Analytical Profiles of Drug Substances and Excipients Volume 15

Analytical Profiles of Drug Substances Volume 15 EDITORIAL BOARD Abdullah A. Al-Badr Steven A. Benezra Gerald S. Br...

Author: Klaus Florey

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Analytical Profiles

of Drug Substances Volume 15

EDITORIAL BOARD

Abdullah A. Al-Badr Steven A. Benezra Gerald S. Brenner Glenn A. Brewer, Jr. Nicholas DeAngelis John E. Fairbrother

Lee T. Grady Eugene Inman Joseph Mollica James W. Munson Milton D. Yudis John Zarembo

Academic Press Rapid Manuscript Reproduction

Analytical Profiles

of Drug Substances Volume 15 Edited h y

Klaus Florey The Sqiiil)t> Institute lor Medical Research New Birinswick, New Jersey

Contribu ti rig Editors

Abdullah A. Al-Badr Glenn A. Brewer, Jr. Gerald S. Brenner Nicholas J. DeAngelis Joseph A. Mollica

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

Orlando Sail Diego New York Austin Boston London Sydney Tokyo Toronto

COPYRIGHT @ 1986 BY THE AMERICAN PHARMACEUTICAL ASSOCIATION. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED I N ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY. RECORDING. OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM. WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER

ACADEMIC PRESS, INC. Orlando, Florida 32887

United Kingdom Edition published by ACADEMIC PRESS INC. (LONDON) LTD. 24-28 Oval Road, London N W I 7DX

Library of Congress Cataloging i n Publication Data (Revised for vol. 15) Analytical profiles o f drug substances. Compiled under the auspices o f the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences. Includes bibliographical references and indexes. 1. Drugs-Analysis. 2. Chemistry, PharmaceuticalCollected works. I. Florey, Klaus. II. Brewer, Glenn A. I l l . Academy o f Pharmaceutical Sciences. Pharmaceutical Analysis and Control Section. [DNLM: 1. DrugsAnalysis-Yearbooks. QV740 A A 1 A551 RS189.A58 615l.1 70-187259 ISBN 0-12-260815-1 (v. 15) PRINTED IN THE l l N l T t O STATtS OF AMERICA

CONTENTS

Affiliutiotis of Editors Preface

uiitl

uii

Contrihutors

ix

1

Amiloride Hydrochloride David J . Mazzo

35

Aminoglutethimide

Hassan Y. Aboul-Enein 71

Caffeine

Mohnmmad Uppal Zubair, Mahmoud M . A . Hassan, and Ibrahim A . Al-Meshal Cocaine Hydrochloride Farid J . Muhtadi and Abdullah A . Al-Badr

151

Ephedrine Hydrochloride

233

Syed Laik Ali E stradiol Eugene G . Salole

283

Guanabenz Acetate

319

Charles M . Shearer 337

Iodamide

Davide Pitrb

V

CONTENTS

vi

Lithium Carbonate

367

Henry C . Stober Maprotiline Hydrochloride

393

Soonhee K . Suh and James B . Smith Penicillin G, Potassium

427

Joel Kirschbaum Piroxicam

509

Mladen MihaliC, Hrvoje Hofmun, Josip Kuftinec, Branku Krile, Vesna Capler, Franjo KujfeZ, and Nikola BlaieviC Ranitidine

533

Marijan Hohnjec, Josip Kuftinec, Miuenka Malncir, Milivoj Skreblin, Franjo KujfeZ, Antun Nagl, and Nikola BlaZeviC Strychnine Farid J , Muhtadi and Mohumed S. Hifnawy

563

Vidarabine

647

Wen-Hai Hong, Tsun Chang, and Robert E. Duly Zomepirac Sodium

673

Mladen ZiniC, Josip Kuftinec, Hrvoje Hofman, Franjo KajfeZ, and Zlatko MeiC PROFILE SUPPLEMENTS Chloramphenicol

70 1

Abdullah A. Al-Badr and Humeida A. El-Obeid Lidocaine and Lidocaine Hydrochloride

761

Michael F. Powell Sodium Nitroprusside

78 1

Auke Bult, Oscar R. Leeuwenkamp, and Wouter P. van Bennekom Cumulative Index Erratum to Volume 14

793 796

AFFILIATIONS OF EDITORS AND CONTRIBUTORS

Hussun Y. Ahoul-Enein, King Faisal Hospital, Riyadh, Saudi Arabia Abdulluh A . Al-Badr, King Saud University, Riyadh, Saudi Arabia Syed Laik Ali, Zentrallaboratorium Deutscher Apotheker, Eschliorn, Federal Republic of Germany Ibrcihim A . Al-Meshul, King Saud University, Riyadh, Saudi Araliia S . A. Renezru, Wellcome Research Laboratories, Research Triangle Park, North Carolina Nikolu Bluieuic', Chromos-Aroma, mirisi, Zagreb, Yugoslavia G. A . Rrenner, Merck Sharp and Dohnie Research Laboratories, West Point, Pennsylvania G. A . Brewer, The Squibb Institute for Medical Research, New Brunswick, New Jersey Auke Bult, Rijksuniversiteit Utrecht, Utrecht, The Netherlands Vesnu capler, Podravka Institute, Zagreb, Yugoslavia Tsun Chung, Warner-Lambert, Morris Plains, New Jersey Robert E . Duly, Warner-Lambert, Morris Plains, New Jersey N . J. DeAngelis, Wyeth Laboratories, Philadelphia, Pennsylvania Humeicla A. El-Obeid, King Saud University, Riyadh, Saudi Arabia J. E . Fairbrother, E. R. Squibb & Sons, Moreton, England Klaus Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey L. T. Grudy, The United States Pharmacopeia, Rockville, Maryland Muhmoud M . A . Hussun, King Saud University, Riyadh, Saudi Arabia Mohumed S. Hvnuwy, King Saud University, Riyadh, Saudi Arabia Wruoje Hofmun, Podravka Institute, Zagreb, Yugoslavia Murijun Hohnjec, Podravka Institute, Zagreb, Yugoslavia

vii

viii

AFFILIATIONS

Wen-Hai Hong, Warner-Lambert, Morris Plains, New Jersey E. L. Znman, Lilly Research Laboratories, Indianapolis, Indiana Frunjo Kujfea, Podravka Institute, Zagreb, Yugoslavia Joel Kirschbaum, The Squibb Institute for Medical Research, New Brunswick, New Jersey Brunku Krile, Podravka Institute, Zagreb, Yugoslavia Josip Kuftinec, Podravka Institute, Zagreb, Yugoslavia Oscar R. Leeuwenkamp, Rijksuniversiteit Utrecht, Utrecht, The Netherlands Miljenka Malnar, Podravka Institute, Zagreb, Yugoslavia David J . Muzzo, Merck Sharp & Dohme, West Point, Pennsylvania Zlatko MeiC, Institute Ruder Boscovii., Zagreb, Yugoslavia Mluden Mihalid, Podravka Institute, Zagreb, Yugoslavia J . A. Mollica, CIBA-GEIGY Corporation, Summit, New Jersey Farid J . Muhtadi, King Saud University, Riyadh, Saudi Arabia J . W . Munson, T h e Upjohn Company, Kalamazoo, Michigan Antun Nagl, University of Zagreb, Zagreb, Yugoslavia Davide Pitrb, Bracco Industria Chimica, Milan, Italy Michael F. Powell, Syntex Research, Palo Alto, California Eugene G. Salole, University of Strathclyde, Glasgow, Scotland Charles M . Shearer, Wyeth Laboratories, Philadelphia, Pennsylvania Milivoj Skreblin, Podravka Institute, Zagreb, Yugoslavia James B. Smith, CIBA-GEIGY Corporation, Suffern, New York Henry C. Stober, CIBA-GEIGY Corporation, Suffern, New York Soonhee K. Suh, CIBA-GEIGY Corporation, Suffern, New York Wouter P. van Bennekom, Rijksuniversiteit Utrecht, Utrecht, The Netherlands M . D . Yudis, Schering-Plough, Kenilworth, New Jersey J. E. Zarembo, Revlon Health Care, Tuckahoe, New York Mladen ZiniC, Podravka Institute, Zagreb, Yugoslavia Mohammad Uppal Zubair, King Saud University, Riyadh, Saudi Arabia

PREFACE

Although the official compendia define a drug substance as to identity, purity, strength, and quality, they normally d o not provide other physical or chemical data, nor do they list methods of synthesis or pathways of physical or biological degradation and metabolism. Such information is scattered through the scientific literature and the files of pharniaceiitical laboratories. I perceived a need to supplement the official cornpendial standards of drug substances with a comprehensive review of such information, and fifteen years ago the first volume of Anulyticul Profiles of Drug Substances was 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 articles and even more so to the enthusiastic response of our authors, an international group associated with pharmaceutical firms, academic institutions, and cornpendial authorities. I would like to express my sincere gratitude to them for making this venture possible. 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 articles 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. Kla~isFlorey

ix

This Page Intentionally Left Blank

AMILORIDE HYDROCHLORIDE

David J. Mazzo

1. Introduction 1.1 Therapeutic Category 1.2 History 2.

Description 2.1 Chemical Name, Formula, Molecular Weight 2.2 Definition 2.3 Appearance, Color, Odor

3. Synthesis 4. Physical Properties 4.1 Infrared Spectrum 4.2 Nuclear Magnetic Resonance Spectrum 4.2.1 Proton NMR 4.2.2 Carbon-13 NMR 4.3 U1 traviolet Spectrum 4.4 Mass Spectrum 4.5 Optical Rotation 4.6 Thermoanalytical Behavior 4.6.1 Melting Point 4.6.2 Differential Thermal Analysis Behavior 4.6.3 Thermogravimetric Analysis Behavior 4.7 Solubility 4.8 Crystal Properties 4.9 Hygroscopicity 4.10 Dissociation Constant 5. Methods of Analysis ANALYTICAL PROFILES O F DHUG SUBSTANCES VOLUME 15

1

Copyright 8 1986 by the American Pharmaceutical Association A11 rights of reproduction in any form reserved.

DAVID J. MAZZO

2

5.1

5.2

5.3

5.4

Identification Tests 5.1.1 Ultraviolet Spectrophotometry 5.1.2 Infrared Spectroscopy 5.1.3 Elemental Analysis Spectrophotometric Analysis 5.2.1 Direct Ultraviolet Spectrophotometry 5.2.2 Ultraviolet Spectrophotometry via Flaw Injection Analysis 5.2.3 Liquid-Liquid Extraction with Ultraviolet Spectrophotometry or Spectrofluorimetry Chromatographic Analysis 5.3.1 Thin-layer Chromatography 5.3.2 High Performance Liquid Chromatography Non-Aqueous Titration

6. Stability

- Degradation

6.1 Solid State Stability 6.2 Solution Stability

7. Biopharmaceutics and Metabolism 7.1 Absorption and Bioavailability 7.2 Metabolism 7.3 Pharmacokinetics 8.

Determination in Biological Matrices

9.

Determination in Pharmaceuticals 9.1 Dissolution Testing 9.2 Assay, Dosage Uniformity and Stability Testing

10. References

AMILORIDE HYDROCHLORIDE

1.

3

Introduction

1.1 Therapeutic Category (1) Amiloride hydrochloride dihydrate, hereafter referred to as amiloride hydrochloride, is a potassium-conserving diuretic with relatively weak natriuretic and antihypertensive activity. It is not an aldosterone antagonist and, therefore, is effective in the absence of aldosterone. Amiloride hydrochloride is indicated as adjunctive treatment with thiazide diuretics or other kaliuretic-diuretic agents in congestive heart failure or hypertension to aid in the restoration of normal serum potassium levels and/or to prevent the development of hypokalemia. Amiloride hydrochloride is available for oral dosing as tablets, is usually well tolerated, and except for hyperkalemia, has had significant adverse effects reported infrequently (1). 1.2 History Amiloride hydrochloride, a substituted (pyrazinecarbonyl)guanidine, was first synthesized in the Merck, Sharp and Dohme Research Laboratories ( 2 ) . The first non-patent literature reference to ami.10ride appeared in 1966 ( 3 ) and the first structureactivity relationship study was published in 1967 (4). Amiloride hydrochloride has gained steadily increasing popularity as a therapeutic drug as well as a pharmacological tool and its actions and effects have k e n the subject of at least three international synposia (5,6,7). A search of Chemical Abstracts from 1966 to 1985 produced 490 bibliographic citations for works dealing with amiloride hydrochloride. 2.

Description 2.1

Chemical N a m e , Formula, Molecular Weight The current accepted Chemical Abstracts name for amiloride hydrochloride (MK-870) is 3,5-diaminoN-(diaminomethylene)-6-~hloropyrazinecarboxamide monohydrochloride dihydrate. The CAS registry no. is 17440-83-4.

4

DAVID J. MAZZO

Other names which have been used for amiloride hydrochloride include 3,5-diamino-y-(aminoiminomethyl)-6-chlorpyrazinecarboxamide monohydrochloride dihydrate, N-amidino-3,5-diamino-6chloropyrazinecarbox%nide monohydrochloride dihydrate, N-amidino-3,5-diamino-6-chloropyrazinamide Gnohydrochloride dihydrate, 1-( 3,5-diamino6-chloropyrazinecarboxy1)guanidine monohydrochloride dihydrate, 1-(3,5-diamino-6-chlorpyrazinoy1)guanidine monohydrochloride dihydrate, as well as the monohydrochloride dihydrated salts of guananprazine, amiprddin and amipramizide

0 CI

II

NH2 I

C N=CN H2 .HCI 02H20

H2N

2.2

NH2

Definition Amiloride Hydrochloride, unless specifically stated otherwise, is defined as the crystalline, monohydrochloride dihydrated salt form of the conpound. Its tradename is M I W R @ . Amiloride, when referred to, indicates the free base.

2.3

Appearance, Color, Odor Amiloride hydrochloride is a yellow to greenish yellow crystalline powder which is odorless or practically odorless.

3.

Synthesis Amiloride hydrochloride, essentially a substituted guanidine, has been prepared through a series of synthetic steps beginning with methyl cyanoacetate and urea (4,8-14). The synthetic route is presented in

AMILORIDE HYDROCHLORIDE

5

Figure 1. The starting materials (I and 11) are reacted in sodium isopropoxide and subsequently nitrosated. The product (111) is reduced to the amino compound (IV), treated with glyoxal to form a pteridine intermediate (V) and hydrolyzed in base which upon acidification gives 3-aminopyrazinoic acid (VI). This substituted pyrazinoic acid is then esterified with methanol (VII) in the presence of sulfuric acid, chlorinated (VIII) and converted to the 5-amlnO ester with ammonia in DMSO. The product (IX) is then reacted with guanidine to form amiloride which upon reaction with hydrochloric acid in water forms amiloride monohydrochloride dihydrate (X). 4. Physical Properties 4.1

Infrared Spectrum The infrared spectrum of amiloride hydrochloride taken in a KBr pellet is Shawn in Figure 2 (15). A Digilab Model FTS-15C fourier transform infrared spectrophotometer was used to acquire the spectrum. Frequency assignments for some of the characteristic bands are listed in Table I. Table I Infrared Spectral Assignments for Amiloiide Hydrochioride Frequency (cm-l) 3250-3500 3150 1680 1640 1600 1240 770

Assignment N-H stretch (NH ) N-H stretch (NHf C=O stretch H2 deformation mode H2 deformation mode N-(C&) Stretch C-H out-of-plane mode

The infrared spectrum of amiloride hydrochloride taken in a mineral oil mull is sham in Figure 3 (15)

-

u u

+ I

P

0

$1 +

Id-

I

i; 5

I

-+

f z

+

4

41 44 t

I"

-0-0

X

t

rB

+

z--0-2

3"

220"

0--0--0

6

W

a

J

a 0

!4

*d

a

a h .c

a tfiu 0

aJ

U

0

1

u

85.0

I

I

I

I

I

I

I

I

1

I

1

I

I

I

I

I

I

1

1

1

1

I

I

1

42.:

\ C

J

I

I

I

I

I

3500 3300 3100 2900 2700 2500 2300 2100 1900 1700 1500 1300 1100 900

Figure 2.

700 I

500 1

I n f r a r e d spectrum of a m i l o r i d e h y d r o c h l o r i d e t a k e n i n a KBr p e l l e t .

9c

I

I

1

I

I

I

I

I

I

1

1

I

I

d

a

0

c

0 c e .-

E

g!

I

4!

2

I-

s

Frequency (cm-’ 1 Figure 3 .

I n f r a r e d spectrum of a m i l o r i d e h y d r o c h l o r i d e t a k e n i n a m i n e r a l o i l mull.

AMILORIDE HYDROCHLORIDE

4.2

Nuclear Magnetic Resonance Spectrum (16) 4.2.1

Proton NMR Spectrum The proton magnetic resonance spectrum of amiloride hydrochloride was obtained using a JEOL(USA) C-60HL spectrometer. The spectrum was acquired from a 1.0 M drug solution in fully deuterated dimethylsulfoxide (d6-DMSO). The spectrum is shown in Figure 4 and the spectral assignments are listed in Table I1 (17). Table I1 Proton NMR Spectral Assignments for Amiloride Hydrochloride

Integral (mm) 3.8

Relative No. Protons Assignments

52

4.4(b)

48.5

4.1

H20

7.4 Aromatic -NH2

7.7

NH2

9.8

57(c) 11.1

4.9

NH2 N -H

Notes: (a) All protons are bound to nitrogen or oxygen (H20), therefore chemical shift will be strongly dependent upon sample concentration, temperature and/or solution pH. (b) This si.gnal includes a small contribution due to water originally present in the d6-DMS0. (C)

The total integral resulting from these signals is reported since the appearance of the spectrum suggests intermediate kinetic exchange between the two types of sites.

(CH314-S

1

I

1

8.0

7.0

Figure 4 .

I

6.0

I

I

I

5.0

4.0

30

1

2.0

1

1.o

Proton NMR spectrum of amiloride hydrochloride.

I

0

AMILORIDE HYDROCHLORIDE

4.2.2

$3

11

NMR Spectrum

The CI3 NMR spectrum shown in Figure 5 was obtained on a Varian Associates CFT-20 spectrometer using a 1.0 M amiloride hydroytjloride solution in d6-DMS0 (17). The C spectral assignments are listed in Table 111. Table I11 Cl3 NMR Spectral Assignments for Amiloride Hydrochloride

0

Assignments

109.3 119.9 154.2 155.3 155.9 165.3

c2 ‘6 c3 C9 c5 c7

0 CI

II

NH2 I 9

HCI 2H20 H2N

NH2

Both the proton and Cl3 NMR spectra are consistent with the amiloride hydrochloride structure.

c9

W

6-DMSO

(CH 3)4Si

I

200

F i g u r e 5.

I

150

I

100

I

50

CI3 NMR spectrum of a m i l o r i d e h y d r o c h l o r i d e .

1

0

AMILORIDE HYDROCHLORIDE

4.3

13

Ultraviolet Spectrum The ultraviolet absorption spectrum of amiloride hydrochloride in 0.01 N aqueous hydrochloric acid is characterized by maxima at approximately 212 mm, 285 nm and 362 MI. The absorbance in absorban?$ units of a 1% drug solution in a 1 cm cell (A1 ) is 642, 555 and 617 for the wavelenggs of 212 nm, 285 nm and 362 nm, respectively. The W spectrum shown in Figure 6 was obtained using a Cary Model 18 ultraviolet spectrophotomter (18).

4.4

Mass Spectrum Figure 7 is a depiction of the low resolution mass spectrum of amiloride hydrochloride obtained using a LKB nrodel 9000 mass spectrometer in the electron impact mode with an ionization energy of 70 eV and a probe temperature sufficiently high to produce sanple vaporization (19). The spectrum is dominated by the molecular ions of the free base (m/e = 229, 231). Masses of m/e = 43, 187, 189, 171, 173 and 212, 214 characterize the guanidino substituent. Loss of the entire guanidino group leads to m/e = 144, 146 and m/e = 86. The remaining less abundant ions are most likely due to losses of CH from the pyrazine ring, although the loss o CO cannot be totally excluded on the basis of low resolution spectra. Figure 8 schematically presents the fragmentation of amiloride hydrochloride.

i?

4 - 5 Optical Rotation

Amiloride hydrochloride is not optically active.

14

DAVID J. MAZZO

8 c 0

e

s

n

a

I

I

I

I

200

250

300

350

L

Wavelength (nm)

Figure 6.

U l t r a v i o l e t spectrum of a s o l u t i o n of a m i l o r i d e h y d r o c h l o r i d e i n 0.1N HC1. 229

loo]

43

40L L

60

144

I

86

116

20

37

Y’

171

143

101

53

09

0

40

60

Figure 7.

80

100

120

140

160

180

200

220

Low r e s o l u t i o n mass spectrum of a m i l o r i d e h y d r o c h l o r i d e t a k e n i n t h e e l e c t r o n impact mode.

240

X

z

E

I Q

a

o=y

R1

z

I

R I t u

I -0

\ /

z I

X

0

z “ 0;;

+

U

* E

Q

DAVID J. MAZZO

16 4.6

Thermoanalytical Behavior 4.6.1

Melting Point The melting point (with decomposition) of anhydrous amiloride hydrochloride is 293.5OC. The dihydrate melts with decomposition at approximately 288OC (20).

4.6.2

Differential Thermal Analysis Behavior ( 1 7 ) The differential thermal analysis (DTA) curve of amiloride hydrochloride is characterized by a water loss endotherm with a peak teprature of ca. 136OC. An endotherm-exotherm combination is observed at ca. 300OC. Actual peak temperatures are 298OC and 304OC for the endotherm and exothem respectively. This thermal event corresponds to a melting plus deconposition

4.6.3

Thermogravimetric Analysis Behavior (17) Thermogravimetric analysis of amiloride hydrochloride indicates no weight loss until 90OC. From 90°C to 17OoC, there is an approximately 12% weight loss due to dehydration of the dihydrate. No further weight loss occurs until sublimation/deconposition begins at 280OC.

-

-

-

-

4.7

Solubility The solubility of amiloride hydrochloride in a variety of solvents at room temperature (- 25OC) is presented in Table IV ( 1 8 ) . Note that these solubilities are stated in terms of the current U.S.P. definitions (21).

AMILORIDE HYDROCHLORIDE

17

Table IV Solubility of Ami1ori.de Hydrochloride at Room Temperature

Solubi1ity

Solvent

Practically insoluble Practically insoluble Practically insoluble Freely soluble Very slightly soluble Practically insoluble Slightly soluble Sparingly soluble Slightly soluble

Acetone Chloroform Diethylether Dimethylsulfoxide Ethanol Ethylacetate Isopropanol Methanol Water

The solubility of amiloride hydrochloride in water is typical of an organic base with limited aqueous solubility and increases with a decrease in pH (Table V) ( 1 8 ) . Table V Aqueous Solubility of Amiloride Hydrochloride As a Function of pH

PH

Solubility (mg/mL)

4.8 7.6 9.4

10.0 4.8

5.2

5.1 0.5 0.3

Crystal Properties Amiloride hydrochloride dihydrate exists as a crystalline powder. Variations encountered in the X-ray powder diffraction pattern suggest the existence of at least two polymorphic forms ( 2 2 ) . Polymrphism of amiloride hydrochloride has not been detected by other physical and/or chemical measurement techniques. Figures 9 and 10 show the X-ray powder diffraction patterns for the two

DAVID J. MAZZO

18 XI02 5.00 4.50 4.00 3.50 3.00 2.50 2.00 1.50 1 .OO

0.50

0.0

5.0

Figure 9.

10.6

15.0

20.0

25.0

30.0

35.0

40.0

X-ray powder d i f f r a c t i o n p a t t e r n of polymorph A of a m i l o r i d e h y d r o c h l o r i d e .

XI02 3.50 3.00 2.50 2.00 1.50

1 .OO 0.50

0.0

5.0

F i g u r e 10.

10.0

15.0

20.0

25.0

30.0

35.0

40.0

X-ray powder d i f f r a c t i o n p a t t e r n of polymorph B of a m i l o r i d e h y d r o c h l o r i d e .

AMILORIDE HYDROCHLORIDE

19

polymorphs (A and B) seen to date. The patterns were obtained using a Phillips Electronics model APD3720 powder diffractometer using copper Ka radiation. Tables VI and VII list the interplanar distances and the relative intensities of the major lines in the X-ray powder diffraction patterns of polymorphs A and B, respectively. Table VI X-Ray Powder Diffraction of Amiloride Hydrochloride Polymorph A Peak Angle (28)

I/Imx ( % )

7.6725 8.9150 10.2425 12.8400 15.3800 15.6100 17.1975 17.8950 19.3425 20* 0200 20.5925 23.1600 24.9000 25.6375 26.0275 26.4375 27.0325 28.0650 29.1425 29.8300 30.4700 31.0600 32.4275 33.3500 34.8350 35.4775 36.4200 37.9675

29.03 22.55 15.06 2.14 30.02 14.71 16.89 7.76 4.41 4.22 6.08 20.85 5.86 15.42 23.43 23.87 5.86 100.0

7.51 14.02 11.11 10.22 9.08 5.64 20.44 12.04 4.61 5.01

DAVID J. MAZZO

20

Table V I I

X-Ray Pawder Diffraction of Ami lor ide Hydrochloride P o l p r p h B

Peak Angle (28)

i

6.7350 7.5025 8 -8375 9.4075 10.2150 12.5850 13.6050 15.0775 15.3400 16.1350 17.1500 18.6225 18.9125 19.7450 20.4850 22 * 1000 22.7050 23.1300 24.2150 25.4975 26.2200 26.5000 27.1625 28.0425 28.4525 29.7150 30.4400 31.0225 31.7100 32.4225 33.2050 34.5275 35.2825 36.2325 36.4600 37.7200 38.7850

I/Imax ( % )

6.49 31.95 14.36 100.00 7.13 5.57 5.57 59.32 14.83 73.47 10.43 11.67 17.84 25.31 5.87 7.47 29.20 13.43 14.83 23.47 63.21 65.20 14.36 44.17 42.53 40.14 14.36 12.10 7.13 34.09 19.45 37.81 12.98 7.47 3.95 6.17 10.84

AMILORIDE HYDROCHLORIDE

4.9

21

Hygroscopicity The monohydrochloride salt of amiloride forms a stable dihydrate and amiloride hydrochloride, unless stated otherwise, is supplied as such. Amiloride monohydrochloride dihydrate may be converted to the anhydrous form of the sa t by drying at 100°C at pressures 6.6 x lo-’ atm for 3 hours (23). No other stable hydrates of amiloride hydrochloride have been reported (24).

4.10 Dissociation Constant The dissociation constant of amiloride hydrochloride as derived from aqueous titration (18) indicates that amiloride is a moderately strong organic base with a pKa of approximately 8.7 at 25OC (amidino nitrogen).

The pKa of amiloride hydrochloride and, in fact, m n y pyrazinylguanidine derivatives has also &en determined using gas-phase proton affinities, enthalpies of solution and semi-empirical calculations (25, 26). These theoretically derived pKa values agree well with the experimentally determined value of pKa = 8.7. 5.

Methods 5.1

Of

Analysis

Identification Tests 5.1.1

Ultraviolet Spectrophotometry Ultraviolet spectrophotometry is used to identify amiloride hydrochloride. A solution in 0.1 N HC1 scanned from 200 nm to 400 nm qualitatively exhibits the same absorbance characteristics at identical wavelengths as does a similarly prepared and concomitantly measured solution of an amiloride hydrochloride standard. Quantitatively, equimlar sample and standard solutions will exhibit ahorbances at 360 nm (Amx) which differ by no more than 3%.

-

DAVID J. MAZZO

22

5.1.2

Infrared Spectroscopy Infrared spectroscopy may also be used to identify amiloride hydrochloride. The infrared absorption spectrum prepared as a potassium bromide disk or a mineral oil dispersion compares qualitatively (with maxima only at the same frequencies) to the spectrum of a similarly prepared amiloride hydrochloride standard.

5.1.3

Elemental Analysis Elemental analysis has been employed to identify amiloride hydrochloride. The results of a weight percent determination of carbon, nitrogen, hydrogen and chloride are compared to the respective theoretical values of 28.85%, 32.45%, 4.34% and 23.47%. Amiloride hydrochloride will respond positively to the test for chloride described in the United States Pharmacopeia (27).

5.2

Spectrophotometric Analysis 5.2.1

Direct Ultraviolet Spectrophotometry Amiloride hydrochloride exhibits a W absorption band near 360 nm attributed to the substituted pyrazine ring system. This absorption is the basis for the quantitative determination of the drug. Assay of the conpound is based on a conparison of the net absorbance at 360 nm of a sample in 0.1 N HC1 with a standard in 0.1 N HC1 of known concentration. The net absorbance is calculated by subtracting the drug free matrix contribution to absorbance at the wavelength of determination from the absorbance of the drug solution at the same wavelength.

AMILORIDE HYDROCHLORIDE

5.2.2

23

Ultraviolet Spectrophotometry Via Flow Injection Analysis Ultraviolet absorbance at 360 nm is also used as the detection mode for Amiloride hydrochloride determinations by flow injection analysis (FIA) (28). In the case of FIA assay, amiloride hydrochloride sample and standard solutions are periodically injected into a flowing stream. The resulting changes in W absorbance at 360 nm of the stream are measured relative to the drug free stream. Calculation of the concentration of amiloride is made in an identical fashion as direct W spectrophotometry.

5.2.3

Liquid-Liquid Extraction with Ultraviolet Spectrophotometry or Spectrofluorimetry Amiloride hydrochloride may be determined in the presence of its degradation products by a liquid-liquid extraction technique followed by quantitation by ultraviolet spectrophotometry or spectrofluorimetry (18). An alkaline aqueous solution of amiloride hydrochloride is extracted with tributyl phosphate. Amiloride is partitioned into the organic layer while its degradates remain in the aqueous phase. Amiloride is then determined by ultraviolet spectrophotometry at 360 nm or fluorimetry with an excitation wavelength of ca. 360 nm and an emission wavelength of ca. 420 nm. Sensitivity is 0.2 ppm.

-

5.3

Chromatographic Analysis 5.3.1

Thin Layer Chromatography Normal-phase thin layer chromatography on silica gel using one of two developing solvent systems has been employed for amiloride hydrochloride. In the first system, a developing solvent of 10% n-propanol in chloroform is used to develop a spot resulting from 5 pL of a 1% aqueous solution of

DAVID J. MAZZO

24

-

the drug. Rf for the analyte in this 0). system is approximately zero (R Detection is by W absorbance at 254 nm or 360 nm (most sensitive). A sensitivity of 0.01% has been reported. In the second TLC system, 4 parts 3 N aqueous a m n i u m hydroxide are mixed with 30 parts of tetrahydrofuran to form the developing solvent (23). A 1 pL spot of a 0.1% aqueous solution of the drug is developed and detected by W absorbance at 254 nm or 360 nm. Rf for the analyte is approximately 0.7. Sensitivity under these conditions is in the range of 0.1% to 0.5% of the analyte concentration. 5.3.2

High Performance Liquid Chromatography Reversed-phase HPLC is routinely used to determine amiloride hydrochloride (29). The method employs an ES Industries C-2(300 mm x 4.6 mm i.d., 10 pm particle size) HPLC column operated at ambient room temperature (- 25OC). A mobile phase consisting of 85% aqueous 0.01M sodium hexane sulfonate (pH = 3.0) in acetonitrile is used to elute amiloride. Flow rate is 2.0 mL/min and detection is by W absorbance at 280 nm. Under these conditions, amiloride elutes in less than 8 minutes (k' 2). Typically 20 pL injections of an approximately 200 pg/mL drug solution are made.

-

5.4

Non-Aqueous Titration Amiloride hydrochloride can be determined by nonaqueous titration with perchloric acid (23). The assay involves dissolution of an appropriate amount (- 450 mg) of amiloride hydrochloride in 100 mL of glacial acetic acid to which is then added 10 mL of mercuric acetate, 15 mL of dioxane and an appropriate amount of crystal violet indicator. The well mixed solution is titrated with 0.1N perchloric acid to a blue endpoint. Assay results must be corrected for a blank titration. Each equivalent of perchloric acid is equivalent to 26.61 mg of amiloride hydrochloride.

AMILORIDE HYDROCHLORIDE 6.

Stability 6.1

-

25

Degradation

Solid State Stability Amiloride hydrochloride is a stable compound at room temperature in the solid state, i.e. no significant degradation has been observed in samples exposed to no excessive humidity for seven years. Some darkening of amiloride hydrochloride powder has been observed upon exposure to intense ultraviolet light (24 times the strength of direct sunlight) but no degradation products were detected by thin layer chromatography, even after 7 days of exposure (18). Elevated temperatures as high as 100°C for 1 week in the absence of excessive humidity do not produce degradation. Only prolonged (> 1 week) exposure to very high humidity (> 90% RH) and temperatures greater than 6OoC will produce significant degradation in the solid state.

6.2 Solution Stability

Aqueous solutions of amiloride hydrochloride are stable at usual ambient temperatures. A study of the stability of amiloride hydrochloride in aqueous solution at elevated temperatures at various pH levels resulted in the identification of three degradation products (Figure 11). The relative amounts of the three degradates vary with pH. At pH < 1, compound I dominates, at pH -5 both I1 and I11 are present and in alkaline solution (pH > 1 3 ) , compound 111 predominates. All three potential degradation products are acidic in comparison to the parent compound. The degradation of amiloride hydrochloride in alkaline solution (pH > 13) follows first-order degradation kinetics (18). The chemical transformation of arniloride hydrochloride in solution formulation with other pharmaceutical agents and excipients has been shown to be more conplex than that observed in simple aqueous solution (30).

DAVID J. MAZZO

26

1 ’ 0

0

,NH2

II

NH2 2

It

L“‘x;I&c’xlI 0

0

c-o-

HeN

OH-

H2N

F i g u r e 11.

II C-OH NH2

II

pH> I3

NH2

m

S o l u t i o n d e g r a d a t i o n p r o d u c t s of a m i l o ri d e h y d r o c h l o r i d e

.

AMILORIDE HYDROCHLORIDE

7.

27

Biopharmaceutics and Metabolism 7.1

Absorption and Bioavailability It has been shown, using radiolabelled (CI4) amiloride hydrochloride, that approximately 50% of an oral dose administered to man is absorbed from the gastrointestinal tract ( 3 1 , 3 2 ) . The remainder of the dose is unabsorbed and can be found in the feces (31-34). Human bioavailability studies comparing 5 mg tablets of amiloride hydrochloride to an aqueous reference solution showed that the pharmaceutical dosage form was fully bioequivalent ( 3 3 ) . Absorption of amiloride hydrochloride is reduced when administered with food ( 3 3 , 2 5 ) .

7.2

Metabolism Amiloride hydrochloride which has been absorbed in man is excreted without metabolism in the urine ( 3 1 , 3 3 , 3 4 , 3 6 ) . No metabolic products have been detected and/or identified. Since the drug is not metabolized it probably can be administered to patients with hepatic dysfunction, providing that renal excretory function is normal ( 3 4 ) . Amiloride hydrochloride is cleared from the body by tubular transport (5,7,31-34,36) and, in combination with hydrochlorothiazide, does not alter the normal renal potassium excretion or the rate of urinary variable excretion ( 3 7 ) .

7.3

Pharmacokinetics Amiloride hydrochloride is rapidly absorbed from the gastrointestinal tract (31,32,38-40). Onset of the physiological effect of the drug in man is usually noted within two hours ( 3 4 , 3 6 ) with peak serum levels being achieved in between 3 and 4 hours (31,321. Typical pharmacokinetic half-life values (T/2) range between 6 and 10 hours Considerable prolongation of the half( 31,32,36!. life has been noted in patients with chronic renal failure ( 3 3 , 3 4 , 3 6 , 3 9 ) . Effects of the drug generally subside within 24 hours resulting in urinary levels of less than 0.5% of the administered dose ( 3 1 , 3 6 ) . Urinary amiloride concentrations range from 4 pg/mL to 30 pg/mL, during diuresis ( 3 1 , 3 2 ) with peak renal clearance of

28

DAVID J. MAZZO

amiloride ranging from 400 mL/min to 600 mL/min (33). A dose-response relationship has been observed in man (33,34,36). Progressively increasing effects of single doses have been noted from 1 mg to 40 mg with a plateau being reached above 40 mg

-

8.

Determination in Biological Matrices Several techniques have been used to determine amiloride in biological fluids. Among them, liquid scintillation counting for radiolabelled compound, liquid-liquid extraction with measurement of ultraviolet absorbance or fluorescence, thin-layer chromatography with W or fluorescence detection and high performance liquid chromatography (HPLC)with fluorescence detection have been the most widely used. Liquid scintillation counting of C14 labelled ami1ori.de in urine, serum, plasma, tissue and feces has been performed routinely (31,32,41) usually with an internal standard used for quantitation of the analyte. Liquid-liquid extraction of amiloride from serum, plasma and/or urine has been performed with a 7% saturated aqueous sodium carbonate solution in ethyl acetate (1 part drug containing fluid to 27 parts extracting solution). The analyte is back-extracted from the ethyl acetate phase into 0.1 N HC1 and then determined by spectrofluorimetry (Aexcite = 365 nm, 'emit. = 420 nm) (40). In matrices containing minimal interferents, ultraviolet absorbance at 360 nm may also be used. Normal-phase thin-layer chromatography (TLC)on silica has also been employed for the determination of amiloride from biological fluids (31,41,42). Generally, an extraction technique similar to that described above is employed for drug isolation prior to TLC. A common developing solvent consists of butanol-acetic acid-water (100:27:73) (41). Other extraction techniques and developing solvents have been reported (42). Detection of TLC spots is accomplished by fluorescence with long wavelength W excitation and emission at ca. 425 nm.

AMILORIDE HYDROCHLORIDE

29

Finally, reversed-phase high performance liquid chromatography with fluorescence detection has become popular for the determination of amiloride in biological fluids. Two HPLC systems have been reported (43,44) with large numbers of samples being processed by one of these systems ( 4 4 ) . A C18 HPLC column (300 mm x 3.9 mm i-d., 10 pm particle size) is employed with a mobile phase of 60:40 [0.1M NaP04(pH = 4.0)l:CH OH flawing at 1.0 mL/min. Column temperature is 35OC and detection is by 'fluorescence with an excitation wavelength of 368 nm and an emission wavelength of 417 nm. Under these conditions, amiloride elutes in 6 minutes. Quantitation is performed using an internal standard, 3,5-diamino-N-(aminoiminomethyl)-6-fluoropyrazinecarboxamide.

-

9. Determination in Pharmaceuticals 9.1 Dissolution Testing The determination of amiloride hydrochloride in samples resulting from dissolution testing of tablets containing the drug can be accomplished by ultraviolet absorbance at 360 nm. Either direct W spectrophotometry or W spectrophotometry visa-vis flow injection analysis may be used (45). 9.2

Assay, Dosage Uniformity and Stability Testing High performance liquid chromatography is the technique of choice for the determination of amiloride hydrochloride in tablets for release and/or stability purposes although ultraviolet spectrophotometry may be used for the determination of dosage uniformity. Reversed-phase HPLC with W detection at 360 nm has been performed using a c18 HPLC colum (300 mm x 3.9 mn i.d., 10 pm particle size) with a mobile phase of 25% methanol in 0.05 M phosphate buffer (pH = 3.0). Flow rate was 1.0 mL/min (30). Recently, a fastHPLC method was developed (46) which employs a c18 mini-colurm (50 mm x 4.6 mm i.d., 5 pm particle size). Using a mobile phase of 20% methanol in 0.02M phosphate buffer pH = 2 at a

DAVID J. MAZZO

30

flow rate of 4.0 mL/min amiloride is eluted in approximately 0.5 minutes (k' 1). Detection is again by W absorbance at 360 nm. This method, because of its speed, has also been used routinely for dosage uniformity samples.

-

Acknowledgement Special thanks go to Mrs. Elizabeth Moyer for typing the manuscript. Thanks are also in order to Mr. James Ryan and Dr. James McCauley for obtaining the IR spectra and X-

ray diffraction patterns, respectively. The assistance of Ms. Florence Berg for literature searching and Mr. William Shearin for background information retrieval is also acknowledged. Finally, sincere gratitude is expressed to Dr. Gerald Brenner for his review of the manuscript.

AMILORIDE HYDROCHLORIDE

31

10. References

Physician's Desk Reference, 36th edition, Charles E. Baker, Jr., Publisher, Medical Economics Company, Inc., Oradell, NJ, U.S.A., 1982, w. 1268. Cragoe, E. J., Jr., Department of Medicinal Chemistry, Merck, Sharp and Dohme Research Laboratories, West Point, PA, U.S.A., internal communication. Glitzer, M.S.; Steelman, S. L.; Nature (London) 212 (1966), 191. Cragoe, E. J., Jr.,; Woltersdorf, O.W., Jr.; Bicking, J. B.; Kwong, S. F.; Jones, J. H.; J. Med. Chem. 10 (1967), 66-75. Amiloride and Epithelial Sodium Transport, A. W. Cuthbert, G. M. Fanelli, Jr. and A. Scriabine, Editors, Urban and Schwarzenberg, Inc., Baltimore, MD, U.S.A., 1979. International Symposium: Diuresis, Kaliuresis, and Hypertension, J. E. Salvioli, Chairman, Biomedical Information Corporation Publications, Inc., New York, NY, U.S.A., 1981. Hyams, D. E., in Arrhythmias and Myocardial Infarction: The Role of Potassium, C. W o o d and W. Somerville, Editors, Academic Press, London, England, U.K., 1981, pp. 65-73. Cragoe, E. J., Jr., Belgium Patent 639,386 (to Merck & Corrpany, Rahway, NJ, U.S.A., 1964). Cragoe, E. J., Jr., United States of America Patent 3,313,813 (to Merck & Campany, Rahway, NJ, U.S.A.), 1967. (10) Cragoe, E. J., Jr., Canada Patents 765823 and 7644963 (to Merck & Co., Rahway, W , U.S.A.), 1967. (11) Cragoe, E. J., Jr., England Patent 1066855 (to Merck & Co., Rahway, W, U.S.A.), 1967.

32

DAVID J. MAZZO

(12) Cragoe, E. J., Jr., France Patent 1563612 (to Merck & Co., Rahway, NJ, U.S.A.), 1969. (13) Cragoe, E. J., Jr., Federal Republic of Germany Patents 1470053.9 and 1795438.0 (to Merck & Co., Rahway, NJ, U.S.A.), 1963. (14) Cragoe, E. J., Jr., Japan Patents 498389, 502839 and 508194 (to Merck & Co., Rahway, NJ, U.S.A. ), 1963. (15) Ryan, J. A., Department of Pharmaceutical Research and Development, Merck, Sharp and Dohme Research Laboratories, West Point, PA, U.S.A., personal communication. (16) Smith, R. L.; COchran, D. W.; Gund, P.; Cragoe, E. J., Jr.; J. Am. Chem. SOC. 101 (1979), 191.

(17) Hitchings, W. S. and O'Brien, M. J., Department of Control and Manufacturing Data, Merck, Sharp and D0hn-e Research Laboratories, West Point, PA, U.S.A., personal communication. (18) Rogers, D. H e , Department of Pharmaceutical Research and Development, Merck, Sharp and Dohme Research Laboratories, West Point, PA, U.S.A., internal communication. (19) Smith, J. L., Department of Analytical Natural Product Chemistry, Merck, Sharp and Dohme Research Laboratories, Rahway, NJ, U.S.A., personal communication. (20) The Merck Index, 10th Edition, Martha Windholz, Editor, Merck and Company, Rahway, NJ, U.S.A, 1983, pg. 403. (21) The United State Pharmacopeia, 21st Revision, Mack Publishing Company, Easton, PA, U.S.A., 1984, pg. 7.

(22) McCauley, J. A., Department of Analytical Research, Merck, Sharp and Dohme Research Laboratories, Rahway, NJ, U.S.A., personal communication.

AMILORIDE HYDROCHLORIDE

33

The United States Pharmacopeia, 21st Revision, Mack Publishing Company, Easton, PA, U.S.A., 1984, pg. 35. Giuliano, J., Department of Chemical Data, Merck Sharp and Dohme Research Laboratories, Rahway, NJ, U.S.A., personal comnunication. Aue, D. H.; Webb, H. M.; Bowers, M. T.; Liotta, C. L.; Alexander, C. J.; Hopkins, H. P., Jr.; J. Am. Chm. SOC., 98(1976), 854-856. Bock, M. G.; Schlegel, H. B.; Smith, G. Org. Chem. 46(9) (1981), 1925-1927.

M.;

J.

The United States Pharmacopeia, 21st Revision, Mack Publishing Company, Easton, PA, U.S.A., 1984, . pg. 1185. Bigley, F. P., Department of Pharmaceutical Research and Development, Merck, Sharp and Dohme Research Laboratories, West Point, PA, U.S.A., personal camrmnication. Martin, M., Department of Chemical Manufacturing, Quality Control, Merck, Sharp and D o h , Ltd., Ballydine, Kilsheelan, Clonmel, County Tipperary, Ireland, personal communication. Roman, R., Department of Pharmaceutical Research and Development, Merck, Sharp and Dohme Research Laboratories, West Point, PA, U.S.A., internal communication. Weiss, P.; Hersey, R. M.; Dujovne, C. A.; Bianchine, J. R.; Clin. Pharmacol. Ther. lO(3) (1969), 401-406. Smith, A. J.; Smith, R. N.; Brit. J. Pharmacol. 48(4) (1973), 646-649. Davies, R. O., in International SyIposium: Diuresis, Kaliuresis and Hypertension, J. E. Salvioli, Chairmn, Biomedical Information Corporation Publications, Inc., New York, NY, U.S.A., 1981, pp. 43-69.

34

DAVID J. MAZZO

(34) Vidt, D. G.; Pharmacotherapy l(3) (1981), 179-187. (35) Hyams, D. E., in Arrhythmias and Myocardial Infarction: The Role of Potassium, C. Wood and W. Somerville, Editors, Academic Press, london, England, U.K., 1981, pg. 68.

(36) Laragh, J. H.; Curr. Ther. Res. 32(2) (1982), 173188. (37) Leary, W. P.; Reyes, A. J.; Curr. Ther. Res. 32(3) ( 1982), 432-438. (38) Baba, W. I.,; Lant, A. F.; Smith, A. J.; Townshend, M. M.; Wilson, G. M.; Clin. Pharmacol. Ther. 9L (1968), 318-327. (39) George, C. F.; Brit. J. Pharmacol 9 (1980), 94-95. (40) Lant, A. F.; Smith. A. J.; Wilson, G. M.; Clin. Pharmacol. Ther., 10 (1969), 50-63. (41) Baer, J. E.; Jones, C. B.; Spitzer, S. A.; RUSSO, H. F.; J. Pharmacol. Exp. Ther. 157(2) (1967), 472485. (42) Reuter, K.; Knauf, H.; Mutschler, E.; J. Chromatogr. 233(1982), 432-436. (43) Yip, M. S.; COates, P. E.; Thressen, J. J.; J. Chromatogr. 307 (1984), 343-350. (44) Vincek, W. C.; Hessey, G. A., 111; Constanzer, M. L.; Bayne M. W.; Pharm. Res. 3 (1985), 143-145. (45) Konieczny, J. M.; Department of Pharmaceutical Research and Development, Merck, Sharp and Dohme Research Laboratories, West Point, PA, U.S.A., personal cmnication. (46) Mazzo, D. J.; Department of Pharmaceutical Research and Development, Merck Sharp and Dohme Research Laboratories, West Point, PA, U.S.A. Chemical Abstracts was searched for amiloride hydrochloride citations from 1966 to 1985.

AMINOGLUTETHIMIDE

HASSAN Y ABOUL-ENE I N

1

DESCRIPTION

1.1

Introduction

1.2

Nomencl a t u r e

1.3

1.2.1

Chemical Names

1.2.2

Generic Names

1.2.3

Trade Names

Formulae 1.3.1

Empirical

1.3.2

Structural

1.3.3

Chemical A b s t r a c t R e g i s t r y Number

1.4

M o l e c u l a r Weight

1.5

Elemental Composition

1.6

Appearance, Color, Odor and Taste

1.7

p~ (0.1% S o l u t i o n )

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 15

35

Copyright 0 I986 by the American Pharmaceutical Association All rights of reproduction in any form reserved.

HASSAN Y. ABOUL-ENEIN

36

2

PHYSICAL PROPERTIES 2.1

M e l t i n g Range

2.2

Solubility

2.3

D i p o l e Moment

2.4

O p t i c a l A c t i v i t y and Absolute C o n f i g u r a t i o n

2.5

Spectral Properties 2.5.1

U1t r a v i o l e t Spectrum

2.5.2

I n f r a r e d Spectrum

2.5.3

Nuclear Magnetic Resonance 2.5.3.1 2.5.3.2

2.5.4.

PMR 1% NMR

Mass Spectrum

3

SYNTHESIS

4

METABOLISM AND PHARMACOKINETICS

5

METHODS OF ANALYSIS

5.1

T i t r i m e t r i c Non-Aqueous A n a l y s i s

5.2

Spectrophotometic A n a l y s i s

5.3

Chromatographic A n a l y s i s 5.3.1

Paper Chromatography

5.3.2

Thi n-Layer Chromatography (TLC)

5.3.3

Gas-Liquid Chromatography (GLC)

5.3.4

High-Performance L i q u i d

6

REFERENCES

7

ACKNOWLEDGEMENT

(HPLC)

37

AMINOGLUTETHIMIDE

ANALYTICAL PROFILE 1.

Description 1.1

Introduction: Aminoglutethimide was i n i t i a l l y developed as an a n t i c o n v u l s a n t i n t h e 1950's b u t was l a t e r withdrawn from c l i n i c a l use a f t e r r e p o r t s of adrenal i n s u f f i c i e n c y (1).

It was subsequently shown t o suppress

adrenal s t e r o i d synthesis by i n h i b i t i n g t h e demolase enzyme system which i s

sion o f cholesterol t o

responsible f o r t h e conver-

(2).

a5 -pregnenolone

It

has been used i n t h e treatment o f a d r e n o c o r t i c a l tumours and Cushing's syndrome ( 3 ) . Aminoglutethimide i n h i b i t s s t e r o i d aromatase enzyme i n v o l v e d i n t h e b i o s y n t h e s i s o f estrogens ( 4 ) , e.g. conversion o f androstenedione t o esterone. c u r r e n t l y used

It i s

as an e f f e c t i v e agent f o r t h e t r e a t -

ment o f advanced b r e a s t cancer i n post-menopausal women (5,6,7).

1.2

Nomenclature:

1.2.1

Chemical Names : CY,

2

3

-

-a(p-Aminophenyl) - 2 (4-Aminopheny1)- 3 (p-Aminophenyl)

p i p e r i d i nedi one

Ethylglutarimide Ethy g l u t a r i m i d e Ethy

-

2,6

-

HASSAN Y. ABOUL-ENEIN

38

3

- Ethyl - 3 -

( p . aminophenyl)

-

2,6

-

d i oxopi p e r i d i ne 1.2.2

Generic Names : Aminoglutethimide, Ciba 16038

1.2.3

Trade Names:

E l ipten, Cytadren, Orimeten 1.3

Formula 1.3.1

Empirical : C13H16N202

1.3.2

Structural : CH2CH 3

H

1.3.3

Chemical Abstract R e g i s t r y Number:

1.4

Molecular Weight:

1.5

Elemental Composition:

125-84-8

232.27 C 67.22%; H 6.94%; N 12.06%;

0 13.78% 1.6

Appearance, Color, Odor and Taste:

a white t o

creamy w h i t e c r y s t a l 1i n e powder, odor1 ess and possessing a b i t t e r t a s t e (8,9,10).

1.7

pH (0.1% s o l u t i o n ) : I n t r o d u c e 200.0 mg

6.2-7.3

2 0.2

mg of aminoglutethimide

i n t o a 200 m l v o l u m e t r i c f l a s k , d i s s o l v e i n 10.0 m l o f methanol and add approx. 180 m l o f C o p f r e e water

AMINOGLUTETHIMIDE a t 50'C.

39

A f t e r shaking w e l l , cool down t o room tem

p e r a t u r e and f i l l up t o t h e mark w i t h C02-free water a t room temperature, then determine t h e pH potent i o m e t r i c a l l y (8).

2. P h y s i c a l P r o p e r t i e s 2.1

M e l t i n g Range: The f o l l o w i n g m e l t i n g range has been r e p o r t e d f o r aminoglutethimide: M.p,

'C

Reference

151

9

149-150 ( f r o m MeOH o r EtOAc)

10

152-154

11

The h y d r o c h l o r i d e s a l t [CAS 31075-85-11 has a m e l t i n g range of 223-225'

2.2

(9).

Solubility: V i r t u a l l y i n s o l u b l e i n water, f r e e l y s o l u b l e i n most o r g a n i c s o l v e n t s e.g. and chloroform.

methanol, methylene c h l o r i d e

Poorly s o l u b l e i n e t h y l a c e t a t e , 0.1N

HC1 and absolute ethanol, r e a d i l y s o l u b l e i n acetone and 100% a c e t i c a c i d ( 8 , 9 ) .

2.3

D i p o l e Moment: Lee and Kuml e r ( 12) determi ned t h e d i p o l e moment and s t r u c t u r e o f t h e imide group i n f i v e and s i x membered c y c l i c imides.

Aminoglutethimide has a d i p o l e moment

o f 2.83 )I i n D u n i t determined a t 30'

i n dioxane.

HASSAN Y . ABOUL-ENEIN

40

2.4

O p t i c a l A c t i v i t y and Absolute C o n f i g u r a t i o n s : e t a1 (13) resolved t h e o p t i c a l enantiomers of Finch -v i a r e c r y s t a l l i z a t i o n o f the t a r aminoglutethimide t a r a t e s a l t from methanol.

The d e x t r o r o t a t o r y a n t i -

pode (+) aminoglutethimide has a m.p. [,ID25

114-115' and

= t 163.1 (MeOH).

The 1evorotatorY antipode ( - ) aminoglutethimide( 11) has a m.p.

114-115'

and [a]D25 = -163.6'

The absolute c o n f i g u r a t i o n o f (t)

-

(MeOH).

isomer was d e t e r -

mined t o have R-configuration w h i l e ( - )

-

isomer has

S - c o n f i g u r a t i o n around t h e assymmetric carbon as shown i n Figure 1.

It i s o f i n t e r e s t t o mention

t h a t t h e (+)-isomer ( I ) had t h e most o f t h e s t e r o i d s y n t h e s i s i n h i b i t i o n a c t i v i t y (2-3 more p o t e n t than t h e racemate), w h i l e t h e ( - ) isomer had very l i t t l e a c t i v i t y a t dose l e v e l s 1 0 - f o l d h i g h e r . 2.5

Spectral P r o p e r t i e s 2.5.1

U l t r a v i o l e t Spectrum The u l t r a v i o l e t spectrum o f aminogl u t e t h i m i d e i n n e u t r a l methanol i s shown i n Fig. 2.

It

e x h i b i t s a maximum a t about 242 nm and a shoulder a t approximately 282 nm.

The maxima

a t 242 and 282 nm do n o t change o r s h i f t i n a c i d i c (0.1N H2SO4) o r b a s i c (0.1N NaOH) media as shown i n Figures 3 and 4.

The u l t r a v i o l e t

AMINOGLUTETHIMIDE

41

":

2

H

H

I

II (-) Aminoglutethlmlde

Fig. 1

-

(t) Amlnoglutethlmlde

The absolute c o n f i g u r a t i o n o f ( - ) hinogl u t e t h i m i d e

-

S and (t)

-R-

o ~ , l , , l , l , l , , , , , , , , , ~ 210 220

Fig. 2

-

240

260

280

300

320

340

360

380

400

U l t r a v i o l e t spectrum o f aminoglutethimide i n n e u t r a l methanol.

HASSAN Y. ABOUL-ENEIN

42 0.8

-

0.7

0.6

-

0.5

-

0.4

0 210 220 240

-

F i g 4.

260

280

300

320 340

360

380

400

U l t r a v i o l e t spectrum o f aminoglutethimide i n 0.1N NaOH.

0.8

-

0.7

-

0.6

0.5

-

0.4

-

0.3

0.1

0 210 220 240

Fig. 3

260

- Ultraviolet 0.1NH2S04.

280

300

320

340

360

380

0 400

spectrum o f aminoglutethimide i n

AMINOCLUTETHIMIDE

43

spectra are recorded on a V a r i a n AG UV-VIS

-

spectrophotometer

Model DMS-90. These data

are i n agreement w i t h p r e v i o u s l y pub1 ished i n f o r m a t i o n (14). 2.5.2

I n f r a r e d Spectrum The i n f r a r e d o f aminoglutethimide i n Nujol m u l l i s presented i n F i g u r e 5 and i s recorded on Perkin-Elmer spectrophotometer model 5808. The frequencies and t h e i r s t r u c t u r a l assignments are as f o l l o w s : Frequency (cm-1)

3470 3480

t

3 180 3080

Assignment

3 asym

NH2 s t r e t c h i n g v i b r a t i o n

3 sym-NH-imide

group

CH aromatic s t r e t c h

C=O imide carbonyl s t r e t c h 1690

o f glutarirnide r i n g C=C aromatic f o r phenyl

1520

skeletal vibrations

830 & 700

CH-out-of-plane bending vibration characteristic for p-substi t u t i o n

Other c h a r a c t e r i s t i c f i n g e r p r i n t bands are 1260, 1200 cm-1.

These data a r e i n agreement

w i t h p r e v i o u s l y reported r e p o r t s (8,14).

MCRONS 2.5

4.0

3.0

5.0

6.0

7.0

8.0

9.0 10

*

12

14 16 1820 25 30 4050

100

100

80-

60-

40-

20-

0 4Mx)

3;oo

I

3ooo

I

2500

2doo

1

lsbo

1800 W A -

Fig. 5

-

14bo

12bo

I

lo00

I

800

I

600

(an-')

I n f r a r e d spectrum o f aminoglutethimide i n Nujol mull.

400

)O I 200

AMINOGLUTETHIMIDE 2.5.3

45

Nuclear Magnetic Resonance Spectra 2.5.3.1

PMR Spectrum

The 60MHz PMR spectrum o f aminoglut e t h i m i d e i n DMSO-d6 i s shown i n F i g u r e 6.

The spectrum was recorded

on a Varian T60-A NMR spectrometer u s i n g TMS as t h e i n t e r n a l standard. The f o l l o w i n g s t r u c t u r a l assignments have been e l i c i t e d from F i g u r e 6:

Ass ig nment s

Chemical S h i f t (ppm) 0.77 t

CH3-CH2

1.80 q

CH3-CH2

2.27 m

CHz-CHz-CH o f t h e g l u t a r i m i d e

r i n g system Hz

aromatic H3 8 H5

6.95 d J=8.5 Hz

aromatic H2 8 H6

6.53 d J=8.5

AB system c h a r a c t e r i s t i c o f p heny 1 p -5 ubs t it ut ion 5.0

s

NH2 exchangeable w i t h D20 NH exchangeable w i t h D20

9.33 s s-singlet,

d=doublet,

m-multiplet,

q=quartet

t=triplet

Defay and D o r l e t (15) r e p o r t e d t h e assignments o f NMR s p e c t r a o f some glutarimide derivatives including

I ' " " ' ' " " ' ' l " " ' l ' " ' ~ " I " " ~ " ' ' I '

I

'

I

1

I

'

I

I

i I

. .O

Fig. 6

-

. . . .

I 70

!

i

1

. . . .

i .o

. . . .

I

I

I0

.

.

. . . . i

. . . I .

4 0

PMR spectrum o f aminoglutethimide in DMSO

i . . . . i 3 0

-

LO

. I . . .

i . . . , 1 0

!

4 '

d 6 using TMS as the internal standard.

47

AMINOGLUTETHIMIDE aminoglutethimide and i s i n agreement w i t h t h e assigned peaks. 2.5.3.2

13C-NMR Spectrum The 13C-NMR spectrum o f aminogl u t e t h i m i d e has been determined on a V a r i a n FT80 spectrometer a t ambient temperature. The sample was d i s s o l v e d i n DMSO-d6 i n a t u b e w i t h l O m m diameter. 5000 Hz, a c q u i s i t i o n t i m e :

Spectral width: 1.638

seconds, p u l s e w i d t h 6 y second and numb e r o f d a t a p o i n t s 16384.

The n o i s e

decoupled and t h e complete o f f - r e s o n a n c e s p e c t r a a r e shown i n F i g u r e s 7 and 8, respectively.

The s p e c t r a l assignments

a r e 1 is t e d be1 ow:

6

7

H

Chemical Shi f t

Assignment

( i n ppm r e l a t i v e t o TMS) 176.23 s

C=O a t C5

172.92 s

C=O a t C 1

147.46 s

c11

126.66 d

c9

c13

48

HASSAN Y. ABOUL-ENEIN

11 Fig. 7

-

Fig. 8

-

13C-NMR off-resonance spectrum o f aminogl utethimide I n DMSO-d6.

13C-MNR off-resonance spectrum o f aminoglutethimde i n DMs0-d~.

AMINOGLUTETHIMIDE

s=singlet, 2.5.4

49

126.11 s

c8

114.04 d

c10 & c12

49.27 s

c4

32.35 t

c3

29.17 t

C2

26.04 t

c6

8.86 q

c7

d=doublet,

t=triplet,

q=quartet

Mass Spectrum The mass spectrum o f a m i n o g l u t e t h i m i d e o b t a i n e d by chemical i o n i z a t i o n ( C I ) on a s o l i d probe u s i n g CH4 as i o n i z i n g gas i s shown i n F i g u r e 9.

The ( M

t

1 ) peak i s a t m/z 233

(100%) which corresponds t o i t s m o l e c u l a r weight, a small peak ( M a t m/z 261 (5%).

t

29) i s a l s o p r e s e n t

The mass spectrum o f ami-

nogl u t e t h i m i d e o b t a i n e d by he1 ium charge exchange (HCE) w i t h d i r e c t i n l e t probe i s shown i n F i g u r e 10. The s p e c t r a were recorded on a F i n n i g a n 3200 GC/MS connected t o an Incos 2300 d a t a system.

The spectrum ( F i g u r e 10) shows a m o l e c u l a r i o n peak (Mt) as m/z 232 and a base peak a t m/z

HASSAN Y. ABOUL-ENEIN

50

132.

The most prominent fragments a r e l i s t e d

i n Table 1 .

TABLE 1. Mass m/z

Most Prominent Fragments o f Aminoglutethimide Relative Intensity, %

232

31.2

203

45.2

175

34.4

160

14

147

11.4

Fragment

C13H16N2Ot2s Mt

ClOHllN20+

ClOH13i 7+

51

AMINOGLUTETHIMIDE 2 I

loo

707504 354

Sample: Aminogknhethimide conds.: cH4 CI sdid Probe

50

73 MI2

94. ,182, 100

Fig. 9

149. 150

-

205

.I

175187

26 1 2$o 'T3

1

219

200

.

.304,

300

250

,

,

3 I

Chemical i o n i z a t i o n (CI) mass spectrum o f aminoglutethimde u s i n g CH4 as i o n i z i n g gas. 1

100

1 16992 117

Sample: Aminogkrthethimide Conds.: Helium Charge Exchange

50 117

175

232

144 190

187 L

MI2

_"217

.

26 1 D

100

Fig. 10

-

Helium charge exchange (HCE) mass spectrum aminoglutethimide by d i r e c t i n l e t probe.

Of

HASSAN Y. ABOUL-ENEIN

52

132

100

117

37.6

115

30.5

+ C10H12

c9Hf7

+ + Other peaks appear a t m/z 131 and 130. Rucker and Bohn discussed t h e f r a g m e n t a t i o n process o f g l u t e t h i m i d e and aminoglutethimide u s i n g t h e deutrium the

3.

l a b e l e d analogs o f these drugs t o c o n f i r m

s t r u c t u r e o f t h e fragments (16).

Synthesis Aminoglutethimide i s synthesized as shown i n scheme 1 (17).

N i t r a t i o n of 2-phenyl b u t y r o n i t r i l e g i v e s t h e

p-nitro derivative.

Conjugate Michael a d d i t i o n o f t h e

carbani on of p-ni trophenyl b u t y r o n i t r i 1e t o methyl acry-

1a t e g i v e s methyl 4-cyano-4 ( p - n i t r o p h e n y l ) hexanoate. The

AMINOGLUTETHIMIDE

53

l a t t e r intermediate i s c y c l i z e d i n t h e presence o f a c e t i c a c i d and 90% s u l f u r i c a c i d [ s i m i l a r t o t h e s y n t h e s i s o f glutethimide ( 1 8 ) l t o give 2-ethyl-2(p-nitrophenyl) tarimide.

-

C a t a l y t i c r e d u c t i o n of t h e n i t r o group a f f o r d s

(t) ami nogl u t e t h i m i d e

4.

glu-

( 17).

Metabol ism A f t e r an o r a l dose o f 250 o r 500 mg o f aminoglutethimide i n man, t h e drug i s absorbed and excreted unchanged t o a c o n s i d e r a b l e e x t e n t i n t h e u r i n e a f t e r 48 hours (19), along w i t h t h e N-acetyl aminoglutethimide which i s cons i d e r e d t h e major m e t a b o l i t e (20).

It has been suggested

t h a t t h i s m e t a b o l i t e may c o n t r i b u t e t o t h e o v e r a l l pharmacological a c t i v i t y o f t h e parent drug (20).

However,

t h e N-acetyl m e t a b o l i t e has been shown t o be l e s s than h a l f as e f f e c t i v e i n reducing g l u c o c o r t i c o i d p r o d u c t i o n as t h e p a r e n t drug (21).

Also, i t lacked t h e a n t i f e r -

t i l i t y a c t i v i t y o f aminoglutethimide (22).

Sheets and

V i c k e r y ( 2 3 ) showed t h a t t h e a b i l i t y o f aminoglutethimide t o i n h i b i t c h o l e s t e r o l conversion t o pregnenolone was l o s t upon a c e t y l a t i o n o f arylamine n i t r o g e n . The authors e x p l a i n e d t h i s f a c t due t o t h e f a i l u r e o f Nacetylami nogl u t e t h i m i d e t o b i n d t o cytochrome P45Oscc. Aminogl u t e t h i m i d e i s among those drugs t h a t a r e p o l y m o r p h i c a l l y a c e t y l a t e d i n humans (24).

Thus, i t s e f f e c t ,

as w e l l as some s i d e e f f e c t s , may be r e l a t e d t o t h e acety-

HASSAN Y. ABOUL-ENEIN

54

l a t o r phenotype o f a p a r t i c u l a r p a t i e n t t r e a t e d w i t h t h i s drug (25).

The serum h a l f l i f e was found t o be about 7

hours f o r aminoglutethimide (25).

The pharmacokinetics,

b i o a v a i l a b i l i t y and b i n d i n g t o blood c o n s t i t u e n t s was e t a1 (26). s t u d i e d by Thompson -Other minor m e t a b o l i t e s o f t h e drug have been i d e n t i f i e d such as N-formylaminoglutethimide and n i t r o g l u t e t h i m i d e (27).

N-Hydroxyaminoglutethimide has been r e c e n t l y

described as an auto-induced-metabol i t e t h a t appears i n t h e u r i n e on c h r o n i c dosing w i t h aminoglutethimide (28). The metabolism o f aminoglutethimide was s t u d i e d i n t h e r a t by use o f t h e 14C-labeled compound (29).

Following oral

doses o f 5 and 50 mg/kg, t h e drug was almost completely e l i m i n a t e d w i t h i n 48 h r i n t o u r i n e and feces, mostly i n t h e form of metabolites.

I n b i l e duct-cannulated r a t s ,

b i l i a r y e x c r e t i o n of r a d i o a c t i v i t y amounted t o about 52% w i t h i n 24 h r o f an o r a l l y administered 50 mg/kg dose, w i t h t h e remainder o f t h e dose being e l i m i n a t e d i n t o urine. The major u r i n a r y metabolites r e s u l t e d from a c e t y l a t i o n o f t h e a n i l i n e moiety, h y d r o x y l a t i o n o f t h e g l u t a r i m i d e r i n g a t p o s i t i o n s 3 and 4, and o x i d a t i v e e l i m i n a t i o n o f t h e e t h y l sidechain.

The p o l a r m e t a b o l i t e s a r e accounted f o r

by aromatic h y d r o x y l a t i o n w i t h subsequent s u l f a t e conj u g a t i o n and by a g l u t a r i m i d e ring-opened compound.

In

AMINOGLUTETHIMIDE

55

a d d i t i o n , a gamma-butyrolactone d e r i v a t i v e was a l s o ident i f i e d ( F i g u r e 11). Murray -e t a1 (30) reported t h a t t h e h a l f - l i f e o f aminoglut e t h im i de admi n i s t e r e d t o p a t i e n t s w i t h mest a s t a t ic breast carcinoma was 13.3 t 2.65 hours f e l l s i g n i f i c a n t l y t o 7.3

-t 2.14

hours a f t e r 6 t o 32 weeks o f therapy.

Recently,

f o u r new m e t a b o l i t e s o f am n o g l u t e t h i m i d e have been ident i f i e d i n t h e u r i n e o f pat ents being t r e a t e d c h r o n i c a l l y w i t h t h e drug (31).

These were products of h y d r o x y l a t i o n

o f t h e g l u t a r i m i d e r i n g system, namely 3-( 4-aminophenyl) -3-ethyl-5-hydroxypiperidine-2,

6-dione and i t s a c e t y l a t e d

analog, 3-( 4-aminophenyl) -3-( l - h y d r o x y e t h y l ) p i p e r i d i n e - 2 , 6-di one and 3-( 4-ami nophenyl)3-( 2-carboxami do-ethyl ) t e t r a hydrofuran-2-one,

a l a c t o n e formed by rearrangement of

3-(4-ami nopheny1)-3-( 2-hydroxyethyl) p i p e r i d i ne -2,6-dione.

These metabolites were minor c o n s t i t u e n t s

compared w i t h aminoglutethimide and t h e major metabolites.

There were marked species d i f f e r e n c e s between

r a t and human inasmuch as almost a l l t h e u r i n a r y metab o l i t e s o f t h e r a t were N-acetylated whereas most of t h e human m e t a b o l i t e s were not (31).

However,

5-hydro-

c i s isomer being x y l a t i o n occurs i n both species, t h e formed e x c l u s i v e l y .

A l l i s o l a t e d m e t a b o l i t e s o f amino-

g l u t e t h i m i d e a r e b i o l o g i c a l l y i n a c t i v e as compared t o

HASSAN Y. ABOUL-ENEIN

56

I

CH,=CH- COOCH, Basic catalyst

Cyclization

SCHEME I

oso; &NH; O

N

I

-

0

-

N I

’

oc r c Qco -N%

&NH,

O

0

H

H H,C,

SYNTHESIS OF AMINOGLUTETHIMIDE

( !)AminOglutc?himida

OH

&NHcocH3-

c&

’1 ‘

NHCOCHg-*

cp

*

Ho+NHcocH3

0

o Hy o

0

X=OH,Y=NH, OR X=NH,,Y=OH

I

H

“3

NHCOCHS

OJ 3H NIP N H C O C H 3

O

N I H

FIG.11 PROPOSED METABOLIC PATHWAYS OF AMINOGLUTETHIMIOE IN THE RAT

AMINOGLUTETHIMIDE t h e p a r e n t drug (31,32),

57

The s t r u c t u r e of t h e i d e n t i f i e d

human u r i n a r y m e t a b o l i t e s o f a m i n o g l u t e t h i m i d e a r e shown i n Table 2. Table 2.. Identified Human Urlnary Metaboiltes of Aminoglutethlmide

Compound Amlnoglutethimide Acet yiaminogiutethimide p-Nitroglutethimlde Formylaminoglutethimide Hydrox yiaminoglutethlmide p- Amino-5-hydrox yglutethimide p-Acetylamino-5-hydroxygiutethlmide Lactone from p-amlno-2'-hydroxygiutethimlde p-Amino-1'-hydroxyglutethimide

R, NH2 H H NHCOCH, H H NO2 H H NHCHO H H NHOH H H NH2 OH H NHCOCH, OH H Structure shown above NH2 H OH

5, METHODS OF ANALYSIS 5.1

T i t r i m e t r i c Non-aqueous Aminoglutethimide has been assayed i n bu k and pharmaceutical f o r m u l a t i o n s by non-aqueous t t r a t i o n u s i n g 0.1 N p e r c h l o r i c a c i d i n g l a c i a l a c e t i c a c i d

(8).

The end p o i n t is determi ned p o t e n t iomet r i c a l l y

u s i n g a g l a s s e l e c t r o d e and a calomel e l e c t r o d e as a reference electrode with a saturated solution o f potassium c h l o r i d e i n methanol as t h e b r i d g e f l u i d . Agarwal and Blake(33) determined a m i n o g l u t e t h i m i d e ,

HASSAN Y. ABOUL-ENEIN

58

g l u t e t h i m i d e and bemegride by d i s s o l v i n g t h e sample i n ( e l m i l l i e q u i v a l e n t o f t h e powdered drug o r t a b l e t s ) i n dimethylformamide (40 m l ) using 0.1N sodium methoxide i n benzene-methanol as a t i t r a t i n g The end-poi n t i s determi ned poten-

agent.

t i o m e t r i c a l l y ( w i t h calomel and p l a t i n u m e l e c t r o d e s ) , o r v i s u a l l y w i t h a z o v i o l e t as i n d i c a t o r .

The coef-

f i c i e n t of v a r i a t i o n i s N 0.7%. 5.2

Spectrophotometric Analysis B u l t and Klasen(34) determined am1 nogl u t e t h i m i d e among o t h e r drugs c o l o r i m e t r i c a l l y through i t s react i o n w i t h cobaltous amine reagent ( P a r r i r e a c t i o n ) . The v i o l e t c o l o r produced c o u l d be measured a t 555 and 530 nm, r e s p e c t i v e l y .

The general procedure i s

described as f o l l o w s : About 1 mg o f t h e drug i s d i s s o l v e d i n 1.6 m l ethanol.

A f t e r a d d i t i o n o f 0.2 m l cobaltous s a l t

s o l u t i o n (cobaltous) n i t r a t e 6 H20: 120 mg/20 m l methanol o r cobaltous acetate 4 H20:100 mg/20 m l methanol) and 0.2 m l cyclohexylamine s o l u t i o n (6 9/20 m l methanol) t h e produced c o l o u r i s compared w i t h

t h a t o f t h e blank (reagents). Another method used t o assay aminogl u t e t h i m i d e

AMINOGLUTETHIMIDE

59

colorimeterically i n b ological f u i d (urine) i s based on t h e f o r m a t i o n o f c o l o r e d Sch if f ' s base w i t h p-dimethylaminobengaldehyde ( E h r l i c h ' s reagent). nm.

The y e l l o w c o l o r formed was measured a t 440

A l i n e a r r e l a t i o n s h i p between c o l o r i n t e n s i t y and

c o n c e n t r a t i o n o f t h e drug was o b t a i n e d f o r t h e range

A m o d i f i e d method was a p p l i e d t o

o f 1 - 1 5 ~g/ml (19).

determine aminoglutethimide serum l e v e l s u s i n g E r l i c h ' s reagent (30).

The a u t h o r s r e p o r t e d t h a t

a l t h o u g h several substances c o u l d p o t e n t i a l l y i n t e r f e r e w i t h t h e blood assay o f a m i n o g l u t e t h i m i d e , none was a c t u a l l y found t o do so t o any s i g n i f i c a n t degree.

5.3

Chromatography 5.3.1

Paper Chromatography Douglas and N i c h o l l s (19) d e s c r i b e d

a method

f o r i d e n t i f i c a t i o n and s e p a r a t i o n o f aminoglut e t h i m i d e i n u r i n e o f man by paper chromatography.

The chromatograms r u n on Whatman No.

1 paper by ascending t e c h n i q u e u s i n g s o l v e n t systems shown i n Table 3. Table 3.

Solvents Systems used f o r I d e n t i f i c a t i o n o f

Ami nogl u t e t h i m i d e by Paper Chromatography

a)

Sol v e n t System

Rf

Local iz i n g Agent

n-Bu0H:HOAc :H20 12: 3:5 v/v

0.73

a)

N,N-dimethyl ami n o c i nnaml dehyde spray (permanent r e d c o l o r )

(35)

HASSAN Y. ABOUL-ENEIN

60 b)

CHC13: MeOH 1:l v/v

0.93

b)

UV l i g h t

c)

CCl4: HOAc: H20 1: 2: 1 v/v

0.34

c)

E r l i ch ' s reagent (ye1 1ow (color)

d)

Aqueous NaCl 10% w/v

0.74

d)

N i t r o u s acid-naphthylethyl e n e diamine(35)

e)

NaOCl - K I - s t a r c h ( b l ue b l a c k c o l o r ) (36)

Dav es and Nicho l s ( 3 8 ) r e p o r t e d a study o f t h e chromatographic behavior o f n i n e g l u t a r i m i d e s i n c l u d i n g aminoglutethimide using Whatman No. 1 paper impregnated w i t h l i q u i d p a r a f f i n (4% i n hexane), o l i v e o i l (20% i n acetone) o r t r i b u t y r i n (10% i n acetone).

The most u s e f u l solvent system used were: 10:5:4

a)

toluene: a c e t i c a c i d : water

b)

carbon t e t r a c h l o r i d e : a c e t i c a c i d : water

c)

10% w/v aqueous sodium c h l o r de

d)

0.066M sodium phosphate (pH7 3)

1:2:1

The compounds were detected by hypochl o r i t e reagent o r a1 k a l ine hydroxyl ami ne reagent. 5.3.2

Thin Layer Chromatography (TLC) Several procedures had been p u b l i s h e d f o r detect i o n o f aminoglutethimide by t l c which i s summarized

i n Table 4.

Table 4.

Thin Layer Chromatography o f A m i nogl utethimide

Sol vent System

Visual iz i ng Agent

Comnents/Reference

CHCl :C6H6

Hydroxyl ami ne reagent

Alumina 50 t h i c k p l a t e ac i v t e d a t 150° f o r v ? hour was used t377

C H CH3:CH$OCH3 9&:20

Ehrl ich ' s reagent

used i n determ na i o n o f aminoglutethimide i n serum 130j

i s 0 PrOH:CHC13:25%NH40H 9:9:2 CHCl :CH3COCH3

D i c h l o r o f 1uoroscei n or Mercuric n i t r a t e

CHC13 :CH3C002CH5

UV l i g h t a t 254 nm

used i n i d n t ' f i c a t i n o f amino l u t e t h i mide i n buyk {Rf:0.47 and t o d e f e c t t h e by-product m-aminoglutethimide (Rf:0.35) MNSi 1 625 HR/UV p l a t e (Macherey-Nagel & Co. was 8 used. (8)

UV l i g h t a t 254 nm or by d i a z o t i zat ion and spraying w i t h napthyl e t h y l enedi ami ne d i hychloride.

a m l i e d t o i d e n t i f y aminoqlutethimide i n t a b l e t s (Rf:O 6 ) - S i l i c a " g e l 60F 254 p l a t e used. (8)

1:l

35 :15

CH3COOC2Hg:CH30H: 100%CH3COOH

17 :3:O. 1

HASSAN Y. ABOUL-ENEIN

62

5.3.3

Gas-Liquid Chromatography (GLC) Adams and Roger(39) r e c e n t l y p u b l i s h e d a rapid, s e n s i t i v e and s e l e c t i v e GLC assay f o r aminoglutethimide i n biological fluids.

The method i s

s u i t a b l e f o r t h e study o f t h e pharmacokinetics o f t h e drug and i t s N-acetyl m e t a b o l i t e (which gave an assymmetric peak shape on t h i s system). The c o n d i t i o n s are as f o l l o w s : Column: glass column (1.5in x 0.4mm I.D.) packed w i t h 2% CDMS (cyclohexanedimethanol succi n a t e ) on Chromosorb W80-100 mesh ( a c i d washed &

dichloromethylsalisilane t r e a t e d ) . W80-100 mesh ( a c i d washed & d i c h 1oromethyl s a l is i lane t r e a t e d ) . Column temperature

: 240'

D e t e c t o r temperature:

350'

C a r r i e r gas

: Nitrogen

F1ow r a t e

: 100ml/min

Detector

: Nitrogen s e l e c t i v e

D u t t reported a gas chromatographic method f o r t h e i d e n t i f i c a t i o n o f 116 common drugs by t h e i r m u l t i p l e peaks o f t h e parent and t h e i r

AMINOGLUTETHIMIDE

63

t r i m e t h y l s i l y l y l d e r i v a t v e s i n c l u d i n g amin o g l u t e t h i m i d e (40).

The study was done u s i n g

3% OV-17 Gas-Chrom Q 100-120 mesh g l a s s column (2m x 3mm I.D.)

(FID).

and flame i o n i z a t i o n d e t e c t o r

The c a r r i e r gas was n i t r o g e n a t a f l o w

r a t e o f 30 ml/min and t h e hydrogen and a i r f l o w r a t e s were 30 and 450ml/min, tively.

respec-

The i n j e c t i o n p o r t and d e t e c t o r tem-

p e r a t u r e s were 300' and t h e oven temperature was programmed from 120" t o 270' a t lO"/min. o r a t an isothermal temperature o f 300'. 5.3.4

High-Performance L i q u i d Chromatography (HPLC) Several HPLC procedures have been p u b l i s h e d f o r t h e d e t e r m i n a t i o n o f aminoglutethimide and i t s m a j o r m e t o b o l i t e (N-acetylaminoglutethimide) i n biological fluids.

Table 5 summarises some o f t h e

HPLC systems used f o r t h e a n a l y s i s o f t h e drug.

Table 5 HPLC Systems Aminoglutethimide Column

Mobile Phase

Detection

Spherisorb ODS column (30cm X 4mm I.D., 5 g particle size

MeCN: 0.01 M phosphate b u f f e r pH 6.8 (22:68), f l o w r a t e 1.5 m l /mi n

UV a t 234 nm Applied f o r simple determination o f ami nogl utethimide & i t s m e t a b o l i t e (41)

r

Octadecyl (C18) s i l i c a MeCN: t e r t . butylamnonium reversed phase uncapped phosphate:H20 (100:3: l o o ) , column (10cm X 0.8cm adjusted t o pH 6.320.2 w i t h I.D. w i t h 10 m parorthophosphori c a c i d , f 1ow t i c l e size). r a t e 2 ml/min.

Comnent/Reference

UV a t 254 nm Applicable f o r assay o f t h e drug i n plasma (42)

Y

ODS H y p e r s i l (10 cm long, 3 m p a r t i c l e s i z e

Y

11%MeCN i n lOOmM ammonium phosphate b u f f e r , pH 3.5 8-20% MeCN i n 15mM also acetate b u f f e r pH 4.5 or lOmM phosphate b u f f e r , pH 6.0, f l o w r a t e 2 ml/rnin. E l u t i o n o f n i t r o g l utethimide r e q u i r e d a step wise increase i n MeCN conc. from 11 t o 23% a f t e r 16 min.

-

-

Applicable f o r a n a l y s i s o f t h e drug and i t s meta b o l i t e s i n plasma. Ret e n t i o n times were approx.: 4.5 min aminoglutethimide, 14 m i n . N formylmetabolite, 17 min N-acetylmetabol it e , 28 m i n. n i t r o g l u t e t h i m i de (43)

Table 5 HPLC Systems Aminoglutethimide Column

Mobile Phase

Detection

Comment/Reference

Nucleosil - 10 (C18) stainless steel column (20 cm x 4.8 mm I.D.)

MeCN:HzO:Et3N (25:75:0.05) flow rate 2 ml/min

UV at 254 mm

Applied as stabi 1 i ty i ndi cati ng assay (8)

Nucleosil - C18 (!Iyn particle size)

MeCN: MeOH:H20 (5:20:75)

UV at 235 mn Appl ied for simultaneous

Reversed phase Magnusphere C18 (7pm particle size 15 cm x 4.6 cm 1.0.) stainless steel column

C &rate bu fer pH 3-4: MeOH (500: 280), flow rate 1.2 ml/min

determination of the drug and its major metabolite in human plasma ( 44 9451 Retention times f o r aminoglutethimide = 8.9 min N-acetylmetabol i te = 12.1 min I

UV at 254 mn

Suitable for pharmacokinetic study of aminogl utethimide and its N-acetyl metabol i te in biological f1 uids (plasma, urine saliva), (39)

HASSAN Y. ABOUL-ENEIN

66

R E F E R E N C E S

A.M.

Camacho, A.J.

Brough, R. Cash and R.S. (1966).

Wilroy,

A.M.

Camacho, R. Cash, A.J. Brough and R.S. Assoc., 202, 20 (1967).

Wilroy,

3. Paediat., 68, 852 -J. -Am. Med.

R. Cash, A.J. Brough, M.N.P. Cohen and P.S. Satoh, J. C l i n . Endocrinol. Metab. 27, 1239 (1967).

-D.F. T.R.

Child, C.W. Burke, D.M. Burley, L.H. Rees and Fraser, Acta Endocrinol., 82 300 (1976). 7

R. J. Santen, S. Santner, B. Davis, J. Veldhuis, E. Samojlik and E. Ruby, J. C l i n . Endocrinol. Metab., (1978).

47, 1257 5

R. J. Santen, T.J. Worgul, A. Lipton, H. Harvey, A. Boucher, E. Sarnojlik and S. A. Wells, Ann. I n t e r n . Med ,96, 94, ( 1982).

-. 6

R. J. Santen, E. Badden, S. Lerman, H. Harvey, A. A. Lipton, A. G. Boucher, A. Manni, H. Rosen and S.A. Wells, ----Breast Cancer Res. Treat, 2, 375 (1982).

S. A. Wells, R. J. Santen, A. Lipton, D.E. Haagensen Jr., E. J. Ruby, H. Harvey and W.G. D i l l e y , Ann. Surg ,187, 475 (1978)

-

.

.

-

E d i t o r i a l N. Engl. J. Med.,

305, 575 (1981).

Ciba-Geigy, A n a l y t i c a l Department, Base1 , Switzerland, P r iv a t e Communi c a t ions

.

9

E.G.C. Clarke, "The I s o l a t i o n and I d e n t i f i c a t i o n o f Drugs", The Pharmaceutical Press, London, 1969, p 187.

.

10

Merck Index, 10th E d i t i o n , Merck & Co. Inc., N.J., Compound No. 443.

11

A l d r i c h Catalog Handbook o f Fine Chemicals, A l d r i c h Chemical Co., Milwaukee, Wisconsin, U.S.A., p. 61, 1984-1985.

12

C.M. Lee and W.D. 4586 (1961).

Kumler, ----J. Amer. Chem. SOC.,

Rahaway,

83,

67

AMINOGLUTETHIMIDE

13

N. Finch, R. Dziemian, J. Cohen and B.G. Experi e n t i a, 31, 1002 ( 1975).

14

C.M.

15

N. Defay and C. D o r l e t , J. Pharm. Belg.,

16

G. Rucker and

-

Steinetz,

Lee e t a l , ----J. Amer. Chem. SOC. 83, 4586 (1961).

--

27 335 (1972).

G. Bohn, Archive der Pharmazie, 302, 204

(1969).

17

K. Hoffman and E. Urech, U.S.

P a t i e n t 2, 848, 455

(1958). 18

E. Tagmann, E. Surry and K. Hoffmann, -Helv Chim.

-Acts., 35, 1541 (1952).

Douglas and P. J. N i c h o l l s , J. Pharm, Pharmacol.

J.S.

Douglas and

17, 115 S (1965).

20

24, (Suppl .) , 150 P

21

22

--

J.S.

19

P. 3. N i c h o l l s , 3. Pharm. Pharmacol. (1970).

R.C. Coombes, M. Jarman, S. Harland, W.A. R a t c l i f e , T.J. Powles, G.N. Taylor, M. O'Hare, E. Nice, A.B. F o s t e r and A.M. N e v i l l e , J. Endocrinol, 87, 31P (1980). R. Paul, R.P.

17, 539

---

Williams and E. Cohen, J. Med. Chem.,

(1974).

23

5.3. Sheets and L.E. Vickery, Naunyn-Schmied. Pharmacol , 321, 70 (1982).

24(a)

R.C. Coombes, A.B. Foster, S.J. Harland, M. Jarman Cancer, 46,340 (1982) and and E.C. Nice, Br. J. 7 references c i t e d t h e r e i n .

24(b)

.

-

Arch.

--

A.M.

Adam, H.J. Rogers, S.A. Amiel and R.D. Pharmac. , 495 (1984).

--Br. 3. C1 i n ,

18,

Rubens,

25

J.S. Schanche, P.E. Lonning, P.M. Ureland and S. K r i n n s l and, Therapeutic Moni t o r i ng , 6, 221 (1984)

26

T.A. A.R.

Thompson, J.D. Vermeulen, W.E. Wagner, Le Sher, J. Pharm. Sci., 700 1040 (1981).

M.H.

Baker, A.B. Foster, S.J. Harland and M. Jarman, Pharmacol , 74, 243 P (1981).

27

-B r i t . J,

HASSAN Y. ABOUL-ENEIN

68

28

M. Jarman, A,B. Foster, P.E. Gross, L.J. Griggs, 1. Howe and R.C. Coombes, Biomed Mass Spectrum, 620( 1983).

29

H. Egger, F. B a r t l e t t , W. I t t e r l y , R. Rodebaugh, C. C. Shimanskas, Drug Metab. Dispos., 10, 405 (1982).

30

F.T. Murray, s. Santner, E. Samojlik and R. J. R.J. Santen, J. Clin. Pharmacol 19,704 (1979).

31

A.B. Foster, L.J. Griggs, I.Howe, N. Jarman, C.S. Leung, 0. Manson, and M.G. Rowlands Drug. Metab. 12, 511, (1984). Dispos. -

32

A.B. G.N.

33

S.P. Agarwal and M.I. 1668 (1965).

34

109, 389

35

J.W. Bridges and R. T. Williams, J. Pharm, Pharmacol , 15,565 (1963).

36

J.V. Jackson and M.S. Moss , "Chromatographic and E l e c t r o h o r e t i c Techniques" e d i t o r I.Smith, Vol. I, p 406, London, Heinemann.

37

D. Davies and P.J. (1965).

38

H.J. Uhlmann, Pharm. Z t Ver A otheker 1998, ( 1964) : - 3 k f . k t - 8 - & 6 8

Zt

39

A.H. Adam and H.J. (1984).

307, 129

40

M.C.

Outt , J. Chromatogr.

M.O.

Kamblawi, R.G.

41 42

--

.

--

Foster, M. Jarman, C-S. Leung, M.G. Rowlands and Taylor, J. Med. Chem. 26, 50 (1983) Blake, ---J. Pharm. Sci,,

54,

A. B u l t and H.B. Klasen, Pharmaceutisch Weekblad, (1974).

.

-

Nicholls, J. Chromatogr. 17 416

--Rogers, J. Chromatogr.

-

248,

115 (1982).

Stevens and P.J. (1984).

J. Chromatogr. 309, 431 B.A. Robinson and F.N. 1104 (1983).

Nicholls,

Cornell, --2 C l i n . Chem.,

29

69

AMINOGIXTETHIMIDE

.

44

P.E. Lonning, J.S. Schance, S. Kvinnsland and P.M. Uel and, GI in Pharmacoki n e t i cs , 10, 353 ( 1985)

45

6. Menge and J.P. Dubois, J. Chromatogr.,

.

310 431

(1984).

A c k n o w l e d g e m e n t s

The Author wishes t o thank Dr. K. Scheibli o f Ciba-Geigy Limited, Basle, Switzerland, for donatf ng a sample of Aminogl utethimide, batch 9614586, used i n t h i s profile.

This Page Intentionally Left Blank

CAFFEINE

l c l b h a d Uppat Zubair

khmoud M.A. Hassan,

and

Ibrahim A . AZ-Meshat

1. Description 1.1 Nomenclature 1.1.1 Chemical Names 1.1.2 Generic Names 1.2 Formulae 1.2.1 Empirical 1.2.2 S t r u c t u r a l 1.2.3 S t r u c t u r a l Confirmation by Degradation 1.2.4 CAS Registry Number 1.2.5 Wiswesser Line Notation 1.3 Molecular Weight 1 . 4 Elemental Composition 1.5 Appearance, Colour, Odor and Taste 2. Physical P r o p e r t i e s 2.1 Melting Range ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 15

71

Copyright Q 1986 by the American Pharmaceutical Association All rights of reproduction in any form reserved.

MOHAMMAD UPPAL ZUBAIR ET AL.

72 2.2

Sublimation Density 2.4 Solubility 2.5 Crystal Structure 2.6 Spectral Properties 2.6.1 Ultraviolet Spectrum 2.6.2 Infrared Spectrum 2.6.3 Nuclear Magnetic Resonance Spectra 2.6.3.1 Proton Spectrum 2.6.3.2 l 3 C - m Spectra 2.6.4 Mass Spectrum 3. Isolation of Caffeine 3.1 From Tea Leaves 3.2 From Green Coffee 3.3 Process for the Extraction of Caffeine from Coffee beans 3.4 Isolation of Natural Caffeine from the decaffeination process. 3.5 Isolation of Caffeine from Various Sources 4. Synthesis 4.1 Route 1 4.2 Route 2 4.3 Route 3 4.4 Route 4 4.5 Route 5 4.6 Route 6 5. Biosynthesis 6. Pharmacokinetics and Metabolism 7. Methods of Analysis 7.1 Elementa1 Analysis 7.2 Identification Tests 7.3 Titrimetric 7.4 Spectrophotometric 7.4.1 Colorimetric 7.4.2 Ultraviolet 7.4.3 Infrared 7.4.4 Nuclear Magnetic Resonance 7.4.5 Mass Spectrometric 7.4.6 Photo-nephlometric 7.5 Coulometric 7.6 Polarographic 7.7 Ring Oven (Micro) 7.8 Radioactive Isotopes 7.9 Radiochemical 7.10 Chromatographic Methods 7.10.1 Paper Chromatography 7.10.2 Column Chromatography 7.10.3 Thin-Layer Chrmakxagraphy 2.3

CAFFEINE

73

7.10.4 Gas Liquid Chromatography 7.10.5 High Performance Liquid Chromatography.

8.

References

9. Acknowledgements

MOHAMMAD UPPAL ZUBAIR ET AL.

74

1. Description

1.1 Nomenclature 1.1.1 Chemical Names

a) lH-Purine-2,6-dione trimethyl.

3,7-dihydro-1 3,7-

b) 1,3,7-Trimethylxanthine.

c ) Methylt heobromine

.

d) 1,3,7-Trimethyl-2,6-dioxo-l,2,3,6-tetrahydropurine.

e) 3,7-Dihydro-l,3 ,T-trimethyl-lH-purine-2,6dione. f) 1,3,7-Trimethyl-4,6-dioxopurine. 1.1.2 Generic Names

Anhydrous caffeine; Caffeine; Coffeniwn; Coffeine; Coffeina; Cuavanine; Methyltheobromine; No-Doz; Thiene. 1.2 Formulae 1.2.1 Empirical

C8H10N402 1.2.2 Structural

CAFFEINE

75

Discovered by Robiquet i n coffee i n 1821, while searching f o r quinine which he believed t o be present. I n 1827, Oudry found an a l k a l o i d i n tea and c a l l e d it t h i e n e . I n 1838, J o b s t and Mulder proved t h e i d e n t i c a l c h a r a c t e r of t h e two p r i n c i p l e s (1). 1.2.3 S t r u c t u r a l Confirmation by Degradation ( 2,3) Fischer has reported t h a t c a f f e i n e on o x i d a t i o n gave dimethylalloxane and methyl urea, i n d i c a t i n g s i m i l a r i t y with t h e skeleton s t r u c t u r e of u r i c acid.

With t h e above information it becomes evident t h a t : t h e t h i r d methyl group i s e i t h e r a t p o s i t i o n 7 o r 9 and t h e remaining oxygen atom i s e i t h e r at p o s i t i o n 6 o r 8. P o s i t i o n of t h e methyl group; Fischer a l s o i s o l a t e d another oxidation product which on hydrolysis, gave N-methylglycine, carbondioxide and ammonia. Thus, t h i s t h i r d oxidation product must be N-methylhydantoin.

co

-

+ NH3 + C02 C02H

N-methylhydantoin Therefore, it follows t h a t c a f f e i n e contains two r i n g s t r u c t u r e s t h a t of dimethylalloxane and t h a t o f methylhydantoin. The following two skeleton

MOHAMMAD UPPAL ZUBAIR E T A L .

76

s t r u c t u r e s were proposed:

F i n a l l y Fischer i s o l a t e d f o u r t h oxidation product,

N , N ' -dimethyloxamid@ CH3NHCOCONHCK3. Examination of ( I ) and (11) showed t h a t only ( I ),

could give r i s e t o t h e formation o f t h e oxamide. Therefore ( I ) must be t h e skeleton of c a f f e i n e . P o s i t i o n of oxygen atoms: t h e following two s t r u c t u r e s were p o s s i b l e with regard t o t h e p o s i t i o n of oxygen atoms.

0

a3

">

1% 0

y3

I

N/

I

CH3 I11

IV

By analogy, it would appear t h a t t h e more l i k e l y s t r u c t u r e o f I11 would be t h a t of u r i c a c i d .

CAFFEINE

77

F i s c h e r , f u r t h e r confirmed t h e c a f f e i n e s t r u c t u r e with t h e following degradation s t u d i e s : Caffeine > - *

c12

CH30H Chlorocaffeine

'l

Met hoxy c a f f c i n e

NaoH

dilute IlCl, boil

Oxycaf feine+CII Cl

3

1.2.4 CAS Registry Number

a ) Anhydrous c a f f e i n e [ 58-08-21 b ) Monohydrat e [ 5743-12-4

I

1.2.5 Wiswesser Line Notation

T 56 BN DN FN VNVJ B F H 1 . 3 Molecular Weight Anhydrous

C8HloN4O2

Monohydrate C8HloN402.H20

=

194.19

=

212.21

1 . 4 Elemental Composition C,

49.48% ; H , 5.13%

; N , 28.85% ; 0,

16.48%.

1 . 5 Appearance, Color, Odor and Taste White powder o r white, g l i s t e n i n g n e e d l e s , u s u a l l y matted t o g e t h e r . It i s o d o r l e s s and has a b i t t e r taste. 2. Physical P r o p e r t i e s 2 . 1 Melting range Between 235' and 237.5'. f o r 4 hours.

determined a f t e r drying at 80'

Hexagonal prisms by sublimation, m.p. 238?

(4)

2.2 Sublimation Caffeine sublimes a t 178? Fast sublimation i s obtained a t 160-165' under 1 mm p r e s s u r e a t 5 mm d i s t a n c e .

MOHAMMAD UPPAL ZUBAIR ET AL.

78

2.3 Density d i 8 1.23 ( 5 ) .

2.4 S o l u b i l i t y (1) 1 gm of anhydrous c a f f e i n e dissolves i n about 50 ml water, 6 ml water at 8 0 ° , 75 m l alcohol, about 25 m l alcohol a t 6 0 ° , about 6 m l chloroform, 600 ml e t h e r , 50 m l acetone, 100 ml benzene and 22 m l b o i l i n g benzene. Freely soluble i n pyrrole; i n tetrahydrofuran containing, 4% water, a l s o soluble i n e t h y l a c e t a t e and s l i g h t l y i n petroleum e t h e r .

Being a weak base, c a f f e i n e does not form s t a b l e salts, and even i t s salts of strong a c i d s , such as t h e hydrochloride o r hydrobromide, are r e a d i l y hydrolysed by water. The s o l u b i l i t y of c a f f e i n e i n water i s increased by t h e presence o f organic a c i d s o r t h e i r a l k a l i salts, e.g., benzoates, s a l i c y l a t e s , cinnamates, o r c i t r a t e s and this i s t h e reason f o r t h e use of s e v e r a l such preparations. 2.5 Crystal S t r u c t u r e Sutor ( 6 ) has determined t h e c r y s t a l s t r u c t u r e of c a f f e i n e , 1,3,7-trimethyl-2,6-dihydroxy purine (Fig. 1). Crystallographically, it is nearly isomorphous with theophylline (Fig.. 2) (7), and a comparison of bond lengths i n t h e two has yielded information concerning t h e e f f e c t s of a s u b s t i t u e n t i n t h e imidazole r i n g on t h e purine ring. The c r y s t a l s of c a f f e i n e are monoc l i n i c , space group P21/a, w i t h a = 14.8 ? 0.01, b = 16.7 ? 0.1, C = 3.97 ? 0.03 A'; 8 = 97.0 ? 0.5'. Systematic absences are hoc with h = 2n + 1, OK0 w i t h K = 2n + 1; space group ~ 2 1 / a' q h . Its f i n a l Fourier projection i s shown i n Fig. 3. Table 1 shows t h e f r a c t i o n a l coordinates r e f e r r e d t o t h e monoclinic c r y s t a l axes. The c r y s t a l s t r u c t u r e w a s solved by an application of t h e isomorphus-replacement method and a consideration of t h e possible hydrogen bond system i n t h e c r y s t a l . Bond lengths and bond angles within t h e molecule are shown i n Fig. 4, Fig. 5 and Table 2.

79

CAFFEINE

Fig. 2:

Crystal structure of theophylline.

10

15

Fig. 1:

Caffeine, showing the numbering system used.

MOHAMMAD UPPAL ZUBAIR ET AL.

80

Fig. 3:

The f i n a l UKO Fourier projection of c a f f e i n e , contours are arbitrary but equal i n t e r v a l s ; ZUO contour broken.

81

CAFFEINE

The F r a c t i o n a l C o o r d i n a t e s Referred t o t h e Monoclinic C r y s t a l Axes.

Table 1.

c2

c4 c5 '6 '8

clo c12

1' 4 N1

N3 N 7

N9 O11

1 ' 3 1 ' 5

H1 H2

H3 H4 H5 H6 H7 H8 H9 H1O

X

Y

0.2h.6 0.1003 0.0835 0.1456 0.4798 0.2879 0.1960 0.4533 0.2193 0.1796 0.ooog 0.0407

0.2225 0.2533 0.1769 0.1151 0.2481

0.0841

0.3641 0 3950

0.1418 0.2764 0.1749 0.3008

0.3057 0.1374 0.0166 0.413 0.487

0.4790

0.435 0.395 0.263

0.437 0.363 0.362

0.228

0.396 0 * 377

0.142

0.348 0.300 0.257

0.2397 0.0404

0.239 0.438

0,100

0.033 0.060

Z

0.9002 0.1295 0.1944 0.1155 0.3638 0.8790 0.9209 0.4584

X*

Y"

0.1019 0.0841 0.1463

0.2225 0.2541 0 1759 0.1143

0.4801

0.2480

0.2891

0.0832 0.3638 0.3947 0.1415 0.2769 0.1749 0.3008

0.2414

0 * 1959 0.4536 0.2196

0.9735 0.9848

0.1801

0.3376

0.0020

0.2440

0.0403 0.3063 0.1363

0.0404

0.0184

0.4705

0.7614 0.1616 0.2705 0.474 599 0.278 0.510 0

0.857 0.105 0.783 0.772 0.022

0.676

-

-

-

0.2400

-

-

x* and y* represent t h e weighted x and y coordinates from t h e hKO and hKL projections.

MOHAMMAD UPPAL ZUBAIR ET AL.

82

Fig. 4:

Bond angles and C-H bond lengths in the caffeine molecule.

10

13

Fig. 5:

14

Bond lengths in the caffeine molecules.

83

CAFFEINE

Table 2.

Bond Lengths and t h e i r Standard Deviation Bond length ( A )

Standard deviation ( A )

C2-N1

1.42

0.014

C -N 2 3

1.35

0.016

C4-N3

1.42

0.021

c4-c5

1.32

0.014

'5-'6

1.44

0.015

C6-N1

1.36

0.021

c4 -N9

1.31

0.019

'8"g

1.34

0.019

C8-N7

1.32

0.012

C -N

1.41

0.021

C1O-N1

1.48

0.016

C12-N3

1.50

0.013

C14-N7

1.47

0.018

c2-011

1.19

0.023

'6"13

1.26

0.013

Bond

5 7

A comparison is made of t h e intramolecular d i s t a n c e s with t h o s e i n o t h e r purines and a pyrimidine,Table 3, and i n d i c a t e s t h a t s t e r i c hindrance must be allowed for, i n a t h e o r e t i c a l c a l c u l a t i o n of bond l e n g t h i n r e l a t i v e l y complicated molecule of t h i s t y p e . Evidence f o r and a g a i n s t t h e e x i s t e n c e o f a s h o r t hydrogen bond between water molecules i s given.

C r y s t a l s t r u c t u r e of 2 : l molecular complexes of c a f f e i n e w i t h hexaaquamagnesium (11) bromide and hexaaquamanganese (11) T r i i o d i d e i o d i d e c r y s t a l s t r u c t u r e s of

MOHAMMAD UPPAL ZUBAIR ET AL.

a4

Table 3.

S i g n i f i c a n t l y D i f f e r e n t Bond Lengths i n a Comparison of C a f f e i n e w i t h Theophylline, Adenine Hydrochloride and U r a c i l . Caffeine with theophylline

Bond

Si g n i f ic a n t d i f f e r e n c e (A')

Actural d i f f e r e n c e (A')

c4-c5

0.04

0.05

C -N

0.05

0.07

5

7

Caffeine with u r a c i l Bond

S i g n if i ca n t difference (Ao)

Actual d i f f e r e n c e (A')

c4 -N3

0.06

0.08

c4-c5

0.05

0.09

Caffeine w i t h adenine h y d r o c h l o r i d e Bond

Significant d i f f e r e n c e (Ao)

Actual d i f f e r e n c e (A')

C2"1

0.05

0.05

C -N 2 3

0.05

0.05

C4-N3

0.06

0.06

c4-c5

0.05

0.05

C4"g

0.05

0.05

( C ~ H ~ O N ~ O ~ ) ~ M)@r2 ~ - ( Oand H~ (C~H~ON~O~)~M~(OH,)~I.I have been determined by X-ray d i f f r a c t i o n methods (83. The c r y s t a l S t r u c t u r e s O f (C8H10N402)2Mg(OH2)6Br2 ( I ) and (C8HloN402)2Mn( OH2 161. I 3 (I1) have been determined by X-ray d i f f r a c t i o n methods; c r y s t a l s o f I are tric l i n i c , s p a c e group P1, w i t h Z = 1, i n a u n i t c e l l of dimension: a = 9 . 6 2 0 ( 7 ) , b = 1 0 . 7 7 9 ( 8 ) , c = 7.645(6) Ao, a = 1 0 7 . 0 3 ( 7 ) , 8 = 1 0 8 . 8 8 ( 7 ) , y 72.71(8)O; c r y s t a l s o f

CAFFEINE

85

II are monoclinic, s p a c e group P2 / n , w i t h Z = 4 , i n a u n i t c e l l of dimensions a = 1 2 . 4 0 k ( 8 ) , b = 2 9 . 6 5 2 ( 1 2 ) , c = 9.419(6) Ao, B = 108.39(7)O. The s t r u c t u r e s of I 3nd I1 have been s o l v e d from d i f f r a c t o m e t e r d a t a by P a t t e r s o n and F o u r i e r methods and r e f i n e d by f u l l m a t r i x l e a s t - s q u a r e s t o R = 0.046 f o r I and 0.090 f o r 11. Both compounds c o n t a i n o c t a h e d r a l hexaaquametal (11) c a t i o n s , uncoordinated c a f f e i n e molecules and bromide a n i o n s i n I and t r i i o d i d e and i o d i d e a n i o n s i n 11, h e l d t o g e t h e r by a network o f hydrogen bonds. The t r i i o d i d e a n i o n s are unsymmetrical [ 1 ( 1 ) - 1 ( 2 ) = 2.89, 1 ( 2 ) - 1 ( 3 ) = 2.95A0, I ( 1 ) - I ( 2 ) - I ( 3) = 1 7 8 O ] , a r r a n g e d i n l i n e a r systems w i t h a weak ' h e a d - t o - t a i l ' i n t e r a c t i o n , t h e d i s t a n c e being o f 3.62 A O . 2.

6 Spectral Properties 2.6.1 U l t r a v i o l e t Spectrum The W spectrum of c a f f e i n e i n methanol (Fig. 6 ) was scanned from 190 t o 440 nm, u s i n g DMS 9 0 . Varian Spectrophotometer. It e x h i b i t e d a Amax at 270 nm. Other r e p o r t e d W s p e c t r a l d a t a are shown below: Solvent

2.6.2

'max

nm

E l % , 1 cm

Ref. -

278

-

10

Ethanol

273

5 19

11

0.1N H C 1

272

470

11

0.1N H C 1

27 5

490

12

0.1N NaOH

275

490

12

Methanol

272

Trichloroethylene

9

I n f r a r e d Spectrum The I R spectrum o f c a f f e i n e as KBr d i s c ( F i g . 7 ) w a s recorded on a P e r k i n E l m e r - 580 B I n f r a r e d spectrophotometer t o which an I n f r a r e d d a t a s t a t i o n is a t t a c h e d . The s t r u c t u r a l assignments

m Q)

Fig.6 UV

Spectrum

of'

CnFFeirle in /Ye!~anbl

2.5

4.0

3.0

5.0 MICk&’5

zo

6.0

80

9.0

10

f2

14

100

I

wavenumber

lob0

2500

.

2000

1

f800

f60d

1

f/oO

fOm

8W 1

s50

MOHAMMAD UPPAL ZUBAIR ET AL.

88

have been c o r r e l a t e d with t h e following frequencies Table 4 ) . Table 4 .

I R C h a r a c t e r i s t i c s of Caffeine.

Frequency cm 3110,

-1

Assignment

2950

and

CH3

1700

c=o

1650

C = N

CH s t r e t c h

Other c h a r a c t e r i s t i c bands are: 1600, 1550, 1480, 1455, 1430, 1400, 1360, 1285, 1235, 1185, 1020, 970, 860, 760 and 745. The i n f r a r e d d a t a f o r c a f f e i n e were also reported and are a s follows: 3100, 2970, 1700, 1660, 1550, 1480, 1360, 1240, 1020, 980, 750, and 610 cm-1 ( 9 ) . P r i n c i p a l peaks are 1658, 1695 and 745 cm-I (11). Major peaks 747, 1454, 1480, 1548, 1658 and 1698 cm-1 ( 1 2 ) . The region from 19-22 p i s important f o r i d e n t i f i c a t i o n of c a f f e i n e (13). 2.6.3 Nuclear Magnetic Resonance Spectra 2.6.3.1 Proton Spectrum The PMR spectrum of caffeine i n CDCl3 (Fig. 8 ) was recorded on a Varian ~ 6 0 - A , 60 MHz NMR spectrometer using TMS ( t e t r a m e t h y l s i l a n e ) a s an i n t e r n a l reference. The following s t r u c t u r a l assignments have been made (Table 5 ) . Table 5.

PMR C h a r a c t e r i s t i c of C a f f e i n e

Chemical S h i f t ( 6 ) ppm

Croup

1-N-CH3

3.53

s

3-N -CH

3.33

s

7-N-CH3

3.98

s

7.54

s

8-H ~~

s

= singlet

c

A -

_

I

I

3.0

-

.

I

1

.

.

.

.

l

.

2.0

.

I

.

.

I

.

1.0

.

.

I

11 27,87 CH3

I

The structure o f c a f f e i n e f o r carbon c h e m i c a l s h i f t s .

53

91

CAFFEINE

O t h e r r e p o r t e d d a t a are 3 . 4 , 3 . 6 , 4.0 and

7.6 ppm (7,14). Stamm(15) has i n v e s t i t h e a s s o c i a t i o n o f c a f f e i n e with sodium benzoate and o t h e r compounds and r e p o r t e d a l a r g e chemical s h i f t of t h e NMR s i g n a l s o f t h e t h r e e methyl Kroups. Also metal p o r p h y r i n / c a f f e i n e complexes have been i n v e s t i g a t e d ( 1 6 ) . &ed

2.6.3.2

13C-NMR

Spectra

The n a t u r a l abundance C-13 NMFf n o i s e decoupled and s i n g l e frequency o f f resonance decoupled (SFORD) s p e c t r a ( F i g s . 9 and 1 0 ) were o b t a i n e d at 20 M l z on a Varian FT-80A, 80 MHz F o u r i e r t r a n s f o r m NMR s p e c t r o m e t e r u s i n g a broad band 1 0 mm probe. The sample w a s run a t c o n c e n t r a t i o n C a 1-2 M i n d e u t r a t e d chloroform w i t h t e t r a m e t h y l s i l a n e as an i n t e r n a l r e f e r e n c e s t a n d a r d . The chemic a l s h i f t s were measured at 5 KHz s p e c t r a l width. The carbon chemical s h i f t s are a s s i g n e d on t h e basis o f t h e t h e o r y o f chemical s h i f t and SFORD s p l i t t i n g p a t t e r n and i s shown i n Table 6. Table 6. Carbon

No.

Carbon Chemical S h i f t s o f Caffeine

.

Chemical S h i f t ( 6 ) ppm

Multipli-

151.69 148.73

S

107.55

S

155.35

S

141.53

d

C-10-CH3

29.70

9

C-11-CH3

27.87

q

C-12-CH3

33.54

9

c-2

C-4

c-5 C-6 C-8

S

s = s i n g l e t ; d = doublet; q = quartet.

1000

8d 0 EbO

1

Fly.9

'3C -NMR

Norse -decoupled Spectrum oP Cp.Lf'eine in CU c/jr

93

h

MOHAMMAD UPPAL ZUBAIR ET AL.

94

2.6.4 Mass Spectrum The mass spectrum of c a f f e i n e obtained by e l e c t ron impact i o n i z a t i o n ( E I ) i s shown i n Fig. 11. It was recorded on Finigan-Mat 1020 GC/Mass spectrometer. The spectrum w a s scanned from 40 upto 440 a.m.a. Electron energy was 70 e V . The mass s p e c t r a l data are shown i n Table 7. Table 7 .

The Most Prominent Fragments of Caffeine.

m/e

Relative i n t e n s i t y

194

I loo

109

66

82

37

67

54

55

80

Fragment Base peak (M+) C5H7N3

-

Other mass s p e c t r a l d a t a were a l s o reported ( 9 $171 19411001 67" 661 , 1091661 , 821 391 42[ 281 , 40[18] and 41[16]. A comparison of mass s p e c t r a l d a t a o f theobromine, theophylline and c a f f e i n e w a s reported by S p i t e l l e r and Friedmann (18). Dunbor and Wilson have published an isotope mass spectrum method f o r i d e n t i f i c a t i o n of t h e geograp h i c a l o r i g i n of c a f f e i n e (19).

3. I s o l a t i o n of C afPeine 3.1 From Tea Leaves : Finely powdered t e a leaves (100 g ) are e x t r a c t e d with ethanol i n a soxhlet apparatus f o r 3 h r s (20). The c a f f e i n e so e x t r a c t e d i s then adsorbed on magnesium oxide. It i s t h e n desorbed after treatment with 10% H2SO4 and i s e x t r a c t e d i n t o chloroform and i s r e c r y s t a l l i s e d . 3.2 From Green Coffee : A procedure f o r t h e e x t r a c t i o n of c a f f e i n e from green coffee has been reported ( 2 1 ) . Green c o f f e e beans were steamed f o r 2 t o 3 hours t o e q u i l i b r a t e t h e water content between 40 and 50%.

100.0

1

55

1- 1

1 I

J

50.0

67

-60

100 F i g . 11.

120

140

160

EX-fiass Spectrum of C a f f o i w .

180

-1J4-

203 I -200

- - - - .222- . 1.

220

.I

- - II 240

96

MOHAMMAD UPPAL ZUBAIR ET AL.

Caffeine was then e x t r a c t e d with CHC13 and c a f f e i n e upto 95% of t h e t h e o r e t i c a l amount was recovered. 3.3 A process f o r t h e e x t r a c t i o n of c a f f e i n e from c o f f e e beans by leaching w i t h water has been developed ( 2 2 ) . The highest y i e l d o f 90.4% was obtained when t h e average c o f f e e p a r t i c l e s i z e w a s 1.4095 mm, t h e water/ coffee r a t i o of 9:1, a t 7 5 O C and t h e e x t r a c t i o n was c a r r i e d out f o r t h i r t y minutes.

3 . 4 Natural c a f f e i n e is a l s o obtained as a by-product, from t h e d e c a f f e i n a t i o n process of coffee and t e a . Some of t h e s e method a r e summarised and presented i n Table 8.

3.5 Caffeine can a l s o be obtained from various sources. Table 9 summarises t h e source, b o t a n i c a l c h a r a c t e r i s t i c s , occurrance and t h e caffeine/theobromine content i n each case ( 3 7 ) .

CAFFEINE

97

Table 8.

I s o l a t i o n o f C a f f e i n c by Decaf f e i n a t i o n Processes.

Material

Solvent

Ref. -

Green c o f f e e beans*

F l u o r i n a t e d hydrocarbons

23

Coffee* and t e a extracts (also o i l caffeine).

With o i l s such as corn o i l , o l i v e o i l , safflower o i l .

24

Coffee base*

Benzyl a l c o h o l

25

-

26

With supercr it carbon dioxide.

27

-

28

Green coffee*

Alcohols

29

Caffeine"

S u p e r c r i t carbon dioxide with water

30

Coffee and t e a

With s u p e r c r i t carbon dioxide.

31

Roast ed co f f ee* (aqueous extracts of)

With super c ri t carbon dioxide and recovery of aroma.

32

Raw coffee*

Superc rit carbon dioxide.

33

Coffee*

Propane and butane

34

Coffee*

Aqueous ( s o l u t i o n s )

35

Coffee"

Aqueous (steam)

36

I n s t a n t coffee Black tea* Tea waste

.

Table 9.

Plant Sources of Caffeine

Caffeine

Source Seeds (beans) of

Information about t h e p l a n t (theobromine) Native region con%ent ( 4 % ) Botanical c h a r a c t e r i s t i c s

0,l-0.8 (0.2-2.7)

Tree, 4-15 m i n height when growing w i l d , but pruned t o 6 m under c u l t i v a t i o n . F r u i t s furrowed l e a t h e r y pods, shaped l i k e l a r g e cucumbers and cont a i n i n g 20-25 cream coloured almond-shaped seeds.

Mexico, Central and South America

West Africa

0.8-2.4

C. arabica: Shrub o r s m a l l t r e e , 4-6 m i n height when growing wild but pruned t o 2-3 m under c u l t i v a t i o n . Fruits ellipsoidal berries ( c h e r r i e s ) , r e d t o . dark purplish, containing 2 s i l v e r y skinned seeds (Beans) within t h e pulp.

E a s t Africa

South America

Thdobroma cacao (stercdiaceae )

.

Seeds (beans) of

Coffea arabica, C. Ziberica, C. excelsa or C. robusta (Rubiaceae)

Main region of c u l t i v a t i o n

Continued Table 9 .

Source

Caffeine Information about t h e p l a n t (theobromine) content ( % ) Botanical c h a r a c t e r i s t i c s Native region

Seeds (beans) of

2-6

Large, climbing o r creeping p l a n t with smooth stem. F r u i t ovoid, nut-like, about as l a r g e as a grape and u s u a l l y containing 1 seed, t h e s i z e o f a hazel-nut, with white, mealy covering.

Amazon v a l l e y of Brazil

S t a t e of Amazons, Brazil.

2.7

Extensive, woody l i a n a . Stem s t o u t , up t o 12 cm i n diameter a t t h e base, with a milkywhite a s t r i n g e n t sap.

Colombia, Equador, Peru

Not c u l t i vated.

Tree, 20-30 m i n height when growing w i l d , 4-6 m under c u l t i v a t i o n . Leaves Persist e n t a l t e r n a t e , f i n e l y toothed at t h e margin and dark green i n colour.

Large a r e a i n v a l l e y s of t h e Parana, Paraguay and Upper Uruguay r i v e r s .

Paraguay and South of Brazil

Paul linia cupana (Sapindaceae)

Bark of Pau l linia yoco (Sapindaceae)

Leaves of 1.1-1.g I l e x paraguayensis ( Aqui f o l i a c e a e )

Main region of c u l t i v a t i o n

Continued Table 9.

Source

Caffeine Information about t h e p l a n t (theobromine) content ( I ) B o t a n i c a l c h a r a c t e r i s t i c s n a t i v e region

Leaves and shoots of

0.1-1.6

E a s t c o a s t of North America from V i r g i n i a t o Mexico.

Not c u l t ivat ed .

Shrub o r s m a l l t r e e , 9-15 m i n height when growing wild, about 1.5 m under c u l t i v a t i o n . Leaves a l t e r n a t e , e l l i p t i c a l on s h o r t s t a l k s , l e a t h e r y and w i t h t o o t h e d margins.

A s s a m , China, Japan.

S r i Lanka, I n d i a , China Jaoan .

C. acwninuta: Slender t r e e , 6-9 m t a l l , t r u n k commonly

Southern Nigeria

Xest Africa

( Aqui f o l i a c e a e )

Leaves of

3-4

(Theaceae)

Seeds ( n u t s ) 1.5-3.5 of Cola n i t i a k and C. acwninata (Stercdiaceae). Most o f t h e caffeine i n kolas not derived from t h e k o l a nut but added i n t h e form o f s y n t h e t i c caffeine.

of c u l t i v a t i o n

Shrub o r s m a l l tree, c l o s e l y r e l a t e d t o I. paraguayensis

IZex vomitoria

Camellia sinensis

Kain region

branching near t h e base. Bark rough and corky, grey i n colour. F o l i a g e s p a r s e , confined t o t h e t i p s o f branches. F r u i t c o n s i s t s of up t o f i v e f o l l i c l e s borne at r i g h t angles t o t h e s t a l k or s l i g h t l y downwards. F o l l i c l e s , russ e t , rough t o t h e touch. Each f o l l i c l e c o n t a i n s up t o 14 pink o r r e d seeds, covered with white s k i n . Cotyledons 3-6.

Continued Table 9.

Source

Caffeine Information about t h e p l a n t (theobromine) Native r e g i o n content ($) Botanical c h a r a c t e r i s t i c s

I n Africa, t h e seeds a r e used

+

0,

inainly as a mssticatory , but t h e y . Kay &so

be used

f o r preparat i o n of a drink (Cola).

c. nitida:

More r o b u s t t r e e ,

9-12 m h e i g h t , t r u n k unbranched for at least 1 m. Bark smooth w i t h f i n e l o n g i t u d i n a l cracks. Foliage dense, n o t c o n f i n e d t o t h e t i p s o f t h e branches. F r u i t c o n s i s t s o f up t o f i v e f o l l i c l e s , u s u a lly bent upwards. F o l l i c l e s g r e e n , smooth t o t h e t o u c h . Seeds up t o 1 0 i n number, w i t h 2 cotyledons and of t h e shape and s i z e of horse chestnuts

.

S i e r r a Leone, Ivory coast, Ghana

Main r e g i o n of c u l t i v a t i o n

West A f r i c a , West I n d i e s South America

MOHAMMAD UPPAL ZUBAIR ET AL.

102

4 . Synthesis 4.1 Route 1 Uric acid with methyliodide gave 1,3,7-trimethyluric acid, which was converted into 8-chloroderivative and its subsequent dehalogenation afforded caffeine

(38-40).

0

0

L% H

H N

CH31

)co

N H

Uric acid

0

C'H3 1,3,7-Trimethyluric acid

NaOH

>

CAFFEINE

103

I + . 2 Route 2

C a f f e i n e h a s a l s o been s y n t h e s i s e d from uric a c i d v i a a n o t h e r r o u t e (41-42).

0

~

~

0

;

>

~

~

~

)

;

H

;

c

H

o

-~2~~~ >L20

H

N H

H

H

0

0

NHCHo

HCOM12

0

H

hYICOFHCH0

MCONHCHO

0

H

0

0

I cH3

Caffeine

MOHAMMAD UPPAL ZUBAIR ET AL.

104 4 . 3 Route 3

A d i f f e r e n t r o u t e f o r t h e p r e p a r a t i o n of c a f f e i n e from

u r i c a c i d has been r e p o r t e d ( 4 3 ) . Uric a c i d is f i r s t c o n v e r t e d i n t o 8-methylxanthine by t h e a c t i o n o f a c e t i c anhydride (44-46), a p r o c e s s which i n v o l v e s t h e i n t e r m e d i a t e formation o f a d i a c e t y l d e r i v a t i v e . The secondary amino groups are t h e n methylated i n an a l k a l i n e s o l u t i o n t o g i v e 1,3,7,8-tetramethylxanthine. F i n a l l y , t h e methyl group o r i g i n a l l y i n t r o d u c e d i n t h e 8 - p o s i t i o n i s e l i m i n a t e d ( 47 )

.

I

H

I 0 -co*

I

CH3

Caffeine

105

CAFFEINE

4 . 4 Route 4 A l k y l a t i o n o f x a n t h i n e d e r i v a t i v e s has a l s o been c a r r i e d o u t f o r t h e s y n t h e s i s of c a f f e i n e . M e t h y l a t i o n o f theobromine g i v e s c a f f e i n e (48-50), w h i l e 1methylxanthine h a s a l s o been c o n v e r t e d t o c a f f e i n e v i a t h e o p h y l l i n e (48,49,51,52). 1,7-Dimethylanalogue ( p a r a x a n t h i n e ) on a l k y l a t i o n a f f o r d s c a f f e i n e ( 5 3 ) , w h i l e x a n t h i n e on t r e a t m e n t w i t h a n e x c e s s o f methylat i n g agent g i v e s r i s e t o a ' c a f f e i n e d e r i v a t i v e

(41,49,54,55). 4.5 Route 5 C a f f e i n e i s u s u a l l y commercially s y n t h e s i z e d by T r a u b ' s methods; ( A and B, g i v e n below: ) ( 56) (A)

CH3

o=

/N H - C H 3

HOC c + %I$ 'NH-CH~ CN'

k-.--.@ i%

NaN1i2

-

~

~

~

Z

n

/

H

2

i i . HCOOH, A

0

1h3 CH31/E t OH CH

a

NH2

1 a3

0

0

H C

0

> N

Caffeine

S

~

MOHAMMAD UPPAL ZUBAIR ET AL.

106

(B)

In this method 4-amino-5-formamido-l,3-dimethyluracil is cyclised and methylated in one step in sodium ethoxide containing methyliodide to afford initially theophylline and subsequently caffeine (57).

0

I cH3

0

CHz

L

(33

4.6 Route 6 It involves ring closure of 5-ethoxycarbonyl-1-methyl-

4-( N-methyl) ureidoimidazole, and subsequent methylation in alkaline medium gives caffeine (58).

(33 Caffeine

CAFFEINE

107

5. B i o s y n t h e s i s It h a s been r e p o r t e d ( 5 9 - 6 2 ) t h a t b i o s y n t h e s i s o f p u r i n e r i n g system probably proceeds i n n e a r l y t h e same manner i n a l l organisms as shown i n Scheme I . I n i t i a l l y , t h i s pathway was proposed f o r micro-organisms and animals b u t r e c e n t evidence s u g g e s t s t h a t it may a l s o hold good f o r h i g h e r p l a n t s . I n o s i n e monophosphate i s t h e f i r s t compound in t h i s b i o s y n t h e t i c pathway t o c o n t a i n a complete p u r i n e r i n g system (59) and it h a s a c e n t r a l p o s i t i o n i n It can be converted t o x a n t h o s i n e t h e purine m e t a b o l i s m . monophosphate i n an " I + dependent r e a c t i o n as shown below:

OH

RIBOSE

I n o s i n e monophosphate

R I BOSE-P

Xanthosine monophosphate

Xanthine, which o r i g i n a t e s from x a n t h o s i n e monophosphate by e l i m i n a t i o n o f phosphate and r i b o s e , i s t h e s t a r t i n g material f o r formation o f c a f f e i n e and o t h e r p u r i n e s . I n t h e p l a n t Coffea arabica, t h e x a n t h i n e is c o n v e r t e d , t o theobromine and t o c a f f e i n e , e i t h e r v i a N3-methylxanthine

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p-o'it=? OH

OH

5-Phosphoribosyl -- pyropkosphate

-1

H

H272 ,Cao ,!j

o=c, NH

5-Phosphoribosyl1- amine

-

a-N-Formylglycinamideribonucleotide

P

P-Ribose Glycinamide ribonucleotide

H H2r"'$ NH=C,

; .

NH

ZHN

I P-Ribose

I P-Ribose

H:'x:)

OH

dH

1 : )I

-

P-Ribose

N-Formylglycinamide 5-Aminoimidazoribonucleotide le-ribonucleotide

+

I

P-Ribose

I

P-Ribose

P-Ribose

5-Aminoimidazole- 5-Aminoimidazole-44-carboxylic acid N-succinocarboxamide ribonucleotide ribonucleotide

5-Aminoimidazole-4 -carboxamide ribonucleotide OH

P-Ribose 5-Formamidoimidazole-4carboxamide-ribonucleotide Scheme I.

Inosine-5-monophosphate

Biosynthesis of inosine monophosphate.

CAFFEINE

109

o r N 7-methylxanthine Scheme 11. The methyl groups o r i g i n a t e from methionine ( 6 3 - 6 5 ) .

0

I 0

3 H

CH

CHJ

N -Methylxanthine

CH3

R

I

3

0

I

H

Xanthine

\

The0bromin e

I CH3 Caffeine

N 7 -Methylxanthine Scheme 11. Formation of theobromine and c a f f i n e from xanthine.

6 . Pharmacokinetics and Metabolism The absorption of c a f f e i n e from t h e g a s t r o i n t e s t i n a l t r a c t i s r a p i d b u t i r r e g u l a r (67,68). It i s d i s t r i b u t e d i n various t i s s u e s of t h e body i n approximate proportion t o t h e i r water content ( 6 3 ) . ‘The absorption o f c a f f e i n e is pH-related, an i n c r e a s e i n pH increases i t s absorption ( 6 7 ) . Within t h e t i s s u e , c a f f e i n e i s r a p d i l y broken down (69), involving metabolite r e a c t i o n s , such as N-demethylat i o n and oxidation, and r i n g cleavage ( 6 7 ) . The drug metabolising enzymes of t h e l i v e r a r e stimulated following t h e ingestion o f l a r g e amount o f c a f f e i n e ( 6 7 ) . The degree o f c a f f e i n e degradation and degradation products excreted i n t h e u r i n e o f d i f f e r e n t species seems t o vary

4;

As pa rt ic acid

\

0 C

\

N/

C 1 Fragments

Fig. 12:

Fragments

H1-CHp-C 0 0 H Glycine

Amide N of g lut amin e

Origin of various atoms in the biosynthesis of caffeine.

CAFFEINE

111

considerably ( 7 0 ) . Blood l e v e l c o n c e n t r a t i o n i n four a n i m a l s p e c i e s , followinr: o r a l a d m i n i s t r a t i o n o f 25 mg/kF: [1-C1’I] c a f f e i n e , have been reported by Burger ( T O ) , (Table 1 0 ) . The plasma h a l f - l i f e f o r humans i s between 4 t o 10 hours ( 6 7 ) . Table 1 0 .

Plasma Concentration o f [1-C D i f f e r e n t A n i m a l Species.

14

] Caffeine i n

Rat

Hamster

Rabbit

Radioactivity

5.4

3.5

11.0

19.0

Caffeine

2.8

3.1

3.7

2.8

Radioac t i v i t y

0.1

0.5

0.7

0.9

Caffeine

0.1

0.1

0.7

-

18

18

22

Rhesus monkey

Half-life ( h )

Absorption h a l f time ( h )

Peak plasma conc e n t r a t i o n of c a f f e i n e (pg/ml).

13

Caffeine metabolism i n t h e rat l i v e r s l i c e s and p o s t n a t a l developing rats has been s t u d i e d (71). Rao e t al, (1973) have proposed a b i o t r a n s f o r m a t i o n r o u t e f o r c a f f e i n e i n rats (72) (Scheme 111).

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>--

Hydroxylationl

Caffeine ( unchanged, 9%)

Reduct'on

N-demet hylat ions

1,3,7-Trimethyldihydrouric acid (11.4%)

I.

dehydrogenation N-demethylations

1,3,7-Trimet hyluric acid (trace)

I

J. Theobromine

( 5.1%)

Paraxanthine (8.a%)

I

>

3-Methyluric acid (trace)

Oxidat ion

Theophylline (1.2%)

3,6,8-Trimethylallantoin (1.3%)

I

I I Oxidat ion

I I

V 1,6,8-Trimethylallantoic acid

I hydrolysis

I J.

Glyoxylic acid Met hylur ea 1,3-Dirnethylurea

Scheme 111.

Biotransformation of caffeine in rat.

113

CAFFEINE

An i n t e r e s t i n g sulphur containinE metabolite o f c a f f e i n e was i s o l a t e d from t h e u r i n e of mouse, r a t , r a b b i t and horse ( 7 3 ) , and i s shown i n Scheme I V .

0

ru

0

n

U

Caffeine

H3Nx-3

II

0

CH S-CHij 2-11

“i /

0’

N

I I1 Scheme I V .

I11 The sulphur metabolite of c a f f e i n e .

I n case o f humans, u s u a l l y 45% o f a dose is excreted i n u r i n e i n 48 hours as 1-methylxanthine (67) and 1-methyluric a c i d (67,74). Other breakdown products excreted i n t h e u r i n e include, t h e o p h y l l i n e , lY7-dimethylxanthine, 7-methylxanthine and 1,3-dimethyluric a c i d , along with some unchanged c a f f e i n e ( 6 7 ) , Further s t u d i e s on human metabolism of (1-methyl-14C) and (2-14C)caffeine have been reported (75,76). Radiolabelled c a f f e i n e was administered ( 7 5 ) o r a l l y a t 5 mg/kg t o a d u l t , male volunteers. Blood, s a l i v a , expired C02, u r i n e , and feces were analysed f o r t o t a l r a d i o l a b e l l e d equivalents of

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c a f f e i n e and i t s metabolites. High-performance l i q u i d chromatography (HPLC) was t h e main technique used f o r t h e separation of c a f f e i n e and i t s metabolites with quantitat i o n by l i q u i d - s c i n t i l l a t i o n counting. The h a l f - l i f e of c a f f e i n e i n both serum and s a l i v a was approximately 3 h r s . , with t h e amount of c a f f e i n e i n s a l i v a samples almost 65 t o 85% of t h a t found i n serum samples. The main metabolites found i n serum and s a l i v a were t h e dimethylxanthines, paraxanthine, theophyllinc and theobromine ( 7 7 ) . It has been reported (76) t h a t demethylation of t h e 3-methyl groups seems t o be t h e most important pathway i n man, and t h e plasma concentration of t h e dimethylxanthines depend on t h e i r urinary excretions a s w e l l as t h e i r own metabolism (76). Effect of c a f f e i n e on alcohol metabolism(78), basal metabolism (75) and i t s e f f e c t on exercise performance have a l s o been s t u d i e d (79). Caffeine metabolism i n sheep h a s a l s o been studied (80-82). 7. Methods o f Analysis 7 . 1 Elemental Analysis The elemental composition of c a f f e i n e i s :

Element

(83)

% Theoretical

C

49.48 %

H

5.19 %

N

28.85

0

16.48 %

7.2 I d e n t i f i c a t i o n T e s t s 1. Addition of t a n n i c a c i d s o l u t i o n t o c a f f e i n e

s o l u t i o n gives a p r e c i p i t a t e , which dissolves on f u r t h e r addition of t h e reagent ( 8 4 ) . 2. Addition of iodine s o l u t i o n and hydrochloric a c i d t o c a f f e i n e s o l u t i o n gives a brown p r e c i p i t a t e , which n e u t r a l i s e s on addition of sodium hydroc h l o r i d e (84).

3 . Reaction of c a f f e i n e w i t h potassium c h l o r a t e i n hydrochloric a c i d , and subsequent exposure t o ammonia, gives purple colour, which disappears on addition of a s o l u t i o n of a fixed a l k a l i (85).

115

CAFFEINE

4 . Addition of gold c h l o r i d e s o l u t i o n t o c a f f e i n e s o l u t i o n a f f o r d s small rods ( 8 6 ) .

5 . Addition of mercuric c h l o r i d e s o l u t i o n t o c a f f e i n e s o l u t i o n gives long needles ( 8 6 ) .

6. Comparison of t h e i n f r a r e d spectrum of t h e c a f f e i n e sample with t h a t of a reference s t a n d a r d i s a l s o employed ( 8 5 ) .

7.3 T i t r i m e t r i c Several methods have been reported f o r t h e analyses of c a f f e i n e by acid-base t i t r a t i o n procedures , e i t h e r by using d i f f e r e n t i n d i c a t o r s (87-100) o r by potentiometric methods (101-108), f o r t h e end-point d e t e c t i o n . An iodometric t i t r a t i o n method (88) w a s developed f o r

t h e determination of c a f f e i n e . It i s mixed with H2SO4 (1:l) and I B r s o l u t i o n , and t h e mixture i s d i l u t e d t o 100 m l . After f i l t r a t i o n , t o a p o r t i o n of t h e f i l t r a t e i s added K I s o l u t i o n , and t h e equivalent amount of iodine l i b e r a t e d i s t i t r a t e d with Na2S204, using s t a r c h s o l u t i o n as i n d i c a t o r . A blank is a l s o c a r r i e d out a t t h e same t i m e . Collado e t a l . , have reported a method (87) f o r t h e determination o f c a f f e i n e , phenazone, phenacetin and phenobarbitone, i n analgesic t a b l e t s . For t h e estimat i o n of c a f f e i n e , phenazone is p r e c i p i t a t e d with p i c r i c a c i d and f i l t e r e d o f f , and c a f f e i n e i s determined i o d i m e t r i c a l l y i n a c i d s o l u t i o n . Caffeine can be determined i o d i m e t r i c a l l y from icecream and cocoa admixtures (109,110). 40 g of t h e sample is heated with 1 0 m l o f water, and 1 ml of 15% aqueous NaOH, on a b o i l i n g water bath f o r 30 minutes. The mixture i s cooled and 3 m l of 30% Pb(N03)2 i s added. It i s d i l u t e d t o 100 m l with water and i s f i l t e r e d . 1 0 M l o f t h i s sample i s used t o determine c a f f e i n e t i t r imet ri c a l l y

.

The U.S. Pharmacopoeia1 method (108) c o n s i s t s i n dissolving about 400 m g of f i n e l y powdered c a f f e i n e i n 40 m l of a c e t i c anhydride. After mixing with 80 m l of benzene it i s t i t r a t e d with 0.1N p e r c h l o r i c a c i d , determining t h e end-point potentiometrically.

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Caffeine has been assayed f o r another non-aqueous t i t r a t i o n procedure ( 9 3 ) . It is dissolved i n w a r m benzene and a f t e r cooling, couple o f drops of a s u i t able i n d i c a t o r , such as Sudan I V o r Nile b l u e A , are added before t i t r a t i o n with 0.1N HCL04 i n g l a c i a l a c e t i c acid. Caffeine i n cacao s h e l l s has a l s o been determined (100) by non-aqueous t i t r i m e t r i c method. About 2 g of t h e ground and de-fatted sample i s mixed with magnesium oxide and water. The mixture i s heated on a water bath f o r 30 minutes and then it i s e x t r a c t e d i n t o chloroform i n a soxhlet apparatus. The chloroform e x t r a c t is d r i e d at lO5'C f o r 25 minutes and t h e r e s i d u e is first t i t r a t e d f o r t o t a l theobromine and c a f f e i n e , and t h e n f o r theobromine only.

7.4 Spectrophotometric 7.4.1 Colorimetric A sample containing 100 mg i s dissolved i n 100 m l o f water and i s f i l t e r e d (111). A n a l i q u o t o f 5 m l i s d i l u t e d t o 25 m l and 1 ml of d i l u t e hydrochloric a c i d , and 1 m l of 10% molybdophos-

phoric a c i d are added t o it. The mixture after heating on a water b a t h i s c h i l l e d i n i c e and centrifuged. The p r e c i p i t a t e is washed and dissolved i n 25 ml o f acetone and t h e e x t i n c t i o n is measured a t 440 mu. The r e s u l t s with compounded tablets show recoveries o f c a f f e i n e , 100.2% o f t h e o r e t i c a l amount. C o r t e ' s method (112,113) c o n s i s t s i n l i b e r a t i n g c a f f e i n e w i t h concentrated H2SO4, followed by e x t r a c t i o n w i t h CHC13. It i s then estimated by a modified c o l o r i m e t r i c method involving formation o f i t s periodide ( 1 1 4 ) . I n another method (115) c a f f e i n e (110 t o 400 pg) i s dissolved i n a f r e s h l y prepared s o l u t i o n of 0.5 m l of acetylacetone and 5 m l o f 2N NaOH. T h i s mixture i s heated at 80°C, and a f t e r cooling, a s o l u t i o n o f p-dimethylaminobenzaldehyde and 20 m l o f conc. H C 1 are added. The mixture i s f u r t h e r heated at 8OoC, and after cooling, 10 m l o f water i s added and e x t i n c t i o n of t h e r e s u l t i n g blue s o l u t i o n i s measured a t 615 mu.

117

CAFFEINE

E f f o r t s t o optimise conditions (116) have r e s u l t e d i n a n improved colorimetric method (117) f o r t h e estimation of c a f f e i n e . A small amount of c a f f e i n e is dissolved i n a mixture of 3% aqueous a c e t i c a c i d s o l u t i o n , and 10% aqueous pyridine s o l u t i o n . The mixture i s then made up t o 18 m l with water and 2 ml of N a O C l s o l u t i o n i s added. Add 2 m l of 0.1N Na2S203, followed by 3 m l of 1 N N a O H , s o l u t i o n and d i l u t e t o 50 ml with water. The e x t i n c t i o n , i n 4 cm c e l l i s measured a t 460 mp.

Iodine has a l s o been employed as a c o l o r i m e t r i c reagent f o r t h e determination of c a f f e i n e (118). 3N i o d i n e (1 ml) s o l u t i o n i s mixed with 1% s o l u t i o n of c a f f e i n e ( 5 m l ) followed by 50% H2SO4 ( 0 . 5 m l ) and i s set a s i d e f o r 1 0 minutes. The p r e c i p i t a t e i s f i l t e r e d and washed before dissolving it i n acetone. Its e x t i n c t i o n i s measured a t 525 mp. A colorimetric method f o r t h e determination of c a f f e i n e i n pharmaceutical preparations has been developed (119-121). The t e s t s o l u t i o n i s t r e a t e d with an equal volume of 10N NaOH a t 12OoC t o convert c a f f e i n e i n t o c a f f e i d i n e . It i s t h e n coupled with d i a z o t i s e d s u l p h a n i l i c a c i d and t h e e x t i n c t i o n is measured a t 451 mp. Other drugs present i n usual c a f f e i n e pharmaceutical preparat i o n s do not i n t e r f e r e .

Caffeine present i n beverages can a l s o be estimated c o l o r i m e t r i c a l l y (121-122). It i s e x t r a c t e d from t h e beverage by t h e known (123) method and is t r e a t e d with 296 s o l u t i o n o f malonic a c i d i n a c e t i c anhydride. The mixture i s heated a t 90°C, and a f t e r cooling it i s made up t o 25 m l with methanol. The e x t i n c t i o n of t h e r e s u l t i n g greenish-yellow s o l u t i o n i s measured a t 430 nm.

7. 4.2 U l t r a v i o l e t Spectrophotometric methods f o r t h e estimation of c a f f e i n e i n pharmaceutical preparations and i t s mixtures with o t h e r drugs and compounds have been developed (124-129). These i n v a r i a b l y involve

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preliminary separation of c a f f e i n e , before i t s spectrophotometric estimation. An i n d i r e c t spectrophotometric method f o r t h e

determination of c a f f e i n e i n quaternary mixtures such as NaOBz, p-EtoC6H)+NHAc, aminopyrine and c a f f e i n e has been reported (1.29). The percentage of these components can be estimated by a compact c a l c u l a t i o n scheme a f t e r determination of e x t i n c t i o n c o e f f i c i e n t s a t t h e given wavelengths. Several u l t r a v i o l e t spectrophotometric methods have been developed f o r t h e estimation of c a f f e i n e i n coffee and decoffeinated coffee ( 130-136), t e a ( 137-141) and beverages (142-146). Caffeine can a l s o be determined i n b i o l o g i c a l f l u i d s spectrophotometrically (147). O.1N-NaOH i s used t o e x t r a c t c a f f e i n e , and a mixture of s o l i d N a C l and Na2SOb is added t o enhance t h e e x t r a c t i o n o f c a f f e i n e i n t o ether-chloroform. The c a f f e i n e i s then t r a n s f e r r e d t o an aqueous a c i d i c s o l u t i o n , t h e s o l u t i o n i n t h e reference c e l l i s made strongly a c i d , and t h e contents of t h e sample c e l l are adjusted t o pH 1.3.

7.4.3 Infrared I n f r a r e d spectrophotometric method has been used (148,149) f o r t h e a n a l y s i s of caffeine. A procedure f o r t h e determination of c a f f e i n e i n pharmaceutical preparation containing aminopyrine and phenacetin has been reported (148), and t h e region 650 t o 400 cm-l o f t h e spectrum, i n KBr d i s c , w a s used f o r r a p i d and simultaneous determination of t h e t h r e e components.

7.4.4 Nuclear Magnetic Resonance NMR method f o r t h e a n a l y s i s of a s p i r i n , phenacetin and c a f f e i n e i n pharmaceutical preparations has been reported (150). The method involves comparing t h e i n t e g r a l s of t h e CH2-signal ( a t 6 ppm) of piperonaldehyde, used as an i n t e r n a l standard, w i t h t h o s e of a s p i r i n methyl s i n g l e t (2.3 ppm) , A simultaneous q u a n t i t a t i v e

119

CAFFEINE

phenacetin, e t h y l t r i p l e t ( a t 1 . 3 ppm) and c a f f e i n e methyl s i n g l e t ( a t 3.4 ppm). The average percent recoveries and standard deviations were 95.61 f 0.37, 96.34 k 0.47 and 101.26 -+ 1.46 f o r a s p i r i n , phenacet i n and c a f f e i n e respectively. MMR-shift technique i n forensic chemistry f o r t h e analyses of mixed samples of c a f f e i n e has been reported (151). NMR s t u d i e s o f metal porphyrin c a f f e i n e complexes (151) and associat i o n o f c a f f e i n e with sodium benzoate have a l s o been reported (152,153).

7.4.5 Mass Spectrometric A new and simple a n a l y t i c a l method using d i r e c t -

i n l e t chemical i o n i s a t ion mass spectrometry, has been reported (154) f o r simultaneous determinat i o n of c a f f e i n e and o t h e r components of a n a n t i cold drug. These components were detected as quasimolecular i o n s , and were q u a n t i t a t e d by using an accumulation program. 2

I n another method (155) B E = constant, l i n k e d scan has been used as a t o o l f o r q u a n t i f i c a t i o n s with reversed-geometry mass spectrometers. Dl a b e l l e d analogs may be used as i n t e r n a l standards, t h u s providing very simple clean-up procedures. The method i s applicable t o c a f f e i n e present i n beverages.

Caffeine w a s analysed together with o t h e r drugs l i k e a c e t y l s a l i c y l i c a c i d and phenacetin by mass spectrometry (156).

7.4.6 Photo-nephlometric Caffeine can be estimated i n t e a by using photonephlometer (157). . A c a l i b r a t i o n curve i s obtained by mixing 2 ml o f 0.01M sodium tungstophosphate, 4 m l of 2.5M HNO3 and 0.5 t o 2.5 m l of 0.001M c a f f e i n e s o l u t i o n . It i s t h e n d i l u t e d . t o 50 m l and examined i n a photo-nephlometer. Other compounds present i n tea do not cause any i n t e r f e r e n c e . With some modifications, t h i s

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120

procedure can be used f o r t h e determination of c a f f e i n e i n presence of various d r u g s .

7.5 Coulometric Kalinowska has used coulometric technique f o r t h e of estimation of c a f f e i n e (158-162). About 0.05 c a f f e i n e i s dissolved i n hot water, and then mixed w i t h 6 m l o f 30% KOH. The mixture i s f u r t h e r heated i n a b o i l i n g water b a t h , a f t e r cooling and d i l u t i o n w i t h water, it i s n e u t r a l i s e d with hydrochloric a c i d . About 1 m l o f t h i s s o l u t i o n i s used f o r t h e determinat i o n o f c a f f e i n e with coulometrically generated c h l o r i n e , with a c u r r e n t o f 5 mA. The end-point i s determined by t h e dead-stop end method. The c o e f f i c i e n t o f v a r i a t i o n w a s about 0.5%. 7.6 Polarographic Caffeine, although not reducible a t dropping-mercury e l e c t rode can be determined polarographically a f t e r i t s oxidation w i t h bromine ( 1 6 3 ) . The wave h e i g h t s of t h e r e s u l t i n g s u b s t i t u t e d parabanic a c i d s are then evaluated. This method i s q u i t e s e n s i t i v e and t h e results obtained are within +- 3%. B a r b i t u r a t e s , a c e t y l s a l i c y l i c a c i d , ephedrine, codeine, papaverine and d i g i t o x i n do not i n t e r f e r e ; however, phenazone, phenacetin and amidopyrine should be removed b e f o r e t h e reaction. In another method (164), t h e c a f f e i n e i s analysed by anodic d i f f e r e n t i a l phase voltametry a t a glassy C e l e c t r o d e , t h e a e t e c t i o n l i m i t i s 0.5 ppm at pH 1.2. Oxidation products o f c a f f e i n e are i d e n t i f i e d by cathodic d i f f e r e n t i a l pulse polarography.

7.7 Ring-Oven

(Micro)

A r e l a t i v e l y s p e c i f i c colour r e a c t ion between chloro-

form e x t r a c t o f c a f f e i n e , a l k a l i n e acetylacetone s o l u t i o n and a c i d p-dimethyl aminobenzaldehyde solut i o n , has been used for t h e micro determination o f air-borne p a r t i c l e s of c a f f e i n e . It i s possible t o determine 0.5 pg of c a f f e i n e by t h i s method with a mean e r r o r of -+ 3% (165).

Table 11. Parameters Used f o r Paper Chromatography of Caffeine

No.

support

Developing Solvent

Detection

Ref.

1.

S & S. 2043

Consisted of s a l i c y l i c a c i d ( 0 . 3 g) dissolved i n butanol ( 30 m l ) ; t h e solut i o n being t r e a t e d dropwise with H20, t o t h e f i r s t turbidity.

Chromatogram immersed i n 1% AgNO3 d r i e d and immersed i n 0.5% aqueous K 2 C r 2 0 p .

169

W light

170

--

171

Paper impregnated with ethanolic s a l i c y l i c acid ( 0 . 3 g i n 30 ml) and dried. 2.

Whatman DE20 Anion-exchange paper.

Develop with 0.2N-aq. ammonia f o r 105 minutes.

3.

Whatman No. 4 paper

Mobile phase w a s a xylenetetralin-n-amyl alcohol mixture and t h e s t a t i o n a r y phase w a s water a c i d i f i e d t o pH 3.2.

MOHAMMAD UPPAL ZUBAIR ET AL.

122

7.8 Radioactive Isotopes Caffeine can be estimated (166) by means of isotoped i l u t i o n a n a l y s i s with [1-14C] c a f f e i n e . It i s prepared by methylation o f theobromine with [ C14]methyliodide. About 1 0 mg o f t h e p u r i f i e d sample is subjected t o combustion i n Baker's apparatus, using t h e Van Slyke-Folch reagent. The l i b e r a t e d C02 i s absorbed i n 0.25N-NaOH and 1.88% BaC12 s o l u t i o n is added. The p r e c i p i t a t e d Ba2CO3 i s f i l t e r e d o f f and i t s s p e c i f i c r a d i o a c t i v i t y i s measured with a Geiger-Muller tube. 30-80 Mg o f c a f f e i n e , even i n presence of a c e t y l s a l i c y l i c a c i d , theobromine, aminophenazone o r phenacetin give s a t i s f a c t o r y r e s u l t s . Noakes has reported (167) another radiocarbon measurement method f o r t h e estimation of natural-product p u r i t y i n case of c a f f e i n e .

7.9 Radiochemical Caffeine was a l s o analysed by using r a d i o a c t i v e t r a c e r techniques (168). It i s p r e c i p i t a t e d from t h e s o l u t i o n of t h e sample i n hydrochloric a c i d with P3*-labelled phosphomolybdic a c i d , and after f i l t r a t i o n , t h e t r a c e r I-phosphomolybdate i s dissolved i n acetone and c o l l e c t e d i n a sample g l a s s holder. The a c t i v i t y of each p r e c i p i t a t e i s determined and t h e amount calcul a t e d from a standard curve. The method i s simple and gives reproducible results with s m a l l amount of samples. 7.10 Chromatographic Methods

7.10.1 Paper Chromatoaraphy Some paper chromatographic systems used f o r t h e determination o f ' c a f f e i n e have been summarised i n Table 11. 7.10.2 Column Chromatography Various column chromatographic systems used (175-182) f o r t h e determination o f c a f f e i n e are presented i n Table 12. I n another method (182) column chromatographic technique has been employed i n i t i a l l y f o r t h e

Table 12.

Summary of Conditions Used f o r t h e Column Chromatography of Caffeine ~

support I.

Eluent

Celite 545 and 4 N &SO4 covered by a l a y e r of a mixture of c e l i t e 545 and N aq. NaHCO3.

11. Basic aluminuim oxide

111.

~~

~~

-~

Detect ion

~~

Sample

Ref.

Pharmaceuticals and c o f f e e

173

( i ) Extinction o f t h e e l u e n t i n C H C l measured a t 257, 27? and 297 m u . ( i i ) For determination i n aqueous s o l u t i o n t h e e x t i n c t i o n i s measured a t 250, 273, and 296 mu.

Coffee and i t s mixtures

174

276 mu

Beverage and tablets.

175

-

Mixture o f C H C 1 3 and diethyl ether.

Alumina (previousl y a c t i v a t e d at 800°C f o r 6 h r s . ) i n a 100 m l burett e

( i ) CHC13

( i ) Celite and ( i i )Column of c e l i t e containi n g 4N-H2S04.

( i ) Etyyl ether ( i i )CHC13

( i i ) H20

.

IV.

~~

-

Continued Table 1 2 .

V.

VI. VII.

VIII. I-

N l h

support

Eluent

Detection

Mgo-Celite 545 (l:l,w/w)

H2°

272

Celite 545

CHC13

Dowex 50W-X2

40% aqueous methanol

Multiple column containing segment s , ( a ) C e l i t e 545 ( 3 g) and 1% tartaric acid soh. (b) Celite 545 ( 3 g) and 12% H2SO4 s o l n . ( 3 ml), ( c ) C e l i t e 545 ( 2 g) and 8.4% NaHCO3 s o l n . ( 2 ml) and

Elute phenacetin with ether-CHClg (8:l) 50 m l . Dismantle t h e column e l u t e a s p i r i n from segment ( c ) and determine caffeine

(a)

Celite 545 ( 3 g) and 22.1% K3P04 soln. ( 3 m l ).

.

Sample

Ref.

Caffeine-containing and caffeine-free coffee

176

276.5 my

Coffee and decaffeinated c o f f e e

177

273

Pharmaceuticals

178

Pharmaceutical combin a t i o n of a s p i r i n , phenacetin, and caffeine

179

--

my

my

Continued Table 12.

IX.

support

Eluent

Detection

Sample

Ref.

Polyamide (0.5 g)

A f t e r passing perculate, t h e caffeine i s washed w i t h H20, 2 x 3 10 ml.

272 nm

Tea

180

H20-methanol (19:l)

254 nm

S o f t d r i n k s and food

181

x

X.

S i l i c a gel (partic l e s i z e 0.04 to 0.063 mm) i n

column (30 cm X 1 cm)

126

MOHAMMAD UPPAL ZUBAIR ET AL.

i s o l a t i o n of c a f f e i n e from a sample of decaffeinated c o f f e e , followed by i t s t i t r a t i o n w i t h 0.01M H C l O 4 using methyl v i o l e t as indicator. 7.10.3 Thin-Layer Chromatography A summary of some of t h e TLC systems i n v e s t i -

gated f o r t h e a n a l y s i s of c a f f e i n e a r e given i n Table 13. Combined TLC colorimetric/spectrophotometric methods f o r t h e determination of c a f f e i n e i n pharmaceutical preparations and beverages, have been reported (183,184). The method i s based on t h e spectrophotometric estimation of t h e q u a n t i t a t i v e l y e l u t e d c a f f e i n e from TLC p l a t e s a f t e r separation from accompanying substances. TLC and high-performance TLC (1985) followed by densitometry has a l s o been employed f o r t h e determination of c a f f e i n e i n drugs ( 186,187 ) , beverages (185,188,189) and c o l a seeds (189). The s o l u t i o n from the aspirin-phenacetin-caffeine t a b l e t s i s chromatographed on Whatman chemically bonded K C 1 8 reversed-phase p l a t e s containing fluorescent phosphor and using 1:l MeOH-O.5M N a C l system (186). Caffeine w a s then determined by using a densitometer.

-

7 .lo.4 Gas Liquid Chromatography Gas l i q u i d chromatographic methods have been

employed f o r t h e estimation of c a f f e i n e , and v a r i a b l e parameters used f o r some o f these a r e summarised i n Table 1 4 . 7.10.5 High Performance Liquid Chromatography High pressure l i q u i d chromatography HPLC method has wide a p p l i c a t i o n f o r t h e estimation o f c a f f e i n e i n host o f d i f f e r e n t samples. A summary o f v a r i a b l e parameters i n a few cases is given i n Table 15.

Table 13. Summary of Conditions Used f o r t h e TLC o f Caffeine -

support

Mobile phase

Detection

Sample

Ref.

Siluf ol

W 254 nm

Drugs

190

K i e s e l g e l 60F254 ( l a y e r t h i c k n e s s 0.2 mm) or C e l l d o s e Fpj4 ( l a y e r t h i c k n e s s 0.1 mm)

Spect rophotometrica l l y

Caffeine

191

Coffee

192

-

K i e s e l g e l GF 2 54

CHC13-cyclohexanea c e t i c a c i d (8:2:1)

Silica gel G

Spray w i t h 2% HgC12 s o l u t i o n containing 1 0 mg of methyl r e d p e r 100 m l . or

W 254 nm

193

Sprayed w i t h 2% HgC12 s o l u t i o n d r i e d and sprayed w i t h K I s o l u t i o n . K i e s e l g e l 60

Chloroform-acetone (9:l)

Chromatograms evalua t e d by remission dens i t o m e t r y at 273 nm w i t h a double beam

Blood

194

Continued Table 13 support

Mobile phase

Detect ion

Sample

Ref.

Cyclohexane-acet one

Determined by t h e i r quenching e f f e c t on t h e background fluorescence.

Drugs

195

Drugs

196

~

Kieselgel F

254

Silica gel G

(4:5)

--

(powders)

Table 1 4 .

Summary of Conditions Employed f o r t h e GLC o f Caffeine

Column

support

31,

Chromosorb W

OV-17

Mesh

-

Chromosorb WHP

SE-30

Chromosorb G

-

Temp. 210oc

Flow (ml/min) C a r r i e r gas

Sample

Ref.

197

Argon Flavor a n a l y s i s and beverages

198

N2(30 ml/min) (flame ionisation detector).

Drug

199

2.0m

280Oc o r N2(16 ml/min) (flame ( 310°C ionisation detector). i f sample contains papaverine)

Drug

200

1.5 glass

190°C

Drug b i o l o g i c a l material

201

0.3 m

25OoC

1.0 m

2 0 0 ~ ~

80-100

2.0 m

180Oc

-

Tenax GC o r

5% OV17

Length

c

Varaport 30

Chromosorb W

3% Dexsil 300

3.5% s i l i cone SE-30

N2(55 ml/min) H-flame i o n i s a t ion

0 (u

rl I 0

0 rl

L-

I

d

i3 m

bp

m

0 N

I

u

0

0 a3 rl

I

I

130

Table 15.

~

W,

Summary o f HPLC Conditions f o r t h e Determination o f Caffeine

Column

Mobile phase

Flow (ml/min)

Partisif 10 SCX

Aqueous s o l u t i o n of 15 mM potassium c i t r a t e , pH 3.0 and 10% (v/v) methanol

1.1 d m i n

lJ Bondapak C18, 1 0 pm

MeOH+Wat er+HOAc ( 20+79+1)

Retent i o n time (m,in)

-

Detect i o n

Sample

Ref.

U.V.

Tea and c o f f e e

204

U.V.

Cocoa and chocolate products

205

U.V. (254 nm)

B 1ack t e a infusion

206

U.V.

Purine alkaloids

207

(270 nm)

U.V.

Animal d i e t s

208

(280 nm)

particle size MeOH-O.lM, pH 7:0, citratephosphate b u f f e r (20 :80)

2a h i n

Corasil I or C o r a s i l I1

Hept ane+ethanol

300 p . s . i .

Cl8 ( R a d i a l Pak A ) c a r t ridge

Wat er+MeOH+HOAc (74 :25: 1)

Bondapak

C18

(10:l).

3.0 ml/min

10

(280 nm)

Continued Table 15.

Mobile phase

Bondapak

0.025 M NaH2P04 i n MeOH+wat e r (2:3) t o PH 7 using NaOH

2 mUmin

Spherosil XOA 600 and XOA 800

Iso-octane+diisopropyloxide+MeOH+ t r ie thylamine+ water (34.93+49.51+ 14.58+0.20+0.78)

1d/min

Silica gel (M&N Nucleosil 50-5.5 IJM)

Methylene chloride: ethanol : water

50ml h-l

PhenylfCoras i l and C o r a s i l I1

Acetonitrile-water; 1,4 dioxan-wat er ; methanol-water; 1-4-dioxan-ac e t on i t r i l e - w a t e r and various mixtures of chloroform with methanol

C18

+,

Flow (ml/min)

Column

to 0

Retention time (min)

Detect ion

Sample

Ref.

0.48

U.V.

Drugs

209

Drugs

210

Caffeine

211

Drugs

212

(254 and

280 nm) Aprox 4 min

-

.

-

U.V. (280 nm)

(936:47:17)

-

-

-

Continued Table l>.

Colunln

Mobile phase

Zipax SCX

O . O M HNO

--

~

Flow ( d / m i n )

4 d/min

3

5% G l a c i a l a c e t i c acid Chloroform

1.1 Bondapak

Methanol-vat er

U.V.

Coffee

213

u-Y.

Beverages

214

-

-

-

Beverages

215

1.0 d / m i n

-

Plasma

216

Beverages

217

Decaffeinated , i n s t an t coffee and o t h e r beverages

218

Appetite/suppr e s s a n t formulations

219

U.V. (273 nm)

(8:92)

-

-

c18 SpherisorbODS w i t h gradiant elution 10-pM P a r t i s i l ODS-2

Ref.

-

(30:70) MeCN-Water

Sample

-

0 0

Bondapak

Detection

(254 nm)

Bond e l u t , C 1 8 bonded silica

c18

Retention time (min)

MeCN-H20 ( 3 0 : 7 0 )

-

-

U.V.

-

U.V.

(272 nm)

(254 nm)

Continued Table 15.

Column

Mobile phase

Flow (ml/min)

YWGCH200x

50% s o h . of MeOH i n 0.002M K2HPO4 a d j u s t e d t o pH 8.0

1.0 ml/min

5mm

Retention time (min)

Detection

Sample

Ref.

U.V.

Drugs

220

-

221

-

222

(254 nm)

by H3P04. Hypersil +

x

5% Isopropanol dichloromethane

Ion 25% aqueous e t h a n o l exchange r e s i n , DowexAG 50w-x8( H+) Nucleosil-

5 C18 Finepak gel-110 or Finepak SIL-CIS

-

U.V. (280 nm)

15ml/hour

U.V. (254 nm)

MeCN-H20-HOAc ( 13:87 :1)

223

MeOH-NH4OH ( 99 :1 and MeCN-NH40H ( 99 :1)

224

Continued Table 15.

c

Column

Mobile phase

Bondapak C18/corasil and LI Bondapak

0.01M sod. acetate buffer, pH 5.0-MeOHt et r ahydro f u r an

C18

(95:h:l)

Radial-Pak C 1 8 reversed phase column

Acetonitrile i n 0.1 mol/L potassium phosphate b u f f e r , pH 4.0 (9.5/90.5 by vol. )

Flow ( d / m i n )

3 mUmin

Retention time (min)

-

Detection

Sample

Ref.

U.V.

Umbil i c a 1 cord plasma

225

Biological fluids ( neonates )

226

Various drugs sample s including caffeine

227

(254 nm)

-

.

Containing H20, acet o n i t r i l e H3PO4 and NaOH with o r without 5C8RAC ( Radial-PAK LI hexylamine Bondapak C 1 8 cartridge, HS/5C18 o r HS/3C18 o r partisil 1O-ODS- 3 Part i s i l 50DS-3RAC o r

-

-

Continued Table 15. Column

Mobile phase

Shodex

Aqueous 70% methanol at 4OoC.

D-814

Flow (ml/min)

Retention time (min)

-

(50 cm X

8 mm) Radial-

PAK C18

0.M-phosphat e b u f f e r (pH 4) acetonitrile

Detection

Sample

Ref.

270 nm Horse u r i n e and by d i f ferent i a l refr a c t ometr y

228

254 nm

Caffeine and t heophylline r eonat e s

229

U.V.

Caffeine , a s p i r i n and phenacet i n

230

254 nm

Metabolites i n Umbilical cord plasma o f bovine

2 31

(181:19). Micro packed fused-silica, used 1 0 and 5 cm columns were used. p Bondapak C 1 8 (30 cm x 3.9 cm) with Bondapak C18/ Corasil guard column

-

1 0 m M-Na a c e t a t e buffer o f pH 5 methanol-t e t rahydrofuran

(95:h:l).

-

3

137

CAFFEINE

8.

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Acknowledgements The authors would l i k e t o thank M r . Mohammad Saleem Mian of t h e Pharm. Chem. Dept., f o r h i s a s s i s t a n c e i n t h e l i t e r a t u r e review and Mr. Khalid N.K. Lodhi of t h e Central Laboratory of t h e College of Pharmacy, King Saud University f o r h i s t e c h n i c a l a s s i s t a n c e .

COCAINE HYDROCHLORIDE

FAR10 J. MUHTAUZ and ABDUllAH A. Al-BAUR

1.

2.

3. 4.

5. 6.

7. 8.

Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Oder and Taste Physical Properties 2.1 Melting Range 2.2 Solubility 2.3 Optical Rotation 2.4 X-rays Diffraction 2.5 Spectral Properties Isolation of Cocaine Synthesis of Cocaine Biosynthesis of Cocaine Metabolism of Cocaine Pharmacokinetics Drug Stability

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 15

151

Copyright 8 1986 by the American Pharmaceutical Association All rights of reproduction in any form reserved.

FARID J. MUHTADI AND ABDULLAH A. AL-BADR

152 9.

Methods of Analysis 9.1 Identification Tests 9.2 Microcrystal Tests 9.3

Titrimetric Methods

9.4 9.5 9.6

Spectrophotometric Methods Counter-Current Extraction Chromatographic Methods

9.7

Radio-immunoassay

References

COCAINE HYDROCHLORIDE

153

1. Description 1.1 Nomenclature

1.1.1 Chemical Names

-

[ 1R - (exotexo ) 1-3- (Benzoyloxy) -8-methyl-8azabicyclo [ 3.2.11 octane-2-carboxylic acid methyl ester.

-

( - ) 3-(Benzoyloxy)-8-methyl-8-azabicyclo [ 3.2.11 octane-2-carboxylic acid methyl ester [l(R), 2(R), 3(s)1.

-

3 &Hydroxy-1 a H, 5 a H-tropane-2%-carboxylic acid methyl ester benzoate.

-

8-Azabicyclo [3.2.1] octane-2-carboxylic acid 3-(benzoyloxy)-8-methyl-methyl ester hydrochloride [lR-(exo-exo)l.

- 2 f3-Carbomethoxy-3~-benzoxytropane. - Methyl-3 B-hydroxy-1 a H, 5 a-tropane-2 6carboxylate, benzoate (ester) hydrochloride.

-

(lR, 2R , 3 S , 5S)-3-Benzoyloxy-2-methoxy carbony1 tropanium chloride.

-

(lR, 2R , 3s , 5S)-2-Methoxycarbonyl-tropane-3yl-benzoate

.

- 2(R) -Carbomethoxy-3( S) - ( - )-benzoxy-1 ( R ) tropane. 1.1.2 Generic Names Cocaine; 1-Cocaine; %-Cocaine; Benzoylmethylecgonine; Methylbenzoylecgonine; Ecgonine methyl ester benzoate. Cocaine hydrochloride; Cocaine muriate; N6urocaine hydrochloride.

FARID J. MUHTADI AND ABDULLAH A. AL-BADR

154 1.2 Formulae

1.2.1 Empirical C17H21N04

for cocaine.

C H NO C1 for cocaine hydrochloride. 17 22 4

1.2.2 Structural

/CH3

H The structure of cocaine was confirmed by the total synthesis of cocaine which was achieved by several authors (1-3). 1.2.3 CAS Registry Numbers

[50-36-2] for cocaine. 153-21-41 for cocaine hydrochloride.

1.2.k Wiswesser Line Notations

T 56 A ANTJ A - F V O l GOVR - & GH LV (Cocaine hydrochloride)

(4).

1.2.5 Stereochemistry The ecgonine moiety of cocaine possesses four

155

COCAINE HYDROCHLORIDE d i s s i m i l a r c h i r a l c e n t r e s a t C 1 , C2, C 3 and c5 and so t h e r e a r e e i g h t p a i r s of enantiomers p o s s i b l e .

H Since, however, only t h e c i s f u s i o n of t h e n i t r o g e n bridge i s p o s s i b l e i n p r a c t i c e , C 1 and C5 t h e r e f o r e have only one c o n f i g u r a t i o n ( t h e c i s form), and so t h e r e are only f o u r p a i r s of enantiomers a c t u a l l y p o s s i b l e , t h r e e p a i r s of which have been prepared s y n t h e t i c a l l y (5). The r e l a t i v e c o n f i g u r a t i o n s of cocaine and Ycocaine have been e x c l u s i v e l y determined by chemical methods (6-11).

N

J

%

COOCH3

CH 65 Cocaine

(-1

Cocaine

H

0 If

O-C-%H

5

Y -Coc a i n e

The a b s o l u t e c o n f i g u r a t i o n of (-)-cocaine w a s e s t a b l i s h e d by t h e following methods. a ) C o r r e l a t i o n w i t h L(+)-glutamic a c i d through ecgoninic a c i d ( 1 2 ) . N-CH3

L

( - ) Ecgonine COOH

(-IEcgoninic a c i d

FARID J. MUHTADI A N D ABDULLAH A. AL-BADR

156

L(+) glutamic acid H

H C02H

COOH

H

CONH2 OH-

(-1

CN

CN

ecgoninic acid

b ) X-ray crystallographic study of 1-cocaine hydrochloride (13). c) The stereoselective synthesis of dl-cocaine (3).

From the above data the absolute configuration of (-)-cocaine is presented in Fig. 0.

157

COCAINE HYDROCHLORIDE

Fig.0 Perspective view of the molecule of cocaine ( 1 3 )

.

158

FARID J. MUHTADI AND ABDULLAH A. AL-BADR

1.3 Molecular Weight 303.35 (cocaine) 339.81 (cocaine hydrochloride )

1.4

Elemental Composition C , 67.30%; H , 6.98%; N, 4.62%; 0 , 21.10%. (cocaine) C , 60.08%; H , 6.53%; N, 4.12%; 0 , 18.83%; C 1 , 10.43%. (cocaine hydrochloride)

1.5

Appearance, Color, Odor and Taste Colorless c r y s t a l s o r a white c r y s t a l l i n e powder, odorless, s l i g h t l y v o l a t i l e . It has a b i t t e r t a s t e , numbs tongue and l i p s . (Cocaine). Colorless granule c r y s t a l s , or a white c r y s t a l l i n e powder, odorless and hygroscopic. It has a s l i g h t l y b i t t e r t a s t e , numbs tongue and l i p s . (Cocaine hydrochloride).

1.6 Dissociation Constant pKa a t 15' = 5.59(cocaine); pKa 8.4 (cocaine H C 1 )

1.7 Loss

on Drying

When d r i e d t o constant weight a t 80' l o s e s not more than 0.5% of i t s weight. ( 1 4 ) . 2.

Physical Properties 2.1

Melting Range

lg70

About 195' 2.2

(14

(15

] cocaine hydrochloride

Solubility

One gram dissolves i n 600 m l water, 270 m l water a t 80°, 6.5 ml alcohol, 0.7 m l chloroform, 3.5 m l e t h e r , 12 ml o l i v e o i l , a l s o soluble i n acetone, e t h y l acet a t e and carbondisulfide (Cocaine).

COCAINE HYDROCHLORIDE

159

One gram d i s s o l v e s i n 0.4 m l w a t e r , 3.2 a l c o h o l , 2 m l hot a l c o h o l , 12.5 m l chloroform, a l s o s o l u b l e i n g l y c e r o l and acetone. (Cocaine hydrochloride) 2.3

O p t i c a l Rotation Cocaine [a]D20

-

[aID

-

16' (C=4 i n C H C l ) ; [a]D18 -35'(50% alcohol). 3 79' t o -81° (0.6 g i n 2.5 m l of M hydroc h l o r i c and s u f f i c i e n t water t o produce 25 m l ) ( 1 4 ).

Cocaine hydrochloride

[aID [alD 2.4

-

72'

(C=2 aqueous s o l u t i o n pH

4.5)

70' t o -73O (2.5% w/v s o l u t i o n )

(14

)

X-ray D i f f r a c t i o n The c r y s t a l and molecular s t r u c t u r e of 1-cocaine w a s determined by X-ray d i f f r a c t i o n . Babe and Barnes ( 1 3 ) have achieved t h i s . Three dimentional study of t h e hydrochloride and t h e hydrobromide s a l t s revealed t h e c r y s t a l s t o be orthorombic with space group P21 21 21; f o r t h e hydrochl o r i d e , a = 7.633, b = 10.300, c = 21.459 A'; for t h e hydrobromide, a = 7.68, b = 10.68, c = 21.65 R o . The study a l s o revealed t h a t t h e stereochemical configurat i o n of cocaine molecule agrees with t h a t deduced from chemical evidence. The p i p e r i d i n e r i n g of t h e tropane nucleus has t h e c h a i r form, with C 3 d i s p l a c e d l e s s , and N displaced more than u s u a l from t h e plane of t h e r i n g . The benzoxy s i d e chain on C 3 i s equator i a l and t h e carbomethoxy s i d e chain on C2 i s a x i a l . Intramolecular bond l e n g t h s , bond angles and t o r s i o n angles of cocaine hydrochloride a r e t a b u l a t e d i n t a b l e s 1, 2 and 3 r e s p e c t i v e l y .

160

1.494 1.382

1.406 1.380 1.370

1.360 1.384

COCAINE HYDROCHLORIDE Table 2. Bond Angles ( ” ) of Cocaine Hydrochloride

C1-N-C

5

103.6

C5 c3 -

N

-

C17

112.4

N

-

C17

112.5

01-

c8

‘15-

‘4-

‘16

117.4 118.1

C1

N N

- C1- C1-

c2

c7

c2 - cl- c7 c1 - c2- c3

109.1

101.1 112.5 109.1

161

FARID J. MUHTADI A N D ABDULLAH A. AL-BADR

162

c1

- c2-

‘9

121.4

108 102.3

- ‘15- ‘15-

‘4

C4 C5- c6

‘3 ‘3

‘2

122.3

‘4

- ‘15-

‘2

115 9

5 -N-C1-

c2

c1

72 -47 -166

75 -72 46 166 -75 77 -157 1

179 8 ‘16- ‘4- ‘15- ‘3 c16’ 04- C l 5 - c2 -178 N C1- C2 - C3 -59

-

- Cl- ‘2 - ‘15 - cl- c2 - c3 - cl- c2 - ‘15 N - C1- C7 ‘6 c2 - cl- c7 - c6 c1 - c2- c3 - o1 c1 - c2- c3 - c4 Cl5c2- c3 - o1 c7 c7

50-

‘13 ‘13- ‘14

‘11- ‘12-

121.1

‘12-

120.7 118.6

- ‘14-

‘13

Torsion Angles ( ” ) of Cocaine Hydrochloride (16).

- c7 C17N - C1 - C2 C17N - c7 C1 - N - C5 - C4 C1-N‘5 - ‘6 C17N - C5 - C4 C17N - c5 - c6 c8 - 01- c3 - c2 c8 - 01- cg - c4 c3 - 01- c8 - O2 c3 - ol- C8 - c9 C5-N-

N

119.6

110.4

- C5-

Table 3.

C

5 0 - cll-5 2

112.8

- c3-

-

N

120.2

c4 c4- c5

- ‘2- c3- c2 o1 - c3- c4

N

51

Cg

‘3 o1

c2 c3

-

109.3 114.7 114.5 108.3

‘15 ‘15

67 52 178 28

-88

172 47 49

163

COCAINE HYDROCHLORIDE 2.5

Spectral Properties 2.5.1

U l t r a v i o l e t Spectrum The UV spectrum of cocaine hydrochloride i n methanol (Fig. 1) was scanned from 190 t o 400 nm using DMS 90 Varian Spectrometer. It exhib i t e d t h e following UV d a t a (Table 4 )

.

Table 4.

W C h a r a c t e r i s t i c s of Cocaine Hydrochloride

hmax. nm

-

202 2 30 272 282

4375 7136 433.3 390.8

A(%%,

E

1 em)

128.75 210.0 12.75 11.50

Other reported UV s p e c t r a l d a t a f o r cocaine i n ethanol hmax. 230, 274, 2 8 1 mu (17) , f o r cocaine hydrochloride i n water h m a x . 233 and 274 mu ( 1 7 ) . 2.5.2

I n f r a r e d Spectrum The I R spectrum of cocaine hydrochloride as KBr-disc was recorded on a Perkin E l m e r 580 B I n f r a r e d spectrophotometer t o which an i n f r a red d a t a s t a t i o n i s attached (Fig. 2 ) . The s t r u c t u r a l assignments have been c o r r e l a t e d with t h e following frequencies (Table 5 ).

Table 5 .

I R C h a r a c t e r i s t i c s of Cocaine Hydrochloride

-1 Frequency em 3020 2780-2500

A s s i gnment CH ( s t r e t c h ) H3C

- N+H

0 I1

1730

C-OCH3

(ester)

0 II

1715

C-0-

1600

C=C (aromatic )

1155,1028 730

C-0-C

(ester)

(ether )

monosubstituted aromatics

I

190

140

wavelength

340

440

!

: 5 d

c

a

C

c

t

Y

c

c

ci

:

*9

3

T------

E

165

FARID J. MUHTADI A N D ABDULLAH A. AL-BADR

166

The I R exhibited t h e following other c h a r a c t e r i s t i c bands :-

1488, 1455, 1430, 1395, 1375, 1365, 1315, 1300, 1268, 1250, 1232, 1210, 1168, 1135, 1108, 1072, 1010, 980, 950, 855, 830, 815, 795, 755, 682 cm-l, Other I R data f o r both cocaine and cocaine hydro-

chloride have been a l s o reported ( 4,17).

2.5.3

Nuclear Magnetic Resonance Spectra

2.5.3.1

Proton Spectra (PMR) The PMR s p e c t r a of cocaine hydrochlor i d e i n D20 and cocaine i n CDC13 and i n TFA ( T r i f l u o r o a c e t i c a c i d ) were recorded on a Varian T ~ O A ,60 MHZ NMR Spectrometer using TMS (Tetramethylsilane) as an i n t e r n a l reference. These a r e shown i n Fig. 3 and Fig. 4 respectively. The following s t r u c t u r a l assignments have been made (Table 6 ).

PMR w a s used as a t o o l t o confirm

the

configuration of both cocaine and pseudococaine (18 ) and provided evidence of t h e p r e f e r r e d confirmation (19).

I C

1

I

I

8.0

I

I

1

*

1

I

I 1

1

1

1

1

6.0

ZO

FIG. 3.

1

1

1

1

5.0

1

.

l

l

PPM(6J

l

l

tO

l

l

l

l

I

I

3.0

THE PMR OF COCAINE HYDROCHLORIDE IN D20

I

I

I

I

2.0

I 1

,

I

,

I

,

1.0

,

,

1

168

m -J u

9 E w

f

V 0

c 0

u.

u

a Y

z

X I-

c

=r

v

2 LL

I

!-

FIG. 4(B) THE PflR

OF COCAINE

IN TFA

FARID J. MUHTADI AND ABDULLAH A. AL-BADR

170 Table 6.

PMR C h a r a c t e r i s t i c s of Cocaine and Cocaine Hydrochloride.

Chemical Shift (Ppm) Group

Cocaine hydrochloride

Cocaine CDC13

TFA

Aromatics 13, 17 HS

1 4 , 15, 16 HS 3 H 1 s 5 HS 0-CH3 N-CH3

s = s i n g l e t , d = doublet, m = m u l t i p l e t , q = q u a r t e t bs = broad s i n g l e t , s i = s i x t e t .

Other PMR data f o r cocaine ( 3,18,20 ) and cocaine hydrohave a l s o been reported. chloride ( 21) 2.5.3.2

13C-NMR

The 13C-NMR noise decoupled and off resonance s p e c t r a are presented i n Fig. 5 and Fig. 6 respectively. Both were recorded over 4000 Hz range i n D20 and i n Dioxan on a Varian FT 80 A-80 MHz spectrometer, using LO mm. sample tube and dioxan a s a reference standard a t 22O. The carbon chemical s h i f t s a r e assigned on t h e bases 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 resonance s p l i t t i n g p a t t e r n (Table 7 ).

t

'

I

I

1

'

I

'

I

'

I

'

I

'

I

'

I

I

I

I

FIG. 5 . THE I3C-NMR NOISE DECOUPLED

l

'

l

'

l

'

SPECTRUM OF

l

'

l

'

l

I

l

'

l

~

COCAINE HYDROCHLORIDE

l

'

l

'

I

I

l

~

I

~

I

FIG. 6. THE I3C-NHR

~

OFF

l

RESONANCE

~

l

~

SPECTRUM OF

~

COCAINE

~

l

~

HYDROCHLORIDE

l

~

~

~

~

~

l

COCAINE HYDROCHLORIDE

Table 7.

Carbon no.

173

Carbon Chemical S h i f t s of Cocaine H C 1

Chemical S h i f t s [ PPm 1

Carbon no.

Chemical S h i f t s [ PPm 1

~~

C

9

5 2 ‘13’

‘17

‘15

‘14’ ‘16 c3

c5 s = s i n g l e t , d = doublet,

Other l3C-NMR reported.

t = t r i p l e t , q = quartet

d a t a f o r cocaine ( 2 0 ~ 2 2) have a l s o been

FARID J. MUHTADI AND ABDULLAH A. AL-BADR

174 2.5.4

Mass Spectrum The mass spectrum of cocaine hydrochloride i s This was obtained by presented i n Fig. 7 e l e c t r o n impact i o n i z a t i o n on a Varian MAT 1020 by d i r e c t i n l e t probe a t 270'~. The e l e c t r o n energy w a s 70 eV. The spectrum scanned t o mass 310 mu. The spectrum (Fig. 7 ) shows a molecular ion peak # a t m/e 303,with a r e l a t i v e i n t e n s i t y of 15.60%. The base peak i s 82 with a relat i v e i n t e n s i t y 100%. The most prominent fragments 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 some proposed ion fragments are given i n t a b l e 8.

.

Table 8.

Elk 30 3

Mass Fragments of Cocaine H C 1

Relative i n t e n s i t y 15.60

%

Ions -

M+ (cocaine)

-

272

6.75

198 183

11.37 9.26

-

182

84.84

183-H

122

9.85

105

29.97

97

10.03

96

24.06

94

35.19

83

35 83

-

96-2H

DATI: KSU a1396

1n.e

1

176

m/e

FARID J. MUHTADI AND ABDULLAH A. AL-BADR Relative i n t e n s i t y

82

100.00

81

10.63 36.66

77 68

55 51 42 41

%

Ions -

e

C

H

82-H

-

6.39 8.29 14.54 32.43 9.45

3

[ CH2--N=CH2

42-H

Other reported mass s p e c t r a of cocaine has been a l s o reported ( 23,24 ).

1+

COCAINE HYDROCHLORIDE

177

Isolation of Cocaine

3.

Cocaine occurs in the leaves of ErythroqZon coca (Bolivian coca leaves), ErythroqZon truxiZZense (Peruvian and Javanese coca leaves) and other species of ErythroqZon family Erythroxyzaceae ( 25,26). Javanese leaves are usually the richest in total alkaloids, of which, the chief alkaloid is cinnamylcocaine, while the South American leaves contain less total alkaloids but higher percentage of cocaine ( 2 7 , 2 8 ) . Java coca leaves are used to isolate cocaine commercially. The method depends upon isolating the total alkaloids including cocaine, hydrolyzing these alkaloids either to (-)-ecgonine or to ecgonine methyl ester, ( - ) cocaine is then synthesized by methylation and benzoylation or by simple benzoylation (Fig. 8). The Procedure

-

-

-

-

-

-

Powdered coca leaves are moistened with sodium carbonate solution and percolated till exhaustion with benzene. The benzene extract is shaken with dilute sulfuric acid. The collected acid extract is rendered alkaline with an excess of sodium carbonate. The resulting precipitated total alkaloids are extracted with ether. The ether extract is dried with anhydrous sodium sulfate, filtered and the ether is distilled off. The residue consisting of the total crude alkaloids is dissolved in methyl alcohol and the resulting solution is heated with sulfuric acid or with alcoholic hydrogen chloride (this treatment splits off any acids from ecgonine and simultaneously esterfies the carboxyl group to carboxymethyl group). After dilution, with water, the organic acids which have been liberated are removed with chloroform. The aqueous solution is then concentrated, neutralized and cooled with ice, whereupon methylecgonine sulfate crystallizes out and collected. This is now benzoylated by heating with benzoyl chloride or benzoic anhydride at about 150°C. Upon adding water and sodium hydroxide, cocaine is precipitated and extracted with ether. The ether extract is concentrated to crystallization, the crystallized cocaine is collected and recrystallized from

~

FARID J. MUHTADI AND ABDULLAH A. AL-BADR

178 Fig. 8.

The Commercial Preparation of Cocaine (27).

,CHI COOCH,

J

COCAINE RELATED ALKALOIDS

H PARTIAL HYDROLYSIS

COMPLETE HYDROLrSlS

,CHI

/

CHaOH HCI

H METHYLATION AND BENZOYLATION

/ BENZoYLAT1oN

J H

COCAINE HYDROCHLORIDE

179

a mixture of acetone and benzene to give colorless prisms (Fig. 8 ) . Cocaine hydrochloride This is prepared by adding cocaine to an alcoholic solution of hydrochloric acid and the resulting salt is purified by subsequent recrystallization.

4. Synthesis of Cocaine 4.1

Partial Synthesis Cocaine can be synthesized by methylation and benzoylation of (-) ecgonine. Thus upon heating a mixture of (-)-ecgonine, benzoic anhydride and methyl iodide at looo, (-)-cocaine is resulted (29). (-)-Ecgonine is esterified with methanol to yield (-)-ecgonine methyl ester and this upon simple benzoylation with benzoyl chloride gives (-)-cocaine (30-32).

4.2 Total Synthesis Since cocaine is an ester alkaloid consists of ecgonine methyl ester and benzoic acid, schemes for the total synthesis of both are required. 4.2.1

Total Synthesis of Ecgonine Two schemes for the total synthesis of ecgonine are known. Scheme I: Willstatter's total synthesis of ecgonine (1) . Suberone (cycloheptanone) [ 11 is reduced to suberol which is treated with hydrogen iodide to give suberyl iodide [ 2 ] . This is treated with potassium hydroxide in ethanol to give Cycloheptene [ 3 ] . Cycloheptene is brominated to give 1,2-dibromocycloheptane [4] which is treated with dimethylamine to yield dimethylaminocyclohept-2-ene [5]. The latter is converted to cyclohepta-193-diene[6] by exhaustive methylation. [6] is brominated at 1,4-positions to give 1,4-dibromocyclohept2-ene [7]. Elimination of two moles of the

FARID J. MUHTADI AND ABDULLAH A. AL-BADR

180

SCHEME

I;

Willstatter s t o t a l s y n t h e s i s of cocaine

-Q exhaust

(Me2NH

QBr

methyln.

[5l N(CH3)2

quinoline

150°C

[41

Br

Br

COCAINE HYDROCHLORIDE

181

182

FARID J. MUHTADI AND ABDULLAH A. AL-BADR

hydrogen bromide of [7] i s e f f e c t e d by quinoline t o give cycloheptatriene [8]. Substance [8] i s t r e a t e d with hydrogen bromi d e t o give bromocyclohepta-3,5-diene [9] which i s reacted with dimethylamine t o give dimethyl aminocyclohepta-2,4-diene [ l o ] . The l a t t e r is t r e a t e d with sodium i n ethanol f o l lowed by bromination t o give 1,2-dibromo-5dimethylamino-cycloheptane [ l l ] This i s warmed i n e t h e r when intramolecular alkylat i o n occurs t o give 2-bromotropane methobromide [12]. Hydrogen bromide i s eliminated from [12] by t h e a c t i o n of a l k a l i t o y i e l d t r o p i d i n e methobromide [13]. This i s t r a n s formed t o t r o p i d i n e methochloride [14] by t h e action of potassium iodide followed by t h e a c t i o n of s i l v e r chloride. Substance [ 141 is pyrolized t o give tropidine [15]. Hydrogen bromide i s added t o an acetic a c i d s o l u t i o n of t r o p i d i n e [15] t o y i e l d 3-bromotropane [16] which i s hydrolysed with 10% s u l f u r i c a c i d a t 200-210' t o give pseudot r o p i n e [17]. $-tropine [17] i s oxidized with chromium t r i o x i d e t o give tropinone [18]. This ketone upon treatment w i t h sodium, (Kolbe-Schmitt type of reaction) gives t h e intermediate [19] which i n the presence of carbon dioxide gives sodium tropinone carbox y l a t e [20]. Upon reduction of [20] followed by acid treatment y i e l d s (+)-ecgonine [21]. (+)-Ecgonine [21] i s resolved with ( + ) - t a r t a r i c a c i d t o furnish (-) -ecgonine.

.

Scheme 11: Total synthesis by adaptation of Robinson's tropinone synthesis (2). Succindialdehyde [ 11 i s condensed with methylamine [2] t o give biscarbinolamine [3]. This i n t u r n condensed with acetondicarboxylic acid monomethyl e s t e r [4] t o give t h e condens a t e [5]. The l a t t e r i s decarboxylated t o y i e l d ecgoninone methylester [6]. This 8k e t o e s t e r i s reduced with sodium amalgam t o give ecgonine methyl ester [ 7 ] , which i s hydrolyzed t o (2) -ecgonine [8]. [8] i s resolved with ( + ) - t a r t a r i c t o render (-) ecgonine.

-

COCAINE HYDROCHLORIDE

SCHEME

I I : Total

(+

-(7"'

183

synthesis by adaption of Robinson's method CH-OH

H

\NCH3 /

H

\

CH2COOCH3

t

CH-OH

CHO

[I1

[21

\c=o

I

C H2-COOH

C 31

[41

COOCH3

COOCH3

COOH

[61

151

COOH

( - 1-ecgonine

(-)-cocaine

184

FARID J. MUHTADI A N D ABDULLAH A. AL-BADR

Synthesis of Cocaine [-I -Ecgonine i s e s t e r i f i e d first with methanol and hydrochloric a c i d , then with benzoyl c h l o r i d e t o y i e l d (-) -cocaine. An a l t e r n a t e s y n t h e s i s of tropinone has been reported (33). Tropinone can be converted i n t o ecgonine by Willstatter’ s method (Scheme I ) .

Diyne d i e s t e r [l] i s condensed with methylamine [2] t o give t h e p y r r o l i d i n e d i e s t e r [3]. C a t a l y t i c hydrogenat i o n gives [4] which on Dickmann c y c l i z a t i o n , hydrolysis and decarboxylation y i e l d s tropinone [S].

0

COCAINE HYDROCHLORIDE

4.2.2

185

Total Synthesis of Benzoic Acid Benzoic acid is known acid and occurs in several sources in nature. It can be prepared by heating gum benzoin when benzoic acid sublimes (34). It can also be prepared from hippuric acid which on boiling with mineral acids is hydrolysed to glycine and benzoic acid (34). It is prepared exclusively from toluene as follows:Toluene is converted into benzotrichloride by treatment with chlorine and this is hydrolysed by lime water to calcium benzoate, from which benzoic acid is precipitated by the addition of hydrochloric acid and purified by recrystallization from water. Benzoic acid is prepared in large quantities by catalytic oxidation of toluene (34). Benzoyl chloride required to prepare cocaine is prepared by warming benzoic acid with phosphorus pentachloride or preferably thionyl chloride or by the action of chlorine on benzaldehyde (34). C6H5. COOH + S0Cl2 = C6H5. COCl + HC1 + SO2 = C6H5. COCl + HC1. C6H5. CHO + C12

4.3 Stereoselective Synthesis of dl-Cocaine Stereoselective synthesis of dl-cocaine was described (3). Mesylate olefin [I] is brominated to the bromo-ester [Z] which is converted into the nitroester [3]. This is condensed with acrolein [4] in the presence of methanol containing sodium methoxide to give the dimethylacetal [S]. The nitrogroup of [5] is reduced with zinc and aqueous ammonium chloride solution to produce the hydroxylamine acetal [6] which upon acidification generates the hydroxylamine aldehyde [7]. [7] is cyclized to the nitron [8] which is converted into the cycloadduct [9]. The cycloadduct [9] is methylated with methyliodide in methylene chloride affords methiodide [9] which is treated with activated zinc in 50% aqueous acetic acid at 7Ooc in order to effect the scission of the nitrogenoxygen bond to provide ecgonine methylester, which is then benzoylated to afford dl-cocaine [lo].

186

FARID J. MUHTADI AND ABDULLAH A. AL-BADR

Stereoselective Synthesis of dl-Cocaine

c;

NQcH3

H

C02Me

N-CH3

COOCH3

OCOC6Hg

[lo1

H

COCAINE HYDROCHLORIDE 4.4

187

Synthesis of Radioactive Cocaines

"-methyl -14C] Of

cocaine i s prepared by N-methylat ion norcocaine with [ ~ n e t h y l - ~ ~iodide C] (35).

[Ester methyl-14C] cocaine i s prepared by methylation of benzoylecgonine with diazomethane-l4C ( 3 5 ) .

H [ B e n z ~ y l - a - ~ ~ cocaine C] i s prepared by benzoylation of e cgon i n e met hy 1e s t e r with benz oy l c h l o r ide -a- l 4 C ( 351.

H M e t h y l - t r i t i a t e d d e r i v a t i v e s of (-)-cocaine and (+) $-cocaine were prepared ( 3 6 ) .

-

188

5.

FARID J. MUHTADI AND ABDULLAH A. ALBADR Biosynthesis of Cocaine It has been assumed t h a t t h e t r o p i n e and ecgonine moieties of hyoscyamine and cocaine r e s p e c t i v e l y a r i s e d from t h e aminoacid o r n i t h i n e (37,38). The aromatic carboxylic a c i d s of both a l k a l o i d s are b u i l t up from phenyl a l a n i n e which i s formed i n p l a n t s from shikimic a c i d ( 38 ,39 ). Using r a d i o a c t i v e t r a c e r technique, it w a s found t h a t t h e administration of [ 3' -14C3 phenylalanine t o Erythroxy Zrm novogranatense yielded r a d i o a c t i v e cocaine i n which a l l t h e a c t i v i t y resided i n t h e carboxyl group of t h e benzoic acid moiety of cocaine (40 ) Feeding sodium [ L-14C 1 a c e t a t e and [methyl-14C J methionine i n t o t h e same species, r e s u l t e d i n t h e i s o l a t i o n of l a b e l l e d cocaine ( 4 1 ) . It w a s a l s o found t h a t feeding sodium [l-C] a c e t a t e t o E. coca p l a n t s , radioactive cocaine was i s o l a t e d i n which about 60% of t h e a c t i v i t y were l o c a t e d i n t h e e s t e r methyl group, 30% i n t h e carboxyl group of t h e benzoic a c i d moiet y and 8.7% i n t h e ecgonine residue (42). Leete ( 43 ) has f e d t h e following radioactive precursor t o Erythoxy Zon coca p l a n t s : DL-[ 5-14C1 o r n i t h i n e hydrochloride , DL-[2,3-13C ,5-14C1 ornithine hydrochloride , DL-[ 2-14CI o r n i t h i n e hydrochlor i d e , [ 2-13C , 1 4 C I-N-methyl-Al-pyrrolinium a c e t a t e , [ 2-14CI -N-methyl-Al-pyrrolinium chloride , sodium [ l - k ] a c e t a t e and [ carboxyl-14C 1 n i c o t i n i c acid. Radioactive cocaine r e s u l t e d from each o f t h e above precursors with v a r i a b l e l e v e l of r a d i o a c t i v i t y . Cocaine containing a s i g n i f i c a n t l e v e l of r a d i o a c t i v i t y was obtained by p a i n t i n g t h e leaves of t h e Erythroxylon coca with an aqueous s o l u t i o n of DL-[5-14C] o r n i t h i n e hydrochloride ( 43 ). A systematic degradation of t h i s cocaine indicated a l l t h e a c t i v i t y was l o c a t e d a t t h e brigehead carbons ( C 1 and C 5 ) of i t s tropane moiety and equally divided between t h e s e p o s i t i o n s (43).

.

-

Leete t h e r e f o r e , proposed t h e following biosynthetic pathway of cocaine (43). Ornithine [11 i s incorporated i n t o cocaine via B-Nmethylornithine [ 2 ] which i s formed by N-methylation of [I]. [ 2 ] w a s i s o l a t e d i n radioactive form a f t e r feeding [ 5-14C 1 or [ 5-3HI o r n i t h i n e t o Atropa belladonna (44). Decarboxylation of [ 2 1 y i e l d s N-methylputrescine [ 3 1 , an e s t a b l i s h e d precursor of t h e tropane nucleus of hyoscyamine and scopolamine (45-47). Oxidation of [31 affords k-methylaminobutanal [4]. This i s cyclized t o give

COCAINE HYDROCHLORIDE

Leete’s Scheme:

189

Biosynthesis of Cocaine COOH

COOH

COOCH3 /

CH3

I

[81

COOCH3

-kH

COOCH3

/

0- 0 II

FARID J. MUHTADI AND ABDULLAH A. AL-BADR

190

N-methyl-Al-pyrrolinium salt [ 51 which is condensed with acetoacetate [61 to afford hygrine-1'-carboxylic acid [ T I . 171 is dehydrogenated and esterified to give dehydrohygrine-1'-carboxymethylester [ 81. The latter is cyclized to yield ecgoninone methylester [ 91. Stereospecific reduction of [ g ] affords ecgonine methylester [ l o ] . Ecgonine methylester [lo] is finally esterified with benzoic acid [Ill to give cocaine [121. Benzoic acid is formed from the aminoacid phenylalanine ( 39 ) which is formed in plants from shikimic acid

shikimic acid

*CH 1 2 CH-NH* I COOH

*COOH

COCAINE HYDROCHLORIDE

6.

191

Metabolism of Cocaine Cocaine i s w e l l absorbed from a l l s i t e s of a p p l i c a t i o n s including mucous membranes and t h e g a s t r o i n t e s t i n a l mucosa (48-49). Absorption i s enhanced i n t h e presence of an inflammation (48,49). After absorption, cocaine i s degraded by plasma e s t e r a s e s ( 5 0 ) and i n some animals by hepatic enzymes ( 5 1 ) t o a number of metabolites (52-63). Some cocaine i s excreted unchanged i n t h e u r i n e (51,52). The major metabolites of cocaine i n man and animals a r e benzoylecgonine, ecgonine, ecgonine methylester and norcocaine (52-57). Minor metabolit,es. i n man a r e ecgonine e t h y l e s t e r , cocaethylene, m-hydroxycocaine and ecgonidine methylester ( 5 6 , 5 7 ) , t h e s e metabolites have been i d e n t i f i e d i n multiple i n t o x i c a t i o n and overdoses of cocaine ( 5 7 ) . Other metabolites which can be detected i n animals are benzoylnorecgonine and norecgonine ( 58-60). The metabolism of cocaine i n man and animals i s presented i n Fig. 9, and t h e s t r u c t u r e s of cocaine and it.s metabol i t e s a r e shown i n Fig. LO ( 5 2 ) .

7.

Pharmacokinetics The pharmacokinetics of cocaine have been reported by s e v e r a l authors. Peak serum l e v e l s occur i n 3-5 minutes following intravenous administration; i n 20-60 minutes following i n t r a n a s a l administration and i n 60-90 minutes after oral administration (64). Plasma l e v e l s of cocaine 30 and 45 minutes post-administrat i o n of 1 . 5 mg/kg of a t o p i c a l i n t r a n a s a l 10% cocaine s o l u t i o n , were 331 and 320 n g / d r e s p e c t i v e l y ( 6 5 ) . Following i n t r a n a s a l a p p l i c a t i o n of 1-5 mg/kg of cocaine t o 1 3 s u r g i c a l p a t i e n t s , plasma l e v e l s reached peak conc e n t r a t i o n s of 120-474 ng/ml a t 15-60 minutes, and then decreased overthe next 3-5 hours (66). Plasma l e v e l of cocaine a f t e r smoking one c i g a r e t t e (75 mg of cocaine) i n 3 minutes w a s 251 ng/ml with a range of 91-462 ng/ml (67). The average plasma l e v e l of cocaine a f t e r smoking 3 cigare t t e s for 1 5 minutes was 478 ng/ml with a range of 226684 ng/ml (67). The h a l f - l i f e of cocaine i n t h e plasma a f t e r o r a l or n a s a l administration i s approximately one hour (68). The mean h a l f - l i f e of cocaine f o r intravenous i n j e c t i o n s i n four human s u b j e c t s was 41.4+-8.2 minutes and t h e range was 19 t o 64 minutes (69).

FARID J. MUHTADI AND ABDULLAH A. AL-BADR

192

Ecgonine (mn, r u t , dogl

(man, r u t , dogl

Ec gonine ethy les t er

(man)

m-Hydroxycocaine

(man) Benzoylnorecgonine

Norcocaine

(rat, dog)

(man, r u t , dog, monkey)

Norecgonine (dog) Fig. 9.

The Metabolism of Cocaine (Cocaine Metabolites).

COCAINE HYDROCHLORIDE

193

P

3~~woJJ

Fig.10. The Structures of Cocaine Metabolites.

N

n COCAINE

BENZOVLECCONINE

ECGONINEi3HYLESTER

BEWZOVLNORECGO)(INE

ECGONINE UETHVLESTEA

/"I

N

8=Tmo+Q H

OH

m-HVOROXYCOCAlNE

ECQONIMNE METHYLESTER

H

ECGONINE

NORECGONINE

194

8.

FARID J. MUHTADI A N D ABDULIAH A. AL-BADR Drug S t a b i l i t y Cocaine hydrochloride i n t h e dry state, s t o r e d i n a well closed container a t room temperature, showed no decompos i t i o n a f t e r f i v e years when examined by d i f f e r e n t physic a l and chemical d a t a including spectrophotometrical evidence (70). Solution of cocaine hydrochloride i s l i a b l e t o develope fungus growths and should contain a preservative. Stored i n a i r t i g h t containers, protected from l i g h t ( 4 9 ) . Aqueous s o l u t i o n s containing cocaine hydrochloride 5% and phenol 0.5% remained clear and c o l o r l e s s f o r a year a t 0' t o 4', room temperature and 37'. A f a l l i n pH from 4.6 t o 3.9; 2.7 and 2 . 1 respectively, suggesting a chemic a l change, such s o l u t i o n should therefore be s t o r e d i n a cool place (49). Factors a f f e c t i n g t h e s t a b i l i t y of cocaine i n s o l u t i o n s were studied. No hydrolysis of cocaine was observed a t pH values below 4.0, but hydrolysis w a s r a p i d when pH w a s g r e a t e r than 5.5. Decreasing i o n i c s t r e n g t h a l s o increased t h e rate of hydrolysis. With proper pH conditions, solutions of cocaine were s t a b l e a t temperatures up t o 24' f o r up t o 45 days (71). Brompton's c o c k t a i l containing morphine, cocaine and alcohol has a l i m i t e d s h e l f l i f e of about 3 weeks. This cockt a i l should be s t o r e d i n well-closed l i g h t - r e s i s t a n t containers (72).

195

COCAINE HYDROCHLORIDE 9.

Methods of Analysis

9.1

I d e n t i f i c a t i o n Tests

-

-

-

-

-

-

-

-

The following i d e n t i f i c a t i o n t e s t s are mentioned under cocaine i n t h e B r i t i s h Pharmacopoeia ( 1 4 ) . The l i g h t absorption i n t h e range 230 t o 3501x11 of a 2-cm l a y e r of 0.001% w/v s o l u t i o n i n 0 . 0 1 M hydroc h l o r i c a c i d e x h i b i t s a w e l l defined maximum only a t about 233 nm; with absorbance about 0.86. 0 . 1 g of cocaine i s heated w i t h 1 ml of s u l f u r i c a c i d f o r f i v e minutes a t looo, upon c o o l i n g and c a u t i o u s l y mixing with 2 ml of water; t h e aromatic odor of methyl benzoate i s p e r c e p t i b l e , and when t h e s o l u t i o n i s cooled and allowed t o s t a n d f o r some hours, c r y s t a l s of benzoic a c i d s e p a r a t e . 50 mg of cocaine i s d i s s o l v e d i n 1.65 m l of 0.1 M hydrochloric a c i d , 8.5 ml of a 5% w/v s o l u t i o n of alum and 5 m l of potassium permanganate s o l u t i o n are added t o t h e cocaine s o l u t i o n w i t h s t i r i n g f o r s e v e r a l seconds; c h a r a c t e r i s t i c r e c t a n g u l a r v i o l e t p l a t e s are formed. A s a t u r a t e d s o l u t i o n i s a l k a l i n e t o phenolphthalein solution. The followings a r e i d e n t i f i c a t i o n t e s t s mentioned under cocaine hydrochloride (14 ) To 0.5 m l of a 2% w/v s o l u t i o n , 0.5 m l of water and 0 . 1 m l of a 3% w/v s o l u t i o n of chromium t r i o x i d e a r e added; a yellow p r e c i p i t a t e i s formed which r e d i s s o l v e s on shaking. Upon a d d i t i o n of t h e same reagent or hydrochloric a c i d , t h e p r e c i p i t a t e reappears. 0 . 1 g of cocaine hydrochloride i s heated with 1 m l of s u l f u r i c a c i d f o r f i v e minutes on a water b a t h , 2 m l of water a r e added c a r e f u l l y , methyl benzoate recognisable by i t s odor i s produced. On c o o l i n g t h e s o l u t i o n , c r y s t a l s a r e deposited. 50 mg a r e d i s s o l v e d i n 1 . 5 ml of water, 8.5 ml of a 5% w/v s o l u t i o n of alum and 5 m l of a 1% w/v s o l u t i o n of potassium permanganate a r e added and t h e r e s u l t i n g mixture i s shaked f o r a few seconds; a c r y s t a l l i n e p r e c i p i t a t e i s slowly formed, which when examined under a microscope, can be seen t o c o n s i s t of c h a r a c t e r i s t i c rectangular v i o l e t c r y s t a l s . S p e c i f i c o p t i c a l r o t a t i o n of a 2.5% w/v s o l u t i o n , -TO0 t o -73', c a l c u l a t e d w i t h r e f e r e n c e t o t h e und r i e d substance. Yields t h e r e a c t i o n s c h a r a c t e r i s t i c of a l k a l o i d s and t h e r e a c t i o n s c h a r a c t e r i s t i c of c h l o r i d e s mentioned i n (14).

.

FARID J. MUHTADI AND ABDULLAH A. AL-BADR

196

-

-

-

-

9.2

The following i d e n t i f i c a t i o n t e s t s a r e mentioned under cocaine i n t h e United S t a t e s Pharmacopeia (73). The u l t r a v i o l e t absorption spectrum of a 1 i n 75,000 s o l u t i o n i n d i l u t e hydrochloric a c i d (1:120 ) e x h i b i t s maxima and m i n i m a a t t h e same wavelengths a s t h a t of a similar s o l u t i o n of USP Cocaine Hydrochloride RS, concomitantly measured, and t h e r e s p e c t i v e molar a b s o r p t i v i t i e s , c a l c u l a t e d on t h e d r i e d b a s i s , a t t h e wavelength of maximum absorbance a t about 233 nm do not d i f f e r by more than 3.0%. It meets t h e requirements under i d e n t i f i c a t i o n Organic Nitrogenous Bases (181) , USP Cocaine Hydrochloride RS. 100 g a r e dissolved i n a mixture of 0.4 m l of d i l u t e hydrochloric a c i d (1i n 1 2 ) and water t o make 5 m l , upon adding 5 drops of chromium t r i o x i d e solution (1:20); a yellow p r e c i p i t a t e i s formed, and it quickl y r e d i s s o l v e s when t h e mixture i s shaken. Upon addition of 1 m l hydrochloric acid; a permanent orange-colored c r y s t a l l i n e p r e c i p i t a t e i s formed. 1 0 mg a r e dissolved i n 1 m l d i l u t e hydrochloric a c i d (1:600) and evaporated on a steam bath j u s t t o dryness. The residue i s dissolved i n 2 drops of water, 1 m l of potassium p e m n g a n a t e s o l u t i o n (1i n 300) i s added; a v i o l e t c r y s t a l l i n e p r e c i p i t a t e i s formed, and it appears brownish v i o l e t when c o l l e c t e d on a f i l t e r paper, and shows c h a r a c t e r i s t i c violet-red c r y s t a l l i n e aggregates under t h e low power of a microscope, similar t o those obtained from USP Cocaine Hydrochloride RS. 1 0 mg of t h e s a l t i s dissolved i n water (1:20), s i l ver n i t r a t e TS i s added dropwise t o t h e s o l u t i o n , a white p r e c i p i t a t e i s Tormed which i s i n s o l u b l e i n n i t r i c acid.

-

Microcrystal Tests

-

-

30 mg of cocaine hydrochloride dissolved i n 25 m l of water. The following microcrystal t e s t s were performed and microscopically examined. Mercuric chloride s o l u t i o n gives with cocaine after 1 5 minutes, c l u s t e r c r y s t a l s which are shown i n Fig. 11 (74). M a r m ' s reagent gives with cocaine feathery needles which formed a f t e r 1 5 minutes, these are presented i n Fig. 12 (74). A concentrated s o l u t i o n of cocaine hydrochloride (30 mg i n 12.5 ml water) gives with potassium permanganate s o l u t i o n (1%), sharp jagged irregular blades

COCAINE HYDROCHLORIDE

197

Q

Q @ @@

FIG. 13 : MIcXCCFWTAKS OF COCAINE WIlM

pd.

PBWJGUU'IE.

198

FARID J. MUHTADI AND ABDULLAH A. AL-BADR

FIG. 14 : YICIMCRYSTALS CW CXXAINE WI?H LJNl ICDIDE.

COCAINE HYDROCHLORIDE

-

199

a f t e r 7-10 minutes as i n Fig. 1 3 ( 7 4 ) . Lead i o d i d e reagent y i e l d s w i t h cocaine immediately, dentated i r r e g u l a r r o s e t t e s as presented i n F i g . 14

(17,74).

- Gold

c h l o r i d e reagent y i e l d s with cocaine immediately, comb-shaped r o s e t t e s as shown i n Fig. 15 (17,74).

The a n a l y t i c a l methods of determination of cocaine and i t s m e t a b o l i t e s i n b i o l o g i c a l materials , have been reviewed by s e v e r a l a u t h o r s (52, 75-78). 9.3

T i t r i m e t r i c Methods 9.3.1

Non-aqueous T i t r a t i o n B r i t i s h Pharmacopeia 1980 (14) and U . S . Pharmacopeia XX ( 7 3 ) described a non-aqueous t i t r a t i o n f o r cocaine and i t s hydrochloride. The following a r e t h e procedures described i n t h e US Pharmacopeia XX:Cocaine Dissolve about 600 mg o f cocaine, p r e v i o u s l y d r i e d and a c c u r a t e l y weighed, i n 50 m l of g l a c i a l a c e t i c a c i d , add 1 drop of c r y s t a l v i o l e t TS, and t i t r a t e with 0.1N p e r c h l o r i c acid V ! : t o a green end-point. Perform a blank determination, and make any necessary c o r r e c t i o n . Each m l of 0.1N p e r c h l o r i c a c i d i s equivalent t o 30.34 mg of C17H21N04. Cocaine hydrochloride Dissolve about 500 mg of cocaine hydrochloride, a c c u r a t e l y weighed, i n a mixture of 40 m l of g l a c i a l a c e t i c a c i d and 1 0 m l of mercuric a c e t a t e TS. Add 2 drops of q u i n a l i d i n e r e d TS, and t i t r a t e with 0.1N p e r c h l o r i c a c i d VS. Perform a blank determination, and make any necessary c o r r e c t i o n . Each m l of 0.1N perc h l o r i c a c i d i s equivalent t o 33.98 mg o f C17H21N0h. H C 1 .

9.3.2

Potentiometric T i t r a t i o n Diamandis and Hadjiioannou (79) published a p o t e n t l o m e t r i c method f o r t h e determination

FARID J. MUHTADI A N D ABDULLAH A. AL-BADR

200

of cocaine and some common a l k a l o i d s w i t h a p i c r a t e - s e l e c t i v e e l e c t r o d e . The method i s based on formation o f t h e i n s o l u b l e p i c r a t e s i n = 1 M c o n c e n t r a t i o n and were determined by d i r e c t potentiometry or by potentiometric t i t r a t i o n w i t h 8 mM- or 40 t o 80 mM sodium p i c r a t e a t pH 6 , a p i c r a t e - s e l e c t i v e e l e c t r o d e being used f o r i n d i c a t i o n . End-point s were determined with use o f Gran p l o t s , f o r which t h e mean e r r o r w a s ? 2%.

9.4

Spectrophotometric Methods

9.4.1

Infra-red Spectrometry Moss et a1 ( 8 0 ) had analysed t h e i n f r a - r e d s p e c t r a of s e v e r a l drugs for s i m i l a r i t i e s by techniques of numerical taxonomy including cocaine. Preliminary r e s u l t for t h i s s e t of drugs i n d i c a t e t h a t an expanded m u l t i - v a r i a t e approach t o drug c l a s s i f i c a t i o n may be u s e f u l . Moore (81) have described i . r . d a t a for t h e i d e n t i f i c a t i o n of cis and t r a n s - cinnamoyl cocaine i n i l l i c i t cocaine s e i z u r e s .

9.4.2

Ultraviolet Kolosova ( 8 2 ) r e p o r t e d t h e use o f u l t r a v i o l e t spectrum for t h e d e t e c t i o n of a l k a l o i d s ( i n c l u d i n g cocaine) and b a r b i t u r a t e s i n o b j e c t s of l e g a l chemical examination. The drugs can be d e t e c t e d i n b i o l o g i c a l m a t e r i a l s by u l t r a v i o l e t absorption i n t h e region 200-400 nm.

C i s and

t r a n s - cinnamoyl cocaine i n i l l i c i t s e i z u r e s were i d e n t i f i e d (81). The isomeric a l k a l o i d s were found i n t h e l e v e l of = 1% in over h a l f of t h e i l l i c t cocaine samples i n v e s t i g a t e d . The author have presented t h e UV d a t a as w e l l as some o t h e r s p e c t r a l d a t a and g.1.c. c o n d i t i o n s o f a n a l y s i s . L-cocaine had been determined by c i r c u l a r dichroism ( 8 3 ) . The samples a r e d i s p e r s e d i n water o r methanol, and, a f t e r removal of

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20 1

i n s o l u b l e material, t h e p r i n c i p l e a b s o r p t i o n bands o f L-cocaine a r e measured a t 275 or 245 nm i n a s p e c t r o p o l a r i m e t e r . The l i m i t o f d e t e c t i o n i s 1 0 uM ( a s c a l c u l a t e d from t h e 245 nm b a n d ) . I n term o f a n a l y s i s t i m e , t h e method compares f a v o u r a b l y w i t h o t h e r methods now i n u s e ; however, any DL-racemate w i l l remain u n d e t e c t e d . R e s u l t s can b e a c c e p t e d a s proof o f t h e presence o f L-cocaine i n samples. T o f f o l i and Avico ( 8 4 ) have r e p o r t e d chemical e v a l u a t i o n o f c r u d e c o c a i n e and o f t h e r e sidue a f t e r i n d u s t r i a l e x t r a c t i o n o f c o c a i n e t h e r e f r o m . The a u t h o r s have d e s c r i b e d a procedure f o r t h e d e t e r m i n a t i o n o f c o c a i n e i n c r u d e c o c a i n e and a n o t h e r procedure f o r t h e d e t e r m i n a t i o n o f ecgonine and anhydroecgonine i n t h e residue a f t e r e x t r a c t i o n of cocaine. The procedure f o r d e t e r m i n a t i o n of c o c a i n e i n crude c o c a i n e i s as follows:D i s s o l v e 0.5 t o 1 g o f sample i n 3% o f H2SO4 (10 m l ) a t 0'. S t i l l a t O o , add 6% potassium permanganate s o l u t i o n ( 8 m l ) and 10% H2SO4 (1m l a t a t i m e , and a t 5 minutes i n t e r v a l s u n t i l t h e solution i s decolorised. Set aside f o r 30 minutes, t h e n add f i n e l y powdered o x a l i c a c i d u n t i l t h e p r e c i p i t a t e formed has dissolved. Extract t h e resulting c l e a r colorless solution t w i c e w i t h e t h e r and d i s c a r d t h e e x t r a c t s . Mzke t h e s o l u t i o n a l k a l i n e w i t h aqueous ammonia and e x t r a c t f o u r times w i t h e t h e r . Dry t h e combined e x t r a c t s , e v a p o r a t e and d r y t h e r e s i d u e f o r two hours a t 60°, t h e n for 1 2 hours over H2SO4 and weigh. D i s s o l v e t h e r e s i d u e i n e t h a n o l ( 2 0 m l ) i n t h e same c o n t a i n e r , add w a t e r ( 2 0 m l ) and re-weigh. From t h e weight of s o l u t i o n , calculate t h e dilution ( w a t e r ) required) t o g i v e a d e n s i t y o f 0.926 a t 20°, i . e . , 54% ( v / v ) of e t h a n o l c o n t e n t . Measure t h e o p t i c a l r o t a t i o n and hence c a l c u l a t e t h e amount o f cocaine. D i l u t e an a l i q u o t o f t h e s o l u t i o n 1:lOOO w i t h 0.1N H C 1 and measure t h e e x t i n c t i o n a t 274 nm; f o r c o c a i n e i n 0.1N H C 1 , EiZm = 38.2 2.0. Two maxima are i n f a c t

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observed ( a t 274 and 233 nm), w i t h molecular e x c i t a t i o n c o e f f i c i e n t o f 1130 and 13,500, r e s p e c t i v e l y . The d i r e c t weighing method t h e most r e l i a b l e of t h e s e methods. Chromatography and e l e c t r o p h o r e s i s o f t h e sample b e f o r e and a f t e r o x i d a t i o n w i t h potassium permanganate shows t h a t o n l y cocaine remains after t h i s treatment. Spectrophotometric a s s a y o f c o c a i n e and some n a r c o t i c s and a l k a l o i d s i n g a l e n i c a l composit i o n s have been r e p o r t e d ( 8 5 ) . To a s s a y c o c a i n e i n aqueous s o l u t i o n of i t s s a l t , t h e e x t i n c t i o n o f t h e d i l u t e d sample i s d e t e r mined a t t h e wavelength f o r maximum a b s o r p t i o n (257 t o 286 nm) and compared w i t h t h a t of p r o g r e s s i v e l y d i l u t e d samples o f s t o c k solut i o n s . The method i s c h i e f l y designed f o r u s e on aqueous p r e p a r a t i o n s ( e . g . from ampoules).

9.4.3

Fluorescence Analysis Shih-Chung Tu (86) have r e p o r t e d f l u o r e s c e i n chloride spot t e s t f o r t h e determination of c o c a i n e and some o t h e r drugs c o n t a i n i n g amino and h e t e r o c y c l i c n i t r o g e n . The d e t e r m i n a t i o n i s based on t h e formation of thodamine dyes. One mg f l u o r e s c e i n c h l o r i d e and 1 drop o f t h e drug (0.01-0.1%)were evaporated t o d r y n e s s , fused w i t h one drop of ZnC12; t h e melt w a s d i s s o l v e d i n 2 drops 10% a l c o h o l i c H C 1 solut i o n . The f l u o r e s c e n c e was determined w i t h u l t r a v i o l e t l i g h t ; t h e c o l o r depends on t h e molecular s t r u c t u r e . The t e s t (2-4 y ) was 2-5 times more s e n s i t i v e t h a n t h e F e i g e l t e s t t u b e method.

9.4.4

Phosphorimetry Harbaugh e t a1 ( 87) have r e p o r t e d q u a l i t a t i v e and q u a n t i t a t i v e a n a l y s i s of cocaine. The phosphorescence emission s p e c t r a , l i f e times, and r e l a t i v e s i g n a l s (peak e m i s s i o n s ) of c o c a i n e and some o t h e r drugs were determined w i t h use o f t h e a p p a r a t u s and procedures prev i o u s l y d e s c r i b e d ( 8 8 ) . For a mdticomponent m i x t u r e , t h e parameters c i t e d i n d i c a t e which

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o f t h e drugs can be s e p a r a t e d s p e c t r a l l y o r t e m p o r a r a l l y o r by a combination o f t h e two t e c h n i q u e s . Some mixtures, however , cannot b e s e p a r a t e d . Examples are given f o r t h e d e t e r mination o f t h e components o f s e v e r a l synthet i c b i n a r y m i x t u r e s o f t h e drugs. I n some i n s t a n c e s where t h e emission s p e c t r a s e v e r e l y o v e r l a p . Time-resolved phosphorimetry prov i d e s a u s e f u l means f o r i d e n t i f y i n g t h e s e drugs even i n some o f t h e i r m i x t u r e s . E x t e r n a l heavy-atom e f f e c t on d e t e c t i o n l i m i t s and l i f e times o f phosphoresence i n t i m e r e s o l v e d l a s e r - e x c i t e d phosphorimetry have been r e p o r t e d by B o u t i l i e r and Winefordner (89). I n t h e instrument d e s c r i b e d , r a d i a t i o n from a p u l s e d n i t r o g e n o r f l a s h lamp-pumped dye laser w a s d i r e c t e d v i a a f i l t e r t o t h e sample c o n t a i n e d i n a q u a r t z c e l l ( 3 0 cm X 2 mm i . d . ) mounted i n an n.m.r. s p i n n e r assemb l y . The e m i t t e d r a d i a t i o n was examined i n a system i n c o r p o r a t i n g a monochromator, photomultiplier, gated amplifier, signal generator, boxcar i n t e g r a t o r , c h a r t r e c o r d e r , o s c i l l o scope, s i g n a l o v e r a g e r , t a p e punch and minicomputer. Phosphoresence l i f e - t i m e and detect i o n l i m i t s were determined a t 77 K f o r solut i o n s o f phenanthrene and o f s e v e r a l drugs ( i n c l u d i n g c o c a i n e ) i n ethanol-HzO (1:9). I n g ' , d e t e c t i o n l i m i t s were t h e presence o f A o f t en improved.

9.4 . 5

Colorimetry Tomasch and Majer ( 9 0 ) have e s t i m a t e d some pharmaceutical e s t e r s which a r e used i n pharmacy. The method o f H i l l ( 9 1 ) w a s modif i e d and adopted f o r t h e d e t e r m i n a t i o n o f t h e e s t e r s . F i v e m l o f t h e e t h a n o l i c s o l u t i o n of t h e e s t e r (1mg/ml) i s added t o 1 ml o f 10% hydroxylamine h y d r o c h l o r i d e i n 80% e t h a n o l , 2 m l o f 10% sodium hydroxide i n 90% e t h a n o l and 5 m l p e r o x i d e - f r e e e t h e r ; t h e m i x t u r e i s shaken w e l l , a f t e r 20 minutes t r e a t e d w i t h 2 ml HC1 d i l u t e d 1 : 3 and 1 ml o f 10% o f f e r r i c c h l o r i d e (FeC13) i n 0.1N H C 1 , d i l u t e d t o 25 ml, f i l t e r e d from sodium c h l o r i d e and d e t e c t e d c o l o r i m e t r i c a l l y a t 320 nm.

204

FARID J. MUHTADI A N D ABDULLAH A. AL-BADR The u s e o f c o b a l t t h i o c y a n a t e i n t h e c o l o r i m e t r i c a n a l y s i s of c o c a i n e and some o t h e r o r g a n i c b a s i s have been r e p o r t e d ( 9 2 ) . The a u t h o r s have d i s c u s s e d e a r l i e r unpublished work by o t h e r a u t h o r s on t h e p r e c i p i t a t i o n and s e p a r a t i o n of a l k a l o i d s and o r g a n i c b a s e s w i t h t h i o c y a n a t e s some metals and w i t h c o b a l t i n p a r t i c u l a r . The p r e c i p i t a t i o n o f such compounds w i t h CO(SCN)z from aqueous medium r e s u l t s i n two t y p e s o f complexes, v i s , a b l u e t y p e ( t h e more common) which t h e c o b a l t forms a complex anion w i t h SCN-, and a r e d , brown o r v i o l e t t y p e i n which t h e c o b a l t forms a comp l e x c a t i o n w i t h o r g a n i c base. Condition for t h e d e t e r m i n a t i o n of v a r i o u s q u a t e r n a r y ammonium compounds and of c o c a i n e i n nonaqueous medium by e x t r a c t i o n w i t h benzene, chloroform o r dichloromethane a r e d i s c u s s e d ; a s u i t a b l e r e a g e n t i s prepared by d i s s o l v i n g CO(SCN)p i n methanolic HSCN ( o b t a i n e d by c a t i o n exchange) t o form a s o l u t i o n of H2CO(SCN)2. This i s d i l u t e d w i t h chloroform o r dichloromethane t o g i v e 0.2N s o l u t i o n , which i s added t o a s o l u t i o n o f t h e b a s e i n t h e same s o l v e n t . The e x t i n c t i o n o f t h e b l u e s o l u t i o n i s measured a t 625 nm. Other c o l o r i m e t r i c methods have been r e p o r t e d ( 93-95 1 *

9.4.6 Mass Spectrometry The mass a n a l y s e d ion-kinetic-energy s p e c t r o meter w a s a p p l i e d t o t h e d e t e r m i n a t i o n o f cocaine i n coca l e a v e s and u r i n e by u s e o f s i n g l e o r m u l t i p l e - i o n monitoring t e c h n i q u e (96); s e n s i t i v i t y w a s maximized by u s e o f s i n g l e - i o n monitoring ( w i t h i s o b u t a n e as reagent g a s ) . The d e t e c t i o n l i m i t f o r c o c a i n e i n 1 pg of coca l e a f b e i n g < 1 ng. Multipler e a c t i o n monitoring provided similar s e n s i t i v i t y t o , and was more s e l e c t i v e t h a n s i n g l e i o n monitoring. The e f f e c t o f t h e method o f sample i n t r o d u c t i o n w a s important. For q u a n t i t a t i v e analysis, isotope dilution with analyte l a b e l l e d w i t h s t a b l e i s o t o p e was p r e f e r r e d ; a l t e r n a t i v e l y , s t andard-addit i o n , e x t e r n a l -

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s t a n d a r d or c a l i b r a t i o n graph methods could be used. The graph ( b a s e d on s i n g l e - r e a c t i o n monitoring) on peak a r e a v s amount f o r solut i o n o f c o c a i n e i n methanol w a s r e c t i l i n e a r f o r 1 t o 1 5 0 ng. S i n g l e r e a c t i o n monitoring permitted detection of cocaine i n unreacted samples ( c o n t a i n i n g l a r g e q u a n t i t i e s of many o t h e r d r u g s ) and o f benzoylecgonine i n u r i n e . Cooks e t a 1 ( 9 7 ) have reviewed mass analyzed i o n k i n e t i c energy spectrometry ( M I K E ) and p r e s e n t e d i t s a p p l i c a t i o n t o t h e d i r e c t analys i s o f cocaine and cinnamoylcocaine i n coca p l a n t (Erythroxylum c o c a ) t i s s u e s . Moore (81) have p r e s e n t e d m . s . d a t a for t h e i d e n t i f i c a t i o n o f cis and t r a n s - cinnamoylc o c a i n e i n i l l i c i t c o c a i n e s e i z u r e s . The n.m.r. d a t a and some o t h e r s p e c t r a l d a t a have a l s o been p r e s e n t e d .

9.5

Counter-Current E x t r a c t i o n The s e p a r a t i o n o f t h e m i x t u r e s o f o r g a n i c s u b s t a n c e s by a s i m p l i f i e d method o f c o u n t e r - c u r r e n t e x t r a c t i o n had been r e p o r t e d ( 9 8 ) . The o p t i m a l c o n d i t i o n s were found f o r t h e s e p a r a t i o n o f two component a l k a l o i d and o t h e r o r g a n i c compound m i x t u r e s by t h e method o f c o u n t e r - c u r r e n t e x t r a c t i o n i n f u n n e l s . The dependance o f t h e e x t r a c t i o n w i t h chloroform for q u a n t i t a t i v e s e p a r a t i o n o f c o c a i n e , and o t h e r a l k a l o i d s , on t h e pH v a l u e s o f t h e b u f f e r s o l u t i o n i s p r e s e n t e d .

9.6

Chromatographic Methods

9.6.1

Thin Layer Chromatography Clarke (17) d e s c r i b e d t h e f o l l o w i n g system: Glass p l a t e s , 20 X 20 cm, c o a t e d w i t h a slurry c o n s i s t i n g o f 30 gm o f s i l i c a g e l G i n 60 m l o f water t o g i v e a l a y e r 0.25 mm t h i c k and d r i e d a t 110' f o r 1 hour. A sample 1 . 0 p 1 o f a 1% s o l u t i o n i n 2 N a c e t i c a c i d , t a k e n by a micro d r o p , i s used. The s o l v e n t system cons i s t s of s t r o n g ammonia s o l u t i o n : methanol (1.5 : 1 0 0 ) . It should be changed a f t e r two r u n s . Solvent i s allowed t o s t a n d i n t h e t a n k

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f o r one hour. The assending chromatogrem i s developed i n a t a n k 21 X 21 X 10 cm, t h e end of t h e t a n k being covered with f i l t e r paper t o a s s e s t evaporation. T i m e of run 30 minutes. The l o c a t i o n reagent i s an a c i d i f i e d iodoplat i n a t e spray, and t h e R f value is 0.60. Several solvent systems have been used f o r t h i n - l a y e r chromatographic s e p a r a t i o n of cocaine andareshown i n Table ( 9 ) . Table ( 9 )

Thin-Layer Chromatography of Cocaine

Absorbent and Solvent Systems

Visualizing agent

Rf

Ref.

--

W a t 230 nm a f t e r e x t r a c t i o n i n t o chloroform

0.73

102

W a t 230 nm a f t e r e x t r a c t i o n i n t o chloroform.

0.31

102

Sprayed by 5% H2SO4 a ) Chloroform : methanol conc ammonia ( 9 :10 :1) followed by iodoplat i nat e

0.90

103

Sprayed by 5% H p O 4 followed by iodoplat i nat e

0.87

103

0.79

105

S i l i c a g e l F 1500 LS 254 a c t i v a t e d a t 110' f o r 2 hours methanol : aqueous ammonia (100:1.5) ethanol : chloroform (1:l

S i l i c a g e l 60 F254 0.25 m

.

b ) Ethyl a c e t a t e : methanol : water : conc. ammonia ( 8 5 :10 :3 :1)

.

Activated precoated s i l i c c Acidic i o d o p l a t i n a t e ( AIPA) g e l p l a t e s of 0.25 mm t h i c k n e s s (Merck) E t h y l a c e t a t e : n propanol: 28% ammonium hydroxide ( 40: 30: 3) Precoated s i l i c a g e l G , 26 X 20 cm, 0.25 mm l a y e r *ethyl a c e t a t e : methanol 17:2 and 20 m l of 50% ammonia i n a beaker.

Dragendoff t h e n iodoplat i n a t e

0.73

207

COCAINE HYDROCHLORIDE Continued Table ( 9 )

Absorbent and Systems

Solvent

Visualizing agent

Chloroform : methanol (1:l) and 20 m l o f 50% ammonia i n a beaker. The above two system are placed i n t h e center of a tank.

Other t h i n l a y e r chromatography systems have a l s o been r e p o r t e d ( 1 7 , 99-101, 104, 106-109,

111-119). 9.6.2

Paper Chromatography Clarke

(17)d e s c r i b e d

t h e f o l l o w i n g system:

Whatman No. 1, s h e e t 1 4 X 6 i n c h , i s b u f f e r e d by d i p p i n g i n a 5% s o l u t i o n o f sodium dihydrogen c i t r a t e , b l o t t i n g , and d r y i n g a t 25' f o r one hour. It c a n b e s t o r e d i n d e n i n i t e l y . A sample o f 2.5 1~.1 o f a 1% s o l u t i o n ; i n 2 N a c e t i c a c i d i f p o s s i b l e , o t h e r w i s e i n 2N h y d r o c h l o r i c a c i d , 2 N sodium hydroxide, or e t h a n o l s o l v e n t , 4.8 g o f c i t r i c a c i d i n a m i x t u r e o f 130 m l of water and 870 m l o f n( T h i s s o l v e n t may be used for butanol. s e v e r a l weeks i f w a t e r i s added from t i m e t o t i m e t o keep t h e s p e c i f i c g r a v i t y a t 0.843 t o 0.844). The chromatogram i s ascending, i n a t a n k 8 X 11 X 15% i n c h , 4 s h e e t s being run a t a t i m e . Time o f run, f i v e h o u r s , R f v a l u e 0.38, l o c a t i o n i s done under u l t r a v i o l e t l i g h t , s t r o n g a b s o r p t i o n and t h e l o c a t i o n reagent i s iodoplatinate spray; strong react ion. Bastos e t a1 ( 1 2 0 ) r e p o r t e d a method f o r r o u t i n e i d e n t i f i c a t i o n of c o c a i n e m e t a b o l i t e s i n human u r i n e . The sample ( 1 0 m l ) w a s a d j u s t e d t o pH 8 t o 9 w i t h NaHC03 and e x t r a c t e d

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chloroform : e t h a n o l ( 3 : 2 ) ( 5 m l ) ; t h e lower l a y e r ( c o n t a i n i n g , e.g. unmetabolized c o c a i n e ) was r e j e c t e d . The upper l a y e r w a s s a t u r a t e d w i t h K2CO3 and c e n t r i f u g e d , and t h e pH o f t h e e t h a n o l i c phase was a d j u s t e d t o < 7 with d i l . H C 1 . The e x t r a c t e d metab o l i t e s were converted i n t o t h e i r b u t y l d e r i v a t i v e s w i t h butanol-H2S04 and t h e solut i o n w a s washed w i t h t o l u e n e . The s o l u t i o n was t h e n s a t u r a t e d w i t h NaHC03 and t h e d e r i v a t i v e s were e x t r a c t e d w i t h cyclohexane ( 1 0 m l ) . The e x t r a c t w a s e v a p o r a t e d , and t h e r e s i d u e w a s d i s s o l v e d i n chloroform and a p p l i e d t o a polygram s i l i c a g e l MN s h e e t . The chromatogram w a s developed w i t h e t h y l acetate-methanol-water ( 7 : 2 : 1 ) , t h e n f o r a s h o r t d i s t a n c e i n t h e same d i r e c t i o n w i t h chloroform-acetone-aqueous ammonia( 5 :94 :1) A l t e r n a t i v e l y , t h e s e two s o l v e n t s were used f o r t w o - d i r e c t i o n a l s e p a r a t i o n . S p o t s were d e t e c t e d by s p r a y i n g w i t h i o d o p l a t i n a t e r e a g e n t . El-Darawy and Mobarak ( 1 2 1 ) s t u d i e d t h e chromatographic s e p a r a t i o n o f some a l k a l o i d s i n c l u d i n g c o c a i n e , on carboxymethylcellulose cation-exchange paper. From 5 t o 8 p1 of methanolic s o l u t i o n (1mg p e r m l ) o f each a l k a l o i d were a p p l i e d t o t h e paper and a f t e r development o f t h e chromatogram, t h e s p o t s were d e t e c t e d by viewing under 254 nm r a d i a t i o n or by s p r a y i n g w i t h Dragendorff r e a g e n t .

.

9.6.3

Gas Chromatography

Clarke ( 1 7 ) d e s c r i b e d t h e f o l l o w i n g system: Column: 5% SE-30 on 60-80 mesh chromosorb W AW. 5 ft X 118 i n c h i n t e r n a l diameter s t a i n l e s s s t e e l column. Column t e m p e r a t u r e 230°, Carrier g a s : Nitrogen g a s flow: 30.7 m l p e r minute, D e t e c t o r : flame i o n i s a t i o n , hydrogen 22 ml p e r minute. R e t e n t i o n time i s 0.57 r e l a t i v e t o codeine. S e v e r a l r e p o r t s had been p u b l i s h e d concerning t h e chromatographic i d e n t i f i c a t i o n and separat i o n of c o c a i n e and i t s m e t a b o l i t e s as shown i n Table 1 0 .

Table (10) G a s Chromatography of Cocaine

Column

U- shaped column ( 6 f t x 0.25 in. ) 2.5% pf SE-30 on G a s Chrom Q (100-120 mesh)

C a r r i e r gas Nitrogen 25 ml/min. operated a t 2000.

Remarks

Detector Flame i o n i s a t ion

-

-

-

3% of

ov-1

-

-

-

The benzoyl ecgonine ( a cocaine m e t a b o l i t e ) i s t h e d e t e c t e d substance which w a s o b t a i n e d from u r i n e . The m e t a b o l i t e i s s e p a r a t e d by TLC t h e n w a s s u b j e c t e d t o GLC a f t e r being converted i n t o i t s methyl d e r i v a t i v e s by t r e a t m e n t w i t h 1,ldimethoxytrimethylamine at 70' t o 80° f o r 1 hour. Down t o 1 g o f t h e m e t a b o l i t e p e r m l of u r i n e w a s e a s i l y detected. Recovery of t h e m e t a b o l i t e a t t h e e x t r a c t i o n s t a g e w a s = 100% but only 30 t o 40% w a s recovered by t h e prepared TLC.

Ref.

99

Tetracosane w a s used as i n t e r n a l standard. 122 The cocaine H C 1 c o n t e n t s of 6 samples analysed ranged from 6 t o 100%. Recoveries were 98.7 t o 103%. C o e f f i c i e n t of v a r i a t i o n were 0.89 t o 3.16. The method w a s adopted as o f f i c i a l f i r s t a c t ion.

-

Continued Table (10 )

.

Carrier gas

Column 2-M g l a s s column packei! with 5% of polyoxyet hylene glycol 20 M on Chromosorb W (100-120 mesh)

-

4 f t X 0.125in packed with Chromosorb W (100-120 mesh) coated with 3% of ov-1.

Helium 30 ml/min-l temperature 230'.

Glass column (20 m X 0.35 mm i . d . 1 wall-coat ed with SE-30.

1.4 nWmin-1

Detector

Remarks

-

Nitrogen 30 ml/min-: Oven temp e r a t u r e i: programmed t o 230' at ao min-1.

-

t o r 0

Nitrogenphosphorous detector

-

Nitrogen

Flame i o n i s a , tion

Used f o r t h e i d e n t i f i c a t i o n n a r c o t i c s and psychotropics through p y r o l y t i c GC. The sample i n aqueous s o l u t i o n (1t o 2 ~1 containing 50 t o 100 pg o f salt of t h e d r u g ) , i s placed on t h e f i l t e r e d w i r e and d r i e d i n a stream o f hot a i r (100'). Pyrolysis i s c a r r i e d out a t 610' for 1 0 s , and t h e product i s t h e n s e p a r a t e d i n t h e column. S a l t a n a l y s i s i s p r e f e r r e d (because o f reducec volatility). Blood + i n t e r n a l standard i s b u f f e r e d a t pH 9 and e x t r a c t e d w i t h 1-chlorobutane and t h e e x t r a c t ( p l u s H C 1 ) i s evaporated a t 60' under nitrogen C a l i b r a t i o n graph a r e r e c t i l i n e a r f o r 0 . 2 t o 1 Ug m l - 1 f o r t h e drugs (cocaine and o t h e r basic drugs).

.

- Ethyl morphine i s used a s t h e i n t e r n a l standard.

Ref.

Continued Table ( 1 0 )

Column A Fye 104

Chromatograph w i t h a column (1.5 m X 4 mm) of Gas-Chrom Q (100-120 mesh) s u p p o r t i n g 3%

Carrier g a s

Remarks

Detector

Ref.

-

Nitrogen temperature 2200.

Rapid method f o r t h e d e t e r m i n a t i o n of c o c a i n e .

Flame i o n i s a Helium t ion 30 ml/min-' The column w a s programmed ( 2 O min-1 f o r 190' t o

- Used f o r t h e d e t e r m i n a t i o n o f t h e drug, h e r o i i 127

126

of ov-17 N ~1 C1

Glass column ( 6 f t x 2 mm) packed w i t h a 1:l m i x t u r e o f 3% o f OV-17 on Varaport 30 (80 t o 1 0 0 nesh and 5% o f SE-3C on Chromosorb W (80 t o 1 0 0 mesh

24O0.

and morphine.

- The powdered sample w a s e x t r a c t e d w i t h chloroform : methanol ( 3 : l ) c o n t a i n i n g r e s m e t h r i n as i n t e r n a l s t a n d a r d .

Cont h u e d Table (10 ) ~

Column

Carrier gas

A silanised Nitrogen g l a s s column 25 ml/min-' (2.4 m X 2 mm) 2200 packed w i t h 6% of OV-1 on Chromosorb W AW - DMCS (100 t o 200 mesh).

Detector Flame i o n i s a t ion

~~

-

~~

Remarks

-

Ref.

-

Used f o r t h e chromatographic a n a l y s i s of t h e cocaine o f Erythroxylum coca from t h r e e l o c a t i o n i n Peru. Androst-4-ene-3,17-dione w a s used as i n t e r n a l standard

128

.

-

~~

Conventional OV-1 column

(74 cm

x

2 mm) column packed 2% of OV-101 on Gas-Chrom-Q AW DMCS (100-120 mesh)

H e l i u m (205')

Helium ( 20 min-l) ( temp-programming f r o n 140° t o 240' a t 100 min-1)

-

Nitrogen phosphorous detector

- Cocaine and benzoyl ecogonine were q u a n t i t a -

-

130

t e d a f t e r JETUBE e x t r a c t i o n and d e r i v a t i s a tion. The propyl e s t e r of benzoyl ecgnonine i s used as t h e i n t e r n a l s t a n d a r d .

-

- Used t o determine ecgnonine methyl e s t e r , a

-

major m e t a b o l i t e of cocaine, i n u r i n e , a f t e r o r a l a d m i n i s t r a t i o n of cocaine. Phencyclidine i s t h e i n t e r n a l standard. The e f f l u e n t i s monitored by 70 e V m . s . a t m/e 82 f o r cocaine and i t s e s t e r and a t m / e 200 for t h e i n t e r n a l standard ( p h e n c y c l i d i n e )

131

I

COCAINE HYDROCHLORIDE

213

Other g a s chromatographic procedures f o r t h e s e p a r a t i o n and i d e n t i f i c a t i o n have been p u b l i s h e d (53,56,81,99,129,132-150).

9.6.4

Gas Chromatography-Mass Spectrometry (GC-MS) Cocaine h a s been determined by GC-MS by several authors:-

a ) J i n d a l and Vestergaard ( 1 5 1 ) have r e p o r t e d a method f o r q u a n t i t a t i o n of c o c a i n e and i t s p r i n c i p a l m e t a b o l i t e , benzoylecgonine by glc-ms u s i n g s t a b l e i s o t o p e - l a b e l l e d analogues as i n t e r n a l s t a n d a r d s . E x t r a c t t h e c o c a i n e from u r i n e a t pH 9 i n t o chloroform and t h e n i n t o 0.1N H C 1 , a d j u s t t h e s o l u t i o n t o pH 9 w i t h aqueous ammonia, r e - e x t r a c t i n t o chloroform, e v a p o r a t e t h e e x t r a c t t o d r y n e s s a t 40' under n i t r o g e n , and d i s s o l v e t h e r e s i d u e i n benzene. Analyse a p o r t i o n of t h e s o l u t i o n of glc-ms on a g l a s s column ( 1 . 8 m X 2 mm) c o n t a i n i n g 1 . 5 % of OV-1 on Gas Chrom 9, ( 1 0 0 t o 200 mesh) and o p e r a t e d a t 205' w i t h helium a s c a r r i e r g a s ( 2 0 m l m i n - l ) . Use N-( t r i d e u t e r o m e t h y l ) c o c a i n e as t h e i n t e r n a l s t a n d a r d , and compare t h e i o n i n t e n s i t i e s a t m / e 303 and 306. E x t r a c t benzoylecgonine and t h e i n t e r n a l s t a n d a r d , N-( trideuterornethy1)-benzoyecogonine, from u r i n e a t pH 7 i n t o chloroform : i s o p r o p y l alcohol ( 4 : 1 ) , evaporate t h e e x t r a c t t o dryness a t 60° under n i t r o g e n , convert benzoylecgonine i n t o t h e e t h y l e s t e r w i t h e t h a n o l i c diazomethane, e v a p o r a t e a t 40' under n i t r o g e n , and d i s s o l v e t h e r e s i d u e i n chloroform. E x t r a c t t h e s o l u t i o n w i t h 0.1N - H C 1 , a d j u s t t h e aqueous phase t o pH 9, and e x t r a c t w i t h benzene, e v a p o r a t e t h ? e x t r a c t t o dryness a t 40' under n i t r o g e n , d i s s o l v e t h e r e s i d u e i n benzene, and submit on a l i q u o t t o glc-rns as f a r c o c a i n e , but compare i n t e n s i t i e s a t rn/e 317 and 320. About 2 ng m l - I o f c o c a i n e and 5 ng m l - 1 of benzoylecgonine can b e determined w i t h a p r e c i s i o n o f about 5%.

2 14

FARID J. MUHTADI A N D ABDULLAH A. AL-BADR Lawry e t a1 ( 5 7 ) have i d e n t i f i e d two novel c o c a i n e m e t a b o l i t e s i n b i l e by g a s chromatography and by g a s chromatographymass spectrometry i n a c a s e of a c u t e i n t r a v e n o u s c o c a i n e overdose. The drug i s e x t r a c t e d from t h e sample i n t o 1c h l o r o b u t a n e and back-extracted i n t o weak a c i d . The a c i d e x t r a c t i s made a l k a l i n e and t h e compound i s e x t r a c t i n t o c h l o r o form f o r g l c w i t h 3% of OV-1 o r OV-17 as s t a t i o n a r y phase. For g l c , m s , t h e OV-1 column i s used, w i t h t e m p e r a t u r e programmi n g and electron-impact m.s. The compounds i d e n t i f i e d i n c l u d e c o c a i n e , t h e known m e t a b o l i t e s , methylecgonine, norc o c a i n e , and benzoylecgonine, and a l s o a hydroxycocaine and methylecgonidine. Chinn e t a1 (152) have d e s c r i b e d a g a s chromatography-chemical i o n i s a t i o n mass spectrometry of c o c a i n e and i t s metabol i t e s i n b i o l o g i c a l f l u i d s . The sample, e.g. blood, u r i n e , aqueous t i s s u e homogen a t e o r v i t r e o u s humour, i s t r e a t e d w i t h sodium f l u o r i d e t o i n h i b i t enzymic hydrol y s i s o f cocaine. The one p o r t i o n ( p l u s t r i d e u t e r a t e d c o c a i n e as i n t e r n a l standard) i s made a l k a l i n e w i t h K2HP04 and e x t r a c t e d w i t h toluene-heptane-isoamyl a l c o h o l ( 7 : 2 : 1 ) ; t h i s e x t r a c t c o n t a i n s c o c a i n e and norcocaine. A second p o r t i o n ( p l u s t r i d e u t e r a t e d benzoylecgonine as i n t e r n a l s t a n d a r d ) i s s a t u r a t e d w i t h sodium chlor i d e and e x t r a c t e d ( a t pB 7 ) w i t h c h l o r o form-isopropyl a l c o h o l (9:l); t h e e x t r a c t i s evaporated a t 60°, and t h e r e s i d u e i s t r e a t e d by R method similar t o t h a t o f J a i n et al(115) t o form t h e propyl e s t e r of benzoylecgonine which i s p u r i f i e d by e x t r a c t i o n . The f i n a l e x t r a c t c o n t a i n s c o c a i n e and t h e ester o f benzoylecgonine. Each e x t r a c t i s a n a l y s e d by g l c a t 205' [column 1 . 2 m X 2 mm; 3% of OV-1 on Gas Chrom Q (100 t o 1 2 0 mesh); methane (=20 m l min-l) as c a r r i e r g a s and r e a g e n t gas f o r t h e subsequent 120 e V m . s . 1. The mass s p e c t r a are monitored a t m / e 304 f o r

COCAINE HYDROCHLORIDE c o c a i n e , 307 ( f o r d e u t e r a t e d c o c a i n e ) ; 290 for norcocaine and 332 and 335 for t h e d e r i v a t i v e s o f benzoylecgonine and d e u t e r a t e d benzoylecgonine r e s p e c t i v e l y

2 15

.

Jindal _ et a1 (153) have a l s o determined c o c a i n e and i t s b i o l o g i c a l l y a c t i v e metab o l i t e , n o r c o c a i n e , i n human u r i n e . The sample (1m l ) w a s t r e a t e d with ['H3]norcocaine ( 5 6 n g ) and, a f t e r adjustment t o pH 8.5, w a s e x t r a c t e d w i t h cyclohexane. The r e s i d u e from e v a p o r a t i o n of t h e e x t r a c t was t r e a t e d w i t h t r i f l u o r o a c e t i c anhydride, t h e m i x t u r e was evaporated and an a l i q u o t o f a s o l u t i o n o f t h e r e s i d u e i n benzene w a s s u b j e c t e d t o g.c.-m.s. w i t h u s e o f a g l a s s column (1.8 m X 2 mm) packed w i t h 1.5% o f OV-1 on Gas Chrom Q (100 t o 120 mesh) and o p e r a t e d a t 205O. The mass s p e c t r o m e t e r was o p e r a t e d i n t h e s e l e c t e d - i o n monitoring mode a t 70 e V , and t h e i n t e n s i t i e s o f t h e i o n s a t m/e 303 and 306 and m / e 263 and 266, were used t o measure c o c a i n e and n o r c o c a i n e , respectively. J i n d a l e t a1 (154) have a l s o p u b l i s h e d a g a s - l i q u i d chromatographic - mass s p e c t r o m e t r i c d e t e r m i n a t i o n o f Lidococaine ( L i g n o c a i n e ) i n an i l l i c i t sample o f c o c a i n e . The sample, i n chloroform, w a s i n j e c t e d on t o a g l a s s column ( 1 . 8 m X 2 mm) s i l a n i s e d w i t h 5% of d i c h l o r o d i m e t h y l s i l a n e i n t o l u e n e , packed w i t h 3% o f OV-17 on Gas Chrom Q (100 t o 200 mesh) and o p e r a t e d a t 200°, w i t h flame i o n i s a t i o n d e t e c t i o n . Two peaks were r e s o l v e d , one w a s due t o c o c a i n e and t h e o t h e r t o l i g n o c a i n e a s w a s shown by combined g . c . 70 e V m . s . ( t h e molecular i n n w a s a t m / e 234 and t h e b a s e peak w a s a t m/e 8 6 ) . Clark ( 1 5 5 ) has d e s c r i b e d a masss p e c t r a l q u a n t i t a t i o n method for t h e a n a l y s i s of cocaine hydrochloride i n powders. A s o l u t i o n c o n t a i n i n g 4 mg each o f cocaine h y d r o c h l o r i d e and [ 2Hs]-cocaine

FARID J. MUHTADI A N D ABDULLAH A. AL-BADR

216

i n 25 m l of methanol i s a n a l y s e d by g.1.c.-m.s. on Finnigan model 9500 and model 3300 i n s t r u m e n t s l i n k e d by a j e t s e p a r a t o r . A 0.2 1-11p o r t i o n o f s o l u t i o n i s i n j e c t e d ( v i a a p o r t a t 240') i n t o a g l a s s column (120 cm X 2 mm) packed w i t h 3% of OV-1 on Gas Chrom Q (100 t o 120 mesh) and o p e r a t e d a t 190°, w i t h helium as c a r r i e r g a s ( 4 0 ml m i n - l ) . The spectrometer i s operated i n r e p e t i t i o n scan mode ( e v e r y 2s) from m / e 75 t o 310. Lewin e t a1 (156) r e p o r t e d a combined g.c.-m.s. o f c o c a i n e and i t s t h r e e i s o mers ( f o u r i s o m e r s ) and of t h e correspondi n g ecgonine methyl e s t e r s , w i t h u s e o f a column (1.8 m X 2 mm) o f 2% of OV-17 and chemical i o n i s a t i o n , w i t h i s o b u t a n e o r NH3 as r e a g e n t as, i s s t u d i e d ; f r a g mentation p a t t e r s are d i s c u s s e d i n d e t a i l . Other g.c.-m.s. methods have a l s o been p u b l i s h e d (62, 81, 157-159).

9.6.4

High-pressure Liquid Chromatography (HPLC) S e v e r a l HPLC systems for t h e i d e n t i f i c a t i o n and a n a l y s i s of c o c a i n e have been r e p o r t e d in the literature:-

Olieman e t a1 ( 1 6 0 ) r e p o r t e d a method f o r a n a l y s i s o f c o c a i n e , pseudococaine, a l l o c o c a i n e and allopseudococaine by ionp a i r reverse-phase high-performance l i q u i d chromatography. The compounds can be s e p a r a t e d by l i q u i d chromatography on a column o f o c t a d e c y l s i l y l - s i l i c a w i t h t h e a d d i t i o n t o t h e e l u e n t of n-heptanesulfon a t e and i d e n t i f i e d by peak area measurements at d i f f e r e n t u l t r a v i o l e t wavelength.

-

Masoud and Krupski (161) have d e v i s e d an HPLC method for t h e a n a l y s i s of c o c a i n e i n human plasma a f t e r being i s used as a n a n a e s t h e t i c for n a s a l s u r g e r y . Blood i s c o l l e c t e d i n a t u b e c o n t a i n i n g sodium f l u o r i d e and D-glucose c i t r a t e phosphate

COCAINE HYDROCHLORIDE

217

s o l u t i o n (pH 5 . 7 ) , and t h e plasma ( w i t h amethocaine H C 1 added as i n t e r n a l s t a n d a r d i f d e s i r e d ) i s made a l k a l i n e w i t h sodium c a r b o n a t e s o l u t i o n and e x t r a c t e d w i t h e t h e r . The drugs are back-extracted i n t o acetic acid, then t h e acid layer i s made a l k a l i n e and t h e drugs are e x t r a c t e d i n t o hexane. T h i s e x t r a c t i s evaporated under n i t r o g e n a t 40' and t h e r e s i d u e i s d i s s o l v e d i n t h e mobile phase [methanol : 0.05 phosphate b u f f e r (pH 6 . 6 ) (3:1)] f o r h . p . 1 . c . a t 40' on a column (25 cm X 2.6 mm) o f Perkin-Elmer ODs-HC SIL-X-1 f i t t e d w i t h a brownless RP-18MPLC guard column's e l u t i o n i s a t 0.8 m l min-l and d e t e c t i o n i s at 232 nm. R e t e n t i o n times are 5.2 min f o r c o c a i n e and about 8 min. f o r amethocaine. Poochikian and Cradock ( 1 6 2 ) have d e t e r mined c o c a i n e i n t h e p r e s e n c e o f i t s h y d r o l y s i s p r o d u c t . The drugs were s e p a r a t e d from each o t h e r and from t h e i n t e r n a l s t a n d a r d ( 4 - c h l o r o p y r i d i n e ) by h . p . 1 . c . on a column (30 cm X 4.6 mm i . d . ) of p Bondapak C 1 8 ( 1 0 pm) o p e r a t e d a t ambient t e m p e r a t u r e w i t h 1 5 mm phosphate b u f f e r (pH ? , ) - a c e t o n i t r i l e ( 3 : l ) as t h e mobile phase (0.8 ml min-1) and d e t e c t i o n a t 235 nm. The d e t e c t i o n l i m i t s were 3 ng f o r cocaine. e t_a 1 (156) have i d e n t i f i e d and Lewin q u a n t i t a t e d isomeric c o c a i n e s by HPLC. Cocaine i s s a t i s f a c t o r i l y s e p a r a t e d from i t s t h r e e isomer by h . p . 1 . c . on a column o f p a r t i s i l - 1 0 PXS o p e r a t e d w i t h i s o propyl alcohol-hept ane-diethylamine (25:75:0.1) as mobile phase at a flow r a t e i n c r e a s i n g ( d u r i n g 1 2 m i n u t e s ) from 0.48 t o 4 m l min-1; t h e e l u t e i s monitored a t 230 nm. C a l i b r a t i o n graphs are based on peak a r e a s r e l a t i v e t o t h o s e of N Ndibenzylbenzamide ( t h e i n t e r n a l s t a n d a r d ) . Evans and Morarity ( 1 6 3 ) have r e p o r t e d t h e a n a l y s i s of cocaine and i t s m e t a b o l i t e s

218

FARID J. MUHTADI AND ABDULLAH A. AL-BADR HPLC. The plasma or t i s s u e homogenate i s mixed w i t h aqueous i n t e r n a l s t a n d a r d ( L i g n o c a i n e ) and s o l i d sodium f l u o r i d e ( t o i n h i b i t enzymic h y d r o l y s i s o f c o c a i n e and n o r c o c a i n e ) , t h e pH i s a d j u s t e d t o 9 ( c a r b o n a t e b u f f e r s o l u t i o n ) and t h e mixt u r e i s e x t r a c t e d w i t h chloroform-isop r o p y l a l c o h o l (3:2). The e x t r a c t i s evaporated under n i t r o g e n a t 40' and a s o l u t i o n o f t h e r e s i d u e i n Ij20 i s subm i t t e d t o h.p.1.c. on a column ( 3 0 cm X 4 mm) o f p Bondapak C 1 8 , w i t h water a c e t o n i t r i l e - methanol ( 8 : l : l ) c o n t a i n i n g 1% o f a c e t i c a c i d and 0.3M i n EDTA as mobile phase ( 2 ml min-1) and d e t e c t i o n at 235 nm. R e t e n t i o n t i m e f o r cocaine 9.7 , benzoylecgonine 2 . 9 , Lignocaine 4 . 2 and norcocaine 11.1 min. F l e t c h e r and Hancock (164) have r e p o r t e d p o t e n t i a l e r r o r s i n benzoylecgonine and c o c a i n e a n a l y s i s . Cocaine H C 1 s o l u t i o n ( 5 0 pg m l - l ) were a d j u s t e d t o pH v a l u e s between 2 and 9.4 and were analysed immediately, or a f t e r being set a s i d e f o r u p t o 6.75 hours by h.p.1.c. on a column ( 1 0 c m X 4.6 mm) of H y p e r s i l 5-ODs ( 5 pm) w i t h aqueous 55% methanol a d j u s t e d t o pH 3.8 w i t h H3PO4 as mobile phase Benzoylecgonine ( r e t e n t i o n ( 2 m l min-l) t i m e 1 . 4 m i n u t e s ) and c o c a i n e ( r e t e n t i o n t i m e 3.4 m i n u t e s ) were d e t e c t e d a t 232 nm.

.

Noggle e t a1 (165) have p u b l i s h e d a l i q u i d chromatographic procedure f o r i d e n t i f i c a t i o n o f cis and t r a n s - cinnamoylcocaine i n i l l i c i t cocaine. A methan o l i c s o l u t i o n o f t h e sample w a s a n a l y s e d by h.p.1.c. w i t h u s e o f a s t a i n l e s s s t e e l column ( 3 0 cm X 4 mm) o f 1.1 Bandapak-C18, w i t h phosphate b u f f e r (pH 3)-methanol (2:l) as mobile phase ( 2 ml min-1) and w i t h two u l t r a v i o l e t d e t e c t o r s , a t 254 and 280 nm, r e s p e c t i v e l y , i n series. The r e l e v a n t f r a c t i o n s were c o l l e c t e d , made a l k a l i n e w i t h aqueous ammonia and e x t r a c t e d w i t h dichloromethane. Residues o b t a i n e d on e v a p o r a t i o n were d i s s o l v e d

219

COCAINE HYDROCHLORIDE

separatdyin methanol for the analysis by ultraviolet spectrophotometry and by mass spectrometry. Development of a standardized analysis strategy for basic drugs using ion-pair extraction and high-performance liquid chromatography, philosophy and selection of extraction technique have recently been reported (166)

.

Others HPLC methods have also been reported (167,168).

9.7 Radio-immunoassay Mule' -et a1

(169) reported the evaluation of the radio-immunoassay for benzoylecgonine (a cocaine metabolite) in human urine. The 251-radio-immunoassay (RIA) for benzoylecgonine in urine was evaluated by comparison with gas liquid chromatograph and thin-layer chromatography and the enzymemultiplied immunoassay technique. By radio-immunoassay, a statistically significant concentration, 2 pgllitre, was observed for urinary benzoylecgonine. The coefficient of variation for the radio-immunoassay was 2.58 0.38% interassay and 2.20 f. 0.14% interassay. There was cross-reactivity with cocaine (more reactive than benzoylecgonine and other members of the tropane family of alkaloids. There was agreement between results by radio-immunoassay and gas liquid chromatography in 95.5% of the samples, between radio-immunoassay and thin-layer chromatography in 87.0% and between radio-immunoassay and enzyme-multiplied immunoassay technique in 84.5%. The percentage of true false-positive was 3.5% for the radio-immunoassay in comparison to gas-liquid chromatography, 8.8% in comparison to thin-layer chromatography and 9.1% in comparison to enzyme-multiplied immunoassay technique. True false-negative were insignificant (0. to 1 . 0 % ) . Gas liquid chromatography and radio-immunoassay results correlated highly ( 4 = 0.908). Gas-liquid chromatography, therefore, was the best comparison method for the evaluation study. Radio-immunoassay for benzoylecgonine is sensitive, reproducible and reliable for the detection of cocaine in urine.

*

220

FARID J. MUHTADI A N D ABDULLAH A. AL-BADR Budd (170) r e p o r t e d a c o c a i n e radio-immunoassay-

s t r u c t u r e v e r s u s r e a c t i v i t y . He t e s t e d s e v e r a l a l k a l o i d s f o r benzoylecgonine antibody-binding a c t i v i t y i n t h e Roche r . i . a k i t s . Benzoylecgonine has t h e optimum antibody-binding a c t i v i t y ; change of any o f t h e s u b s t i t u e n t s ( e x c e p t e s t e r i f i c a t i o n of t h e carboxy-group) reduced t h e b i n d i n g , cocae t h y l e n e w a s t h e only drug t h a t i n t e r f e r r e d w i t h t h e Roche a s s a y a t t h e r a p e u t i c o r overdose l e v e l s , but it was seldom encountered under t h e s e conditions. Thus t h e k i t was c o n s i d e r e d t o be s u i t a b l e f o r a s s a y i n g cocaine and i t s m e t a b o l i t e s . Baum-gartner et a1 (171) p u b l i s h e d a method f o r r a d i o - i m u n o a s s a y of c o c a i n e i n h a i r . The drug was detected i n h a i r o f 13 p a t i e n t s from a drug-abuse c l i n i c who acknowledged having used c o c a i n e i n v a r y i n g amounts d u r i n g t h e l a s t s i x months. A c o r r e l a t i o n was observed between t h e amount of c o c a i n e used and t h e q u a n t i t y t r a p p e d i n t h e i n t e r i o r o f h a i r grown d u r i n g t h e s i x month p e r i o d . I n c o n t r a s t t o h a i r a n a l y s i s , u r i n a l y s i s by t h i n l a y e r chromatography w a s n e g a t i v e i n a l l c a s e s . I n d i c a t i n g t h a t , c o c a i n e h a s not been used by t h e p a t i e n t s w i t h i n 48-72 hours b e f o r e t h e u r i n e c o l l e c t i o n . Hair a n a l y s i s t h u s appears t o be f a r superior t o urinalysis for establishing h i s t o r i e s of drug use. Other immunoassay methods have a l s o been r e p o r t e d (110, 172-174).

ACKNOWLEDGEMENT The a u t h o r s would l i k e t o thank Mr. Uday C . S h a n a and Tanvir A . B u t t , b o t h o f College of Pharmacy, King Saud h i v e r s i t y €or t h e i r v a l u a b l e s e c r e t a r i a l a s s i s t a n c e i n t y p i n g o f t h i s manuscript,

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N.C. J a i n , D.M. Chinn, R . D . Budd, T . S . Sneath and W . J . Leung, J . F o r e n s i c S c i . , 2, 7 (1977).

140.

P. 1. J a t l o w and D.N. B a i l e y , C l i n . Chem. , 21, 1918, (1975).

141.

J . I . J a v i a d , H. Dekirmenjian, E.G. Brunngraber and J.M. Davis, J . Chromatogr., 1 1 0 , 1 4 1 (1975).

142.

J.I. J a v a i d , H . Dekirmenjiab,: J.M. Davis and C.R. S c h u s t e r , J . Chromatogr. , 105 (1978).

143.

J.I. J a v a i d , M.W.

Panzik; J . Pharm.

z,

and J . M .

Fishman, C . R .

Davis, S c i e n c e ,

S c h u s t e r , H . Dekirmenjian

202, 227

(1978).

144.

M . J . Kogan, K.G. Verebey, A.C. de Pace, R . B . S . J . Mule', Anal. Chem., 1965 ( 1 9 7 7 ) .

145.

S.H. M i l l e r , B. Dvorchik and T.S. Davis, P l a s t i c R e c o n s t r u c t i v e S u r g e r y , @, 566 ( 1 9 7 7 ) .

146.

D.L.

147.

J.M.

Moore, J . Chromatogr.,

148.

J.M.

Moore, C l i n . Chem.,

149.

J . C . Valentour, V . Aggarwal, M.P. McGee and S.W. Goza, J . Anal. T o x i c o l . , 2, 134 (1978).

150.

J.E. Wallace, H.E. Hamilton, D.E. King, D. J . Bason, H.A. S c h u e r t n e r and S.C. Harris, Anal. Chem., 34 (1976) .

9,

von Minden and N.A. ( 1977

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Resnick and

D'Amato, Anal. Chem.,

49,1974

101,215 ( 1 9 7 4 ) .

21,

1538 ( 1 9 7 5 ) .

48,

FARID J. MUHTADI A N D ABDULLAH A. AL-BADR

230

151.

67,811

S.P. J i n d a l and P. Vestergaard, J . Pharm. S c i . ,

(1978) 152.

D.M. Chinn; D . J . Grouch; M.A. P e a t ; B.S. F i n k l e ; and T.A. J e n n i s o n , J . Anal. T o x i c o l . , k, 37 ( 1 9 8 0 ) .

153.

S.P. J i n d a l ; T. Lutz; and P. Vestergaard, Biomed. Mass Spectrom, 5, 658 (1978).

154. 155.

S.P. J i n d a l ; T. Lutz; and Per. Vestergaard, J . Chromat., 357 (1979)-

179, C.C.

C l a r k , J . Assoc. O f f . Anal. Chem.,

6 4 ( 4 ) , 884

(1981)

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A.M.

Lewin; S.R. P a r k e r , and F . I . C a r r o l l , J . Chromat.,

193, 371 ( 1 9 8 0 ) . 157.

J.E.

Grass and E. Watson, J. A n a l . T o x i c o l . ,

(1978) 158.

2,

80

9

B. Holmstedt, J . E . Lindgren, L. R i v i e r and T. Plowman, B o t a n i c a l Museum Leaflets Harward U n i v e r s i t y , 26, 199

(1978) 159.

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C. Olieman; L. Maat; H . C . Beyerman, R e d . Trav. Chim. 501 (1979). Pays-Bas ,

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98,

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65,

321 (1983).

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Massart, J. Pharm. Biomed. Anal.,

1,

COCAINE HYDROCHLORIDE

167. 168.

A.P. G r a f f e o , D.C.K.

126,717 (1976).

23 1 Lin and R . L .

F o l t z , J . Chromatogr.,

P . I . J a t l o w , C . van Dyke, P. Barash and R. Byck, J. Chromatogr., 152, 115 (1978).

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Jukofsky; M. Kogan; A . D e Paco; and K. Verebey, C l i n . Chem. 23, 796 (1977).

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R.D.

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S . J . Mule', M.L. 20,

(1981). Blahd,

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This Page Intentionally Left Blank

EPHEDRINE HYDROCHLORIDE SYED LAIK ALI

1. 2. 3. 3.1. 3.2. 4. 5. 5.1. 5.2. 5.3. 5.4. 5.5. 5.6. 5.7. 5.8. 5.9. 5.10. 5.11. 5.12. 6.

7. 8. 9. 9.1. 9.2. 9.3. 9.4.

9.5. 9.6. 9.7. 10. 11.

History Nomenclature Description Name, Formula, Molecular Weight Appearance, Colour, Odour S ynthesis Physical Properties Solubility Loss on Drying Melting Point Specific Optical Rotation Sulphates Sulphated Ash pH Value Dissociation Constant Ultraviolet Spectrum Infrared Spectrum Nuclear Magnetic Resonance Spectrum Mass Spectrum Stereochemistry Colour and Identification Reactions Stability and Degradation Methods of Analysis Ti trimetry Visible and UV Spectrophotometry Fluorimetry Chromatographic Methods Thin Layer Chromatography , Paper Chromatography, Gas-Liquid Chromatography, High Performance Liquid Chromatography NMR Assay Radioimmunoassay Isotachophoresis Drug Metabolism and Pharmacokinetics Acknowledqement References

ANALYTICAL PROFILES O F DRUG SUBSTANCES VOLUME 15

233

Copyright 0 1986 by the American Pharmaceutical Association All rights of reproduction in any form reserved.

SYED LAIK ALI

234

Ephedrine Hydrochloride Ephedrine and ephedrine hydrochloride have been mostly used synonymously in this monography. Ephedrine is quite often cited in literature but it is presumed to be valid also for ephedrine hydrochloride. Aqueous or acidic solutions of ephedrine hydrochloride are often converted into ephedrine before other chemical or chromatographic operations and measurements. Casually ephedrine sulfate has been also included in this monograph. Physical properties, spectra etc. are given for ephedrine hydrochloride. 1.

History Ephedra is a Phanerogame-Gymnosperme from the family of Gnetaceen. There are 30 different types of this plant-species known which grow in Asia, mediteranian countries and America. Specially ephedra vulgaris, ephedra equisetina and ephedra sinica contain ephedrin with its other isomers. A certain ephedra species has been used in ancient Chinese medicine since ages. Already in 5 0 0 0 B.C. an ephedraplant was widely used in China under the name Ma-Huang. Ma-Huang has been mentioned as medicine of moderate therapeutic range in the first Chinese pharmacopoe, published under the government of Shen Lung in 1 7 6 0 B.C. A detailed description of the plant and its pharmacological action is given in the Chinese pharmacopoe of modern times in 1 5 9 6 . According to this pharmacopoe Ma-Huang was applied as Diaphoreticum, Antipyreticum and Sedativum for the treatment of coughs and colds as well as a stimulant for blood-circulation ( 1 ) . These therapeutic effects were mostly confirmed later for ephedrin which was discovered and isolated in pure form by Nagai (2) in 1 8 8 7 as a main alkaloid of ephedra from ephedra sinica.

EPHEDRINE HYDROCHLORIDE

2.

235

Nomenclature Ephedrine hydrochloride is ( I R , 2 . S ) - ( - ) - / 3 hydroxy-N d-dimethyl-phenethylammonium chloride. The formula is illustrated at the next Page

3.

Description

3.1. Name, Formula, Molecular weight Ephedrine Hydrochloride CloH15NO HC1 201.70 3.2.

Appearance, Colour, Odour Colourless crystals or white, crystalline, orthorohombic needless or powder, odourless, with a bitter taste.

4.

Synthesis Ephedrin was synthesised for the first time by Spzth ( 3 ) as illustrated in Fig. 1. Racemic ephedrin was then further split up into its components. Addition of methylamin to I-phenylpropylenoxyd leads also to the synthesis of ephedrin (4). Ephedrine could further be synthesised through amination of A-Brompropiophenon with methylamine or benzylmethylamine. A-Brompropiophenon was synthesised through bromination of propiophenon and propiophenon itself was prepared with Friedel-Crafts reaction using benzene and propoyl chloride (5). The resulting product methylaminoketon or benzylmethylaminoketone was then hydrogenated in presence of palladium as catalyst and a racemic mixture of ephedrin was obtained (Fig. 2). This was further reacted with sodium dibenzoyl tar-

SYED LAIK ALI

236 Ephedrine

0

CHOH-

Hydrochloride

I CH - C H 3

STRUCTURAL FORMULA

FIG, 1

CH3

y 3

I

FIG, 2

CI

-

EPHEDRINE HYDROCHLORIDE

237

trate to convert ephedrine into its optical isomers. Dibenzoyl tartrate of (-)ephedrine was then treated with hydrochloric acid to give (-)ephedrine hydrochloride in 60 % yield. This process was used on an industrial scale by C-H-Boehringer Company in Germany during second world war (5). Later on this method was further modified by using dibenzoyl(+)-tartaric acid or mandelic acid for the resolution of racemic o( -methylaminopropiophenone into its optical isomers in 90 % yield. The resulting product was then catalytically hydrogenated to (-)ephedrine (6). A biochemical process for the production of optical active (-)ephedrine-isomer is applied by Knoll Company in Germany. L-phenyl acetyl carbinol is obtained through an acyl condensations with benzaldehyde during the fermentation process of a Melasse-solution. This compound is then reacted with methylamine and simultaneously reduced to (-)ephedrine in presence of active aluminium or a platinum catalyst (Fig. 3 ) . Ephedrine is then converted into ephedrine hydrochloride which is recrystallised from water into optically pure isomer ( 7 , 8 ) . Upon heating ephedrine hydrochloride is decomposed into phenyl ethyl ketone and methyl amine. 5.

Physical properties

5.1. Solubility It is freely soluble in 4 parts of water, and in 17 parts of alcohol ( 9 6 % ) , very slightly soluble in chloroform and practically insoluble in ether (9). 5.2. Loss on Drying

Not more than 0.5 %, determined with 1.00 g by drying to constant weight in an oven at 100" - 105°C (9).

1. H2N -CH3(Amination)

Q

2. HZ (Reduction) 3

- CH ~ C - H N - C I- H

HO - C - H

HO

I

c =o I

I

CH3

H3

FIG, 3

H

239

EPHEDRINE HYDROCHLORIDE

5.3.

Melting point 217O - 22OOC (9)

5.4.

Specific optical Rotation to -35.5O, determined by dissolving 5.00 g in sufficient water to produce 50.0 ml, diluting 1 0 ml to 20 ml with water and calculated with reference to the dried substance (9). -33.5O

5.5.

Sulphates 1 0 0 ppm with limit test for sulphates (9).

5.6. Sulphated Ash Not more than 0.1 (9). 5.7.

determined with 1 .O g

pH-Value The pH of a 0.5

5.8.

%,

%

aqueous solution is 5.9.

Dissociation constant The Pk, of ephedrine hydrochloride in water at 2 O o C is reported to be 9.68 ( 1 1 ) . This value is attributed to the ammonium cation of the molecule.

5.9.

Ultraviolet Spectrum The ultraviolet spectra of ephedrine hydrochloride were taken with a Perkin-Elmer UV spetrophotometer 5 7 1 at a concentration of 238

SYED LAIK ALI

240

0 . 5 mg/ml in methanol, 0.1N HC1 and 0.1N

In all three solvents the spectra show absorption bands at almost identical wave lenghts of 2 5 0 , 2 5 6 and 2 6 2 nm.

NaOH.

The molecular extinction coefficients and A 1 % of ephedrine hydrochloride are reported Icm to be ( 1 2 ) Absorption Maximum

Methano 1 2 5 0 nm 2 5 6 nm 2 6 2 nm 8.4 10.8 8.2 170 220 165

A1 %'

1 cm

€

0.1N HC1 2 5 0 nm 2 5 6 nm 2 6 2 nm 7.4 9.4 7.2 150 190 145

0.1N N a O H 2 5 1 nm 2 5 7 nm 2 6 3 nm 7.8 9.7 7.4 160 195 150

The UV spectra are shown in Fig. 4. 5.10.

Infrared Spectrum The infrared spectrum of ephedrine hydrochloride is given in Fig. 5 . The spectrum was obtained with a Perkin-Elmer 1 4 2 0 Ratio Recording Infrared Spectrophotometer from a KBr pellet. The structural assignments may be correlated with the following band frequencies: Frequency

( cm-11

3330 2700-2840 2480 1 4 5 0 and 1 4 9 0 7 5 0 and 6 9 8

Assignments Stretching vibrations of OH Amine halide salt stretching bands NH2+ stretching Characteristic vibrations of the aromatic ring C-H out of plane deformation, monosubstitued benzene

EPHEDRINE HYDROCHLORIDE

24 1

- Methanol - - 0,lN - HCI ---- 0,l N - Na OH FIG, 4 'UV SPECTRUM OF EPHEDRINE I N DIFFERENT SOLVENTS

HYDROCHLORIDE

3

4

I

coo0

3000

MlKR ONS 5 6

.

7

8

.

91012U1

2OOO 1800 1600 UOO 1200 loo0 800 625 I

WAVE NUMBER

1

CM

O1

FIG, 5 IR

SPECTRUM OF EPHEDRINE HYDROCHLORIDE,

1420 S PECTROPHOTOMETER

O R PELLET,

PERKIN-ELMER

243

EPHEDRINE HYDROCHLORIDE

5.11. Nuclear Magnetic Resonance Spectrum The nuclear magnetic resonance spectrum of ephedrine hydrochloride was obtained with a Varian T-60 NMR spectrometer in D20. The following spectral assignments are made for the spectrum reproduced in Fig. 6. Chemical shift 1.20 doublet 2.90 singlet 3.60 multiplet 5.28 doublet 7.53 singlet

Assiqnment CH3 at CH CHS at NH2 CH at NH2 CH at OH aromatic protons

OH and NH2 deuterated. 5.1 2. Mass spectrum In the highest mass regions molecular ion peak ist not observed. Prominent ion-peaks observed are m/e, 31, 59, 78 and 108. The mass spectrum of ephedrine hydrochloride is given in Fig. 7. Instrument: Sample temperature: Source temperature: Electron energy:

Varian Mat 44 (direct inlet) 5OoC - 25OOC in 5 min 25OOC 80eV

Some ions of this spectrum can be correlated to the structure as following: NH CH3 M-C6H5CHOHHCl = 59; M-CHOHCHCH3HC1 = 78 M-CHCH3HC1 = 108 NHCH3

1

a: W

IW

r 0

a: Iu W a cn 0

Lo I-

z c (

a cc

%

a > W

U

n a: 0

-1

u

I

0

a: n

>

I W

U

z a: n W

I

I I W LL

0

a:

r 3 I-

u

W

a v)

cc

z

I

EPHEDRINE HYDROCHLORIDE

FIG, 7 MASS SPECTRUM

OF

EPHEDRINE HYDROCHLORIDE

245

SYED LAIK ALI

246

6.

Stereochemistry Ephedrine has two asymmetric centres; there exist four steroisomers and two inactive forms 5 ephedrin and 2 pseudoephedrin. The erythro-configuration of asymmetric centres in (-)ephedrine was confirmed by Freudenberg (13) through the synthesis from D(-Imandelic acid and L(+)-alanin. According to the CahnIngold-Prelog nomenclature the absolute configuration of asymmetric carbon atoms in (-)ephedrine could be defined as 1R,2S. Hyne has found with nmr measurements the dieder angle of 90' between OH and NHCH3 in ephedrine base (14). Gauche-Confirmation A and B are strongly favoured in free ephedrine base and its salts (15). Ephedrine hydrochloride dissolved in D20 lies 90% in A and B forms and 10% in trans-form C (Fig. 8). Testa (16) found good agreement of CD and ORD results with nmr values.

7.

Colour and Identification Reactions To 10 mg ephedrine hydrochloride in 0.1 ml water when 0.2 ml of copper sulphate solution (12.5%) and 1 ml of strong sodium hydroxide solution (10 N) are added, a violet colour is produced. When this solution is shaken with 2 ml ether, the ether layer turns purple and the aqueous layer remains blue (17). 50 mg of the substance dissolved in 1 ml water is mixed with 4 ml 0.1N NaOH, shaken with 3 ml CCl4 for ten seconds and then allowed to stand for 2 minutes. The organic layer is separated and treated with few copper turnings. A turbidity appears rapidly which turns in few minutes into a copious precipiate (18, 19). L- and D,L-ephedrines could be distinguished through their crystalline modification. An aqueous ephedrine salt solution is acidified with 3 drops of 15%

I

I " u Ln

I,

m

0

I i i

I

I

0

I

Xi

x

0

-

w

n CI

CK 0

o

0

0 0 - I I

w

>-

- C K L L

I w

~

SYED LAIK ALI

248

H2SO4, 2 ml potassium iodobismutate solution (prepared by mixing 2 parts of 7 % basic bismuth nitrate in nitric acid, 1 part potassium iodide and making upto volume of 1 0 ml with water) are added and left to stand overnight. L-ephedrine is identified under microscope with needle-like crystals, whereas D,L-ephedrine is visible as dark-red prisims (20). Ephedrine hydrochloride gives in weak alkaline solutions with ninhydrine a violett colour (21 1 . 8.

Stability and Degradation Decomposition of (-)-ephedrine was less than 1 % after the prolonged passage of air through cold (2OOC) or refluxing neutral or basic aqueous solutions (0.2% W / V ephedrine hydrochloride in phosphate buffer pH 7 . 4 or in 1% sodium hydroxide). On exposure to heat ephedrine hydrochloride is decomposed in phenyl ethyl ketone and methyl amine. GLC-MS provided almost the sole means of identifying some of the breakdown products of ephedrine due to their similar GLC properties and lability on tlc. Significant losses occurred during the extraction of small quantities of ephedrine from aqueous media using either regular or analytical grades of diethyl ether. The losses were, at least in part, caused by reaction of the ehedrine with aldehydic impurities in the ether. The addition of n-butanol to the ethereal extract before evaporation reduced the "breakdown" that occurred if the extracts were allowed to boil dry in the water bath. For this reason the routine use of aldehyde-free n-butanol is of value. Different substituted oxazolidines were identified through GLC-MS as break-down products. Consistent low levels of ephedrine "breakdown" were achieved by prior washing of the

EPHEDRINE HYDROCHLORIDE

249

ether with 1 0 % sodium metabisulphate solution followed by IN hydrochloric acid and finally with sodium hydroxide,5N. Negligible decomposition was observed on refluxing ephedrine ( 0 . 5 % ) in ether sturated with aqueous 20% NaOH for 8h or ephedrine (8%) in ethanolic sodium hydroxide for 3h. Ephedrine base stored in ether ( 1 0 0 mg/ml) at room temperature in light for several weeks decomposed to give oxazolidines. This is in contrast to the small amount of decomposition that occurred upon ultraviolet irradiation of aqueous solution of ephedrine. Solutions of ephedrine base (3% W / V ) in ether or benzene were extensively degraded by ultraviolet light over 18h. The major decomposition products yielding peaks upon glc examination after extraction of ephedrine solutions with ether and concentration of these extracts arise from addition and condensation of ephedrine with acetaldehyde, propionaldehyde and formaldehyde impurities in the solvent. Oxidation of (-)-ephedrine base with nickel peroxide, active silver carbonate and with active manganese dioxide gave benzaldehyde, a mixture of oxazolidines and 2-methyl-amino-I-phenyl-I-propanone as oxidation products. The degradation products were identified through glc and glc-ms analysis ( 2 2 ) . 9.

Methods of Analysis

9.1. Titrimetry

The assays of halogen salts of organic bases can be carried out either by a two-phase-titration or through non-aqueous titration of the substance. The two-phase titration applies ethanol 9 6 % and chloroform as solvent, phenolphthalein as an indicator and 0.1N sodium hydroxide as the titrant. The deter-

250

SYED LAIK ALI

mination of ephedrine hydrochloride in this medium is rather uncertain and gives about 3 % lower results ( 2 3 ) . Ephedrine hydrochlo-. ride is determined in german pharmacopoeia (DAB 7 ) through dissolving it in water, acidifying with 3 N nitric acid, additon of excess of O . l N silver nitrate solution and back-titration of silver nitrate with 0 . 1 N ammonium thiocyanate using ferric ammonium sulphate as an indicator ( 2 4 ) . In non-aqueous medium either ephedrine hydrochloride is dissolved in glacial acetic acid, mercuric acetate and then few drops of crystal violet indicator solution are added. The solution is then subsequently titrated with 0 . 1 N perchloric acid to an emerald-green end-point (USP XXI). Another method of non-aqueous titration is to dissolve the substance in warm mercuric acetate solution, to add acetone and then to titrate it against 0 . 1 N perchloric acid using saturated solution of methyl orange in acetone as an indicator until a red colour is obtained (26, 27). An indirect non-aqueous titrimetric method was devised for the determination of hydrochlorides of nitrogen-bases. The chloride-ion interference was prevented without the use of mercuric acetate reagent. The method depends on the treatment of a solution of the hydrochloride of the organic base with an excess of standard perchloric acid solution in acetic acid and the hydrogen chloride displaced is removed by boiling. The excess of perchloric acid is determined by titration against the basic titrant sodium acetate in g.lacia1 acetic acid using either potentiometric or visual end-point detection with a crystal violet indicator. Potentiometric titrations showed that the point of the maximum inflection in the titration curve coincided with the appearance of the violet colour of the indicator. The mean percent recovery of 99.66 % obtained indicates that the proposed method is equivalent in accuracy and precision to the non-aqueous titrimetric

EPHEDRINE HYDROCHLORIDE

25 1

method most commonly used by official compendia (28). Sanchez determined ephedrine by its reaction with alkaline iodine solution at 5OoC to form iodoform and titration of the excess of iodine in acid solution. Application of this method to standard solutions of ephedrine hydrochloride has given recoveries within 2 3% of the theoretical values. However, this method could not be applied to the determination of ephedrine HC1 in cough mixtures due to interferences from other constituents (29). Horak and Gasperik used a method for the determination of ephedrine based on the liberation of methylamine by alkaline hydrolysis. Good results were obtained for the determination of ephedrine HC1 in pharmaceutical injections and tablets (30). 9.2. Visible and UV-Spectrophotometry UV spectrophotometric determination of benzaldehyde or substituted benzaldehydes formed by periodate oxidation of ephedrine and other drugs with vicinal hydroxyl and amine functions has provided simple and sensitive assay methods for them. Ephedrine hydrochloride in dilute HC1 solution exhibits a molar absoptivity of 190 at its about 258 nm maximum. Oxidation of it to benzaldehyde affords an €-value of about 14400 at its maximum at about 241 nm in hydrocarbon solvents, about a 75-fold again in sensitivity (31). Periodate oxidations are among the most elegant reactions used in organic chemistry, because they are often quantitative within minutes at room temperature in aqueous media. Malaprade introduced periodic acid as a reagent for the oxidation of 1,2-glycols in 1928 (32). Nicolet and Shinn first reported the use of periodate for oxidation of ethanolamine derivatives. They found that ethanolamines with primary or secondary amine functions were rapidly and quantitatively cleaved to al-

252

SYED LAIK ALI

dehydes and ammonia or a primary amine (33). Wickstrom studied the rate of periodate oxidation of ephedrine as a function of pH, titrating excess of oxidant iodometrically. He found that periodate consumption was too slow to measure at pH 3.0, very slow at pH 6.0 and stoichiometric within 10 min at pH 7.5 or higher ( 3 4 ) . Oxidation potential of periodateiodate couple is -1.6V in acid solution and about 0.7 V in alkaline solution (35). Wickstrom found that the reaction products of periodate oxidation of ephedrine are benzaldehyde, acetaldehyde and methylamine. The determination of ephedrine via colorimetry of acetaldehyde distilled from the reaction mixture was suggested ( 3 4 ) . Chafetz studied among others the periodate oxidation of ephedrine (36). It was quantitatively oxidised to benzaldehyde in 1 0 min in a bicarbonate medium. Extraction of benzaldehyde in n-hexane and its spectrophotometric determination was recommended. Spectrophotometry of the benzaldehyde produced by periodate oxidation will not distinguish between compounds such as ephedrine and phenylpropanolamine. It will further not discriminate steroisomers such as ephedrine (erythro-configuration) and pseudoephedrine (threo-configuration) ( 3 1 ) . Wallace conducted a reaction for the determination of ephedrine in biological samples at the temperature of boiling hexane, about 69OC, with a reaction time of 3 0 min. The hexane layer was separated, washed with dilute acid and benzaldehyde determined spectrophotometrically either as such or after conversion to its semicarbazone (37, 38). Chafetz ( 3 1 ) further showed that the carbonyl compounds analogs of ephedrine are oxidised to benzoic acid by periodate and do not interfere in the assay of ephedrine. It is further demonstrated that N-acetylephedrine does not react with periodate under the assay conditions.

EPHEDRINE HYDROCHLORIDE

253

An application of orthogonal functions to the UV spectrophotometric determination of ephedrine hydrochloride in tablets is reported (39). The method is applicable for the determination of a single substance in the presence of irrelevant absorption from excipients such as lactose, starch, sucrose, gelatin, talc, stearic acid and magnesium stearate. The choice of polynomial, number of points, wavelength range and intervals are illustrated. Glenn's method of orthogonal functions proved to be powerful in discounting irrelevant absorption contribution ( 4 0 ) . A colorimetric method for the quantitative determination of ephedrine hydrochloride in presence of chlorpheniramine maleate and guaiacolsulfonate potassium in a cough syrup containing colouring agents is described ( 4 1 ) . Ephedrine hydrochloride is assayed using bromothymol blue as a dye in which interference from chlorpheniramine maleate is taken into consideration. The extinction of the chloroform extract is measured at 4 2 0 nm against the reagent blank. A collaborative study for the on-column periodate reaction method for analysis of ephedrine in solid dosage forms is reported. Ephedrine is separated from water-soluble impurities and strong acids by elution from a weakly basic celite column, and further cleaned up by retention on a weakly acidic column while the weak acids, weak bases and organic-soluble neutrals are eluted. Ephedrine is eluted from the column after neutralisation with N H 3 and is converted to benzaldehyde via on-column periodate reaction and determined spectrophotometrically ( 4 2 , 43). An UV spectrophotometric determination of ephedrine hydrochloride in an antiasthma capsule preparation containing aminophylline and amobarbital is reported ( 4 4 ) . On a prepared column containing alginic acid ephedrine HC1 is retained from an ethanolic solution; aminophylline and amobarbital pass through the column. Subse-

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quently ephedrine HC1 is eluted with 0.1N HC1 and determined spectrophotometrically at 2 5 7 nm. A rapid second and fourth derivative UV spectrophotometric assay procedure is described for the determination of ephedrine or pseudoephedrine in pharmaceutical formulations. The method has been applied successfully to Ephedrine Elixir BP, Ephedrine HC1 tablets BP, Ephedrine Nasal Drops, Paedriatric Belladonna and Ephedrine mixture, tablets, capsules containing aminophylline and amobarbital and coloured syrups containing tripoldine hydrochloride and codeine phosphate. A simple extraction procedure avoids interference from colouring agents in certain formulations. Specificity, accuracy and precision of the method has been assessed. The second derivative absorption spectrum of ephedrine HC1 shows enhanced resolution of the fine structure. Discrimination against broad spectral bands in favour of narrow bands is one of the advantages in derivative spectroscopy. The second derivative assay of Ephedrine Elixir BP eliminates the interference of the broad absorption bands of excipients ( 4 5 ) . Colouring agents or other coformulated drugs are removed from an ephedrine formulation by a simple solvent extraction procedure before the derivative spectroscopy. The recovery of ephedrine by the extraction method was 99.2 % ( 4 5 ) . Ephedrine hydrochloride has been determined directly or after separation on cellulose thin layer plates photometrically using p-dianisidine in aqueous solutions at pH 7. The yellow colour has been measured at 4 2 4 nm. The method permits the determination of ephedrine in the range of 5 0 - 2 0 0 pg with a relative standard deviation of 2 4 % (46). Spectrophotometric determination of ephedrine is done at 629 nm after reacting it with periodic acid 3-methylbenzthiazolin-2-on-hydrazon (47). Organic bases like ephedrine are reacted with

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bromthymol blue, subsequently extracted with chloroform or methylene chloride and the extinction determined at 4 2 0 nm. The dyestuff itself does not dissolve in organic phase. An alkaline reagent like tetraethyl ammonium hydroxide solution gives a blue coloured organic phase which could be determined at 6 2 0 nm ( ( 4 8 , 4 9 ) . The coloured reaction product of bromcresol green with ephedrine can either be extracted at pH 5-6.2 with chloroform and the extinction is measured at 4 2 0 nm or the organic phase is further treated with an alkaline reagent and the extinction of the coloured aqueous solution is determined at 6 2 0 nm ( 5 0 ) . Secondary alkyl amines react with carbon disulphide to give dithiocarbamates which form with cupric salts coloured chelates. Ephedrine hydrochloride has been determined spectrophotometrically at 4 3 7 after extracting its coloured chelate with chloroform ( 5 1 , 52, 5 3 ) . 9.3.

Fluorimetry A fluorimetric method has been developed for the determination of ephedrine hydrochloride as its dansyl derivative in ephedrine tablets. After dansylation the dansyl derivative is separated on a thin layer plate and determined directly densitometrically at 5 2 6 nm ( 5 4 ) . In another method the dansyl derivative of ephedrine hydrochloride is eluted from the plate with ethanol and determined at its fluorescence maximum of 5 0 5 nm ( 5 5 ) . Dansyl derivative of ephedrine has its excitation maximum at 3 5 4 nm and emission maximum at 4 7 6 nm ( 5 6 ) .

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9.4. Chromatographic Methods Thin layer chromatoqraphy Beckett and Choulis have separated ephedrine from other compounds such as adrenalin, noradrenalin etc., on cellulose, silicagel G and aluminium oxide plates using n-butanol + glacial acetic acid and water (40 + 1 0 + 50) or with water saturated with n-butanol as mobile phases. On cellulose tlc plates ephedrine and its salts give two spots. The use of alkaline mobile phases leads to only one spot (57). Choulis has used the mobile phases n-butanolacetic acid-water (4+1+5) or phenol-0.1N HC1 (85+15) with cellulose tlc plates for separation of ephedrine (58). Waldi recommended the converting of ephedrine to its corresponding triacetylderivative before applying it on silicagel G plates. Having decreased its hydrophilic character it was possible to develop the chromatogram with chloroform - methan o l (9+1) as mobile phase. The Rf-value of ephedrine was found to be 0.51 ( 5 9 ) . Ephedrine could be detected by spraying tlc plates with a 0.2 % ninhydrin solution in ethanol or n-butanol and heating the chromatogram to 13O-14O0C ( 6 0 ) . The best modification with ninhydrin reagent is to use 0.3 % solution in n-butanol + 3 m l acetic acid ( 6 1 , 6 2 ) . A similar reaction can be carried out with Folin's reagent which re- sults in the formation of light rose-coloured spot for ephedrine ( 6 1 ) . A spectraldensitometric method is described for the determination of ephedrine in ephedra herb and ephedrine extracts. After t l c separation on silicagel G plates with the mobile phase n-butanol-glacial acetic acid-water (4+1+5) the chromoplate was dried, subsequently immersed in a 0.2 % ninhydrine solution in ethanol and finally dried at llO°C.

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The violet spots of ephedrine at Rf-value 0.3 were evaluated densitometrically at a measuring wavelength of 540 nm and a reference wavelength of 680 nm ( 6 3 ) . Ephedrine hydrochloride in tablets along with other components has also been determined densitometrically after its conversion to the dansyl derivative and chromatographic separation on at 105'C activated silicagel 60 (Merck) tlc plates using benzene + ethanol + glacial acetic acid (90 + 10 + 1 ) as a mobile phase. Spectrodensometric measurement was carried on with a 526 nm filter. This procedure was on account of its great sensitivity and good reproducibility very useful in content uniformity determinations of ephedrine H C 1 in tablets and capsules (54). A tlc separation of ephedrine after its conversion to the corresponding dansyl derivative was carried on silica gel tlc plate with toluene + methanol + acetone (9+1+1) as mobile phase. The Rf-value for ephedrine was found to be 0.50. The spots were evaluated using spectrofluorimeter equipped with a tlc scanning attachment; excitation wavelength was 360 nm and the emission wavelength was set between 500-510 nm using a U V filter. The improved selectivity and sensitivity have permitted an analysis in 10-100-fold excesses of other drugs. Detection limits are reported in the range of 1-10 ng or better. The reproducibility of the method is limited by the derivatization step, but a relative standard deviation of less than 2 % could be obtained (56). Lang has applied cellulose tlc plates and mobile phases of n-butanol-glacial acetic acid-water (4+1+5) or n-propanol+benzene+glacia1 acetic acid + water (40+30+10+1) for the separation of ephedrine HC1 in pharmaceuticals. The ephedrine spot was localised, scratched from the tlc plate, extracted with the solution of diazotized p-dianisidine

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reagent, the solution was filtered and then measured in a spectrophotometer at 424 nm against corresponding blank reagent (46). Waldi has separated ephedrine from alkaloids of various other groups on a silica gel tlc plate with the mobile phase chloroform-diethyl-amine, (19+1), Rf:0.29 (64). Simultaneous detection of ephedrine along with a wide variety of commonly abused drugs in a urine screening program using tlc techniques has been reported. A detailed extraction procedure for the urine samples is given. Gelman precoated silica gel glass microfilter sheets with a layer thickness of 250 pm were used. Different solvent systems and detection reagents were used. The specific colour reactions obtained there may not be achieved on glass plates coated with silica gel. Solvent systems ethyl acetate-cyclohexane-methanolammonia (70+15+10+5)and ethyl acetate-cyclohexane-ammonia (50+40+0.1) were found suitable for the separation of ephedrine. Microgram amounts of ephedrine could be detected with the detection reagents 0.5 % ninhydrin in n-butanol, mercury(II)sulfate, iodoplatinate and iodine-potassium iodide solutions (65). NBD-C1 derivative of ephedrine was separated along with the corresponding derivatives of other drugs on silica gel G plate with solvent systems diethyl ether-benzene ( l + l ) , ethyl acetate-cyclohexane ( 2 + 3 ) and ethyl acetate-cyclohexane ( 3 + 2 ) with Rf-values 0.42, 0.18 and 0 . 3 3 respectively. The spots showed yellow fluorescence in UV light 254 nm (66). Separation and determination of ephedrine

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along with other doping agents has been obtained through overpressurised TLC and HPTLC silica gel 60 F254 plates using an eluent n-butanol-chloroform-methyl ethyl ketonewater-acetic acid ( 2 5 + 1 7 + 8 + 4 + 6 ) with an external membrane pressure of 1 . 0 mPa. In comparison with the classical TLC, the resolution was improved, the development time was shorter and detection limit was lower. The quantitative evaluation was carried out with a scanner at 210 nm ( 6 7 ) . Ephedrine hydrochloride has been determined densitometrically at 270 nm after its separation on silica gel 60 HPTLC plates with the mobile phase ethyl acetate-glacial acetic acid-water ( 2 7 + 6 + 4 ) and nitration with nitrous gases ( 1 0 0 % nitric acid). After chromatography the HPTLC plate is heated in a drying chamber for 1 5 min at 16OoC and then exposed while still hot to nitrous gases for 1 0 min. The relative standard deviation for ephedrine hydrochloride was found to be 1.75 8 ( 6 8 ) .

Paper Chromatography By using papers impregnated with alkaline buffers, such as boric acid-NaOH, pH 1 0 , and ether saturated with water as mobile phase it is possible to separate (+)ephedrine (Rf: 0 . 7 0 ) from (+)pseudoephedrine (Rf: 0 . 3 8 ) ( 6 0 ) . Ephedrine and pseudoephedrine can be estimated after elution in the form of copper dithiocarbaminate complexes ( 6 0 ) . Dittrich quantitates ephedrine in paper chromatography by using the iodine fixation properties of the ephedrine followed by iodometric determination after elution with KI solution ( 6 9 ) . Other solvent systems for the paper chromatographic separation of ephedrine are n-butanol-acetic acid-water ( 4 + 1 + 5 ) , Rf: 0 . 7 3 ( 6 1 )

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isobutanol+formic acid+water (100+12+10), Rf: 0.50 (70), n-butanol saturated with water, Rf: 0.64 (71), cyclohexane-diethylamine (9+1), Rf: 0.47 (72). Gas Liquid Chromatography The separation of optical amines by GLC can be achieved by using either an optically active stationary phase after making derivatives with a suitable optically inactive reagent or an optically active reagent such as N-trifluoroacetyl-L-prolylchloride (TPC) to form diastereoisomers followed by chromatography on an optically inactive stationary phase. TPC has been used for the resolution of numerous asymmetric amines, including ephedrine ( 7 3 , 74, 75). TPC-derivative of ephedrine was prepared through addition of 0.1 M TPC solution in chloroform to the amine in chloroformic solution. After 10-15 min the solution was injected into a gas chromatograph with FID detector on a 3 % SE 30 packed column on Chromosorb G (AW, DMCS treated, 100-120 mesh), at 17OOC oven temperature isotherm and 22OoC injection block temperature using nitrogen (25 ml/min) as carrier gas. A quantitative determination of the enantionmetric percentages of ( - ) ephedrine, ( + ) and ( - 1 norephedrine-TPC derivatives was possible. The method was found to be suitable for biological studies (76). Ephedrine was determined by treating the sample with sodium periodate to form benzaldehyde and gaschromatographing a solvent extract of this mixture. The oxidation step was included because ephedrine did not give a well-resolved peak (77). rapid GLC method for the determination of ephedrine hydrochloride in suspension formulation along with other components such as A

EPHEDRINE HYDROCHLORIDE

26 1

theophylline and phenobarbital with A-naphthylamine as an internal standard is described. The analysis was performed on a 3 % OV 1 7 on Gaschrom Q 1 0 0 - 2 0 0 mesh packed column using a flame ionisation detector. The injection port, column and detector temperatures for the assay of ephedrine hydrochloride were maintained at 200, 150 and 2 0 0 ' C respectively. For the assay of ephedrine hydrochloride the sample was diluted with water, the pH was adjusted to pH 1 1 with 2 0 8 NaOH and the solution was extracted twice with chloroform. The chloroform solution was injected directly into gas chromatograph. Several additional substances such as flavouring or colouring agents were extracted by chloroform along with ephedrine, but they did not interfere with ephedrine assay. The meanrecovery in the assay of synthetic mixtures for ephedrine hydrochloride was 99.2 2 0.6 % and for commercial suspension 95.8 0.8 % ( 7 8 ) . A method for the assay of ephedrine hydrochloride is recommended by the joint committee of the pharmaceutical society and the society for analytical chemistry of Great Britain ( 7 9 ) . The assay procedure is a modification of the method proposed by Beckett and Wilkinson ( 8 0 ) . Ephedrine hydrochloride tablets, elixir, syrup and nasal drops have been analysed. An internal standard phendimetrazine bitartrate was added to the sample prior to its extraction. The aqueous solution is made alkaline with 20 % NaOH and the liberated ephedrine base is extracted with diethylether, the ether extract dried with anhydrous sodium sulphate, evaporated and made up to volume with ether. This solution is injected into gaschromatograph with a FID. The packing used in the 1 m, 4 mm i.d. glass column was 8 0 - 1 0 0 mesh, acid washed, silanised Chromosorb G impregnated with 2 % carbowax 6000 and 5 % of potassium hydroxide. The oven, injector and detector temperatures

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maintained were 150, 200, 2OO0C, respectively with nitrogen as carrier gas (35 ml/min). The retention times for phendimetrazine bitartrate base and ephedrine were found to be 3 and 5 min respectively. The collaborative study carried on found this procedure adequate for the determination of ephedrine in certain pharmaceutical preparations. An examination by mass spectrometry and IR analysis of the ephedrine peak confirmed that ephedrine base was eluted intact. Some tailing of the ephedrine peak was noted by some collaborators. In general coefficients of variation within laboratories were not greater than 3 % (80). An electron-capture GLC procedure for determination of plasma ephedrine concentrations is described (81). The procedure is capable of determining 2 ng/ml of ephedrine. Pentane extraction of the drug and the internal standard 3,4-dimethoxyamphetamine and formation of the N-pentafluorobenzyl derivatives were followed by GLC determination. The analysis was performed on a 0.9 m x 2 mm i.d. glass column filled with 3 % OV 225 on Chromosorb W, AW-DMCS, 100-120 mesh. The injection port, oven and detector temperatures were 250, 235 and 325OC respectively. An electron-capture 63 Ni detector was operated with a standing current of 3.0 n amp. Argon-methan (95:5) as a carrier gas was maintained at 93 ml/min. The retention times of N-pentafluorobenzoyl derivatives of ephedrine and internal standard were reported to be 1.40 and 3.82 min respectively. Different GLC stationary phases such as OV-7, OV-17 were tried but were not suitable for quantitation. The drug and internal standard were poorly resolved with the former and broad peaks were obtained with the later. A OV-25 column gave sharp peaks for ephedrine and internal standard derivatives, but the norephedrine, the major metabolite of ephedrine, could not be separated. Formation of N-trifluoroacetyl, N-pentafluoropropionyl,

EPHEDRINE HYDROCHLORIDE

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N-heptafluorobutryl and N-pentafluorbenzoyl derivatives and their glc-mass spectrometric identification are discussed together with comparative electron-capture sensitivities of these derivatives with Nickel-63 detector. The detection of the N-pentafluorobenzoyl derivative of ephedrine is at least 100-fold greater in sensitivity than detection of the N-trifluoroacetyl derivative (81). Heptafluorobutyryl ephedrine derivatives following benzene extraction of alkaline serum were used for electron-capture analysis of blood levels at therapeutic dosages (82). The procedures reported earlier were found insufficently sensitive for clinical use (83). The determination of ephedrine plasma levels were performed through GLC with FID using a 8 % carbowax 20 M + 2 % KOH on Chromosorb W column. Ephedrine was extracted from plasma with diethylether after alkalising the sample with NH40H (84). GLC with FID determination of ephedrine was performed on a 2.0 % Carbowax 20 M and 5 % KOH on Chromosorb G (100-120 mesh), AW, DMCS column at oven temperature of 100°C (22). Gas chromatography has been further applied for the specific quantitative determination of ephedrine and its metabolite norephedrine in urine (85, 86). A 1.83 m, 4 mm i.d. 3 % OV 1 on Gaschrom Q, 100-200 mesh column at 14OOC oven, 140' injection block and 23OOC detector temperatures was used to monitor urinary excretion of ephedrine in man through gas chromatography with FID (87). Derivatives of ephedrine with trialkylsilyl groups attached to the hydroxyl function of the molecule were synthesised and tested gas chromatographically on a 2 m 3 % OV 17 on chromosorb G, AW, DMCS glass column at oven temperatures between 175-185OC (88). N-TFA-L-alanine and N-TFA-L-alanyl chloride were used for the preparation of high volatile diastereomeric derivatives of ( - ) and (+)-ephedrine which were separated along with

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the derivatives of other chiral amino alcohols and amines on a 30 m glass capillary column OV-17 or SE 30 at a column temperature of 2OO0C with FID (89). The separation of the N,O-pentafluoropropionyl derivatives of the enantiomers of ephedrine and of some analogues has been carried out using chirasil-val (90). N,O-Bis-heptafluorobutyryl derivatives of ephedrine and other analogues were separated on a 18-m glass capillary column coated with XE-60-L-valine-(R)- d-phenylethylamide (91). Excellent separations of diastereomeric derivatives of several amino alcohols of the ephedrine type have been obtained on a column with a chiral stationary phase after N-acylation with L- A-chloroisovaeryl chloride and o-trimethylsilylation (92). KGnig and Benecke reported the resolution of a number of amino alcohols including the N-demethylated analogues of ephedrine and norephedrine on a GLC chiral modified OV-225 phase (93). High Performance Liquid Chromatoqraphy The separation and quantitation of ephedrine is carried out by means of HPLC after its conversion into 4-nitrobenzamide with 4-nitrobenzoyl chloride. A procedure for the preparation of derivatives is given. The determination was carried out on a 20 cm spherisorb 5 pm column using a solvent mixture of isooctane methylene chloride + ethanol + water (400+87+8+5)as a mobile phase with 1.8 ml/min flow-rate and the ephedrine derivative was detected at 337 nm. The detection limit is given as about 5 ng per 20 pl injected pure derivative with a relative standard deviation of less than -f 6 %. The method could also be applied for the analysis of plasma samples (94). The quantitative determination of combination of antihistamine and decongestant drugs inclu-

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EPHEDRINE HYDROCHLORIDE

ding phenylepherine, dl-ephedrine, 1-ephedrine, chlorpheniramine etc. contained in solid and liquid dosage forms are described. All active ingredients except the ephedrine optical isomers were separated from other ingredient with ion-paired HPLC. Elixirs, syrups, tablets and timed-release capsules or tablets were analysed. The chromatographic separation was done on a 4 mm i.d. x 30 cm p-Bondapak phenyl column with a mobile phase water-methanol-glacial acetic acid (55+44+1 V / V ) containing enough heptanesulfonic acid sodium salt to yield a 0.005 M solution. The flow rate was 2.0 ml/min and the detection wavelength 254 nm. The column used is capable of resolving almost all of the compounds except the stereoisomers 1-ephedrine and d-ephedrine. The reproducibility of the method was excellent with a coefficient of variation of 0.9 % (95). Simultaneous determination of ephedrine sulphate, hydroxyzine hydrochloride and theophylline in tablets by HPLC involved a 1 0 m Bondapak c18 column with acetonitrile-aque us ammonium carbonate solution (50+50) at pH 7.0 as the eluent and UV detection at 254 nm. 0.1 % aqueous ammonium carbonate buffer was prepared and adjusted to pH 7.0 with acetic acid (96). The chiral forms of ephedrine were analysed as the corresponding oxazolidines formed by reaction between the propanolamine and 2-naphthaldehyde. HPLC separation was carried out on a column of 25 cm x 4.6 mm i.d., 5 pm aminopropyl-bonded silica gel modified with (R)-N-(3,5dinitrobenzoyl)phenylglycine using a mobile phase n-hexaneisopropanol (99.5+0.5) with a flow rate 1.0 ml/min at 254 nm ( 9 7 ) . Following their conversion to dithiocarbamate ligands and subsequently to nickel complexes, the separation and quantitation of enantiomeric mixtures of ephedrine and pseudoephedrine were accomplished by liquid chromatography with

6"

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ternary solvent mixtures. Formation of nickel complexes prior to chromatography and on-column formation using nickel(I1)ions in the mobile phase has been studied ( 9 8 ) . Ephedrine and pseudoephedrine in formulated products were determined on a 1 0 pn alkylphenylp-Bondapak column with acetonitrite-water-monobasic sodium phosphate ( 1 % acetonitrile in 0.05 M monobasic sodium phosphate aqueous solution) as the eluent and UV detection at 2 1 0 nm ( 9 9 ) . A HPLC method is described in which ephedrine hydrochloride is measured after its oxidation to benzaldehyde through periodate simultaneously with theophylline and phenobarbital in tablets with butabarbital as the internal standard. A pH of 7.8 was selected for rapid oxidation of ephedrine and a detection wavelength of 2 4 1 nm was chosen which is near to the maximum for benzaldehyde and barbiturates and to the minimum for theophylline. Chromatographic column was a reversed phase C18 phase bonded on silica and the mobile phase consisted of acetonitrile ( 2 4 0 ml) mixed with 0.01 M phosphate buffer, pH 7.8 ( 7 6 0 ml). Benzaldehyde obtained from ephedrine had a retention time of 1 1 . 7 min. The chromatogram showed no interference from the excipients and other oxidation products. Procedures are provided for the assay of conventional and sustained-action tablet formulations ( 1 0 0 ) . In another HPLC method the simultaneous assay of ephedrine hydrochloride, theophylline and phenobarbital in tablets is reported. 2 5 cm x 4.5 mm i.d., 1 0 pm Partisil ODS I1 column and a methanol-0.007 M monobasic potassium phosphate ( 3 7 + 6 3 , pH 2 . 3 ) as mobile phase was used at detection wavelength 2 5 4 nm. A methanolic extract of the powdered sample containing salicylamide as the internal standard was injected into the chromatograph ( 1 0 1 ) . In the determination of ephedrine using reversed-phase ion-pair liquid chromatography, a chromatographically pure sample

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was observed to give three peaks under certain mobile phase condition. The peak due to ephedrine was found to vary from symmetrical to almost completely resolved split peaks in mobile phases containing only P I C B7 (heptane sulfonic acid). When sodium sulphate was included in the mobile phase peak-splitting was more pronounced. A proposal, that peak splitting was the result of the composite interplay of two discrete chromatographic mechanism, was investigated. The results of analysis by GC/MS confirmed that each peak was due to ephedrine, however, only one of the three split peaks was found to contain ionpairs. It is postulated that peak splitting is a physical phenomenon on reversed-phase column and the separation of these drugs by ion-pair HPLC is based on a mixed rather than a single mechanism ( 1 0 2 ) . Derivatization of ephedrine with dansyl chloride and a sensitive, specific HPLC method for its determination in complex pharmaceutical dosage forms is reported. A 25 cm x 2 . 8 m i.d. column SI 1 0 0 , 1 0 pm filled with Merck silica ge! and diisopropyl ether saturated with conc. Ammonia-isopropanol ( 9 9 + 1 ) were used for separation. The detection was carried out simultaneously with a fluorescence detector, 3 5 4 nm excitation, 4 7 6 nm emission, and a fixed-wavelength 2 5 4 nm UV detector (56). Determination of ephedrine sulfate in coughcold mixtures along with various other analgesic and antihistamine compounds was performed on a Corasil C 1 8 column with the mobile phase acetonitrile-water ( 6 0 : 4 0 ) with 1 % ammonium acetate and the pH adjusted to 7.40

(103).

HPLC retention characteristics of ephedrine have been measured along with 8 4 other basic drugs of forensic interest. Chromatography was carried on using 250 x 5 mm i.d. column packed with Spherisorb S5W at 2 5 4 nm. The

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eluent consisted of methanol-aqueous ammonium nitrate buffer (9+1). The buffer was prepared by adding 94 ml ammonia (35 % ) and 21.5 ml nitric acid (70 % ) to 884 ml water and then adjusting the pH to 10.1 with ammonia. The flow-rate was 2 ml/min. Because of the alkaline nature of the eluent, a short column, dry packed with silica (40 pm) was included between the pump and injector to minimise dissolution of the analytical packing material (104). Resolution of the enantiomers of ephedrine and other related compounds through a simple HPLC method is described. A 150 mm x 4.6 mm column was packed with Ultrasphere O D s , 5 pm particle size and the mobile phase was prepared by mixing 400 ml acetonitrile with 600 ml water containing 1.4 g of monobasic ammonium phosphate. The flow-rate was 1.0 ml/min and the column eluent was monitored at 254 nm. Ephedrine or ephedrine hydrochloride is derivatised with the chiral reagent 2,3,4,6-tetra0-acetyl-P-D-glucopyranosyl isothiocyanate (GITC) and the separation of the resulting diastereomeric thioureas is performed by reversed phase HPLC. The derivatisation method is extremely simple, the chiral reagent is commercially available, chemically and stereochemically stable. The resolution of ephedrine, pseudoephedrine and norephedrine is considerably better (105). R-4-Methylbenzyl isothiocyanate, a commercially available chiral compound, was evaluated as a chiral derivatizing agent for the separation of among others ephedrine's enantiomers through HPLC on a 150 x 4.6 mm Ultrasphere ODS 5 pm column with a water-acetonitrile (50:50) mobile phase at 1.0 ml/min flow-rate (106). The enantiomers of ephedrine were resolved as their cyclic oxazolidine derivatives which

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were produced by the condensation of the amino alcohol with 2-naphthaldehyde. The enantiomeric resolution of ephedrine was performed on an ionically bonded chiral stationary phase. The column was a stainless steel Regis-packed pirkle type I-A (250 x 4.6 mm) with an d-aminopropyl packing of 5 pm spherical particles modified with (R)-N-(3,5dinitrobenzoy1)phenyl-glycine. The mobile phase consisted of hexane-isopropanol (99.5+0.5), the flow-rate was 1.0 ml/min and the detection wavelength was set at 254 nm (107). Ephedrine is converted to metal (copper and nickel) dithiocarbamate complexes by means of a pre-column derivatisation method. Chromatography is done on a Lichrosorb RP 18 column with mixtures of acetate buffer (pH 5.8) and organic solvents like methanol, acetonitrile or ethanol as mobile phases. The complexes were detected amperometrically (applied potential + 0.7 V VS SCE) using a thin-layer electrolytic cell fitted with glassy carbon working and auxiliary electrodes. The spectrophotometric detector was set at 325 nm for nickel chelates and at 270 nm for copper chelates. The procedure is described to have a great sensitivity (about -1 2 10 M) and good selectivity for the more substituted amino drugs (108). 9.5. NMR-Assay rapid NMR method is described for the determination of ephedrine hydrochloride in tablets. The NMR spectrum of ephedrine hydrochloride in D20 displays chemical shifts for methyl proton doublet at 1.16 ppm, N-methyl proton singlet at 2.83 ppm and aromatic protons singlet at 7.43 ppm. The doublet at 1.16 ppm was chosen for quantitative work. Acetamide was used as an internal standard on A

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the basis of its methyl three proton singlet at 2 ppm, which is sufficiently separated from the 1.16 ppm doublet of ephedrine hydrochloride and the solvent signal at 4.75 ppm to allow satisfactory determination. The results for synthetic mixtures and tablets are comparable to those obtained by other methods. The relative standard deviations were 0.5 and 1.2 % for the pure drug and tablets respectively. In addition the NMR method furnishes a specific means of identification of ephedrine (109). 9.6. Radioimmunoassay

Stereospecific radioimmunoassay for l-ephedrine and d-ephedrine in human plasma after administration of a single 50 mg oral dose of dl-ephedrine hydrochloride has been reported (110). Separate RIAS developed for d-ephedrine and 1-ephedrine were used to measure the concentrations of the enantiomers of ephedrine in the blood of two volunteers dosed with racemic ephedrine. The RIAS were validated by comparing the sum of the concentrations of the enantiomers with total ephedrine concentrations determined by a nonstereoselective GLC-ECD method. Fig. 9 shows graphically plasma concentrations of dand 1-ephedrine (110). Budd has presented a comparison of GC and EMIT (enzyme multiplied immunoassay technique) methods for the analysis of ephedrine and other related drugs. For GC analysis an Apiezon-KOH column was used ( 1 1 1 ) . 9.7. Isotachophoresis

The utility of isotachophoresis has been examined for the simultaneous determination of all the six ephedrine alkaloids in ephedra

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HOURS

FIG, 9 (4) AND L-EPHEDRINE ( A ) CONCENTRATIONS I N THE PLASMA OF A HEALTHY HUMAN VOLUNTEER FOLLO-

D-EPHEDRINE

WING A S I N G L E

50 MG ORAL DOSE OF DL-EPHEDRINE

HYDROCHLORIDE

(110)

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herb extracts. Ephedra herb was extracted with 50 % ethanol. All analysis were carried out on a Shimadzu model Ip-2A Isotachophoretic analyzer equipped with a Shimadzu potential gradient detector with a 4 0 mm PTFE capillary ( 1 . 0 mm i.d.1 as the pre-column and a 1 5 0 mm FEP capillary (0.5 mm i.d.) connected in series. The leading electrolyte was prepared by adding p-alanine or histidine to 0.005 M barium hydroxide to adjust the pH. 0.1 % Triton X-100 was added to improve separation by enhancement of viscosity and reduction of electrodenosmosis. 0.01 M Ammediol was used as the terminating electrolyte. The analysis was first proceeded at 2 0 0 pA for 1 2 min and was then continued at 1 0 0 PA. Calibration curves for the six ephedrine alkaloids were constructed in the range of 0.2 - 2.0 mg/ml. The ephedra extract (20.0 mg) was dissolved in water ( 1 . 0 ml) and an aliquot ( 0 . 0 1 0 ml) was injected directly (112). 10.

Drug Metabolism and Pharmacokinetics The uses of ephedrine indicate that the drug has sympathomimetic actions grossly resembling those of epinephrine. It is readily absorbed from the upper gastrointestinal tract. Good effects have also been achieved from the lower tract also through its employment in a retention enema. The gastrointestinal absorbability and the long duration of action has been shown to be associated with the attachment of a methyl group on the alpha carbon. Ephedrine is not oxidized by enzymatic action, but conjugation occurs in some species ( 1 1 3 ) . Ephedrine is readily and completely absorbed from the GI tract, peak plasma concentrations being achieved about an hour after oral administration. It is resistant to metabolism by

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monoamine oxidase and is largely excreted unchanged in the urine, with some deaminated and N-demethylated metabolites. Half-life lies between 3-6 h. Elimination is enhanced and half-life is accordingly shorter in acid urine ( 1 1 4 ) . The relative metabolism and urinary excretion of the ephedrines are dependent upon the urinary pH and in certain circumstances the volume also (115). Intrasubject variation was observed in both the lag time and the rate constant for absorption in case of (-)ephedrine. Ephedrines were readily absorbed within 3 hours of administration. Mean overall elimination t 1/2 value for unchanged (-)ephedrine was 3 . 0 3 h. It appears that ephedrine and norephedrine, when present in the body as metabolites, are eliminated faster than when they are administered per se. A possible explanation for this phenomenon is that the kinetics of elimination of the ephedrines is dose-dependent; elimination is faster at low body drug levels than at high levels. The kinetics of absorption, metabolism and excretion of (-)-ephedrine has been elucidated using analog computer analysis of urinary excretion data from three male subjects under constant acidic urine control ( 1 1 6 ) . In a feasibility study 25 mg single oral doses of ephedrine sulfate in the form of a commercial syrup and two commercial capsules were administered in crossover fashion to three non-smoking subjects. Urinary pH was not controlled, but adequate urinary flow rates were maintained by regulated water intake. There were no significant differences in average amounts of ephedrine excreted during any of the 1 1 sampling intervals, in average peak excretion rates, nor in average times of occurrence of the peak excretion rate. The average elimination half-life of ephedrine was 5.99 h and average urinary pH 6.30. The urinary excretion data was adequately described by the two-compartment open model with first-order

HOURS F I G , 10 EPHEDRINE CONCENTRATIONS I N THE PLASMA OF A HUMAN VOLUNTEER (67.6 KG) FOLLOWING A S I N G L E 24 MG DOSE OF EPHEDRINE HYDROCHLORIDE I N A COMBINATION TABLET

(81)

EPHEDRINE HYDROCHLORIDE

275

absorption and lag time. The baseline concentration of ephedrine in plasma 1 5 hours after the night dose was 20 ng/ml. Following administration of one tablet containing 1 5 mg ephedrine sulphate the concentration rose to 9 5 ng/ml after 4 h, falling to 6 5 ng/ml at 6 h (84). Ephedrine is readily absorbed after oral or percutaneous administration; gastrointestinal absorption is increased by antacids but decreased by kaolin. After an oral dose of 22 mg of ephedrine hydrochloride plasma concentrations of 40-140 ng/ml are obtained and after an oral dose of 3 3 mg peak plasma concentrations of 6 5 - 120 ng/ml are attained during therapy: effective bronchodilator plasma concentrations are in the range of 35 - 80 ng/ml and plasma half-life lies between 3 - 1 1 hours. Metabolic reactions are N-demethylation and oxidative deamination followed by conjugation. Upto about 9 5 % of a dose may be excreted in the urine in 24 hours, 55 - 7 5 % as unchanged drug, 8 20 % as the N-demethylated metabolite and 4 - 1 3 % as deaminated metabolites such as benzoic acid, hippuric acid and l-phenylpropane-lI2-diol. The rate of urinary excretion of ephedrine is pH-dependent and is increased in acidic urine ( 1 1 7 ) . A combination tablet containing ephedrine hydrochloride (24 mg) theophylline (130 mg) and phenobarbital (8 mg) was administrated and aliquots of plasmas collected showed peak plasma concentration of over 100 ng/ml. Plasma concentrations over 24 h in the subject fit a one-compartment model from which the elimination half-life was calculated as 4.8 h. Fig. 1 0 graphically illustrates this phenomenon (81 )

.

11.

Acknowledgement Mrs. Monica Hediger has put a lot of efforts

276

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and taken great pains in typing this manuscript. Dr. Wolfgang Stcber has kindly performed the mass spectrometric measurements. The author is greatful to them for their assistance.

EPHEDRINE HYDROCHLORIDE

277

Ref e re nc e s G u i d i , V., E f e d r a e d E f e d r i n a , Corriere d e i F a r m a c i s t i , Anno X X X I I 1 2 4 ( 1 9 4 0 ) , Milano, I t a l y . N a g a i , J. Pharm. SOC. J a p a n 120 109; 1 2 1 181 ( 1892). 4 1 319 ( 1 9 2 0 ) ; S p Z t h a n d G 8 h r i n g , Monatsh. Ber. 58 1 9 7 ( 1 9 2 5 ) . Rabe, B e r . 44 827 ( 1 9 1 1 ) . B u d e s i n s k y , Z. and M. P r o t i v a , S y n t h e t i s c h e A r z n e i m i t t e l , p. 24, Akademi V e r l a g , B e r l i n , ( 1 9 6 11. Takamatsu, J. Pharm. SOC. J a p a n 76 1 2 7 9 ( 1 9 5 6 ) ; C.A. 4303 a n d 4304 (1957). Blanke, H.-J., Ullmans E n c y k l o p a d i e d e r Techn i s c h e n C h e m i e , Bd. 18, P a g e 1 4 0 , V e r l a g C h e m i e , Weinheim ( 1 9 7 9 ) . G r B g e r , D . and D. E r g e , P h a r m a z i e 20 9 2 (1965). E u r o p e a n P h a r m a c o p o e i a , Volume 111, Page 2 1 3 , Maisonneuve S . A., F r a n c e ( 1 9 7 5 ) . E u r o p a i sches A r z n e i b u c h Bd. 111, Kommentar , p a g e 434, w i s s e n s c h a f t l i c h e V e r l a g s g e s e l l s c h a f t , S t u t t g a r t 1979. E v e r e t t , D.H. a n d J . B . Hyne, J. C h e m . SOC., 1636 ( 1 9 5 8 ) . D i b b e r n , H.W. , W and I R s p e c t r a o f some important drugs, E d i t i o contor, Aulendorf (1979). F r e u d e n b e r g , K . , J. Am. Chem. SOC. 54 234 (1932). Hyne, J . B . , Canad. J. Chem. 39 2536 ( 1 9 6 1 ) ; C.A. 56 5556 ( 1 9 6 2 ) . P o r t o q e s e , J., Med. Chem. 10 1057 ( 1 9 6 7 ) T e s t a , B., Pharm. A c t a Helv: 48 3 8 9 ( 1 9 7 3 ) Chen, K . K . a n d C.H. Kao, Pharm. Zentralhalle 70 27 ( 1 9 2 9 ) . P e t e r s o n , J. B., Ind-Eng. Chem. 20 388 (1928). Lohmann, K. and K . H a r t k e , D e u t s c h e Apothek e r Z e i t u n g 1 2 3 105 ( 1 9 8 3 ) . B a u e r , K.H. and H. Moll i n " D i e o r g a n i s c h e Analysell , p a g e 577, A k a d e m i s c h e V e r l a g s g e s e l l s c h a f t , L e i p z i g 1967. VQgh, A, A c t a Pharmac. Hung. 33 1 0 5 ( 1 9 6 3 ) .

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B e c k e t t , A.H., G.R. Jones and D.A. Hollingsbee, J. Pharm. Pharmac. 30 1 5 ( 1 9 7 8 ) . O t t o , H.H. and c o w o r k e r s , Deutsche A p o t h e k e r Zeitung 418 (1977). D e u t s c h e s A r z n e i b u c h 7 , Kommentar, p a g e 785, w i s s e n s c h a f t l iche V e r l a g sgesell s c h a f t S t u t t g a r t 1968. U n i t e d S t a t e s Pharmacopeia XXI page 372 R o c k v i l l e , Md. 2088 USA ( 1 9 8 5 ) . B r i t i s h pharmacopeia 80 page 1 7 3 , H e r Majesty's S t a t i o n a r y o f f i c e , London ( 1 9 8 0 ) . European Pharmacopeia Volume 111 p a g e 213, Maisonneuve S.A., F r a n c e ( 1 9 7 5 ) . Soliman, S-A., H. Abdine and N.A. Z a k h a r i , J. Pharm. S c i . 63 1767 ( 1 9 7 4 ) . J. Pharm. C h i m . 22 489 (1935). Sanchez, A . J . , Horak, F. a n d J . G a s p e r i k , C h e m T Z v e s t i 11 558 ( 1 9 5 7 ) . C h a f e t z , L., J. Pharm. S c i . , 2 291 ( 1 9 7 1 ) . M a l a p r a d e , L., B u l l . SOC. C h i m . F r a n c e 43 683 ( 1 9 2 8 ) . N i c o l e t , B.M. and L.A. S h i n n , J. h e r . Chem. SOC. 6 1 1615 ( 1 9 3 9 ) . WicksEom, A., Ann. Pharm. F r a n c e 8 86 (1950). L a t i m e r , W.L., O x i d a t i o n P o t e n t i a l e 2nd e d i t o n , p a g e 6 6 , P r e n t i c e H a l l , N e w York 1952. C h a f e t z , L., J. Pharm. S c i . 52 1193 ( 1 9 6 3 ) . Wallace, J . E . , J. Pharm. S c i F 5 8 1 4 8 9 ( 1 9 6 9 ) . Wallace, J . E . , Anal. Chem. 39 531 ( 1 9 6 7 ) . A b d i n e , H . , A.M. Wahbi and K A . Korany, J. Pharm. Pharmac. 23 444 ( 1 9 7 1 ) . G l e n n , A.L., J. Pharm. Pharmac., 15 s u p p l . 123 T ( 1 9 6 3 ) . D a s Gupta, V. a n d A. J. L. d e Lara, J. Pharm. S c i . 64 2001 ( 1 9 7 5 ) . C l a r k , C.C., J. A s s o c . o f f . Anal. chem., 58 852 ( 1 9 7 5 ) . C l a r k , C.C., J. Assoc. O f f . Anal. C h e m . , 63 692 ( 1 9 8 0 ) D e F a b r i z i o , F., J. Pharm. S c i . , 66 811 ( 1977 1. Davidson, A.G. and H. E l s h e i k h , A n a l y s t 107 879 (1982). Lang, E. Z . Anal. Chem. 253 32 ( 1 9 7 1 ) .

117

24 1

27 1

33 1

34 1 35)

41) 42 1 43 1 44 1 45 1 46)

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P a y s , M. a n d 0. Danlos, Ann. Pharm. F r a n c e 533 ( 1 9 6 7 ) . S c h i l l , G . , A c t a Pahrm. S u e c . , 2. 1 3 , 9 9 , 1 0 9 ( 1965). F r e n c h , W.N. and B.A. R i e d e l , Can. J. Pharm. Sci. 1 80 (1966). McMarxin, C . , P. Simpson and N. Thorpe, J. Chromatog. 43 7 2 ( 1 9 6 9 ) . P h i l l i p s , B.M., P. Y. K r a u s a n d M. E . S t r a t m e y e r , J. Pharm. S c i . 5 4 803 ( 1 9 6 5 ) . Karawya, M.S. and C o w o r k e r s T J . Pharm. Pharmac. 20 650 ( 1 9 6 8 ) S t e i n i g e n T M . , Pharm. Z t g . 1185 ( 1 9 6 8 ) . D e r t i n g e r , G., Deutsche Apotheker Z e i t u n g 113 1 7 3 5 ( 1 9 7 3 ) . K a u k i n e n , A., J. H a l m e k o s k i and H. A u t e r h o f f , Arch. Pharm. 476 ( 1 9 7 8 ) . F r e i , R.W., W. S a n t i and M. Thomas, J. Chromatog. 116 365 ( 1 9 7 6 ) . B e c k e t t , A. H. and N . H. C h o u l i s , J . Pharm. Pharmac. 15 236 ( 1 9 6 3 ) . C h o u l i s , N. H., J. Pharm. S c i . 2 1 9 6 , 904 (1967). W a l d i , D., M i t t . Deut. Pharm. G e s . 32 1 2 5 ( 1 9 6 2 1. H i l l e r , K., P h a r m a z i e 16 600 ( 1 9 6 1 ) . P o h l o u d e k - F a b i n i , R. a 2 K . Kbnig, P h a r m a z i e 13 135 ( 1 9 5 8 ) . WickstrZjm, A . a n d B. S a l v e s e n , Pharm. Pharmac. 5 6 3 1 ( 1 9 5 2 ) . S c h i l c h e r , H., Pharm. A c t a . Helv. 5 3 288 (1978). W a l d i , D., K. S c h n a c k e r z and F. M u n t e r , J. Chromatog. 61 ( 1 9 6 1 ) . Kaistha, K.K., R. T a d r u s and R. J a n d a , J. Chromatog. 359 ( 1 9 7 5 ) . Hudson, J. C. and W. P. R i c e , J. Chromatog. 117 449 ( 1 9 7 6 ) . G u l a y a s , H. and Coworkers, J. Chromatog. 2 9 1 471 (1984) B o t t l e r , R. and Th. Knuhr, Z. A n a l . Chem. 302 286 ( 1 9 8 0 ) . D i t t r i c h , S.,' J. Chromatog. 1 2 4 7 ( 1 9 6 3 ) . S k a l i k s , H., Arzneim. F o r s c h T 7 3 8 6 ( 1 9 5 7 ) . Wagner, G . , Sci. Pharrn. 23 148-(1955). W a l d i , D . , A r c h . Pharm. 292 206 ( 1 9 5 9 ) .

25

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Halpern, B. a n d J. W. Westley, C h e m . Commun. 34 ( 1 9 6 6 ) . K a r g e r , B.L. a n d Coworkers, Anal. Chem. 228 ( 1 9 6 9 ) . Westley, J. W., B. Halpern and B.L. K a r g e r , Anal. C h e m . 40 2046 ( 1 9 7 0 ) . B e c k e t t , A.HTand B. Testa, J. Pharm. Pharmac. 25 3 8 2 ( 1 9 7 3 ) . E l e f a n t , M . , L. C h a f e t z and J. M. Tolmage, J. Pharm. S c i . 56 1181 ( 1 9 6 7 ) . S c h u l t z , H. W. and C. Paveenbampen, J. Pharm. S c i . 62 1 9 9 5 ( 1 9 7 3 ) . D e a v i n , J. C y Analyst 1 0 0 1 3 6 ( 1 9 7 5 ) . B e c k e t t , A. H. and G. R. W i l k i n s o n , J. Pharm. Pharmac. 17 1 0 4 S ( 1 9 6 5 ) . Midha, K.K., J.K.Cooper and I.J. M c G i l v e r a y , J. Pharm. S c i . 6 8 557 ( 1 9 7 9 ) . Cummins, L.M. and M . J . F o u r z r , A n a l . L e t t . 2 403 ( 1 9 6 9 ) Bye, C. a n d Coworkers, E u r . J. Chin. Pharmacol. 8 4 7 ( 1 9 7 5 ) . P i c k u p , M.ET a n d J. W. P a t e r s o n , J. Pharm. Pharmac. 26 5 6 1 ( 1 9 7 4 ) . W i l k i n s o n 7 G . R . and A.H. B e c k e t t , J. P h a r m a c o l . Exp. T h e r . 139 (1968). B e c k e t t , A.H. a n d G.R. W i l k i n s o n , J. Pharm. Suppl. 104 S (1965). Pharmac. W e l l i n g , P.G. a n d C o w o r k e r s , J. Pharm. S c i . 60 1 6 2 9 ( 1 9 7 1 ) . B e c k e t t , A.H., D.C. Taylor and J. W. Gorrod, J. Pharm. Pharmac. 27 5 8 8 ( 1 9 7 5 ) . K r u s e , K., W. F r a n c k e a n d W.A. Konig, J. Chromatog. 423 ( 1 9 7 9 ) . F r a n k , H., G . J . N i c h o l s o n and E. Bayer, J. Chromatog. 146 1 9 7 ( 1 9 7 8 ) . Kbnig, W.A. a n d K. E r n s t , J. Chromatog. 280 135 (1983). KEnig, W.A., K . S t b l l i n g and K. K u r s e , Chrom a t o g r a p h i a 10 444 ( 1 9 7 7 ) . KEnig, W.A. E d I. Benecke, J. Chromatog. 209 9 1 ( 1 9 8 1 ) . TroschGtz MerzhBuser, J. and E. R i j d e r , 2. Anal. Chem. 2 8 5 377 ( 1 9 7 7 ) . Koziol, T.R., J . T . Jacob and R. G. Achari, J. Pharm. S c i . 68 1 1 3 5 ( 1 9 7 9 ) .

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Roberts, S . E and M.F. D e l a n e y , J. Chromatog- 242 364 ( 1 9 8 2 ) . W a i n e r , I. W. and C o w o r k e r s , J. Chromatog. 261 123 (1983). Low, G.K. C . , P.R. Haddad and A. M. D u f f i e l d , J. L i q . Chromatog. 6 3 1 1 ( 1 9 8 3 ) . 99 1 B a r k a n , S., J . D . Weber and E. S m i t h , J. Chromatog. 219 81 ( 1 9 8 1 ) . 70 1 0 0 ) Chen, T.M. and L. C h a f e t z , J. Pharm. S c i . 804 ( 1 9 8 1 ) . P.C. Booncong and S . L . F i n e , J . 1 0 1 ) Tan, H.S.I., Pharm. S c i . 70 783 ( 1 9 8 1 ) . A.M. D u f f i e l d and P.R. Haddad, 1 0 2 ) Low, G.K.C., C h r o m a t o g r a p h i a 1 5 289 ( 1 9 8 2 ) . 1 0 3 ) H o n i g b e r g , I . L . , J.I. S t e w a r t and A.P. S m i t h , J. Pharm. S c i . 63 766 ( 1 9 7 4 ) . G i l l , B.L.R. and A.C. M o f f a t , J. Chromatog. 104 301 165 (1984). 307 220 ( 1 9 8 4 ) . 1 0 5 ) G a l , J . , J. Chromatog. 1 0 6 ) G a l , J . a n d A . J . Sedman, J. Chromatog. 314 275 ( 1 9 8 4 ) . and C o w o r k e r s , J. Chromatog. 1 0 7 ) Wainer, I . W . 261 123 (1983). 317 1 0 8 ) L e r o y , P. a n d A. N i c h o l a s , J. Chromatog. 513 ( 1 9 8 4 ) . M.U. Z u b a i r a n d J.S. Mossa, 1 0 9 ) H a s s a n , M.M.A., T a l a n t a 28 1 8 7 ( 1 9 8 1 ) . 72 1 1 0 ) Midha, K X . and C o w o r k e r s , J. Pharm. S c i . 736 ( 1 9 8 3 ) . J. Chromatog. 245 1 2 9 ( 1 9 8 2 ) . 111) Budd, R.D., 1 1 2 ) Kasahara, Y. and H. H i k i n o , J. Chromatog. 324 503 ( 1 9 8 5 ) . Beckman, V. : Pharmacology-The N a t u r e , A c t i o n 1 1 3) and U s e of Drugs, P a g e 434, W.B. S a u n d e r s C o m p . P h i l a d e l p h i a and London ( 1 9 6 1 ) . 1 1 4 ) M a r t i n d a l e , The E x t r a P h a r m a c o p o e i a , P a g e 11, 28th E d i t o n , The P h a r m a c e u t i c a l P r e s s , London ( 1 9 8 2 ) . 115 ) B e c k e t t , A.H. a n d G.R. W i l k i n s o n , J. Pharm. Pharmac. 107 S ( 1 9 6 5 ) . 1 1 6 ) W i l k i n s o n , G.R. and A.H. B e c k e t t , J. Pharm. S c i . 57 1 9 3 3 ( 1 9 6 8 ) . 117 ) The P G r m a c e u t i c a l C o d e x , 11th E d i t i o n , P. 3 2 5 , T h e P h a r m a c e u t i c a l P r e s s , London ( 1 9 7 9 ) .

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ESTRADIOL E u g e n e G.

Salole

1.

Foreword

2.

De s c r i p t i o n

3.

Synthesis

4.

Physical Properties 4.1 I n f r a r e d Spectrum 4.2 N u c l e a r M a g n e t i c Resonance S p e c t r a 4.3 U l t r a v i o l e t Spectrum 4.4 Mass S p e c t r u m 4.5 D i p o l e Moment, O p t i c a l R o t a t i o n a n d pKa 4.6 S o l u b i l i t y , C o m p l e x a t i o n and Distribution Ratios 4.7 Crystal Properties

5.

Methods o f A n a l y s i s 5.1 E x t r a c t i o n and P u r i f i c a t i o n 5.2 A b s o r p t i o n and F l u o r e s c e n c e Spectrophotometry 5.3 Chromatography 5.4 P r o t e i n - b i n d i n g Assays 5.5 Bioassay

6.

S t a b i 1i t y

7.

Metabolism, availability

8.

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

9.

Determination i n Pharmaceuticals

Pharmacokinetics

and

Bio-

Acknowledgements References ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 15

283

Copyright 0 1986 hy the American Pharmaceutical Association All rights of reproduction in any lorn1 reserved.

EUGENE G. SALOLE

284 1.

Foreword

E s t r a d i o l i s t h e most p o t e n t e s t r o g e n i c hormone, s e c r e t e d i n n o r m a l p r e - m e n o p a u s a l women m a i n l y b y the ovaries. The l a s t o f t h e m a j o r n a t u r a l h o r m o n e s t o b e i s o l a t e d (12mg w e r e e x t r a c t e d f r o m 4 t o n o f p o r c i n e o v a r i e s i n 1935, a l t h o u q h i t h a d p r e v i o u s l y been s y n t h e s i z e d f r o m e s t r o n e ) , i t s m a i n c l i n i c a l u s e i s i n p o s t - c l i m a c t e r i c replacement therapy, f o r w h i c h i t i s a d m i n i s t e r e d ( a l o n e and i n c o m b i n a t i o n w i t h o t h e r hormones) by t h e o r a l , t r a n s d e r m a l , s u b c u t a n e o u s and i n t r a v a g i n a l r o u t e s i n a v a r i e t y o f d o s a g e f o r m s 1 92. The i n t e n s e i n t e r e s t i n e s t r o g e n s s i n c e t h e t u r n o f the c e n t u r y has generated a vast, and s t i l l r a p i d l y e x p a n d i n g , l i t e r a t u r e ; t h i s modest p r o f i l e t h e r e f o r e a i m s p r i n c i p a l l y t o c o l l a t e some o f t h e p h y s i c o c h e m i c a l d a t a on e s t r a d i o l w h i c h w o u l d be o f i n t e r e s t t o pharmaceutical s c i e n t i s t s . Reviews o f t h e b i o c h e m i s t r y , m o l e c u l a r p h a r m a c o l o q y and o t h e r b i o l o g i c a l a s p e c t s o f e s t r a d i o l may b e f o u n d i n t h e b o o k s e d i t e d by Chaudhury3 and M a k i d ; i t s c h e m i s t r y i s c o v e r e d b y F i e s e r and F i e s e r 5 and i n B e i l s t e i n ' s Handbook6. 2.

Description

Estra-1,3,5(lO)-triene-3,17~-diol; 17p-estradiol; E2; obsolete terms include dihydrofollicular hormone, dihydrofolliculin, d i h y d r o t h e e l i n and dihydroxyestrin ('17a-estradiol' i s a misnomer i n the literature prior to ca. 1952); Chemical A b s t r a c t s S e r v i c e r e g i s t r y number 5 0 - 2 8 - 2 . '1 EH24'2 M -r

HO

A white,

272.3E7

(see Sect. 4.7)

odorless,

tasteless,

c r y s t a l l i n e powder.

ESTRADIOL 3.

285

Synthesis

The t o t a l s y n t h e s i s o f e s t r a d i o l f r o m a c y c l i c p r e c u r s o r s h a s b e e n a c c o m p l i s h e d 6 , 7, b u t i t i s u s u a l l y p r e p a r e d by t h e sodium b o r o h y d r i d e r e d u c t i o n o f e s t r o n e , i t s e l f o b t a i n e d by t h e p y r o l y s i s o r reductive aromatization o f androstenedione8. Syntheses o f i s o t o p i c a l l y - l a b e l l e d e s t r a d i o l h a v e been described9. 4.

Physical Properties

4.1 I n f r a r e d Spectrum I R spectrum o f estradiol hemihydrate is The i l l u s t r a t e d i n F i g . 1; d e t a i l e d a s s i g n m e n t s h a v e b e e n p r o p o s e d by Smakula e t a l l 0 . The s p e c t r u m i n F i g . 1 is i d e n t i c a l t o t h a t r e p o r t e d by N e u d e r t and R o p k e l l and Hayden e t a l l 2 . , and t o one s u p p l i e d by t h e MRC S t e r o i d R e f e r e n c e C o l l e c t i o n , London, b u t q u i t e d i f f e r e n t f r o m t h e spectra in the Sadtler and Sammul et al., c ~ m p i l a t i o n s l ~w, h i c h a l s o d i f f e r f r o m e a c h o t h e r ( s e e S e c t . 4.7). 4.2 N u c l e a r M a g n e t i c Resonance S p e c t r a I H - and I3C-NMR spectra o f e s t r a d i o l hemihydrate i n a c e t o n e - d 6 w e r e o b t a i n e d a t 2 5 0 . 1 3 a n d 62.9MHz, respectively. Assignments f o r the p r o t o n spectrum (Fig. 2 ) are:

b/PPm ( I H , d, J = 8.3Hz, H - I ) ( I H , ad,-J = 8 . 3 , 2.8Hz, H - 2 ) ( I H , d, J-= 2.8Hz, H - 4 ) (3H, 6r.s, 2xOH & l / 2 H 2 0 ) ( I H , dd, J = 8.8, 8.1Hz, H - 1 7 a ) (2H, ( I H , K) (12H,-m)(3H, 2, M e - 1 8 ) .

7.09 6.59 6.52 4.10 3.67 2.76 2.28 2.20-1.10 0.77

The following assignments for the I3C-NMR spectrum (Fiq. 3) concur w i t h the l i t e r a t u r e 1 4 :

d/ppm 155.89 138.38

d/PP

(5,C-3)

-

(5,

C-5)

44.04 40.03

(5,

C-13)

(7, - C-8)

100w

I

4000 cm-1 Fig. 1

I

I

3000

I

1

I

I

2000

1800

I R spectrum o f e s t r a d i o l hemih d r a t e : ( p o l y s t y r e n e marker a t 1602cm- ).

T

I

1400

I

I

I

1000

Pye-Unicam SP3-200 spectrometer,

KC1 disc

J 600

. Fig. 2

'H-NMR spectrum o f e s t r a d i o l hemihydrate: Bruker WM250 spectrometer a t 250.13MHz, acetone-d s o l u t i o n , TMS i n t e r n a l standard. 6

-%-

Fig. 3

I3C-NMR spectrum o f e s t r a d i o l hemihydrate: Bruker WM250 spectrometer a t 62.9MHz, acetone-d s o l u t i o n , TMS i n t e r n a l standard. 6

289

E STRADIOL 132.09 126.94 115.96 113.58 81.86 50.96 44.90

(s,

C-IO)

(7,C - 1 ) (3,C-4) (Ti, C-2) (3,C-17) (3,C-14) (7, - C-9)

(t,

C-12) (T, C-16) (T, C-6) C-7) ( t , C-11) ( f , C-15) (4, - C-18).

37.76

31.02 30.30 28.13 27.26 23.80 11.63

(5,

4.3 U l t r a v i o l e t Spectrum I n 2Xv/v m e t h a n o l , e s t r a d i o l h e m i h y d r a t e e x h i b i t s U V m a x i m a a t 221nm ( A l # l c m 2 8 9 ) a n d 280nm ( A I X l c m 751, w i t h a s h o u l d e r a t 287nm ( F i g . 4 ) . Absorbance values quoted f o r e s t r a d i o l i n o t h e r media are:

0.1M h y d r o c h l o r i c a c i d q 5 80Xv/v methanol, alkalinifiedll 0.1M s o d i u m h y d r o x i d e A 5 6M ammonia16

max/nm

A 1X 1 c m

2 78

76

285.6 238 296 240 297

69 34 1

102 309 98.

The a b s o r p t i o n s p e c t r u m o f e s t r a d i o l u n d e r g o e s b a t h o c h r o m i c s h i f t s w i t h r i s e i n pH ( i s o s b e s t i c point at 285.6nmll); spectra i n concentrated s u l f u r i c a c i d a r e i l l u s t r a t e d i n R e f . 17. 4.4 Mass S p e c t r u m T h e f o l l o w i n g a s s i g n m e n t s o f a h i q h - r e s o l u t i o n mass spectrum, o b t a i n e d u s i n g an A E I (Kratos) MS9 spectrometer ( e l e c t r o n impact i o n i s a t i o n , d i r e c t i n s e r t i o n ) agree t o s i x s i g n i f i c a n t f i g u r e s w i t h t h e e x p e r i m e n t a l mass d a t a f o r e s t r a d i o l h e m i h y d r a t e :

m/z

Rel.Int.

2 72

100 46 14 13 27 43 29 13 28 18 23.

213 186 185 172 160

159 158 146 145 133

nm Fig. 4 UV spectrum o f estradiol hemihydrate: in Z.OXv/v methanol.

Cecil CE505 spectrophotometer, 0.33mg d1-I

ESTRADIOL

29 1

A simulated low-resolution

p l o t o f the spectrum i s i l l u s t r a t e d i n F i g . 5. The c h e m i c a l i o n i z a t i o n mass s p e c t r u m has been d e s c r i b e d 1 8 . 4.5 D i p o l e Moment, O p t i c a l R o t a t i o n a n d pKa T h e d i p o l e moment o f a n h y d r o u s e s t r a d i o l i n d i o x a n e was m e a s u r e d a t 2.330 ( 7 . 7 7 2 ~ 1 0 - j ~C m ) l l . S p e c i f i c o p t i c a l r o t a t i o n values reported are:

[ a l ~( 2 2 - 2 4 ' C , (IE'C,

dioxane) ethanol)

+76'11 +78"19.

Values a t d i f f e r e n t w a v e l e n g t h s and t e m p e r a t u r e s , and i n o t h e r s o l v e n t s , a r e q u o t e d i n B e i l s t e i n 6 . Spectrophotometrically determined apparent v a l u e s f o r t h e p h e n o l i c OH a r e : 10.12

2 0.025

10.30

;t

10.71

pKa

SD20 0.10 M S E ~ ~ 2 0.02 S D I 6 .

4.6

S o l u b i l i t y , Complexation and D i s t r i b u t i o n Ratios T h e s o l u b i l i t i e s o f e s t r a d i o l i n some a q u e o u s a n d o r g a n i c media a r e l i s t e d i n Tables 1-3. These v a l u e s , p a r t i c u l a r l y f o r aqueous s o l u t i o n s , s h o u l d be accepted c a u t i o u s l y , because the apparent s o l u b i l i t y o f e s t r a d i o l depends upon t h e d e t e r m i n i n q procedure. For i n s t a n c 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 s a c h i e v e d b y u n s a t u r a t e d s o l u t i o n s may d i f f e r f r o m t h o s e a t t a i n e d by i n i t i a l l y s u p e r saturated solutions26, and whereas s h a k i n q an aqueous s u s p e n s i o n for 48h r e s u l t e d i n s o l u b i l i t y o f 0.319mg d l - I a t 25'C, u l t r a - s o n i c a t i o n f o r 0.5h i n s t e a d p r o d u c e d a c o n c e n t r a t i o n o f 0.613mg d l - I 2 8 ; t h e f i l t e r s u s e d t o c l a r i f y s u p e r n a t a n t s may adsorb e s t r a d i o l t o a degree dependent on i t s c o n c e n t r a t i o n and t h e i r c o m p o s i t i o n 2 9 . A l t h o u g h t h e 25mg d l - I c o n c e n t r a t i o n r a p i d l y achieved by a 1 : 9 s o l i d c o - p r e c i p i t a t e o f e s t r a d i o l i n p o l y v i n y l p y r r o l i d o n e 4 0 0 0 0 was a t t r i b u t e d t o t h e s t e r o i d b e i n g in m o l e c u l a r d i s p e r s i o n 2 5 , t h e a q u e o u s solubility o f estradiol may be modified by a s s o c i a t i o n w i t h a v a r i e t y o f compounds. The a m i n o a r g i n i n e and l y s i n e acids tyrosine (Table I ) ,

t9

C D I

W

N T E N

S

I T Y

Fig. 5

Simulated (low-resolution)

mass s p e c t r u m o f e s t r a d i o l h e m i h y d r a t e .

TABLE 1 S o l u b i l i t y (mg d1-I) o f E s t r a d i o l i n Aqueous S o l v e n t s Temperature/OC

20

25

0.17

0.30

Water

0.02 M Sodium c h l o r i d e 0.20 M Sodium c h l o r i d e 0.40 M Sodium c h l o r i d e Phosphate b u f f e r ,

pH7.2, 0.05 I pH7.2, 0.10 I pH7.2, 0.20 I 0.005 M L - t y r o s i n e i n

35

37

0.399 0.56 0.38 0.34 0.28

42.5

50

Ref.

0.77

0.97

22 23 24

0.38f0.046 0.36+0.016 0.34-0.024

phosphate b u f f e r ,

pH7.2, 0.10 I

0.6620.049

Phosphate b u f f e r ,

pH7.4, 0.15 I 0.02 M Sodium deoxycholate i n phosphate b u f f e r , pH 7.4, 0.15 I

0.512

3.92

25

TABLE 2 Solubility (mg d1-I) o f Estradiol in Organic Solvents26 Temperature/OC ~

0

Acetone Benzene Chloroform Cyclohexane Dichloromethane Dioxane Ethanol 95% v/v Ethanol Diethyl ether Hexane Methanol Tetrahydrofuran Toluene

2594.1

-

140.5

-

45.1 1541.8 1060.4 533.2 < 0.5 862.1 25447.2 4.3

15 4291.2 21.7 251.2 0.5 108.4 7075.2 2387.2 1606.9 700.8 < 0.5 1811.3 28006.4 14.8

~

25 7068.8 46.7 411.4 1.3 192.6 12085.6 3134.4 2908.8 754.5 < 0.5 2548.8 29222.4 33.1

40 8914.8 80.1 642.7 2.2 267.1 19868.4 3727.4 4186.3 836.7 0.9 3525.6 33788.8 51.5

13715.2 198.3 762.0 7.6

-

30968.0 4890.7 4805.3

5342.4 43313.6 95.0

TABLE 3 Solubility (mg g-')

o f Estradiol in Organic Solvents at 2 2 O C

1 -Decanol Dimethyl sulfoxide Ethyl oleate Ethylene glycol Ethylene glycol: polyethylene glycol 4 0 0 , 1:l Glycerol Polyethylene glycol 400 Polysorbate 80 (Tween 80) Propylene glycol 80% w/w Propylene glycol 60% w/w Propylene glycol 40% w/w Propylene glycol 20% w/w Propylene glycol Propylene glycol: glycerol, I :I w/w

*

2

167x10 rngdl-A at 35OCZ2

28

> 500 16 16 W/W

78

1.5 105" 36 75 2.8 0.52 0.10

0.023 25

27

EUGENE G. SALOLE

296

p r o m o t e s o l u b i l i t y 2 4 , a s do p r ~ g e s t e r o n ea~n d~ human serum a l b u m i n 3 0 ( w h i c h admixed w i t h p r o p y l e n e g l y c o l has been used t o p r e p a r e i n t r a v e n o u s i n j e c t i o n s o f 100mg d l - 1 3 1 ) . P o l y s o r b a t e 2 0 ( a t 20'C) a n d s o d i u m d o d e c y l s u l f a t e ( a t 40'C) m a x i m a l l y s o l u b i l i z e d e s t r a d i o l a t 1 3 a n d 25mmol p e r m o l s u r f a c t a n t , r e s p e c t i v e l y 3 2 ; w i t h egg l e c i t h i n v e s i c l e s t h e r a t i o was f o u n d t o c h a n g e f r o m 0 . 0 1 7 6 t o 0 . 0 4 2 2 m m o l p e r mol l i p i d depending on their method of preparationz8. p - c y c l o d e x t r i n was f o u n d t o c o m p l e x w i t h e s t r a d i o l i n aqueous s o l u t i o n ; the white amorphous powder isolated by interfacial co-precipitation exhibited a rapid rate o f d i s s o l u t i o n , w i t h a s o l u b i l i t y a t 25'C o f 12mg d l - 1 33. I n c o n t r a s t , b o t h u r e a and d i q i t o n i n f o r m o n l y s p a r i n g l y s o l u b l e complexes w i t h e s t r a d i o l : columnar c r y s t a l s ( o f orthorhombic symmetry) o f t h e 1:l complex p r e c i p i t a t e f r o m a s o l u t i o n i n b e n z e n e o f e s t r a d i o l and urea i n 1 : l O mole r a t i o 3 4 , whereas n e e d l e s o f t h e d i g i t o n i d e c o m p l e x ( m . p s 2 6 5 " C ) may b e o b t a i n e d by m i x i n g s o l u t i o n s o f t h e s t e r o i d w i t h 1- 4 7 i W / v d i g i t o n i n i n 8 0 X v / v e t h a n o l 3 5 . Apparently t h e u r e a complex i s n o t o f t h e c l a t h r a t e t y p e 3 4 , and l i k e the d i g i t o n i d e i s r e a d i l y cleaved (by warming i n w a t e r and d r y p y r i d i n e , r e s p e c t i v e l y ) . Estradiol i s s u f f i c i e n t l y s o l u b l e i n peanut and sesame o i l s f o r them t o b e u s e d as v e h i c l e s f o r intramuscular injections36. D i s t r i b u t i o n r a t i o s o f e s t r a d i o l (and i t s e s t e r s ) i n s e v e r a l dozen systems have been c o m p i l e d by E n g e 1 1 7 ; a s e l e c t i o n o f v a l u e s a t room t e m p e r a t u r e a r e l i s t e d i n T a b l e 4. Lundberg23 has determined t h e thermodynamic parameters a s s o c i a t e d w i t h t h e p a r t i t i o n i n g o f e s t r a d i o l b e t w e e n o c t a n o l and w a t e r . TABLE 4 Distribution Ratios o f Estradiol17 So 1v e n t s y s t e m

Benzene/Wat e r Benzene/l.54M H y d r o c h l o r i c a c i d Benzene/O.lOM Sodium h y d r o x i d e Benzene/l.OM Sodium h y d r o x i d e Benzene:petroleum e t h e r , l:l/Water 50Sv/v M e t h a n o l / C a r b o n t e t r a c h l o r i d e Water/Carbon t e t r a c h l o r i d e

L cc #: 0.23 0.04 24 2.10 0.08

ESTRADIOL

297

D i e t h y l ether/Water D i e t h y l ether/l.6M Hydrochloric a c i d D i e t h y l ether/O.lOM Sodium h y d r o x i d e D i e t h y l e t h e r / l . O M Sodium h y d r o x i d e E t h y l acetate/Water Hexane/Water Petroleum e t h e r (35-6O0)/Water

'

55 50 2.0 0.7 28 1.07 0.79

4.7 Crystal Properties E s t r a d i o l e x h i b i t s a v a r i e t y o f s o l i d - s t a t e phases and t r a n s f o r m a t i o n s , w h i c h h a s l e d t o a d e q r e e o f c o n f u s i o n i n t h e somewhat f r a g m e n t e d l i t e r a t u r e , e.g. d i f f e r e n t I R s p e c t r a i n r e f e r e n c e w o r k s ( S e c t . 4.1). This section attempts t o put the c r y s t a l properties o f estradiol into perspective by p r e s e n t i n g a r e s u m e o f p u b l i s h e d w o r k , some o f w h i c h i s reinterpreted i n the l i g h t o f a close comparison o f d a t a a n d some u n p u b l i s h e d r e s u l t s . The m o s t n o t e w o r t h y p r o p e r t y o f e s t r a d i o l i s i t s t e n d e n c y t o a d o p t t h e h e m i h y d r a t e d form, i n which phase i t c r y s t a l l i s e s f r o m n o t o n l y p a r t i a l l y aqueous s o l u t i o n s b u t a l s o f r o m e t h 1 a c e t a t e l o , and o t h e r chloroform12, absolute e t h a n o l 2 5 9 r7 a p p a r e n t l y anhydrous solvents38. In ignorance o f t h i s c h a r a c t e r i s t i c some c r y s t a l l o g r a p h i c d a t a h a s b e e n e r r o n e o u s l y a s c r i b e d , e.g. t h e X-ray powder d i f f r a c t i o n d a t a o f P a r s o n s a n d B e h e r 3 9 ( T a b l e 51, c i t e d i n t h e J o i n t Committee o n Powder D i f f r a c t i o n Standards File, properly refers to estradiol hemihydrate, similarly the 'anhydrous' single c r y s t a l a n a l y s i s r e p o r t e d b y N o r t o n e t a140., and t h e ' m o n o h y d r a t e ' d a t a c i t e d i n S t r u c t u r e ReportsLL1 are erroneous. TABLE 5 X-ray Powder D i f f r a c t i o n D a t a f o r E s t r a d i o l H e m i h ~ d r a t e ~ ~ d/i -

1/10

d/i

1/10

7.50 6.71 6.03 5.64 5.00 4.78 4.63

4 9 4

2.90 2.80 2.65 2.56 2.49 2.40 2.32

2 4 1 2 4 2

10" 4 9 4

1

EUGENE G . SALOLE

298 2.27 2.21 2.14

4.32 4.08 3.92 3.72 3.35 3.24 3.13 3.02

2.08 2.00 1.93 1.86 1.79

* These d a t a were o b t a i n e d by v i s u a l a n a l y s i s o f Debye-Scherrer photographs; t h e d i f f r a c t o m e t e r c u r v e p r e s e n t e d b y R e s e t e r i t s e t a l Z 5 . ,o i n d i c a t e s a d o u b l e t ( s t r o n g p e a k s a t 5.64 a n d 5.69A). The crystallo raphic h e m i h y d r a t e a r e 37 : a

5 c

7 -

=

= =

12.055

19.280 6.632 4

orthorhombic,

parameters

o f

estradiol

2 0.003 f 0.003 f

I,

= 1.21q I I ~ 1.209 space group P21212.

-

cm-3 cm-3

The w a t e r m o l e c u l e s a r e l o c a t e d on t h e l a t t i c e b i n a r y axis, i n a s s o c i a t i o n with t h e D-rings o f s t e r o i d a l molecules packed 'head-to-tail', and p a r t i c i p a t e i n the hydrogen b o n d i n g which s u p p o r t s the lattice. The d i f f e r e n t i a l t h e r m a l a n a l y s i s (DTA) c u r v e o f e s t r a d i o l h e m i h y d r a t e e x h i b i t s e n d o t h e r m i c p e a k s b e g i n n i n q a t 112 a n d 174'C p r i o r t o t h e m e l t i n g e n d o t h e r m a t 179°C ( F i g . 6a)42; s i m i l a r c u r v e s have been o b t a i n e d u s i n q d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y (DSC)25,38,43. Simultaneous e f f l u e n t g a s a n a l y s i s o f DSC s p e c i m e n s i n d i c a t e d t h a t t h e t w o p r e - m e l t i n q endotherms were a s s o c i a t e d with solvent 1 0 ~ ~ 3 8 ~ 4 3 T h. e r m o g r a v i m e t r i c a n a l y s i s was i n c o n c l u s i v e ( d u e t o t h e s m a l l m a s s c h a n g e s i n v o l v e d ) , b u t K a r l F i s c h e r t i t r a t i o n o f a sample w h i c h had b e e n m a i n t a i n e d f o r I h a t 148'C ( i . e . a b o v e t h e t e m p e r a t u r e o f t h e f i r s t DTA p e a k , w h i c h was a b o l i s h e d ) i n d i c a t e d 1.8Xw/w r e s i d u a l m o i s t u r e (cf. 3.5#W/w initially). Thermomicroscopic examination o f hemihydrated c r y s t a l s had suggested t h a t some s t r u c t u r a l r e a r r a n q e m e n t o c c u r r e d p r i o r t o melting30; the pre-melting endotherm-exotherm d o u b l e t on DTA ( F i g . 6 b ) a n d DSC25,43 curves o b t a i n e d a t l o w h e a t i n g r a t e s would s u p p o r t t h i s

I

l-

a

I

80 Fig. 6.

1

1

120

1

1

160

1

1

L

2 O O 0 C 160

190 O C

DTA curve of estradiol hemihydrate at ( a ) loo C. min.-l and ( b ) 2 O C. min.-l heating rate: Stanton Redcroft 671B analyser, alumina reference, 8 mg. samples, open cups, static ambient atmosphere.

EUGENE G . SALOLE

300

view. I t appears t h e n t h a t e s t r a d i o l hemihydrate d e s o l v a t e s i n t w o s t a g e s , a t a b o u t 1 1 2 a n d 174'C, t h e complete l o s s o f l a t t i c e water r e s u l t i n g i n s i m u l t a n e o u s t r a n s f o r m a t i o n t o an a n h y d r o u s p h a s e . Smakula e t a l l 0 . , f i r s t reported t h e apparent polymorphism o f e s t r a d i o l ; on t h e b a s i s o f X-ray powder diffraction and I R spectroscopy they i d e n t i f i e d four c r y s t a l l i n e m o d i f i c a t i o n s (Forms A t o 0) a n d a n a m o r p h o u s , g l a s s y p h a s e , a l l o f w h i c h t r a n s f o r m e d when s u b j e c t e d t o h e a t a n d / o r g r i n d i n g , a s s u m m a r i s e d i n t h e f o l l o w i n g scheme: Form

B

b -.

(m.p.

\

\ \

Amorphous f o r m

\

Form C 4 ,178-9'C)

11

/

I

\

L

- - -b

Form D 0

/

heating grinding

r//

Form A

-

---

Independently, Kuhnert-Brandstatter44 concluded (from the thermomicroscopic examination o f melts, l a r q e l y ) t h a t e s t r a d i o l was d i m o r p h i c : Form I 1 (m.p, 169°C) b e i n g t h e u n s t a b l e monotrope o f Form I (m.p, 178"C), t h e two m o d i f i c a t i o n s d i f f e r i n g i n h a b i t , b i r e f r i n g e n c e and I R s p e c t r u m 4 5 . Close s c r u t i n y o f i s t h e r e p o r t s shows t h a t Form A o f S m a k u l a e t a l . , i n f a c t e s t r a d i o l h e m i h y d r a t e and Forms C and D correspond t o Kuhnert-Brandstatter and c o l l e a g u e s ' F o r m s I a n d 11. It appears then t h a t anhydrous e s t r a d i o l i s d i m o r p h i c and t h a t i n t h e p r o c e s s o f d e s o l v a t i o n t h e hemihydrate transforms t o anhydrous F o r m I, t h e a p p r o x i m a t e i n t e r p l a n a r - s p a c e s a n d corresponding r e l a t i v e i n t e n s i t i e s (extrapolated f r o m t h e X - r a y powder d i f f r a c t o m e t e r c u r v e p r e s e n t e d b y R e s e t a r i t s e t al25., f o r a dehydrated sample) o f which are:

7.63 6.44 5.77 5.55 5.26 4.86 4.62 As n o t e d a b o v e ,

3 5 10 4 3

4 2

4.23 4.06

3 3

4.01 3.88 3.71 3.32

3 1 1 2.

g r i n d i n g transformed t h e anhydrous

E STRADIOL

30 1

m o d i f i c a t i o n s o f e s t r a d i o l t o the hemihydrate, which i s i t s e l f a f f e c t e d by comminution. Grinding c r y s t a l l i n e e s t r a d i o l hemihydrate r e s u l t e d i n no change i n I R spectrum, b u t t h e D T A c u r v e e x h i b i t e d o n l y a s i n g l e , e n l a r g e d , p r e - m e l t i n g peak a t about lZO'C42; s i m i l a r changes i n DSC c u r v e s were r e p o r t e d f o r samples which had been m i l l e d ( t h e s e a l s o exhibited diffuse X-ray powder diffraction p a t t e r n s ) 4 3 o r o b t a i n e d by r a p i d p r e c i p i t a t i o n f r o m ethanolZ5. These changes suggest t h a t comminution can s t r u c t u r a l l y deform t h e c r y s t a l l i n e h e m i h y d r a t e t o t h e e x t e n t t h a t d e h y d r a t i o n and s i m u l t a n e o u s t r a n s f o r m a t i o n t o a n h y d r o u s Form I a r e f a c i l i t a t e d , o c c u r r i n g a t a t e m p e r a t u r e a b o u t 60'C l o w e r t h a n usual. ( I t has been noted that commercial ' m i c r o n i z e d ' e s t r a d i o l a p p a r e n t l y v a r i e s i n degree o f c r y s t a l l i n i t y , some b a t c h e s , e v e n f r o m t h e s a m e s u p p l i e r , e x h i b i t i n g 'deformed' DTA c u r v e s . ) E s t r a d i o l a l s o forms solvates with organic solvents: a hemisolvate w i t h methanol38 and a m o n o s o l v a t e with ethanolz5, both o f which desolvate ( a t 1 5 5 a n d 1 1 9 " C , r e s p e c t i v e l y ) t o F o r m 138. The c r y s t a l l o g r a p h i c parameters o f the monosolvate w i t h p r o p a n o l are46:

-a = c = z- =

12.215i

26 4 *. 62 75 1 ! 4

space group P212121.

The p r e s e n c e o f p r o p a n o l i n t h e l a t t i c e was f o u n d t o only very s l i g h t l y perturb the conformation o f estradiol molecules46, which may explain the a p p a r e n t f a c i l i t y w i t h w h i c h some o r g a n i c s o l v e n t s s u b s t i t u t e f o r water o f c r y s t a l l i s a t i o n . Estradiol crystals precipitate i n a variety o f h a b i t s (Table 6); i n view o f i t s tendency t o incorporate solvent o f c r y s t a l l i s a t i o n , these very p r o b a b l y r e p r e s e n t s o l v a t e d forms, t h e hemihydrate i n particular. TABLE 6 E s t r a d i o l C r v s t a l Habits26947 Habit Amorphous

Crystallisation solvent Acetone, dioxane,

benzene, ether.

EUGENE G. SALOLE

302 Bladed Platy

P r isma t i c

Dioxane, e t h a n o l , aq.ethano1, e t h e r , tetrahydrofuran. Chloroform, dichloromethane, hexanol, isopropanol, methanol Benzene, c h l o r o b e n z e n e , methanol.

.

The o u t s t a n d i n g s o l i d - s t a t e c h a r a c t e r i s t i c o f estradiol i s the tenacity with which i t adopts the h e m i h y d r a t e form. I n t h e experience o f t h i s author ( a n d other^^^,^^) c o m m e r c i a l s a m p l e s a r e i n v a r i a b l y composed o f t h i s p h a s e a n d , a s d e s c r i b e d a b o v e , i t has been demonstrated t h a t water i s s e l e c t i v e l y incorporated i n t o the l a t t i c e when estradiol crystallises from solution, that anhydrous c r y s t a l l i n e and amorphous phases t r a n s f o r m t o t h e h e m i h y d r a t e ( i t was i n t e r e s t i n q t o n o t e t h a t t h e D T A c u r v e o f s h a v i n g s from t h e s u r f a c e o f a p r o p r i e t a r y i m p l a n t p r e p a r e d b y f u s i o n was t y p i c a l o f t h e h e m i h y d r a t e ) and t h a t d e h y d r a t i o n i s d i f f i c u l t , u s u a l l y o c c u r r i n q c o m p l e t e l y o n l y a t temperatures close t o melting. C l e a r l y t h e presence o f water molecules i s a crucial requirement for the crystallographic stability of estradiol under normal conditions; on t h i s b a s i s i t has been s u g g e s t e d t h a t w a t e r may e v e n p l a y a s i g n i f i c a n t part i n the hormone's interactions with i t s receptors48. However, p r o p e r c o g n i s a n c e o f t h i s c h a r a c t e r i s t i c has n o t been taken by t h e major p h a r m a c e u t i c a l c o m p e n d i a , a l t h o u g h t h e NF X I V 4 9 s t a t e d t h a t e s t r a d i o l i s ' h y g r o s c o p i c ' and t h e USP X X I 5 0 i m p o s e s a 3.5% l i m i t o n w e i q h t l o s s o n d r y i n q a t 105'C ( a t w h i c h t e m p e r a t u r e t h e r e may n o t b e a n y d i s c e r n a b l e l o s s 3 8 o r s u b s e q u e n t a l t e r a t i o n i n DTA curve). The e v i d e n c e s u m m a r i s e d h e r e p r e s e n t s a s u b s t a n t i a l case f o r t h e m o l e c u l a r formula and r e l a t i v e m o l e c u l a r mass o f e s t r a d i o l t o b e p r o p e r l y acknowledged as :

5.

Methods o f A n a l y s i s

5.1 E x t r a c t i o n and P u r i f i c a t i o n In biological fluids estradiol exists i n the

'free'

ESTRADIOL

303

f o r m and c o n j u g a t e d as s u l f a t e s and glucuronides. B e i n g an e s t r o g e n o f r e l a t i v e l y l o w p o l a r i t y , e s t r a d i o l can be q u a n t i t a t i v e l y e x t r a c t e d w i t h diethyl ether or benzene:petroleum ether, the o r g a n i c p h a s e washed w i t h p H 1 0 . 5 c a r b o n a t e b u f f e r t o remove non-steroidal acidic impurities, then back-extracted i n t o 0.1M sodium h y d r o x i d e and re-extracted into organic solvent after a c i d i f i c a t i o n w i t h s u l f u r i c acid. I t s highly polar c o n j u g a t e s may b e e x t r a c t e d w i t h e t h y l a c e t a t e ( a f t e r s e p a r a t i o n o f t h e u n c o n j u g a t e d f r a c t i o n ) and hydrolysed by e i t h e r r e f l u x i n g w i t h h y d r o c h l o r i c or incubation with sulfatases or acid p - g l u c u r o n i d a s e enzymes4. S e v e r a l techniques have been used t o p u r i f y e s t r a d i o l e x t r a c t s , i n c l u d i n g p r e c i p i t a t i o n as a complex with u r e a or d i g i t o n i n ( t h e l a t t e r i s - e p i m e r ; c o m p l e x e s may b e c l e a v e d selective for the w i t h warm w a t e r a d p y r i d i n e , r e s p e c t i v e l y , a n d t h e s t e r o i d e x t r a c t e d ) , c o u n t e r - c u r r e n t d i s t r i b u t i o n and v a r i o u s c h r o m a t o g r a p h i c methods.

I?

5.2

A b s o r p t i o n and F l u o r e s c e n c e Spectrophotometry S e v e r a l methods f o r t h e c o l o r i m e t r i c a n a l y s i s o f e s t r a d i o l have b e e n d e v e l o p e d on t h e b a s i s o f t h e Kober r e a c t i o n , where a sample i s h e a t e d w i t h a phenol (e.g. p-naphthol) and sulfuric acid ()60Xw/v), t h e y e l l o w s o l u t i o n d i l u t e d t o an a c i d c o n c e n t r a t i o n o f 30-50%W/v and r e - h e a t e d t o q i v e a p i n k s o l u t i o n which absorbs maximally a t around 530nm. I n t h e more s e n s i t i v e and a c c u r a t e I t t r i c h modification, the re-heating a f t e r d i l u t i o n i s o m i t t e d a n d t h e c o l o u r e d complex i s e x t r a c t e d i n t o 2Xw/v p-nitrophenol i n chloroform, giving a solution with yellow-green fluorescence. The n a t u r a l f l u o r e s c e n c e o f e s t r a d i o l is e n h a n c e d i n c o n c e n t r a t e d s u l f u r i c and o t h e r m i n e r a l a c i d s q 7 . B a r t o s and Pesez51 c o m p r e h e n s i v e l y d i s c u s s t h e c o l o r i m e t r i c and f l u o r i m e t r i c methods f o r e s t r a d i o l . 5.3 Chromatography The e n t i r e r a n q e o f c h r o m a t o g r a p h i c t e c h n i q u e s has been a p p l i e d t o t h e a n a l y s i s o f e s t r a d i o l ; paper c h r o m a t o g r a p h i c s e p a r a t i o n s have been d i s c u s s e d a t l e n g t h by Bush52 and E n q e l l 7 , and Stah153 l i s t s a number o f t h i n - l a y e r s y s t e m s , e.g. adsorbent

-

s i l i c a q e l G,

250pm l a y e r ,

304

EUGENE G. SALOLE

s o l v e n t system

-

location

-

a c t i v a t e d a t 150'C f o r 3h; (a) ch1oroform:ethyl acetate, 80:20, ( b ) acetone: d i c h l o r ome t h a n e 20: 8 0 ; 1OO%W/w a n t i m o n y t r i c h l o r i d e i n g l a c i a l a c e t i c a c i d spray, d e v e l o p e d a t 95'C f o r 5 m i n t o b r i g h t r e d s p o t s a t ( a ) Rf0.32, ( b ) Rf0.56.

,

The d e v e l o p m e n t o f m o d e r n c h r o m a t o g r a p h i c m e t h o d s f o r e s t r a d i o l c o n t i n u e s apace. The p o w e r f u l c o m b i n a t i o n o f g a s o r l i q u i d c h r o m a t o q r a p h y and mass s p e c t r o m e t r y i s u s e d t o d e t e c t and a s s a y e s t r a d i o l i n b i o l o g i c a l f l u i d s 5 4 , and s e v e r a l h i g h - p e r f o r m a n c e (HPLC) systems for the l i q u i d chromatographic steroid and i t s metabolites have been d e v e l o p e d 5 5 , 56. 5.4 P r o t e i n - b i n d i n g Assays A v a r i e t y o f t h e s e h i g h l y s e n s i t i v e and ( w i t h c a r e ) s p e c i f i c methods have been developed f o r e s t r a d i o l . S e v e r a l r a d i o i m m u n o a ~ s a y s 5 ~a r e a v a i l a b l e a n d d e v e l o p m e n t s i n n o n - i s o t o p i c t e ~ h n i q u e s ~e.g. ~ , c h e m i l u m i n e s c e n c e i m m u n o a ~ s a y s ~h~a ,v e made t h e m v i a b l e a l t e r n a t i v e s f o r r o u t i n e analyses. 5.5 Bioassay Initially, e s t r a d i o l i n b i o l o g i c a l f l u i d s was assayed by b i o l o g i c a l m e t h o d s based o n changes i n the vaginal epithelium or uterine mass of oophorectomized rodents60. Although often hiqhly s e n s i t i v e and s p e c i f i c (even i n t h e presence o f i m p u r i t i e s ) , t h e precautions necessary f o r p r e c i s i o n make t h e m e x p e n s i v e a n d l a b o r i o u s a n d t h e y a r e therefore now consigned to validating the p h y s i c o c h e m i c a l methods which have superseded. Modern c y t o c h e m i c a l bioassays61, though s e n s i t i v e and e l e g a n t , have a l s o n o t been a d o p t e d f o r r o u t i n e use. 6.

Stability

E s t r a d i o l c a n w i t h s t a n d b o i l i n g i n d i l u t e a c i d s and a l k a l i , and s o l i d m a t e r i a l r e m a i n s c h e m i c a l l y s t a b l e for at least five years under temperate conditions6*. However, i t has been r e p o r t e d t h a t e s t r a d i o l a p p l i e d t o s i l i c a g e l TLC p l a t e s a n d exposed to the atmosphere for Ih underwent

ESTRADIOL s u b s t a n t i a l decomposition, o x i d a t ion63. 7.

305 b u t n o t p r i m a r i l y due t o

M e t a b o l i s m , P h a r m a c o k i n e t i c s and Bioavailability

E s t r a d i o l is b i o s y n t h e s i s e d i n n o r m a l p r e - m e n o p a u s a l women p r i m a r i l y b y c o m p o n e n t s o f t h e o v a r i e s ( i . e . f o l l i c l e s , c o r p u s l u t e u m and s t r o m a ) , f o l l o w i n q t h e classical route for s t e r o i d s from acetate through cholesterol, pregnenolone and a n d r o s t e n e d i o n e (Fig. 7). S e c r e t i o n v a r i e s w i t h t h e phase o f t h e m e n s t r u a l c y c l e , i s e p i s o d i c and t o a n y c t e r o h e m e r a l r h y t h m , so p l a s m a l e v e l s f l u c t u a t e r a p i d l y ; t y p i c a l s e c r e t i o n r a t e s d u r i n g t h e f o l l i c u l a r , m i d c y c l e and l u t e a l p h a s e s a r e 8 0 , 4 0 0 a n d 200pq d - 1 , p r o v i d i n q t o t a l e s t r a d i o l plasma l e v e l s o f about 6 , 30 and 15ng d l - l , r e ~ p e c t i v e l y ~ ~O.n l y 1-37; o f ' f r e e ' ( i . e . unconjugated) hormone c i r c u l a t e s unbound t o protein, about 40% b e i n g bound t o sex hormone bindin g l o b u l i n a n d 6 0 % t o a l b u m i n (K=6.4x108 and 1.8x1O2 M - I , r e s p e c t i ~ e l y ) ~ ~ T h. e e x t r a g l a n d u l a r ( p r i n c i p a l l y i n the l i v e r , adipose t i s s u e and s k i n ) a r o m a t i s a t i o n o f androgens i s v i r t u a l l y t h e s o l e s o u r c e o f e s t r a d i o l i n p o s t - m e n o p a u s a l women a n d a c c o u n t s f o r a b o u t 70% o f t h e s t e r o i d i n males64. The p r o d u c t i o n o f e s t r a d i o l b y t h e f e t o p l a c e n t a l u n i t d u r i n g pregnancy h a s been r e v i e w e d 4 . A m u l t i p l i c i t y o f transformations are involved i n the catabolism o f estradiol, estrone (the subject o f a recent Analytical Profile66) beinq a principal metabolite (Fig. 8). I n humans, e s t r a d i o l is e x c r e t e d m a i n l y i n u r i n e as g l u c u r o n i d e and s u l f a t e conjugate^^^, i n w h i c h f o r m s i t a l s o u n d e r g o e s e n t e r o h e p a t i c r e c i r c ~ l a t i o n s~o ~ t h a t a l t h o u g h 2 3 - 6 8 % o f a d o s e may b e r e c o v e r e d f r o m b i l e a s conjugates (mostly sulfoqlucuronides), these are normally hydrolysed by mucosal enzymes and i n t e s t i n a l f l o r a ( m a i n l y i n t h e l o w e r i l e u m and u p p e r c o l o n ) and t h e f r e e s t e r o i d m o s t l y r e a b s o r b e d , l e a v i n g o n l y 10-30% t o be e x c r e t e d i n faeces ( c f . 50-80% as conjugates i n urine). Due t o enterohepatic c y c l i n g the complete elimination o f e s t r a d i o l i s delayed for 3-6d; the metabolic c l e a r a n c e r a t e i n women i s t y p i c a l l y 1 3 0 0 1 d - l 64. The d e c l i n e i n b l o o d l e v e l s a f t e r i n t r a v e n o u s i n f u s i o n i n d i c a t e d t h a t e l i m i n a t i o n was b i p h a s i c , w i t h a h a l f - l i f e o f 20 and 70min68; however t h e r e s u l t s a f t e r b o l u s i n j e c t i o n showed t h a t t h e c u r v e

EUGENE G. SALOLE

306

7% A4 pathway

A5 pathway I40

y 3

1

p r egnenolone

C=O

no

~.

17a-hydroxypregnenole

&

no 17a- hydroxyprogesterooe

dehydroeplandrosterone

oestradiol

Fig. 7

Ovarian b i o s y n t h e s i s o f e s t r a d i o l

64

.

cH30no &

' 2-methox yoestrone

2-hydroxyoestrone

4 -hydroryot?slfone

im Ga-hydroxyOBJtm

Fig. 8 Metabolism o f estradiol 6 4

.

EUGENE G. SALOLE

308

was b e s t d e s c r i b e d b y t h e sum o f t h r e e e x p o n e n t i a l s , t h e i n i t i a l v o l u m e o f d i s t r i b u t i o n b e i n g 1 0 . 9 ( * 1.1 S E M I 169. To e l i c i t a c l i n i c a l r e s p o n s e i n r e p l a c e m e n t therapy ( a s s o c i a t e d w i t h plasma l e v e l s i n t h e p r e - m e n o p a u s a l f o l l i c u l a r phase range, c f . I n g d l - 1 post-menopausally64) e s t r a d i o l administered o r a l l y must be f i n e l y - d i v i d e d , i.e. w i t h an a v e r a g e p a r t i c l e s i z e o f o n l y a f e w pm70. However, a l t h o u q h capsules, aqueous suspensions and t a b l e t s o f m i c r o n i z e d m a t e r i a l ma be absorbed as r a p i d l y as ethanolic solutions71-~3, the bioavailability o f e s t r a d i o l by t h e o r a l r o u t e i s v a r i a b l e ( w i t h plasma c o n c e n t r a t i o n s p e a k i n g a t 0.5-8h), incomplete (due t o e x t e n s i v e i n t e s t i n a l and h e p a t i c m e t a b o l i s m ) a n d affected by diet and medication74. Oral a d m i n i s t r a t i o n a l s o r e s u l t s i n a s u b s t a n t i a l and sustained increase i n c i r c u l a t i n g l e v e l s o f estrone, an u n d e s i r a b l e f e a t u r e because o f t h e apparent association with endometrial carcinoma75. Notwithstanding these disadvantages, t h e o r a l r o u t e remains c l i n i c a l l y useful. The i n t r a n a s a l i n s t i l l a t i o n o f a n e s t r a d i o l suspension i n s a l i n e r e s u l t e d i n r a p i d a b s o r p t i o n , b u t t h e r i s e i n p l a s m a l e v e l s was s h o r t - l i v e d a n d accompanied by an i n c r e a s e i n c i r c u l a t i n g e s t r o n e , suggesting local catabolism76. Similarly, a b s o r p t i o n f r o m t a b l e t s p l a c e d s u b l i n g u a l l y was rapid, a 0.5mg dose producing serum levels e q u i v a l e n t t o 2mg o r a l l y , b u t a g a i n e s t r o n e l e v e l s were r a i s e d , p e r h a p s due t o c a t a b o l i s m w i t h i n the r e ticuloendoth e l i a l ~ y s t e m ~ ~ - ~ ~ . E s t r a d i o l i s w e l l absorbed from s o l i d p e l l e t s p r e p a r e d by f u s i o n and i m p l a n t e d s u b c u t a n e o u s l y i n t o t h e abdominal w a l l or buttocks, and has been administered i n this form since the late 1 9 3 0 ' ~ ~ ~ , * P~l a.s m a l e v e l s c a n i n c r e a s e b y 5 0 % w i t h i n an h o u r o f i m p l a n t a t i o n E 2 and r e m a i n i n t h e pre-menopausal r a n g e f o r s e v e r a l months83,84 w i t h o u t large increases i n c i r c u l a t i n g estrone occurring concomitantly, since first-pass metabolism i s avoided. However, a l t h o u g h e s t r a d i o l i m p l a n t s a r e w e l l t o l e r a t e d ( c f . progesterone, implants o f which a r e e x p e l l e d o r form s t e r i l e abscesses i n a b o u t 20% o f p a t i e n t s ) and can remain c l i n i c a l l y e f f e c t i v e f o r over three years85, inter-patient variation in a b s o r p t i o n i s s u b s t a n t i a l E 3 and t h e occasionally f a i l t o p r o d u c e a s u s t a i n e d response52,84 perhaps due t o e n c a p s u l a t i o n i n f i b r o u s tissue8°,8 since

,

I,

ESTRADIOL

309

sometimes i m p l a n t s e x c i s e d a f t e r s e v e r a l months appear i n p r i s t i n e c o n d i t i o n 8 z ) . The v a g i n a l r o u t e o f a d m i n i s t r a t i o n h a s b e e n a d v o c a t e d as t h e most a p p r o p r i a t e f o r replacement therapy because, apart from beneficial local e f f e c t s , s y s t e m i c a b s o r p t i o n occurs within minutes and, s i n c e t h e p o r t a l c i r c u l a t i o n i s bypassed, l e v e l s o f estrone a r e r e l a t i v e l y low. However, d e s p i t e e a r l y work on m i c e showing t h a t e s t r a d i o l was an order of magnitude more potent when a d m i n i s t e r e d i n t r a v a g i n a l l y i n aqueous g l y c e r i n t h a n i n o l i v e o i 1 8 6 , d e l i v e r y by t h i s r o u t e has y e t t o be optimised: n e v e r t h e l e s s a v a r i e t y o f f o r m u l a t i o n s have been used, i n c l u d i n g s a l i n e s ~ s p e n s i o n s ~ ~ , ~ 7 , creams88 polysiloxane rings89, hydrophilic pessariesbO and even o r a l t a b l e t s 9 ' . E s t r a d i o l is r e a d i l y a b s o r b e d t h r o u q h t h e s k i n ; some p a r a m e t e r s m e a s u r e d i n v i t r o a r e 9 z ; 9 3 : 106k,/cm h-' permeability constant ' 3888 h y d r a t e d whole s k i n hydrated dermis 55080 h y d r a t e d s t r a t u m corneum 300 distribution ratio s t r a t u m corneum/water 46.

-

Despite the mediocre partition coefficient, e s t r a d i o l p e n e t r a t e s t h e s t r a t u m corneum w i t h i n minutes but is only slowly, variably and i n c o m p l e t e l y absorbed i n t o t h e systemic c i r c u l a t i o n a f t e r t o p i c a l a p p l i ~ a t i o n ~ 4 , ~T ~h i. s is b e c a u s e t h e percutaneous absorption o f e s t r a d i o l i s h i g h l y dependent on t h e v e h i c l e z 7 (perhaps also the s i t e a n d mode o f a p p l i c a t i o n ) a n d i t i s t o s o m e d e g r e e c a t a b o l i s e d by t h e s k i n 9 6 . Nevertheless, although maximum p l a s m a l e v e l s a r e a c h i e v e d o n l y a f t e r several hours, they are sustained (perhaps f o r days) due t o t h e ' r e s e r v o i r e f f e c t ' o f t h e s t r a t u m corneum and l o c a l r e t e n t i o n i n u n d e r l y i n g dermal t i s s u e , with t h e a d d i t i o n a l advantage o f c i r c u l a t i n q e s t r o n e b e i n g maintained a t low, pre-menopausal l e v e l s . Replacement t h e r a p y has been s u c c e s s f u l l y a c h i e v e d in by the topical application o f estradiol hydro-aldoholic e1s94,95 and transdermal therapeutic

system^^^,^^.

8.

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

The e n t i r e a n a l y t i c a l

a r m a m e n t a r i u m is a p p l i e d t o

EUGENE G. SALOLE

310

t h e a n a l y s i s o f e s t r a d i o l i n body f l u i d s and t i s s u e s , and more s p e c i f i c m e t h o d s a r e c o n t i n u a l l y being developed. C o l o r i m e t r i c and f l u o r i m e t r i c methods based o n t h e K o b e r r e a c t i o n r e m a i n u s e f u l f o r r o u t i n e assay o f t h e r e l a t i v e l y l a r g e amounts o f e s t r a d i o l i n u r i n e 9 9 , b u t f o r b l o o d and o t h e r f l u i d s more s p e c i f i c and s e n s i t i v e t e c h n i q u e s a r e a p p l i e d , n o t a b l y radioimmunoassay, w h i c h has been a p p l i e d t o p l a s m a l o o , s a l i v a 1 Ol a n d f a e c e s l o 2 , a n d HPLC55956. A l t h o u g h gas c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y h a s b e e n u s e d t o a s s a y b o d y f l u i d s , e.g. semenqo3, b e i n g the definitive i t i s usually reserved f o r s p e c i a l circumstances. The r e v i e w b y B u s h 1 0 5 i s recommended a s a n i n t r o d u c t i o n t o t h e c o m p l e x a n d delicate business o f e s t r o g e n e x t r a c t i o n and analysis. 9.

Determination i n Pharmaceuticals

P h y s i c o c h e m i c a l methods a r e u s u a l l y chosen f o r t h e a n a l y s i s o f e s t r a d i o l i n medicines. Thin-layer chromatography remains useful for routine i d e n t i f i c a t i o n , as i n s t a b i l i t y t e s t i n q o f s o l i d dosage forms62. Creams h a v e b e e n a s s a y e d b y q a s c h r o m a t o g r a p h y l o 6 and t h e h i g h l y f l u o r e s c e n t d a n s y l d e r i v a t i v e o f e s t r a d i o l has been used t o a n a l y s e s o l i d a n d p a r e n t e r a l f o r m u l a t i o n s d i r e c t l y l 0 7 and a f t e r HPLC s e p a r a t i o n l o f l . HPLC w i t h a n o v e l light-scatterin d e t e c t o r may f i n d a p p l i c a t i o n t o p h a r m a c e u t i c a l s ?09

.

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P.G. L.M.

Ryan,

M a r t i n . S.S.C. Yen. A.M. Burnier Hermann, J.Am.Med.Assoc. 242,

H.

2699-700 89.

(1940)

(1979).

Stumpf, Demers,

208-10

J.

Maruca,

R.J.

S a n t e n and

J.Clin.Endocrinol.Metab.

54,

(1982). Maturitas 6,

90.

H. K a l u n d - J e n s e n a n d C.J. 3 59-67 ( 19 8 4 ) .

91.

P.L. M a r t i n , M . O . G r e a n e y , A.M. Burnier, P.M. Brooks, S.S.C. Yen a n d M.E.T. Quiqley, Obstet.Gyneco1. 63, 4 4 1 - 4 ( 1 9 8 4 ) .

Myren,

-

92.

R.J. S c h e u p l e i n , I . H . B l a n k , B r a u n e r a n d D.J. MacFarlane, D e r m a t o l . 52, 6 3 - 7 0 ( 1 9 6 9 ) .

-

G.J. J.Invest.

93.

W.R. G a l e y , H.K. L o n s d a l e a n d S. N a c h t , 67, 713-7 ( 1 9 7 6 ) . J.Invest.Dermato1.

94.

M.I. Whitehead, P.T. Townsend, Y. K i t c h i n e t a l . , i n : P. M a u v a i s - J a r v i s , C.F.H. V i c k e r s a n d J. W e p i e r r e , e d s . Percutaneous Absorption o f Steroids, pp. 231-48, Academic P r e s s , London ( 1 9 8 0 ) .

95.

L. F a h r a e u s a n d U . L a r s s o n - C o h n , 101, 592-6 Endocrinol.(Copenh.)

96.

Acta.

(1982).

C. L o n q c o p e , i n : P. M a u v a i s - J a r v i s , C.F.H. V i c k e r s a n d J. W e p i e r r e , eds. Percutaneous A b s o r p t i o n o f S t e r o i d s , pp.89-98, Academic P r e s s , London ( 1 9 8 0 ) .

EUGENE G . SALOLE

318

J.K.H.

Lu e t

97.

L.R. L a u f e r . J.L. DeFazio, a l . , Am. J . O b s t e t . G y n e c o l . 533-8 ( 1 9 8 3 ) .

98.

L. S c h e n k e l , J. B a l e s t r a , L. S c h m i t t and J. Shaw, i n : L.F. P r e s c o t t and W.S. Nimmo, eds. R a t e C o n t r o l i n Drug Therapy, ~ ~ . 2 9 4 - 3 0 3 ,C h u r c h i l l L i v i n q s t o n e , E d i n b u r q h ii985).

99.

R . S c h o l l e r , P. L e y m a r i e , M. H e r o n and M.F. J e y l e , A c t a E n d o c r i n o l . [ S u p p l . ] (Copenh.) 107 7-71 ( 1 9 6 6 ) .

-146,

-’

100.

R . M e r t e n s , R.J. L i e d t k e a n d J.D. B a t j e r , C l i n . C h e m . 2, 1 9 6 1 - 3 ( 1 9 8 3 ) .

101.

J. V i t t e k . S. K i r s c h , S.C. Bergman and A.L. Southren, 1 9 , 5 4 5 5 5 ( 1 984). Res. --

102.

H. A d l e r c r e u t z a n d P. J a r v e n p a a , J . S t e r o i d Biochem. 17, 639-45 ( 1 9 8 2 ) .

103.

A. R e i f f s t e c k , L. D e h e n n i n a n d R . S c h o l l e r , J . S t e r o i d Biochem. 17 567-72 ( 1 9 8 2 ) .

104.

S.J. G a s k e l l , i n : D. G l i c k , ed. M e t h o d s o f B i o c h e m i c a l A n a l y s i s , Vo1.29, p p . 3 8 5 - 4 3 4 , Wiley-Interscience, New Y o r k ( 1 9 8 3 ) .

105.

I.E. flush, Adv.Clin.Chem. 57-139 ( 1 9 6 9 ) .

106. 107.

108. 109.

R a p p a p o r t , M. J.Periodont.

-

-’

G.K.

71,

P i l l a i and K.M. 583-5 ( 1 9 8 2 ) .

Fishman, (1975).

S.

R.W.

74,

P.A.

316,

McErlane,

J.Pharm.Sci.

Roos a n d C.A. 201-4 ( 1 9 8 5 ) .

12,

64,

Lau-Cam,

Asmus a n d J.B. 461-72 ( 1 9 8 4 ) .

Landis,

J.Pharm.Sci.

674-80 J.Pharm,Sci. J.Chromatoqr.

GUANABENZ ACETATE

Charles M. Shearer

1. Description 1.1 Name, Formula, Molecular Weight 1 . 2 Appearance, Color, Odor 2 . Physical Properties 2.1 Infrared Spectrum 2 . 2 Nuclear Magnetic Resonance Spectrum 2 . 3 Ultraviolet Spectra 2 . 4 Mass Spectrum 2 . 5 Melting Range 2 . 6 Differential Scanning Calorimetry 2.7 Solubility 2 . 8 Crystal Properties 2 . 9 Dissociation Constants 2 . 1 0 Electrochemical Properties 3 . Synthesis 4 . Stability and Degradation 5 . Metabolism and Pharmacokinetics 5.1 Metabolism 5 . 2 Pharmacokinetics 6. Identity 7 . Methods of Analysis 7.1 Elemental Analysis 7 . 2 Phase Solubility Analysis 7.3 Direct Spectrophotometric Analysis 7 . 4 Titrimetric Analysis 7.5 Chromatographic Analysis 8. References ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 15

319

Copyright 0 1986 by the American Pharmaceutical Association All rights of reproduction in any form reserved.

320

CHARLES M. SHEARER

1. Description 1.1 Name, Formula, Molecular Weight The name used by Chemical Abstracts for guanabenz is hydrazinecarboximidamide, 2-[(2,6-dichlorophenyl)methylene]. It is also called (2,6-dichlorobenzylidene)aminoguanidine and N-(2,6-dichlorobenzylidene)-N'aminohydraz~ne ( 1 ) . The drug code number (1) is NSC-68982 and the Chemical Abstracts Registry number is 5051-62-7 for guanabenz and 23256-50-0 for guanabenz acetate. This compound can exist (2) as the E-isomer (CAS-60329-04-6) or the Zisomer (CAS:60329-05-7). Guanabenz acetate is the Eisomer.

c1OH 12N402C

2

Mol. Wt. = 291.14

1.2 Appearance, Color and Odor Guanabenz acetate is'a white to off-white practically odorless crystalline powder.

2. Physical Properties 2.1 Infrared Spectrum An infrared absorption spectrum of a potassium bromide dispersion of guanabenz acetate (Wyeth Reference Lot C-12258) is presented as Figure 1. The spectral band assignments are listed in Table I. Table I IR Spectral Band Assignments Wave number (cm-l) 3400 3000 to 2600 1685 1600 1600 and 1395 780

Vibration Mode NH2 stretch protonated nitrogen stretch C=N stretch NH' and C=C stretch COO- stretch three adjacent ring hydrogen deformations

WAVELENGTH (MICRONS1 2.5 100

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10

12

16

20

25

30 35 4045

80 Y

V

5 E

60

I z

vl

2 +

40

7 s

20

0 4000

3500

3000

2500

1800 1600 FREOUENCY (CM-lI

1400

1200

1000

F i g u r e 1 - I n f r a r e d Spectrum of Guanabenz Acetate, (Wyeth Reference S t a n d a r d , L o t C-12258) KBr p e l l e t

600

4w

200

322

CHARLES M . SHEARER

The UV absorptivities found for guanabenz acetate (Wyeth Reference Standard, Lot C-12258) are given in Table 111. Table I11 Ultraviolet Spectra Characteristics Solvent 0.1N HC1 0.1N NaOH absolute ethanol

Max (nm) 270 295 312

Absorptivity 44.5 43.4 45.2

2.4 Mass Spectrum The mass spectrum of guanabenz acetate (Wyeth Reference Standard, Lot C-12258) was obtained (5) by direct injection of the sample into a MS-25 spectrometer. The ionizing beam energy was 70 eV. Figure 5 is a bar graph of the mass spectrum with the molecular ion at m/e 230. Identification of the pertinent masses is presented in Table IV. Table IV Mass Spectrum Fragmentation Pattern m/e 230 195 153 123

Species M+ M+ - C1 M+ - C1 - CN2H2 M+ - C1 - CNqHq

2.5 Melting Range Wyeth Reference Standard, Lot C-12258 of guanabenz acetate melts at 188 - 190°C (dec) by USP Class 1 ( 6 ) . The Merck Index ( 1 ) reports a value of 192.5OC (dec).

323

GUANABENZ ACETATE 2.2

Nuclear Magnetic Resonance Spectrum The nuclear magnetic resonance spectrum sample (Wyeth Reference Standard, Lot C-12258) was prepared in deuterated dimethylsulfoxide containing tetramethylsilane as internal reference ( 3 ) . The spectrum was obtained on a 300 MHz Varian XL-300 spectrometer and is presented as Figure 2 . The spectral assignments are listed in Table 11. Table I1 NMR Spectral Assignments Ass igned Chemical Proton Shift (ppm) 02CCH3 1.88 Aromatic 7 . 2 5 to 7 . 6 0 Methine 8.26 exchangeable 6 . 0 0 to 9 . 5

Type Singlet Multiplt-t Singlet Broad

Number of Protons 3 3 1

OH

2.3

Ultraviolet Spectra The ultraviolet spectra of the neutral guanabenz species (in 0.1N NaOH) and the protonated species (in 0.1N HC1) are presented in Figure 3 . In nonaqueous solvents such as methanol or absolute ethanol (see Figure 4 ) guanabenz forms another tautomer and the spectrum is considerably changed ( 4 ) . This tautomer is the more conjugated species with the following structure.

Cf I

H I

It was demonstrated that the ultraviolet spectra of an N-methylated guanabenz, which could not undergo such a tautomeric shift, had a maximum in either water or methanol near that shown by guanabenz in water.

I

10

a

9

8

6

7

1

5

4

3

I

I

2

1

PPm

Figure 2

-

NMR Spectrum of Guanabenz Acetate (Wyeth Reference Standard, Lot C-12258) in deuterated dimethylsulfoxide

1

0

W

Figure 3

0.7

-

0.6

-

0.5 -

- U l t r a v i o l e t S p e c t r a of Guanabenz A c e t a t e (Wyeth Reference Standard, Lot C-12258) NaOH and ( B ) 0 . 1 N H C 1

i n (A) 0 . 1 N

0.8 0.71

0.6

8 z

0.5 -

a

2

0.4-

0 VI M

a

0.3 -

0.2 0.1 -

0.0;

1

1

I

I

I

I

,

I

,

I

,

I

,

,

,

,

,

, ,

Figure 5

-

Mass Spectrum of Guanabenz Acetate (Wyeth Reference Standard, Lot C-12258)

GUANABENZ ACETATE

327

2.6 Differential Scanning Calorimetry The DSC thermogram of guanabenz acetate (Wyeth Reference Standard, Lot C-12258) is shown in Figure 6. The thermogram was obtained (7) at a heating rate of 10°C/min using a Perkin-Elmer DSC-2. The thermogram shows no endotherm or exotherm other than that associated with the decomposition melt. 2.7

Solubilit d i n g approximate solubilities at room temperature have been reported. Solvent Solubility (mg/mL) Reference Water 11 8 Alcohol 8 50 Propylene Glycol 100 8 Chloroform 0.6 9 Ethyl Acetate 1 9 2.8 Crystal Properties The X-ray powder diffraction pattern of guanabenz acetate (Wyeth Reference Standard, Lot C-12258), obtained (7) with a Phillips diffractometer using CuKq radiation is presented as Figure 7. The calculated "d" spacings are given in Table V. Table V X-Rav Powder Diffraction Pattern d 15.0 18.5 25.6 27.7 29.1 31.0 31.3 33.1 34.2 35.8 40.2

1/10

-

9 38 L

2 4

11 14 1 I10

30 90 81;

d 41.8 42.7 43.3 b5.3 46.9 47.7 51.2 53.5 55.7 56.3 58.3 58.6

1/10

8 26 5 15 57 1[ I 10 7 3 3

10 10

I

0

n

z W

0

15

I

40 Figure 6

I

80

-

I

1

120

1

oc

I

160

1

I

200

I

I

240

D i f f e r e n t i a l Scanning C a l o r i m e t r i c Thermogram of Guanabenz Acetate (Wyeth Reference S t a n d a r d , Lot C-12258)

I

, ze Figure 7

- X-Ray D i f f r a c t i o n P a t t e r n of Guanabenz Acetate (Wyeth Reference S t a n d a r d , L o t C-12258)

CHARLES M. SHEARER

330

The single crystal X-ray crystallographic structure determination of guanabenz acetate indicates that guanabenz (base) and acetic acid are hydrogen bonded and that the entire salt lies substantially within a single plane (10). On the basis of bond lengths and angles the bonding for guanabenz acetate in the crystal may be represented as given below.

.

.

The crystal system is orthorhombic, space group of 2Pbca-D15 (NO. 61). The lattice constants are A = 2R 17.326(3) .f, b=13.436(2) 6: and c=10.999(2) 6: with eight molecules per unit cell. 2.9 Dissociation Constants The ionization constants for guanabenz acetate were determined by potentiometric titration [method of Parke and Davis (1111 in 40% ethanol/water to be 5.8 (acetate) and 8.1 (guanidinium). Spectrophotometric determination in water for the guanidinium ion also gave 8.1 (12). 2.10 Electrochemical Properties Guanabenz acetate at a concentration of 0.1 mg/mL is reduced with a half-wave potential of about -0.9V vs SCE in 0.1M acetic acid and about -1.1V vs SCE in 0.1M NaH2P04. The half-wave potentials and the limiting diffusion currents are concentration dependent in these solvents (12).

GUANABENZ ACETATE

33 1

3.

Synthesis Guanabenz can be synthesized (13) as the acetate by the condensation reaction of 2,6-dichlorobenzaldehyde with aminoguanidine in the presence of acetic acid. 4.

Stability and Degradation Guanabenz acetate can decompose by to - hydrolysis the starting materials in its synthesis, aminoguanidine and 2,6-dichlorobenzaldehyde. It can also hydrolyze forming 2,6-dichlorobenzaldehyde semicarbazone. These reactions occur very slowly even under reflux in strong acid or base (2). It has been shown by spectral evidence that guanabenz acetate exists in the more stable E-configuration (2). The Z-isomer is formed from guanabenz by irradiation in solution with ultraviolet light (2,14). The reaction is reversible in the presence of acid (14) and an equilibrium between the two isomers is formed (2).

5.

Metabolism and Pharmacokinetics 5.1 Metabolism In man the major metabolite of guanabenz is (E)-p-hydroxyguanabenz and its glucuronide. Other identified metabolites are Z-guanabenz, (Z)-p-hydroxyguanabenz, 2,6-dichlorobenzyl alcohol glucuronide and guanabenz glucuronide. These metabolites and those reported below for studies in the Rhesus monkey were characterized by comparison of their Rf's in thinlayer chromatography to that of authentic sample (15). The following metabolites have been identified (16) following administration of guanabenz to Rhesus monkeys: Z-guanabenz, 2,6-dichlorobenzyl alcohol, 2,6dichlorobenzaldehyde, p-hydroxyguanabenz and its glucuronide conjugate and 2,6-dichlorobenzaldehyde azine. After oral or i.v. administration of guanabenz acetate to rats, thin-layer chromatographic analysis of urine and bile revealed the metabolites: Z-guanabenz, 2,6-dichlorobenzyl alcohol, p-hydroxyguanabenz, 3-hydroxyguanabenz, and sulfates and/or glucuronides of these (17).

332

CHARLES M. SHEARER

5.2 Pharmacokinetics Meacham et al. (15) reported on the disposition of 14C guanabenz in hypertensive patients. Maximum concentrations of guanabenz were reached at 2 to 5 hours after dosing. The major route of elimination was into urine (80%), but guanabenz itself accounted for less than 1% of the total. Kinetic parameters for guanabenz were estimated by fitting the plasma and urine data to a 2compartment model.

A study similar to the one described above, but using Rhesus monkeys was also described by Meacham et al. (18). The results of this study, which were similar to those obtained in human patients, indicates that the Rhesus monkey may serve as a satisfactory model for man in disposition studies of guanabenz. The pharmacokinetics of guanabenz in rats were reported by De Marchi (19) and by Yokozama (20,211. 6.

Identity Infrared spectroscopy can be used directly upon the drug substance for its identification. Thin-layer chromatography, ultraviolet and nuclear magnetic resonance spectrophotometry will readily distinguish guanabenz from the 2-isomer (2).

7.

Methods of Analysis 7 . 1 Elemental Analysis The elemental analysis of guanabenz acetate (Wyeth Reference Standard, Lot C-12258) is presented below. % Calculated % Reported (22) Element C 41.26 41.00 H 4.15 4.05 N 19.24 19.34 c1 24.35 24.50 7.2 Phase Solubility Analysis Phase solubility analysis (23) on guanabenz acetate (Wyeth Reference Standard, Lot C-12258) using 12% methanol in acetone as the solvent gave a purity of 100

+

- 0.4%.

GUANABENZ ACETATE

333

7.3 Direct Spectrophotometric Analysis A stability-indicating assay by ultraviolet spectrophotometry has been reported (2). It will assay intact guanabenz in the presence of its degradation products. 7.4 Titrimetric Analysis Guanabenz acetate can be titrated with perchloric acid in acetic acid after dissolving the sample in glacial acetic acid (24,251. 7.5 Chromatographic Analysis 7.51 Thin-Layer Chromatography The following systems using silica gel plates have been reported: Solvent chloroform/methanol (10:l) chloroform/methanol/ ammonium hydroxide (48.5:50:1.5) ethyl acetate/methanol/ water/ammonium hydroxide (68:26 :4:2 ) benzene/acetone/acetic acid (70:10:20) chloroform/methanol/ ammonium hydroxide (60:40:1)

Rf 0.7

Reference

0.71

18

-

14

15 15

25

7.52 Gas Chromatography Knowles et al. (26) describe a sensitive gas chromatographic assay for guanabenz in urine and plasma. The method involves the acid hydrolysis of guanabenz to 2,6-dichlorobenzaldehyde which is chromatographed on a 2 mm i.d. x 3 m column packed with 1% neopentylglycol succinate on 60/80 mesh Chromosorb G AW-DMCS at 200' and detected by an electron capture detector.

CHARLES M . SHEARER

334

7.53 High Performance Liquid Chromatography Guanabenz can be separated (2,271 from its degradation products by an eluant consisting of 430 mL methanol/570 mL distilled water13 mL phosphoric acid on a octadecyl reverse-phase column. 7.6 Radioligand Binding Analysis Fluck et al. (28) describe a radioligand assay specifically for guanabenz. It is based upon its differential binding of guanabenz and its metabolite to cerebral4 2 receptors. It can be used in analysis of guanabenz in plasma and urine. It will distinguish guanabenz from the 2-isomer.

8. References 1. Merck Index, 10th ed., Merck and Co., Rahway, N.J., (1983) page 656. 2.

C.M. Shearer and N.J. DeAngelis, J. Pharm. Sci.,

68, 1010 (1979). 3. B. Hofmann, Wyeth Laboratories, Inc., personal communication.

4. R. Rees, Wyeth Laboratories, Inc., personal communication. 5. C.A. Hetzel, Wyeth Laboratories, Inc., personal communication. 6. J.T. Stoklosa, Wyeth Laboratories, Inc., personal communication. 7. L. Sivieri, Wyeth Laboratories, Inc., personal communication. 8. J.M. Bissinger, Wyeth Laboratories, Inc., personal communication.

9. D.N. Gutekunst, Wyeth Laboratories, Inc., personal communication. 10. R. McCaully, Wyeth Laboratories, Inc., personal communication. 26, 642 11. T.V. Parke and W.W. Davis, Anal. Chem., (1954).

GUANABENZ ACETATE

335

12. C.M. Shearer, Wyeth Laboratories, Inc., unpub 1ished work. 13. W.F. Bruce and T. Baum, Ger. Offen. 1802394 14. T. Tsujikawa, E. Mizuta and M. Hayashi, J. Pharm. SOC. (Japan), 96, 125 (1976). 15. R.H. Meacham, M. Emmett, A.A. Kyriakopoalos, S.T. Chiang, H.W. Ruelius, B.R. Walker, R.G. Narins and M. Goldberg, Clin. Pharmacol. Ther., 27, 44 (1980). 16. R.H. Meacham, C. Kick, H. Ruelius and S. Sisenwine, Fed. Proc. Fed. Am. SOC. Exp. Biol., 39, 850 (1980). 17. A. Mizamoto, Y. Soda, J. Mori, N. Yokozama, H. Nobuharu, K. Horisaka, F. Shichino and H. Tatsumi, 13, 1214 (1982). C.A. Iyakuhin Kenkyu, 98:83214v (1983). 18. R.H. Meacham, S.T. Chiang, C.J. Kick, S . F . Sisenwine, W.J. Jusko and H.W. Ruelius, Drug Metab. Disp., 9, 509 (1981). 19. G. DeMarchi. P. Gomarasca, E. Marmo and 113, 225 C. Scolastico, Boll. Chim. Farm., (1974). 20. N. Yokozama, K. Horisaka, Y. Soda, I. Mori, H. Sakamoto, K. Ohata, A. Shimada, F. Shichino, K. Murrai and H. Tatsumi, Iyakuhin Kenkyu, 13, 1190 (1982). C.A. 98:83212t (1983). 21. N. Yokozama, K. Horisaka, Y. Soda, I. Mori, A. Shimada, F. Shichino, K. Murai and H. Tatsumi, Iyakuhin Kenkyu, 13, 1207 (1982). C.A. 98:83213u ( 1983). 22. M. Politowski, Wyeth Laboratories, Inc., personal communication. 23. E. DiEgidio, Wyeth Laboratories, Inc., personal communication.

CHARLES M. SHEARER

336

24. P.E. Leander, Wyeth Laboratories, Inc., personal communication. 25. United States Pharmacopeia/National Formulary XXI, Supp. 1, page 1.725, U.S. Pharmacopeia, Rockville, Md. (1984). 26. J . Knowles, G.R. White, C.J. Kick, T.B. Spangler and H.W. Ruelius, J . Pharm. Sci., 71,710 (1982) 27.

Snodgrass-Pilla, Wyeth Laboratories, Inc., personal communication.

C.

28. E . R . Fluck, C . A . Homon, J . A . Knowles and H.W. 3, 91 (1983). Ruelius, Drug. Dev. Res., -

IODAMIDE Davide Pitre 1.

Description 1.1 Nomenclature 1.1.1 Chemical Names 1.1.2 Generic Names 1.1.3 Trade Names 1.2 Formula, Molecular weight 1.3 Appearance, Color, Odor

2.

Physical Properties 2.1 Spectra 2.1.1 Infrared Spectrum 2.1.2 Nuclear Magnetic Resonance Spectra 2.1.2.1

'H-NMR

2.1.2.2 2.1.3 2.1.4 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.3 2.3.1 2.3.2 2.3.3 2.3.4 2.3.5 2.3.6

13 C-NMR Mass Spectrum Ultraviolet Spectrum Physical Properties of Solid State Differential Thermal Analysis X-Ray Powder Diffraction Melting Range Eutectic Temperature Solution Data Solubility PKa Partition Coefficients Index of Refraction Specific Gravity Osmotic Properties

3.

Manufacturing Procedures 3.1 Synthesis

4.

Stability

ANALYTICAL PROFILES OF DRUG SUBSTAKCES VOLUME 15

337

Copyright 0 1986 by the Anirrirari Pharmaceutical Association All rights of reproduction in any furin reserved.

DAVIDE PITRE

338 5.

Analysis of impurities 5.1 Free aromatic amine 5.2 Free Iodine and free Halides 5.3 TLC of free amino compounds

6.

Metabolism and Pharmacokinetics 6.1 Metabolism 6.2 Pharmacokinetics 6.3 Protein Binding 6.4 Acute toxicity

7.

Polarography 7.1 Elemental composition 7.2 Identification tests 7.3 Official methods 7.3.1 Organically bound iodine 7.3.2 Titrimetry 7.4 Chromatography 7.4.1 Paper chromatography 7.4.2 Thin layer chromatography 7.4.3 High pressure Liquid Chromatography

8.

Determination of Iodamide in Body fluids and tissue

339

IODAMIDE

1.

Description

1.1

Nomenclature

1.1.1

Chemical Names acetylamino) meBenzoic acid, 3-( acetylamino)-5-[( thyl] -2,4,6-triiodo n-Toluic acid,&, 5-diacetamido-2,4,6-triiodo CAS Reg. N. (240-5857

1.1.2

Generic Names Iodamide (BAN, DCF, USAN) Iodamidum (NFN) Ametriadinic acid Deriv. Iodamide Meglumine (USAN)

1.1.3

Trade Names Angiomiron (Schering AGBerlin) Contraxin (Takeda - Osaka) Isteropac (Bracco-Milano) Opacist (Bracco-Milano) Uromiro ( Bracco-Milano) Uromiron (Schering A.G.-Berlin) Urombrine (Dagra-Dienmarc Trade names of 1-deoxy-1-(methylamino)-D-glucitol salt Renovue (Squibb) Jodomiron (Erco-Vedback)

-

DAVIDE PITRE

340

1.2

Formula, Molecular weight COOH

i

CH COHN 3

CH NHCOCH3

2

1 C

1.3

H

I N 0

12 11 3 2 4

Mol.wt. 627.93

Appearance, Color, odor

White, fine, crystalline powder, odorless and with a very bitter taste (2). 2.

Physical Properties

2.1

Spectra

2.1.1

Infrared Spectrum

The infrared spectrum in potassium bromide pellet was reported by E. Felder et a1 ( 3 ) . The spectral band assignments are reported in Table I.

34 1

IODAMIDE Table n. 1

Values of i n f r a r e d a b s o r p t i o n ~

-r

Freq ency (cm 1

Type of vibration

Assignment

3380 3200

~ N H

-CONH groups

2980+2500

4 C H and
-CH

1728

qc

= 0

-COOH group

1660 1635

qc

= 0

-CONH

"Amide I band" groups

1525

$NH

+ (CN

-CONH

"Arnide I1 band"groups

1425

&OH

+

-COOH group

1460

6CH

-CH

1370

bCH

-CH

1296 1280

VCN + SNH

-CONH

1210 1193

60H

985

758 738 695

685 650

+

'fC-0

((2-0

2'

2 3

-CH

3

and

-COOH groups

group group "Amide I11 band'lgroups

-COOH group

DAVIDE PITRE

342 2.1.2

Nuclear Magnetic Resonance Spectra

2.1.2.1

'H-NMR 1

The H-NMR spectrum of Iodamide, recorded in DMSO solution with a EM 390 Varian spectrometer at 90 MHz, is reported in Fig.1 (4). The spectral assignment is shown in Table 4. Table N.' 2 H-NMR data

1

Multiplicity

n. Protons

Assignment

10.00

S

1, exch.

+NHCO-

7.93

t

1, exch.

4- CHgHCO

4.67

d

2

$-CH2

N1

-COOH

3.0+4.0

v.1.

2.01

S

3

&NHCOCH3

0.90

S

3

B-CH2-NHCOCH -3

LOCI PO5 L

p

LOCKPOWER

amx5

p

m

SPECTRUM

AMPL

FILTER

o.05

boo

5

rrun

NUCLEUS

-do

ppn

ZERO REF

SWEEP

TIME

sec

SWEEP

WIDTH

WG

END OF SWEEP^.^^

mG

, '

SAMWE

caiu;r (yr&h

'(WF

OAT€

DECOUPLE 415 DECOUPLING

P

O

w

E

R

L

Fig. 1

m

G

RF POWER

- 'H-NMR

6.41

Spxtrun o f

lodanide i n DMSO

- d6

SAMPLE TEMP

,/

OPERATOR

o

SOLVFN~

)M%

9

5!Jz!%

SPECTRUM

No=

DAVIDE PITRE

344 2.1.2.2

13C-NMR

The 13C-NMR spectrum, recorded in DMSO solution with a XL-100 Varian spectrometer at 25.2 MHz, is shown in Fig.2. The assignments are presented in Table 3 ( 4 ) . p b l e n. 3 C-NMR data

Line n.

Assignment

Intensity

1

84

169.6

2

100

168.8

3

86

167.5

4 5 6

27 77 104

149.0 143.7 143.6

C -C and C aromatil 1 3 5

7

107.5 97.0 95.8

C -C and C aromatir 2 4 6

9

39 35 47

10

32

56.1

$-g2

11

69

22.8

&CH2NHCOCH3

12

74

22.0

$-NHCOCH,

-COOH

-+ ~CH~NHCO

$NHCO

8

2.1.3

carbon

carbon

Mass Spectrum

The main fragmentation patterns observed in EI/MS were reported by E. Felder et a1 ( 3 ) . In order to better characterize the molecular ion which in the EI spectrum is very scarcely abundant (0.001%), the mass spectrum of iodamide was recorded using a Fast Atom Bombardment (VG Micromass 70-70E, thioglycerol mixture). The normalized spectrum is shown in Fig. 3 from which it appears that the pseudomolecular ion fi + +H at m/z = 629 represents the base peak.

C .-

. ?

00

t

68

I

N

N

88

21

m VI

48

m

28

I

m m Ln

E

m

IODAMIDE

2.1.4

347

Ultraviolet Spectrum

The ultraviolet absorption spectra of Iodamide in methanol, 0.1N NaOH and 0.1N HC1 were reported by E. Felder et a1 ( 3 ) . The relevant spectrophotometric data are reported in Table 4. Table n. 4

2.2

Physical Properties of Solid State

2.2.1

Differential Thermal Analysis

Differential thermal analysis was carried out in a Perkin-Elmer model DSC-1 calorimeter(heating rate of 4O/min). In the temperature range from 40°-350° only one endothermic transition was seen at 284O. A thermogravimetric analysis carried out under nitrogen with RH Cahn electrobalance and a Stanton-Redcroft temperature programmer showed decomposition with loss of iodine at 280° ( 3 ) . 2.2.2

X-Ray Powder Diffraction

The X-ray powder diffraction pattern of Iodamide was determined with a Philips Powder Diffractometer PW 1710 using nichel-filtered copper radiation with the following instrumental conditions: tube LFF, Cu, 40 KV, 40 mA; slits lo0.1 mm-lo; detector PW 1171 proportional counter plus dis3 criminator; scale 2x10 cps; time constant 1"; scanning speed 0.004°/sec; paper speed 1 crn/lo 28; specimen holder Niskanen plus internal standard.

DAVIDE PITRE

348 Interplanar distances (d(8)=1.54051/2d Sin intensities are reported in Table 5.

4

and relative

Table n. 5 X-Ray Powder Diffraction Pattern of Iodamide

N

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 22 23 24 25 26 27 28 29 30

10.52 8.91 7.11 6.32 6.04 5.75 5.70 5.43 5.26 5.14 5.02 4.64 4.21 4.14 4.07 4.03 3.86 3.81 3.77 3.65 3.50 3.43 3.43 3.31 3.22 3.19 3.12 3.07 3.03 2.98 2.96

70 2 6 5

4 2 3 2 100 8 12 6 11 2 3 4 14 6 4 5 12 6 6 8

3 9 4 4 4 2 3

d

(A)

2.92 2.91 2.86 2.81 2.75 2.69 2.65 2.63 2.59 2.56 2.54 2.48 2.43 2.40 2.38 2.35 2.31 2.29 2.28 2.20 2.13 2.11 2.01 1.96 1.93 1.75 1.72 1.71 1.67 1.61 PLUS OTHER LINES

31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

I/I,xlOO

11 10 12 6 3 4 2 11 4 4 10 3 3 3 3 3 4 3 3 3 3 7 5 4 3 5 2 2 2 2 :2

IODAMIDE 2.2.3

349 Melting Range

Iodamide shows in thermal microscopy a melting range between 200° and 270°, with decomposition (3). M.p. 255-7O (5). 2.2.4

Eutectic Temperature

The following melting points of Iodamide, that have the advantage of being very sharp, have been reported (3) Mixture

M.p.

N-Phenylacetamide

110.10

p-Ethoxyacetamide

131.8O

Salophen

186.7O

1-Cyanoguanidine

183. Oo

N-Phenylbenzamide

158.8O

DAVIDE PITRE

350 2.3

Solution Data

2.3.1

Solubility

Iodamide is sparingly soluble in methanol, slightly soluble in water and in ethanol and practically insoluble in ether and in chloroform (2). Water solubilities, expressed in g/100 ml, are 0.30 at 20°, 0.32 at 40° and 0.39 at 60°. Water solubilities, as a function of pH, are reported in Table n. 6 (3). Table n. 6 Solubility in water as a function of pH at 20° PH

+0.05)

Solubility g/100 ml

0.03 0.14 1.16 5.41 11.1 18.7

1.0 2.0

3.0 3.6 3.8 4.0 4.5 5.0

2.3.2

52.9

73.?

PKa

The apparent ionization p y t a n t , determined in methylcellosolve/water (W/W) was pK = 4.15; extrapolation MCS

Other Authors titration.

25

= 1.88 (7). *2O (6) reported a pKa = 3.7,

to aqeuous solution gave a pK

determined by

IODAMIDE 2.3.3

351

Partition Coefficients -

The n-octanol/buffer partition coefficients at pH between 1 and 8 are listed in Table 7 (3). Table n. 7 n-octanol/aqueous phase partition coefficients Part. Coeff.

Aqueous phase

0.1 M Citrate buffer pH 1

2.71

0.1 M Citrate buffer pH 2

1.02

0.1 M Citrate buffer pH 3

0.12

0.1 M Citrate buffer pH 4

0.02

0.1 M Citrate buffer pH 5

40.01

0.1 M Phosphate buffer pH 6

QO.01

0.1 M Phosphate buffer pH 7

60.01

0.1 M Phosphate buffer pH 8

so. 01 -3

A partition coefficient of 0.9 x 10 pH 3.3 was reported (6). 2.3.4

in chloroform/water at

Index of Refraction

The refraction index (n2') of the meglumine and sodium salts Table 8.

of the aqueous solutions of Iodamide is given in

DAVIDE PITRE

352 2.3.5

Specific Gravity

(4

20 The specific gravity ) of water solutions of 0 the meglumine and sodium salts of Iodamide is reported in Table 8. 2.3.6

Osmotic Properties

Osmolalities of aqueous solutions of the sodium and meglumine salts of Iodamide are reported in Table 8 (4).

353

IODAMIDE Table n. 8 Osmotic properties Sodium salt of Iodamide (M.W. = 650.0)

Iodamide-Na g/100 m l

29.3 58.6 117.2 175.8 234.3 293.0 351.5 410.1 468.6

5.00 10.00 20.00 30.01 40.00 50.01 60.00 70.01 a0

mg I/ml

.oo

20 d20

n

1.0282 1.0595 1.1217 1.1850 1.2467 1.3092 1.3707 1.4347 1.4979

1.3410 1.3493 1.3658 1.3822 1.3989 1.4152 1.4314 1.4488 1.4645

20

Osmol/kg

D

0.1464 0.2869 0.5668 0.8740 1.202 1.591 2.025 2.527 3.092

Meglumine salt of Iodamide (M.W. = 823.2)

Iodamide-MGA g/100 m l

10.09 20.17 30.24 40.33 50.42 60.48 70.57 80.63

mg I/ml

46.6 93.1 139.6 186.2 232.8 279.2 325.8 372.3

20 d20

20 n

Osmol/kg

1.0499 1.1052 1.1593 1.2133 1.2671 1.3202 1.3749 1.4297

1.3491 1.3655 1.3819 1.3989 1.4158 1.4325 1.4490 1.4652

0.2234 0.4346 0.6532 0.8955 1.165 1.490 1. a84 2.370

D

DAVIDE PITRE

354 3.

Manufacturing Procedures

3.1

Synthesis

The starting step in the synthesis of Iodamide (1) (1) is the introduction of an acylaminomethyl group in position 5 of a benzoic acid molecule, either substituted or not in position 2 and/or 4 with a chlorine atom, followed by the introduction of an amino group in position 3 by means of nitration and reduction. The compound thus obtained is subsequently iodinated and acetylated. If the starting product is benzoic acid ( x = H, X = 1 H) the synthesis has no industrial value because the reaction leads mostly to an undesired compound with the nitro group in position 4. The synthesis was therefore studied using 2-chloro benzoic acid (X = C1, X = H), 4 chlorobenzoic acid (X = 1 H, X = C1) and 2,4-dichlorobenzoic acid (X = X = C1) as a 1 starting products in which the introduction of the chain is easier. Nowdays the industrial synthesis is performed starting from 2-chlorobenzoic acid using the scheme through 3-acetylaminomethyl-6-chlorobenzoic acid (IVb). This product is nitrated to (Vb), reduced to (VI) and iodinated to 3-acetylaminomethyl-5-amino-2,4,6-triiodobenzoic acid (VII) which is finally acetylated to Iodamide.

x

U

z rN

“

X

P

!

r z

50 urn r U

1:” rN

B

N

4

X

U

U

11

u

11

4 v

I1 4

.x r

r

4

L r

4

rN

r

=N

r

I N

5

z u

4.4

I1

x

DAVIDE PITRE

356 4.

Stability

Iodamide, stored in the solide state at room temperature (150-25OC) and at 40°C/75%/RH for 12 months, is physically and chemically stable. Injectable solutions of Iodamide Sodium salt or Meglumine salt stabilized with sodium edetate, have not shown any evidence of chemical change after storage for 60 months at room temperature (15O-25OC). 5.

Analysis of impurities

5.1

Free aromatic amine

For the determination of free aromatic amine an official method (2), based on the Bratton-Marshall colorimetric reaction, was reported. The procedure has been automated (9) for bulk product and injectable solutions. 5.2

Free Iodine and free Halides

The method reported by Jap.P. IX for detection of free iodine consists in the extraction with chloroform of a suspension of Iodamide obtained by acidification of an aqueous solution of the sodium salt. Chloroform must be colorless. Free halides are tested with silver nitrate after acidification with nitric acid of an aqueous solution of the ammonium salt of Iodamide and filtration. 5.3

TLC of free amino compounds

The following de-acetylation products may be considered as by-products of Iodamide ( A ) : 3-amino-5-aminomethyl-2,4,6-triiodobenzoic acid (B) 3-amino-5-acetylaminomethyl-2,4,6-triiodobenzoic acid (C) 3-acetamido-5-aminomethyl-2,4,6-triiodobenzoic acid (D) The Rf values of these compounds in the solvent system namylalcoho1:acetic acid:water 4:1:5, using Silica-gel 60 F 254 pre-coated plates (Merck), are summarized in Table 9 (10).

IODAMIDE

357

Table 9 Rf of de-acetylation products of Iodamide Compound

Rf 0.14 0.08 0.3 0.03

6.

Metabolism and Pharmacokinetics

6.1

Metabolism

Chromatographic studies on urine of normal subjects showed that Iodamide was excreted unchanged in the first 4 hours after administration, as unchanged compound together with amounts of an unidentified metabolite from 4 to 12 hours and mostly as the metabolite after 12 hours. However, the total amount of excreted metabolite is quite small (less than 1.5 per cent of the injected dose) (13). 6.2

Pharmacokinetics

Urinary excretion in rabbits after i.v. administration was found to be (14): Sodium salt of Iodamide: 84.9% after 195 min. Meglumine salt of Iodamide: 79.6 after 180 min. Plasma concentrations in rabbits, after i.v. administration of the meglumine salt of Iodamide, were the following Minutes % dose

7

42.3+11.3 -

10 24.8+9.1 -

15

-

18.1+2.0

20

11.9+1.9 -

27 3.56+1.37 -

The same Authors report a 97% excretion in 24 hours after i.v. administration to rats and give the content in blood, liver, kidney, spleen and thyroid after 30 min., 1 hour, 2 and 24 hours.

DAVIDE PITRE

358

Urinary e x c r e t i o n i n mouse is 0.6% a f t e r 4 hours when Iodarnide is a d m i n i s t e r e d o r a l l y (300 mg/kg). As r e g a r d s humans, urinary excretion after i.v. administ r a t i o n , is o f 84.7% a f t e r 4 hours and 94.2% a f t e r 72 h o u r s while f e c a l e x c r e t i o n is 0.5%. Pharmacokinetic parameters f o r i . v . a d m i n i s t r a t i o n and f o r i n f u s i o n a r e s i m i l a r and w e have T 1 j o f about 3 min f o r t h e 2, d i s t r i b u t i o n h a l f - l i f e and T 1 o f about 69 minutes f o r t h e 2~ disposition h a l f - l i f e (16).

P

6.3

I

P r o t e i n Binding

Plasma p r o t e i n binding o f Iodamide i n 2 p a t i e n t s v a r i e d between 4 and 13 p e r c e n t (17). Other a u t h o r s r e p o r t t h a t t h e b i n d i n g t o serum p r o t e i n is n e g l i g i b l e (13). 6.4

Acute t o x i c i t y

........................................... Animal

i.v.

i.p.

----------.--------------_.----------------. Mause

9

(16)

11

(18)

Rat

11.4

(16)

17.5

(16)

Rabbit

13.2

(16)

15.5

(16)

Dog

.

dose of 1 5 ml/kg of a 50% solut i o n do n o t cause l e t h a l i t y

.........................................

The a c u t e t o x i c i t y o f t h e sodium s a l t of Iodamide i n mouse was found t o be 17.3 g/kg ( 1 9 ) . H.J. Herms r e p o r t s a DL = 16.5 g/kg a f t e r i . v . adrninis50 t r a t i o n o f Iodamide ( c a l c u l a t e d as a c i d ) t o r a t s ( 1 9 ) and on i n t r a c e r e b r a l t o x i c i t y o f 0.2 g/kg i n mice a f t e r a d m i n i s t r a t i o n of t h e meglumine s a l t .

IODAMIDE

7.

359 Polarography

Iodamide exhibits a wave at 0.6 volt in 0.1M phosphate buffer at pH 9.5 (10). 7.1

Elemental composition

% Calc.

Element C H

% Found

22.95 1.77 60.63 4.46 10.19

I N 0

22.93 1.78 60.75 4.42 10.12

Identificatipn Tests

7.2

Iodamide may be identified by the following methods (2) (13): a) dissolve 0.01 g in 5 ml of hydrochloric acid and heat in a water bath for 5 minutes. The solution must respond to the qualitative test for aromatic primary amines. b) Heat strongly: violet vapours of iodine are evolved

c) Infrared spectrum recorded on 3 m g , previously dried at 105O for 4 hours, by the PO assium bromi e disk meth d exhibits maxima at 3 85 cm , 1369 cm , 1269 cm , 1210 cm-l and 1191 cm

-? .

-1

-P

-P

d) The UV spectrum of a solution, containing about 10 a buffer at pH = 9 must show a maximum at 238 mg/ml,

&

nm and E of about 526. lcm

DAVIDE PITRE

360

7.3

Official methods

7.3.1

Organically bound iodine

The reductive dehalogenation method with zincsodium hydroxide is used to free organically bound iodine. The iodide thus formed is determined by titration in acidic solution with standardized silver nitrate in presence of tetrabromophenolphthalein ethyl ester until the color of the precipitate turns from yellow to ireen (2). The determination can be carried out in a simpler way through a reduction with sodium borohydride (10) according to the method first proposed by Egli (11). 7.3.2

Titrimetry

Iodamide can be titrated directly with 0.1N NaOH, after dissolving it in CH OH and adding the same volume of H 0, using phenol red (0.02% of sodium salt) as an indicator, 2 or potentiometrically using a glass electrode (10). 7.4

Chromatography

7.4.1

Paper chromatography

Ascending paper chromatography was performed using Whatman N1 filter paper and the following systems: a) n-C H OH : CH COOH : H 0 = 3:1::2 4 9 3 2

b)

CH CHOHCH3 : NH40H 25% = 8:2 3

The product was visualized as a blue spot on a white background (Rf 0.67 (a), 0.63 (b)/ after spraying with soluble stark, exposing to UV light (254 nm) and again spraying with 2N HC1.

IODAMIDE

7.4.2

36 1

Thin layer chromatography.

Thin layer chromatographic methods for identification and separation of Iodamide and related compounds are summarized in the following Table 10. Table 10 Thin layer systems of separation of Iodamide (12)

a) b)

-

Solvent system

Rf a

Rf b

I I1 I11 IV V VI VII VIII

0.43 0.34 0.70

0.59 0.55 0.47 0.74

0.51 0.44 0.24

0.59 0.74 0.33

0.28

0.50

0.50

(Merck) pre-coated TLC plates Silica gel 60 F 254 Cellulose F (Merck) pre-coated TLC plates

Solvent system:

-

3:l:l

I1 = iso-C H OH : CH CHOHCH : NH40H 25% 49 3 3

-

2:5:3

I11 = iso-C H OH : CH CHOHCH3 : NH40H 25% 49 3

-

5:2:2

-

3:2:2

V = n-C H OH : NH40H 25% 3 7

-

7:3

VI = CHCl ' CH COOH : H2° 3' 3

-

5:5:1

I = n-C4H9OH : CH3COOH : H2°

IV = CH COOC H : CH3COOH 3 2 5

: H20

VII = CHCl ' CH OH : NH40H 25% 3' 3 VIII = n-C H OH : CH3COOH : H2° 49

- 6:3:1 -

4:1:5

DAVIDE PITRE

362

-

Detection system a) 0.5% aqueous starch solution with subsequent exposure of the plates to UV light (254 nm): formation of brown spots; b) 1:l aqueous solution of 10% cerium sulfate and 5% sodium arsenite: white spots on a yellow background.

7.4.3

High pressure Liquid Chromatography

A HPLC method for quantitative determination of Iodamide and for separation of possible impurities was developed (10). Instrumental conditions:

-

Apparatus:

Hewlett-Packard, mod. 1084B

-

Column:

25 cm x 4 mm Hibar(R), RP-18 (5 um)

packed with Lichrosorb

/

-

Injection:

10 ul of a 01 mg/ml aqueous solution (PH 7.5)

-

Eluant A:

0.076 M phosphate buffer, pH 7.5, in water

-

Eluant B:

1:l (v/v) methano1:phosphate buffer

-

Flow:

1.2 ml/min

-

Gradient prophile :

/

minutes 0 5 15

16 17 22

pH 7.5)

% eluant B

15 15 40 40 15 stop

IODAMIDE

363

-

Column temperature:

4OoC

-

Detection

UV, 240 nm

: :

- tR

@

7.2 min

Determination of Iodamide in Body fluids and tissue

8.

The following methods are used for this determination: a) Total iodine determination, after decomposition with perrnanganate in hot acid or alkaline medium (16)(23) according to the methods of White and Rolf (22); b) Total iodine (13) (14) determination with a PBI Technicon Autoanalyzer (23);

c) Spectrophotometric determination in diluted urine of dog (6);

d) X-Ray Fluorescence analysis after irradiation with 241Am (15); e) Total radioactivity of gamma counter (17).

131

I material determined in a

DAVIDE PITRE

364

References 1) E. Felder, D. PitrB, L. Fumagalli, Helv. Chim. Acta, 259 (1965)

48,

2) Jap. P., IX (1976) 3) E. Felder, D. Pitr&, M. Grandi, I1 Farmaco Sc. ed., 755 (1977)

32,

4 ) M. Grandi, Bracco SPA, Personal communication 5) Index Merck 6) A.J.M. Engelen, J.F. Rodrigues de Miranda, E. J. Ariens, Invest. Rad. g, 210 (1973)

7) E. Felder, D. Pitrb, M. Grandi, I1 Farmaco Sc. ed., 485 (1973) 8)

28,

M. Svoboda, I. Fiala, Rontgenbl., 19, 273 (1966)

9) E. Felder, D. PitrB, M. Grandi, J. Pharm. Sc., 64, 684 (1975) 10) U. Tiepolo, Bracco SPA, Personal communication

11) R. Egli, Z. Anal. Chem.,

247,

39 (1969)

12) M. Brocchetta, Bracco SPA, Personale communication 13) L.T. Di Fazio and coll., J. Chem. Pharm. 14) D. PitrB, M. Fonberg-Broczek, Rad. Med.,

35 (1978)

3, 729

(1970)

15) V . E . Zajcik, I.S. Arnosov, E.I. Frolova, V.I. Jakoder, Radiol. diagn. , 24, 429 (1983) 16) F. Bonati, G.F. Rosati, M.G. Poletto, Arzneim. Forschr., 15, 222 (1965)

-

IODAMIDE

365

Bollerup, B. Hesse, E. Sciven, Europ. J. Clin. Pharmacol., 2, 63 (1975)

17) A.C.

18) F. Bonati, G.F. Rosati, Riv. Rad., 19) H.J. Herms, Rontgenbl.,

21,

12, 66

(1972)

3 (1958

20) F. Bonati, G.F. Rosati, V. Zanichelli, Radiol. Med., 52, 577 (1966)

-

21) H.L. White, D. Rolf, Proc. SOC. Exp. Biol. Med., (1940) 22

H.L. White, D. Rolf, Proc. SOC. Exp. Biol. Med.,

2,1 45,

433 (1940) 23) M. Riley, N. Grochman, "A fully automated method for the

determination of serum protein-bound iodine", Technicon Symposium, N.Y., 1964

This Page Intentionally Left Blank

LITHIUM CARBONATE Henry C. Stober 1.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor 1.3 Drug Properties

2.

Physical Properties 2.1 Infra-red Spectroscopy 2.2 Raman spectroscopy Atomic Emission and Absorption Spectroscopy 2.3 2.4 Melting Point 2.5 Thermogravimetric Analysis 2.6 Dissociation Constant 2.7 Conductivity 2.8 Microscopy 2.9 Index of Refraction 2.10 Density 2 . 1 1 Crystal Structure 2.12 X-Ray Powder Diffraction 2.13 Polymorphism 2.14 Solubility 2.15 Dissolution

3.

Preparation of Lithium Carbonate

4.

Stability 4.1 Solution 4.2 Solid State (Light, Thermal, Humidity)

5.

Methods of Analysis 5.1 Identification Test for Lithium 5.2 Identification Test for Carbonate 5.3 Microchemical Test for Lithium 5.4 Microchemical Test for Carbonate 5.5 Volumetric Analysis Atomic Emission and Absorption Spectroscopy 5.6 Other Spectrometric Techniques 5.7 5.8 Conductivity 5.9 Ion Selective Electrodes 5.10 Ion Chromatography

6. Medicinal History

7. Pharmacology ANALYTICAL PROFII.ES OF DRUG SUBSTANCES VOLUME 15

367

Copyriglit Q 1986 hy the American Pharmaceutical Association All rights o!' reproduction i i i any form reserved.

HENRY C.STOBER

368

LITHIUM CARBONATE 1.

Description 1.1 Name, Formula, Molecular Weight Chemical Name Lithium Carbonate Nomenclature The following nomenclature is used in Chemical Abstracts: Carbonic Acid, Dilithium Salt [ 554-13-21 Trademarks The following trademarks are listed in the Merck Index (1): Camcolit; Candamide; Carbolith; Ceglution; Eskalith; Hypnorex; Lithane; Lithobid; Lithonate; Lithotabs; Plenur; Priadel; Quilonum retard. Molecular Formula and Weight (1) Li2CO3 73.89 0: 6 4 . 9 6 % C: 16.25% Li: 18.78% 1.2 Appearance, Color, Odor White, granular, odorless, light alkaline powder (1,2) * 1.3 Drug Properties Lithium Carbonate, by virtue of the therapeutic properties of lithium, is used for the treatment of manic depressive psychoses. The drug is listed in the United States Pharmacopea (21, the British Pharmacopoeia (3)) and the Modern Drug Encyclopedia (4), as well as Remington's Pharmaceutical Sciences (5).

2.

Physical Properties 2.1 Infra-red Spectroscopy The infra-red spectrum of lithium carbonate dispersed in KBr is shown in Figure 1 (6). Literature sources have demonstrated that the infra-red spectrum of lithium carbonate is consistent with its known crystal structure ( 7 , 8). The infra-red spectrum of lithium carbonate

n UI

n d

U

E

I W

0 0 03

0 0 0 rl

0 0 hl rl

0 0

4

-3

0

3 rl

\D

0 0

rl

c3

0 0 0 hl

0 0 Ln N

3 3 3

m

3 3

n

m

HENRY C.STOBER

370

has also been obtained as a film on sodium chloride plate and as Vaseline and flurolub suspensions ( 7 , 9 ) . A summary of the characteristic infra-red bands for lithium carbonate is presented in Table I. TABLE I Characteristic Infra-red Bands of Lithium Carbonate (7) Frequency (cm-l) 2558 2494 1842 1806 1495 1437 1088 866 846 741 7 12

Wavelength (p) 3.91 4.01 5.43 5.54 6.69 6.96 9.19 11.55 11.82 13.50 14.04

Relative Intensity

W W W W

vs vs M S

W W

vw

The infra-red absorption band at 1088 cm-l can be used to uniquely quantitate lithium carbonate in the presence of other alkali carbonates ( 1 0 ) . The observed frequencies related to the isotopic species 6LizC03 and 'Li~C03 have been reported by Tarte ( 1 1 ) . 2.2

Raman Spectroscopy The Raman spectra of crystalline and molten lithium carbonate have been reported by Brooker and co-workers ( 1 2 , 1 3 ) . Major bands are observed at 1091 and 1459 cm-l.

2.3 Atomic Emission and Absorption Spectroscopy Lithium carbonate can be made to exhibit the characteristic emission spectrum of lithium. Three typical analytical emission lines are obtained for lithium containing aqueous solutions. These are summarized along with their relative intensities in Table I1 ( 1 4 ) .

LITHIUM CARBONATE

371

The line at 670.8 nm is particularly intense and imparts a deep red color to an oxidizing flame. TABLE I1 Lithium Analytical Emission Lines Wavelength (nm) 670.8 323.3 610.4

Relative Intensit9 1

235 3600

+'relative amount of lithium required for 1% response Typical sources of excitation include air-acetylene and nitrous oxide-acetylene flames. More recently electrothermal and argon plasma excitation techniques have become available. Because of its greater sensitivity the line at 670.8 nm is most often used for the analysis of lithium by flame emission spectroscopy, atomic absorption spectroscopy and plasma spectroscopy (14, 15). 2.4

Melting Point The melting behavior of lithium carbonate has been evaluated by DTA using both heating and cooling programs. Lithium carbonate has been reported by various sources to melt in the temperature range of 714O to 733OC (16-25) depending on the atmosphere employed (ie. C02 or air) and the degree of dissociation of Li2CO3 to Liz0 and C02 that occurs as the melting point is approached (16, 18, 20, 21). Typical thermal properties reported in the literature for lithium carbonate are summarized in Table 111.

HENRY C. STOBER

372

TABLE I11 Thermal Properties of Lithium Carbonate

Melting Point (23) Heat Capacity (23) Specific Heat (24) Heat of Fusion (24) 2.5

993OK; 72OOC 23.2 cal/mole /deg 0.315 cal/g 10.7 kcal/mole

Thermogravimetric Analysis Data obtained for a typical lot of lithium carbonate (H2O < 0.5%)- by thermogravimetric analysis is presented in Table IV. Under the conditions employed the compound is essentially weight stable up to 200OC with only water loss. Above 200OC a gradual continuing weight loss is observed. This behavior is consistant with that reported by Machaladze and co-workers (21). TABLE IV

Lithium Carbonate:

Thermogravimetric Behavior (26)

TGA (N2 atmosphere) Scan rate 10°C/minute

RT to 90°C 900 to 200oc ZOOo to 45OOC above 45OOC 2.6

Perkin-Elmer TGS-1

0.11% weight loss 0.08% weight loss 0.63% weight loss continuing weight loss

Dissociation Constant The pKa's for the first and second ionization steps of the conjugate acid of the carbonate ion are reported in the literature to be 6.38 and 10.25 respectively (27).

373

LITHIUM CARBONATE

2.7

Conductivity The relationship between the equivalent conductance (4) and the concentration of lithium carbonate is typical of a strong electrolyte. A plot of& versus JC yields straight line for concentrations less than 0.01N. The equivalent 4 ' )for lithium conductance at infinite dilution ( carbonate was determined to be 110.2 R/cm2/Eq at approximately 25OC, from this plot (6).

2.8 Microscopy USP Lithium Carbonate is a microcrystalline solid that is birefringent under crossed polars. The solid has been observed to exist as polycrystalline aggregates ranging from approximately 10 to 85 micrometers in diameter (6).

2.9 Index of Refraction (ND25) The refractive indices of Lithium Carbonate have been reported as 1.428, 1.567 and 1.572 (25). Lithium Carbonate is biaxial and is optically negative. 2.10 Density The density reported for Lithium Carbonate is 2.11 g/cc 125): 2.11 Crystal Structure The crystal structure of Lithium Carbonate is monoclinic w'th unit cel dimensions of a = 8.39 A, b = 5.00 c = 6.21 , and f3 = 114.5+ (28). The crystal lattice belongs to the space group Cg - C 2/C and there are four lithium carbonate mofecules per unit cell (29, 7).

k,

2.12 X-Ray Powder Diffraction Major lines present in the x-ray powder diffraction pattern of USP grade lithium carbonate are presented in Table V. Strong lines are observed at 31.6, 21.4, and 30.6 degrees 28 for copper Ka radiation. These values are in good agreement with the literature values of 28 = 31.4, 21.0 and 30.3 [ASTM Data (30)] and 28 = 31.8, 21.3, and 30.6 [JCPDS Data (3111.

HENRY C. STOBER

374

T y p i c a l i n s t r u m e n t a l and experimental c o n d i t i o n s used t o o b t a i n t h e x-ray powder d i f f r a c t i o n p a t t e r n of l i t h i u m carbonate d e p i c t e d i n F i g u r e 2 (32) a r e p r e s e n t e d below. I n s t r u m e n t a l Conditions Spectrometer: Generator: Tube T a r g e t : Radiation: Optics :

Diano 8535 D i f f r a c t o m e t e r 30 KV, 13 mA cu 0 Cu, N i F i l t e r e d , Ka = 1.542 A lo Beam S l i t , MR S o l l e r S l i t , 0.1' D e t e c t o r S l i t , 3 O Take-Off Angle Scan Rate: 2 degrees 29/minute SPG-10 D e t e c t o r Rate meter 2500 cps f u l l s c a l e P u l s e Height S e l e c t i o n , EL = 0.2V

Goniometer: Detection:

-

Sample Preparation:

10

Sample was ground, s i e v e p t E r o u g h a No. 100 US Standard Sieve, and backpacked i n t o an aluminum sample h o l d e r . X-Ray Powder D i f f r a c t i o n P a t t e r n o f Lithium Carbonate (USP)

MAJOR LINES 28 Degrees 21.4 23.5 29.5 30.6 31.6 34.1 36.1 36.9 39.7 48.8

f

*k

*

1/10

dJ;J, 4.15 3.79 3.03 2.92 2.83 2.62 2.49 2.43 2.27 1.87

84 18 24 81 100 31 19 40 19 15

28 degrees read t o n e a r e s t 0 . 1 degrees 28

Interplaner Distance

(1):

nh d = 2 Sin 8

Hdc

Figure 2:

X-Ray Powder Diffraction Pattern of Lithium Carbonate

m

. h

u

m

I

2 .

u

C

H

$

-74

u cd

4

d b

s

ry *

. 7

U

1

I

15

19

23

27

31

35

39

43

47

Degrees 28

HENRY C . STOBER

376 +d** Relative

Intensity in percent based on strongest signal. Under the experimental conditions employed the relative intensities are subject to change due to variations in sample handling and particle size and are only included as a guide for identifying strong lines. The Hanawalt indices for lithium carbonate are 2.8lX, 4.168 and 2.928 (33). 2.13 Polymorphism Recent literature indicates that up to its fusion temperature lithium carbonate exists as only one crystal form (18). An earlier report in the literature indicated the presence of a polymorphic transition at 166OC (17). The possibility of lithium carbonate existing as a stable c1 and a metastable f3 phase, under atmospheric conditions, also has been proposed (34). It should be noted, however, that in the crystalographic sources consulted (30, 31, 33) only one form of lithium carbonate is listed, implying that other crystalline forms are uncommon. Impurities present in lithium carbonate may account for some of the thermal effects noted. In this respect, Reisman reported an anomalous transition at -410OC and an additional heat effect at 35OOC (18). The later was suspected to be due to the presence of Li20. Impurities such as Li20, Na2C03 and K2CO3 are known to form eutectics with lithium carbonate and may account for some of the anamolies observed. The ternary eutectic with Na2C03 and K2CO3 melts at 397O (19) 2.14 Solubility The following equilibrium solubility data was obtained either experimentally at 37OC (6) or from the literature as indicated in Table VI.

LITHIUM CARBONATE

377

TABLE VI Solubility of Lithium Carbonate in Common Solvents Solvent

Solubility (g/lOO ml)

1.5 Water, O°C 1.0 Water, 37OC 0.7 Water, 100°C 0.1N NaOH, 37OC 1.1 1.ON NaOH, 37OC 1.7 0.05M tris-buffer, 37OC 1.1 Ethanol Insoluble Acetone Insoluble

Source

(25) experimental (25) experimental experimental experimental (25) (25)

Lithium carbonate decomposes in strong mineral acids to yield carbonic acid, carbon dioxide and the conjugate salt. The solubility of lithium carbonate in water has been extensively studied as a function of temperature and has been observed to have a negative temperature coefficient of solubility; the solubility decreasing significantly with increasing temperature (35, 36). The heat of solution of lithium carbonate in water at 25OC has been reported as -14,800 20.021 kJ mol-' (37) * When considering the solubility of lithium carbonate in aqueous solutions, it should also be noted that lithium forms insoluble salts with several common anions including phosphate, fluoride and the carboxylate anion of the C14 - C ~ Bfatty acids (25). The solubility product of lithium carbonate in water at 25OC is 1.7 x (38) when the concentration of the lithium and carbonate ions are expressed in moles/liter. 2.15 Dissolution The intrinsic dissolution rate of lithium carbonate in aqueous solutions was reported by Wall and co-workers (39) using the rotating disc method. They found linear dissolution rate profiles for

HENRY C. STOBER

378

lithium carbonate in water, simulated gastric fluid and tris buffer. Dissolution studies in simulated intestinal fluid containing phosphate were complicated by the precipitation of trilithium phosphate onto the disc. Dissolution rate determinations for various experimental and commercial lithium carbonate preparations have been reported in the literature (40 4 4 ) . Ritschel and Parab ( 4 3 ) evaluated seven (six conventional and one sustained release) lithium carbonate commercial preparations. For the conventional preparations they found a good correlation between the ENSLIN number and t ( 5 min.), t (10 min.) and MRT (mean residence time). The ENSLIN number (amount of water in mL absorbed by 1 g of powdered substance) is a measure of the hydrophilicity, or wetting, of the formulation.

-

The wetting of pharmaceutical powders, including lithium carbonate, has also been evaluated by Lerk and co-workers ( 4 5 ) using contact angle measurements. The contact angle 8 obtained for lithium carbonate by these workers was SO0 indicating hydrophilicity. 3.

Preparation of Lithium Carbonate Lithium carbonate is primarily prepared from the mineral spodumene, LiAlSizOs ( 4 6 ) . Other mineral sources of lithium include petalite (LiAlSi4010), amblygonite (LiAl[F,OH]PO4) and lepidolite Spodumene is the most commer(K2Li3A14Si702[0H,F]3). cially important o f the lithium ores because of its relative abundance and its relatively high lithium content (3.757,).

In the manufacturing process employed by the Lithium Corporation ( 4 7 ) spodumene crude ore, which also contains such components as mica, quartz and feldspar, is crushed to a fine sand. The crushed spodumene is separated from the other components by flotation. The “purified” spodumene is subjected to intense heat (approximately llOO°C) and milled to a fine powder to increase its surface area and reactivity. The powdery

LITHIUM CARBONATE

379

spodumene is treated with strong sulfuric acid at The lithium sulfate produced is separated from the residual insoluble components of the ore by aqueous dissolution. The resulting lithium sulfate solution is reacted with sodium carbonate to produce lithium carbonate, which remains in solution. Impurities yielding insoluble carbonates are precipitated in this step. The lithium carbonate solution is further purified by pH adjustment and filtration and concentrated by evaporation. Lithium carbonate is precipitated from the concentrated solution by further treatment with sodium carbonate. Pharmaceutical grade material is also further processed to meet compendia1 and special requirements such as particle size and bulk density. The flow chart depicted in Scheme I summarizes the process just described. 25OoC to produce lithium sulfate (LiZSO4).

Other processes reported to utilize spodumene involve treatment with limestone to produce lithium hydroxide ( 4 6 ) and direct recovery of lithium carbonate by digestion with aqueous sodium carbonate at 200°C ( 4 8 ) . The production of lithium carbonate from lithium hydroxide has been accomplished using carbonization (COz) ( 4 9 ) and treatment with urea (50). A procedure for purifying lithium carbonate by suspen-

sion in boiling water is described in Volume I of "Inorganic Synthesis" ( 5 1 ) . The procedure is based on the fact that lithium carbonate is less soluble in hot water than in cold water, in contrast to the salts that are present as impurities. A zone melting procedure for producing single crystals of lithium carbonate has also been described ( 5 2 ) . 4.

Stability 4.1

Solution The predominant stability problem for lithium carbonate in aqueous solutions occurs in acid solutions, in which it decomposes to yield the lithium salt of the acid, bicarbonate, carbonic acid and ultimately, carbon dioxide. Solutions of lithium carbonate are also incompatible with a variety of cations and anions.

HENRY C . STOBER

380 SCHEME I

Preparation of Lithium Carbonate from Spodumene

I

I

1

Mined Spodumene Ore

Powdery Spodumene

Crushina Milling

I

'

Fine Spodumene Ore

Sintering (1lOO"C)

I

Size Reduction

1 (250OC)

Solution

Solution

I

Filtration Evaporation Sodium Carbonate

Lithium Carbonate Solid

LITHIUM CARBONATE

38 I

Cations such as calcium and barium, whose carbonate salts are much less soluble than lithium carbonate, and anions such as phosphate, that form less soluble lithium salts, should be excluded from lithium carbonate preparations. 4.2 Solid State Light Pharmaceutical grade lithium carbonate was subjected for one week to visible light, whose intensity was 600 foot candles (6). No decomposition as noted by changes in physical appearance (color, texture), weight and titrimetric assay for carbonate was observed.

-

Therma1 Samples of lithium carbonate were stored in open weighing dishes at 25OC and 105OC, respectively, for one week (6). A small increase in the titrimetric assay for carbonate from 99.4% to 99.6% was observed for the sample stored at 105OC. No change in the assay was noted for the sample stored at 25OC. No measureable changes in weight or physical appearance were observed for either sample. This stable behavior for lithium carbonate in the solid state is confirmed by the literature (16, 17, 21). Accordingly, the lowest temperature at which lithium carbonate has been reported to begin to dissociate to lithium oxide and carbon dioxide is 2OOOC (21). Other workers have reported that dissociation occurs only near the melting temperature (15, 16). Humidity Samples of lithium carbonate stored for one week at 25OC/85% RH, 35OC/lO% RH and 35OC/85% RH were essentially weight stable (6). This is consistent with the literature (8, l o ) , which indicates that lithium carbonate is not very hygroscopic. 5.

Methods of Analysis 5.1

Identification Test for Lithium (2) When lithium carbonate is moistened with hydrochloric acid, it imparts an intense crimson color to a non-luminous flame.

HENRY C. STOBER

382

5.2

Identification Test for Carbonate (2) Lithium carbonate effervesces upon the addition of an acid, yielding a colorless gas, which when passed into a solution of calcium hydroxide, immediately causes a white precipitate to form.

5.3

Microchemical Test for Lithium The microscopic identification of lithium is practical oniy in materials in which lithium is present in relatively high concentrations. Identification has been achieved by preparation of the tri-lithium phosphate salt, which forms star-like clumps (53), the pyroantimonate salt, which gives hexagons (53) and an orange-brown aurate salt (54). In the absence o f other alkali metals lithium reacts with zinc uranylacetate to yield regularly developed octahedra (55).

5.4

Microchemical Test for Carbonate Absorption of carbonate, evolved upon acidification of a lithium carbonate preparation, by a hanging drop of lead acetate, gives rise to acicular crystals occurring singly or in irregular aggregates. Both bicarbonate and carbonate give a positive response under these conditions. When added directly, thallus acetate does not precipitate with bicarbonate, but forms colorless, long, slender needles with carbonate (56).

5.5

Volumetric Analysis Lithium carbonate is analyzed in the USP ( 2 ) , BP ( 3 ) and ACS Reagent Chemicals (57) by titration of the carbonate anion. Excess strong acid is added to a solution of lithium carbonate and the residual acid remaining after neutralization is back-titrated with sodium hydroxide. Differences exist between these methods with respect to the indicators used and the emphasis placed on expulsion of carbon dioxide after acidification. Alternatively, potentiometric end point detection can be accomplished using glass and calomel electrodes.

LITHIUM CARBONATE

383

5.6

Atomic Emission and Absorption Spectroscopy Solutions of lithium carbonate are frequently analyzed for lithium using emission techniques such as flame photometry, plasma spectroscopy and by atomic absorption. The methods typically employ analysis at a wavelength of 670.8 nm, which is the most sensitive specific lithium line (2, 1 4 ) . These techniques are particularly effective for the analysis of lithium in dosage forms and biological fluids (2, 40, 4 4 , 58). A detailed discussion of the analysis of lithium in biological fluids and tissues by flame photometry and atomic absorption is presented in the text "Lithium Research and Therapy" (59).

5.7

Other Spectrometric Techniques Lithium carbonate has been determined spectrophotometrically in tablets using the color formed upon complexation of lithium with a "crowned" dinitrophenylazophenol ether (60). The purple color produced is stable and the absorbance of the solution measured at 560 nm is linear over the concentration range from 25 to 250 ppb. Field desorption mass spectrometry (FD-MS) in conjunction with a multichannel analyzer has proven to be a useful tool for trace analysis of lithium (61). The method also allows for the determiation of the isotopic distribution of the lithium salt analyzed.

5.8

Conductivity Although nonspecific, conductimetric methods have been used for the analysis of strong electrolytes The methods tend to be easily automated. (62). The technique is comparatively simple and applicable to the dissolution rate determination of dosage forms providing that the other conducting components of the formulation are not present in significant amounts. It has been used successfully in the authors laboratory for the determination of the dissolution rate of lithium carbonate sustained release formulations in water (63). Precautions were required against absorption of atmospheric Cop. This was accomplished by performing the dissolution rate studies with the vessels under a steady stream of nitrogen.

HENRY C. STOBER

384

5.9 Ion Selective Electrodes Several workers have explored the usefulness of ion selective electrodes for the analysis of solutions of lithium (64, 65, 66). The electrodes are limited by comparatively poor Li+/Na+ selectivity. This deficiency is especially apparent in biological fluids where comparatively high levels of Na+ are encountered. Using elecrodes based on PVC membranes containing ETH 1810 as a neutral carrier, Metzger and co-workers (64) obtained recoveries of lithium in serum to within +_lo% over the clinically relevant concentration range. 5.10 Ion Chromatography Aqueous solutions of lithium carbonate can be readily analyzed for lithium by ion chromatography. A polystyrene-divinyl benzene sulfuric acid cation exchanger in the hydrogen form (Waters, IC-PAC-C) and 2 mM nitric acid eluent is employed (67). Under these conditions lithium at the ppm level is readily separated from sodium and potassium at comparable concentrations. The carbonate anion can also be determined using a polymethacrylate anion exchanger in the quaternary ammonium ion form (Waters, IC-PAC-A).

6.

Medicinal History (68, 69) Lithium, the lightest of the alkali metals, was discovered in 1817 by Arfwedson. Its salts were later found in the spa waters of Germany and England, where it was believed that it was therapeutic in the treatment o f rheumatoid arthritis and gout. Its usefulness in the treatment of gout was attributed to the high solubility of lithium urate. The use of lithium as a uricosuric prompted J. F. Cade in Australia to give animals a lithium salt to decrease the nephrotoxicity of uric acid. He noted that the lithium salt produced a calming effect in the animals and proceeded to use lithium salts clinically as a sedative. During the 1950's and 1960's the clinical use of lithium

LITHIUM CARBONATE

385

salts was intensely investigated in Europe, where it became accepted as an effective and safe treatment for manic depressive illness. Lithium salts were not accepted in the United States until 1970. This was due in part to concerns about the safety of lithium salts after several deaths were reported (1949-50) among patients using large amounts of lithium chloride as a salt substitute. Although several lithium salts such as the chloride and bromide have been used, lithium carbonate is preferred because of its relative stability (it is less hygroscopic than the halogen salts) and it is less irritating to the gastrointenstinal tract. 7.

Pharmacology (69, 70) Lithium carbonate is usually administered as 300 mg tablets or capsules. Each 300 mg of the carbonate contains 8.12 mEq of lithium. The usual daily dose of lithium carbonate for prophylactic therapy is 600 mg to 1200 mg. Lithium is readily absorbed after oral administration in the gastrointestinal tract, peaking in the plasma within 1 to 3 hours and tends to distribute evenly throughout the total body water space. Lithium is not bound to plasma proteins. There is some lag in penetration into the cerebrospinal fluid, but there is no absolute barrier to its entry into the brain. Equilibration of lithium between the blood and the brain is almost complete within 24 hours. The "metabolism" of the lithium ion is almost entirely via the kidneys. About half of a single dose of lithium is excreted in 24 hours. An important feature of the renal excretion of lithium is that its rate is not typically increased by the administration of most diuretics. Increased administration of sodium appears to have minimal effect on the normal excretion of lithium, while depressed in-vivo levels of sodium facilitate lithium retention. These physiological factors have important implications for the management of lithium intoxication. The therapeutic plasma range (0.5 1.5 mEq/L) and the toxic plasma levels (>1.6 mEq/L) are very close and monitoring is performed at

-

386

HENRY C. STOBER

the onset of therapy. The mechanism of action of lithium ion in manic depression is not clearly understood. Lithium interferes with the action of the catecholamines in the brain. This supports the popular hypothesis that in mania catecholamines may be functionally overactive in the brain. The role played by lithium may also be related to competition with sodium ions in body sites, such as electrolyte balance across cell membranes, including those of the neurons.

Acknowledgement The typing assistance o f Mrs. Frances Ghiazza in the preparation of this manuscript is appreciated.

LITHIUM CARBONATE

387

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The United States Pharmacopoeia XXI, Mack Publishing Co., Easton, PA (1985) page 602.

3. British Pharmacopoeia 1980, University Press, Cambridge, England (1980) page 259. 4. Modern Drug Encyclopedia and Therapeutic Index, G. D. Gonzales, ed., York Medical Books, New York, NY 14, (1977) page 492.

5. Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, PA (1985) page 1088. 6. G. Dolch and H. Stober, CIBA-GEIGY Corp., Internal Communication, ARD 77121.

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15. M. Thompson and J. N. Walsh, A Handbook of Inductively Coupled Plasma Spectrometry, Blackie (1983) London, England, page 102. 16. T. V. Rode, Chem. Abstr.,

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17. M. N. Sermenov and T. V. Zabolotskii, Chem. Abstr., 48, 8697 (1954). 18. A . Reisman, J. Am. Chem. SOC.,

80, 6853 (1962).

19. G. J. Janz, E. Neuenschwander and F. J. Kelly, Trans. Faraday SOC., 59, 841 (1963). 20.

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21. T. E. Machaladze, G. D. Chachanidze and R. N. Pirtskhalava, Chem. Abstr., 2,1214112 (1973). 22.

G. Papin, Chem. Abstr., 80, 53148n (1974).

23. Comprehensive Inorganic Chemistry, Vol. 1, Pergamon Press Ltd., Oxford, Great Britain (1973) page 335.

24. Data Sheet €or Lithium Carbonate Pharmaceutical, Lithium Corporation of America, North Carolina, USA. 25. Handbook of Chemistry and Physics, 66th ed., The Chemical Rubber Co., Cleveland, Ohio (1985) page B108. 26. R. Morris, CIBA-GEIGY Corp., Personal Communication. 27. Handbook of Chemistry and Physics, 66th ed., The Chemical Rubber Co., Cleveland, Ohio (1985) page D163. 28. Crystal Data Determination Tables, 3rd. ed., Vol. 2. Inorganic Compounds, JCPDS (1973). 29. J. Zemann, Acta. Cryst.,

lo, 664 (1957).

30. Index to Powder Diffraction, ASTM (1963). 31. Powder Diffraction Data, JCPDS, 1st ed. (1976) 349.

,

LITHIUM CARBONATE

389

32. L. Busch, CIBA-GEIGY Corp., Personal Communication. 33.

Inorganic Phases Powder Diffraction File, JCPDS (1982),

34. A . N. Khlapova and E. S. Koraleva, Chem. Abstr.,66, 230492 (1967). 35. S. H. Smith, Jr., D. D. Williams and R. R . Miller, J. Chem. Eng. Data, 16,74 (1971). 36. V. M. Elenevskaya and M. I. Ravich, Chem. Abstr., 8702q (1962). 37. R. L. Montgomery, R . A. Melaugh, C. C. Lau, G. H. Meier, R. T. Grow and F. D. Rossini, Chem. Abstr. 4969b, (1978).

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38. Handbook of Chemistry and Physics, 66th ed., The Chemical Rubber Co., Cleveland, Ohio (1985), page B222 39. B. P. Wall, J. E. Parkin and V. B. Sunderland, J. Pharm. Pharmacol., 37, 338 (1985). 40. B. P. Wall, J. E. Parkin, V. B. Sunderland and A . Zorbas, J. Pharm. Pharmacol., 34, 601 (1982). 41. R. E. Shepard, J. C. Price and L. A. Luzzi, J. Pharm -9 Sci. -361 1152 (1972).

42. H. C. Caldwell, J. Pharm. Sci., 63, 770 (1974).

43. W. A. Ritschel and D. Parab, Drug Dev. and Indus. Pharm

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-9

147 (1988).

44. T. E. Needham, D. David and W. Brown, J. Pharm. Sci., 68, 952 (1979). 45. C. F. Lerk, M. Lagas, J. P. Boelstra and P. Broersama, J. Pharm. Sci., 66, 1481 (1977).

46. W. A . Hart and 0. F. Beumel, Jr. Comprehensive Inorganic Chemistry, Vol. 2 , Chapt. 7, A. F. TrotmanDickensen ex. ed., Pergamon Press, Elmsford, NY (1973).

47. Lithium, Lithium Corporation of America, Gastonia, North Carolina (1985).

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48.

C. A. Oliver, E. H. Nenniger, Chem. Abstr., 12451h (1979).

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49. E. B. Gitis, F. I. Strigunov, I. D. Solyanik, G. A. Trutnev, Chem. Abstr., 97, 130000P (1982). 50. Chem. Abstr.,

101,93728k

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51. F. R. Coley and P. J. Elving, Inorganic Synthesis, Vol. 1, Chapter 1, H. S. Booth, ed., McGraw-Hill, New York, NY (1939). 52. P. Ramasamy, G. S . Laddha, Cryst. Res. Technol., l7, 911 (1982). 53. E. M. Chamot and C. W. Mason, Handbook of Chemical Microscopy, Vol. 2 , John Wiley, New York, NY (1931)) 87. 54. C. F. Fulton, Modern Microcrystal Tests for Drugs, John Wiley, New York, Ny (1969)) 73. 55. A. A. Benedetti-Pichler, Identification of Materials via Physical Properties, Chemical Tests and Microscopy, Springer Verlog, New York, NY (1965)) 325. 56. E. M. Chamot and C. W. Mason, Handbook o f Chemical Microscopy, Vol. 2, John Wiley, New York, NY (1931) , 296. 57. Reagent Chemicals, 6th ed., American Chemical Society, Washington, D.C. (1982), 321. 58. A . Palladino, Jr., R. G. Longenecker, L. J. Lesko, J. Clin. Psychiatry, 5, 7 (1983). 59.

Lithium Research and Therapy, F. N. Johnson, ed., Chapters 10 and 11 , Academic Press, New York, NY (1975).

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LITHIUM CARBONATE

39 1

62. H. H. Willard, L. L. Merrit, J. A. Dean, Instrumental Methods of Analysis, 4th ed., Chapter 2, D. Van Nostrand Co. Inc., New York, NY (1965). 63. G. Dolch and H. Stober, CIBA-GEIGY Corp., Internal Communication, ARD 77135. 64.

E. Metzer, D. Ammann, R. Asper and W. Simon, Anal. Chem. -958 132 (1986).

-9

65. S. Kitazawa, K. Kimura, H. Yano and T. Shono, Analyst, 110, 295 (1985). 66.

A. F . Zhujow, D. Erne, D. Ammann, M. Guggli, E. Pretsch and W. Simon, Anal. Chem. Acta., 131, 117 (1985).

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68. Lithium Research and Therapy, F . N. Johnson, ed., Chapters 1 and 2. Academic Press, New York, NY (1975). 69. Handbook o f Psychopharmacology, L. L. Iversen, S. D. Iversen and S. H. Snyder, ed., Chapter 6 , Plenem Press, New York, NY (1978). 70.

Lithium Research and Therapy, F . N. Johnson, ed., Chapters 16, 17, and 18, Academic Press, New York, NY (1975).

This Page Intentionally Left Blank

MAPROTILINE HYDROCHLORIDE

Soonhee K. Suh and James B. Smith

1.

Description 1.1 1.2

2.

Name, Formula, Molecular Weight Appearance

Physical and Chemical Properties 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12

Infrared Absorption Spectrum Nuclear Magnetic Resonance Spectrum Ultraviolet Absorption Spectrum Mass Spectrum Polymorphism Melting Range Differential Scanning Calorimetry X-Ray Diffraction Thermogravimetric Analysis Dissociation Constant Solubility Partition Coefficient

3.

Synthesis

4.

Stability

5.

Drug Metabolism and Pharmacokinetics 5.1 5.2 5.3

-

Degradation

Metabolism Methodology Pharmacokinetics

ANALYTICAL PROFILES OF DRUG SUBSTANCES

VOLUME 15

393

Copyright 0 1986 by the American Pharmaceutlcal Association All rights of reproduction in any form reserved.

SOONHEE K. SUH AND JAMES B. SMITH

394

6.

Toxicity

7.

Methods of Analysis

7.1 Identification 7.2 Elemental Analysis 7.3 Nonaqueous Titration 7.4 Phase Solubility Analysis 7.5 Thin-Layer Chromatography 7.6 Gas Chromatography 7.7 High Pressure Liquid Chromatography 7.8 Gas Chromatography Mass Spectrometry 7.9 Double Radio-Isotope Derivative Method 7.10 Colorimetric Assay

-

References

MAPROTILINE HYDROCHLORIDE

1.

395

Description 1.1 Name, Formula, Molecular Weight Maprotiline hydrochloride is 1-(3-methylarninopropyl)-dibenzo[b,e]bicyclo[2.2.2.]octadiene

hydrochloride. CH2 CH 2 CH2NHCH 3 *HC1

I

C20H23N0HC1

Molecular Weight 313.87

1.2 Appearance Maprotiline hydrochloride occurs as a white to off-white, odorless, fine, crystalline powder.

2.

Physical and Chemical Properties 2.1

Infrared Absorption Spectrum The infrared absorption spectrum obtained from a mineral oil dispersion of maprotiline hydrochloride on a Perkin Elmer Model 281B IR spectrophotometer is shown in Figure 1. The spectral assignments are listed in Table I. Table I -1

Wavenumber, cm

Assignment

2720-2780

N-CH3

2455

-NH2

1596

Aromatic stretch

746-755

o-substituted benzene ring

-

Percent transmission 1O(

80

60

40

20

4000

3500 I

W ’ 2500 ,

3000

2000 1800 1600 1400 1 I I I

1200 I 1000 I

800 I

600 I

MAPROTILINE HYDROCHLORIDE

2.2

397

Nuclear Magnetic Resonance Spectrum (NMR) The IH-NMR spectrum of maprotiline hydrochloride is shown in Figure 2. The spectrum was determined on a Varian CFT-20 NMR instrument at ambient temperature. The sample was dissolved in deuterated dimethylsulfoxide containing tetramethylsilane as an internal standard. The spectral assignments are shown in Table 11. Table I1 No. of Chemical Shift Multiplicity Protons ~. (PPm)

Assignment

1.65

m

g, h

2.45

m

e,

2.66

S

d

3.19

t

C

4.24

t

b

-

m

7.0

7.4

9.7

f

aromatic H

s (broad)

a

e f c a d a H~-CH~-CH~-NH-CH~OHC~ H *

2.3

H

H

Ultraviolet Absorption Spectrum The ultraviolet absorption spectrum of maprotiline hydrochloride in methanol (0.1 mg/ml) exhibits two maxima at 265 and 272 with A(l%,lcm) values of 40.0 and 49.7, respectively. A typical spectrum obtained from HP 8450A W/Visible spectrophotometer is shown in Figure 3 .

SOONHEE K. SUH AND JAMES B. SMITH

398

I

Id.0

910

I

I

8.0

7.0

I

I

I

I

I

I

6.0

5.0

4.0

3.0

2.0

1.0

Figure 2 NMR Spectrum of Maprotiline Hydrochloride

I

0 PPm

MAPROTILINE HYDROCHLORIDE

399

Absorbance

0.70

0.60

0.50

0.40

0.30

0.20

0.10

1

% I240

260

280 300 Wavelength (nm)

320

340 350

Figure 3 Ultraviolet Absorption Spectrum of Maprotiline Hydrochloride

SOONHEE K. SUH AND JAMES B. SMITH

400

2.4 Mass Spectrum Figure 4 shows the mass spectrum of maprotiline hydrochloride obtained at 70 ev on a Kratos MS25 spectrometer using solid probe insertion. An interpretation of the fragmentation ions is given in Table 111. Table I11

m/z

Species

277

M+

247

M

-

NHCH3

218

203

CH=CH~ 19 1

178

70

44

MAPROTILINE HYDROCHLORIDE

Relative Intensity

1 Ot

9c

80

70

60

50

40

30

20.

10-

0-

r' MassKharge

Figure 4 Mass Spectrum of Maprotiline Hydrochloride

401

SOONHEE K. SUH AND JAMES B. SMITH

402

2.5 Polymorphism Maprotiline hydrochloride is polymorphic and known to exhibit several crystal forms (1, 2, 3 ) . A typical production batch is a mixture with varying ratios of crystal form A and B. The two crystal forms show no difference in solubility characteristics at room and physiological temperatures. In the solid state, they show differences in IR, DSC and x-ray powder diffraction patterns. Crystal form A is thermodynamically stable under 104OC, while the B form is metastable. Under conditions such as storage and tablet compression, no transformation of form B to form A, or the reverse, has been observed. However, a transformation of form B to form A has been observed upon melting.

2.6 Melting Range MaDrotiline hvdrochloride melts between 237 246OC with partial decomposition.

-

2.7 Differential Scanning Calorimetry (DSC) The existence of polymorphism has also been shown by differential scanning calorimetry. A typical batch of maprotiline hydrochloride frequently exhibits two melt endotherms indicating the presence of two crystal forms. The thenogram shown in Figure 5 was produced on a Perkin Elmer DSC-2, scanning 10°C per minute. Crystal forms A and B show extrapolated melting points at 242OC and 239OC, respectively. 2.8

X-Ray Diffraction The x-ray powder diffraction pattern obtained for a typical batch of maprotiline hydrochloride, as shown in Figure 6, is relatively weak with the strongest reflection occurring at approximately 16.4 k 0.2 degrees 28. The characteristic reflection patterns attributable to crystal form A and B , although very weak signals, appear at 3.85 and 3.60 angstrom, respectively. They are the basis for quantitative determination of a mixture by x-ray diffraction (4).

2.9 Thermogravimetric Analysis The TGA of maprotiline hydrochloride typically exhibits a mean weight l o s s of about 0.2% between

MAPROTILINE HYDROCHLORIDE

0 ; 310

403

I

I

I

1

I

I

I

I

I

340

370

400

430

460

490

520

550

580

TemperatureOK

Figure 5 DSC Scan of Maprotiline Hydrochloride

I

I

1

I

I

1

I

I

5

10

15

20

25

30

35

40

Degrees Two Theta

Figure 6 X-ray Powder Diffraction Pattern of Maprotiline Hydrochloride

SOONHEE K. SUH AND JAMES B. SMITH

404

room temperature and 175OC. Above 175OC a r a p i d weight l o s s due t o decomposition and/or s u b l i m i n a t i o n i s observed. 2.10

D i s s o c i a t i o n Constant The pKa v a l u e determined i n water a t 25OC i s 10.5 Also, v a l u e s of 9.0 k 0 . 1 and 9.4 k *0.2-(5). 0.1 determined i n 80% e t h y l e n e g l y c o l monomethyl e t h e r i n water have been r e p o r t e d (1, 6 ) .

2.11 S o l u b i l i t y Maprotiline hydrochloride i s f r e e l y soluble i n methanol and chloroform, s l i g h t l y s o l u b l e i n wat e r , and p r a c t i c a l l y i n s o l u b l e i n i s o o c t a n e . S o l u b i l i t i e s i n some commonly used o r g a n i c solv e n t s and aqueous s o l u t i o n s a r e shown i n Table I V . Table I V Solubility (mg/ml)

--

Chlorof orm

25

Chloroform

30

Methano 1

25

--

Dimethylformamide

25

Acetone

25

Ether

25

Water

25

Water

30

Water

Refer-

ence

100

7

142

9

100

7

--

30

7

---

0.1

7

0.02

7

10.3

8

11.1

9

37

5.6 5.7 5.6

13.2

8

0.1N H C 1

25

1.0

3.1

8

0.1N H C 1

37

1.0

4.4

8

Gastric Fluid (simulated)

37

1.1

3.7

1

I n t e s t i n a l Fluid (simulated)

35

7.5

5.3

1

Phosphate B u f f e r

RT

7.4

1.3

1

Phosphate B u f f e r

RT

11.0

0.29

10-4

1

MAPROTILINE HYDROCHLORIDE

405

2.12 Partition Coefficient The partition coefficient data shown in Table V were obtained at 25OC as the ratio of the concentration found in the organic phase to that of in the aqueous phase (5, 9 ) . Table V Aqueous Phase

Chlorof o rm Chloroform Hexane n-Octanol

Water 0.1N HC1 Water 0.1 Hydrochloric Acid 0.1M Glycine Buffer + HC1 1/15M Phosphate Buffer 1/15M Phosphate Buffer 0.1M Glycine buffer + NaOH

n-Octanol n-Octanol n-Octanol n-Octanol 3.

@

Organic Phase

--

---

Partition Coefficient 0.30

4.7

1.1

0 15.0

3.0

3.51

5.2

1.25

7.4

27.3

9.0

708

Synthesis Maprotiline hydrochloride is synthesized by the following route shown below (10,ll): CH=CHCHO

CH=CHCH0

CH=CHCH=NCH3 I

CH~CHZCH~NHCH~*HC~ I

SOONHEE K. SUH AND JAMES B. SMITH

406

4.

-

Stability Degradation Maprotiline hydrochloride is a very stable compound and resists degradation even in very harsh environments (9, 12). When maprotiline hydrochloride was refluxed for 94 hours in 0.1N HC1 and 0.1N NaOH, no degradation product was observed in the sample refluxed in acid, while one trace impurity was detected in the sample refluxed in base. An aqueous solution (0.5%) stored at 100°C for one week showed no decomposition. Maprotiline hydrochloride is stable after storage for 5 years at room temperature, one year at 5OoC, 12 weeks at 40°C/70% relative humidity and 600 f.c. light.

5.

Drug Metabolism and Pharmacokinetics 5.1

Metabolism The lipophilic properties of maprotiline produced by the tetracyclic-propyl portion of the molecule account for the in vivo distribution behavior of this antidepressant. In rodents, following intravenous administration, maprotiline is quickly distributed into organs, with the highest concentrations found in the lungs, adrenal and thyroid glands, and heart. The lowest concentrations are found in the blood. High tissue levels of maprotiline as compared to blood apparently also occur in man, as indicated in a postmortem study of a suicide from an overdose of maprotiline ( 1 3 ) . Following intravenous administration of 14C-labeled maprotiline in man, 57% and 30% of the dose, respectively, were found in urine and feces after 21 days. Maprotiline is excreted in urine predominantly (90%) as metabolites, 75% of which are glucuronides. Principal metabolic transformations involve N-demethylation, deamination, aromatic as well as aliphatic hydroxylations and aromatic methoxy derivative formation ( 1 3 ) .

5.2 Methodology The method most often used in the determination of the pharmacokinetic parameters of maprotiline has been a double radio-isotope derivative technique coupled with thin layer chromatography for specificity ( 4 8 , 14). A similar double radio-isotope method, with a gas chromatographic

MAPROTILINE HYDROCHLORIDE

407

determination for specificity, has been described for the tricyclic nortriptyline (15). Using an internal standard and a nitrogen detector, maprotiline in plasma has been determined by gas chromatography as the free base (33). When compared to the double radio-isotopic technique, this procedure was found to be comparable in the range of 20 to 150 ng/ml (16). Nitrogen detection by gas chromatography after reaction with acetic anhydride was sensitive to 50 ng/ml for the determination of maprotiline and its N-desmethyl metabolite in blood (30). Sensitivity to 10 ng of maprotiline in biological samples was achieved using gas chromatography and electron capture detection. Maprotiline was chromatographed as the heptafluorobutyramide after reaction with heptafluorobutyric anhydride (17, 34). Using the same anhydride with either N-desmethylclomipramine or isotope labeled maprotiline as the internal standard, maprotiline can be determined with the combination of gas chromatography and chemical ionization mass spectrometry in biological fluids at concentrations of 0.5 to 150 ng/ml (46). Reversed phase high perfomance liquid chromatography on a Nucleosil C-18 column with 35% acetonitrile, 65% 0.05 M phosphate buffer (pH 2.7) . has also been utilized for the determination of maprotiline in human plasma. Ultraviolet absorbance at 214 run provided sensitivity to 2 ng/ml of plasma after isolation of maprotiline as the free base (41). 5.3

Pharmacokinetics The biological half life of maprotiline following a single intravenous dose of 50 mg to 6 healthy volunteers was found to be 43 hours (13). The half life of 40 hours reported in a similar study using normals and a 75 mg intravenous dose, is in excellent agreement (14). Availability of an oral dose of 50 mg of maprotiline compared to an equal intravenous dose was comparable as determined by AUC measurements (13). Peak levels of maprotiline appear in blood or plasma relatively slowly following oral admin-

SOONHEE K. SUH AND JAMES B. SMITH

408

istration. Maximum maprotiline levels have been found to occur within about 8-24 hours post administration (13, 18, 19). The relatively large apparent volume of distribution (13, 14, 19) indicates maprotiline is highly tissue-bound, as the aforementioned post-mortem study indicated (13). When normal subjects received simultaneously a 50 mg maprotiline tablet and 50 mg of tridueterated maprotiline hydrochloride in an aqueous solution, blood levels for the isotope labeled and unlabeled drug were essentially superimposable. Peak blood level averaged about 50 ng/ml (19).

In five depressive patients receiving seven day incremental doses of 25 mg, 50 mg, and 75 mg, plasma levels of maprotiline were clearly dose related. At the end 7, 14, and 21 days, maprotiline plasma levels were respectively, 18-46 ng/ml, 64-103 ng/ml and 96-155 ng/ml. Although therapeutic activity was observed at all plasma levels (18-155 ng/ml), the incidence of side effects appeared to be related to the 75 mg twice daily dosage regimen (20). Proportional blood levels have also been reported under steady state conditions (about 7 days) for maprotiline doses of 50 mg, 100 mg, and 150 mg (13). Several reviews and summaries of the pharmacology, pharmacokinetics, toxicity, and efficacy of maprotiline are available (21, 22). 6.

Toxicity The standard intravenous LD50 established for maprotiline hydrochloride in male rats is 27.3 mg/kg. Maprotiline hydrochloride is therefore classified as moderately toxic when administered intravenously to rats (23).

7.

Methods of Analysis 7.1

Identification Maprotiline hydrochloride is best identified by: 1)- infrared absorption; 2) ultraviolet absorption; 3 ) a test for chloride.

MAPROTILINE HYDROCHLORIDE 7.2

409

Elemental Analysis The elemental composition determined for a typical sample of maprotiline hydrochloride on a Perkin Elmer Model 240 CHN Analyzer is given below. Element Carbon Hydrogen Nitrogen

Theory, % 76.53 7.71 4.46

Found, % 76.71 7.78 4.44

7.3

Nonaqueous Titration Maprotiline hydrochloride may be titrated with acetous perchloric acid in glacial acetic acid containing mercuric acetate. The titration can be carried out potentiometrically using a glass electrode and a calomel electrode containing glacial acetic acid saturated with lithium chloride.

7.4

Phase Solubility Analysis Phase solubility analysis of maprotiline hydrochloride has been carried out using the following systems: Solvent: n-propanol Approximate Solubility: 1 6 . 4 mg/g Temperature: 25OC

7.5

Thin-layer Chromatography

A number of thin-layer chromatographic systems have been developed for the identification and the determination of the drug and compounds related to the drug. System I Adsorbent:

Silica Gel G or GF254, 2501.1 thicknes s

Mobile Phase:

sec-Butyl Alcohol/Ethyl Acetate12N Ammonium Hydroxide ( 1 2 0 : 6 0 : 2 0 ) saturated with ammonia vapor

Detection:

a ) After exposing the plate under hydrogen chloride vapor for 30 minutes, irradiate it with high intensity W light for 5 minutes. Observe under longwave W source. b) Iodine vapor

SOONHEE K. SUH AND JAMES B. SMITH

410

System I1 Adsorbent :

Silica Gel G or GF254 2 5 0 ~ thickness

Mobile Phase: sec-Butanol/Ethyl Acetate/5N Ammonium Hydroxide ( 1 4 : 4 : 5 ) Detection:

System I11 Adsorbent :

After exposing the plate under hydrogen chloride vapor for 30 minutes, irradiate with high intensity W light for 5 minutes. Observe under longwave W source. Silica Gel GF254, 250p thickness

Mobile Phase: n-ButanolIAcetic Acid/Water (70:10:20) Detection:

System IV Adsorbent:

a) Iodine vapor b) After exposing the plate to chlorine vapor for 1-2 minutes, spray with sodium hypochlorite (2%), followed by a second spray of a mixture of 10 ml of KI (1 in loo), 10 ml of soluble starch ( 3 in 100) and 3 ml of methanol. c) W 254nm Silica Gel GF254, 250p thickness

Mobile Phase: Chloroform/Methanol (95:5) saturated with ammonia gas Detection:

Same as System I11

System V Adsorbent :

Silica Gel HF, 250p thickness

Mobile Phase: n-Butanol/acetic acidlwater (70:10:20)

Detection:

Spray the plate with sodium hypochlorite (2%) in 0.125N sodium hydroxide solution. Dry the plate at 4OoC for 1 hour. Spray the plate with a mixture (1:l) of KI (0.83%) and o-tolidine (0.2% in 2% acetic acid).

MAPROTILINE HYDROCHLORIDE

System VI Adso rbent :

411

Silica Gel HF, 250p thickness

Mobile Phase: BenzeneIDiethylamine (9:l) Detection:

Same as System V

System VII Adsorbent :

Silica Gel HF, 250p thickness

Mobile Phase:

Chloroform/Methanol/Acetic Acid (7:2:1)

Detection:

Same as System V

System VIII Ad so rbent :

Silica Gel GF2s4, 250p thickness

Mobile Phase: Methanol/Ethylacetate/l,ZDichloroethane/water (3:3:3:1) Detection: 7.6

Same as the detection method b ) in System 111.

Gas Chromatography System I:

The following system has been used to determine the drug substance for routine toxicological analysis (24).

Column:

SE-30 coated fused silica capillary, 3.5 to 15m x 0.25 mm ID

Temperature:

Column programmed from 100°C to 295OC at 5OC/min. Injector & detector temperatures not reported

Carrier:

Helium, linear velocity p = 45 cm/second at 100°C

Detection:

Flame Ionization

System I1

The following system has been used f o r the determination of the drug substance and other basic drugs in liver (25).

SOONHEE K. SUH AND JAMES B. SMITH

412

Column :

AR-glass capillaries coated with 0.3% (W/V) SE-52, 15 m x 0.26 mm ID

Temperature:

Injector at 25OoC, detector at 3OO0C, column temperature programmed by S°C/min. from 190 to 22OOC followed by 15OC/min. to 28OOC and then by 8OC/min. to the final temperature of 3OO0C, at which it was held for 5 min.

Carrier:

Helium, linear velocity 35-40 cm/sec.

Detection:

Nitrogen Phosphorus Detector

System I11

The following system has been used for the identification of the drug and its silylated derivatives along with a number of other drug substances and their silylated derivatives (26).

Column:

3% OV-17 on Gas Chrom Q (100-120 mesh) Glass, 2 m x 3 mm ID

Temperature:

Injection port and detector at 3OO0C, column at 3OO0C for isothermal run or 120 to 27OOC at 10°C/min., hold at 27OOC for 16 min. for programmed run.

Carrier:

Nitrogen, 30 mllmin.

Detection:

Flame Ionization

System IV:

The following system has been employed for the determination of the drug substance in plasma, derivatizing it with acetic anhydride (27).

Column :

5% OV-17 on Gas Chrom Q (100-120 mesh), 1.83 m x 2 mm ID glass

Temperature:

Injector and detector at 3OO0C and column at 255OC

Carrier:

Helium, 30 ml/min.

MAPROTILINE HYDROCHLORIDE

413

Detection:

Alkali Flame Ionization

System V:

The following system has been used to determine the drug substance and a number of other neutral and basic drugs in blood ( 2 8 ) .

Column :

a) 3% OV-101 on Chromosorb (100-120 mesh), 2 m x 0.6 cm ID glass. b) 3% OV-17 on Chromosorb (100-120 mesh) 2 m x 0.6 cm ID glass.

Temperature:

Injector and detector at 3OO0C and column from 170 290°C programmed at a rate of 4OC/min.

Carrier:

Helium, 30 ml/min.

Detection:

Nitrogen Phosphorous Detector

System VI

The following system has been used for the assay of the drug substance in serum (29).

Column:

1.4% Carbowax 20M plus 1.4% KOH on Gas Chrom Q (60-80 mesh), 1.8 m x 2 mm ID glass

Temperature:

Injection port and detector at 25OoC and column at 210°C

Carrier:

Nitrogen, 30 ml/min.

Detection:

Flame Ionization

System VII

The following system has been employed for the assay o f the drug substance in biological fluids derivatizing it with acetic anhydride (30).

Column:

3% HI-EFF-8BP on Chromosorb W HP (100-120 mesh), 0.30 m x 3 mm ID.

Temperature:

Injector and detector at 3OO0C and column at 245OC

Carrier:

Nitrogen, 40 ml/min.

-

414

SOONHEE K. SUH AND JAMES B. SMITH Detector:

Nitrogen S e l e c t i v e Flame I o n i z a t i o n

System VIII

The following system h a s been employed f o r t h e d e t e r m i n a t i o n of t h e drug s u b s t a n c e i n plasma, d e r i v a t i z i n g it w i t h h e p t a f l u o r o b u t y r i c anhydride ( 3 1 ) .

Column

a ) 3% SP-2250 (OV-17) on Supelcoport 80-100 mesh, 2 m x 4 mm I D

b) Open t u b u l a r column coatded w i t h SE-30, 25 q x 0.37 mm I D Temperature : Carrier:

I n j e c t o r and d e t e c t o r a t 3OO0C and column a t 26OoC a)

b)

Helium, 50 ml/min. H e l i u m , 1 . 5 ml/min.

Detection:

Nitrogen S e l e c t i v e D e t e c t o r

System I X

The f o l l o w i n g system has been used f o r t h e d e t e r m i n a t i o n o f t h e drug s u b s t a n c e and o t h e r drugs i n serum, d e r i v a t i z i n g it w i t h t r i f l u o r o a c e t i c anhydride (32).

Column:

3% OV-17 on Gas Chrom Q (100-120 mesh), 138 cm x 2 mm I D g l a s s

Temperature:

I n j e c t o r a t 23OoC, d e t e c t o r a t 27OOC and column from 220 - 275OC programmed a t 8OC/min.

Carrier:

N i t r o g e n , 35 ml/min.

Detection:

Nitrogen Phosphorous D e t e c t o r

System X

The f o l l o w i n g system h a s been used t o determine t h e drug s u b s t a n c e i n plasma (33).

Column

3% OV-17 on gas chrom Q (100-120 mesh), 2 m x 2 mm I D g l a s s

Temperature :

I n j e c t o r and d e t e c t o r at 3OOOC and column a t 265OC

MAPROTILINE HYDROCHLORIDE

415

Carrier:

Not r e p o r t e d

Detection:

N i t r o g e n Phosphorous D e t e c t o r

System X I :

The f o l l o w i n g system has been used f o r t h e a s s a y of t h e drug substance i n biological f l u i d s d e r i v a t i z i n g with heptafluorobut y r i c anhydride (34).

Column:

3% JXR ( m e t h y l s i l i c o n e ) on Gas Chrom Q , 4 ' x 3 mm I D

Temperature:

I n j e c t i o n p o r t a t 25OoC, column a t 23OoC, d e t e c t o r a t 28OOC

Carrier:

N i t r o g e n , 40 ml/min.

Detection:

6 3 N i E l e c t r o n Capture

System XII:

The f o l l o w i n g system has been used t o determine t h e drug s u b s t a n c e and i t s t r i f l u o r o a c e t y l a t e d d e r i v a t i v e i n b i o l o g i c a l sample (35).

Column:

2% D e x s i l 300 on Chromosorb W , AW-DMCS (80-100 mesh), 2 m g l a s s column (column I D n o t r e p o r t e d )

Temperature:

I n j e c t o r a t 3 1 O o C , column a t 28OOC and d e t e c t o r t e m p e r a t u r e n o t reported

Carrier:

N i t r o g e n , 30 ml/min.

Detection:

Flame I o n i z a t i o n

System X I 1 1

The f o l l o w i n g system has been used t o determine t h e drug s u b s t a n c e i n plasma and u r i n e a s h e p t a f l u o r o b u t y r i c d e r i v a t i v e (36).

Column:

a ) 0.75% OV-17 o r b) 0.5% XE-60 and 0.25% DC LSX-3-0295 on s i l a n i z e d Chromosorb G (80-100 mesh), 320 cm x 1.8 mm I D g l a s s column

Temperature:

I n j e c t o r a t 255OC, d e t e c t o r a t 21OOC and column a t 245OC

SOONHEE K. SUH AND JAMES B. SMITH

416

7.7

-

Carrier :

Nitrogen a t 10

Detect i o n :

3H E l e c t r o n Capture

System XIV:

The following system h a s been used t o determine t h e drug s u b s t a n c e i n blood a s h e p t a f l u o r o b u t y r i c d e r i v a t i v e (17).

Column:

1.5% 08-225 on Supelcoport packed i n a s i l a n i z e d g l a s s column, 1.8 m x2mmID

Temperature :

I n j e c t o r a t 250 - 26OoC, column a t 23OOC and d e t e c t o r a t 200 320 330OC.

Carri.er:

Nitrogen

Detection:

63Ni Electron capture

15 ml/min.

-

High P r e s s u r e Liquid Chromatography System I

The following system h a s been employed € o r t h e a n a l y s i s of t h e p u r e drug s u b s t a n c e and dosage forms (37).

Column:

ZorbaxB CN (DuPont) 250 mm

Mobile Phase:

Methanol/O.O8M sodium a c e t a t e (95:5) w i t h t h e a p p a r e n t pH adjusted t o 7.5 8.0

, 4.6

mm I D x

,.,

Flow Rate:

2 mljmin.

Detection:

U l t r a v i o l e t a b s o r p t i o n a t 272 nm

System I1

The f o l l o w i n g system h a s been employed f o r t h e a n a l y s i s of t h e pure drug s u b s t a n c e and dosage forms ( 3 7 ) .

Column:

Zorbaxb CN (DuPont), 4.6 mm I D x 250 mm

Mobile Phase:

Tetrahydrofuran/O.O25M sodium acet a t e (9O:lO) w i t h t h e apparent pH adjusted t o 7.4 8.0

-

MAPROTILINE HYDROCHLORIDE

417

Flow Rate:

1 ml/min.

Temperature:

4OoC

Detection:

Ultraviolet absorption at 272 nm

System I11

The following system has been employed for the analysis of the drug substance and a number of other basic drugs (38).

Column:

Spherisorb 5 Silica, 4.9 mm ID x 250 mm

Mobile Phase: a) Methanol/Hexane (85:15) with 0.02 v/v% (1.85 mM) Perchloric Acid b) Same as the above except 0.05 v/v% (4.63 mM) Perchloric Acid c) Same as the above except 0.1 v/v% (9.25 mM) Perchloric Acid Flow Rate:

2.0 ml/min.

Detection:

Ultraviolet Absorption at 215 nm

System IV

The following system has been employed for the analysis of the drug substance and a number of tricyclic antidepressants (39).

Column:

Hypersil 5 I.rm ODs, 4.6 mm ID x 70 or 100 mm

Mobile Phase:

a) Acetonitrile/lO mM Sodium Dihydrogen Phosphate (50:50) at pH 2 with 80 mM of sodium lauryl sulfate b) Same a s the above except the addition of 5 mM of tetrabutylammonium bromide

Flow Rate:

2.0 mllmin.

Detection:

Ultraviolet Absorption at 254 nm

System V

The following system has been employed for the determination of the drug substance from biological fluids (40).

SOONHEE K. SUH AND JAMES B. SMITH

418

Column:

VBondapak

CIS,

3.9 mm ID x 300 mm

Mobile Phase: Acetonitrile/O.l M Potassium Phosphate, pH 2.5 (30:70)

Flow Rate:

2 ml/min.

Detection:

Ultraviolet Absorption at 205 nm

System VI

The following system has been used for the assay of the drug substance in human plasma (41).

Column:

Nucleosil

c18

Mobile Phase: Acetonitrile/O.O5M Phosphate Buffer, pH 2 . 7 (35:65) Flow Rate:

Not indicated

Detection:

Ultraviolet Absorption at 214 nm

System VII

The following system has been used for the qualitative identification of the drug substance and other basic drugs (42).

Column:

Three types o f columns have been used. a) 3-7 pm silica b) mercaptopropyl bonded-phase silica c) aliphatic strong cation (n-propylsulphonic acid) bondedphase silica, 5 mm ID x 250 m m

Mobile Phase : Methanol/ZM Ammonium Hydroxide/lM Ammonium Nitrate (27:2:1) Flow Rate:

1 ml/min.

Detection:

Ultraviolet Absorption at 254 nm

System VIII

The following system has been used for the assay of biological samples by derivatizing maprotiline with p-nitroazobenzenecarbonyl chloride ( 4 3 ) .

MAPROTILINE HYDROCHLORIDE

419

Column:

Lichrosorb SI 100 (5p), 3 mm ID x 100 mm and 3 mm ID x 300 mm connected by two-way switching valve

Mobile Phase:

1.1% t-Butanol, 23% Dichloromethane, 0.05% Morpholine, 0.05% water and make to 100% with Hexane

Flow Rate:

Not indicated

Detection:

Ultraviolet Absorption at 337 nm

System IX

The following system has been used for the analysis of the drug substance and a number of tricyclic antidepressants (44)

Column:

Partisil 5 pm, 3.0 mm ID x 150 mm

Mobile Phase: Dichloromethane/Methanol/Acetate Buffer pH 3.2 (90+10+0.15) Flow Rate:

1.0 ml/min.

Detection:

Ultraviolet Absorption at 254 nm

7.8 Gas Chromatography

-

Mass Spectrometry (GC-MS)

System I

The following system has been used for the determination of the drug substance and other neutral and basic drugs in blood (28).

Column:

3% 33-30 on Chromosorb (100-120 mesh), 2 m x 0.6 cm ID glass

Temperature:

Injector at 270OC and column from 270OC programmed at a rate of 10°C./min.

220

-

Carrier:

Helium, 30 ml/min.

MS EI and CI Source:

70 eV

CI Reagent Gas :

Methane

Detection:

EI selected ion-monitoring at m/e = 59 and 191 CI selected ion-monitoring at m/e = 278

SOONHEE K. SUH AND JAMES B. SMITH

420

System I1

The following system has been used for the determination of the drug substance and its major metabolite derivatized with trifluoroacetic anhydride using stable isotopelabeled analog as internal standard (45).

Column:

1% OV-17 on Gas Chrom Q (100 - 120 mesh), 1.8 m x 2 mm ID glass

Temperature:

Injector at 24OoC, column at 255OC, separator at 25OoC, and ion source at 275OC

Carrier:

Helium, 20 ml/min.

MS EI Source: 70 eV Detection:

EI selected ion-monitoring at m/e = 345

System 111

The following system has been used for the determination of the drug substance in biological fluids, derivatizing it with heptafluorobutyric anhydride. Isotope labeled analogs of the drug substance were used as internal standard (46).

Column:

1.5% Poly 5-179 on Chromosorb W AW DMCS (80-100 mesh), 1 m x 2 mm ID glass

Temperature:

Injector at 250 - 26OoC, column at 240 - 25OoC, GC-MS interface at 245 - 27OoC and ion source at 245 - 265OC

Carrier:

Not reported

MS CI Source: 40 CI Reagent Gas : Detection:

-

100 eV

Methane CI selected ion monitoring at m/e = 474

MAPROTILINE HYDROCHLORIDE

42 1

System I V

The f o l l o w i n g system has been used f o r t h e d e t e r m i n a t i o n o f t h e drug s u b s t a n c e and o t h e r t r i c y c l i c ant i d e p r e s s a n t s i n blood plasma, using deuterated i n t e r n a l stand a r d s (47)

Column:

3% OV-17 on chromosorb HPW-DMCS, (80-100 mesh), 40 cm x 2 mm I D glass

Temperature:

I n j e c t o r a t 3OO0C, column a t 24OoC, s e p a r a t o r a t 35OOC and i o n s o u r c e a t 24OOC

Carrier:

H e l i u m , 15 m l / m i n .

MS C I Source:

70 eV

C I Reagent Gas :

.

Methanol Vapor

Detection:

C I s e l e c t e d ion-monitoring a t m/e = 378/286 + [ (M+1) Ludiomil/ (M+l) d e u t e r a t e d Ludiomil]

System V

The f o l l o w i n g system has been used f o r t h e d e t e r m i n a t i o n of t h e drug s u b s t a n c e and i t s t r i f l u o r o a c e t y lated derivative i n biological sample (24).

Column:

1%OV-17, 2 m g l a s s ( s o l i d s u p p o r t and column I D n o t r e p o r t e d )

Temperature:

Column a t 15O-32O0C, programmed a t a r a t e of 15OC/min. Temperature f o r i n j e c t o r and i o n s o u r c e n o t reported.

Carrier:

Not r e p o r t e d

MS E I Source:

70 eV

Detection:

E I s e l e c t e d ion-monitoring a t m / e = 345

SOONHEE K. SUH AND JAMES B. SMITH

422 7.9

Double Radio-Isotope Derivative Method The double radio-isotope derivative technique for the assay of the drug substance in biological material has been employed by Riess ( 4 8 ) . A biological sample spiked with a definite amount of 14C-labeled maprotiline standard was extracted with heptane containing 1% amyl alcohol. The extracted sample ( 14C-standard plus unknown) was derivatized with 3H-acetic anhydride. After the addition of an amount of unlabeled acetyl derivative of maprotiline as carrier, the sample was purified by two-dimensional thin-layer chromatography. The purified sample was analyzed for both 3H activity and 14C activity by liquid scintillation counting with the discriminatorratio method. Appropriately prepared standard samples allow calculation of the overall individual yield of the 14C-internal standard in each sample. B means of the 14C-yield found, the recovered H activity value can be corrected to the equivalent of a theoretical 100% yield. The resultant 3H activity value can be converted to the concentration units by the 100% 3H activity value resulting from an appropriate number of tests samples which have been treated in exactly the same way as the unknown and contain a definite, known amount of trial compound.

41

7.10 Colorimetric Assay Maprotiline hydrochloride can be assayed by the dithiocarbamic acid copper complex method. Maprotiline, a secondary amine, undergoes the following reaction with carbon disulfide and cupric ion in an alkaline medium.

0

0

KOH

0

0

cu 2

The absorbance of the colored complex is measured at 437 run. The method is specific to the secondary aliphatic amine.

423

MAPROTILINE HYDROCHLORIDE

Acknowledgement The authors express appreciation to Frances Ghiazza and Richard Brown for their help in the preparation of this manuscript.

References 1.

Stahl, P., CIBA-GEIGY, Ltd., Personal Communication.

2.

Marti, E. and Stahl, P . , CIBA-GEIGY, Ltd., Personal Communication.

3.

Kuhnert-Brandstaetter, M., Wurian, I., and Geiler, M., Sci. Pharm. , 50(3), 208-16 (1982).

4.

Mfiller, R., CIBA-GEIGY, Ltd., Personal Communication.

5.

Jakel, K., Moser, P., and Stahl, P., CIBA-GEIGY, Ltd., Personal Communication.

6.

Smith, J., Hagman, H., and Mollica, J., CIBA-GEIGY, Personal Communication

7.

Steiner, H., CIBA-GEIGY, Ltd., Personal Communication.

8.

Stahl, H . , CIBA-GEIGY, Ltd., Personal Communication.

9.

Mollica, J., Pucket, J., and Rhem, C., CIBA-GEIGY, Personal Communication.

10.

Beck, D., Bernasconi, R., Schenker, K., and Wilhelm, M. (CIBA-GEIGY A.-G.) Ger. Offen. 2, 207, 111 (C1. C 0 7 C ) , 07 Sep., 1972, Swiss Appl. 257671, 23 Feb. 1971.

11.

Wilhelm, M., and Schmidt, P . , Helv. Chim Aceta

52,

1385-95 (1969) 12.

Cohen, J., CIBA-GEIGY, Personal Communication.

13.

Riess, W., Dubey, L., Fcnfgeld, E. W., Imhof, P., Hfirzeler, H., Matussek, N., Rajagopalan, T. G., Raschdorf, F., and Schmid, K., J. Int. Med. Res., 3, Supplement (2)16 (1975).

SOONHEE K. SUH AND JAMES B. SMITH

424 14.

Maguire, K. P., Norman, T. R., Burrows, G. D., Scoggins, B. A., Eur. J. Clin. Pharmacol. g, 249

- 254

(1980).

15.

Maguire, K. P., Burrows, G. D., Coghlan, J. P . , Scoggins, B. A., Clin Chem., 22/6, 761 764 (1971).

16.

Witts, D. J., Proceedings of an International Symposium Verdala, Malta, November, 1976, Edited by Jukes, A., Published by CIBA Laboratorie, Horsham, 173 (1977). England, 169

-

-

-

17.

Alkaxay, D., Khemani, L., Volk, J. Analytical Letters,

15(B18), 1493 18.

-

1503 (1982).

Jones, R. B., Luscombe, D . K., Proceedings of an Intefnational Symposium, Verdala, Malta, November, 1976, Edited by Jukes, A., Published by CIBA Laboratories, 143, (1977). Horsham, England, 135

-

19.

Alkalay, D., Wagner, W. E., Carlsen, S., Khemani, L., Volk, J., Bartlett, M. F., LeSher, A., Clin. Pharmacol. 703, (1980). Ther., 11, 5, 693

-

20.

Mathur, G. N., Jones, R. B., Luscombe, D. K., Proceedings of an International Symposium, Verdala, Malta, November, 1976, Edited by A. Jukes, Published by CIBA Laboratories, Horsham, England, 157 - 168 (1977).

21.

Wells, B. G., Gelenberg, A. J., Pharmacotherapy, (11, 2, 121

-

139

1981).

22.

Stimmel, G. L , Drug Intelligence and Clinical PharmaCY, 14,585 - 590 (1980).

23.

Huber, K., CIBA-GEIGY, Personal Communication.

24.

Anderson, W. H., and Stafford, D. T., J. High Resolut. 6, 247-54 (1983). Chromatogr. Chromatogr. Commun. -

25.

Eklund, A., Jonsson, J., and Schuberth, J., J. Anal. Toxicol. 7 , 24-28 (1983).

26.

Dutt, M. C., J. Chromatogr.;

27.

Charette, C., McGilveray, I. J., and Midha, K. K., J. Chromatogr., 224, 128-132 (1981). L

3, 115-24

(1982).

MAPROTILINE HYDROCHLORIDE

425

28.

Cailleux, S., Turcant, A., Premel-Cabic, A., and Allain, P., J. Chromatogr. Sci., 2 , 163-176 (1981).

29.

KErkkainen, S . , and SeppPlE, E., J. Chromatogr.,

221,

319-326 (1980). 30.

Sioufi, A., and Richard, A . , J. Chromatogr.

221,

393398 (1980). 31.

Rovei, V., Sanjuan, M., and Hrdina, P. D., J. Chromatogr., 182, 349-357 (1980).

32.

Conner, J.,. Johnson, G., and Solomon, H., J. Chromatogr. 143, 415421, (1977).

33.

Witts, D., and Turner, P., Br. J. Clin. Pharmacol.,

4,

249-52 (1977). 34.

Geiger, U. P., Rajagopalan, T. G., and Reiss, W., J. Chromatogr. 114, 167-173 (1975).

35.

Meinhart, K., Nikiforov, A., and Vycudilik, W., Arch. 33, 6571 (1974). Toxicol. -

36.

Borga, O., and Garle, M., J. Chromatogr.,

68,

77-88

(1972). 37.

Smith, J. B., and Suh, S., CIBA-GEIGY, Personal Communication.

38.

Flanagan, R., Storey, G., and Bhamra, R., J. Chromatogr., 247, 15-37 (1982).

39.

Hung, C. T., and Taylor, R. B., J. Chromatogr.,

240,

6173 (1982). 40.

Salonen, J., and Scheinin, M., J. Anal. Toxicol.,

1,

175-7 (1983). 41.

KUSS, H. J., and Fleistenauer, E., J. Chromatogr., 204, 349-353 (1981).

42.

Wheals, B. B., J. Chromatogr.,

43.

Hesse, C., proceedings of the 9th Materials Research Symposium issued April, 1979.

44.

Uges, D. R. A., and Bouma, P., Pharmaceutisch Weekblad 1, 41724 (1979). Scientific Edition -

187,65-85

(1980).

426

SOONHEE K. SUH AND JAMES B. SMITH

45. Jindal, S. P., Lutz, T., and Vestergaard, P . , J. Pharm. Sei. 69(6), 6847 (1980). 46.

Alkalay, D., Carlsen, S., Khemani, L., and Bartlett, M. F., Biomed. Mass Spectrom. 6(10), 4358 (1979)

47.

Lapin, A., Eur. J. Mass Spectrom. Biochem., Med. Environ. Res. I,1217 (1980).

48.

Riess, W., Anal. Chim Acta 68, 363-76 (1974).

PENICILLIN G , POTASSIUM

(POTASSIUM BENZYLPENICILLIN) JOEL KIRSCHBAUM 1.

Introduction History Names, Formula and Molecular Weight 1.3 Appearance, Color, Odor and Precautions 1.4 Biosynthesis, Synthesis, and Commercial Production 1.5 Reactions, Stability, Degradation and Polymerization 1.6 Mode of Antibacterial Action 1.7 Non-Microbiological Effects 1.8 Pharmacokinetics and Metabolism 1.1 1.2

2.

Physical Properties 2.01 Physical Properties of Crystalline 2.02 2.03 2.04 2.05 2.06 2.07 2.08 2.09 2.10

3.

Benzylpenicillin, Potassium X-Ray Powder Diffraction Mass Spectrometry Infrared Spectrometry Thermal Analysis Microscopy and Particle Size Surface Area Cohesion (Stickiness) Hydration Polymorphism

Spectrometry in Solution 3.1 3.2 3.3

Nuclear Magnetic Resonance Spectrometry (NMR) Ultraviolet Spectrometry Optical Rotatory Dispersion and Circular Dichroism Spectrometry

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 15

427

Copyright 0 1986 by the American Pharmaceutical Association A11 rights of reproduction in any Corm reserved.

JOEL KIRSCHBAUM

428

4.

Bulk Solution Properties 4.1

Solubilities in Aqueous and Nonaqueous Solvents 4.2 Partition Coefficients 4.3 Ionization 4.4 Molecular Aggregation and Critical Micelle Concentration 5.

Methods of Analysis 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9

Compositional Analysis Titration Colorimetric and Spectrophotometric Methods Chromatographic Methods of Analysis Electrochemical Methods of Analysis Biologically-Based Assays Isotopic Assays Contamination Methods Comparison of Methods

6.

Possible Future Analytical Problems

7.

Acknowledgements

8.

References

PENICILLIN G, POTASSIUM

1.

429

Introduction 1.1

History

In 1928, Alexander Fleming at St. Mary's Hospital in London noticed the partial lysis of colonies of staphylococci on a plate that had been contaminated by PeniciZZium notation. Previous observations of the antagonism of the growth of bacteria by fungi of the genus PeniciZZiwn had been made between 1870 and 1895, however Fleming further found that this fungus, when cultured, gave a "mould broth filtrate" that was relatively nontoxic, possessed selective antibacterial activity and could be used as a local antiseptic. He believed that "the trouble of making it seemed not worthwhile". For the next few years, little new information on the properties and purification of this substance was obtained. As the second world war approached, sulfa drugs were the only known useful way to treat infections chemically. Exploratory research by E.B. Chain, Howard Florey, N.G. Heatley and E.P. Abraham, all at Oxford University, yielded small quantities of impure penicillin. Because of the bombings of Britain, Florey and Heatley went to the United States to obtain aid, after a close escape in Lisbon from being arrested for swimming topless. Work in the United States with deep fermentation (rather than surface growth) and corn steep liquor as a growth promoter, with the efforts of personnel at Squibb, Pfizer, Merck and several other organizations, resulted in the eventual production of sufficient antibiotic for both battle casualties, and, subsequently, civilians. The Germans concentrated on sulfa drugs, although there was sufficient fermentation capacity, considering beer production. Penicillin G was first crystallized at Squibb (as a sodium salt) by MacPhillamy, Wintersteiner and Alicino, where the existance of sulfur in the molecule was first noted by Alicino due to its characteristic odor. The B-lactam structure was supported by R.B. Woodward, especially since many other possible reactive groups could be eliminated by such evidence as potentiometric titrations that indicated the absence of a basic functionality.

JOEL KIRSCHBAUM

430

The structure of benzylpenicillin was established by Dorothy Hodgkin and Barbara Lois in May, 1945, ' using three-dimensional x-ray crystallography. Thus, it was shown that penicillin contained a previously unknown ring system. The B-lactam ring is the source of antibacterial activity. Fleming, Chain and Florey shared the Nobel prize in 1945. Fleming received popular honors for the discovery of penicillin. For example, in the fishing village of Gijon, Spain, the aggrandized photograph of Sir Alexander was paraded about at the yearly fiesta with the caption, "To the Holy Virgin we pray: for us, many Sardines; for the wizard who gave us penicillin, Glory". 1.2 Names, Formula and Molecular Weight Penicillin G Potassium is the United States adopted name (10). The preferred chemical names are (1) 4-Thia-l-azabicyclo[3.2.Olheptane-2carboxylic acid,-3,3-dimethyl-7-0~0-6-[(phenylacetyl)amino]-,monopotassium salt , [ 2S-(2a, Sa, 68)]-; and (2) Monopotassium (2S,5R,6R)-3,3dimethyl-7-oxo-6-(2-phenylacetamido)-4-thia-lazabicyclo[3.2.0]heptane-2-carboxylate. The chemical abstracts systematic number for potassium penicillin G is CAS-113-98-4; CAS-61-33-6 is for penicillin G itself. Below is the structure, C H KN 0 S, with molecular weight of 372.48 dAftA&s f l f ) . Other names include benzylpenicillin potassium,

H

0l6 22 23

i

H I I H aC-C-N-C, H17 15 14

H

PU-9

I / :

c 5

8

potassium benzylpencillinate, benzylpenicillinic acid potassium salt, Cilloral, Cosmopen, Cristapen, Crystapen, Eskacillin, Forpen, Hipercilina, Hydsorb, Hylenta, Liquapen, M-Cillin, Megacillin tablets, Monopen, Notaral, Penisem, Pentid, Scotil, SK-Penicillin G, Sugracillin and Tabilin. Benzyl-

PENICILLIN G, POTASSIUM

431

penicillin has been formulated in tablets (12), ointments (13,14), lotions (15) and intravenous solution (16). It has been added to drinking water (17) and sutures (181, and conjugated to lysine to minimize immune reactions (19). 1.3

Appearance, Color, Odor and Precautions

Potassium benzylpenicillin is a white, freeflowing crystalline powder with a faint pungent odor characteristic of penicillins. The compound itself should not be mixed with acids, alcohol, bases, ephredrine, glycerol, iodine and iodides, naphthalene oils, oxidizing agents, resorcinol, salts of heavy metals, vitamin B 1 and zinc oxide (cf. Section on Stability). The significant adverse reaction to benzylpenicillin is the induction of an allergic reaction as servere as anaphylactic shock leading to death. Numerous studies (20) have shown that high and low molecular weight compounds were responsible (21); including penicillin itself, penicilloyl derivatives (such as penicilloyl-lysine and penicilloyl-albumin), oligomers and degradation products (such as penicilloic acid, benzylpenicilloate, monopenicilloyl amides, Dpenicillamine and D-penicillamine-Lcysteine) (22, 23) which can elicit an immediate reaction in patients allergic to penicillin. As expected, different commerical preparations of benzylpenicillin vary in their ability to evoke allergic reactions (24). 1.4

Biosynthesis, Synthesis, and Commercial Production 1.41 Biosynthesis

Beta-lactam compounds are formed in nature as secondary metabolites of both eukaryotic and prokaryotic organisms. The penicillins, cephalosporins and Ja-methoxycephalosporins are products of biosynthetic pathways with many identical steps (26). The reaction of an activated form of L-a-aminoadipic acid (11, Fig. 1 ) with L-cysteine to form a common precursor

Activated Form of L-u-Aminoadipic Acid II

L-ct-Aminoadipic Acid I

+(L-o-Aminoadipy1)-L-Cysteinyl-D-Valine IV

8-(L-a-Aminoadipy1)-L-Cysteine Y = OH (Activated Form: Y = SR or OR) 111

lsopenicillin N V

F i g u r e 1. B i o s y n t h e s i s of P e n i c i l l i n G.

Penicillin G

PENICILLIN G, POTASSIUM

433

6-(L-a-aminoadipyl)-L-cysteinyl-D-valine (IV, 27). Cyclization proceeds via an enzyme, cyclase, dependent on ferrous ion and ascorbate oxidation to yield isopenicillin N (28). Isopenicillin N is converted to penicillin N through the action of an epimerase which converts the L- to a D- form, leading to both penicillins and cephalosporins (29), Fig. 3. Interconversions between penicillins are possible using enzymes (30) in the reactions shown below: 6-APA is 6-aminopenicillanic acid,

35 SlPenicillin V Penicillin G 6-APA [

+ +

6 5 Y A ~ P 4 V y+ [35S] 6-APA [ Sl6-A A d [ SIPenicillin G +

35 SIPenicillin V+ Penicillin G [

a

Penicillin V+[ 35S] Penicillin G

A chemical synthesis similar to the biosynthetic sequence involving the carbonhydrogen bond-breaking steps of IV at the cysteinyl-6- and valinyl-&positions has been devised (31).

1.42 Synthesis The first successful synthesis of penicillin (25) is shown in Figure 2 and, when modified, can be used for related penicillins. 1.43 Commercial Production Originally, penicillin G was so rare that it was repurified from the urine of patients. Now, with large-scale production (32), the cost is 2 0 ~(U.S.) a day based on a four tablet-a-day regimen and a 100% profit margin. Penicillin G is also used to synthesize other B-lactam antibiotics. Radioayhive penicillin G has been produced from L[1- C]-valine by mycelia of PeniciZZiwn chrysogenwn immobilized in an alginate gel (35).

-

NaOH

Phenoxylacetyl

HCI

carbodiimide

chloride

Na+ C02H

C02H

Figure 2 .

Synthesis of a Penicillin..

Penicillin Salt

PENICILLIN G , POTASSIUM

1.5

435

Reactions, Stability, Degradation and Polymerization 1.51 Reactions, Potassium penicillin G can be converted to the sodium salt using cation exchange resin (36,37) and to other salts (38,39). Antimicrobial activity is lost by hydrolysis of the 8-lactam ring, which is catalyzed by various 8-lqctamases induced or naturally occurring i n V Z V O (40-42); cf. stability section V . i . for studies of hydrolysis i n VitrO. Other reactions of the 8-lactam group include aminolysis (43,44) and amino group conjugation (45,46). Benzylpenicillin has been converted to penicillanic acids (47) and penicillamine $48,491 enzymatically, to 5,5-dimethyl4 -thiazoline-4-carboxylic acid (50) and, via an oxygen bridge at C-6, to a dimer (51). Removal of the acyl side chain, either chemically (52,53) or enzymatically (54-56) leaves an active nucleus, 6-aminopenicillanic acid, that can be reacted with other groups to create various semi-synthetic penicillins. The side chain can undergo various reactions (57-59). The carboxylic acid moiety may readily be converted to esters (60,61). The 8-lactam can be reduced (62) to an amino alcohol and the sulfur oxidized (63-65). Penicillin sulfoxide can be converted to cephalosporins (66) as well as the protected ester (67). To investigate monocyclic 8-lactams, penicillin was desulfurized with Raney nickel (68). Benzyl-penicillin reacts with metals like mercury (11) acetate to form the azetidinone (69) via 1,5-bond scission (70), cf. the stability section above for less lucid metallic effects. A penicillin antigen can be formed by the conversion of benzylpencillin to D-benzyl-

JOEL KIRSCHBAUM

436

penicillenic acid followed by u-ipling with 8-alanine (71). Other artificial antigens were created via coupling with lysine (72), polyacrylamide (73) and diaminocarboxymethylIevan (74). Noncovalent bonding of benzylpenicillin was studied in microorganisms (75), to serum albumin by NMR (76) with the binding sites localized (77), to serum (78) and to tissues (79) as well as bacterial proteins (cf. section on mode of action).

1.52 Stability and Degradation The kinetics and stability of penicillin G have been studied extensively. Even the effect of high concentrations of potassium benzylpenicillin was investigated, since it was found that micellar penicillin G is 2.5 times as stable as the nonmicellar solution under the conditions of constant pH and ionic strength ( 8 0 ) . Effect of pH The stability of a commercial preparation of potassium penicillin G in water, 0.1 M NaOH and 0.1 E HC1 was studied using ultraviolet sgectrophotometry (81). Solvent and Concentration

Wavelength (nm)

Water 2.7 x 10-3M

262 256

Molar Abs.

164 163 163 164 164 251 250 250 251 250

0.1 HCA4 1.4 x 10 M

273

Molar Abs.

1435 1406 1385 1392 1343

0.1 M NaOF 2.9 10- M

263* 257 251, 246

Molar Abs.

x

Time (hr)

0 1 3-5 -2 4 -

160 223 218 211

160 223 217 209

162 222 215 205

164 223 214 203

* Shoulder, Instrument: Perkin-Elmer Model

178 234 219 200

320

PENICILLIN G, POTASSIUM

437

From these data, and especially from the studies discussed below, benzylpenicillin is shown to be most stable at neutral pH. Low temperature also enhances stability. The activation energies were reported to be 17.6, 21.0 and 22.7 kcal/mole at pH 1.2, 4 . 5 4 and 9.57, respectively. Several pathways have recently been proposed for the degradation of benzylpenicillin (I) at low pH. Evidence for scheme 1 is based on kinetics and the existance of penicillamine as demonstrated by HPLC and differential pulse polarography ( 8 2 ) . An alternative route to benzylpenillic acid involves intermediate I1 depicted in scheme 2 ( 8 3 ) . Evidence is based on (a), the increased stability of benzylpenicillins by the incorporation of electron-withdrawing suhstituents in the a-position of the amide side-chain and (b), that benzylpenicillinic acid in ethanol yields benzylpenillic acid in 25% yield. Scheme 3, showing the conversion of benzylpenicilloic acid to benzylpenillic acid via a carbinolamine intermediate (IV), is based on HPLC data ( 8 4 ) showing such a degradation at ,pH 2.5 of benzylpenicilloic acid to benzylpenillic acid. Another alternative scheme (851, not shown here, lacks such a pathway for the formation of benzylpenillic acid from benzylpenicilloic acid, although the kinetics are supported by NMR.

JOEL KIRSCHBAUM

438

Bsnzylpenlclllln

P " y ' )HOpC =\,y

Bsnzylpm;;~

Ph

COpH

H

Bsnzylpinamaldlc Acld

Acld

JYZCOzH Banzylpenllllc Acld

BenzylpenlcIIIolc Acid

Penlclllarnlna + Benzylpenllloildehyde

COzH

+cop

Bsnzylpsnlllolc Acld

8.nzylpenllllo Acld

111

Scheme 2 H

ti+

:0:

ti'

0enzylpenkillolc Acld

'H

J

0~nzylp.nklllln

cozn H

0mqlpenolllc Acld

IV

Scheme 3

Figure 3. Possible Pathways for the Degradation of Benzylpenicillin

PENICILLIN G , POTASSIUM

439

Below are tabulated other selected references describing the stability of benzylpenicillin under various conditions. Stability Condition Bulk (Pure) P e n i c i l l i n

Comments

pH, ionic strength, temp. Study side-chains Acid Study mechanism Acid Study kinetics Kinetics PH Ionic strength, temp. Effect ionic strength Acids, bases, temp. Determine best stability Buffers Effect phosphate buffers Intravenous Solutions Dextrose ti Sucrose, Inactivated In 10 Solutions In 7 Solutions In 4 Solutions In 3 Solutions In 2 Solutions In dextrose + 10 drugs In minibags In dextrose Hyperalimentation Y-Injection Unit Doses

Used HPLC

Reference 86 87 88 89,90 91 92 93

94

Hydroxylamine assay 95 Retained activity 96 Student experiments 97 Study effect time 98,99 and temperature 6-8 hr stability 100 Predict stability 101 Effect temp. 102 Effect pH 103 Effect temp. 104-107 and nutrients Study 108-110 incompatibilities Effect time 111

Other Fomulations Sensitivity discs Topical Suppositories

Effect temperature Metals degrade Effect excipients

112 113 114

440

JOEL KIRSCHBAUM

Temperature Heat Heat Heat Freezing

Study kinetics solids Effect water Predict stability Effect dextrose, NaCl with time Effect mode freezing Effect various thaws Study phase transitions

Freezing Freezing Freez ing

Chemicals

115 116 117 118 119 120 121

(also see Intravenous Solutions)

Alcoho1s Amikacin Ascorbic Acid Broth

Little effect Little effect Little effect High productivity = low stability Broth Production depends on syn. rate and pencillin stability Magnesium Sulfate Depends on pH Metals Penicillin + penicilloic acid Metals In culture medium Preservatives Crystallize Silica Surface acidity causes degradation Sucrose 1:l Molar complex Sucrose Study kinetics pH 6-10 Surfactants Stability depends on ionic type Surfactants Micelles-penicillin 3 1pplex Tobramycin [ 8-methyl- HI [ Clbenzylpenicillin Hydroxyl.amineprevents loss Tromethamine Aminolysis Water Causes degradation

122 123 124

125 126 127 128 129 130 131 132 133 134 135 136 137 138

44 1

PENICILLIN G , POTASSIUM

Radiation Decomposition & dimerization Bacterial inhibited none

Solid State

uv

139,140 141

Th deute ium isotope effect with pH (pH 4-10) was studied (142) and demonstrated a water-catalyzed rearrangement of penicillin G to benzylpenicillenic acid (scheme 1). A microcomputer was used to help predict the stability of penicillin formulations (143). De rada on products of double-labeled [B-methyl- HI [ C]benzylpenicillin were separated (144) using a cation-exchange column (ef. thin-layer and high-performance liquid chromatography section for additional references) after storage under various conditions. Of particular importance in the treatment of infection with penicillin G is the complex interplay possible with certain other drugs (145).

3

f.i

1.53 Polymerization Polymers of such 8-lactam antibiotics as benzylpenicillin elicit a passive cutaneous anaphylactic reaction in sensitized animals. Various methods have been used to clarify the antigenic determinants relative to the allergic reactions evoked by the administration of penicillin G containing polymers and 8-lactam reaction products with proteins. Dextran gels Sephadex G-10 and G-25 (size-exclusion chromatography) was used to separate various reaction products of penicillin G ( 1 4 6 ) . Aged, 25% solutions of penicillin G were chromatographed on G2000SW and G-25 columns (147). Polymer contents increased with duration of storage. These major fractjons were found after 14 days storage in the dark, following chromatography on Sephadex G-25. Two fractions consisted of equimolar phenylacetyl and thiazolidine moities and had C:N:S ratios similar to that of penicillin G. These polymers appeared to be trimers and decamers. The third fraction was mainly N-formylpenicillamine, benzylpenilloic acid, benzy penicilloic acid and benzylpenillic acid, using H NMR and IR spectra, and thin-layer chromatography (148). These appear to be

1

JOEL KIRSCHBAUM

442

products resulting from the opening of the 8-lactam ring, as was previously suggested in a prior study of penicillin polymers (149) using gel exclusion separations and I R , W and NMR techniques. 1.6

Mode of Antibacterial Action

Penicillin enabled the cure of many common, often serious infections such as meningococcal meningitis, pneumococcal pneumonia, group A streptococcal pharyngitis as well as most staphlococcal and many skin infections. Prior to the advent of penicillin, bacterial endocarditis was almost uniformly fatal, with less than a 1%rate of spontaneous healing; however, treatment with penicillin resulted in cures. Unfortunately, resistance to penicillin G emerged rapidly. However, synthetic modifications to the 8-lactam structure preserved many of the therapeutic gains and, in some compounds, produced new advantages. The targets of 8-lactam antibiotics are the enzymes involved in synthesizing the cell wall surrounding most bacteria. The cell wall of grampositive bacteria consists of 5 0 to 100 molecular layers of peptidoglycan, which is a long linear polysaccharide chain of alternating N-acetylglucosamine and N-acetylmuramic acid extending in one direction, and cross-linked by short peptides in a second dimension. These peptidoglycan strands have a dipeptide terminus, acyl-D-alanyl-D-alanine, that may assume a similar conformation to penicillin (150). The peptide bond cleaved during transpeptidation (which leads to incorporation and insolubilization into the peptidoglycan cell wall) would be in the same position as the highly reactive CO-NH bond in the 8-Iactam ring of penicillin. Normally, the transpeptidase is thought to react with its R-Dalanyl-D-alanine substrate to form an acyl-enzyme intermediate as the terminal D-alanine is simultaneously eliminated (151). Cross-links are formed as a free amino group from an adjacent glycan strand reacts with an amino acceptor to yield the transpeptidase product and concurrently regenerate the enzyme. Penicillin inhibits this reaction, presumably by acting as a structural analog of the substrate, via reaction of the 8-lactam with the active site, to form an inactive penicilloyl-enzyme

PENICILLIN G, POTASSIUM

443

(Fig. 4 ) . Inhibition of peptide cross-linking results in the production of linear, soluble, peptide-substituted glycan chains that are not linked to the insoluble cell wall, but instead are excreted as peptideglycan. This often results in the lytic death of the microorganism. (Except when betalactamases enable cell wall synthesis to proceed and thus account for penicillin resistance). Evidence for the peptidoglycan hypothesis is 1) the existance of proteins that covalently bind penicillins ( 1 5 2 ) , 2) the reversal by hydroxylamine of penicillin binding to membranes to give a penicilloylhydroxamate with the concomitant restoration of cell wall biosynthetic activity, and 3 ) the binding of both a penicilloyl moiety and an acyl moiety (derived from substrate) to the same amine acid residue at the active site of sensitive enzymes in stoichiometric quar.tities ( 1 5 3 , 1 5 4 ) . Additional evidence is from the evolutionary relationship between 8-lactamases and penicillin-sensitive enzymes of cell wall biosynthesis, as shown by sequence homology and similar secondary structure. When 4 8 strains of the soil bacterium Rhizobium japonicwn were screened for their responses to several widely used antibiotics, 25% were found to be resistant to penicillin G, tetracycline, neomycin, chloramphenicol and streptomycin. The occurence of multiple drug resistance in a soil bacterium that is not a vertebrate pathogen suggested that the therapeutic use of antibiotics may not be required to develop multiple drug resistance ( 1 5 5 ) . Resistance appears to depend ( 1 5 6 ) on 8-lactamase activity and penicillin-binding protein alterations. Evidence against this theory is the lack of analogy to the dipeptide of portions of both rings of penicillin and the slight activity of some penicillins and cephalosporins with structural fyalogies to acy1-Dalanyi-D-alanine. Solid state N-NMR studies using L- [ E- 5N]lysine and benzylpenicillin indicated that disruption of the mechanism for control of peptidoglycan synthesis probably is a contributing factor in the death of bacteria ( 1 5 7 ) . 1.7 Non-Microbiological Effects In addition to the antimicrobial activity of potassium benzylpenicillin, in rabbits penicillin

\

CT

\

0

a

.... I

rz CT

6 I

0=0

a

IZ I

Z X' "-0 I

0

+

I

:X

I

w

z0

I U

I

N C

w

c

rd .rl

c 0

.d

PENICILLIN G, POTASSIUM

445

administered intramuscularly increased cytochrome C oxidase activity and decreased the concentrations of free SH and -S-S- (158). Red blood cells in healthy children showed higher potassium concentrations after the addition of therapeutic concentrations of benzylpenicillin (159). Sodium excretion in urine was significantly higher (160). A diet containing 4% potassium penicillin G resulted in the inhibition of collagen biosynthesis and cross-linking (161). Suppression of intestinal bacteria by benzylpenicillin resulted in changes in the concentrations of amino acids in tissues (162). Lipid metabolism in microorganisms was also affected by penicillin G (163). Symptoms of Lyme arthritis (spirochete infection-caused) are relieved by penicillin (164). 1.8

Pharmacokinetics and Metabolism

In man, 15-30% of the total oral dose is absorbed, due, to a great extent, to the low stability of penicillin G in stomach acid. Lower percentages are absorbed if benzylpenicillin is Disappearance administered during a meal (165-167). of drug from the gut lumen depends on the intrinsic absorption rate and the non-enzymatic rate of degradation (168). Approximately 60% of the antibiotic is bound to serum proteins. Peak serum concentrations after a 500 mg oral dose (fasting) are 1.5-2.7 pg/mL of total drug and 0.6-1 pg/mL of free drug. This concentration can inhibit streptococci, staphylococci and pneumococci. Infusion of 500 mg/kg/hour gave 16 pg/mL total drug and 5.6 pg/mL free drug (165). For potassium penicillin G , the half-life is 5 min. (Orally active penicillins typically give values of 180 and 1160 min.), with the reaction following pseudo-first order kinetics (169). The effect on bacteria of this rapid decline in concentrations was studied (170). Distribution of injected potassium benzylLower penicillin depends on the dose (171). concentrations are found in cerebrospinal fluid than in serum (172), due to the blood brain barrier. High doses of penicillin provide a minimum inhibitory concentration. Bone shows no detectable penicillin even when serum concentrations are as high as 9 pg/d

JOEL KIRSCHBAUM,

446

(173). Alveolar macrophages also show restricted entry (174). Penicillin is principally excreted in the urine (175), usually in unmetabolized form (10-30%). Biliary excretion also occurs (176,177) but is almost negligible. Biotransformation results mainly in inactive penicilloic acids. Pharmacokinetics were also studied in turkeys (178), horses (179), swine (180), and rabbits (181). A synthetic, oxygen carrying, resuscitation fluid (perfluorocarbon emulsion Fluosol DA 20X) showed no significant difference in pharmacokinetic parameters (182). 2.

Physical Properties 2.01 Single Crystal X-Ray Diffraction The original three-dimensional structure was obtained by Dorothy Crowfoot Hodgkin and coworkers (1831, which served to establish the structure of the B-lactam ring and the location of the sulfur atom. However, due to the large fluctuations in the background intensity of electron density and the abnormal interatomic distances, additional refinements were required to define the benzene ring (184). Fig. 5 depicts the structure obtained using MO-:~ radiation measuring 1875 reflections using 1' min 0-20 scans (185). The bond distances and angles are tabulated in Table 1. Crystal data a e as follows: P2 2 2 U/A' = 1,770.9, D /g cm-5 = 1.359; a = 9.103; (!!;, b =36.342 (3) andmc = 30.015 ( a ) , and 2 = 4. Of greatest interest is that four of the five atoms of the thiazolidine ring tend to be co-planar, the conformation of the thiazolidine ring depends on the local environment and the interior angle of the S atom is 94.7". The potassium ion is co-ordinated by seven oxygen atoms. Biological activity correlates with the conformation of the thiazolidine ring (186). The three-dimensional structure was compared to the spatial characteristics of glycyglycine, a model of the D-alanyl-D-alanine terminus of the precursors of bacterial cell-wall peptidoglycan cross-links (187) and to acyl-D-alanyl-D-alanine (188) itself. Structures were obtained for the related 6-aminopenicillanic acid (189), benzylpenicillin 1 'diethyl carbonate ester (190) and phenoxy- methylanhydropenicillin (191), whose stereochemical structure was related to other penicillins.

447

PENICILLIN G, POTASSIUM

These data may be used to calculate a conformational energy map depicting the conformations possible for the 8-lactam-thiazolidine ring system with respect to the side-chain (192).

Figure 5 . Three-Dimensional, X-Ray Derived, Structure of Potassium Penicillin G. Table 1.

Bond distances and angles

Distances

s ( 1)-c (2) s (1) -c (5) C (2) -C (3)

c (3) -N (4) N(4) -C (5) N (4) -C (7) C (5 1-C (6) C (6) -C (7) C (7) -0 (8) c (2) -c (9) c (2)-C( l o ) C (3)-C (11) c ( 11)-0 (12) C (1 1)-0( 13) C (6) -N( 14) N( 14) -C (15) C ( 15) -0 ( 16) C(15)-C(17) C (17) -C (18) C ( 18)-C (23) C (23) -C (22) c (22) -c (2 1) C(2 1)-C(20) C(20) -C( 19)

(A) 1.84 7 ( 10) 1.818 (9) 1.57(1) 1.46(1) 1.45(1) 1.38(1) 1.56(2) 1.52 1.21 1.52 1.53 1.55 1.23 1.25 1.43 1.34 1.22 1.51 1.45 1.33 1.37 1.28 1.32 1.41

448

JOEL KIRSCHBAUM

Angles (")

Degrees

c (2)-S (1)-c (5) s (1) -c (2) -c (3) c (9)-c (2) -c (10)

95.2(4) 106.2(6) llO(1) 105.2( 7) 113.9 (7) 111.3 (7) 119.4(7) 125(1) 94.1(8) 105.1(6) 119.7 (6) 88.3(7) 84.1(7) 117.3(7) 115(1) 93(1) 131 (1) 137(1) 117(1) 116(1) 127(1) 121.1(1) 117(1) 122(1) 121(1) 118(1) 124(1) 122(1) 128(2) 115(2) 125(2) 118(2) 120(2) 114(1)

C (2) -C (3)-N( 4) C(2)-C(3)-C (1 1) N(4)-C(3)-C(ll) C (3)-N(4)-C (5) C (3) -N(4)-C (7) C (5) -N(4) -C( 7) s(1)-c(5)-c( 7) S (1) -C (5) -C( 6) N(4) -C (5)-C (6) C (5) -C (6)-C( 7) C(5)-C(6)-N(14) C(7)-C (6)-N( 14) N(4) -C (7)-C (6) N (4)-C (7)-0 (8) C (6)-C (7)-0 (8) C (3) -C ( 11) -0 ( 12) C(3)-C(11)-0(13) 0 (12) -C (11)-0 (13) C (6)-N ( 14)-C ( 15) N( 14)-C (15) -C (17) C (17) -C (15) -0( 16) C (14)-C (15)-0 (16) C( 15) -C( 17)-C (18) C (17) -C (18)-C (23) C( 17)-C( 18)-C( 19) C (18)-C (23)-C (22) c (23) -c (22) -c (21) C(20) -C(21)-C(22) c (21) -c (20) -c ( 19) C (20) -C (19)-C (18) C(23)-C (18)-C( 19)

2.02 X-Ray Powder Diffraction To observe x-ray diffraction patterns, a Philips APD 3720 Automated Powder Diffractometer unit emitting CrK radiation at 2.2897"A was used (193). The upper poQtion of Figure 6 shows the powder x-ray diffraction pattern for potassium penicillin G, the lower portion a stick plot of the crystalline peaks, using the same data, where the y-axis (height) represents intensity and the x-axis represents the diffraction angle at the peak maximum in degrees 20. Below are the data for the most intense peaks.

PENICILLIN G, POTASSIUM 1.00 - P E N I C I L L I N G

449

POTASSIUM

- DEG

0.81 . 0.64. 0.49 .

213 [ C R )

->

T

I

N

0.36. 0.25 . 0.16. 0.09 0.04 0.01

1.00 0.81

T E

; I

.

T

A"r

. .

.

0.64. 0.49 . 0.36. 0.25 .

25.0 9 .OO

30.0

PENICILLI N G

35.0

40.0

P O T A S S IU M

45.0

-DEG

20

(CR)->

?.

8.10

I

6.30

N T E N S

5.40

T Y

4.50

I

7.20

I

3.60 2.70 1.80 0.90

0.0

10.0

20.0

36.0

40 .6

Figure 6. Powder X-Ray D i f f r a c t i o n P a t t e r n s ; Upper P o r t i o n s , Conventional P l o t ; Lower P o r t i o n , S t i c k P l o t .

56 .0

JOEL KIRSCHBAUM

450

Peak Angle 8.6250 26.1050 28.7600 44.6600 29.9850 36.1800 28.4400 40.2250 29.7400 19.2250

D Spacing (A) 15.2248 5.0692 4.6098 3.0132 4.4255 3.6870 4.6606 3.3294 4.4611 6.8561

111 Max

(X)

100.0 20.9 10.08 9.74 8.95 8.63 5.54 5.09 4.81 4.39

2.03 Mass Spectrometry Positive and negative mass spectra (Fig. 7) of potassium benzylpenicillin were obtained (194) from a thioglycerol matrix using a 8 KeV Xe fast atom beam. The resulting secondary ions were mass analyzed using a VG-ZAB-2F mass spectrometer. Fig. 7 shows fast atom bombardment MS/MS spectra of the potassiated parent (top) and of the deprotonated molecule (bottom). The suggested fragmentation is shown below, which is general for many penicillins and highly diagnostic.

367'

I I I

C02K

PENICILLIN G, POTASSIUM

45 1

1 1

2 8

2 2

I

.

-

POTASSIUM POSITIVE

POTASSIUM NEGATIVE

Figure 7.

PENICILLIN G FA8

PENICILLIN FAB

Y l

MASS SPECTRUM

MASS

G SPECTRUM

Mass Spectroscopy of Potassium Benzylpenicillin Positive (top) and Negative (bottom) Fast Atom Bombardment Mass Spectra.

JOEL KIRSCHBAUM

452

+

Decarbgxylation from the (M+K) parent accounts for the 367 fragment observed in its MS/MS spectrum (top). The dipotassiated fragment at m/z 270 is analogous to its protonated counterpart (195) as shown below. This intense daughter ion is formed by cleavage of the B-lactam ring with oxygen migrqtion. A similar relationship exists fgr the 236 dipotassiated fragment with its (160 ) protonated analog (196). This daughter ion arises from a reverse 2+2 Diels-Alder cleavage. The remainder is observed in the negative FAB MS/MS spectrum at 174-. Decarboxylation from the (M-H)- parent yields the 327- fragment. Although the 192- daughter ion is the base peak in the mass spectrum, its intensity is small in the MS/MS spectrum (bottom). This fragment was assigned (197) as C H CH2CONHCHCHS, which originates predominantlg 5rom the deprotonated molecule of the free acid rather than from the monopotassium salt. Using electron-impact mass spectrometry, the ion at m/z 334 was found to correspond to the free acid of potassium penicillin G (198). Pyrolysis mass spectrometry was used to characterize some penicillins and cephalosporins (199). Fragments were assigned the following structures: m/e 92, d-CH ; 117, ,&CH2CZN, and 104, 8'-HC=CH2. Benzylpenicihn was reacted with BF3-methanol to yield a derivative amenable to gas-liquid chromatography (see section 5.43). The product was characterized by MS. 2.04 Infrared Spectrometry Fig. 8 shows the infrared spectra of a commercial preparation of potassium benzylpenicillin using mineral oil and potassium bromide (200). The instrument used was a Perkin-Elmer Model 983 ratio recording (dispersive) infrared spectrometer. Below are the interpretations of these spectra (200,201). Absorption (Scm-') 1772 1666 1486 1606

Assignment B-lac tam hide, I hide, I1 Carboxylate as "2-

-

PENICILLIN G , POTASSIUM

453

Mineral O i l Mull

in=e.ee ie

T

3eee

35'ee

me

me

me

ieee

cn-i

see

KBr P e l l e t

I

reee

35ie

3iee

F i g u r e 8.

nie

2eie

me

ieee

cn-i

5ee

I n f r a r e d S p e c t r a of Potassium B e n z y l p e n i c i l l i n .

JOEL KIRSCHBAUM

The results are similar to those found in a The IR study of the IR spectra of penicillins (202). spectra of various derivatives and 6-aminopenicillin were obtained and correlated with structure (203). 2.05 Thermal Analysis A sample of a commercial preparation of potassium penicillin G started melting with decomposition at 218OC (204) using the USP procedure for the melting range of class IB compounds (205). Thermogravimetric analysis (206) of potassium benzylpenicillin, using a heating rate of 20°C/min, showed no loss in weight up to 15OoC.

I 50

I

100

I 150

I

I

I

1

200

250

300

350

TEMPERATURE, OC

Differential thermal analysis of potassium penicillin G gave an exotherm with decomposition at This is 235-240°C, using a heating rate of 15'min. in good agreement with differential scanning calorimetry which also yielded an exotherm with decomposition at 237O-242"C (206). Thermograms of penicillin and stearic acid, alone and mixed together, were used to demonstrate the formation of an unstable mixture (207). Thermal conductivity and Prandtyl numbers (208,209) for water-butanol solutions of benzylpenicillin were calculated.

PENICILLIN G, POTASSIUM

455

2.06 Microscopy and Particle Size A commercial preparation of potassium penicillin was found to contain rectangularly-shaped crystals 1 to 3 pm wide and 4 to 25 pm long (206). There was no visual evidence of polymorphism. Particle size was determined after dispersal in acetonitrile using a Malvern Model 3600E particle size analyzer. The table below gives the percentage of penicillin U S size ranges. G

Microns

-

564 262 262 - 160 160 - 113 113 84 84 65 65 50 50 - 39 39 30 30.3- 23.7 23.7- 18.5 18.5- 14.5 14.5- 11.4 11.4- 9.1 9.1- 7.2 7.2- 5.8

-

Percent Penicillin

1.6 4.4 6.3 7.5 8.8 9.8 10.4 11.3 11.8 8.8 5.9 5.3 3.4 1.9 1.4

2.07 Surface Area A s measured by nitrogen gas adsorption (206), the surfac5 area of one lot of potassium penicillin G was 0.78 m I g . 2 The surface area was determined to be 0.5 m /g €or potassium benzylpenicillin (2102. After grinding in a jet mill it increased to 2.1 m Ig, using gas permeability with a PSKh-4 instrument.

2.08 Cohesion Cohesion (stickiness) was measured by determining the resistance to breakdown of a cylinder of compressed antibiotic powdes (210). Potassium benzylpenicillin was 0.52 g/cm before grinding and 11.7 after grinding in a jet mill. The respective measurement errors are 11%and 20%.

JOEL KIRSCHBAUM

456

2.09 Hydration The crystals are not solvated with water, based on the thermogravimetric and differential thermal analyses previously described. 2.10 Polymorphism There is weak evidence for polymorphism from infrared spectrometry and no evidence from x-ray diffraction studies. 3.0

Spectrometry in Solution 3.1

Nuclear Magnetic Resonance Spectrometry (NMR) 3.11 1H-NMR Figure 9 is the 270 MHz proton NMR spectrum (211) of potassium benzylpenicillin in D20 obtained on a J E O L FX-270 NMR spectrometer using a 5 rnm C/H dual probe at 30". The HDO peak at 4.70 ppm from DSS was used as reference. The interpretation of the spectrum is given below.

Chemical shift (PPM)

Number of Protons Assignment

6 1.47 ( s ) , 1.50 (8) 6 3.47, 3.56(ABq, J=14 Hz) 6 4.22 (s) 6 5.43 (d, J = 4 Hz) 6 5.47 (d, J = 4 Hz) 6 7.2 (m)

6 2 1

CH3's CH N-&I-COO' -

lactam C-H's 5

Aromatic

The results are similar to those found in a study of the identification of penicillins and cephalosporins (212). The nuclear Overhauser effect helped elucidate the three-dimensional conformation. Stereof hemist13 of various derivatives were studied using H and C NMR spectroscopy (213) and with lanthanide-induced shifts (214). Metal complexation depends on pH (215). A study of the 100 MHz proton magnetic resonance spectra of benzylpenicillin in various solvents that cleave non-covalent bonds (216) showed significant shifts, supporting the hypothesis of self-association previously introduced to explain concentration dependences of H NMR spectra (217).

PENICILLIN G, POTASSIUM

457

Penicillin G was found to interact with guanosine using 250 MHz NMR and various solvents (218). Binding to bovine serum albumin was studied by 100 MHz N M R , and the 1 terature summarized (219). Spin-echo 400 MHz H NMR was used to study the metabolism (220). The kinetics of spontaneous first order degradation of benzylpenicillin in aqueous -2 h-l solution to penicilloic acid gave k = 0.7 x 10 (221). The conversion of penicillofc acid to labile seconda$y Efoducts followed first kinetics (k2 = 6 x 10- h ) using computer simulation.

1

3.12 13C-NMR Figure 10 depicts the I3C-NMR spectrum of benzylpencillin in D 0 also using a JEOL FX-270 Fourier transform NMl? spectrometer (211). The reference was p-dioxane at 67.6 ppm from TMS for the assignments tabulated below (cf. reference 222). Chemical Shift (PPM)

Assignment

175.6, 175.1, 174.5

c

= 0's

135.5 130.4, 129.9 128.3 8

74.2

N-?H-COO

67.7

N-CH-CH-S

65.5

\b

59 .O

N -tH-CH-S

43.1

CH2

31.9, 27.6

CH3 ' s

I

/

CH

. C H : -I

I

I

JOEL KIRSCHBAUM

458

I

1:

........................................................

7

.I

5

6

3

.

2

I,,,

,

L 0

I

1 Figure 9. 270 MHz H-NMR spectrum of Potassium Benzylpenicillin in D20. See text for details.

J u I

m no

kD

u(0

Itp

100

eb

1. W

Figure 10. 13C-NMR Spectrum of Potassium Benzylpenicillin. See text for details.

c

*

. . M y I

to

PENICILLIN G , POTASSIUM

459

13C-NMR spectrometry was used to study the structures of a series of penicillins (223), penicillins and cephalosporins (224,225) and penicillin derivatives (226). These papers also summarize prior NMR studies. 3.13 I5N-NMR Although 15N-NMR spectrometry produces sharp resonances, because of it -3 spin of 1/2 its sensitivity is 1.04 x 10 that of the proton. In addition, its lower natura abundance (0.37%) results in a sensitivity 3.8 x 10 that of the proton. To optimize signal strength, a 2.5 M concentration of the soluble methyl ester was used. (Other penicillin and cephalosporin derivatives were also studied.) Two thousand transients were obtained in 1.8 hours to give f5S/N ratio of 10. Natural abundance spectra gave N chemical shifts relative to an ammonium chloride reference of 134.30 ppm for the lactam and 85.13 ppm for the amide ('fZ6). These results are in exact agreement with the N-NMR chemical shifts that were obtained in 1,4-dioxane (224). The potassium salt o f benzylpenicillin gave shifts of 143.7 and 90.7, respectively, in water, and 142.6 and 86.6 in dimethylsulfoxide using 1.2 to 2 second repetition rates over 6 to 16 hours, also using ammonium chloride.

-A

3.2

Ultraviolet Spectrometry

Figure 11 shows the ultraviolet spectrum of a commercial preparation of potassi m benzylpenicillin at a concentration of 2.738 x 10 M in water, obtained with the aid of a Perkin-Elmer Model 320 Spectrophotometer (227). At 262 nm the E value is 164 and at 256 nm, E = 251. Using traditional nomenclature, the E (l%, 1 cm) values are 4.41 and 6.73, respectively. These data are in generally good agreement with previous results (228).

-Y

1

JOEL KIRSCHBAUM

460

240

2g01 290 300

p .

.

-

310 310.

320

-

330

-

Potassium Pen G

H20 H20 00510g/50 ml-Sol A 5 ml Sol A/100 ml-Sol 8 Initial

340 r I

I

n

-1

-

I

I

I

I

I

I

1

1

I

I

I

I

200

250

300

350

400

I

WAVELENGTH, nm

and Figure 12. The optical rotatory dispersion (-) circular dichroism (---) spectra of potassium benzylpencillin in water at pH 6.5. Reprinted here with the permission of the authors and Academic Press.

PENICILLIN G, POTASSIUM

3.3

46 1

Optical Rotatory Dispersion and Circular Dichroism Spectrometry

Figure 12 shows the ORD and CD spectra of potassium benzylpenicillin in water at pH 6.5 ( 2 2 9 ) as reproduced with the permissions of the author and publisher. Two maxima are visible, one at 230 nm and one below 200 nm. ORD is more informative than CD. The B-lactam functionality produces the 230 nm positive Cotton effect, because 6-aminopenicillanic acid exhibits a similar spectrum, and hydrolysis of the B-lactam bond leads to the loss of this absorption (cf. Methods of Analyses, Section 5 ) . The B-lactam (amide) function lacks the ground-state symmetry of the ketone groups. A further complication of the spectra was induced by interaction with the n-electrons of the sulfur atom and lack of x plagarity of the blcyclo ring system. Tge [.0Imax 10- was 2.92 at 243 nm and [OIma x 10- nm was +3.14 at 234 nm in 0 . 1 M citrate guffer, pH 6.5 (229,230). 4.0

Bulk Solution Properties 4.1

Solubilities in Aqueous and Nonaqueous Solvents

Solubilities of a commercial preparation of potassium penicillin G were determined ( 2 3 1 ) at room temperature in various solvents with about one minute of mixing. Solvent

Solubility (mg/mL)

Acetonitrile

0.1

Chloroform

0.2

Dimethylsulfoxide

>loo

Ethanol

10

Hexanes

0.2

Isopropanol

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Table Continued Solvent

Solubility (mg/mL)

Methanol

> 100

Methylene chloride

Methanol-water ( 1 : l )

>loo

0.9 M NaCl

>loo

0.1 M HC1

>loo

Aqueous buffer, pH 2

>loo

Aqueous buffer, pH 4

>loo

Aqueous buffer, pH 7

>loo

Aqueous buffer, pH 10

>loo

0.1 M NaOH

>loo

4.2 Partition Coefficients Apparent partition coefficients P were determined (232) in octanol-water at v%Pous pH values at 37' with the help of spectrophotometry at 260 nm, and are summarized below. The intrinsic partition coefficient for the unionized form, P was U

5.07 5.15

P -aPP 0.37 0.32 0.23

5.47

0.15

E 4.86

1.70 in octanol. In 2-methylpropanol, the intrinsic partition coefficient for the ionized form was -0.30, The salt form effects apparent partition coefficients. Using thin-layer chromatography, partition coefficients were calculated for benzylpenicillin

PENICILLIN G , POTASSIUM

463

distributed in various amounts of acetone in an aqueous, sodium acetate - veronal buffer, pH 7.4 using siliconized silica plates (233). Benzylpenicillin was visualized using an alkaline solution of KMn04. Hansch ZIT values were also obtained. However, the silica gel plates may have exerted a buffering effect on the system. Instead, n-octanol-impregnated microcrystalline cellulose tlc plates were used with a developing solvent of 0.5 M 6-aminohexanoic acid (pH 3.5) saturated with n-cotanol (234). Benzylpenicillin was visualized on wet plates using NH3 vapor, and spraying with 10% acetic acid in acetone followed by starch-iodine solution. Log P, the partition coefficient of the organic acid was calculated to be 1.76. This value agrees well with the calculated log P results of 1.72 obtained using highoctanol performance liquid chromatography with an octadecylsilane column, a mobile phase of 0.035 ammonium chloride-aqueous methanol solution adjusted to an apparent pH of 7.4, and detection at 254 nm (235) for benzylpenicillin partitioned between n-octanol and water. Serum protein binding was found to correlate to partition coefficients of the penicillins (236). Partition coefficients were determined in an organic phase of chloroform and an aqueous phase containing tetrab'utylammonium ion and n-dodecylamine to form ion pairs (237). 4.3

Ionization

Almost all pencillins and cephalosporins are ionized at physiological pH, making knowledge of the predominant species important in the design of suitable 8-lactam antibiptics and their dosage forms. In addition, binding of C-benzylpenicillin to such micro organisms as Staphylococcus aureaus is a function of pH (238). The pK of benzylpenicillin at 25" and in water at a concentration of 0.0099 M was 2.73 2 0.03 and for 0.0093 M was 2.71 f 0.05 using titration with 0. M HC1 (239). Titration with 0.01 N iodine of benzylpenicillin in aqueous solution at 60"

JOEL KIRSCHBAUM

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gave a pK of 2.78 ( 2 4 0 ) . Potentiometric titration was used to determine that the apparent pK value for penicillin G, at 20" and ionic strength 0.15, in 20% methanol was 3.82 and in 30% methanol was 4.10 ( 2 4 1 ) . 4.4

Molecular Aggregation (Self-Association) and Critical Micelle Concentration (CMC)

Potassium penicillin G is capable of selfassociation in aqueous solution via non-covalent bond formation ( 2 4 2 ) . Light-scattering properties of aqueous and 0.15 M and 0.5 M potassium chloride solutions indicated the existance of dimers and trimers at critical micelle concentrations, respectively, of 0.32 and 0.30 mollkg. (The a values of 0.7 imply that most of the monomers constituting the micelle are not in close association withltheir countkions.) These results agree well with H nmr studies ( 2 1 7 ) from 0.01 M to 1 M at 30" that indicated a CMC of 0.275 and 0.251 M, respectively, for the aromatic and methylene protons. The benzylpenicillin ions appear to aggregate in aqueous solution primarily through hydrophobic interactions of the benzyl side chains. A CMC of 0.25 M w a s also found ( 2 4 3 ) using cryoscopic and dye-solubilization methods. Unpublished studies using an analytical ultracentrifuge also showed aggregation ( 2 4 4 ) . Potassium penicillin G also interacts with lipids, such as phosphatidylcholine and lysophosphatidylchloride, as shown by viscosity studies ( 2 4 5 ) . This also indicates that hydrophobic interactions can exist. 5.1

Methods of Analysis 5.1

Compositional Analysis 5 . 1 1 Elemental Analysis

Elemental analysis ( 2 4 6 ) of a commercial preparation of potassium penicillin G gave the following contents, in percent, C, 51.43 ( 5 1 . 5 9 ) ; H, 4.62 ( 4 . 6 0 ) ; N, 7.52 ( 7 . 5 2 ) S , 8.44 The value for ( 8 . 6 1 ) and K, 10.54 ( 1 0 . 5 0 ) . sulfur is in excellent agreement with the previously determined content of 8.47 ( 2 4 7 ) .

PENICILLIN G , POTASSIUM

465

5.12 Water Content

Water content was determined ( 2 4 8 ) by mixing 5 to 200 mg with 1 to 5 mL of dehydrated isopropanol and centrifuging. The supernatent (3 ~ 1 ) was injected into a gas chromatograph. Potassium benzylpenicillin yie ded a value of 0.11% (detection limit 3 x 10 g, linear range 0.20 to 0 . 2 % ) . In another laboratory, automated stop-flow Karl Fischer titrimetry ( 2 4 9 ) gave a value of 0.42% (relative standard deviation 0.034%, n=5). These differences probably reflect differences between samples. When gas chromatography ( 2 5 0 ) was used to analyze residual acetic acid, 0.003% was found (recovery of 9 9 % ) .

-+

5.2

Titration

The analysis of penicillins and their formulations using titration methods has been known and practiced since 1946 ( 2 5 1 ) when Alicino utilized the unreactivity of intact penicillin to iodine, while the hydrolysis product, the penicillinoate, reacts with iodine. Titriant PH PH

Comments

References

252 pH-Stat alkalimetric method Alkalimetric and complexometric 253

25 1 Iodometric Measure iodine consumption before and after hydrolysis 254 Iodometric In broth, uses penicillinase 255,256 Iodometric Automated, broth 25 7 Iodometric Automated, sensitive 258 Iodometric Dosage forms 259 Iodometric Na metaperiodate and arsenite treatment 260 Uses untreated and hydrolysed Bg (11) penicillin Complexes with penicillamine 261,262 Hg (11) 263 Fe (111) Tablets

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Titriant

Comments

References

p-Chloromercuri- After penicillinase cleavage 264 -benzoate 3-Bromo-4,4Brominating agent, comparison 265 dimethyl-2with N-bromosuccinimide oxazolidinone Penicillin Used to determine Ag and Cu (11) 266 using ion specific electrodes 5.3 Colorimetric and Spectrophotometric Methods 5.31 Colorimetric and W Spectrophotometric Analysis Potassium benzylpenicillin has been determined using ultraviolet spectrophotometry virtually from the first production of purified antibiotic (267,268). The technique advanced to the point of using orthogonal polynomials (269) to determine benzylpenicillin in the presence of its degradation products. Penicillin G was also indirectly quantified by W after reaction with thiols (270). Penicillins in deuterium oxide or dimethylsulfoxide solution were quantified on the basis of the 6-lactam band at of the IR ahso -f bance Accuracy was claimed to be 22% about 1760 cm (201) and stability in solution can be monitored. Below is a tabulation of colorimetric assays €or benzylpenicillin. Note that benzylpenicillin has been shown to interfe.rewith the determination of other substances (271,272).

.

Method or Reagent

Comments

Reference

Hydroxylamine Hydroxylamine Hydroxylamine

Automated, compendia1 Bulk drug + broths In broths

273 274 275

Hydroxylamine Hydroxylamine Hydroxylamine

In broths Automated, dissolution Automated, tablets + capsules Automated, liquid formulations

276 277 278

Hydroxylamine

279

PENICILLIN G, POTASSIUM

467

Hydroxylamine Chromotropic acid

Intravenous solutions Estimation, identification Chromotropic acid Characteristic colors H2S04-HCH0 for different penicillins Copper Sulfate, UV In broths Copper Sulfate, UV Various penicillins Copper Acetate, UV Tablets Arsenomolybdate In broths, automated Mercuric Chloride Penicillin + penicilloic acid Mercuric Chloride Also analyze degradants Mercuric Chloride Automated, sensitive Mercuric Chloride

280 281 282,283 284 285 286 287 288 289 290

Mercuric Chloride Starch-Iodine Rhodamine 6

Stabilized assay, sensitive + Other penicillins In sensitivity discs And other compounds

29 2 293 294

Azure R

Via solvent extraction

295

Methylene Blue

Quantify at 6 4 0 nm t Penicillamine

296 297

2-(N-methylamino) phenyl sulfate + Cr (VI) Ferrous Nitrate

Penicillin as reagent

29 1

298-300

5.32 Optical Rotatory Dispersion and Circular Dichroism Methods

Changes in optical activity of benzylpenicillin due to hydrolysis of the B-lactam ring by penicillinase were measured using a Cary 60 recording spectropolarimeter with a Cary Model 6 0 0 1 circular dichroism accessory (301). Since the change in -1 spe ific rotation at 255 nm, [ A a ] 2 5 deglml g -!i dm , is 5053.9 and the precision e%? instrument is 0.22, the assay has the potential of being precise. Actual precision was found to be 0.12, with pH and temperature control. Accuracy was verified using microbiological and iodometric assays (302). The method is limited by the error introduced by mutarotation of penicilloic acids,

02

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5.33 Fluorescence Methods Although penicillin G is only weakly fluorescent after treatment with 0.1 M sodium hydroxide (303), it can be derivatized with 5-dimethylaminonaphthalene1-sulphonylhydrazine. This permits the assay of both the intact penicillin and its biotransformation product, penicilloic acid (304). 5.4 Chromatographic Methods of Analysis 5.41 High-Performance Liquid Chromatography (HPLC) This author prefers chromatographic method6 since similar compounds are usually resolved from each other due to selective interactions of the analyte with the mobile and stationary phases via weak, non-covalent, bonds. HPLC is preferred by many analysts and regulatory agencies because of its specificity, ease of use and high sample capacity. Fig. 13 shows a chromatogram of potassium penicillin G and potassium phenylacetate isolated from a fermentation broth (305). An octadecylsilane column (12% loaded or covered) was used with a mobile phase of 0.1 M phosphate-methanol-acetonitrile (60:40:5) apparent pH 4.15, flowing at 1 mL/min. into an ultraviolet detector set to 220. Penicillins V, F, and K and 6-aminopenicilloic acid do not interfere. Table 2 , below, summarizes HPLC methods. Partition coefficients were determined by HPLC (cf. section 4.2). 5.42 Thin Layer Chromatography Table 3 , below, summarizes thin-layer chromatographic methods for potassium benzylpenicillin arranged in chronological order. Use of a-acceptors as spray reagents for the detection of penicillin was described (359). 5.43 Gas-Liquid Chromatography Table 4, below, summarizes the GLCsystems developed for bulk benzylpencillin.

PENICILLIN G, POTASSIUM

469

Figure 13. Chromatogram of broth sample with detector set at 220 nm. A, potassium phenylacetate; B, potassium penicillin G. See text for further details.

Table 2.

High-Performance Liquid Chromatography of Potassium Benzylpenicillin

Column

Mobile Phase

Detection

Comments

Reference

Bulk o r Pure Material

!A

4

C18

MeOH-0.5% ammonium carbonate (3:7)

254 nm

Resolves Pen. V.

306

C18

MeOH-0.05 M KH2P04 (35:65) + 0.1% tetrabutylammonium bromide, apparent pH 3.35

254 nm

Resolves other penicillins + degradation products

307

C18

M H 0-MeOH (3:7) containing 0.02 2dicyclohexyl-18-crown 6

220 nm

Describes effects of 308 varying mobile phase. Resolves other B-lactams

C18

CH30H-H20

+ PIC Reagent

254 nm

Resolves Pen. V + 309 dibenzylethylenediamine

C18

Acetonitrile-MeOH-0.01 MKH2P04 (19:11: 70)

256 nm

Resolves other penicillins

C18

311 MeOH-H,O (2:l) containing 1mM each Postcolumn Sensitive, also resolves penicillioates Na2H604 and NaH2P04 Rxn

C8

MeOH-0 01 M NaH2P04 (7 :13)

0

.

B-7

225 nm

Resolves other penicillins

310

312

Table 2. Column

HPLC (Continued)

Mobile Phase

Detection

Comments

Reference

C8

Resolves Pen V. MeOH-0.05 M phosphate buffer (53:47) 274 nm apparent pH 3.5-3.3 or 254 nm preservatives

Anion

0.02 M NaN03 in 0.01 M borate, pH 9.15

254 nm

Resolves ampicillin & degradation products

315

C18

1% KH2P04-acetonitrile (4:l) pH 4.15

254 nm

Resolves degradation products

3 16

C18

Acetonitrile-phosphate buffer 0.008 tetrabutylammonium chloride, pH 7.5 (3:7)

254 nm

Resolves deg. pro. Mobile phase optimized

317

C18

Acetonitrile-0.01 M KH2P04 (1:4), pH 4.1

254 nm

Resolves deg. pro. & other penicillins

3 18

Anion

0.03 M citric acid-0.0067 M sodium phosphate buffer, pH 2.7, 37"

254 nm

Resolves deg. pro.

319

Anion

15.4 mL 0.1 M citric acid + 7 mL 0.2 M Na2HP04 diluted to 650 mL, pE 3.8

RI

&

313,314

Stability

+

Study stability in acid 320

Table 2. HPLC (Continued) Column Anion

Mobile Phase

Detection

Comments

+ 0.1 g

254 nm

Resolves deg. pro.

0.68 g KH2P04

NaN03/liter,

Reference 321

pH 5.1

Formu Zations C 18

0.01 M ammonium acetate-acetonitrile 254 nm

Penicillin in infusates 322

( 4 : 1)

C18

0.01 M KH2P04-acetonitrile ( 4 : l )

240 nm

Effect of excipients 323 (dextrose and sucrose adverse)

C18

0.42% KH2P04, 0.6% sodium heptane1-sulfonate, pH 3.17-acetonitrile (71.5 : 28.5)

258 nm

Controlled-release products

324

C18

MeOH-H20-acetic acid (40:60:0.5)

232 nm

Study kinetics decomp. eyedrops

325

220 nm

0.05 ppm in tissues

326

Biological Matrices C18

0.01 M H PO acetonitrile 3 4(4:l to 2:3)

Table 2. HPLC (Continued) Column

Mobile Phase

Detection

Comments

Reference

C18

0.01 M NaH2P04, 0.01 M EDTA325 nm acetonitrile (72:28), pH 6.5 (NaOH)

Derivatize with imidazole-HgC1 2 reagent, broths

327

C18

acetonitrile-pH 7 buffer (23:77)

230 nm

In body fluids

328

C 18

phosphate buffer (ionic strength 0.1)-methanol (6:4) pH optimum

Derivatization

In body fluids

329

C18

Gradient: 90% 0.05 M Na formate, pH 5-102 formate-acetonile (5:3) to 75:25

240 nm

In urine

220

Pen. G in broth & 6-aminopenicillanic acid

330

le

4

w

Misc. and Related Projects

C8

phosphate buffer, pH 7-MeOH (6:4)

220 nm

Anion Cation

0.003 M NaHC03/0.0024 M Na2C03 0.005 M HN03

Conduc- Cation analysis & tivity Pen. G

C 18

50X MeOH in 0.1 M phosphate buffer, pH 4.8; 0.1 MH3P04 to 40% acid in 9 min.

254 nm

331

Sep. procaine, 332 benzathine and Na pen.

Table 2. HPLC (Continued) Column

Mobile Phase

Detection 230 nm

Comments

Reference

Prep. LC of Derivatives 333

Silica

Acetonitrile- CC12H2 (1:199)

C18

15-20% MeOH-0.01 M potassium phosphate, pH 7

C18

Acetonitrile-0.01 M sodium phosphate 325 nm buffer, pH 6.5 (1:4) containing 0.01 M EDTA

Derivatization with imidazole plus metal salts

C18

Acetonitrile-0.05 M KH PO (17:83) adjusted to pH 4.0 (43P64)

Study epimerization of 336 benzylpenicilloic acid

220, 254 nm

254 nm

Sep diastereoisomers of 334 penicilloic acids (allergens) 335

Table 3.

Thin-Layer Chromatography of Potassium Penicillin G

Coating

Developing Solvent

Silica

Org. Phase isoamyl acetate-CH3OHHCO H-H20 (65:20:5:10) or Acegone-acetic acid (95:5)

Visualization

Comments

+

Ref.

For 10 penicillins

337

iodine-azide

Sep. most penicillins

338

Bioautography

Sep. other antibiotics

339

10% Aq. FeC13 (20 mL) 5% aq. potassium ferricyanide (10 mL) + 20% H2S04 (70 mL)

Cellulose 0.1 M NaCl or 0.3 M citric acid MW300 saturated with n-butanol Silica

CHCl :isopropanol-H20 (60:40:4) 3

Sephadex 0.25 M phosphate buffer, pH 6 G-15 containing 0.5 M NaCl Acetone-acetic acid (19:l) or isoamyl acetate-CH OH-HCO H-H20 3 2 (13:4:2:1) (Org.)

UV. then Ref 337 reagent Resolves 340 or Bratton-Marshall degr. products reaction

Polyamide H20-HOAc-isopropanol (50:15:8.5) or H 0-HOAc-isoprOH-MeOH (50: $6 :7.1 :5)

Detects 341 0.5% Br solution or 0.25% Na fluorescein other penicillins

Silica

Table 3. TLC (Continued)

A -4

Coating

Developing Solvent

Silica

n-BuOH-H20-EtOH-AcOH (5:2:1.5:1.5) ~-BU~H-H,O-ACOH(4:1: 1) Acetone-&OH (95:5) 85% aq. acetone

Cellulose BuOH-MeOH-HOAc-H20 (45:30:9:36)

Visualization

Comments

Ref.

First 2 M NaOH, then Sep. and 342 iodine-azide, followed detection of penicillins and by 1% starch cephalosporins and their degradation products. Detects as little as 0.001 pg in feed and food

Bioautography

Q,

Silica

Acetone-HOAc (95:5)

See reagent ref 337

Silica

Acetone-CHC13-HOAc (10:9:1)

W densitometry

230 nm

For degradation 344 products Quantify in 345 syrups and tablets

Silica

Acetone-CHC13-HOAc (10:g:l)

Ferricyanide (ref 337) also degr. products then I2

Silica

n-BuOH-HOAc-H20 (12:3:5)

1 mL 2% PtC14, 0.1 mL 20% KI, 0.1 mL 44: HCl

and 20 mL acetone

343

0.05 pg limit

346 347

Table 3. Coating

TLC (Continued)

Developing Solvent

Visualization

Ref.

Comments

Silica

CHC13-MeOH-H 0 (80:20:2.5) or Citrate buf ger

UV, 254 nm

Resolves many antibiotics

Silica

Plates impregnated with 2% NaAc, adjusted to pH 7.4 barbital. Barbital acetate buffer-acetone (94:6)

Bioautography

as contaminant

Silica

BuOAc-AcOH-MeOH-n-BuOH-phosphate buffer, pH 7.3 (80:4:5:15:20)

Silica

BuOH-MeOH-AcOH-H20(37.5:25:7.5:16)

10% Fe C1 -2% hexaFor various 3 cyanoferratepenicillins HC1 (1:2:6) Chloroplatinate In chicken muscle

Glass Fiber

Si

BuOH-H20-HOAc (4:l:l) or hexaneBuOH-H20-EtOH-HOAc (5:10:4:3:3) or hexane-acetone-H0Ac (1:9:1) acetone-CHC1 HOAc (10:9:1) 3Benzene (caution; replace, if possible, with toluene)-CH OH3 acetone ( 15 :3 :2)

348

-

Sep. various penicillins

Spray of 50% H SO Seperate and followed by 126'C4 identify various or 5% FeCl in 0.5 M HC1 or 0.13 aq. fast green (diazotized), drying, then 0.5 M NaOH spray

349, 350

35 1 352

353

354

Table 3. Coating

TLC (Contined)

Developing Solvent

Visualization

Comments

pef.

Silica

CHC13:MeOH:HOAc (90:8:2) or Bioautography or CHCl :MeOH:H20 (20:4:5) or colorimetry 3 Potassium phosphate, pH 3-washed plates with BuOH-HOAc-H20 (2:l:l) or H 0:Na citrate:citric acid 2 (100:20:5) or MeOH or EtOH or H20

A classification 355 and identification system for 45 antibiotics is given.

Silica

Ethyl acetate

W, 254 nm

Identify 10 penicillins

356

Silica

Acetone-CHC13-HOAc (10:g:l)

10% FeC13-5% K ferricyanide-20% H2S04 (2: 1:7)

Contamination

357

Silica

0.5 M NaCl or acetonitrile-water (4:l) or EtOAc-CH30H-HOAc(20:10:1)

I2 vapor

18 penicillins 358 studied in various normal-and reversedphase mobile phases

or CHC13-EtOH-HOAc(100:50:7.5) or EtOAc-acetone-H 0 (1:2:1) or isoamylacetate-6H3OH-HC02H-H20 (13:4:1:2) (upper layer) or acetoneHOAc (95:5) or EtOAc-acetone-HOAcH 0 (5:2:2:1) or n-BuOH-HOAc-H20 (2 :1 :1) or ~ - B ~ o A ~ - ~ - B ~ o H - H o A ~ 0.066 M phosphate, pH 6.0 (90:9:25:15) or n-BuOAc-n-BuOH-HOAc-O.l% EDTA, diNa in 5% NaH2P04 (10:1:6:2)

Table 3. Coating

Developing Solvent

TLC (Continued) Visualization

Silica Buffers are 2 M NH40Ac adjusted to I2 vapor Silanized pH 5.0 or 6.2 or buffer pH 6.2-MeOH (17:3) or buffer pH 6.2-MeOH-acetonitrile (7:1:2) or buffer pH 6.2-acetone-EtOH(7:2:1) or buffer pH 6.2-acetone-EtOH(6:3:1) or buffer . pH 6.2-MeOH-EtOH(5:4:1) or buffer pH 5acetonitrile (17:3) or buffer pH 5.0ethylene glycol monoethyl ether (4:l) or buffer pH5-acetonitrile-ethylene glycol monoethylether (8:l:l) or buffer pH 5THF(3:l) or buffer pH 5-acetone-ethyleneglycol monoethyl ether (15:3:2) or buffer pH 5-EtOH(3:1) or buffer pH 5-EtOH-ethylene glycol monoethyl ether (15:3:1) or buffer pH 5-acetone (7:3) or buffer pH 5-EtOH-ethylene glycol monoethyl ether (7:1:2) or buffer pH 5-EtOH (13:7) or buffer pH 5-MeOH-acetonitrile (12:3:5) or buffer pH 5-MeOH-EtOH (5:4:1) or 0 . 3 M NaCl in 0.05 M potassium phosphate buffer pH 5.6acetone (2:l) or 0.1 M NaC1-acetone (2:l) or 0.05 M potassium phosphate buffer pH 6.0acetone (4:1)

Comments

Ref.

18' 358 penicillins studied in various normal- and reversedphase mobile phases.

Table 4 .

Gas-Liquid Chromatography of Benzylpenicillin

Column

T(Co1umn)

Detection

Reference

240°C

Strontium, 800V

360

2% OV-17 on Supelcoport He, H2, (80-100 mesh); 66Omm x 4 mm, Air conditioned with HMDS to silylate reactive sites. Penicillins silylated within 10 min. with HMDS in pyridine.

275°C

Flame Ionization

361

3% XE-60 on Gas Chrom Q (80-100 mesh) 2 m x 0.32 cm. Pyrolysis unit 2OoC/ millisec to 875°C.

100°C

Flame Ionization

362

190°C or Flame Ionization 14O"-23O0C at 16" min.

363

0.4% SE-52 on acid-washed, silanized Gas-Chrom I (100-120 mesh) ; 130 cm x 4 mm, Penicillin converted to methyl ester by reaction with diazomethane.

5% FFAF' on Chromosorb W (AW-DMCS, 80-100 mesh), 3 m x 0.32 cm. Penicillin reacted with BF in 3 methanol

Carrier Ar

He

Air,

.

3% OV-1 on Gas-Chrom Q (60-80 mesh), 2.75 m x 0.63 cm glass. Benzylpenicillin was derivatized with BF 3 in methanol.

He

50" to 200°C at 16"/min.

MS

363

PENICILLIN G, POTASSIUM

481

5.44 Paper Chromatography

Details of several methods for resolving potassium benzylpenicillin are to be found in ref. 338 and 364-366. 5.5

Electrochemical Methods of Analysis 5 . 5 1 Electrophoresis

Electrophoresis was used to separate and detect benzylpenicillin mixed with other penicillins in various electrolytes ( 3 6 7 , 3 6 8 ) in animal tissues ( 3 6 9 ) and feedo ( 3 7 0 ) and in food ( 3 7 1 ) . 5.52 Polarography and Related Techniques

Penicillin G was analzyed using potentiometric ( 3 7 2 , 3 7 3 and references therein), polarographic (374-379 and references therein), voltammetric ( 3 8 0 ) and coulometric ( 3 8 1 ) methods of analysis. For other electrochemical methods see the following sections on enzyme electrodes and electrophoresis. 5.53 Enzyme Electrodes and Flow Injection

Penicillin-sensitive electrodes were developed with high selectivity based, in general, on the immobilization of a penicillinase on an electrode. Potassium penicillin G could be quantified in fermentation broths and capsule formulations from 3.5 to 1100 pg/mL, with a precision of +3%, measuring changes in pH as the penicillin was converted to penicilloic acid ( 3 8 2 ) . Changes in pH were measured and shown t o be linear with increasing concentrations of penicillins ( 3 8 3 , 3 8 4 ) in buffered solutions using penicillinase-coated pH electrodes. Care must be taken to allow equilibration to occur to achieve reproducibility and linear s nsitivity to concentrations as low as lo-' M ( 3 8 5 ) . An enzyme electrode sensitive to sodium and potassium ions was used to analyze for potassium benzylpenicillin ( 3 8 6 ) , with generally non-linear results, Using a co-cross-linked penicillinase-albumin layer over a pH-sensitive, field effect transister gave an enzyme electrode generally insensitive to

JOEL KIRSCHBAUM

482

variations in temperature and ambient pH v riation. -8 The sensitivity is approximately 2.5 x 10 international units ( 3 8 7 ) . As many as 150 analyses per hour could be performed using flow injection ( 3 8 8 ) equipment in conjunction with penicillinase immobilized on a glass surface with glutaraldehyde, and pH monitoring. Penicillin G was determined in pharmaceutical samples in the range of 0.05-0.50 mM after dilution. 5.6

Biologically-Based Assays 5.61 Microbiological Assays

The original assays for benzylpenicillin were , predominately microbiological ( 3 8 9 , 3 9 0 ) . More recently, microbiological tests were developed to assay penicillin G in broths ( 3 9 1 ) , bulk material (392-395), formulations ( 3 9 6 - 3 9 8 ) , and tissues and body fluids (399-402). There have been many publications describing assays for residual penicillin G in animal tissue and milk (403-410). The rate of microbial growth was investigated in the presence of various B-lactam antibiotics ( 4 1 1 , 4 1 2 ) , with the resistant organism followin apparent first order regrowths. Inhibition of 44 c02 release by bacteria was studied ( 4 1 3 ) . Technical advances resulted in rapid assays based on light scattering ( 4 1 4 ) and changes in pH ( 4 1 5 , 4 1 6 ) . The factors affecting accuracy were studied ( 4 1 7 - 4 2 3 ) , as was susceptibility ( 4 2 4 - 4 2 8 ) , and the effect of infection ( 4 2 9 ) . Bioassays can be automated ( 4 3 0 , 4 3 1 ) . Unfortunately, space limitations preclude a more complete discussion of this once-essential method. 5.62 Immunoassay

Antibiotic immunoassays ( 4 3 2 ) were applied to potassium penicillin G to analyze bulk penicillin ( 4 3 3 ) , penicilloyl derivatives ( 4 3 4 , 4 3 5 ) and penicillins for allergy producing compounds (436,437). 5.63 Enzyme-Aided Assays (Hydrolysis)

Penicillins in mixtures can be quantified on the

PENICILLIN G, POTASSIUM

483

basis of different rates of hydrolysis at acid pH with the final measurements performed by UV spectrophotometry (438). 8-lactam antibiotics, including potassium benzylpenicillin, could be estimated in biological fluids on the basis of these antibiotics' ability to inactivate the R39 DD-carboxypeptidase (4391, a rather expensive enzyme. Penicillin acylase was used to distinguish penicillin G from ampicillin or cloxacil.lin in bovine urine (440). The rate of reduction of cytochrome c linked to an enzyme system containing benzylpenicillin and B-lactamase from Bacillus cereus was measured at 550 nm to determine activity (441). Differential UV spectrometry of various penicillins in the presence of B-lactamases, a convenient and widely used method, showed that the changes in absorption were linear with time (442). Kinetic properties of the 8-lactamases, like the 1, could Michaelis constant and rate constant (V max- induced also be measured. The rate of 8-lactamase penicillin hydrolysis was also followed by the starch-iodine reaction (443) as well as by iodometric titration (444,445), and acidimetric titration (446). B-lactam-containing antibiotics could be determined within 20 min. using the "Penzym assay" (447). Hydroxylamine assays were once used routinely for B-lactamase studies; cf section 5.31 and reference (448).

5.7

Isotopic Assay

14C-Labeled potassium penicillin G was added t o bone cement, pellets molded and the rate of release into aqueous liquids measured using a scintillation counter (449). Subsequent, in V ~ V Oexperiments involved measuring concentrations of antibiotic in the bone adjacent to the antibiotic-impregnated cement. B-Lactam antibioticslyere screened in milk based on a competition between C-penicillin and betalactam antibiotics for sites on a microbial cell wall that specifically binds B-lactam (450).

JOEL KIRSCHBAUM

484

5.8

Contamination Methods

Assays were developed to examine benzylpenicillin €or contamination, as summarized below. Contaminant

Comment

Reference

Ethylene oxide

In powders & liquids

45 1

Cephalosporins

1-2 uglg

452

Pyrogens

Rabbit test

453

Pyrogens

Limulus (LAL)

454

Particulates

Microscopy

455

Particulates

Scanning EM

456

5.9

Comparison of Methods

Presently, most reliance is placed on HPLC. One company virtually ceased all microbiological testing of B-lactam antibiotics, because of their relatively uncomplicated structures, in favor of chemical testing. However, in the opinion of the author, an HPLC specialist, at least one biological use test is mandatory prior to human use, and safety tests ensure lack of adverse reactions. Various assays involving titration, spectrophotometry with Hg (11) solution and other non-selective methods ( 4 5 7 ) can only give maximum contents. In the secondary literature may be found assays based on iodometry, hydroxylamine colorimetry, sterility, identity, safety, pyrogenicity, moisture, pH, content, crystallinity, heavy metals content and residue on ignition ( 4 5 8 , 4 5 9 ) . 6.

Possible Future Analytical Problems

The rapid excretion and facile inactivation in vivo of henzylpenicillin decreases its usefulness as a therapeutic agent. However, these difficulties can be overcome. The rapid renal excretion (tl of 30 min.) can be obviated by large, frequent or coatinuous intravenous or intramuscular infusion doses. A less wasteful alternative involves formulating potassium penicillin G in waxes or

PENICILLIN G, POTASSIUM

485

oil.-like liposomes (460) from which, after injection, the penicillin is slowly released. [Slightly soluble salts of penicillin G, like benzathine and procaine are varients of this approach (461)l. Renal excretion can be blocked 9 0 % by administering probenecid [(p-(dipropylsulphamoyl) benzoic acid] which inhibits active tubular secretion. Concentrations of both drugs need to be monitored. Inactivation in V ~ V Oof benzylpenicillin is due principally to the hydrolytic action of B-lactamases (462). The B-lactam clavulanic acid, found by the systematic screening of fermentation products of Streptomyces cZavutigerus actinomyces, possesses little antibacterial activity but is a potent inhibitor of some 6-lactamases of plasmid and chromosomal origin. Administration in combination with clavulanic acid lowers the minimum inhibitory concentrations of many penicillins and cephalosporins. This is an alternative t o using synthetic and semi-synthetic @-lactam antimicrobial agents, like methicillin, moxalactam, cefotaxime and the monobactams, which are resistant to hydrolysis by 6-lactamases. The amoxacillin-clavulanic acid combination product is called Augmentin, and the potassium clavulanate-ticarcillin injectable is Timentin. Some other B-lactamase inhibitors currently being investigated ( 4 6 3 , 4 6 4 ) are sulbactam, mecillinam, halopenicillanic acids 6-B-sulfonamidopenicillanic acid sulfones 6-(methoxymethy1ene)penicillanic acid, acetylmethylene penicillanic acid and 6-aminopenicillanic acid (465-467). These suicide or "Kamikaze" substrates lead to a "dead1' 6-lactamase (468) via an acyl enzyme intermediate, Fig. 14. The sulbactam-ampicillin combination product is sultamacillin (469). Such formulations of two ingredients require monitoring of bulk, formulations, and body tissue and fluid concentrations similar to those needed for penicillin itself, at least with respect to pharmacokinetic studies. The recent isolation of biosynthetic genes from Streptomyces through molecular cloning techniques should make possible improvements in fermentation yields of penicillin, and, via gene transfer between organisms, novel antibiotics which are "hybrid" compounds (470). 7.

Acknowledgements

The author gratefully acknowledge the assistance of the many contributors from the Squibb Institute for Medical Research cited as "personal communication". In

p’, 0

‘coo-

I

OH

sulbactam

Acyl Enzyme,_l,

-

0

deacylation and turnover

/

coo -

p+-]

.eNq7 coo -

sop , -

0

malonic semialdehyde

+

coo -

sop H

&

y

t

Ser

A

Ser

“waiting room”

n.L

Figure 14. Inhibition of a B-Lactamase by Sulbactam (Redrawn from Ref. 4 6 8 )

coo -

PENICILLIN G, POTASSIUM

487

many instances, the information was especially obtained for this summary. Special thanks go to Dr. G. Brewer, Dr. B. Kline and S . Perlman for their critical reading of this MS, to Mrs. M. George for several computer-assisted literature searches, to Robert Bragalini for programming, to P. Moebus, and L. Moore for obtaining many of the references, to Ms. Linda J. Boor f o r typing the manuscript, and to Mr. J. Alcantara for drawing the figures. General review articles and books are listed The literature was reviewed to below (Ref. 1-9). August, 1985. 8.

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5.

6.

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"Automation in Microbiology and Immunology, Ed. by C.-G. Hedkn and T. Illkni, John Wiley, 1975, New York, p. 307. 380. U. Forsman, Anal. Chim. Acta, 1 4 6 , 7 1 (1983). 381. K. Kalinowski and F. Czlonkowski, Acta Pol. Pharm., 2 4 , 3 1 (1967); CA, 6 7 , 676325 (1967). 382. L.F. Cullen, J.F. Rusling, A. Schleifer and G.J. Papariello, Anal. Chem., 4 6 , 1955 (1974).

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383. S.O. Enfors and H. Nilsson, Enzyme Microb. TechzoZ., 1 , 260 (1979). 384. S . O . Enfors and N. Molin, Process Biochem., 1 3 , 9 (1978). 385. G.J. Papariello, A.K. Mukherji and C.M. Shearer, Anal. Chem., 45, 790 (1973). 386. C.J. Olliff, R.T. Williams and J.M. Wright, J . Pharm. Pharmacol., Suppl. 30, 45P (1978). 387. S . Caras and J. Janata, Anal. Chem., 5 2 , 1935 (1980). 388. R. Gnanasekaran and H.A. Mottola, Anal. Chem., 57, 1005 (1985). 389. E.P. Abraham and E. Chain, Nature, 196, 837 (1940). 390. J.W. Foster and H. Boyd Woodruff, J . Bacter., 46, 187 (1943). 391. T.P. Verkhovtseva, L.M. Lurie and M.M. Levitov, A n t ib i o t i k a , 15, 323 (1970). 392. D. Greenwood, Chemother., 23, 11 (1977). 393. H. Hariharan and D.A. Barnum, Can. J . C o p . Med., 38, 437 (1974). 394. R.A. Rippere, J . Assoe. Off. Anal. Chem., 6 2 , 951 (1979). 395. A. Kabay, AppZ. Microbiol., 2 2 , 752 (1971). 396. H. Kubin, AutoAnal. Innovationen, Problemloesun Med. Forsch. Ind. Dot. Vortr. Technicon Symp. 7 , 1978, 2 , 133 (1979); CA, 9 3 , 210363s (1980). 397. A. von Graevenitz, M. Heitz, R. Lffthy, J. Meyer and W. Vischer, Schweiz Med. Wschr., 1 1 4 , 1079 (1984). 398. J. Dony, J. Pijck, I. Boudru and A. De Roeck, J . Pharm. Belg., 28, 29 (1973). 399. S.A. Strog and D.A. Preston, A p p l . MicrobioZ., 2 1 , 1002 (1971). 400. J.V. Bennett, J.L. Brodie, E.J. Benner and W.M.M. Kirby, AppZ. MierobioZ., 1 4 , 170 (1966). 401. L.R Peterson, D.N. Gerding, C.E. Fasching and C . Costas-Martinez, Minn. Med., 6 6 , 321 (1983). 402. H.J. Simon and E.J. Yin, A p p z . Microbiol., 1 9 , 573 (1970). 403. R.B. Read, Jr., J.G. Bradshaw, A.A. Swartzentruber and A.R. Brazis, AppZ. MierobioZ., 2 1 , 806 (1971). 404. P. Baldini, G. Frati and G. Pezzani, Ind. Conserve, 4 8 , 135 (1973). 405. H.J. Langner, H. Weiss and U. Teufel, Zentralbl. Veterinaermed., Reiheb, 2 0 , 435 (1973); CA, 80, 58457 (1974). 406. L.A. Ouderkirk, J . Assoc. Off. Anal. Chem., 6 0 , 1116 (1977). 407. I b i d , 6 2 , 985 (1979).

FRn

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Y.S. Nimbkar, B.B. Gaitonde, R.A. Dudani, S.K. Jadav and V.N. Acharya, J . NucZ. Med., 2 1 , 135 ( 1 9 8 0 ) . 414. R.M. Berkman, P.J. Wyatt and D.T. Phillips, Nature, 228, 458 ( 1 9 7 0 ) . 415. T.M. Cogan and P.F. Fox, J . Dairy Res., 3 7 , 165 (1970). 4 1 6 . I.G. Rosen, J. Jacobson and R. Rudderman, A p p l . MicrobioZ., 2 3 , 649 ( 1 9 7 2 ) . 417. F. Kavanagh and H.S. Ragheb., J . Assoc. Off. AnaZ. Chem., 6 2 , 943 ( 1 9 7 9 ) . 418. J. de Louvois, J . Antimicrob. Chemother., 9 , 259 (1982). 419. K.S. Kim, R.N. Yoshimori, D.T. Imagawa and B.F. Anthony, Antimicrob. Agents Chemother., 1 6 , 214 (1979). 420. T.A. Pursiano, M. Misiek, F. Leitner and K.E. Price, Antimicrob. Agents Chemother., 3 , 33 ( 1 9 7 3 ) . 421. S.G. Pathak and D.R. Reese, J . Pharm. S c i . , 5 8 , 505 (1969). 422. S.L. Hansen and P.K. Freedy, J . Clin. MicrobioZ., 2 0 , 494 ( 1 9 8 4 ) . 423. J. Billow and T.J. Speaker, J . Chromatogr., 6 7 , 191 (1972). 424. T.A. Kurzynski, J.W. Yrios, A.G. Helstad and C.R. Field, Antimicrob. Agents Chemother., 10, 727 (1976). 425. T.J. Cleary and D. Maurer, Antimicrob. Agents Chemother., 1 3 , 837 ( 1 9 7 8 ) . 426. D.L. Gibbs and C. Thornsberry, Curr. Microbiol., 2, 239 ( 1 9 7 9 ) . 427. G. Foulds, J.P. Stankewich, D.C. Marshall, M.M. O’Brien, S . L . Hayes, D.J. Weidler and F.G. McMahon, Antimicrob. Agents Chemother., 2 3 , 692 (1983).

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New York. 465. Anonymous, Med. WorZd News, Sept. 10, 1984, p. 10. 466. D.G. Brenner, J . Org. Chem., 5 0 , 18 ( 1 9 8 5 ) . 467. G.I. Drnitrienko, C.R. Copeland, L. Arnold,

M.E. Savard, A.J. Clark and T. Viswanatha, Bioorg. Chem., 1 3 , 34 ( 1 9 8 5 ) . 468. C.T. Walsh, Ann. Rev. Biochem., 5 3 , 493 ( 1 9 8 4 ) . 469. A.R. English, D. Girard and S.L. Haskell, Antimicrob. Agents Chemother., 2 5 , 599 ( 1 9 8 4 ) . 470. D.A. Hopwood, F. Malpartida, H.M. Kieser, H. Ikeda, J. Duncan, I. Fujii, B.A.M. Rudd, H.G. Floss and S . Omura, Nature, 3 1 4 , 642 ( 1 9 8 5 ) .

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PIROXICAM

Mladen Mihalib, Hrvoje Hofman, Josip Kuftinec, Branka Krile, Vesna Caplar, Franjo Kajfe2, N i k o l a Blaievi6

1. Foreword, History, Therapeutic Category 2. Description 2.1. Name, Formula, Molecular Weight 2.2. Appearance, Color, Odor, Taste 3 . Synthe6 is 4. Physical Properties 4.1. Spectra 4.1.1. Infrared 4.1.2. Nuclear Magnetic Resonance 4.1.3. Ultraviolet 4.1.4. Mass 4.2. Solid Properties 4.2.1. Polymorphism 4.2.2. Crystal Properties 4.3. Solution Properties 4.3.1. Solubility 4.3.2. Acidity /pK,/ 4.3.3. Partition Coefficient 4.3.4. Dipole Moment 5. Methods of Analysis -5.1. Elemental 5.2, Chromatographic Methods 5.2.1. Thin Layer 5.2.2. High Performance Liquid 6. Stability Degradation 6.1. Thermal Stability 6.2. Photostability 7. Drug Metabolic Products, Pharrnacokinetics, Bioavailability

-

ANALYTICAL PROFILES OF VOLUME 15

m u e SUBSTANCES 509

Copvnght 0 1986 hy the American Pharmaceutical Association All rights of reproduction in any form reFerved

510

7.1.

MLADEN MIHALIC ET AL.

Pharmacokinetics Metabolism 8, Toxicity 9. Identifieation and Determination in Body Fluids and Tissues lo. Identification and Determination in Pharmaceuticals 11. References

7,2.

PIROXICAM

511

1. Foreword, History, Therapeutic Category Piroxicam belongs to the class of acidic, non-steroid anti-inflammatory (NSAI) drugs. The compound is quite efficient in the treatment of rheumatoid arthritis and other inflammatory disorders in humans /1,2/. The drug is highly potent, has a long half-life of over 30 hours which makes it suitable for once daily dosage /2-4/. It does not have unwanted cardiovascular or central nervous effects /4/. Piroxicam was first developed by Pfizer and Co. about fifteen years ago / 5 / and in seventies it entered into medicinal praxis. Piroxicam is used as an effective analgesic and anti-inflammatory agent in rheumatoid arthritis, osteoarthritis, ankylosing spondylitis and acute pain in musculoskeletal disorders and acute gout / 6 / . It has been shown to be an effective analgesic in fracture, dental, postoperative and postpartum pain. 2. Description 2.1. Name, Formula, Molecular Weight Piroxicam is 4-hydroxy-2-methyl-N-(2-pyridyl)-2H-1,2-benzothiazine-3-carboxamide-l,l-dioxide. There are two possible tautomeric forms:

H

-

2

C

H N O S 15 13 3 4

‘CH3 Mol

. wt . 331.36

2.2. Appearance, Color, Odor, Taste Piroxicam is an odorless, colorless crystalline povder of a bitter taste.

3. Synthesis The most important synthetic pathways to piroxicam are outlined in Scheme 1. The first one /5, 7 - 9 / starts with sodium saccharin which is alkylated at nitrogen using chloroacetic acid esters /lo/. 3-Acyl-2H-1,2-benzothiazin-4(3H)-one-l, 1-dioxide is prepared by the base catalysed

SCHEME 1.

9 W N N a + ClCH&OOR 02

SNHCH2C OOR C HzC 0OH

d

02

A 2eq.RONa

ROH

02

PIROXICAM

513

r e a r r a n g e m e n t of t h e 5-membered i n t e r m e d i a t e c o m p r i s i n g a l k o h o l y s i s followed by a Dieckmann r i n g c l o s u r e /11-13/. N-Methyl d e r i v a t i v e i s r e a d i l y o b t a i n e d by r e a c t i o n w i t h methyl i o d i d e /11, 14/. The p r o d u c t i s t h e n t r e a t e d w i t h a p p r o p r i a t e amine i n r e f l u x i n g x y l e n e t o o b t a i n t h e c o r r e s p o n d i n g carboxamide.

4. P h y s i c a l P r o p e r t i e s k h ? % % r e d Piroxicam e x i s t s i n two d i f f e r e n t i n t e r c o n v e r t i b l e c r y s t a l polymorphs. T h e i r i n f r a r e d s p e c t r a d i f f e r o n l y s l i g h t l y i n f i n g e r p r i n t r e g i o n , b u t t t e band o f -NH and -OH s t r e i c h i n g which l i e s a t 3385 cm- i n n e e d l e form and 3330 cm- i n c u b i c form is s i g n i f i c a n t /l5/. The o t h e r c h a r a c t e r i s t i c bands /Fig.l./ may be a t t r i b u i e d t o t h e f o l l o w i n g group v i b r a t i o n s : 1635,or 1625 cm- ( s t r e t c h i n g ( s t r e t c h i n g o f t h e seof t h e amide c a r b o n y l ) , 1525 cm cond ami e b a n d ) , 1440 cm- ( 3 a s . - C H Af-C=Cstretching), 1322 cm-'( 3 sym.-CH ), 1155 and 10703'c y o r 1050-1070

-

-

cm ( -SO 2- N - ) , 770 3and 730 o r 740 cm- ( o r t h o - d i s u b s t i t u t e d p h e n y l ) . The s p e c t r a a r e t a k e n i n K B r d i s c s a t Pye-Unicam SP3-200 i n f r a r e d s p e c t r o p h o t o m e t e r . 4.1.2.

Nuclear Magnetic Resonance P r o t o n magnetic r e s o n a n c e spectrum was r e c o r d e d i n C D C l s o l u t i o n by Bruker WP8oPS s p e c t r o m e t e r . It i s char a c t z r i z e d by one s h a r p s i n g l e t a t 2.93 ppm ( 6 s c a l e ) f o r t h e N-methyl group, two broad s i n g l e t s , one a t 9.00 ppm (amide h y d r o g e n ) , and a n o t h e r a t 13.20 ppm ( h y d r o x y g r o u p ) . Both hydrogens a r e exchangeable by a d d i t i o n o f D20. A m u l t i p l e t a t 6.93-8.50 ppm b e l o n g s t o a r o m a t i c prot o n s and c o r r e s p o n d s t o f o u r p r o t o n s from t h e benzene, and f f y r p r o t o n s from t h e p y r i d i n e r i n g , s e e F i g . 2 /15/. C-Nuclear magnetic spectrum was r e c o r d e d i n C D C l s o l u t i o n by J e o l FX-loo s p e c t r o m e t e r . It i s r a t h e r com- 3 p l e x and d i f f i c u l t t o a s s i g n p r o p e r l y . The s i g n a l s of t h e b e n z o t h i a z i n e moiety i n t e r f e r e w i t h t h o s e from t h e p y r i d i n e r i n g ( F i g . 3 ) . The v a l u e s f o r chemical s h i f t s a r e p r e s e n t e d i n Table 1. Note t h a t a s s i . g n a t i o n s of s i g n a l s t o C-5 and C-6 might be i n v e r t e d /15/.

4.1.3.

Ultraviolet The u l t r a v i o l e t s p e c t r a o f piroxicam i n 0.1 M H C 1 and methanol r e c o r d e d by SP8-loo U V / V I S Pye Unicam s p e c t r o photometer a r e shown i n Fig. 4 /l5/. These s p e c t r a a r e c h a r a c t e r i z e d by two maxima i n 0.1 M H C 1 : a t 242 nm ( & = = W o o ) and 339 nm ( € = 2 2 5 0 0 ) , and t h r e e i n methanol: a t

-

I

4

LOO0

3500

3000

2500

2000

1800

1600

1400

1200

1000

F i g . 1. Infrared s p e c t r a o f piroxicam i n K B r p e l l e t s , n e e d l e form /A/ /B/. Instrument: Pve-Unicam sP3-200.

800

-1

cm

600

and cubic form

PIROXICAM

I

14

515

I

12

f

10

I

8

1

6

I

4

2

PPmO

Fig. 2. '€I-Nuclear magnetic resonance spectrum of piroxicam in CDCl Instrument: Bruker WP 80PS at 80 Mz. 3*

I

I

I

200

I

180

I

I

160

I

t

140

I

I

120

I

1

100

1

I

80

I

I

I

60

LO

2o

Fig. 3. 13C-Nuclear magnetic resonance spectrum of piroxicam in CDCl 3. Instrument: Jeol FX-loo at 25.05 MHz.

PPm 0

PIROXICAM

517

1.0.

- 0.1M HCI

A

---- MeOH

0.8 I I I

I I

I I

I

I

0.6

I

I I I

I

I I I I

I

I

I I

0.4

0.2

Fig. 4. Ultraviolet spectra of piroxicam. Instrument: Pye-Unicam sPS-loo.

MLADEN M I H A L I ~ET AL.

518

T a b l e 1. f 2 e v a l u e s f o r chemical s h i f t s of piroxicam i n C-NMR spectrum i n CDCl

3

OH 0

3 C-atom

Chemical s h i f t (ppm)

c- 1 c- 2 c- 3 c- 4 c- 5 c- 7 C- 8 c- .9

117 7 159.3 128.8 126.6 132.9 133-3 124.2 135.2 167 5

c-lo

150-5

C- 6

c-11

c-12

c-13 C-14 c-15

115.8 13% 0 120.4 147.2 39.2

Multiplicity

s S

6

d d

d d 6

6 6

d d

d d 9

256 nm (~=12700), 290 nm ( f = l o o o o ) and 358 nm (~=14600). The second maximum i n 0.1 M H C 1 i s s u i t a b l e f o r s p e c t r o photometric d e t e r m i n a t i o n of piroxicarn i n p h a r m a c e u t i c a l dosage forms. Mass The f r a g m e n t a t i o n was s t u d i e d by K r a t o s M-25 s p e c t r o m e t e r l i n k e d t o d a t a system DS+5oS, and i t i s shown i n Fig. 5 /15/. A m o l e c u l a r i o n M ~ 3 3 1i s p r e s e n t w i t h 39% i n t e n s i t y r e l a t i v e to t h e b a s i c i o n m/e=l73. The pathway of f r a g m e n t a t i o n i s shown i n Scheme 2. 4.1.4.

Solid Properties Polymorphism Piroxicam e x i s t s i n two d i f f e r e n t i n t e r c o n v e r t i b l e c r y s t a l polymorphs w & t h m e l t i n g p o i n t s of 196-198"C /needl e form/ and 199-201 C / c u b i c form/. The two forms a r e 4.2.

4.2.1.

519

PIROXICAM

100 73

120

C.....

80 117. 78

60

11

s

21

% CI

331

'3i L O c

2 7

Q,

CI

.-c !

d

20

0

100 Fig.

5. Mass spectrum of piroxicam at 2300 C. Instrument: Kratos M-25.

SCHEME 2.

H

-121

1

1-94

OH

m / e 173

+

I

m / e 117

1m / e 146

CH3]

m / e 104

m/e 76

521

PIROXICAM

distinguished by infrared absorption and, even more, by X-ray powder diffraction (refer to the corresponding chapters). There are no data about a possible different activity of the particular form.

4.2.2. Crystal Properties When allowed to crystallize from an ethanolic solution by fast cooling, piroxicam precipitates as needles, while by slow cooling from the same solution precipitates in cubic form. By crystallization from an aqueous ethanol or aqueous acetone piroxicam crystallizes with one Bolecule of water which disappears at approximately 120 C and the melting point does not change. The measurements of a sample obtained by grinding the crystals of piroxicam at room temperature were taken by a General Electric XRD-6 spectrogoniometer. The value of 2 0 , interplanar distances d=nh/2 sin@, and relative intensities I/Io based on highest intensity of loo, for cubic and needle form of piroxicam, respectively, are given in Tables 2 and 3. The data are obtained with a rate meter T.C. 0.5, lo 0 0 0 cps. The corresponding spectra of the X-ray diffraction pattern are presented on Figs. 6 and 7. The space group of piroxicam (monoclinic, P2 /c) is determined using Weissenberg photographs taken wigh CuK& radiation /16/. The cell dimensions were defined from diffractometer measurement~:~a=7.127(2), b=15.136(7), c = =&3.949(6) 8, 4~97.3 (4) , Z=4, Vdt91.15 8 , Dd.481 mg m , MoKu//h.=o.7107 , &=0.244 mm- /; final h0.050 for 2289 observed reflexions [ I 7 2 C?‘( I)]. The piroxicam moleTable 2. X-Ray characteristics of cubic form of piroxicam

11.74

24

12.30

37 87 19 46

14.62 16.25 16.70 17.70 2i.0 0

22.56 26.72 27.40 27.92 34.26

loo 21 20

72 99 34 19

7 5387 7 1967 6 0595 5 4552 5.2041 4.9091 4.0773 3.9416 3.3367 3.2554 3.1959 2.6176

MLADEN MIHALIC ET AL.

522

l

25

.

.

.

.

,

.

l

,

.

,

15

20

,

,

,

.

,

10

,

,

,

.

,

5'

l

e

Fig. 6. X-Ray diffraction pattern of piroxicam cubic form. Instrument: General Electric XRD-6 spec trogoniom8t er

.

20

15

10

5' 0

Fig. 7. X-Ray diffraction pattern of piroxicam needle form. Instrument: General Electric XRD-6 spectrogoniometer.

PIROXICAM

523

Table 3. X-Ray characteristics of needle form of piroxicam 2 0"

9.18 10.60 15-78

I/Io

loo

18.16 19.64

17

20.20 21.90

lo

15

9

25.22

24 34

26.90 30.24

8

22.85

9.6345 8.3468 5.6166 4.8855 4.5206 4.3965 4.0443 3.8923 3 5316 3*3051 2.3539

23 7

0

cule is not far from being planar (-bc plane). The thiazine ring exhibits a half-chair conformation. An amide group is involved in an intramolecular hydrogen bond with the hydroxy group. It also forms an intermolecular hydrogen bond with the oxygen atom bonded with the sulphur atom, connecting piroxicam molecules in an infinite chain along axis. The molecular packing is also influenced by van der Waals interaction. Piroxicam monohydrate, unlike the piroxicam structure, exists in a zwitterionic form /17/, the enolic hydrogen having been transferred to the pyridine nitrogen. Two intramolecular hydrogen bonds are formed by an internal rotation of the neutral structure (between enolate oxygen and hydrogen on amide nitrogen, and between carbonyl oxygen and the hydrogen on pyridine nitrogen). The side chain and the atoms in the thiazine ring are planar. The crystals are prismatic (space group P1) with following cell dimensions: b=12.909( 4) c=10.481( 3) 2 d = =99.31( 2), f'=102.64(2j0, V-1 37.9(7)8 , -1 Z=4, taken with CuKa radiation, hd.5410 /lc=21.3 cm

;:$78';1:\:

z,

/17/*

4.3. Solution Properties 4.3.1. Solubility Piroxicam is not soluble in water and cyclohexane, sparingly soluble in diisopropyl ether and in toluene, and only slightly more soluble in lower aliphatic alcoIt is soluble hols methanol, ethanol and isopropanol in some polar organic solvents such as dimethylformamide (1 g/lo dimethylsulphoxide ( 1 g/lo n i l ) , chloroform (1 g/20 ml , and somewhat less soluble in dioxane ( 1 g/

mil,

.

MLADEN MIHALIC ET AL.

524

/40 ml), acetone (1 g / 5 0 ml) and ethyl acetate (1 g/80 m l ) /15/*

1

Acidity (PK Potentiometrit titration of piroxicam solution in a mixture of dioxane and water (2:l) gave a pK value of 6.3, effected by the enolic hydroxyl group a? C-4 /4,7, 18/. 4.3.2.

4.3.3. Partition Coefficient Piroxicam has a partition coefficient of 1.8 between n-octanol and aqueous buffer pH=7.4 /4/.

4.3.4. Dipole Moment The dipole moment+of giroxicam was determined in dioxane solution at 20.0-0.1 C, using a Dipolmeter DM 01 ( Wiss.-Techn. Werkstatten, D 812 Weilheim). The value found was 3.6820.06 D /15/.

5 . Methods of Analysis 5.1. Elemental Analysis Piroxicam C H N 0 S (331.36) calc.: C 54.37 15 13 3 4 H 3.96 N 12.68 0 19.31 s 9.68 100.00

% % % %

% %

3 . 2 . Chromatographic Methods

3.2.1. Thin Laser The identity and purity of piroxicam may be checked and by the use of 0.2 mm thick Fertigplatte Merck F 254 with corsuitable solvents or solvent mixtures, which are responding Rf values of piroxicam listed in Table 4 /15/. Table 4. TLC

characteristic6 of piroxicam

Solvent chloroform acetone-cyclohexane dichloromethane-ethanol 20:l chloroform-ethanol 1o:l acetone

Rf 0.1 0.56

0.58 0.64 0.68

High Performance Liquid The chromatogram was made by a Pye-Unicam LC-3-XP chromatograph with UV-LC-3 detector. The column used was

5.2.2.

03-

b-

N-

3O-.

I

d

526

MLADEN

MIHALICET AL.

Whatman-Partisil PXS l0/25 PAC. The applied mobile phase was acetonitrile-water-acetic acid 25-75-5 ml, 1.2 ml/ /min The sample of piroxicam was dissolved in 0.1 N NaOH in concentration of 1.0 mg/ml and gives one peak on HPL chromatogram /19,20/, Fig. 8.

.

6, Stability - Degradation 6-1. Thermal Stability The sample of pirox'cam was gept in a brown powder-glass in the dark at 20bC and 40 C. After two years no change in color, smell, taste and shape of crystals could be observed in samples kept at either temperature. TL and HPL chromatograms did not show degradation products. Contents in piroxicam obtained by analytical determinations at various times of exposure are shown in Table 5. /15/. Table 5. Content of piroxicam kept at two temperatures at various times of exposure Content of piroxicam inosample % Time months 2ooc 40 C 0

99.9

3

loo. 0 loo. 1

6 9

98.8

12

loo. 1

24

99.8

100.1

99.9 99.8 99-6 99.7 99.5

6-2. Photostability Samples of piroxicam 0.5 g were filled into colorigradiated for 72 hr with less 50 m l bottles and we:e light of 300-830 nm at 30-0.5 C. At 24 hr intervals, some of the samples were examined for changes in appearance, smell and taste. Piroxicam contents were determined by HPLC /15/. The results are presented in Table 6. Table 6. Photostability of piroxicam after irradiation Time hours 0

24 48 72

Content of piroxicam

%

99.8 99.7 99.6 99-6

7. Drug Metabolic Products, Pharmacokinetics, Bioavailabili ty Piroxicam's pharmacological and pharmacokinetic profile'has been rationalised in terms of chemical groupings

L

I

t

'0

ZI I"

Q 0

0 c

\ /

L .-Q

I

0

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-

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"8: 0

528

MLADEN

MIHALICET AL.

of the molecule. The enolic pdrtion has a relatively low pKa which leads to virtually complete ionizations and prolongs the activity of the drug. The group is also necessary for prostaglandin biosynthetase inhibition, the proposed mode of action of piroxicam. The sulphoxide group is lipophilic, enhancing absorption and facilitating the passage of piroxicam across the gut-blood barrier. The heterocyclic side-chain increases acidity and lipophilicity, it slows hydroxylation and therefore prolongs the half-life. The N-methyl group increases efficacy by a greater inhibition of PG synthetase than that provided by a larger chain.

7.1.

Pharmacokinetics Piroxicam is readily ab6orbed after oral or rectal administration and accumulates after repeated doses to reach steady-state after about 7 days. The drug is extensively metabolised to apparently inactive metabolites and has a half-life of about 40 hours in man. Peak plasma concentrations are attained about 2 hours after a single oral dose and at about 5.5 hours after rectal administration as suppositories /2U. Due to the extended plasma half-life of piroxicam, plasma concentrations remain very stable over the next 24-48 hr. Mean peak plasma concentrations are roughly related t o dosage betand 13.5,bg/ml after ween lo and loo mg, being 0.85,l,i,g/ml a single lo or loo mg dose, respectively /22/. At concentrations of between 5 and 3o&g/ml, piroxicam is 99.3% bound to plasma proteins /21,22/. Piroxicam penetrates into the synovial fluid of patients with rheumatoid arthritis and attains concentrations of about 40% of those in plasma. Piroxicam is highly protein-bound and thus might be expected to displace other protein-bound drugs. Its absorption and disposition are unaffected by concomitant administration of aspirin and antacids.

7.2. Metabolism Available data sumest that piroxicam is extensively metabolised in man /237-by the routes of 5' -pyridine ring hydroxylation, glucuronide formation, cyclodehydration a n d amide hydrolysis leading to decarboxylation, ring contraction and N-dealkylation /24/. The metabolic pathways recognized for piroxicam are shown in Scheme 3. The principal metabolite in man is that produced by hydroxylation of the pyridyl ring (metabolite B ) and exists either free or conjugated with glucuronic acid. The metabolite is at least 1000 times less active than piroxicam in inhibiting

PIROXICAM

529

prostaglandin synthetase /25/. About 10% of a single oral dose of piroxicam is recovered unchanged in the urine within the first 8 days after administration. The elimination of half-life of piroxicam is extended due to a low clearance rate and has most often been calculated at 38 hours range 31 to 56.7 hours in healthy subjects /21-26/. Half-life appears not to be related to dose or plasma concentration. The formation of the cyclodehydrated metabolite ( A ) is a major pathway in the dog, while the rat seems to hydroxylate the pyridyl ring in the para-position to form metabolite B. The latter compound, either free or conjugated with glucuronic acid, is the major human metabolite, accounting for up to 60% of a daily dose in urine and feces. Laboratory animals also deamidate, decarboxylate, and cause ring contraction, all in varying amounts. These latter processes seem to be of minor importance in man

/n/.

None of the metabolites of piroxicam have significant antiinflammatory activity in laboratory animals / 2 8 / . The lack of activity for the metabolites, together with their very low concentration in plasma [at or below the limits of analytical detection), leads to the hypothesis that piroxicam is intrinsically active, and its metabolites do not contribute to that activity. Apparently, piroxicam embodies a unique combination of functional groups which are all required for its action.

8. Toxicity Acute toxicity of piroxicam is low: the LD for orally applied piroxicam is 360 mg/kg in the mo?ge, 270 mg/kg in the rat and over 700 mg/kg in the dog. When administered intraperitoneally, the LD values are 360 and 220 mg/kg in the mouse and rat, respzgtively / 6 , 2 9 / . 9. Identifioation and Determination in Body Fluids and Tissues Piroxicam in plasma was assayed spectrophotometrically at 355 nm. The sensitivity limit of-the assay was approxirnFtely 0.5/Ccg/ml /22/. As well, piroxicam in plasma was assayed fluorometricakly after hydrolysis with 6N H SO4 for 20 hours at lo5 C. The released 2-amino-pyridine wis examined in an Xminco Bowman spectrofluorimeter with excitation at 310 nm and measurement at 380 nm. A Gilford Model 410 digital absorbance meter and a Gilford Model 4006 data lister were interfaced with the spectrofluorimeter for recording of the fluorescence values. The assay was linear and sensitive down to 0 . 5 /ccg/ml. High concen-

MLADEN MIHALICET AL.

530

t r a t i o n s of s a l i c y l a t e were found n o t t o i n t e r f e r e i n t h e a s s a y of piroxicam /22/.

l o . I d e n t i f i c a t i o n and D e t e r m i n a t i o n i n P h a r m a c e u t i c a l 6 Piroxicam i n c a p s u l e s was determined s p e c t r o p h o t o m e t r i c a l l y by measuring t h e e x t i n c t i o n of t h e s o l u t i o n a t 340 nm, 1 mg/loo m l , a g a i n s t R s u i t a b l e b l a n k , and t h e c o n t e n t of piroxicam i n 1 c a p s u l e was c a l c u l a t e d /15/. Acknowledgements. The a u t h o r s a r e t h a n k f u l t o d r . A. Nagl, L a b o r a t o r y of General and I n o r g a n i c Chemistry, F a c u l t y of S c i e n c e , U n i v e r s i t y of Zagreb, f o r X-ray d i f f r a c t i o n ~ p e c t r a l d a t a , and t 9 d r . Z. Meib, ”Ruder Bogkovib” I n s t i t u t e , Zagreb, f o r C n u c l e a r magnetic resonance d a t a . 11. References 1. N.E. P i t t s and R.R. P r o c t o r , i n W.M. O’Brien and E.H. Wiseman / E d i t o r s / flPiroxicamlf Academic P r e s s , London 1978, P 97-108. 2. C.M. Williamson, Curr. Med. Res. Opin. 8, 622 /l9U3/. 3. E.H. Wiseman, D.C. Hobbs, Am. J. Med. 72, 9 /1982/. 4. E.H. Wiseman, Y.-H. Chang, J.G. Lombardino, Arzneim.-Forsch. /Drug Res./ 26, 1300 /1976/. 5. P f i z e r and Co., Ger. Offen. 1,943,265 /1969/, C.A. 2, 1 2 0 6 4 7 ~/1970/. 6. R.N. Brogden, R.C. Heel, T.M. S p e i g h t , G . S . Avery, Drugs 22, 165 /1981/. 7. J . G . Lombardino, E.H. Wiseman, W.M. McLamore, J. Med. Chem. 14, 1171 /1971/. 8. J.G. L G b a r d i n o P f i z e r Inc. U.S. Pat. 4,289,879 /1981/, C.A. 96, 20llow /1982/. 9. H, Zinnes, N . T Lindo, J.C. S i r c a r , M.L. Schwartz, J. Shavel, Jr., G. D i Pasquale, J. Med. Chem. l6, 44

/1973/ l o . H. Zinnes, R.A. Shavel, J. Org. 11. C.B. S c h a p i r a , Chem. 17,1 2 8 1 12. H. Zinnes, N.A. U.S. P a t . Co.

Comes, F.R. Z u l e s k i , A.N. Caro, J. Chem. 2,2241 /1965/. I . A . P e r i l l o , S. Lamdan, J. H e t e r o c y c l .

/1980/. Lindo, J. Shavel, Jr. 4,074,048 /1978/, C.A.

Warmer-Lambert

88, 190868b

/1976/. 13. I.A. P e r i l l o , C.B. S c h a p i r a , S. Lamdan, J. H e t e r o c y c l . Chem. 20, 155 /1983/. 14. H.L. Rice, G.R. P e t t i t , J. Amer. Chem. SOC. 76, 302 /I 954/ 15. M. Mihalib, H. Hofman, F. KajfeB, J. K u f t i n e c , N. B l a i e v i b , M. Z i n i b , Acta Pharm. Jugoslav. 2,13 /1982/. 16. B. Kojib-Prodib, 2. Ruii&-ToroH, Acta C r y s t . B 38, 2948 /1982/.

PIROXICAM

53 1

17. J. Bordner, J.A. Richards, P. Weeks, E.B. Whipple, Acta Cryst. C 40, 989 /1984/. 18. J.G. Lombardino, E.H. Wiseman, J. Chiaini, J. Ned. Chem. 2,493 /1973/.

19. T.M. Twomey, S. R. Bartolucci, D.C. Hobbs, J. Chromatog. 104 /1980/. 20. K.D. Riedel, H. Laufen, J. Chromatog. 276, 243 /1983/.

a,

21. P. Schiantarelli, D. Acerbi, c). Bovis, Arzneim.-Forsch. (Drug Res.) 2,92 /1981/. 22. D.C. Hobbs, T.M. Twomey, J. Clin. Pharmacol. 2,270

/1979/23. T. Ishisaki, T. Nomura, T. Abe, J. Pharmacokinet. Biopharm. 2, 369 /1979/. 24. T.M. Twomey, D.C. Hobbs, Fed. Proc. Fed. Am. SOC. Exp. Biol. 271 /1978/. 25. E.H. Wiseman, J.A. Boyle, Clinics in Rheumatic Diseases 5, 585 /1980/. 26. J.G. Lombardino, E.H. Wiseman, Med. Res. Rev. 2, 127 /1982/. 27. D . C . Hobbs, T.M. Twomey, Drug. Metab. Dispos. 4, 114

x,

/1981/. 28. JOG. Lombardino, J. Med. Chem. 24, 39 /1981/. 29. E.H. Wiaeman, Royal Society of Medicine International Cougress and Symposium Series, No. 1, pp. 11-23 /1978/.

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RANITIDINE

Marijan Hohnjec, Josip Kuftinec, Miljenka Malnar, Milivoj gkreblin, Franjo Kajfei, Antun Nagl, Nikola Blaievid

1. Foreword, History, Therapeutic Category 2, Description 2.1. Nomenclature 2.1.1. Chemical Name 2.1.2. Generic Name 2.1.3. Trade Name 2.2. Formula 2.3. Molecular Weight 2.4. Appearance, Color, Odor 3. Synthesis 4. Physical Properties 4.1. Spectral Properties 4.1.1. Infrared Spectra 4.1.2. Ultraviolet Spectrum 4.1.3. f'jjoton Magnetic Resonance 4.1.4. C-Nuclear Magnetic Resonance 4.1.5. Mass Spectrum 4.2. Solid Properties 4.2.1. Melting Characteristics 4.2.2. X-Ray Diffraction 4.3, Solution Properties 4.3.1. Solubility 4.3.2. Acidity (pKa) 5. Methods of Analysis 5.1. Chromatographic Methods 5.1.1. Thin Layer 5.1.2. High Pressure Liquid 5.2. Spectrophotometric Determination ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 15

533

Copyright Q 1986 by the American Pharmaceutical Association All rights of reprodiirtion in any form reserved

MARIJAN HOHNJEC ET AL.

534

-

6. Stability Degradation 7. Drug Metabolism, Pharmacokinetics, Bioavailability 8. Identification and Determination in Body Fluids and Ti 6 sues

9. Identification and Determination in Pharmaceuticals lo. References

RANITIDINE

535

1. Foreword, History, Therapeutic Category Ranitidine is a new histamine H -receptor antagonist which, unlike cimetidine that contaizs an imidazole ring, has a furane ring structure /1,2/. This substituted amino-alkyl-furane derivative is more potent than cimetidine in inhibition of gastric acid secretion induced by various stimuli and lacks cimetidine's anti-androgenic and hepatic microsomal enzyme inhibiting effects. The drug has been used in the treatment of duodenal and gastric ulceration. In the recommended dosage of 150 mg twice daily, ranitidine is as effective as cimetidine and has therefore the advantages of less frequent doeing and fewer side effects. Ranitidine appears to be the drug of choice in the treatment of the Zollinger-Ellison syndrome because of its increased potency and lesser effect on endocrine function compared to cimetidine. The compound is given orally a6 a tablet (150 m a of ranitidine base) and as an injection Eolution ( 5 0 mg/5 ml). The first synthesis of ranitidine was reported in 1973 /3/ followed by pharmacological and clinical studies in 1979 /1,4/ and 1980 /5-7/. Finally, ranitidine was introduced on to the market in 1981.

2. Description 2.1. Nomenclature 2.1.1. Chemical Name The Chemical Abstracts name for ranitidine is N-[2-1 [ 5-1:(dimethylamino)methyl] -2-furanyl]methyl]thiolethyl] -N'-methyl-2-nitro-l,l-ethene diamine. The CAS registry No. is 66357-35-5. The other name is 2-[ [ [ 5-(dimethylamino)-

-

methyl-2-furanyl]-methyl]thio]-ethylamino-2-methylamino-

-1-nitroethene. 2.1.2. Generic Name Ranitidine

2.1.3. Trade Name Zantac 2.2. Formula

CHN02

!

0 \

NCH2 CH3

0

-HCI

CH2SCH2CH2NH NHCH3 C13H22N,+03Se HC1

MARIJAN HOHNJEC ET AL.

536

2.3. Molecular Weight 350.869 2.4. Appearance, Color, Odor Ranitidine is marketed only as the hydrochloride salt. It is a white to yellowish solid with little or no odor. A slight sulfur-mercaptan odor may be present.

3.

Synthesis The first synthesis of ranitidine, as shown in Scheme I, started with the reaction of 5-dimethylaminomethyl-2-furanyl-methanol (I)with 2-mercaptoethylamine by means of aqueous hydrochloric acid to give 2-[[(5-dimethylaminomethyl-2-furanyl)methyl] thiolethaneamine ( 11) This intermediate is then condensed with N-methyl-1-methBlthio-2-nitro etheneamine (IV)by heating in water at 50 C for 4 hours. Compound IV is obtained in the reaction of 1,l-bis(methylthio)-2-nitroethene (111)with methylamine in refluxing ethanol /3/. Alternatively, ranitidine ( V ) may be prepared by condensation of 5-dimethylaminomethyl-2-furanyl methyl mercaptan (VI) with aziridine derivative VII /8/ and by the reaction of amine I1 with 1-nitro-3-methyl ketene imine (1x1 /9/ Scheme 11. Other patented methods for ranitidint preparation are also shown in Scheme 11. The reaction of nitromethane with pseudothioureido compound VIII /lo/ and carbodiimide XI /11/ as well as the condensation of aziridine with nitroethene intermediate X /12/ and mercapto dfamino nitroethene XI1 with 5-dimethyl-aminomethyl-2-furanylmethanol (I)/3/ leads to ranitidine in moderate yields.

-

4. Physical Properties spectral ropert ties

4.1.

The IR absorption spectrum of the crystal form 1 ranitidine hydrochloride is shown in Figure 1. Figure 2 is the infrared spectrum of the form 2 ranitidine hydrochloride. These spectra were recorded with KBr-pelleted Barnples using a Pye Unicam SP-200 Infrared Spectrophotometer /13/. Our results are in good agreement with the published data for the cryeta1 form 2 ranitidine hydrochloride /14/. In the IR spectrum of the crystal form 1 (Fig. 1) the well-known bandrs characteristics of a nitro group attached to a saturated carbon atom.!:i stretching vibra/15/, are hardly tion frequencies at 1554 and 1382 cm vi_efble as well as the band6 in the range of 1505-1540 cm characteristic of a nitro group attached to a substi-

cv

0

7

= 0

11

0

cv

0

7

0

f

m

I

cv

I 0

I"

0 I/ \=

-2

0

I"

I"

0

I

*

0

7

I"

cv

0

I

m

I / \ '2

0

II

I w >

z m

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o

0

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m

=:I

0

m' m ' c*) m X I 0

I

0 cv

I

m I

U

>

c L

538

0 4000

3500

3000

2500

2om

1600

1200

cm-1

800

Fig. 1. Infrared spectrum of ranitidine hydrochloride form 1 in KBr pellet. Instrument: Pye-Unicam SP3-200.

0

10 0 r

m o

0

D

8 0 0

9

0

2 D 0

5 0

z r

0

8 N

0

ul N

D D D n

8 D

D D Y

c, al

rl d

k f4

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k4 .rl

ru

E

al

0 k

k

2. . s

0 d 0

k

0

.c

A

T¶

N M .rl

k

RANITIDINE

54 1

tuted ethenf group /16/. There is, however, a strong band at 1620 cm- which corresponds to the stretching vibration of the C=N double bond in an aci-nitro group of nitronic acid /l7(. Markedly 6trong bands appearing at 2640 and 2560 cm- are characteristic of the N+-H bond which exists in the protonated tertiary arnine group H-N’R in V hydrochloride. The properties of the IR spectrum Juggest that ranitidine hydrochloride exists mostly in the tautomeric form denoted by B in Scheme 111. Thic c o n c l u sion is in agreement with other authors’ findings for similarly substituted nitroethenes in which nitronic acid forms are stabilized by conjugation /18/. Other spectroscopic data also suggest that ranitidine hydrochloride exists predominantly in the form represented by formula B. These conclusions were also confirmed by single crystal X-ray diffraction studies reported by B. Kojit-ProdiE et a1 /19/. Namely, the bond lengths in the 2-ethyl-2-methylamino-1-nitroethene residue are in agreement with the structure B.

SCHEME I11

CHN02

II

C RNH HNCH3

&\

0\

RN

HNCH3

A

R=

B

‘k

CH3 ‘NCH2 ‘ 0 /

CH2SCH2CH2-

CH3 4.1.2. Ultraviolet Spectrum The ultraviolet spectrum of ranitidine hydrochlorlge was recorded in an aqueous solution, concentrations lo g / l , using a Pye Unicam sP8-loo UV-spectrophotometer, and is shown in Figure 3 /13/. In the ultraviolet spectrum of V hydrochloride an expected bathochromic shift of the abolefin sorption maximum, characteristic for nitro group

-

MARIJAN HOHNJEC ET AL.

542

0.7 0.6

0.5

g 0.4 c d a L 0

0.i

0.'

nm Fig.

3. Ultraviolet spectrum of ranitidine hydrochloride in an aqueous solution. Instrument: Pye-Unicam SP8-loo.

RANITIDINE

543

conjugation, is present. The spectrum shows two absorption maxima, at 228 nm (&=23,485) and at 313 nm ( & = =16.030). The measurement of the absorption at 313 nm is very convenient for quantitative determination of ranitidine hydrochloride even in the presence of intermediates in the ranitidine synthesis.

4.1.3. Proton Magnetic Resonance The proton magnetic resonance spectra of ranitidine base (Figure 4) and ranitidine hydrochloride (Figure 5 ) were recorded with a WP 80PS instrument by Bruker /13/. The spectra illustrated were obtained using deuterated chloroform and deuterated dimethyl sulfoxide solutions containing approximately 80 mg/ml of the compound with tetramethylsilane as the internal standard. The curve denoted by B in both spectra represents the case when D 0 2 was added to deuterated organic solvent solutions, The spectrum of ranitidine base shows a singlet at 2.24 ppm (due to six protons of the N ( C H ) group) and a broad 2 doublet at 3.00 ppm (due to prozons of the CH NH group) which, upon addition of D 0, contracts into a3singlet. 2 Spin decoupling at 3-40ppm - the position of a multiplet causes due to the methylene protons of the NHCH group a contraction of the upfield triplet at ?.84 ppm to a singlet. As the coupling constant J ( C H 2 S , C H N ) is 7.5 H e , the triplet at 2.84 ppm sensitive to decoupfing influences must correspond to the two protons of the methylene group connected with the sulfur atom, which points away from the ring. Further signals may be assigned as follows: the singlet at 3.50 ppm ( 2 H ) corresponds to protons of the C H N group in 5-position of the farane ring. The next 2 downfield singlet at 3.72 ppm ( 2 H ) corresponds to the protons from the methylene bridge linking the ring to the S-atom. The pair of doublets at 6.22 ppm ( 1 H ) and 6.26 ppm (1H) aDrresponds to ring protons in 3- and 4-positions, respectively (J 4=3,75 H e ) . The nearest singlet potiitioned at 6.56 ppJ'[lH) corresponds to the vinyl proton of the nitroethene group CHNO In the spectrum of ranitidine hydrochloride (Figure 53 the signal for this proton is situated between signals for the furane ring protons. This proton is in both cases displaced by a deuteron upon addition of D 0, which indicates a tautomeric prototropic shift in $he nitroethene terminal. This observation has been confirmed by A. Sega, et al. /20/ in their investigation of the H/D exchange in 2,2-disubstituted nitroethenes. Finally, the broad singlet centered at 10.33 ppm (1H) cannot be definitely assigned to either the proton NH, or that of the nitronic acid group =N-OH. The contrac-

-

.

L

*

B

A

11

10

I

I

9

8

I

7

I

I

6

5

L

3

2

1 PPm

Fig. 4. Proton magnetic resonance spectrum of ranitidine base in CDCl

Instrument: Bruker WP8oPS

0

3.

I

I

10

Fig.

I

I

9

I

l

8

l

I

I

7

I

6

I

I

5

1

I

4

I

I

3

I

1

2

1

I

1

I

PPm

I

0

5. Proton magnetic resonance spectrum of ranitidine hydrochloride in DMSQ-d Instrument: Bruker w P ~ o P s .

6'

MARIJAN HOHNJEC ETAL.

546

t i n g e f f e c t of D 2 0 a d d i t i o n upon t h e C H NH- m u l t i p l e t conf i r m s t h a t t h e n e i g h b o u r i n g N-atom b i n d 2 a p r o t o n . The i n f l u e n c e of D 0 d o e s n o t extend t o t h e CH N a m u l t i p l e t 2 2 which c o n f i r m s t h e a b s e n c e of a p r o t o n from N-atom. One s h o u l d e x p e c t , t h e r e f o r e , t h a t r a n i t i d i n e i s more l i k e l y t o b e r e p r e s e n t e d by formula B (Scheme 111) t h a n by f o r m u l a A.

4.1.4.

13C1Nucl e a r MaRnet i c Resonance The &'C-NMR s p e c t r a o f r a n i t i d i n e h y d r o c h l o r i d e were r e c o r d e d w i t h a J e o l FX-loo s p e c t r o m e t e r a t 25.05 MHz. The samples were d i s s o l v e d i n DMSo-d6 and s p e c t r a were r e c o r d e d i n broad-band, o f f - r e s o n a n c e and NOE modes /l3/. The broad-band and o f f - r e s o n a n c e s p e c t r a were shown i n F i g . 6 . I n b o t h modes i t was p o s s i b l e t o a s s i g n a l l carbon atoms. The d e t e r m i n a t i o n s of C-H c o u p l i n g c o n s t a n t s were d i f f i c u l t f o r groups N ( C H ) and NHCH because t h e r e s p e c t i v e s i p n a l s were s i t u a ? e g w i t h i n tAe r a n g e of t h e s e p t e t c o m e s T h e r e f o r e , t h e y were ponding t o t h e s o l v e n t DMSO-d6 determined u s i n g NOE measurements i n CD OD a t 20.1 MHz.

,

.

3

T a b l e I. I3C-NMR spectrum of r a n i t i d i n e h y d r o c h l o r i d e i n CD OD a t 20.1 MHz; c h e m i c a l s h i f t s ( d ) and C-H coapling c o n s t a n t s (J C-H C-atom

C-5 f u r a n e C-2 f u r a n e C-4 f u r a n e C-3 f u r a n e CH=N02H N-CH, NHCH -CH

-sc6

-CH,S-

4.1.5.

3-

-

c3

(ppm)

158.0 155. o 145. o 116.6 110.2 99.3

53.8 42.8 42.3

31.7 28.9 28.6

JC,H

doublet doublet doublet quartet quartet doublet triplet quartet quartet triplet triplet triplet

( Hz)

6.3 12.6 12.6 128.1 and 5.0 123.9 and 5.0 189.2 148 3 126.3

.

139.1 150.1 142.2 139 1

Mass Spectrum The mass spectrum was r e c o r d e d w i t h a n MS-25 mass s p e c t r o m e t e r w i t h d a t a s y s t e m DS 50s from K r a t o s , Manches t e r . Samples f o r mass s p e c t r a were d i r e c t l y i n t r o d u c e d i n t o t h e i o n source. The e l e c t r o n impact i o n i s a t i o n was a p p l i e d a t 70 eV ( 4 7 3 K s o u r c e t e m p e r a t u r e ) and s p e c t r a were r e c o r d e d a t a s c a n n i n g speed of 3 sec/scan. The spe-

I

I

1

16 0

1

1LO

I

120

100

I

I

80

60

LO

8

I

*O

PPm

0

Fig. 6. 13C-NMR broad-band ( A ) and off-resonance ( B ) spectra of ranitidine hydrochloride in DMs0-d~. Instrument: Jeol FX-loo at 25.05 MHz.

MARIJAN HOHNJEC ET AL.

548

58

138

224

176

NHCH?

I

loo

297 269

224 1

I

281

.dl

100 7 8

1

42 cI

z L $50-

.-

I

d

8

169

1 II,

0 40

60

80

100

120

140

1

160

m/e

180

200

Fig. 7. Mass spectrum and fragmentation of ranitidine. Instrument: Kratos MS-25.

RANITIDINE

549

Table 11. X-Ray d i f f r a c t i o n d a t a of r a n i t i d i n e hydrochlor i d e form 1

8 ("I 7-02 7.21 7.59 7.76 8.43 8.66 lo. 36 10.81 11.16

InterDlanar distance d* ( 8 )

6.31 6.14 5.84

11.34 12.12

13- 07 14.24 14.49 14.18

I/I22

5.71 5.26

34 39 79 loo

5.12

34

4.29 4.11

21

3.98 3.72 3.61 3.40

11 97

Relative intensity

3. 17 3-13 3.08 2.04

87 64 24 76 59 73 45 42 21

*d = n h / 2 s i n e **Based

on t h e h i g h e s t i n t e n s i t y a d j u s t e d t o 1.00.

**

MARIJAN HOHNJEC ET AL.

550

L

25

I

1

.

1

20

r

l

'

'

.

15

"

'

'

'

.

10

. " .

5'

.

8

Fig. 8. X-Ray diffraction pattern of ranitidine hydrochloride form 1. Instrument: General Electric XRD-6 spectrogoniometer.

RANITIDINE

551

Table 111. X-Ray diffraction data of ranitidine hydrochloride form 2

Q

In t e rplana r distance ( O )

d*

4.2 793 7.7 8-3 9.1 9.6 lo. 2 10.5

11.4 11.8 12.1

12.4 12.9 13.8 14.4 14.8 16.0

l o . 47

20

6.08

13

5.75 5.34 4.87 4.62 4.36 4.23 3.88

21

37 12

11

loo

15 14

3.77 3.67 3.60 3.44

58 23

3.23 3. l o 3.02

21

2-79 2.46

18.2 it d =

(8)

Relative intensity** I/In-

12

15

19

7 20

9

n h / 2 sin@

**Based

on the highest intensity adjusted to 1.00.

Fig.

8.

X-Ray diffraction pattern of ranitidine hydrochloride form 2. Instrument: General Electric XRD-6 spectrogoniometer.

RANITIDINE

553

ctra were evaluated using the attached data system /l3/. The mass spectrum of ranitidine and its main fragments are shown in Figure 7. The molecular ion on the ranitidine spectrum could not be detected. The abundance of the ion m/e 297, which differs by 17 mass units from the proposed molecular ion, suggests the presence of a hydroxyl group in the structure of ranitidine. Loss of OH from the molecular ion was confirmed by exact mass measurements of the m/e 297 by high resolution mass spectrometry. This fragmentation is in accordance with our proposal that the structure of ranitidine is described by formula B (Scheme 111). According to Martin, et al. /21/ the ion m/e 297 was generated by loss a molecule of water from the protonated molecular ion. It should be noted that this mass spectrum was obtained by the chemical ionization mode, while we used an electron impact technique.

4.2. Solid Properties 4.2.1. Melting Characteristics The ranitidine base is difficult to crystallize, but its hydrochloride can be conveniently crystallized, particularly from isopropanol. The melting range of ranitidine hydrochloride depends on the polymorphic form in which this compound is crystallized. When ethylacetate is added to an ethanolic solution of ranitidine hydrochloride the crystalAine form 1 is obtained gith the melting range of 135-136 C /ref. 4: m.p. 133-134 C/. The form 2 is cryatallized from isopropanol-HC1 solutionowith the melting range of 143-144OC /ref. 14: m.p. 141-142 C/. X-Ray Diffraction The X-ray diffraction patterns were determined with a Mod. XRD-6 spectrogoniometer from General Electric, Schenectady. The spectra were taken with a monochromatic radiation which was obtained from the CuKw line (15.42 nm) excited at 35 kV and 20 mA /13/. The X-ray powder diffraction spectra for both forms of ranitidine hydrochloride are given in Figures 8 and 9. Tables I1 and I11 list the interplanar distances, the diffraction angle and the relative peak intensities. 4.2.2.

4.3. Solution Properties -4.3.1. Solubility The solubility of ranitidine hydrochloride in various solvents at room temperature is summarized in Table IV.

4.3.2. Acidity ( P K For the detergination of the pKa value the spectro-

554

MARIJAN HOHNJEC ET AL.

T a b l e IV. S o l u b i l i t i e s o f r a n i t i d i n e h y d r o c h l o r i d e Solvent

Solubility freely soluble very f r e e l y soluble soluble sparingly soluble very s l i g h t l y soluble v e r y s l i g h t l y sOlublt? insoluble insoluble

acetic acid water methanol ethanol ethylacetate isopropanol dioxane chloroform

p h o t y n e t r i c method was used /22/. 2.19-0.04 /13/.

The o b t a i n e d v a l u e wa8

5. Methods of A n a l y s i s 5.1. Chromatographic Methods Thin Layer The p u r i t y of r a n i t i d i n e h y d r o c h l o r i d e can b e quickl y a s s e s s e d by TLC o v e r s i l i c a g e l . Table V shows i t s R f - v a l u e s w i t h s e v e r a l s o l v e n t systems /13/. S p o t s were

7.1.1.

Table V, R f - v a l u e s S o l v e n t s and Rf-values A

B

C

D

E

F

0.50

0.44

0.36

0.64

0.73

0.39

A= EtOAc/MeOH/Et NH (3:3:1), B= CHC13/i-PrOH/Et NH ( 4 : 3 : :2 ) , C= dioxane/$eOH/DMF ( 6 :3 :2 1, D= MeCN/MeOH/$% NH OH ( 5 : 2 : 1 ) , E= EtOAc/MeOH/25% NH40H ( 1:5:1), F= EtOAc/i-hOH/ /25% NH40H (4:3: 1). l o c a t e d e i t h e r under a n UV lamp, o r by s t a i n i n g t h r o u g h exposure t o i o d i n e vapors. Suggested p r o c e d u r e f o r t h e i d e n t i f i c a t i o n of r a n i t i d i n e by t h i n - l a y e r :romatography: f i v e , 1 5 and 30 /u1 samples of a 20 mg.cm m e t h a n o l i c s o l u t i o n , and t h e same volume of a m e t h a n o l i c s o l u t i o n o f e q u a l c o n c e n t r a t i o n s of t h e s t a n d a r d s u b s t a n c e a r e a p p l i e d t o a s i l i c a g e l p l a t e and chromatograms developed w i t h e t h y l a c e t a t e h e t h a n o l / d i e t h y l a m i n e (3:3:1). The s p o t s must have t h e same f l u o r e s c e n c e i n t e n s i t y under UV-radiation, and t h e same shade of s t r a i n subsequent t o exposure t o i o d i n e vapor. All R v a l u e s must be c l o s e t o 0.5. f

’

RANITIDINE

555

5.1.2. High Pressure Liquid The HPLC analyses were performed with a HPLC apparatus, LC-3-XP with an UV-LC detector. The HPL chromatogram is shown in Figure lo /13/. The chromatograms were run through a column filled with Li-Chromosorb RP-8 (5,Um) using a mixture of acetonitrile, methanol, w ter, and concentrated ammonia (250, 20, 6 and 0 . 0 5 cm3, respectively) having a pH-value of 7.4. The elution was carried out under 13.78 to 17.22 MPa (20.000- 5.000 p.s.i,) pressure, maintaining a flow rate of 1 cm$.min-l. The effluent was monitored optically at 217 nm. 2.2.

Spectrophotometric Determination Ten mg of "unknown" ranit'dine hydrochloride is acvolumetric flask, dissolcurately weighed into a lo ved by swirling with 50 c water and the solution made up to the mark Ten cm' of the resulting solution is diluted to loo cm3 in another volumetric flask, and the absorbance of the final dilution is measured at 313 nm against water. The same procedure is carried out with a standard sample of known ranitidine concentration /U/. Calculation: AUMsP C H C1N 0 S in unknown, % = A loo 13 23 4 3 s u A = absorbances, M = masses [ g), u = unknown, s = standard, P = % of C13H,JC1N 0 S in the standard.

6. Stability

' zy

-

43

Degradation The stability of ranitidine hydrochloride in tablets was t % sted in two ways: one series of samples was kept at 40 C and 50-6096 relative humidity f o r five days, and the other at 6ooC and loo% relative humidity during the same period. According to the spectrophotometric determination, the degradation amounted to -5% (under the first conditions) and 11.5%, respectively. The thin-layer chromatographic analysis on silica gel shows five different degradation products in both cases.

7. Drug Metabolism, PharmacQkinetics, Bioavailability Metabolic studies of "C-ranitidine in rat and dog showed that ranitidine was mainly metabolised by oxidation to give N-oxide X I I I , S-oxide XV, and desmethyl ranitidine X I V . The relative amount of each metabolite formed was found to vary with the species /23/. Thin-layer chromatographic analyses of the urine collected from volunteers given oral and intravenous doses of ranitidine

556

MARIJAN HOHNJEC ET AL.

Fig. lo. HPL chromatogram of ranitidine hydrochloride. Instrument: Pye-Unicam LC-3-XP.

RANITIDINE

557

CHN02

II

2SC H2CH2 NHCNHCH3

t 0

H H 3c)NCH2

Xlll

XIV

CHN02

II

CH2SCH2CH2NHCNHCH3

i 0 xv

showed that ranitidine was the major component present. Compound XI11 was the major metabolite, and small quantities of compound XIV and XV were also detected / 2 4 / . Ranitidine is a potent inhibitor of gastric secretion after oral administration. It is four to seven times more potent than cimetidine. In doses of 20, 40 and 80 mg, ranitidine reduces hydrogen output by 29%, 50%, and 70%, and gastric secretion volume by 2196, 37%, and 47%. With the same doses it also reduce6 pepsin activity by 8%, 50% and 49%. Serum concentration of 0.08 ,ug/ml of ranitidine reduces gastric acid output by 50% /25,26/. Following oral and parenteral administration ranitidine blood concentration curve has a pronounced secondary peak. After oral administration ig the first peak in the plasma occurs within 1.1+humans, - 0 . 4 h, and the second peak within 3:o h. These peak plasma concentrations are not influenced by food /27/. Following intravenous administration there is a biexponential decline in the plasma levels from 576556 ng/ml after 4 min to 1022 ng/ml after 8 h. In healthy subjects the cere-

MARIJAN HOHNJEC ET AL.

558

brospinal fluid concentration of ranitidine is one twentieth, to one-thirtieth of that in the plasma sampled at the same time. The distribution half-life of ranitidine + is 6.1-0.9 min., elimination half-life being 1.9-0.1 h, the volume of distributfon is 96-115+7 1, and systemic plasma clearance is 709-62 ml/min. The reported bioavailability after a single dose was between about 40 and 88%, but mostly around 50% /28,29,30/. In the research of the effects on hepatic drug metabolism, it has been found that ranitidine does not enhibit the microsomal drug oxidative function /31/. Ranitidine is 15% protein bound. Half of the oral dose of ranitidine is readily absorbed, and half of the+absorbed amount is found unchanged in the urine. Only 1.3-0.3% of the intravenous dose and 2.6toe2% of the oral dose is converted into the desmethyl metabolite.

8. Identification and Determination in Body Fluids and Tissues Ranitidine may be determined in the serum, plasma, and urine by high pressure liquid chromatographic analysis /32,33,24/. Martin, et al. /21/ used the on-line high performance liquid chromatography mass spectrometry for the identification and structure determination of ranitidine and its metabolites in the urine. The separation of ranitidine and its metabolites is usually carried out by extraction of the biological medium with methylene chloride from an aqueous alkaline solution ( 2 M NaOH or 5M KOH), followed by mixing, addition of an internal standard and centrifugation /33/. The addition of 10% isopropanol to methylene chloride increased the recovery of ranitidine in this extraction procedure /32/. The organic gayer was then evaporated to dryness under nitrogen at 45 C. When treateg in this way, the dry residue was stable for 7 days at -20 C. Before chromatography, the residue was dissolved in a methanol-dibasic ammonium phosphate mixture /33/. Ranitidine was also assayed in the urine by HPLC procedure using direct injection and no internal standard /24/.

-

XVI

RANITIDINE

559

During usual HPLC analyses metiamide /32/ and N-methyl-N' -[ 3-[ ( 3-dimethylaminomethyl)phenoxy] propyl] -2-ni tro-1,l-ethenediamine hydrochloride XVI /33/ were used as internal standards. The columns used for HPLC were the reverse phase p Bondpak c-18 (Waters Associates), Spherisorb ODs (Phase Separations, Clwyd, Great Britain) and Spherisorb S5 CN. An example of the conditions of analysis is shown in Table VI /32/. Table VI. Conditions of HPLC analysis Parameter

Assay conditions required

Mobile phase 92/8 mixture Reagent A/Reagent B U , Bondpak c-18 Column Temperature ambient temperature ( 20-25OC) 1000 - 3 0 0 0 psi Pressure 0.005 Absorbance full scale Flow rate 2 ml/min 228 nm Wavelength Internal standard met iamid e Chromatography time 8 min A

-

ranitidine, B

- metiamide

9. Identification and Determination in Pharmaceuticals For the determination of ranitidine hydrochloride content in a tablet dosage form we recommend the following procedure /13/; Crush 20 tablets in a mortar, Quantitatively transfer the mass of powder equiv lent to lo mg of ranitidine hydrochloride into R 250 cm3 volumetric flask. Add loo cm3 of water and sheke the resulting suspension automatically for 20 minutes. Make up to volume, mix well and centrif ge 20 ml aliquot at 2000 G for 5 minute Pipet lo cm' of the clear supernatant into a loo cm3*volumetric flask and make up to volume. Measure the absorbance of the solution at 313 nm against water and compare it to that of an appropriate standard solution. Calculation : AU*MS ut C H C1N 0 S contents, mg per tablet = 13 23 4 3 ut the average mas6 of one tablet For other symbols see chapter 5.

.

-

Acknowledgements. The authors would like to thank Dr B. RugEi6 and Z. Boii6evi6 of the Institute "Ruder Bo5kovib1I, Zagreb, for the pK determination, and to Dr M. Milun of "CHROMOS", Researca and Development, Zagreb, for NMR

MARIJAN HOHNJEC E T A L .

560

measurements

.

l o . References I. N.R, Peden, J.H.B.

-

79, 3* 4.

,

m

5. 6. 7.

8.

. . 13. 9.

lo 11. 12

14. 15 16. 17

.

19 20. 21

Wormsley, Lancet

1,

Domschke, G. Lux, S. Domschke, G a s t r o e n t e r o l o g y 1-26? /1980/* B. J. P r i c e , J, W. C l i t h e r o w , J. W. Bradshaw ( A l l e n and Hanburys Ltd.) Ger. Offen. 2,734,070; C O A O 88, 190580b /197a/. H. J. Ruoff, U. Gladziwa, K.F. Sweng, G a s t r o e n t e r o l o g y 76, 1230 /1979/* R. S t a b l e s , M.J. Daly, Agents A c t i o n s lo, 191 /1980/. K. K e t t , F. Aadland, A. B e r s t e d , Scan. J. G a s t r o e n t e r o l . 2,249 /i980/. E.P. Woodings, G.T. Dixon, C. H a r r i s o n , P. Carey, D.A. Richard?, Gut 21, 187 /1980/. R. Toso, V. s u n j i 6 ( C R C ) , Ger. Offen. 3,118,813 /1982/; C.A. 96, 181127~/1982/. Lek, Pa$, Appl. 2608/82. Glaxo, Eur. Pat. 55,626 /1982/; C.A. 97, 1 9 8 0 9 4 ~/1982/. Evers, Neth. Pat. 8303-4354 /1984/. Glaxo, Eur. Pat. 59082 /1982/. M. Hohnjec, S. Rendit, T. Alebit-Kolbah, F. K a j f e i , N. B l a i e v i t , J. K u f t i n e c , Acta Pharm. J u g o s l . 2,131 11981~ D.L. Crookes ( G l a x o ) , Ger. Offen. 3,139,134; C.A. 97, 61014g /1982/. L.J. Begiamy "The I n f r a r e d S p e c t r a of Complex Moleculestt, 2 Ed., Methuen and Co., London 1957, p 333. R. Gompper, H. S c h a e f e r , Chem. Ber. loo, 591 /1967/. H. Feuer, "The Chemistry of t h e N i t r o and N i t r o s o Groups1!, John Wiley, N e w York 1969, p 383. W.L.F. Armarego, J.T. Batterham, K. S c h o f i e l d , R.S. Theobald, J. Chem: SOC. ( C ) , 1969, 1433. B. Koji6-Prodi6, 2. RuiiE-Toro5, R. Toso, Acta C r y s t . B 1837 /1982/* A , Sega, R. TOSO, V. g u n j i 6 , L. K l a s i n c , A. S a b l j i 6 , D. S r z i 6 , Gazz. Chim. I t a l . 111,217 /1981/. L.E. Martin, J, Oxford, R. J.N. Tanner, Xenobiotica 11, 831 /1981/. N,N. Ferguson i n "Advances i n A n a l y t i c a l Chemistry and I n s t r u m e n t a t i o n 1 t Ed. C.N. R e i l l e y , Vol. 4, I n t e r s c i e n c e Publ., N e w York 1965, p. 411. J.A. B e l l , F.A.A. Dallas, W.N. J e n n e r , L.E. Martin, Biochem. Sac. Trans. 8 , 93 /1980/. P.F. Carey, L.E. Martin, P.E. Owen, J. Chromatog. 225,

2. W.

18

Saunders, K.G.

690 /1979/*

xg.

2,

.-

22. 23.

24.

161 /1981/.

56 I

RANITIDINE 25. P.A, H.M.

L e b e r t , S,M. 'Mac Leod, W.A. Mahon, S. J. S o l d i n , Vandenberghe, C l i n . Pharmacol. Ther. 2, 539

/1981/.

26. D.C. B r a t e r , C l i n . Pharmacol. Ther. 32, 484 /1982/. 27. A.M. Van Hecken, T.B. Tjandramaga, A. M u l l i e , R. Verb e s s e l t , P.J. De Schepper, B r . J. C l i n . Pharmacol, 14, 195 /1982/. 28. T J . McNeil, G.W. Mihaly, A. Anderson, A.W. M a r s h a l l , R.A. Smallwood, W . J . Louis, B r . J. C l i n . Pharmac. 12, 411 /19a1/. 29. T N . Brogden, A.A. Carmine, R.C. Heel, T.M. S p e i g h t , G.S. Avery, Drugs &, 267 /1982/. 3 0 . D.A. Henry, G.KStchingman, M.S.S. Langman, B r i t . Med. J. 281, 775 /1980/* 31. S. Rendi;, F. K a j f e i , H.H. Ruf, Drug Metabolism and D i s p o s i t i o n 2,137 /1983/.

32. H. M. Vandenberghe, S. M. Mac Leod, W. A. Mahon, P.A. L e b e r t , S.J. S o l d i n , T h e r a p e u t i c Drug Monitoring 2,

379 33. G.W. W.J.

/ww. Mihaly,

O.H. Drummer, A. Louis, J. Pharm. S c i .

Marshall, R.A,

69,1155 /1980/.

Smallwood,

This Page Intentionally Left Blank

STRYCHNINE

Farid J. Muhtadi

and

Mohamed S. Hifnawy

Introduction 1. Description 1.1 1.2 1.3 1.4 1.5

Nomenclature Formulae Molecular Weight Elemental Composition Appearance, Color, Odor and Taste

2. Physical Properties

2.1 2.2 2.3 2.4 2.5

Melting Range Solubility Optical Rotation Crystal Structure Spectral Properties

3. Isolation

4. Total Synthesis and Degradation 5. Biosynthesis

6. Metabolism and Toxicity ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 15

563

Copyright 0 1986 by the American Pharmaceutical Association All rights of reproduction in any form reserved.

FARID J. MUHTADI AND MOHAMED S. HIFNAWY

564

7. Methods of Analysis 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8

Identification Tests Microcrystal Formation Titrimetric Methods Compleximetric Determinations Spectrophotometric Methods Chromatographic Methods Radiometric Determinations Polarographic Determination.

Acknowledgements References.

565

STRYCHNINE

Introduction Strychnine is an indole alkaloid occurs in numerous strychnos plants ofthe family Loganiaceae. The most important of which are the seeds of strychnos nux-uomica L. and the beans of strychnos ignatii Berg. These species contain up to 5.3% of the total alkaloids of which approximately one-half is strychnine. Strychnine was first discovered in 1817 by the French Pharmacists, Pierre Pelletier and Joseph Caventou, and these workers were also responsible for the first isolation of brucine in 1819. Structural investigations of strychnine were begun by Hanssen and Tafel and continued by Leuchs, Perkin, Robinson, Wieland and Woodward. Finally the structure of strychnine was established by Robinson and co-workers in 1946 and confirmed by X-ray crystallographic analysis and total synthesis by Woodward. The alkaloid has no important therapeutic use as it is highly toxic. However, the crude drug nux-vomica is used as a bitter tonic and stimulant (as it is official in certain pharmacopoeias including the B. P. of 1980). Strychnine is used as a rodenticide for destroying agricultural rodents and predatory animals. Occasionally, domestic animals and man are poisoned by this agent. Strychnine is CNS stimulant, it stimulates the spinal cord. It is also a powerful convulsant which produces characteristic convulsions. Sometimes, strychnine is used for the adulteration of street drugs. 1.

Description 1.1 Nomenclature 1.1.1 Chemical Name Strychnidin-lO-one 1.1.2 Generic Name 1.2

Strychnine Formulae 1.2.1 Empirical C21H22N202 C42H46N408S

(strychnine) (strychnine sulfate)

FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

566 1.2.2

Structural

5

10

11

'19

Several structures have been proposed for strychnine, these include, Perkin and Robinson structure of 1910(1), Perkin and Robinson structure of 1929(2), Robinson structure of 1932 (3,4), Leuchs structure of 1932 (5). The currently accepted structure of strychnine was finally established in 1946 by Robinson et al. (6) and was confirmed by the total synthesis of strychnine which was carried out by Woodward et a1 (7-9). The absolute structure was deduced by extensive X-ray crystallographic studies which were achieved by several authors (10-13 ) . 1.2.3 CAS Registry No. [ 57-24-91 strychnine

[60-41-31 strychnine sulfate 1.2.4

Wiswesser Line Notation T6 G656 B 7 C6 E5 D 5ABCEF - 0 VN

1.2.5

AGFX MQ

NH-UHJ

114)

Stereochemistry The absolute stereochemistry has been deduced

STRYCHNINE

567

from combinations o f massive chemical d e g r a d a t i o n s and c o r r e l a t i o n s (15-18), X-ray c r y s t a l l o g r a p h i c s t u d i e s (10-131 9 lH-and carbon-13 n u c l e a r magnetic resonance s p e c t r a l d a t a (19,20). From t h e s e s t u d i e s , i t h a s been e s t a b l i s h e d t h a t D and G r i n g s have t h e b o a t conformation, w h i l e E and F r i n g s have t h e c h a i r conformation. H atom a t C 2 and carbon 7 are above t h e p l a n e o f t h e molecule (8-atoms), H atoms a t C3, Cis, c16 and C 1 7 l i e below t h e p l a n e of t h e molecule (a-atoms). Hydrogens a t C3 and t h a t a t C15 are equat o r i a l , w h i l e H a t C2 and t h a t a t C16 are a x i a l w i t h i n t h e c h a i r conformation o f r i n g E. The conformation of s t r y c h n i n e molecule i s p r e s e n t e d i n I . Recent s t u d i e s (20) h a s f a v o r e d a c h a i r c o n f o r mation f o r t h e seven-membered t e t r a h y d r o o x e p i n r i n g F (11).

H

7 \

Strychnine

II 1.3

9

"W

I

Molecular Weight 334.40 766.92

1.4

a

II

(strychnine) (strychnine s u l f a t e )

Elemental Composition C , 75.42%; H, 6.63%; N , 8.38 %; 0 , 9.57%. (strychnine) C , 65.78%; H , 6.05%; N , 7.31%; 0, 16.69%; S, 4.18%. ( s t r y c h n i n e s u l f a t e )

1.5

Appearance, C o l o r , Odor and Taste Orthorhombic, s p h e n o i d a l p r i s m s (from a l c o h o l ) ( 2 1 ) , o r translucent c o l o r l e s s c r y s t a l s o r white c r y s t a l l i n e powder ( 2 2 ) , o d o r l e s s and h a s a v e r y b i t t e r t a s t e . (strychnine)

FARID J. MUHTADI AND MOHAMED S. HIFNAWY

568

Colorless c r y s t a l s o r white c r y s t a l l i n e powder, odorless and has a ' b i t t e r t a s t e , e f f l o r e s c e s i n a dry a i r (strychnine s u l f a t e ) . 1.6

Dissociation Constant pK1 a t 20'

1.7

: 6.0; pK2 : 11.7

(21)

PH Range pH of a s a t u r a t e d s o l u t i o n of strychnine i s 9.5, pH of a strychnine s u l f a t e s o l u t i o n (1:lOO) is 5.5 (21)

2.

Physical Properties 2.1

Me 1t ing Range 268-290'

Depending on the speed of heating (21)

270-271O

Slow heat ( 2 3 )

strychnine s u l f a t e (anhydrous) : about 200' 2.2

(21).

Solubi 1i t y One gram d i s s o l v e s i n 6400 m l water, 3100 m l b o i l i n g water, 150 m l alcohol, 35 m l b o i l i n g alcohol, 5 m l chloroform, 180 m l benzene. (strychnine) One gram d i s s o l v e s i n 35 m l water, 7 m l b o i l i n g water, 81 m l alcohol, 26 m l alcohol a t 60', 220 m l chloroform and i n 6 m l glycerol (strychnine s u l f a t e ) .

2.3

ODtical Rotation [ a ] D18

-

139.3'

(chloroform)

[a] D20

-

104'

(c = 0.5 absolute alcohol)

109.9'

(80% ethanol)

[a] D

(21) (23).

( a l l above d a t a a r e f o r strychnine) The s p e c i f i c r o t a t i o n s f o r both strychnine i n chloroform and strychnine s u l f a t e i n water were d e t e r mined as 20 mg/ml s o l u t i o n s using a Perkin Elmer Polarmatic model 241 MC and found:[a]D2'

-

142.6' 25.1'

(strychnine) (strychnine s u l f a t e )

STRYCHNINE 2.4

569

Crystal Structure The c r y s t a l s t r u c t u r e of s t r y c h n i n e was determined by X-ray d i f f r a c t i o n , which was achieved by s e v e r a l a u t h o r s (10-13). The c r y s t a l s of s t r y c h n i n e hydrobromide, C21H22N202. H B r . 2H20 are orthorhombic w i t h space group P212121 and w i t h c e l l dimensions a=7.64, b=7.70 and c=33.20Ao ( 1 0 , l l ) . Each bromine atom is bonded t o a s t r y c h n i n e molecule through t h e b a s i c n i t r o g e n atom (N2). While t h e c r y s t a l s o f s t r y c h n i n e s u l f a t e p e n t a h y d r a t e are monoclinic with space group C2 and c e l l edges a=35.85+0.05, b=7.56+0.01, c=7.84+0.01A0 (12). I t h a s been shown t h a t t h e r e are f o u r molecules of s t r y chnine i n t h e u n i t c e l l (11,lZ). The s t u d y r e v e a l e d t h a t t h e s t r y c h n i n e molecule has a c o n f i g u r a t i o n i d e n t i c a l w i t h t h a t deduced by o t h e r methods. Peerdeman (13) by r e c a l c u l a t i n g v a l u e s of t h e i n t e n s i t i e s of r e f l e x i o n s o f s t r y c h n i n e hydrobromide dihyd r i d e , h a s deduced t h e a b s o l u t e c o n f i g u r a t i o n o f n a t u r a l s t r y c h n i n e as p r e s e n t e d i n Fig. 1. The c r y s t a l s t r u c t u r e o f s t r y c h n i n e s u l f a t e p e n t a h y d r a t e is p r e s e n t e d i n Fig. 2 (12). I n t r a m o l e c u l a r bond l e n g t h s of s t r y c h n i n e hydrobromide (11) a r e t a b u l a t e d i n t a b l e 1 and shown on s t r y c h n i n e molecule.

570

FARID J. MUHTADI AND MOHAMED S. HIFNAWY

F i g . 1: The Absolute C o n f i g u r a t i o n o f Strychnine.

F i g . 2: The C r y s t a l S t r u c t u r e of Strychnine Sulfate.

STRYCHNINE

571

Table 1. C

- N

c8

-

1

1

N1

c21- N1

0

I n t r a m o l e c u l a r Bond Lengths ( A ) of S t r y c h n i n e Hydrogen Bromide

1-37 C2 - C3 1.46 C3 - C4 1-50 C4 -

1.34 1.29 1.30

‘15- N2

c5 1.59 c - c6 5 1.44 c6 - c7 1-55 c7 - c8

1.59 1.49

c21-

0 1

1.15

‘18- ‘2

1.45

C7

-

c9

-

N2

5 0 - N2

-

1.30

1.56

‘13- ‘14

1.51

‘13- ‘16

1.58

‘14- ‘19

1.36

‘15- ‘16

1.65

‘16- ‘17

1.16

c9 Cll

1.41

c17- c 18

1.54

1.53

‘19- ‘20

1.56

c20- c21

1.49

2 - Br

3-17

c1

-

c2

1.31

‘8 C9

‘14 C12

1.55 1.64

‘1

- ‘6

1.40

Cl0- Cll

1.54

‘19- ‘2

‘12- ‘13

N

FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

572

2.5

Spectral Properties 2.5.1

Ultraviolet Spectrum (UV) The UV spectrum of strychnine in methanol (Fig. 3) was scanned from 190 to 400 nm using DMS Varian Spectrometer. It exhibited the following UV data (Table 2). Table 2.

Amax. nm 205 254 280 290

UV Characteristics of Strychnine log

E

-

A (l%, 1 cm)

-

4.10 3.64

377 131.4

3.54

104

Other reported W spectral data for strychnine in ethanol Xmax. 255 nm (E 1%, 1 cm = 377) (24 ) ; in sulfuric acid hmax. at 255 nm (E 1%, 1 cm = 315) (24 > ; Amax. for strychnine in ethanol, 254, 278, 288 mu (log~4.10, 3.63, 3.53 (21 ) . 2.5.2

Infrared Spectrum (IR) The IR spectrum of strychnine as KBr disc was recorded on a Perkin Elmer 580 B Infrared Spectrometer to which an infrared data station is attached (Fig. 4). The structural assignments have been corelated with various frequencies (Table 3). Table 3.

Frequency cm-1 2950-2800 1672 1600, 1485 1150, 1110, 1055 770

IR Characteristics of Strychnine As signment

CH stretch C=O substituted amide C=C aromatic ether 1inkage'O' 4-adjacent aromatic hydrogens

V

F i g . 3 : The W s p e c t r u m of s t r y c h n i n e i n methanol

0-

40 wavrnum&r(c~-1)3ooo FIG. 4 .

2500

2000

1CW

ItiE 1R SPECTRUM OF STRYCHNINE

Hoo

KBR. DISC

toe0

&O

coo

4

575

STRYCHNINE

The I R e x h i b i t e d t h e f o l l o w i n g o t h e r c h a r a c t e r i s t i c bands : - 1465, 1450, 1395, 1365, 1350, 1295, 1280, 1240, 1190, 1170, 1000, 990, 950, 925, 860, 780, 540, 470 cm-1. Other I R d a t a f o r s t r y c h n i n e have been r e p o r t e d ( 9 ,14,24)

.

2.5.3

Nuclear Magnetic Resonance S p e c t r a 2.5.3.1

Proton S p e c t r a (PMR) The PMR s p e c t r a o f s t r y c h n i n e i n CDC13 and i n TFA ( t r i f l u o r o a c e t i c a c i d ) were r e c o r d e d on a Varian T60A, 60 MHz NMR S p e c t r o m e t e r u s i n g TMS ( T e t r a m e t h y l s i l a n e ) as an i n t e r n a l r e f e r e n c e . The s p e c t r a are shown i n Fig. 5 and 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 a l assignments have been made (Table 4 ) .

22

The PMR of s t r y c h n i n e i n CDC13 u s i n g 250 MHz s p e c t r o m e t e r h a s been p u b l i shed e a r l i e r ( 1 9 ) . The spectrum o b t a i n e d by t h i s i n s t r u m e n t (Fig. 7 ) afforded b e t t e r resolution f o r s t r u c t u r a l assignment p a r t i c u l a r l y i n t h e r e g i o n 1.2 - 4 . 3 ppm. The r e p o r t e d PMR d a t a of s t r y c h n i n e (19) are a l s o p r e s e n t e d i n t a b l e 4 a l o n g w i t h o u r found PMR c h a r a c t e r istics.

I ,

*

l

I

8.0

,

,

,

I I

,

70

I

I ,

200

300

400

500

,

,

,

6.0

FIG. 5. THE PMH

I l

l

,

5 0 PPM(6)

,

4.0

,

l

# ~

~

3.0

SPECTRUM OF STRYCHNINE IN C D C L ~ ,

~

I

I

~

l

2.0

~

l

1.0

~

~

~

0

'

"

'

~

~

577

T

Fig.7: The PMR of strychnine in CDC13 using 250 MHz spectrometer.

579

.. M

580

FARID J. MUHTADI AND MOHAMED S. HIFNAWY

Table 4. PMR Characteristics of Strychnine Chemic 1 Shift (ppm) Proton at

CDC13 Reported

4

TFA

7.20(m)

7.40 8.13

3.84

8.07 (2q) 3.88(s)

3.10 2.65

3.08(s) 2.60(s)

4.23

4.26

4.27(q)

4.76

‘13

1.25

1.25(t)

1.74

‘14

3.12

-

‘15

1.43 2.33

1 .52(d) 2.35(t)

‘16

3.92

3.88(s)

7.14,7.07,7.23 C

CDC13 Found

(aromatic) ‘8

cll c12

‘17 ‘18

c20 CZ2 (vinylic)

‘23

8.08

1.86, 1.87 2.86 3.18 2.71, 3.69

2.03 2.76

1.85 (m)

2.38

2 83(q) 3.161s)

3.66

3.6 ( s )

5.88

5.87(bt)

6.53

4.04 4.12

4.04( s ) 4.13(s)

4.40

s = singlet, d = doublet, t = triplet, m = multiplet, q = quartet, 2q = 2 quartets, bt = broad triplet.

Some stereochemical correlations in strychnine molecule were deduced from the coupling constants of its protons (19,ZO).

58 1

STRYCHNINE 2.5.3.2

"C-NMR

The 13C-NMR n o i s e - 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 are p r e s e n t e d i n Fig. 8 and Fig.9 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 5000 Hz i n CDC13 on a J o e l FX-100 NMR S p e c t r o m e t e r , u s i n g a 10 mm sample t u b e and t e t r a m e t h y l s i l a n e (TMS) as an i n t e r n a l reference s t a n d a r d a t 20'. The carbon chemical s h i f t s are a s s i g n e d on t h e b a s i s of 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 tern (Table 5 ) . Assignments of a l l 21 c a r b o n s of strychnine are consistent with those

o f Wenkert e t a l . (20) and Verpoovte e t a1 (26). T a b l e 5.

Carbon Chemical S h i f t s of S t r y c h n i n e ~~

Carbon No

Chemical S h i f t [PPml 122.17 (d)

c2 c3 c4

124.05 (d) 128.39 (d) 116.06 (d)

Carbon

No '13

'14 15 '16

-

Chemical S h i f t

[PPml 48.14 (d) 31.52 (d) 26.77 ( t ) 60.00 (d)

I

I

582

I

d

583

c.( ... a

LL

f

0

wl &

w

U

c I-

v) wl

5

4

FARID J, MUHTADI A N D MOHAMED S. HIFNAWY

584

Carbon No

Chemical Shift: [PPml 142.08 (s)

c5

132.68 (s)

‘6

51.84 (s)

c7

Carbon No ‘17 ‘18 ‘20

60.11 (d)

‘8

Chemical S h i f t PPml

r

42.80 ( t ) 50.25 ( t ) 52.40 ( t ) 140.49 (s)

‘10

169.14 (s)

c22

127.04 (d)

cll

42.38 ( t )

‘23

64.52 ( t )

77.49 (d)

‘12

M u l t i p l i c i t y symbols, s = s i n g l e t , d = d o u b l e t , t = triplet. Other 1 3 C - M s p e c t r a l d a t a o f s t r y c h n i n e have been a l s o r e p o r t e d (20,25-27). 2.5.4

Mass Spectrum The mass spectrum of s t r y c h n i n e i s p r e s e n t e d i n Fig. 10. T h i s was o b t a i n e d by e l e c t r o n impact i o n i z a t i o n ( E I ) on a Finnigan - Mat 1020 by d i r e c t i n l e t probe a t 2 7 0 ’ ~ . The e l e c t r o n energy was 70 eV. The spectrum scanned t o mass 550 amu. The spectrum (Fig. 10) shows a molecular i o n peak M+ a t m / e 334 w i t h r e l a t i v e i n t e n s i t y of 100% which corresponds t o t h e base peak. The most prominent fragments 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 are p r e s e n t e d i n t a b l e 6 .

585

STRYCHNINE

Table 6.

Mass Fragments of Strychnine

m/ e

Relative Inten. %

m/e

335

24.65 (M”)

130

30.50

334

100.0 (M’)

129

12.79

333

16.20 (M-l)

120

20.40

167

13.11

115

14.74

162

12.41

108

11.13

144

13.90

107

19.42

143

18.17

94

11.00

134

11.76

Relative Inten. %

m/e

Re 1a t ive Inten. %

91

17.07

81

11.18

80

11.10

79

23.50

77

19.13

71

14.55

69

19.99

Other mass spectrum d a t a been a l s o r e p o r t e d (28).

of s t r y c h n i n e have

586

3.

FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

Isolation of Strychnine Strychnine occurs along with brucine in several species of Strychnos particularly S. nux-vomica and S. ignatii ( F d Z y Loganiaceael which contain varying quantities of both alkaloids (0.5 to 5.3%). Finely powdered nux-vomica is thoroughly moistened with lime water and extracted with hot chloroform till exhausion. The alkaloids are removed from the solvent by shaking with successive portions of diluted sulfuric acid. The combined acid solution is concentrated. Strychnine can be isolated from brucine by one of the following steps:1- The less soluble brucine bisufate crystallizes from the acid concentrated solution first and removed by filteration. Upon neutralization of the mother liquor, strychnine sulfate crystallizes out, and purified. The alkaloid is made from it by precipitation with ammonia solution ( 2 9 ) . 2- The concentrated acid solution is rendered alkaline with excess ammonia solution, where strychnine and brucine precipitated together. The precipitate is extracted with 25% ethanol which dissolves the brucine, and leaves the strychnine as insoluble residue. The undissolved strychnine is filtered off (30). - Strychnine is purified by repeated crystallization from ethanol.

4. Total Synthesis of Strychnine

The total synthesis of strychnine was achieved in 1954 by Woodward and his associates ( 7, 8 ). Before this several attempts have been made, but these were unsuccessful. Woodward’s synthesis of strychnine stands out as a major synthetic achievement (17,31). Woodward’s total synthesis of strychnine ( 7-9):Acetoveratrone [l] and phenylhydrazine are added to polyphosphoric acid and the mixture is warmed to give 2veratrylindole [ 21. 2-veratrylindole [2] is condensed with formaldehyde and aqueous dimethylamine in dioxan and acetic acid to give 2-veratrylgramine [3]. The methiodide of [3] is converted by sodium cyanide in dimethylformamide to the nitrile, which in turn is reduced by lithium aluminium hydride in hot tetrahydrofuran to 2-veratryltryptamine [4]. This is condensed with ethyl glyoxylate in warm benzene to give

STRYCHNINE

587

t h e corresponding S c h i f f ' s base [5]. The l a t t e r i s treat e d w i t h p - t o l u e n e s u l f o n y l c h l o r i d e i n p y r i d i n e t o produce t h e i n d o l e n i n e [6]. The i n d o l e n i n e [6] i s reduced by sodium borohydride i n e t h a n o l t o t h e corresponding i n d o l i n e which i s converted i n t o t h e N - a c e t y l - i n d o l i n e d e r i v a t i v e [7] by t h e a c t i o n o f a c e t i c anhydride and p y r i d i n e . [7] i s t r e a t e d with ozone i n aqueous a c e t i c a c i d ( o z o n o l y s i s ) t o y i e l d t h e t r i e s t e r [8]. The t r i e s t e r [8] i s r e a c t e d with b o i l i n g methanolic hydrogen c h l o r i d e , cleavage o c c u r s t o g i v e t h e pyridone ester [ 9 ] . T h i s i s t r e a t e d with h o t h y d r i o d i c a c i d i n t h e presence of r e d phosphorus, followed by e s t e r i f i c a t i o n , N - a c e t y l a t i o n and t r e a t m e n t w i t h sodium methoxide i n h o t methanol t o y i e l d t h e c y c l i z e d product [ l o ] . The l a t t e r i s t r e a t e d w i t h p-toluene s u l f o n y l c h l o r i d e i n p y r i d i n e t o y i e l d 0 - t o s y l d e r i v a t i v e which i s react e d w i t h sodium benzylmercaptide t o g i v e t h e 8-benzylmerc a p t o e s t e r . T h i s upon t r e a t m e n t with d e a c t i v a t e d Raney n i c k e l i n h o t e t h a n o l followed by r e d u c t i o n with hydrogen i n t h e p r e s e n c e of palladium on c h a r c o a l g i v e s t h e c i s s a t u r a t e d ester, a l k a l i n e h y d r o l y s i s i s e f f e c t e d followed by a c i d i f i c a t i o n t o give t h e t r a n s a c i d [ l l ] . The a c i d [ l l ] i s r e s o l v e d with q u i n i d i n e and ref luxed w i t h acetic anhydri d e and p y r i d i n e t h e n hydrolysed w i t h aqueous h y d r o c h l o r i c and a c e t i c a c i d s t o produce t h e aminoketone [ 1 2 ] . Oxidat i o n of [12] with selenium d i o x i d e i n e t h a n o l y i e l d s dehydrostrychninone [13]. The l a t t e r i s r e a c t e d w i t h sodium a c e t y l i d e i n t e t r a h y d r o f u r a n followed by r e d u c t i o n w i t h hydrogen i n t h e p r e s e n c e of L i n d l a r palladium ( 3 2 ) to g i v e t h e corresponding v i n y l compound [ 1 4 ] . T h i s i s t r a n s formed by l i t h i u m aluminum hydride i n e t h e r i n t o t h e b a s e [15]. The base [15] i s s u b j e c t e d t o b o i l i n g a t 120' w i t h hydrogen bromide i n a c e t i c a c i d i n t h e presence o f red phosphorus followed by h y d r o l y s i s with b o i l i n g aqueous s u l f u r i c a c i d t o y i e l d i s o s t r y c h n i n e [16]. T h i s upon t r e a t m e n t w i t h e t h a n o l i c potassium hydroxide i s converted i n t o s t r y c h n i n e [ 171.

FARID J. MUHTADI AND MOHAMED S. HIFNAWY

588

The Total Synthesis of Strychnine

O

a

o

C

HOCH3 3

Fischer reaction

*

0ch3

0ch3

[I1

[51

COOCH3

c =o 1

0ch3

STRYCHNINE

589

OH

590

FARID J. MUHTADI AND MOHAMED S. HIFNAWY

LiAIH4

1141

i) H Br ii) H2S04

1171

1151

1161

Degradation Strychnine molecule can be degraded into several products. The most interesting degradative products are those obtained by alkaline degradation and by the action of 20% nitric acid. Alkaline degradation generate seven simple isolable products of known structures (15,16,17, 31,33,34), while 20% nitric acid treatment affords 5,7dinitroindole-2,3-dicarboxylic acid (35). The degradative products are shown below.

STRYCHNINE

59 1

Products of the Alkaline Degradation of Strychnine and the effect of 20% nitric acid.

Qjr$'.". N

\

N02 5,7-dinitroindole,2,3dicarboxylic acid

H

Tryptamine

20%

Alcoholic

HN03

4 -

H/

STRYCHNINE

B-picoline

-+ B-Collidine

Zn dust.

A

H

H

3-ethylindole

H

3-methyl indol e

FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

592

5.

Biosynthesis of Strychnine It has long been assumed that the indolic moiety of the indole alkaloids is derived from the aminoacid "tryptophan" (36-38). Thomas (39) and Wenkert (40) have independently predicted that the non-tryptophan portion of these alkaloids is formed from two mevalonate units to afford a cyclopentane monoterpene. Thomas (41) has further suggested that the glucoside "loganin" could be a key intermediate in the biosynthetic pathway to indole alkaloids. This suggestion has been established upon experimental evidence by several authors (42-50). It is now known that loganin arises in the plants from mevalonate which is transformed by a series of steps to isopentenyl diphosphate (51) and dimethylallyl diphosphate (52). Combination of these leads to geraniol (53,54), then to loganin and finally to secologanin which condenses with tryptophan to afford intermediate condensate to many indole alkaloids (55). Many radioactive precursors were fed into the plants of Stryehnos nm-vornica. Several of those were incorporated into strychnine. These include [2-14C] -tryptophan (47), [l-15N]-tryptophan (56,57), [1-14C]-acetate, [2-14C]~] acetate (47 , [2-14~]-glycine(57-59), [ ~ - 1 4-meValonate ( 47 ) , [2-l C] -geraniol (47), and [2-14C]-geranylpyrophosphate (47). While other precursors were not incorporated into strychnine and among these are [~P~H]. WeilandGumlich aldehyde and [2-14C]-1-acetyl Weiland-Gumlich aldehyde (47). The biosynthetic pathway leading to strychnine can be visualized from two different systems ( 55 ) . The first system involves condensation of tryptamine and secologanin to form a Schiff base which could be attacked intramolecularly by the @-position of the indole nucleus to afford the corresponding spiroindolenins (55 ) . This system is presented in Scheme I. The other system involves condensation of tryptophan and secologanin at the a-position of the indole neucleus to afford 8-carboline system which leads to strictosidine (55 ) . This system is presented in Scheme I1 ( 55,60).

a

Scheme I Tryptomine [l] is condensed with secologanin (Mannich condensation) [Z] to give the spiroindolenine [3]. This undergoes ring opening to give the intermediate [4]. The latter is transformed into the intermediate [S]. Acetate unit is incorporated into [5] to afford [6]. Intramolecular cyclization gives [7], which upon further cyclization and decarboxylation yields strychnine [8].

STRYCHNINE

593

Biosynthesis of Strychnine (Scheme I).

['I

I

o*c

,

H3C0

CH20H CHO OH

FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

594

Biosynthesis of Strychnine (Scheme 11) H

k

OTJH'

OCH3

OCH3

STRYCHNINE

595

J

[91

OCH3

FARID J . MUHTADI AND MOHAMED S. HIFNAWY

596

Scheme I1 Tryptophan [ l ] is condensed with secologanin [2] t o give the condensate [3]. Cyclization of [3] y i e l d s isovincos i d e ( a l s o known as s t r i c t o s i d i n e ) [4]. Further c y c l i z a t i o n and r i n g opening of [4] a f f o r d s pregeissoschizine [S]. This is transformed i n t o preakuammicine [6] which i s converted by a r e t r o - a l d o l process t o akuammicine [7]. Oxidat i o n a t t h e a l l y l i c carbon of [7] a f f o r d s [8]. Incorporat i o n of a c e t a t e u n i t t o [8] y i e l d s [9] which undergoes reductive c y c l i z a t i o n t o produce strychnine [ l o ] . 6.

Metabolism of Strychnine Strychnine i s very r a p i d l y absorbed from the gastroi n t e s t i n a l t r a c t , through mucous membranes, i n t a c t s k i n and from an i n j e c t i o n s i t e (22,24,61). Strychnine t r a v e l s i n both plasma and erythrocytes and approximately 50% of t h e agent w i l l d i f f u s e i n t o t i s s u e s i n f i v e minutes (62). The CNS does not contain higher concentrations than do o t h e r t i s s u e s (61). Strychnine i s r e a d i l y metabolised i n t h e l i v e r by microsoma1 enzymes (61,63), where up to 80% of t h e dose i s oxidized and approximately 10 t o 20% of t h e dose w i l l appear i n t h e 24 hour u r i n e unchanged (24,62), Strychnine i s cumulative and very s t a b l e and may be found i n cadavers exhumed many years a f t e r death (24). Toxi c i t v Poisoning by strychnine s t i l l occurs p a r t i c u l a r l y from rodenticides o r from t h e a d u l t e r a t i o n of s t r e e t - d r u g s with strychnine. Many cases of a c c i d e n t a l poisoning a r e i n c h i l dren who may swallow o r suck strychnine b a i t s . The f a t a l adult o r a l dose is about 50 t o 100 mg of strychnine, but 30 mg has been found l e t h a l (61). The main e f f e c t of strychnine is on t h e s p i n a l cord where i t i n h i b i t s post-synaptic i n h i b i t o r y c o n t r o l , leading t o t e t a n i c convulsions. Death occurs from c e n t r a l r e s p i r a tory failure.

STRYCHNINE

7.

597

Methods of Analysis 7.1

Identification tests

-

-

-

-

-

7.2

The following c o l o r t e s t s a r e used t o i d e n t i f y strychnine. I f a t r a c e (few c r y s t a l s ) of strychnine is dissolved i n few drops of concentrated s u l f u r i c acid and s t i r red with a c r y s t a l of potassium dichromate, a deep b l u i s h v i o l e t c o l o r develops, which gradually changes t o red and f i n a l l y t o orange yellow color. When a t r a c e of strychnine i s t r e a t e d with Mandalin's reagent (Ammonium vanadate i n s u l f u r i c a c i d ) , a blue c o l o r is formed which changes first t o purple then t o red c o l o r ( s e n s i t i v i t y 0.05 ug). Few drops o f fuming n i t r i c a c i d a r e added t o a t r a c e of strychnine, a yellow c o l o r is produced. Brucine gives with n i t r i c acid an i n t e n s e orange red color. Strychnine gives with V i t a l i ' s t e s t a brown c o l o r ( s e n s i t i v i t y 0.5 ug). The t e s t can be performed as follows: Few c r y s t a l s of strychnine a r e dissolved i n few drops of n i t r i c a c i d and the yellow s o l u t i o n is evaporated t o dryness on a water bath, when a l c o h o l i c potash i s added t o the residue, a brown t o purple c o l o r i s developed. When 0- Tosyl-p-phenolsulfonic acid i s added t o c e r t a i n a l k a l o i d s including strychnine, a p r e c i p i t a t e i s obtained which i s c r y s t a l l i z e d from water t o give c r y s t a l s with m.p. of 242-244' (64). When 3-nitro-4-chlora-5-methylbenzene s u l f o n i c a c i d i s added t o some a l k a l o i d s including strychnine, a p r e c i p i t a t e of the s a l t is formed, upon c r y s t a l l i z a t i o n from water, the c r y s t a l s melt a t 265-266' (64). A simple, rapid and s e n s i t i v e technique f o r t h e d e t e c t i o n of strychnine among o t h e r a l k a l o i d s and psychotropic amines i n physiological f l u i d s such as s a l i v a and u r i n e was described, pot. a c i d p h t h a l a t e b u f f e r 0.1 M, pH 4.5, CHC13, and NaOH 0.05 N were used f o r e x t r a c t i o n and bromophenol blue 0.04% i n MeOH f o r c o l o r production. The blue c o l o r i n a l k a l i n e medium was very v i s i b l e . The method was recommended f o r toxicology (65). Microcrystal Formation Strychnine hydrochloride was dissolved i n water (10 mg i n 10 ml), 1 t o 2 drops of t h i s s o l u t i o n was treat e d with t h e reagent on a microscopical g l a s s s l i d e . After s p e c i f i c time, the c r y s t a l s were microscopicall y examined. The c r y s t a l formation i s presented i n t a b l e 7.

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Table 7. Microcrystal Formation of Strychnine

Time

Plate

Shape of t h e Crystal

1

Mayer s

2 - 3 min.

minute p l a t e s i n s t a r l i k e arrangement

2

P 1a t e n i c ch 1o r i d e

2-3 min.

broad p l a t e s , r o s e t t e arrangement

3

Mercuric chloride

10 min.

f i n e curved h a i r y with specif i c arrangement

4

Wagner s

15 min.

f i n e s h o r t rods

5

Dragendor f f

6

Marm s

7

Sodium carbonate

8

Sodium phosphate

10-15 min, c l u s t e r of l a r g e p l a t e s

9

Pot a s sium chromate

immediate

10

Potassi um iodide

3-5 min.

7.3

2-3 min. immediate 10 min.

rods i n c l u s t e r form r o s e t t s of p l a t e s , enlarged by time long rods i n r o s e t t e shape and f r e e arrangement

t h i n r a d i a t i n g rods broad p l a t e s , r a d i a t i n g form

T i t r i m e t r i c Methods The o f f i c i a l methods of determination of s t r y c h nine i n formulations o r i n powdered nux vomica and i t s preparations, by t i t r i m e t r i c methods a r e described i n t h e Egyptian Pharmacopoea (E.P.) 1963 and 1972 (66,67). 7.3.1

Aqueous T i t r a t i o n The E.P. 1963 method f o r determination of strychnine i n nux vomica g a l e n i c a l s is based on t h e oxidative d e s t r u c t i o n of brucine with HN03 and t h e a c i d base t i t r a t i o n of strychnine. Transfer accurate volume (20 ml) of nux vomica e x t r a c t t o a s e p a r a t o r , d i l u t e with about 20ml H20, a c i d i f y with d i l u t e H2SO4 (about 5 ml) and e x t r a c t with CHC13 (2x20 ml). Receive t h e

STRYCHNINE

Platc 1 : hlicrocrystalr of strychninc w i t h Hayer’s rcagcnt

Plate 2 : Microcrystals of strychnine with I’latinic c h l o r i d e

Plate 4 : Microcrystals of strychnine with Wagner‘s reagent

599

600

FARID J. MUHTADI AND MOHAMED S. HIFNAWY

Plate 5 : Microcrystals o f strychnine with Uragendorff's reagent

Plare 7 : Flicrocrystals of strychnine with Sodium carbonate

I'latc 6 : L1icrocryst;ils o f strychninc with Llnrm's rcngent

STRYCHNINE

P l a t e 8 : M i c r o c r y s t a l s of s t r y c h n i n e w i t h Sodium phosphate

P l a t c 9 : b l i c r o c r y s t a l s of s t r y c h n i n e n i t h P o t a s s i u m chromatc

60 1

602

FARID J. MUHTADI AND MOHAMED S. HIFNAWY CHC13 i n t o another separator containing 10 m l N H2SO4, shake, r e j e c t t h e CHC13 and add t h e a c i d washings t o t h e first separator. Render alkaline with ammonia and e x t r a c t the l i b e r a t e d a l k a l o i d s completely with successive port i o n s of CHC13 (3x20 ml). Wash t h e CHC13 e x t r a c t with 5 m l H20, dehydrate over anhydrous Na2S04 and d i s t i l l t h e CHC13 completely. Dissolve the residue i n 15 m l of 3%w/v H2SO4, warm i f necessary, cool, then add 2 m l concd. HNO3 and few c r y s t a l s of NaN02, allow t o s t a n d f o r 30 min. a t ordinary temperature. Transfer the contents t o a separator containing 30 m l of 10%NaOH, r i n s e t h e container with 2 successive portions each of 5 m l H20, add t h e r i n s i n g t o the separator and shake f o r about 2 min. Extract strychnine with CHC13 (3x10 ml). Wash t h e combined CHCl3 e x t r a c t with 2x10 m l H2O. Dehyd r a t e the CHC13 over anhydrous Na2S04. Evapor a t e the CHC13, add t o t h e residue 5 m l n e u t r a l 95% alcohol, evaporate and leave t h e residue on a b o i l i n g water bath f o r f u r t h e r 15 min. Dissolve t h e residue i n about 2 m l CHC13, add 20 m l N/20 H2SO4, evaporate CHC13 completely, then t i t r a t e excess a c i d with N/20 NaOH using methyl red as i n d i c a t o r . Each 1 m l N/20 H2SO4 E 0.01672 g strychnine. The r e s u l t i s multip l i e d by 1.02 t o c o r r e c t f o r the l o s s of s t r y chnine during oxidative d e s t r u c t i o n of brucine. Another method based on the same p r i n c i p l e was a l s o reported (68). A method f o r simultaneous determination of strychnine and brucine was presented (69). A s u i t a b l e vol. of an aq. soln. of the 2 a l k a l o i d s , made a l k . with N a O H , i s e x t r a c t e d first with E t 2 0 , then with CHC13, and t h e combined e x t r a c t s , d r i e d with anhydrous Na2S04, is f i l t ered i n t o a t a r e d f l a s k . After evaporating t h e s o l u t i o n , the combined weight of brucine and strychnine i s determined, following which t h e residue is dissolved i n alcohol and H20 and t i t r a t e d with 0.01 N HC1, Another method (70) f o r determination o f micro amounts of strychnine using heteropoly a c i d s was described. T i t r a t e 20 m l of 0.001 - 0.002 M s o l u t i o n of strychnine,containing 1 m l of 1 M HC1 (or 2 m l of M NaOAC) with 0.00125 - 0.005 M t u n g s t o s i l i c i c , tungstophosphoric o r molybdophosphoric

STRYCHNINE

603

acids. The molar ratios of the stoichiometric compounds formed in pH 1 (HC1) o r pH 7 (NaOAC) solutions are respectively : with tungstosilicic acid 4, 4; with tungstophosphoric and molybdophosphoric acids 3, A method for determination of strychnine nitrate by titration with silicatungstic acid was also reported (71). Another method (72) for rapid titrimetric determination of strychnine with 2 1% accuracy is based on addition of CHC13 and a buffer solution to the base dissolved at pH 2 . 8 . Titrate with Na dioctylsulfosuccinate (dimethyl yellow screened with oracet blue as indicator). The color change is from green to pink. Satisfactory results were obtained for the determination of strychnine nitrate by using K-Bi complex; the liberated EDTA is titrated with 0.01 M ZnSOq with the Nag B03 buffer (73). A direct amperometric titration of amines with sodium tetraphenylborate (Na B Ph4) solution, based upon anodic depolarization at a dropping Hg electrode (74), was found suitable for determination of strychnine. A micro-procedure, permitting determination of strychnine in mg rang was described ( 7 5 ) . A small separatory funnel ($ 50 ml capacity) is used to shake out the sample with 2 ml of 10% NH40H, then with CHC13. The CHC13 extract is transferred to a second small separatory funnel, washed with H20, placed in a 50 ml (or less) Erlenmeyer, dried, treated with 0.5 ml of 95% alcohol and with 2.0 ml of 0.01 N H2SO4, heated gently to aid solution and the excess acid titrated against 0.01 N NaOH from a 2 ml microburet, using 1 drop of methyl red solution as indicator. Good accuracy was obtained with strychnine - HC1 and a mixture of strychnine salt with Na methyl arsenate. The use of 2 , 5-dimethylbenzene sulfonic, 3, 4-dichlorobenzene sulfonic and 4-(benzene sulfonyloxy) benzene sulfonic acids (76) and 3 methyl-6-nitrobenzene sulfonic acid, 2 , 5dichlorobenzene sulfonic acid (77 ) were used for titrimetric determination of strychnine in injection solutions, giving reproducible results and lower mean standard deviation.

-.

FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

604

Another method depends on the p r e c i p i t a t i o n of strychnine from an a c i d i f i e d d i l u t e s o l u t i o n with K3 C r (SCN)6. The excess reagent hydrolyzed i n t h e f i l t r a t e by a l k a l i n i z a t i o n and SCN determined by t i t r a t i o n with Br03 The reagent/strychnine r a t i o i n t h e ppt. was 1 : 3 , r e l a t i v e deviation f 0.3 - f 0 . 7 % (78). Other method f o r t i t r i m e t r i c determination of strychnine a t p o l a r i z a t i o n p o t e n t i a l s of up t o 1000 mv was reported ( 7 9 ) A more recent method f o r photometric t i t r a t i o n of strychnine n i t r a t e was described ( 8 0 ) . The a l k a l o i d a l s a l t i s mixed with 1 N HC1, d i l u t e d with H20, and t i t r a t e d with tungstophosphoric a c i d with photometric end p o i n t indication. 7.3.2

Non-Aqueous T i t r a t i o n The Egyptian Pharmacopoeia (1972) describes the following method: Dissolve 0.5 g strychnine hydrochloride i n 30 m l a c e t i c anhydride. Add 10 m l HgC12 and 20 m l dioxane. Using 2-3 drops of c r y s t a l v i o l e t reagent, t i t r a t e with 0.1 N HClO4. Carry a blank experiment and c a l c u l a t e the mls of HC104 consumed; l m l 0.1 N HC104 5 0.03343 g strychnine. Another method based on t i t r a t i o n of s t r y c h nine with 0.1 N of hydrochloric a c i d complex of chloroaluminium isoproxide i n CHC13 ( 81 ) The deviation was f 1 % i n the range of 38-245 mg of a l k a l o i d . A t h i r d method f o r determination of s t r y c h nine n i t r a t e i n j e c t i o n s i n an anhydrous medium was presented (82 ) . Adjust t h e pH of the i n j e c t i o n s o l u t i o n t o 8-9 with NaHC03. Extract 3 times with CHC13, f i l t e r the e x t r a c t and titrate i n t h e presence of dimethylyellow by 0.005 N p-toluenesulfonic a c i d i n dioxane. Alkaloids i n t i n c t u r e of nux vomica were determined i n non aqueous media, by t h e i r l i b e r a t i o n with NH40H, e x t r a c t i o n with CHC13 and t i t r a t i o n with 0.02 N HClo4 i n dioxane i n t h e presence of methyl red i n PhCl ( 83) ; 1 m l 0.02 N H C 1 a i s equivalent t o 6.688 mg a l k a l oids (strychnine + brucine). Another method f o r non aqueous determinat i o n of strychnine was reported ( 8 4 ) . The

.

STRYCHNINE

605

following a c i d s i n 0.005 N dioxane s o l u t i o n s a r e used as t i t r a n t s : naphthalene s u l f o n i c , naphthalene-2-sulfonic, 5-nitro-naphthalene-6s u l f o n i c , and 2-propoxy~1aphthalene-6-sulfonic. The t i t r a n t s contained 1%phOH and were standardized against atropine o r brucine dissolved i n CHC13 using 0.1% dimethyl yellow a s i n d i c a t o r . For the determination, 5 m l of a s o l u t i o n t o be analyzed were taken (containing about 5 mg alkal o i d a l s a l t ) . i t s pH adjusted t o 8-9 with s a t u r a t e d NaHC03 s o l u t i o n o r 5% NaOH, and then e x t r a c ted 4-5 times with 5-10 m l CHC13 each. The combined e x t r a c t s a r e f i l t e r e d and t i t r a t e d ; t h e e r r o r was <-I 1%. T i t r a t i o n i n non aqueous media was a l s o adopted f o r determination of strychnine and other a l k a l o i d s i n e l i x i r t o n i c and Cola e x t r a c t ( 8 5 ) . The preparations a r e e x t r a c t e d with CHC13 - E t O H (5:1), and the CHC13 phase i s separ a t e d by TLC on s i l i c a gel DG p l a t e s using E t O H CHC13 (88:12) f o r development. The strychnine spot was i d e n t i f i e d by sequential treatment with alcoholic KI, 25% HC1 - 95% E t O H ( l : l ) , and Dragendorff reagent. For q u a n t i t a t i v e determination, t h e drug i s e x t r a c t e d with CHC13 - E t O H (20:3) and the CHC13 phase was mixed with A C 2 0 , d i l u t e d with CgHg and t i t r a t e d with 0.1 N H C l O 4 , using c r y s t a l v i o l e t i n d i c a t o r , The r e l a t i v e standard deviation i s 3%. 7.3.3

Potentiometric T i t r a t i o n s Strychnine among o t h e r a l k a l o i d s was investo t i g a t e d i n t h e concentration range M with potentiometric t i t r a t i o n ( 8 6 ) . An adsor p t i o n microelement Sb/gel/K C l (0.1 N ) , Hg2 C 1 2 /Hg connected with a lindemann quadrant e l e c t r o meter was used. The g e l layers contacting t h e i n d i c a t i n g e l e c t r o d e with the reference one were made of acacia gum i n 0.05 N KC1. The t i t r a t i o n was c a r r i e d out i n petroleum e t h e r , C C 1 4 , cyclohexane, CgHg, and acetone. A s a t i t r a n t , C13 C C02H o r p i c r i c a c i d i n t h e same solvent as t h e t i t r a t e d a l k a l o i d was used. The experimental conditions a r e most favorable when the d i f f e r ence between t h e d i e l e c t r i c constants of t h e solvent and t h e gel adsorption l a y e r i s large.

-

FARID J. MUHTADI AND MOHAMED S. HIFNAWY

606

Another method described t h e determination of strychnine n i t r a t e potentiometrically by t i t r a t i o n with 2.5% Na tetraphenylborate using valinomycin ion s e l e c t i v e e l e c t r o d e ( 8 7 ) . A new e l e c t r o d e was developed which was s e n s i t i v e and reasonably s e l e c t i v e f o r s t r y c h nine ( 8 8 ) . I t is based on the use of t h e strychnine-picrolonate ion-pair complex i n n i t r obenzene a s a l i q u i d membrane. The e l e c t r o d e shows Nernstian response down t o lO-5M s t r y c h nine s o l u t i o n , over a pH range of 2-7 with a c a t i o n i c slope of 57 mV/concn. decade from 5x10-2 t o 5x10-5M. Direct poteniometric d e t e r mination of samples down t o 5 ppm shows an average recovery of 101.3% and a mean standard devia t i o n of 2.4%. Poteniometric t i t r a t i o n with e i t h e r p i c r o l o n i c a c i d o r Na tetraphenylboron shows an average recovery of 99.1% and a mean standard deviation of 1.2%. No i n t e r f e r e n c e s were caused by o t h e r a l k a l o i d s and many of t h e substances normally occurring as n a t u r a l cont aminant s with strychnine Another method (89 3 f o r the potentiometric determination of strychnine was presented. I t is based on the formation of insoluble a l k a l o i d p i c r a t e s a l t using a p i c r a t e ion-selective i n d i c a t i n g electrode. The average e r r o r was about 4%. The method was s u c c e s s f u l l y applied t o pharmaceutical preparations. Determination of strychnine by a s e l e c t i v e l i q u i d membrane e l e c t r o d e was a l s o described ( 90 ) The electrode s t r y c h n i n e - t e t r a (m-methyl phenyl) borate was prepared from strychnine and the borate compound. The c a l i b r a t i o n p l o t was l i n e a r i n t h e range 1 ~ 1 0 t- o~ 5 ~ 1 0 ' ~ M and , the lowest d e t e c t i o n l i m i t was 1x10-6M. Optimal pH f o r t h e determination of strychnine a t 10-5 t o 10-2M was 2-6.

.

.

7.4

Compleximetric Determinations A method f o r t h e gravimetric determination of s t r y chnine s u l f a t e was described ( 9 1 ) . The method based on conversion of the a l k a l o i d a l s a l t t o t h e corresponding hexabromotellurate by r e a c t i o n with hexabromotellu r i c acid. A second method based on the same p r i n c i p l e was described f o r strychnine a n a l y s i s and c o n t r o l ( 9 2 ) .

STRYCHNINE

607

The s t r y c h n i n e s u l f a t e i s d i s s o l v e d i n HC1 and p r e c i p i t a t e d as h e x a c h l o r o t e l l u r a t e s w i t h H2 ( T e c l g ) . The ppt. i s washed with HC1, d r i e d a t 50°C and weighed. The e r r o r of t h e g r a v i m e t r i c method was 2 0.75%. Other methods f o r g r a v i m e t r i c and t i t r i m e t r i c d e t e r m i n a t i o n s o f s t r y c h n i n e a r e p r e s e n t e d and d i s cussed (93). 7.5

Spectrophotometric Methods 7.5.1

Colorimetry A c o l o r i m e t r i c procedure was d e s c r i b e d f o r t h e a s s a y of s t r y c h n i n e n i t r a t e i n t a b l e t s containing codeine base and o t h e r i n g r e d i e n t s (94 ) . The a s s a y based on t h e i n t e r a c t i o n o f s t r y c h n i n e reducing p r o d u c t s and HN02 y i e l d i n g a measurable r e d c o l o r . The t a b l e t a l k a l o i d s a r e E t 2 0 - e x t r a c t e d and s t r y c h n i n e i s d e t e r mined c o l o r i m e t r i c a l l y . The accuracy was 1%. A second c o l o r i m e t r i c method f o r determinat i o n o f s t r y c h n i n e and b r u c i n e i n t h e i r m i x t u r e s was p r e s e n t e d ( 9 5 ) . Strychnine was determined a f t e r t h e s e p a r a t i o n o f b r u c i n e . Add H2SO4 (3%, 7.5 ml) t o t h e mixture a l k a l o i d s '(- 1 mg), c o o l , add 1.5 m l o f a mixture of HNO3, d . 1.42 - H20 (1: 1) , make t h e mixture a l k a l i n e w i t h NaOH a f t e r 10 min, e x t r a c t s t r y c h n i n e with CHC13, (3x10 ml), e v a p o r a t e t h e CHC13 e x t r a c t t o d r y n e s s , add 4 m l H20, 6 m l HC1 (d. 1.12), and 1 g Zn d u s t t o t h e r e s i d u e , h e a t t h e mixt u r e on a b o i l i n g water b a t h f o r 20 min. and c o o l . Add NaN02 ( l o % , 1 drop) t o t h e mixture, d i l u t e t o 50 m l and measure t h e o p t i c a l d e n s i t y of t h e s o l u t i o n on a p h o t o e l e c t r i c c o l o r i m e t e r u s i n g a green f i l t e r . The e r r o r o f t h e method was 2 0.76%. A t h i r d method f o r s e p a r a t i o n and colorimet r i c e s t i m a t i o n o f s t r y c h n i n e , among o t h e r s t i mulants, i n doping h o r s e u r i n e was r e p o r t e d ( 9 6 ) . S t r y c h n i n e i s s e p a r a t e d from o t h e r i n g r e d i e n t s by TLC on s i l i c a g e l G p l a t e s u s i n g MeOH as a developer. The corresponding s p o t i s e l u t e d and determined c o l o r i m e t r i c a l l y . 18% o f t h e a d m i n i s t e r e d s t r y c h n i n e , was d e t e c t e d i n t h e u r i n e o f t h e doped h o r s e s w i t h i n 48 h after a d m i n i s t r a t j o n .

608

FARID J. MUHTADI A N D MOHAMED S. HIFNAWY A f o u r t h method was described f o r c o l o r i metric estimation of strychnine i n b i o l o g i c a l preparations ( 9 7 ) . Strychnine i s separated by ascending paper chromatography by using BuOH-ACOH-H20 (4:1:5) a s a solvent and BiI3 as a developer. The a l k a l o i d i n t h e s p o t s i s separated by a c i d i f y i n g with 0.1 N HC1 and d e t ermined c o l o r i m e t r i c a l l y with bromothymol blue. After i n v e s t i g a t i n g 13 l e t h a l cases of poisoning, i t was found t h a t i n organs preserved with HCOzH, the amounts of strychnine a r e f a r lower than i n the same organs which were not preserved. A c o l o r i m e t r i c method f o r determination of t h e sum of strychnine and brucine i n seeds and galenic preparations of nux vomica was discussed ( 9 8 ) . A 0.01 g sample of f i n e l y ground seeds a r e shaken, 30 min, with 5 g CHC13, 10 g Et20, and 2 m l 10% Na2C03. The organic solvent evaporated t o dryness, the residue dissolved i n phosphate b u f f e r , pH 5.5, t o make 100 m l . A 1 m l a l i q u o t shaken 5 min with 20 m l C6&, 2 m l phosphate b u f f e r , pH 5.5, and 3 m l s a t u r a t e d s o l u t i o n of bromothymol blue. The developed c o l o r i n t h e organic l a y e r i s measured c o l o r i m e t r i c a l l y . The same procedure was used f o r determination of t h e a l k a l o i d s i n dry e x t r a c t s and t i n c t u r e s prepared from nux vomica. Two o t h e r methods f o r colorimetric determination of strychnine were developed; one (99) was a modification f o r t h e o t h e r (100). The modification r e s u l t e d i n increasing the s e n s i t i v i t y of t h e assay. Evaporate 10 m l nux vomica t i n c t u r e o r 1 m l of i t s e x t r a c t t o dryness. Extract residue with 1%aqueous H2SO4. Transfer the aqueous e x t r a c t i n t o separatory funnel t h r ough f i l t e r paper and wash w i t h 2 m l d i l H2SO4. Add aqueous washings t o mother l i q u o r and render a l k a l i n e t o litmus with 10% NH40H s o l u t i o n (Ca 5 ml). Shake with successive portions of CHC13 u n t i l a l k a l o i d s a r e completely extracted. Evaporate combined CHC13 e x t r a c t (3x20 ml) and d i l u t e t o 25 m l with the same solvent. Take 0.5 m l a l i q u o t s f o r t h e proposed c o l o r i m e t r i c method a s follows: Evaporate t h e CHC13 solut i o n t o dryness on a water bath, add 5 m l HC1 and 1 g granulated zinc, heat 10 min i n a b o i l ing water bath o r l e t stand 20 min a t room t e m p e r a t u r e , and then d i l u t e with 5 m l d i s t i l l e d

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STRYCHNINE

609

water. Cool to room temperature, transfer quantitatively to 25 ml volumetric flask, rinse container several times with small portions of distilled water, and dilute to volume with distilled water. To different aliquots of the reduced solution (containing -1OOpg strychnine) add 3 ml nitroprusside-acetyldehyde reagent (10% acetaldehyde in 1% sodium nitroprusside) and adjust the volume to 12 ml with distilled water. Let mixture stand 15 min and measure color at 530 nm against blank of 9 ml water and 3 ml nitroprunide-acetaldehyde reagent. The percentage was calculated from a standard curve of strychnine. The developed color obeyed Beer's law in the range of 10-320,ug/12 ml. The standard deviation was 1.02. A fast identification and determination method for strychnine in heroin specimen was reported (101). It depends on measuring the pink color developed after boiling with HgClZ and Zn and addition of NaNo2. Two spectrophotometric procedures depending on complexation of strychnine with hexabromotelluric acid (91 ) or hexachlorotelluric acid ( 9 2 ) were presented. The ppt. is dissolved in MeOH and strychnine determined photometrically at 450 nm for the first complex and at 420 nm for the second complex. The error of the second process was < 1.6%. A method forthe extractive spectrophotometric determination of strychnine with Solochrome Green V150 was presented (102). The method depends on extracting strychnine 1:2 ion pair with Solochrome Green V150 into CHC13. The absorbance of the organic phase is measured at 520 nm (molar absorptivity = 3.7200~104). Beer's law was obeyed for 1-300,ug strychnine; the error is f 1.5%. The differential acid dye method for determination of different pharmaceutical compounds was successfully applied for the determination of strychnine (103). The method involves extraction of a mixture of 5 ml pH 4.2 K H phthalate buffer, 5 ml Methyl orange, and 3 ml of the alkaloidal solution of a suitable concentration, with CHC13, followed by absorbance measurement of the pooled extract against a reference extract. It was reported that the results obtained

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FARID J. MUHTADI AND MOHAMED S. HIFNAWY

by traditional and differential acid dye method were comparable and the reproducibility of the latter method was higher. A method for spectrophotometric determination of strychnine nitrate and other alkaloids was presented (104). Strychnine nitrate is dissolved in acetate buffer pH 4.1-4.2, Chromazurol S is added, after 30 min the reaction mixture is extracted with CHC13 and optical density is measured at 510 nm. Relative error of the determination was ? 1-3%. Chromazurol S reacted also with 34 other alkaloids. A spectrophotometric titration method was described for the determination of some alkaloids and their dosage forms using 0.005 M chloranilic acid solution in 1, 4 dioxane as the titrant (105). The end point is determined by measuring the change in absorbance of the sample at 535 nm. Quantitative recoveries with good reproducibility were reported for strychnine. Comparati.ve study on various sulfonic azodyes for alkaloids analysis (106) showed that Litol red was the most suitable for colorimetric determination of strychnine by ion-pair extraction in CHC13. Seven azo dyes including orange I1 were evaluated for the spectrophotometric determination of alkaloids and compared with the two most common dyes for such determinations, methyl orange (11) and bromothymol blue (107). Orange I1 was more sensitive for strychnine determination in blood and urine and tolerated greater amounts of extraneous ions (such as , C~04~-). Mg2+, Co2+, h 2 +F-, Another method for colorimetric determination of strychnine with chloranilic acid was described (108): in dioxane - CHC13 medium, the acceptor chloranilic acid forms purple 1:l mol. complexes with the alkaloid which gave maximum absorption at 535 nm. Strychnine was determined in 4 pill and powder preparations by TLC-Colorimetry (109). As an example, a pill preparation is powdered, extracted with CHC13-EtOH (10 : 1) and NH40H and the extract is chromatographed on a silica gel G plate (toluene-acetone-EtOH-NH40H, 8:6:0.5:2 as eluent). Spots are visualized with iodine

STRYCHNINE

611 vapor and e x t r a c t e d t o g i v e a f r a c t i o n , which i s t r e a t e d w i t h bromothymol b l u e and benzene. The o r g a n i c phase t r e a t e d w i t h KOH-EtOH i s analyzed a t 610 nm f o r t h e d e t e r m i n a t i o n o f s t r y c h n i n e . The c a l i b r a t i o n p l o t was l i n e a r up t o 4 Q y g and t h e recovery was 89.84 - 93.96%; r e l a t i v e s t a n d a r d d e v i a t i o n s were 2.59-4.80%. A method €or s e m i 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 s t r y c h n i n e i n 10-50 y q u a n t i t i e s by t h e Weisz r i n g oven c o l o r i m e t r i c procedure was proposed (110). Another c o l o r i m e t r i c d e t e r m i n a t i o n o f s t r y chnine was r e p o r t e d . I t i s based on t h e d e v e l opment o f i n t e n s e l y c o l o r e d r a d i c a l i o n s due t o the reaction of the alkaloid with s e l e c t e d polyhaloquinone and polycyanoquinane n- a c c e p t o r (111).

7.5.2

U l t r a v i o l e t Spect ropho t omet ry . (UV) The B.P. (1973, 1980) (112,113)describe a method o f a s s a y i n g s t r y c h n i n e i n nux vomica s e e d s and g a l e n i c f o r m u l a t i o n s a s f o l l o w s : Mix 0.4 g, i n f i n e powder, with 2 m l of a l c o h o l (70%). Add 5 m l of d i l u t e ammonia s o l u t i o n and t r a n s f e r with t h e a i d of 25 m l of H20 and 20 m l o f CHC13 t o a 60-ml ground g l a s s , downward d i s placement, l i q u i d - l i q u i d e x t r a c t o r c o n t a i n i n g 60 m l of CHC13, t h e e x t r a c t o r being equipped with a d i s t r i b u t o r of f o u r b a f f l e d i s c s and a 100-ml short-necked f l a s k . Ensure t h a t a l l lumps a r e broken down and t h a t no powder adher e s t o t h e t o p of t h e d i s t r i b u t o r . Attach a r e f l u x condenser and e x t r a c t f o r 4 h w i t h occa s i o n a l s w i r l i n g of t h e c o n t e n t s of t h e e x t r a c t o r . T r a n s f e r t h e CHC13 i n t h e f l a s k t o a s e p a r a t i n g f u n n e l and e x t r a c t w i t h 4 q u a n t i t i e s , each o f 20 m l , o f 0.5M H2SO4. Wash t h e combined a c i d e x t r a c t s w i t h 10 m l of CHC13. F i l t e r t h e aqueous s o l u t i o n through a small wet p l u g of c o t t o n wool i n t o a shallow d i s h and w a r m g e n t l y with s t i r r i n g t o remove t r a c e s o f CHC13. Cool and d i l u t e t o 100 m l with N H2SO4. D i l u t e 20 m l t o 100 m l w i t h N H2S04 and measure t h e e x t i n c t i o n of a 1-cm l a y e r a t 262 nm and 300 nm. C a l c u l a t e t h e % o f s t r y c h n i n e from t h e e x p r e s s i o n S(0.321 a - 0.467 b)/w, where a i s t h e e x t i n c t i o n a t 262 nm, b i s t h e e x t i n c t i o n a t

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300 nm, and w i s the weight of t h e powder taken. For t h e assay of nux vomica l i q u i d e x t r a c t The method mentioned f o r t h e powder was adopted, e x t r a c t i n g 2.0 m l of t h e l i q u i d e x t r a c t , omitting the 2 m l of alcohol 70%, and t a k ing 5 m l of t h e acid e x t r a c t i v e f o r t h e preparat i o n of t h e f i n a l s o l u t i o n . Calculate the % w/v of strychnine from t h e expression : 20(0.321 a - 0.467 b ) / v , where a i s the e x t i n c t i o n a t 262 nm, b i s t h e e x t i n c t i o n a t 300 nm, and v i s t h e volume of l i q u i d e x t r a c t . For nux vomica t i n c t u r e c a r r y out t h e assay as described under t h e powder, e x t r a c t i n g 5.0 m l of the t i n c t u r e and omitting t h e 2 m l of alcohol 70%. Calculate the percentage w/v of strychnine from t h e expression : S(0.321 a 0.467 b ) / v , where a i s the e x t i n c t i o n a t 262 nm, b i s t h e e x t i n c t i o n a t 300 nm, and v i s t h e volume of t h e t i n c t u r e . A second method making use of the absorbance a t t h e forementioned wave lengths was presented (114). I t depends on complexation of both strychnine and brucine with methyl orange a t pH 5.0, and a f t e r treatment with 0.1 N NaOH, t h e l i b e r a t e d a l k a l o i d s a r e determined spectrophotometrically. The method was c a r r i e d out as follows : Dilute 10 m l t i n c t u r e nux vomica (0.125% w/v) t o 100 m l with Mcllvaine's b u f f e r , (prepared by a d d i t i o n of 48.50 m l 0.1 M c i t r i c a c i d + 51.50 m l 0.2 M Na2HP04. 2H20 and a d j u s t i n g pH t o 5 with a s u i t a b l e pH meter). Accurately t r a n s f e r 2 m l d i l u t e d sample t o 50 m l separatory funnel containing 10 m l dye s o l u t i o n i n b u f f e r . Shake 1 min with 10 m l CHC13; l e t l a y e r s separate completely. Transfer CHC13 layer t o another 100 m l separatory funnel cont a i n i n g 10 m l 0.1 N NaOH. Extract aqueous l a y e r 3 times each with 10 m l CHC13. Shake combined CHC13 e x t r a c t s 15 s e c t o l i b e r a t e t h e dye. Transfer CHC13 layer t o 100 m l separatory funn e l containing 10 m l 0.1 N H2SO4. Shake, transf e r a c i d e x t r a c t of t h e a l k a l o i d s t o 25 m l volumetric f l a s k , and d i l u t e t o volume. Measure absorbance of 1 cm pathlength a t 262 and 300 nm. Calculate concentration (mg %) of strychnine, Ca, and brucine, Cb i n the t i n c t u r e from t h e following equation:

STRYCHNINE

613 Ca =[(0.200 A 1 - 0.290 A2)/0.5955]X (1/4) x (100/2) x (10) Cb =[(0.305 A2 - 0.005 A1)/0.05955] x (1/4) x (100/2) x (10) where A 1 and A2 a r e absorbances of f i n a l solut i o n ; 0.305 and 0.005 a r e absorbances of 1 mg % strychnine; 0.290 and 0.200 a r e absorbances of 1 mg % of brucine, a t 262 and 300 nm, respec t i v e l y . The mean percentage recovery was 100.7+0.93 f o r strychnine. Different o t h e r spectrophotometric procedu r e s have been reported f o r the UV spectrophotometric determination of strychnine i n crude drugs o r formulations. An o l d method makes use of t h e absorption band a t 254 nm shown by strychnine i n absolute alcohol (brucine shows maxima a t 264 nm) (115). The a l k a l o i d e x t r a c t , containing both s t r y c h nine and brucine, i s t r e a t e d with 0.5 g of potassium p e r s u l f a t e i n the presence of 3% H2SO4 f o r 1 h, thereby brucine i s destroyed while strychnine i s not. The s o l n . i s then made a l k a l i n e and t h e strychnine e x t r a c t e d with CHC13. The residue l e f t a f t e r evaporation of CHC13 i s dissolved i n absolute a l c . and t h e e x t i n c t i o n (E) a t 254 nm of t h e s o l n . i s measured. This E value is divided by E 1%a t 254 nm (which i s equal t o 390) gives the concentrat i o n , as % of strychnine i n t h e a l c o h o l i c soln. being measured. Constituents which may absorb a t 254 nm, i f not properly removed would of course i n t e r f e r e with accurate q u a n t i t a t i v e estimation. An ammoniacal suspension of t h e drug i s e x t r a c t e d with CHC13 i n a downward displacement l i q u i d - l i q u i d e x t r a c t o r . Strychnine and brucine a r e then e x t r a c t e d from t h e CHC13 with N H2SO4 and the strychnine determined spectrophotometrically (116). I t was reported t h a t t h e obtained r e s u l t s a r e i n agreement with t h e o f f i c i a l methods but saving h a l f t h e determinat i o n time. The two wave length procedure of a n a l y s i s was a l s o applied f o r determination of mixtures of strychnine and brucine a t 252 and 262 nm (117) and a t 255 and 264 nm i n nux vomica t i n t u r e (118). The l a t t e r method involves

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p u r i f i c a t i o n by adsorption on alumina and by c a t i o n exchange r e s i n . A method f o r t h e q u a n t i t a t i v e determination of strychnine i n brucine o r brucine s u l f a t e was described (119). Brucine i s n i t r a t e d and s t r y chnine i s e x t r a c t e d with CHC13. The CHC13 is then evaporated and strychnine i s determined spectrophotometrically by d i s s o l v i n g t h e residue i n 0.1 N H2SO4 and measuring absorbances a t 254 and 275.5 nm. The d i f f e r e n c e between these absorbances, compared with s i m i l a r d i f f erences a t these wave lengths f o r standard strychnine s o l u t i o n s i s a measure of t h e s t r y chnine content; standard deviation was l e s s than 0.03%. A combined chromatographic and spectrophotometric method f o r determination of strychnine t i n c t u r e was described (120). After paper chromatography and development with the Dragend o r f f reagent, t h e spot i s e x t r a c t e d from a p a r a l l e l undeveloped chromatogram with 0.1 N HC1 and t h e amounts of strychnine present determined using t h e absorption of l i g h t a t 300 nm. An u l t r a v i o l e t determination of strychnine i n commercial b a i t formulations was developed (121). The strychnine i s e x t r a c t e d i n 0.5% (vol.) H2SO4 and the r e s u l t i n g e x t r a c t i s cleaned up. Strychnine i s determined by t h e d i f f e r e n c e i n absorbance a t 254 and 287 nm. I t was reported t h a t t h e r e s u l t s obtained by t h i s method c o r r e l a t e well with those obtained by a gravimetric determination of strychnine as t h e p i c r a t e. Spectrophotometric determination of s t r y c h nine and brucine (100-500,ug) from a c i d i c s o l u t i o n s was c a r r i e d out a f t e r p r e c i p e t a t i o n with the Mayer-Valser reagent. The ppt. a l k a l o i d H I + HgI i s dissolved i n 3 M AcOH and t h e a l k a l o i d determined i n d i r e c t l y over Hg-dithizon (122). UV spectrophotometry was a l s o used t o follow t h e decomposition of c e r t a i n a l k a l o i d s , including strychnine, (123) , on s t e r i l i z a t i o n of t h e i r 1%aqueous s o l u t i o n s by autoclaving 20 min and 2,5,7 and 10 h a t 120'. Two-dimensional t h i n l a y e r chromatography i s used as a reference check. UV spectrophotometric d e t e r mination was accurate with samples heated up

STRYCHNINE

615 t o 2 h ; prolonged h e a t i n g r e s u l t e d i n t h e formation o f p p t s . i n t e r f e r r i n g w i t h t h e d e t e r minat i o n . S t r y c h n i n e was determined p h o t o m e t r i c a l l y a t 365 nm by r e a c t i o n with I2 i n C2HqC12 (124). The r e s u l t s are r e p r o d u c i b l e and a c c u r a t e . A method f o r t h e q u a n t i t a t i o n o f s t r y c h n i n e depends on t h e s p e c t r o s c o p i c c h a r a c t e r i s t i c s o f t h e i n t e r a c t i o n o f a l k a l o i d s with p i c r o l o n i c a c i d i n s o l v e n t of low and i n t e r m e d i a t e p o l a r i t y was s t a t e d (125). The p r e s e n c e o f a l k a l o i d s caused a r e d s h i f t of t h e 322 nm band o f t h e nonionized p i c r o l o n i c a c i d t o 355 nm, c o r r e s ponding t o t h e a n i o n i c resonance band. Conside r a b l e i n c r e a s e i n a b s o r b t i v i t y which was dependent on b o t h t h e b a s i c i t y and molar c o n c e n t r a t i o n o f a l k a l o i d s p r e s e n t was used f o r t h e d e t e r m i n a t i o n of v a r i o u s a l k a l o i d a l s a l t s i n c l u d i n g s t r y c h n i n e s u l f a t e . The method was s e n s i t i v e t o 2,ug/ml with accuracy of 21.5% and s t a n d a r d d e v i a t i o n k1.05-1.31. Quantitative determination of strychnine n i t r a t e and codeine i n p i l l s c o n t a i n i n g e x c i p i e n t s of p l a n t o r i g i n was r e p o r t e d (126). I t depends on e x t r a c t i o n of t h e a l k a l o i d w i t h CHC13, p u r i f i c a t i o n by p a s s i n g through A1203 column and r e - e x t r a c t i o n o f s t r y c h n i n e from C H C 1 3 i n t o 0.25% H2SO4. Chromatography on s i l i c a gel p l a t e s u s i n g CHC13 - a c e t o n e - NH3 (12:24:1) was t h e f i n a l s t e p i n p u r i f i c a t i o n of t h e a l k a l o i d . The s u i t a b l e wave l e n g t h f o r d e t e r m i n a t i o n of s t r y c h n i n e was 254 nm. S t r y c h n i n e n i t r a t e was determined i n c a p s u l e s w i t h & 21.52% e r r o r by UV spectrophotometry i n 0.1 N H2SO4 ( 127). Using t h e same p r i n c i p l e , a s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n of s t r y c h n i n e n i t r a t e i n p i l l s c o n t a i n i n g o t h e r i n g r e d i e n t s such a s p h e n o b a r b i t a l , bromcamphor, Ca glycero-phosphate, and As203 was proposed (128). To 15 m l 0 . 1 N H2SO4 i s added powdered p i l l c o n t a i n i n g -0.002g s t r y c h n i n e n i t r a t e , followed by 30 m l E t 2 0 s a t u r a t e d with 0 . 1 N H2SO4 and e x t r a c t e d f o r 3 min The aqueous phase i s r e e x t r a c t e d , t h e e x t r a c t s are washed with 10 m l 0 . 1 N H2SO4 a f t e r combini n g , f i l t e r e d , brought t o a d e s i r e d volume and t h e absorbance determined a t 254 nm. The proposed procedure e l i m i n a t e s p h e n o b a r b i t a l and bromcamphor i n t e r f e r e n c e .

616

FARID J. MUHTADI A N D MOHAMED S. HIFNAWY An improved spectrophotometric determinat i o n of teriary amine drugs, including strychnine, using malonic a c i d / a c e t i c anhydride reage n t was reported (129). The method involves t h e condensation of malonic acid and AC20 under t h e c a t a l y t i c e f f e c t of the t e r t i a r y amine function, e i t h e r f r e e o r i n the form of s a l t . The condensation product has 2 prominent absorption peaks a t 333 and 400 nm. The apparent molar a b s o r p t i v i t y of t h e 333 nm band was i n the range of 1.37~105t o 5 . 5 ~ 1 0 5 . Absorbance vs. concn. was l i n e a r up t o 1.2,ug/ml of a l l drugs studied and t h e s e n s i t i v i t y was O.l,ug/ml. The method was reported t o be highly s e n s i t i v e and s p e c i f i c f o r t e r t i a r y amine drugs without i n t e r f e r e n c e from primary and secondary amines o r quaternary ammonium s a l t but does not d i s t i n g u i s h between t e r t i a r y amines. A method f o r determination of strychnine i n nux vomica preparations, involving modification of t h e French Pharmacopoeia method was d i s c u s s ed (130). The modifications proposed a r e : maceration of t h e nut powder f o r 30 min i n d i l u t e ammoniacal s o l u t i o n ; e x t r a c t i o n with CHC13 - d i l . NH40H f o r 2 h; evaporating t h e CHC13 e x t r a c t t o dryness, d i s s o l v i n g t h e r e s i due i n MeOH and examining the s o l u t i o n d i r e c t l y with UV spectrometry. Strychnine was assayed i n the seeds of strychnos pierriama by dual - wavelength spectroscopy (131) as follows: The seeds a r e d r i e d , powdered and t h e powder (-0.4g) i s treated with 2 N NaOH (1 ml) and mixed with -80 m l CHC13. h. cooled, f i l The mixture i s refluxed f o r - 1 t e r e d and t h e f i l t r a t e i s evaporated, t r e a t e d with 1 N H2SO4 on water bath and d i l u t e d with 1 N H2SO4 t o 100 m l . A 5-ml s o l u t i o n d i l u t e d with d i s t i l l e d H 2 0 ( t o 25 ml) and analyzed by dual-wavelength spectrophotometry a t 255 and 278 nm f o r t h e determination of strychnine ( r e l a t i v e standard deviation = 1.1%,r e c o v e r i e s were 98.9%). First d e r i v a t i v e spectrophotometric d e t e r mination of strychnine and brucine i n nux vomica l i q u i d e x t r a c t was reported (132). Strychnine and brucine a r e e x t r a c t e d from nux vomica e x t r act, d i l u t e d with H20 and NWOH by using CHC13 and a r e then back-extracted with 0.1 N H2SO4. ,

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617 The 1st d e r i v a t i v e s p e c t r a are measured a t 2 6 2 . 8 m f o r s t r y c h n i n e . The limits of d e t e c t i o n were 3,ugg/ml and t h e mean r e c o v e r i e s were 101.1%. They r e p o r t e d t h a t t h i s method was much faster than t h e B r i t i s h Pharmacopoea method. Other methods f o r s p e c t r o p h o t o m e t r i c a s s a y o f s t r y c h n i n e i n d i f f e r e n t f o r m u l a t i o n s and b i o l o g i c a l f l u i d s and t i s s u e s were a l s o d i s cussed (133-137).

7.S.3

InfraredSpectrophotometry (IR) Small amounts o f s t r y c h n i n e and o t h e r organ i c b a s i s , i n u r i n e could be i d e n t i f i e d by I R (138). The b a s i s are e x t r a c t e d from u r i n e with CHC13, t r a n s f e r r e d t o s p o t s o f d i l . H2SO4 supp o r t e d on s t r i p s of Whatman 3 MM paper and chromatographed, a f t e r n e u t r a l i z a t i o n o f t h e a c i d s p o t , u s i n g e i t h e r BuOH-HC1 o r ISo-Bu Me Ketone - ACOH as s o l v e n t . Detection of t h e a l k a l o i d is achieved by an i o d o p l a t i n a t e s p r a y , o r a bromocresol green spray. S t r y c h n i n e may t h e n be recovered f o r spectrophotometry by e x c i s i n g t h e s p o t , making a l k a l i n e w i t h NH3 and e x t r a c t i n g w i t h CHC13. Spectrophotometry is b e s t c a r r i e d o u t i n a s o l u t i o n i n an o r g a n i c s o l v e n t i n m i c r o c e l l s w i t h N a C l windows. Another method i n v o l v e s t h e i d e n t i f i c a t i o n and e s t i m a t i o n o f s t r y c h n i n e by IR spectrometry a f t e r i t s e x t r a c t i o n and s e p a r a t i o n by TLC on s i l i c a g e l (139). The chromatogram e l u a t e s are c o n c e n t r a t e d on K B r m i c r o p e l l e t s . S p o t s c o n t a i n i n g about 10 Y of s t r y c h n i n e could be e s t i m a t e d with a p r e c i s i o n o f 8-15%.

7.5.4

Nuclear Magnetic Resonance: NMR S t r y c h n i n e was determined i n nux vomica powder u s i n g NMR (140). The f o u r a r o m a t i c p r o t o n s of s t r y c h n i n e e x h i b i t s i g n a l s a t 7.20 ( p r o t o n s 9,10,11), and a t 8.20 ppm (proton 1 2 ) , t h e l a t t e r being i n f l u e n c e d by t h e carbonyl group of t h e lactam. The 2 aromatic p r o t o n s of b r u c i n e form well d e f i n e d s i g n a l s a t 6.73 (proton 9), and 7.88 ppm (proton 1 2 ) , t h e l a t t e r a l s o i n f l u e n c e d by t h e lactam carbonyl. The h e i g h t t o t h e s t r y c h n i n e peak a t 7.17 ppm, o v e r t h e h e i g h t t o t h e b r u c i n e peak a t 6.73 ppm, i s a f u n c t i o n of t h e p e r c e n t s t r y c h n i n e i n

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t h e sample, The NMR method was checked by v o l u metric a n a l y s i s of t h e mixtures. 7.5.5

Atomic Absorption An i n d i r e c t d e t e r m i n a t i o n of a l k a l o i d s by atomic a b s o r p t i o n spectrometry was a p p l i e d f o r d e t e r m i n a t i o n o f s t r y c h n i n e (141). Strychnine i s r e a c t e d with Reinecke s a l t s o l u t i o n , PhN02 i s added and t h e mixture i s shaken f o r 2 min. The PhN02 l a y e r is s e p a r a t e d and dehydrated and t h e C r i n t h e Ph NO2 s o l u t i o n i s measured by atomic a b s o r p t i o n spectrometry. Cu2+, Co2+ Mg2+, Ca2+, Na+, N i 2 + , K+, Pb2+, Cd2+, Fe3+, NH4+, C 1 - , Po$-, Br-, NO3-, Na2S203, monosodium glutamate, s t a r c h , and s u c r o s e d i d n o t i n t e r e f e r e

7.6

Chromatographic Methods 7.6.1

Paper Chromatography R e s u l t s o f s e p a r a t i o n of i n d o l e a l k a l o i d s i n c l u d i n g s t r y c h n i n e by paper chromatography (Whatman No. 1) i n v a r i o u s s o l v e n t s , were given (142). Petroleum e t h e r s a t u r a t e d w i t h formamide-benzene-Et0H (100:25:25) , BuOH-HC1 (conc.)H20 (50:7.5: 17.5), BuOH-ACOH-HzO (4 :1:5) were s u i t a b l e . Impregnating t h e paper w i t h formamide + 8% NH4 formate gave round s p o t s . Using Whatman No. 1, b u f f e r e d by d i p p i n g i n a 5% s o l u t i o n of sodium dihydrogen c i t r a t e , b l o t t i n g , and d r y i n g a t 25OC f o r 1 h , a s o l v e n t system was given f o r t h e d e t e c t i o n o f o r g a n i c b a s i s (143,144). For s t r y c h n i n e , a s o l v e n t composed o f 4.8 g of c i t r i c a c i d i n a mixture o f 130 m l o f H20 and 870 m l of n. b u t a n o l i s used; i t s Rf = 0.30 u s i n g UV and i o d o p l a t i n a t e s p r a y reagent f o r location. A method f o r q u a n t i t a t i v e s e p a r a t i o n o f s t r y chnine by paper chromatography on 'segment s t r i p ' and i t s c o l o r i m e t r i c e v a l u a t i o n by adduct format i o n with dyes was d i s c u s s e d (145).

7.6.2

Thin Layer Chromatography (TLC) The s o l v e n t system, ( s t r o n g NH3 : MeOH, 1.5:100), used f o r g e n e r a l s c r e e n i n g o f n i t r o g e nous bases,was used f o r d e t e c t i o n o f s t r y c h n i n e on s i l i c a g e l G l a y e r s (Rf=0.22) (146). Locat i o n was under UV and w i t h a c i d i f i e d i o d o p l a t i n a t e spray r e a g e n t . TLC u s i n g Dragendorff's o r t h e previous r e a g e n t was a l s o r e p o r t e d f o r

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619

separation and detection of nux vomica alkaloids (147,148). A method for the identification of strychnine in biological material, using TLC was presented (149). The alkaloid is extracted with 0.02 N H2SO4 at pH 2.5-3.0. The extract is alkalinized with 20% NaOH to pH 8-9, extracted with CHC13 and chromatographed on silica gel KSK layers using CHC13 - Me2CO - NH3 (30: 30:2) for development. The use of TLC layers containing adsorbents, binders, fluorescent substances and colloidal silica for separation and identification of strychnine was discussed (150). C6H6-CHC13Et2NH (9:4:1) was the developer and spots were visualized under W. Application of different chromatographic techniques (TLC, GLC and HPLC) for rapid detection of drugs, intoxicants and related compounds, including strychnine was investigated (151). An improved TLC method €or detection and identification of basic drugs extracted from tissues was reported (152). This was achieved by a double development technique using CH2C12 : Me Et ketone: NH3 (90:10:1.5 and 30:70:2). Identification was achieved after the traditio. nal detection spray sequence with successive topical applications of : Marquis reagent. Mandelin reagent and a special NH4 molybdate fuming H2S04 reagent. TLC was also applied for the detection and determination of strychnine and brucine (153). The two alkaloids are first separated by TLC and then determined photometrically with 0.5% picric acid in HOAC. Another method for the determination of strychnine in Chinese medicines by dual wave length TLC-densitometry was also reported (154). 7.6.3

Adsorption Chromatography A method for separation of strychnine from nux vomica seeds by adsorption chromatography was described (155). A 2 g sample of powdered seed material, made alkaline, is extracted with CHC13 and the solvent evaporated to dryness. The residue is dissolved in trichloroethylene and chromatographed on alumina (log) column.

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Strychnine i s then e l u t e d with 70 m l of CCl4 containing 9% acetone. The solvent i s then removed by d i s t i l l a t i o n and t h e a l k a l o i d a l r e s i d u e t i t r a t e d with a standard acid. 7.6.4

Ion Exchange Chromatography A procedure was described f o r separation of strychnine from nux vomica using ion exchange resin followed by i t s t i t r i m e t r i c determination 0 5 6 ) . 0.3 g sample of the d e f a t t e d powdered nux vomica seed material i s e x t r a c t e d with a mixture of 5 g CHC13, 15 g e t h e r and 1 m l of 10% ammonia. The CHC13/Et20 e x t r a c t i s then shaken f o r 30 seconds with 5 m l H20 and t h e solvent l a y e r i s then evaporated t o almost dryness, 1 m l of alcohol added, and evaporation i s c a r r i e d out t o dryness. To t h e residue i s then added 3 drops of 2% H2SO4, followed by 10 m l of 90% alcohol and t h e s o l u t i o n i s passed through ion exchange r e s i n (Amberlite IR-4B) t o remove i m p u r i t i e s , and t h e e l u a t e i s t i t r a t e d with 0.01 N HC1, using t h e antimony e l e c t r o d e f o r e l e c t r o m e t r i c t i t r a t i o n , and the q u a n t i t y of strychnine calculated. Another method f o r t h e separation of s t r y chnine from e x t r a c t s of animal t i s s u e s using cation-exchange r e s i n s was reported (157). Liver t i s s u e (100 g) i s macerated i n 500 m l of H20, and100.ml 0.01N HC1 i s added. The mixture is heated t o b o i l i n g and held i n a b o i l i n g water bath f o r 1 h; then the mixture i s d i l u t e d t o 1 1 with H20 and f i l t e r e d . The f i l t r a t e i s put through a column of Dowex SOW x 12 (200400 mesh) cation-exchange r e s i n . Strychnine i s e l u t e d with 8 N H C 1 a f t e r removal of o t h e r a l k a l o i d s and the e l u a t e i s n e u t r a l i z e d by NaHC03 before i t s q u a n t i t a t i v e determination. A t h i r d method involves t h e use of a c a t i o n exchange paper f o r determination of strychnine was described (158). Although simpler than t h e c o l o r i m e t r i c methods, but it ndeds a l a r g e r amounts of substance f o r analysis. In a f o u r t h a r t i c l e , s t r i p of Amberlite SA-2 (RH-form) and Amberlite SB-2 (R OH form) a r e used f o r determination of strychnine (159).

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621 A f i f t h method d e s c r i b e d t h e use of s y n t h e t i c i o n exchange r e s i n f o r compleximetric d e t e r m i n a t i o n o f s t r y c h n i n e i n pharmaceuticals (160). The s o l u t i o n c o n t a i n i n g a l k a l o i d s a r e passed through cokumn packed w i t h Wofatite KPS s a t u r a t e d with Zn2+ and Zn2' i s determined i n t h e e l u a t e by t i t r a t i o n with e d e t i c a c i d .

7.6.5

Gas Liquid Chromatography

(GLC )

Although non v o l a t i l e , numerous methods f o r GLC a n a l y s i s o f s t r y c h n i n e were r e p o r t e d . A system u s i n g 1%SE-30 on 100-120 mesh Anakrom ABS 6 f t x 4 mm I . D . b o r o s i l i c a t e g l a s s columns was d e s c r i b e d f o r GLC a n a l y s i s o f s t r y c h n i n e (161). The f o l l o w i n g o p e r a t i n g c o n d i t i o n s were adopted : column temperature, 25OoC; c a r r i e r g a s , argon; flow r a t e , 80 ml/min; d e t e c t o r , argon i o n i z a t i o n o r FID (Hz, 50 ml/min; a i r , 300 ml/min). The r e l a t i v e r e t e n t i o n time o f s t r y c h n i n e ( r e l a t e . t o codeine) is 4.83. A method i n v o l v i n g t h e use of g l a s s columns ( 3 f t x 0.07 i n ) packed w i t h Gas Chrom. P was d e s c r i b e d (162), f o r s e p a r a t i o n of d i f f e r e n t a l k a l o i d s i n c l u d i n g s t r y c h n i n e . The packing material i s washed w i t h concd. HC1, d r i e d , t r e a t e d w i t h h e x a m e t h y l d i s i l i z a n e and c o a t e d with 1%o f q a n o s i l i c o n e , a p o l y e s t e r methyl s i l i c o n e copolymer and cyclohexane dimethanol succ i n a t e p o l y e s t e r A t h i r d method f o r GLC d e t e r m i n a t i o n o f d r u g s , i n c l u d i n g s t r y c h n i n e i n body f l u i d s was r e p o r t e d ( 163). Four columns are used (2.5% Marlophene 87 (M 8 7 ) , 1%Neopentylglycol sebacate (NGSB), 2.5% S i l i c o n Rubber SE 52 and 1% Versamid 900 (SE 52/V 900).N2 i s used as a c a r r i e r gas and Me m y r i s t a t e , Me stearate and Me behenate are used as s t a n d a r d s . Best r e s u l t s are o b t a i n e d w i t h a low t e m p e r a t u r e , a l i g h t l y packed column, and a s e n s i t i v e d e t e c t o r . Afourthmethod which a l l o w s GLC d e t e c t i o n and q u a n t i t a t i v e d e t e r m i n a t i o n o f s t r y c h n i n e i n b r u c i n e and v i c e v e r s a , and allowed s e p a r a t i o n of t h e a l k a l o i d b a s e s i n e x t r a c t s o f S t r y chnos nux vomica and S. icaja was p r e s e n t e d (164) A FID,used f o r d e t e c t i o n , N2, a s c a r r i e r gas (35 ml/min), and a 2 - f t s t a i n l e s s steel column 1/8 i n d i a m e t e r , packed with Aeropak

.

622

FARID J. MUHTADI A N D MOHAMED S. HIFNAWY 30 (100-200 mesh) impregnated with 5% SE-52 a s s t a t i o n a r y phase. To determine strychnine i n brucine, d i s s o l v e 100 mg of t h e sample i n a mixture of 5 m l 5% H2SO4 and 15 m l H20. Heat i f necessary. After cooling t o room temperat u r e , added 20 m l 10% NaOH and e x t r a c t with successive 20 m l portions of CHC13. F i l t e r the combined CHC13 f r a c t i o n , Add 5 m l 95% E t O H ; and evaporate t o dryness. Dissolve t h e residue i n successive 10-ml p o r t i o n s 95% E t O H , and d i l u t e t o 100 m l with EtOH. I n j e c t 0 . 4 ~ 1 s o l u t i o n i n t o t h e gas chromatograph system described. Percent of strychnine = 84 A1/A1+ A2 where A 1 i s the a r e a of strychnine peak and A2 i s a r e a of brucine peak. Retention time of strychnine was 5.85 min with column temperat u r e , 25OoC and i n j e c t o r temperature 28OoC. A f i f t h GC method described determination of strychnine i n b i o l o g i c a l materials (165). Tissue homogenate a t pH 8.6 i s s t i r r e d with acetone f o r 15 min. The mixture i s heated and the acetone volatized. The mixture i s a c i d i f ied t o pH 3.3 with 5% C C13C02H, s t i r r e d f o r 30 min., and f i l t e r e d through sand. The f i l t r a t e i s e x t r a c t e d with CHC13 which i s concent r a t e d and an a l i q u o t i s i n j e c t e d on a s t a i n l e s s s t e e l column packed with 30% SE-30 on Chromosorb W a t 250'. Recovery from l i v e r i s 90%. Another method presented a GLC procedure f o r determination of strychnine i s grain b a i t s (166), using glass column (6 f t x i n . ) packed with 5% SE-30 on Chromosorb W (DMCS) and F I D f o r detection. The g r a i n b a i t s containing strychnine are ground, mixed, and e x t r a c t e d by shaking with CHC13 containing 1,3,5-triphenylbenzene as i n t e r n a l standard. Without f u r t h e r cleanup, e x t r a c t f i l t r a t e s a r e i n j e c t e d d i r e c t l y i n a gas chromatograph. Peak height r a t i o s a r e used f o r q u a n t i t a t i o n of strychnine with 2,ug s e n s i t i v i t y and recoveries ranging from 89.9 t o 91.7%. The r e t e n t i o n index IR a n d d IR were considered f o r the gas chromatographic assay of d i f f e r e n t compounds including strychnine (167). Another method f o r GLC a n a l y s i s of s t r y c h nine a f t e r i t s s i l y l a t i o n with N-methyl-N-(trimethylsilyl) hepta fluorobutyramide I)- BuOAC

STRYCHNINE

623

(40:1:59) was d i s c u s s e d (168). An a l k a l i flame i o n i z a t i o n d e t e c t o r (AFID), and t e m p e r a t u r e programming w i t h lS°C/min from 150-260°C are adopted. A gas chromatographic d e t e r m i n a t i o n of b a s i c d r u g s i n u r i n e o r blood and i t s a p p l i c a t i o n f o r doping a n a l y s i s was r e p o r t e d f o r s t r y c h n i n e ( 169). A r a p i d gas chromatography method f o r t h e q u a l i t a t i v e detection of b a s i c drugs, includi n g s t r y c h n i n e , i n postmortem blood, u s i n g a n i t r o g e n phosphorous d e t e c t o r was d e s c r i b e d (170). The method i n v o l v e s a 1 - s t e p e x t r a c t i o n from 1 . 0 m l of postmortem blood b u f f e r e d t o pH 9.0-9.5 i n t o n-Bu c h l o r i d e . No back e x t r a c t i o n or c l e a n up s t e p s a r e r e q u i r e d . E x t r a c t s are i n j e c t e d on 3% OV-1 and 3% OV-17 columns coupled t o NP d e t e c t o r s . S e n s i t i v i t y l i m i t s are drug dependent and range from 200-500 ng/ml f o r the various drugs l i s t e d i n the report. Combined u s e of f u s e d s i l i c a c a p i l l a r y c o l u mns and nitrogen-phosphorus d e t e c t i o n f o r t h e t o x i c o l o g i c a l a n a l y s i s of i l l i c i t h e r o i n samples was s u c c e s s f u l l y a p p l i e d f o r d e t e c t i o n o f nanogram amounts of s t r y c h n i n e (171). The method i n v o l v e s t h e u s e of c a p i l l a r y columns o f CP-Sil 5 o r C P - s i l 8 f u s e d s i l i c a , d e a c t i v a t e d w i t h a p o l y s i l o x a n e a t high t e m p e r a t u r e and d i a c e t y l n a l o r p h i n e a s t h e i n t e r n a l s t a n d a r d The same column was a l s o used i n t h e a n a l y s i s o f an e x t r a c t o f t h e small i n t e s t i n a l c o n t e n t i n an overdose c a s e (172). Drug s c r e e n i n g v i a gas chromatography, f u s e d s i l i c a c a p i l l a r y columns c o a t e d w i t h a 0.25,um f i l m o f SE 30, y i e l d e d r e p r o d u c i b l e r e t e n t i o n i n d e x e s f o r s t r y c h n i n e (173). The s c r e e n i n g was performed on a t e m p e r a t u r e programmed columns a t 100°-2950C a t S°C/min w i t h t h e c a r r i e r v e l o c i t y o f 45 cm/s a t 100°C. The c a p i l l a r y column p r o v i d e s s u p e r i o r r e s o l u t i o n a n d / o r g r e a t l y reduced a n a l y t i c a l time r e l a t i v e t o packed columns and i s t h u s well s u i t e d t o emergency t o x i c o l o g i c a l s i t u a t i o n s i n f o r e n s i c laboratories. The v a r i o u s s u b s t a n c e s p r e s e n t i n s t r e e t samples of n a r c o t i c s ( i n c l u d i n g s t r y c h n i n e ) were d e t e c t e d i n a s i n g l e a n a l y s i s by means of s i l y l a t i o n and h i g h - r e s o l u t i o n gas chromato-

624

FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

graphy 0 7 4 ) . This technique allows t h e detect i o n of n a r c o t i c s , of most common a d u l t r a n t s , and of organic d i l u e n t s a t the same time. The method i s highly s e n s i t i v e and has high r e s o l v ing power. The use of t h e combined technique GC/MS f o r i d e n t i f i c a t i o n of strychnine among o t h e r compounds e x t r a c t e d from b i o l o g i c a l m a t e r i a l s i n a v e t e r i n a r y toxicology laboratory was d i s cussed (1 75). Gas chromatography employing 3%OV-1 on chromosorb W-HP f o r r a p i d d e t e c t i o n of drugs, i n t o x i c a n t s and r e l a t e d compounds , including strychnine, was a l s o reported (151). Another GLC method f o r separation of some strychnos a l k a l o i d s was discussed (176). An experiment was c a r r i e d out by us on t h e e f f e c t of s i l y l a t i o n on GLC c h a r a c t e r s of s t r y chnine. Under our operating conditions, the non d e r i v a t i z e d base showed a r e t e n t i o n t i n e 6.3 min. Although t h e peak was f a i r l y well defined and sharp, but i t s base l i n e was spread out over a period of approximately 6 min. The s i l y l a t e d strychnine, on the o t h e r hand, showed a sharp well defined peak a t 6.0 min r e t e n t i o n time with a highly reduced baseline spread. The s e n s i t i v i t y of d e t e c t i o n was much higher (Fig. 11). The following operating conditions were adopted. Column, 3% OV 1 on Gas Chrom, S O / l O O mesh; temperature, isothermal a t 25OOC; d e t e c t o r F D H2 flow, 16 ml/min; a i r flow, 160 ml/min); c a r r i e r gas, N2 with a flow r a t e , 40 ml/min; i n j e c t o r and d e t e c t o r temperature, 25OoC. Si Yl a t i o n was c a r r i e d out by using Tri-Sil/BSA reagent and a small amount of pyridine as a solvent. The apparatus used: Gas Chromatograph Varian Model 3700 with Dual FID.

STRYCHNINE

625

Fig. 11. GLC o f s t r y c h n i n e

(a)

Free base

(b)

S i l y l a t e d base

a

7.6.6

b

High Performance Liquid Chromatography (HPLC) HPLC o f f e r s obvious advantages by combining a s e p a r a t i v e power s u p e r i o r t o t h a t o f TLC with a means o f a c c u r a t e q u a n t i t a t i o n and e a s y t r a p ping o f e l u t e d m a t e r i a l f o r subsequent c o n f i r mation of s t r u c t u r e . T h i s technique i s moving f a s t f o r t h e a n a l y s i s of many components. Becau s e a l l t h e experimental work i n HPLC i s p e r f ormed a t ambient temperature, t h e problem o f thermal i n s t a b i l i t y i s e l i m i n a t e d . Using C o r a s i l I with 1.1%Poly G 300 as a s t a t i o n a r y phase and heptane - E t O H r a n g i n g from 100:s t o 100:10, v/v a s mobile phase, q u a n t i t i e s i n nanogram l e v e l o f s t r y c h n i n e can be s a t i s f a c t o r i l y analyzed with r e a s o n a b l e accuracy ( 177). A paper d e s c r i b i n g t h e high-speed l i q u i d chromatography o f some a l k a l o i d s i n c l u d i n g s t r y c h n i n e , u s i n g l i q u i d - s o l i d chromatography was published (178). The wave l e n g t h o f d e t e c t i o n was 255 nm. The column was a s t a i n l e s s s t e e l t u b e , 30 cm x 2 mm I.D., f i l l e d with Merckosorb S i 60 (5,um). The column temperat u r e was maintained a t 20'. The r e t e n t i o n time (RT) o f s t r y c h n i n e i n f i v e d i f f e r e n t mobile phases and t h e i r flow r a t e s (FR) are as

626

FARID J. MUHTADI AND MOHAMED S. HIFNAWY follows: CHC13 - MeOH (9:1), RT, 6.9, FR 0.81; CHC13 - MeOH (8:2), RT 3.9, FR 0.81; CHC13 MeOH (7:3), RT 4 . 7 , FR 0.75; E t Z O - MeOH (7:3) RT 9.4, FR 1.43; E t 2 0 - MeOH (6:4), RT, 7.3, FR 1.34. Using t h e same column and as mobile phases, E t 2 0 containing 1%of diethylamine a t a flowr a t e of 2.0 ml/min a t 205 kg/cm2 and E t 2 0 MeOH ( 1 : l ) a t a flow-rate of 1.15 ml/min a t 200 kg/cm*, strychnine and o t h e r r e l a t e d a l k a l oids were analyzed (179). RT of strychnine was 7.2 and 12.4 min i n the two systems respectively. A simple and r a p i d HPLC procedure i s described f o r t h e determination of strychnine i n grain b a i t s (180). The b a i t s are ground, mixed, and e x t r a c t e d by shaking with CHC13. Without f u r t h e r cleanup, e x t r a c t f i l t r a t e s a r e i n j e c t e d d i r e c t l y i n t o a l i q u i d chromatograph. The analy s i s i s completed within 7 min and peak heights are used f o r q u a n t i t a t i o n . Separations were made on a 30 cm x 4 mm I . D . , s t a i n l e s s s t e e l column packed w i t h p p o r a s i l (8-12,um s i l i c a ) . The e l u t i n g solvent was MeOH-CHC13 (10:90) a t a flow rate of 2 . 0 ml/min. Recovery of spiked samples ranged from 91.5 t o 95.2%. Confirmat i o n of strychnine from a commercial sample was made by high r e s o l u t i o n mass spectrometry with mass agreement t o 1 . 2 ppm. A procedure is described f o r t h e q u a l i t a t i v e d e t e c t i o n of strychnine among other contaminants i n an i l l i c i t diamorphine s e i z u r e s by means of high-pressure c a t i o n exchange chrnmatography (181). The i l l i c i t diamorphine sampl e s a r e homogenised by grinding i n a mortar and 20-25 mg, accurately weighed, are dissolved i n 50% aqueous methanol, The s o l u t i o n is made up t o 2 m l i n a volumetric f l a s k and 2 p l a r e taken f o r an i n i t i a l q u a l i t a t i v e a n a l y s i s using t h e following chromatographic conditions: column, 120 cm x 2.1 mm. Zipax SCX; e l u e n t s : (a) H3B03 (0.2 M y aqueous), adjusted t o pH 9.3 with 40% NaOH, (b) H3B03 (0.2 M y aqueous) - acetonitrile-n-propanol (86: 12:2) adjusted t o pH 9.8 with NaOH (40% aqueous); flow r a t e , 2 m l / min; l i n e a r gradient, 0-100% (b) i n 6 min; UV absorbance detector, 270 nm, 0.2 absorption u n i t s f u l l scale. Strychnine was found i n

-

627

STRYCHNINE

many of the analyzed samples, r e t e n t i o n time, 6 . 3 min and r e t e n t i o n volume 12.8 m l (Fig. 12).

3

4

7

1

I

I

0

5

10

(rnin.)

F i g . 1 2 . HPLC of some c o n s t i t u e n t s of i l l i c i t diamorphine s e i z u r e s . For column conditions, see t e x t . 1 = Barbitone; 2 = c a f f e i n e ; 3 = morphine; 4 = monoacetylmorphine; 5 = strychnine; 6 = diamorphine; 7 = quinine; 8 = cocaine.

628

FARID J. MUHTADI AND MOHAMED S. HIFNAWY The r e t e n t i o n of strychnine r e l a t i v e t o morphine, when analyzed by HPLC among a wide range of drugs of abuse, was 1.57 (182). The following conditions were adopted: column, s t a i n l e s s s t e e l , 25 cm x 4.6 mm I.D. f i l l e d with small s i z e s i l i c a ' f i n e s ' ; solvent, MeOH2 N ammonia s o l u t i o n - 1 N ammonium n i t r a t e s o l u t i o n (27:2:1); flow-rate, 1 ml/min; p r e s s ure, 1500 p . s . i , room temperature; d e t e c t o r , UV a t w , l . 278 nm. A weighed amount of sample is made up i n e i t h e r water o r d i l u t e HC1 t o known concentration, usually 10 mg/ml, and d i s s o l u t i o n i s a s s i s t e d by immersion f o r about 1 min i n an u l t r a s o n i c bath. An a l i q u o t of 1-5,ul of t h i s sample i s i n j e c t e d on t o t h e chromatographic column. Peak h e i g h t s a r e used f o r q u a n t i t a t i o n . A simple, rapid e x t r a c t i o n and subsequent determination f o r strychnine, using HPLC with a reverse-phase solvent system was described (183 ) . Stomach contents o r g r a i n b a i t cont a i n i n g strychnine a r e made a l k a l i n e with NaOH and e x t r a c t e d with CHC13. Extract f i l t r a t e s a r e i n j e c t e d d i r e c t l y i n t o a l i q u i d chromatograph without f u r t h e r preparation, except f o r d i l u t i o n , i f necessary. Peaks are resolved within 3.5 min and peak heights a r e used f o r q u a n t i t a t i o n . HPLC of strychnine was c a r r i e d out on a 30 cm x 4 mm I.D. s t a i n l e s s s t e e l column packed with Bondapak C18. The solvent program was 0.005 M phosphate buffer-MeOH (60 + 40) a t a flow r a t e of 1.5 ml/min. Recovery from spiked stomach contents was 93.9%. The d e t e c t i o n limits, using t h e 254 nm UV d e t e c t o r , was 5 mg. The strychnine peak was c o l l e c t e d and subjected t o TLC with standard f o r confirmation. Reversed-phase HPLC employing octadecyls i l a n i z e d columns (RP 18) was described f o r d e t e c t i o n of strychnine (151). An HPLC method was developed f o r t h e quant i t a t i o n of strychnine i n u r i n e of children with nonketotic hyperglycinemia and o t h e r development d i s o r d e r s t r e a t e d with t h e a l k a l o i d (184). Mobile and s t a t i o n a r y phases were p o l a r , i . e . MeOH-H20-330 g/kg NH3 ammonia (vols., 85

STRYCHNINE

629 m l + 14.2 m l + 0.8 ml) and LiChrosorb S i - 6 0 , 7,um. Brucine was t h e i n t e r n a l s t a n d a r d .

E x t r a c t i o n was performed by t h e E x t r l u t t e c h n i que. A t s t r y c h n i n e n i t r a t e c o n c e n t r a t i o n s i n u r i n e of 2 1 , 126 and 760,ug/L, recovery was 92.1, 98.1 and 102.5. A c h i l d with n o n k e t o t i c hyperglycinemia under continued s t r y c h n i n e t r e a t m e n t e x c r e t e d 1-13.6% o f t h e d a i l y dose unmetabolized i n u r i n e . The method was a l s o s u i t a b l e f o r the estimation of unreacted s t r y chnine i n t i s s u e e x t r a c t s . The d e t e r m i n a t i o n of s t r y c h n i n e i n s e e d s o f S. piersirma o r t h e i r p r e p a r a t i o n s , i n c l u d e d e x t r a c t i o n with CHC13 - NH40H ( 9 : 1 ) , s e p a r a t i o n of t h e o r g a n i c phase, and i n j e c t i o n of an a l i quot i n t o a HPLC column (4 mm diam. x 250 mm) c o n t a i n i n g s i l i c a g e l (3-5 i r r e g u l a r ) and Et20-MeOH-NHqOH (80:20:1 v/v) a s mobile phase (flow r a t e 2 ml/min.) f o r a n a l y s i s . D e t e c t i o n was a t 250 nm. The r e l a t i v e s t a n d a r d d e v i a t i o n was 1.29% (measured by t h e peak h e i g h t method) and t h e recovery was 99.3%. The c a l i b r a t i o n p l o t was l i n e a r t o 0.75,ug. The method was simple and r a p i d , and may be used i n r o u t i n e a n a l y s i s (185,186). Measurement o f s t r y c h i n e by HPLC i n b i o l o g i c a l media was a l s o r e p o r t e d (187). CHC13 a t b a s i c pH was used t o extract s t r y c h n i n e and t h e i n t e r n a l s t a n d a r d q u i n i n e from plasma. HPLC was c a r r i e d o u t on a s i l i c a g e l column u s i n g as e l u e n t c o n c e n t r a t e d NH4OH i n MeOH a t a flow r a t e of 1.1 ml/min and d e t e c t i o n a t 254 nm. Recovery from plasma was 83%, t h e lower l i m i t o f s e n s i t i v i t y was 0 . 6 2 5 ~ g / m l , and i n t r a - a n d i n t e r - a s s a y v a r i a n c e was 3-5% and ~ 1 0 %res, pectively. A method f o r i d e n t i f i c a t i o n and determinat i o n of s t r y c h n i n e and b r u c i n e i n nux vomica e x t r a c t s and o t h e r pharmaceuticals u s i n g HPLC was d e s c r i b e d (188 ) . The e x t r a c t s were f i r s t p u r i f i e d by p a s s i n g through a Sep Pak C18 c a r t r i d g e column and HPLC c a r r i e d o u t on a reversed-phase ODS column with MeCN-H2O (25:75) as t h e mobile phase c o n t a i n i n g 1 - h e p t a n e s u l f o n i c a c i d as t h e c o u n t e r i o n . The recovery was S98.1. Another method f o r r a p i d e s t i m a t i o n of s t r y c h n i n e i n t i n c t u r e nux vomica BP was r e p o r t e d (189 ). The method was a m o d i f i c a t i o n o f

v,

FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

630

a previous procedure (182 ) . This involves the use of a s h o r t e r column (12.5 cm x 4.6 mm I.D.), changing t h e s t a t i o n a r y phase t o s p h e r i c a l p a r t i c l e s (Hypersil), increasing the flow rate (2 ml/min) ,and changing the d e t e c t o r wave length from 278 t o 254 nm; t h e n e t e f f e c t being t o decrease t h e r e t e n t i o n time of strychnine from 14 t o 3.6 min and 6 min were s u f f i c i e n t t o s e p a r a t e strychnine, brucine and the minor a l k a l o i d s of nux vomica. Concentrations were c a l c u l a t e d using e i t h e r peak a r e a s o r h e i g h t s and comparing against an e x t e r n a l standard (0.15% w/v strychnine base i n 45% ethanol). Other a r t i c l e s discussing t h e a p p l i c a t i o n s of d i f f e r e n t HPLC techniques f o r a n a l y s i s of strychnine among drugs of f o r e n s i c i n t e r e s t , i n v e t e r i n a r y toxicology, i n pharmaceutical formulations, i n mixture with o t h e r b a s i c drugs and f o r screening of drugs and i n s e c t i d e s were a l s o reported (190-194). 7.6.7

Electrophoresis Electrophoretic determination of s t r y c h nine, brucine and quinine i n t i n c t u r e mixtures was developed (195). The separation was done i n 2 N HC02H s o l u t i o n (pH 1.5) a t 0 . 5 ma./cm and 10 v./cm. The s p o t s were developed with Dragendorff reagent, and t h e concentration determined photometrically o r by planimetry. Errors, were within 5-12%.

7.7

Radiometric Determinations A radiometric t i t r a t i o n method has been developed t o enable microdetermination of many a l k a l o i d s , including strychnine (196 ) i n t h e form of t h e i r s a l t s (hydrochloride, phosphate o r s u l f a t e ) . The method was c a r r i e d out a s follows: For hydrochlorides: Add a known volume of concd. HNO3 t o 0.5-3 m l of t h e a l k a l o i d a l s a l t i n a c e n t r i f u g e test tube, t o r e s u l t i n 0.7-0.8 M HNO3. Then t i t r a t e with 0.01 M AgN03 (approx. 3000 c.p.m./cc). After each addition of AgN03 s o l u t i o n , a g i t a t e t o homogen i z e and c e n t r i f u g e with complete sedimentation of the ppt., t r a n s f e r a known volume of the c l e a r solut i o n t o t h e Y-counter, and a f t e r counting, r e t u r n t o the t e s t tube, and repeat sequence. I t i s necessary

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63 1

t o make a t l e a s t 2 a d d i t i o n s o f t i t r a t i n g agent b e f o r e t h e e q u i v a l e n t p o i n t and a t l e a s t 3 a d d i t i o n s a f t e r it. The e q u i v a l e n t p o i n t i s determined g r a p h i c a l l y by e x t r a p o l a t i o n , with an e r r o r o f ?0.4%. For phosphates : b r i n g t h e a l k a l o i d a l s a l t with d i l . NaOH t o pH 8 and t i t r a t e w i t h AgN03 as b e f o r e ; b u t s t r o n g e r a g i t a t i o n and b e t t e r c e n t r i f u g a t i o n are r e q u i r e d , t h e e r r o r being 20.8%. For s u l f a t e s : t i t r a t e i n a pH 7 a l c o h o l i c medium i n which t h e Ag s a l t is i n s o l u b l e , t h e e r r o r being 0.8%. The method can be adapted t o mixtures of a l k a l o i d a l s a l t s by t i t r a t i n g a t d i f f e r e n t pH v a l u e s . Another method f o r r a p i d d e t e r m i n a t i o n o f s t r y c h n i n e w i t h Mayer's r e a g e n t was based on r a d i o m e t r i c t i t r a t i o n ( 197 ) . Mayer r e a g e n t was p r e p a r e d by irra d i a t i o n of 1,358 g HgC12 i n a n e u t r o n f l u x o f 1012 n e u t r o n s / s q . cm. sec. f o r 10 h and a d d i t i o n of t h e s a l t t o 50 m l H20. A s o l u t i o n of 4.9 g K I i n 20 m l H 2 0 was prepared and added with s t i r r i n g t o t h e above. The s o l u t i o n was d i l u t e d t o 100 m l . D i l u t i o n o f 5 m l of t h e s o l u t i o n t o 100 m l gave 0.0025 M o f Mayer reagent. The pH o f a known volume o f t h e a l k a l o i d s o l u t i o n was made n e u t r a l o r s l i g h t l y a c i d i c . Mayer r e a g e n t was added i n small increments, and t h e mixt u r e was s t i r r e d f o r 20-30 s e c . and c e n t r i f u g e d . A known volume of t h e c l e a r l a y e r i n t h e c e n t r i f u g a t e was t r a n s f e r r e d i n t o a s p i r a l surrounding a c y l i n d r i c a l c o u n t e r and t h e r a d i o a c t i v i t y was determined. The procedure was r e p e a t e d s e v e r a l times t o allow a p l o t o f c o u n t s p e r min. vs. m l o f Mayer r e a g e n t t o be made. The equivalence p o i n t was s h a r p l y d e f i n e d . N i t r a t e o r s u l f a t e i o n s do n o t i n t e r f e r e . S t r y c h n i n e can a l s o be determined i n a l c o h o l i c s o l u t i o n s and i n t h e presence o f s 0.5 g b r u c i n e . S t r y c h n i n e and b r u c i n e , 5-30 Y each, a r e determined r a d i o m e t r i c a l l y i n t i n c t u r e of nux vomica ( c o n t a i n i n g 0.224% t o t a l a l k a l o i d s ) by measuring t h e p - a c t i x v i t y of iodinated a l k a l o i d - I ppts, after separati n g s t r y c h n i n e and b r u c i n e by TLC on p l a t e s c o a t e d with A 1 2 0 3 without a b i n d i n g agent (198). The method was c a r r i e d out as f o l l o w s : Apply t h e s o l u t i o n c o n t a i n i n g s t r y c h n i n e and b r u c i n e , develop t h e chromatogram with Et20 - E t O H ( 9 5 : 5 ) , d r y a t 2 S 0 , s p r a y t h e chromatogram with 1%i o d i n e i n CHC13, mark t h e s p o t s , and remove excess i o d i n e by h e a t i n g t h e p l a t e a t 105'. Remove t h e A1203 c o n t a i n i n g t h e marked s t r y c h n i n e and b r u c i n e s p o t s w i t h a m i c r o p i p e t by a p p l y i n g g e n t l e s u c t i o n . Convert t h e a l k a l o i d s

632

FARID J. MUHTADI AND MOHAMED S. HIFNAWY t o t h e r e s p e c t i v e a c e t a t e s a l t s by warming t h e A1203 containing spot with 5 m l o f 2% HOAC, f i l t e r the A1203 on a s h l e s s f i l t e r paper. Ppt strychnine and brucine s e p a r a t e l y with 2 . 5 m l of a c i d i f i e d aqueous "1 s o l u t i o n , f i l t e r t h e p p t s , measure t h e p - a c t i v i t y of t h e p p t s , and f i n d the concentration of both a l k a l oids from the r e s p e c t i v e c a l i b r a t i o n curve. The ,&count vs. concn. curve is l i n e a r f o r 5-30 Yof s t r y chnine and brucine. A method f o r determination of strychnine i n blood by double i s o t o p i c l a b e l i n g was described (199 ) . Double-labeled (3H, 14C) strychnine methiodide was prepared. For t h e assay, add strychnine stock solut i o n and (or) water t o 1 m l human c i t r a t e d blood t o make t o t a l volume of 2 m l , then add 0 . 2 m l (9600 counts/min) of a strychnine s o l u t i o n i n absolute E t O H and 0.2 m l of absolute EtOH. Evaporate a t 60' i n vacuo nearly t o dryness. Add 20 m l E t O H and repeat the evaporation. Add 1 m l of methyl iodide - 3H s o l u t i o n (absolute EtOH) (-30 c./ml), s e a l t h e mixture i n a g l a s s tube, heat a t 60' f o r 4 h , open t h e tube, and evaporate t o 0 . 1 m l . This was paper chromatographed by a descending technique using EtOAC-MeOH-28% NH40H (100:10:15); unlabeled s t r y c h nine and double-labelled strychnine (for quenching corrections) were a l s o chromatographed simultaneous l y with the sample, The amount of 14C and 3H i n each spot was determined, using t h e percent recovery of 14C t o c o r r e c t f o r l o s s e s during work up, t h e amount of strychnine - 3H and thus t h e amount of strychnine i n t h e o r i g i n a l sample was calculated. I t was reported t h a t strychnine can be analyzed more a c c u r a t e l y by t h i s method than by more convent i o n a l procedures. Another method €or determination of strychnos a l k a l o i d s by radiometric t i t r a t i o n s with potassium thallium iodide (K204TLI)reagent was discussed (200). The sample s o l u t i o n containing 0.01 N acid was react e d with excess t i t r a n t within a t o t a l s o l u t i o n volume of 5 m l . The ppt of (Alkaloid H) TLI4 was separat e d by centrifuging and t h e p a c t i v i t y of a supernat a n t a l i q u o t was measured by using a Geiger counter. The s e n s i t i v i t i e s were 5 ~ 1 0 '-~ 2x10-3 M f o r s t r y chnine and brucine. The e r r o r s were within +2.0% f o r determining 4-6 mg a l k a l o i d alone and they were within 23.2% f o r determining 7-15 rng a l k a l o i d i n pharmaceutical preparations.

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The c a t a l y t i c p o l a r o g r a p h ic waves o f s t r y c h n i n e i n b u f f e r s pH 8 were used f o r i t s d e te r m in a tio n i n 10-4 - 10-5 M i n pharmaceutical p r e p a r a t i o n s , i n s o l u t i o n s ( 1 ml) and t a b l e t s ( a f t e r paper chromatog r a p h i c s e p a r a t i o n from v i t a m in s and s u g a r s (201).

ACKNOWLEDGEMENT The a u t h o r s would l i k e t o thank M r . A m i r Shehata (B.Pharm.) of t h e Pharmacognosy Dept. f o r performing t h e m i c r o c r y s t a l t e s t s . Our thanks a r e a l s o due t o Mr. Uday C. Sharma of Pharmacognosy Dept., C o lle g e of Pharmacy, King Saud U n i v e r s i t y f o r h i s secretarial a s s i s t a n c e i n t h e r e p r o d u c t i o n of t h e m a n u sc r ip ts.

FARID J. MUHTADI AND MOHAMED S. HIFNAWY

634

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118. M. S c o t t ; A. Taub and C. P i a n t a d o s i , J. Am. Pharm. 45, 232 (1956). Assoc. S c i . Ed. 119. Burton W. Rehn, J AOAC, 5 0 ( 3 ) , 660 (1967). 120. V. Vukcevic - Kovacevic and Z. Bozin, S c i . Pharm. 3 6 ( 3 ) , 180 (1968) (Ger).

121. Lawrence A. Wapensky, JaAOAC 5 2 ( 5 ) , 1015 (1969). 1 2 2 . Milorad Dugandzic; B r a n i s l a v a Stankovic; L j i l j a n a Milovanovic and Zagorka Koricanac, Acta Pharm. Jugoslav. z(1), 25 (1971). 123. S t e f a n i a Kanafarska-Mlotkowska, Acta Pol. Pharm. 28(1), 23 (1971) ( P o l ) . 124. U. M a r a l i k r i s h n a ; N. Somenswara Rao and G.V. Curr. S c i . , 44(15), 534 (1975). 125. A.M.

Ramandham,

Taha and C. Gomaa, J-AOAC -5 9 ( 3 ) , 683 (1976).

126. V.V. Drozhzhima, V . E . C h i c h i r o and T . I . 25 (5), 44 (1976). Formatsiya (Moscow) , -

Bulenkov,

127. V.V. Drozhzhina and V . E . 2 8 ( 2 ) , 5 7 (1979).

Chichiro, Farmatsiya (Moscow),

128. V.V. Drozhzhina and V.E. (Russ).

Chichiro, i b i d , 29(5), 44 (1980)

129. A.M. Taha, S.R. E l Shabouri and A. I. Rageh, Egypt. J. Pharm. S c i . g ( 3 - 4 ) , 363 (1980). 130. A. Puech, D. Gaudy; C. Duru and M. Jacob, Ann. Pharm. Fr. 4 0 ( 2 ) , 167 (1982). 131. M. Q i and K. Hu, Yaowu Zazhi, 3 ( 3 ) , 170 (1983) (Ch). 132. Mohamed A. Korany and Ahmed A. S a i f El-Din, J - A O A C , 6 7 ( 1 ) , 138 (1984).

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133. Z. K l e f l i n , Acta Pharm. Jugoslav. 11, 23 (1961). 134. J. Bogan; E. Rentoul; H. Smith and W.P. 6 , 166 (1966). S c i . SOC. -

Wein, J. Forens.

135. V . E . C h i c h i r o ; Z.N. Kostennikoiva and V.V. Drozhzhina, Tr. V N I I Farmatsii, 236 (1977) (Russ); C.A. 89; 220948 t , (1977).

g,

136. Z.P. Kostennikova, I . V . Avdyukevich and V.E. L i v s h i t s , T r . VNII F a r m a t s i i l4, 195 (1977) (Russ.); C.A. 89: 22094 P (1977). 137. M.P. Lirova, L . I . Naumenko, and V.E. C h i c h i r o , Nauch. T r . VNII Farmatsii, 18, 36 (1980) (Russ.) 86, 631 (1961). 138. P . J . Morgan, Analyst 139. H. Corneteau; R. Monnet and H.L. Domm. Corpor. 1 ( 4 ) , 352 (1968).

Boiteau

, Med.

Leg.

140. M. P l a t and J. Poisson, Ann. Pharm. Franc. 22(10), 603 (1964). 141. Y. Minami; T. M i t s u i and Yoshikazu Fujimura, Bunseki Kagaku, 3 0 ( 1 2 ) , 811 (1981) (Japan) ,; Through-C.A. 96: 62356 m, 1981).

142. I.R.M.

Pinyazhko, Farmatsevt. Zh (Kieu) 1 7 ( 3 ) , 26 (1962).

143. A.S. Curry and H. Powel, Nature 173, - 1143 (1954). 144. E.G.C. C l a r k e , "Methods o f F o r e n s i c Science, ed", Frank Lundquist, New York, I n t e r s c i e n c e P u b l i s h e r s , v o l 1, p. 31 (1962). 1 9 ( 2 ) , 111 (1964). 145. J. Zarnack and S. P f e i f e r , Pharmazie 146. I . Sunshine, American J. o f C l i n i c a l Pathology, 40, 576 (1963). 147. J . D . P h i l l i p s o n and N.G. 493 (1970). 148. S.N. Tewari and A.K.

Bisset, J. Chromatogr. 48,

Singh, Chem. Era. 1 4 ( 9 ) , 360 (1978).

149. O.A. Akopyan; B . I . Shvydkii;S.L. B a i k ; D. Yu. Rogovskii; Z.S. Rokach; A. I . Shakadova, and O.M. Shcherbina, Farm. Zh (Kiex), 4, 49 (1979).

STRYCHNINE

643

150. Shinonogi and Co. Ltd. Jpn. Kokai Tokai Tokkyo, Koho J P 58, 27, 061, (17 Feb. 1983); C.A. 98: 166991 X , (1983).

-

151. T. Daldrup; F . 'Susanto and P. Michalke, F r e s e n i u s Z. Anal. Chem. 3 0 8 ( 5 ) , 413 (1981); C.A. 96: 29498 m (1981). 152. L. G a l a n t e ; J. Bonventre; Henry S a l v i o n e and M i l t o n L . Bastos, J. Anal. T o x i c o l . 6 ( 5 ) , 262 (1982). 153. W. Poethke and W. Kinze, Pharm. Z e n t r a l h a l l e , 489 (1965) (Ger)

.

154. M. Hong; S. Gao and Baogin Wang, Yaowu Fenxi Zazhi, 3 ( 3 ) , 155 (1983) (Ch) 155. F i s c h e r and Buchegger, i n "Modern Methods of P l a n t A n a l y s i s "By K. P a e t b and M. Tracy - Vol. IVY pp. 480-87, S p r i n g e r B e r l i n (1955). 156. A. J i n d r a and J. Pohorski, J . Pharm. Pharmacol. 3 , 444 (1951). 157. S.L. Tompsett, Acta Pharmacol, T o x i c o l ,

18,414

(1961).

158. H. Witkowski, Bull. SOC. A m i s . S c i . Lettres Poznan, S e r . B. 18, 29 (1964/65) (Ger.) 159. T. Yoshino and M. S u g i h a r a , Kagaku To Kogyo (Osaka) 4 0 ( 1 0 ) , 471 (1966) (Japan);C.A. 88666 t , (1966). 160. J. J a r z e b i n s k i , Acta Pol. Pharm. 33, 4 , 493 (1976); C.A. 86: 127355 A (1977). 161. L. Kazyak and E . C .

Knoblock, Analyt. Chem. 35, 1448 (1963).

162. E. Brochmann-Hanssen and C.R. 296 (1965). 163. H. Kolb and P.W. (1965) (Ger)

Fontan, J . Chromatog. 1 9 ( 2 ) , I

Patt, Arzneimittel-Forsch 1 5 ( 8 ) , 924

164. N . G . B i s s e t ; M. Dejestret and P. Fouche, Ann. Pharm. Fr. 2 7 ( 2 ) , 147 (1969). 165. N . Platonow; H.S. F u n n e l l ; W.T. O l i v e r , J . F o r e n s i c 1 5 ( 3 ) , 443 (1970). Sci. -

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166. A.R.

58(5),

Hanks; B.S. Engdahl and B i l l y M. Clovin, J. AOAC 961 (1975).

167. E. Marozzi; V. Gambaro; F. Lodi and A. P a r i a l i , Farmaco, Ed. P r a t . 31(5) , 253 (1976). 168. M. Dorike (Macherey Nagel and Co) US 3, 954, 651, (1976); C.A. 85; 103518 (1976). 169. M. Auer; G.L. Dadisch; B. Kolb; G. Machata, P. P o s p i s i l and W. Vycudilik, Angew Chromatogr. 30, 1 2 pp (1977) (Ger) ; C.A. 91 : 169305 t (1977). 170. W.O. Pierce; T.C. Lamoreaux; F.M. Urry, L. Kopjak and B.S. F i n k l e , J. Anal. Toxicol. 2 ( 1 ) , 26 (1978). 171. P. Demedts; M. Vanden Heede; J. Van d e r Verren and A. Heyndrickx, J . Anal. Toxicol, 6 ( 1 ) , 30 (1982). 172. P. Demedts; J. Van d e r Verren and A. Heyndrickx, F o r e n s i c Sci Int. 23(2-3), 137 (1983). 173. W.H. Anderson and D.T. S t a f f o r d , J . High Resolut. Chroma t o g r . Chromatogr. Commun. 6(5), 247 (1983).

278(2) , 174. F.T. Delbeke and M. Debackere, J. Chromatogr. 418 (1983). 175. A.C. Ray; L.O. P o s t ; Tracy P. Hewlett and John C. Reagor, Vet. Hum Toxicol , 2 3 ( 6 ) , 418 (1981). 176. N.G.

Bisset and P. Fouche', J. Chromatogr., 37, 172 (1968).

u and S. S i g g i a , A n a l y t i c a l Chemistry, 44(8), 177. C. - Y i W 1499 (1972). 178. R. Verpoorte and A. Baerheim Svendsen, J. Chromatogr. 100, 227 (1974). 179. R. Verpoorte and A. Baerheim Svendsen, J. Chromatogr. 109, 441 (1975). 180. R . J . Bushway; C.V. Cramer; A.R. J-AOAC, 58(5), 957 (1975).

Hanks and B.M.

181. P. James T w i t c h e t t , J. Chromatogr.,

104,205

Clovin, (1975).

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Crouch and C . R .

S h o r t , J. AOAC, 6 1 ( 3 ) , 612 (1978).

184. T. E g l o f f ; A. Niederwieser; K. P f i s t e r ; A. Otten; B. Steinmann; W. S t e i n e r and R. Gitzelmann, J. C l i n . Chem. 20(4), 203 (1982). C l i n . Biochem. 2 ( 6 ) , 326 (1982);. 185. H. L i and J . Zhou, Yaowu Fenxi Zazhi, C.A. 98: 166957 t” (1982).

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186. H. L i and J. Zhou, i b i d , 2 ( 6 ) , 330 (1982); C.A. 98 : 204487X (1982). 187. L. A l l i o t ; G. Bryant and P. S. Guth, J. Chromatogr. 2 3 2 ( 2 ) , 440 (1982). 188. J. Hayakawa; N. Noda; S. Yamada and K. Uno, Yakugaku Zasshi, 104(1), 57 (1984); C.A. 100: 180156 v (1984).

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189. R. Dennis, J. Pharm. Pharmacol, 36, 332 (1984). 190. I . L u r i e , J . AOAC 6 0 ( 5 ) , 1035 (1977). 191. A.C. Ray; S.H. Tamulinas and J . C . Reagor, Proc. Annu. Meet. - Am. Assoc. Vet. Lab. Diagn., 21, 185 (1978). 192. R. Gimet and A. F i l l o u x , J. Chromatogr. 177(2), 333 (1979). 193. B.B.

Wheals, J. Chromatogr. 187(1), 65 (1980).

194. T. Daldrup; P. Michalke and W. Newsl. 1 0 ( 1 ) , 1 (1982).

Bolehme, Chromatogr.

195. A. Constantinesou and T. S t a n , Farmacia l l ( 6 ) , 379 (1963). 196. I. S i r b u and P. Bebesel, Rev. Chim. (Bucharest) 10, 641 (1959)

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197. P. Bebesel and I . S i r b u , Acad. Rep. Populare Romine, S t u d i i Cercetari Chim. 9, 351 (1961). 198. M. Sarsunova, J. Tolgyessy and M. H r a d i l , Pharmazie 1 9 ( 5 ) , 336 (1964).

-

199. R.A. Wiley and J . L . 144 (1967).

Metzger, J. Pharm. S c i . , = ( 1 ) ,

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201. W. Manikowski and Z. Kedziora, Ann. Pharm. 131 (1967).

(Poznan),

6,

VIDARABINE

Wen-Hai Hong, 'bun Chang and Robert E. Daly

1.

Introduction 1.1 History

1.2 Name, Formula, Molecular Weight 1.3 Appearance, Color, Odor

2.

Physical Properties 2.1 Melting Range 2.2 Infrared Spectrum 2.3 Ultraviolet Spectrum

2.4 Nuclear Magnetic Resonance Spectrum 2.5 Optical Rotation 2.6 Solubility 2.7 pKa Value

2.8 Thermal Analysis (DSCand TGA)

3.

Synthesis and Production

4.

Impurity

5.

Stability

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 15

647

Copyright 0 1986 by the American Pharmaceutical Association A11 rights of reproduction in any form reserved.

WEN-HA1 HONG ET AL.

648 6.

Metabolism and Disposition

7.

Pharmacokinetics

8.

Methods of Analysis 8.1 Elemental Analysis 8.2 Identification

8.3 Loss on Drying 8.4 Specific Rotation 8.5 Ultraviolet Spectrophotometric Analysis 8.6 Thin-Layer Chromatography

8.7 High-pressure Liquid Chromatography 8.8 Microbiological Assay 9.

References

VIDARABINE

1.

649

Introduction 1.1 History

Vidarabine is a purine nucleoside which does not occur naturally. It was first synthesized by Lee, et. al., as a potential anticancer agent in 1960 (1.2). Its antiviral activity against herpes simplex virus (HSV) and vaccinia virus (VV) in cell culture was first demonstrated by Privat de Garilhe and DeRudder in 1964 (3). Vidarabine has subsequently been obtained from fermentation cultures of Streptomyces antibioticus, tested and developed by Warner-Lambert/Parke-Davis Company ( 4). It has been used for the treatment of herpes simplex virus types 1 and 2, varicellazoster, and vaccinia viruses infections ( 5). 1.2 Name, Formula, Molecular Weight

Vidarabine is a U.S. Adopted Name (USAN). Its chemical name is 9 4 8 -D-arabinofuranosy1)adenine monohydrate. It is also called adenine arabinoside, spongoadenosine, ara-A, vira-A and CI-673 ( Parke-Davis).

C10H13N504 H 2 0

MW: 285.27

1.3 Appearance, Color, Odor

Vidarabine is an odorless, white crystalline solid. 2.

Physical Properties 2.1 Melting Range

Vidarabine melts in the range of 262O and 27OOC.

WEN-HA1 HONG ET AL.

650

2.2 Infrared Spectrum

The infrared spectrum of vidarabine presented in Figure 1 w a s taken as a 0.5% dispersion of vidarabine in potassium bromide using a Perkin-Elmer Model 6 2 1 infrared spectrophotometer. Table I gives the infrared assignments which are consistent with the structure of

vidarabine. TABLE I INFRARED SPECTRAL ASSIGNMENTS FOR VIDARABINE FREQUENCY (em-1)

ASSIGNMENTS

3550 and 3450

NH stretch OH stretch NH2 bending various C-0-C stretchings

3400

- 3200

1640 1140 2.3

- 1040

Ultraviolet Spectrum The ultraviolet spectrum of vidarabine in water is shown in Figure 2. Table I1 summarizes the ultraviolet spectral data for vidarabine ( 6). TABLE II ULTRAVIOLETSPECTRAL DATA FOR VIDARABINE

PlI

1 MAX(nm)

E

1.0

257.5

12,700

7.0

259.0

13,400

13.0

259.0

14,000

2.4

Nuclear Magnetic Resonance Spectrum The NMR spectrum of vidarabine is shown in Figure 3. The spectrum was obtained with a Varian Model 60 MC spectrometer. Deuterated dimethylsulfoxide was used as the solvent with tetramethylsilane as an internal standard. The assignments, chemical shifts and multiplication are described in Table I11 and are consistent with the structure of vidarabine.

VIDARABINE

65 1 WAVELENGTH MICRONS

2.5

100

3

$

f,

5

8 ?lF

1214 1822 35 3

Figure 1 Infrared Spectrum of Vidarabine

w 0 z U

m

a 0 v)

m U

220

260

WAVELENGTH(nm)

Figure 2 Ultraviolet Spectrum of Vidarabine in Water

rr 0

m

VIDARABINE

653

TABLE IIl NUCLEAR MAGNETIC RESONANCE ASSIGNMENTS FOR VIDARABINE CHEMICAL SHIFTS ppm

MULTIPLICITY

NO. OF PROTONS

8.27, 8.21

Doublet

2

Two aromatic protons, one at C-2, and one at C-8 of adenine

7.29

Singlet

2

Two protons of the 6amino group

6.38, 6.31

Doublet

1

Anomeric proton at C-1 split by the cis proton at C-2 with a J of 4.2 cps

5.63

Triplet

2

A superposition of two doublets due to the hydroxyl protons of (7-2 and Ct-3

5.18

Triplet

1

Ct-5-h ydroxyl proton being split by the Ct-5 protons

4.22

Multiplet

2

Two of the five protons at C'-2, C'-3, (7-4 and C'-5 being split by the neighboring protons

3.8, 3.71

Multiplet

3

Three of t h e five protons a t C'-2, (7-3, C'-4 and C'-5

ASSIGNMENTS

WEN-HA1 HONG ET AL.

654

2.5 Optical Rotation The specific rotations of a 1% (w/v) vidarabine solution in dimethylformamide are presented in Table IV. TABLE IV SPECIFIC ROTATIONS OF A 1%(w/v) VIDARABINE SOLUTION IN DIMETHYLFORMAMIDB

I

WAVELENGTH (nm)

589 (NaD) 578

-2.5' -2.9'

546 436 365

-12.5' -30.4' -76.2'

Vidarabine Lot RxX41064 was used The specific rotations of a 1% (w/v) solution of vidarabine and its optical enantiomorph, 9 4 B -L-arabinofuranosyl) adenine, in dimethylsulfoxide are presented in Table V. As shown, the rotations for the enantiomorph are opposite in sign. The enantiomorph was prepared according to the procedure given by Glaudemans and Fletcher ( 7). TABLE V SPECIFIC ROTATIONS OF A 1%(w/v) VIDARABINE AND ITS OPTICAL ENANTIOMORPH 9-( bL-ARABINOFURANOSYL) ADENINE, SOLUTIONS IN DMSO [ a1

WAVELENGTH

(nm) 589 (NaD) 578 546 436 365

VIDARABINE -9.7'

-10.5' -12.5' -30.4' -76.2'

25

OPTICAL ENANTIOMORPH +9.2'

+9.8' +12.0° +30.2' +76.4'

VI DAM B I N E

655

2.6 Solubility

Since vidarabine behaves as both a weak acid and a weak base, its solubility in water depends on pH as well as other factors such as temperature and state of hydration. The aqueous solubility of vidarabine, expressed in terms of the hydrate, at various pHs is shown in Figure 4. As seen, vidarabine behaves as a neutral compound over the pH region 5 to 10.5. Over this pH region the solubility is invarient and equal to 0.44 mg per m l (23OC). Also, as seen, rather acidic or basic pHs are required to significantly increase its solubility. Hydration influences vidarabine solubility as shown in Figure 5. The solubility of the anhydrate has not been accurately determined since conversion of anhydrate to hydrate occurs very rapidly as shown in Figure 5. However, the anhydrate is at least 5 times more soluble than the hydrate in water. Solubilities of vidarabine at various temperatures are given in Figure 6. Since the van't Hoff relationship is applicable, these data indicate the monohydrate is the stable solid phase in equilibrium with solution at temperatures in the range of 25' to 100°C. In regard to nonaqueous solvents, the solubility of vidarabine in such commonly used organic solvents as alcohols, esters, chlorinated hydrocarbons, etc., is very low. Polar solvents such as dimethylsulfoxide, and dimethylformamide are good solvents for vidarabine as is indicated by the data given in Table VI. TABLE VI SOLUBILITY OF VIDARABINE IN DIMETHY LFORMAMIDE, DIMETHY ISULFOXIDE AND DIMETHYISULFOXIDE - WATER MIXTURE AT 25OC SOLVENT Dimethylf ormamide

Dimethylsulfoxide DMSOI Water (v/v) 90/10

SOLUBILITY (mg/ml) 20 250 150

75/25

20.5

50150

3.4

25/75

1.2

WEN-HA1 HONG ET AL.

656

4 7

04

I

I

2

4

6

. 10

8

12

14

PH

Figure 4 Solubility Profile of Vidarabine

0

E

\

2.0

-

E"

Y

1.0-

a

A I

Y

m

0 . 0

c

-

I

10

20

30

40

50

60

70

80

TIME (min.)

Figure 5 Dissolution of Vidarabine in Water at Room Temperature

VIDARABINE

657

1OOOO( 1IT" K)

Figure 6 A van't Hoff Plot of Vidarabine Solubility Data

WEN-HA1 HONG ET AL.

658

2.7 pKa Value Vidarabine is an amphoteric compound which can act as both a weak acid and a weak base, The protonated form of vidarabine has a pKa of 3.5. This value is similar to that reported for adenosine, (3.6) as would be expected ( 8 - 12). The protonation site for vidarabine has not been established but anology with adenosine suggests the nitrogen in position 1 is involved ( 13).

Vidarabine

+

H+

Vidarabine's second pKa ( 11.4) also approximates that reported for adenosine ( 12.34). This ionization is likely associated with a hydroxyl group (position unknown) contained in arabinofuranosyl moiety. 2.8 Thermal Analysis (DSC and TGA) A differential scanning calorimetry curve was obtained (Figure 7) on a Perkin-Elmer DSC-2C differential calorimeter. Using nitrogen as the purge gas, the scan was performed at a rate of 10°C/minute from 50' to 300OC. The DSC curve reveals a broad endothermic dehydration peak (onset 123.1OC) and a single sharp endothermic melting peak (onset 263.9OC) followed by decomposition of the sample.

5.00

sample wt : 2.69 mg scan rate : 10.00 degfmin

3.25

0 \

0 Q

-m

m

\

2.50

0

E max:140.91 125

I

0

50.0

80.0

110.0

140.0

170.0

TEMPERATURE

2OOx)

230.0

260.0

('GI

Figure 7 Differential Scanning Calorimetry Curve of Vidarabine

290.0

WEN-HA1 HONG ET AL.

660

A thermogravimetric analysis curve was obtained using a

Perkin-Elmer TGS-2 thermogravimetric analyzer. The analysis was performed at a rate of lO'C/minute from 35.8' to 230'C. The thermogravimetric analysis curve (Figure 8) shows a weight loss of 6.32% from 30' t o llO°C, which conforms the presence of one mole of water. 3.

Synthesis and Production Vidarabine has been synthesized by two different methods. The first involves a cleavage reaction of 94 2,3-anhydro- I3 -Dlyxofuranosyl)adenine, which is derived from 9- B -Dxylofuranosyladenine ( 1). The second is a condensation of benzoyladenine with 2,3,5-tri-O-benzyl- 6 -D-arabinosylchloride ( 7). Both methods involve laborious condensation of adenine with a sugar derivative. Ikehara $ & (14) synthesized vidarabine from naturally occurring adenosine employing a selective cleavage reaction of 8,2'-anhydroadenosine by hydrogen sulfide, and desulfurization of the resulting 6-amino9- I3 -D-arabinof uranosylpurine-8-thiol, Production of vidarabine by fermentation was conducted until the late 1960's by Parke Davis and Company using a strain of Streptomyces Antibioticus (4). The harvested beer was filtered, concentrated under reduced pressure and cooled at 2 - l0'C to effect optimal precipitation of vidarabine. The deposited crystals were filtered off and recrystallized from boiling water. The thrice crystallized vidarabine was filtered off, washed with cold water, and cold acetone, and then dried in vacuo at room temperature.

4.

Impurity The impurity most likely in vidarabine is the deaminated analog, 94 B -D-arabinofuranosyl) hypoxanthine. This impurity can be detected by TLC using silica gel plate F-254 with the

solvent

system

ethylacetate-isopropyl

alcohol-water The Rf values will vary depending on temperature, TLC plates, etc., but t h e separation of the two components is consistent. The chloroform-methanol system provides the most reproducible results.

(195:70:30) or chloroform-methanol (100:30).

VIDARABINE

66 1

Rf VALUE SOLVENT SYSTEM

Ethylacetate-isopropyl alcohol-w ater Chlorof or m-methanol

VIDARABINE DEAMINATED ANALOG 0.42

- 0.58

0.33

0.38

- 0.39

0.14

- 0.49 - 0.17

sample wt : 2.8917 mg

scan rate

TEMPERATURE ( "C)

Figure 8 Thermogravimetric Analysis Curve of Vidarabine

: 10.00 deglmin

WEN-HA1 HONG ET AL.

662

Inspection of the developed plates under short wavelength UV light reveals blue spots for either vidarabine or the deaminated analog; a minimum of 0.25 pg of either compound per spot is necessary t o detect the purine derivatives. Elution of each spot or zone from the TLC plate with aqueous methanol (50%) and examination of each eluate in the UV spectrophotometer gave the respective UV spectra for vidarabine (1 max at 260 nm) or the deaminated analog (A max at 249 nm). A minimum of 6 pg of material is required to obtain the spectrum. 5.

Stability

The solution stability of vidarabine in 0.1 N hydrochloric acid a t 100°C was followed by spectrophotometric and polarimetric methods (15). Both methods gave the same results indicating the hydrolysis only involved the cleavage of the N-glycoside linkage without racemization or other side reactions. This result was also confirmed by TLC analysis. 6.

Metabolism and Disposition The disposition of vidarabine has been studied in laboratory animals and in man following parenteral administraiton of tritium labeled drug. Vidarabine is rapidly and extensively deaminated to the less active metabolite, ara-hypoxanthine (ara-Hx) ( 16-18) followed by subsequent oxidation of the purine ring (19) (Figure 9). A small fraction of vidarabine is phosphorylated by cellular enzymes to yield ara-ATP (20). Ara-Hx represents the major metabolite in plasma, urine, erythrocytes, and tissues after the administration of tritium labeled drug.

7.

Pharmacokinetics Animal Studies Following intravenous administration of tritium labeled vidarabine to dogs and monkeys, plasma nonvolatile tritium (NV-H3) declined rapidly with estimated half life of 30 minutes and 2 to 4 hours, respectively. Loss of the tritium label resulted from further oxidation of ara-Hx which was evident from the presence of tritiated water in plasma and urine. Application of a specific HPLC assay revealed that greater than 90% of the plasma NV-H3 could be accounted for by ara-Hx. Only traces of vidarabine was detected 5 minutes after iv injection. Existing evidence also indicate that vidarabine or metabolites equilibrated rapidly between plasma and erythrocytes followed by slow incorporation of drug

VIDARABINE

663

Ara-A

Adenine I

I

I Ara-AMP

I

c

Ara-ADP

6 Ara-ATP

H

arabiise

Ara-Hx Nucleotides

Hypoxanthine

I

I

I

I

I I

I I

I ~ i n o s e

( * 3H)

Ara-X

HO

I H Xanthine

Figure 9 Pathways of Metabolic Disposition of Vidarabine

WEN-HA1 HONG ET AL.

664

related tritium label into the erythrocytes. Urinary excretion accounted for 20% and 90% of the administered dose in dogs and monkeys, respectively, with less than 1% of the dose recovered in feces. Chronic once daily iv doses failed to show any evidence of drug (or metabolite) accumulation (17). Absorption following intramuscular administration of vidarabine suspension was slow and prolonged resulting in lower plasma levels in both animal species. Half life values of 12 to 24 hours were reported (17). Vidarabine w a s extensively distributed to tissues. Following parenteral administration of 3H-ara-A, highest concentration of tritium were found in the kidneys with progressively lower concentrations in the spleen, liver, heart, skeletal muscle, lung, brain, and blood plasma ( 17). Clinical Studies Glazko et al (17) reported that following iv administration of tritium labeled vidarabine, the plasma NV-3H half life ranged from 3 to 5 hours. Among infants and children receiving unlabeled iv doses of vidarabine, the maximal plasma level was on t h e order of 1-2 pg/ml and the half life was about 1.5-2.5 hr, shorter than in the adult. In adults receiving im vidarabine, the maximal plasma level was about 0.2-0.3 pg/ml and the plasma half-life about 10-16 hr. With iv administration of vidarabine to humans, the maximum urinary excretion rate of 3H occurred in the first 4 hr. after dosing with mean recovery of about 45 percent within 24 hr. Again as in animals, maxima were delayed after intramuscular adm inistra t ion.

LePage et al (21) studied the excretion of 3H-labeled vidarabine and ara-Hx in human patients receiving rapid intravenous infusions ( 1/2-2 hr), continuous intravenous drips ( 2 4 hr), or intramuscular injections. They reported that in the two patients receiving rapid infusions, 90 percent of the drug was recovered in the urine in 24 hr. In the two patients receiving im injections, about 5 percent was recovered in 24 hr.

Kinkel and Buchanan (22) studied the plasma, cerebrospinal fluid, erythrocyte, and urine levels of vidarabine and ara-Hx Vidarabine was following iv infusion in seven patients. administered by either iv drip or by a constant rate infusion pump over a range of 4-10 days.

VIDARABINE

665

The levels of vidarabine appearing in the plasma were just barely above the limits of the HPLC assay. The levels of araHx, however, rose promptly during the infusion period and then declined promptly when infusion w a s stopped. The elimination half lives in the patients observed were between 3 1/2 and 4 hr. The levels found on day 1 and day 5 were similar, indicating no accumulation or change in metabolism, distribution, or excretion over that period. 8.

Methods of Analysis 8.1 Elemental Analysis A typical elemental analysis of a sample of vidarabine lot RxX41064 is presented in Table VII.

TABLE M ELEMENTAL ANALYSIS FOR VIDARABINE % AS MONOHYDRATE

ELEMENT

% AS ANHYDRATE

C H N

THEORY 42.10 4.60 24.55

FOUND 42.76 4.64 25.09

THEORY 44.94 4.90

FOUND 45.10 4.89

26.21

26.46

H20

6.32

5.19

0.00

0.00

8.2 Identification

The identity of vidarabine is established by infrared spectroscopy. The infrared absorption spectrum of a 0.5% dispersion in potassium bromide exhibits maxima only at the same wavelengths as that of a similar preparation of USP Vidarabine Reference Standard. Ultraviolet spectroscopy is also used to identify vidarabine. The spectrum of a sample dissolved in water (approximately 15 pg per ml) scanned from 320 - 220 nm compares qualitatively to that of a vidarabine standard similarly prepared. 8.3 Loss on Drying

Dry about 100 rng of vidarabine in vacuum a t 100°C and at a pressure not exceeding 5 m m of mercury for 4 hours; i t loses between 5.0% and 7.0% of its weight (23).

666

WEN-HA1 HONG ET AL. 8.4 Specific Rotation

Using a solution containing 10 mg of anhydrous vidarabine per m l in dimethylformamide and a polarimeter tube 1.0 decimeter in length, determine the specific rotation at 25'C at 365 nm. The value lies between -56.0' and 65.0' (23). 8.5 Ultraviolet Spectrophotom etric Analysis

Vidarabine exhibits a UV absorption band with a maximum near 260 nm in water. Quantitation of the vidarabine content is based on the net absorbances at max of the assay preparation as compared to that of the standard preparation. The method is not stability indicating since adenine, a hydrolytic product, possesses a similar UV spectrum. However, separation of adenine from vidarabine prior to UV analysis can be achieved on an anionic ion exchanger using 0.1 M pH 10 borate buffer as the eluent ( 15). 8.6 Thin-Layer Chromatography

A number of thin-layer chromatographic systems have been reported for the identification of vidarabine and for the separation from its mostly likely impurities, adenine The and 9- B-D-arabinofuranosylhypoxanthine ( 24,25). systems are described below and their respective Rf values are tabulated.

System I Plate: Silica Gel GF 254 plate, 250 p m Mobile phase: isopropyl alcohol/ammonia/water ( 65: 10:25)

Detection: shortwave UV System II

Plate: Silica Gel GF 254 plate, 250 p m Mobile phase: lower phase of chloroform/methanol/ 3% acetic acid (3:2:1) Detection: shortwave UV

VIDARABINE

667

8.6 Thin-Layer Chromatography (continued) System I11

Plate: Silica Gel GF 254 plate, 250 p m Mobile phase: Lower phase methanol/l5% ammonia (3:2:1)

of

chloroform/

Detection: shortwave UV System IV

Plate: Plastic s h e e t s (20 x 20 c m ) coated with 10 p m of PEI-impregnated MN300 cellulose with fluorescent indicator Mobile phase: 0.1 M boric acid Detection: shortwave UV

TABLE MI THIN-LAY ER CHROMATOGRAPHIC SEPARATION OF MDARABINE, 9- 8-D-ARABINOPURANOSYLHYPOXANTHINE AND ADENINE

Rf VALUE 9- B-D-ARABINOFURANOSYL

SYSTEM I I1

YIDARABINE 0.67 0.26

I11

IV

0.44

HYPOXANTHINE 0.60 0.16 0.18

ADENINE 0.74 0.43 0.60

0.57

0.49

0.40

8.7 High-pressure Liquid Chromatography (HPLC) The following systems have been reported f o r t h e analysis of vidarabine in dosage formulations and biological fluids. System 1(23,26)

Column: 30 cm x 4 mm i.d., c 1 8 column 5 or 10 p m Mobile phase: Docusate sodium/glacial acetic acid/ methanol (2.2:10:500), 4.s. to 1000 with water.

WEN-HA1 HONG ET AL.

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8.7 High-pressure Liquid Chromatography (HPLC) (cont.) System I (23,26) (continued) Temperature: ambient Detection: 254 nm

Flow rate: 1.5 ml/ml System I1 (17) Column: 60 c m x 1.8 mm i.d., Aminex A-28 column Mobile phase: 0.2 M pH 7.4 acetate buffer Temperature: 65OC Detection: 254 nm Flow rate: 1 5 - 20 ml/hr. System I11 (27) Column: 1 5 c m x 3.7 mm i.d., Aminex A-28 column Mobile phase: 0.01 M pH 6.3 borate buffer Temperature: 6OoC Detection: 254 nm Flow rate: 0.5 ml/min. System IV (28) Column: 15 cm x 4.6 mm i.d., Ultrasphere ODS 5 p m column Mobile phase: 0.01 M potassium dihydrogen phosphate (Solution A) and 30% methanol in 0.01 M potassium dihydrogen phosphate ($elution B). Linear gradient of 10 - 30% of Solution B in Solution A, achieved in 1 5 minutes. Temperature: Ambient Detection: 254 nm Flow rate: 1.0 ml/min.

VIDARABINE

669

8.7 High-pressure Liquid Chromatography ( HPLC): (cont.) System V (29) Column: column

25 c m x 4.6 mm i.d., Li-Chrosorb RP-8

Mobile phase: 20 m l of acetonitrile in 480 m l of 0.005 M sodium pentanesulfonate buffer, pH 7.2 Temperature: 4OoC Detection: 250 nm Flow rate: 1.0 ml/min. System VI (30) Column: 25 c m x 4.6 m m i.d., Whatman Partisil 10/ODS-3 column, preceded by a Brownlee C-18 guard catridge system Mobile phase: 0.01 M potassium dihydrogen phosphate, pH adjusted to 3.25 + 0.005 with acetic acid/acetonitrile (95:5) Temperature: Ambient Detection: 254 nrn Flow rate: 1.0 ml/min. 8.8 Microbiological Assay Vidarabine is assayed microbiologically with Pichia membranae-faciens as the test organism using the agar diffusion method. The potency of a sample of vidarabine is then obtained by comparing the zone of inhibition with those of the standard preparation (31). The sensitivity of this method is approximately 20 vg per ml of the test solution.

WEN-HA1 HONG ET AL.

670

9.

References

1. W.W. Lee, A. Benitez, L. Goodman, and B.R. Baker, JAm. Chem, Soc., 82, 2648 (1960). 2. E.J. Reist, A. Benitez, L. Goodman, B.R. Baker, and W.W. Lee, J. Ow. Chem., 27, 3274 (1962).

3. M. Privat de Garilhe and J. DeRudder, C. R. Acad. Sci. (D), (Paris), 259, 2725 (1964). 4. Brit, Pat. 1,159,290 (1969 to Parke Davis Company). 5. Physicians Desk Reference Medical Economics Company, 38th Edition, p 1530, 1532 (1984). 6. The Merck Index, Merck ti Co., Inc. 10th Edition, 9779 1983). Glaudeman and H.G. Fletcher, Jr., J. Org. Chem., x 3 0 0 4 (1963).

7. C.P.J.

8. H.A. Sober ed., l'Handbook of Biochemistryf1, The Chemical Rubber Company, Cleveland, Ohio, 2nd edition, 5-65, 1970. 9. E.P. Serjeant and B. Dempsey "Ionization Constants of Organic Acids in Aqueous Solutionf1, p. 561, Pergamon Press, 1979. 10. R.B. Martris and Y.H. Mariam, Met. Ions Biol. Syst., 8, 57 (1979)

11. A.E. Martell and J. Schwarzenbach, Helv. Chim. Acta, 39,653 (1956). 12. K. Wallenfels and H. Sund, Biochem. Z., 329, 41 (1957). 13. N.C. Gonnella, H. Nakanishi, J.B. Holtwick, D.S. Horowitz, K. Kanamori, N.J. Leonard, and J.P. Roberts, J. Am. Chem. Soc., 105, 2050 (1983). 14. M. Ikehara, Y. Ogiso, T. Maruyama, and M. Kaneko, Nucl. Acid Chem., 2, 485 (1978). 15. W.H. Hong and D.H. Szulczewski, J. Pharm. Sci., 68, 499 (1979). 16. G.A. LePage and I.G. Junfa, Cancer Res. 25, 46 (1965).

VIDARABINE

9.

67 1

References (continued) 17. A.J. Glazko, T. Chang, J.C. Drach, D.R. Mouger, P.E. Borondy, H. Schneider, L. Croskey, and E. Maschewske, In Adenine Arabioside: An Antiviral Agent, pp 111-133, Pavan Langston, D. Buchanan, R.A., and Alford, C.A., Jr (eds). Raven Press, N.Y (1975).

.,

18. F.A. Miller, G.J. Dixon, J. Ehrlich, B.J. Sloan and I.W. McLean, Jr., Antimicrob. Ag. Chemother., 8, 136-147 (1968). 19. M.E. Cowen, T.R. Oegema and J.C. Drach, Fed. Proc. 33, 471 (1974). 20. J.C. Drach, J.S. Bus, S.K. Schultaz and J.N. Sandberg, Biochem. Pharm., 23, 2261 (1974). 21. G.A. LePage, A. Khaliq and J.A. Gottlieb, Drug Metab. Dispos. 1, 756 (1973). 22. A.W. Kinkel and R.A. Buchanan, In Adenine Arabinoside: An Antiviral Agent, pp 197-204, Pavan-Langston, D., Buchanan, R.A., and Alford, C.A., Jr (eds), Raven Press, N.Y. (1975). 23. USP XXI, p. 1115 (1985), 21 CFR 436, 200 (e). 24. J.C. Drach and J.M. (1973).

Novack, Anal. Biochem., 52, 633

25. P.M. Schwartz and J.C. Drach, Nucl. Acid Chem., 2, 1061 (1978). 26. 21 CFR 436, 325. 27. H.G. Schneider and A.J. (1977).

Glazko, J. Chrom.,

139, 370

28. R.P. Agarwal, J. Chrom., 231, 418 (1982). 29. D.B. Bowman and R.E. Kauffman, J. Chrom., 229, 487 ( 1982). 30. W. Schroeder 111, T.L. Cupps, and L.B. Chrom., 254, 315 (1983).

Townsend, J-

31. Dr. S. W. Mamber, Warner-Lambert/Parke-Davis, Personal Com -m unica t ion.

WEN-HA1 HONG ET AL.

672

ACKNOWLEDGEMENT

We thank Mrs. Patricia A. Estwin for excellent secretarial assistance.

ZOMEPIRAC SODIUM

Mladen iiniE, Josip Kuftinec, Hrvoje Hofman, Franjo KajfeZ, Zlatko Meit

1. Foreword, History, Therapeutic Category 2. Description 2.1. Name, Formula, Molecular Weight 2.2. Appearance, Color, Odor 3. Synthesis 4, Physical Properties 4.1. Spectra 4.1.1, Infrared 4.1.2. Ultraviolet 4.1.3. Mass 4.1.4. f3oton Magnetic Resonance 4.1.5. C-Nuclear Magnetic Resonance 4.2. Solid Properties 4.2.1. Melting Characteristics 4.2. X-Ray Diffraction 4.3. Solution Properties 4.3.1. Solubility 4.3.2. Acidity (pKa) 5. Methods of Analysis 5.1. Elemental Analysis 5.2. Chromatographic Methods 5.2.1. Thin Layer 5.2.2. Gas 5.2.3. High Performance Liquid 5.3. Titrat ion 6. Stability and Degradation 7. Drug Metabolism, Pharmacokinetics, Bioavailsbilitp ANALYTICAL PROFILES O F DRUG SUBSTANCES VOLUME 15

673

Copyright 0 1986 by the American Pharmaceutical Association All rights of reproduction in any form reserved.

MLADEN 2INIC ET AL.

674

8. I d e n t i f i c a t i o n and D e t e r m i n a t i o n i n Body F l u i d s and Tissues

9. D e t e r m i n a t i o n i n PharmaceiiticRls l o . References

ZO M E PI RAC S OD1U M

675

l * Foreword, History, Therapeutic Category Zomepirac is a benzoylpyrrole acetic acid derivative which is chemically closely related to the non-steroidal anti-inflammatory agent tolmetin /I/. It is a potent analgesic with a predominantly peripheral action mediated through prostaglandin synthetase inhibition. Zomepirac sodium has been shown to be effective in many chronic and acute pain conditions; it is at least as effective as usual therapeutic doses of aspirin, codein alone or with aspirin, phenacetin and caffein, dextropropoxyphene with paracetamol or orally administered pentazocine. Additionally, zomepirac sodium may provide analgesia comparable to that of standard doses of intramuscular morphine. However, serious side effects have been found during the therapy with zomepirac sodium which resulted in withdrawal pf this drug by the manufacturer (McNeil Lab. USA) in 1983. It is expected that zomepirac sodium will be again available but only for those patients where treatment with other non-steroidal anti-inflammatory agents is not successfull.

2. Description 2.1. Name, Formula, Molecular Weight Chemical Name: sodium salt of 1,4-dimethyl-5-( p-chlorobenzoyl)-1H-pyrrole-2-acetic acid. Generic name: Zomepirac sodium Trade name: Zomax

H ClNNaO 15 13 3

C

I CH3

Mol. wt. 313.56

2.2. Appearance, Color, Odor Zomepirac is marketed as the sodium salt dihydrate; pale yellow crystalline, odorless powder. Free acid appears as white, odorless crystalline powder.

3. Synthesis The first description of a synthesis of zomepirac I) was published by Carson and Wong /1,2/ in 1973. The first step in this procedure is a modified Hantzsch pyrrole synthesis from chloroacetone ( I), diethyl 3-oxoglutarate I1 and 40% aqueous methylamine which gave the diester 111. Saponification of this product gave ( see Scheme

40% a q . C H 3 h J ~ ! 5 c 0 0 E t

1. NaOH/

H2yv:H)y

CHzCOOEt 2. H+/H20 CHzCOOEt I

I1

I CH3

SCHEME 1.

111

OOHEtOH/ HCL,

I CH3

IV

ZOM EPIRAC SODIUM

677

the diacid IV which was selectively esterified to give the monoester V. The latter was decarboxylated by heating at 190-2oo0C. The resulting pyrrole derivative VI was aroylated with N,N-dimethyl-p-chlorobenzamide (VII) and phosphoryl chloride ( Vilsmeier aroylation) to give compound VIII, which was finally saponified to zomepirac. The overall yield is about 6%. Recently, two modifications of this synthetic route appeared in the literature describing selective one pot decarboxylation /3/ and decarbethoxylation /4/ of intermedate 111 to VI. The syntheses of deuterium /5/ and tritium /6/ labelled zomepirac were also published.

4. Physical Properties The IR absorption spectra of zomepirac and its sodium salt dihydrate are shown on Fig. 1. Spectra were recorded from KBr-pelleted samples on the Pye-Unicam SP-3 infrared spec trophotometer. On the spectrum of zomepirac ( A ) , 4pe following characteristic bands should be noted (cm ) : 3100 ( C - H stretching, phenyl and pyrrole rings), 2500-3000 (carboxylic 0-H stretching), 1720 and 1700 [ketone and carboxylic C=O strzfching respectively). Further bands at 1604 and 1475 cm can be attributed to the pyrrole ring ske--l leton vibrations, and the strong bands at 760 and 750 cm to the pyrrole C-H out-of-plane vibrations. On the spectrum of zomepirac sodium salt dihydrate (BJ1 the 0-H stretching vibration band appears at 3500 cm , due to the pre ence of crystal water. Strong bands at 1580 and 1370 cm-f are caused by symmetric and asymmetric stretching vibrations of the carboxylate anion.

4.1.2. Ultraviolet Fig. 2 shows the UV spectra of zomepirac measured in methanol, 0.1 M hydrochloric acid and 0.1 M sodium hydroxide solution. Slight shift of the longer wavelength maxima toward higher values should be noted by the change of the medium from acidic (321 nm) over neutral (322 nm) to basic (328 nm). Shorter wavelength maxima at 252 nm is also shifted to 258 nm in basic solution and 261 nm in hydrochloric acid.

4.1.3. Mass The mass spectra of zomepirac Fig.(3A) and its sodium salt dihydrate (Fig. 3B) were recorded on a Kratos

Fig. 1. A-

Infrared spectrum of zomepirac i n KBr p e l l e t . Instrument: Pye-Unicam SP 3-200 spectrophotometer.

04

3500

3000

2500

2000

1600

1200

-f

cm

800

Fig. 1. B- Infrared spectrum of zomepirac sodium salt dihydrate in KBr pellet. Instrument: Pye-Unicam SP3-200 spectrophotometer.

680

MLADEN ZINIC ET AL.

0.7

0.6

0.5

0.4 Q,

0

c

5 0.3 0

v)

a 4

0.2

0.1

0

Fig. 2. Ultraviolet spectra of zomepirac in: 0.1 M sodium hydroxide, 0.1 M hydrochloric acid, and methanol solution.

......

------

Instrument: Pye-Unicam sP8-loo.

ZOMEPIRAC SODIUM

68 1

60-

6020-

0

40

60

II

80

I

’

I

100

120

*

llrrrllllllrllrl

160

Fig. 3. A- Mass spectrum of zomepirac. Instrument: Kratos MS-25.

mle

160

MLADEN ZINIC ET AL.

682

100

-

80

-

60-

-

4 0-

-

$20h

-

.CI

I-

.-a 0 0 .-5a> 80-E -

d

60-

40 20-

Fig. 3. B- Mass spectrum of zomepirac sodium salt dihydrate. Instrument: Kratos MS-25.

ZOMEPIRAC SODIUM

683

MS-25 s p e c t r o m e t e r l i n k e d t o DS 50s d a t a system u s i n g d i r e c t i n s e r t i o n probe. I n t h e mass spectrum of zomepirac, t h e m o l e c u l a r i o n a p p e a r s a t m/e 291 w i t h 51% of r e l a t i v e i n t e n s i t y . The b a s e peak a t m/e 246 i s a p p a r e n t l y formed by d e c a r b o x y l a t i o n . Other prominent peaks a r e m/e 139 ( C H 4 0 C l ) and m/e 111 ( C H C 1 ) formed by f r a g m e n t a t i o n of t h z c a r b o n y l group. .Smhft p e a k s a t m/e 276 and m/e 256 r e s u l t e d from N- d e m e t h y l a t i o n and t h e c l e a v a g e of c h l o r i n e atom i n t h e p - p o s i t i o n of t h e phenyl r i n g r e s p e c t i v e l y . The main c h a r a c t e r i s t i c s of t h e mass spectrum shown on Fig. 3 B i s t h e a b s e n c e of m o l e c u l a r i o n . The b a s e peak a t m/e 247 r e s u l t s from t h e d e c a r b o x y l a t i o n p r o c e s s . Furt t e r f r a g m e n t a t i o n can+be i n t e r p r e t e d a s f o l l o w s : m/e 231, (M C O +O), m/e 212, ( M C O + C 1 ) , m/e 184, (M+- C 0 2 + C 1 + C O ) , m/e 136 ( C 7 H 4 0 C 1 ) and m/e ( c ~ H1. ~ c ~

-

-

lfl

4.1.4,

P r o t o n Magnetic Resonance 'H-NMR spectrum of zomepirac o b t a i n e d from t h e d e u t e r a t e d DMSO s o l u t i o n i s shown on Fig. 4. The spectrum was r e c o r d e d on JEOL FX-loo s p e c t r o m e t e r u s i n g TMS a s i n t e r n a l s t a n d a r d . Assignements, c h e m i c a l s h i f t s and r e l a t i v e i n t e n s i t i e s of s i g n a l s a r e p r e s e n t e d on Table 1.

-

Table 1. 'H-NMR spectrum of zomepirac chemical s h i f t s , r e l a t i v e i n t e n s i t i e s and a s s i g n e m e n t s

1

L

0 II C

Cl

3

HzCOOA 1 2

cH3 _-I.

chem. s h i f t

i n t ens.

1.67 30 67 3.74 5.97 7.59

3 H 3 H

12.66

1 H

2 H 1 H

4 H

multiplicity singlet singlet singlet singlet mult i p l e t broad

assignement 1 2

3 4 5 6

I 5

I

3

L

I

I

'I

1

12

10

8

6 4 Fig. 4. Proton magnetic resonance spectrum of zomepirac in DMSO-d6.

2 PPm 0 Instrument: Jeol FX-loo.

ZOMEPIRAC SODIUM

685

4.1.5, ,"C-Nuclear

Magnetic Resonance &'C-NMR spectrum of zomepirac shown on Fig. 5 was obtained from the deuterated DMSO solution using TMS as internal standard. It was recorded on JEOL FX-loo spectrometer at 25.05 MHz. Chemical shifts and coupling constants are given in Table 2. The assignements for the two methyl, methylene, carboxyl and carbonyl carbons are straightforward because of their well defined regions of chemical shifts. On the other hand, resonances due to benzene and pyrrole rings give a rather complicated pattern in the appropriate region. The additivity rules for substituted benzenes /7/ have

6 and 13C-'H Table 2, 13C-NMR c$eEical shifts ' constants JCH for zomepirac

C-atom

6 (PPd

2'

136.25

3' 4' 5' 2

112.15

a

127.89

s

1 N-CH CH C=a 1'I 21'

3 4I f

s

128.62 s 32.11 t

170.78 s 32.80 q 14.13 q 185.03 s 134.81 s 130.30 d 128.45 d 139.34 6

1 JC H

171.2 129.4 139 8 127. o

164.8 168.5

2 JCH

7.9 c-2

8.i c-3

3.3 7.6

-

2.4

3.7

coupling

3 JC H

-

4.3 7.1

-

1.8

5.5 4,o

-

1.0

6.1

a &in ppm downfield from the internal TMS. Off-resonance multiplicities are given an a second dolumn. J in HZ.

m

v

r i

cy

u

i..

m

I 0

I1

0 0

-0 hl

U

-0

(D

- 0

aD

.o

0

'I!

0

-e

-5 0

-z

0

-2

0

i:' .#-I

ZOMEPIRAC SODIUM

687

h e l p e d t o a s s i g n t h e s i g n a l s f o r C - l " , C-2", C-3I1 and C-411 a t 134.81, 130.30, 128.45 and 139.34 ppm, r e s p e c t i v e l y . The o f f - r e s o n a n c e spectrum, and t h e comparison w i t h t h e r e l a t e d spectrum of p-chloroacetophenon /8/, c o n f i r med t h i s assignement. However, t h e p y r r o l e "C-resonances were n o t e a s y t o a s s i g n b e c a u s e o f t h e complex s u b s t i t u e n t e f f e c t s . I n a g a t e d decoupled spectrum t h e s i g n a l a t 112.33 pfm was a d o u b l e t w i t h a c h a r a c t e r i s t i c f i r s t - o r der C- H c o u p l i n g c o n s t a n t a t 171.2 Hz. The o t h e r t h r e e c a r b o n s were a s s i g n e d from t h e i n t r i c a t e long-range coupl i n g e f f e c t s . It should be n o t e d t h a t t h e C-5' r e s o n a n c e i s , s u r p r i s i n g l y , s i t u a t e d a t h i g h e r f i e l d (128.62 ppm) t h a n s h o u l d be e x p e c t e d f o r a p y r r o l e r i n g carbon c a r r y i n g a c a r b o n y l group /9/. 4.2. S o l i d P r o p e r t i e s 4.2.1. M e l t i n g C h a r a c t e r i s t i c s Zomepirac can be c o n v e n i e n t l y c r y s t a l l i z e d from i s o propanol. The c r g s t g l l i n e p r o d u c t n e e d l e s m e l t s and decomposes a t 178-179 C . The sodium s a l t of zomepirac c r y s t a l l i z e s a s t h e d i h y d r a t e f r m a n i s o p r o p a n o l / w a t e r mixture. The s a l t m e l t s a t 295-296 C.

a

4.2.2.

X-Ray D i f f r a c t i o n

X-Ray d i f f r a c t i o n p a t t e r n s f o r zomepirac and i t s sodium s a l t - d i h y d r a t e a r e - g i v e n i n T a b l e s 3 and 4, r e s p e c -

t i v e l y . D i f f r a c t i o n s p e c t r a were prod ced by monochromatic r a d i a t i o n from t h e CuK, l i n e (1.542 ) which wa6 o b t a i n e d by e x c i t a t i o n a t 35 kV and 20 mA. Recording c o n d i t i o n s were a8 fo11ows. O p t i c s : d e t e c t o r s l i t 0.2 , M.R. s o l l e r s l i t 3 , beam s l i t O . O O G ? " , I I i f i l t e r 3' t a k e o f f a n g l e . 0 Goniometer: s c a n a t 2 , 2o/min. D e t e c t o r : a m p l i f i e r g a i n 16 c o a r s e , 9.1 f i n e . S c i n t i l l a t i o n c o u n t e r t u b e and DC v o l t a g e a t p l a t e a u . P u l s e l i g h t s e l e c t i o n EL 9V, En o u t . Rate m e t e r : T.C. 1.0, 1000 c p s f o r zomepirac and 1.0, 2000 f o r sodium s a l t .

Y?

4.3.

Solution Properties Solubilitx Zomepirac i s f r e e l y s o l u b l e i n methanol, e t h a n o l and h o t i s o p r o p a n o l . It i s s o l u b l e i n a c e t o n e and c h l o r o f o r m , s l i g h t l y s o l u b l e i n e t h e r and p r a c t i c a l l y i n s o l u b l e i n m e t h y l e n c h l o r i d e , t o l u e n e and hydrocarbon s o l v e n t s .

3.3.1.

4.3.2.

Acidity pK pK Value of &mepirac was determined by p o t e n t i o m e t r i c ' E i t r a t i o n u s i n g a n a u t o m a t i c b u r e t t e , Model ABU 13,

MLADEN ZINIC ET AL.

688

Table 3. X-Ray d i f f r a c t i o n d a t a of zomepirac interplanar d i s t a n c e rel. i n t e n f i t y 0" da ( 8 ) I/I, 10.63 8.62 7.40 6.07 5.38 4.30 4.18 3.93 3.63 3.59 3- 50 3.41

4.16 5913 5.08 7.30 8.24 10.33 10.63 11.32

11.60 12.40 13-7 1 13.08 13.31 13.98 14.32 15.99 16.17 16.58 17-38 18.42 19.79 21.77 a

3*53 3.19 3.12 2.80

2.77 2.70 2.58 2.44 2.28 2.08

6 7 15 50

57 75

loo

75 49 16 21

19 51 25 9 6 lo 17 12

14

8 lo

~~

a=nh/2 sin

0

Based on t h e h i g h e s t i n t e n s i t y which i s s e l e c t e d a s u n i t y . coupled w i t h t h e r e c o r d i n g u n i t T i t r i g r a p h SBR-2C of a T i t r a t o r TTT2 ( a l l equipment from Radiometer-Copenhagen). The g l a s s e l e c t r o d e G-202 c , was used a g a i n s t a calomel K 401 r e f e r e n c e e l e c t r o d e . Zomepirac a c c u r a t e l y weighed and d i s s o l v e d i n an ethanol/water m i x t u r e 1:1(v / v ) , was t i t r a t e d w i t h 0.1 M NaOH s o l u t i o n . P o t e n t i o m e t r i c c u r v e s were recorded between pH 3.45-12.0 and t h e pKa v a l u e of 4.75 was obtained by c a l c u l a t Y o n a c c o r d i n g t o r e f . /lo/.

5. Methods o f A n a l y s i s Elemental A n a l y s i s P e r c e n t a g e s of e l e m e n t a l c o n t e n t of a zomepirac mol e c u l e and i t s sodium s a l t d i h y d r a t e a r e given i n Table 5.

5.1.

ZOMEPIRAC SODIUM

689

Table 4. X-Ray d i f f r a c t i o n d a t a of zomepirac sodium s a l t dihydrate

0"

interplanar distance da ( 8 )

6.07 7.00 7.64

rel. i n t e g s i t y I/Io

7.29 6.33 5.80 5.48 5-31 4.87 4.49 4.23 4.07 3.82 3-75 3.51

8.10 8.34 9-11 9.89 10.50 lo. 92 11.64 11.85 12.68 13.90 14.23 15.37 15.60 16.45 16.06 17-31 17 59

11 32

84 65 28 31 32

71 loo 20

30 44 48 30 16

3.21

3.14 2.91 2.87 2.72 2.64 2.59 2-55

12

19 lo 20 21

14 13 25 14

2,15 2.11

20.00

21.41 21.90 24.91

2-07 1.83

~~

a'b

See f o o t n o t e s i n Table 3.

Table 5. Elemental c o m p o s i t i o n of zomepirac and i t s s o d i um s a l t d i h y d r a t e

Mol. formula zomepirac

C15H14C1N03

96 of t h e element ( t h e o r . ) H

C1

N

61.79 4.89 12.16 4.80

3.2.

Na

-

0

16.41

Chromatographic Methods Thin Layer R - v a l u e s and s o l v e n t s y s t e m s f o r e l u a t i o n of zomes i l i c a g e i p l a t e s a r e g i v e n i n Table p i r a c &n Merck F 6. S p o t s a r e 1oc&d under a n UV lamp.

5.2.1.

254

MLADEN ZINIC ET AL.

690

T a b l e 6. R v a l u e s and s o l v e n t systems f o r TL chromatof graphy of zomepirac s o l v e n t system Me OH C H C1 /conc.

HOAc 3:l ~ t 6I c; f. conc. HOAc 6 : l ether/conc. HOAc 1 o : l C H C l /conc. HOAc 1 o : l cyclahexane/conc. IIOAc 3:l

Rf

0.78 0.80 0.82

0.84 0.62 0.12

5.2.2.

Gas Zomepirac w a s chromatographed on c o i l e d g l a s s column 1 m x 4 mm i . d . f i l l e d w i t h G a s Chrom Q 80-100 mesh i m p r e g n a t e d w i t h 3% 0V;jlol. Temperature maintenagce wafi a s f o l l o w s : oxen a t 200 C , i n j e c t i o n b l o c k a t 3 0 0 C , d e t e c t o r a t 300 C. The g a s r a t e s (ml/min) were: c a r r i e r g a s ( N ) and hydrogen 40, a i r 400, The column e f l u e n t was mon i $ o r e d w i t h a f l a m e - i o n i s a t i o n d e t e c t o r . Zomepirac was i n j e c t e d i n t h e form of i t s methyl e s t e r which was o b t a i n e d by t h e f o l l o w i n g p r o c e d u r e : l o mg o f zomepirac was mixedowith 0.3 m l of 10% m e t h a n o l i c BF s o l u t i o n , h e a t e d a t 60 C f o r l o min and allowed t o c o o l ? The r e s u l t i n g liq u i d was mixed w i t h 3 m l o f hexane, t r a n s f e r e d t o a s e p a r a t o r y f u n n e l and washed t h r e e t i m e s w i t h a s a t u r a t e d aqueous sodium s u l p h a t e . The d r y l i q u i d was used d i r e c t l y f o r GC. Gas chromatogram of zomepirac methyl e s t e r i s shown on Fig. 6. A l t e r n a t i v e l y , zomepirac can b e e s t e r i f i e d b y d i a z o methane s o l u t i o n ; i f i t i s a n a l y s e d w i t h o u t e s t e r i f i c a t i on d e c a r b o x y l a t i o n t a k e s p l a c e d u r i n g t h e GC a n a l y s i s /ll/.

5.2.3.

High Performance L i q u i d Zomepirac can be a n a l y s e d by HPLC on a column f i l l e d w i t h S u p e l c o s i l LC-8 ( 5 p m ) . A s a mobile p h a s e t h e mixt u r e of a c e t o n i t r i l e , 0.05 M KH PO and phosphoric a c i d (85%, d = 1 . 7 1 ) , 60, 40 and 2 m l , 2 r e i p e c t i v e l y , was used; t h e m i x t u r e had pH v a l u e of 3.5. The mobile phase was f o r c e d t h r o u g h t h e column a t 40-50 b a r ; t h i s p r e s s u r e m a i n t a i n e d a flow r a t e of 1.7 ml/min. The e f l u e n t was mon i t o r e d a t 323 nm. HPL chromatogram i s shown on Fig. 7.

5.3. T i t r a t i o n

T h i r t y mg of zomepirac was weighed (20.1 mg) i n t o a t i t r a t i o n v e s s e l an(? d i s s o l v e d i n a m i x t u r e of 1.0 p.1 o f e t h a n o l and l o m l of water. The r e s u l t i n g s o l u t i o n was

69 1

ZOMEPIRAC SODIUM

1

'

0

2

1

4

1

6

1

8

.

8

1

10 min.

Fig. 6. Gas chromatogram of zomepirac methyl ester (peak 21 and internal standard methyl heptanoate,(peak 1). Instrument: Pye-Unicam 204 chromatograph.

MLADEN ZINIC ET AL.

692

F i g . 7. HPL chromatogram of

zomepirac. I n s t r u m e n t : Pye-Unicam LC-3-XP.

J t

l

l

l

N

l

6 4 2 0 min.

ZOMEPIRAC SODIUM

693

t i t r a t e d p o t e n t i o m e t r i c a l l g u s i n g t h e g l a s s and calomel e l e c t r o d e p a i r . The c o n t e n t of zomepirac was c a l c u l a t e d from t h e q u a n t i t y o f consumed 0.1 M NaOH s o l u t i o n u s i n g t h e e q u a t i o n (1): c o n t e n t of zomepirac % =

V x Nf x E x l o o

( 1)

where V = volume of 0.1 M N a O H s o l u t i o n consumed ( m l ) f = n o r m a l i t y f a c t o r of 0.1 M NaOH s o l u t i o n E = 29.16 e q u i v a l e n t of zomepirac f o r 1 m l of 0.1 M NaOH s o l u t i o n W = mass of t h e sample (mg)

-

Using t h e above p r o c e d u r e , t h e s o l u t i o n of 50 mg of zomepirac sodium s a l t d i h y d r a t e i n 20 m l of w a t e r was t i t r a t e d w i t h 0.1 M h y d r o c h l o r i c a c i d s o l u t i o n . The c o n t e n t of t h e zomepirac sodium s a l t d i h y d r a t e was c a l c u l a t e d u s i n g t h e e q u a t i o n (1) where V = volume of 0.1 M hydroc h l o r i c a c i d consumed ( m l ) , f = n o r m a l i t y f a c t o r of 0.1 M h y d r o c h l o r i c a c i d s o l u t i o n and E = 33.96, e q u i v a l e n t of zomepirac sodium s a l t d i h y d r a t e f o r 1 m l of 0.1 M h y d r o c h l o r i c acid s o l u t i o n . 6. S t a b i l i t y and D e g r a d a t i o n Zomepirac and i t s sodium s a l t d i h y d r a t e , powdered, and t a b l e t s c o n t a i n i n g sodium s a l t d i h y d r a t e were k e p t a t 40-50 Sh r e l s t i v e humidity, a t t e m p e r a t u r e s w i t h i n t h e r a n g e of 45-50 C f o r seven days. The samples were a n a l y s e d by HPLC. T a b l e t s and powdered s a l t d i h y d m t e d i d n o t show any change b u t 7.6% o f n o n i d e n t i f i e d .i.mpurity appea r e d i n t h e powder of zomepirac.

7. Drug Metabolism, F h a r m a c o k i n e t i c s , B i o a v a i l a b i l i t y Because of i t s l i p o p h i l i c i t y , zomepirac may owe a t l e a s t p a r t of i t s a n a l g e s i c a c t i v i t y t o an i n c r e a s e d a b i l i t y t o p e n e t r a t e i n t o t h e s p i n a l c o r d and b r a i n and subs e q u e n t l y t o t h e i n h i b i t i o n of p r o s t a g l a n d i n s y n t h e s i s w i t h i n t h e c e n t r a l n e r v o u s system /l.2/. On t h e b a s i s of u r i n a r y r e c o v e r y d a t a , zomepirac i s a l m o s t absorbed a f t e r o r a l a d m i n i s t r a t i o n of d o s a g e s of 25-200 mg t o h e a l t h y s u b j e c t s . Mean peak plasma concent r a t i o n s of 2.47, 4.42 and 7.94,&g/ml were a t t a i n e d 44, 57 and 80 min a f t e r a s i n g l e o r a l dose of 50, l o o o r 200 mg, r e s p e c t i v e l y . B i o a v a i l a b i l i t y of zomepirac i s t h e same a f t e r i n g e s t i o n of t a b l e t s , c a p s u l e s o r a n aqueous sol u t i o n . Zomepirac i s e x t e n s i v e l y bound t o plasma albumin (98.5%) i n man. Maximal plasma c o n c e n t r a t i o n s ( 4000 ng/ml) a r e r e a c h e d i n 1 hour a t d o s e of l o o mg. T i s s u e c o n c e n t r a t i o n s o f r a d i o l a b e l l e d zomepirac i n

0

d

Q 0

t Q

0

E" m

N -r

/

I

0

d

0

I

0 0

I

%

d

u

ZOMEPIHAC SODIUM

695

r a t s a r e i n t h e stomach, k i d n e y s , i n t e s t i n e , and l i v e r , 2.9, 2.2, 1.7, and 0.5 t i m e s , r e s p e c t i v e l y , plasma conc e n t r a t i o n 6 h o u r s a f t e r o r a l a d m i n i s t r a t i o n of a 6 mg/ /kg dose. The r a t plasma c o n c e n t r a t i o n s of zomepirac and m e t a b o l i t e s a r e about 5 t i m e s t h e t i s s u e c o n c e n t r a t i o n s of h i g h l y p e r f u s e d o r g a n s such a s t h e h e a r t and l u n g s , and about 50 t i m e s t h a t o f t h e b r a i n . A f t e r 48 h o u r s a b o u t 0.3% of t h e dose r e m a i n s i n t h e c a r c a s s of t h e r a t , 0.1% of t h e d o s e b e i n g c o n c e n t r a t e d i n t h e l i v e r /13/. Zomepirac i s d e t e c t e d i n t h e c e r e b r o s p i n a l f l u i d o f c a t s l o m i n u t e s a f t e r i n t r a v e n o u s a d m i n i s t r a t i o n of a 3 mg/kg dose. C e r e b r o s p i n a l c o n c e n t r a t i o n s were a p p r o x i m a t e l y 7% of plasma c o n c e n t r a t i o n s 24 h o u r s a f t e r d o s i n g /14/. The e l i m i n a t i o n h a l f - l i f e of zomepirac i s a b o u t 4 h o u r s f o l l o w i n g a s i n g l e dose, b u t may be i n c r e a s e d f o l lowing m u l t i p l e d o s e s . Plasma c l e a r a n c e a f t e r d o s e s of 40 mg/kg i n r h e s u s monkeys i s d e p r e s s e d t o l e s s t h a n one h a l f t h a t observed a f t e r d o s e s of 5 and l o mg/kg. A u t h o r s consider t h a t the reason f o r t h i s nonlinear k i n e t i c s is s a t u r a t i o n o f m e t a b o l i c c o n j u g a t i o n /15/. Zomepifac i s e x c r e t e d a l m o s t e x c l u s i v e l y i n t h e u r i n e , t h e major m e t a b o l i t e b e i n g t h e g l u c u r o n i d e c o n j u g a t e , which a c c o u n t s f o r a b o u t 57% of r a d i o a c t i v i t y a f t e r a 25 mg dose. Hydroxyzomepirac and 4-chlorobenzoic a c i d a r e minor m e t a b o l i t e s /Fig. 8/ and t h e y a r e a p p r o x i m a t e l y 200-300 t i m e s l e s s a c t i v e t h a n zomepirac a s i n h i b i t o r s of human p l a t e l e t a g g r e g a t i o n i n v i t r o . About 22% of t h e d o s e i s e x c r e t e d i n u r i n e unchanged.

8. I d e n t i f i c a t i o n and D e t e r m i n a t i o n i n Body F l u i d s and Tissues The d e t e r m i n a t i o n of zomepirac i n t h e b l o o d , plasma and u r i n e i s p o s s i b l e by HPL and g a s chromatographic meHPL chromatographic d e t e r m i n a t i o n of t h o d s /11,16,17/. zomepirac i n t h e human plasma a l l o w s i t s d e t e r m i n a t i o n a t c o n c e n t r a t i o n s down t o l o ng/ml f o r 2 m l plasma sampSchutz l e s and 5 0 ng/ml f o r 1 m l plasma samples /16,17/. and Suphachearabhan /11/ r e p o r t e d t h a t UV photometry seemed t o b e t h e b e s t method f o r t h e u r i n e s c r e e n i n g . The d e t e r m i n a t i o n of zomepirac and i t s m e t a b o l i t e s by R cont i n o u s body f l u i d m o n i t o r i n g system based on HPLC was d e s c r i b e d by Muller and Z u l l i g e r /la/. T h e i r method a l lows a d e t e r m i n a t i o n of zomepirac down t o 50 ng/ml i n t h e 50 s / m l plasma samples; i n t h e c o n c e n t r a t i o n r a n g e o f 0.5-100 P g / m l o f t h e plasma a f u l l l i n e a r i t y was a c h i e ved w i t h a r e c o v e r y of 97% f o r t h e plasma samples a n d 95% f o r t h e u r i n e samples. I n t h e r e f e r e n c e s /11/ and /19/ r e v e r s e d phase HPLC

696

MLADEN ZINIC ET AL.

was used under t h e f o l l o w i n g o p e r a t i n g c o n d i t i o n s : (125 m m x 4.9 mm), Column: S p h e r i s o r b O D s , 5,&m Kontron AG Mobile p h a s e : a c e t o n i t r i l e , water, o r t h o p h o s p h o r i c a c i d 50 : 49.5 : 0.5 ml, r e s p e c t i v e l y . Flow r a t e : 2 ml/min. D e t e c t o r : UV, 313 nm. Sensitivity: variable P. more s e n s i t i v e and p r e c i s e method f o r t h e d e t e r m i n a t i o n I n t h i s meof zomepirac i n t h e human plasma i s GC /20/. thod, zomepirac m u s t be t r a n s f o r m e d i n i t s p e n t a f l u o r o b e n z y l e s t e r p r i o r t o GC a n a l y s i s . The u s e o f a n e l e c t r o n c a p t u r e d e t e c t o r ( E C D ) a l l o w s t h e d e t e r m i n a t i o n of zomep i r a c p e n t a f l u o r o b e n z y l e s t e r even i n t h e Dicogram range. The l o w e s t a c c u r a t e c o n c e n t r a t i o n i s 5 ng/ml f o r 2 m l p l a sma samples and 25 ng/ml f o r 0.1 m l plasma samples. Column f i l l i n g and o p e r a t i n g maintenance were a s f o l l o w s : Column: 122 cm x o.It cm i . d . , s i l i l a t e d g l a s s f i l l e d w i t h a Gas Chrom 8, (60-83 mesh) impregnated w i t h 3% OV-17. Temp. 230 C. C a r r i e r gas: a r g o n w i t h 546 o f mgthane. D e t e c t o r : ECDb 63 N i . Temp. 295 C. I n j e c t o r : 200 C. A combination of GC and MS a l l o w s i d e n t i f i c a t i o n and det e r m i n a t i o n o f zomepirac and i t s m e t a b o l i t e s i n body f l u i d s /11,20/.

9. D e t e r m i n a t i o n i n P h a r m a c e u t i c a l s P r e p a r i n g a n a n l y t i c a l s o l u t i o n : t e n t a b l e t s , each c o n t a i n i n g 119.89 mg of zomepirac sodium s a l t d i h y d r a t e , a r e powdered i n a m o r t a r and a mass of powder e q u i v a l e n t t o 150 mg of zomepirac i s weighed i n t o a l o o m l v o l u m e t r i c f l a s k . F i f t y m l of w a t e r a r e added, t h e c o n t e n t shaken f o r 30 min, and t h e volume made up t o mark. An a l i q u o t of 15 m l i s c e n t r i f u g a t e d ( 3 0 0 0 g, l o m i n ) , and t h e s u p e r n a t a n t taken f o r a n a l y s i s ( s o l u t i o n A ) . Standard s o l u t i o n : i n t o a l o m l v o l u m e t r i c f l a s k 18.0 mg of zomepirac sodium s a l t d i h y d r a t e a r e weighed i n , d i s s o l v e d i n w a t e r and t h e volume made up. Work up and measurement: 2 m l of c l e a r s o l u t i o n A a r e p i p e t t e d i n t o a l o m l t e s t tube. To each t u b e a r e added: 1 m l of 0.1 N H C 1 s o l u t i o n and 5 r n l of e t h e r c o n t a i n i n g 0.5 mg/ml of methyl h e p t a n o a t e a s a n i n t e r n a l s t a n d a r d . Both t u b e s a r e s t o p p e r e d , shaken f o r 5 min. and l e f t t o s t a n d f o r a n o t h e r 5 min. T h e r e a f t e r 2 m l of t h e e t h e r e a l l a y e r s were t r a n s f e r e d i n t o two c l e a n t e s t - t u b e s and t h e e t h e r was c o m p l e t e l y e v a p o r a t e d i n a s t r e a m o f a i r . To e a c h r e s i d u e were added 2 m l of 10% m e t h a n o l i c boron tri-

ZOMEPIRAC SODIUM

697

f l u o r i d e s o l u t i o n . Both t u b e s were t h e n p l a c e d i n block- t h e r m o s t a t , k e p t a t b o i l i n g t e m p e r a t u r e f o r 5 min, t h e n l e f t t o c o o l down t o room temperature. A f t e r a d d i t i o n of 8 m l of water and 1 m l of hexane t h e methyl e s t e r of zomepirac i s e x t r a c t e d i n t o t h e hydrocarbon l a y e r and t h e e x t r a c t i n j e c t e d i n t o a g a s chromatograph. D e t e r m i n a t i o n of t h e d e t e c t o r r e l a t i v e weight r e s p o n s e (RWR) : o n e p of t h e hexane e x t r a c t from t h e s t a n d a r d was i n j e c t e d i n t o t h e chromatograph. The RlrlR v a l u e was c a l c u l a t e d a c c o r d i n g t o equation ( 2 ) : *Z cI.s. RWR = (2) AI.s. cz = where A z = peak a r e a of zomepirac methyl e s t e r , A = peak a r e a of i n t e r n a l s t a n d a r d methyl h e p t a n o a h Cz= = c o n c e n t r a t i o n 0 % zomepirac methyl e s t e r i n m g / m l , cI. s. = c o n c e n t r a t i o n of methyl h e p t a n o a t e i n mg/ml Content of zomepirac i n one t a b l e t : t h e hexane e x t r a c t of t h e sample s o l u t i o n was i n j e c t e d i n t o t h e g a s chromatograph and t h e d e s i r e d c o n t e n t was c a l c u l a t e d u s i n g equation (3): X 50 X Wa

:

mg of zomepirac p e r t a b l e t =

AZ

cI.s. x RWR x W AI.s.

(3)

have t h e same meaning as i n eq. and C I where AZ, A 2 , WA is &ig'average*m&ss of a t a b l e t a n d W = mass of t h e powdered sample /21/. Acknowledgement The a u t h o r s a r e t h a n k f u l t o d r A. Nagl, L a b o r a t o r y of General and I n o r g a n i c Chemistry, F a c u l t y of S c i e n c e , U n i v e r s i t y of Zagreb, f o r X-ray d i f f r R c t i o n data.

lo. References 1. J.R. Carson and S. Wong, J. Med. Chem. 16, 172 /1973/. 2. J.R. Carson, U.S. Pat. 3,752,826 /1973/I3 . G.P. S t a h l y , E.M. Marlett and G.E. Nelson, J. Org. Chem. 48, 4423 /1973/. 4. M. i i n i 6 , J. K u f t i n e c , H. Hofman, F. K a j f e i and Z. Mei6, J. Org. Chem.. 2,697 /1985/. 5. T.H. Sun, F.S. Abbott, R. Burton and J. O r r , J. Labell e d , Compd. Radiopharm. 2,1043 /1982/. 6. D.C. Hoerr, A.T. S c h a r f , J.R. Carson and L.E. Weaner, J. L a b e l l e d Compd. Radiopharm. 20, 1383 /1983/. 7. E. P r e t s c h , T. C l e r c , J. S e i b l and W. Simon, "Tabellen z u r S t r u k t u r a u f k l a r u n g o r g a n i s c h e r Verbindungen m i t s p e k t r o s k o p i s c h e n Methoden" Springer-Verlag, B e r l i n 1976, p C120. 479 8. K.S. Dhami and J.B. S t o t h e r s , Can. J. Chem.

9,

MLADEN AINIC ET AL.

698

/1965/.

9. E B r e i t m a i e r and V o e l t e r , lo, 11.

12.

13. 14.

tr13C NMR Spectroscopy", Zhd ed. V e r l a g Chemie, Weinheirn 1978, pa 199. C. Tenford and S. Wawzanek i n l f P h y s i c a l Methods of Organic Chemistry", A. Weissberger e d i t o r , I n t e r s c . Publ. Co., v o l . I, Bd. 4, p. 2915. H, Schutz and Suphachearabhan, Arzneim.-Forsch. Drug Ri&. 293 /1984/. P.A. Morley, R.N. Brogden, A.A. Carmine, R.C. Heel, T.M. Speght and G.S. Avery, 2,250 /1982/. J.M. G r i n d e l , P.J. O ' N e i l l , K.A. Yorsey, M.H. Schwartz, L.A. Mckown, B.H. Migdalof and W.N. Wu, Drug Metab. and Disp. 8, 343 /1980/. S. Divinetz-Romero, T.L. Yake, L,D. Muschek and P.J. O ' N e i l l , Ann. Meet. SOC. Neurosci. L.A. USA, Abs. -9286

,

2,

1s /1981/. 15. P.J. O ' N e i l l , J. Pharm. S c i . 70, 818 /1981/.

178,

16. K.T. Ng and T. Snyderman, J. Chromatog. 241 /1979/* 17. C.L. Welch, T.M. Annesley, H.S. L u t h r a and T.P. Moyer, C l i n . Chem. 28, 481 /1982/. 18. H, Miiller and H.W. Z u l l i g e r , Arzneim.-Forsch. ( Drug Res.) 152 /i985/. 19. P.J. O ' N e i l l , K.A. Yorsey and L.A. Mckown, J. Pharmac o l . Exp. Ther, 219, 665 /1981/. 20. K.T. Ng and J.J. Kalbron, J. Chromatog. 311 /1983/. 21. M. i i n i 6 , J. K u f t i n e c , H. Rofman, B. B e l i n , F. KajfeZ, N. B l a i e v i 6 and 2. Meit, Acta Pharm. Jugosl. 281 11982/

z,

*,

.

z,

PROFILE SUPPLEMENTS

This Page Intentionally Left Blank

CHLORAMPHENICOL

AbduReah A. M-Badh and Hurneida A. El-Obeid

1. D e s c r i p t i o n 1.1 Nomenclature

1.2

Formulae

1 . 3 Molecular Weight

1 . 4 Elemental Composition 1.5 2.

Appearance, Color, Odor and Taste

Physical Properties 2.1

Melting P o i n t

2.2

Solubility

2.3

Spectral Properties

3.

Synthesis

4.

Methods of A n a l y s i s

4.1

I d e n t i f i c a t i o n Tests

4.2

Q u a n t i t a t i v e Analysis

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 15

701

Copyright Q 1986 by the American Pharmaceutical Association All rights of reproduction in any form reserved.

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

702

5.

Pharmacokinetics 5.1

Absorption and Distribution

5.2

Excretion

5.3

Half-life

5.4 Metabolism Acknowledgement References

CHLORAMPHENICOL

703

1. Description

1.1 Nomenclature 1.1.1 Chemical Names D(-)-threo-2-Dichloroacetamido-l-p-nitro-

phenyl-1,3-propanediol.

D( - ) threo-N-Dichloroacetyl-1-p-nit rophenyl2-amino-1,3-propanediol.

D(-) threo-2,2-Dichloro-N-[B-hydroxy-a(hydroxymethyl)-p-nitrophenethyl]acetamide. D( - ) threo-2,2-Dichloro-N- [ 2-hydroxy-l( hydroxymethyl) -2-( 4-nitrophenyl)ethyl] acetamide. 1.1.2 Generic Names

Chloramphenicol, Chloramfenicol, Chloramphenic o l m , Chloramphenicolo, Laevomycetin. 1.1.3

Trade Names (1) Alficetyn, Ambofen, Amphicol, Anacetin, Aquamycetin, Bemacol, Berlicetin, Biocetin, Biophenicol, Cafenolo, Cebenicol, Chemicetina, Chemyzin, Chlomin, Chloramex, Chloramol, Chloramphenicol-POSY Chlorasol, Chlora-tabs, Chloricol, Chlornitromycin, Chlorocid, Chloromycetin, Chloronitrin, Chloroptic, Chloro-25 Vetag, Chlorsig, Cloramidina, Clorbiotina, Clorof'enicina, Cloromicetin, Cloromycetin, Clorosintex, Comycetin, Cylphenicol, Desphen, Detreomycine, Devamycetin, Dextromycetin, Doctamicina, Duphenicol, Econochlor , Erbaplast , Ert ilen, Farmicetina, Fenicol, Globenicol, Glorous, Halomycetin, Hortfenicol , Isicetin, Ismicetina, Isophenicol, Isopto Fenicol, Kamaver, Kemicetin, Kemicetine, Kloromisin, Labamicol, Leukomycin, Levomycetin, Lomecitina, Loromisin, Mammaphenicol, Pledichol, Micochlorine, Misetin, Mycetin, Mychel, Mycinol, Neocetin, Novochlorocap, Nova-Phenicol, Novophenicol, Oftakloram, Oftalent , Oleomycetin, Opclor, Ophtaphenicol, Ophtochlor, Oralmisetin, Otachron, Otomycin, Otophen, Pantovernil, Paraxin,

CH,- OH I

H,N@ I

VH-CH-NHCOCHCl, OH

O,N-@

YH2 OC 6 H9 7 FH-CH-NHCOCHC1 OH

,

CH OH FH-;H-NH-CCOOH 11 OH 0

o,N@

FH2OH FH-CH-NHCOCH2 OH OH CH, OH FH-CH-NH, I

O,N@

dog.

O,N@

CHO

OH O2N

0

yH2 OC 6 H9 7 FH-CH-NHCOCHC12

-0OH

0

II

, O,N

f--+ OH-CH,CH,-NHCOCHCl, 02N@

yH20H ~H-CHNHCOCH2C1 OH

FH2OH

yH2OH

H-C-CH,NHCOCHC~,--€

-@ YH-CH-NHCOCHC1, OH

H,N

@-F H - c H - ~ ~ o c H c ~ , OH yH2OH

0

CH,?HN @ : C H NO C H ~ ,

-

R a t hepato-

.*

yH2 OC 6 H9

0,N -@:H-CH-NIICOCHCl

OH 0,N

CH, OH -@FH-CH-NH, I

OH Scheme

4.

Metabolites o f chloramphenicol i n i n vivo and i n v i t r o s t u d i e s .

7

705

CHLORAMPHENICOL 1.2.3

CAS R e g i s t r y No.

[ 56-75-7 1.2.4

1

Wiswesser Line Notation WNR DYQYIQ MVYGG ( 2 ) .

1.3

Molecular Weight 323.13,

1.4

322.01 ( 2 )

Elemental Composition C 40.88%,

H 3.74%, C 1 2-1.95%,

N

8.67%, 0 24.76%.

1 . 5 Appearance, Color, Odor and T a s t e Fine white t o greyish white o r yellowish white c r y s t a l s , n e e d l e s o r e l o n g a t e d p l a t e s from water o r e t h y l e n e d i c h l o r i d e w i t h very b i t t e r t a s t e . 2.

Physical Properties 2.1

Melting P o i n t

2.2

Solubility S o l u b l e (25') i n water : 2.5 mg/ml , i n propylene g l y c o l : 150.8 mg/ml, v e r y s o l u b l e i n methanol, ethanol, butanol, e t h y l a c e t a t e , acetone. F a i r l y s o l u b l e i n e t h e r , i n s o l u b l e i n benzene, petroleum e t h e r , v e g e t a b l e o i l s . S o l u b i l i t y i n 50% acetamide s o l u t i o n i s 5%. Aqueous s o l u t i o n s are n e u t r a l . N e u t r a l and a c i d s o l u t i o n s are s t a b l e on h e a t i n g .

2.3

Spectral Properties 2.3.1

U l t r a v i o l e t Spectrum The u l t r a v i o l e t a b s o r p t i o n spectrum o f chloramphenicol i n n e u t r a l methanol w a s obtained on a Cary 219 spectrophotometer. The spectrum, shown i n F i g . 1, i s c h a r a c t e r i z e d by a maximum a t 274 nm and a minimum a t 235 nm. The spectrum o f chloramphenicol i n

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

706

w u z 4 m

L

0

In m 4

-

- -

~'

1- * - . * . 220 230 240 250 260 270280290 300 30 320330340 WAVELENGTH Figure 1.

Ultraviolet epectnuP of chlormphenicol in neutral amthmol.

707

CHLORAMPHENICOL

water showed a maximum a t 278 nm (E 1%, 1 cm 2 9 8 ) ; i n 0.1N NaOH, a maximum a t 276 nm (E l%, 1 cm 2 0 0 ) ; i n 0.1N H SO4, a maximum at 278 nm ( E 1%, 1 cm 284) 73). 2.3.2

I n f r a r e d Spectrum The i n f r a r e d a b s o r p t i o n spectrum o f chloramp h e n i c o l o b t a i n e d from a potassium bromide d i s p e r s i o n i s shown i n F i g u r e 2. The spectrum w a s recorded on a Pye Unicam SP 1025 i n f r a r e d spectrophotometer. The c h a r a c t e r i s t i c bands o f t h e spectrum w i t h t h e a s s i g n ments are l i s t e d below: Frequency ( cm-l)

Assignment

3230, 3520

Broad H-bonded OH and NH stretch

3100

Aromatic C-H s t r e t c h

1700, 1570

C = 0 s t r e t c h amide 1 band, amide I1 band.

1530, 1360

N

1070

C

0

-

s t r e t c h (ArN02)

0 s t r e t c h (primary

alcohol) 850

C

-N

s t r e t c h (ArN02)

The p r i n c i p a l peaks as r e p o r t e d by Clarke ( 3 ) are 1682, 1061 and 1351 or 1526 cm'l

2.3.3

I

H-Nuclear Magnetic Resonance Spectroscopy

H ' NMR s p e c t r a o f chloramphenicol i n DMSO-d6 ( F i g u r e 3 ) and i n DFlso-dg i n t h e p r e s e n c e of D20 ( F i g u r e 4) are o b t a i n e d on a Varian-T6OA NMR s p e c t r o m e t e r . The band assignments are r e f e r e n c e d t o TMS and are l i s t e d below:

f

WAVEL NGTH Urn4

01

5

7

6

8

9

10 11 12 U 14151 i

100 90 80 70

$'

-

3500 3WO 2500 2000

1800

1600

1400

1200

lo00

WAVENUMBER Figure 1.

Infrared e p e c t w of chloramphenicol from IEBr dime.

800

60 50 40

30 20 10 0 62

CHLORAMPHENICOL

709 0

100

500

m

TM!

,......... 8.0

1.

.

.

.

6.0

7.0

.

.

.

,

5.0

.

.

.

.

...........

I

co

30

.

.

2.0

.

a

.

. . . *

0

1.0

PPM ( 6 ) Ii#uro 3.

500

'H-NMR

400

apectrum of chloramphenicol i n DWO-de.

0

100

300

patcr

TM!

I , . . - . . " . . ~ . . . . . . ' . . . . ' . . . ' ' . . . . ' . . . . . . .

8.0

7.0

'

6.0

50

GO PfW ( 8 )

3.0

2.0

Figure 4. lH NHU spectrum of chloramphenicol in WSO-d, and D1O.

1.0

0

710

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

Chemical s h i f t *

(6)

Proton Assignment

Multiplicity

No. of Protons

a.

3.43

b.

3.47

a.

3.97

b.

3.98

a.

4.93

b.

Exchanged

a.

Triplet

Cg-OH

1

b.

5.12 ) ) 5.08 )

a.

5.97

Doublet

CH-OK

1

b.

Exchanged

-

-

a.

6.47 )

b.

6.40 )

a.

7.60 )

M u l tiplet

-CH20H

2

M u l tiplet

Cg-N-

1

Triplet

-CH2-Og

1

-

-

-

-cocgc1

Singlet

1 )

Doublet

1 b.

7.58

a.

8.15 ) 1

OpN

q

Doublet

8.13

a.

8.25

b.

Exchanged

a, i n DMSO-d6 ;

2

-H -NH

Doublet

b , i n DMSO-d6

1

-

~

*

1

2

) )

b.

-

+ D20.

711

CHLORAMPHENICOL

2.3.4

1 3 C Nuclear Magnetic Resonance ( 1 3 C NMR)

The 1 3 C NMR s p e c t r a of chloramphenicol a r e obtained i n DMS-d6, containing a drop of CDC13, a t ambient temperature with 'Hdecoupling (Figure 5 ) and off-resonance (Figure 6 ) . The s p e c t r a are recorded using TMS as i n t e r n a l standard on a JEOL FXlOO MHz instrument. The chemical s h i f t s , m u l t i p l i c i t i e s and s p e c t r a l assignments a r e given below: Chemical s h i f t

(6)

Multiplicity

56.71

Doublet

60.18

Triplet

66.35

Doublet

68.93

Doublet

122.76

Doublet

127.17

Doublet

146.31

Singlet

151.12

Singlet

163.33

Singlet

Carbon assi nment* c2

c3

c21

c31

c4'

*Refer t o s t r u c t u r e i n Figure 5 f o r carbon numbering. 2.3.5

Mass Spectrum

The combined gas-chromatographic/massspectrophotometric technique w a s used f o r t h e i d e n t i f i c a t i o n and a n a l y s i s o f chloramphenic o l i n aqueous s o l u t i o n s ( 4 ) and f o r chloramphenicol and i t s metabolites i n animal t i s s u e s and body f l u i d s ( 5 , 6 ) . Becker et a1 ( 7 ) and Krueger ( 8 ) s t u d i e d t h e s p e c t r a of nonv o l a t i l e substances by f i s s i o n fragment

712

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

NH c"0 6H CI, 4N

3

Figure 5.

Proton decoupled 13C lsyR rpwtrwn of chlorunphoniool in DYBO-dg + a drop of CDClg.

DMSO

Figure 6. Off-resonance 13C NMR spectrum of chlorsmphenicol In DYBO-dg + a drop of CDC13.

CHLORAMPHENICOL

713

d e s o r p t i o n mass spectrometry. In t h e s p e c t r a o f chloramphenicol b o t h quasimolec u l a r i o n s are p r e s e n t e d w i t h t h e i r t y p i c a l two-chlorine i s o t o p i c p a t t e r n . The p o s i t i v e i o n s p e c t r a show v a r i o u s d i f f e r e n t subgroups. Although t h e r e i s v e r y l i t t l e f r a g m e n t a t i o n i n t h e n e g a t i v e i o n spectrum, a s t r o n g peak group arises n e a r m/e 152. E l spectrum o f chloramphenicol w a s shown (2) t o p o s s e s s t h e f o l l o w i n g peaks: 153(100%),

60(99%), 70(85%) 15508%), 170(70%), 106(46%), 77(40%).

P r e s e n t e d i n F i g u r e 7 i s t h e 70 e V e l e c t r o n impact ( E I ) mass spectrum o f chloramphenicol o b t a i n e d on Varian MAT 311 mass s p e c t r o m e t e r u s i n g i o n s o u r c e p r e s s u r e o f 10-6 T o r r , i o n s o u r c e t e m p e r a t u r e of 18OoC and a n emission c u r r e n t o f 300 PA. No molecular i o n i s d e t e c t e d and t h e spectrum i s dominated by m / e 153 i o n (base peak) r e s u l t i n g from t h e l o s s o f 02NC6H4CHO and H20. A proposed mechanism o f f r a g m e n t a t i o n and t h e mass/ charge r a t i o s o f t h e major fragments i s given i n Scheme 1. The chemical i o n i z a t i o n (CI) spectrum ( F i g u r e 8) w i t h methane g a s as a r e a g e n t i s o b t a i n e d on a Finnigan 4000 mass s p e c t r o m e t e r w i t h i o n e l e c t r o n energy o f 100 eV, i o n s o u r c e p r e s s u r e o f 0.3 T o r r , i o n s o u r c e t e m p e r a t u r e o f 150°C and emission c u r r e n t of 300 PA. The spectrum shows no p a r e n t molecular i o n b u t a pronounced peak r e s u l t i n g from t h e l o s s o f water (MH+-18) c o n s t i t u t e t h e b a s e peak a t m / e = 305. A quasi-molecular i o n ( M + 1) i s a l s o prominent. Two peaks a p p e a r i n g at m / e = 351 and m / e 363 are a t t r i b u t e d t o t h e t r a n s f e r o f c a r b o c a t i o n s from t h e c a r r i e r g a s . The mass s p e c t r a l assignment o f t h e prominent i o n s under CI c o n d i t i o n s i s g i v e n i n Table I .

lW.0-

-

€0

a.070

.

170

Sl

-

1W.O-

-

a.0-

200

220

Figure 7.

2w

2 40

280

300

320

Yaee spectrum of chlorunphenicol (EI).

SO

I

MIL

2M

200

Figure 8 .

$00

PO

YO

960

300

LOQ

420

Haem epectrum of chloramphenicol (CI).

440

28416.

$5

02N@

CH20H YH-CH I - NH-C-CHClp OH m/e

322 (0%)

--*

[

02N@

CH2 OH I FH-CH - NH-C E

3

+-+ 02N-@!€$H ,

OH

+I

-NHC=g 0 -HCH20H

m/e 239

m/e 239

NHCOCHC12

I J

1

m/e 77

m/e 152

CH,= CHNHCOCH = C1+ m/e 118 CH, = CH-NH-C +

0

1

CH = C1

+

1

c1

+

CH, = CH-NH-C = O m/e 70 Scheme 1. Mechanism of chloramphenicol fragmentation.

m/e 83

Scheme I (continued) CH2 OH ~

02N

1

+.

y-&-NH-COCHClp

d

0

2

.

9

+

-2P+

CH-OH

HOCH,CH = NH-CO-CC1,

U

+

OH

+ 0,N

CH = OH

+

m / e 152 HOCH2CH =

i

NH2

m / e 60

m / e 106

+

r

L

OH

m / e 291

J

+ O=C=C

/

\

c1 c1

717

CHLORAMPHENICOL Table 1. Mass S p e c t r a l Assignments of Chloramphenicol Using C I with Methane as Reagent Gas.

m/e

3.

Species

363

[M + C3H51+

351

[M + C2H51

32 3

MH+

305

[MH

- H20]+

287

[MH

-

HCl]'

27 5

[MH

-

(H20

+

+ CH2 =

O)]'

Synthesis a.

The a n t i b i o t i c w a s i n i t i a l l y i s o l a t e d from c u l t u r e s o f v a r i o u s Streptomyces s t r a i n s . The s t r u c t u r e s i m p l i c i t y of chloramphenicol made it amenable t o p r e p a r a t i o n by t o t a l s y n t h e s i s both i n t h e l a b o r a t o r y and on commercial s c a l e . One method o f s y n t h e s i s involves a base-catalysed condensation of benzaldehyde with n i t r o e t h a n o l t o a f f o r d t h e a l d o l product as a mixture of stereoisomers (Scheme 2 ) . C a t a l y t i c r e d u c t i o n g i v e s an aminodiol whose threo-isomer i s s e p a r a t e d and resolved i n t o t h e o p t i c a l isomers. The ( - ) isomer i s t r e a t e d with d i c h l o r o a c e t y l c h l o r i d e followed by treatment w i t h base t o remove t h e 0a c y l a t e d products t o a f f o r d t h e amide. The hydroxyl groups are t h e n p r o t e c t e d by means of a c e t i c anhydr i d e . The product i s n i t r a t e d t o produce t h e pn i t r o d e r i v a t i v e . Removal of t h e p r o t e c t i n g groups i s achieved by treatment with a base t o g i v e chloramphenicol ( 9 ) .

b.

'H-Chloramphenicol and i t s erythro-diastereomer with high s p e c i f i c a c t i v i t i e s were prepared by o x i d a t i o n of t h e nonlabeled a n t i b i o t i c t o i t s 0x0 d e r i v a t i v e (Scheme 3 ) , which upon reduction with 3 H - s o d i ~borohydride w a s converted t o t h e corresponding d i a s t e r e o mers. The diastereomers were s e p a r a t e d by HPLC. 2HChloramphenicol diastereomers can b e s y n t h e s i s e d s i m i l a r l y using 2H-sodium borohydride ( 1 0 ) .

X"

8 u I N

0"

!3-E I

8-3

8 x" u 1

x

I

V-Z

8-3

6 X

0

+

I

B-3

718

0

k

O2N

a

CH2OH YH-CH-NHC-CHC12 I

I

I

OH

It

0

Oxidation

-4 c ED

0

C - CH

Scheme 3

.

Synthesis of labeled chloramphenicols.

-

NH

-

It C

-

CHC12

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

720

4.

Methods of Analysis

4.1

I d e n t i f i c a t i o n Tests Dissolve about 10 mg i n 1 ml of alcohol (50%), add 3 m l o f a 1% of calcium c h l o r i d e and 50 mg o f zinc powder, heat on a water b a t h for 1 0 minutes, cool and f i l t e r ; t o t h e f i l t r a t e add 100 mg of anhydrous sodium a c e t a t e and 2 drops o f benzoyl c h l o r i d e , shake f o r 1 minute and t h e n add 0.5 ml of f e r r i c c h l o r i d e s o l u t i o n and 3 ml o f d i l u t e hydrochloric a c i d and mix; a reddish-violet or purple color i s produced. No such c o l o r i s produced when t h e t e s t i s repeated without zinc powder (11). To 5 ml of 0.1% s o l u t i o n add a few drops of s i l v e r n i t r a t e s o l u t i o n , no p r e c i p i t a t e i s produced. Heat about 50 m g with 2 ml of alcoholic potassium hydroxide s o l u t i o n on a water bath for 1 5 minutes, add a s m a l l q u a n t i t y o f decolorizing charcoal, shake, f i l t e r and t o t h e f i l t r a t e add s i l v e r n i t r a t e s o l u t i o n ; a white p r e c i p i t a t e i s produced which i s i n s o l u b l e i n n i t r i c a c i d but s o l u b l e a f t e r washing with water, i n d i l u t e ammonia s o l u t i o n . (11)

.

Dissolve about 1 0 mg i n 2 m l of alcohol ( 5 0 % ) add 4.5 m l o f d i l u t e s u l f u r i c a c i d and about 50 mg zinc powder, allow t o stand f o r 10 minutes and decant t h e supernatant l i q u i d ; cool t h e supernatant l i q u i d i n i c e ; add 0.5 ml sodium n i t r i t e s o l u t i o n , allow t o stand f o r 2 minutes and t h e n add 1 gm of urea followed by 1 ml of 2-naphthol s o l u t i o n and 2 ml of sodium hydrox i d e s o l u t i o n ; a red c o l o r i s produced (11). The n i t r o group i s reduced t o an amino group by zinc-HC1 and t h e amine i s caused t o r e a c t with dimethylaminobenzaldehyde , t h e r e s u l t i n g Schif f base gives a colored s a l t i n a c i d medium. The method can be applied as a spot t e s t . Chloramphenicol i n ointments i s e x t r a c t e d i n t o 96% ethanol. Riboflavine does not i n t e r f e r e i n t h e d e t e c t i o n o f chloramphencol (12).

CHLORAMPHENICOL

721

An orange-red c o l o r i s produced and ammonia i s evolved when chloramphenicol i s heated with 50% NaOH s o l u t i o n ( 3 ) . Chloramphenicol g i v e s a p o s i t i v e r e a c t i o n t o Fujiwara's T e s t ( 3 ) as follows: a l i t t l e s o l i d o r drop of a t e s t s o l u t i o n i s added t o a mixt u r e of p y r i d i n e (1m l ) and 20% NaOH s o l u t i o n The mixture i s heated on a b o i l i n g (2 ml) water b a t h f o r 3-5 minutes with vigorous p e r i o d i c shaking. A c o n t r o l t e s t must be c a r r i e d o u t . A red c o l o r appears i n t h e p y r i d i n e l a y e r .

.

Ammonium molybdate t e s t for micro q u a n t i t i e s g i v e s f a i n t b l u e color ( 3 ) . 4.2

Q u a n t i t a t i v e Analysis 4.2.1

B i o l o g i c a l Methods 4.2.1.1

Microbiological Methods Chloramphenicol bioassay have been r e p o r t e d by Bannatyne and Cheung ( 1 3 ) . The a u t h o r s described an accurate p l a t e diffusion bioassay f o r t h e drug, i n which t h e f a s t r e p l i c a t i n g Beneckea n a t r i e g e n s and 1.5% s a l t a g a r are used. Zone of i n h i b i t i o n were w e l l defined a f t e r 3 hours and t h e l i m i t of s e n s i t i v i t y o f t h e method w a s around 2 Llg/ml.

Fabiansson and Rut egaerd ( 1 4 ) have reviewed t h e b i o l o g i c a l methods i n c u r r e n t use f o r t h e d e t e c t i o n of a n t i b i o t i c r e s i d u e s i n s l a u g h t e r anim a l s and reported a modified method i n which t h e c o n d i t i o n s f o r t h e c o n t r o l were standardized. The standardized c o n d i t i o n s i n c l u d e t h e use of a s p o r u l a t i n g organism, B a c i l l u s s u b t i l i s , an inoculum s i z e of 0 . 5 x 105 spores/ml medium, add 5 m l o f medium (pH 6.0) p e r p l a t e . A preincubation d i f f u s i o n t i m e of

722

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

1 hour at room temperature i s recommended before incubat ion.

Horng and KO (15) have reported systematic a n a l y s i s of a n t i b i o t i c s via agar g e l electrophoresis and antimicrobial spectrum of methodology. A n t i b i o t i c s are detected i n food and drugs, by a method which employed both agar g e l electrophoresis and antimicrobial spectrum. Horng e t a 1 (16) have also reported systematic a n a l y s i s of a n t i b i o t i c s via agar g e l electrophoresis and antimicrobial spectrum-candidacy f o r detecting residual a n t i b i o t i c s i n foods. The method i s a modification of t h e method of Horng and KO (15) t o increase i t s r e s o l u t i o n power and s e n s i t i v i t y . Modification included t h e amount of agar g e l and t h e height of t h e t e s t organism s t r i p and t h e width of sample s l i t and enabled t h e a n a l y s i s of a n t i b i o t i c s at food residue concentrations (1 pg/ml).

4.2.1.2

Enzymatic Methods Smith and Smith (17) have reported an improved enzymic assay of chloramphenicol. R-factor-incoded chloramphenicol a c e t y l t r a n s e f e r a s e , from an Escherichia & mutant t h a t was highly r e s i s t a n t t o chloramphenic o l was p a r t l y p u r i f i e d and used f o r t h e assay. Only a n t i b i o t i c a l l y a c t i v e chloramphenicol i s attached by t h e enzyme. C r y s t a l l i n e chloramphenicol i s d r i e d t o constant weight a t 60" and dissolved i n serum, and 5 0 - ~ 1portions (as standards) a r e Serum samples a r e s t o r e d at -70'. and standards a r e heated a t 60° f o r 1 5 minutes bef re addition of 1 0 pl t o 50 p1 of ['&I acetylcoenzyme A

CHLORAMPHENICOL

723 s o l u t i o n (pH 7.8) and 25 p 1 o f enzyme source ( 0 . 1 spectrophotomet r i c u n i t ) . A f t e r incubation a t 37' f o r 60 minutes, t h e d i a c e t y l a t e d product i s s e l e c t i v e l y absorbed on micropore f i l t e r s and t h e n assayed by s c i n t i l l a t i o n counting

.

Robison e t a1 (18) have developed a s i m p l i f i e d radio-enzymatic assay f o r chloramphenicol , by elminat i n g t h e need for cumbersone e x t r a c t i o n procedure. A f t e r t h e a c e t y l a t i o n of chloramphenicol w i t h 14C-labeled a c e t y l CoA i n t h e presence of chloramphenicol ac e t y l t r a n s f e r a s e t h e r e a c t i o n mixture w a s added t o a toluene-based s c i n t i l l a t i o n f l u i d . Since 14C-ac e t y l a t ed c h l o r amphenicol i s more s o l u b l e t h a n 14C-labeled a c e t y l CoA i n t o l u e n e , t h e radioa c t i v e product could be counted directly. Detection and q u a n t i t a t i o n of chloramphenicol by competitive enzymel i n k e d immunoassay w a s r e p o r t e d by Campbell e t a1 ( 1 9 ) . The a s s a y f o r t h e drug i n meat involves competit i v e i n h i b i t i o n , by free chloramphenic o l i n t h e sample, of t h e binding of s p e c i f i c r a b b i t antibody t o solidphase-bound chloramphenicol. The antibody not d i s p l a c e d w a s measured by using a commercially a v a i l a b l e enzyme-linked a n t i - r a b b i t 1 gm preEnzyme p r a t ion and added s u b s t r a t e a c t i v i t y , measured spectrophotometric a l l y , w a s inversely proportional t o t h e c o n c e n t r a t i o n of chloramphenic o l i n t h e sample.

.

4.2.2

Chemical Methods 4.2.2.1

T i t r i m e t r i c Methods Navik and Polyakova ( 2 0 ) have r e p o r t e d t h e a n a l y s i s of some multi-

724

ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID component w a t er-alcohol mixtures where aqueous ethanol s o l u t i o n of chloramphenicol was determined by a tit rimet r i c method. El-Sebai e t a 1 (21) have described new i n t e r n a l i n d i c a t o r s f o r t h e determination of primary aromatic amines (chloramphenicol y i e l d s an amino group on reduction with zinc dust and hydrochloric a c i d ) . Various diazo- compounds were synthes i z e d and t e s t e d a s an i n d i c a t o r s i n t h e t i t r a t i o n of such amines with NaN02 solution. Talegaonkar e t a1 (22) have described an a l k a l i m e t r i c determination of chloramphenicol i n dimethylformamide. A solution of t h e drug i n dimethylformamide i s t i t r a t e d with sodium methoxide s o l u t i o n i n benzene-methanol 4:1, with 2 drops 1% 2-nitroaniline s o l u t i o n i n benzene a s i n d i c a t o r , t h e color change i s from yellow t o red. The r e a c t i o n involved i n t h e t i t r a t i o n i s r e p l a c e ment of one chlorine atom i n t h e drug by a methoxy group. Koka (23) has described an iodimetric method f o r t h e determination of chloramphenicol i n some medicinal mixtures. The sample i s boiled with sodium hydroxide s o l u t i o n , cooled, d i l u t e d , t r e a t e d with 0.1N iodine, s e t a s i d e for 1 0 t o 1 5 minutes i n t h e dark, and then t r e a t e d with potassium iodide s o l u t i o n and d i l u t e sulphuric acid, and t h e l i b e r a t e d iodine i s t i t r a t e d w i t h sodium thiosulphate s o l u t i o n using starch a s indicator.

-

Koka and Koltun (24) have reported another iodimetric method f o r t h e determination of chloramphenicol i n

CHLORAMPHENICOL

725 some o t h e r medicinal forms.

4.2.2.2

Polarographic Methods Chloramphenicol [ i n milk] w a s d e t e r mined by Fossdal and Jacobsen ( 2 5 ) p o l a r o g r a p h i c a l l y . The e l e c t r o n r e d u c t i o n o f t h e drug w a s s t u d i e d by polarography of 0.5 mM s o l u t i o n of v a r i o u s e l e c t r o l y t e s and of 1.7 mM t o ~.IM s o l u t i o n i n 0.5M-acetate b u f f e r a t pH 4.7 and by c y c l i c v o l t ammetry chronopot ent iomet r y and coulometry; t h e r e a c t i o n s involved a r e discussed. A well-defined polarographic wave was obtained which even i n t h e presence of 50% of milk i s a n a l y t i c a l l y u s e f u l over t h e range of 0.3 t o 60 pg of a n t i b i o t i c per m l . Chloramphenicol and it h y d r o l y s i s product 2 amino-1-( p-nitropheny1)1,3-propanediol were determined p o l a r o g r a p h i c a l l y i n pharmaceutical formulations ( 2 6 ) a f t e r s e p a r a t i o n by t h i n - l a y e r chromatography on s i l i c a g e l GF254 using a 4 : l : l nbut anol-acet i c ac id-wat e r or 2 :2 :4 ac et one-benz ene-pet roleum e t h e r solvent mixtures. The s e p a r a t i o n of t h e above two compounds by highp r e s s u r e l i q u i d chromatography (Perkin-Elmer r e v e r s e phase C l 8 column with 45:55:1 methanol-watera c e t i c a c i d o r 30:70:1 isopropanolwater-acetic a c i d ) made p o s s i b l e simultaneous determination o f t h e above two compounds. Polak e t a1 ( 2 7 ) have determined chloramphenicol i n body f l u i d s by d i f f e r e n t i a l p u l s e polarography Reduction of t h e drug a t a droppingmercury e l e c t r o d e a t -0.4 V ( v s t h e s . c . e ) a t pH 4.5 ( B r i t t o n Robinson b u f f e r ) i s used f o r t h e determination

.

726

ABDULLAH A. AL-BADR AND'HUMEIDA A. EL-OBEID of t h e drug i n such samples a f t e r p r e c i p i t a t i o n of p r o t e i n s w i t h a c e t o n i t r i l e or methanol. The c o e f f i c i e n t of v a r i a t i o n w a s 4% f o r determination of 1 5 pg/ml of chloramphenicol i n serum. Convent i o n a l d.c. plarography i s s u i t a b l e f o r determining t h e drug only i n urine. 4.2.2.3

Colorimetric Methods Several colorimetric methods f o r t h e determination of chloramphenicol have been reported i n t h e l i t e r a t u r e . Plourde and Braun (28) have described a colorimetric procedure f o r t h e determination of t h e drug i n t a b l e t s and capsules. For t a b l e t s , powder t h e m a t e r i a l and dissolve a sample containing 20 mg of chlorphenicol i n 50 ml of 1,2-dichloroethane a t 60°, cool and d i l u t e t o 100 m l w i t h dichloroethane. F i l t e r t h e s o l u t i o n and r e j e c t t h e f i r s t few m l s . For s o f t capsules, s e c t i o n longitudinaly, and dissolve i n dichloroethane (150 ml) at 60°, cool and d i l u t e t o 250 ml w i t h dichloroethane. F i l t e r , r e j e c t t h e first few m l s . and d i l u t e an a l i quot containing 20 mg of chloramphenicol t o 100 ml w i t h dichloroethane. To t h i s d i l u t e s o l u t i o n (4.5 m l ) add dichloroethane ( 6 . 5 r n l ) and 'piperidine-8-hydroxyquinoline vanadate' reagent ( 4 m l ) and l e a v e f o r 30 minutes a t room temperature. Extract excess reagent w i t h M-NaOH ( 1 0 m l ) for 30 seconds, s e t aside f o r 30 seconds and f i l t e r t h e organic phase over Na2S04 i n t o 1 m l of 5% dichloroacetic a c i d s o l u t i o n i n a c e t i c a c i d then measure t h e extinction of the r e s u l t i n g blue s o l u t i o n a t 625 nm within 2.5 hours.

CHLORAMPHENICOL

727 A c o l o r i m e t r i c method using pdimethylaminobenzaldehyde i s desc r i b e d ( 2 9 ) f o r t h e determination of chloramphenicol and o t h e r compounds. I n an a c i d medium p-dimethylaminobenzaldehyde produced r e a c t i o n prod u c t s varying i n c o l o r from yellow t o deep r e d . The r e a c t i o n products showed maximud absorption spectrum peaks between 425 and 450 nm. Ivakhnenko et a1 ( 3 0 ) described a procedure f o r absorbtiometric d e t e r mination of chloramphenicol. D i l u t e t h e r e d u c t i o n product of 0.5 gm of chloramphenicol t o 100 ml. To 1 ml of t h e s o l u t i o n add 5 ml of N-HC1, 2 ml of 0.01M - NaNO and, a f t e r 4 t o 5 minutes, 5 m l of 0.3% s o l u t i o n of a diaminoacridine reagent ( e t h a c r i d i n e l a c t a t e or p r o f l a v i n e ) ; after a f u r t h e r 2 minutes d i l u t e t h e s o l u t i o n t o 50 ml and measure t h e e x t i n c t i o n of t h e r e s u l t i n g diazocompound a t 508 nm for t h e f i r s t reagent o r a t $84 nm for t h e o t h e r a g a i n s t water. The determination of chloramphenicol i n pharmaceuticals ( s u p p o s i t o r i e s and coated t a b l e t s ) have been r e p o r t e d by Cieszynski e t a1 ( 3 1 ) using colorimetry. The product of t h e r e d u c t i o n of chloramphenicol r e a c t s w i t h guaiacol i n a l k a l i n e medium (pH 9.6) t o form a b l u e complex, which i s s t a b l e f o r up t o 5 hours, with measurement of t h e e x t i n c t i o n , a t 610 nm, 30 minutes a f t e r t h e s o l u t i o n s a r e mixed. Przyborowski ( 3 2 ) described a method for t h e determination of t h e drug and i t s p a l m i t a t e i n pharmaceuticals. The method involves t h e h y d r o l y s i s of chloramphenicol by NaOH i n a medium containing hydroxylammonium c h l o r i d e , and r e a c t i o n of t h e result-

728

ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID ing 2,2-dichloroacetohydroxamic a c i d with Fe3+. The coloured complex produced i s determined by spectrophotometry at 505 nm. A modified method f o r t h e determination of chloramphenicol palmitate and t h e contents of chloramphenicol and chloramphenicol palmitate i n s e v e r a l preparation i s a l s o given. Krezk and Lechniak (33) have reported t h e a p p l i c a t i o n of copper (11) t o t h e colorimetric determinat i o n of chloramphenicol i n ointments. The method i s based on t h e formation of a complex a f t e r mixing methanolic s o l u t i o n s o f chloramphenicol, copper (11) and methanol; t h e p r e c i p i t a t e of Cu(OH)2 i s f i l t e r e d and t h e absorbance o f t h e f i l t r a t e i s measured a t 550 nm. The composition o f t h e complex corresponds t o copper: chloramphenicol molar r a t i o = 1:2. Catechol and iodine have r e c e n t l y been used f o r t h e spectrophotometric determination of aromatic m i n e s ( 34). The method, involves mixing 15 m l of potassium a c i d p h t h a l a t e buffer s o l u t i o n (pH 3.1) , 1 m l of aqueous 0.1% catechol, 1 ml of 0.01Niodine and 1.5 ml of t h e m i n e s o l u t i o n , d i l u t i o n of t h e mixture t o 25 m l with water, and, after 5 t o 30 minutes (depending on t h e m i n e ) , spectrophotometry a t 500 t o 520 nm ( v s a reagent blank). A modification of t h i s procedure i s described f o r compounds t h a t y i e l d primary arylamino-groups on reduction (chloramphenicol)

.

Yang ( 3 5 ) has described a simult aneous determination of chloramphenicol and i t s metabolite D-threo2-amino-1-( 4-nitrophenyl )propane-l,3d i o l i n i n j e c t i o n s . A 1 0 m l sample

729

CHLORAMPHENICOL

i s d i l u t e d t o 25 ml w i t h anhydrous ethanol and a p o r t i o n i s sampled f o r s p e c t r o p o l a r i m e t r i c determinat i o n of t h e s p e c i f i c o p t i c a l r o t a t i o n a t 418 and 589 nm for t h e drug, and i t s m e t a b o l i t e s , r e s p e c t i v e l y . Divakar e t a1 ( 3 6 ) have used b r u c i n e and sodium metaperiodate f o r t h e c o l o r i m e t r i c e s t i m a t i o n of chloramphenicol. A 10-ml p o r t i o n o f t h e t e s t s o l u t i o n was b o i l e d under r e f l u x f o r 45 minutes w i t h 1 0 ml of 2M-HC1, and t h e excess of H C 1 was removed i n vaccu. The r e s i d u e was d i s s o l v e d i n 20 m l of w a r m water, and t h e s o l u t i o n was d i l u t e d t o 50 m l with water. This s o l u t i o n i n t e s t tubes w a s then t r e a t e d with 3 ml of 5 mM-brucine, 1 . 5 m l of 5 mM-NaIO4 and 2 m l of 2.3 M-H2S04 and t h e s o l u t i o n w a s d i l u t e d t o 1 0 m l with water. The t e s t t u b e s were shaken and heated on a boiling-water b a t h f o r 15 minutes. A f t e r cooling t h e cont e n t s of each t u b e were d i l u t e d t o 25 m l w i t h water and t h e absorbance of t h e s o l u t i o n w a s measured at 500 nm. 4.2.2.4

U l t r a v i o l e t Spectrophotometric Methods Chloramphenicol w a s determined i n pharmaceutical p r e p a r a t i o n s containing b o r i c a c i d , g l y c e r i n , sodium c h l o r i d e , zinc s u l p h a t e , belladonna e x t r a c t , s t r e p t o c i d , glucose, or r esorc i n o l by u l t r a v i o l e t spectrophotometric a n a l y s i s a t 278 or 315 nm. The drug w a s determined w i t h a r e l a t i v e e r r o r of f 3.46 ( 3 7 ) . Buryak ( 3 8 ) r e p o r t e d t h e a n a l y s i s of multicomponent medicines w i t h t h e a i d of a computer. The sample i s

730

ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID b o i l e d f o r 5 minutes w i t h 95% ethanol, and t h e e x t r a c t i s f i l t e r e d and d i l u t e d as necessary with ethan o l . The absorbance i s measured a t various wavelengths between 226 and 330 nm vs ethanol. The concentr a t i o n of individual components are c a l c u l a t e d from a matrix of equations r e l a t i n g t h e t o t a l absorbance t o t h e sum of t h e p a r t i a l absorbances of t h e components. The method i s s u i t a b l e f o r lanolin-based ointment s containing chloramphenicol. 4.2.2.5

I n f r a r e d Spectrophotometric Methods I n f r a r e d spectroscopy had been used for t h e determination o f t h e a n t i b i o t i c s t a b i l i t y ( 3 9 ) . The e f f e c t of e x t e r n a l f a c t o r s such as h e a t , a c i d i t y and hydrolysis on c r y s t a l l i n e a n t i b i o t i c s was examined spectroscopically. Chloramphenicol r e s i s t e d dry heat at 80' f o r 24 hrs, but was unstable at 100' f o r 2 h r s . Namigohar e t a1 (40) reported an i n f r a r e d spectrophotomet r i c determinat ion of chloramphenicol. The drug was e x t r a c t e d from capsules, creams and eye drops, with ethanol or e t h y l acetate-chloroform and chloramphenicol palmitate was extracted with chloroform. Chloramphenicol and chloramphenicol palmit a t e were then determined by i n f r a r e d spectrophotometry, i n KBr and i n chloroform, r e s p e c t i v e l y .

4.2.2.6

Proton Magnetic Resonance Spectrometric Methods Chloramphenicol i n pharmaceutical preparation has been determined using proton magnetic resonance spectrometry (41). The drug was dissolved i n dimethyl sulfoxide containing

731

CHLORAMPHENICOL

maleic a c i d as i n t e r n a l standard. The n.m.r spectrum o f t h e s o l u t i o n was recorded, and t h e peaks f o r t h e aromatic protons o f chloramphenicol at 7.6 and 7.5 ppm and t h e v i n y l i c protons of maleic a c i d a t 6.25 ppm were i n t e g r a t e d ; t h e amount of chloramphenicol w a s c a l c u l a t e d from t h e i n t e g r a t i o n r a t i o and t h e known amount of maleic a c i d . For t e n samples c o n t a i n i n g 100 t o 150 mg of pure chloramphenicol, t h e average recovery w a s 100.22% ( standard d e v i a t i o n 1.37%). The method has been a p p l i e d t o t h e a n a l y s i s of commercial capsules and o r a l suspens i o n of chloramphenicol p a l m i t a t e ; it i s r a p i d and simple and can a l s o b e used t o check t h e p u r i t y of t h e drug.

Mass Spectrometric Methods

4.2.2.7

Mass spectrometric methods have been described f o r t h e a n a l y s i s of chloramphenicol i n aqueous s o l u t i o n s (4) and i n animal t i s s u e s and body f l u i d s ( 5,6 ,52)

.

4.2.3

Chromatographic Methods A multitude of t h i n l a y e r , paper, column, gas and l i q u i d chromatographic methods have been developed f o r t h e d e t e c t i o n and d e t e r mination of chloramphenicol i n pharmaceutical formulations, b i o l o g i c a l f l u i d s and animal t i ssues

.

4.2.3.1

Thin Layer, Paper and Column Chromatography Various t h i n l a y e r , paper and column chromatographic methods used for t h e a n a l y s i s of chloramphenicol a r e o u t l i n e d i n Table 2.

732

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

4.2.3.2 Gas Chrmatographic Methods Gas chromatographic methods have been used for the determination of chloramphenicol in dosage forms and biological fluids and tissues. Table 3 summarises some of these methods.

4.2.3.3

High Performance Liquid Chromatography-(HPLC) HPLC has been extensively usea f o r the determination of chloramphenicol in pharmaceutical formulations and biological fluids as well as for the detection and determination of the drug residues in animal tissues. Some of these methods are outlined in Table 4.

Table 2.

Thin Layer, Paper and Column Chromatographic Methods for t h e A n a l y s i s o f Chloramphenicol.

Support

Solvent System

Detect i o n

Ref.

Silanized s i l i c a g e l (reverse phase)

Mixture of s o l v e n t s e .g . Dioxane , a c e t o n e , i s o p r o p y l a l c o h o l , methanol, t e t r a h y d r o f u r a n or e t h y l methyl k e t o n e w i t h c i t r a t e - p h o s p h a t e b u f f e r (pH 3, 5 or 7 ) .

-

42

Silica gel

C H C l -methanol-2.5% a q . NH3 (60:6:1)

Spray with SnC12 solut i o n , h e a t t o llO'for 7 min and s p r a y w i t h 4-dimethylaminobenzaldehyde

43

3

.

S i l u t o l W 254 Sheet

Ethyl ether.

Fluorescence quenching

44

S i l i c a gel

CH C 1 - e t h y l a c e t a t e ( 1 : b ) . 2 2

15% SnC12 i n aq. H C 1 , t h e n W.

46

High p e r f o r mance TLC cont a c t spotter

Chloroform-heptane-methanol ( 4 :2: 1)

W a t 280 nm.

46

S i l i c a g e l GF 254

n-Butanol-acetic

acid-water

( 4 :1:1)

or Acetone-benzene,

petroleum e t h e r ( 2 : 2 : 4 )

26

Table 2 (Continued)

Support

Solvent System

Detect i o n

Ref.

Whatman No. 1 Paper

2.5% Acetic a c i d i n butanol water ( 2 2 : 3 ) .

4-Dimethylaminobenzaldehyde

47

17.5 cm x 2 cm

Ethanol-ethylacetate-aq. NH3 (50: 50:l)

Biological assay

48

column of neutral alumina

.

735

(u In

m

Ln

Table 4.

Column

Mobile Phase

Detect or

Micropak CN

Hexane-CH C 1 -methanol. 2 2

W at 254 nm

A r e v e r s e phase column

A c i d i f i e d ethanol-water

w

55

DVB-MCL-0

Methanol-ammonia.

w

56

w

57

W a t 254 m

58

RP-2 (10 4 0

HPLC Methods f o r t h e Determination of Chloramphenicol

m)

0.01M K2HP04 -methanol ( 29 :21 )

.

4.5).

Ref.

P a r t i s i l - 1 0 ODs

50 mM KH2P04 (pH

1.1 Bondapak phenyl

0.05M H P O - a c e t o n i t r i l e 3 4

1-1 Bondapak C18

20% A c e t o n i t r i l e i n 0.05M N a a c e t a t e b u f f e r (pH 5.3).

W at 278 nm

60

Sep-Pak C18

Ethyl e t h e r and e t h a n o l .

w

61

Nucleosil C18

Wat er-methanol

W at 278 and 350 nm

62

W a t 276 nm

63

w

64

6,

( 7 :3 )

( 5 Fun). Hypersil H

ODS

5

1-1 Bondapak C

18

(3:l)

.

Wat e r - a c e t o n i t r i l e - a c e t a t e b u f f e r (80:20:1). Aqueous methanol

.

59

Table

4.

( Continued)

Column

Mobile Phase

Detector

Ref.

A c e t o n i t r i l e - p h o s p h a t e b u f f e r (1:3 )

W a t 278

52

RP-18

35 t o 40% a q . methanol c o n t a i n i n g 1 0 0 mg/L o f K2HP04.

w

45

Radial-PAK c18 w i t h RCSS GuardPAK precolumn.

Methanol-0.75% a c e t i c a c i d ( 3 : 7 ) a d j u s t e d t o p H 5.5 w i t h t r i e t h y l a m i n e .

W at 280 nm

65

c18 Varian Micropak MC H ( 1 0 urn)

Phosphate b u f f e r (pH 3.25)-methanola c e t o n i t r i l e (27:9:4).

w

66

Perkin-Elmer Reverse Phase c18 column.

Methanol-water-acetic a c i d ( 4 5 : 5 5 : l ) o r Isopropyl alcohol-water-acetic a c i d (30:70:1).

Polarographic

26

1 ~ - Bondapak C18

Wat er-methanol-acet i c a c i d

w

67

1-1 Bondapak C

4

w

4

18

at 278 nm

a t 280

738

5.

ABDULLAH A. AL-BADR A N D HUMEIDA A. EL-OBEID

Pharmacokinet i c s

5.1 Absorption and D i s t r i b u t i o n Oral doses o f 1 gm chloramphenicol produce peak l e v e l s of 10-20 pg/ml at 2 t o 4 h r . (68, 69). The serum l e v e l peak following o r a l a d m i n i s t r a t i o n i s approximately t h e same as t h a t o b t a i n e d following I V a d m i n i s t r a t i o n , although peak l e v e l s are reached slower by t h e former r o u t e . Yogev e t a1 ( 7 0 ) conducted a s t u d y i n which 39 c h i l d r e n w i t h H i n f l u e n s a e m e n i n g i t i s were t r e a t e d f o r 5 days w i t h o r a l chloramphenicol. All p a t i e n t s responded w e l l t o t h e r a p y and no r e l a s p s e developed. The r o u t e of a d m i n i s t r a t i o n had l i t t l e impact on t h e p e d i a t r i c p a t i e n t s t r e a t e d and t h e serum l e v e l s a f t e r o r a l a d m i n i s t r a t i o n w a s equal t o o r g r e a t e r t h a n I V f o r m u l a t i o n s . Chloramphenicol p a l m i t a t e administ e r e d t o c h i l d r e n between t h e age o f 2 months and 14 y e a r s o r a l l y every 6 h r i n doses o f 60-70 mg/kg/day r e s u l t e d i n a serum c o n c e n t r a t i o n ( a t s t e a d y s t a t e ) r a n g i n g from 1 5 . 5 t o 29.0 pg/ml w i t h a mean o f 20.2 ug/ml a f t e r 90 min from a d m i n i s t r a t i o n (71). Chloramphenicol s u c c i n a t e a d m i n i s t e r e d every 6 h r by IV r o u t e t o 18 c h i l d r e n between t h e age o f 2 months and 14 y e a r s i n doses o f 60 t o 109 mg/kg/day r e s u l t e d i n a serum c o n c e n t r a t i o n ( a t s t e a d y state) ranging from 1 2 . 5 t o 43.1 pg/ml a f t e r 90 min. from administr a t i o n (71). Lower blood l e v e l s are produced by 1M chloramphenicol sodium s u c c i n a t e t h a n by i d e n t i c a l I V doses ( 7 2 ) and about 50% t h e serum l e v e l o b t a i n e d a f t e r i d e n t i c a l doses given by o r a l r o u t e (5-6 g m / m l ) ( 6 9 ) . Oral a d m i n i s t r a t i o n o f chloramphenicol r e s u l t e d i n about 75-90% a b s o r p t i o n w i t h peak l e v e l s o c c u r i n g 0 . 5 t o 2 h r f o l l o w i n g a d m i n i s t r a t i o n (73, 7 4 ) . The p a l m i t a t e ester, however, must be hydrol y s e d b e f o r e a b s o r p t i o n . Hydrolysis may be inadeq u a t e i n newborns, i n f a n t s and c h i l d r e n and absorp. t i o n delayed and u n r e l i a b l e ( ~ 5 ~ 7 6 )Chloramphenicol b a s e a d m i n i s t e r e d o r a l l y produces peak serum l e v e l s equivalent t o o r higher than I V administration (77, 78). Peak l e v e l s o f 10-13 pg/ml are o b t a i n e d i n about 2 h r a f t e r t h e a d m i n i s t r a t i o n o f 1 gm o r a l d o s e , and s u s t a i n e d a d m i n i s t r a t i o n every 6 h r provides cumulative e f f e c t w i t h somewhat h i g h e r peak l e v e l s (68,79). Chloramphenicol sodium s u c c i n a t e , u s i n g 1 gm I V dose, produces similar blood pe& l e v e l s

CHLORAMPHENICOL

739

o n l y o c c u r i n g immediately. Blood l e v e l s o f t h e same o r d e r are o b t a i n e d i n c h i l d r e n w i t h an e q u i v a l e n t s i n g l e o r a l or I V dose ( 7 9 ) . I n newborn i n f a n t s 2 , h , or 1 2 h r a f t e r chloramphenicol s u c c i n a t e administr a t i o n ( 1 2 . 5 mg/kg, I V ) serum chloramphenicol concentr a t i o n s were < 1 0 mg/L a t each t i m e s t u d i e d . A f t e r l o a d i n g dose o f 20 mg drug/kg t h e mean serum drug c o n c e n t r a t i o n s were h i g h e r i n i n f a n t s I 2 day old t h a n i n i n f a n t s 2 3 days old ( 8 0 ) . The e f f e c t of dosing methods on chloramphenicol a b s o r p t i o n was s t u d i e d (81) u s i n g young y e l l o w t a i l s . The a b s o r p t i o n i s found t o b e g r e a t e r i n p r p o r t i o n t o t h e i n c r e a s e i n t h e drug c o n t e n t i n t h e f e e d . Of s e v e r a l d i e t s t e s t e d with varying concentration of t h e drug, a d i e t c o n t a i n i n g 50% o f t h e drug and 0.2% of a b i n d e r produced t h e h i g h e s t drug l e v e l i n t h e fish. The a b s o r p t i o n o f chloramphenicol w a s s t u d i e d ( 8 2 ) i n 6 normal v o l u n t e e r s by v a r i o u s r o u t e s of administr a t i o n . Peak serum l e v e l s were maximum w i t h t h e o r a l r o u t e o f a d m i n i s t r a t i o n . With t h e 1M r o u t e t h e average peak l e v e l w a s o n l y 70% of t h a t of t h e o r a l r o u t e . The a b s o r p t i o n of t h e drug w a s minimum and v a r i a b l e a f t e r r e c t a l a d m i n i s t r a t i o n . Animal s t u d i e s were conducted for improving t h e r e c t a l a b s o r p t i o n of chloramphenicol ( 8 3 ) . The drug r e c t a l a b s o r p t i o n was found t o b e s t r o n g l y species-dependent. In r a b b i t s t h e drug a b s o r p t i o n w a s g r e a t l y i n c r e a s e d a f t e r r e c t a l a d m i n i s t r a t i o n o f chloramphenicol erythromycin m i x t u r e and chloramphenicol - oleandomycin m i x t u r e , compared t o a b s o r p t i o n a f t e r administr a t i o n of o t h e r chloramphenicol m i x t u r e o f t h e drug e s t e r s . The i n c r e a s e i n a b s o r p t i o n w a s not observed i n people, r a t s and g u i n e a p i g s . Chloramphenicol e s t e r s e . g . chloramphenicol p a l m i t a t e must be hydrol y s e d by p a n c r e a t i c l i p a s e s i n t h e duodenum b e f o r e a b s o r p t i o n t a k e s p l a c e . Accordingly t h e r a t e o f h y d r o l y s i s o f t h e p a l m i t a t e i s a major f a c t o r i n determining t h e u l t i m a t e blood l e v e l s achieved. That t h e a b s o r p t i o n o f t h e p a l m i t a t e i s slower has been shown by Weiss e t a1 ( 7 5 ) i n t h e i r s t u d i e s i n newborn i n f a n t s . I n o l d e r c h i l d r e n , it i s a l s o r e p o r t e d (76), up t o 50% o f an a d m i n i s t e r e d dose o f p a l m i t a t e may be l o s t i n t h e f e c e s . The dose of t h e p a l m i t a t e must be h i g h e r t h a n w i t h t h e c r y s t a l l i n e chloramphenicol base (100-200 mg/kg/day) This i s i n agreement w i t h t h e r e s u l t s o f e a r l i e r s t u d i e s (84, 8 5 ) .

.

740

ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID The slow h y d r o l y s i s r e s u l t i n g i n lower blood l e v e l s r e p o r t e d by some i n v e s t i g a t o r s i s due t o t h e s e p a r a t e polymorphic s t a t e s i n which t h e p a l m i t a t e can e x i s t . One c r y s t a l l i n e form i s s u b s t a n t i a l l y more hydrolysed t h a n t h e o t h e r and t h e blood l e v e l s observed are d i r e c t l y r e l a t e d t o t h e p r o p o r t i o n o f t h a t form which i s p r e s e n t i n t h e p r e p a r a t i o n ( 8 6 ) . Subsequent t o t h e s e r e p o r t s , however, Park-Davis Company has i n d i c a t e d t h a t t h e l e s s h y d r o l y s a b l e polymorph of t h e p a l m i t a t e h a s been removed from t h e preparat i o n and t h e a b s o r p t i o n o f chloramphenicol from t h e p a l m i t a t e i s now complete and r e l i a b l e , producing blood l e v e l s e q u i v a l e n t t o I V a d m i n i s t r a t i o n ( 7 8 ) . A p r o s p e c t i v e , randomized e v a l u a t i o n o f o r a l chloramp h e n i c o l a d m i n i s t r a t i o n f o r completion of t h e r a p y o f H i n f l u e n z a e t y p e b m e n i n g i t i s i s conducted (87) i n 44 c h i l d r e n : 21 p a t i e n t s r e c e i v e d t h e drug o r a l l y a f t e r t h e second day of t h e r a p y , t h e remainder c o n t i nued t o r e c e i v e t h e drug v i a I V r o u t e . There was e q u i v a l e n t b i o a v a i l a b i l i t y o f chloramphenicol. I n 43 p a t i e n t s t h e r e s o l u t i o n o f c l i n i c a l m a n i f e s t a t i o n s and CSF a b n o r m a l i t i e s of m e n i n g i t i s w a s e q u i v a l e n t with b o t h r o u t e s . These f i n d i n g s i n d i c a t e t h a t higher t h a n normal doses of chloramphenicol p a l m i t a t e a r e not n e c e s s a r y . A 6 h o u r l y devided dosage o f 1 0 0 mg/kd/day w i l l produce blood l e v e l s e q u i v a l e n t t o t h o s e o b t a i n e d by I V o r o r a l a d m i n i s t r a t i o n o f t h e base. Adequate CSF l e v e l w i l l be maintained s i n c e c o n c e n t r a t i o n s o f chloramphenicol i n t h e CSF r e a c h l e v e l s as high as 50% of t h a t o b t a i n e d i n t h e blood and a r e w e l l above t h e M I C f o r H i n f l u e n z a e . The a b s o r p t i o n o f chloramphenicol a f t e r o r a l a d m i n i s t r a t i o n i n s e v e r e l y malnourished c h i l d r e n was found t o be e r r a t i c . T h i s r o u t e should b e avoided i n such patients (88). The b i o a v a i l a b i l i t y o f t h e o r a l chloramphenicol p a l m i t a t e salt w a s i n v e s t i g a t e d ( 7 1 ) and compared t o t h a t o f I V s u c c i n a t e s a l t i n 18 c h i l d r e n , age 2 months t o 14 y e a r s . The b i o a v a i l a b i l i t y of t h e o r a l dose w a s found t o b e g r e a t e r t h a n t h e I V p r e p a r a t i o n . The r e l a t i v e b i o a v a i l a b i l i t y o f t h e s u c c i n a t e compared t o t h e p a l m i t a t e w a s 7 0 % . T h i s can be explained by t h e prominent i n t e r p a t i e n t v a r i a t i o n i n t h e e x t e n t of h y d r o l y s i s of t h e I V s u c c i n a t e s a l t t o t h e b i o l o g i c a l l y a c t i v e chloramphenicol when compared t o t h e o r a l s a l t form. I n t h i s s t u d y a mean of 36% o f t h e a d m i n i s t e r e d I V dose w a s e x c r e t e d

CHLORAMPHENICOL

74 1

unchanged i n u r i n e . T h i s v a l u e probably a c c o u n t s f o r t h e 30% r e d u c t i o n i n b i o a v a i l a b i l i t y of t h e I V dosage form when compared t o t h e o r a l form which i s more completely hydrolysed t o t h e a c t i v e form i n t h e GIT

.

Simultaneous a d m i n i s t r a t i o n o f v i t a m i n s w i t h t h e a n t i b i o t i c has been found t o reduce chloramphenicol blood l e v e l s ( 8 9 ) . Chloramphenicol i s widely d i s t r i b u t e d i n t h e body w i t h t h e r a p e u t i c l e v e l s o c c u r i n g i n most body c a v i t i e s , t h e eye and CSF. Chloramphenicol i s 60 t o 80% p r o t e i n bound ( 7 3 ) . Other s t u d i e s , however, suggest t h a t it i s about 36% p r o t e i n bound ( 9 0 ) . There i s no l i t e r a t u r e r e p o r t on complete pharmacokin e t i c p r o f i l e o f chloramphenicol i n t h e c e r e b r o s p i n a l f l u i d . Although many s t u d i e s have documented t h e achievement o f t h e r a p e u t i c l e v e l s i n t h e CSF, o n l y one has measured s e q u e n t i a l CSF l e v e l s over an e n t i r e dosing p e r i o d ( 9 1 ) . A male a d u l t p a t i e n t w i t h H i n f l u e n z a e m e n i n g i t i s r e c e i v e d o r a l chloramphenicol 1 2 . 5 mg/kg every 6 h r . Serum and CSF l e v e l s were measured a f t e r t h e 7 t h dose t o e n s u r e s t e a d y s t a t e k i n e t i c s . The CSF l e v e l s were 5.4 pg/ml a t 0 h r , 5.7 at 30 min, 6 . 3 a t 1 h r , 7.4 a t 2 h r , 7 . 2 a t 3 h r and 7.9 a t 6 h r . The mean CSF/serum r a t i o w a s 36%. R e s u l t s o f i s o l a t e d experiments i s i n c l u d e d i n Table 5. The r e p o r t e d results emphasise t h e consid.erable i n d i v i d u a l v a r i a t i o n s , probably due t o v a r y i n g d e g r e e s o f miningeal inflammation, t h e drug a p p e a r s t o produce v e r y good CSF l e v e l s when s u f f i c i e n t doses are a d m i n i s t e r e d . With I V a d m i n i s t r a t i o n t h e CSF/serum r a t i o s range from 22.5-99%, w i t h s t e a d y s t a t e CSF l e v e l s from 4-23.3 pg/ml. Oral dosages produce s i m i lar and p o s s i b l y h i g h e r CSF l e v e l s w i t h CSF/serum r a t i o from 20-60% and CSF l e v e l s o f 4-32 pg/ml. A comparative s t u d y ( 1 0 0 ) o f t h e s t e a d y - s t a t e CSF l e v e l s a f t e r I V or o r a l doses of 1 0 0 ug/kg/day o f chloramphenicol i n 1 4 p a t i e n t s showed t h a t serum l e v e l s a f t e r I V a d m i n i s t r a t i o n occured a t 45 min. With mean corresponding t o CSF l e v e l s of 4.2 pg/ml. Peak serum l e v e l s r e s u l t i n g from o r a l dosing occured

Table

5.

Dose (mg/kg/D)

7 7 9

- 15

- 15 - 45

Summary of CSF Levels Reported i n I s o l a t e d Determinations

Dosage form

Oral

50

4

;P

28 - 66 50 - 68 18 - 26

Rectal Or& ( s i n g l e ) Or& ( m u l t i p l e ) I M (single)

Ref.

0

92

0

4 - 32 20- 64

32- 40

6 - 20 20-40

32

128

25

0

2 - 3.6 12.2 - 34 1 - 42 5 . 1 - 23

IM

66

O r a1

2-

50

IV

25 - 35

IV

18 - 72

CSF/Serum r a t i o (%)

4 8

4.- 10.3 o - 16.7 0 - 4.2 3.2 - 9.9

unknown

*

0

93

24.8 - 74.4

25-42 0 - 39 o - 28 6 - 27

3 - 21.8

50

94

14

25

56

95

mean

14.1 - 54.4

50 - 87

96

8.9

23.3 +/- 7.7 "Through Drugdex, Microfiche System, Micromedex, I n c . , Englewood, Colorado, U . S . A .

Table 5 (Continued)

... Serum l e v e l (U d m l )

CSF/Serum ratio (%)

eak a f t e r 3 hr) 11.5(peak a f t e r 3 hr) (Ventricular fluid)

1 6 . 5 ( peak a f t e r

22.5

IV

4 - 18

8

100

IV

5.5 - 1 3

10

100

IV

100

Oral

mean 4.2 (1 - 7 . 5 ) mean 6.6 (1.5 - 1 1 . 5 )

Dosge (mg/kg/D)

Dosage form

60

IV

30 m i n )

4(p

75

Ref.

57.5

97

45 - 99

98

29.5

23 - 85

99

mean peak 1 5 a t 45 min. mean peak 81.5 a t 2-3 hr.

mean 65

2 O ( peak a f t e r 30 m i n )

~

- 25 (neonates) 40 - 100

12.5 -4 0

-

40

(Older

c h i 1d r en )

-

mean 60

100

ABDULLAH A. AL-BADR A N D HUMEIDA A. EL-OBEID

744

a t 2-3 h r w i t h CSF l e v e l s o f 6.6 pg/ml. Four premature i n f a n t s under 5,500 gm were t r e a t e d w i t h p a r e n t e r a l chloramphenicol f o r c e n t r a l nervous system i n f e c t i o n due t o organisms r e s i s t a n t t o t h e p e n i c i l l i n s . Serum, c e r e b r o s p i n a l f l u i d (CSF) and v e n t r i c u l a r f l u i d c o n c e n t r a t i o n of t h e drug were measured f r e q u e n t l y d u r i n g t h e r a p y and were used t o m a i n t a i n drug dosages i n t h e safe and t h e r a p e u t i c range. Concentration of t h e drug i n t h e lumbar CSF and v e n t r i c u l a r f l u i d had a mean o f 23.3 vg/ml, c o n s i s t e n t l y g r e a t e r t h a n 45% of peak serum l e v e l s ( 9 6 ) . The d a t a show t h a t chloramphenicol e n t e r s t h e CSF i n b o t h v e n t r i c u l a r and lumbar r e g i o n s i n t h e r a p e u t i c c o n c e n t r a t ' o n s when a d m i n i s t e r e d I . V . The d i s t r i b u t i o n of ltC-labeled D( -)-threo-chloramphenic o l was s t u d i e d ( 1 0 1 ) i n newborn p i g s by whole-body autoradiography The amount o f r a d i o a c t i v i t y i n t h e l u n g , l i v e r , a d r e n a l c o r t e x , kidney, myocardium, Pancreas, t h y r o i d , s p l e e n and s k e l e t a l muscles w a s higher t h a n t h a t i n t h e blood s h o r t t i m e a f t e r t h e i n j e c t i o n and remained h i g h e r upto 8 r . A f t e r 4 and 8 h r t h e b r a i n c o n c e n t r a t i o n o f 1CC was a l s o h i g h e r t h a n t h a t o f t h e blood. I n t h e bone marrow, however, t h e c o n c e n t r a t i o n d i d not r e a c h t h a t o f t h e blood d u r i n g t h e whole experiment. I n t h e organs > 90% of t h e r a d i o a c t i v i t y was r e p r e s e n t e d by unchanged chloramphenicol; t h e e x c r e t o r y o r g a n s , t h y r o i d s and a d r e n a l s being e x c e p t i o n s . I n t h e s t u d y (81) u s i n g c u l t u r e s y e l l o w t a i l , S e r i o l a q u i n q u e r a d i d a , t h e d i s t r i b u t i o n o f chloramphenicol f o l l o w s t h e o r d e r : l i v e r > muscle > b l o o d . The volume o f d i s t r i b u t i o n f o r chloramphenicol was r e p o r t e d t o b e about 40 l i t r e s ( 7 4 ) .

.

5.2

Excretion Chloramphenicol i s r e p o r t e d t o b e 5 - 15% e x c r e t e d unchanged (73,102) w i t h r e p o r t e d r e n a l c l e a r a n c e o f 13-36 ml/min ( 7 3 ) . Renal l e v e l s may b e i n a d e q u a t e t o treat urinary t r a c t i n f e c t i o n s e s p e c i a l l y i n t h e presence o f moderately t o s e v e r e l y impaired r e n a l f u n c t i o n ( 1 0 3 ) . Some nom-al p a t i e n t s and p a t i e n t s w i t h impaired r e n a l f u n c t i o n e x h i b i t impaired f r e e drug e l i n i n a t i o n ( 1 0 4 ) . The recovery o f f r e e drug from t h e u r i n e i s d i r e c t l y p r o p o r t i o n a l t o c r e a t i n i n e c l e a r a n c e . With c r e a t i n i n e c l e a r a n c e s o f l e s s t h a n

CHLORAMPHENICOL

745

20 ml/min, l e s s t h a n 1% o f t h e a d m i n i s t e r e d doses t h a t are recovered i n t h e i n e r t i n a c t i v e form. With c r e a t i n i n e c l e a r a n c e s of < 40 m l / m i n , u r i n a r y c o n c e n t r a t i o n s o f t h e drug a r e g e n e r a l l y not h i g h enough t o t r e a t s u s c e p t i b l e organisms ( 1 0 5 ) . The s t e a d y s t a t e k i n e t i c s o f t h e o r a l p a l m i t a t e vers’is t h e I V s u c c i n a t e s a l t w a s s t u d i e d (71). No s i g n i f i c a n t c o r r e l a t i o n between t h e dose of chloramphenic o l s u c c i n a t e and serum c o n c e n t r a t i o n s o f ”free” chloramphenicol o r t h e average u r i n a r y c o n c e n t r a t i o n were found. However, t h e average u r i n a r y concentrat i o n and serum c o n c e n t r a t i o n of o r a l chloramphenicol p a l m i t a t e c o r r e l a t e s w e l l w i t h dose i n d i c a t i n g more complete and p r e d i c t a b l e h y d r o l y s i s . V a r i a b l e f r a c t i o n s of t h e dose ( a mean of 36%) w a s e x c r e t e d i n u r i n e unchanged and w a s t h e r e f o r e n o t b i o a v a i l a b l e i n a c t i v e form. Both v a r i a b l e h y d r o l y s i s and r e n a l e l i m i n a t i o n o f t h e nonhydrolyzed chloramphenicol s u c c i n a t e seems t o reduce t h e b i o a v a i l a b i l i t y of t h e a n t i b i o t i c and a p p e a r s t o c o n t r i b u t e s u b s t a n t i a l l y t o t h e wide v a r i a t i o n s i n serum c o n c e n t r a t i o n s produced f o l l o w i n g an I V dose. I n premature i n f a n t s , a n i n c r e a s e d b i o a v a i l a b i l i t y o f chloramphenicol was a result o f decreased r a t e of clearance of t h e succinate salt causing a g r e a t e r f r a c t i o n o f t h e s a l t dose t o be hydrolysed t o chloramphenicol (106). With t h e d a t a c u r r e n t l y availa b l e , chloramphenicol i s not a d v i s e d d u r i n g t h e b r e a s t f e e d i n g p e r i o d . Chloramphenicol i s e x c r e t e d i n t o breast m i l k , i n some i n s t a n c e s i n c o n c e n t r a t i o n s which are 50% of blood l e v e l s ( 1 0 7 ) . S i n g l e 1 gm o r a l doses produce peak milk l e v e l s at 3 h r , which are u n d e t e c t a b l e a t 6 h r ( 1 0 8 ) . S i n g l e dose of 500 mg o r a l l y given every 6 h r produced serum l e v e l s of 0.98 - 3.5 pg/ml i n b r e a s t milk ( 1 0 9 ) . Most i n f a n t s do not have developed h e p a t i c c o n j u g a t i o n system f o r g l u c u r o n i d a t i o n which could r e s u l t i n t o x i c i t y . Although chloramphenicol milk l e v e l s a r e not s u f f i c i e n t t o induce t h e grey-baby syndrome, t o x i c i t y t o t h e bone marrow may occur ( 1 1 0 ) . Toxic e f f e c t s i n i n f a n t s had been r e p o r t e d (111) d u r i n g t h e b r e a s t feeding period.

5.3

Half-Life I n normal, o t h e r w i s e h e a l t h y a d u l t s , t h e h a l f - l i f e o f chloramphenicol r a n g e s from 1.6 - 3 . 3 h r w i t h an

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

746

average v a l u e o f 2.7 h r (102,112). I n r e n a l and l i v e r f a i l u r e s t h e h a l f - l i f e i s a p p r e c i a b l y prolonged. I n i n f a n t s and c h i l d r e n between t h e age o f 1 month and 11 y e a r s , a mean apparent h a l f - l i f e w a s r e p o r t e d (113) t o b e 5.94 h r . A n a l y s i s o f v a r i a n c e r e v e a l e d a sample v a r i a n c e o f 21.85 i n c h i l d r e n l e s s t h a n 4 months o f age compared t o 5.87 i n c h i l d r e n g r e a t e r t h a n 4 months o f age r e f l e c t i n g a h i g h l y v a r i a b l e h a l f - l i f e i n t h e younger i n f a n t s . A s t u d y ( 1 1 4 ) o f t h e pharmacokinet i c parameters o f chloramphenicol s u c c i n a t e i n i n f a n t s and young c h i l d r e n r e v e a l e d t h a t h a l f - l i f e o f serum chloramphenicol s u c c i n a t e d i d not c o r r e l a t e with t h e h a l f - l i f e o f serum chloramphenicol. Chloramphenicol s u c c i n a t e i s l o s t i n t o t h e u r i n e i n s i g n i f i c a n t q u a n t i t y and t h i s u r i n a r y loss must b e t a k e n i n t o account i n t h e e s t i m a t i o n o f chloramphenic o l pharmacokinetic parameters. One d u r a t i o n o f i n f u s i o n o f chloramphenicol s u c c i n a t e does not a f f e c t t h e amount e x c r e t e d i n t h e u r i n e .

5.4

Metabolism I n e a r l y s t u d i e s by Glazko e t a1 (73,115,116) d a t a on t h e f a t e o f chloramphenicol i n d i f f e r e n t s p e c i e s were produced. A major m e t a b o l i c r o u t e i n v o l v i n g g l u c u r o c o n j u g a t i o n as w e l l as t h e r e d u c t i o n o f t h e n i t r o group by t h e i n t e s t i n a l f l o r a and conjugat i o n o f t h e r e s u l t i n g m i n e s were d e s c r i b e d i n r a t s , guinea p i g and dog. I n man, 90% o f a s i n g l e dose o f o f t h e drug a p p e a r s i n t h e u r i n e w i t h i n 24 h r , c h i e f l y as chloramphenicol-3-glucuronide ( 1 1 7 ) . The l i v e r i s t h e main s i t e o f g l u c u r o n i d a t i o n . With t h e development o f more s e n s i t i v e a n a l y t i c a l t o o l s o t h e r m e t a b o l i t e s of chloramphenicol have been sugggest ed (Scheme 4 ) . I n t h e i r s t u d y o f chloramphenicol metabolism i n i s o l a t e d rat h e p a t o c y t e s S i l i c i a n o e t a1 (118) chloramphenicol-3-glucuronide w a s found t o b e t h e major m e t a b o l i t e t o g e t h e r w i t h a minor m e t a b o l i t e b e l i e v e d t o b e D( -) threo-2-amino-l-( p-nitropheny1)1,3-propanediol, The formation o f t h e 3-glucuronide w a s l i n e a r w i t h r e s p e c t t o b o t h t h e c e l l concentrat i o n and t o t h e t i m e o f t h e f i r s t hour o f i n c u b a t i o n . The Ic, and Vmax v a l u e s f o r t h e g l u c u r o n i d a t i o n o f chloramphenicol were 6.4 x 10-6 M and 420 pmol/min/l08 c e l l s r e s p e c t i v e l y . The k i n e t i c s o f t h e glucuronidat i o n r e a c t i o n i n r a t hepatocytes suggest a low hepat i c e x t r a c t i o n r a t i o o f chloramphenicol ,

CH,- OH I

H,N@ I

VH-CH-NHCOCHCl, OH

O,N-@

YH2 OC 6 H9 7 FH-CH-NHCOCHC1 OH

,

CH OH FH-;H-NH-CCOOH 11 OH 0

o,N@

FH2OH FH-CH-NHCOCH2 OH OH CH, OH FH-CH-NH, I

O,N@

dog.

O,N@

CHO

OH O2N

0

yH2 OC 6 H9 7 FH-CH-NHCOCHC12

-0OH

0

II

, O,N

f--+ OH-CH,CH,-NHCOCHCl, 02N@

yH20H ~H-CHNHCOCH2C1 OH

FH2OH

yH2OH

H-C-CH,NHCOCHC~,--€

-@ YH-CH-NHCOCHC1, OH

H,N

@-F H - c H - ~ ~ o c H c ~ , OH yH2OH

0

CH,?HN @ : C H NO C H ~ ,

-

R a t hepato-

.*

yH2 OC 6 H9

0,N -@:H-CH-NIICOCHCl

OH 0,N

CH, OH -@FH-CH-NH, I

OH Scheme

4.

Metabolites o f chloramphenicol i n i n vivo and i n v i t r o s t u d i e s .

7

748

ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID The u s e o f chloramphenicol h a s been shown t o cause bone marrow d e p r e s s i o n . T h i s t o x i c i t y i s u s u a l l y r e v e r s i b l e i f t h e drug i s d i s c o n t i n u e d , b u t i n r a r e c a s e s (1 i n 20,000) p a t i e n t s develop a p l a s t i c anemia a bone marrow d i s e a s e which i s o f t e n i r r e v e r s i b l e and f a t a l ( 1 1 9 ) . Many mechanisms have been suggested (120-126) t o account f o r t h i s chloramphenicol-induced t o x i c i t y , but have not been unequivocally e s t a b l i s h e d . T h i s rare i n c i d e n c e o f chloramphenicol t o x i c i t y suggested t o Pohl and Krishna (127) t h a t a minor a c t i v e m e t a b o l i t e may be involved i n i t s i n d u c t i o n . Using a cytochrome P-450 enzyme system i n l i v e r rnicrosomes o f r a t s , t h e y s t u d i e d t h e mechanism o f t h e metabolic a c t i v a t i o n o f chloramphenicol by measuring t h e c o v a l e n t b i n d i n t o microsomal p r o t e i n s of s p e c i f i c a l l y l a b e l l e d [l C ] and [ 3 H ] d e r i v a t i v e s o f chloramphenicol. The l a c k o f b i n d i n g o f d i c h l o r o a c e t i c a c i d , chloramphenicol base (2-amino-l-( pnitrophenyl)-1,3-propanediol), and t h e acetamido and t r i f l u o r o a c e t amido d e r i v a t i v e s of chloramphenicol i n d i c a t e s t h a t t h e dichloroacetamido group is required f o r a c t i v a t i o n . The b i n d i n g o f dichloroacetamide support t h i s c o n c l u s i o n . Moreover, t h e C-H bond o f t h e dichloromethyl carbon of chloramphenicol a p p e a r s t o be broken i n t h e a c t i v a t i o n p r o c e s s s i n c e t h e hydrogen i s l o s t i n c o v a l e n t b i n d i n g . Accordingly, a mechanism (Scheme 5 ) i s proposed i n which chloramp h e n i c o l i s a c t i v a t e d by h y d r o x y l a t i o n o f t h e dichloracetamido group followed by spontaneous deh y d r o c h l o r i n a t i o n t o a n oxamyl c h l o r i d e which a c y l a t e s microsomal p r o t e i n s . R e c e n t l y , it has been shown t h a t cytochrome P-450 i s t h e predominant p r o t e i n i s l i v e r microsomes t h a t i s a c y l a t e d by t h e oxamyl c h l o r i d e ( 1 2 8 ) . Moreover , t h e c o v a l e n t l y modified cytochrome P-450 a p p e a r s t o b e i r r e v e r s i b l y i n a c t i v a t e d as a mixed-function o x i d a s e ( 1 2 9 , 1 3 0 ) .

,f

Morris e t a1 ( 1 3 1 ) have f u r t h e r c h a r a c t e r i z e d t h e o x i d a t i v e metabolism o f chloramphenicol and have found a new pathway f o r t h e o x i d a t i v e metabolism of chloramphenicol i n a d d i t i o n t o t h e o x i d a t i v e dehalog e n a t i o n r e a c t i o n o u t l i n e d above. According t o t h e s e a u t h o r s , when chloramphenicol was incubated w i t h r a t l i v e r microsomes, four p r e v i o u s l y u n i d e n t i f i e d metab o l i t e s were d e t e c t e d and i d e n t i f i e d . They i n c l u d e chloramphenicol aldehyde, p-nitrobenzyl a l c o h o l , N(2-oxoethyl) d i c h l o r o a c e t a m i d e , and N-( 2-hydroxyethyl)

0,@TI

a

CH, OH Nucleophil i c fH-CH-NHCOCONu-P I 0, N

H:

OH

OH p H C 1

P r o t e i n ( P-Nu)

O,N

-

OH Reduction

Scheme 5.

OH

-@

CHzOH 102NaFH-!!H OH

I 02N@

CH, OH I

CH,OH OH I I CH-CH- NHCO-C-C1 I I OH c1 P-450, NADPH,

1 ozN*EH2

CH, OH CH I -

CHO +

-

NHCOCHCl,

Retroaldol condensat i o n

OHC-CH2NHCOCHC12

+ -*

Reduct i o n

HO-CH2CH2NHCOCHC1,

Mechanism o f chloramphenicol o x i d a t i v e pathways by r a t l i v e r microsomes (127, 131).

750

ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID dichloroacetamide. The formation of t h e s e metabol i t e s was dependent upon t h e presence of NADPH and 02 and w a s uninhibited when SKF' 5258 or CO/O2 (8 : 2, v / v ) were present i n t h e r e a c t i o n mixture. Moreover, t h e metabolites were formed by l i v e r microsomes from phenobarbital-treated rats but not by microsomes from untreated rats or rats t r e a t e d with 6-naphthoflavone. The formation of t h e s e metab o l i t e s i s c o n s i s t e n t with a mechanism t h a t involves an i n i t i a l oxidation of chloramphenicol t o chloramphenicol aldehyde by cytochrome P-450. The metabolite, being a 0-hydroxyaldehyde, can chemically undergo a retro-aldol cleavage t o p-nitrobenzaldehyde and N( 2-oxoethyl)dichloroacetamide. Enzymatic reduction of t h e s e aldehyde intermediates would y i e l d p-nitrobenzyl alcohol and N-( 2-hydroxyethyl) dichloroacetamide, r e s p e c t i v e l y (Scheme 5 ) . I n t h e above study by Pohl and Krishna (127) it w a s observed t h a t only a 58% decrease i n covalent binding of chloramphenicol metabolites t o microsomal p r o t e i n occurred when r e a c t i o n s were conducted i n an atmosphere of nitrogen. A t t h e time, it was f e l t t h a t t h e reason t h e covalent binding w a s not decreased t o an even g r e a t e r extent w a s because of i n s u f f i c i e n t deoxygenat i o n of t h e incubation mixtures. I n a f u r t h e r study by Morris et a1 (132), however, it i s shown t h a t a t low oxygen t e n s i o n , chloramphenicol i s a c t i v a t e d by reductive dechlorination pathways of metabolism. Thus, when chloramphenicol was incubated with rat l i v e r microsomes anaerobically, it was metabolized predominently t o deschloro-chloramphenicol and products t h a t become i r r e v e r s i b l y bound t o microsomal p r o t e i n . Cytochrome P-450 induced by phenobarbital appeared t o c a t a l y s e t h e s e r e a c t i o n s 'most e f f e c t i v e l y . Glutathione increased t h e formation of deschlorochloramphenicol by 13%and decreased t h e amount of i r r e v e r s i b l y bound product by 18%. Only small amount of t h e nitroaromatic-reduced product, chloramphenicol m i n e , w a s detected by HPLC. The r e s u l t s a r e consist e n t with t h e drug being biotransformed and a c t i v a t e d by cytochrome P-450 anaerobically through predominently reductive dechlorination. A proposed mechanism f o r t h e reductive dechlorination i s shown i n Scheme 6. Bories e t a1 (133) developed a simple and ion-pair reverse phase high performance l i q u i d chromatographic separations combined with s e l e c t i v e e x t r a c t i o n i n

L-450,

Fe ++

pc1Covalent b i n d i n g 4 . -

Scheme

6.

NHZCOCH 02N@CH-CH-CH20H I I OH

A proposed mechanism for t h e r e d u c t i v e d e c h l o r i n a t i o n o f c h l o r m p h e n i c o l by rat l i v e r microsomes ( 1 3 2 ) .

75 2

ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID order t o achieve an improved a n a l y t i c a l t o o l for chloramphenicol metabolic p r o f i l i n g . Q u a n t i t a t i o n w a s achieved by summation of r a d i o a c t i v i t y values f o r f r a c t i o n s belonging t o t h e same peak. Metabol i t e s of chloramphenicol were i d e n t i f i e d by electronimpact and chemical i o n i z a t i o n mass spectrometry. The study l e a d t o t h e confirmation 'of previously suggested chloramphenicol metabolites as well as t h e i d e n t i f i c a t i o n of new metabolites.

Acknowledgement t h e authors would l i k e t o thank M r . Tanvir A . Butt for typing this manuscript.

753

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3.

E . G . C . Clarke, " 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", (19 ) . The Pharmaceutical P r e s s , London p.

4.

A B r i t h w a i t e and C. Wilson, Dyn. Mass Spectrom., $, 1 2 1 (1978).

5. 6.

R . K u t t e r , D. J a h r and H . S t r i t z i n g e r , F l e i s c h w i r t s c h a f t ,

62, 515 (1982). T. Nakagawa

and

M. Masoda, J . Chromatogr.,

111,355

(1975) ' 7.

0. Becker, N. Furstenav, W. Knippelberg and F.R. Krueger, Org. Mass Spectrom. , 12, 461 (1977).

8.

F.R. Krueger, Chromatographia,

9.

J. C o n t r o u l i s , M.C. Rebstock and H.M. h e r . Chem. SOC., 71,2463 (1949).

10,151

(1977).

Crooks, J r . ,

2.

Hansen, B. John, P.E. Nielsen, V. Leik and 0. Buchardt , Hoppe-Seyler I s 2 . Physiol. Chem, 721

10. J . B .

(1979)

m,

-

11. " B r i t i s h Pharmacopoeia", Vol. I , Her Majesty's S t a t i o n e r y O f f i c e , London, U.K.

p. 95 (1980).

12. L.A. Chekryshkina, L . I . Parfenova and N.K. Farmatsiya, 32, 39 ( 1 9 8 3 ) .

Evich,

13.

R.M. Nannatyne and R. Cheung; Antimicrob. Agents 43 (1979). Chemother. ,

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LIDOCAINE AND LIDOCAINE HYDROCHLORIDE Michael F. Powell

1.

Introduction

2.

Description 2.1

3.

Nomenclature, Formulas, M o l e c u l a r Weights and CAS Numbers

P h y s i c a l , Chemical and S p e c t r a l P r o p e r t i e s 3.2 3.3 3.5 3.6 3.9 3.10 3.11 3.1 2

Solubility D i s s o c l a t l o n Constant I n f r a r e d A b s o r p t i o n Spectra N u c l e a r Magnetic Resonance S p e c t r a X-ray C r y s t a l S t r u c t u r e X-ray D i f f r a c t i o n Hygroscopicity Self Association

6.

Stability 6.2 P h y s i c a l S t a b i l i t y i n P a r e n t e r a l and I V S o l u t i o n s 6.3 Photochemical S t a b i l i t y

7.

Pharmacokinetics 7.1

7.3 8.

Plasma C o n c e n t r a t l o n s a f t e r D i f f e r e n t Routes o f Admi n i s t r a t ion B l o t r a n s f o r m a t i o n and E l i m i n a t i o n

Methods o f A n a l y s i s 8.1 8.2 8.3

Reverse Phase HPLC Normal Phase HPLC T h i n Layer Chromatography

10. References ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 15

761

Copyright Q 1986 by the American Pharmaceutical Association All rights of reproduction in any form reserved.

MICHAEL F. POWELL

762

I nt roduct 1on

1.

Lidocaine i s widely used as a local anaesthetic and for the management of cardiac arrhythmas, particularly those assoclated with acute myocardlal infarction. The following supplement contains updated Information pertalning to the analytical chemlstry of lidocaine free base and lldocalne hydrochloride. A llterature survey was conducted and i s complete up to December 1985. The numbering system for topics dlscussed i s the same as that I n the original profile1 (see Volume 14, pages 207-243). 2.

Description

2.1

Nomenclature. Molecu'lar Weights and CAS Numbers

The structural formula for lldocalne i s given below.

names for lldocalne have been used In the recent literature, for example: llgnocalne, 2-(diethylam1no)-N-( 2,6-dlmethylphenyl) -acetamlde, 2-(diethylamino)2',61-acetoxyidide, N,N-diethyl-(2,6-xylylcarbamoyl)methylamine and 2-diethylaminoaceto-2 I ,6 I-xyl idide. The hydrochloride salt i s named similarly but with the added suffix I'hydroch 1 or1 dell or "hydrochl or1 de monohydrate". Trade names for lidocaine are: Lidocitin, Leostesin, Xylocalne, Xylotox, Xylestesin, Xylocitln, Rucaina, Duncalne, Islcalne and Anestacon. A plethora of

Table 1. FORM Formula

Descrlptlon o f Lldocalne and its Hydrochloride Salts Free Base

1' 4H22N20 MW 234.34 %C,H,N 71.75,9.46,11.76 CAS Number 137-58-6

Salt

Salt Monohydrate

C14H22N20*HC 1 270.80 62.09,8.56,l0.34 73-78-9

288.81 58.22.8.72.9.70 61 08-05-0

763

LIDOCAINE AND LIDOCAINE HYDROCHLORIDE 3.

P h y s i c a l and Chemical P r o p e r t i e s

3.2

Solubility

The s o l u b i l i t y o f l i d o c a i n e f r e e base i n aqueous s o l u t i o n i s unusual i n t h a t i t decreases as t h e temperature i n c r e a s e s ( T a b l e 2) . 2 T h i s i n v e r s e temperature - s o l u b i l i t y r e l a t i o n s h i p has a l s o been v e r i f i e d by others.3-5 The pH dependence on t h e aqueous s o l u b i l i t y o f l i d o c a i n e can be c a l c u l a t e d f r o m t h e e q u a t i o n ST = S t b ( 1 + lO(PKa-PH)) where ST and Sfb a r e t h e t o t a l and f r e e base s o l u b i l i t i e s , r e s p e c t i v e l y . 6 The c a l c u l a t e d s o l u b i I i t y o f l i d o c a i n e a t 14.9", 25" and 37°C a r e shown i n F i g u r e 1. The s o l u b i l i t y o f l i d o c a i n e and l i d o c a i n e h y d r o c h l o r i d e 7 i n v a r i o u s s o l v e n t s a r e g i v e n i n Table 3. Table 2. Free Base S o l u b i l i t y and pK, a t Various Temperatures Temperature ( " C )

Values o f L i d o c a i n e

S o l u b i l i t y (mg/mL)

10.0 14.9 25.0 34.5 37.0 38.0

2

8.24 4.33 0.12 3.81 + 0.02 3.42 2 0.02 3.36 5 0.02

7.92 7.57

I

7.5

I

I

0

I

I

0.5

1

I

9

I

I

4

9.5

PH

F i g u r e 1. C a l c u l a t e d pH dependence o f t h e aqueous s o l u b i l i t y o f l i d o c a i n e a t 1 4 . 9 " , 25" and 37°C. A t pH 7.4, t h e c a l c u l a t e d s o l u b i l i t i e s o f l i d o c a i n e a t 25" and 37°C a r e 16.4 and 8.7 mg/mL, r e s p e c t i v e l y .

764

MICHAEL F. POWELL

Table 3. S o l u b i l i t y of Lidocalne Free Base and Lldocaine Hydrochloride a t 25°C i n Various Solvents

so Iu b i 1 it y (mg/mL) So 1 vent

Methanol Ethanol Acetone Chloroform Ether Carbon t e t r a c h l o r i d e

3.3

Free Base

Lidocaine HC I 67 11 1.8 4
>500 >500 >500 -500 >so0

D i s s o c i a t i o n Constant

The pKa o f l i d o c a i n e has been measured by p o t e n t i o m e t r i c t i t r a t i o n a t several temperatures a t i o n i c s t r e n g t h 0.05 M (KC1),8 and t h e data are shown i n Table 2. The i o n i c strength, when adjusted w i t h KC1 from 0.005 t o 0.077 M, showed l i t t l e e f f e c t on t h e a c i d i t y c ~ n s t a n t . ~The pH o f a 5% (w/v) s o l u t i o n o f l i d o c a i n e hydrochloride i s approximately 4.0 t o 5.5. 3.5

I n f r a r e d Absorption Spectra

The I R spectra o f l i d o c a i n e and l i d o c a i n e h y d r o c h l o r i d e were measured i n chloroform using a Sargent Welsh Model S-200 I R spectrometer (Figure 2 ) . Note t h a t most o f t h e s t r e t c h i n g frequencies are s h i f t e d upwards than those obtained i n K B r discs’. The s t r u c t u r a l assignments 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 assignments (Table 4). Table 4. I R Band Frequencies (cm-’) f o r Lidocaine and Lidocaine Hydrochloride Measured i n CDC13. Assignment N-H S t r e t c h C-H S t r e t c h C-H S t r e t c h Amide I,C=O Amide 11, C-N Fingerprint region

Lidocaine 3290 2820 2970 1670 1495 81 2 900

L i d o c a i ne Hydrochloride 3180

-

2978 1682 1525 949 978

765

LIDOCAINE AND LIDOCAINE HYDROCHLORIDE

3000

2500

2000

I

1600 Wave Number, cm

I

-

1200

800

l

I

T

I

B

3000

2500

2000

1600 Wave Number, cm

-

1200 l

F i g u r e 2 . I R s p e c t r a o f a) l i d o c a i n e and b) l i d o c a i n e h y d r o c h l o r i d e i n CHC13.

800

MICHAEL F. POWELL

766

3.6

Nuclear Magnetic Resonance Spectra

The 90 MHz p r o t o n and 75.5 MHz 13C-NMR s p e c t r a f o r l i d o c a i n e h y d r o c h l o r i d e i n CDC13 a r e shown i n F i g u r e 3. A sumnary o f t h e p r o t o n chemical s h i f t s as r e f e r e n c e d t o TMS a r e g l v e n i n Table 5 and t h e 1%-chemical s h i f t s and r e l a x a t i o n times a r e g i v e n i n Table 6. The numbering i s as shown e a r l i e r i n t h e s t r u c t u r a l formula. The 14N-NMQR spectrum has a l s o been reported. Table 5. Summary o f Proton Chemical S h i f t s and Assignments f o r Lidocaine H y d r o c h l o r i d e i n CDC13a ~~

Chemical S h i f t (ppm)

Assignment

7.02 (m) 7.02 (m) 2.21 ( s ) 10.24 (s-broad) 4.22 ( 5 ) 3.60 ( 9 ) J=7.20 HZ 1.42 (t) 5-7.20 HZ

C3-H C4-H

Ar-Cb N-tj

N-C( 0) -CHz-N N-CHz-CHs N-CHz-CH3

as = s i n g l e t , t = t r i p l e t , q = q u a r t e t , m = m u l t i p l e t Table 6.

Surmary o f 13C-NMR Data f o r L i d o c a i n e H y d r o c h l o r i d e

Assignment

Re1a x a t i on Timea (s)

Chemlcal S h i f t (PPN

N-C( 0) -CHz-N c2, c6 C1 c3, c 5 c4 N-C( O)-CHz-N N-CH2CH3 Ar-CH3 N-CHzCH3

11.8 20.1 20.1 1.4 1 .o 1 .o 1.2 3.1 2.0

162.9 135.1 133.1 128.1 127.5 51 .O 49.1 18.7 10.0

a L i d o c a i n e i n CDC'l3 a t 32°C I n undegassed s o l u t i o n , Reference 11.

LIDOCAINE AND LIDOCAINE HYDROCHLORIDE

10

8

4

6

767

0

2

PPm

I

I

I

I

I

I

I

150

120

90

60

30

0

PPm

Figure 3 . Proton NMR and 13C-NMR spectra o f lidocaine hydrochloride in C D C 1 3 .

MICHAEL F. POWELL

768

3.9

X-Rav Crvstal Structure

The crystal structure of lidocalne free base was determjned

from a single crystal grown from dlmethylformamlde - water solution at -2OOC.12 Cell dimensions were a = 13.24 A, b = 14.06 A, c = 19.25 A and U = 123.7" at 20°C. Both of the independent molecular conformations observed were I n the trans-amide configuration.

X-ray crystal analysis of lidoca determined from a single crystal ethy1a~etate.l~ The translucent mosaicity and gave the followlng b = 7.11 A, c = 27.58 A and U =

ne hydrochloride was grown in acetoneprisms were of high cell parameters: a = 8.49 A , 06.87".

3.10 X-Ray Diffraction The X-ray powder diffraction patterns of lidocalne and lidocaine hydrochloride are shown in Figures 4a and 4b, respectively, and a sunmary of the scannlng angles and relative Intensities are given in Table 7. The data were collected uslng a Nlcolet X-ray dlffractometer equipped with a fine focus X-ray tube and a diffracting beam monochromator. The scanning angle was from 3" to 30" 28 at O.O5"/second.

Table 7. X-Ray Powder Dlffractlon Data - Scanning Angles and Relative Intensities for Lidocaine and Lidocaine Hydrochloride Lldocai ne

Lidocaine Hydroch lorlde

Degrees 28 Relative Intensity 10.5 12.7 12.5 10.0 8.0 14.3 15.1

100 100 46 37 13 12 12

Degrees 28 Relative Intensity 20.2 13.5 16.5 25.4 27.1 6.7 14.3

100 97 92 66 55

49 44

LIDOCAINE AND LIDOCAINE HYDROCHLORIDE

I

I

5

10

I

769

I

I

15 20 Degrees 20

25

B

I

I

5

10

I

I

15 20 Degrees 20

I

25

Figure 4. X-ray diffraction pattern of a) lidocaine and b ) lidocaine hydrochloride.

770 3.11

MICHAEL F. POWELL Hysroscopicity

L i d o c a i n e f r e e base i s n o t hygroscopic. F o r example, l i d o c a i n e f r e e base does n o t absorb w a t e r even a t 92% RH a t room temperature. The anhydrous HC1 s a l t however, does adsorb 1 mole o f w a t e r p e r mole o f l i d o c a l n e below 80% A t 93% r e l a t i v e h u m i d i t y , more r e ' l a t l v e h u m i d i t y a t 25°C. t h a n two moles a r e a d ~ 0 r b e d . l ~L i m i t e d h y g r o s c o p i c i t y d a t a f o r l i d o c a i n e h y d r o c h l o r i d e f r o m 22% t o 90% r e l a t i v e hum1d i t y has a 1 so been r e p o r t e d . 3.12

Self Absociation

L i d o c a i n e i n s o l u t i o n depresses o n l y s l i g h t l y t h e s u r f a c e t e n s i o n o f w a t e r and thus, does n o t f o r m mice'l'les t o any measurable e x t e n t . 1 6 However, l i d o c a i n e has been r e p o r t e d t o f o r m charge t r a n s f e r complexes, f o r example w i t h t r i n i t r o b e n z e n e o r ch l o r o p r o m a ~ i n e . ~ ~

A c o n t r o v e r s y r e g a r d i n g i n t e r versus i n t r a m o l e c u l a r hydrogen bonding i n l i d o c a i n e emerged i n t h e e a r l y 7 0 ' s and appears now t o be r e s o l v e d I n f a v o r o f t h e l a t t e r . An e a r l i e r p u b l i c a t i o n l a p o s t u l a t e d t h e a c y l i c cis-amide c o n f i g u r a t i o n based on I R s t u d l e s , however, subsequent I R s t u d i e s 1 9 and NMR r e s u l t s 2 0 r e b u t t e d w i t h s t r o n g evidence f o r i n t r a m o l e c u l a r trans-amide hydrogen bonding as shown i n F i g u r e 5.

N E< 'Et

F l g u r e 5.

CIS

TRANS

Cis- and trans-amide c o n f i g u r a t i o n s o f l i d o c a i n e .

77 1

LIDOCAINE AND LIDOCAINE HYDROCHLORIDE

6.

Stability

6.2

P h v s l c a l S t a b l l l t y I n P a r e n t e r a l and I V S o l u t i o n s

Some I i d o c a i n e p r e p a r a t i o n s a r e p h y s i c a l l y u n s t a b l e r e s u l t i n g i n s o l u t i o n c l o u d i n e s s , p r e c i p i t a t i o n o f drug, o r l o s s o f d r u g potency i n s o l u t i o n . P r e c i p i t a t i o n o r c l o u d i n e s s i s u s u a l l y caused by o t h e r a d d i t i v e s which may complex w i t h l i d o c a i n e o r change t h e s o l u t i o n pH. Loss o f d r u g potency i n s o l u t i o n i s n o t due t o chemical I n s t a b i l i t y , b u t i s caused by a d s o r p t i o n o f l l d o c a i n e t o t h e c o n t a l n e r s u r f a c e , e s p e c i a l l y when p a r e n t e r a l o r I V s o l u t l o n s a r e s t o r e d i n p l a s t i c containers (Table 8). Table 8.

S t a b i l i t y of Lidocaine i n Parenteral Solutions

So 1 u t ion

Container

5% d e x t r o s e

plastic plastic

120 120

4 30

99.0 2 1.6 100.2 2 1 . 7

21

0.9% sodium chloride i n j e c tiona

glass plastic

14 14

25 25

100.9 5 0.8 101.9 5 0.8

22

0.45% sodium c h l o r i d e b and 5% d e x t r o s e b

glass plastic

14 14

25 25

94.3 2 0.3 98.6 2 0.9

Lactated r i n g e r s glass solutiona plastic

14 14

25 25

101.9 2 0.9 101.9 2 1.7

5% d e x t r o s e and lactated ringers solutiona

glass plastic

14 14

25 25

94.7 f. 0.8 101.9 2 0.9

Cardioplegic solutionb

glass plastic glass plastic

21 21 21 21

22 22 4 4

90.1 25.6 100.0 89.1

Time (days)

Temp. ("C)

% Reference Remaining

f. 2.0

5 1.1 f. 2.2 2 5.8

I1

II

II

I1

II II

I1

II

23 I1

II I1

aAdmixture w i t h a m l n o p h y l l i n e , b r e t y l ium t o s y l a t e , c a l c i urn gluconate, d i g o x l n , dopamine h y d r o c h l o r i d e , r e g u l a r I n s u l i n , p h e n y t o l n sodium and procainamide h y d r o c h l o r i d e . bWith l i d o c a i n e h y d r o c h l o r l d e , K C I , NaHC03, d e x t r o s e and NaCl.

MICHAEL F. POWELL

772

The f i r s t comprehensive paper on t h e c o m p a t i b l l l t y o f l i d o c a i n e i n glass and p l a s t i c containers c o n t a i n i n g 5% dextrose i n s a l i n e , normal s a l i n e , o r 'lactated r i n g e r s s o l u t i o n reported a s l i g h t loss o f l i d o c a i n e concentration a f t e r o n l y twenty-four hours; t h i s decrease was a t t r i b u t e d t o experimental error.24 Subsequent r e p o r t s showed t h a t lldocaine i s stable i n parenteral solutions21 providing c e r t a i n admixtures are n o t includedZ2. It has a l s o been demonstrated r e c e n t l y t h a t adsorption t o p o l y v i n y l c h l o r i d e bags may occur, e s p e c i a l l y a t room temperature. 21

6.3

Photochemical S t a b i l i t y

The weak UV-visible absorption o f l i d o c a i n e a t A = 262 nm has o n l y a small molar a b s o r p t i v i t y o f 470 t4-l cm-l and so l i d o c a i n e i s n o t photoreactlve. In our l a b o r a t o r y , Rayonet i r r a d i a t i o n o f a 5 x loW5 H aqueous s o l u t i o n of l i d o c a i n e f o r 10 days r e s u l t e d I n o n l y 5% l o s s o f drug. Since t h e Rayonet r e a c t o r (Rayonet Model No. RPR-100) has been shown t o a c c e l e r a t e most r e a c t i o n s by a f a c t o r of 100-200 times over i n d i r e c t window ' l i g h t , t h e c a l c u l a t e d s h e l f - l i f e o f l l d o c a i n e exposed t o i n d i r e c t window l i g h t I s approximately e i g h t years. 7.

Pharmacokinetics

7.1

Plasma Concentrations a f t e r D i f f e r e n t Routes o f Admi n i s t r a t 1on

Lidocaine e x h i b i t s r a p i d and r e l a t i v e l y h i g h absorption a f t e r o r a l a d m i n i s t r a t i o n . However, i t I s n o t c l i n i c a l l y u s e f u l when taken o r a l l y due t o extensive f i r s t - p a s s metabolism and I t s s h o r t b i o l o g i c a l h a l f - l i f e . I t was reported t h a t a f t e r o r a l a d m i n i s t r a t i o n o f l i d o c a i n e t o r a t s , dogs and man, o n l y 0.2%, 2.0% and 2.8%, r e s p e c t i v e l y , was recovered unchanged i n t h e urine25 (Table 9 ) . The mean apparent o r a l absorption o f l i d o c a i n e i n normal subjects was approximately 35%,26 i n c l o s e agreement w i t h t h e reported hepatic e x t r a c t i o n r a t i o . 2 7 This i s c o n t r a r y t o an e a r l i e r r e p o r t i n which t h e c o l o r i m e t r i c method used f o r a n a l y s i s may n o t have been s p e c i f i c f o r unmetabolized lidocaine.28 A p r e d i c t i o n o f t h e b i o a v a i l a b i l i t y using o r a l clearance data has a l s o been offered.29 Plasma l e v e l s obtained from i n g e s t i n g 500 mg o f l i d o c a i n e appeared t o be less than t h e l e v e l s r e q u i r e d t o cause an antiarrhythmic e f f e c t .

773

LIDOCAINE AND LIDOCAINE HYDROCHLORIDE

Table 9. T i s s u e D i s t r i b u t i o n A f t e r L i d o c a i n e p.0. 10 mg/kg t o Rats.

Tissue

Total Radioactivity 0.5 h

Stomach Intestine B 1 ood Feces Brain Liver Spleen Kidney Heart Lung Carcass U r i ne

13.7 27.9 2.8 0.0 0.6 11.1 0.4 2.0 0.2 1.1 40.4 6.1

Dosing o f

Unchanged L i d o c a i n e a

2 h

24 h

0.5 h

2 h

24 h

3.3 45.3 2.1 0.2 0.3 3.4 0.0 0.8 0.1 0.3 24.2 24.6

0.0 6.9 1.6 0.5 0.1 1.1 0.0 0.2 0.5 0.3 20.8 73.0

14.1 2.4 0.2 n.d. 0.0 0.3 0.1 1.3 0.0 0.2 b 0.3

2.7 0.3 0.0 n.d. 0.0 0.0 0.0 0.1 0.0 0.0 b 0.5

0.0 0.0 0.0 n.d. 0.0 0.0 0.0 0.0 0.0 0.0 b 0.3

aUnchanged l i d o c a i n e as c a l c u l a t e d f o r t h e h y d r o c h l o r i d e s a l t . h o t determined. Intravenous a d m i n i s t r a t i o n o f l i d o c a i n e t o r a t s r e s u l t s i n r a p i d uptake by t h e h i g h l y p e r f u s e organs such as t h e l i v e r (which i s c o n s i d e r e d t h e p r i m a r y m e t a b o l i c s i t e ) , h e a r t , lungs, b r a i n and kidneys ( T a b l e 9 ) .25 The e l i m i n a t i o n h a l f - l i f e o f l i d o c a i n e i n r a t s i s 30 minutes and i n dogs i s 45-60 minutes. A f t e r I V i n j e c t i o n , t h e mean h a l f - l i f e i n plasma o f normal s u b j e c t s I s a p p r o x i m a t e l y 6 minutes and t h e e l i m i n a t i o n h a l f - l i f e is -100 minutes.30 A r a p i d I V i n j e c t i o n of 160 mg ' l i d o c a i n e i n t o normal s u b j e c t s f o l l o w e d by a 4 mg/min i n f u s i o n r e s u l t e d i n a mean i n i t i a l plasma l e v e l of' 2.6 ug/mL which decreased t o a steady s t a t e plasma l e v e l o f 2-4 ug/mL a f t e r a p p r o x i m a t e l y 20 minutes. It has been shown u s i n g averaged d a t a f r o m s e v e r a l p a t i e n t s t h a t an i n i t i a l b o l u s o f 125 mg and an i n f u s i o n o f 0.8 mg/mL m a i n t a i n e d plasma l e v e l s o f 1 pg/mL i n a 70 kg man. When i n f u s i o n was g i v e n alone, plasma l e v e l s o f l i d o c a l n e i n c r e a s e d u n t i l a p l a t e a u was reached, 1.e. when i n p u t equaled o u t p u t . T h i s u s u a l l y t o o k t h r e e t o f o u r h a l f - l i v e s o r f r o m f i v e t o seven hours. E f f e c t i v e c o n c e n t r a t i o n s o f l i d o c a i n e f o r l o c a l anaesthesia were achieved f o r a p p r o x i m a t e l y one h o u r by a s i n g l e 200 mg i n t r a m u s c u l a r i n j e c t i o n . 31

MICHAEL F. POWELL

774

L i d o c a i n e proved t o be s l i g h t l y more b i o a v a i l a b l e when a d m i n i s t e r e d r e c t a l l y r a t h e r t h a n o r a l l y . 3 2 T h i s drug I s a l s o absorbed w e l l through i n t a c t mucous membranes, s k i n and damaged t i s s u e . 3 3 The s k i n p e r m e a b i l i t y was g r e a t l y enhanced i n i n - v i t r o experlments by t h e a d d i t i o n o f N ,N-di ethyl-m-to1 uamide .34

A r e v i e w o f t h e c l i n i c a l harmacokinetics o f l i d o c a i n e has been p u b l i s h e d r e ~ e n t l y ,and ~ ~ a synopsls o f e f f e c t s w i t h r e s p e c t t o pregnancy and t h e neonate appears r e g u l a r l y I n M a r t i n d a l e , The E x t r a P h a r m a ~ o p o e i a . ~ 7.3

B l o t r a n s f o r m a t i o n and E l i m i n a t i o n

Two o f t h e f i r s t - f o r m e d m e t a b o l i t e s , e t h y l g l y c l n e x y l i d i d e and g l y c i n e x y l i d i d e , showed 83% and 10% of t h e a n t i a r r h y t h m i c a c t i v i t y o f 1i d o c a i ne, r e s p e c t i v e l y 36 The a r y l - h y d r o x y l a t e d analogues o f these compounds were a I s 0 formed and were u s u a l l y found as acld-hydro1 zab'le ' c o n j u g a t e s ' .25 It was o r l g l n a l l y proposed3r t h a t N-deethyl a t 1on preceded hyd r o x y l a t l on b u t r e c e n t stud1 es i n r a t s have shown t h a t 1) b o t h N,N-diethyl and N-monoethyl g l y c l n e a r e formed, 11) l i d o c a i n e and m o n o e t h y l g l y c l n e x y l l d l d e show c o m p e t i t i v e and, Ill)t h e 3 - h y d r o x y l a t l o n and N - d e e t h y l a t i o n a r e c a t a l y z e d by d i f f e r e n t P450-dependent enzymes ,39940 The m e t a b o l i c p r o d u c t d i s t r l b u t i o n s i n r a t s , guinea p i g s , dogs, and man a r e shown i n Table 10.

.

Table 10.

Summary o f M e t a b o l i c Products o f L i d o c a i n e

Compound

% Dose Recovered I n 24 h Urinea

Lidocalne Ethylglycinexylldlde Glyclnexylidide 3-Hydroxy-1 i d o c a i ne

3-Hydroxy-ethylglyclnexylidide 2,6-Xyl i d i n e 4-Hydroxy-2,6-xy.l i d i n e Total

Rat

G. P i g

0.2

0.5 14.9 3.3 0.5 2.0 16.2 16.4 53.8

0.7 2.1 31.2 39.6 1.5 12.4 85.0

Dog

Man

2.0 2.3 12.6 6.7 3.1 1.6 35.2 65.5

2.8 3.1 2.3 1.1 0.3 1.0 72.6 83.8

".O. doses g l v e n t o r a t , guinea p i g , dog and man were 20, 20, 10 and 3 mg/kg, r e s p e c t i v e l y . Because of' t h e r e ' l a t l v e l y l a r g e d i f f e r e n c e s i n m o l e c u l a r weights o f t h e ' l i d o c a i n e m e t a b o l i t e s , doses and r e c o v e r i e s were determined as molar c o n c e n t r a t i o n s o f t h e f r e e bases.

LIDOCAINE AND LIDOCAINE HYDROCHLORIDE

8.

Methods o f A n a l y s i s

8.1

Reverse Phase HPLC

77s

Although t h e a n a l y s i s o f i o n i c and s t r o n g l y b a s i c drugs by RP-HPLC i s n o t w i t h o u t problems such as 'long s o l u t e r e t e n t i o n o r p e a k - t a i l i n g , i t was e s t i m a t e d some t i m e ago t h a t a p p r o x i m a t e l y e i g h t y p e r c e n t o f a'I1 l i q u i d chromatography analyses a r e c a r r i e d o u t u s i n g r e v e r s e phase methods.41 The f o u r examples o f f e r e d h e r e were chosen because o f t h e i r a b i l i t y t o separate ' l i d o c a i n e f r o m i t s d e g r a d a t i o n p r o d u c t s o r common pharmaceuticals under a variety o f conditions. System 142 Column : Detection: Temperature: Flow Rate: M o b i l e Phase:

R e t e n t i o n Time: Sepa r at 1 on : System 1143 Column: Detection: Temperature : M o b i l e Phase: R e t e n t i o n Time: Sepa r a t 1on : System 11144 Column: Detection: Temperature: Flow Rate: M o b i l e Phase: Separation:

30 cm x 4 mn I D p-Bondapak-CN, 10 urn Dual channel, f i x e d wavelength, Waters Model 440 Ambient 2 mL/min 0.01 M O c t a n e s u l f o n i c a c i d sodium s a l t , 0.01 M e d e t a t e disodium, 2% ( v / v ) a c e t i c a c i d , 2% aceton i t r i l e and 1% methanol i n d i s t i l l e d water 7 min From d e g r a d a t i o n p r o d u c t s and e p i n e p h r i ne Nuc 1 e o s i 'I 100, 1Opm F i x e d wavelength, 254 nm 25.0 _t_ 0.1"C Sodium phosphate b u f f e r , pH 2.2, p = 0.1 and 1% ( v / v ) l - p e n t a n o l 1 min From o t h e r l o c a l a n a e s t h e t i c s L i Chromsorb RP-18 F i x e d wavelength, 254 nm 25°C Not g i v e n 0.01 M Tetramethylammonium bromide, 20% water, 80% methanol, pH 3.6 w i t h H3PO4 From pharmaceuticals h a v i n g b a s i c f u n c t i o n a l groups

MICHAEL F. POWELL

776

System IV45 Columns :

Detection: Flow Rate: Mobile Phase:

Retentlon Time: Sepa r a t ion : 8.2

p-Bondapak C1 , p-Bondapak Phenyl, V-Bon apak-CN, p-Bondagel, Chromegabond C8 and Chromegabond C6H11 UV-Vis d e t e c t o r 1.5 mL/min Methano 1 :water:acetic a c i d (29:50:1) c o n t a i n i n g .005 M heptane s u l f o n i c a c i d sodium s a l t , pH 4 Retention volumes f o r t h e various columns given i n Reference 45 From o t h e r common pharmaceuticals

!

-

Normal Phase HPLC System 146

Columns : Detection: Temperature : Flow Rate: Mob1 l e Phase: Retention Time: Separation: 8.3

125 o r 250 x 4.9 mm I D column packed w i t h Spherisorb 5 5 W s i l i c a , S y l o i d 74 s i l i c a 215 nm Ambient 2 mL/min Methanol c o n t a i n i n g 1 .85 mM HC 104 7 min From o t h e r l o c a l anaesthetics

Thin Laver ChromatonraPhv (TLC)

TLC and h i g h performance TLC are o f t e n used f o r drug a n a l y s i s because they are r a p i d , Inexpensive, and o n l y r e q u i r e small amounts o f sample. Lidocalne i s i d e n t i f i e d on t h e TLC p l a t e by e i t h e r s h o r t wavelength UV l i g h t o r by a p o s i t i v e t e s t w i t h a c i d i f i e d l o d o p l a t i n a t e spray. For example, t h e f o l l o w i n g systems have been used: System 147 Plate: Temperature: Detection: Mob1l e Phase: Rf :

S l l l c a g e l GF-254, 0.25 mm Ambient

uv

CHC13/ether/MeOH/conc. (15:25: 5 : l ) 0.80

NH40H

LIDOCAINE AND LIDOCAINE HYDROCHLORIDE

System 1147 Plate: Temperature : Detection: M o b i l e Phase Rf :

System 11147 Plate: Temperature: Detection: M o b i l e Phase: Rf : System IV47 Plate: Temperature: Detection: M o b i l e Phase: Rf :

System ~ 4 8 Plate: Temperature: Detection: M o b i l e Phase: Rf :

777

S i l i c a g e l GF-254, Ambient

0.25 mm

uv

Ethyl acetateh-propanol/conc. NHqOH (40:30:3) 0.82 S l l l c a g e l GF-254, 0 . 2 5 m Ambient

uv

MeOH/conc 0.84

. NH40H

( 100 :1 .5)

S i l i c a g e l GF-254, 0.25mm Ambient

uv

Alcohol USP/acetic a c i d / w a t e r (60: 30: 10) 0.60 S i l i c a g e l G60 F254 ( u n t r e a t e d ) Ambient

uv

0.01 M K B r I n methanol 0.84 (0.54, p l a t e t r e a t e d w i t h 0.1 M KHS04)

Acknowledgements T h i s p r o f i l e supplement was made p o s s i b l e w i t h t h e h e l p o f Drs. M. Mattox and L. P a r t r i d g e , and t h e i r colleagues a t t h e I n s t i t u t e o f Organic Chemistry a t Syntex, and by my colleagues i n t h e P r e f o r m u l a t l o n Department a t Syntex, D r . D . Johnson and W. Tamraz.

MICHAEL F. POWELL

778 10.

References

1.

K. Groningsson e t a l . Anal. P r o f i l e s Drug. Sub. l 4 , 207 (1984).

2.

N.I.

3.

I.S t e t n i k a . J. Pharm. S c i .

4.

A. B r o d i n e t a l .

5.

N.I.

6.

The D e t e r m i n a t i o n o f I o n i z a t i o n Constants by A. A l b e r t and E.P. S e r j e a n t . ChaDman and H a l l Ltd.. London (1971).

7.

M a r t i n d a l e . The E x t r a PharmacoDoeia. Ed. by J.E.F. Reynolds. The Pharmaceutical Press (1982).

8.

H. Kamaya

9.

R.H. Levy and M. Rowland. ( 1 972).

J. Pharm. Pharmacol. 24, 841

10.

M.L. Buess and P.J. Bray.

Org. Mag. Reson. 22, 233 (1984).

11.

S.P.

12.

A.W. Hanson and D.W. (1974).

13.

A.W.

14.

H.M. Koehler and J . J . H e f f e r r e n . 1126 (1964).

15.

M.H.

16.

K. Thoma and C.D. H e r z f e l d t . Expo. Congr. I n t . Technol. Pharm. 1, 157 (1977).

17.

G. E c k e r t and F. Gutman.

18.

G.A.

19.

R.L. Jones.

J . Pharm. S c i .

20.

J.P.

J. Pharm. S c i . 53, 1524 (1970).

21.

F.M. Smith and N.O. 1745 (1981).

22.

H.L. Kirschenbaum e t a l . (1982).

23.

T.E.

Nakano.

J . Pharm. S c i . 68, 667 (1979).

55, 1190

(1966).

J. Pharm. S c i . 73, 481 (1984).

Nakano e t a'l.

Chem. Pharm. B u l l . 26, 936 (1978).

-

fi &IAnesth. .

Singh e t a l .

Analg. 62, 1025 (1983).

Spectros. L e t t . l2, 95 (1979). Banner.

Hanson and M. R o h r l .

Lannung.

Acta C r y s t .

Acta C r y s t .

830, 2486,

828, 3567

J. Pharm. S c i .

(1972).

53,

Arch. Pharm. Chem. 72, 703 (1965)

E l e c t r o a n a l . Chem. I n t e r f a c i a l Electrochem. 6 2 , 267 (1975). J. Pharm. S c i . !XI, 636 (1969).

N e v i l l e and D. Cook. Chupp.

Lackner e t a l .

Nuessle.

63,

1170 (1974).

Am. J . Hosp. Pharm. ja,

Am. J . Hosp. Pharm.

39, 1013

Am. J . Hosp. Pharm. 40, 97 (1983).

779

LIDOCAINE AND LIDOCAINE HYDROCHLORIDE

24.

S.L. M i l l s e t a l .

I R C S Med. S c i . L i b r . Compend.

3, 592

(1975). 25.

J.B.

Keenaghan and R.N.

180, 454

J. Pharm. Exp. Ther.

Noyes.

(1972).

26. R.N. Boyes e t a l . (1971).

C l i n . Pharmacol. Ther. l 2 , 105

27. R.E. Stenson e t a.1. C i r c u l a t i o n 40, 195 (1969). 28. P . 1 . Parkinson e t a l . B r i t . Med. J . 2, 29 (1970). 29. A. Somogyi e t a l . Eur. J . C l i n . Pharmacol. 22, 85 (1 982). 30. M. Rowland. Ann. N.Y. Acad. S c i . 179, 383 (1981). 31. V . B e r n s t e i n e t a l . J. Amer. Med Ass. 219, 1027 (1972). 32. A.G. de Boer e t a l . C l i n . Pharmacol. Ther. 26, 701 (1979). 33. J. Thomas e t a l . B r . J. Anaesth. 4 l , 442 (1969). 34. T . L o f t s s o n e t a l . I n t . J . Pharmacol. 11 345 (1984). 35. N.L. Benowitz and W. M e i s t e r . C l i n . Pharmacokinet. 3, (1 978). 36. R.G. Burney e t a l . Am. H e a r t J. 88, 765 (1974). 37. S.D. Nelson e t a l . J. Pharm. S c l . 66, 1180 (1977). 38. T. Suzuki G gl-. J. Pharm. S c l . 73, 136 (1984). 39. C. von Bahr e t a l . Acta Pharmacol. T o x i c o l . 4 l , 39 (1977). 40. R . Kawai e t a l . J. Pharm. Dyn. 6 , 5-79 (1983). 41. C. Horvath and W . Melander. Amer. Lab. lo, 17 (1977). 42. S. M . Waraszkiewicz e t a l . J . Pharm. S c i . 7 0 , 1215 (1981).

m,

705 (1979). 43 * J. Crommen. J . Chromatog. 44. M. Wolff Pharmazie 38, 891 (1983). 45. R.G. A r c h a r i and J.T. Jacob. J. L i q u i d Chrom. 9, 81 (1 980). 46. R.J. Flanagan e t a l . J. Chromatog. 247, 15 (1982). 47. A.N. Masoud. J. Pharm. S c i . 65. 1585 (1976).

c.

48.

E.G.

Sundholm.

J. Chromatog.

265. 285 (1983).

This Page Intentionally Left Blank

SODIUM NITROPRUSSIDE

Auke B u l t , Oscar R. Leeuwenkamp and Wouter P. van Bennekom

1. 2.

3.

4. 5.

6.

History Description 2.1 Name, Formula, Molecular Weight Physical P r o p e r t i e s 3.1 13C-NMR Spectrum 3.2 Massbauer Spectrum 3.3 Mol ecul a r Orbital Diagram S t a b i l i t y and Degradation Pharmacology and In-Vitro Degradation 5.1 Pharmacology 5.2 In-Vitro Degradation Methods o f Analysis 6.1 I d e n t i f i c a t i o n Tests 6.2 P u r i t y Tests 6.3 Colorimetry 6.4 High-Performance Liquid Chromatography 6.5 Pol arography 6.6 Application of N i t r o p r u s s i d e as Analytical Reagent 6.7 Miscellaneous Methods o f Analysis References

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 15

781

Copyright 0 1986 by the American Pharmaceutical Association All rights of reproduction in any form reserved.

AUKE BULT ET AL.

782

1.

HISTORY

N i t r o p r u s s i d e was f i r s t described i n 1849 by P l a y f a i r (1). The compound has a t t r a c t e d considerable i n t e r e s t a t v a r i o u s periods i n chemical h i s t o r y . A t present, t h e r e a r e s t i l l c o n t r o v e r s i e s on v a r i o u s p o i n t s o f i t s physical and (photo)chemical p r o p e r t i e s . The pharmaceutical i n t e r e s t i n n i t r o p r u s s i d e i s due t o i t s a p p l i c a t i o n s as a n a l y t i c a l reagent and as s t r o n g hypot e n s i v e agent. Boedeker ( 2 ) i n t r o d u c e d i n 1861 n i t r o p r u s s i d e as reagent f o r t h e d e t e c t i o n o f s u l p h i t e . L a t e r on, Legal ( 3 ) extended i t s use as reagent t o t h e d e t e c t i o n o f ketone bodies i n u r i n e o f d i a b e t i c p a t i e n t s . Simon ( 4 ) and Rimini ( 5 ) employed t h e substance f o r d e t e c t i o n o f secondary and primary a1 i p h a t i c amines, r e s p e c t i v e l y . The blood-pressure l o w e r i n g e f f e c t o f n i t r o p r u s s i d e was already reported i n 1887 by Davidsohn ( 6 ) , w h i l e t h e f i r s t c l i n i c a l t r i a l was described i n 1928 by Johnson (7). This p r o f i l e i s supplementary t o t h e p r o f i l e o f Rucki (1 i t e r a t u r e was surveyed through October 1976) (8).

2.

DESCRIPTION 2.1 Name, Formula, Molecular Weight

-

Generic name Nomenclature

-

Synonyms

-

-Trade Names -

Sodium n i t r o p r u s s i d e The f o l l o w i n g nomenclature i s used i n Chemical Abstracts: F e r r a t e ( 2 - ) , pentaki s (cyano-C) n i t r o s y l , d i sodi urn ( OC-6-22) C144O2-89-21 Sodium n i t r o f e r r i c y a n i d e , sodium n i t r o p r u s s i a t e , d i sodi um pentacyanonitrosyl f e r r a t e ( I I ) d ihyd r a t e N i pride. (Roche) , Nipruss. (Cedona)

-

2-

0

111, N

I I '-* c

.Fe''2+

.2H2O

111 N

CgN60FeNa?*%H20

Mol ecul a r Weight : 297.95

SODIUM NITROPRUSSIDE

3.

783

PHYSICAL PROPERTIES

3.1 13C-NHR Spectrm The 13C-NMR spectrum o f n i t r o p r u s s i d e i n D20 i s simple. The e q u a t o r i a l and a x i a l CN appear w i t h chemical s h i f t s o f 104.2 and 102.2 ppm r e f e r r e d t o t - b u t a n o l , r e s p e c t i v e l y (9,lO). These s h i f t s a r e about 40 ppm l o w e r as compared t o Fe(CN)5X compounds (X = NH3, H20, CN-) and i n d i c a t e s a s t r o n g ds-backdonation t o t h e NOt l i g a n d (10).

3.2 Mssbauer Spectrum Data on t h e MZissbauer spectrum o f n i t r o p r u s s i d e were presented by Danon (11). The isomer s h i f t ( u ) and quadrupole c o u p l i n g ( A E ) have v a l u e s o f -0.012 cm s - l and 0.185 cm s - l , respectively.

3.3 Molecular Orbital Diagram The MO diagram o f n i t r o p r u s s i d e i s i m p o r t a n t f o r t h e i n t e r p r e t a t i o n o f i t s p h y s i c a l and photochemical p r o p e r t i e s . F i g . 1 p r e s e n t s t h e p a r t i a l MO-energy l e v e l diagram as c a l c u l a t e d w i t h t h e SCCC-MO method ( 1 2 ) . A l l d - e l e c t r o n s a r e p a i r e d and t h e compound i s t h e r e f o r e diamagnetic. The d i s t r i b u t i o n o f t h e e l e c t r o n s i m p l i e s t h a t t h e n i t r o s y l group has a formal charge t 1 and i r o n +2 (d6). The p o s i t i v e charge o f NO e x p l a i n s t h e r e l a t i v e l y h i g h v a l u e of v(N0) = 1940 cm-l and t h e r e a c t i v i t y o f t h i s group towards a wide v a r i e t y o f n u c l e o p h i l i c agents as w e l l (13). The MO diagram i s a l s o i l l u s t r a t i v e f o r t h e assignment o f t h e U V - V I S spectrum as presented i n Table 1 ( 1 2 ) . R e c e n t l y , a r e - i n t e r p r e t a t i o n o f t h e MO diagram based on Scaled INDO c a l c u l a t i o n s has been p u b l i s h e d (14). Now bands I t o 111 t r a n s i t i o n s a r e c o n s i d e r e d t o be due t o d-d and i n t e r n a l t r a n s i t i o n s i n t h e l i g a n d s , w h i l e band I V i s an a1 lowed c h a r g e - t r a n s f e r band.

8e

(TI*-

CN I

7e (n*-NO, d y z ,d,,

_ld_

2bz(dxy) 6e ( d y z , d , , , ~ f N O )

3

I occupied

Fig. 1. Energy l e v e l diagram o f t h e n i t r o p r u s s i d e i o n (12).

784

AUKE BULT ET AL.

Table 1. Assignment o f t h e U V - V I S spectrum

A remarkable phenomenon o f n i t r o p r u s s i d e i s t h a t exc i t a t i o n w i t h a s u i t a b l e l a s e r a t T < 150 K r e s u l t s i n t h e formation o f an extremely l o n g - l i v i n g metastable s t a t e (15). 4.

STABILITY AND DEGRADATION

Two important pathways o f photochemical degradation o f nitroprusside are ( 8 ) : [Fe(CN)5N0J2-

t

H20

t

H20

A nd

[Fe(CN)5N072-

hv

hv

[FeI1(CN)5H20l3-

[FeIII(CN)tjH20]2-

NOt

t

t

NO

[:A 1

r B1

Recently t h e mechanism o f photodecomposition has been re-examined w i t h continuous and f l a s h p h o t o l y s i s (16,17). The conclusions are: 1 ) band I i s p h o t o - i n a c t i v e , 2) band I 1 i r r a d i a t i o n r e s u l t s i n pathway r A ] a n d 3 ) bands I V and V i r r a d i a t i o n g i v e s pathway r B 3 ( f o r bands see Table 1). Owing t o t h e o v e r l a p o f band I 1 1 w i t h bands I 1 and I V , b o t h pathways can occur simultaneously. The conclusions are c o n s i s t e n t w i t h t h e MO diagram o f Golebiewski and Wasielewska (14). For c l i n i c a l use sodium n i t r o p r u s s i d e has t o be recons t i t u t e d w i t h a s t e r i l i z e d aqueous s o l u t i o n r e s u l t i n g i n a concentrated stock s o l u t i o n (ca. 25 g 1-1 i n 0.94: s a l i n e o r 5% glucose), which i s d i l u t e d t o an i n f u s i o n s o l u t i o n (ca. 50-200 mg 1-1 i n 5% glucose). Both s o l u t i o n s have been s t u d i e d f o r thermal and photochemical s t a b i l i t y . Protected from l i g h t , t h e concentrated s o l u t i o n i s s t a b l e a t room temperature and 4°C f o r more than two years (18). Autoclaving (15 min, 121OC) o f n i t r o p r u s s i d e s o l u t i o n s i n water and i n 0.9% s a l i n e (50 mg 1 - l ) gives e s s e n t i a l l y no degradation, whereas s t e r i l i z a t i o n o f n i t r o p r u s s i d e i n 5% dextrose s o l u t i o n r e s u l t s i n about 40% l o s s (19). N i t r o -

785

SODIUM NITROPRUSSIDE

prusside s o l u t i o n s i n l i g h t - p r o t e c t e d g l a s s o r p l a s t i c cont a i n e r s and i n p l a s t i c i n f u s i o n s e t s remain s t a b l e f o r a t l e a s t two days (20). I n s o l u t i o n n i t r o p r u s s i d e i s h i g h l y 1 i g h t - s e n s i t i v e and decomposes r a p i d l y ( 8 ) . Modern photodegradation s t u d i e s are based on " s t a b i l i t y - i n d i c a t i n g " assay methods f o r n i t r o prusside, v i z. c o l o r i m e t r i c measurement o f i n t a c t n i t r o prusside w i t h sulphide (18) and HPLC (19-22). The r a t e o f degradation upon exposure t o d a y l i g h t and t o l i g h t o f 350 nm i s e s s e n t i a l l y t h e same i n water, i n 0.9% s a l i n e and i n 591 dextrose s o l u t i o n s (19). The degradation i s a n o n - l i n e a r process, i.e. an i n i t i a l r a p i d l o s s i s f o l l o w e d by a phase o f slower decomposition (19). Th n o n - l i n e a r i t y i s due t o t h e f o r m a t i o n o f [Fe(CN) H2OI5-, a y e l l o w compound a c t i n g as a l i g h t - f i l t e r . I n a d i t i o n , t h e back-formation o f n i t r o p r u s s i d g from i t s degradation products n i t r i t e and CFe(CN)5H201 occurs according t o ( 1 9 ) :

a

-

[Fe(CN)5H20l3- t NOp- t 2 Ht-

[Fe(CN)rjNOl2-

t

2 H20

The degradation y i e l d s n i t r i t e , n i t r a t e , CFe(CN)5H20I2-, CFe(CN)5H20l3-, Fe(CN)63- and Fe(CN)64- (19). Contrary t o previous r e p o r t s , t h e a d d i t i o n o f c i t r i c a c i d o r sodium edet a t e ( b o t h 0.25 mg 100 m l ) t o 5% dextrose s o l u t i o n o f n i t r o prusside (50 mg 1 - ) does not improve t h e s t a b i l i t y , w h i l e cyanocobalamin (10 mg 1 - l ) s i g n i f i c a n t l y increases t h e p h o t o - s t a b i l i t y (19). Before t h e i n t r o d u c t i o n o f t h e s t a b i l i t y - i n d i c a t i n g assay o f n i t r o p r u s s i d e , t h e degradation s t u d i e s were based on t h e spectrophotometric measurement o f t h e increase i n absorbance a t 395 nm. However, many degradat i o n products o f n i t r o p r u s s i d e e x h i b i t h i g h molar absorpt i v i t i e s i n t h a t region, r e s u l t i n g i n c o n t r a d i c t o r y i n t e r p r e t a t i o n s o f t h e r e s u l t s (23-26). Very r e c e n t l y a study on t h e photodegradation o f n i t r o p r u s s i d e was pub1 ished ' n which were t h e release o f HCN and t h e f o r m a t i o n o f Fe(CN)6 I measured c o l o r i m e t r i c a l l y w i t h d e t e c t i o n l i m i t s o f 10-12 M and 2-3 IM, r e s p e c t i v e l y (27). Dimethyl s u l f o x i d e (10% v / v ) was l a t e l y found t o be an e f f e c t i v e p h o t o p r o t e c t i v e agent f o r s o l u t i o n s o f n i t r o p r u s s i d e (28,29).

!i

3-

5.

PHARUACOLOGY AND

IN-VITRO DEGRADATION

5.1 Pharmacology The main pharmacological and t o x i c o l o g i c a l f e a t u r e s o f n i t r o p r u s s i d e have been discussed b e f o r e (8). Recent r e p o r t s i n t h i s f i e l d are i n d i c a t i v e f o r t h e continued i n t e r e s t i n t h i s compound (30-37).

AUKE BULT ET AL.

786

5.2 In-Vitro Degradation I n - v i t r o s t u d i e s have revealed t h a t cyanide i s released from n i t r o p r u s s i d e on i n c u b a t i o n w i t h serum, plasma, whole blood, 1 iver homogenate , haemogl o b i n and e r y t h r o c y t e s (30). For instance, n i t r o p r u s s i d e incubated w i t h blood releases 50% o f t h e t o t a l amount o f cyanide w i t h i n 20 min and more than 90% over 2 h (38). Upon i n f u s i o n , i n - v i v o cyanide l e v e l s up t o 3.2 IIM and 45.5 IIM were found i n plasma and blood o f p a t i e n t s , r e s p e c t i v e l y (26). The number o f methods s u i t a b l e f o r measurement o f low concentrations o f i n t a c t n i t r o p r u s s i d e and i t s degradation products (cyanide, n i t r i c o x i d e / n i t r i t e ) i n body f l u i d s i s very l i m i t e d (26,39-41). Arnold e t a l . (26) described an i n d i r e c t c o l o r i m e t r i c method, based on a d i a z o coup1 i n g r e a c t i o n , f o r measurement o f n i t r i c oxide released from n i t r o p r u s s i de. The cyanide d e t e r m i n a t i o n i s based on c o l o u r f o r m a t i o n w i t h pyridine/pyrazolone (39). This method i s s u i t a b l e f o r t h e determination o f f r e e cyanide (released from n i t r o prusside) and i n t a c t n i t r o p r u s s i d e v i a q u a n t i t a t i v e conv e r s i o n t o cyanide by i n c u b a t i o n w i t h cysteine. The detect i o n l i m i t amounts t o about 3 pg m l - l o f n i t r o p r u s s i d e (39). Recently Alkayer e t a l . ( 4 0 ) and Leeuwenkamp e t a l . (41) developed pol arographic methods f o r t h e d i r e c t d e t e r m i n a t i o n o f nitroprusside i n i o l o g i c a l m a t r i es w i t h d e t e c t i o n l i m i t s o f 450 ng m l - ) and 15 ng m l - ? , r e s p e c t i v e l y ( f o r d e t a i l s see Section 6.5). E s p e c i a l l y t h e last-mentioned procedure enables measuring o f therapeut 'c l e v e l s o f n i t r o prusside, ranging from 100 t o 1000 ng ml-! (41). Leeuwenkamp ( 4 2 ) employed t h e polarographic method i n a study o f t h e i n - v i t r o degradation o f n i t r o p r u s s i d e (200 ng m l - l ) i n v a r i o u s media a t 37"C, v i z . s o l u t i o n s o f albumin, cysteine, g l u t a t h i o n e and (met)haemoglobin; human p l a s ma, e r y t h r o c y t e suspensions and blood, and i n 100,000 g crude a o r t i c - s o l u b l e f r a c t i o n s . A h a l f - l i f e t i m e ( t l 2) of about 2.6 min was found i n t h e s o l u b l e f r a c t i o n s , w i c h i s comparable t o t h e i n - v i v o t l 2. The t 1 / 2 i n human blood was about 15 min and i n t h e o t e r mentioned media considerably h i g h e r values o f t 1 / 2 were observed.

I

6

6.

METHODS OF ANALYSIS

6.1 I d e n t i f i c a t i o n Tests The i d e n t i f i c a t i o n t e s t s f o r sodium n i t r o p r u s s i d e and i t s dosage form " s t e r i l e sodium n i t r o p r u s s i d e " are t h e same i n USP X X I as i n USP X I X (8). Recently a new p r e c i p i t a t i o n reagent f o r t h e n i t r o p r u s s i d e anion has been described (43).

787

SODIUM NITROPRUSSIDE

6.2 P u r i t y Tests The p u r i t y t e s t s i n USP X X I f o r sodium n i t r o p r u s s i d e comprise c h l o r i d e and sulphate; b o t h t e s t s a r e based on t u r b i d i m e t r i c measurements. The i m i t t e s t f o r d e t e r m i n a t i o n o f t h e hexacyanoferrates Fe(CN)6 - and Fe(CN)64- a t 0.059: l e v e l can be performed by chromatographic s e p a r a t i o n o f sodium n i t r o p r u s s i d e f r o m t h e s e substances, followed by spectrophotometric measurement a t 415 nm (44). Moreover, t h e chromatographic procedure d e s c r i b e d by Leeuwenkamp e t a l . (21) enables o n - l i n e d e t e r m i n a t i o n o f b o t h hexacyanoferrates

I

.

6.3 Colorimetry Determinations o f n i t r o p r u s s i d e , based on a q u a n t i t a t i v e r e l e a s e o f n i t r i c o x i d e and c o l o r i m e t r i c measurement o f t h e n i t r i t e formed by a d i a z o c o u p l i n g r e a c t i o n have a l r e a d y been described i n S e c t i o n 5.2 (26). S i m i l a r procedures have r e c e n t l y been r e p o r t e d (45,46). N i t r o p r u s s i d e d e t e r m i n a t i o n , based on a q u a n t i t a t i v e re1 ease o f cyanide and subsequent c o l o r i m e t r i c measurement o f cyanide has been d e s c r i b e d i n S e c t i o n 5.2 (39). Burger (47) r e p o r t e d t h e d e t e r m i n a t i o n o f c y a n o f e r r a t e compounds, e.g. n i t r o p r u s s i d e , by complete d e g r a d a t i o n o f these compounds i n a m i x t u r e o f phenanthrol i n e / a s c o r b i c acid/mercury( I I ) c h l o r i d e / a c e t a t e b u f f e r (pH = 3.5) and q u a n t i t a t i o n o f t h e r e l e a s e d HCN by t i t r a t i o n w i t h s i l v e r n i t r a t e and c o l o r i m e t r i c measurement o f Fe2+ as f e r r o i n a t 510 nm.

6.4 High-Performance Liquid Chromatography Up t o now t h r e e HPLC procedures have been described i n t h e l i t e r a t u r e (20-22). Baaske e t a1 ( 2 2 ) used a reversedphase i o n - p a i r system w i t h a s t a t i o n a r y phase o f m i c r o p a r t i c u l a t e 10 Ilm phenyl-bonded s i l i c a gel and a m o b i l e phase c o n s i s t i n g o f acetonitrile/phosphate/tetra-n-butylammonium hydroxide b u f f e r o f pH = 7.1 ( 3 0 t 7 0 ) ; UV-detection a t 210 nm; l i n e a r c a l i b r a t i o n c u r v e i n t h e c o n c e n t r a t i o n range: 10-50 p g m l - 1 ; c o e f f i c i e n t o f v a r i a t i o n (CV) < 3.1%. The system separates n i t r o p r u s s i d e from i t s d e g r a d a t i o n p r o d u c t Fe(CN)64-, b u t n i t r o p r u s s i d e and Fe( CN)63- a r e n o t separated. A s i m i l a r system was d e s c r i b e d by Leeuwenkamp e t a1 (21). They used p-Rondapack phenyl-bonded pel 1 i c u l a r s i l i c a g e l (10 p m ) as s t a t i o n a r y phase and a m o b i l e phase cons i s t i n g o f water-methanol (65+35) c o n t a i n i n g 5 mM t e t r a n-butyl-ammonium phosphate, 1.1 mM n-octylamine and 6.5 mM potassium dihydrogen phosphate (pH = 7.0); UV-detection a t 220 nm; l i n e a r c a l i b r a t i o n c u r v e i n t h e c o n c e n t r a t i o n range:

.

.

AUKE BULT ET AL.

788

10-120 pg m l - 1 . This system enables t h e separation of n i t r o p r sside from ‘ t s photodegradati n products NOp-, NO Fe(CN)$, Fe( CN)6 -, [Fe( CN)5H201a- and [Fe(CN)5H20j3’ (19). Most r e c e n t l y an HPLC procedure has been published (20) i n which P a r t i s i l 10-SAX anion-exchange m a t e r i a l i s used as s t a t i o n a r y phase w i t h a m o b i l e phase c o n s i s t i n g o f 0.5 M potassium dihydrogen phosphate b u f f e r (pH = 3.0) ; UV-detection a t 230 nm; l i n e a r c a l i b r a t i o n curve i n t h e c o n c e n t r a t i o n range 1-200 119 m l - 1 ; CV < 1%. However, w i t h t h i s system no degradation products were detected.

-

4

6.5 Pol arography The key f e a t u r e s o f t h e pol arographic behaviour o f n i t r o p r u s s i d e have been discussed b e f o r e ( 8 ) . I n t h e pH range 4-9 a t t h e dropping mercury e l e c t r o d e , t h r e e r e d u c t i o n processes (1-111) were observed w i t h d i f f e r e n t i a l p u l s e pol arography (DPP) and high-performance d i f f e r e n t i a l p u l s e polarography (HPDPP) w i t h peak p o t e n t i a l s a t -360 ( I ) , -610 (11) and -1500 (111) mV vs. s a t u r a t e d sodium c h l o r i d e c a l o mel e l e c t r o d e (SSCE) (48). A t pH > 10 t h e polarographic d i f f u s i o n - c u r r e n t d creases most probably because o f format i o n o f [Fe(CN) NO21 - (49). A t pH = 6 peak I 1 s t a r t s t o s h i f t anodical y and increases i n i n t e n s i t y w i t h a decrease i n pH (48). Peak I 1 superimposes peak I a t pH < 1.5 and t h e r e s u l t i n g peak s h i f t s a n o d i c a l l y i n a l i n e a r way down t o pH = 0 (48). A t pH = 0 (1 M HCl04) t h e combined r e d u c t i o n peak appears a t -270 mV vs. SSCE and maximum s e n s i t i v i t y i s obtained ( l i n e a r c l i b r a t i o n curve i n t h e c o n c e n t r a t i o n range 4-1000 ng m l - ) (48).

f

7

f

An extensive a n a l y s i s o f t h e r e d u c t i o n processes i s g i v e n e l sewhere (23,48-51). L a t e l y , t h e one-el$ctron reduct i o n product o f n i t r o p r u s s i d e , v i z . [Fe(CN)5N0l3- ( I ) , has a t t r a c t e d considerable a t t e n t i o n because o f t h e r a p i d equi l i b r i u m (50-53):

-

The r e d u c t i o n product I decomposes t o a r e a c t i v e pentacoordinate complex (11) due t o r e l e a s e o f cyanide, w h i l e t h e unpaired e l e c t r o n t r a n s f e r s from t h e n i t r o s y l N atom t o t h e i r o n atom. Compound I 1 forms t h e hexacoordinate complex CFe(CN)3(chel)NOJ- w i t h s u i t a b l e b i d e n t a t e l i g a n d s ( c h e l ) , e.g. 2,2’ - b i p y r i d i ne, 1,lO-phenanthrol ine (54). Polarography i s used t o q u a n t i t a t e n i t r o p r u s s i d e i n dosage form (USP XXI) and i n body f l u i d s (40,41). Alkayer e t

SODIUM NITROPRUSSIDE

789

a l . (40) developed a d e t e r m i n a t i o n method f o r n i t r o p r u s s i d e i n human serum based on phase-sensitive sine-wave p o l a r o graphy a t pH = 7.4 a f t e r p r o t e i n e l i m i n a t i o n w i t h p e r c h l o r i c a c i d (1 i n e a r c o n c e n t r a t i o n range 2-24 119 ml-1). Leeuwenkamp e t a l . (41) based t h e i r method on HPDPP and DPP a t pH = 0 (1 M p e r c h l o r i c a c i d ) . The l i n e a r c a l i b r a t i o n range i n plasma, serum and blood ( p r o t e i n e l i m i n a t i o n by p e r c h l o r i c a c i d ) i s 30-1000 ng m l - l . 6.6 Application o f Nitroprusside as Analytical Reagent N i t r o p r u s s i d e i s a v a l u a b l e reagent f o r t h e d e t e c t i o n and determination o f a wide v a r i e t y o f n u c l e o p h i l i c agents, e.g. primary and secondary a1 i p h a t i c amines, aldoximes, amino acids, a n i l i n e s , i n d o l s , ketones, n i t r i l s , phenols, p y r r o l e s , quinones, s u l p h i t e , t h i o l s , t h i o u r e a s and u r a c i l s (13,23,55-58). I t s r e a c t i v i t y i s based on t h e p o s i t i v e n i t r o s y l group, r e a c t i n g w i t h n u c l e o p h i l i c species ( m o s t l y i n a l k a l i n e medium) (13). Most a d d i t i o n compounds formed are h i g h l y coloured and s u f f i c i e n t l y s t a b l e t o be used as a b a s i s f o r spectrophotometric determination. The r e a c t i o n mechanisms are (sometimes) r a t h e r complicated (23).

6.7 Miscellaneous Methods o f Analysis Low concentrations o f n i t r o p r u s s i d e ( d e t e c t i o n 1i m i t ca. 3 ng ml-1) have been assayed by v i r t u e o f i t s c a t a l y t i c 620 nm) e f f e c t on t h e f o r m a t i o n o f indophenol b l u e (Am from ammonia, phenol and h y p o c h l o r i t e i n a l k a q i n i medium ( r e a c t i o n o f B e r t h e l o t ) (59). N i t r o p r u s s i d e can be determined by f l o w - i n j e c t i o n analys i s w i t h amperometric d e t e c t i o n ( o x i d a t i o n a t a g l a s s y carbon e l e c t r o d e o r r e d u c t i o n a t a sessi e mercury drop ~ C V < 2% e l e c t r o d e ) ; c o n c e n t r a t i o n range 10-6 t o 5 0 1 0 - M;

(60) References ( t h e 1it e r a t u r e i s c i t e d through 1985)

1 2 3 4 5 6

7

L. P l a y f a i r , Proc. R. SOC. London 5, 846 (1849). C. Boedeker, L i e b i g s Ann. Chem. 117, 193 (1861). E. Legal, Breslauer A e r z t l Z. 5 3 8 (1883). L. Simon, C.R. Acad. Sci. 125, T105 (1897). E. Rimini, Ann. F a r m a c o t e r x h i m . 27/28, 193 (1898). K. Davidsohn, Versuche cber d i e Wirkung des Nitroprussid-natriums. Kb'nigsburg ( P r u s s i a ) : A l b e r t u s - U n i v e r s i t x t , D i s s e r t a t i o n (1887). C.C. Johnson, Proc. SOC. Exp. R i o l . Med. 26, 102 (1928).

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11 12 13 14

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R. Rucki , A n a l y t i c a l P r o f i l e s o f Drug Substances, Vol. 6, Ed. K. Florey, Academic Press, New York (1977), p. 487. A.D. James and R.S. Murray, Inorg. Nucl Chem. L e t t . 12, 739 (1976). K A . Narayanan and P,T. Manoharan, J. Inorg. Nucl. Chem. 40, 1993 (1978). 3. Danon, J. Chem. Phys. 41, 3378 (1964). P.T. Manoharan and H,B. Gzy, J. Am. Chem. SOC. 87, 3340 (1965). J.H. Swinehart, Coord. Chem. Rev. 2, 385 (1967). A. Golebiewski and E, Wasielewska, J. Mol. S t r u c t u r e 67, 183 (1980). Woike, W. Krasser, P.S. Bechthold and S. Hauss'u'hl, S o l i d S t a t e Commun. 45, 499 (1983). T. Jarzynowski, T. Senkowski and Z. Stasicka, Roczniki Chem. 51, 2299 (1977). T. Jarzynowski ,7. Senkowski and Z. Stasicka , Pol. J. Chem. 55, 3 (1981). C.J. Vesey andG.A. B a t i s t o n i , J. C l i n . Pharmacol. 2, 105 (1977). O.R. Leeuwenkamp, E.J. van der Mark, W.P. van Bennekom and A. B u l t , I n t . J. Pharm. 24, 27 (1985). C. Mahony, J.E. Brown, W.W. S t a r g e l , C.P. Verghese and T.D. Bjornsson, J. Pharm. Sci. 73, 838 (1984). O.R. Leeuwenkamp, E.J. van der Mark, P.M. van der Klauw, W.P. van Bennekom and A. B u l t . Anal. Chim. Acta 161, 211 (1984). D.M. Baaske, M.D. Smith, N. Karnatz and J.E. Carter, J. Chromatogr. 212, 339 (1981). O.R. LeeuwenkamcW.P. van Bennekom, E.J. van der Mark and A. B u l t , Pharm. Weekbl. [ S c i l 6, 129 (1984). A.C. van Loenen and W. Hofs-Kemper, Pharm. Weekbl. 114, 424 (1979). K C h a b r e l , M. M o l l e t , F. Puisieux, T.X. Thao, P. Canivet, L. Assamoi, C. Hazebroucq and M. Hamon, Ann. Pharm. Fr. 38, 307 (1980). W.P. Arnold, D.ETLongnecker and R.M. Epstein, Anesthesiology 61 , 254 (1984). T.F. H a r t l e y , JX. P h i l c o x and J . Willoughby, J. Pharm. Sci. 74, 668 (1985). A.F. Asker and R. Gragg, Drug Dev. Ind. Pharm. 8, 837 (1983). A.F. Asker and D. Canady, Drug Dev. Ind. Pharm. 10, 1025 (1984). J.H. Tinker and J.D. Michenfelder, Anesthesiology 45, 340 (1976). L. Greiss, N.A.G. Tremblay and D.W. Davies, Canad. Anaesth. SOC. J. 23, 480 (1976).

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32 A. Raithelhuber, Pharm. Ztg. 124, 593 (1979). Kreye, Sodium N i t r o p r u z d e , Pharmacology o f 33 V.A.W. A n t i h y p e r t e n s i v e Drugs , Ed. A. S c r i abine , Raven Press , New York (1980), p. 373. 34 L. Dumont, C. Lamoureux, 3. L e l o r i e r , P. Stanley and C. Chartrand, Arch. I n t . Pharmacodyn. 261, 109 (1983). 35 S.A. Napoli , C.A. G r u e t t e r , L.J. I g n a r F a n d P.J. Kadowitz, J. Pharmacol. Exp. Ther. 212, 469 (1980). 36 L.J. Ignarro, M. L i p p t o n and J.C. E d w a r d c J. Pharmacol. Exp. Ther. 218, 739 (1981). Kreye and S.N. Reske, Naunyn-Schmiedeberg's Arch. 37 V.A.W. Pharmacol 320, 260 (1982). 38 C.J. V e s e y , T Cole and P. Simpson, B r . J. Anaesth. 54, 791 (1982). 39 F.L. Rodkey and H.A. C o l l i s o n , C l i n . Chem. 23, 1969 (1977). 40 % A1 kayer, J.J. Val l o n and Y. P6gon, Anal. L e t t e r s 14, 399 (1981). 41 O.R. Leeuwenkamp, E.J. van der Mark, H. Jousma,

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54 J. Fiedler and J. Mazek, Inorg. Chim. Acta 81, 117 (1984). 55 Kak6E and J. Vejdglek, Handbuch der photometrischen Anal y s e organi scher Verbi ndungen , Band I und I I , Verl ag Chemie, Weinheim (1974), pp. 285,487,537. 56 M.K. Gadia and M.C. Mehra, Microchem. J. 22, 561 (1977). 57 M.R. Prasad, M. Sugumaran and C.S. Vaidyanathan, Anal. Biochem. 80, 483 (1977). 58 M. Sugumaran an?rC.S. Vaidyanathan, J. Indian Inst. Sci. 60, 51 (1978). 59 A. Sanchez Perez, J. Hernandez Mendez and J. Montero Garcia, Cienc. Ind. Farm. 1, 114 (1982). 60 A.G. Fogg, M.A. Ferngndez-Arciniega aiid R.M. Alonso, Analyst 110,345 (1985).

CUMULATIVE INDEX Bold numerals refer to volume numbers

Acetaminophen. 3, I ; 14, 551 Acetohexamide. 1, I ; 2, 573 Allopurinol, 7, 1 Alpha-tocopheryl acetate, 3, I I I Amantadine, 12, I Amikacin sulfate, 12, 37 Amiloride hydrochloride, 15, I Aminoglutethimide, 15, 35 Aminophylline, 11, I Aminosalicylic acid. 10, I Amitriptyline hydrochloride. 3, 127 Amoxicillin. 7, 19 Amphotericin B. 6, I; 7, 502 Ampicillin. 2, I ; 4, 518 Ascorbic acid, 11, 45 Aspirin. 8, I Atenolol. 13, I Atropine, 14, 32 Azathioprine. 10, 29 Bacitracin, 9, I Baclofen, 14, 527 Bendroflumethiazide. 5, I ; 6, 597 Benperidol 14, 245 Benzocaine. 12, 73 Benzyl benzoate. 10,55 Betamethasone dipropionate, 6, 43 Bretylium tosylate, 9, 71 Bromocriptine methanesulfonate. 8,47 Caffeine, 15, 71 Calcitriol, 8, 83 Camphor. 13, 27 Captopril. 11, 79 Carbamazepine, 9,87 Cefaclor. 9, 107 Cefamandole nafate. 9, 125; 10, 729 Cefazolin, 4, I Cefotaxime, 11, 139 Cefoxitin. sodium, 11, 169 Cephalexin, 4, 21 Cephalothin sodium. 1, 319

Cephradine. 5, 21 Chloral hydrate, 2, 85 Chloramphenicol. 4,47. 518; 15, 701 Chlordiazepoxide. 1, 15 Chlordiazepoxide hydrochloride, 1, 39; 4, 518 Chloroquine, 13, 95 Chlyroquine phosphate, 5, 61 Chlorpheniramine maleate. 7, 43 Chlorprothixene, 2, 63 Chlortetrdcycline hydrochloride, 8, 101 Chlorthalidone, 14, I Cholecalciferol, see Vitamin D, Cimetidine. 13, 127 Cisplatin. 14, 77: 15, 796 Clidinium bromide. 2, 145 Clindamycin hydrochloride. 10, 75 Clofibrate, 11, 197 Clonazepam, 6, 61 Clorazepate dipotassium. 4, 91 ClotrimaLole, 11, 225 Cloxacillin sodium, 4, I13 Cocaine hydrochloride. 15, I S 1 Codeine phosphate, 10, 93 Colchicine. 10, 139 Cyanocobalamin. 10, 183 Cyclizine. 6, 83; 7, 502 Cycloserine, 1, 53 Cyclothiaz.ide.1, 66 Cypropheptadine. 9, 155 Dapsone, 5, 87 Dexamethasone, 2, 163: 4, 519 Diatrizoic acid. 4, 137; 5, 556 Diazepam. 1, 79; 4, 518 Dibenzepin hydrochloride, 9 , 181 Dibucaine and dibucaine hydrochloride. 12, 105 Diflunisal, 14, 491 Digitoxin. 3, 149 Digoxin, 9 , 207 Dihydroergotoxine methanesulfonate. 7, 81 Dioctyl sodium sulfosuccinate. 2, 199; 12, 713

793

794 Diperodon. 6 , 9 9 Diphenhydramine hydrochloride, 3, 173 Diphenoxylate hydrochloride, 7, 149 Disopyramide phosphate. 13, 183 Disulfiram. 4, 168 Dobutamine hydrochloride, 8, 139 Dopamine hydrochloride. 11, 257 Doxorubicine, 9, 245 Droperidol, 7, 171 Echothiophate iodide, 3, 233 Emetine hydrochloride, 10, 289 Ephedrine hydrochloride. 15, 233 Epinephrine, 7, 193 Ergonovine maleate, 11, 273 Ergotamine tartrate, 6, I13 Erythromycin, 8, 159 Erythromycin estolate, 1, 10 I ; 2, 573 Estradiol. 15, 283 Estradiol valerate. 4, 192 Estrone, 12, 135 Ethambutol hydrochloride, 7, 231 Ethynodiol diacetate, 3, 253 Etomidate, 12, 191 Fenoprofen calcium 6, 161 Flucytosine. 5, I15 Fludrocortisone acetate, 3, 281 Flufenamic acid, 11, 313 Fluorouracil, 2, 221 Fluoxymesterone, 7, 251 Fluphenazine decanoate, 9, 275; 10, 730 Fluphenazine enanthate, 2, 245; 4, 524 Fluphenazine hydrochloride, 2, 263; 4, 519 Flurazepam hydrochloride, 3, 307 Gentamicin sulfate, 9, 295; 10, 731 Glibenclamide, 10, 337 Gluthethimide. 5, 139 Gramicidin, 8, 179 Griseofulvin, 8, 219; 9, 583 Guanabenz acetate. 15, 319 Halcinonide, 8, 251 Haloperidd, 9, 341 Halothane, 1, 119; 2,573; 14,597 Heparin sodium, 12, 215 Heroin. 10, 357 Hexestrol, 11, 347 Hexetidine, 7, 277 Hydralazine hydrochloride, 8, 283 Hydrochlorothiazide. 10, 405 Hydrocortisone, 12, 277 Hydroflumethiazide. 7, 297 Hydroxyprogesterone caproate. 4, 209 Hydroxyzine dihydrochloride, 7, 319

CUMULATIVE INDEX Imipramine hydrochloride, 14, 37 Indomethacin, 13, 21 I lodamide, 15, 337 lodipamide. 2, 333 lopanoic acid, 14, 181 Isocarboxazid, 2, 295 Isoniazide, 6, 183 Isopropamide, 2, 315; 12, 721 lsoproterenol, 14, 391 lsosorbide dinitrate, 4, 225; 5, 556 Kanamycin sulfate, 6, 259 Ketamine. 6, 297 Ketoprofen, 10,443 Ketotifen, 13, 239 Khellin, 9, 371 Leucovorin calcium, 8, 315 Levallorphan tartrate, 2, 339 Levarterenol bitartrate, I, 49; 2, 573; 11, 555 Levodopa, 5, 189 Levothyroxine sodium, 5, 225 Lidocaine base and hydrochloride, 14, 207: 15, 761 Lithium carbonate. 15, 367 Lorazepam, 9, 397 Maprotiline hydrochloride. 15, 393 Mefloquine hydrochloride, 14, 157 Melphalan. 13, 265 Meperidine hydrochloride, 1, 175 Meprobamate, 1, 209; 4, 520; 11,587 6-Mercaptopurine, 7, 343 Mestranol. 11, 375 Methadone hydrochloride, 3, 365; 4, 520; 9,601 Methaqualone, 4, 245. 520 Methimazole, 8, 351 Methotrexate, 5, 283 Methoxsalen, 9,427 Methyclothiazide, 5, 307 Methylphenidate hydrochloride, 10, 473 Methyprylon, 2, 363 Metoprolol tartrate, 12, 325 Metronidazole, 5, 327 Minocycline, 6, 323 Moxalactam disodium, 13, 305 Nabilone, 10,499 Nadolol, 9,455; 10, 732 Nalidixic acid, 8, 371 Naloxone hydrochloride. 14,453 Natarnycin, 10, 513 Neomycin, 8, 399 Nitrazepam, 9,487 Nitrofurantoin. 5, 345 Nitroglycerin, 9, 519 Norethindrone, 4, 268

CUMULATIVE INDEX Norpestrel. 4, 294 Nortriptyline hydrochloride. 1, 233: 2, 573 Noscapine, 11, 407 Nystatin, 6, 341 Oxazepam. 3,441 Oxyphenbutazone. 13, 333 Oxytocin. 10, 563 Penicillamine, 10, 601 Penicillin-G benzothine, 11, 463 Penicillin G. potassium. 15, 427 Penicillin-V, I, 249 Pentazocine, 13, 361 Phenazopyridine hydrochloride, 3,465 Phenelzine sulfate, 2, 383 Phenformin hydrochloride, 4, 319; 5,429 Phenobarbital. 7, 359 Phenoxymethyl penicillin potassium, 1, 249 Phenylbutazone, 11, 483 Phenylephrine hydrochloride, 3, 483 Phenylpropanolamine hydrochloride, 12, 357; 13, 77 I Phenytoin, 13, 417 Pilocarpine, 12, 385 Piperazine estrone sulfate, 5, 375 Piroxicam, 15, SO9 Primidone, 2, 409 Probenecid, 10, 639 Procainamide hydrochloride, 4, 333 Procarbazine hydrochloride. 5 , 403 Promethazine hydrochloride, 5 , 429 Proparacaine hydrochloride. 6, 423 Propiomazine hydrochloride, 2, 439 Propoxyphene hydrochloride, I, 301; 4, 520; 6, 598 Propylthiouracil. 6,457 Pseudoephedrine hydrochloride, 8,489 Pyrazinamide, 12, 433 Pyridoxine hydrochloride, 13, 447 Pyrimethamine, 12,463 Quinidine sulfate, 12, 483 Quinine hydrochloride, 12, 547 Ranitidine, 15, 533 Reserpine, 4, 384: 5,557; 13, 737 Rifampin, 5 , 467 Rutin, 12, 623 Saccharin, 13, 487 Salbutamol, 10, 665 Salicylamide, 13, 521

795 Secobarbital sodium, 1, 343 Silver sulfadiazine, 13, 553 Sodium nitroprusside. 6, 487; 15, 781 Spironolactone, 4, 431 Strychnine. 15. 563 Succinycholine chloride. 10, 691 Sulfadiazine. 11, 523 Sulfamethazine. 7, 401 Sulfamethoxazole, 2, 467: 4, 521 Sulfasalazine, 5 , 515 Sulfisoxazole, 2, 487 Sulindac. 13, 573 Sulphamerazine, 6, 515 Terpin hydrate, 14, 273 Testolactone, 5 , 533 Testosterone enanthate, 4, 452 Tetracycline hydrochloride, 13, 597 Theophylline, 4, 466 Thiostrepton, 7, 423 Tolbutamide, 3, 513; 5, 557; 13, 719 Triamcinolone. I, 367: 2, 571; 4, 521, 524; 11, 593 Triamcinolone acetonide, 1, 397, 416; 2, 571; 4, 521; 7, 501; 11, 615 Triamcinolone diacetate, 1, 423; 11, 651 Triamcinolone hexacetonide, 6, 579 Triclobisonium chloride, 2, 507 Trifluoperazine hydrochloride, 9, 543 Triflupromazine hydrochloride, 2, 523; 4, 521; 5, 557 Trimethaphan camsylate, 3, 545 Trimethobenzamide hydrochloride, 2, 551 Trimethoprim, 7, 445 Trimipramine maleate, 12, 683 Trioxsalen. 10, 705 Tripelennamine hydrochloride, 14, 107 Triprolidine hydrochloride. 8, 509 Tropicamide, 3, 565 Tubocurarine chloride, 7, 477 Tybamate, 4,494 Valproate sodium and valproic acid, 8, 529 Vidarabine. 15, 647 Vinblastine sulfate. 1, 443 Vincristine sulfate. 1, 463 Vitamin D,, 13, 655 Warfarin. 14, 243 Xylometazoline hydrochloride, 14, 135 Zomepirac sodium, 15, 673

ERRATUM TO K. Florey ( E d . ) . vol. 1 4 .

(1985). Analytical P r o f i l e s of Drug Substances,

Page 82, T a b l e 1:

The molar e x t i n c t i o n c o e f f i c i e n t a t 285 sun i s 109, not 190.

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