdate solution. Alkaline tetrazolium salts.

39

Method f o r de- 40 tection of lfpg quantity.

ASCORBIC ACID

75

the reverse phase ion-pairing mode to determine ascorbic acid in food with tridecyl ammonium formate as counter-ion. 9.5 Enzymatic Method A new enzymatic method based on the oxidation of the ascorbic acid to dehydroascorbic acid with ascorbic oxidase permit assaying for ascorbic acid and dehydroascorbic acids in vegetable extracts (45). 9.6

Polarographic Method Ascorbic acid can be analysed in a variety of mixtures and multivitamins preparations by cathode ray reverse sweep polarography (46).

9.7 Chronometric Method The reduction oxidation couple between ascorbic acid oxidation and reduction of the semiquinoid form pphenylenediaimine is the basis of a new chronometric assay of vitamin C. Ascorbic acid interrupts formation of the colored product by coupling its oxidation to reduction of the semiquirioid proceeds to dye form (47).

76


10. References

1. G.C.G. Grasselli and N.M. Ritchey "Atlas of Spectral Data and Physical Constants for Organic Compounds" 2nd Ed. , Vol. 2, CRC Press Inc., Cleveland, Ohio, 1975. 2. R.W. Herbert, E.L. Hirst, E.G.U. Percival, R.J.W. Reynolds and Smith, J. Chem. SOC., 1270 (1933). 3. J.E. Hoover "Remington's Pharmaceutical Sciences", 15th Ed., Mack Publishing Company, U.S.A., (1975). 4. M. Windholz "The Merck Index" 9th Ed. Merck and Co., Inc., Rahaway, U.S.A. (1976). 5. "The British Pharmacopeia", Her Majesty's Stationary Office, Cambridge, (1973). 6. "The British Pharmacopeia", Her Majesty's Stationary Office, Cambridge, (1980). 7. "The United States Pharmacopeia", XX, Mack Publishing Co., Easton, Pa (1980). 8. R.C.R. Barreto, Rev. Quim. Ind., Rio de Janeiro, 24, 13, (1955). Through, Anal. Abstract , 3, 567 (1956). 9. E. Stahl , "Thin Layer ChromatograpFy", 2nd Ed. , SpringerVerlag., p. 304, (1966). 10. C.J. Pouchert and J.R. Campbell "The Aldrich Library of NMR and Spectra" Vol XI (1974). 11. "High Resolution NMR Spectra Catalog", Varian Analytical Instrument Division, Vol. 2, The National Press, U.S.A. (1963) . 12. D.T. Sawyer and J.R. Brannan, Anal. Chem., 38 (2), 192, (1966). 13. S . Berger, Tetrahedron, 33, 1587 (1977). 14. R.D. King "Developments in Food Analysis Technique-1" Applied Science Publishers, 18 (1978). 15. C.B. Bourgeois, P.R. Mainuy, R. George and A.M. Czornomaz, Analusis, 2, 556 (1973). Through Reference 14. 16. V.G. Randall, E.L., Pippen, A.L. Potter and R.M. McCready, J. Fd. Sci., 40, 894. Through Reference 14. 17. G, Enachescu,%al. Inst. Cerc. Agron. Romn., 22, 463, (1955) . Through Anal. Abstract , 3, 3474 (1956): 18. T.A. Geissman, "Principles of Organic Chemistry", 3rd. Ed., W.H. Freeman and Company, p. 497, (1968). 17, 311, 19. T. Reichstein and A. Grussner, tlelv. Chim. Acta., (1934). 20. J. Bakke and 0. Theander Chemical Comm. , 175 (1971). 21. M. Luckner "Secondary Metabolism in Plants and Animals", Science Paperbacks, p. 74, (1977). 22. E.G.C. Clarke "Isolation and Identification of Drugs", The Pharmaceutical Press, London, (1971).

.

ASCORBIC ACID

77

23. T. Masaru, W. Sanae, M. Katsuko, T. Sachiko, F. Akji (Biochem. Lab. Kagawa Nutr. Coll., Tokyo, Japan). Vilamins 45(3), 136, 1974, fJapan). Through 24. C.D. Wilson, 0. Gisvold and R . F . Doerge "Textbook o f Organic Medicinal and Pharmaceutical Chemistry", 7th Ed., J.B. Lippincott Co., Philadelphia, (1977). 25. E . Piyanowski, Prezem. Rony Spozywezy, 11,410 (1954). 26. G.A. Vaisman and S.G. Rozhntskaya Aptechnoc Delo. (3), 16. Through: Anal. Abstract, 2, 563 (1955). 27. S.R. Mohanty, K.R.K. Rao and L.V. Kannan, Anal. Chim. Acta, 14 (6) 587 (1956). Through: Anal. Abstract, 2, 347, (1956); 28. G.S. Deshmukh and M.G. Bapat, Z. Anal. Chem. 145(4), 256 (1955). 29. (a) M. Schmall, C.W. Pifer and E.G. Wollish, Anal. Chem. 25(10), 1486 (1953). Through Anal. Abstract, 1,370, (1954). (b) M.H. Hashmi, M.A. Shahid, M.A. Akhtar and N.A. Chugtani, Mikrochim Acta, 5, 901 (1973). 30. H.. Freytag, Z. anal. Chem., 139(4), 263, 1953. Through. Anal. Abstract, 1, 371, (1954). 31. J.A. Owen and B.Iggo, Biochem. J., 62(4), 675 (1956). 32. C. Mike, J. Deutch and C.E. Weeks, J. Assoc. Office Agr. Chemists, 48(6), 1248 (1965). Through Chem. Abstract, 64, 72840 (1966). 33* H. Imai and T. Fugitani J. Chem. SOC., Japan, Pure Chem. Sect., s(11), 1212 (1955). Through: Anal. Abstract 2, 2567 (1956). 34. M.X. Sullivan and H.G.N. Clarke, J. Ass. Off. Agric. Chem., 38(2), 514 (1955). Through: Anal. Abstract, 2, 258 (1956). 35. J.H. Roc and C.A. Kuether, J. Biol. Chem. 147, 399 (1943). Through: Chem. Abstract, 37, 31185 (1943). 36. W.J. Ralls, J. Agric. Food Chem. 23(3), 609, (1975). Through: Chem. Abstract, 83, 41610 F (1975). 37. Yu-Tuan Cheng., F.A. Isherwood and L.W. Mapson, Biochem. J., 55(5), 821 (1953). Through: Anal. Abstract, I, 372, (1954). 38. V. Sanda Ceskosl. Farmac., 2(3), 79 (1954). Through: Anal. Abstract, 2, 2563, (1955). 39. W. Hermann, R. Strohecker and F. Matt., Z. Lebensmitt. Untersuch. U. Forsch., 97, 263, (1953). Through: Anal. Abstract, 1, 369, 1954. 40. 2. Padre, E. Smid and V. Sicho, Naturwissenschaften, 42(8) , 210, (1955). Through: Anal. Abstract, 2, 853 (1956). 41. J.E. Schlack, J. Ass. Office, Analyt. Chemist., 57, 1346, (1974). Through: Reference 14. 42. R. Gerstl and K. Ranfft, Z. Lebensmitt-elunters. U. Forsch., 154, 12, (1974). Through Reference 14.

78


43. L . A . Packla and P.T. Kissinger, Anal. Chem., 48, 3 6 4 , ( 1 9 7 6 ) . Through Reference 1 4 . 4 4 . S.P. Sood, L.E. Sartori, D.P. Wittmer and W.G. Haney, Anal., Chin., 4 8 , 796, ( 1 9 7 6 ) . Through Reference 1 4 . 4 5 . A. Marchesini, F . , Montauori, D. Muffats, D. Maestri, J. Food Sci., 3 9 ( 3 ) , 5 6 8 , ( 1 9 7 4 ) . Through Chem. Abstract, 8 2 , 2717 F ( 1 9 7 5 ) . 4 6 . K S . Owen, F.W. Franklin, J. Food Technol., E ( 3 ) , 2 6 3 , ( 1 9 7 5 ) . Through Chem. Abstract, 83, 1 3 0 1 0 2 J ( 1 9 7 5 ) . 4 7 . B. Roe, J . H . Bruemmer, Proc. Fla. State Hortic. SOC., 87, 210, ( 1 9 7 5 ) . Through Chem. Abstract, 83, 130100 G (1975).

CAPTOPRIL Harold Kadin

1.

2. 3. 4.

5.

6.

7

8.

9. 10. 11.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor History Synthesis Physical Properties 4.1 Spectral Properties 4.2 Solid State Properties 4.3 Solution Data Stability 5.1 Solid State Stability 5.2 Solution Stability Analytical Tests and Methods 6.1 Elemental Analysis 6.2 Spectrophotometric Methods 6.3 Chromatographic Methods 6.4 Titrimetric Methods Analysis in Biologic Fluids and Tissues and in Animal Rations 7.1 Thin Layer Radiochromatography (TLRC) 7.2 Gas Chromatography-Mass Spectroscopy (GC-MS) 7.3 Gas Chromatography-Flame Photometric Detection (GF-FPD) 7.4 High Performance Liquid Chromatography with UV Detection (HPLC-UVD) 7.5 Spectrofluorometry 7.6 Radioimmunoassay (RIA) 7.7 Semiautomated Ellman Colorimetry 7.8 High Performance Liquid Chromatography with Electrochemical Detection (HPLC-ECD) 7.9 High Performance Liquid Chromatography with Fluorescence Detection (HPLC-FD) Drug Metabolism-Pharmacokinetics 8.1 Blood Level Studies 8.2 Urinary Excretion Studies 8.3 Miscellaneous Distribution Studies Acknowledgments References Review Coverage Dates

Analylical Profilca of Drug Substances Volume 11

79

80 80 80 80 84 84 84 97 106 107 107 108 112 112 112 113 120 120 120 122 123 124 124 124 125 126 128 128 128 129 134 131 131 137

Copynghi 0 1982 by The American Pharmaceutical Association ISBN 0-12-260811-9

80

1.

HAROLD KADIN

Description

1.1 Name, Formula, Molecular Weight Captopril, Capoten@, or Lopirins is l-(3-mercapto-2-D-methyl-l-oxopropyl)-L-proline (S,S) with Chem. Abstr. Registry Number 62571-86-2.

The asterisks indicate the two S,S optically active centers. 1.2

Appearance, Color, Odor

Captopril is a white to off-white crystalline powder with a slight mercaptan odor. 2.

History

The captopril story began in 1971 with a report (1) on the isolation and synthesis of teprotide, an antihypertensive nonapeptide from the venom of a Brazilian Pit Viper. This venom peptide was hypotensive through inhibition of an exopeptidase, known as the angiotensin converting enzyme (ACE). The latter performs dipeptide scission at the carboxyl end of the decapeptide, angiotensin I to yield angiotensin 11, the most powerful natural vasoconstrictor known. ACE further potentiates hypertension through scission-inactivation of the nonapeptide vasodilator, bradykinin. Clinical investigations with both teprotide (2) and captopril ( 3 ) have implicated ACE as the key enzyme in human hypertensive diseases. The first ACE inhibitor shown to be clinically efficacious against hypertension was the synthetic venom peptide, teprotide ( 2 ) .

CAPTOPRlL

81

However teprotide was expensive and only effective parenterally. A simpler, orally effective ACE inhibitor was desired. Analogs of the snake venom hypotensive peptides were quantitatively rated for their in vitro inhibition of ACE (4) and for their effect o n e contractile properties of guinea pig ileum ( 5 ) . The ratings aided construction of a hypothetical active site for ACE (Figure 1). This model was based primarily on the similarity of the enzymatic properties of ACE to those of carboxypeptidase A (CASE A) even though the latter yields amino acids rather than the dipeptides of the former. Further, the active site of CASE A, like ACE contains zinc but unlike that of ACE had been structurally characterized, in the crystalline state, by x-ray diffraction (6). Thus the elaboration of the hypothetical ACE active site and the possibility of a simple ACE inhibitor were stimulated by a 1973 report (7) on, D-2-benzylsuccinic acid, a simple inhibitor, of CASE A. Specific points of inhibitor attachment within the real CASE A site were extrapolated to that within the hypothetical ACE site ( 8 ) . On the basis of these extrapolations, synthesis of simple ACE inhibitors were initiated in 1974. As each of a great number of candidates was quantitatively rated in the aforementioned -in vitro screens (previously developed for the venom peptide studies), the hypothetical site was verified and refined. In the refined model schematically represented in Figure 1, three dimensional amino acid configurations within the catalytic site provided suitably spaced subsites or multiple points of attachment to substrate or inhibitor. The semi-circular clefts in the figure represent hydrophobic subsites which may interact with the lipophilic side chains of inhibitor or substrate. The latter are also putatively bound to the ACE site via the X-H group at their nonscissile terminal peptide bond. As indicated, one of the first simple ACE inhibitors was patterned after all of the venom peptide inhibitors in sharing a terminal proline (succinyl-L-proline). The choice of proline was also influenced by the superior biological stability exhibited by teprotide with

82

HAROLD KADIN

ENZYME

INHIBITOR

RELATIVE IN VlTRO INHIBITION

CAABOXYPEPTIDASE A

D-2-EENZYLSUCCINIC ACID

-7 FH2 ?-

0 = CCH,-CH-C -C = 0

ANGIOTENSIN-CONVERTING ENZYME

SUCC1NYL.L-PROLINE

1

D-2-METHYLSUCCINYL.L-PAOLI"

15

CAPTOPRIL

14000

F i g . 1. Key steps i n t h e d e s i g n o f a s p e c i f i c i n h i b i t o r of t h e a n g i o t e n s i n c o n v e r t i n g enzyme.

CAPTOPRIL

its two terminal prolines (9). In an emulation of D-2-benzylsuccinic acid, but with appropriate lengthening of the chain, the nitrogen and carbonyl of the penultimate "venom peptide" amide linkage were substituted, respectively, with a peptidase-inhibitory methylene and a highly anionic, zinc binding carboxyl. As indicated in Figure 2 the analogy was then extended to the better snake venom inhibitors bearing a penultimate alanine in addition to the terminal proline (D-2-methylsuccinyl-L-proline) . The largest increase in inhibitory activity (about 14 thousand fold over succinyl-L-proline) was obtained when the zinc binding carboxyl was replaced with a thiol which has considerably greater affinity for zinc. The snake venom peptide, teprotide, does not have comparable affinity for zinc, however its binding is enhanced by additional interactions of its amino acid side chains with "clefts" on the enzyme beyond the active peptidase site (10). In short, the orally active antihypertensive drug, captopril, was announced to the scientific community in 1 9 7 7 (11). It was made generally available for treatment of hypertensive diseases in 1 9 8 1 (12,841. Clinical investigations (13) suggest it is also highly efficacious in the treatment of congestive heart failure. Its uniquely designed highly specific affinity for the active site of ACE has resulted in a high ratio of clinical success with a relatively low index of side effects. It has been effective where conventional antihypertensive therapies fail or have an untoward number of side effects.

83

84

3.

HAROLD KADIN

Synthesis

A process (14) is presented (Figure 2) in a chemical reaction sequence which follows this brief description. Methacrylic acid (I) is condensed with thiolacetic acid (11) to give racemic 2-methyl-3acetylthiopropionic acid (111). L-proline is then acylated with the acid chloride (IV) of the thioester (111). The resulting proline thioester (VI) is resolved from its R,S - isomer by aqueous crystallization. Saponification of compound VI with sodium hydroxide affords the sodium salt of captopril which after acidification yields captopril (VII)

.

4.


Spectral Properties 4.11

Infrared Spectra

The infrared spectrum of captopril in chloroform is presented in Figure 3 and as a KBr pellet in Figure 4. The infrared spectrum in the latter indicates the presence of the following frequencies and their structural assignments (15). cm-1 1750 1725 1640 2560

Assignment C = 0 (COOH group) C = 0 (amide) S - H

DiffereQfes in the fingerprint regions (1350-900 cm ) of the mineral oil mull infrared spectra of batches 3 and 4 (Figure 5 and 6, respectively) indicate that the low melting batch 3 and the high melting batch 4 are polymorphs (see Section 4.21)

.

85

CAPTOPRIL

Figure 2 Chemical Reaction Schematic Diagram

+

I

CH2 = CCOOH MW = 86.01 Methacrylic

I1

I11

+

AcSH MW 76.11 Thiolacetic

AcSCH2CHCOOH

MW = 162.20

-+

Acetylthioisobutyric

IV

AcSCH2CHC

\

c1

COOH

COOH

MW = 180.65

MW = 259.32 Proline Thioester

VI

VI I

MW = 115.13 Acid Chloride" L-Proline

"'?

H3C 0

AcSCH2CHCN

d)

COOH

COOH MW = 217.28

Proline Thioester

Captopril

0

II

Ac = CH3C(a) Reflux (b) SOCl , DMF, Distillation Ci C12 Wash, HC1, (c) H 0 + NaHCO ci!ystallizaiG.on (d) H20 + NaOH, HC1, CH2C12 extraction

41.

86

Ll

0 0

c

rl

c

u

.ti

k ld

a fd

c

a 4J

ul ro

a,

5 0

X

Ll

.ti

d

a 0

4J

a Id u 0

w

5 Ll

LI

a

a,

i, m

a 0

a c

m k

Q)

c

4J

4J

c

ro

H

a,

4J rl r(

k

a, PI

m

c

M -4

ld

ak ld

c

a &

a, [o

7

0

X Li

a 0

ld

PI

4J

u u-l 0

k

4J 0

a,

a

a,

a

5

2 k

2 rl

wI

PI

a,

k

2

c

v)

4

a

a, k

id k

44

..

G

lJ

H

k

(I)

&

c

H

WAVELENGTH (MICRONS)

3500

F i g u r e 5. Instrument:

2500

2Ooo

lsbo

1600

1400

FREQUENCY (CM-’)

1200

I n f r a r e d S p e c t r u m of C a p t o p r i l , B a t c h 3 , Mineral O i l M u l l Perkin-Elmer,

Model 6 2 1

200

WAVELENGTH (MKRONS)

F R E w € w (CM-')

Figure 6. Instrument:

Infrared Spectrum of Captopril, Batch 4, Mineral Oil Mull Perkin-Elmer, Model 621

HAROLD KADIN

90

4.12

Nuclear Magnetic Resonance Spectra 1

The 270 MHZ H-NMR spectrum of captopril in CDCl is shown in Figure 7. The spectrum was obtained from the University of Chicago courtesy of Professor Josef Fried. Spectral assignments are shown in Table 1. The 13C-NMR of captopril in CD OD is shown in Figure 8. The spectrum was obtained on a Varian Associates XL-100 NMR spectrometer equipped with a Nicolet TT-100 data system. Major peaks are assigned in Table 2 . Minor peaks arise from the presence of cis-trans isomerism at the amide bond (16).

Table 1 Proton-NMR Data for Captopril Proton COOH CL-CH B-CH2 Y-CH,L 6 -CH2 9 -CH-C -STHA -CH3 L SH

'

Chemical Shift ( 6 )'PPM

from TMS (ext.

9.81 4.60 2.03 2.07 3.63

(m) (m)i 2.25 (m) (m) (m)

2.44 2.82 1.17 1.53

(d,q) J=6,9 (m) (d) J=6 (ad) J=9,8

(s)

multiplicities: d=doublet; q=quartet; m=multiplet. J=proton-proton coupling constants in Hertz.

0 F

I

CJ

k

X 5:

a, ZJ

x

..k m

c

4J

$ (II

k 4J

c

H

f *

4

92

i

0 0 4 I I 4

X

c a

L! rd

-d

3

.. c, d

$ k

m

4J

c

H

93

CAPTOPRIL

4

Table 2 Carbon-13 NMR Data f o r C a p t o p r i l i n CD30D. Carbon #

Chemical S h i f t ( 6 )

1 ppm from TMS

175.69 59.84 30.03 25.49 48.24 174.91 43.1 17.07

28.1 R e f e r e n c e d from c e n t e r peak o f t h e C D 3 0 D m u l t i p l e t a t 4 9 . 0 ppm 4.13

Ultraviolet Spectra

S p e c t r a of c a p t o p r i l i n a q u e o u s m e t h a n o l , ( F i g . 9 ) w a t e r , 0.1M sodium h y d r o x i d e and 0 . 1 M h y d r o c h l o r i c a c i d ( F i g . 101, a r e p r e s e n t e d (17). These s p e c t r a d e p i c t a n end a b s o r p t i o n s l o p e w i t h o u t peak o r s h o u l d e r . The s l o p e s p e c t r u m i n 0 . 1 M sodium h y d r o x i d e was s h i f t e d c o n s i d e r a b l y towards h i g h e r wavelengths. S i n c e weak s u l f h y d r y l a b s o r p t i o n i s r e p o r t e d ( 1 8 ) i n t h e 220-230 nm r e g i o n , t h i s a b s o r p t i o n s h i f t may b e d u e t o i o n i z a t i o n o f t h e s u l f h y d r y l f u n c t i o n by t h e a l k a l i . This s h i f t towards higher wavelengths w i t h i n c r e a s e i n pH h a s been u s e d by O n d e t t i ( 1 9 ) t o d e t e r m i n e t h e pK o f t h e s u l f h y d r y l i n captopril (Section 8.32).

HAROLD KADIN

. I

;

. !. !. .. . . ./ i ! I

. . . . . .

0 1

. . . .

.

.

.

.

.

.0,

'

.a,*

,

,

,

4

,

-

Fig. 9. U l t r a v i o l e t absorption s p e c t r a of C a p t o p r i l i n 10%aqueous methanol s o l u t i o n . Instrument: Cary 15.

Fig. 10. U l t r a v i o l e t absorption s p e c t r a of C a p t o p r i l i n H20, 0.1E H C 1 and 0.1N NaOH Instrument: Cary 15

CAPTOPRIL

95

The spectra suggest that there is a maximum at about 200 nm, attributable to the thiol function. However, precise determination of the peak absorbance was difficult because the extremely large blank absorbances prevented maintenance of a stable balance at this wavelength. Consequently the peak maximum is uncertain in Figure 9. However the peak absorbance is valid in Figure 15 in which the solvent was an HPLC mobile phase (System 4 - Table 8 - Section 6.32). 4.14

Mass Spectrum

The mass spectral pattern indicated in Figure 12 was obtained (20) on an AEI MS-9 Mass Spectrometer. The fragmentation, responsible for the spectrum in Figure 12+ is depicted schematically in Figure 11. The M of m/z 217 and the other fragments are consistent with a sulfur-containing compound of the composition C 9 H1 5NO 3 S (Section 1.1). Fiaure 11 Mass Spectral Fragmentation Schematic Captopril

170

103

m/z 199

+

m/z 202

+ CH3

F

M+ m/z 2 1 7 , 1 m / z 1{3

I

J

L m / z 171

H

172+m/z

m/z 70

r m / z 140

+

b m / z 126

+ CH2SH

H20

+ C02

SH

+ S C H 2 4 m/z 127 + C02

HAROLD KADlN

96

6640

SQ14225 BFlTCH ttNNQ23NB

90.> F U

II)

Z W

t-

Z

H

W

>

80-

78.-

7 50

H

t-

a _I

w

L l

Figure 12. Instrument:

Mass Spectrum of Captopril AEI MS-902 Spectrometer Equipped with Frequency Modulated Tape Recorder, Spectrum Processed o n Digital Equipment Corporation PDP-11 Computer

CAPTOPRIL

4.2

97

Solid State Properties 4.21

Polymorphism

An unstable, low (86OC) melting and a stable, high (106OC) melting form of captopril have been observed. These forms exhibited different unit cells (Section 4.26) on single crystal X-ray examination, differences in their powder X-ray (Section 4.27), and differences in the solid state infrared spectra (Figures 5 and 6). Agreement of their optical rotations, infrared in solution and bioassays established them as polymorphs.

4.22 Differential Thermal Analysis (D.T.A. 1 DTA of the high melting polymorph ( 2 2 ) yielded a sharp, well-defined endotherm at 106OC whereas the low melting polymorph produced a sharp endotherm at 86OC. When the low melting polymorph was allowed to resolidify and the DTA repeated, the endotherm at 86OC had disappeared and an endotherm at 106OC appeared. The latter suggests that the high melting form is the stable polymorph. A DuPont 900 Thermoanalyzer programmed for a temperature rise of 15' per min was utilized for these thermograms. 4.23

Melting Range

The U.S.P. (Class 1) melting range for the high melting polymorph was 105.2 - 105.9' ( 2 1 ) . This agrees well with its D.T.A. endotherm of 106OC. The low melting polymorph has a melting range of 87-88', in agreement with its DTA endotherm of 86O. 4.24

Differential Scanning Colorimetry (D.S.C.)

Use of DSC as a purity index for captopril is supported by titrimetric assays (17) of the carboxyl function (alkalimetry) and of the sulfhydryl function (iodimetry). For instance for batch 4 these yielded 99.6% for the carboxyl and 99.2% for the sulfhydryl in very good agreement with the DSC of 99.7% mole 8 ( 2 2 ) .

HAROLD KADIN

98

4.25

Hygroscopidity

Under ordinary conditions captopril is not hygroscopic. Equilibrium moisture studies (23) indicate no moisture pickup by captopril up to 50% relative humidity at room temperature. Above 50% R.H. it shows a tendency to cake after one to two days. Captopril did not exhibit any visual physical changes and remained dry from 0 to 67% R.H. on exposure for 14 days. Samples exposed to 81% R.H. for 14 days appeared moist (24). 4.26

Single Crystal X-ray Diffraction

Single crystal X-ray analyses have been completed (25) for both the low (melting range 86-87OC) and high (melting range 105-106O) melting polymorphs. Both forms are orthorhombic with the following crystal data: High melting polymorph a = 6.834(2), b = 0 8.821(2), c = 17.982(4)A; V = 1084A, " 3 space group P2 2 2 wiFh four molecules3per unit cell; calRefined to R = 0.04 culakeh density = 1.33 gcmfor the 745 observed single crystal intensities.

A.

.

Low melting polymorph a = 9.496(3), b -= 0 03 space group 12.304(3), c = 19.282(5)A; V = 2253A; P2 2121 witF eight molecules egr unit cell; Refined to R = calculated density = 1.28 gcm 0.06 for the 1093 observed single crystal intensities. B.

.

The structure in both has the S,S absolute configuration with a 2 ( T r a n s ) conformation about the N-C(0) amide bond (the O-C-N-C(2) dihedral angles vary from -4 to +6O). The molecular conformation differ in detail, most notably in the conformation about the (S)C-C(C0) bond. Atomic coordinates relative to orthogonal axes for the high melting form are:

99

CAPTOPRIL

S

N1

c2 c3 c4 c5 C6 06A 06B c7 07 C8 c9 c10

-7.010 -6.499 -5.799 -6.274 -7.067 -7.511 -6.188 -7.165 -5.394 -6.215 -5.3 08 -7.041 -6.163 -8.052

1.595 -1.729 -2.755 -2.492 -1.228 -1.024 -4.132 -4.364 -5.106 -1.576 -2.253 -0.624 0.238 -1.432

-5.766 -1.938 -1.152 0.282 0.252 -1.144 -1.626 -2.286 -1.204 -3.227 -3.754 -4.050 -4.947 -4.821

It was predicted that salt formation with resultant dissociation to a carboxylate anion would influence Capoten to crystallize in its less common E ( c i s ) conformation. This prediction was tested (25) by performing single crystal analysis on the dicyclohexylamine salt of captopril. The analysis indicated that the salt was indeed in the E conformation, i.e. the carbonyl groups of the amide and carboxyl functions are cis to each other. 4.27

Powder X-ray Diffraction

The stable, higher melting polymorph and the lower melting, metastable polymorph are shown in the powder X-ray patterns Figures 13 and 14 respectively (26). The values given in the patterns are also listed in Tables 3 and 4 for the high and low melters respectively. The tables also show the relative intensities (based on peak areas) of the various peaks. A powder X-ray pattern taken on the low melting polymorph after it was heated to about 95OC showed conversion to the stable form.

The X-ray pattern was taken with copper Ka, nickel filtered X-radiation.

100

a a u 0

w

k a, 4J

0

E

a,

P I

L

I !

'

i

I

i

101

0

PI

Table 3 Powder X-Ray Diffraction Data f o r Figure 13 (High Melting Polymorph) 2-13 (DEG.)

9.99 11.35 14.24 16.45 17.21 17.98 19.25 19.85 20.78 22.23 24.52 25.03 25.97 26.56 28.26 29.79 30.81 31.66 33.45 34.28

D(ANGSTROMS)

8.85 7.80 6.22 5.39 5.15 4.93 4.61 4.47 4.27 4.00 3.63 3.56 3.43 3.36 3.16 3.00 2.90 2.83 2.68 2.61

PEAK

22.7 37.9 15.2 21.0 57.1 47.5 34.4 109.1 37.7 49.8 24.1 15.3 62.6 13.7 68.7 10.6 8.4 9.1 8.3

17.9

REL. PEAK

0.208 0.347 0.139 0.192 0.523 0.435 0.315 1.000 0.346 0.456 0.221 0.140 0.574 0.126 0.630 0.097 0.077 0.083 0.076 0.164

AREA

88.1 138.4 122.3 77.5 169.6 166.8 146.3 324.5 130.1 161.7 86.4 60.2 326.6 68.2 243.1 102.0 42.5 43.9 47.9 98.2

REL. AREA

0.270 0.424 0.374 0.237 0.519 0.511 0.448 0.994 0.398 0.495 0.265 0.184 1.000 0.209 0.744 0.312 0.130 0.134 0.147 0.301

Table 3 (Continued) ~ - ~ ( D E G . ) D(ANGSTROMS)

PEAK

36.17 36.34 37.53 38.63

14.8 13.3 9.3 15.3

2.48 2.47 2.40 2.33

REL.

PEAK

0.136 0.122 0.085 0.140

AREA

62.3 37.5 64.9 73.3

REL.

AREA

0.191 0.115 0.199 0.224

Sorted Data (Highest Peak First) z-O(DEG.) 19.85 28.26 25.97 17.21 22.23 17.98 11.35 20.78 19.25 24.52 9.99

D(ANGSTR0MS) 4.47 3.16 3.43 5.15 4.00 4.93 7.80 4.27 4.61 3.63 8.85

PEAK

109.1 68.7 62.6 57.1 49.8 47.5 37.9 37.7 34.4 24.1 22.7

REL.

PEAK

1.000 0.630 0.574 0.523 0.456 0.435 0.347 0.346 0.315 0.221 0.208

AREA

324.5 243.1 326.6 169.6 161.7 166.8 138.4 130.1 146.3 86.4 88.1

REL.

AREA

0.994 0.744 1.000 0.519 0.495 0.511 0.424 0.398 0.448 0.265 0.270

Table 4 Powder X-Ray Diffraction Data f o r Figure 1 4 (Low Melting Polymorph) 2 - 8 (DEG.) 8.71 9.31 11.77 12.71 13.13 15.00 17.21 17.89 18.23 18.40 19.51 20.10 20.61 20.87 21.38 22.14 23.25 23.50 24.10 24.86 25.20

D (ANGSTROMS) 10.15

9.50 7.52 6.96 6.74 5.91 5.15 4.96 4.87 4.82 4.55 4.42 4.31 4.26 4.16 4.02 3.83 3.79 3.69 3.58 3.53

PEAK

24.5 13.7 14.3 14.0 16.6 31.8 14.7 14.0 39.5 34.8 16.9 13.1 30.6 13.1 13.0 62.5 13.9 12.1 16.8 12.5 17.4

REL. PEAK 0.392 0.219 0.229 0.224 0.266 0.509 0.235 0.224 0.632 0.557 0.270 0.210 0.490 0.210 0.208 1.000 0.222 0.194 0.269 0.200 0.278

AREA

68.7 26.3 89.1 43.0 69.4 142.4 43.2 36.8 101.0 77.2 91.2 57.3 112.0 34.8 33.8 265.5 69.1 34.4 113.4 43.4 83.3

REL.

AREA

0.259 0.099 0.336 0.162 0.261 0.536 0.163 0.138 0.380 0.291 0.343 0.216 0.422 0.131 0.127 1.000 0.260 0.130 0.427 0.163 0.314

Table 4 (Continued) ~-WDEG.)

25.88 27.33 28.35 28.94 30.05 34.47

D(ANGSTROMS)

PEAK

3.44 3.26 3.15 3.09 2.97 2.60

17.8 14.6 12.8 14.4 14.0 16.6

REL. PEAK

0.285 0.234 0.205 0.230 0.224 0.266

AREA

105.0 90.5 33.3 104.1 72.8 99.3

FEL.

AREA

0.395 0.341 0.125 0.392 0.274 0.374

Sorted data (Highest Peak First) 2-B(DEG.) 22.14 18.23 18.40 15.00 20.61 8.71 25.88 25.20 19.51 24.10 34.47 13.13 17.21

D(ANGSTR0MS) 4.02 4.87 4.82 5.91 4.31 10.15 3.44 3.53 4.55 3.69 2.60 6.74 5.15

PEAK

62.5 39.5 34.8 31.8 30.6 24.5 17.8 17.4 16.9 16.8 16.6 16.6 14.7

REL.

PEAK

1.000 0.632 0.557 0.509 0.490 0.392 0.285 0.278 0.270 0.269 0.266 0.266 0.235

AREA

265.5 101.0 77.2 142.4 112.0 68.7 105.0 83.3 91.2 113.4 99.3 69.4 43.2

REL.

AREA

1.000 0.380 0.291 0.536 0.422 0.259 0.395 0.314 0.343 0.427 0.374 0.261 0.163

HAROLD KADlN

106

4.3

Solution Data 4.31

Solubility

Captopril at 25OC is freely soluble (1 to 10 parts solvent to 1 part solute) in water, methanol, ethanol (SD3A), isopropanol, chloroform, or methylene chloride. However, it is only soluble (10-30 parts solvent to 1 part solute) in ethyl acetate (27). The solubility of captopril in water, at 250CI is 160 mg/ml (28). A solubility-temperature profile of captopril in water obeyed a linear equation up to 4OoC (28). Beyond this temperature captopril showed extraordinarily high water solubility. Solubility in sesame and corn oils was less than 1 mg/ml at 25OC, whereas the solubility in the synthetic oil triacetin (glyceryl triacetate), at 25OC, was greater than 20 mg/ml (29). 4.32

pKa

-

The pK of the carboxyl of captopril (pK ) is reported (23) to be 3.7. Whereas a carboxyl break was readily observed with alkali potentiometry, the sulfhydryl break could not be detected (17). Therefore, the pK of the sulfhydryl in captopril (pK ) was not estfmated by classical potentiometgy. It was, however, estimated at 9.8 (pK2) by Ondetti (19) and Weiss (30) using sulfhydryl U.V. shifts to higher wavelengths with increase in pH (Section 4.13). The method utilized was adapted from Benesch and Benesch (31). 4.33

Metal Complex Formation

Captopril was modelled (11) as a selective and competitive inhibitor of the angiotensin converting enzyme (Section 2 ) . Part of this inhibition resides in the binding of the zinc cofactor within the enzyme's active site by captopril's thiol function. Constrained within the active site by the multiple interactions of site and inhibitor, captopril bars entry of angiotensin I and thus prevents its conversion to the most powerful natural pressor, angiotensin 11. Since captopril lacks an amino group, it does not

107

CAPTOPRIL

complex metals in solution with the well-documented avidity (32,33) of amino group bearing thiols like cysteine, glutathione, and in vivo metal depletors like 2 - m e r c a p t o p r o p i o n y ~ g l y c i n e and penicillamine. Indeed, Weiss (30) reported that an alkali potentiometric study of the extent of zinc ion complexation with cysteine (I), 2mercaptopropionyl glycine (11), and captopril (111) indicates the order of binding, at pH 7.4, to be I > I1 > 111. Captopril binds mercuric ion (Section 7 ) to block its colorimetric reaction with Ellman's reagent, thus allowing a measurement of non-sulfhydryl colorimetric interferences. 4.34

Optical Rotation

The optical rotation of the captopril in absolute ethanol (34), using the Perkin-Elmer 155 Automatic Polarimeter, was determined to be: a = -127.8'. The R,S - isomer rotates at about +5'? 4.35 Partition Coefficients A partition ratio (solvent/aqueous) after shaking equal volumes of cosaturated aqueous (pH 2) and methylene chloride was 1.39 ( 2 1 ) . A comparably determined partition ratio between equal volumes of cosaturated 0.1M HC1 and octanol was 1.9 (30). When utilizing salcing out partition from aqueous acid into methylene chloride at the captopril concentrations prevalent in the urine analysis (about 25-50 mcg/ml for a 100 mg dose) NaCl but not Na2S04 was found to enhance captopril oxidation (Section 7). This has been attributed to trace chlorine generation from acidic chloride plus oxygen.

5.

Stability 5.1

Solid State Stabilitv

No significant decomposition was detected (35) in SQ 14,225 bulk samples, stored at +5'c, +33'C and +5OoC f o r up to 6 months or exposed to 900 foot-candles in a light box f o r 30 days, when compared to -2O'C samples which served as the

HAROLD KADIN

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c o n t r o l . Samples were examined f o r a p p e a r a n c e , c o l o r , o d o r , LD s a f e t y and by q u a n t i t a t i v e TLC and IlPLC , i o d i m 2 p r i c t i t r a t i o n , i n f r a r e d , and optical rotation. 5.2

Solution S t a b i l i t y

C a p t o p r i l i n aqueous s o l u t i o n undergoes an oxygen f a c i l i t a t e d , f i r s t o r d e r , f r e e r a d i c a l oxidation a t its t h i o l to yield captopril d i s u l f i d e ( 2 8 ) . H y d r o l y s i s a t t h e amide l i n k a g e o c c u r s o n l y u n d e r f o r c i n g c o n d i t i o n s (see S e c t i o n 5 . 2 5 ) . O x i d a t i o n w a s d e l a y e d by a d j u s t m e n t t o lower pH, a d d i t i o n o f c h e l a t i n g a g e n t s , i n c r e a s i n g captopril concentration, u t i l i z a t i o n of nitrogen o r l o w oxygen h e a d s p a c e , and i n c o r p o r a t i o n o f a n t i o x i d a n t s . O x i d a t i o n seems t o o c c u r l e s s r e a d i l y i n methanol ( 3 6 ) . N o d e g r a d a t i o n o f c a p t o p r i l w a s o b s e r v e d ( 4 0 mcg/ml) i n t h i s s o l v e n t f o r up t o 2 weeks a t 5OC. 5.21

S t a b i l i t y and S o l u t i o n pH

O x i d a t i o n r a t e c o n s t a n t s a t v a r i o u s pli v a l u e s ( 2 8 ) i n Table 5 , s u g g e s t t h a t c a p t o p r i l i s o p t i m a l l y s t a b l e below pH 3.5, t h e o x i d a t i o n r a t e b e i n g e s s e n t i a l l y c o n s t a n t from pH 2 t o 3. The r a t e c o n s t a n t s i n c r e a s e r a p i d l y above pH 4 . Using HPLC and c o l o r i m e t r y ( 3 8 ) , c a p t o p r i l a q u e o u s s t a b i l i t y w a s s t u d i e d , a t 50 mcg/ml, i n a r o t a t i n g b a s k e t d i s s o l u t i o n a p p a r a t u s f o r up t o 1 8 0 m i n u t e s a t 37OC i n d i s t i l l e d water, and a t pH 1, 2 and 3 . E x c e l l e n t s t a b i l i t y a t pH 1 and 2 b u t a p p r e c i a b l e d e g r a d a t i o n a t pH 3 , and i n d i s t i l l e d w a t e r w a s observed. S u r p r i s i n g l y , t h e r a t e of d e g r a d a t i o n a t pH 3 exceeded t h a t i n d i s t i l l e d water. The more r a p i d o x i d a t i o n a t pH 3 w a s a t t r i b u t e d t o c a t a l y s i s v i a g r e a t e r t r a c e m e t a l s o l u t i o n from the dissolution baskets.

109

CAPTOPRIL

Table 5 Oxidation Rate Constant for Captopril (5 mg/ml) in Citrate-Phosphate Buffers at Various pH Values at 5OoC PH

Rate-Eonstan (day ) x 10f 8.38 9.01 8.22 8.31 9.92 9.13 12.94 19.43 28.93 42.03

2.13 2.59 2.89 3.13 3.53 3.88 4.23 4.67 5.16 5.63 5.22

Solution Stability, Metal Ions and Chelating Agents

Transition metal ions most effectively catalyze oxidation of captopril through a recycling of oxygen free radicals (28). The most effective of these catalysts are ubiquitous copper and iron, in given order. As little as 1 ppm of copper has been observed to catalyze captopril oxidation in solution (28). As has been demonstrated with cysteine (39) lower levels of disodium edetate (EDTA Na2) may enhance metal ion catalyzed thiol oxidation, whereas higher levels retard oxidation. Disodium edetate 0.1% (Na2EDTA 0.1%) best stabilized 0.5 mg captopril per ml (of citrate-phosphate buffer at pH 4, p = 0.5) in Teflon-faced rubber sealed vials (37). Analysis of urinary captopril was necessary for dosage form bioavailability and dose titration studies. The necessity for long term storage of samples prior to analysis required development of an acid-chelate stabilization (40). This stabilization utilized diethylenetriamine pentaacetic acid (DTPA) reputed (40) to be a more effective metal chelator than Na2EDTA. A

HAROLD KADIN

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relatively large amount of DTPA (about 4 0 0 mg in 5 ml) was mixed into the periodic urine voiding. This was followed by an acidification to about pH 2 with a mixture of citric and oxalic acids and a rapid refrigeration. DTPA, citric and oxalic have been reported ( 4 1 ) to be effective sequestering agents in the pH range 2 to 5 for Al, Cu, Fe, Ni and Zn. Analysis of captopril in urine samples stabilized in this manner, before and after 90-120 days of refrigeration, agreed remarkably well(40). 5.23

Concentration and Solution Stability

The greater the captopril concentration, the slower the oxidation ( 2 8 ) . For example, no significant degradation (within + 3 % ) could be detected by automated Ellman colorimgtry ( 4 2 ) at a concentration of 2 5 0 mg captopril per ml of solution at pH 1 2 . 5 - 1 4 after overnight room temperature storage in uncovered beakers. At the opposite extreme, captopril at an analysis concentration of 2 5 - 5 0 micrograms per ml of solution at pH 1 3 . 5 stored overnight at room temperature in open tubes lost 8 4 % of its sulfhydryl activity ( 2 1 ) . 5.24

Oxygen Tension and Solution Stability

The following 2 1 / 2 hour accelerated stability at 4OoC on 2 5 ml of 1 mg captopril per ml solutions in unstoppered 100 ml volumetric flasks using air, oxygen, or nitrogen purging, where indicated, was carried out ( 2 8 ) . Table 6 Oxygen Tension and Solution Stability M

PH

1 2 3

0.08 0.08

4

0.16 0.16

5 6

0.08 0.04

7.9 7.9 7.5 7.5 7.8 7.8

Code

Na2HP04

(KH2P041

PPm Cu 2 2 10

10

Purge None Air None Air

2

N2

1

O2

8 Captopril Recovered (HPLC) 73 58 88 62 96 0

111

CAPTOPRIL

Under these conditions it is apparent that oxygen causes rapid, complete degradation, air facilitates degradation, and nitrogen protects captopril in solution. 5.25

Amide Hydrolysis and Solution Stability

Captopril solutions at 5 mg per ml in 0 . 5 M HC1, containing 0.1 mg EDTANa /ml to minimize oxidation , were heated at elegated temperatures. The rate of hydrolysis was monitored by HPLC. The data yielded first order linear plots from which Wang (43) calculated rate constants (Table 7). An Arrhenius plot yielded a heat of activation for amide hydrolysis of 21.4 kcal/mole, comparable to other amides. Table 7 Rate Constants for Captopril Hydrolysis

4OoC 4OoC 22oc 22oc

PH PH PH PH

3

4 3 4

k -

t90%

-1 5.5 x lo,*-7 hr-l 5.5 x hrml 6.7 x 10-lOhr -1 6.7 x 10 hr

10 10 86 86

years years years years

It is clear from these t ' s (times for 90% amide hydro1ysis1 , that hydro188f s contributes insignificantly to degradation.

HAROLD KADIN

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6.

Analytical Tests and Methods 6.1

Elemental Analysis

The following results were obtained: Element Calculated C H N S

49.78 6.96 6.45 14.77 6.2

Found for Batch 5 49.97 6.84 6.50 14.63

Spectrophotometric Methods 6.21

Colorimetry

Spectrophotometry for captopril has included the widely utilized Ellman's reaction (44) in which sulfhydryl reduction of 5,5'-dithiobis-2nitrobenzoic acid yields a mole of intensely yellow 2-nitro-5-thiobenzoate anion per mole of captopril. Manual (40,451 and automated colorimetries have been utilized for captopril analysis in chows (Section 7.71), urine (Section 7.72) and in pharmaceutical formulations by TLC (Section 6.31). Kinetic colorimetry vs pH (from pH 5 to 10) studies (46) of a manual version of Ellman's colorimetry of captopril established that the pH optimum was at pH 7 (1 M phosphate, 0.05 M EDTA) Maximum color was attained within 2 min. and stability was maintained for at least 45 min.

.

The S-nitroso-Bratton-Marshall colorimetry (47) has been applied to analysis for captopril in various formulations (48). It was adapted from a method (47) for cysteine in biological materials. In this method the thiol reacts with nitrous acid to form a relatively stable S-nitroso derivative. Excess nitrous acid is destroyed by sulfamic acid. The S-nitroso derivative is then hydrolyzed by mercuric ions to release nitrous acid. The latter diazotizes sulfanilamide, presumably at a faster rate than destruction of the nitrous acid by

CAPTOPRIL

113

excess sulfamic acid. The diazotized sulfanilamide is then coupled to N-(1-naphthy1)ethylenediamine to yield a stable measureable red azo dye. The method was found to be less suitable for analysis of captopril in urine than Ellman's Reaction (Section 7.72). Five simple, spectrophotometric identity tests for captopril reported by Valatin (49) include the observation of an evanescent red when captopril reacts with nitrous acid. In addition captopril color tests were described yielding purple with nitroprusside, blue with ferric chloride, red on addition of neutral N-ethylmaleimide followed by strong alkali, and finally orange-yellow (specific for proline) on acid hydrolysis followed by neutralization then reaction with ninhydrin. 6.22

Fluorometry

Captopril was reacted with N-[p-(2-benzoxazoyl)- phenyl] maleimide in pH 6.85 buffer and the fluorescence of the captopril-maleimide adduct was then measured at 310 nm excitation and 365 nm emission (49). For coupling of captopril to N-(7-dimethylamino-4-methyl coumarinyl) maleimide to yield a highly fluorescent derivative see Section 7.5. 6.3

Chromatosraphic Methods 6.31 Thin Layer Chromatography (TLC)

For quantitation of captopril (purity assay) standard and sample solutions are each chromatographed at 100 pg and 200 pg levels on Analtech silica gel G plates using the solvent system benzene-acetic acid (75:25). The captopril zones on the plate are located by spraying a guide-channel with a basic methanolic solution of 5,5'-dithiobis-2-nitrobenzoic acid. Captopril on the guide-channel appears as a yellow zone. The silica gel containing captopril zones on the untreated portion of the plate are removed from the plate, eluted with 5% aqueous trichloroacetic acid, and reacted with a methanolic solution of 5,5'-dithiobis-2-nitrobenzoic acid, at an alkaline

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pH, to form an intensely yellow 2-nitro-5thiobenzoate anion that is measured at 412 nm in a spectrophotometer (50)

.

In a thin-layer semi-quantitative procedure for measuring the individual impurities in captopril, samples are chromatographed at 100 pg and 200 pg levels and standards are chromatographed at concentrations ranging from 0.5 pg to 4.5 pg. TLC separation is carried out on Analtech Silica Gel G plates, developed in conventional and in continuous development chambers, using the solvent system benzene-acetic acid (75:25). After development, the plates are air dried and placed in a chamber saturated with iodine vapors. A semi-quantitative estimate of the concentration of each impurity in the sample is made based on a visual comparison of the size and intensity of each impurity zone with the appropriate standard zones (50). These TLC procedures, after appropriate initial extractions, have been adapted for formulations. They have also been applied to semi-quantitative analysis of captopril disulfide in tablets and as a TLC identity test for captopril in tablets (50). Streaked or spotted plates should be introduced into the developing chamber (2 per chamber) immediately after applying the solutions to the plates. This minimizes the conversion of captopril to its disulfide. Placebo powders were spiked with known amounts of captopril and assayed using the TLC procedure. The results of 15 assays gave a recovery of 100.6%, a standard deviation ( s ) of 1.39 and a coefficient of variation (C.V.) of 1.38 (50)

6.32

High-Performance Liquid Chromatography ( HPLC)

Three HPLC systems were investigated (51) for selective separation of captopril from pharmaceutical excipients, synthetic intermediates, degradation products and impurities. These included anion exchange, amino, and

CAPTOPRIL

115

octadecylsilane (ODS) systems. The first and second appeared not to be the systems of choice when they did not achieve necessary separations of pharmaceutical excipients from captopril. A heavily loaded version of the third system ( 1 5 % O D s , monolayer bonded on silica, Partisil ODS-2) was selected as optimum for bulk and tablet analysis. The third system was clearly superior. Nevertheless, the first two systems had very occasional usage when formulations contained excipients which interfered with captopril or its disulfide analysis in the heavily loaded C18 system. The characteristics of these systems and of several others investigated are summarized in Table 8 below. Since captopril has a U.V. absorption maximum at about 2 0 0 nm with broad end absorption (see Figure 1 5 ) U.V. detection was at 230 nm or lower, compatible with a good baseline and low solvent interference inherent in a good signal to noise ratio. An alternative to U.V. detection, for captopril per se (see Section 7.4 for captopril U.V. derivative HPLC) is the electrochemical detector (systems 8 and 9) introduced for sulfhydryl analysis by Saetre and Rabenstein ( 5 2 ) . The electrochemical detector's near neutral detection potential makes it especially thiol selective. It has been reported (53) to have a sensitivity at least 200 fold over that of U.V. detection.

Fig. 15. U.V. Spectrum of C a p t o p r i l in Mobile Phase System 4 (Table 8)

Instrument: Beckman Acta C I11

116

-

Table 8

C a p t o p r i l HPLC S y s t e m s System No.

1

2

Flow Rate ml/min 0.6

1.0

Loop Injection Volume p 1 20

20

S t a t i o n a r y Phase

Mobile Phase

250 x 4 . 6 mm P a r t i s i l SAX 10um S t r o n g a n i o n Whatman

U.V. 5 2 5 mg c i t r i c acid. H20 220 nm +37.7mg N H 4 C I T to 1 liter with CH30H a d j u s t t o p H 3.30 w i t h U.V. 0.1 N HC1 220 nm

300 x 3 . 9 mm u Bondapak NH2 1 0 um a m i n o

Detection

Remarks and Reference 51

51

0 . 0 1 M N a EDTA i n 0.05% SACCH3CN, 9 5 : 5

U.V. 2 2 0 nm

51

CH,OH-H,O-85% ~ ~ $ 20 7 : 7~ 5: '

U.V. 2 2 0 nm

51

Waters 3

1.0

20

250 x 4 . 6 mm P a r t i s i l ODS 5% 10um ODS monol a y e r Whatman

0.1

System No.

Flow Rate ml/min

Loop Injection Volume p 1 Stationary Phase 250 x 4 . 6 mm Partisil ODS 15% lOpm ODS mono layer Whatman

Mobile Phase

Detection

CH OH-H 0-85% H3$04, 3 0 :50: 0.05

U.V. 220 nm

4

1.0

20

5

1.0

20

6

1.0

1.0 (ml)

CH OH-H 0 - 1 8 H3$04, $3 :57 : ODS + trimethyl- 1.0 silylimidazole monolayers Shandon

7

1.0

20

2 5 0 x 4 . 6 mm Partisil ODS 5 % 10pm ODS mono layer Whatman

200

mm Hypersil

+

TSIM 5um + trimethylsilylimidazole monolayers Shandon ODS ODS

H go4, 47.5: 52.5:l. 0

200 mm Hypersil ODS + TSIM 5pm

CH30H-(0.1 M KH PO +

152mMfi2P04) ,

55% :456

U.V. 230 nm

U.V. 230 nm

Remarks and Reference 51 Optimum resolution system

54

54 As say disulfide in low potency captopril formulations

Electrochem., 53 + 0.10 V , Hg Pool vs Ag/AgCl

8

0.5

20

300 x 4.6 mm MC Chromegabond 209 10um heavily loaded ODS ES Industries

CH30H-(0.05 M KH PO + 30mM H $0 53% :15%

Electrochem., 55 + O.lOV, Adapted Hg film for vs. Ag/AgCl "total" urinary captopril analysis

250 x 4.6 mm Partisil ODS 5% 10pm ODS Whatman

CH OH-H 0-85% H3d04, ?0:80: 0.1

U.V. 214 nm

f,

9

1.0

10

1-2

Waters 300 x 3 . 9 mm p Variable Porasil 1Opm Volume silica gel normal phase Waters

CH C1 : HAc, 9 l2

U.V. 240 nm

11

0.5-1

Waters 250 x 4.6 mm Variable Lichrosorb RP18 Volume 5pm "capped" ODS Merck

H O(to pH 3.0 z6.5 M H3P04) - CH3CN, 65 : 35

U.V. 220 nm

12

100

--

c

W

20

ODS = Octadecylsilyl

T

30 x 5.7 cm Prep- CH OH-H20-0.05 PAK C18 ODS M PO 45 : Waters 55 ? 0?65

d

RI

56

57 Qua1 Sepn. of captopril from its R,S isomer

.

58

59 Prep. Sepn. captopril impurities

HAROLD KADIN

120

6.33

Gas Liquid Chromatography (GLC)

A c a p t ~ p r i l - ~synthesis ~c separated captopril from its R,S optical isomer by GLC of its methyl ester (after CH2N2 treatment) on 1.5% OV-17 at 18OOC (60). The gas chromatogram of the pre-resolution isomer mixture showed two peaks of almost the same peak area. After a dicyclohexylamine resolution, GLC of the desired captopril (S,S fraction) indicated a single peak at the retention time of the second peak in the chromatogram of the mixture. The method is recommended, over the more conventional, appreciably less sensitive, measurement of optical rotations (Section 4.34), for process control of the isomer separation. G.C.-mass spectrometry (Section 7.2) and G.C.-flame photometry (Section 7.3) of the N-ethyl maleimide adduct (Section 7 ) of captopril as methyl and heptafluoroisopropyl esters, respectively, have also been reported. 6.4

Titrimetric Methods

Acid iodate (Iodine) titration, before and after chromatography through a Jones Reductor Column was used to measure disulfide in captopril (61). For small amounts of disulfide this technique measures, relatively inaccurately, small differences between two large numbers. Therefore the subsequently developed, more accurate TLC (Section 6.31) and HPLC (Section 6.32) direct assays are preferred. However the iodine titration, with starch indicator, is useful as a thiol purity assay (61). A "Dead Stop" end point indicator (62) makes the titration more amenable to automation. 7.

Analysis in Biological Fluids and Tissues and in Animal Rations

Extreme instability of captopril in biological media necessitated development (63) of an immediate, quantitative thiol derivatization with N- ethylmaleimide (NEM) followed by freezing. Studies (64) with radioactive captopril in whole blood had indicated that a 5 min delay before addition of NEM resulted in a 10% l o s s of

CAPTOPRIL

121

captopril whereas a 30 min delay yielded a 65% loss. The NEM derivative was found (65) to be stable in frozen whole blood or in blood stored at 5OC for at least 3 months: it was unstable in a frozen or 5OC phosphate buffer (pH 6 . 8 5 ) when stored for more than 4 weeks. Antioxidants and metal chelators were ineffective (63) as captopril stabilizers in whole blood. Long term stabilization, at the relatively high concentrations of 1 to 100 mcg per ml prevalent in urine after captopril dosage, has been achieved (40) with metal chelators, acid pH, and 5 O C storage (see Section 5.22). Stability and homogeneity analysis of captopril in animal feeds was required for multiple long range toxicological studies. Despite a high proportion of thiol reactive air, inorganics, and proteins in the feeds, stabilization during the analysis was achieved (46) by an isopropanolic extractant fortified with trichloracetic acid and metal chelators (see Section 7.71). 7.1

Thin Layer Radiochromatography (TLRC)

NEM stabilization allowed TLRC of captopril in whole blood (63). Aliquots of whole blood were analyzed for total radioactivity and NEM- treated aliquots were extracted with methanol. Reconstituted residues of the extracts were applied to silica gel GF plates, developed with chloroform/ethyl acetate/glacial acetic acid (4:5:3) and analyzed for radioactivity associated with captopril and its disulfide by zonal analysis. The limit of detection was about 10 ng captopril/ml of blood. In TLRC analysis of the highly radioactive captopril preparations used for drug metabolism studies the thiol of captopril is particularly susceptible to free radical degradation (Section 4.2). Reaction of thiol across the highly active carbonyl double bond of formaldehyde (incorporated in the mobile phase) protects the radioactive captopril during the chromatography (66).

HAROLD KADIN

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When dilute aqueous solutions of captopril and an excess of formaldehyde are used the product of the reaction has been shown by NMR and IR to be the captopril hemithioacetal.

cp

COOH

The hemithioacetal can be reverted to captopril, for sulfhydryl analysis, by acid, base, or bisulfite. 7.2

Gas Chromatography-Mass Spectroscopy (GC-MS)

NEM-Captopril has been determined by GC-MS in the selected ion mode (67) in whole blood, urine (68), amniotic fluid (691, milk (69), and tissues (68) (liver, kidney, lung, and placenta). For determination in whole blood, proteins are precipitated from vortexed whole blood plus internal standard with freshly prepared 10% metaphosphoric acid. The NEM-captopril is absorbed from 0.45 pM membrane filtrates on purified suction-dried Amberlite XAD-2 resin and eluted with a freshly purified (neutral alumina chromatography) ethyl acetate. The drug is extracted from the ethyl acetate into 5% sodium bicarbonate. After acidification and saturation of the aqueous layer with sodium chloride, the NEM derivative is extracted with ethyl acetate. After evaporation of the solvent extract, samples are methylated for GC-MS. The G.C. was carried out with dried helium gas at 6 ml/min on a 3 % OV-101 column with injector at 28OOC and column temperature programming at 200-280°C at 20°C/min. All glass surfaces had been previously silanized with Sylon-CT. The spectrometer was a modified Electronic Associates Quad 300 quadropole. The samples (1 p l ) were coinjected with 0.5 p 1 of N,O-bis(trimethylsily1) trifluoroacetamide. The latter was used to prolong column life. The peak height intensity

CAPTOPRIL

123

data of the m/z 230 and 248 ions was collected by selected ion monitoring. The limit of detection and the practical detection limit with a 90% confidence limit are 5.5 and 16.5 ng per ml of blood, respectively. The internal standard, an NEM blocked, deuterated or fluorinated derivative of captopril, compensates for extraction and other possible losses. The procedure described (67) could be applied to most of the biological media mentioned with but slight revision. However milk required a greater modification. The high fat content of milk made XAD-2 chromatography unfeasible. Therefore NEM-captopril was instead extracted from sodium chloride saturated milk samples with ethyl acetate. 7.3

Gas Chromatography-Flame Photometric Detection (GC-FPD)

A GC method for captopril in blood and urine utilizing a sulfur selective dedicated FPD has been reported (70). Blood is treated with a s o h . of N-ethylma1eimi.de in phosphate buffer s o h (pH 7.4) and with metaphosphoric acid (10% soln); after centrifugation, the supernatant soln. is extracted with ethyl acetate, and, after further purification, the extract is evaporated, the captopril-N-hexylmaleimide adduct is added as internal standard, and the mixture is treated to convert the two adducts into their hexafluoroisopropyl esters for analysis by g.1.c. at 215O on a glass column (1 m x 3 mm) packed with 2 % of OV-210-yn Gas-Chrom Q (80 to 100 mesh), with N (50 ml min ) as carrier gas and flame-photometric detection. Urine is analysed similarly, but without deproteinisation. The calibration graph (peak-height ratio vs. concn.) is rectilinear for 1 to 5 ug ml- of I.

The sensitivity though not specified appears to be in the microgram range. It is therefore not satisfactory for human blood studies which are in the nanogram range.

HAROLD KADIN

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7.4

High Performance Liquid Chromatography with U.V. Detection (HPLC-UVD)

An HPLC procedure for captopril in whole blood and urine has been reported (71). Captopril is thiol-blocked with p-bromophenacyl bromide (~BPAB)or N-(4-dimethylamino-3,5-dinitrophenyl) maleimide (DDPM), then the addition products were separated and determined by HPLC on a reversed phase column. Captopril in blood or urine could be derivatized with pBPAB then excess pBPAB removed by hexane extraction. The captopril disulfides in the same samples were then reduced by tributylphosphine to captopril which in turn were thiol-blocked with DDPM. HPLC of the adduct mixture thus allowed a separate analysis of captopril or disulfides, before and after reduction. The method is reported to be sensitive, and precise down to 5 ng per ml of whole blood and 0.1 mcg per ml of urine.

7.5

Spectrofluorometry

The drug, stabilized with dithioerythritol is absorbed on a Brinkmann XAD-2 column from an acidified diluted blood and eluted with pH 6.9 phosphate-30% dimethylformamide buffer. Captopril in the eluate is reacted with N-(7-dimethylamino-4-methyl coumarinyl) maleimide (DACM) to form a fluorescent derivative (Section 6.22). After acidification, the derivative is extracted into toluene. The fluorescence is measured at 375 nm/425 nm-excitation/fluorescence. The analysis was validated by the GC-MS procedure described above in the range of 0.5 mcg to 10 mcg per ml of blood (72). 7.6

Radioimmunoassay (RIA)

Development and application of a simple, highly sensitive RIA for NEM-captopril in blood plasma has been described (89). The lower detection limit is 2 ng per ml of plasma.

125

CAPTOPRIL

7.7

Semiautomated Ellman Colorimetry, (Figure 16) 7.71

Semiautomated Sulfhydryl Analysis in Laboratory Animal Rations

Feeds were extracted with isopropanol fortified with trichloroacetic, ethylenediamine tetraacetic, and diethylenetetramine pentaacetic acids. Pigments in the initial extract and turbidity on aqueous dilution necessitated a cleanup using isooctane after aqueous dilution of the extract. Further aqueous dilution of the isopropanol-water extract insolubilized trace isooctane and more polar lipids. Finally centrifugation in open top tubes removed the last traces of isooctane. In the design of the colorimetry from manual colorimetry ( 7 3 ) the sequence of adding Ellman's reagent before the alkali was reversed to maintain solubility of Ellman's within the autoanalyzer lines. In addition, methanol and aqueous buffer were added to Ellman's reagent to enhance both stability and solubility. Finally the pH of Ellman's reaction was maintained at pH 8.5 rather than at its optimum pH 7 (Section 6 . 2 1 ) to dissolve trace protein haze. Due to the limited selectivity of the colorimetry it was necessary to analyze for and subtract the response of drug- unfortified (blank) feed. The cleanup served to minimize this blank such that captopril could be analyzed down to 0.03% in a variety of animal feeds with good accuracy. 7.72

Semiautomated Ellman Colorimetry for Urinary Captopril and Its Disulfides

The necessity for long term storage of samples prior to analysis and the presence of an oxidation-prone thiol of captopril required development of an acid-chelate stabilization method for urinary captopril (Section 5.22). Thinlayer radiochromatography (Section 7 . 1 ) indicated that human urinary captopril was primarily free (unchanged) and, in almost equal proportion,

HAROLD KADIN

126

disulfide-conjugated with cysteine ( 7 4 ) . Relatively small amounts of captopril disulfide were observed. An electrochemical reduction (52) was utilized to release disulfide-conjugated captopril for thiol colorimetry. Of several rugged reduction cells evaluated, one with a VycorB Disc separating the anode and the mercury pool cathode was preferred. Methylene chloride partitions from acidified salt-saturated urines, before and after reduction, allowed the measurement of free and disulfide-conjugated captopril. The drug partitioned into the solvent whereas the aqueous phase retained acid protonated, amino group-bearing thiols like cysteine. Subsequent solvent evaporation volatilized other potential colorimetric interferences. An automated colorimetry of 25 samples per hour was developed by utilizing relatively thiol selective Ellman's Reagent, 5,5'-Dithio-Bis-(2-Nitrobenzoic Acid) (Sections 6.21 and 7.71). Results were confirmed by an HPLC method with electrochemical detection, developed while this method was in use (42). 7.8

High Performance Liquid Chromatography with Electrochemical Detection (HPLCECD)

-

Captopril in urine has been determined by an HPLC-ECD procedure in which the drug is separated from interferences on a monolayer bonded, heavily loaded (about 20% carbon) octadecylsilane reversephase column with a mobile phase of 55% methanol and 45% aqueous phosphate buffer (55). A commercially available thin layer mercury film detector was used (Section 6.32). The anodic current, resulting from the oxidation of Hg to Hg-captopril complex, was a linear function of captopril concentration. The peak height was reproducible with a relative standard deviation of 0.54% (n=5). Captopril concentration as low as 0.5 mcg per ml of urine can be quantitated. Sample treatment involved simply centrifugation, filtration, and deoxygenation with nitrogen. Recovery of captopril from urine spiked at 2.0 mcg per ml was better than 95%.

CAPTOPRIL

127

TECHNICON AUTOANALYZER FOR ELLMAN COLORIMETRY WASTE

P r RECORDER

IFLOW CELL I

COLOR I M ETER

WASTE

-

FUNCTION

NOMINAL ML/MIN ~~

2 MIN

MIXING c 01LS

YELLOW

HCI WASH

1.2

YELLOW'

CELL

1.2

YELLoW'

J

GREY

4

,TEA-EDTA

1.2

, AIR

1.o

HCI DIL

0.8

ELLMANS

0.8

WASTE * = SOLVAFLEX SOLVENT

RESISTANT TUBING, OTHER TUBING TYGON TEA-EDTA = 1.86% EDTA Nao 2H20 + 2Ooh TRIETHANOLAMINE + 0.02% TWEEN 80

ELLMAN'S =

0.08% ELLMAN'S

IN 50% MEOH (COLD) 0.01 M ACETATE pH 4.7 +

Figure 16

TECHNICON SAMPLER I I WITH 50 2/1 CAM

HAROLD KADIN

128

7.9

High-Performance Liquid Chromatography with Fluorescence Detection (MPLC-FD)

Jarrott et a1 (75) describe an assay for quantitating plasma captopril levels. Blood from patients taking this drug was collected into tubes containing edetate and ascorbic acid, and the plasma was separated by centrifugation. After addition of an internal standard, the plasma was deproteinized and the supernate was adjusted to pII 6.5. N-(l-Pyrene)-maleimide was added to derivatize captopril and an internal standard to fluorescent adducts. These derivatives then were extracted into ethyl acetate-benzene (1:l) and separated from other derivatized thiols by highperformance liquid chromatography. The sensitivity of the assay was 150 pmoles/ml. The HPLCFD uses a mobile phase of methanol-potassium phosphate buffer (5 mM, pH 6.5) (52:48) and the flow rate was 2 ml/min through a radially compressed octadecylsilane 10 cm x 8 mm (i.d.1 column and a spectrofluorometer with a 20 p 1 flow cell. Excitation was at 340 nm with emission taken at 390 nm. 8.

Drug Metabolism

-

Pharmacokinetics

Two features salient to the metabolism and pharmacokinetics of captopril include a) the reversible transformation of its thiol function to dimer and mixed disulfide (74,76-79) and b) the biological stability of its amide linkage (80, Section 3). The following will emphasize these aspects. 8.1

Blood Level Studies

Captopril is absorbed rapidly as indicated by measurable blood levels of the drug 15 min after ingestion (81). These findings are compatible with reports of the onset of antihypertensive activity as soon a s 15 min after a single oral dose (3) in hypertensive patients and with the rapid onset of blockade of angiotensin I-induced increases in blood pressure in healthy subjects (91). Other studies revealed that the captopril metabolites included its dimer disulfide and the mixed disulfides with glutathione, cysteine, and serum albumin (78,811

.

129

CAPTOPRIL

After single oral dosage with 100 mg radioactive captopril, ten healthy subjects attained a mean maximal concentration ( C ) in blood of 800 + 76 ng/ml at a mean time ) of 0.93 + 0.08 hours (8l,82). Methanol extr%%.on, ultrariltration, and dithiothreitol reduction studies (77) coupled with TLRC (Section 7.1) established that about 5 0 % of the total blood radioactivity was unchanged captopril, about 10% dimer disulfide and the remainder other polar metabolites including protein and non-protein mixed disulfides (81,82). Thereafter the captopril fraction of total radioactivity declined rapidly with time whereas the mixed disulfide fraction increased. The curvilinearity of semilogarithmic plots of these captopril blood levels with time indicated that half-life (t 1 , values could not be accurately calculated. #or unchanged captopril levels, curvilinearity may be due to the mixed disulfide fraction acting reversibly to replenish captopril, possibly extending the duration of pharmacologic activity (34,76-79). However plots of total radioactivity allowed calculation of t of 4.3 + 0.2 hours for the 4-12 hour time inter$al and 1g.4 + 2.6 hours Mean for the 12-48 hour time interval (81): concentrations of total radioactivity in blood (expressed as captopril equivalents) were 41 + 7 ng/ml at 24 hours and 20 + 5 ng/ml at 48 fiours after drug administration.

(Fax

Blood level and urinary excretion studies in

6 hypertensive patients, dosed over a 10 day

perigd, indicated that the metabolic disposition of C-captopril was the same at the beginning and end of the study, and was comparable to the disposition in healthy subjects (83). Radiolabeled captopril was administered on days 1 and 10, and blood and urine samples were analyzed for captopril and its metabolites by radiometric assay procedures. Non-radioactive drug (100 mg t.i.d.1 was used for dosage on Days 3 to 9. 8.2

Urinary Excretion Studies

In the fasting state, absorption averaged

7 0 - 7 5 % of an oral dose, as evidenced by human

urinary excretion studies (81,82,84). The presence of food in the gastointestinal tract

HAROLD KADIN

130

reduces absorption of an oral dose by about 35 to 40% (84).

At least 95% of the radioactivity recovered in urine after a 100 mg oral dose of radiolabelled captopril to mildly hypertensive patients was accounted for as captopril and specific urinary metabolites. Captopril and captopril-cysteine mixed disulfide each accounted for about 45% of the urinary radioactivity (about 3 3 % of the dose) and the disulfide dimer accounted for about 5% of the radioactivity in urine (about 4% of the dose) (74) Additional minor urinary metabolites, in rats and dogs, are S-methyl captopril, captopril-S-methyl sulfoxide, N-acetyl cysteine-captopril and glutathione-captopril mixed disulfides (80). These minor metabolites have not, as yet, been reported in man, but appear to be present in monkeys (90).

.

Renal dysfunction, as measured by creatinine clearance, decreased the excretion rate of captopril after a single 100 mg oral dose (85). The authors suggest a method of dosage reduction, based on creatinine clearance measurements, in cases of moderate to severe renal dysfunction. 8.3

Miscellaneous Distribution Studies

Biological stability of the amide function was confirmed by studies (80) on rat dermal collagen; uptake of radioactive proline was considerable, whereas insignificant uptake of captopril labelled in the proline moiety was observed. Whole body radioautography of rats indicated that captopril ( 5 0 mg/kg-intravenously) did not readily enter the central nervous system whereas it readily entered the placenta of pregnant rats (86) Captopril did not readily enter the breast milk of lactating women dosed with captopril at 100 mg t.i.d. for 7 days (87,88). Relatively insignificant milk peak levels were found (4.7 ng/ml)

131

CAPTOPRIL

9.

Acknowledgments

I would like to express my appreciation to individuals who have been very helpful for the contributions indicated: Drs. J. Fried and M. Porubcan - NMR, Drs. Y.J. Wang and T. Prusik Stability, Mr. A. Restivo and Mr. D. Domina Synthesis and Solubility, Drs. A. Cohen and P. Funke - MS and GC-MS, Ms. M. Malley and Dr. J. Gougatas - Single Crystal X-Ray, Mr. F. Dondzila, Mr. S. Perlman and Dr. J. Kirschbaum - HPLC, Mr. H. Roberts - TLC, Mr. R. Poet and Dr. G. Brewer Proof Reading and Manuscript Clarity, Ms. D. Walker - Word Processing, Dr. D. Cushman History, Drs. B. Migdalof and D. McKinstry - Drug Metabolism - Pharmacokinetics. 10.

References

1.

Ondetti, M.A., Williams, N.J., Sabo, E.F., Pluscec, J., Weaver, E.R.; and Kocy, O., Biochemistry, 10, 4033 (1971).

2.

Gavras, H., Brunner, H.R., Laragh, J.H., Sealey, J.E. Gavras, I., and Vukovich, R.A., New Eng. J. Med., 291, 817 (1974).

3.

Case, D.B., Atlas, S.A. Laragh, J.H., Sealey, J.E., Sullivan, P.A., and McKinstry, D . N . , Progr. Cardiovasc. Dis.; 21, 195 (1978).

4.

Cushman, D.W. and Cheung, H.S., Biochem. 20, 1637 (1971). Pharmac., -

5.

Rubin, B., Laffan, R.J. Kotler, D.G., O'Keefe, E.H., DeMaio, D.A., and Goldberg, 204, 271 M.E., J. Pharmacol. Exp. Therap., (1978).

6.

Quiocho, F.A. and Lipscomb, W.N., Adv. Protein Chem., 25, 1 (1971).

7.

Byers, L.D. and Wolfenden, R., Biochem.,

8.

Cushman, D.W., Cheung, H.S., Sabo, E.F. and Ondetti, M.A., Biochemistry, 16, 5484 (1977).

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2070 (1973).

12,

132

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10.

HAROLD KADIN

Cushman, D.W., Cheung, H.S., Sabo, E.F., and Ondetti, M.A., Fed. Proc., 38, 2778 (1979). Cushman, D.W., Pluscec, J., Williams, N.J. Weaver, E . R . , Sabo, E.F., Kocy, O., Chueng, H.S., and Ondetti, M.A., Experientia, 29, 1031 (1973).

11.

Ondetti, M.A., Rubin, B., and Cushman, D.W., 196, 441 (1977). Science, -

12.

New Drug Application No. 18-343 Submitted by E . R . Squibb & Sons Inc., Approved April 6, 1981.

13.

Levine, T.B., Franciosa, J.A., Cohn, J.N., 62, 35 (1980). Circulation, -

14.

Ondetti, M.A. and Cushman, D.W., United States Patent 4,105,776, August 8, 1978.

15.

Toeplitz, B., Personal Communication, April,

16.

Porubcan, M.A., Personal Communication, June,

17.

Whigan, D.B., Personal Communication, April,

18.

Karchmer , J H. , "Treatise on Anal. Chem. ' I , Part 11; Volume 13, Page 410 (1966), Edited By Kolthoff, I.M. and Elving, P.J., John Wiley Publishers.

19.

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20.

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21.

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22.

Valenti, V., Personal Communication, March,

23.

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1976.

1981. 1976.

.

1976.

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1976.

1976.

CAPTOPRIL

133

24.

Karten, J. and Kabadi, B., Personal Communication, July, 1980.

25.

Malley, M. and Gougatas, J.Z., Personal Communication, May, 1976.

26.

Jacobson, H. and Ochs, Q . , Personal Communication, May, 1976.

27.

Weiss, A.L. and Wang, Y.J., Personal Communication, February, 1980.

28.

Wang, Y.J., Personal Communication, December, 1980.

29.

Weiss, A.L., Personal Communication, March, 1980.

30.

Weiss, A.L. Personal Communication, May, 1976.

31.

Benesch, R. and Benesch, R., J. Am. Chem. 5877 (1955).

SOC. , 77, -

32.

Funae, Y., Toshioka, N., Mita, I., Sugihara, T., Ogura, T., Nakamura, Y., and Kawaguchi, S., Chem. Pharm. Bull., 19, 1623 (1971).

33.

Jocelyn, P.C. , "Biochemistry of the Sulfhydryl Group", Page 344 (19721, Academic Press.

34.

Dondzila, F., Personal Communication, May, 1981.

35.

Shipkowski, E.R., Personal Communication, October, 1980.

36.

Roberts, H.R., Personal Communication, May, 1977.

37.

Timmins, P., Personal Communication, January, 1980.

38.

Poet, R., Personal Communication, May, 1980.

39.

Hanaki, A. and Kamide, H., Chem. Pharm. Bull., 26, 325 (1978).

134

HAROLD KADIN

40.

Kadin, H., Poet, R.B., "Sequential Electrochemical Reduction, Solvent Partition and Automated Thiol Colorimetry for Urinary Captopril and its Disulfides", J. Pharm. Sci., In Press.

41.

Chas Pfizer and Company, New York, "Chelating Agent Usage Calculator".

42.

Kadin, H., Personal Communication, March,

43.

Wang, Y.J., Personal Communication, September, 1979.

44.

Ellman, G.L. and Lysko, H., J. Lab. and Clin. Med., 70, 518 (1967).

45.

Poet, R., Personal Communication, February, 1978, July 1979.

46.

Kadin, H., Personal Communication, October,

47.

Lidell, H. and Saville, B., Analyst, 84, 188

48.

Whigan, D.B., Personal Communication, January, 197 9.

49.

Valatin, P., Personal Communication, July, 1980, Sept., 1980.

50.

Roberts, H., Personal Communication, January, 1978, August, 1979.

51.

Perlman, S. and Kirschbaum, J., J. Chromat., 206, 311 (1981).

52.

1979.

1978.

(1959).

Saetre, R. and Rabenstein, D.L., Anal. Chem.,

50,

276 (1978).

53.

Yeh, P., Abstracts, No. 60, Page 54, Eastern Analytical Symposium, New York, N.Y., November, 198 0.

54.

Tenneson, M.E., Personal Communication, February-March, 1980.

135

CAPTOPRIL

55.

Yeh, P., Personal Communication, October,

56.

Perlman,

57.

Dondzila, F., Personal Communication, May,

58.

Dondzila, F., Personal Communication, May,

59.

Dondzila, F., Personal Communication, December-January, 1980.

60.

Hasegawa, M., Ohyabu, S., Ueda, T., and Yamaguchi, M., J. of Labelled Compounds and 18 I 643 ( 1981). Radiopharmaceuticals, -

61.

Whigan, D.B., Willis, S.L., and Jenkins, E.J., Personal Communication, October, 1976.

62.

Rohmer, M., Personal Communication, February,

63.

Migdalof, B.H., Singhvi, S.M., and Kripalani, K.J., J. Liquid Chromat., 3(6), 857 (1980).

64.

Yeh, P. and Roberts H., Personal Communication, July, 1979.

65.

Ivashkiv, E. and Roberts, H., Personal Communication, July, 1978.

66.

Egli, P., Personal Communication, October,

67.

Funke, P.T., Ivashkiv, E., Malley, M.F., and Cohen, A.I., Anal. Chem., 52, 1086 (1980).

68.

Ivashkiv, E., Personal Communication, March-April, 1980.

69.

1980. 1980.

S.,

Personal Communication, April,

1979. 1981.

1980.

1979.

Ivashkiv, E., Personal Communication, August,

1979.

136

70.

HAROLD KADIN

Matsuki, Y., Fukuhara, K., Ito, T., Ono, H., 188, 177 Ohara, N . , and Yui, T., J. Chromat., (1980).

71.

Kawahara, Y., Hisaoka, M., Yamazaki, Y., Inage, A., and Morioka, T., Chem. Pharm. Bull., - 29(1), - 150 (1981).

72.

Ivashkiv, E., Personal Communication, August,

73.

Kadin, H., Personal Communication, January,

74.

Kripalani, K.J., Meeker, F.S., Dean, A.V., McKinstry, D . N . , and Migdalof, B.H., Fed. 39, 307 (1980). Proceedings, -

75.

Jarrott, B., Anderson, A., Hooper, R. and 70, 665 (1981). Louis, W.J., J. Pharm. Sci., -

76.

Komai, T., Ikeda, T., Kawai, K., Kameyama, E., Shindo, H., J. Pharm. Dyn., 4, 677

1980.

1977.

(1981).

77.

Wong, K.K., Lan, S.J., and Migdalof, B.H., Biochem. Pharmacology, 30, 2643 (1981).

78.

Migdalof, B.H., Wong, K.K., Lan, S.J., Kripalani, K.J. and Singhvi, S.M., Fed. Proc., - --39, 757 (1980).

79.

Shindo, H., Komai, T., Ikeda, T., Kawai, K., Kawamata, W., and Kameyama, E., J. Pharmacobiodyn., 30, S-9 (1980).

80.

Ikeda, T., Komai, T., Kawai, K., and Shindo, 29(5), 1416 (1981). H., Chem. Pharm. Bull., -

81.

Kripalani, K.J., McKinstry, D . N . , Singhvi, S.M., Willard D . A . , Vukovich. R.A. and Migdalof , B. H. , Clin. Pharmacol. and Therap. , 27, -

82.

636 (1980).

McKinstry, D . N . , Singhvi, S.M., Kripalani, K.J., Willard, D.A., and Migdalof, B.H., "Recent Advances in Hypertension Therapy: Captopril", Excerpta Medica, Page 3, (1981), Amsterdam-Oxford-Princeton.

CAPTOPRIL

137

83.

McKinstry, D.N., Kripalani, K.J., Migdaloff, B.H., Willard, D.A., Clin. Pharmacol. & Ther.. 2 7 . 2 7 0 ( 1 9 8 0 ) .

84.

Singhvi, S.M., McKinstry, D.N., Shaw, J.M., Willard, D.A., and Migdalof, B.H., J. Clin. Pharmacol. (in press).

85.

Rommel, A. J. Pierides, A.M., and Heald, A. Clin. Pharmacol. Ther., 27, 282 ( 1 9 8 0 ) .

86.

Heald, A.F. and Ita, C.E., Pharmacologist, 19, -

129 (1977).

87.

Devlin, R.G. and Fleiss, P.M., Clin. Pharmacol. & Ther., 27, 2 5 0 ( 1 9 ' 8 0 ) .

88.

Devlin, R.G. and Fleiss, P.M., J. Clin 21, 1 1 0 ( 1 9 8 1 ) . Pharmacol. , -

89.

Pearson, F.M., Martin, V.I., Aldujaili, E.A.S., Williams, B.C., and Edwards, C.R.W., 97, 5 2 4 3 , 1 4 8 ( 1 9 8 1 ) . Acta Endocrinologica, -

90.

Migdalof, B.H., Personal Communication, January, 1 9 8 2 .

91.

Ferguson, R.K., Brunner, H.R., Turini, G.A., Gavras, H. and McKinstry, D.N., Lancet, I, 775 I

11.

(1977)

.

Review Coverage Dates

This review summarizes communications up to June 10, 1 9 8 1 . However Sections 2 and 8 are' updated to about January, 1 9 8 2 .

CEFOTAXIME Farid J . Muhtudi and Mahmoud M . A. Hassan

1

2.

3. 4.

5. 6.

7.

8.

140 140 140 142 142 142 142 142 142 142 142 142 152 152 156 156 159 159 159 159 165 166 167

Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 ElementalComposition 1.5 Appearance, Color, Odor, and Taste Physical Properties 2.1 Solubility 2.2 Moisture Content 2.3 pH Range 2.4 Optical Rotation 2.5 Spectral Properties Synthesis of Cefotaxime Metabolism Pharmacokinetics Microbiological Activity Methods of Analysis 7.1 Identification Tests 7.2 Non-Aqueous Titration 7.3 Chromatography 7.4 Spectrophotometry 7.5 Microbiological Assay References

Analytical Profiles of Drug Substances Volume I I

139

Copyright 0 1982 by The American Pharmaceutical Association

ISBN &12-260811-9

140

1.

FARID J . MUHTADI AND MAHMOUD M. A. HASSAN Description

1.1.

Nomenclature

1.1.1 Chemical Names (a) (b)

Sodium 7-[2-(2-amino-4-thiazolyl)-2m e t hox y iminoa c e tam i d o ] c epha 1o s po r a n a t e Sodium 3-acetoxymethyl-7-[

2-(2-amino-

4- t h i a z o 1y 1) -2- m e t hox y i m i n o ] a c e t am id0 ] -3-cephem-4-carboxyla

1.1.2

te

.

(c)

(6 R-trans)-3-[ (Acetyloxy) methyl]-7-[ [ (2-amino-4-thiazolyl) (methoxy- imino) a c e t y 11 amino]- 8-0xo-5 - t h ia-1 -azab i c yclo [ 4 . 2 . 0 ] oct-2-ene-2-carboxylic acid monosodium s a l t .

(d)

(6-R, 7R)-7-[ 2-(2-Amino-4-thiazolyl) glyoxylamido] -3-(hydroxy methyl) -8-0x05-thia-1-azabicyclo [ 4 , 2 , 0 ] oc t-2-ene2-carboxylic a c i d a-(O-methyloxime), a c e t a t e ( e s t e r ) monosodium s a l t .

(e)

5-Thia-1-azabicyclo [ 4.2.01 oc t-2-ene 2-2 c a r b o x y l i c a c i d , 3 - [ ( a c e t y l o x y ) methyl] -7-[ [ (2-amino-4-thiazolyl) (me thox y i m i n o ) y a c e t y l amino ] -8 -0xo- , [6R-[6a,78(Z)II-

Generic Name Cefotaxime sodium; HR 756; RU-24,662; RU-24,756

1.1.3

P r o p r i e t a r y Names C l a f o r a n ; Primafen; Z a r i v i z ; T a r i v i d .

1.2.

Formula

1 . 2 . 1 Empirical

C16H16N507S2Na

.

1.2.2 Structural

m ?2

I

V I

0 =. v 0 I hl X

V

142

FARID J . MUHTADI AND MAHMOUD M. A. HASSAN 1 . 2 . 3 CAS no.

[ 611846-23-31 (1) [ 60846-21-11 (1) [63527-52-61 1.3.

C16H16N507S2Na C H N 0 S (as f r e e acid) 16 1 7 5 7 2

(2)

M o l e c u l a r Weight 477.23

1.4.

E l e m e n t a l Composition C, 40.23;;

H , 3.38%; N , 14.67%;

0, 23.47%; S , 13.44%; Ma 4.82%.

1.5.

Appearance, C o l o r , Odor and T a s t e : White t o creamy w h i t e c r y s t a l l i n e powder, o d o r l e s s and h a s a s a l t y t a s t e a t the b e g i n n i n g , f o l l o w e d by b i t t e r n e s s

.

2.

Physical Properties

2.1.

Solubility F r e e l y s o l u b l e i n water ( 0 . 5 g s o l u b l e i n 5 ml) ( 3 ) , s l i g h t l y s o l u b l e i n alcohol ( a b s o l u t e , 95%), i n s o l u b l e i n chloroform (4)

2.2. M o i s t u r e C o n t e n t Not more t h a n 6 p e r c e n t , d e t e r m i n e d by t h e Karl F i s c h e r method, u s i n g a 0.2 g sample d i s s o l v e d i n 2 m l methanol ( 3 ) . 0

Loss on d r y i n g a t 60 C u n d e r vacuum i n t h e p r e s e n c e o f phosphorus p e n t o x i d e f o r 4 h r . s h o u l d n o t exceed 6 % ( 3 ) . 2.3. pH r a n g e The pH of c e f o t a x i m e a s a 10%a q u e o u s s o l u t i o n i s 4 . 5 t o 6.5 d e t e r m i n e d p o t e n t i o m e t r i c a l l y ( 3 ) . 2.4. O p t i c a l R o t a t i o n

[ a]D ( c = l % aqueous s o l u t i o n ) (on d r i e d b ases) (3).

+

58 t o

+

64

0

The o p t i c a l r o t a t i o n of c e f o t a x i m e ( c = l % aqueous s o l u t i o n ) was d e t e r m i n e d u s i n g a P e r k i n E l m e r 2 4 1 MC P o l a r m a t i c and found t o be:

[ a~D240

+

59.30.

143

CEFOTAXIME 2.5.


Ultraviolet Spectra The UV s p e c t r a o f c e f o t a x h e i n a n aqueous s o l u t i o n and i n 0 . 1 N H C 1 a r e p r e s e n t e d i n Fig. 1 and F i g . 2 r e s p e c t i v e l y . These were scanned from 200 t o 400 nm u s i n g a PyeUnicumSP 8-100 U l t r a v i o l e t s p e c t r o photometer. The f o l l o w i n g t a b l e 1 shows t h e UV d a t a .

Table 1.

UV c h a r a c t e r i s t i c s of cefotaxime

c onc en t r a t i o n

solvent

water

0.5 mg/ml 0.05 mg/ml 0.01 mg/ml

0.1N R C l 0.1N HC1

2.5.2

.

E (lX, lcm)

max nm 234 205,262 263

420 (5)

Infrared Spectra The I R s p e c t r a of c e f o t a x i m e a s KBr-disc and a s n u j o l mull a r e shown i n F i g . 3 and The KBr-disc w a s Fig. 4 r e s p e c t i v e l y . recorded on a P e r k i n E l m e r 58OB i n f r a r e d s p e c t r o m e t e r . The s t r u c t u r a l a s s i g n m e n t s have been c o r r e l a t e d w i t h t h e f o l l o w i n g band f r e q u e n c i e s (Table 2).

Table 2.

I R c h a r a c t e r i s t i c s of c e f o t a x i m e

Frequency Cm

-1

Assignment

3420

-NH2

3340 (broad)

-NH,

2940

-S-CH2

1 7 60

-C=O

-NH2

lactam

0 II

1730

-C=O

e a r b o x y l i c , O-C-CH3

1650

0 -C-NH

1620

0 -C-NH,-C=N-,-C=C-

1540

0 -C-N-

1385-13 55

-0-CO-CH3

1180 1050

C=O i n e s t e r

11

C-0

stretching

230 240 250

304

350

400

Fig.1

The UV spectrum of

Cefotaxtme

in

water

200

210

250 2 60 270

300

350

400

Fig.2 The UV spectrum o f C e f o t a x i m e in O . I N H C I .

144

3.0

4000

40

5.0

2500

2000

MICRONS

70

6.0

I

3500

3000

1800

1600

1400

8.0

1200

9.0

10

1000

12

1)oo

14

8 CD 0 0

a0

0 0 0 4

2

0 0

0

$

0

0

0

'0, 0

0

2 0

cu

0

0

0 v)

0

cy

a 0

0

c3

0 0

In rn

CEFOTAXIME

147

2.5.3

Nuclear Magnetic Resonance S p e c t r a

2.5.3.1

Proton SDectra

The PMR s p e c t r a of c e f o t a x i m e i n d e u t e r a t e d d i m e t h y l s u l f o x i d e (DMSOD6) and d e u t e r i u m o x i d e w e r e r e c o r d e d on a v a r i a n T-60A, 60-MHZ NMR s p e c t r o m e t e r u s i n g sodium -2, 2-dimethyl-2-silapentane-5-sulfonate (DSS) a s a n i n t e r n a l r e f e r e n c e . These a r e shown i n F i g . 5 and F i g . 6 r e s p e c t i v e l y . The f o l l o w i n g s t r u c t u r e a s s i g n m e n t s h a v e been made ( T a b l e 3 ) ( 6 ) . T a b l e 3.

PMR c h a r a c t e r i s t i c s of c e f o t a x i m e

Group

Chemical Sh DMSQD6

Et ( P P ) D2°

-0co CH,

2.00(s)

2.12(s)

2-G2

3 . 3 3 (9)

3 . 5 3 (9)

N - E 3

3 . 8 3 (s)

4.00(s)

4.97(9)

4.83 ( d )

4.97(q)

5.20(d)

7-H

5.60(2d)

5.82 ( d )

5-H

6.70(s)

6.97 (s)

2-Nl12

7.22 (bs)

CONH -

9.47 ( d )

0-CH -3 6-H

AC

.

-

s = s i n g l e t , d=doublet, q = q u a r t e t , 2d=doublet of d o u b l e t s , bs=broad s i n g l e t . 13 2.5.3.2 C-NMR 13 C-NMR c o m p l e t e l y d e c o u p l e d and o f f r e s o n a n c e s p e c t r a a r e p r e s e n t e d i n F i g . 7 and F i g . 8 respectively. Both were r e c o r d e d o v e r 5000 H, r a n g e i n d e u t e r i u m o x i d e ( c o n c . 100 m g / l m l D20)

148

0

149

FARID J. MUHTADI AND MAHMOUD M . A. HASSAN

150

4

a

1 I

-J

-TTJ_LLl-

F i g . 7. spectrum

.

'_J _ ' I ' -I

'

' '-11

I

rI ' I '

The 1 3 C NMR of Cefotaxime, completely decoupled

'I I

I

II

Fig. 8 . The 1 3 C NMR of Cefotaxime, o f f resonance (Proton coupled) spectrum

151

CEFOTAXIME

on FT-80A-8OMHz NMF. s p e c t r o m e t e r , u s i n g 1 0 mm sample t u b e and d i o x a n e a s a r e f e r e n c e s t a n d a r d a t 2OoC. T h e c a r b o n chemical s h i f t a r e a s s i g n e d on t h e b a s i s of t h e a d d i t i v i t y p r i n c i p a l s and t h e o f f r e s o n a n c e s p l i t t i n g p a t t e r n ( T a b l e 4) ( 6 ) . 12 NOCH3

!?1 6 OCCH3 15

H2N

COONa

13

T a b l e 4. Carbon no. 8-C=0

Carbon Chemical s h i f t s o f c e f o t a x i m e Chemical s h i f t [ PPm 1 174.69(s)

Carbon no. C-3

Chemical s h i f t [ PPm 1 117.37 ( S )

1o-c=0

171.25(s)

C-5

113.64 (d)

13-C=0

168.78 ( s )

c-7

65.07 (d)

15-C=0

165.08( s)

C-6

59.59(d)

c-2

164.65(s)

C-14

58.23 ( t )

c-11

148.62 (s)

c-2

26.35( t )

c-4

141.40( s)

C-12

63.55 ( 4 )

c-4

132.42 (s)

C-16

21.22 ( 4 )

,

,

s=singlet, d=doublet, t = t r i p l e t , q=quartet.

FARID J. MUHTADI AND MAHMOUD M. A. HASSAN

152

3.

S y n t h e s i s of Cefotaxime Cefotaxime i s a s e m i - s y n t h e t i c c e p h a l o s p o r i n . The s t a r t i n g material f o r s u c h c e p h a l o s p o r i n s i s 7-aminocepha l o s p o r a n i c a c i d (7-ACA) which o b t a i n e d e i t h e r by mild a c i d o r enzymatic h y d r o l y s i s of c e p h a l o s p o r i n C . ( 7 , 8 , 9 , 1 0 )

P r e p a r a t i o n of t h e S i d e Chain:

(5)

The s t a r t i n g m a t e r i a l e t h y l a c e t o a c e t a t e [ l ] w a s o x i mated t o produce oximinoethylacetoacetate [ 21. Methylat i o n of [ 21 followed by bromination y i e l d e d syn-methoxyiminobromoketone [ 3 ] . Condensation of [ 3 ] w i t h t h i o u r e a [ 4 ] i n aqueous medium and a t a low t e m p e r a t u r e gave syna m i n o t h i a z o l y l d e r i v a t i v e [ 51. The a m i n o t h i a z o l y l r i n g w a s p r o t e c t e d by t r i t y l a t i o n t o g i v e t h e N - t r i t y l d e r i v a t i v e [6]. S a p o n i f i c a t i o n of t h e l a t t e r by b o i l i n g w i t h NaOH s o l u t i o n a f f o r d e d t h e c o r r e s p o n d i n g a c i d [ 7 ] . Acyl a t i o n of t h e amino group of 7-ACA w i t h t h e r e s u l t i n g a c i d [ 7 ] proved d i f f i c u l t . T h i s h a s been overcome by t h e u s e of symmetrical a n h y d r i d e [ 8 ] , which w a s o b t a i n e d by condensing two m o l e c u l e s of [ 7 ] i n t h e p r e s e n c e of d i c y c l o h e x y l c a r b o d i i m i d e [ a ] . 7-ACA w a s a c y l a t e d by compound [8] t o g i v e [ 91. Removal of t h e t r i t y l r e s i d u e under a c i d i c c o n d i t i o n gave t h e f r e e a c i d form (R=H) of c e f o taxime [ l o ] . P r e p a r a t i o n of a S t a b l e Sodium S a l t : T h i s w a s prepared by adding t h e s o l i d f r e e a c i d t o a n aqueous a l c o h o l i c s o l u t i o n of a n o r g a n i c sodium s a l t t o g i v e t h e s t a b l e a c t i v e D-form. (5) The s y n t h e s i s of c e f o t a x i m e i s p r e s e n t e d i n Scheme 1. I t i s i n t e r e s t i n g t o n o t e t h a t t h e syn-isomer of cefotaxime i s u p t o 100 times more a c t i v e a g a i n s t c e r t a i n organisms t h a n t h e a n t i - i s o m e r .

4.

Metabolism The metabolism of c e f o t a x i m e i n r a t , dog and man u s i n g 14C-labelled c e f o t a x i m e h a s been s t u d i e d by Chamberlain e t a l . (11). Cefotaxime i s w e l l absorbed i n t h e t h r e e species a f t e r intramuscular administration. It i s e l i m i n a t e d mainly v i a t h e u r i n e . The major m e t a b o l i t e being d e s a c e t y l c e f o t a x i m e . The amount of unchanged c e f o -

153

CEFOTAXIME OH

Scheme 1:

S y n t h e s i s of Cefotaxime

N’

I

methylation bromination

then

N,0CH3

+

H2N R r i j 9 9 > T w e e n 40 > M y r j 53. T h e e f f e c t of u r e a , a m i d o p y r i n e , p h e n a z o n e a n d p a r a c e t a m o l on t h e s o l u b i l i t y of f l u f e n a m i c a c i d a n d o t h e r a n t i r h e u m a t i c d r u g s w a s s t u d i e d b y D a a b i s e t al. ( 2 2 ) . 1- O c t a n o l / w a t e r p a r t i t i o n c o e f f i c i e n t w a s e s t i m a t e d b y Dunn (2 3 ) t a k i n g a d v a n t a g e of t h e a d d i t i v e - c o n s t i t u t i v e n a -

ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

324

TABLE VIII Solubility of F l u f e n a m i c Acid in Water a t v a r i o u s pH V a l u e s PH

Solubility , mg/ml

Ref,

3 7 7 8

0. 003a 1. 8a 1 4. oa

13 13 20 13

a : a t 37OC t u r e of l o g P, as follows: = log 'anthranilic =

1. 21

acid

+ 2 . 60 + 1 . 0 7

=

+

nphenyl

+

n3-cF3 =

4. 88

T e r a d a e t al. ( 2 4 ) have d e t e r m i n e d the t r u e p a r t i t i o n coefficient, P, a n d the a p p a r e n t p a r t i t i o n coefficient, P', of f l u f e n a m i c acid. P w a s m e a s u r e d by e q u i l i b r a t i n g a 1-octanol solution of the d r u g , the i n i t i a l c o n c e n t r a t i o n , C,, of which w a s 1 0 - 3 - 1 0 - 2 m o l / l , with 0. 01 N HC1: u n d e r t h i s condition, d r u g m o l e c u l e s e x i s t e x c l u s i v e l y a s the unionized f o r m . A f t e r e q u i l i b r i u m w a s a t t a i n e d , c o n c e n t r a t i o n i n the a q u e o u s p h a s e , Cw, was m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y and P w a s c a l c u l a t e d b y the equation: P = C,/Cw, s i n c e t h e c o n c e n t r a t i o n change in t h e o r g a nic p h a s e c a n b e n e g l e c t e d f o r s u c h a highly hydrophobic compound, obtaining l o g P = 5 . 62. PI w a s d e t e r m i n e d with the 1- o c t a n o l / p h o s p h a t e b u f f e r (pH 8. 0) s y s t e m , u s i n g the equation:

w h e r e C a n d V a r e t h e e q u i l i b r i u m c o n c e n t r a t i o n and v o l u m e of a q u e o u s ( s u b s c r i p t w) and o r g a n i c ( s u b s c r i p t 0 ) p h a s e s , r e s p e c t i v e l y . Ci is t h e i n i t i a l c o n c e n t r a t i o n i n the a q u e o u s p h a s e . L o g PI w a s found t o b e 1 . 7 4 .

FLUFENAMIC ACID

325

Lombardino et al. (25) have a l s o determined the p a r t i tion coefficient of flufenamic acid with some 1-octanol/ buffer s y s t e m s . 2. 8. Crystal P r o p e r t i e s , Polymorphism Flufenamic acid can exist a s s e v e r a l crystalline modifications. Kuhnert-Brandstatter et al. have d e s c r i bed five different modifications and have reported t h e i r melting points and infrared s p e c t r a , a s well a s the t h e r mogram obtained by differential scanning c a l o r i m e t r y f o r the f i r s t four f o r m s (16, 26). According to K r c flufenamic acid can exist a s at l e a s t seven crystalline modifications with different melting points (27). K r c has reported the f r e e energy vs. t e m p e r a t u r e plot of seven crystalline f o r m s of flufenamic acid. Modifications I, I1 and I11 were described in detail in t e r m s of c r y s t a l morphology, optical p r o p e r t i e s , X - r a y diffraction powder data and infrared s p e c t r a . Other authors a l s o studied the polymorphism of flufenamic acid. Galdecki et al. (28) investigated the c r y s t a l l i zation of the d r u g f r o m boiling solvents, B u r g e r and Ramberger ( 2 9 ) examined the applicability of s o m e t h e r modynamic r u l e s to the polymorphic s y s t e m of flufenamic acid. These r u l e s c o r r e l a t e the heats of transition o r fusion, IR s p e c t r a and densities of the modifications with t h e i r stability behavior. In this study flufenamic acid w a s investigated mainly by quantitative DSC and qualitative s o lubility determination (by thermomicroscopy) a s well a s by IR spectroscopy to differentiate eight crystalline modifications (Table IX). It w a s pointed out ( 2 9 ) that modifications I, I1 and I11 investigated by the various authors a r e identical, whereas modification IV studied by Kuhnert ( 2 6 ) coincides with modification V by K r c ( 2 7 ) , who did Since the l a t t e r not describe Kuhnert's modification V. f o r m had the lowest melting point of all eight modifications, it was indicated by B u r g e r and R a m b e r g e r a s modification VIII. F r o m the practical point of view, modifications I and I11 a r e the most important, because they can be present in the c o m m e r c i a l product. The transition point of these two f o r m s is a t 42°C: modification I11 i s the stable f o r m

326


at room t e m p e r a t u r e ( b e l o w 42" C) , w h e r e a s m o d i f i c a t i o n I is t h e s t a b l e form a b o v e 42OC ( 2 7 ) . M o d i f i c a t i o n I11 w a s o b t a i n e d by B u r g e r a n d R a m b e r g e r by s t i r r i n g f o r 1 2 h o u r s a t 2OoC a x y l e n e s u s p e n s i o n of a commercial p r o d u c t f o r m e d by m o d i f i c a t i o n I ( 2 9 ) . T A B L E IX M e l t i n g P o i n t s of c r y s t a l l i n e M o d i f i c a t i o n s of F l u f e n a m i c A c i d Mo d ific a t i on

M. P . , O C

Ref.

I

133 134

16,26 27, 2 9

I1

128

1 6 , 26, 27, 2 9

I11

125 126 126. 5

16,26 29 27

IV

124

27

Va

122 122.5

1 6 , 26, 29 27

VI

120

27

VII

118

27

VIIIb

100-110 108 2 5

26 29

a : T h i s m o d i f i c a t i o n w a s i n d i c a t e d a s IV i n t h e p a p e r s 1 6 a n d 26. b : T h i s modification was indicated as V in the p a p e r s 1 6 a n d 26.

2. 9. D i s s o c i a t i o n C o n s t a n t T h e pKa of f l u f e n a m i c a c i d w a s r e p o r t e d to b e 3 . 9 by A g u i a r a n d F i f e l s k i ( 2 0 ) a n d 4 . 5 by F r e y a n d ElS a y e d (30). Terada et al. ( 2 4 ) h a v e found a v a l u e of 3 . 8 5 u s i n g t h e pH -dependent s o l u b i l i t y m e t h o d ( 3 1 ) ; t h i s value is c o n s i d e r a b l y d i f f e r e n t from t h e c o r r e s p o n d i n g

FLUFENAMIC ACID

327

value obtained by p o t e n t i o m e t r i c t i t r a t i o n i n 5 - 10% a q u e o u s acetone ( 3 2 ) . In T a b l e X a r e l i s t e d s o m e pKa v a l u e s obtained by p o t e n t i o m e t r i c t i t r a t i o n in v a r i o u s a q u e o u s s o l vent s y s t e m s . TABLE X pKa V a l u e s of F l u f e n a m i c Acid obtained bv P o t e n t i o m e t r i c T i t r a t i o n Solvent Water 75% Aqueous methanol 50Vn Aqueous ethanol 80% Aqueous 2-methoxyethanol Dioxane: w a t e r ( 2 : 1)

pKa

Ref.

7. 5 5.75 5. 94

33 3 33

6. 0 6. 8

33 25

3 . SYNTHESIS Wilkinson and F i n a r ( 1 2 ) f i r s t s y n t h e s i z e d flufenamic a c i d by r e a c t i n g o-iodobenzoic a c i d with m - t r i f l u o r o m e thylaniline in p o t a s s i u m c a r b o n a t e a q u e o u s solution, i n t h e p r e s e n c e of c o p p e r b r o n z e . T h e c r u d e p r o d u c t w a s p u r i fied via i t s a m m o n i u m s a l t . Moffett and A s p e r g r e n (19) p r e p a r e d f l u f e n a m i c a c i d s t a r t i n g f r o m o - c h l o r o b e n z o i c a c i d which w a s r e a c t e d with m-trifluoromethylaniline i n 85070 aqueous p o t a s s i u m hydroxide and a m y l alcohol with c o p p e r p o w d e r . Some patented synthetic m e t h o d s follow the l a t t e r s c h e m e , as i l l u s t r a t e d in F i g u r e 5.

Figure 5 Synthesis of F l u f e n a m i c Acid F l u f e n a m i c a c i d w a s a l s o obtained via the r e a c t i o n b e t ween o-iodobenzoic a c i d and m-trifluoromethylphenylhydroxylamine ( 3 4 ) . Another method involves the r e a c t i o n of

328


m e t h y l o - c h l o r o b e n z o a t e with N-(3-trifluoromethylphenyl)f o r m a m i d e (35). F l u f e n a m i c a c i d w a s a l s o p r e p a r e d by photolysis of the c o r r e s p o n d i n g b e n z o t r i a z i n o n e ( 3 6 ) . 4. DRUG METABOLISM AND PHARMACOKINETICS 4. 1. M e t a b o l i s m T h e m e t a b o l i c t r a n s f o r m a t i o n s which f l u f e n a m i c a c i d u n d e r g o e s in m a n and a n i m a l s a r e d e p i c t e d in F i g u r e 6 , a c c o r d i n g t o Glazko ( 3 7 ) and O b e r e t al. (38), which d e m o n s t r a t e d that t h e d r u g is e x c r e t e d m a i n l y i n f o r m of i t s m e t a b o l i t e s . T h e i r s t u d i e s w e r e c a r r i e d out by t r a c e r m e t h o d s u s i n g [14C] - c a r b o x y l - l a b e l l e d f l u f e n a m i c acid. T h e 4'-hydroxy- and 5 - h y d r o x y - d e r i v a t i v e and f l u f e n a m i c a c i d itself a r e e l i m i n a t e d in u r i n e chiefly i n conjugated f o r m , w h e r e a s t h e 4', 5 - d i h y d r o x y - d e r i v a t i v e is not conjugated. A l l f o u r compounds h a v e b e e n found i n t h e s t o o l i n unconjugated f o r m . 4'-Hydroxy- and 5-hydroxyflufenamic a c i d s w e r e s y n t h e s i z e d by Bowman et al. (39). 4. 2. Absorption and E x c r e t i o n 4. 21.

In A n i m a l s T h e r a t e of p e r m e a t i o n of f l u f e n a m i c a c i d t h r o u g h the gut m e m b r a n e and the amount of d r u g a b s o r bed w e r e studied i n v i t r o on s e g m e n t s of t h e small i n t e s t i n e of golden h a m s t e r s ( 2 0 ) . T h e p e r m e a t i o n of flufenam i c a c i d w a s pH-dependent, a c c o r d i n g t o t h e postulation t h a t i t is only the unionized m o l e c u l e of t h e d r u g t h a t pass e s through a cell, due t o i t s lipoid solubility. T h i s s t u d y showed t h a t a t pH 2. 5 the p e r m e a t i o n of f l u f e n a m i c acid, which is a p p r o x i m a t e l y 96% unionized, is 20 t i m e s f a s t e r t h a n a t pH 7. 2, at which only 0. 05% of t h e d r u g is i n the unionized f o r m . T h e a b s o r p t i o n and t h e u r i n a r y e x c r e t i o n of f l u f e n a m i c a c i d w a s s t u d i e d i n r a b b i t s following both c u t a n e o u s and o r a l application ( 4 0 ) of t h e same d o s e ( 3 0 m g / k g ) . A f t e r 4 8 h o u r s from cutaneous application, 5. 9% of t h e applied d o s e w a s found i n the u r i n e ; t h e blood level of flufenam i c a c i d r e m a i n e d c o n s t a n t o v e r the f i r s t s i x h o u r s at about 3 pg/ml, o v e r c o m i n g t h e blood l e v e l obtained o r a l l y a s f r o m the 4th hour.

m

f 4 R Erc

m

u.

0

r

I

R

q Erc

&

0 I

u3

/ \ 4

0 c k I V

0

I

CD

a,

lil

.rl~

R

c

a,

I

rcl

Erc

rl

0

+I

E

(I)

0

d .rl

Y

lu

e

3 30


Rosenberg and Bates ( 4 1 ) compared the blood concentrations produced in r a t s following o r a l administration of f l u fenamic acid alone o r together with cholestyramine. F l u fenamic acid strongly bound to the r e s i n , s o that a 6 0 70% d e c r e a s e in both the r a t e and the extent of absorption of the d r u g was observed: following an o r a l dose of 5 0 m g / k g a peak plasma concentration of 8 9 . 2 p g / m l w a s reached at 1 hour, whereas when cholestyramine and the d r u g were coadministered the peak l e v e l dropped to 3 2 . 3 F u r t h e r studies on pharmacokinetics of flufenapg/ml. m i c acid in the r a t a r e reported. F r e y and El-Sayed ( 3 0 ) determined the flufenamic acid concentrations in s e r u m and g a s t r i c mucosa a f t e r o r a l and subcutaneous administration. Lin et al. ( 4 2 ) determined p l a s m a l e v e l s a f t e r intravenous administration, Cotellessa e t al. ( 4 3 ) d e t e r mined plasma and u t e r u s levels a f t e r intravenous and o r a l administration. A s r e g a r d s the excretion, Glazko ( 3 7 ) showed that dogs eliminated only 2-770 of an o r a l dose in the urine and 537 9 % in the f e c e s , w h e r e a s the corresponding values f o r monkeys were 45-8070 and 12-2170, respectively, S i m i l a r r e s u l t s were obtained by Ober et al. ( 3 8 ) in e x p e r i m e n t s with dogs. Lombardino et al. ( 2 5 ) r e p o r t e d that the s e r u m half life a f t e r o r a l administration of the aluminium salt of flufenamic acid w a s 3 h o u r s f o r rats and dogs, and 4 hours f o r rabbits.

4 . 2 2 . In Humans In addition to the r e s u l t s obtained in r a b bits ( 4 0 ) , P a n s e e t al. have a l s o studied the absorption of flufenamic acid through the human skin ( 4 4 ) . Glazko ( 3 7 ) reported that n e a r l y 100% of an o r a l dose of flufenamic acid w a s absorbed; the r e n a l elimination of the drug and i t s metabolites was 5170, of which only 2 . 6% w a s unaltered drug. Such r e s u l t s were m o r e r e c e n t l y confirmed by Dell e t al. ( 4 5 , 4 6 ) with two different methods for the determination of all fluorine-containing c o m pounds a s a group in the urine, which showed that the r e nal elimination of flufenamic acid and i t s metabolites w a s 4 9 . 470 within t h r e e days a f t e r o r a l administration. The peak p l a s m a level w a s reached a f t e r two hours, and the p l a s m a elimination half life w a s found t o be approximate-

FLUFENAMIC ACID

331

l y 3 h o u r s . D e l l e t al. ( 4 5 ) a l s o r e p o r t e d t h a t 3. 670 of a n o r a l d o s e of flufenamic a c i d was e x c r e t e d unconjugated into the u r i n e within 6 d a y s : however, f e m a l e s u b j e c t s e l i m i n a t e d only 1. 9% and the m a l e o n e s 5. 370. On the c o n t r a r y , no difference w a s o b s e r v e d between m e n and women in the t o t a l amount of all m e t a b o l i t e s e x c r e t e d by the r e n a l route. Another study on t h e sex-dependence of the r e n a l e x c r e t i o n of flufenamic a c i d and o t h e r f e n a m a t e s in m a n and a n i m a l s was r e p o r t e d by L o r e n z a n d D e l l (47). T h e bioavailability of o r a l 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 of flufenamic a c i d w a s investigated by A r i a s a n d Cadorniga ( 4 8 ) and Angelucci e t al. (49). 4. 3 . P r o t e i n binding T h e bovine and human s e r u m a l b u m i n (BSA and HSA, r e s p e c t i v e l y ) binding affinity of flufenamic a c i d w a s investigated by Chignell b y c i r c u l a r d i c h r o i s m s t u d i e s (50-52). T h e r o l e of hydrophobicity f o r the binding affinit y w a s investigated by Dunn on HSA (23) and by T e r a d a e t al. on BSA (24). T h e hydrophobicity a s well a s the withd r a w i n g ability of the - C F 3 substituent c o n t r i b u t e significantly t o the binding affinity, which w a s d e t e r m i n e d f o r BSA by m e a s u r i n g the ability of flufenamic a c i d t o d i s p l a c e 2 - (4' - hydroxypheny1azo)benzoic a c i d c o m p e t i t i v e l y u n d e r conditions of pH 7 . 0 and 25OC (24). T h e binding constant, K, w a s d e t e r m i n e d : the value obtained f o r BSA by T e r a d a e t a l . , 6 . 5 l~o 5 1. m o l - l , s e e m s t o c o n f o r m t o a v a lue, 1. 3 x l o 6 1. m o l - l , obtained by Chignell with HSA at pH 7 . 4 (51). T h e i n t e r a c t i o n between BSA and s e v e r a l c a t i o n i c and anionic d r u g s including flufenamic a c i d w a s studied by B l a n c h a r d e t al. (53) u s i n g the e l e c t r o n s p i n r e s o n a n c e s p i n labeling technique. T h e binding of flufenamic a c i d t o HSA w a s s t u d i e d by O t a g i r i e t al. ( 5 4 ) v i a m i c r o c a l o r i m e t r i c i n v e s t i g a t i o n s . T h e h e a t flux g e n e r a t e d by the binding is p r o p o r t i o n a l t o the amount of the d r u g bound t o the p r o t e i n . If only one binding s i t e on t h e d r u g m o l e c u l e c o n t r i b u t e s t o the h e a t flux, then the d a t a can r e a d i l y be i n t e r p r e t e d i n t e r m s of the binding constant, AG, AH, and A S f o r binding t o that site. If m a n y s i t e s a r e involved having d i f f e r e n t e n t h a l -

332


pies of binding, then unambiguous interpretation of the data may be impossible. T h i s is the c a s e of flufenamic acid, which h a s been reported t o have t h r e e v e r y high affinity s i t e s f o r HSA and other s i t e s of lower affinity(51). Sudlow et al. (55, 56) have c h a r a c t e r i z e d two distinct binding s i t e s ( I and 11) f o r anionic d r u g s on HSA by the u s e of fluorescent probes. Flufenamic acid binds s e l e c t i vely to s i t e 11, a s well a s ibuprofen, flurbiprofen and ethacrynic acid , whereas phenylbutazone and warfarin bind to s i t e I. Drugs which bind t o s i t e I1 a r e all a r o m a t i c carboxylic acids, which would be l a r g e l y ionized a t physiological pH. Kaneo et al. (57) examined the binding t o BSA of s i x nonsteroidal antiinflammatory d r u g s including flufenamic acid by the u s e of dialysis a t pH 7 . 4 and 37OC. It was found that flufenamic acid strongly binds t o BSA and the f r e e fraction of t h i s d r u g e x i s t s within 1% over the t h e r a peutic range. 5. METHODS OF ANALYSIS 5. 1. Identification T e s t s Flufenamic acid can be identified by virtue of i t s U V , IR, N M R , m a s s and fluorescence s p e c t r a (see Section 2). Various chromatographic methods a r e a l s o s u i table f o r purposes of identification ( s e e Section 5 . 7 ) . Devaux e t al. (58) described two color r e a c t i o n s and one fluorescent reaction. The color r e a c t i o n s a r e due t o the diphenylamine s t r u c t u r e , w h e r e a s the fluorescent r e a ction was explained by the formation of substituted a c r i dones ( s e e Section 5.5). T h e s e reactions can be c a r r i e d out a s follows: a ) Flufenamic acid (at l e a s t 1 m g ) and about 0. 5 g of oxalic acid a r e heated into an oil bath at 180-200°C 4-5 minutes. After cooling the r e s i d u e is dissolved in 95% ethanol t o obtain a stable, intense blue color. The a b s o r ption maximum is at 585-590 nm. b ) Flufenamic acid ( a t l e a s t 100 p g ) is added in a mixt u r e of 1 m l CH3COOH:H2S04 (d.1.83)(98:2), 5 rnl CH3CO0H:HCl (d. 1.18)(50:50), and 1 m l 0. 10% aqueous levulose. The mixture is heated a t 100°C 25 minutes t o ob-

FLUFENAMIC ACID

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tain a violet color. The absorption maximum is at 597nm. c ) Flufenamic acid is dissolved in conc. H2SO4 and heated 10 minutes at 100°C: the solution exhibits an intense green fluorescence when excited by white light, and blue when excited by UV light. 5. 2. T i t r i m e t r i c Analysis Flufenamic acid can be titrated in acetone with 0 . 1 N aqueous potassium hydroxide in the p r e s e n c e of phenolphtaleine (3). A nonaqueous t i t r i m e t r i c method w a s described by Walash and Rizk (59), which used 0 . 1 N s o dium methoxide as the t i t r a n t and DMF o r tetramethylu r e a as the solvent, m e a s u r i n g the end point e i t h e r with a thymol blue indicator o r potentiometrically. Various potentiometric methods in aqueous solvent s y s t e m s a r e cited in Section 2. 9. 5. 3. Colorimetric Analysis Flufenamic acid and i t s metabolites w e r e d e t e r mined a s a group colorimetrically in urine a f t e r alkaline hydrolysis and fusion with sodium peroxide: fluorine was then distilled a s H2SiF6 in the p r e s e n c e of H2S04 and Si02. A. solution of alizarine-3-methylamino-N, N-diacetato-cerium(II1) was added to the distillate to obtain a col o r reaction, and a c o l o r i m e t r i c determination w a s effected at 617 nm. Measurements in the range h l p,g a r e possible (46). The color reactions cited in Section 5. 1 can a l s o be used for quantitation. 5.4. Spectrophotometric Analysis The f i r s t UV spectrophotometric method f o r fenam a t e s analysis w a s described by Carey (60). The W absorbance of flufenamic acid ( s e e Section 2. 3 ) e i t h e r in methanol a t 288 nm (3,40,44) o r in 0. 1 N NaOH at 287290 nm (4, 30,411 can be used f o r quantitative analysis. Beltagy (61) described a spectrophotometric method f o r the determination of s e v e r a l acidic d r u g s including flufenamic acid which w a s determined obtaining the ion-pair association complex of the d r u g and safranine in a pH 7. 4 buffer, then extracting the complex with chloroform and measuring the absorbance of the e x t r a c t .

334


5. 5. F l u o r o m e t r i c A n a l y s i s Mehta and Schulman ( 9 ) a f f i r m e d t h a t t h e native f l u o r e s c e n c e exhibited by f l u f e n a m i c a c i d i n o r g a n i c s o l v e n t s (see Section 2. 5) could b e u s e f u l f o r i t s d e t e c t i o n and d e t e r m i n a t i o n , N e v e r t h e l e s s the m e t h o d m o s t corn monly u s e d involves the f l u o r o m e t r i c d e t e r m i n a t i o n of fluf e n a m i c a c i d i n c a r b o n t e t r a c h l o r i d e a f t e r addition of a CC14 solution of t r i c h l o r o a c e t i c a c i d which s t r o n g l y i n c r e ases the f l u o r e s c e n c e i n t e n s i t y (11). T h e f l u o r e s c e n c e m a x i m a u n d e r t h i s condition a r e r e p o r t e d i n Section 2. 5. T h i s method w a s u s e d by a n u m b e r of a u t h o r s t o a s s a y flufenamic a c i d i n body f l u i d s and t i s s u e s (8, 37, 40, 49, 62, 63, 64). T h e r e a c t i o n of f l u f e n a m i c a c i d with f o r m a l d e h y d e gives 1-(m-trifluoromethylphenyl)-4-oxo-1,2 - d i h y d r o - 3, 1- b e n z o xazine (4), which is s u i t a b l e f o r f l u o r o m e t r i c d e t e r m i n a tion of the p a r e n t d r u g (63). T h e r e a c t i o n s c h e m e is d e picted in F i g u r e 7. T h e m e t h a n o l i c s o l u t i o n of t h e b e n z o xazine d e r i v a t i v e s h o w s two e x c i t a t i o n m a x i m a at 278 and COOH

NH I

0

HCHO

Figure 7 Reaction of F l u f e n a m i c Acid with F o r m a l d e h y d e 342 n m and a n e m i s s i o n m a x i m u m a t 440-450 n m ( 4 ) . T h e c o r r e s p o n d i n g v a l u e s r e p o r t e d by D e l l e t al. ( 6 3 ) a r e 346 and 458 nm. Another fluorogenic r e a c t i o n of f l u f e n a m i c a c i d , a l r e a d y c i t e d i n Section 5. 1, w a s s t u d i e d by D e l l and K a m p ( 4 ) . F l u f e n a m i c a c i d w a s h e a t e d with c o n c e n t r a t e d s u l f u r i c a c i d to give a m i x t u r e of two i s o m e r i c a c r i d o n e s , I and 11, as i l l u s t r a t e d i n F i g u r e 8. T h e f l u o r e s c e n c e f e a t u r e s of t h e s e compounds, which had b e e n p r e v i o u s l y s y n t h e s i zed by Wilkinson and F i n a r (12), a r e v e r y s i m i l a r s o that

FLUFENAMIC ACID

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Reaction of F l u f e n a m i c Acid with conc. H2S04 the quantitative d e t e r m i n a t i o n c a n b e m a d e on t h e mixtur e . In T a b l e XI a r e l i s t e d the wavelengths of excitation and e m i s s i o n m a x i m a i n n e u t r a l , a c i d i c and a l k a l i n e m e thanol ( 4 ) . T A B L E XI F l u o r e s c e n c e Data of Trifluoromethylacridones Solvent system Methanol Methanol- H C1 Methanol - NaOH

E x c i t a t i o n / e m i s s i o n m a x i m a , n m , of: 4 - C F 3 - a c r i d o n e (I) 2 - C F 3 - a c r i d o n e (11) 400 / 420 400/440 400 / 4 5 5 - 4 6 2

400 / 4 2 1 400/440 400/460

H a t t o r i e t al. ( 6 5 ) p r o p o s e d a f l u o r o m e t r i c method which involves the t r e a t m e n t of a n ethanolic solution of flufenamic a c i d with 0. 5% A1C13 solution i n a b s o l u t e e t h a nol t o obtain a n a l u m i n i u m c h e l a t e , which f l u o r e s c e s a t 440 n m following activation a t 358 n m . T h e m a x i m u m f l u o r o m e t r i c s e n s i t i v i t y of flufenamic a c i d c l a i m e d f o r t h i s method is 4 n g / m l .

5 . 6. I n d i r e c t Atomic Absorption A n a l y s i s It w a s found that flufenamic acid, c o p p e r and 2-(2-hydroxyethyl)pyridine combined i n the r a t i o 1:1:1 t o f o r m a c h e l a t e complex (18). T o obtain t h i s r e s u l t the s a m p l e containing the d r u g w a s t r e a t e d with a r e a g e n t p r e p a r e d adding 9. 0 m l of 0. 1% c u p r i c s u l f a t e solution t o

336


1.5 m l of 2-(2-hydroxyethyl)pyridine. The complex w a s then extracted with propyl acetate, and the amount of flufenamic acid p r e s e n t in the s a m p l e was obtained i n d i r e c tly f r o m the amount of copper determined in the organic solvent by atomic absorption analysis. 5. 7. Chromatographic Analysis 5.71. P a p e r Chromatography Flufenamic acid can be detected by p a p e r chromatography (66) using the following solvent s y s t e m s : a ) Methyl isobutyl ketone : f o r m i c acid : w a t e r (10 p a r t s of ketone s a t u r a t e d with 1 p a r t of 470 f o r m i c acid); Rf = 0.95. b ) Chloroform : methanol : f o r m i c acid : w a t e r (a mixtur e of 1 p a r t of methanol and 1 p a r t of 470 f o r m i c acid used t o s a t u r a t e 10 p a r t s of chloroform); Rf = 0. 95. c ) Benzene : methyl ethyl ketone : f o r m i c acid : water ( a mixture of 9 p a r t s of benzene and 1 p a r t of ketone saturated with 1 p a r t of 2’70 f o r m i c acid); Rf = 0. 94. d ) Benzene : f o r m i c acid : water (10 p a r t s of benzene s a t u r a t e d with 1 p a r t of 270 f o r m i c acid); Rf = 0. 91. e ) Methyl ethyl ketone : diethylamine : w a t e r (921:2:77); Rf = 0. 83. f ) Methyl ethyl ketone : acetone : f o r m i c acid : water (40:2:1:6); Rf = 0. 95. Flufenamic acid can be visualized by UV light, or s p r aying the p a p e r with 0.470 p-nitrobenzenediazonium fluoborate solution in 1:2 dioxane:water o r with 2’70 aqueous phosphomolybdic acid solution. Schmollack and Wenzel (67) developed a method f o r the detection and quantitative determination of flufenamic acid using a c h a m b e r p a p e r a n a l y s i s apparatus. Flufenamic acid w a s then determined fluorometrically a f t e r t r e a t m e n t with formaldehyde vapor t o obtain the strongly fluorescent benzoxazine derivative. 5.72. Thin L a y e r Chromatography Several thin l a y e r chromatographic methods have been developed f o r identification and quantitative determination of flufenamic acid. Some d e t a i l s of these methods a r e s u m m a r i z e d in Table XII. It h a s t o point out that in all c a s e s s i l i c a g e l plates w e r e used.

T A B L E XI1 T h i n L a y e r C h r o m a t o g r a p h y of F l u f e n a m i c Acid Solvent S y s t e m

Ref. 3

Cyclohexane:CHC13:CH30H:CH3COOH (60:30:5:5) B e n z e n e : e t h e r : CH3 COOH: CH3 OH ( 120 : 60: 18: 1)

Plates

Rf

D e t e c t ion

S. G. G F

0. 54

W(254nm)

0. 7 8 NR

id. UV following h e a t i n g with HCHO W ( 3 5 6 nm)

3 4

Benzene:C2H50H:CH3COOH (20:2:1)

id. S. G. H F

8

Cyclohexane :e t h y l acetate : CH3 COOH ( 2 0 :30 :2 )

S. G. F 6 0

0.58

8

Cyclohexane: CHC13:CH3COOH (40:50: 10)

S. G . H F

NR

id.

0.43

W

S. G. H F

NR

UV

id.

0. 58

40

id.

S.G. H F F

44

id.

63

id.

U V ; heat. HCHO + U V ; iodine id.

63

B e n z e n e : m e t h a n o l (9: 1)

id.

63

Cyc1ohexane:ethyl acetate (1:1 0 )

id.

0. 23

id.

41

1sopropanol:ammonia:water (20: 1 : 2 )

NR

0. 64

NR

68

To1uene:acetic a c i d ( 9 : l )

S. G. G

0.73

68

To1uene:acetic a c i d (97. 5:2. 5 )

69

Chloroform:methanol(7:3) i n NH3 atm.

S. G . : silica gel.

NR : not r e p o r t e d .

,

0. 23

id.

0. 60

S.G. 60

0. 37

H N 0 2 spray id.

HCHO/HCOOH at

loooc + w

338


Unterhalt ( 3 ) h a s identified f l u f e n a m i c a c i d i n a m i x t u r e with o t h e r n o n s t e r o i d a l a n t i i n f l a m m a t o r y d r u g s : the s p o t v i s u a l i z e d by UV light w a s e l u t e d with m e t h a n o l and flufen a m i c a c i d w a s 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 c a l l y a t 288 n m with a m e a n r e c o v e r y of 88%. Dell and K a m p ( 4 ) a s s a y e d f l u f e n a m i c a c i d in u r i n e and s e r u m : t h e i r method involved a n e t h e r e a l e x t r a c t i o n f r o m t h e biological fluids followed by T L C . T h e p l a t e s w e r e then exposed t o f o r m a l d e h y d e v a p o r ( 2 h o u r s at 8OoC) and t h e UV-fluorescent spot w a s eluted with m e t h a n o l and f l u f e n a m i c a c i d d e t e r m i n e d f l u o r o m e t r i c a l l y . An a l t e r n a t i v e method p r o p o s e d by the s a m e a u t h o r s involved t h e v i s u a lization of flufenamic a c i d by UV light and a t r e a t m e n t of the s c r a p e d off s i l i c a g e l with conc. H2SO4 followed by f l u o r o m e t r i c d e t e r mi nat ion. T h e method d e s c r i b e d by P a n s e e t a l . ( 4 0 ) f o r t h e d e t e r m i n a t i o n of flufenamic a c i d in u r i n e and p l a s m a w a s s i m i l a r , involving e x t r a c t i o n f r o m b i o l o g i c a l m a t e r i a l , T L C and elution of the d r u g , followed by q u a n t i t a t i v e d e termination ei t h e r spectrophotometrically in methanolic solution o r f l u o r o m e t r i c a l l y in a CC14 solution i n the p r e s e n c e of t r i c h l o r o a c e t i c a c i d . T h e l a t t e r m e t h o d w a s a p plied by s e v e r a l a u t h o r s ( a l r e a d y c i t e d in Section 5. 5) f o r t h e d e t e r m i n a t i o n of f l u f e n a m i c a c i d in b i o l o g i c a l fluids and t i s s u e s . P a n s e e t al. ( 4 4 ) d e s c r i b e d a l s o a method f o r t h e d i r e c t d e n s i t o m e t r i c d e t e r m i n a t i o n of f l u f e n a m i c a c i d on the s i l i c a gel thin l a y e r s . F l u f e n a m i c a c i d w a s d e t e r m i n e d d i r e c t l y in p l a s m a by G e i s s l e r e t a l . ( 6 9 ) by addition of m e t h a n o l t o p r e c i p i t a t e p r o t e i n s , T L C , t r e a t m e n t of t h e d r i e d p l a t e with f o r m a l dehyde v a p o r in t h e p r e s e n c e of f o r m i c a c i d a t 100°C f o r 45 m i n u t e s t o f o r m t h e benzoxazine d e r i v a t i v e , and d i r e c t f l u o r i m e t r y of t h e plate. Use of f o r m i c a c i d s h o r t e n s t h e r e a c t i o n t i m e and e n h a n c e s t h e f l u o r e s c e n c e i n t e n s i t y and the sensitivity (quantities I 2 ng/spot m a y be detected). A method involving r e v e r s e d - p h a s e thin l a y e r c h r o m a t o g r a p h y w a s r e p o r t e d by B o l t z e and K r e i s f e l d (2), which u s e d s i l i c a gel p l a t e s i m p r e g n a t e d with a 10'70 solution of Dow-Corning 200 i n e t h e r . F l u f e n a m i c a c i d w a s c h r o m a t o g r a p h e d u s i n g buffer : dioxane : a c e t o n e (2: 1 : l ) a s the s o l vent s y s t e m , in which t h e buffer w a s a t pH 5. 2 , 6. 2, and

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339

7. 2, r e s p e c t i v e l y . In T a b l e XI11 a r e l i s t e d Rf and R M v a l u e s found f o r flufenamic a c i d . T A B L E XI11 Reversed-Dhase T L C of F l u f e n a m i c Acid pH of the buffer 5. 2 6. 2 7. 2

-

Rf

RM= l o g ( l / R f - l )

0. 78 0. 76 0. 69

- 0.55 0.5 - 0. 350 -

5.73. Gas Chromatography Roseboom and Hulshoff ( 7 0 ) h a v e developed a r a p i d and s i m p l e c l e a n - u p and d e r i v a t i z a t i o n p r o c e d u r e that c a n be g e n e r a l l y applied t o the g a s c h r o m a t o g r a p h i c d e t e r m i n a t i o n of a c i d i c d r u g s including f l u f e n a m i c a c i d i n p l a s m a s a m p l e s . T h e d r u g w a s e x t r a c t e d f r o m acidified p l a s m a with c h l o r o f o r m : i s o p r o p a n o l (95:5), which w a s then e v a p o r a t e d . T h e r e s i d u e w a s d i s s o l v e d i n toluene, then the d r u g w a s b a c k - e x t r a c t e d with a small v o l u m e of a methanolic 20% t e t r a m e t h y l a m m o n i u m hydroxide solution (TMAH). T h e solution obtained w a s added t o N , N - d i m e t h y l a c e t a m i d e . A f t e r t r e a t m e n t with n-butyl iodide the d r u g w a s c h r o m a t o g r a p h e d a s i t s n-butyl ester. A gas c h r o m a t o g r a p h equipped with a flame ionization d e t e c t o r w a s u s e d . T h e g l a s s c o l u m n s (150 c m x 2 mm I. D. ) w e r e packed with 3% OV-1, 3% OV-17 o r 3y0 SP-1000, all on 100-120 m e s h C h r o m o s o r b W HP. T h e c a r r i e r gas ( n i t r o g e n ) f l o w - r a t e w a s mantained a t 20 m l / m i n . T h e r e c o v e r y of flufenamic a c i d in the first e x t r a c t i o n s t e p with ch1oroform:isopropanol w a s 69%, but with toluene, which c a n a l s o be u s e d f o r the e x t r a c t i o n f r o m p l a s m a , a r e c o v e r y of 95% w a s achieved. Toluene h a s the advantage that no e v a p o r a t i o n of the e x t r a c t is n e c e s s a r y , and it can b e e x t r a c t e d d i r e c t l y with the TMAH solution. In t h e b a c k - e x t r a c t i o n with TMAH a r e c o v e r y of 62Y0 w a s obtained. T h e r e t e n t i o n t i m e s of the n-butyl e s t e r of flufenam i c a c i d with v a r i o u s s t a t i o n a r y p h a s e s a r e l i s t e d i n Table XIV.

340


TABLE XIV G a s ChromatoaraDhic Data f o r Flufenamic Acid (701

Stationary phase 370 ov-1 370OV-17 370 sP-1000

Column temperature, OC

Re tent ion time, sec

2 00 210 230

2 34 271 218

Another g a s chromatographic method f o r quantitative determination of flufenamic acid in p l a s m a w a s reported by Cotellessa et al. (43). Flufenamic acid w a s extracted f r o m plasma with benzene, a f t e r dilution of p l a s m a s a m ple with an equal volume of 0. 25 M acetate buffer (pH 4.35). The benzene e x t r a c t w a s evaporated to d r y n e s s under vacuum. The r e s i d u e w a s dissolved in methanol and methylated with diazomethane. The s a m e procedure w a s a l s o applied t o r a t u t e r u s homogenates. The methylated sample w a s dissolved in a n acetone solution of the i n t e r n a l standard 3-chloro-6-aminobenzophenone. G a s chromatographic analysis w a s c a r r i e d out using a gas chromatograph equipped with a flame ionization detector. The stationary phase w a s 370 OV-17 on Gas-Chrom Q (100-120 m e s h ) packed into a g l a s s column ( 3 m x 2 m m I. D. ). The column t e m p e r a t u r e w a s 23OoC and the c a r r i e r g a s (nitrogen) flow-rate w a s 40 m l / m i n . F I D s e n s i tivity was 1 p g / m l p l a s m a and 5 ,ug/g u t e r u s . A m a s s s p e c t r o m e t e r coupled with the g a s chromatograph was employed to a s c e r t a i n the identity of the m e thyl e s t e r of flufenamic acid with the GC peak. A m a s s s p e c t r u m of the methyl e s t e r is presented. The recover i e s f r o m plasma in the range 10-100 ,ug/ml and f r o m u t e r u s homogenates were 98. 270 and 9070, respectively. A g a s chromatographic method f o r the detection of nons t e r o i d a l antiinflammatory d r u g s including flufenamic acid in urine collected f r o m h o r s e s that had received these compounds orally h a s been developed by Hunt et al. (71). T h i s procedure involves the isolation of the d r u g s f r o m

FLUFENAMIC ACID

341

urine by solvent extraction and on-column methylation of the carboxylic acid group. 5. 74. High P e r f o r m a n c e Liquid Chromatography A method f o r the separation and d e t e r m i n a tion of some nonsteroidal antiinflammatory d r u g s including flufenamic acid w a s described by Dusci and Hackett ( 7 2 ) . This procedure, which can be applied to s e r u m s a m p l e s of s m a l l volume (100 pl), involved the extraction of the drugs with acetonitrile. The e x t r a c t w a s taken to d r y n e s s at 5OoC under a s t r e a m of nitrogen. The r e s i d u e w a s redissolved in 1 0 0 pl of the elution solvent (60% acetonitrile in 45 mM KH2P04 adjusted to pH 3. 0 with H 3 P 0 4 ) . An aliquot of 10-20 pl was injected in a high performance liquid chromatograph equipped with a variable wavelength U V detector. The column (30 c m x 3. 9 m m I. D. ) w a s packed with pBondapak c18. The conditions f o r individual analysis of flufenamic acid were as follows: flow-rate of the elution solvent 2. 0 ml/min, wavelength 282 nm. F o r the separation of flufenamic acid f r o m the mixture of antiinflammatory drugs (flufenamic and mefenamic acid, naproxen , ibuprofen , indomethacin, phenylbutazone, oxyphenbutazone) a flow-rate of 0 . 8 m l / m i n and a wavelength of 225 nm was used: under these conditions the elution t i m e of flufenamic acid w a s 10. 5 min. Using the above -mentioned elution solvent , flufenamic and mefenamic acid were not separated. A modified elution solvent (3570 acetonitrile in 0. 7% NHqC1 buffered to pH 7. 8 with ammonia) allowed to obtain the separation of all the drugs investigated. Using a flow-rate of 1. 0 m l / min, the elution time of flufenamic acid w a s 10. 2 min (mefenamic acid 7. 8 min). The r e c o v e r y of flufenamic acid was 92t370 in a s e r i e s of ten plasma s a m p l e s e x a m i ned, in the range 1. 0-20 p g / m l . Lin et al. ( 4 2 ) have developed a H P L C procedure f o r the determination of flufenamic acid and mefenamic acid in plasma, A single extraction s t e p i s followed by r e v e r sed-phase chromatography. Flufenamic acid and mefenamic acid can be internal standards f o r each other during e i t h e r assay. The extraction of flufenamic acid f r o m a c i dified plasma s a m p l e s (1 m l ) , t o which 4 p g of mefenamic acid had been added, w a s accomplished with carbon t e t r a -

342


chloride. T h e e x t r a c t w a s e v a p o r a t e d t o d r y n e s s u n d e r a s t r e a m of nitrogen and t h e r e s i d u e w a s r e d i s s o l v e d i n t o 0. 5 m l of methanol, and a n aliquot w a s i n j e c t e d in t h e c h r o m a t o g r a p h , which w a s equipped with a s t a i n l e s s steel column ( 3 0 c m x 4 m m I. D. ) packed with a s t a b l e r e v e r s e d - p h a s e s t a t i o n a r y p h a s e of p o r o u s silica b e a d s c o a t e d with c h e m i c a l l y bonded cyanopropylsilane m o n o l a y e r s . T h e elution solvent w a s w a t e r : a c e t o n i t r i l e : a c e t i c a c i d (60:30: 10). T h e f l o w - r a t e w a s 1 m l / m i n with a n o p e r a t i n g p r e s s u r e of 1000 p s i at r o o m t e m p e r a t u r e . T h e effluent w a s m o n i t o r e d continuously at 254 n m . Under t h e s e conditions flufenamic a c i d had an elution t i m e of 10. 4 min, with a m e a n r e c o v e r y f r o m p l a s m a of 100.7?3.4% in t h e 1-10 p g r a n g e . T h e s e n s i t i v i t y l i m i t w a s 1 p g / m l of p l a s m a . A method f o r t h e d e t e r m i n a t i o n of flufenamic a c i d a n d i t s m a j o r m e t a b o l i t e s , 4 ' - h y d r o x y - and 5-hydroxyflufenam i c a c i d , w a s d e s c r i b e d by Kubo e t al. ( 7 3 ) : t h e a c i d i fied s e r u m w a s e x t r a c t e d with ethyl a c e t a t e . T h e o r g a n i c e x t r a c t was e v a p o r a t e d and the r e s i d u e w a s r e d i s s o l v e d i n ethanol and c h r o m a t o g r a p h e d , u s i n g a c o l u m n packed with Bondapack c 1 8 . T h e m o b i l e p h a s e c o n s i s t e d of wat e r : ethanol (52:48) containing 0. 1% N a 2 H P 0 4 and 0. 5% t e t r a b u t y l a m m o n i u m b r o m i d e , a d j u s t e d t o pH 7. 81. T h e r e c o v e r i e s f r o m p l a s m a w e r e 98. 870 f o r f l u f e n a m i c a c i d , 97.0% f o r 4 ' - h y d r o x y - d e r i v a t i v e J a n d 98.070 f o r 5-hydroxy-derivative. 6. DETERMINATION IN BODY FLUIDS AND TISSUES Many m e t h o d s a m o n g t h o s e outlined in t h i s a n a l y t i c a l p r o f i l e have been applied t o t h e detection a n d quantitative d e t e r m i n a t i o n of flufenamic a c i d in biological s a m p l e s of a n i m a l o r human origin. Such a p p l i c a t i o n s w e r e r e p o r t e d i n the p a p e r s l i s t e d below : Colorimetry 46 Spe c t rophot o m e t r y 4, 30, 40,41, 44, 60, 61 Fluorimetry 4, 8, 11, 37, 40, 45, 47, 49, 62, 63, 64, 65, 67, 69 18 Indirect A A A n a l y s i s PC 67 4, 8,40, 41, 44,45, 63, 64, 68, 6 9 TLC

FLUFENAMIC ACID

343

43, 70, 7 1 42, 72, 7 3

GC HPLC 7. AC KNOWL ED G EM EN T

T h e a u t h o r s would l i k e t o t h a n k D r . M i c h e l e L i g u o r i and M r . Vincenzo Migliaro, Ente F a r m a c o l o g i c o Italiano, N a p l e s , f o r p r o v i d i n g IR, NMR a n d UV s p e c t r a of f l u f e namic acid. 8. R E F E R E N C E S

1. M e r c k Index, 9th e d . , 1976, M e r c k a n d C o . , R a h w a y , N. J . ; p. 4028. 2. K. H. B o l t z e a n d H. K r e i s f e l d , A r z n e i m . - F o r s c h . 27, 1 3 0 0 (1977). 3. B. U n t e r h a l t , A r c h . P h a r m . 303, 4 4 5 (1970). 4. H. D. D e l l a n d R. K a m p , A r c h . P h a r m . 303, 785 (1970). 5. K. Ikeda, K. U ekam a, M. O t a g i r i a n d M. H a t a n o , J. P h a r m . Sci. 63, 1168 ( 1 9 7 4 ) . 6. J. K r a c m a r , M. A l v a r e z s o t o l o n g o , J. K r a c m a r o v a , B. M o r a v c o v a a n d H. D osl ova, P h a r m a z i e 33, 659 (1978). 29, 1 9 7. J. K r a c m a r and J. K r a c m a r o v a , C e s k . F a r m . (1980). C . A . 93, 192094 ( 1 9 8 0 ) . D o e r s i n g , W. F i s c h e r , J. F i e d l e r , 8. H. D. Del l , H. J a c o b i a n d R. K a m p , A r z n e i m . - F o r s c h . 31, 9 (1981). 9. A. C. M e h t a a n d S. G. S c h u l m a n , T a l a n t a 20, 702 (1973). 10. J. N . M i l l e r , D. L. P h i l l i p s , D. T. B u r n s a n d J. W. B r i d g e s , T a l a n t a 25, 4 6 (1978). 11. H. D. D e l l a n d B. K u x c h b a c h , F r e s e n i u s ' Z.Ana1. Chem. 262, 356 (1972). 12. J. H. Wi l ki nson a n d I. L. F i n a r , J. C h e m . S o c . , 3 2 ( 1948). 1 3 . P a r k e , D a v i s & Co. , F r . P a t e n t M1341, J u l y 2, 1962. C . A . 58, 10130a (1963). B r i s t o c M y e r s C o . , Neth.App1. 6, 604, 860, O c t . 13, 14. 1966. C . A . 66, 55513 ( 1 9 6 7 ) . 15. P. F. J uby, T. W. H u d y m a a n d M. B r o w n , J . M e d .

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M.

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16. 17. 18.

C h e m . 11, 111 ( 1968). M. K u h z r t - B r a n d s t g t t e r , A . K o f l e r a n d G. K r a m e r , S c i . P h a r m . 42, 150 ( 1 9 7 4 ) . C . A . 82, 4 7 6 8 1 ( 1 9 7 5 ) . E . M. J o n e s T P a r k e , D a v i s & C o . , B r i t . P a t e n t 953, 741, A p r . 2, 1964. C. A . 60, 1 5 6 8 8 b ( 1 9 6 4 ) . T. Minamikawa, K. Sakai, N. H a s h i t a n i , E . F u k u s h i m a and N. Y a m a g i s h i , C h e m . P h a r m . B u l 1 . 21, 1 6 3 2 (1973). R . B. Moffett and B. D. A s p e r g r e n , J. Am. C h e m . SOC. 82, 1600 (1960). A . J . A g u i a r a n d R. J. F i f e l s k i , J . P h a r m . S c i . 55, 1 3 8 7 (1 9 6 6) . A . H. Ghanem, H. E l - S a b b a g h a n d H. A b d e l - A l i m , P h a r m . Ind. 42, 854 (1980). N. A . D a a b i r S. A . K h a l i l a n d V. F. N a g g a r , Can. J. P h a r m . Sci. 11, 1 1 4 ( 1 9 7 6 ) . W. J. Dunn, J-Med.Chem. 16, 4 8 4 ( 1 9 7 3 ) . H. T e r a d a , S. M u r a o k a a n d T. F u j i t a , J . M e d . C h e m . 1 7 , 330 (1974). G. L o m b a r d i n o , I. G. O t t e r n e s s a n d E. H. W i s e m a n , A r z n e i m . - F o r s c h . 25, 1 6 2 9 ( 1 9 7 5 ) . M. K u h n e r t - B r a n d s t a t t e r , L. B o r k a a n d G. F r i e d r i c h - S a n d e r , A r c h . P h a r m . 307, 8 4 5 (1974). J. K r c , J r . , M i c r o s c o p e 25, 31 ( 1 9 7 7 ) . Z . Galdecki, M. L. G l o w k a a n d Z . G o r k i e w i c z , A c t a P o l . P h a r m . 35, 77 (1978). C.A. 89, 1 5 5 7 6 3 ( 1 9 7 8 ) . A . B u r g e r a n d R. R a m b e r g e r , M i k r o c h i m . A c t a 1, 17 (1980). H. H. F r e y a n d M. A . E l - S a y e d , A r c h . I n t . P h a r m a codyn. T h e r . 230, 300 (1977). A . A l b e r t a n d E . P. S e r j e a n t i n "The D e t e r m i n a t i o n of Ionization Constants", C h a p m a n a n d Hall, London, 1971, p. 72. H. T e r a d a a n d S. M u r a o k a , M o l . P h a r m a c o 1 . 8, 9 5 (1 9 7 2 ). U. Jahn and T. W a g n e r - J a u r e g g , A r z n e i m . - F o r s c h . 24, 4 9 4 (1974). H. M o r i y a m a , H. N a g a t a and T. T a m a k i , S u m i t o m o C h e m i c a l Co. , L t d . , J a p a n . P a t e n t 7 1 14, 656, A p r . 20, 1971. C.A. 75, 48703 (1971 ) . S. K o m u r a , K. K k a m u r a and M. T a k e n a k a , O t s u k a

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19. 20. 21. 22.

23. 24. 25. 26. 27. 28. 29. 30. 31.

32. 33. 34.

35.

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7

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-

FLUFENAMIC ACID

36.

37. 38. 39.

40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52.

53. 54. 55.

345

C h e m i c a l D r u g s C o . , L t d . , J a p a n . P a t e n t 7 3 26, 744, A p r . 9, 1973. C.A. 79, 31680 ( 1 9 7 3 ) . G. R . Allen, J r . , and F. R . R . Church, A m e r i c a n Cyanamid Co., S. A f r i c a n P a t e n t 71 00, 512, Sep. 3, 1971. C.A. 76, 140237 ( 1 9 7 2 ) . A . J. Glazko, Ann. P h y s . Med. , Suppl. 9, 24 ( 1 9 6 7 ) . R . E. O b e r , K. Ritchie and S. F. Chang, F e d . P r o c . 24, 547 ( 1 9 6 5 ) . R. E. Bowman, K. D. Brunt, K. E. Godfrey, L. K r u s z y n s k a , A . A . Reynolds, R . I. T h r i f t , D. Waite and W. R . N. Williamson, J. Chem.Soc. P e r k i n I, 1 (1973). P. P a n s e , P. Z e i l l e r and K. H. Sensch, A r z n e i m . F o r s c h . 21, 1 6 0 5 ( 1 9 7 1 ) . H. A . R o s e n b e r g and T. R . B a t e s , P r o c . S o c . E x p . Riol. Med. 145, 93 ( 1 9 7 4 ) . C. K. Lin, C. S. Lee and .J. H. P e r r i n , J . P h a r m . Sci. 69, 95 ( 1 9 8 0 ) . L. C z e l l e s s a , R . Riva, P. Salva, F. M a r c u c c i and E. Mussini, J. C h r o m a t o g r . 192, 4 4 1 ( 1 9 8 0 ) . p. P a n s e , P. Z e i l l e r and K. H. Sensch, A r z n e i m . F o r s c h . 24, 1 2 9 8 ( 1 9 7 4 ) . H. D. Dell, J. F i e d l e r , H. J a c o b i and B. Wasche, A r z n e i m . - F o r s c h . 27, 1 3 2 2 (1977). H. D. D e l l and J. F i e d l e r , F r e s e n i u s ' Z.Ana1. Chem. 270, 278 ( 1 9 7 4 ) . D. L o r e n z and H. D. Dell, Naunyn-Schmiedeberg's A r c h . P h a r m a c o l . 277, Suppl. , R 4 4 (1973). J. A r i a s and R . Cadorniga, Boll. Chim. F a r m . 112, 804 (1973). L. Angelucci, €3. P i e t r a n g e l i , P. C e l l e t t i and S. F a villi, .J. P h a r m . Sci. 65, 455 ( 1 976). C. F. Chignell, 1 , i f e S c i . 7, 1181 (1968). C. F. Chignell, Mol. P h a r m a c o l . 5, 4 5 5 ( 1 9 6 9 ) . C. F. Chignell and D. K. S t a r k w e a t h e r , M o l . P h a r m a c o l . 7, 229 ( 1 9 7 1 ) . J. B l a n c h a r d , T . N. T o z e r , D. L. S o r b y and L. D. Tuck, Mol. P h a r m a c o l . 11, 1 3 3 ( 1 9 7 5 ) . M. Otagiri, G. E. H a r d e e and J. H. P e r r i n , Bioc h e m . P h a r m a c o l . 27, 1 4 0 1 (1978). G. Sudlow, D. J. B i r k e t t and D. N. Wade, Mol.

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56. 57. 58. 59. 60. 61.

62. 63. 64. 65. 66. 67. 68. 6 9. 70. 71.

72. 73.


P h a r m a c o l . 11, 824 (1975). G. Sudlow, T J . B i r k e t t a n d D. N. W a d e , Mol. P h a r m a c o l . 1 2 , 1052 ( 1976). Y. Ka n e o, A. K ai , S. K i r y u a n d S. I g u c h i , Y a k u g a k u 86, 1 0 0 7 4 7 ( 1 9 7 7 ) . Z a s s h i 96, 1412 (1976). C.A. G. D e v a G , P. M e s n a r d a n d A . M. B r i s s o n , Ann. P h a r m . F r . 27, 239 (1969). M. I. W a l a s r a n d M. R i z k , I nd i a n J . P h a r m . 39, 8 2 (1 9 7 7 ). C . A . 87, 157253 (1977). J. B. C a r e y , J. C l i n . I n v e s t . 40, 1 0 2 8 ( 1 9 6 1 ) . Y. A . B e l t a g y , Z e n t r a l b l . P h a r m . , P h a r m a k o t h e r . Lab o r a t o r i u m s d i a g n . 116, 925 (1977). C. A , 88, 1 5 8 5 4 8 (1978). R. A . B u chanan, C. J. E a t o n , S. T. Koeff a n d A . W. K r i n k e l , C u r r . T h e r . R e s . 11, 5 3 3 ( 1 9 6 9 ) . H. D. Del l , J. F i e d l e r a n T B . W z s c h e , A r z n e i m . F o r s c h . 27, 1312 ( 1977). H. D. Del l , H. Jacobi, R. K a m p a n d J. K o l l e , A r z n e i m . - F o r s c h . 31, 2 1 ( 1981). Y. H a t t o r i , T. Arai, T. M o r i a n d E. F u j i h i r a , C h e m . P h a r m . Bull. 18, 1 0 6 3 (19 7 0 ) . L. R e io , J. C h r o m a t o g r . 68, 1 8 3 ( 1 9 7 2 ) . W. S c h m o l l a c k a n d U. W e n z e l , P h a r m a z i e 29, 5 8 3 (1 9 7 4 ). B. D e m e t r i o u a n d B. G. O s b o r n e , J. C h r o m a t o g r . 90, 4 0 5 (1 9 7 4) . H. E. G e i s s l e r , E. M u t s c h l e r a n d A . S c h u m a c h e r , J. C h r o m a t o g r . 146, 1 6 9 ( 1978). H. R o s e b o o m a n d A . Hulshoff, J. C h r o m a t o g r . 173, 6 5 (1 9 7 9 ) . J. P. Hunt, P. E. Haywood a n d M. S. M o s s , P r o c . Int. S y mp . E q u i n e Med. C o n t r o l , 3 r d , 1979, 9. C. A . 94, 97262 (1981). J. D u s c i a n d L. P. H a c k e t t , J. C h r o m a t o g r . 1 7 2 , 5 1 6 (1 9 7 9) . 0. Kubo, K. N i s h i d e a n d N. K i r i y a m a , J. C h r o m a t o g r . 174, 254 (1979).

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1981.

HEXESTROL Hassan X Aboul-Enein, Essam A. Lo@, and Mohumed E . Mohumed

348 348 348 348 349 349 349 349 349 350 350 350 358 361 361 362 362 362 363 364 370 370 372

1. Description 1 . 1 Nomenclature 1.2 Formulae 1.3 Molecular Weight I .4 Elemental Composition 1.5 Appearance, Color, Odor 2. Physical Properties 2.1 Crystal Properties 2.2 Melting Point 2.3 Solubility 2.4 Identification 2.5 Spectral Properties 3. Synthesis 4. Stability and Decomposition Products 5 . Metabolism 6. Methods of Analysis 6.1 Titrimetry 6.2 Colorimetry 6.3 Ultraviolet Spectrophotometry (Uv) 6.4 Chromatographic Analysis 6.5 Mass Fragmentography 6.6 Biological Assays References

Analytical Profiles of Drug Substances Volume I I

347

Copyright 0 1982 by The American Pharmaceutical hsociation

ISBN 0-12-Mo811-9

HASSAN Y. ABOUL-ENEIN ET AL.

348

ANALYTICAL PROFILE-HEXESTROL

1. Descr i p t ton 1.1 Nomenclature 1.11 - Chemical N a m e s (?)

3 , 4 - Di(p-hydroxy phenyl) n-hexane. 4 , 4 ' - (1, 2 d i e t h y l - 1, 2 - e t h a n e d i y l ) bis-phenol. 4 , 4 ' - (1, 2 d i e t h y l e t h y l e n e ) d i p h e n o l . 4 , 4 ' - dihydroxy - y,b-diphenyl hexane. 4 , 4 ' - dihydroxy - a ,B-diethyldiphenylethane. p, p l - dihydroxy - d i p h e n y l hexane. meso - 3 , 4 - b i s (p-hydroxyphenyl) -nhexane

.

1 . 1 2 - Generic Name H e x e s t r o l , H e x o e s t r o l , Dihydrodiethyls t i l b e s t r o l , Hexanoestrol, Cycloestrol , Hormoestrol, S y n e s t r o l .

1.13 - Trade Name S yn t r o g h e , Fo 11i p l e x , S yn t hovo

1.2

Formula

1 . 2 1 - Emprical

'18 H22 '2 1.22

- Structural C2R5

1.3


.

349

HEXESTROL

1.4

Elemental Composition C , 79.96%; H, 8.2%; 0,11.84%

1.5

Appearance, Color, odor w h i t e , o d o r l e s s , c o l o r l e s s c r y s t a l s or c r y s t a l l i n e powder (1).

2.

Physical properties

2.1

Crystal properties The f o l l o w i n g t a b l e shows t h e c r y s t a l form of h e x e s t r o l d e r i v a t i v e s and t h e i r m e l t i n g p o i n t s . These d e r i v a t i v e s c a n be used f o r i d e n t i f i c a t i o n purposes too ( 2 ) .

Derivative

Crystalization solvent

0

Crystal form

M.P.,

plates crystals crystals crystals crystals

146 127-128 106-107 96-97 150-153

C

Meso Form Di-Me e t h e r Dipropionyl Dibutyryl Dicaproyl Disuccinyl

Me2CO-MeOH Pet. ether Pet. ether

Pet. e t h e r CHC13-pet. e t h e r

DL(+) Form Di-Me

2.2

ether

C&-pet.

ether

56

Melting p o i n t The f o l l o w i n g are t h e m e l t i n g p o i n t s f o r h e x e s t r o l i n i t s meso, DL-forms and t h e i r a n t i p o d e s .

Form M.P.,

0

Ref

C

Meso -

DL (+)Form

185-188 184-185 186

3 4 5

128

2

350

HASSAN Y. ABOUL-ENEIN ET AL. L(-) Form D(+) Form

2.3

80 80

2 2

Solubility Freely soluble i n ether, soluble i n acetone, a l c o h o l , methanol, s o l u b l e i n v e g e t a b l e o i l s upon warmming a l s o s o l u b l e i n d i l u t e s o l u t i o n of a l k a l i h y d r o x i d e . S l i g h t l y s o l u b l e i n c h l o r o f o r m , benzene; i n s o l u b l e i n water (1, 3 ) .

2.4

Identification The f o l l o w i n g i d e n t i f i c a t i o n tests a r e d e s c r i b e d i n t h e P h a r m a c e u t i c a l Codex 1979 ( 5 ) .

1) To a b o u t 25Omg add l m l of a c e t i c a n h y d r i d e and 2 m l of d e h y d r a t e d p y r i d i n e , b o i l u n d e r a r e f l u x c o n d e n s e r f o r 1 5 m i n u t e s , c o o l , add 5Oml of water and s h a k e t h o r o u g h l y u n t i l a p r e c i p i t a t e is produced which, a f t e r washing w i t h water and d r y i n g , m e l t s a t a b o u t 138OC. 2) D i s s o l v e a b o u t lOmg i n 5 m l of H2SO4; t h e s o l u t i o n i s c o l o r l e s s ( d i s t i n c t i o n from s t i l b o e s t r o l , which g i v e s a g o l d e n - y e l l o w color). 2.5

Spectral properties

2.51

Ultraviolet spectra H e x e s t r o l i n e t h a n o l shows maxima a t 230nm ( E l % , l c m , 775) and 280 nm ( E l % , lcm, 1 4 0 ) ; 0.11NaOH h e x e s t r o l g i v e s maxima a t 242 nm ( E l % , l c m , 965) and 295 nm (El%, l c m , 1 7 5 ) . A s shown i n f i g u r e ( 1 ) and i n agreement t o t h e f i g u r e s p u b l i s h e d by C l a r k e ( 3 ) .

2.52

Infrared Spectra The i n f r a r e d spectrum of h e x e s t r o l i n K B r d i s c i s g i v e n i n f i g u r e ( 2 ) . Major band a s s i g n m e n t s are a s follows: -1 Assignment Frequency Cm 3400 P h e n o l i c OH 1610, 1600 Aromatic r i n g C=C s t r e t c h

35 1

HEXESTROL

L 200 nm

F i g . 1.

300 nm

250 nm

350 nm

The u l t r a v i o l e t a b s o r p t i o n spectrum of H e x e s t r o l

i n ethanol. I n s t r u m e n t : Pye Unicam SP8-100

Wavenumber

Figure 2.

I R s p e c t r u m of H e x e s t r o l i n K B r .

Instrument:

P e r k i n Elmer 567


352

Other f i n g e r p r i n t bands c h a r a c t e r i s t i c to-l h e x e s t r o l a r e 1530, 1450, 1300 and 1110 cm 2.53 a)

.

Nuclear Mametic Resonance Spectrum PMR A t y p i c a l PMR spectrum of h e x e s t r o l i s shown

i n Figure (3). The sample was d i s s o l v e d i n d e u t r a t e d CDC13 and a drop of d e u t r a t e d d i m e t h y l s u l f o x i d e (DMSO-db) u s i n g TMS a s t h e i n t e r n a l s t a n d a r d . The spectrum was determined on a V a r i a n T60-A s p e c t r o m e t e r . The f o l l o w i n g s t r u c t u r a l a s s i g n m e n t s have been e l i c i t e d from F i g u r e ( 3 ) . Chemical S h i f t (6)

T r i p l e t a t 0.50

CH CH -3 2

Multiplet centered a t 1.3

C H e 2

M u l t i p l e t c e n t e r e d a t 1.90

b)

Assignment

-CH-CH-

Doublet of d o u b l e t c e n t e r e d a t 6.86

eight aromatic protons character i s t i c f o r para substitution of t h e r i n g .

Broad s i n g l e t exchangable w i t h D20 a t 7.67

p h e n o l i c OH group.

13C-NMR

*

The I3C-NMR of h e x e s t r o l has been d e t e r m i n e d , t h e off-resonance spectrum ( F i g . 4 ) shows seven s i n g l e t s . The complete spectrum i s shown i n F i g . 5. The spectrum was determined on a Varian FT-80A i n DMSO-db a s s o l v e n t , t u b e d i a m e t e r 10 mm, s p e c t r a l width 5000 Hz

*H.Y. Aboul-Enein, unpublished d a t a

I

I

.

1

ao

.

.

F i g u r e 3.

.

.

1

ZO

.

I

I

.

.

.

1

6.0

.

.

.

.

1

5.0

.

.

. . ~ PPM( b) 4.0

.

.

. .

.

3.0

1

~

.

.

.

. ’ 2.0

~

”

. ” 1.0

PMR s p e c t r u m of H e x e s t r o l i n C D C l -DMSO-d w i t h IT% a s i n t e r n a l standard. 3 6

Instrument:

V a r i a n T60-A

”

~

354

355

356

HASSAN Y. ABOUL-ENEIN ET AL. a c q u i s a t i o n t i m e : 1.638 sec.; p u l s e w i d t h : 4 Usec; number of d a t a p r i n t : 16384. S p e c t r a l a s s i g n m e n t s a r e l i s t e d below:

(

Chemical S h i f t i n ppm r e l a t i v e t o TMS) 12.06

CH3

26.97

CH2

52.77

CH-

134. 4 2 115.05 128.82 155.39 2.54

Assignments

c 1- c.1 c 2-c 6 02- c 6 c3-c 5 c--c 3 5 c4-c*4

Mass Spectrum

The mass spectrum of h e x e s t r o l , o b t a i n e d by chemical i o n i z a t i o n w i t h i s o b u t a n e g a s , i s shown i n P i g . 6. The spectrum w a s determined by d i r e c t i n l e t t o Ribermag 10-10R m a s s s p e c t r o m e t e r and e x h i b i t s c o m p a r a t i v e l y l i t t l e fragmentation. The f o l l o w i n g t a b l e g i v e s t h e most prominent i o n s and t h e i r r e l a t i v e i n t e n s i t i e s .

Mass ( d e )

27 0

Relative Intensity % 4.6 (M+)

219

10.5

178

11.8

1 77

93

1 36

9.9

. I

80 90

14011012Q 130 W 1 5 0 1601?01801902002102PO2303+0250260270

F i g u r e 6.

Mass Spectrum of H e x e s t r o l ( C I - i s o b u t a n e ) by d i r e c t i n l e t i n s e r t i o n .

determined


358

100 ( b a s e peak)

135 134

36.9

107

42.6

+'

+.

1

0

-@

CH2CH3

I

CH-CH m/e 177

mfe 178 +*

CH2CH3

O D

CH2CH3

H@ @ H!

LH

m f e 134

m f e 135 3.

CH CH r - C Hl 2

Synthesis S e v e r a l methods have been p u b l i s h e d and p a t e n t e d f o r t h e s y n t h e s i s of h e x e s t r o l . I n 1938 Campbell et-a1 - (6) i s o l a t e d h e x e s t r o l i n p o o r y i e l d , from t h e p r o d u c t s of d e m e t h y l a t i o n of a n e t h o l e . Some of t h e s y n t h e t i c a p p r o a c h e s f o r h e x e s t r o l a r e summerized a s f o l l o w s : -

1) C a t a l y t i c h y d r o g e n a t i o n of p s e u d o d i e t h y l s t i l b e s t r o l g i v e s h e x e s t r o l a l o n g w i t h some d i e t h y l s t i l b e s t r o l (7).

=:

EtEt

H

O

G

@OH

I

catalytic hydrogenat i o n

HO&-

,CH

I

Et

@H+HO

0

Et

I c = c I

Et

+*

359

HEXESTROL Hydrogenation of t h e d i m e t h y l e t h e r s of d i e t h y l s t i l b e s t r o l and pseudodiethylstilbestrol w i t h subsequent d e m e t h y l a t i o n a f f o r d s t h e meso-isomer of h e x e s t r o l which i s more p o t e n t t h a n t h e DL-isomer ( 7 ) . H CH CH

HO

meso-f orm 2) Hydrogenation of 4 , 4 ' dihydroxy y-6 d i p h e n y l y-6 hexadiene g i v e s h e x e s t r o l i n q u a n t i t a t i v e y i e l d ( 7 ) .

CH CH

HOO'L!!

0

Fd Icharcoa 1

>- .

OH

Hexestrol

3) H e x e s t r o l may be prepared by t h e a c t i o n of e t h y l magnesium bromide on a n i s a l d a z i n e w i t h subsequent d e m e t h y l a t i o n (8).

Hexes t r o l The above methods f o r t h e s y n t h e s i s of h e x e s t r o l i n v o l v e t h e u s e of i n t e r m e d i a t e s which a r e d i f f i c u l t t o prepare.

360

HASSAN Y. ABOULENEIN ET AL. 4) B e r n s t e i n and Wallis d e s c r i b e d ( 4 ) a n a l t e r n a t e i n e x p e n s i v e method f o r t h e s y n t h e s i s o f h e x e s t r o l s t a r t i n g from p-hydroxypropiophenone a s shown i n scheme ( 1 )

I I1

I1

-@

-b ,CH30 N a

YHCH

alcohol

OH2

I11 I11

F

HBr C

H

3

@'

0

CH-CH

Br 2 5

IV

Scheme 1

VI

5) Kharasch and Kleiman(9) p r e p a r e d h e x e s t r o l d i m e t h y l e t h e r from a n e t h o l e hydrobromide and G r i g n a r d r e a g e n t i n t h e p r e s e n c e of a h a l i d e of c o b a l t , n i c k e l o r i r o n , t h e f r e e r a d i c l e g e n e r a t e d from t h i s r e a c t i o n dimerizes t o give hexestrol dimethylether i n y i e l d s r a n g i n g from 14-41%. The h i g h e r m e l t i n g p o i n t f o r t h e meso-form a l l o w s i t s s e p a r a t i o n from t h e DLby-product formed i n t h i s r e a c t i o n .

r

1

HEXESTROL

4.

361

S t a b i l i t y and Decomposition p r o d u c t s H e x e s t r o l is a r e l a t i v e l y s t a b l e compound a t room t e m p e r a t u r e ; however i t i s recommended t o b e k e p t i n a w e l l c l o s e d c o n t a i n e r p r o t e c t e d from l i g h t .

5.

Yetabolism I l e x e s t r o l i s m e t a b o l i z e d i n a s i m i l a r way a s d i e t h y l s t i l b e s t r o l , i t i s excreted c h i e f l y as a glucuronide conjugate ( 3 ) . This glucuronide i s mostly excreted i n t o t h e b i l e which i s s u b j e c t e d t o h y d r o l y s i s by i n t e n t i n a l g l u c u r o n i d a s e enzyme d u r i n g i t s p a s s a g e i n t o t h e g u t . T h i s a l l o w s t h e d r u g be r eab so r b ed , r e c o n j uga t e d and r e - e x e r e t e d ( h e p a t i c c i r c u l a t i o n ) ( 1 0 ) . O t h e r p o s s i b l e m e t a b o l i t e i n t e r m e d i a t e s which This should b e i n v e s t i g a t e d a r e shown in scheme ( 2 ) . i s i n a n a l o g y t o t h e p o s s i b l e m e t a b o l i t e s of d i e t h y l s t i l b e s t r o l which h a s r e c e n t l y a t t r a c t e d t h e a t t e n t i o n by b e i n g l i n k e d t o t h e o c c u r a n c e o f v a g i n a l a d e n o c a r c i noma i n a d o l e s c e n t d a u g h t e r s whose m o t h e r s had r e c e i v e d d i e t h y l s t i l b e s t e r a l d u r i n g p r e g n a n c e (11, 1 2 , 13).

HO HO *

H

o

e

o OH

Me0

Me0

HO

Me0

Scheme 2.

Expected M e t a b o l i t e s of H e x e s t r o l

H


6.

Methods of A n a l y s i s

6.1

Titrimetry

- Aqueous (Bromometry) The f o l l o w i n g method h a s been d e s c r i b e d f o r 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 h e x e s t r o l d i a c e t a t e (14) : To 70 mg of sample add 1 0 m l of 0.5N m e t h a n o l i c KOH, and h e a t under r e f l u x f o r 30 m i n u t e s on a water-bath. Cool, add 5Oml of a c e t i c a n h y d r i d e , shake u n t i l t h e h y d r o l y s a t e i s d i s s o l v e d , t h e n add 2 m l of 30% K B r s o l u t i o n , 2 ml of conc. H2SO4 and 20 m l of 0.1N-KBr03, and set a s i d e i n t h e d a r k f o r 1 0 min. Add lgm of K I and 100 m l of H 2 0 , and t i t r a t e w i t h O.lN-Na2S203 u s i n g +1% over t h e r a n g e s t a r c h a s i n d i c a t o r . The e r r o r i s 7 0 t o 90 mg of h e x e s t r o l d i a c e t a t e . 6.2 Colorimetry H e x e s t r o l has been determined i n o i l y s o l u t i o n and t a b l e t form a s f o l l o w s ( 1 5 ) : For t a b l e t : e x t r a c t a sample c o n t a i n i n g 2 t o 3 mg of h e x e s t r o l w i t h methanol, d i l u t e t h e e x t r a c t w i t h water t o 100 m l , and f i l t e r . To a 20 m l a l i q u o t add b o r a t e b u f f e r s o l u t i o n (pH 1 1 ) . For o i l y i n j e c t i o n s : Mix a sample c o n t a i n i n g 0.4 t o 0.6 mg of h e x e s t r o l w i t h l i g h t petroleum (5ml) , shake t h e s o l u t i o n w i t h methanol (6ml) , add b u f f e r s o l u t i o n (13ml), shake, and c o l l e c t t h e aq. methanol phase i n a lOOml f l a s h . Twice r e p e a t t h e e x t r a c t i o n w i t h methanol (6ml) and b u f f e r s o l u t i o n (13ml) and t o t h e combined aq. e x t r a c t s add methanol (2ml). For d e t e r m i n a t i o n of h e x e s t r o l : h e a t t h e prepared s o i u t i o n on a water b a t h f o r 30 min., c o o l i t t o room temperature, add d i a z o t i z e d s u l p h a n i l i c a c i d s o l u t i o n (12ml) and mix. A f t e r 40 minutes d i l u t e w i t h b u f f e r s o l u t i o n t o 100 m l , and measure t h e e x t i n c t i o n a t 495 nm a g a i n s t water. Beer's law i s obeyed f o r 0.5 t o 4 mg of h e x e s t r o l p e r 1 O O m l . R e s u l t s a g r e e to. w i t h i n +lo% w i t h t h e o r e t i c a l values.

HEXESTROL

363

B e l i k o v , d e s c r i b e d a s i m i l a r p r o c e d u r e (16) which depends on d i a z o d i z a t i o n of h e x e s t r o l w i t h d i a z o s u l p h a n i l i c a c i d . However, t h e azo-dye formed i s measured i n a l k a l i n e medium a t 420 nm. Another c o l o r i m e t r i c method f o r d e t e r m i n a t i o n of hexestrol i n feeds i s reported a s follows (17): The ground f e e d i n g s t u f f (40gm) mixed w i t h lOgm of sand i s s e t a s i d e w i t h c h l o r o f o r m o v e r n i g h t , t h e n e x t r a c t e d w i t h CHC13 f o r 6 h o u r s . The e x t r a c t i s made up t o 2 O O m l of CHC13. The r e s i d u e from e v a p o r a t i o n of t h e f i n a l CHC13 e x t r a c t i s d i s s o l v e d i n t r i e t h y l a m i n e t e t r a h y d r o f u r a n - w a t e r (1:5 : 1 4 ) , t h e s o l u t i o n is r e t a i n e d i n an o l e a t e d c e l l u l o s e column f o r 1 h o u r , t h e n t h e impurities a r e eluted with triethylamine - tetrahydrof u r a n - water m i x t u r e . The column i s a c i d i f i e d w i t h N-H2SO4 and e x t r a c t e d w i t h e t h y l e t h e r , t h e e x t r a c t i s evaporated, the r e s i d u e i s dissolved i n ethanol, and t o t h i s s o l u t i o n a r e added water, dil.HC1, a molybdotungstophosphate r e a g e n t and t h e n Na2C03 s o l u t i o n . The e x t i n c t i o n of t h e c e n t r i f u g e d s o l u t i o n i s measured a t 750 run a g a i n s t a r e a g e n t b l a n k and compared w i t h t h a t of s t a n d a r d s o l u t i o n of h e x e s t r o l t r e a t e d s i m i l a r l y .

6.3

U l t r a v o i l e t S p e c t r o p h o t o m e t r y (U.V.)

H e x e s t r o l w a s q u a n t i t a t i v e l y determined i n tablet s p e c t r o p h o t o m e t r i c a l l y ( 1 8 ) . The d r u g was e x t r a c t e d w i t h CHC13 from a n a c i d i f i e d powdered sample ( c o n t a i n i n g a b o u t 5mg of h e x e s t r o l ) . The CHC13 e x t r a c t w a s conc e n t r a t e d and 2 , 2 , 4 t r i m e t h y l p e n t a n e w a s added and h e x e s t r o l w a s e x t r a c t e d w i t h 0.1N NaOH. The combined a l k a l i n e s o l u t i o n w a s a c i d i f i e d and r e - e x t r a c t e d w i t h CHC13. The CHC13 s o l u t i o n was washed w i t h water and d r i e d o v e r Na2S04 and e v a p o r a t e d t o d r y n e s s . The r e s i d u e w a s d i s s o l v e d i n e t h a n o l and measured a t 280nm. The p e r c e n t a g e r e c o v e r y ranged from 94.4-98.9%. Kovalenko r e p o r t e d a q u a n t i t a t i v e method f o r t h e a s s a y of h e x e s t r o l ( 1 9 ) . About 25mg of h e x e s t r o l w a s weighed and d i s s o l v e d i n 25-3Om1 of 0.1N NaOH i n a 5Oml v o l u m e t r i c f l a s k . The volume w a s a d j u s t e d t o 5Oml w i t h 0.1N NaOH s o l u t i o n . Then p i p e t t e 5ml of t h i s s o l u t i o n i n t o a second 5Oml v o l u m e t r i c f l a s k and t h e volume a d j u s t e d


364

t o 5Oml w i t h 0.1N NaOH s o l u t i o n . The same s e r i a l d i l u t i o n was r e p e a t e d a n d t h e c o n c e n t r a t i o n of h e x e s t r o l i n t h e l a s t s o l u t i o n w a s d e t e r m i n e d by m e a s u r i n g t h e a b s o r p t i o n a t 241 nm u s i n g 0.1N NaOH s o h t i o n f o r t h e b l a n k . S i n c e t h e s e s o l u t i o n s f o l l o w t h e Lambert-Beer law 1.8 pg m l , t h e d e t e r m i n a t i o n c a n b e made from a s t a n d a r d c u r v e . 6.4

Chromatographic A n a l y s i s 6 . 4 1 P a p e r Chromatography Tompsett h a s d e s c r i b e d a method f o r d e t e c t i o n of h e x e s t r o l and o t h e r s t i l b e s t r o l d e r i v a t i v e s a l o n g w i t h t h e p - h y d r o x y m e t a b o l i t e s of p h e n o b a r b i t o n e and p h e n y t o i n i n u r i n e u s i n g t h e 2-dimensional paper chromatography ( 2 0 , 2 1 ) . The two s y s t e m s u s e d were i s o p r o p a n o l : NH3 (0.99) :H20 (8 :1:1) and C6H6 :E t O A c :H 2 0 ( 2 :1: 1). The d e t e c t i n g a g e n t s u s e d were P a u l y ' s r e a g e n t (red-brown) , d i a z o t i z e d p - n i t r o - a n i l i n e (brown) , d i a z o t i z e d d i e t h y l a m i n o e t h y l p-aminophenyl s u l p h o n e (brown) and 1 - n i t r o s o - 2 - n a p h t h o l n i t r i c a c i d m i x t u r e (+ve). The s e n s i t i v i t y r a n g e f o r t h i s method i s 5-80ug. 6.42 Column Chromatography Column chromatography h a s b e e n a p p l i e d t o p u r i f y the c a t t l e feed e x t r a c t s containing h e x e s t r o l and o t h e r s t i l b e s t r o l s t o remove i n t e r f e r i n g substances b e f o r e i t s determinat i o n . An example of t h e columns u s e d f o r t h e p u r i f i c a t i o n of t h e e x t r a c t s i s A 1 0 column 2 3 (22). Verbeke ( 2 3 ) used columns c o n t a i n i n g XAD-2, C e l i t e , o r n e u t r a l A1203 (Brockman A c t i v i t y I ) f o r p u r i f i c a t i o n o f t i s s u e e x t r a c t s and u r i n e u s i n g d i s t i l l e d water; 1 5 m l water-washed e t h e r f o l l o w e d b y , lhl CgHg t h e n benzenei s o o c t a n e ( 1 : l ) ; and b e n z e n e : i s o o c t a n e (1:l) a s e l u e n t s r e s p e c t i v e l y f o r d e t e c t i o n of h e x e s t r o l and o t h e r a n a b o l i c s .

365

HEXESTROL 6 . 4 3 Thin Layer Chromatography S e v e r a l r e p o r t s have been published on t h e d e t e c t i o n , 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 h e x e s t r o l and o t h e r c h e m i c a l l y r e l a t e d d r u g s f o r example d i e t h y l s t i b e s t r o l i n f e e d s (meat, milk) and i n b i o l o g i c a l f l u i d s . Table (1) summerizes t h e s o l v e n t systems a n d t h e d e t e c t i n g a g e n t s used i n t h e cited references.

6.44 Gas-Liquid Chromatography Various g a s - l i q u i d chromatographic methods have been developed f o r d e t e c t i o n of h e x e s t r o l i n meat and o t h e r a g r i c u l t u r a l p r o d u c t s . Gain and S c h o l l ( 3 1 ) d e s c r i b e d a method f o r d e t e r m i n a t i o n of h e x e s t r o l i n m o l a s s e s - based l i q u i d feed supplements, a f t e r t h e p r e p a r a t i o n of t h e b i s ( t r i m e t h y l s i l y l ) acetamide d e r i v a t i v e . However t h e method showed i n t e r f e r i n g peaks o r low recovery due t o emulsion f o r m a t i o n . Most of t h e g a s chromatographic d e t e r m i n a t i o n r e q u i r e s d e r i v a t i z a t i o n of h e x e s t r o l b e f o r e i n j e c t i n g i n t o t h e g a s chromatograph. Table ( 2 ) summerizes t h e d a t a o b t a i n e d from t h e l i t r a t u r e s t i l l 1980.

T a b l e 1.

S o l v e n t system

a)

Stationary phase

Detect i n p agent

m m

R-emarks

2 d i m e n s i o n a l TLC

5%H2SO4-

s e n s i t i v i t y 0.5-

CHC13:EtOH:C H 6 6 (36:1:4)

induced f l u o r e

lOppb

Ref.

23

scence a t

n-C H :Et20:CH2C12 6 14 (4:3: 2)

W

(contd.)

366m v a n i l li n

petroleum e t h e r

Silica gel

(40-65OC) :

( a c t i v a t e d a t 120 OC reagent)

a p p l i e d t o animal

27

feeding s t u f f

2 d i m e n s i o n a l TLC using: a)

CHC13:EtOH(9:1)

b)

C 6H :E t OAc ( 3 :1 )

28

Silica gel H

t.1.c CH2 C12:Me2C0(4 :1)

K i e s e l g e l or

ultraviolet a t

s e n s i t i v i t y range

Kieselgel F

254m

0 . 2 . 2 up,

254

( t h e h i g h performance t l c r a n g e 10-200ng

29

T a b l e 1.


Toleune: EtOAc ( 1 9 :1 )

Stationary phase

A l u m i n ium ox i d e

Detecting agent

Ultraviolet a t 254 nm

C6H6 :E t O A c (20: 1)

CH2C12:Me2C0(4: 1)

Remarks

S e n s i t i v i t y 5,lOppb

Ref.

24

( i n f r e s h l i v e r and kidney) Silica gel

U 1 tr av i o l e t a t 254 nm

recommended € o r r o u t i n e test of e s t r o g e n s i n food

25

Silica gel G

ultraviolet

s e n s i t i v i t y of t h e

26

254m. por 20'

test i s 0 . 5 y g

CHC13:EtOAc(4:1)

2 d i m e n s i o n a l TLC w i t h n C6H14:Et20:CH2C12

(4:3:2) i n b o t h d i r e c t i o n or EtOAc:C6H6(l:3) a f t e r s o l v e n t system ( a ) n-C H

6 14 ( 4 :3 :2)

:Et20:CH2C12

T a b l e 1.


Stationary phase

(contd.)

Detecting agent

Remarks

Ref.

h.p. t.1.c.

CH C1 :Ple2C0(4:1) 2

2

Kieselgel F

W

91 W

254

d e n s itome-

(t.1.c.

range

trically at

200-2000ng)

.

287

(h.p.t.1.c

range

10-2 Ong ) C6H14:Me2C0(3 :2 ) o r C6H6:Me2C0(3:1)

Po 1yamid e

t h e i n f l u e n c e s of chemical s t r u c t u r e and m i g r a t i o n r a t e are discussed.

30

T a b l e 2.

Carrier G a s

Column

Detector

Remarks

d er i v a t i z e d by hep t a f l u o r o b u t y r i c a n hyd r i d e

3% OV-1 on Chromosorb

Ar-CH

WHP (80 t o 100 mesh) a t

(19:l)

capture

He

electrolytic conductor

s e n s i t i v i t y €or electron c a p t u r e (40-400 p i c o g ) ; and f o r e l e c t r o l y t i c c o n d u c t o r (1-5 ng)

electron capture

d e r i v a t i z e d by heptafluorobutyric anhydride; s e n s i t i v i t y range 6 t o 262 ng/ml

flame ionizat ion d e t e c to r

i n j e c t e d as a dipropionate derivative; sensitivity 0.05 mg/ml

Electroncapture

d er i v a t i z ed by trifluoroacetic anhydr iod e .

4

electron

Ref.

32

var ious temperature

2% OV-17, 3%0V-1,3%0V-1 5% of n e o p e n t y l g l y c o l s e b a c a t e o r 2% Carbowax 20M on s i l a n i s e d Chromosorb W (80 t o 100 m e s t ) a t var i o u s temperatures

N2

32

33

.

3% QF-1 o n 100 t o 200 mesh Gas. Chrom Q a t 170'. Ap i e z o n L-Ep i k o t e 1001(5 :1) on s i l a n i s e d Chromosorb G a t 160Oc.

N2

34

3 70

HASSAN Y. ABOUL-ENEIN ET AL. 6.45 High Performance L i q u i d Chromatography H e x e s t r o l has been determined among o t h e r a n a b o l i c s y n t h e t i c and n a t u r a l hormones i n meat by HPLC. The columns used were Rp-2 (10 v m ) , Rp-18 ( 5 pm) o r Zorbax CN ( 5 pm) by g r a d i e n t e l u t i o n w i t h a c e t o n i t r i l e : H 2 0 (1:9) p l u s 25%, i n c r e a s i n g r e c t i l i n e a r l y i n 5 m i n u t e t o 45%, of a c e t o n i t r i l e : H20 ( 9 : l ) . The f l o w r a t e w a s 2 m l min.-l, and 100 mg e a c h of L i C l and LiC104 were added t o each 1 0 h l of e l u e n t . The e l u a t e w a s examined by voltammetry w i t h v i t r e o u s carbon e l e c t r o d e s and on silver-AgC1 (3M-KC1) r e f e r e n c e e l e c t r o d e ; t h e p o t e n t i a l sweep r a t e was 5 m ~ s - 1 . Other columns used f o r t h e d e t e r m i n a t i o n of h e x e s t r o l and o t h e r r e l a t e d d r u g s i s L i c h r o s o r b RP-8 w i t h m i x t u r e s (35:13, t o 25:23) of methanol and water ( c o n t a i n i n g i n a l l i n s t a n c e s 2 p a r t s of a c e t o n i t r i l e ) as a mobile phase (0.8 t o 1.6 m l min-l). The v o l t a m m e t r i c c u r v e of t h e e l u a t e was recorded by t h e u s e of a v i t r e o u s - c a r b o n e l e c t r o d e , a p l a t i n u m c o u n t e r - e l e c t r o d e and a silver-AgC1 r e f e r e n c e e l e c t r o d e a t pH 3 , t h e peak p o t e n t i a l ( V ) f o r h e x e s t r o l w a s +.9. Amounts r a n g e from 1-4 ng g-l of h e x e s t r o l i n meat could be determined by HPLC

.

6.5

Mass Pragmentography H e x e s t r o l among o t h e r s t i l b e s t r o l w a s q u a n t i t a t i v e l y determined by combined GC/MS ( 3 8 ) . The s e n s i t i v i t y r a n g e i s 40 p a r t p e r 1 09 , t h e method

is s u c c e s s f u l l y used f o r d e t e c t i o n of e s t r o g e n s and i n meat p r o d u c t s . I t i n v o l v e s e x t r a c t i o n of l i v e r , kidney and muscle t i s s u e i n a c e t o n i t i l e : water ( 9 : l ) . The drug w a s c o n v e r t e d t o i t s t r i m e t h y l s i l y l d e r i v a t i v e f o r a n a l y s i s on a column packed w i t h 2%0V-17 on Chromosorb G and coupled t h r o u g h a Watson-Biemann H e s e p a r a t o r t o t h e mass s p e c t r o m e t e r . 6.6

B i o l o g i c a l Assays Heinert, i d e n t i f i e d hexestrol i n milk using the mouse u t e r i n e weight b i o a s s a y method ( 3 9 ) . Rennet

HEXESTROL

371

c o a g u l a t i o n of t h e m i l k p e r m i t t e d d e t e c t i o n of 0.001 ppm of h e x e s t r o l which could a l s o be d e t e c t e d e ta1 (40) i n c h e e s e a f t e r c o a g u l a t i o n of m i l k . L i e m d e s c r i b e d a b i o l o g i c a l a s s a y of e s t r o g e n i c s u b s t a n c e s i n cosmetic i n c l u d i n g h e x e s t r o l , based on a p p l i c a t i o n of t h e cosmetic p r o d u c t s t o t h e shaven s k i n of c a s t r a t e d female mice; v a g i n a l smears were t a k e n subsequently f o r a n a l y s i s .

Acknowledgements

The a u t h o r s wish t o t h a n k M r . Khalid N.K. Lodhi f o r h i s technical a s s i s t a n c e i n determining t h e NMR s p e c t r a of h e x e s t r o l and M r . Uday C. Sharma f o r t y p i n g t h e manuscript.


372

References

1.

a)

R e m i n g t o n ' s P h a r m a c e u t i c a l S c i e n c e s , #ack P u b l i s h i n g Co., E a s t o n , P a . , p. 920 ( 1 9 7 5 ) .

b)

Merck I n d e x , N i n t h e d i t i o n , Merck & Co. , I n c . , Rahaway, N . J . , U.S.A., p . 615, 4571 ( 1 9 7 6 ) .

2.

D i c t i o n a r y of o r g a n i c compounds Vol. 2 , E y r e S. S p o t t i s woods - (London) P u b l i s h e r s L t d . , E . & F. M. Spon L t d . , New York, p. 1036 ( 1 9 6 5 ) .

3.

E.G.C. C l a r k e , " I s o l a t i o n and I d e n t i f i c a t i o n of Drugs", The P h a r m a c e u t i c a l P r e s s , London, 1 9 6 9 , p. 363.

4.

S. B e r n s t e i n and E.S.

Wallis, J . Amer. Chem. S O C . ,

62, 2871, ( 1 9 4 0 ) . 5.

6.

The P h a r m a c e u t i c a l Codex, E l e v e n t h E d i t i o n , t h e P h a r m a c e u t i c a l Press, London, p . 4 1 1 ( 1 9 7 9 ) . N.R.

Campbell, E . C .

Dodds and W . Lawson, N a t u r e ,

142, 1 1 2 1 ( 1 9 3 8 ) .

7.

E . C . Dodds, R . Robinson, e t a l . ( a ) N a t u r e , 141, 247 (1938), (b) P r o c . Roy. SOC. (London), B 7 , 1 4 0 (1939).

8.

N . R . Campbell, E.C. Dodds and W. Lawson, P r o c . Roy. SOC. (London), B 8 , 253 ( 1 9 4 0 ) .

9.

M.S.

K h a r a s c h and M. Kleiman, J . Amer. Chem. SOC. (1943).

65, 491,

10.

B.N. La Du, H.G. Mandel and E . L . Way, "Fundamentals of Drug Metabolism and Drug D i s p o s i t i o n " , The W i l l i a m s & W i l k i n s Co., B a l t i m o r e , MD., 1 9 7 2 , p.263.

11. A.L. H e r b s t , H. U l f e l d e r and D . C . P o s k a n z e r , N e w Engl. J . Med.,

284,

878 ( 1 9 7 1 ) .

12.

A.L. H e r b s t , R . J . Kurman and R . E . GynecFl. , 9, 287 ( 1 9 7 2 ) .

13.

a)

Scully, Obstet.

J.A. McLachlan and R.L. Dixon, New C o n c e p t s i n S a f e t y E v a l u a t i o n , ( e d i t e d by M . A . Mewhlman, R.E. S h a p i r o and H. B l u m e n t h a l ) , Vol. 1, P a r t 1, p . 423, Hemisphere, Washington ( 1 9 7 6 ) .

HEXESTROL b)

373 M. M e t z l e r , R. G o l t s c h l i c h and J . A . Mc L a c h l a n , E s t r o g e n s i n t h e Environment, ( e d i t e d b y J . A . McLachlan), p. 293, Elsevier (1980).

14.

I . C a l a f e t e a n u , E. Dumitrescu and P. G r i n t e s c u , Revta Chim., 41, (1965); t h r o u g h Anal. A b s t r . 3786 (1966).

15.

261, ( 1 9 6 4 ) , I. K e r e n y l , Acta Pharma. hung., t h r o u g h Anal. A b s t r . 1 3 , 1980, ( 1 9 6 6 ) .

16.

V.G.

17.

The A d d i t i v e s i n Animal F e e d i n g - s t u f f s Subcommittee of t h e A n a l y t i c a l Methods c o m m i t t e e of t h e S o c i e t y f o r A n a l y t i c a l C h e m i s t r y . R e p o r t of t h e Hormones P a n e l . A n a l y s t , 88, 925, ( 1 9 6 3 ) .

18.

G,

13,

34,

B e l i k o v , Med. Prom. SSSR, 1 7 , 32, (1963).

M.J. Matherne, J r . , (1965).

J . A s s . o f f . a g r i c . chem.,

48, 613,

g,

19.

L. I. Kovdlenko, F a r m a t s i y a , 5 4 , (1969) ; t h r o u g h Chem. A b s t r . 7 2 , 59121e, (1970).

20.

S.L.

Tompsett,

21.

S.L.

T o m p s e t t , J . Pharm. Pharmacol. 1 6 , 207,

22.

H. Van Waes, Rev. Agr. ( B r u s s e l s ) , 23, 1 1 3 5 , ( 1 9 7 0 ) ; t h r o u g h Chem. A b s t r . , 74, 632312, ( 1 9 7 1 ) .

23.

R. Verbeke, J . Chromatogr., 1 7 7 , 6 9 , (1979); t h r o u g h Chem. A b s t r . , 21, 206599q, (1979).

24.

H. J . S t a n and F.Y. H o h l s , Z. Lebensm. - Unters F o r s c h . , 1 6 6 , 257, (1978); t h r o u g h Anal. A b s t r . 35, 6F16, (1978).

25.

J. Fharm. Pharmacol. 13, 747, ( 1 9 6 1 ) . (1964).

.

H. Jare, 0. R u t t n e r , W. Kroeza, F l e i s c h w i r t s c h a f t , 86, 3 6 3 1 t , (1977).

56, 1326, ( 1 9 7 6 ) ; t h r o u g h Chem. A b s t r . ,

I . Karkocha, Rocz. Fanstw. Z a k l . H i g . , 26, 497, (1975); t h r o u g h Chem. A b s t r . , 8 4 , ?368w, ( 1 9 7 6 ) . R. F e r r a n d o and A. R e n a r d , B u l l . SOC. Chim. b i o l . , 50, 1855 ( 1 9 6 8 ) .

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314

28. 29.

T. L a r s , J . Chromatgr.,

48,

560, (1970).

H. J a r c , 0. R u t t n e r , and \J. Krocza, J . Chromatogr.,

1 3 4 , 351, (1977). 30.

31.

H.C. H s i n , J a p a n A n a l y s t , 23, 1 2 2 6 , (1974); t h r o u g h Anal. A b s t r . 29, 2E16, (1975).

A.V.

J a i n , E.D.

S c h o l l , J . Assoc. O f f . Anal. Chem.,

58, 818, (1975).

130,

32.

F . J . Lawrence and J . J . Ryan, J . Chromatogr., 97, (1977).

33.

H. Ehrsson, T. Walle and H. B r o e t e l l , A c t a . Pharm. Suec., 8 , 319, (1971).

34.

M. G a b r i e l l a . C. Guido and S. P a t r i z i a , A n n a l i 1st. Sup. Sanita, 5, 586, ( 1 9 6 9 ) ; t h r o u g h Anal. A b s t r . , 2051; (1971).

35.

P . J . Cooper, M.J. d e F a u b e r t Maunder and G . J . McCutcheon, A n a l y s t , 92, 382, ( 1 9 6 7 ) .

36.

C.G.B. F r i s c h k o r n , M.R. Smyth, H.E. F r i s c h k o r n and J . Golimowski. F r e s e n i u s ' Z. Anal. Chem. 300, 407 (1980).

37.

M.R. Smyth, C.G.B. F r i s c h k o r n , F r e s e n i u s ' 2. Anal. Chem. 301, 220 (1980).

38.

G . H o e l l e r e r and D. J a h r , Z. L e b e n s m i t t e l u n t e r s .

39.

H.H. H e i n e r t , Arch. L e b e n s m i t t e l h y g . , 26, 1 5 2 , ( 1 9 7 5 ) ; t h r o u g h Chem. A b s t r . 8 3 , 162274h, (1975).

40.

D.H. Liem, L.G. H u i s i n ' t Veld, G . J . R u n d e r v o o r t , J. R o o s e l a a r , J. Ten Have, J. SOC. Cosmet. Chem., 307 (1976); t h r o u g h Chem. A b s t r . , 8 8 , 41529q (1978).

20,

U. - F o r s c h . , 157, 65, ( 1 9 7 5 ) ; t h r o u g h Anal. A b s t r . 29, - 4 D 83, (1975).

27,

MESTRANOL Hum&

1.

2.

3. 4. 5.

A . El-Obeid and Abdulluh A. Al-Badr

376 316 316 377 377 371 377 377 377 311 311 378 385 387 390 390 391 391 404

Description 1 . 1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance Physical Properties 2.1 Crystal Data 2.2 Melting Point 2.3 Solubility 2.4 Identification 2.5 Spectral Properties Synthesis Absorption, Metabolism, and Excretion Methods of Analysis 5.1 Titrimetric Methods 5.2 Spectrophotometric Methoas 5.3 ChromatographicMethods References

Analytical Profiles 01Drug Subslancea Volume I I

375

Copyright 01982 by The American Pharmaceutical Association ISBN 0-12-260811-9

376

HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR

1. Description

1.1 Nomenclature 1.11 Chemical Names Ethinyloestradiol-3-methyl ether. 1 7 a-Ethnylestradiol-3-methyl ether 3-Plethoxy-19-nor-17 a-pregna-1,3,5(10)-trien-20yn-17-01. 19-Norpregna-1,3,5(1O)-trien-2O-yn-l7-ol,3methoxy- (17a)17a-Ethynyl-3-methoxy-1,3,5(lO)-estratrien-l7~-01. 17~-Ethynyl-3-methoxyoestra-ly3,5(lO)-trien-l7-ol. 17~t-Ethynyl-1,3,5(10)-estratriene-3, 17B-diol-3methyl ether. 1.12 Generic Name Mestranol. 1.13 Trade Names Mestranol is an ingredient of the following propietary oral contraceptive preparations: Conovid, C-Quens, Enavid, Enovid, Metrulen, Norinyl, Previson, Ortho-Novum, Ovanon, Ovulen, Sequens, Syntex Menophase. 1 . 2 Formulae

1.21 Empirical C21H2602 1 . 2 2 Structural

OH

377

MESTRANOL

1.23 CAS No. 72-33-3 1.3 Molecular Weight 310.42

1.4 Elemental Composition C 81.25%, H 8.44%, 0 10.31%. 1.5 Appearance White crystalline powder. 2. Physical Properties 2.1 Crystal Data Crystal data were reported by Ohrt et a1 (1) for some esterone-related compounds. The data given for mestranol was : a 6.998, b 39.737, c 6.8718., B117.58', Pz1, 2 = 4. 2.2 Melting Point Melts between 146' and 1 5 4 O with a range of 4'(2,3). 2.3 Solubility Almost insoluble in water, soluble 1 in 44 of ethanol, 1 in 23 of ether, and 1 in 4.5 of chloroform, 1 in 12 of dioxane, 1 in 23 of acetone, slightly soluble in methanol ( 2 , 4 , 5 ) . 2.4 Identification 2.41 Infrared Spectroscopic Test B.P. (4) and U.S.P. XIX (2) make use of the infrared absorption spectrum of mestranol as me n of identification of the drug. The infrared absorption spectrum of the sample exhibits maxima which are only at the same wavelengths as, and have similar relative intensities to, those in the spectrum of a standard mestranol. The infrared spectrum of mestranol will be discussed later

318


in the spectral properties of the drug. 2.42 Ultraviolet Spectroscopic Test

U.S.P. X I X requires that for the identification o f the drug, the ultraviolet absorption spectrum of methanolic solution exhibits maxima and minima at the same wavelengths as that of a similar solution of a standard mestranol, concomitantly measured. The ultraviolet spectrum of mestranol will be discussed later in the spectral properties of the drug. 2.43 Thin Layer Chromatographic Test

B.P. and U.S.P. XIX describe a thin layer chromatographic method for the identification of mestranol in which the principal spot in the chromatogram of the substance being examined is compared with that of mestranol obtained under identical conditions. 2.44 Color Test

According to B.P. a solution of mestranol in sulfuric acid appears orange-red by transmitted light, shows a yellowish-green fluorescence by reflected light and produces a reddish-brown f l o cculent precipitate after addition of ferric ammonium sulfate solution and a rose-red flocculent precipitate when water is added. 2.45 Solubility Test

B.P. uses a solubility test to distinguish mestranol from ethinylestradiol. The former is insoluble in a 5 % wfv solution of potassium hydroxide. 2.5 Spectral Properties 2 . 5 1 Ultraviolet Spectrum

The ultraviolet absorption spectrum of mestranol obtained from a solution in neutral methanol in the region of 200 to 350 nm using a Varian Cary 219 spectrophotometer is shown in Figure I. Two absorption maxima at about 2 1 8 and 286 nm and

379

MESTRANOL

J c c CI

F i g . 1. methanol.

U l t r a v i o l e t spectrum of m e s t r a n o l i n n e u t r a l

380


two minima at about 246 and 284 nm were observed. Reported (5) ultraviolet absorption spectrum of mestranol in methanol exhibited two maxima at 279 nm (E 1%, 1 cm 82) and 287.5 nm (E 1%,lcm 14.4). 2 . 5 2 Infrared Spectrum

The infrared spectrum of mestranol is presented in Figure 2 . The spectrum was obtained from nujol mull using a Unicam SP 3-300 infrared spectrophotometer. The spectral assignments are presented in Table 1. Table 1. Infrared spectral assignments for mestranol. Band Frequencyl Wavenumber cm

Structural assignment

1460

0-H stretching. ECH stretch. -C=C-stretch of the aromatic ring. C-H Deformation of -CH2-

1380

-C-H Deformation of -C-CH

1300 65 3

C-0-C stretch. 32-H Deformation.

3500 3 300 1510, 1620

3

2 . 5 3 Proton Magnetic Resonance (PIR) Spectrum.

The PMR spectrum of mestranol is presented in Figure 3 . The sample was dissolved in CDCl and 3

the spectrum obtained on a Varian-T60A NMR spectrometer with tetramethylsilane as the internal standard. The spectral assignments shown in Table 2 , are in agreement with reported studies (6) *

W.Vd#@P-

3

4

5

5

6

7

9

10

11

12

13 (4I S U

100

w

u 7u

60 50

44

34 20 10 0

Fig. 2.

I n f r a r e d spectrum of mestranol; Nujol M u l l .

do0

200 axyl -H

ti

F i g . 3. PMR spectrum of m e s t r a n o l i n CDC13 w i t h TMS i n t e r n a l standard

0 wa

MESTRANOL

383

Table 2.

PMR spectral assignments for mestranol.

Chemical Shift. PPd6)

Multiplicity Number of Protons.

Species

0.92

singlet

3

-CH3

2.12

singlet

1

-OH

2.55

singlet

1

acetylenic proton.

3.72

singlet

3

methoxy protons.

6.87

multiplet.

3

Aromatic protons.

2.54 I3C - Nuclear Magnetic Resonance (13C NMR) Spectrum The I3C NMR spectrum of mestranol in CDCl using 3 tetramethylsilane as an internal standard reference was obtained on a Jeol FX 100, 100 MHz instrument at an ambient temperature using a lorn. sample tube. The spectrum is presented in Figure 4 and the chemical shift values, derived from the off-resonance spectrum, is shown in Table 3. Table 3. I3C NMR spectral assignments for mestranol. Carbon

Chemical shift.

1 2 3 4 5 10

Carbon No.

No.

126.2590 113.9788 157.5908 111.5932 137.9037 132.6407

Chemical shift. (ppm)

13 17 18 20 21 CH3-0-

47.2199 79.9181 12.7185 87.7616 73.9221 55.2115

2.55 Mass spectrum and Fragmentometry The mass spectrum of mestranol, obtained by electron impact ionization, using Nermag GC-Mass spectrometer model R 1010, is presented in Figure 5. The spectrum shows a molecular ion PI? at m/e 310 (relative intensity 24.4%) and a base peak

384

J 1 3 C NMR spectrum of mestranol i n CDC13 with TMS Fig. 4. i n t e r n a l reference.

11

Fig.

5.

Mass spectrum of mestranol (EI).

385

MESTRANOL

at mfe 227. Based on the data interpretation and summary (INTSIJM) program presented by Smith et al, (7) for the possible fragmentation of the basic skeleton of estrogenic steroids, a proposed fragmentation pattern of mestranol is shown in Table 4. Table 4 . mle -

Proposed mass fragmentation pattern of mestranol. Relative intensity %

ion -

311

6.3

M+I

310

24.4

M+

242

15.7

C16H18d

228

19

‘1bH20q

227

100

160

10

159

14.6

147

20.5

145

11.9

129

20.7

128

26.8

116

13.9

+ +

C15H150? -t

Cl0H93 +

3. Synthesis Mestranol was prepared by Colton et al. (8) as f o l l o w s : Estrone {I} i s converted to its 3-methoxy analog (11) by reaction with methyl sulfate. The ethynyl group may then be introduced at position 1 7 either through reaction with sodium acetylide in liquid ammonia followed by hydrolysis o f the sodoxy compound, or through Grignardization with ethynyl magnesium bromide. Almost the sole product of the ethynylation reaction is that which results from attack of reagent from the least hindered a-side of the steroid,Fig.6.


3 86

3

t I11

J

0

HCEC N&iq

.NH3

&’ &Br

TECH

CH30

&CH

t

CH30

Figure 6.

Synthesis of mestranol.

ONa

MESTRANOL

387

4. Absorption, Metabolism and Excretion Mestranol is a synthetic estrogen which is more potent than estradiol. It is readily absorbed from the gastrointestinal tract and is slowly metabolized and excreted in urine and feces. The absorption, metabolism and excretion of the drug has been extensively studied in animals and humans I Fig. 7. Wijmenga and Van der Molen (9) reported a biological halflife of mestranol of 50 hours and that a small proportion of the drug was excreted in milk of nursing mothers. Mills et al, (10) showed that after the injection of tritium-labelled mestranol to women, the radioactivity representing the metabolites of the drug in the blood disappeared with an average half-life of 45 hours (range et al, (11) studied the metabolic 37-65 hours). Mahesh clearance rate and blood half-life of mestranol. The metabolic clearance rate of the drug after a single i.v. injection to women was 1265Llday. The radioactivity halflife after injection was 45.1 hours. No change in the metabolic clearance rate of the drug was noticed for upto 7 months of oral contraceptive medication. Using the constant infusion technique, Bird & Clark (12) measured the metabolic clearance rate of mestranol in normal young women and found it to be 1741L124 hours. The mean conversion ratio for mestranol to ethynylestradiol was 0.236 and to ethynylestradiol sulfate 3.369; that of mestranol to the sulfate was 6.476. The mean transfer constant for mestranol to ethynylestradiol was 0.182. The principal circulating form of the drug was ethynylestradiol sulfate. Mills et a1 (13) administered mestranol orally to normal women in order to measure the metabolic clearance rate and to study the urinary excretion of the drug. Mestranol was rapidly cleared from the plasma with an average metabolic clearance rate of 1247Llday. About 30% of mestranol administered was excreted in urine in 5 days, less than 4% in an unconjugated form, 10% as sulfate conjugates and about 52 as glucuronide conjugates. Comparison of the metabolic clearance and urinary excretion rates made before and again after six cycles of treatment with OrthoNovum SQ containing mestranol showed that prolonged administration had no effect on the metabolic clearance rate, rate of urinary excretion or mode of conjugation of the drug or its metabolites.

388


In a study by Bolt and Remmer (14) following the i.v. administration of 14C-Labelled mestranol to female rats; 45 and 3% of the radioactivity were found to be excreted in the feces and urine, respectively, in 3 days. Only 2 . 5 % of the radioactivity was expired as 14CO2 within 4 days indicating no significant degradation of the steroid nucleus. Some metabolites separated from fecal extracts had an unaltered 3-methoxy group. Others, however, were demethylated to derivatives of ethynylestradiol. The chronic i.v. injection of radioactive mestranol into mice resulted, as reported by Bolt and Remmer ( 1 5 ) , in an accumulation of radioactivity in organs to a greater extent than activity accumulated after radioactive estradiol administration. The metabolites of mestranol were tightly bound to the liver tissue and were not removed by solvent extraction or by acid hydrolysis. The high demethylation rate of mestranol in mice as compared to that in rats may be due to a high activity of the microsomal oxidase in the mouse liver. Following a single i.v. injection of the steroid into female rats, 1.5% of the radioactivity was recovered from urine collected in the first 3 days, and 55% of the activity was excreted in the feces within 3 weeks. Hanasono and Fischer (16) studied the excretion of tritiumlabelled mestranol and other contraceptive steriods and the enterohepatic circulation (EHC) of their metabolites in female rats. Mestranol was rapidly and extensively eliminated as metabolites in the bile after a single i.v. dose. The cumulative percentage of administered radioactivity appearing in the bile at the end of 8 hours was 6 9 % . Mestranol appeared in the bile primarily as a glucuronide conjugate and other polar materials which were not sulfate conjugates. Intact female rats given single i.p. doses of the labelled steroid eliminated radioactive metabolites in the urine and feces at a much slower rate than that seen in the bile of animals with biliary fistulas. Fecal excretion was the major route of elimination in intact animals for the steroid and accounted for 80% or more of the radioactive dose by the end of 7 days. Experiments were also conducted to assess the enterohepatic circulation of metabolites of the steroid. 59% of the intraduodenally infused radioactivity associated with the biliary metabolites of mestranol, underwent enterohepatic circulation and appeared in the bile during a 24 hour period. The glucuronide conjugate fraction of biliary metabolites was the most important fraction undergoing enterohepatic circulation. The

389

MESTRANOL

T

Glucuronide and sulfate conjugates

17a-Ethynylestradiol

H O HO

@PCH 1 2-Hydroxy-17a-ethynylestradiol.

OH

0

17a-D-Homoestradiol Figure 7.

D-Homoestrone

Metabolism of mestranol.

390


polar metabolites, which could not be hydrolysed by 6-glucuronidase, were not absorbed from the intestine and re-excreted in the bile. The authors conclude that the enterohepatic circulation is an important feature of the disposition of the contraceptive steroid in rats and is dependent on the excretion of glucuronide conjugates of the steroid metabolites in the bile. In a study using radioactive mestranol, Williams (17) identified 17 a-ethynylestradiol as a urinary metabolite using reverse isotope dilution technique. Abdel-Aziz and Williams (18), studying the urinary metabolites of mestranol in guinea pigs, identified 17 a-ethynylesteradiol conjugates, D-homoestradiol-176 and D-homoestrone, as metabolites. 3

14

In their study of the metabolism of 4- H and 4- C-mestraet a1 ( 1 9 ) , reported that reactions no1 by women, Williams involving position 4 were no greater than 1.7 to 3% of the dose as measured by the liberation of 3H into body water. The extent of deethynylation in vivo was no greater than 1 to 2% of the dose as measured by urinary estrone metabolites. Mestranol (0.7 and 0.32% of the dose), 17a-ethynylestradiol ( 6 . 6 and 11.3%) and 2-hydroxy-17a-ethynylestradiol ( 0 . 6 4 and 0.7%) were identified as metabolite glycons by reverse isotope dilution after ketodase hydrolysis of the urine from two of the women.

5. Methods of Analysis 5.1 Titrimetric Methods

(a)

The B.P. ( 4 ) recommended a titrimetric method in which the drug in tetrahydrofuran is treated with silver nitrate and then titrated with standard sodium hydroxide solution. The end point determined potentiometrically.

(b)

An argentimetric determination of some acetylene steroidal drugs, including mestranol, had been et _ a1 (20). The assay method developed by Roushdi _ depends on the precipitation of the steroid silver salt from alcoholic ammoniacal silver nitrate solution followed by extraction with a suitable organic solvent, evaporation of the solvent and then applying Volhard's method.

MESTRANOL

(c)

391

Roushdi et a1 (21) used a nonaqueous titrimetric method for determination of acetylenic steroids. The method involves the precipitation of the steroid silver salt using ammoniacal silver nitrate solution. The salts extracted will chloroform or ether and titrated with perchloric acid in acetic acid using Gentian Violet as indicator.

5.2 Spectrophotometric Methods 5.21 Ultraviolet Spectrometric Methods a) Bastow (22) deternined mestranol from its absorption at 280 nm after elimination of the interfering ketonic absorption by reduction with borohydride. Residual interference was allowed for by a three point correction. b) Gorog and Csizer (23) described methods for the determination of (i) mestranol as an impurity in norethynodreal and norethisterone and (ii) mestranol in contraceptive tablets. For (i) the sample is dissolved in methanol and treated with sodium borohydride. A solution of mestrano1 is treated sinilarly. The extinctions of the two solutions are measured at 287, 290.5 and 294 nm, and the mestranol content is calculated from a given equation. For (ii) the powdered tablets are heated under reflux with dichloromethane. After filtration and evaporation of the solvent, mestranol is determined as in (i). The mean recovery for the latter method was 98.3 ? 2.3%. c) A specific spectrophotometric method for the determination of 17-ethynyl steroids was published by Szepesi and Gorog (24). 17a-Ethynyl steroids were determined by quantitative conversion to 17-0x0 steroids by sodium t-butoxide at 81°, followed by spectrometric determination of the 16-glyoxalyl derivatives, Anlax. 294 nm, E 10,700, standard deviation 1.1%. 0 By the same principle, at 0 , the 17-0x0 impurity content in 17-ethynyl steroids was determined. The method was not applicable to +OX0 steroids.

392

HUMEIDA A. EL-OBEID AND ABDULLAH A. ALBADR

d) Shroff and Grodsky ( 2 5 ) described an ultraviolet spectrophotometric method for the estimation of mestranol in fresh tablets. In this method the sample is shaken with water and methylcyclohexane, centrifuged and the extinction of the upper phase measured at 287.7 nm. Baseline corrections applied are calculated from (i) the extinction at 302 nm and (ii) the extinctions at 278 nm and 302 nm and the extincfor pure mestranol; tion ratio E to E 278 287.7 for the the two corrected values of E287. 7

sample should agree within 2 0.006%. The contents of mestranol is calculated from the mean corrected value. The coefficient of variation for ten samples is 1.14%. The method is not preferred for aged tablets because on storage an interfering complex is formed between poly (vinyl pyrrolidone) and magnesium stearate excipients. 5.22 Colorimetric Methods

a) Chlorine o-tolidine reagent was used by Huettenrauch ( 2 6 ) for the selective detection of steroids with aromatic ring A and C13-OH group whether this was free, esterified or ether-linked. Mestranol gave positive reaction with sensitivity of 1-2 pg/cc.

b) Shroff and Huettemann ( 2 7 ) developed a colorimetric method for assaying mestranol in tablets. The method is based on the formation of a colored complex with phenol-sulfuric acid reagent. This complex exhibits an absorption maximum of 550 nm, obeys Beer's Law and is stable for reasonable length of time. According to the method the tablets are moistened with water and shaken with methylcyclohoxane. An aliquot of the organic phase is evaporated in an atmosphere of nitrogen and treated with phenol-sulfuric acid reagent and the extinction measured at 5 5 0 nm against a reagent blank. The error and precision are 2 1 . 6 % and 1 . 5 2 % respectively. The presence of norethisterone and excipients do not interfere.

393

MESTRANOL

The method is claimed to be superior to the ultraviolet spectrophotometric method described by Shroff and Grodsky and summarized above. c) A direct colorimetric method for the assay of mestranol in one tablet is reported by Comer et a1 (28). In this method one tablet is dissolved in sulfuric acidlmethanol (7:3) and the extinction measured at 545 nm against a reagent blank. To avoid interference by chlormadinone, the temperature must be kept below The coefficient 5 O during the color reaction. of variation was 1 . 0 5 % . Very similar methods have also been described by Templeton et a1 (29) for one tablet assay and by Wu (30) for mestranol in combination with ethynodiol diacetate. The latter procedure require prior chromatographic separation of mestranol.

d) Beyer (31) developed an automated colorimetric procedure for the quantitation of mestranol in tablets according to which the tablets are suspended in water and extracted with chloroformethanol. In an automatic system, re-extraction is effected with 10% ethanol solution in 90% sulfuric acid. The extinction is measured at 538 nm. The coefficient of variation is 21%. e) Rizk et a1 (32) determined mestranol colorimetrically by means of silver ions. An ethanolic solution is treated with aqueous ammonia and an excess of aqueous silver nitrate and the precipitated silver compound is filtered off. Excess silver ions in the filterate is determined by a dithizone spectrophotometric procedure and the amount of steroid is calculated from the silver ions consumed. 5 . 2 3 Infrared Spectroscopic Methods

a) Mestranol was determined in chloroform solution -1 by infrared spectrophotometry (33) at 3308 cm -1 (aromatic (Ethynyl vibrations) or 1 5 0 2 cm system vibrations).

394


b) Chatten et a1 (34) reported a quantitative assay of antifertility agents by infrared spectroscopy. Two pure estrogenic substances and eight pure progestogens were analysed in the selected wavelength range and shown to obey Beer-Lambert Law. Although progestogen analysis in compound tablets was accomplished in several cases using the technique, the estrogenic content in all compound tablets was too low t o permit analysis. c) Beyermann and Roder (35) analysed mestranol in oral contraceptives by infrared spectrophotometry after separation of the components by thin-layer chromatography. The separation was performed on silica gel with ethyl acetate as a solvent. The appropriate spot extracted with chloroform and the infrared spectrum in liquid paraffin mull was recorded and mestranol determined from its extinction at 7.95 um. 5 . 2 4 Spectrofluorometric Methods

a) Cullen et a1 ( 3 6 ) developed a sensitive procedure for the analysis of norgestrel and structurally related steriods based on sulfuric acid induced fluorescence. The selectivity of the reaction and mechanism of fluorescence formation were studied. The reaction is specific for A4 -3-ketosteroids which have both a 17Bhydroxy and a 17a-alkyl or alkyne substitution and A 1y3y5(10)-triene-3-01 steroids. A twostep mechanism was tentatively explained on the basis of the effects of temperature, time, initial acid concentration and subsequent dilution with water on fluorogen development. The procedure has been automated to permit unit dose analysis. b) A spectrofluorometric procedure for the determination of mestranol in some oral contraceptive tablets had been developed (37) which utilizes the native fluorescence of mestranol. The method is claimed to be rapid, reliable and sensitive. No separations are required and the method is applicable in the presence of norethynodrel, ethynodiol diacetate and norethisterone,

MESTRANOL

395

but chlormadinone acetate interferes. The method is applicable to single-tablet analysis. According to the procedure one powdered tablet is shaken with anhydrous ethanol and centrifuged. An aliquot of the supernatant is diluted with ethanol and the fluorescence at 327 nm (excitation at 284 nm) is compared with that of a standard solution. Anhydrous ethanol is used as a reference solution and a correction applied for its fluorescence. The accuracy under the conditions studied was 2 1.29% and the precision ranged from 2 0.896 to ?r 1.73. c) Mariani and Mariani-Vicari (38) also described a method for the assay of mestranol spectrofluorometrically. A sulfuric acid-methanol (1:l) reagent is used and a previous separation of the two components (ethynylestradiol and mestranol) in the compound preparation is not required. For tablets the hydrolysis of the starch by diastase is followed by extraction into chloroform. The wavelengths of emission and excitation spectra are 515 nm and 475 nm respectively. d) An automated fluorimetric method for one-tablet assay of mestranol was carried out by Comer et a1 (28). One tablet i s dissolved in sulfuric acid-methanol (7:3) and the fluorescence measured with Kodak Wratten N0.58 ,& Corning 1-60 primary filters and a Kodak Wratten 22 secondary filter. A scheme for automation of the fluorimetric method is illustrated. In twelve replicate experiments, the coefficient of variation was 1.30%. The fluorescence reaction is retarded by NO NO2 and H 0 Related ster3’ 2 2’ oids, such as estradiol methyl ether, esterone methyl ether and esterone, cause little interference.

e) Templeton et a1 ( 2 9 ) described a fluorimetric assay method of mestranol in which one tablet is allowed to disintegrate in 20% sodium hydroxide solution and water, then extracted with chloroform and an aliquot of the extract evaporated to dryness. The residue dissolved in 50% methanol solution in concentrated sulfuric acid and the fluorescence measured at 498 nm, with excitation at 468 nm.

396

HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR f> A fluorimetric

assay method for the determination of mixture of mestranol and norethynodrel was reported by Pastor et a1 (39). For mestrano1 alone extracts (in 1 : 5 chloroform-methanol) were evaported to dryness, dissolved in ethanol cooled, and allowed to react with a mixture of 1 : 3 acetic acid-sulfuric acid at room temperature before measuring the fluorescence emission at 560 nm after excitation at 545 nm. When norethynodrel was present with mestranol, the extracts were treated with borohydride in methanol at room temperature before addition of concentrated hydrochloric acid and processing as above to estimate mestranol. Norethynodrel was estimated in the mixture, from borohydride and acetic acid-sulfuric acid treatment, by excitation at 485 nm and emission at 520 nm. Variation in measurements were 3% for mestranol and 2% for norethynodrel.

g) Mestranol in oral contraceptive tablets was also determined fluorirnetrically ( 4 0 ) by disintegrating a tablet in dilute hydrochloric acid, extracting with methylene chloride, treating with 0.2% hydroquinone (in 70:30 sulfuric acidethanol) and measuring the fluorescence.

h) Dusinsky and Radejova (41) reported fluorimetric methods for the determination of mestranol and some other estrogenic hormones. Methods, based on the determination of 'native' fluorescence and that 'induced' by sulfuric acid, for determining mestranol, ethinylestradiol, estradiol benzoate and valerate and the total estrogenic hormone content in mixtures of natural conjugated estrogens are described. The methods are claimed to be simple, highly sensitive and rapid. There is no need €or preliminary clean-up or hydrolysis. 5.25 Nuclear Magnetic Resonance (NMR) Spectrometric

Methods

Avdvich et al(6) used an NMR technique to quantitate mestranol bulk drug. Diphenylacetic acid was used as the internal standard and pyridine as the solvent. The amount of mestranol was calculated from the integrals of the peaks at 6.33 ppm

MESTRANOL

397

(methoxyl protons) and at 6.80 ppm (ethynyl proton de-shielded by pyridine). The average deviation was ? 0.6% and the results were in good agreement with those obtained by an official method. 5.3 Chromatographic Methods 5.31 Thin-Layer Chromatography

The literature describes several thin-layer chromatographic methods for the separation and analysis of mestranol in mixtures and contraceptive tablets. For quantitation, the plate is first scraped and mestranol eluted before treatment with a suitable reagent. Table 5 symmarizes thinlayer chromatographic methods for mestranol. 5.32 Column Chromatography a) Quantitative separation of progestins and es-

trogens from anovulatory formulations had been performed (57) using gel filteration on a synthetic polysaccharide (Sephade LH-20) and the compounds were determined directly by U.V. spectrophotometry.

b) Erunner and Kunze (58) published an analytical method for mestranol in combination with norethisterone (norethinadrone) or norethynodrel by partition chromatography and ultra-violet measurement. According to this procedure oral contraceptive tablets were extracted with dimethylformamide-formamide (1:l) and the steroids separated by partition chromatography with use of this solvent as stationary phase on Celite with heptane as mobile phase. Mestranol, in the relevant fraction, is determined by measuring the extinction at 287 nm with base-line correction obtained from the extinction at 302 and 315 nm. Interference was noticed with ethynodiol diacetate and chlormadinone acetate. The method had been adopted as official first action after a collaborative study was conducted (59). c) A method was described (60) for the separation of steroids in mixtures of pharmaceutical in-

terest by means of silicic acid column chro-

Table 5: Stationary Phase

Thin-Layer Chromatographic systems for mestranol analysis.

Developing solvent

Silica Gel or Neutral Allumina Kieselgel G

Silica Gel G/H

Cyclohexane/EtoAc (7:3) Ether/Cyclohexane

Visualization

Quantitative

0.5% Vanillin H2S04-EtON

-

-

Reference 42

Treat with 40% SbCl in measure extincgion 43 at 570 nm or fluoresence. HOAC

Iodine vabors

(8:2)

Treat with 15% TiCl in 3 RoAc-H2S04, measure extinction at 530 nm.

44

W

W

00

Propylene glycolimpregnated Kaieselguhr G.

Toluene, Cyclohexanetoluene ( 3 : 2 ) or petroleum ether.

Paraffin oil-impregnated -Kieselguhr G.

30,50 or 70% HoAC solutions.

Silica Gel

Benzenelethyl methyl ketone (9:l).

20% p-MeC H4S03H

-

45

-

45

in 947: E t h , heat at 120 for 10 min. Same as above.

-

Treat with H SO -i"ieOH(2:1)46 2 3 measure extinction at 540 run. contd......

Stationary Phase

Developing solvent

Visualization

Quantitative

Reference

EtoAc/cyclohexane/Me2C0 (5:1.5:2 )

85X PhosphoricMeOE (1:1) or SbCl -EoAc (1:l w/v>? heat at 12Oo-13O0. or Iodine vabors.

Use Icieselgel FIR 47,43 Treat with 15% TiCl conc. HC1. Pleasure 3 extinction.

Xieselgel G/HR a) 1-Dimensional

b) 2-Dimensional Above solvent followed by Cyclohexane/EtoAc (23:27). Silica Gel GF

2-Dirfiensional : Chloroform/methanol (9:l) H SO. and heat for 4 36 minutes at 1 00'. or Benzene/acetone(95:1) followed by benzene/ UV irradiation. methanol (95:5) or roethylene chloride/raethanol /water (150:9:0.5)

47,48

Visual comparison of 49 of the spots sizes and color intensities with standard spots.

-

Kiesel G

Benzene

Iodine vabors

Silica gel G

Heptane/acetone(4:1)

EtOII-conc. HCl(49 :1) Extract in propanol 51 plus H SO -H 0(7:3) 2 4 2 measure extinction at 545 nm.

Silica gel

-

HC1 gas and W irradiation.

50

-

52 contd...

....

Stationary phase

Developing solvent

Visualization

Silica gel G, A1 0 GF254 and 2 3 Silica gel GF 254

Petroleum ether/benzene/ He2C0 (5 :4 :1)

Iodine vabors, 2,4dinitro-phenylhydrazine or 2% H SO in 2 4 EtOIi.

Silica gel G-MiJ

Petroleum et'ner/?{e CO 2

50% H2S04

54

Silica gel 60 F254

2-Dimensional: Toluene-95Z/ethanol(9:1) followed by BuOAcfLight petroleum/AcOE(70:30:1)

H SO 2 4

55

Silica gel GF

BenzenefEtoAc (4:l)

0.5% H3 SeO -conc. H SO and Aeat at 04 100 for 15 min.

Quantitative

-

Reference

53

By spectrodensitome- 56 try by scanning at 360 nm by reference to an internal standard.

MESTRANOL

401

matography. Association of estrogenic with progestational steroids, in sone cases extended to androgens, were considered. The elution was performed with a gradient of ether in petroleum ether, obtained by means of two metering pumps. The effluent was monitored continuously by means of a hydrogen flame ionization detector, a part of the effluent being continuously drawn off t o the detector. The quantitative analysis of the steroids separated by the method was performed by a U.V. spectrophotometric or a colorimetric procedure. 5.33 Liquid Chromatography

Hara and Hayashi (61) studied the correlation of retention behaviour of steroidal pharmaceuticals in polar and bonded reverse-phase liquid column chromatography. For the systematization of correlation between the chemical structures of solutes and retention behaviour in liquid column chromatography, the retention volume of the modified steroids on silica (Corasil 11) and chemically bonded reverse-phase columns (Bondapak C18/Corasil) were studied using various binary solvent systems. Retention parameters for the functional groups of the steroids were calculated according to Martin's additive rule. By comparing these values obtained on normal and reverse-phase columns, characteristic features of both packings with regard to solute structures and solvent systems were elaborated. 5 . 3 4 Gas Chromatography

a) A comparison of an W spectrophotometric assay and a gas-liquid chromatographic assay was conducted (25) for mestranol in tablets. Both methods gave results in good agreement and both are suitable for fresh tablets. In the gaschromatographic method the column used was stainless steel (21 in x 0.25 in) of 4% of 0 XE-60 on Diatoport S, maintained at 195 Nitrogen was used as a carrier gas at 70-75 m l / hour. The method is preferred for the assay of aged tablets because on storage a complex between poly (vinylpyrolidone) and magnesium stearate excipients was produced which interfered with the W methods and was identified by

.

402


gas-liquid chromatography. b) Okuno and Higgins (62) reported a method for the determination of residues of mestranol and ethynylestradiol in foliage, soil and water samples. The lower limits for the detection of mestranol and its 3-hydroxy homolog, ethynylestradiol were 0.05, 0.1 and 0.01 ppm for foliage, soil and water respectively. Samples were extracted in an acid medium to free any conjugated ethynylestradiol and then cleaned up by Florisil column chromatography. Water samples were directly analysed by gas chromatography using a flame ionization detector and a column packed with OV 17 on Gas Chrom Q. Operating 0 temperatures were 260 for column, 275'for the inlet and 290' for the detector. Further cleaning up of the soil and vegetation samples was carried out by gel permeation and Florisil chromatography. Analysis was then carried out by gas chromatography. The lower limit of detection was lOOng for each steroid. c) Mestranol and norethisterone were determined in estrogen-progestin contraceptive tablets containing both components (63) by extraction with ethyl acetate and a single gas chromatography, with testosterone propionate as internal standard. The recoveries of mestranol and norethisterone were 98.54 and 99.14% respectively and the precision was 1.40 and 1.74% respectively. The procedure could be applied to single tablets containing 0.05 mg mestranol and 1 mg norethisterone.

d) Templeton et a1 (29) performed a gas-liquid chromatographic assay for mestranol. A tablet is allowed to disintegrate in sodium hydroxide in water, then extracted with chloroform. A dilute cholestane solution is used as an internal standard. The column used is a glass column (2 ft. x 4 mm) containing Diatoport S (80 to 100 mesh) impregnated with 6%0of silicone-gum rubber W-96 operated at 205 , with He (60 to 70 ml per min.) as carrier gas and flame ionization detector.

MESTRANOL

403 ACKNOWLEDGEMENT

The a u t h o r s wish t o t h a n k Mr. A l t a f Hussain Naqvi, f o r t y p i n g t h i s manuscript. A sample of m e s t r a n o l was k i n d l y donated by S e a r l e Research and Development, D i v i s i o n of G.D. S e a r l e and Co., Chicago, I l l i n o i s , USA.

404


REFERENCE 1. J.M. Ohrt, B.A. Haner and D.A. Norton, Acta Cryst., 1 7 ( 2 ) , 1 6 1 1 (1964). 2. The United-States Pharmacopeia, 19th ed., Mack Publishing Co., Easton, Pa., p. 1405 (1975). 3. Remington's Pharmaceutical Sciences, 15th ed., Mack Publishing Co., Easton, Pennsylvania p.920 (1975). 4 . British Pharmacopoeia, Her Majesty's Stationery Office, London, U.K., p. 291 (1973). 5 . E.G.C. Clarke, Isolation and Identification of Drugs, The Pharmaceutical Press, London, p . 4 0 5 (1969). 6 . E.W. Avdovich, M. Bowron and B.A. Lodge, J. Pharm. S c i . , 59(12), 1 8 2 1 (1970). 7. D.H. Smith, B.G. Buchanan, I?.C. White, E.A. Feigenbaum, J. Lederberg and C. Djerassi, Tetrahedron, 2, 3117 (1973). 8 . F.B. Cotton, L.N. Nysted, B. Reigel and A.L. Raynold, J. Amer. Chem. Soc., 79, 1 1 2 3 (1957). 9. H.G. Wijmenga and H.J. van der Molen, Acta endocr.,Copenh., 61, 665 (1969), per , 209, 2106 (1969). 10. T.M. Mills, T.J. Lin, W.E. Braselton, J.E. Ellegood and V.B. Mahesh, Am. J. Obstet. Gynecol., 1 2 6 ( 8 ) , 987 (1976). 11. V.B. Mahesh, T.M. Mills, T . J . Lin, J . O . Elegood and W. E. Braselton, Pharmacol. Steroid Contracept. Drugs, 117,30 (1977). 12. C.E. Bird and A.F. Clark, J. Clin, Endocrinol. Metab. 3 6 ( 2 ) , 296 (1973). 13. T.M. Mills, T.J. Lin, S . Hernandez-Ayub, R.B. Greenblatt, J . O . Ellegood and V.B. Mahesh, Am. J. Obstet. Gynecol., 1 2 0 ( 6 ) , 773 (1974). 1 4 . H.M. Bolt and H. Remmer, Xenbiotica, 20, 77 (1972). 1 5 . H.M. Bolt and H. Remmer, ibid, 2 ( 5 ) , 489 (1972). 1 6 . G.K. Hanasono and L.J. Fischer, Drug Hetab. Dispos., 2 ( 2 ) , 159 (1974). 1 7 . K . I . Williams, Steroids, 1 3 ( 4 ) , 539 (1969). 18. M . T . Abdel-Aziz and K . I . H . Williams, J . Drug Res., 60, 195 (1974). 1 9 . J.G. Williams, C. Longcope and K . I . H . Williams, Steroids, 2 5 ( 3 ) , 343 (1975). 20. I.M. Roushdi, A . I . El-Sebai and S . Belal, Egypt. J. Pharm. Sci., 1 5 ( 1 ) , 8 1 (1974). 21. I.M. Roushdi, A . I . El-Sebai and S. Belal, ibid, 1 5 ( 2 ) , 217 (1974). 22. R.A. Bastow, J. Pharm. Pharmacol., 1 9 ( 1 ) , 4 1 (1967). 23. S. Gorog and E. Csizer, Acta Chim. (Budapest), 6 5 ( 1 ) , 41(1970). 24. G. Szepesi and S. Gorog, Analyst, 9 9 ( 1 1 7 7 ) , 218 (1974).

MESTRANOL

405

25. A.P. S h r o f f and J . Grodsky, J . Pharm. S c i . , 5 6 ( 4 ) , 460 (1967). 26. R. H u e t t e n r a u c h , P h a r m a z i e , 2 0 ( 1 1 ) , 740 (1965). 27. A.P. S h r o f f a n d R.E. Huettemann, J . Pharm. S c i . , 5 6 ( 5 ) , 654 (1967). 28. J . P . Comer, P . Hartsaw a n d C.E. S t e v e n s o n , J . Pharm. S c i . , 5 7 ( 1 ) , 147 (1968). 29. R . J . Templeton, W.A. A r n e t t and I . M . J a k o v l j e v i c , 5 7 ( 7 ) , 1168 (1968). 30. J.Y.P. Wu, J . Assoc. O f f . Anal. Chem., 5 8 ( 1 ) , 75 (1975). 31. W.F. Beyer, J . Pharm. S c i . , 5 7 ( 8 ) , 1415 (1968). 32. M. R i z k , J.J. V a l l o n and A. Badinand, A n a l y t i c a chim.Acta, 6 5 ( 1 ) , 220 (1973). 33. E . Pawelczyk, I. P l u t a and B. M a r c i n i e c , Acta P o l . Pharm., 3 0 ( 3 ) , 289 (1973). 34. L,T.- C h a t t e n , E . J . T r i g g s and S . J . Glowach, J . Pharm. Belg. 3 1 ( 1 ) , 6 3 (1976). 35. K. Beyermann and E. Roder, Z. A n a l y t . Chem., 2 3 0 ( 5 ) , 347 (1967). 36. L . F . C u l l e n , J . G . R u t g e r s , P a . A. L u c c h e s i and G . J . Papar i e l l o , J . Pharm. S c i . , 5 7 ( 1 1 ) , 185 (1968). 37. J . Theron, J . Pharm. S c i . , 4 9 ( 1 1 ) , 1648 (1970). 38. A. M a r i a n i and C. Mariani-Vicari, Z e n t r a l b l . Pharm. Pharmakother. L a b o r a t o r u i m s d i a g n . , 111(1),1 9 (1972). 39. J . P a s t o r , A.M. P a u l i and N. P a p o c c h i a , Trav. SOC. Pharm. i v l o n t p e l l i e r , 3 3 ( 3 ) , 411 (1973). 40. J . H . M . Miller and P. Duguid, P r o c . Anal. Div. Chem. SOC., 1 3 ( 1 ) , 9 (1976). 41. G . Dusinsky and E. R a d e j o v a , Farm. Obz., 4 6 ( 5 ) , 225 (1977). 42. J . S . Hathews, Biochim. Biophys. Acta, 69, 1 6 3 (1963). 43. R. H u e t t e n r a u c h and I. K e i n e r , P h a r m a z z , 2 0 ( 4 ) , 242 (1965). 44. D. H e u s s e r , Dent. Apotheker-Ztg., 1 0 6 ( 1 3 ) , 4 1 1 (1966). 45. D. S o n a n i n i and L. Anker, Pharm. Acta Helv., 4 2 ( 1 ) 54 (1967). 46. P. Horak, Cesk. Farm., 1 6 ( 5 ) , 232 (1967). 47. E. Roder, Dent. Apotheker-Ztg., 1 0 7 ( 2 9 ) , 1007 (1967). 48. I. Szekacs a n d M. Klembala, Z . Klin. Chem. Klin. Biochem., 8 ( 2 ) , 1 3 1 (1970). 49. M.B. Simard and B.A. Lodge, J . Chromatogr., 5 1 ( 3 ) , 517 (1970). 50. H. Sachweh, P r o c . Conf. Appl. Phys. Chem., 2nd., 1,289 (1971). E d i t e d by Buzas, I l o n a . Akad. Kiado: B u d a p e s t ,

u,

51. 52. 53. 54.

Hung. G r o c h u l s k i , Acta P o l . Pharm., 2 8 ( 2 ) , 185 (1971). J e f f e r y , J . Chromatogr., 5 9 ( 1 ) , 216 (1971). Enache, Rev. Chim., 2 4 ( 5 ) , 648 (1973). M i j a t o v i c , L j . R i s t o v i c and J . G r u b o r , Arch. Farm.,

A. J. S. B.

2 3 ( 2 ) , 9 1 (1974).

406

HUMEIDA A. EL-OBEIDAND ABDULLAH A . AL-BADR

55. G. Cavina, G . Moretti and M. P e t r e l l a , J . Chromatogr., 1 0 3 ( 2 ) , 268 (1975). 56. A.P. Shroff and C . J . Shaw, J . Chromat. S c i . , 1 0 ( 8 ) , 509 (1972). 57. A. Alvarez Fernandez and N.V. T o r r e , J . Pharm. S c i . , 5 8 ( 6 ) , 740 (1969). 58. C.A. Brunner and F.M. Runze, J . A s s . O f f i c . Anal. Chem., 53(2). . . _ 234 (1970). 59. C.A. Brunner, J . A s s . O f f i c . Anal. Chem., 54(3), 590 (1971). 60. G . Cavina, G . M o r e t t i , A. M o l l i c a and R. A n t o n i n i , J., Chromatogr., 60 ( 2 ) , 1 7 9 (1971). 61. S. Hara and S. Hayashi, J. Chromatogr., 142, 689 (1977). 62. I. Okuno and W.H. H i g g i n s , B u l l . Environ. Contam. T o x i c o l . , 1 8 ( 4 ) , 428 (1977). 63. G. M o r e t t i , G . Cavina, G . C h i a p p e t t a , I. F a t t o r i , M. P e t r e l l a and V . Pompi, B u l l . Chim. Farm., 1 1 6 ( 8 ) , 463 (1977).

NOSCAPN Mohammed A. Al-Yahya and Mahmoud M . A . Hassan

408

1. Description 1. I Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Odor, and Taste 2. Physical Properties 2.1 X-Ray Diffraction 2.2 Solubility 2.3 Dissociation Constant 2.4 Optical Rotation 2.5 Spectral Properties 3. Preparation 3.1 Isolation from Opium 4. Synthesis of Noscapine 4.1 Tissue Culture Method 4.2 Chemical Methods 5 . Biosynthesis of Noscapine 6. Metabolism 7. Methods of Analysis 7.1 Identification Tests 7.2 Microcrystal Tests 7.3 Titrimetric Methods 7.4 Complexometric Methods 7.5 Spectrophotometric Methods 7.6 Chromatographic Methods 8. References

Analytical Profilesof Drug Substances Volume 1 I

408

408 414 414 414 414 414 415 415 415 416 429 429 429 429 429 429 43 3 436 436 436 439 441 441 445 45 6

407

Copyight 0 1982 by The AmdUn phmn.ce~iiulAmciation ISBN 0-12-260811-9

408

MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN

1. D e s c r i p t i o n 1.1

Nomenclature

1.1.1 Chemical Names a-

(S)-6,7-Dimethoxy-3-(5, 6, 7 , 8tetrahydro-4-methoxy-6-methyl-l , 3-d i o x o l o [ 4,5-g] i s o q u i n o l i n - 5 - y l ) - l (1) (3 H) -isobenzof uranone.

b-

1-a -2-methyl-8-methoxy-6,7-methylene dioxy-1- ( 6 , 7-dimethoxy-3-phthalidyl) 1, 2 , 3 , 4-tetra-hydroisoquinoline.

-

(1)*

1.1.2

c-

1 (3 H) Isobenzofuranone, 6 , 7 dimethoxy-3-(5, 6, 7 , 8-tetrahydro-4methoxy-6-methyl-1, 3-dioxolz-[4, 5-g] isoquinolin-5-y1)-, [s-(R* s ) I . (2)

d-

(3 S)-6, 7-dimethoxy-3-[(5R)-5, 6, 7 , 8-tetra-hydro-4-methoxy-6-methyl-1,3-dioxolo [ 4 , 5-g] isoquinolin-5-y1] phthalide. (3).

Generic Names d-Gnoscapine; 1-6-Narcot i n e ; I-Narcot i n e ; N a r c o t i n e ; Noscapine.

1.1.3

Trade Names Capval; Coscopin; Coscotabs; Keyt u s s c a p i n e ; Longatin; Lyobex; Fethoxyh y d r a s t i n e ; Narcompren; Narcosine; Marcotussin; N e i t a c l o n ; Nicolane; Nipaxon; Noscapal; Noscapalin; NSC 5366; Opian; Opianine; Terbenol; Tusscapine; Vadebex.

1.2

Formulae

1.2.1

Empirical

409

NOSCAPINE 1.2.2

Structural

OCH3

Nos c a p i n e The a l k a l o i d n o s c a p i n e can be c l e a v e d v e r y r e a d i l y i n t o two m o i e t i e s ; w i t h d i l u t e s u l f u r i c a c i d , c o t a r n i n e and o p i a n i c a c i d a r e g e n e r a t e d . Under a c i d i c r e d u c i n g c o n d i t i o n s , e.g., zinc i n hydrochloric acid o r s u l f u r i c a c i d , h y d r o c o t a r n i n e and meconine a r e formed (Scheme 1 ) . With t h e s t r u c t u r a l e l u c i d a t i o n of c o t a r n i n e , o p i a n i c a c i d , h y d r o c o t a r n i n e and meconine, and g i v e n t h e p r e s e n c e of a l a c t o n e r i n g i n n o s c a p i n e , the structure o f t h i s a l k a l o i d was e s s e n t i a l l y e s t a b l i s h e d ( 4 ) . 1.2.3

CAS No

(12 8- 62 - 1) 1.2.4

Niswesser L i n e N o t a t i o n T C566 DO FO K N EH & & T J H 01 K J T56 B VO D H J HOL 1 0 1 "ALPHA" LV.

1.2.5

S t e r e o c h e m i s t r y and A b s o l u t e C o n f i g u r a t i o n T h e s t e r e o c h e m i s t r y of n o s c a p i n e h a s been s t u d i e d by many w o r k e r s (5-9). The prolonged a c t i o n of h o t m e t h a n o l i c p o t a s s i u m h y d r o x i d e on n a t u r a l (-)- a - n a r c o t i n e r e s u l t s i n t h e f o r m a t i o n of a n e q u i l i b r i u m m i x t u r e of t h e o r i g i n a l b a s e and a new o p t i c a l l y

410


'n'

'2

OCH3

Noscapine

OCH 3

OC!I

Hydrocotarnine

Cotarnine

+

+ CRO

OCH

OCH

Meco n ine

Opianic acid Scheme 1

41 1

NOSCAPINE a c t i v e d i a s t er eoisomer , (-) -6 -narc0 t i n e , which can be w r i t t e n a s shown i n Scheme 2. Lithium aluminum h y d r i d e r e d u c t i o n of t h e a- and 8-noscapines r e a d i l y a f f o r d s anarcotinediol and 6 - n a r c o t i n e d i o l respectively

.

1

2

A c e t y l a t i o n of t h e s e d i o l s g i v e s r i s e t o t h e corresponding d i a c e t a t e s 2 and k , b u t subsequent c a t a l y t i c h y d r o g e n o l y s i s y i e l d s one and t h e same d e x t r o r o t a t o r y benzyli s o q u i n o l i n e 2. The f o r e g o i n g sequence c l e a r l y e s t a b l i s h e s t h a t a- and 8-noscapine must d i f f e r from each o t h e r o n l y i n t h e i r s t e r e o c h e m i s t r y a t C-9. The b e n z y l i s o q u i n o l i n e 5 shows a p o s i t i v e Cotton e f f e c t near 295 m u , s o t h a t i t s C-1 hydrogen must b e a l p h a as i n d i c a t e d . I t f o l l o w s t h a t t h e C-1 hydrogen i n (-)-an a r c o t i n e and i n (-)- 6 - n a r c o t i n e must a l s o be a l p h a (Scheme 2 ) .

A 1 t e r n a t i v e l y , a-narco t ined i o l w a s c y c l i z ed v i a i t s monomesylate d e r i v a t i v e t o t h e Nm e t h o t e t ra h y d r o p r o t o b e r b e r i n e s a l t 5. T h i s material underwent N-demethylation on p y r o l y s i s t o y i e l d t h e p r o t o b e r b e r i n e b a s e 7. Reductive removal of t h e hydroxyl group w a s achieved i n e t h a n o l i c p e r c h l o r i c a c i d o v e r a palladium c a t a l y s t . The t e t r a h y d r o p r o t o b e r b e r i n e 8 t h u s o b t a i n e d showed a s t r o n g n e g a t i v e r o t a t i o n , so t h a t i t s C-14 hydrogen must be a l p h a . The i d e n t i c a l sequence w a s c a r r i e d o u t using p n a r c o t i n e d i o l t o y i e l d t h e tetrah y d r o p r o t o b e r b e r i n e b a s e ?. Hydrogenolytic c l e a v a g e of t h i s s p e c i e s t h e n provided t h e same 1evor o t a t o r y t e t r a hyd r o pro t o b er b er i n e 8. The c o n c l u s i o n i s t h a t t h e C-1 hydrogens i n b o t h a- and 6-noscapine a r e a l p h a (Scheme 3 ) . Turning t o t h e s t e r e o c h e m i s t r y a t C-9 f o r (-)-a and (-)- B-narcotine, m o l e c u l a r models i n d i c a t e d t h a t t h e d i h e d r a l a n g l e between t h e p r o t o n s a t C-13 and C-14 of t h e 13-a-hydroxy

-

412

MOHAMMED A. AL-YAHYA AND MAHMOUD M . A. HASSAN

CH3

OCH3 (-) -6-Narcotine

(-)-a-Narcotine ( n a t u r a l isomer)

1

1 --

L1A1H4

LiA1H4

OCH~ a-Narcotinediol

1.

1 -

B-Narcotinediol

1 --

2

-

-

Ac 20 , P Y ~

Ac 0, pyridine

idine

OAc CH20Ac OCH3 0CH3

Scheme 2

413

NOSCAPINE

H

........OH

pyridine

6 @CH3 d-Narcotinediol

7 -

as o u t l i n e d

9 OCH3 B-Narcotinediol Scheme 3


414

b a s e 7 i s a b o u t 160'. On t h e o t h e r hand, f o r the 13-6 -hydroxy b a s e 2 d e r i v e d from 6 - n a r c o t i n e , t h i s a n g l e is o n l y a b o u t 60'. Following exchange of t h e h y d r o x y l i c p r o t o n s f o r d e u t e r i u m , i t w a s determined t h a t t h e s p l i t t i n g c o n s t a n t .J13,14 w a s 9 Hz f o r and o n l y a b o u t 1 . 5 Hz f o r 9. species The l a r g e c o u p l i n g v a l u e of 9 Hz i s accord w i t h a t r a n s arrangement of t h e C-13, 1 4 hydrogens i n I_, and t h e s m a l l c o u p l i n g c o n s t a n t of 1 . 5 Hz a r g u e s f o r a c i s r e l a t i o n s h i p i n 9, t h u s s e t t l i n g t h e s t e r e o c h e m i s t r y a t C-9 f& a- and 8-noscapine.

1,

1.3

iz


1.4

E l m e n t a l Composition C, 63.91%; H, 5.61%; N , 3.39%; 0 , 27.09%

1.5

Appearance, C o l o r , Odor

and Taste

Noscapine o c c u r s i n t h e form of Orthorhombic b i s p h e r o i d a l p r i s m s , t a b l e t s from d i a c e t o n e o r a s f i n e , almost w h i t e c r y s t a l l i n e powder. T r i b o l u m i n e s c e n t d 1.395. I t i s o d o r l e s s and tasteless. 2.

Physical Properties 2.1.1

X-ray d i f f r a c t i o n Crystallographic d a t a f o r noscopine are s c a r c e . The o n l y r e p o r t e d d a t a is due t o Love11 (10) and Steward and P l a y e r (11). These a r e a s f o l l o w s : Long needle-shaped c r y s t a l s were o b t a i n e d by r e c r y s t a l l i s a t i o n of t h e commercial n o s c a p i n e from e t h a n o l o r methanol. Weissenberg photographs t a k e n w i t h Cu Ka (1.5418 A)' r a d i a t i o n revealed the following systematic absences:

hOO, h

= 2n

+

1

OKO, K = 2n -I-1 001, 1 = 2n + 1

415

NOSCAPINE

d e f i n i n g unambiguously t h e space group P212121. C e l l dimensions were o b t a i n e d from 28 v a l u e s of 32 r e f l e x i o n s from n o s c a p i n e u s i n g two a x e s i n each c a s e , measured w i t h a counter diffractometer. The f o l l o w i n g d a t a were o b t a i n e d :

M.W.

413.41

178

M.p. (OC)

C r y s t a l system Space group Cell

Dimensions

(8)

d 3 )

r

P212121 15.398(12) b 32.686(36) c ( p r i m ) 8.022(8) 4037(11)

z

8

Qcalc (g. ~ m - ~ )

1.360

.

Qexp (g ~ m - ~ ) 2.1.2

O r t hor homb i c

1.38

Melting P o i n t 174-176OC(3) 176OC s u b l i m e s a t 15OoC-16O0C under 11 mm p r e s s u r e a t 2 mm d i s t a n c e (1)

2.2

Solubility I t i s i n s o l u b l e i n water; s l i g h t l y s o l u b l e i n a l c o h o l ( 9 5 % ) , i n e t h e r and i n carbon t e t r a c h l o r i d e . S o l u b l e i n chloroform, benzene and v e r y s o l u b l e i n a c e t o n e (12).

2.3

D i s s o c i a t i o n Constant I t i s a v e r y weak b a s e , pKa 7.8 ( 1 ) and 4.85 i n 80% m e t h y l c e l l o s o l v e ( 1 3 ) .

2.4

Optical Rotation

+

42'

to

+

(2% w/v i n 0 . 1 M h y d r o c h l o r i c a c i d ) (3) [ u I D - 198O (1% w/v i n c h l o r o f o r m ) ,

[a],

48'


416

[a],

- 146 (2% w/v i n t o l u e n e ) , - 147' (1.59% i n b e n z e n e ) ,

[a],

+

[a],

2.5

50 (1% w/v i n h y d r o c h l o r i c a c i d ) (14)


U l t r a v i o l e t Spectrum The W spectrum of n o s c a p i n e i n methanol w a s scanned from 200 t o 400 nm u s i n g V a r i a n Carry 119 Spectrophotometer. I t e x h i b i t s a c h a r a c t e r i s t i c UV spectrum ( F i g . 1) w i t h two maxima: Xmax 310.2 290.6

1% cm 114.9 106.4

(C, 9.42 mg p e r 100 ml) (C, 9.42 mg p e r 1 0 0 ml)

Other UV s p e c t r a l d a t a of n o s c a p i n e have a l s o been r e p o r t e d : Xmax 209 291 309-310 Xmax 291 310 291 3 09 2.5.2

Log

E

4.86 3.60 3.69

) ) )

i n e t h a n o l (1, 15)

(E)

a b o u t 1.1 ) a b o u t 1.4 ) 3981 4898

i n a l c o h o l 95% (3)

1 )

i n methanol (12)

I n f r a r e d Spectrum The I R s p e c t r a of n o s c a p i n e a s K B r d i s c and n u j o l m u l l were r e c o r d e d on a P e r k i n E l m e r FT-680B s p e c t r o p h o t o m e t e r and shown in F i g . 2 t?, F i g . 3 respectively. The s t r u c t u r a l a s s i g n e m e n t s have been c o r r e l a t e d w i t h t h e f o l l o w i n g band f r e q u e n c i e s (Table 1)

417

NOSCAPINE

Fig.

1.

W Spectrum of N o s c a p i n e i n M e t h a n o l .

Wavelmgt h F *O

Fig.

2.

5.0

6.0

70

8.0

s.0

I R Spectrum of Noscapine as K B r d i s c .

f0

12

!4

1

2500

Fig. 3 .

.

2000

I R Spectrum of Noscapine as Nujol Mull.

800

700

420

MOHAMMED A . AL-YAHYA A N D MAHMOUD M. A . HASSAN T a b l e 1. I R C h a r a c t e r i s t i c s of Moscapine Frequency c m

-1

Assignement

3000, 2945, 2880, 2845, 2800

Methylened i o x y and C-H and -CH frequencies. 3 ( y - l a c t o n e ) 3-C=O g r o u p

17 60 1625 1600,1505,1480, 1280-1 22 5

-c=cAromatic Aromatic m e t h o x y - a r y l C-0 stretching vibrations.

790, 815, 8 3 5 , 885

2 a d j a c e n t H atoms, i s o l a t e d H atom C-H o u t of p l a n e d e f o r m a t i o n . Tetra and pen t a s u b s t i t u t e d b e n z e n e s

.

Other c h a r a c t e r i s t i c a b s o r p t i o n bands are: 1460, 1430, 1405, 1 3 9 0 , 1 3 8 0 , 1 3 6 5 , 1 3 3 0 , 1310, 1 2 0 0 , 1120, 1 0 8 5 , 1040, 1 0 1 0 , 980, 930, 900, B O O , 765, 750, 735, 725, 715, and 700 cm-'. O t h e r I R d a t a a r e a l s o r e p o r t e d (16) 2.5.3

N u c l e a r Magnetic Resonance S p e c t r a 2 . 5 . 3 . 1 P r o t o n Spectrum The PMR s p e c t r u m of n o s c a p i n e i n d e u t e r a t e d chloroform w a s r eco r d ed on a V a r i a n XL200, 2 0 0 MHz NMR s p e c t r o meter u s i n g t e t r a m e t h y l s i l a n e a s a r e f e r e n c e s t a n d a r d ( F i g . 4 ) . The following s t r u c t u r a l a s s i g n m e n t h a v e been made ( T a b l e 2 ) . 4

I, 0

7

F i g . 4.

6

5

4

PMR Spectrum of Noscapine and T e t r a m e t h y l s i l a n e

3

2

i n Deuterated Chloroform.

MOHAMMED A . AL-YAHYA AND MAHMOUD M.A. HASSAN

422

Table 2.

PMR C h a r a c t e r i s t i c s of Noscapine

Ass ignemen t (Group)

Po s i t i o n

Chemical S h i f t ( 6 )

3 , 4 of i s o q u i n o l i n e

2.32 (m)

N- CH3

2 of i s o q u i n o l i n e

2.53 ( s )

OCH3


3.84 ( s )

OCH3

&'of p h t h a l i d y l

4.02 (s)

OCH3

5'of

4.08 ( s )

-CH2-CH2

phthalidyl

-CH-


4.37 (d)

-CH-

9 of p h t h a l i d y l

5.55 (d)

-CH2-

methylened i o x y

5.92 ( s )

-CH-

2'0f

6.05 (d)

-CH-


6.29 ( s )

-CH-

3'of

6.94 (d)

phthalidyl

phthalidyl

s = s i n g l e t , d = doublet, m = multiplet

Other PMR s p e c t r a l d a t a was a l s o r e p o r t e d ( 1 7 an.d 5 5 ) . 2.5.3.2

I3C-NMR

Spectra

I3C-NMR c o m p l e t e l y decoupled and o f f - r e s o n a n c e s p e c t r a are shown i n Fig. 5 and Fig. 6 r e s p e c t i v e l y . Both were r e c o r d e d o v e r 11001.1 HZ r a n g e , i n d e u t e r a t e d c h l o r o f o r m (CDC13) on XL-200,200 MHz NMR s p e c t r o m e t e r . Using 10 mm sample t u b e and tetramethylsilane as reference standard a t 25OC. The c a r b o n chemical s h i f t s a s s i g n e d on t h e b a s i s of t h e a d d i t i v i t y p r i n c i p a l s and o f f - r e s o n a n c e s p l i t t i n g p a t t e r n (Table 3) (18).

9

a Fig. 5.

7

6

5

4

1 3

2

f

l3C-NMR Spectrum of Noscapine i n Deuterated Chloroform.

0

3

1

424

50

60

1

T

70

90

80

100 110 120 130 1 4 0 150 160

170

180

1

1

190 200 210 220 230 240 250 260 270 280 290 300

310

1

1 2

320 330

340 350 360 370 380 390 400 410 420 430 440 4 5 0 Fig. 7.

EI-Mass

Spectrum of Noscapine.

MOHAMMED A . AL-YAHYA AND MAHMOUD M. A. HASSAN

426

’

22

OCH3

Table 3. Carbon No.

c-1 c- 2

Carbon Chemical S h i f t s of Noscapine

Chemical S h i f t PPm 60.84 49.99 28.03 134.03 117.65 140.45 100.73 141.14 152.18 132.09 59.36

c-3

c-4 c-5 C-6

c-7 C-8 c- 9 c-10

c-11 2.5.4

(d) (t) (t) (s)

(d) (s) (t) (s)

(s) (s) (q)

Carbon No.

Chemical S h i f t Ppm

c-12 C-13 C-14 C-15 C-16 C-17 C-18 c-19 c-20 c-21 c-22

46.29 81.83 120.17 118.19 102.29 147.67 148.37 117.11 168.06 56.78 62.22

(q) (d) (s) (d)

(d) (s) (s) (s) (s) (4)

(q)

Mass Spectrum

The mass spectrum of n o s c a p i n e by e l e c t r o n impact i o n i z a t i o n and recorded on Ribermag R-10-10 mass equibbed w i t h d i r e c t i n l e t probe. ( F i g . 7) shows m o l e c u l a r i o n peak a b a s e peak a t m / e 220.

obtained which w a s spectrometer The spectrum and shows

The mass spectrum of n o s c a p i n e o b t a i n e d by butane chemical i o n i z a t i o n ( F i g . 8) shows a m o l e c u l a r i o n peak PI+ a t m / e 413 w i t h a r e l a t i v e i n t e n s i t y of 2.8% and a b a s e peak a t m / e 220. The most prominent f r a g m e n t s ,

90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 2t

1

290 300 31 0 320 330 340 350 360 370 380 390 4-00410 420 430 440 450 460 470 Fig.

8.

CI-Mass

Spectrum of Noscapine.

428

MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN t h e i r r e l a t i v e i n t e n s i t i e s and s t r u c t u r e s a r e l i s t e d i n Table 4 . Other mass s p e c t r a l d a t a f o r p h t h a l i d e i s o q u i n o l i n e s was a l s o r e p o r t e d (19, 2 0 ) . Table 4 .

m/e 413

Mass Fragments of Moscapine Relative Intensity

96

Fragment

M+

2.8

221

220

195

8.0

OH

193 OCH3

429

NOSCAPINE

3.

P r eDara t i o n 3.1

I s o l a t i o n from Opium Noscapine o c c u r s up t o 11%n a t u r a l l y i n opium (Papaver sornniferum L . (Fam. papaveraceae) I t w a s f i r s t d i s c o v e r e d by Derosne i n 1803 ( 2 0 ) , and i s o l a t e d by Robinquet i n 1817 ( 2 1 ) . Noscapine can be s e p a r a t e d from o t h e r opium a l k a l o i d s by t h e procedure o u t l i n e d i n Scheme 4 (90).

.

Another p a t e n t method h a s been a l s o d e s c r i b e d f o r i t s i s o l a t i o n on a n i n d u s t r i a l scale ( 2 2 ) .

4.

S y n t h e s i s of Noscapine

4.1

By T i s s u e C u l t u r e Method Khanna e t a 1 (23) d e s c r i b e d a method f o r t h e s y n t h e s i s of noscapine a l o n g w i t h o t h e r a l k a l o i d s by t i s s u e c u l t u r e of Papaver somniferum Linn.

4.2

By Chemical Methods P e r k i n and Robinson (24) d i s c o v e r e d t h a t h e a t i n g a m i x t u r e of c o t a r n i n e 1 and meconine 1i n e t h a n o l r e s u l t e d i n a s m a l l y i e l d of noscapine 3 . The expected second isomer of noscapine-(because of t h e presence of 2 a s s y m e t r i c c e n t r e s ) w a s n o t found. The s y n t h e t i c noscapine was t h e n r e s o l v e d and t h e n o s c a p i n e o b t a i n e d shown t o b e i d e n t i c a l w i t h t h e n a t u r a l p r o d u c t . (Scheme 5).

A q u i t e e f f i c i e n t s y n t h e s i s of noscapine w a s developed by Hope and Robinson i n 1914 ( 2 5 ) , i n which c o t a r n i n e i s condensed w i t h iodomeconine 2 and t h e adduct w a s reduced w i t h sodium amalgum t o g i v e t h e d e s i r e d p r o d u c t , corresponding t o t h e n a t u r a l s e r i e s (Scheme 6 ) .

L

5.

B i o s y n t h e s i s of Noscapine I t has been p o s t u l a t e d t h a t t h e ph t h a l i d e i soqu i n o 1i n e s a r e formed i n n a t u r e by o x i d a t i v e m o d i f i c a t i o n of t e t r a h y d r o p r o t o b e r b e r i n e s , and p r e v i o u s work w i t h l a b e l e d precursors supports t h i s hypothesis (26).

430

MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN Powdered Op i um

+

+

Shake w i t h warm calcium c h l o r i d e s o l u t i o n F i 1t e r Insoluble matter C (discard)

F i ltrate (hydroch l o r i des o f a 1ka l o i ds)

4

Reduce volume (evaporate under reduced p r e s s u r e ) t o syrupy 1 i q u i d

4

Add 10% NaOH s o l u t i o n Precipitates p k (noscapine, papaverine, thebaine)

4

a

l ine solution (morphine, codeine,

E x t rn aa c rt cw e i nt he ) ci h l o r o f o r m

Dissolve i n d i l u t e a l c o h o l

$.

Add a c e t i c a c i d t o make s l i g h t l y

0

C h 1 o r o f orm

Aqueous extract alkaline (containsolution i n g codeine) (morphine, narceine)

acidii

Add 3 volumes o f b o i l i n g water

A Solution

Precipitate (papave r ine, noscap ine)

4

(theba ine)

4

4

Further purification

Make a c i d i c

rz 1

\

Further pur i f ic a t i o n

Dissolve i n b o i l i n g 0.33%(aqueous)oxali c a c i d soln.

4

4

Make s l i g h t l y a l k a l i n e w i t h ammonia

A l l o w t o stand

Bring t o b o i l i n g A l l o w t o stand

C rys t a 1 s (papave r ine ac id oxalate)

4

Aqueous a c i d i c s o l n . ( s a l t s of morphine and na r c e i ne)

Sol u t i o n (noscap ine oxa 1 a t e )

+

Repeat

Precipitate (morphine)

I

Further p u r i f ic a t ion

Solution (narceine)

Make a l k a l i n e w i t h ammonia

Precipitate (noscapine)

+

1 i s o l u t on (discard)

+

Dissolve i n b o i l i n g alcohol Crystallization Scheme 4:

i

Further pur i f i cat ion

Isolation of

from powdered op i um .

NOSCAPINE

43 1

OCH

OCH3

2 Plecoriine

I

Cotarnine

D

C2H50H

0

0ch3 3

(2)-a-Narco t ine

Scheme 5

432

MOHAMMED A. AL-YAHYA AND MAHMOUD M . A. HASSAN

Cotarnine 1

Iodomeconine

\

2

\

CH 3

CH 3 CH 3O

Na/Hg H..

... *..s . 0

OCH3

OCIl

3

(+) -a-Narcotine

Scheme 6

433

NOSCAPINE

S e v e r a l f e e d i n g e x p e r i m e n t s ( 6 , 8 , 94 ) have been r u n t o e l u c i d a t e t h e b i o g e n e s i s of n o s c a p i n e i n Papaver somnif erum L . (Papaveraceae) When l a b e l e d (+) t y r o s i n e was f e d t o t h e p l a n t , r a d i o a c t i v e n a r c o t i n e l a b e l e d s p e c i f i c a l l y and e q u a l l y a t C-1 and C-3 was o b t a i n e d . The b e n z y l i s o q u i n o l i n e s y s t e m of n o s c a p i n e i s t h u s d e r i v e d b i o l o g i c a l l y from two Ar-C-C u n i t s which c a n a r i s e from t y r o s i n e .

.

The c a r b o n atoms t h a t a r i s e from t h e S-methyl of m e t h i o n i n e were c l e a r l y p i n p o i n t e d when, a f t e r f e e d i n g radioactive methionine, noscapine labeled a t t h e l a c t o n e c a r b o n y l , t h e m e t h y l e n e d i o x y g r o u p , and t h e N- and 0-methyl c a r b o n atoms w a s o b t a i n e d ( 2 7 , 2 8 ) . Progressing f u r t h e r along the biogenetic locus, t h e b e n z y l i s o q u i n o l i n e (+)- n o r l a u d a n o s o l i n e l a b e l e d C-1 l e d t o n o s c a p i n e a l s o l a b e l e d C-1 ( 2 9 ) . Even more s i g n i f i c a n t l y , when q u a d r u p l y l a b e l e d (+)- and (-) r e t i c u l i n e were f e d s e p a r a t e l y t o P. somniferum, i t w a s found t h a t b o t h enantiomers were i n c o r p o r a t e d i n t o n o s c a p i n e , b u t w i t h t h e (+)-isomer d o i n g so s l i g h t l y more e f f i c i e n t l y . E v i d e n t l y e p i m e r i z a t i o n of t h e wrong b e n z y l i s o q u i n o l i n e p r e c u r s o r must o c c u r , p r o b a b l y by o x i d a t i o n - r e d u c t i o n a t C-1. I n keeping w i t h t h i s c o n c l u s i o n c o n s i d e r a b l e l o s s of t r i t i u m o c c u r e d i n t h e c o u r s e of i n c o r p o r a t i o n of b o t h r e t i c u l i n e s . Another i m p o r t a n t o b s e r v a t i o n i s t h a t t h e l a c t o n e c a r b o n y l of t h e p h t h a l i d e i s o q u i n o l i n e must b e d e r i v e d from t h e N-methyl group of t h e b e n z y l i s o q u i n o l i n e p r e c u r s o r ( 2 7 , 29, 3 0 ) . F i n a l l y , i t h a s been found t h a t t h e f e e d i n g of l a b e l e d ( - ) - s c o u l e r i n e r e s u l t s i n t h e f o r m a t i o n of r a d i o a c t i v e noscapine. Protoberberines are, therefore, t h e precursors f o r t h e phthalideisoquinolines i n plants. S i g n i f i c a n t l y , ( - ) - s c o u l e r i n e , which p o s s e s s e s t h e same a b s o l u t e c o n f i g u r a t i o n a s ( + ) - r e t i c d i n e and ( - ) - a - n a r c o t i n e , w a s more t h a n one hundred t i m e s more e f f i c i e n t t h a n i t s enantiomer a s a p r e c u r s o r f o r ( - ) - a - n a r c o t i n e . The b i o g e n e t i c sequence i n p l a n t s i s , t h e r e f o r e , b e n z y l i s o qu i n o l i n e s +- t e t r a h y d r o p r o t o b e r b e r i n e s + p h t h a l i d e i s o q u i n o l i n e s . The b i o s y n t h e s i s of (-)-cC-narcotine i s shown i n Scheme 7 .

6.

Metabolism The m e t a b o l i s m of n o s c a p i n e was r e p o r t e d


434

&:

Labeled t y r o s i n e

bCH3

*

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