Analytical Profiles of Drug Substances and Excipients Volume 10

Analytical Profiles of Drug Substances Volume 10 Edited by Klaus Florey The Squibb Institute for Medical Research New B...

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Analytical Profiles of Drug Substances Volume 10 Edited by

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

Contributing Editors

Rafik Bishara Lee T. Grady Glenn A. Brewer, Jr. Hans-Georg Leemann John E. Fairbrother Joseph A. Mollica Bruce C. Rudy Compiled under the auspices of the Pharmaceutical Analysis and Control Section Academy of Pharmaceutical Sciences

ACADEMIC PRESS

1981

A Subsidiary of Harcourt Brace Jovanovich, Publishers

New York London Sydney Toronto San Francisco

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

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

Academic Press Rapid Manuscript Reproduction

COPYRIGHT @ 1981, BY ACADEMIC PRESS,INC. 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 I N WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NW1 IDX

Library o f C o y r e s s C a t a l o g i y i n Publication Data Main e n t r y under t i t l e : Analytical p r o f i l e s of drug substances. Compiled under t h e auspices o f t h e Pharmaceutical Analysis and Control Sect ion. Academy o f Pharmaceutical Sciences. Includes bibliographical r e f e r e r c e s and index. 1. Drugs--Analysis--Collected works. 2. Chemistry, Pharmaceutical--Collected works. I. Florey, Klaus. 11. Brewer, Glenn A. 111. Academy of Pharmaceutical Sciences. Pharmaceutical Analysis and Control Section. [ONLM: 1. Drugs--Analysis--Yearbooks. QV740 A A 1 A551 RS189.A58 615' .1 70-187259 ISBN

0-12-260810-0

(v. 10)

AACRl

PRINTED I N THE UNITED STATES OF AMERICA

81 82 83 84

98 76 5 4 3 2 1

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

H . Y. Aboul-Enein, Riyadh University, Riyadh, Saudi Arabia A . A . Al-Badr, Riyadh University, Riyadh, Saudi Arabia N . W. Atwater, E. R. Squibb and Sons, Princeton, New Jersey S. A . Benezra, Burroughs Wellcome Company, Research Triangle Park, North Carolina W. F . Beyer, The Upjohn Company, Kalamazoo, Michigan R . Bishara, Eli Lilly and Company, Indianapolis, Indiana J . I . Bodin, Carter Wallace, Inc., Cranbury, New Jersey G. A . Brewer, The Squibb Institute for Medical Research, New Brunswick, New Jersey H . Brik, Gist-Brocades, Delft, Holland L. W. Brown, The Upjohn Company, Kalamazoo, Michigan L . Chafetx, Warner-Lambert Research Institute, Morris Plains, New Jersey C . C . Chiu, The United States Pharmacopeia, Rockville, Maryland H . P. Deppeler, Ciba-Geigy Ltd., Basel, Switzerland H . A . El-Obeid, Riyadh University, Riyadh, Saudi Arabia J. Fairbrother, Stiefel Laboratories Ltd., Sligo, Ireland L. V. Feyns, The United States Pharmacopeia, Rockville, Maryland K. Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey P. R. B . Foss, Burroughs Wellcome Company, Research Triangle Park, North Carolina S. A . Fusari, Parke-Davis, Inc., Detroit, Michigan L. T . Grady, The United States Pharmacopeia, Rockville, Maryland M . M . A . Hassan, Riyadh University, Riyadh, Saudi Arabia S. E . Ibrahim, Riyadh University, Riyadh, Saudi Arabia A . I . Judo, Riyadh University, Riyadh, Saudi Arabia

X

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

T . Kho, Ayerst Laboratories, Rouses Point, New York J. Kirschbaum, The Squibb Institute for Medical Research, New Brunswick, New Jersey K . Krummen, Sandoz, Basel, Switzerland H . G. Leemann, Sandoz, Basel, Switzerland G. G. Liuersidge, University of Nottingham, Nottingham, England M . A. Loutfy, Riyadh University, Riyadh, Saudi Arabia F . M a d , Sendoz, Basel, Switzerland J. Mollica, Ciba-Geigy Corporation, Suffern, New York 1. S. Mossa, Riyadh University, Riyadh, Saudi Arabia F. J. Muhtadi, Riyadh University, Riyadh, Saudi Arabia F . Nachtmann, Sandoz, Basel, Switzerland G. R . Padmanabhan, Ciba-Geigy Ltd., Suffern, New York G. Papariello, Wyeth Laboratories, Philadelphia, Pennsylvannia E . Riemer, Sandoz, Rasel, Switzerland B . C. Rudy, Mary Kay Cosmetics, Dallas, Texas R. W. Souter, Eli Lilly, Indianapolis, Indiana S. Sun, The United States Pharmacopeia, Rockville, Maryland P . G. Takla, University of Wales Institute of Science and Technology, South Wales, United Kingdom W . P . Wilson, Burroughs Wellcome Company, Research Triangle Park, North Carolina D. K . Wyatt, The United States Pharmacopeia, Rockville, Maryland M . D. Yudis, Schering-Plough, Inc., Rloomfield, New Jersey M . U . Zubair, Riyadh University, Riyadh, Saudia Arabia

PREFACE

Although the official compendia list tests and limits for drug substances related to identity, purity, and strength, they normally do not provide other physical or chemical data, nor do they list methods of synthesis or pathways of physical or biological degradation and metabolism. For drug substances important enough to be accorded monographs in the official compendia, such supplemental information should also be made readily available. To this end the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences, has undertaken a cooperative venture to compile and publish Analytical Profiles of Drug Substances in a series of volumes of which this is the tenth. The concept of analytical profiles is taking hold not only for compendia1 drugs but, increasingly, in the industrial research laboratories. Analytical profiles are being prepared and periodically updated to provide physiochemical and analytical information of new drug substances during the consecutive stages of research and development. Hopefully, then, in the not-too-distant future, the publication of an analytical profile will require a minimum of effort whenever a new drug substance is selected for compendial status. The cooperative spirit of our contributors has made this venture possible. It is gratifying to note that increasingly profiles are being written not only in industrial laboratories but also in academic institutions worldwide. All those who have found the profiles useful are requested to contribute a monograph of their own. The editors stand ready to receive such contributions. The goal to cover all drug substances with comprehensive monographs is still a distant one. It is up to our perseverance to make it a reality. Klaus Florey

xi

AMINOSALICYLIC ACID Mahmoud M . A . Hassan, Ahmad I . Jado, and Muhammad Uppal Zubair 1. Description 1 . 1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Taste, Odor 2. Physical Properties 2.1 Crystal Properties 2.2 Solubility 2.3 Identification 2.4 Spectral Properties 3. Synthesis 4. Metabolism 5. Methods of Analysis 5.1 Nonaqueous Titration 5.2 Diazometric Assay 5.3 Spectrophotometry 5.4 Combined TLC and Colorimetry 5.5 Ultraviolet Method References

2 2 2 3 3 3 3 3 6 7 7 17 19 21 21 22 23 23 23 25

2

MAHMOUD M. A. HASSAN e t a ! .

1. DESCRIPTION 1.1 Nomenclature 1.1 1 Chemical Names a. 4-Amino-2-hydroxybenzoic

acid.

b. 4-Aminosalicylic acid. c. Benzoic acid, 4-Amino-2-hydroxy. The CAS Registry No. is [65-49-61. 1.1 2 Generic Name p-Aminosalicylic acid. 1.1 3 Trade Names Apas, Apacil, Deapasil, Hellipidyl, PAS,. PAS-C, Pamcyl, Pamisyl, Parasil, Pasorbic, Pasolac, Parasalicil, Parasalindon, Pasnodia, Propasa, Rezipas, Sanipir’ol-4,Para-Pas, Pasem. 1.2 Formulae 1.2 1 Empirical

c7 H7 N03 1.2 2 Structural COOH

1.2 3 Wiswesser Line Notation ZR CQ DVQ

AMINOSALICYLIC ACID

3

1.3 Molecular Weight 153.13 1.4 Elemental Composition C,54,90%; H, 4.61%; N, 9.5%; 0, 31.34%. 1.5 Pppearance, Color, Taste, Odor White, o r yellowish white, bulky powder or crystals darkens on exposure to light and air, odorless or has slight acetous odor. 2 ., Physical Properties

2.1 Crystal Properties 2.1 1 X-Ray Diffraction Crvstal data Monoclinic, a = 7.209 (2), b = 3.786 (l), co= 25.109 (9) A o , B = 103.22 (3)O, U = 6.67.14 A 3, Z = 4, Dc = 1.53, F (000) = 320. Cu-Ka radiation, A = 1.5418 A'; u (Cu-Ka) = 1 0 . 2 0 ~ m - ~ . Systematic absences = h01, 1 = 2n + 1, OkO, k = 2n + 1, space g r m p P21/C from systematic absences (1). Optical goniometry It crystallises from ethanol in at least two habits. The interfacial angles of habit I were measured with a Huber two circle optical goniometer and conpared with angels calculated from unit-cell dimentions for all faces having Miller indices between (and including) +2 and -2. A unique set of assignments f o r the faces was obtained and confirmed by precision photography. The h k o net was in approximately reflecting position on the precession camera when the faceassigned indices (001) were approximately normal to X-ray beam. Fig. 1 shows a schematic drawing of habit I with assigned faces. The end faces of habit I1 did not have the indices (011) but precession photography and optical goniometry showed that (001) and (103) were its two largest faces.

MAHMOUD M. A. HASSAN etal.

4

Fig. 1 : Schematic diagram of crystals of p-Aminosalicylic acid in habit I. Crystal Structure Two different crystal structures have been reported for p-aminosalicylic acid. Structure 11 has been reported before the advent of modern computers (2) while structure I has been developed very recently (1). Table 1 and 2 list the bond lengths and angels and Table 3 atom positions. Intramolecular contacts and angels involving the 0(1)-H(21). . .0(2) hydrogen bond are also included. Data for p-aminosalicylic acid are consistant with the idea that resonance structure (Ib) and (Ic) contribute significantly to its structure.

H”

I

P; I1

AMINOSALICYLIC ACID

5

Table 1 Bond l e n g t h s (A) i n p - a m i n o s a l i c y l i c a c i d ( l ) , w i t h s t a n d a r d d e v i a t i o n s i n p a r e n t h e s e s . I n t r a m o l e c u l a r cont a c t s i n v o l v i n g t h e 0 ( 1 ) - H ( 2 1 ) . . 0 ( 2 ) hydrogen bond a r e included.

.

O ( 1 ) -C(2> 0 (2) -c (7) 0 (3) -C (7) O(2). . 0 ( 1 ) N (1 1-c (4 1 0(1)-H(21) O ( 3 ) -H(71) 0 ( 2 ) . .H(21) N-Ff (4 1) N-H(42)

.

.

1 . 3 6 1 (2) 1.243 (2) 1.311(2) 2.620 (2) 1 .364 (2) 0.98 (3) 0.95(3) 1.73(3) 0.91(3) 0 . 8 3 (3)

1.414(2) 1.400 (3) 1.447 (2) 1 .3 7 1 ( 2 ) 1.392 (3) 1 .4 0 6 ( 3 ) 1.362 ( 2 ) 0 .9 8 (2) 0.98(2) 0.94 (2)

Table 2 Bond a n g l e s (") i n p - a m i n o s a l i c y l i c a c i d ( 1 ) , w i t h e s t i m a t e d s t a n d a r d d e v i a t i o n s i n p a r e n t h e s e s . Angles i n v o l v i n g t h e O(1)-H(21). . 0 ( 2 ) hydrogen bond a r e i n c l u d e d .

.

O(2) -C(7)-0(3) 0 (2) -C (7) -C (1) 0 (3) -C(7) - C ( l ) C (7) - C ( l ) -C(2) C(7) -C(1) -C(6) C (2) - C ( l ) -C (6) C (1) -C(2) -0 (1) C(1) -C(2) -C(3) H(71) -0 (3) -C(7) H(21)-0(1)-C(2) O(2). .H(21)-O(1) C ( 7 ) - 0 ( 2 ) . . .H(21) H(3) -C (3) -C (2) H(3)-C(3)-C(4) H(41) -N(l) -H(42)

.

121.1(1) 123 (2) 115.8 (2) 1 2 0 . 8 (2) 121.7(2) 117.4 ( 1 ) 1 2 1 . 3 (1) 120.6 (2) 113 ( 2 ) 107(2) 147(3) lOO(1) 118 (1) 121 (1)

0 (1)-C (2) -C (3)

C(3) -C(4) -C(5) C (3) -C (4) -N(1) C ( 5 ) -C (4) -N( 1 ) C(6)-C(S)-C(4) C(1) -C(6) -C(5)

118.2 (2) 1 2 1 . 1 (2) 1 1 8 .7 ( 1 ) 120.7 (2) 120.6 (2) 1 2 0 .1 ( 2 ) 122.0 (2)

H(4l)-N(l)-C(4) H(42)-N(1) -C(4) H(5) -C(5) -C(4) H(S)-C(S)-C(6) H (6) -C (6) -C (1) H (6) -C (6) -C(5)

120(2) 115 (2) 119 (1) 121(1) 119 (1) 119 (1)

c ( 2 ) -c (3) -c (4)

MAHMOUD M. A. HASSAN e t a [ .

6

Table 3

4 3 F i n a l atomic p o s i t i o n s (x10 ; f o r H x 10 ) f o r p-aminos a l i c y l i c acid ( I ) , with standard deviations i n parentheses. X

6 5 7 13 8 8 10 11 11 10 7 1 1

1 1

882(2) 572(2) 438(2) 290(3) 718(2) 539(2) 041(3) 784(2) 966(3) 474(2) 136(2) 601 (4) 637 (4) 316(4) 427(4) 958 (3) 319(3) 064(2)

Y 3 1 1 7 3 4 5 6

539(4) 178(4) 345(4) 453(5) 353(5) 138(5) 531(5) 175(5) 5 457(5) 4 OS8(5) 1 SSO(5) 241 (9) 37(8) 789 (8) 782(8) 602 (6) 595 (6) 354 (5)

z 1 641.0(5) 65 1 . 0 ( 5 ) 58.2 ( 5 )

2 111.6(8) 946.6 (6) 1 483.1(6) 1 860.5(7) 1 728.6(7) 1 193.8(7) 819.9 (7) 547.1(6) 133 (1) -19(1) 246 (1) 200 (1) 223 (1) llO(1) 47(1)

2 . 1 2 Melting Range The m e l t i n g p o i n t o f 4 - a m i n o s a l i c y l i c a c i d i s u n c e r t a i n (3) : 135'-140° w i t h decomposition ( 4 ) , 148' (dec.) ( 5 ) , 149-151°(dec.) ( 6 ) . 150-151' with e f f e r v e s c e n c e ( 7 , 8 ) , 1 3 9 - 1 4 l 0 ( d e c . ) (9) and 220" (dec.) (10,ll) have been r e p o r t e d . Seaman e t a 1 (3) have concluded t h a t t h e most n e a r l y c o r r e c t melti n g p o i n t i s about 240' and t h e m e l t i n g p o i n t i s n o t a good c r i t e r i o n o f p u r i t y . 2.2 Solubility 1 g i n about 600 m l of water and about 2 1 m l o f a l c o h o l ; s l i g h t l y soluble i n ether; practically insoluble i n benzene. S o l u b i l i t y i s i n c r e a s e d with a l k a l i n e s a l t s o f a l k a l i metals (NaHC03) and i n weak n i t r i c a c i d , t h e amine s a l t s of h y d r o c h l o r i c and s u l p h u r i c a c i d s a r e i n s o l u b l e . The aqueous s o l u t i o n s have a pH o f about 3.2 and when h e a t e d t h e a c i d decomposes ( 1 2 ) .

AMINOSALICYLIC ACID

7

2.3 Identification

1. p-Aminosalicylic acid gives an intense orange-brown color when reacted with potassium ferricyanide in alkaline solution (13). 2. It gives a green color which changes first to orange and then to orange-red on reaction with hexamine and sulphuric acid at room temperature (14). 2.4 Spectral Properties 2.4 1 Infrared Spectrum The infrared spectrum of 4-aminosalicylic acid is recorded as a nujol mull on Unicam SP 1025 Spectrophotometer and is shown in Fig. 2. The assignments €or the characteristic bands in the infrared spectrum listed in Table 4. Table 4 Frequency cm

-1

3520

Assignment NH2

3400

NH2;,OH

1630

bonded

C = 0

890

isolated C-H out of plane deformation.

820 800 770

C-H out of plane deformation.

Other characteristic finger print bands are: 1305, 1230, 1200, 1170, 1110, 970, 725 and 690 cm-'. Other values for PAS in potassium bromide disc (15) are, 3571, 3448, 3030, 1667, 1613, 1515, 1449, 1299, 1220, 1190, 1163, 813 and 775. 2.4 2 Ultraviolet SDectrum IUVI

UV spectrum of PAS in ethanol was scanned using

Cary, 219 spectrophotometer ; from 400 to 200 nm(16), three maxima and two minima were observed. The maxima are located at 235, 274 and 303 nm.

1 ,

AMINOSALICYLIC ACID

9

The minima occur at 252 and 289 nm. The spectrum is shown in Fig. 3. The UV spectral data of PAS have also been reported earlier (17). 2.4 3 Nuclear Magnetic Resonance Spectrum (NMR) PMR The proton NMR spectra of PAS in DMSO-d6 and in acetone-d6 are shown in Fig. 4 and 5 . These were recorded on Varian T-60A, 60 MHz NMR Spectrometer, using tetramethylsilane as internal reference (18). The PMR spectral data of PAS are given in Table 5. Table 5 : PMR Chemical Shifts of PAS

Chemical shifts (6)

~

DMSO-d6

8.07

Acetone-d6

-

8.07

-

6.08

6.08

6.13 7.50

6.10

6.10

6.20 7.56

(s) = singlet, (d) = doublet. Long range coupling between the C(~)-B and C(5)-H is observed in the 200 MHz spectrum irl.DMSO-d6 (Fig. 6) (18). 13C NMR Hassan and Uppal Zubair (19) have investigated the NMR spectrum of PAS, and determined its carbon shifts. The spectrum (Fig. 7) shows seven singlets. The carbon chemical shifts of are as PAS in hexadeuterodimethylsu.1foxide follows : CO : 172.17, C(l) : 100.46, C(2) : 163.56, C(3) : 98.81, C(4) : 155.73, C(5) : 106.34, C(6) : 131.56. The off-resonance decoupled spectrum F i g . 8 and 9 shows four singlets representing COY C(1), C(2)

F i g . 3 : UV Spectrum o f p-Aminosalicylic a c i d i n E t h a n o l .

10

-0

0

-

-0

m

.a 0

8

B

11

s

OD

F i g . 4 : PMR Spectrum of p - h i n o s a l i c y l i c a c i d i n DMSO-d a n d 6 TMS .

12

Fig. 5 : PMR Spectrum of p-Aminosalicylic acid in Acetone-d

6

and TMS.

t

,

m

,

,

r

l

,

l

5.44

I

,

5.20

1

I

I

I

I

I

i

9

8

7

6

5

4

3

I

2

F i g . 6 : 200 Mttz PMR S p e c t r u m of p-Aminosalicylic acid in L)i.!SO-d

I

f

6.

14 m

U

a

4

U

0

s Fig. 7 : 13C

NMR

Spectrum of p-Aminosalicylic acid in DMSO-d6.

15 3

3 N

B

2

is

5

2

>

3

D

s

8

9

0

F i g . 8 : I3C NMR o f f Resonance Decoupled Spectrum o f p - h i n o s a l i c y l i c a c i d i n DMSO-d6.

b 0

c,

V

E 3 k w

c

e,

cn a 4

.I+

u a

c c rdrd c c o x

o c

( c 1 0

I7

AMINOSALICYLIC ACID

and C(4) and three doublets representing C(3), C(5) and C(6). The carbon chemical shifts are as follows:

CO : 172.08, C(1)

: 100.49, C(2) : 163.59, C3 : 99.29, and 98.33, C(4) : 155.77, C(5) : 106.84 and 105.85, C(6) : 132.12 and 131.03.

2.4 4 Mass SDectrum The mass spectrum of PAS obtained by conventional electron impact ionisation shows a molecular in M+ at m/e 153. The base peak is at m/e 135. The MC ion peak has about 62.1% relative intensity (Fig.10). The m/e f o r the most prominent fragments are listed in Table 6. Tateniatsu et al, have also reported the mass spectrometry of mixed drugs including 4-aminosalicylic acid (20). Table 6.

m/e 52 79 107 135 136 153

Relative Intensitv 14.3 14.3 24.6 100.0 15.1 62.1

3. Svnthesis Several synthetic routes to 4-aminosalicylic acid have been reported (21-30). Two of these are illustrated below. Route I : Modified Kolbe-Schmidt Reaction of 4-aminosalicylic acid have been obtained by heating dry finely divided m-aminophenol and potassium carbonate under anhydrous conditions at 150-190' in C02 atmosphere (27). The yield is 90%. Route 11: This route describes the synthesis of C14-carboxyl-labelled 4-aminosalicylic acid by Sandmeyer Reaction

AMINOSALICYLIC ACID

19

using potassium radio-cyanide to synthesise p-nitrosalicylic acid which was then reduced by Catalytic hydrogenation at room temperature. The yiels is 62% (29). COOH

I

I

NH2

NH2

Route I

N02

*

*

COOH

FOOH

NH2

Route I1 4. Metabolism The metabolism of PAS has been studied in both rabbits and humans. Bray et a1 (31)have studied in great detail the metabolism of PAS in the rabbit and found that approximately 50% of a dose of 1-2 gms is excreted unchanged and 50% as 4-acetamido-salicylic acid (m.p. 238-23g0), which has been isolated and characterised. Also they have isolated 4-acetamido-salicylic acid from human urine after oral administration of 3 gms sodium 4-aminosalicylate. Considerable amounts were excreted unchanged. This has also been proved by others (32). Zini (33) has studied the fate of 4-aminosalicylic acid in humans, the

MAHMOUD M . A. HASSAN et al.

20

+;H

Q”

H COOH

H

+ NH2CH2 COOH

_____3

Glycine

NH2

NH2

i

Salicyluric acid

Acetyl at ion

@ +HoQ \ NHCOCH3

00 co -

COOH

--+ OH

H

\

OH Glucuronic acid

OH

NHCOCH Estere?ucuronide COOH

OH

NHCOCH~ Ether-glucuronide Scheme I

+

AMINOSALICYLIC ACID

21

urinary metabolites of PAS were acetylated-PAS, unchanged PAS, glycine-PAS and glucuronic acid-PAS conjugated compounds. Way et a1 (34), have reported the quantitative determination of the various metabolites of PAS excreted in the urine of human subjects by using countercurrent distribution and paper chromatography. They found that of the total dosage o f PAS, 14 to 33% was excreted unchanged, 28 to 63% as acetyl-PAS, 0 to 26% as p-aminosalicyluric acid, 2 to 10% as unknown free amines and 3 to 10% as unknown bound amine. Lehman (35) have reported the occurrance of N-acetyl-PAS and N-(4-aminosalicyloyl) glycine in human blood plasma and urine after oral administration of PAS. He concluded that concomitant administration of high dosage of PAS with isoniazid probably depletes CO-A and thereby inhibits the acetylation of isonizid. Wan et al., (36), have reported that the metabolism of PAS is mainly by acetylation which accounts for 50 to 70% of the absorbed dose and glycine conjugation to p-aminosalicyluric acid accounts for up to 25% of the dose. These two metabolites together constitute greater than 90% of the metabolites found in urine (37, 38). Metabolites of PAS are shown in scheme I. 5. Methods of Analysis 5.1 Non-aqueous titration

Kucharsky et a1 (39) and Chatten (40) have described a non-aqueous titration technique for the determination of PAS and Sodium PAS, both in pure form and in tablet formulation. Determination of pure PAS is based on titration of anhydrous acetone solution of the acid with 0.1N potassium hydroxide solution in anhydrous methanol using 0.5% of thymol blue solution in anhydrous methanol until the color of the indicator changes to blue. For tablet formulation the above determination is preceded by extraction of the specified amount of the tablet powder with anhydrous acetone. For the determination o f Sodium PAS the method is based on dissolving the specified amount of the substance on anhydrous methanol and titrated with 0.05N perchlo-. ric acid solution in dioxane using 0.5% thymol blue solution as indicator, until its color changes to peach. For the same in tablets the above determination is preceded by extraction of the specified amount of the powdered tablets with anhydrous methanol. These methods were reported to be specific even in the

MAHMOUD M. A. HASSAN e t a / .

22

presence of m-aminophenol (MAP). Butter and Ramsay (41) titrated PAS and its sodium salt potentiometrically with perchloric acid in glacial acetic acid and acetic acid. Carbon tetrachloride solvent mixture served as the titration medium. Stockton and Zuckerman (42) determined sodium PAS and its solutions by potentiometric titration with perchloric acid in propylene glycol and isopropyl alcohol (l:l), using the same solvent mixture as the titration medium. The decomposition products MAP and sodium bicarbonate did not interfere. Das and Pate1 (43) employed the same titrant and solvent system. Hunt and Blake (44) have described a non-aqueous titration method for the analysis of PAS and its salts and dosage forms. This method was reported to be specific in the presence of MAP. The method is based on titration with sodium methoxide in benzene-methanol using dimethylformamide as titration solvent. The end point is detected visually using thymol blue as indicator o r potentiometrically. PAS and its decomposition product, m-aminophenol may be differentiated with this titration system. Salts of p-aminosalicylic acid are converted to the acid form by ion-exchanged chromatography prior to titration. 5.2 Diazometric Assay USP XVIII method (45)for the determination of PAS, its salts and dosage forms, involves the diazotisation reaction and is based on procedures developed by Tarnoky and Bews (46) and Pesez (47,48). Blake et a1 (49) have described a method for determination of sodium p-aminosalicylate in the presence of m-aminophenol. m-Aminophenol, the major breakdown produced p-aminosalicylic acid, if present, is also diazotised and constitutes an interference in the official assay procedure. In this method the PAS content and mixtures containing MAP is determined by the modification of the official assay procedure. The MAP is removed by passing the solution of the mixture in dimethylformamide through a column containing a strong cation exchange resin. The elute is then treated according to the official method.

AMINOSALICYLIC ACID

23

5 . 3 Spectrophotometry Coccia (50) has d e s c r i b e d d e t e r m i n a t i o n o f PAS, m-aminophenol and p-aminophenol c o l o r i m e t r i c a l l y by u t i l i s i n g t h e i r r e a c t i o n w i t h sodium n i t r o p e n t a c y a n o c o b a l t a t e t o g i v e an orange compound. The c o l o r p r o duced obeys B e e r ' s law a t 440 nm i n t h e range of 0 t o 0.75 1.18 o f PAS p e r m l . The compound o b t a i n e d w i t h PAS was p r e p a r e d and i t s formula and molecular weight were obtained. Rieder (51) has r e p o r t e d a n o t h e r c o l o r i m e t r i c method f o r d e t e r m i n a t i o n o f f r e e PAS i n blood. The method i s based on t h e coupling o f PAS w i t h 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 i n a s t r o n g l y a l k a l i n e a l c o h o l i c medium The r e s u l t i n g s o l u t i o n shows maximum e x t i n c t i o n a t 600 nm, b u t a n a l y s i s were c a r r i e d out a t 630 nm i n o r d e r t o avoid i n t e r f e r e n c e . The c o l o r i s s t a b l e f o r 30 minutes and t h e maximum e r r o r i s f 5% i n t h e range of 5 t o 20mg of PAS p e r 100 m l . Another c o l o r i m e t r i c method has a l s o been r e p o r t e d (52) u t i l i s i n g r e a c t i o n o f PAS and MAP with n i n h y d r i n solution. 5.4 Combined TLC and Colorimetry Kinze (53) has r e p o r t e d t h e s e p a r a t i o n o f PAS and MAP on l a y e r s o f Alumina oxide by u s i n g e t h a n o l o r methan o l a s a d e v e l o p e r . PAS remains on t h e base l i n e i n b o t h i n s t a n c e s . The s p o t s a r e d e t e c t e d by s p r a y i n g with 1%p-dimethylaminobenzaldehyde s o l u t i o n i n ethanol t r e a t e d w i t h 5% h y d r o c h l o r i c a c i d . After e x t r a c t i o n from t h e p l a t e 2-60 mg of MAP can be determined c o l o r i m e t r i c a l l y a t 420 nm with 1%f u r f u r y l a l c o h o l s o l u t i o n i n anhydrous a c e t i c a c i d . 5 . 5 U l t r a v i o l e t method Moussa (54) h a s r e p o r t e d a U . V . method f o r determinat i o n o f PAS i n t h e p r e s e n c e o f i t s d e g r a d a t i o n product MAP. The f i n e l y powdered t a b l e t s a f t e r e x t r a c t i o n w i t h e t h a n o l i s f i l t e r e d and t h e f i l t r a t e i s d i l u t e d and t r e a t e d w i t h b o r a t e b u f f e r s o l u t i o n o f pH3 and t h e absorbance i s measured a t 300 nm a g a i n s t t h e b u f f e r s o l u t i o n . There i s no i n t e r f e r e n c e from MAP i n amounts u p t o a t l e a s t twice t h a t of PAS.

MAHMOUD M. A. HASSAN et a / .

24

PAS can be analysed spectrophotometrically by dissolving the sample in ethanol (95%) to give a concentration of about 15 Ug/ml and the absorbance of the solution so produced is measured at 303 nm. The log 5 values are given in Table 7 (16). Table 7

X max nm

Log

5

2 35

2.765

274

3.622

303

3.624

AMINOSALICYLIC ACID

25

REFERENCES 1.

Chung-Tang Lin, Pik-Yen Siew and S.R. Byrn, J. Chem.Soc., Perkin 11, 957 (1978).

2.

F. Bertinotti, G. Giacomello and A.M. Liquori, Acta Cryst., - 7, 808 (1945).

3.

W. Seaman, W. Allen, R.L. Pasternak and A. Pollara,

J . Am. Chem. SOC., 71, 2940 (1949).

4.

Remington Pharmaceutical Sciences, Arther Osol et al, XV Edition, Mack Publishing Co., Easton, Pennsylvania, U.S.A. p. 1149 (1975).

5.

Kolb, U.S. Patent 427, 564; German Patent 50, 835.

6.

Erlenmeyer, et al, Helv. Chim. Acta, 31, 988 (1948).

7.

The Merck Index, An Encyclopedia of Chemicals and Drugs, Martha Windholz et al, IX Edition, Merck and Co. Inc., Rahway, N.J., U.S.A., p. 66 (1976).

8.

O'Connor, Lancet, -254, 191 (1948).

9.

Whittel, Lancet, -254, 268 (1948).

Ber.,34, 4351 (1901). 10. Seidel, 11. Seidel and Bittner, Monatsh, 23, 415 (1902). 12. Textbook of Organic, Medicinal and Pharmaceutical Chemistry, C.O. Wilson et al, VII Edition, J.B. Lippincott. Co., Philadelphia, U.S.A. p. 236, 1977. 13. Yu M. Ostroskil, Aptechnoe Delo, 4(6),

10 (1955).

4, No.6, 8, (1955). 14. A.M. Gal'perina, Aptechnoe Delo, 15. I.R. Grating Collection, Sadtler, Research Laboratories, SADG 9560. 16. M.Uppa1 Zubair, M.M.A. Hassan, unpublished results. 17. Spectral Collection data, Sadtler Research Labs.SADG 3162. 18. M.M.A. Hassan and M. Uppal Zubair, unpublished results.

MAHMOUD M. A. HASSAN et al.

26

19.

M.M.A.

20.

A. Tatematsu, T . Nadai, T. Goto, Y . Nakajima, H. 87 ( 4 ) , 329 (1967). Tsuyama and H. Doi, Yakugaku Z a s s h i , -

21.

German P a t e n t 50, 835, F r i e d l a n d e r , 2 , 139 (1887-90).

22.

J . T . Sheehan, J . Am. Chem. SOC. 70, 1665, (1948).

23.

A. Wander, A . G .

24.

D . D . M a r t i n , D . E . Seymour and F.S. S p r i n g , B r i t . P a t . 636, 333, Apr. 26 (1950).

25.

D . D . M a r t i n , D . E . Seymour and F.S. S p r i n g , B r i t . P a t . , 697, 965, Oct 7 (1953).

26.

G . F . Felemons and R . A . Aug 26 (1953).

27.

R . P . P a r k e r and J . M . June 30 (1953).

28.

Brit.,

29.

Hassan and M. Uppal Z u b a i r , unpublished d a t a .

Swiss 265, 516, Dec., 15 (1949).

Wilkinson, B r i t . Pat.696, 132,

Smith. Jr. U.S. P a t . 2 , 644, 011,

P a t . 693, 386, J u l y 1 (1953).

L . C l e r k , A. H e l l e r and L . J .

Roth, J . Am.

Pharm.

Assoc.

4 4 , 328 (1955).

30.

I . H i r a o , Y . Kosugi, T . Matsuura, Y . Hironaka and Y. Gosei, Kagaku Kyokai S h i , 25 ( 5 ) , 417 (1967).

31.

H . G . Bray, B . E . 64 (1948).

32.

A . Venkataraman, P . R . Venkataraman and H . B . J . B i o l . Chem., 173, 641 (1948).

33.

F . Z i n i , Riv. C r i t . C l i n . Med. 5 3 , 308 (1953).

34.

E . L . Way, G . Peng, N . Allawala and T . C . D a n i e l s , J . Am. Pharm. Assoc., 4 4 , 65 (1955).

35.

J . Lehman, Scand. J . Resp. D i s . ,

36.

Ryman and W.V. Thorpe, ~Nature, 162, Lewis,

50 (3), 169 (1969). -

S.H. Wan, P . J . P e n t i k a i n e n and D . L . Azarnoff, J.Pharm. 63, 708 (1974).

Sci.,

AMINOSALICYLIC ACID

27

37.

E . L . Way, P.K. Smith, D . L . Howe, R. Weiss and R . Swanson, J . Pharmacol. Exp. T h e r . , 93, 368 (1948).

38.

J . Kawamata and J . Kashiwagi, Med. J . Osaka Uni., 6 , 119 (1955).

39.

T i t r a t i o n s i n non-aqueous s o l v e n t s , J . Kucharsky and L . S a f a r i c k , E l s e v i e r , New York, N . Y . , p . 182 (1965).

40.

L.G.

41.

A.Q.

B u t l e r and J . C . Ramsay, i b i d , 42, 338 (1953).

42.

J.R.

S t o c k t o n and R . Zuckerman, i b i d , 43, 273 (1954).

43.

M . N . Das and S.R. P a l i t , J . Ind. Chem. S O C . , 31, 34 (1954).

44.

J . Hunt and M.I. Blake,

45.

The Pharmacopeia of t h e United S t a t e s of America, p . 3 6 , 1 8 t h r e v . , Mack Pub.Co.Easton, Pa,USA, p.36 (1970).

46.

A. Tarnoky and B . A .

47.

M. Pesez, B u l l . SOC. Chim. F r . , 30, 918 (1949).

48.

M. Pesez, B u l l . SOC. Chim. B i o l . , 31, 1369 (1949).

49.

M.I. Blake, K . Makris and J . Hunt, J. Pharm. S c i . , 60 ( l l ) , 1695 (1971).

50.

P . A . Cuccia, Anal. Chem., 3 1 ( 8 ) , 1306 (1959).

51.

H . P . R i e d e r , Kiln Wochscher, 39 ( 1 5 ) , (1961).

52.

K . N . Gaind, R . N . Dar and S.C. Bapna, I n d i a n J . Pharm., 26 ( 9 ) , 248 (1964). -

53.

W.

54.

A. Moussa, Pharmzie, 33 ( 7 ) , 460 (1978).

Chattan, (1956) .

J . Am. Pharm. Ass.

,

S c i . Ed.

,45, 556

J . Pharm. S c i . , 5 9 , 683(1970).

Brews, Biochem. J . , 45, 508 ( 1 9 4 9 ) .

Kinze, Pharm. Z e n t r a l h a l l e D t l . ,

105 ( 6 ) , 365 (1966)

AZATHIOPRINE Wendy P . Wilson and Steven A. Benexra 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 Spectra 2.3 Ultraviolet Spectrum 2.4 Mass Spectra 2.5 Melting Point 2.6 Solubility 2.7 Dissociation Constant 3. Synthesis 4 . Stability 5. Metabolism and Pharmacokinetics 5.1 Metabolism 5.2 Excretion 5.3 Tissue Distribution 6. Methods of Analysis 6.1 Elemental Analysis 6.2 Nonaqueous Titration 6.3 Polarography 6.4 Microbiological Assay 6.5 Phosphorescence Spectroscopy 6.6 Fluorimetric Analysis 6.7 Chromatography References

ANALYTICAL PROFILES OF DRUG SUBSTANCES, 10

29

30 30 30 30 30 32 34 37 37 37 39 39 39 39 39 41 42 42 42 43 43 43 44 44 44 51

WENDY P. WILSON AND STEVEN A. BENEZRA

30

1.

Description 1.1

Name, Formula, Molecular Weight

Azathioprine is 6-[(l-methyl-4-nitroimidazol-5-yl)thio]purine

277.3

9H7N702S

1.2 Appearance, Color, Odor Azathioprine is a pale yellow, odorless powder.

2.

Physical Properties 2.1

Infrared Spectrum

The infrared spectrum of azathioprine i s shown in Figure 1. The spectrum was obtained as a 0.4% dispersion of azathioprine in KBr with a Nicolet Model 7199 FT-IR spectrophotometer. The infrared assignments consistent with the structure of azathioprine are given in Table 1 . ' Table I Infrared Spectral Assignments for Azathioprine Band Frequency (Wavenumbers)

921 and 857 831 and 637

Structural Assignment C-H deformation attributable to the purine nucleus. C-H deformation attributable to the imidazole ring.

J

n 3

?! 3

3 3

#. .

D

Ln

N 0

0 Ln I

0 Ln

r I

0

0

0 W

U

NUl

6

>

g :$

0 3

0

Ln N 0

N

r

In 0

0 0

m

0

m

N

In

P7

lP

0 0

0

m

.r

In

0

¶ .

0 '3


32

1233 1470 and 1390 1537 and 1374 1595 and 1570 1893 and 1807

2810 2976 3109 3191

2.2

C-N stretching from a tertiary amine and a purine nucleus. C-H bending from a methyl group. C-NO, stretching (asymmetric and symmetric, respectively). C=N stretching characteristic of the amidine groups in substituted purine and imidazole structures. C-H deformation overtones attributable to the substituted purine and imidazole functions. C-H stretching indicative of a CH3-N group. C-H stretching characteristic of a pyrimidine group. C-H stretching characteristic of imidazole groups. N-H stretching characteristic of a purine function.

Nuclear Magnetic Resonance (NMR) Spectra

The 'H NMR spectrum o f azathioprine is shown in Figure 2 . The spectrum was obtained in deuterated dimethyl sulfoxide with a Varian XL-100A NMR spectrometer at 100 MHz. Chemical shifts referenced to DMSO at 2 . 5 1 ppm and consistent with the structure of azathioprine are presented in Table II.2 Table I1

NMR Assignments for Azathioprine Proton a

No. of Protons 1

Shift (ppm) 8.59. 8.55 8.25 3.70 13.8

Mu1tip licit y singlet singlet quartet doublet broad singlet

14

12

13

I

I '

I

11 1 '

10 I

...

i i

--

...~... ........

!

I

9

I

8

I

7

I

6

1

I

5

4

I

3

PPm

Figure 2 - ' H Nuclear Magnetic Resonance Spectrum of Azathioprine

1

2

I

1

1

0

WENDY P. WILSON AND STEVEN A . BENEZRA

34

C

d

e The 13C NMR of azathioprine, shown in Figure 3 , was obtained with a Varian CFT-20 NMR spectrometer at 80 MHz. Deuterated dimethyl sulfoxide was used as the solvent with tetramethylsilane a s an internal standard. Carbon assignments for the 13C NMR are given in Table III.3 Table I11 Carbon No. 2 4 5 6 8 2' 4' 5'

CH3

Chemical Shift (ppm) 151.6 150.6 130.0 154.6 144.5 139.4 149.7 117.1

32.9

2.3 Ultraviolet (W) Spectrum The ultraviolet spectrum of azathioprine in methanol was obtained with a Beckman ACTA CIII W spectrophotometer and i s shown i n Figure 4 . Table IV gives UV data f o r azathioprine in various solvents.

34

d

C


e

32.9

117.1

Chemical Shift (ppm) 151.6 150.6 130.0 154.6 144.5 139.4 149.7

Table I11

The 13C NMR of azathioprine, shown in Figure 3 , was obtained with a Varian CFT-20 NMR spectrometer at 80 MHz. Deuterated dimethyl sulfoxide was used as the solvent with tetramethylsilane a s an internal standard. Carbon assignments for the 13C NMR are given in Table III.3

Carbon No. 2 4 5 6 8 2' 4' 5'

CH3

2.3 Ultraviolet (W) Spectrum The ultraviolet spectrum of azathioprine in methanol was obtained with a Beckman ACTA CIII W spectrophotometer and i s shown i n Figure 4 . Table IV gives UV data f o r azathioprine in various solvents.

I

7

0

I W

0

9

I

d 0

I

0

I N

0

0 N

-v)

0

E

37

AZATHIOPRINE

Table IV W Spectral Data for Azathioprine

Solvent

’max

methanol 0.1N NaOH 0 . 1 N HC1 2.4

(-1 276 285 280

&

max

1 . 8 2 x 104

1.55 x 104 1.73

x 104

Mass Spectra

The low resolution electron impact’ and field desorption6 mass spectra of azathioprine are shown in Figures 5 and 6 . The electron impact spectrum was obtained with a Varian MAT CH5-DF mass spectrometer. The sample was introduced into the ion source y& direct probe at 285OC. The electron energy was 70 eV. The major fragment ions formed on electron impact are consistent with those found by Brent _ et -a L 7 Loss of NO2 yields CSH7N6S, m/z 231 (100%). Cleavage between sulfur and the purine ring with retention of charge on the purine ring results in (Pur)+, m/z 119 ( 4 2 % ) . The fragment m/z 152 (10%) is formed by fission of the sulfur imidazole bond with fearrangement of a hydrogen to the purine moiety (PurS + H)., and m/z 42 (45%) is C2H4N. The field desorption spectrum was obtained with a Varian MAT 731 mass spectromFter at an emitter heating current of 18 ma. The (M+1) ion (m/z 2 7 8 ) , while absent from the electron impact spectrum, appears in the field desorption spectrum ( 4 . 6 % ) . Other fragments prTsent in the field desorption spectrum are m/z 231 (loo%), M.-NO2 and m/z 2 7 7 . 2.5

Melting Point Azathioprine melts and decomposes at approximate-

ly 240°C.8 2.6

Solubility

Azathioprine is very slightly soluble in water It is also slightly soluble in (-0.01% w/v at 25°).8 chloroform, ethanol and dilute mineral acids. Azathioprine i s soluble in dilute solutions of alkali hydroxides with slow decomposition, dimethyl sulfoxide and poly-


38

1

0

0

1

J

80 -

>

cn 6 0 Z u

5

40-

H

140 180 m/z

220

I

I

260

200

Figure 5 - Electron Impact Mass Spectrum of Azathioprine

m/z

Figure 6 - Field Desorption Mass Spectrum of Azathioprine

AZATHIOPRINE

ethylene glycol 400. 2.7

39

'

Dissociation Constant The pKa2 of azathioprine is 8.2 at 25OC.'

3.

Synthesis

Azathioprine is synthesized by the synthetic route shown in Figure 7 . Diethylsuccinate ( 1 ) is reacted with methylamine (2) to give N,"-dimethylsuccinamide (3) which in turn is reacted with-PCl,/POCl, to ring close to 1-methyl-5-chloroimidazole (4). The imidazole, 4, is converted to its salt with nitric acid to give l-methyl5-chloroimidazole nitrate (5). The imidazole nitrate, 5 , is then converted to l-methyl-4-nitro-5-chloroimidazole (6). Ethyl cyanoacetate (7) is nitrosated, reduced and acetylated to give e t h y l a c e t a m i d o c y a n o a c e t a t e (8). The ring closure of 8 is done with formamide to give hypoxanthine ( 9 ) which is reacted with phosphorous pentasulfide to give 6-mercaptopurine ( 1 0 ) . The imidazole (6) and 6-mercaptopurine (10) are condensed to yield azathioprine. lo 4.

Stabilitv

Bulk samples of azathioprine are stable for at least two years at temperatures between 5OC and 37OC and one year at 5OoC when stored in well closed, light resistant containers.'''I2 The drug develops a dark orange surface after four weeks when exposed to fluorescent or ultraviolet light." Azathioprine is stable in neutral and acidic solutions but is hydrolyzed to 6-mercaptopurine by alkali.13'14 5.

Metabolism and Pharmacokinetics 5 . 1 Metabolism

Azathioprine is initially split by glutathione in the liver to 6-mercaptopurine and l-methyl-4-nitro-5(5-glutathiony1)imidazole. To a much lesser extent azathioprine may be split between the purine ring and the sulfur to yield the metabolite l-methyl-4-nitro-5-thio-

m I V

$1

I

2

A

m

I N

V z

t

I

N

--

N

0

z

i

r-

I

AZATHIOPRINE

imidazole. l5 The metabolism o f the 6-mercaptopurine moiety follows two known pathways. It can be inactivated by xanthine oxidase to 6-thiouric acid or it can be converted to its active form, the ribonucleotide 6-thioinosinic acid, by hypoxanthine-guanine phosphoribosyl transferase in tissues. 16’ l7 ’ l8 The major urinary metabolite of the l-methyl-4nitro-5-(S-glutathionyl)imidazole moiety in man and in dogs is N,N’- [5-(methyl-4-nitro)imidazolyl]cysteine. The major metabolite in the rat, 1-methyl-4-nitro-5-(N-acetylS-cysteinyl)imidazole, accounted for only a small-percentage of the dose in dogs and in man. Other metabolites o f the methylnitroimidazole moiety include several 5-substituted amino-1-methyl-4-nitroimidazoles one of which, a glycine derivative, indicates that 6-mercaptopurine may also be displaced from azathioprine by nucleophilic attack o f amino acids. 19’20’21 5.2 Excretion In a human study using 35S-azathioprine to follow the fate o f the purine moiety, over 50% of the radioactive dose was excreted in the urine in twenty-four hours indicating a good absorption o f the drug. Seventy percent of the 35S had been excreted in forty-eight hours. Twelve percent unabsorbed material was excreted in the forty-eight hour stool specimens. Very little of the drug was eliminated unchanged. The major urinary metabolite was thiouric acid with less than 1%of the dose eliminated as 6-mercaptopurine and from 10% to 20% inorganic ~ u 1 f a t e . l ~ Similar ’~~ studies done in rats and in dogs gave similar results with the exceptions of relatively larger quantities of 6-mercaptopurine being excreted by rats and both rats and dogs excreted slightly more unchanged azathioprine.23’24 Clearance of the methylnitroimidazole portion of the drug is much slower than that of the purine moiety. Following an oral dose of 90 mg o f 14C-azathioprine the patient excreted only 20% of the 14C in the first twentyfour hours. In forty-eight hours only 37% of the 14C had been excreted in the urine in contrast to the 70% excretion of 35S in forty-eight h o u r ~ . ~ Similar ” ~ ~ results were obtained in the rat and dog studies with 14C-azathio~ r i n e . ~ ” ~ Forty-two ’ percent of the 14C had been excret-

41


42

ed by the dogs in 32 hours with very little radioactivity excreted after 32 hours. l 9 5.3

Tissue Distribution

The peak plasma radioactivity of the purine portion of azathioprine occurred at 2 hours in a patient treated with 35S-azathioprine. The half-life of the plasma radioactivity was 4 . 5 to 5 hours and after 10 hours, when most of the remaining 35S was inorganic sulfate, the clearance of radioactivity was much slower.15 Another patient was treated with 14C-azathioprine. Plasma radioactivity of the methylnitroimidazole moiety peaked at 4 hours at which time the plasma radioactivity was twice that found in the blood cells. After 12 hours the radioactivity had equilibrated between the plasma and the cells. At twelve hours the level o f radioactivity was 40% of the peak value and this level persisted for 36 hours.15 The concentration of 35S was determined in several organs of rats treated with 35S-azathioprine. The highest concentration o f 35S was found in the liver 6 hours after administration of the drug. This concentration was five times that found in the blood plasma. Only traces of radioactivity were found in the fat-rich organs.23 Another rat study showed that there is rapid hepatic extraction of azathioprine. After only 5 minutes a high proportion of the radioactive dose was recovered in the liver.25 Radioactivity levels rapidly attained a maximum in the blood cells and then declined rapidly in dogs treated with 14C-azathioprine. The peak plasma radioactivity was reached about 5 hours after drug administration and after 8 hours the radioactivity had equilibrated between the plasma and the blood cells. The radioactivity then declined gradually over 48 hours. l 9 6.

Methods of Analysis

6.1 Elemental Analysis The elemental analysis of azathioprine is given in Table V . 2 6

AZATHIOPRINE

43

Table V Elemental Analvsis of Azathiofirine Element 0

L

H N 0

S

5.2

Theory (%> 38.98 2.55 3.5 .36 11.54 11.57

Nonaaueous Titration

An accurately weighed sample of azathioprine is dissolved in dimethylformamide. The solution is titrated with standardized 0 . 1 N tetrabutylammonium hydroxide to the thymol blue endpoint. Precautions must be taken to prevent the absorption of atmospheric carbon dioxide. Each milliliter o f 0.1 N tetrabutylammonium hydroxide is equivalent to 27.73 mg of azathioprine.' 6.3

Polarography

A differential pulse polarographic analysis is used to assay azathioprine tablets and azathioprine sodium for injection. The samples are dissolved, diluted with 0.1 N sulfuric acid and de-aerated with nitrogen. Using a dropping mercury electrode with a saturated calomel reference electrode, the polarogram is recorded from -0.60 volt to -1.00 volt. The height of the diffusion current is compared to that of a reference standard prepared in a similar manner to obtain the concentration of azathioprine in the formulations. 6.4

Microbiological Assay

Harber and Maddocks described a method o f estimating nanogram quantities of azathioprine by measuring the extent of growth inhibition o f Lactobacillus casei. A modified folic acid assay medium containing between 20 and 200 ng azathioprine was inoculated with 2 drops o f a stock solution of Lactobacillus casei. The cultures were incubated at 37OC for 18 hours and turbidity was then measured at 560 nm. A range of standards were similarly prepared and a standard curve was drawn from which the concentration of azathioprine was read.27


44

6.5 Phosphorescence Spectroscopy Azathioprine has been analyzed phosphorimetrically at -196OC. In alkaline ethanol, with excitation and phosphorescence wavelengths of 311 nm and 451 nm, respectively, azathioprine had a detection limit of 2 . 6 vg/ml and the concentration to phosphorescence relationship was linear over at least two orders of magnitude of concentration. Phosphorescence in neutral ethanol was observed at 442 nm with an excitation wavelength of 300 nm. The detection limit of azathioprine under these conditions was 10 pg/m1.28 6.6 Fluorimetric Analysis Azathioprine and its metabolite 6-mercaptopurine have been successfully quantitated in plasma using a fluorimetric assay. The 6-mercaptopurine was first derivatized with phenyl mercuric acetate. This derivative could then be extracted from the plasma with toluene. The derivative was convert.ed back t o 6-mercaptopurine with 0.1 N hydrochloric acid and the toluene was removed. The 6-mercaptopurine was then oxidized to purine 6-sulfonate with potassium chromate followed by sodium metabisulfate and sodium hydroxide solutions. The fluorescence of the solution was measured at 398 nm with an excitation wavelength of 288 nm. Azathioprine was hydrolyzed to 6-mercaptopurine with 5 N sodium hydroxide. After neutralization with 5 hydrochioric acid the derivatization, extraction, oxidation and fluorimetric analysis steps were followed as for the 6-mercaptopurine. The concentration of azathioprine was calculated from the difference in the 6-mercaptopurine concentration in the hydrolyzed and non-hydrolyzed samples.2 9 6.7

Chromatography 6.71 Column Chromatography

Nelson and coworkers have separated several azathioprine metabolites by column chromatography on DEAESephadex columns. The metabolites were eluted with pH 4.7 triethylammonium acetate buffer. 10 mM f3-mercaptoethanol was added to the mobile phase to prevent oxidation of the thiopurines. Azathioprine was converted to 6-mercaptopurine on the column under these conditions and could not

45

AZATHIOPRINE

be separated. Detection was W at 254 Measurement of azathioprine, 6-mercaptopurine and 6-thiouric acid in urine was achieved on the cation exchange resin Zeo Karb 225. 6-Thiouric acid was eluted first with 30-40 ml water which was then evaported to dryness. 6-Mercaptopurine was eluted next with 15 ml of 20% ammonium hydroxide and then evaporated to dryness. Azathioprine was converted to 6-mercaptopurine by the addition of glutathione to the pH 8.9 adjusted urine, which was then chromatographed as described above. The eluates were dissolved in 5% perchloric acid and concentrations were determined by the decrease in extinction measured after the addition of mercuric chloride. 6-Thiouric acid was measured at 345 nm and 6-mercaptopurine was measured at 330 run. The concentration of azathioprine was determined by the difference in 6-mercaptopurine concentration before and after the addition of glutathione.3 1 Azathioprine has been separated from other purines on Sephadex G-10. The mo.bile phase was 0.05M, pH 7 phosphate buffer. The recovery of the chromatographed purines was quantitative. 3 2 6.72 High Performance Liquid Chromatography (HPLC) Table VI gives various HPLC systems used for azathioprine and its metabolites. 6.73 Paper Chromatography Azathioprine and several methylnitroimidazole metabolites have been separated on Whatman no. 3 filter paper. The two solvent systems used were p-butano1:acetic acid:water ( 4 : 1 : 5 ) , the top layer was used, and p-propanol: water (7:3). The chromatograms were developed for 20 hours and the compounds were detected under ultraviolet light. The R values for azathioprine were 0.75 in the p-butanol f system and 0.86 in the 2-propanol 6.74 Thin Layer Chromatography (TLC) TLC systems used for azathioprine and its metabolites are given in Table VII. Ito and Fujita describe the use of 3,5-di-tertbutyl-1,2-benzoquinone-iron (111) chloride as a TLC spray

reagent for the detection of thiols.

Fifty nanomoles of

Table VI HPLC Svstems for Azathiourine Column

Mobile Phase

Flow (ml/min)

Retention Time (min)

Detection

Ref

PA-38 pellicular anion-exchange resin (3m x lmm i.d.)

gradient from 0 . 0 3 M to 4.0 M ammonium acetate pH 4.7

0.4

AZA- 18 6-MP- 4 6-TU- 78

254 nm or 350 nm

30

PBondapak C 1 8 (30cm x 3.9mm i.d.)

11% Acetonitrile in 0.01 M sodium acetate buffer pH 4.0

2

AZA- 8

280 nm

33

5~ ODs-Hypersil (10cm x 5mm i.d.)

Methanol:25 mM potassium dihydrogen phosphate : glacial acetic acid (20:79.5 :O.5) pH 4.50

1.5

AZA- 4

240 nm

34

PBondapack C18 (30cm x 4mm i.d.)

Water : Methanol (70: 30)

2

AZA- 3 . 2

280 nm

35

VBondapak Cl,

Acetonitri1e:water: glacial acetic acid (15:85:0.02)

AZA- 11

280 nm

36

6-MP- 2 MNTI- 2.5 "HI- 1

Table VI continued Column

Mobile Phase

Flow (ml/min)

Aminex-27 (100cm x 1.24mm i.d.)

gradient from 0.015 M to 6.0 M sodium acetate pH 4.0

0.13

PA-38

0.02 M ammonium formate pH 4.9

0.4

pellicular anion exchange resin (3m x lmm id)

AZA 6-MP MNHI MNT I

N,N'-MNIC 6 -TU

Retention Time (min)

l-methyl-4-nitro-5-thioimidazole _N,N'-[5-(methyl-4-nitro)imidazolyl)cysteine _ 6-thiouric acid

Ref

AZA- 300

254 run or 280 nm

19

AZA- 15 6-MP- 7

280 nm

20

N,N'-MNIC- 925 MNTI- 1020

azathioprine 6-mercaptopurine l-methyl-4-nitro-5-hydroxyimidazole

Detection

Table VII TLC Systems for Azathioprine Adsorbent Silica gel 60 F 254

b

Cellulose

Mobile Phase aceti (1:9)

acid:ethanol

-

6-MP- 0.59 6-TU- 0.30

ammon a :butanol : water (1 :60:39)

6-MP- 0.63

heptane :chlorofo rm : ethanol (1:l:l)

6-MP- 0.58 6-TU- 0.04

0.1 M hydrochloric acid

AZA- 0.66 6-MP- 0.44

6-TU- 0.37

Detection and Comments

Ref -

Azathioprine metabolites were converted to phenyl mercury derivatives before chromatograpy. Following conversion back to the parent thiols by spraying with 2 N HC1, compounds were detected by low temperature (-196OC) phosphorescence at 254 nm and 366 nm. Detection of mercury can also be achieved by spraying chromatogram with 0.1 N acetic acid followed by a dithiozone solution.

39

Low temperature (-196OC) phosphorescence detection was used with excitation and phosphorescence wavelengths of 342 nm and 485 nm respectively and 320 nm and 448 nm respectively.

28

Table V I I continued Mobile Phase

Adso rb ent Cellulose

ECTEOLAcellulose

AZA 6- MP 6- TU

0.1

N hydrochloric acid

Rf AZA- 0 . 7 0 6-MP- 0.43 6-TU- 0.24

water

AZA- 0.70 6-MP- 0.26 6-TU- 0 . 7 5

isopropano1:methanol: water:ammonia (60:20:20:1)

AZA- 0.87 6-TU- 0.25 6-MP- 0 . 5 5

acet0ne:O.l M sulfuric acid:ethyl acetate

AZA- -0.8 6-MP- 0.45

acetone:water (20:80)

AZA- 0.47 6-MP- 0.36

(45 : 10 :45)

azathioprine 6-mercaptopurine 6-thiouric acid

Detection and Comments

Ref ~

low temperature (-196OC) luminescence detection at 366 nm

37

Viewed under an W lamp. 6-Mercaptopurine fluoresced at 254 nm and 366 nm. Azathioprine quenched fluorescence at the same wavelengths.

38


50

6-mercaptopurine was detected on a cellulose TLC plate with this reagent.4 0 6.75

Thin Layer Electrophoresis

The separation of azathioprine from other thiopurine derivatives has been achieved on both silica gel and ECTEOLA-cellulose thin layer chromatography plates with the use of low-voltage thin layer electrophoresis. A 0.7% triethanolamine buffer adjusted to pH 9.5 with acetic acid was used with the silica gel plates and a 5% pyridine buffer adjusted to pH 6.0 with acetic acid was used with the ECTEOLA-cellulose. The electrophoresis w a s carried out at 300 V at 4OC for 3 hours for the silica gel plates and 3.5 hours for the ECTEOLA-cellulose plates. After electrophoresis the plates were air dried then dipped into an ammonia fume chamber for 30 seconds. Low temperature (-196OC) phosphorescence detection was performed at 254 nm and 366

AZATHIOPRINE

51

References 1.

H. Powell, Burroughs Wellcome Co., personal communication, 1980.

2. A. Ragouzeos, Burroughs Wellcome Co., personal communication, 1980. 3.

B.S. Hurlbert, R . Crouch, Burroughs Wellcome Co., personal communication, 1981.

4.

W.P. Wilson, Burroughs Wellcome Go., unpublished data, 1981.

5. R. Johnson, Burroughs Wellcome Co., personal communication, 1980.

6. B. Soltman, Burroughs Wellcome Co., personal communication, 1980. 7.

D.A. Brent, P . de Miranda, H.R. Schulten, J. Pharm. Sci., 6 3 , 1370 ( 1 9 7 4 ) .

8.

G.R. Griffith, Wellcome Foundation Ltd., personal communication, 1980.

9.

U.S.P.

10.

XX, Mack Printing Co., 1 9 7 9 .

H.N. Yeowell, G.B. Elion, J. Heterocyc. Chem.,

lo ,

1017 ( 1 9 7 3 ) . 11.

R.C. Thompson, R.I. Poust, Burroughs Wellcome Co., personal communication, 1 9 7 9 .

12.

R.C. Thompson, S. Cliett, Burroughs Wellcome Go., unpublished data, 1979.

13.

G.B. Elion, Burroughs Wellcome Go., personal communication, 1 9 6 7 .

14.

Medical Department, Burroughs Wellcome Co., unpublished data, 1969.

15.

G.B. Elion, Proc. Roy. SOC. Med.,

16.

G.B. Elion, S . Bieber, G.H. Hitchings, Ann. N.Y. Acad. Sci., 6 0 , 297 ( 1 9 5 4 ) .

9, 257

(1972)


52

17.

P.R.B. FOSS,S.A. Benezra In "Analytical Profiles of Drug Substances", v o l . 7 ; K . Florey, Ed.; Academic Press: New York, 1 9 7 8 ; p. 3 5 5 .

18.

N. Kaplowitz, J. Pharmacol. Exp. Ther.,

200

( 3 ) , 479

(1977). 19.

P. de Miranda, L.M. Beacham, 111, T.H. Creagh, G.B. Elion, J . Pharmacol. Exp. Ther.,

20.

195,50

(1975).

P. de Miranda, L.M. Beacham, 111, T.H. Creagh, G.B. Elion, J. Pharmacol. Exp. Ther., 187, 5 8 8 (1973).

21.

G.B. Elion, F.M. Benezra, L.O. Carrington, R . A . Strelitz, Fed. Proc., Fed. h e r . SOC. Exp. 2 9 , 2027 ( 1 9 7 0 ) . Biol., -

22.

G.B. Elion In "International Symposium on Immunopathology", Vth, Punta Ala, Italy, June 1 9 6 7 ; P.A. Miescher, P. Grabar, Ed.; Grime and Stratton: New York, 1 9 6 8 ; p. 3 9 9 .

23.

U. Bar, H. Becker, B. May, D. Mayer, S . Ohlendorf,

P. Otto, F.W. Schmidt, Verh. Dtsch. Ges. Inn. Med.,

7 9 , 943 ( 1 9 7 3 ) .

24.

Medical Department; Burroughs Wellcome Co., unpublished data, 1 9 6 3 .

25.

N. Kaplowitz, J. Kuhlenkamp, Gastroenterology, 74

,

90 (1978).

26. Merk Index, Ninth Edition, Merck and Co. Inc: Rahway, N.J., 1 9 7 6 , p. 9 1 2 . 27.

M.J. Harber, J.L. Maddocks, J. Gen. Microbiol.,

2,

351 ( 1 9 7 3 ) . 28.

A.I. Al-Mosawi, J.N. Miller, J . W . ~

Bridges, Analyst,

1 0 5 , 448 ( 1 9 8 0 ) .

8,273

29.

J . L . Maddocks, Br. J. Clin. Pharmac.,

30.

D.J. Nelson, C.J.L. Bugge, H.C. Krasny, T.P. Zimmerman, J. Chromatogr.,

31.

E,1 8 1

A.H. Chalmers, Biochemical Medicine,

(1979).

(1973).

12, 2 3 4

(1975).

53

AZATHIOPRINE

32,

32.

L . Sweetinan, W.L. Nyhan, J . C h r o m a t o g r . , (1968).

33.

T . L . Ding, L . Z . B e n e t , 3. Chromatogr., (1979).

34.

A.F. F e l l , S.M. P l a g , J . M . 691 ( 1 9 7 9 ) .

35.

N . Hobara, A . Watanabe, A c t a . Med. Okayama, 239 ( 1 9 7 9 ) .

36.

S . - N . Lin, K . J e s s u p , M. F l o y d , T.-P.F. Wang, C . T . Van Buren, R.M. C a p r i o l i , B.D. Kahan, T r a n s plantation, 2 9 , 290 ( 1 9 8 0 ) .

37.

J . L . Maddocks, B r . J , C l i n . P h a r m a c o l . , (1975).

38.

M . J . H a r b e r , J . L . Maddocks, J. Chromatogr., 231 (1974).

39.

R . C . T h a p l i y a l , J . L . Mahdocks, J. Chromatogr., 239 (1978).

40.

S. I t o , K . F u j i t a , J . C h r o m a t o g r . ,

41.

P.C.-P. Wong, J . L . Maddocks, J. C h r o m a t o g r . , 491 (1978).

662

163,281

N e i l , J . Chromatogr.,

2,

2,

359

187, 418

101,

160,

(1980).

150,

BENZYL BENZOATE Mahmoud M . A. Hassan and Jaber S. Mossa 1. Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Taste, Odor 2. Physical Properties 2.1 Boiling Range 2.2 Melting Point 2.3 Density 2.4 Refractive Index 2.5 Solubility 2.6 Identification 2.7 Spectral Properties 3 . Synthesis 4. Metabolism 5. Methods of Analysis 5.1 Titrimetric Method 5.2 Spectrophotometric Methods 5.3 Spectrotitrimetric Methods 5.4 Gas Chromatographic Method 5.5 Proton Magnetic Resonance Method 6. References

ANALYTICAL PROFILES OF DRUG SUBSTANCES. 10

55

56 56 56 56 56 57 57 57 57 57 57 57 57 58 64 67 68 68 68 68 68 69 73

Copyright l Y X l byAcademicPre,s. Inc All rights of reptoduction in an) h r m rewved. ISBN 0-12-260810-0

56

MAHMOUD M. A. HASSAN AND JABER S. MOSSA

1. Descrivtion 1.1 Nomenclature 1.11 Chemical Names a) Benzoic acid phenylmethyl ester. b) Benzoic acid benzyl ester. c) Benzylbenzenecarboxylate. 1.12 Generic Name Benzyl benzoate. 1.13 Trade Names Ascarbin; Ascabiol; Benylate; Vanzoate; Venzonate. 1.2 Formulae 1.21 Emperical C14H1202 1.22 Structural

1.23 CAS No. 120-51-4 1.24 Wiswesser Line Notation RVOIR. 1.3 Molecular Weight 212 - 2 5 1.4 Elemental Composition C,

79.22%; H, 5.70%;

0, 15.8%.

57

BENZYL BENZOATE

1.5 Appearance, Color, Taste,OdorLeaflets or colorless oily liquid, faint, pleasant aromatic odour, sharp burning taste. 2. Physical Properties 2.1 Boiling Range 323 - 324°C (1) , (2) . bp16 189 - 191°C (2); bpll 170'C bpqe5 156'C

(1)

(2).

2.2 Melting Point 21°C. 2.3 Density di5 1.118 (2) , dt5 1.1121 (1).

2.4 Refractive Index n2' D

1.5681 (2)

nZo

1.5680 (1)

n20

1.568 - 1.570 (3).

D

D

2.5 Solubility Insoluble in water o r glycerol, mescible with alcohol (95%), chloroform, ether, oi.ls, acetone, benzene, methanol, petroleum ether (1-3) . 2.6 Identification Boil 2 g with 25 ml of alcoholic potassium hydroxide solution for 2 hours under a reflux condenser. Remove the alcohol on a water-bath, add 50 ml of water, and distill until the liquid distilling is no longer turbid. The liquid remaining in the flask, after acidification with dilute hydrochloric acid, yields a white crystalline precipitate of benzoic acid.

58


To the distillate add 2.5 g of potassium permanganate and 2 ml of sodium hydroxide solution, boil for 15 minutes under a reflux condenser, cool, and filter. The filtrate after acidification with dilute hydrochloric acid, yields a white crystalline precipitate of benzoic acid (3). 2.7 Spectral Properties 2.71 Infrared Spectrum The infrared spectrum of benzylbenzoate is recorded as a film on a Unicam SP 3-300 Spectrophotometer and is shown in Fig. 1. The assignments for the characteristic bands in the infrared spetrum are listed in Table 1. Table 1

IR Characteristics of Benzvlbenzoate -1 Assignment Frequency CM 1720 1601 1590 1275 1110 710, 700

C = C = C = c c Aromatic

(ester) aromatic aromatic - c 0 - c monosubstitution. 0 C C 0

Other finger print bands characteristic of benzylbenzoate are 3060, 3030, 1500, 1450, 1380, 1315, 1180, 1070, 1025 and 740. The IR spectral data have also been reported (1,4), 2.72 Ultraviolet Spectrum (UV) The UV spectrum of benzylbenzoate in ethanol was scanned from 400-200 nm using Varian Cary 219,six maxima and six minima were observed (Fig.2). The maxima were located at 229, 256, 263, 266, 272 and 280 nm. The minima occur at 215, 254, 260, 265, 270 and 277. The UV spectral data of benzylbenzoate have also been reported (1,s). The El%, 1 cm = 843 at 230 nm (6).

m

0

0 LD

8 ol

0

60

Fig. 2 .


Ultraviolet spectrum of benzyl benzoate in ethanol.

BENZYL BENZOATE

61

2.73 Nuclear Magnetic Resonance Spectrum 2.731 Proton Spectrum The proton NMR spectra of benzylbenzoate in deuterated chloroform and in DMSO-D6 are shown in Fig. 3a and Fib. 3b. These were recorded on a Varian T-60A, 60 MHz NMR spectrometer, using tetramethylsilane as an internal reference. The PMR spectra assignment of benzylbenzoate are given in Table 2. Table 2 PMR Characteristics of Benzylbenzoate Chemical shifts DMS0-D 6 CDCl3

Protons -CH2

5.26

5.39

-2H, 6H (adjacent to C) 7.23 Other aromatic Drotons. 8.00

7.47 8.03

0

Other PMR spectral data have also been reported (7,8). 2.732 I3C Suectrum I3C NMR spectrum of benzylbenzoate in carbon tetrachloride using tetramethylsilaine as an internal standard was recorded using Jeol FX 100 MHz instrument at ambient temperature and using 10 mm sample tube. The data consist of 8192 data points over a 5000 Hz spectral width Fig. 4. The carbon chemical shift values areshown in Table 3. (9-11). 10

4

11

62 F i g . 3 a . PMR s p e c t r u m of b e n z y l b e n z o a t e a n d TMS i n CDCl?.

F i g . 3b. PMR s p e c t r u m of b e n z y l b e n z o a t e a n d TMS i n DMSO-D6.

63

-r

V

4

c

u (u

-4

m

c, 0 a,

c

N

R N

>r

r i

c

a,

0

R w

5 c,

m

u 0) a

za V

m

7.

-r

tn

a

4

64

MAHMOUD M. A. HASSAN A N D JABER S . MOSSA

Table 3 13C NMR Characteristics of Benzylbenzoate Carbon No.

1 2 3 4 5 6

7

Chemical Shift.

Carbon

165.63 130.30 129.56 128.20 132.59 128.20 129.56

8 9 10 11 12 13 14

No.

Chemical Shift. 66.40 136.29 128.40 128.01 128.01 129.01 128.40

2.74 GC/Mass Spectrum The GC/Mass spectrum was recorded on Ribermag R 10-10 GC/Mass spectometer using 3% SE 30, packed glass column. The GC trace shows a retention time o f 6.57 minutes. The mass spectrum was obtained by conventional electron impact ionisation at 70 eV, shows a molecular ion M+ at m/e 212 and shown in Fig. 5. Other prominent fragments and their relative intensity are shown in Table 4. Table 4 m/e

Relative Intensity

77

100.00

91

77.3

105

95.9

65

42.9

51

57.4

Fragment 'gH; C H CH2 6 5 C6H5 CO+

+

The mass spectrum o f benzyl benzoate has also been reported (1, 9). 3. Synthesis Three main methods are used f o r preparation of benzyl benzoate.

B B l SCAN 156 SICMA=S ~ r = 6 : 5 7 BACKGD= 15x100 100t/.=4046848 TITLE: SAHPLE BENZYL BENZOATE: 150-230 DEG(t0 DEG/ M I N I ; 3XSE30;E.I.

Figure 5. Mass spectrum of benzyl benzoate.

MAHMOUD M. A. HASSAN AND JABER S.MOSSA

66

I) Estrification of benzoic acid with benzylalcohol(12,13)

h2504

+

H20

11) Transposition between sodium benzoate and benzyl-

chloride. (12, 13).

Et3N 120

-

14OOC ( 1 hr)?

+ NaCl

BENZYL BENZOATE

67

111) Condensation of two molecules of benzaldehyde in the presence of sodium hydroxide (13).

4. Metabolism Benzylbenzoate is rapidly hydrolysed in vivo to benzoic acid and benzylalcohol. Benzylalcohol in turn is oxidised to benzoic acid which is then conjugated with glycine to form hippuric acid (Scheme 1). , ( 5 , 6 ) .

In V i v o

Oxidation

O

il C

CH2 NH2 COOH

H

I

H

I

-N -C

I H

- COOH


68

5. Methods of Analysis

5.1 Titrimetric Method The U.S.P. XVIII (14) describes a titrimetric method for determination of benzylbenzoate. The method is based on the hydrolysis of a weighed amount of the ester with aknown volume of O . 5 N alcoholic potassium hydroxide by boiling under reflux for an hour. Then the reaction mixture is cooled, phenolphthalin T.S. as indicator is added and the excess alkali is back titrated with 0.5N Hydrochloric acid. A blank determinanation is also performed. 1 ml of 0.5N alcoholic potassium hydroxide Z 106 mg of benzylbenzoate(C 14H 120 2) . 5.2 Spectrophotometric Methods Quantitative determination of benzylbenzoate as pure drug and in benzylbenzoate lotion by a spectrophotometric methods have been reported (15,16). The methods involve heating the sample under reflux with 10% alcoholic potassium hydroxide for 5 minutes and measuring the extinction of the cooled reaction mixture after dilution with water or ethanol at 268 nm. Beer's law is obeyed for up to 250 g ml-1 of hydrolysed benzylbenzoate. Interference from other ingredients of the sample (e.g., oleic acid and triethanolamine) is negligible. 5.3 Spectrotitrimetric Methods

Benzylbenzoate and dibutylphthalate are determined in mixtures by measurement of the absorbancy at 250 nm and the quantity of alkali required to saponify the esters (17). The concentration of the esters are calculated by application of a differential equation for which the absorptivity and the saponification contents of the components are required. Analysis of five know mixtures showed average recoveries of 100.8% for benzylbenzoate and 99.97% for dibutylphthalate. Application to cloth patches impregnated with insect repellents containing these esters gave average recoveries of 101.4% for benzylbenzoate and 99.66% for dibutylphathalate. 5.4 Gas Chromatographic Method

A gas-chromatographic method has been described for the determination of benzylbenzoate as a product of catalytic oxidation of toluene (18). The determination was

BENZYL BENZOATE

69

carried out on a column ( 2 m) of 20% of carbowax 20 M on chromosorb W (60 to 80 mesh) operated at 2000 with N ( 4 4 ml min-l) as carrier gas, flame ionisation detection and acetophenone or-benzylalcohol as nternal standard. 5.5 Proton Magnetic Resonance Method

An accurate, simple and precise PMR procedure has been developed in our laboratory for the quantitat on of benzylbenzoate and benzylcinnamate as pure drugs and in Peru and Tolu balsams (19). The method is based on the integration of the benzylmethylene protons of benzylbenzoate appearing at 5.30 ppm (Fig. 6 ) . In Peru and Tolu balsam the corresponding peak appears at 5.32 ppm (Figs. 7 8). Ethylbenzoate is chosen as the internal standard, since it has methylene protons that provide comparable area of integration. Acetone, rather than acetone-D6, is employed as the solvent, since it is inexpensive and dissolves all balsam constituents as well as the internal standard. The average recovery of pure benzylbenzoate in standard mixture is 100.2 0.38 w/w. This method also offers the advantage of individually quantitating the esters, rather than the total ester contents in the medicinally used balsams. Moreover the spectrum of the balsam provides a useful mean for estimating the exact ratio of benzylbenzoate and benzylcinnamate, by simply measuring their corresponding benzylmethylene protons integrals. A l s o the PMR spectra of the esters and balsams are specific means of identification.

so

7.0

Fig.

6:

6.0

A.

PMR s p e c t r l l m of b e n z y l b e n z o a t e i n a c e t o n e - D 6 .

B.

P a r t of t h e PMR s p e c t r u m of b e n z y l b e n z o a t e a n 6 e t h y l benzoate i n acetone.

M 4

TMS

0

I C

.

I

, .

l

a0

B

.

.

*

i

I-

A

I *

7.0

Fig. 7:

*

I *

A. B.

.

l

6.0

.

.

.

.

l

.

.

.

5.0 P P M ( b )

.

4.0

l

*

.

,

,

I

.

3.0

l

.

.

.

I

.

2.0

l

PMR spectrum of Feru balsam in acetone-D6. Part of the PMR spectrum of Peru balsam and ethyl benzoate in acetone,

.

.

’

1.0

.

l

.

‘

.

-

.

t 3

m

a

-4

Fig.

3:

A.

PMR s p e c t r u m o f T O ~ Jb a l s a m i n a c e t o n e - D 6 .

0.

P a r t ?f t h e PMR s p e c t r l i n of Tolu balsm a n d e t h y - b e n z o a t e i n acetorc.

BENZYL BENZOATE

73

References 1.

Atlas of Spectral data and Physical constants of Organic Compounds, edited by J . G . Grasselli and M 7 . M . Ritchey. Volume 2, CRC Press 1975, page 414.

2.

Merck Index, ninth edition, Merck 6 Co., Inc., Rahaway, N . . J . , {J.S.A., 1976, page 148.

3.

British Pharmacopoeia, London, Her Majesty's Stationery Office, 1973, page 51.

4.

The Aldrich Library of Infrared Spectra, Charles S. Pouchert, Second edition, Aldrich Chemical Company Tnc., 1975, page 90OC.

5.

F.C.G. Clarke "Isolation and Identification of Drugs". The Pharmaceutical Press, London, 1971, page 217.

6.

The Pharmaceutical Codex, Eleventh edition, The Pharma ceutical Press, London, 1979, page 91.

7.

Hi'gher resolution W R Spectra Catalog, Vol. 2, Spectrum No.627 compiled by Y . S . Rhacca, D.P. IJollis, L . F . ,Johnson and F.A. Pier of the Instrument Division of Varinn Associates, 1963.

8.

The Aldrich Library of NFlR Spectra. S. Pouchert and J.R. Compbele, \'olurr.e 7 , Aldrich Chemical Company, 1g7A, page 27D.

9.

F . Stenhagen, S. Ahrahamsson and F.W. Mclafferty, "Req-

10.

Rruker 1.3-CData Rank, Volume 7, Rr000218.

11.

Sadtler Standard Carbon-13 NMR Spectra SAD 02833.

istry of )lass Spectral Data", .John Wiley and Sons, London, 1974, page 1122, A.A. 1511-1.

12. Remington's Pharmaceutical Sciences, Fifteenth edition, Mack Publishing Co., Faston, Pa., 18042, 1975, paeel179 13. L.M. Atherden, "Bentley and Driver's Textbook of Pharmaceutical Chemistry", Eighth edition, London, Oxford University Press, 1969 page 571. 14.

The Pharmacopeia o f the U.S.A., Eighteenth revision, The [J.S. Pharmacopeial Convention Inc., 1970, page 76.


74

15.

V . Das Gupta and Hon. W. No, Am. J . Hosp. Pharm. 33 ( 7 ) , 665 (1976).

16.

V . Das Gupta and Hon. W . Ho, Am. J . Hosp. Pharm. 34 ( 6 ) , 453 (1977).

17.

J.O.

18.

Mager, S o r i n ; Hoparlean, I o n e l ; Toranu, Ruxandra and 22(2), P a i n , F l o r i c a , S t u d . Babes-Bolyai, Ser. Chem. 45 (1977).

19.

A . H . Al-Obeid, M . M . A . Hassan and J . S . Mossa, S p e c t . 1 3 ( 6 ) , page 361 ( 1 9 8 0 ) . Letters, -

Page, Anal. Chem. 27 ( 8 ) , 1233 (1955).

CLINDAMYCIN HYDROCHLORIDE Leo W. Brown and William F. Beyer 1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Taste, Odor 2. Physical Properties 2.1 Approximate Solubility 2.2 Melting Range 2.3 Specific Rotation 2.4 pKa 2.5 Crystal Properties 2.6 Infrared Spectrum 2.7 Nuclear Magnetic Resonance Spectrum 2.8 Mass Spectrum 3. Synthesis and Proof of Structure 4. Drug Metabolites 5. Pharmacokinetics and Toxicity 6. Antibacterial Activity 7. Methods of Analysis 7.1 Microbiological 7.2 Paper Chromatographic 7.3 Gas Chromatographic 7.4 Liquid Chromatographic 7.5 Titrimetric 7.6 Radioimmunoassay 7.7 Thin Layer Chromatographic 8. References

76 76 76 76 76 77 77 77 77 77 79 80 80 80 82 82 83 83 83 84 84 R7

87 89 90

LEO W. BROWN AND WILLIAM F. BEYER

76

1.

Description 1.1


C1 i ndamycin hydrochloride is methyl 71S)-chloro-6,7 ,atrideoxy-6-trans-(1 -methyl-4-propyl-L-2-pyrrolidinecarboxarnido)-1 -thio-L-threo-a-D-galacto-octopyranoside monohydrochl oridel, also 7 C1-7-deoxylincomycin.

CH3

I

HCCl CH3 I

I

HC I

C1gH33C1 N205S.HC1

Mol Wt. 461.44

1.2 Appearance, Color, Taste, and Odor Clindamycin hydrochloride monohydrate is a white or practically white, crystalline powder. It is odorless or has a faint mercaptan-like odor and has a bitter taste. 2.

Physical Properties 2.1 Approximate Sol ubil i ty2 Sol vent

Solubility, mg/ml

Water Pyridi ne Met ha no1 Ethyl Acetate

%?.

CHaO cno'QQ;

7 m / e 246

mle 480

t

m/e 288

Fig. 8

Fragmentation pattern €or emetine

1

EMETINE HYDROCHLORIDE

4.2

305

Melting range

Emetine Hydrochloride contains water of crystallization ranging from 3 to 8 H epending mainly on After drying at the solvent used for crystallization.3':4g 105", most references indicakg a melting range between 235" and 255" with decomposition. One reference uotes a melting point of 269-270" with decomposition.39 The melting point of the amorpho s free base is 74"; crystalline emetine melts at 104-105".

Y

I n a study of isotonic solutions the freezingpoint lowering of a 1% solution of emetine in water was found to be 0.082". 50

4.3

Solubility-Partition

Emetine: Water - O.O02g/lOO m151 Conc. NaOH, KOH - insolublez2 Methanol, ethanol, ether, acetone, acetic acid - very ~ o l u b l e ~ ~ , ~ ~ Chloroform - soluble51 Benzene - sparingly soluble22 Emetine Hydrochloride: Water - 13.1 g/100 ml (18")22352 Diluted HC1 - spa5lngly soluble22 Ethanol - 1 in 12 Chloroform - 1 in 433 Ether - insoluble33 The effect of partition coefficient of partition coefficient of chloroform and a mixtu buffer 1:l was 1252.0.

55

4.4 water:

pH and organic acids emetine was studied.58'5bheThe emetine hydrochloride between water: pH 7.0 0.05 M phosphate

Dissociation Constants The following pKb values have been reported for -at 15" 5.77 and 6.6422,60 -at 40" 5.47 and 6.3461 Other values reported: 5.73 and 6.74 (no

L. VALENTIN FEYNS AND LEE T. GRADY

306

indication on the temperature) 59 4.5 Emetine

Optical Rotation Solvent

g/100 ml

[aID

Ref.

50% EtOH

1.8 4.1

-24.4" -25.8" -32.7"

7 6,16,22 6,22

2 2.8 3

-50" -49.2" -49.7"

35 62 22

0.9 5.0

+11.2" +17.7" +17.8" +20.9"

6.22 79 6,22

1

+50.5" +53"

40 16,22

-

2

-

+25.7" +51" +17"

22 6 6

-

+83 "

6

Chloroform

Eme t ine Water Hydrochloride

8.1 Chloroform

5% HC1 Butanol Benzyl alcohol Bromoform

The study of the optical rotatory dispersion curves of emetine and its salts played a major r le in determining the stereochemistry of the molecule.' Since emetine hydrobromide showed no rotational change in the 300700 nm range, the benzylic centers of asymmetry at C-14 and C-l', the only ones in proximity to W chromophores, had to be antipodal to one another thus canceling each other's contribution. 5.

Methods of analysis 5.1

Identity and Color Tests

Treated with ammonium molybdate or molybdenum oxide in sulfuric acif3 emetine gives a bright green color22 The reaction is used in (sensitivity 0.1 pg). official compendia as an identification test. 63,6Zom;i taliI test yields the following colors: addition of fuming nitric acid - pale yellow; evaporation-pale brown. addition of ethanolic KOH - yellow (sensitivity 1 vg). 33 An orange-color is produced when em tine is treated in an acidic medium with either H20265,65 or barium


307

8ramine,

peroxide.66 When heated in solid phase with ch emetine hydrochloride gives a red-yellow color.

Salts of emetine with arylsulfonic acids having characteristic melting points were prepared for identification purposes.68 Some of the paper and thin-layer chromatographic separations reported under 5.3 were also recommended as identity tests, preferably in conjunction with colors formed with specific visualization reagents. The use of the infrared identification test was suggested. The Identification Tests in the USP-NF monograph of Emetine Hydrochloride call for comparison of IR and UV spectra of the sample with those of USP Emetine Hydrochloride Reference Standard. All the official compendia require also an identification test for chloride. 5.2

Elemental Analysis

The calculated values for the elemental analysis of emetine hydroch ride are: C 62.91%; H 7.65%; C1 12.81%; N 5.06%; 0 11.56%.

is

5.3

ChromatograDhic methods

5.31

Paper chromatography

The paper chromatographic systems have been summarized in Table 111. System No. 1 was reported to separate emetine from some of its stereoisomers, but not from cephaeline. Emetine is visualized by examination under W light or spraying with iodoplatinate, bromocresol green, modified Dragendorff reagent or I/KI solutions. When the dried chromatogram is sprayed with a 10% chlorine solution in water acidified with acetic acid, emetine is oxidized to rubremetine and a strong orange-ye ow W fluorescence is obtained (detection limit - 2 us).

+i

Table I11 Paper Chromatography of Emetine No.

Support

I 1

Paper

2 3

4

w

g

5 6 7 8 9 10 11

12

13 14 15

--

--

__"__ --''--

__"__

__"--- --- --

Developing solvent Ethyl methyl ketone satd. with 2 N HC1 BuOh: 0.1-N HC1 1:l BuOH:MeOH: HTO 45: 5 :50 B ~ O :HAC OH :H;O (various ratios Bu0H:formic acid:H20 120:10:70 BuOH :AcOH :Ac OBu i-Bu0H:toluene (satd. H20) 1:l BuOH:AcOBu:Phenol:H20 BuOH:Toluene:AcOH:H20 10:10:5:2 Acetone:AcOEt :10% NH4OH 2: 20: 80 Bu0H:aq. citric acid 87:13

Paper impregnated with 5% sodium dihydrogen citrate Paper impregn. CHC13 or trichloroethylene or with phosphate BuOH saturated with buffers or citrate buffers Paper impregn. Petr.ether or cyclohexane or CHC13 and with HCONH2 Et2NH ---CHC13 or mixtures with aromatic hydrocarbons ----"---Triple development: 1) cyclohexane: benzene 9:l 2) benzene 3) CHC13 ----'I

Ref. 62.70.71 72,73 74 72,73,75-79 72,73,80 81 74 81 82 83 33

84

85

76,86 87

Table I11 benzene

9 :1

16

Paper impregn with HCONH~/HCOONH~

CHC13 :

17

Paper impregn. with tributyr in Car boxyme t hylcellulose cation exchange paper

pH 7.4 Phosphate buffer

18

w c 0 o

.

(contd.)

aq. NaCl

88,89

33,90

91


310

5.32

Thin-layer chromatography

The chromatographic systems investigated for the analysis of emetine on silica gel plates are presented in Table IV. The spots can be visualized by one of the following methods: -spraying with iodine-chloroform solution and heating to 60" for 10-15 minutes: emetinelemon yellow (under W365nm-7ellow), haelinelight brown (under UV365nm-light blue)

"58

-examination under W365nm (blue) 92 -spraying with 10% ninhydrin in 95% ethanol (no color at room temperature) and heating at: 80" (grey-purpigJ, 120" (red-violet) or 160" (brown-violet) -spraying with 1% chloranil in toluene (brown), heating to 105" fpb315 minutes and spraying with 2N H2SO4 (ochre) -spraying with Dragendorff reagent used also on cellulose or ion-exchange plates) 164-106 -spraying with potassium hexaiodoplatinate (IV) 33,107

(KZPt1(j)

System 12 separates potential impurities present in emetine of natural or synthetic origin: cephaeline, 0methylpsychotrine and isoemetine. System 7 was reported to separate emetine from its thermochemical and photochemical decomposition products. The procedure was developed into an assay by transferring the emetine spot to a column, eluting with 0.2 N - HC1 and determining the emetine concentration at 284 nm. For quantitative purposes, re-washing of the plates with methanol was recommended.9t

T e hromatog53phi behavior of emetine cellulose,lo A1203, 3"' ion-exchangers,105,188 and silica gel-glass powder sintered platesg9 was also investigated.

2 6


311

Tab. IV Thin-Layer Chromatography of Emetine RF

No.

Developing solvent

1

Chlorofor :methanol (85:15) or (9:l) Benzene:toluene:ethyl acetate: diethy1amine:methanol (35:35:20:10:2) To1uene:ethyl acetate:85% formic acid (50:45:5) To1uene:Z-propano1:conc. ammonia (70:29:1) To1uene:ethyl acetate:2-propanol: 2 N AcOH (10:35:35:20) To1uene:dioxane:methanol: conc. ammonia (25:50 :20:10) CC1 :butanol:met ano1:ammonia ($0:30:30: 2) b? Benzene:ethyl acetate:diethylamine (7:2:1) Ch1oroform:diethylamine (9:l) Ethyl acetate:methanol:conc. ammonia (170: 20: 10) Methano1:conc. ammonia (100:1.5)

2 3

4 5

6 7 8

9 10 11 12 13 14 15 16

17 18

19 a) b,

Chloroform:2-methoxyethanol: methano1:water:diethylamine

0.3-0.5 0.54

Ref. 20,64,92 93,94

0

95

0.26

95

0.05

95

0.9

95

-

96 97

0.45-0.64

92,98

0.67 0.45

18,99 100

0.52

-

33 63

0.31 0.52 0.65 0.70

101 101 80 102

0.70

102

0.40

92

0.06

92

(100:20:5:2:0.5)

Ch1oroform:acetone:methanol (5:4:1) Ch1oroform:acetone:dimethylformamide

0.1 N MI3 in Methanol Ch1oroform:ace tone :diethylamine (5:4:1) Methyl ethyl ketone:ethanol:ammonia (5:4:1) Cyc1ohexane:chloroform:diethylamine

(5:4:1)

Cyc1ohexane:diethylamine

(9:l)

Single or double development. Single or followed by two-dimensional development with petroleum ether: Et20:EtOH:Et2NH (4:16:2:1)

L. VALENTIN FEYNS AND LEE T. G W Y

312

A study of the optimization of the dansylation reaction, TLC separation of mono-dansyl-emetine and fluorescence detection was reported by the same authors110 who later reported a similar HPLC procedure (see 5 . 3 4 ) . Emetine and cephaeline were separated by TLC after a preliminary oxidation by Hg(OAfJP to products of characteristic color and fluorescence. 5.33

Gas chromatography

Gas chromatographic methods were described mostly for the s f toxicological extraction residues.SS~fPf'flS A s stationary phase 1-5% SE-30 on silanized Gas Chrom P (100-140 mesh) or Chromosorb W (60-80 mesh) was used, with nitrogen or helium as carrier gas; 5-6 feet stainless steel columns were operated at 170-230". It was reported that apparently emetine hydrochloride dissociated in the injection port ( 3 2 5 " ) since the same retenffpn times were obtained for the salt and the free base. 5.34

High-pressure liquid chromatography

The systems reported in the literature are summarized in Table V. Pre-column derivatization: -dansylation with dansyl chloride; normal phase chromatography (mobile phase--diisopropyl ether:isopropyl alcoho1:conc. ammonia 4 8 : 2 : 0 . 0 3 ) . 121 Post-column derivatization procedures: -fluorescence labeling with dansyl chloride line two-phase "solvent segmentation" flow (reaction time - 16 min at 5 6 " ; excitation emission cut-off filter >450 nm; detection

in an onsystem 365 nm, limit 30 ng)

-air-segmented flow, ion-pairing with 9.10dimethoxyanthracene-2-sulfonate and extraction in chlorinated organic solvents. Excitation at 383 nm, emission - 446 nm. ktqearity range 40-600 ng. Limit of detection 0.2 ng. Used in conjunction with system 3 , the detection limit of capacitance-conductance detector was 500 ng of emetine. l a 6

Table V HPLC Analysis of Emetine

Column Silica Silica Silica LiChrosorb RP 8 u-Bondapak C18 LiChrosorb DIOL Mercaptopropylbonded phase Aliphatic strong cation exchanger

Mobile Phase Ethyl ether (95% water saturated) + 0.5% diethylamine Ch1oroform:methanol or ether:methanol Ch1oroform:methanol:hexane 7:3:10 pH 3.0 0.02 M phosphate buffer:methanol 2:3 Methano1:water ( 5 6 : 4 4 ) or ( 6 0 : 4 0 ) + 0.5% AcOH and 2.5m M octane sulfonate 0.05-0.i M NaHC03:acetonitrile 1OO:O - 70:30 pH 3 . 1 0.0 M phosphate buffer Methanol:2 Mammonium hydroxide:l M ammonium nitrate (27:2:1) M ammonium hydroxide: 1 M ammonium nitrate Methanol:2 (27:2: 1)

Ref. 114

115 116 117 118

119 117 120 120


314

5.35

Electrophoresis

The electrophoretic mobility of emetine on paper in buffers from pH 2.3 to 11.4122-125 and on cellulosecoated glass plates in acidic and alkaline electrolytes126 was studied for separation and identification in alkaloid mixtures. 5.4

Titration

Potentiometric titration of emetine hydrochloride with 0.01 N NaOH avoids the difficulties of the visual determinatTon of thelgyd-point due to the buffering effect of the organic base. Some compendia1 assays63,128 consisted of extraction of an alkaline solution with ether, back extraction of emetine with H C 1 and titration of the excess of acid. It has been reported that products of photochyyical decomposition of emetine interfere with the method. I n other official procedures,18,12' emetine hydrochloride is assayed in glacial acetic acid by titration with 0.1 N perchloric acid in the presence of mercuric acetate with crystal violet indicator.130-132 The end-point can be also determined pp53ntiometrically or using p-naphtholbenzein as an indicator.

Emetine was determined in a two phase chloroformwater system by titration with 0.01 M sodium d ioctylsulfosu,,ina te using dimethylyellow-Oracet blue as the indicator. Emetine was radiopjlrically titrated by I3lIlabeled Dragendorff reagent.

5.5

Colorimetric and Spectrophotometric Methods

Most colorimetric methods for the determination of emetine involve its extraction from aqueous solution into an organic solvent by ion-pairing with a dye anion. Emetine forms a 1:2 complex with bromothymol blue which can be best extracted with chlorofor aqueous solutions buffered in the pH range of 4.0-5.8. ''ff9' Similar procedures were developed using methyl orange, bromocresol purple, bromocresol green, phenol red, Direct Acid Blue, cresol red and bromophenol blue. 5Y:Y35"-3?w


315

Complications related to the stepwise dissociation of diprotic acids such as bromothymol blue are avoided by using the singly charged tetrabromophenolphthalein ethyl ester. The absorbance o f the red extract in 1,2dichloro thane is measured at 5 7 0 nm (linearity range 2-10ft8-' M (1.1-5.5 pg/ml). The method is less pH dependent. The reaction of emetine with sodium 1,2naphthoquinone-4-sulfonate gives a co ound extractable in chloroform and measurable at 4 6 0 nm. 1TP Emetine is precipitated quantitatively from aqueous solutions as a reineckate, which may be di ~ acetone and determined colorimetrically at 525 m. 9 $ 5 ~ P $in The concentration ranges in which Beer's law is valid €or the UV spectrophotometric determinations wa reported for 20 quinoline and isoquinoline alkaloids.f44 Emetine acting as electron donor forms a charge transfer complex with iodine whose absorbance in chloroform at 292 nm is greatly increased over that of the uncomplex and can be used for a spectrophotometric assay. ,

5f 33ka10id

The red shift ( 3 2 2 to 3 5 5 nm) accompanying the ionization of picrolonic acid in the presenc of emetine was developed in an assay sensitive to 2 ug/ml. Emetine forms a colored adduct with picric acid in acetic whose extinction can be measured photometrically.l&ed* The yellow color produced by the oxidation of emetine with ceric ammonium sulfate was measured photometftyally after stabilization with sodium acetate The colored product resulted from the reaction of emetine with benfzjuinone was extracted in CHC13 and measured at 5 4 0 nm. Emetine hydrochloride yields a highly colored condensation complex (Xmax 333 nm) when heated with malonic acid in acetic anhydride. The spectrophotometric method developed on the basi of this reaction has a detection limit of 0.03 bg/ml. 129

5.6

Spectrofluorometric Methods

The fluorescence of emetine has a maximum emission at 318 m with a excitation maximum at 2 8 4 nm. Concentrations in the lo-' M range can be determined (as


316

compared with lo-' M for W determinations) an the decomposition products seem not to interfere." The intensity of fluorescence increases linearly over the concentration range 0.01-1.00 pg/ml; it increases with decreasing pH being maximal in the 1-3 range and it decreases with the increase of temperature (0.5%/degree in the 15-30" range). 150 Emetine Hydrochloride treated with iodine in alcoholic solution gave a gold-colored fluorescence with Xmax at 570 and 620 nm (Xex 436 nm). Fluorescence intensity was lifffr with concentration in the range of 0.05-1.00 p.p.m. 5.7

Polaroeraohic Methods

Emetine yields catalytic waves over the pH range 3 to 10. For quantitative determinations the wave at pH 3 has been employed over the concentration range 0.08-0.25 x M (the limiting current is in linear proport on to the concentration) and at pH 8 f o r l j p 0.01-0.1 x lo-' M ran e (calibration curve necessary). Half-wave potential EB/2 = -1.62V. 153 A polarometric titration of emetine after coupling with p-diazobenzene-sulfonic acid was reported.154

5.8

Thermogravimetric Analysis

Thermogravimetric analysis at 5"/min in a N2 atmosphere showed that emetine hydrochloride forms no stable hydrates, water l o s s takes place even at room temperature (the water content will fluctuate with the relative humidijjy) and a slow l o s s continues at temperatures above 105". 6.

Determination in Bioloeical Fluids and Tissues

In the earlier publications, gravimetric (preci i ation with silicotungstic acid; detection limit 20 ug/ml), colorimetric (methyl orange or brpygph 01 blue extraction) -138 were used. and UV spectrophotometric methods

P5l

Th first spectrofluorometric method was reported in 1961.15' After an extraction procedure adapted to each preparation (plasma, urine, tissue homogenates) a fluorescent compound (rubremetine) is produced by a dehydrogenation reaction with mercuric acetate. (Xex = 365


nm; Xem

=

317

470 nm).

After extraction by benzene or ether of blood or tissue homogenate samples at an alkaline pH, emetine is taken up in an aqueous acidic solution and determined by measuring the fluorescence 287-318 nm (sensitivity threshold: 0.010.02 pg/ml). 1% After extraction with dichloromethane from human plasma, emetine can be analyzed directly at levels above 500 ng/ml by ion-paired reversed-phase chromatography (see 5.34); by introducing an oxidation step with mercuric acetate between extraction and chromatography, the limit of spectrofluoromet detection is lowered to levels of 10 ng/ml of plasma.

€18

Combustion and liquid scint llation counting were used in pharmacoki tics studies of “C-labelled emetine in guinea pigs. 1 8 7.

Determination in Pharmaceutical Preparations Aqueous titration

Depending on the final stage of sample preparation, emetine (or total ether-soluble alkaloids in ipecac) is titrated with 0.01 or 0.1 N EC1 or the excess acid used in the final extraction is titrat with 0.02- .1 N NaOH. The method is used for ti s tablets,16’ ipecac, ipecac powder or extracts.i239sy1g5-165 The buffering effect of the phenolic alkaloids and the yellow color of the extract tending to mask the end-point have been mentioned as disadvantages of the method when applied to ipecac. 166

’‘

Non-aqueous titration in glacial acetic acid with 0.1 N perchloric acid using crystal violet a ator is used for the assay of emetine formulations. s3Si:Pf’ A modified procedure distributes the sample over a mixture of magggsium oxide and Celite and elutes the free base with CHC13. N e p h e l ~ m e t r i c ’and ~ ~ phototurbidimetric168 titrations were also reported. Colorimetric and spectrophotometric methods

In 1942 an author was writing “a satisfactory colorimetric method for the determination of [emetine] could not be found...The reaction with hydrogen peroxide in the

318


presence of hydrochloric acid, which produces an orange colour...is y capable of detecting gross errors in dispensing".

na4

The acid-dye technique has been widely used in assaying the alkaloi i emetin f mulations, ipecac powder and tinctures. 1'8,121,166,1'o-pr2 A s an example of the technique, the complex formed between the alkaloid and methyl orange at pH 5 is extracted with chloroform and treated with 0.1 N NaOH to liberate the dye and extract the phenolic alkaloids. The liberated dye determined at 460 nm in the alkaline extract is a measure of the total alkaloids. The non-phenolic alkaloids are extracted from the chloroform phase with 0.1 NH2SO4 and their concentration in the acid extract is determined at 283 run and calculated as emetine. The procedure is reported as less time-consuming an equiring less sample than the compendia1 procedures.f6f; After separation from phenolic alkaloids, emetine was assayed in liquid extracts of Ipecacuanha and powdered root by determining the W absorbagte (292 nm) of the chargetransfer complex with iodine. The yellow color arising by the action of iodine in the presence of aqueous sodium acetate was used previously for the deter ation of emetine in ipecac and its galenical preparations.rps9 Phosphomolybdic acid was used to precipitate emetine from ipecac extract. The pref+litates were taken in acetone and assayed colorimetrically. After buffering the injection at pH 9.5, emetine was extracted in ethylene dichloride and interacted with picrolonic acid. The absorbance at 362 (anionic band of picrolonic acid) is used for the assay. 1 8 Pharmaceutical preparations were subjected to dialysis across a cellophane membrane and the amounts diffusing after a fixed time interval wefg5determined colorimetrically using Lautenschlager's method. Powdered ipecac was extracted with MeOH/HCl, the extract evaporated, mixed with basic A1203 transferred to a column and eluted with CHC13. Emetine was determined as the difference between the total alkaloid content (measured at 286 run) and the cephaeline content (determined co metrically with 2,6-dichloroquinone-chloroimide).

w-


319

Ultraviolet spectrophotometric assays of emetine and cephaeline in ipecac became possible after an elaborate four-column chromatographic syste ceous earth in different buffers) was developed. The procedure was adopted as an assay for Ipecac and its syrup fluidextract and powdered eparations in the United States Pharmacopeia'" and the Official Methods of Analysis of The Association of Official Analytical Chemists180

'"st''

.

Other attempts to extract, purify and separate the alkaloids in ipecac into phenolic and non-phenolic fractions rri d on Fluorisi 181 by c o l y ~chromatography ~ were A1203, 185ion-exchange resins ,'$3 18' oxycellulose~84 and Celite. After oxidation in aqueous solution with acidic KMn04, emetine in pharmayjgtical preparations was assayed fluorimetrically. Chromatographic procedures Thin-lay r chromatograph e arations followed by den~itometric'~~ or spot area" 9 p88 measurements were used for the quantitative determination of emetine in ipecac and its preparations. Emetine-containing syrups and capsules were subjected to derivatization with dansyl chloride. Separations carried out by TLCljf HPLC were followed by fluorometrical determinations. 8.

Stabilitv - Degradation

Solid forms and solutions of both emetine and the hydrochloride turn yellow on exposure to light and heat. The thermal and photochemical stability of s solutions pH 3 being of emetine hydrochloride are pH dependent,18B"'Y Cysteine, aminoacetic acid, that of maximum stability. 18' thioglycolic acid, D-penicillamine, Na2S204, NaHS03, Na2S03, Pb2+ and jsg8&yy2edetate increased the stability of the solution. The following compounds were identified among the products of the photochemical and thermal decomposition of emetine:emetamine, 3,4-dihydro-6,7-dimethoxyisoquinoline, 0methyl-psychotrine, l-methyl-3,4-dihydro-6,7-dimethoxyisoquinoline, tetradehydroemetinium chloride, rubremetinium chloride, 1' , 2 '-didehydroemetine, 2-methyl-3-ethyl-

320


1,4-dihydro-9,10-dimethoxybenzo[a]-quinolizinium chloride, 3-ethyl-1,4-dihydro-9,lO-dimethoxybenzo[a]quinolizinium chloride and a benzoquinolizinium dimer. The fragment products resulted only by photochemical decomposition.? G O n

One of the compounds isolated in the above mentioned study, didehydroemetine, was synthesized in 1961 by etine with mercuric acegate and designated by oxidation of A recent publication proved the structure A . degradation and synthetic products to be identical, on the basis of their UV, IR and mass spectra and their chromatographic behaviour and assigned to didehydroemetine the structure B. The same paper assigns to O-methylpsychotrine structure C , instead of the previously reported structure B.

A

9.

Toxicitv

-

B

Pharmacokinetics

In mice, the following acute LDs0 values were reported: subc taneous - 35 mg/kg2 (32 mg of base/kg 195 oral - 3 0 mg/kgy3,195 and intraperitoneal - 62 mg/kg. rats, y d e r intraperitoneal administration, LDz0 is 12.1 The therapeutic dose in men is 1 mg/ g body weight mgfkg. dailyigyubcutaneously, a course of treatment lasting ten days. The toxic dose by accumulation is between 1.1 and 1.8 g6. 700 mg is considered to be the fatal dose in humans.’ 33 Emetine is rapidly absorbed and is distributed mainly


321

in the liver (high concentrat ondrial Low concenfraction), kidney and spleen.fs:f56:l%:l%!f?8$ trations were found in the brain, in agreement with unsucces f 1 attempts to treat amebic cerebral abscess with ernet ine f 6y

.

Emetine does not appear to be metabolically transformed and it is slowly excreted, which may account for the cumulative toxicity (dehydroemetine is eliminated more rapidly). In contradiction with previous findings about emetine being primarily excreted in urine, a 1965 study reported th t after intraperitoneal injections of guinea pigs with "C-labelled emetine, 95% of the injected radioactivity was recovered from the feces, while only 5% appeared in the urine. It was suggested that emetine passes into the bowel through the gastro-intestinal wall where rather high concentrations were found rather than through the bile. In humans, excretion all other routes than urine is reported to be negligible. Excretion in urine begins 20 m Utes after injection and continues for as long as 2 months. $3 Acknowledgments The authors wish to thank Dr. J. A. Kelley, Laboratory of Medicinal Chemistry and Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland for the acquisition and help in the interpretation of the mass spectral data, Vivian A. Gray for the technical assistance and Ann K. Ferguson, Barbara Bowman and Patricia Perando for processing the manuscript.


322

10.

References

1.

F. b . Flkkiger, "Pharmacographia. A History of the Principal Drugs of Vegetable Origin, met with in Great Britain and British India," Macmillan and Co., London, 1 8 7 9 , p. 3 7 0 .

2.

E. F. Elslager in "Medicinal Chemistry," Third Edition, Part I, p. 5 3 0 , A. Burger, Editor, Wiley-Interscience, New York, 1 9 7 0 .

3.

W. Lewis, "The Pharmacopoeia of the Royal College of Physicians at Edinburgh," London, 1 7 4 8 , p. 3 7 .

4.

"The Pharmacopoeia of the United States of America," Second Edition, S. Converse, New York, 1 8 3 0 , p. 4 0 .

5.

The Pharmacopoeia of the United States of America," Ninth Decennial Revision, J. B. Lippincott, Philadelphia, 1 9 1 6 , p. 1 3 3 .

6.

M. M. Janot in "The Alkaloids" (R. H. F. Manske and H.

L. Holmes, eds.), Vol. 3 , Chap. 2 4 , Academic Press, New York, 1 9 5 3 , p. 3 6 3 . 7.

G. E. Foster and G. W. Norgrove, J. Pharm. Pharmacol., 480 ( 1 9 5 3 ) .

5, 8.

F. H. Carr and F. L. Pyman, J. Chem. SOC., 1 0 5 , 1593 (1914).

9.

a. E. E. Van Tamelen, P. E. Aldrich and J. B. Hester, J. Amer. Chem. SOC., 81, 6214 ( 1 9 5 9 ) ; b. A . R. Battersby et al. ~J. Chem. SOC. 1 9 5 9 , 2 7 0 4 , 3474, 3512.

10.

S.

C.

Chem., -

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L. VALENTIN FEYNS AND LEE T . GRADY

334

183.

A. J i n d r a and J. Pohorsky, J. Pharm. Pharmacol.,

344 (1951).

3,

184. D. A. E l v i d g e , K. A. P r o c t o r , and C. B. Baines, Analyst, 82, 367 (1956). 185.

S. Kori and M. Kono, Yakugaku Z a s s h i , 82, 1211 (1964); 59, 1945 (1963). -

C.A.

186. H. Wullen, E. S t a i n i e r , and M. Luyckx, J. Pharm. Belg., 66, 22265 (1967). - 21 (7-8), 409 (1966); C.A. 187.

T. Kazunori and M. Ono, E i s e i Shikenso Hokoku, 1979 93, 54055 (1980). (97), 21; C.A. -

188.

M.

S . Habib and K.

J. H a r k i s s , P l a n t a Med.,

18, 270

(1970). 189.

G. Bayraktar-Alpmen, E c z a c i l i k Bul., 13 (l), 1 (1971); 75, 67447 (1971). -

C.A.

190. V. S p r i n g e r , M. S t r u h a r , Z. Zembiakova, and M. Mandak, Farm. Obz., 45 (9), 391 (1976); C.A. 90, 12252 (1979). 191.

M. S t r u h a r , F. Kubek, V. S p r i n g e r , M. C h a l a b a l a , and M. Mandak, Acta Fac. Pharm. Univ. Comeniane, 7 90, 210008 (1979). (1977); C.A. -

192.

C. Schuyt, G. M. J. B e i j e r s b e r g e n van Henegouwen, and K. W. Gerritsma, Pharm. Weekbl., 112 (43), 1125 (1977); C.A. 88, 41601 (1978).

193.

C. S c h u i j t , G. M. J. B e i j e r s b e r g e n van Henegouwen, and K. W. Gerritsma, Pharm. Weekbl., S c i . Ed. 1 (l), 186 91, 57253 (1979). (1979); C.A. -

194.

H. Auterhoff and W. J a c o b i , Arch. Pharm.,

31,

294, 591

(1961). 195.

K. J. C h i l d , B. Davis, M. G. Dodds, and E. G. J. Pharm. Pharmacol., 16, 65 (1964).

196.

H. H. Miller and W.

Tomich,

R. J o n d o r f , J. Pharm. Pharmacol.,

22, 659 (1970). 197. A. Marino, Chemotherapia, 5, 56 (1962).


335

198.

D. E. Schwartz and J. Rieder, B u l l . SOC. Path. e x o t . , 54, 38 (1961). -

199.

P. Synek, Cas. Lek. Cesk., 113 (28), 82, 92862 (1975). -

856 (1974);

C.A.

For t h i s p r o f i l e , the l i t e r a t u r e has been searched through Chemical Abstracts Vol. 93 (1980).

GLIBENCLAMIDE Pamela Girgis Takla 1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Odour, Colour 1.3 Therapeutic Category 1.4 Usual Dose Range 2. Physical Properties 2.1 Melting Range 2.2 Solubility 2.3 Infrared Spectrum 2.4 Nuclear Magnetic Resonance Spectrum 2.5 Ultraviolet Absorption Spectrum 2.6 Mass Spectrum 2.7 pKa 3. Synthesis 4. Stability 5. Drug Metabolism and Pharmacokinetics 6. Methods of Analysis 6.1 Polarography 6.2 Non-Aqueous Titration 6.3 Chromatography 7. Identification and Determination in Pharmaceuticals 8. Identification and Determination in Body Fluids 8.1 Extraction 8.2 Ultraviolet Spectrophotometry 8.3 Colorimetry 8.4 Fluorimetry 8.5 Thin-Layer Chromatography 8.6 Gas-Liquid Chromatography 8.7 High Performance Liquid Chromatography 8.8 Radioimmunoassay 9, References

338 338 338 338 338 339 339 339 339 339 339 343 343 343 344 346 348 348 348 348 348 349 349 349 349 350 350 350 351 35 1 352

PAMELA GIRGIS TAKLA

338

GLIBENCLAMIDE 1.

Description 1.1


G1ibenclamide is 1- (4-(2-(5-chloro-2-methoxybenzamido)ethyl) benzenesulphonyl)-3-cyclohexylurea. It is also known2 as 5-chloro-N- (2- ( 4 - (( ((cyclohexy1amino)carbonyl) amino) sulphonyl)phenyl) ethyl)-2-methoxybenzamide and as 1- ((p- (2(5-chloro-o-anisamido)ethyl)phenyl) sulphonyl)-3-cyclohexylurea.

C 0 N H CH2C H 2

Ooc':?

c1

Molecular Weight

=

494.0

Synonyms: Glybenzcyclamide; Glyburide; HB419; U 26,452. Proprietary Names: Daonil; Euglucon; Diabeta; Maninil; Lisaghicon; Glidiabet; Euclamin; Gilemal. 1.2

Appearance, Odour , Colour

Glibenclamide is a white, crystalline, odourless powder; practically without taste. 1.3

Therapeutic Category Oral hypoglycaemic.

1.4

Usual Dose Range 2.5 to 20mg once daily.

GLIBENCLAMIDE

2.

339


Melting Range

This has been reported as 172-1740’’3 ; 169-170°4; and 168-170°5. 2.2

Solubility’

Glibenclamide is virtually insoluble in water and ether; soluble in 330 parts of alcohol, in 36 parts of chloroform, and in 250 parts of methanol. It forms water-soluble salts with alkali hydroxides. 2.3

Infrared Spectrum

Fig. 1 shows the infrared spectrum of a sample of glibenclamide supplied by Hoechst Pharmaceuticals recorded from a potassium bromide disc using a Perkin-Elmer Model 357 grating spectrometer. The spectrum is in agreement with published spectra’’ ’ The major peaks are at 1163, 1333, 1471, 1515, 1613 and 1724cm-l. According to the findings from a study’ of the infrared spectra of a number of s u l phonylurea derivatives, assignments for the peaks observedfor glibenclamide can be made as follows: 3363 and 3313 cm to urea N-H stretch; 1515 cm-’ to urea, amide 11; 1333 cm-’ to -S02-N-; 1163 cm-’ (split peak) to -S02-. Salt formation has been reportedgto decrease the intensity of many of the absorption maxima.

.

’

2.4

Nuclear Magnetic Resonance Spectrum

The NMR spectrum (Fig. 2) for glibenclamide in dimethylsulphoxide-D6 (DMS) was obtained using a Perkin-Elmer R32 (9OMHz) spectrometer. The assignments made on the figure agree with those published by HajdG et a1.6, who show also the signal produced by the -SOz-NH proton (offset) at 10.27 ppm. The -CO-NH- proton observed in DMS as a doublet at 6.27ppm, disappears when the spectrum is determined using trifluoroacetic acid as solvent. 2.5

Ultraviolet Absorption

lo

The ultraviolet absorption spectra for glibenclamide shown in Fig. 3 were determined in 0.01M methanolic hydrochloric acid using lcm silica cells with a Pye-Unicam SP 1800 spectrophotometer. Absorbance measurements at the wavelengths of maximum absorption were made with a Pye-Unicam

c

9

4

I

a,

a

a,

a k cd k

H

c

rcl

I

I

t-aJ

I z 0" cn-

N

1" I

-u-u-

E : f j

n

aJ

0

v

I

0

V

OCH3 c1

(@

C O N H C H 2 C H 2 0 S02NHCONH

-()

OCH3

4.

Stability

A test is specified in the British Pharmacopoeia 19801, using thin-layer chromatography on silica gel GF254 with chloroform-cyclohexane-ethanol-glacial acetic acid (9:9:1:1) as mobile phase, to limit the amounts of 4-(2-(5-chloro-2-

345

GLIBENCLAMIDE

rnethoxybenzamid0)ethyl)benzenesulphonamide (I), ethyl N-4( 2 - (5-chloro-2-methoxybenzamido) ethyl) benzenesulphony1-N-

methylcarbamate (11) or related substances which may be present as impurities in glibenclamide or glibenclamide tablets. The spots are observed under an ultraviolet lamp at about 254nm.

c1

OMe

c1

Wiseman et a1.I6 in a study of sulphamylurea hypoglycaemic agents have postulated that an initial protonation is probably the rate determining step in the hydrolysis of sulphonylureas as follows: RSO~NH-C-NHR' II 0

H+

+

1 0-

I1

0

+ 0% I RS02NH-G-6H2R'

+

RSO~NH-C-NH~R' ) -H20

+ j

OH2 I RS02NH-C

I

+

NH2R'

PAMELA GIRGIS TAKLA

346

Thus, in the case of glibenclamide, the hydrolysis products have would be (I) and cyclohexylamine. Kuriki et a1 descr bed a procedure for the determination of glibenclamide and i s decomposition products, in which cyclohexylamine is first extracted into isoamylacetate from aqueous alkaline solution, and determined by a colorimetric procedure based on the Spingler method” of assay using 2,4-dinitrofluorobenzene (DNFB). The aqueous solution is subsequently acidified and extracted into organic solvents to allow the determination of glibenclami.deby heating with DNFB to produce a yellow colour, and the determination of glibenclamide plus (I) by ultraviolet spectrophotometric measurement at 299nm. Poirier et al.” have reported that (11) forms gradually from glibenclamide in methanol or chloroformmethanol (1:l) even at room temperature, and observed that the British Pharmacopoeia test for impurities should be completed immediately after the test solution (in chloroformmethanol) has been prepared. The characterization and structure of (11) formed by refluxing Flibenc1ami.de with There are no methanol has been proved by synthesis2 reports that glibenclamide shows instability under normal storage conditions. A report of l o s s of strength in tablets stored for six weeks at 20’ and 75% relative humidity has however been made. 21

.

.

5.

Drug Metabolism and Pharmacokinetics

Since plasma levels of glibenclamide are general1 low, most metabolic studies have been carried out using the y4C labelled drug, although in some recent work radioimmunoassay has been used. Pharmacokinetic parameters have been estimated from a single compartment model from investigations in and in man24’25. The closest similarities with man were observed in the rabbit. In man, 45% of a single oral dose of 5mg was absorbed, and peak blood concentrations of 0.044+0.004ug per ml (0.089+0.008 nanomoles per ml) were attained. Other studiesz6’ 2 7 however revealed a practically complete intestinal absorption, and it has been shown28-30 that the bioavailability of glibenclamide is dependent upon particle size. Fuccella et al. 3 1 reported that absorption was complete within 30 to 60 minutes after administration of a 5mg tablet containing micronized glibenclamide. Maximum plasma concentration^^^-^^' 3 1 ’ 32 are usually attained within 2 to 4 hours, and are in the range 120 to 360ng per ml after a single 5mg oral dose. KO et a1.33 found peak levels to occur within 3 to 8 hours. Dose response curves show that the decrease in blood sugar which occurs is limited, and

GLIBENCLAMIDE

347

higher doses only increase the duration of effect34. After a single intravenous injection, the initial biological halfLife period was 2 3 minutes, but the half-life under steady state conditions was 6.6 hoursz4. The increase in halflife which occurs with time makes it difficult to predict drug levels after multiple doses32. The drug is widely distributed throughout the body, and does not accumulate in the blood. Its apparent volume of distribution, owing to its lipophilic nature, is 10 to 11 litres31. It shows no substantial binding to blood cells, but is more than 99 per cent bound to serum proteinsz4. The binding of glibenclamide to plasma has been studied extensively by equilibrium dialysis3 3 5 and by a fluorescence probe technique36. Brown and Crooks have studied the effect of different salts and buffers37 and of various anionic on the binding, which was found to occur by a non-ionic mechanism. No Cotton effects are generated by glibenclamide bound to albumin39. The absorbed drug is completely metabolized, 95% of a single oral dose being excreted within 5 days in similar amounts in urine and f a e c e ~ ~ Metabolism ~ ’ ~ ~ probably takes place in the liver32. Metabolites of glibenclamide are formed by hydroxylation of the cyclohexyl ring at positions 3 and 4 to give 4-trans-hydroxyglibenclamide (the principal metabolite) Both metabolites have been and 3-cis-hydroxyglibenclamide. identified in blood, but were without hypoglycaemic effect at the levels found. A third metabolite has been found in trace amounts in urine, but not identified24y32. 4-Transhydroxyglibenclamide is about 5 or 6 times less effective than glibenclamide in the rat40-42, and the metabolites are eliminated rapidly with a half-life of 1 2 minutes provided renal function is Attempts have been made to fit the disposition of glibenclamide in man3’ and in the dog44 into a two-compartment model with a first-order absorption rate. In another pharmacokinetic study, Balant et al. 32 have made a detailed comparison o f their findings with other published results, and found that the kinetics involved were too complicated to be resolved adequately by such a model. They suggested instead a third hypothetical slowly equilibrating ”deep” compartment, in which the drug could accumulate during long term therapy. Further evidence in favour of this approach is cited in a later report45 for which radioimmunoassay was used to measure glibenclamide levels. Comparative studies of the metabolic parameters of various sulphonylureas including glibenclamide have been made in rabbits46 and in Happ et al? measured glibenclamide levels by radioimmunoassay in a comparative study in adult diabetics. Some reviews on glibenclamide metabolism have been p~blished~’-~’.

‘,

.

PAMELA GIRGIS TAKLA

348

6.

Methods of Analysis 6.1

Polarography

Procedures have been described by S i l v e ~ t r i ~ ~ ’ ~ and by T a m m i l e h t ~ ~ ~For . quantitative work, an automated system, having a flow through micro cell used with a silversilver chloride reference electrode, has been stated5 to give good reproducibility. 6.2

Non-Aqueous Titration

Tetramethylurea has been used as solvent for the titration of glibenclamide with 0.1 N lithium methoxide in 55 benzene-methanol. The end-point was determined potentiometrically or by using 0.2% azoviolet in toluene as visual indicator. Tablet excipients generally were found not to interfere with the assay. Alternatively, the assay can be performed by titration with 0.1 N potassium hydroxide in dimethylformamide solution with thymolphthalein as indicator56. 6.3

Chromatography

Several procedures6’5 7-62 have been proposed f o r the identification o f glibenclamide by thin-layer chromatography. Among the solvent systems described are butanolmethanol-chloroform-25% ammonia5* , propanol-cyclohexane5 and propanol-benzene-cyclohexane5

.

’,

High-performance liquid chromatography has been recommended by Beyer6 for the quantitative determination of glibenclarni.de in tablets. The column packing used was 1% ethylene propylene copolymer on DuPont Zipax, with 0.01 M sodium borate containing 27.5% vfv methanol as mobile phase. Testosterone serves as internal standard. An impurity, 5-chloro-N-(p-sulphamoyl-phenethyl)-o-anasimide, was eluted as a separate peak.

7.

Identification and Determination in Pharmaceuticals

Identification tests for glibenclarni.de given in the British Pharmacopoeia’ depend upon: a) its infrared absorption spectrum; b) its light absorption in the range 230 to 350nm; c) the evolution of fumes having a pungent, arnine-like odour which change moistened red litmus paper to blue after boiling with 6M sodium hydroxide solution; and d) positive tests for chloride and sulphate in an aqueous

GLIBENCLAMIDE

349

extract of the residue obtained after igniting glibenclamide with anhydrous sodium carbonate and potassium carbonate. The identification tests for glibenclamide in tablets depend upon: a) light absorption measurements in the range 230 to 350nm; and b) thin-layer chromatography on silica gel GF254 with chloroform-cyclohexane-ethanol (96 per cent)glacial acetic acid (9:9:1:1 parts by volume) as mobile phase. Glibenclamide is assayed' by titration in ethanol with 0.1M sodium hydroxide using phenolphthalein solution as indicator, and protecting against exposure to atmospheric carbon dioxide. Glibenclamide tablets are assayed' by a spectrophotometric procedure which depends upon extraction of the tablets with 0.1M methanolic hydrochloric acid, and measurement of absorbance at about 300nm.

8.

Identification and Determination in Body Fluids 8.1

Extraction

Glibenclamide is extracted from aqueous acid solution or acidified plasma or serum by chloroform 6'64,65, ethyl acetate6, amyl acetate66, toluene6 and benzene6'. Alternati~ely~l, plasma can be deproteinized with acetone, the acetone evaporated to small volume and extracted with chloroform after dilution with pH 4.5 buffer solution. Balant et al. 3 2 were not successful in separating glibenclamide from its metabolites using a procedure that they had found applicable to glipizide which involved adjusting the pH of plasma to 4.3 with acetate buffer, and extracting with methylene chloride.

8.2

Ultraviolet Spectrophotometry

A procedure for glibenclami.de in serum has been described by HajdG et a1.6, but is insufficiently sensitive for normal applications. 8.3

Colorimetry

A modification6 of the colorimetric procedure reported by Spingler'' for tolbutamide in serum involves heating glibenclamide in amyl acetate with 2,4-dinitrofluorobenzene to 150' for 5 minutes. Absorbance is measured at 380nm. The method can only be used for glibenclamide when it is present in much higher concentrations than those normally encountered in serum.

PAMELA GIRGIS TAKLA

350

8.4

Fluorimetry

Glibenclami.de when excited by radiation of wavelength 290nm emits a weak fluorescence which can be measured in 0.1 M sodium hydroxide at 350nm. HajdG et a1.6 have described a procedure for serum, but the method has never been successfully applied. The detection limit for glibenclamide in aqueous alkali is about 0.41-1 per ml, and plasma blanks are likely to be high6'. Beckerg6 has reported that his fluorimetric procedure which was developed for glibornuride in plasma can be applied to glibenclamide. The lower limit of detection for glibenclamide is about 40ng per ml. The fluorescence is developed by heating an amyl acetate extract of the drug at 140' for 15 minutes with 7-chloro-4nitrobenzo-2-oxa-1,3-diazole (NBD chloride). The reaction depends upon the degradation of glibenclamide to give 4- (2- (5-chloro-2-methoxybenzamido) ethyl)benzenesulphonamide and cyclohexylamine. The latter compound reacts with NBD chloride present in excess to produce a fluorescent product. The reagent itself is non-fluorescent. There are no other reports of this method having been used for the determination of glibenclamide. 8.5

Thin-layer Chromatography

Balant et al.32 used silica gel plates with benzene-glacial acetic acid-ethyl acetate-acetone (65:6:12:30) for methylene chloride extracts of urine and plasma. 8.6

Gas-Liquid Chromatography

A procedure65 employing a column packed with 5% OV-17 on 80-100 mesh Chrom G-AW-DMCS has been used for the determination of glibenclamide in the plasma of healthy adults after oral administration of 5mg of the drug. The method involves derivatization of the glibenclamide by heating with 2,4-dinitrofluorobenzene in amyl acetate at 13OOC for 1 hour. A 3Ni electron-capture detector was used. The procedure was found to be specific for glibenclamide, and not subject to interference by metabolites. 4-Hydroxyglibenclamide could be determined qualitatively by a slight modification of the gas chromatograph parameters. The quantitative determination of glibenclamide was carried out using tolbutamide as internal standard. The lowest detectable amount of glibenclamide was 1OOpg. Plasma concentrations found 1, 3 and 5 hours after the administration of the drug are reported. They range from 0.05 to 134.76 ng per ml.

GLIBENCLAMIDE

8.7

351

High-Performance Liquid Chromatography

Several procedures have been reported in recent months 67,68,70 which are sufficiently sensitive for clinical 67 assays. Adams and Krueger mix canine serum with monobasic sodium phosphate solution and extract with toluene containing butyl-p-hydroxybenzoate as internal standard. The extract is evaporated to dryness, and the residue is dissolved in the h.p.1.c. mobile phase which is 50mM-NH H PO -acetonitrile 4 2 4 (1:l). Chromatography is carried out on a reversed-phase column of Lichrosorb RP-8. Detection is at 228nm. The lower glibenclamide detection limit wasabout20ng per ml of serum extracted. The major metabolites in the dog, 3-cis(1- [ (4- (2-(2-methoxy-5-chlorobenzamido) ethyl) phenyl) sulphonyl) ureido)cyclohexanol, 1- ( (4-carboxyphenyl)sulphonyl)-3cyclohexylurea, and 2-methoxy-5-chlorobenzamide did not interfere. The main metabolites of glibenclamide in human serum are also stated not to interfere. Another procedure6’ developed for glipizide which has been found applicable also to glibenclamide uses a VBondapak c18 column. Glibenclamide was extracted from serum with benzene after acidification to pH3. The mobile phase was 30% 0.01 M phosphate buffer (pH 3.5) in 70% methanol. Glibornuride served as internal standard. Reinauer et a1.” used an RP18 column with CH3CN-H PO4 (45:55) as mobile phase for the determination of gllbenc?amide in blood serum of diabetics. 8.8

Radioimmunoassay

A number of radioimmunoassays have been developed which have the desired sensitivity for metabolic studies. Some of t h e ~ e ~ ldo- show ~ ~ cross-reaction with the two major metabolites of glibenc1ami.de. The radioimmunoassay developed by .Kawashima et al. 7 4 is however stated not to be subject to interference from these metabolites, although the closely related hypoglycaemic drug, glipizide, does show significant cross reactivity. The antiserum is produced in rabbits immunized with an antigen prepared by conjugating the diazonium salt of N-(p-amino-benzamidoethy1)-benzenesulphonylN’-cyclohexylurea to bovine serum albumin through diazocoupling. Dextran coated charcoal is used to adsorb the free glibenclamide, and separate it from the bound drug. It was found possible, with this procedure, to determine as little as 2.5ng per ml of glibenclamide in plasma by using l o p 1 samples without the need for extraction. Results obtained with dog plasma samples were comparable with those obtained by the less sensitive liquid chromatography method. The paper gives also the results of plasma assay carried out in diabetic

PAMELA GIRGIS TAKLA

352

patients on glibenclamide treatment. A patent has been taken

Lindner et al. 76 ,77 out in connection with this pr~cedure?~. have compared the determination of glibenclamide in the serum of diabetics by radioimunoassay and high-pressure liquid chromatography. References

"British Pharmacopoeia 1980", H.M. Stationery Office, London, 1980, 210, 773. "The Merck Index", Ninth Edition, Merck & Co. Inc. , 2. U.S.A., 1963, 4311. Aumueller, W., Bander, A,, Heerdt, R., Muth, K., 3. Pfaff, W., Schmidt, F.H., Weber, H. and Weyer, R., Arzneim.-Forsch., 16, 1640 (1966) Neth. Pat. Appl. 6,603,398, Sept. 19, 1966; Ger. Appl. 4. March 16 and Aug. 26, 1965 (to C.F. Boehringer and Soehne); Chem. Abstr., 66, 65289h (1967) Weber, H., Aumueller, W., Weyer, R., Muth, K., Schmidt, 5. F.H., Ger. Pat. 1,283,837, Nov. 28, 1968 (to Farbwerke Hoechst A.-G.); Chem. Abstr., 70, 47140f (1969) HajdG, P., Kohler, K.F., Schmidt, F.H. and Spingler, H., 6. Arzneim.-Forsch., 19, 1381 (1969) "The Pharmaceutica~CodeX",Eleventh Edition, The 7. Pharmaceutical Press, London, 1979 Abdel-Wahab, M.F., El-Kinawy, S.A., Farid, N.A. and 8. El-Shinnawy, A.M., Anal. Chem., 38, 508 (1966) Khlapopina, L.N. , Moroz, V.V. , Kgm.-Farm. Zh. , 10. 9. 132 (1976); Chem. Abstr., 85, 68196d (1976) Kuhnert-Brandstatter, M. , Kofler, A. and Kramer, G. , LO. Sci.Pharm., 42, 150 (1974) 11. Neth. Pat. A E l . 6,610,580, Jan. 30, 1967; Ger. Appl. July 27, 1965 (to Farbwerke Hoechst A.-G.); Chem. Abstr., 68, 1273311 (1968) 1.2" Hung. Tei-fes 6063, April 28, 1973, Appl. CI-1112, May 6, 1971 (to Chinoin Gyogyszer es Vegyeszeti Termekek Gyara Rt.); Chem. Abstr., 2, 104974t (1973) 13. Vogt, B.R., Bernstein, J., Weisenborn, F.L., Fr. Demande 2,138,112, Feb. 2, 1973; U.S. Appl. 144,678, May 18, 1971 (to Squibb, E.R. and Sons, Inc.); Abstr., 2, 18452q (1973) 14. Weber, H., Aumueller, W., Weyer, R., Muth, K,, Stach, K., S . African Pat. 68 05, 127, Jan. 20, 1969; Ger. Appl. Aug. 12, 1967 (to Farbwerke Hoechst A.-G.); Chem. Abstr., 71,91085m (1969) 1.5. Kantolahti, E., Malkonen, P . J . , Suom. Kemistilehti A, 46, 75 (1973) -

1.

e.

GLIBENCLAMIDE 16.

Wiseman, E.H., Chiaini, J . , Pinson, Jr. R., J. Pharm. Sci. 53. 766 (1964) Kuriki, T., Tsujiyama, T. and Suzuki, N., Bunseki Kagaku, 2, 872 ( 1 9 7 4 ) ; Chem. Abstr., 82, 35085b (1975) Spingler, H., Klin. Wschr., 1 9 5 7 , 3 5 , 533. Poirier, M.A. , Black, D.B. and Lovzing, E.G., 5 . J . Pharm. Sci., 15, 8 (1980) Chubb, F.L., Simmons, D.L., Can. J. Pharm. Sci., 7 , 28 (1972) Izgu, E. and Kafali, N., Bilim Kongr. Tip Arastirma Grubu Tebligleri, Turk. Bilimsel Tek. Arastirma Kurumu, 6th. 227 (1977) (Pub. 1 9 7 9 ) ; Chem. Abstr., -20601m(1980 j Kellner, H.M., Christ, O., Rupp, W. and Heptner, W., Arzneim.-Forsch., 19, 1388 (1969) Heptner, W., Kellner, H.M., Christ, 0 . and Weihrauch, D. , Arzneim.-Forsch., 1 9 (Suppl.) , 1 4 0 0 (1969) Christ, O.E., Heptner,W. and Rupp, W., Horm. Metab. Res. Suppl., 1_, 5 1 (1969) Rupp, W . , Christ, 0 . and Heptner, W., Arzneim.-Forsch 1 9 . 1 4 2 8 (1969) Rupp, W., Christ, 0. and Fulberth, W., Arzneim.-Forsch., 22, 4 7 1 (1972) Schmidt, H.A.E. and Petrides, Pl., Arzneim.-Forsch., 19, - 1 4 2 2 (1969) Fr. Pat. Appl. 2 , 2 0 4 , 4 2 5 , May 24, 1 9 7 4 ; Ger. Appl. P 22 5 3 3 1 8 . 2 , Oct. 3 1 , 1 9 7 2 , (to Farbwerke Hoechst. A.-G ) ; Chem. Abstr., 83, 33027f (1975) Rothe, W., Heinemann, H., Schmidt, F.H. and Betzien, G . , Ger. Offen. 2 , 3 4 8 , 3 3 4 , March 2 7 , 1 9 7 5 , Appl. P 2 3 48 3 3 4 . 3 , Sept, 2 6 , 1 9 7 3 ; Chem. Abstr., 83, 8 4 8 7 1 j ( 1 9 7 5 ) Borchert, H.H., Mueller, H. and PfeifG, S., Pharmazie, 31, 307 (1976) Fuccella, L.M., Tamassia, V, and Valzelli, G., J. Clin. Pharmacol., 13, 6 8 (1973) Balant, L., Fabre, J. and Zahnd, G.R., Europ. J. clin. Pharmacol.. 8. 6 3 (1975) KO, H., Royer, M.E,, Molony, B.A., Excerpta Med. Int. Congr. Ser. 382, 20 (1975) Bander, A., Pfaff, W., Ritter, K., Wohlfahrt, A. and Schmidt, F.H., Proc. Tegernsee Conference on the New Oral Antidiabetic Agent HB419, 27th-29th Jan. (1969) Crooks, M.J and Brown, K.F. , J. Pharm. Pharmac., 26, 304 (1974) Hsu Par-Lin, Joseph, K.H. and Luzzi, L.L., J . Pharm. Sci., - 63, 570 (1974) Brown, K.F. and Crooks, M.J., Can. J. Pharm. Sci., 9, 7 5 (1974) -

17. 18. 19.

20. 21.

22. 23. 24. 25. 26

I

27. 28.

29.

30. 31. 32.

353

L

I

33. 34.

35. 36. 37.

_

354

PAMELA GIRGIS TAKLA

38.

Brown, K.F.

and Crooks, M . J . ,

Biochem. Pharmacol.,

25, 1175 (1976) 39. 40.

41. 42. 43. 44. 45. 46.

47. 48. 49. 50. 51. 52. 53. 54. 55. .56

.

57. 58 59. 60. 61. 62. 63. 64.

M u e l l e r , W.E., W o l l e r t , U., R e s . Commun. Chem. P a t h o l . Pharmacol., 551 (1976) Samimi, H . , Loutan, L . , B a l a n t , L . , T i l l o l e s , M . , Fabre, J . , Schweiz. Med. Wochenschr., 107, 1291 (1977) Loutan, L . , Samimi, H., B a l a n t , L . , Favre, H . , F a b r e , J . , Schweiz. Med. Wochenschr., 108, 1782 (1978) B a l a n t , L . , F a b r e , J . , Loutan, L . , S a m i m i , H . , Arzneim. -Forsch., 29, 162 (1979) Schmidt, T H . , H r s t k a , V . E . , X I I e Congres I n t e r n a t i o n a l d e Therapeutique, Geneve 1973. C a s t o l d i , D . , Chinea, B . , T o f a n e t t i , O . , Farmaco, 2 7 1 (1978) Ed. P r a t . , B a l a n t , L . , Zahnd, G.R., Weber, F. and Fabre, J . , Europ. J . c l i n . Pharmacol., 11, 19 (1977) Fukuchi, H . , T s u k i a i , S . , K u G g a i , M. and K i t a u r a , T . , Hiroshima J. Med. S c i . , 2, 269 (1977) B i g l e r , F . , Rentsch, G., R i e d e r , J . , Denes, A . , J o u r n e e s Annu. D i a b e t o l . Hotel-Dieu, 333 (1973) Happ, J . , Nest, E., F r o e h l i c h , A . , S c h o e f f l i n g , K . , Beyer, J . , Verh. Dtsch. Ges. Inn. Med., 82, 776 (1976) Bander, A , , A u s t r a l i a n and New Zealand J F M e d . , 1 (Supp. 2 ) , 22 (1971) Chabria, N.L., Proc. Asia Ocean. Congr. E n d o c r i n o l . 5 , 2 , 432 (1974) -t h K a i s e r , D . G . , F o r i s t , A.A., Excerpta Med. I n t . Congr. S e r . 382, 31 (1975) S i l v e s t r i , S., Lucr. Conf. Nat. Chim. Anal., 3 r d , 1, 1 3 (1971) S i l v e s t r i , S . , Pharm. Acta Helv., 47, 209 (1972) Tammilehto, S . , Farm. Aikak., 82, 140 (1973) Agarwal, S.P. and Walash, M . I . , I n d i a n J . Pharm., 34, 109 (1972) Eichhorn, A. and Wagler, M., Z e n t b l . Pharm., Pharmakother. u . Lab.-diagnostik, 111, 1049 (1972) Guven, K . C . , B e r g i s a d i , N . , E c z a c i l i k Bul. 1 2 , 30 (1970) Thielemann, H., S c i . Pharm., 4 1 , 70 (1973) Surborg, K.H. and Roeder, E . , Pharmazie, 28, 485 (1973) Agarwal, S.P., Walash, M . I . , Blake, M . I . , I n d i a n J . Pharm., 35, 181 (1973) Walash, M . I . and Agarwal, S . P . , J . Drug Res., 5, 217 (1973) Schmidt, F., Dtsch. Apoth.-Ztg., 1 1 4 , 1593 (1974) Beyer, W.F., Anal. Chem., 4 4 , 1 3 1 2 1 9 7 2 ) Clarke, E.G.C., " I s o l a t i o n a n d I d e n t i f i c a t i o n o f Drugs" Volume 2 , The Pharmaceutical Press, London, 1975, p . 1045.

2,

33,

_ -

355

GLIBENCLAMIDE

65.

Castoldi, D. and Tofanetti, O., Clin. Chim. Acta, 93,

66. 67.

Becker, R., Arzneim.-Forsch., 27, 102 (1977) Adams, W.J. and Krueger, D.S., J . Pharm. Sci., 2,

1 9 5 (1979)

1138 (1979)

68. 69. 70.

71. 72. 73.

74.

Wahlin-Boll , E. and Melander, A., J. Chromatogr., 164, 541 (1979) Chroneos, I.D., Ph.D. thesis, University of Wales, June 1979 J.. Fresenius 2 . Reinauer, H a , L ndner. G.. Oldendoem. _ , Anal. Chem., 301, 110- (1980) Glogner, P . , Burmeister, P. and Heni, N., Klin. Wochenschr., 51, 352 (1973) Royer, M.E., KO, H., Evans, J.S., Johnston, K.T., Anal. Lett., 9 , 629 (1976) Glogner, P., Heni, N., Nissen, L., Arzneim.-Forsch., 27, 1 7 0 3 (1977) 221 (1979)

75. 76.

77 *

-

Kawashima, K., Kuzuya, T., Matsuda, A., Diabetes, 2 8 , Kawashima, K. and Kuzuya, T., Jpn. Kokai Tokkyo Koho 79 0 5 , 9 5 0 , Jan 17th, 1 9 7 9 , Appl. 77169,048, June llth, 1 9 7 7 ; Chem. Abstr. 9 0 , 1866372 (1979) Lindner, G . , Reinauz, H. , Kontrolle Plasmaspiegel Pharmaka, Workshop Rahmen Kongr. Laboratoriumsmed. 7 5 (1979) (Pub. 1 9 8 0.) ., Ed. Rudolf Sommer. Lindner, G., Herbertz, L., Reinauer, H., Laboratoriumsmedizin. 4. 34 (1980)

Dorothy K . Wyatt and Lee T. Grady 1. Description 1.1 Heroin 1.2 Heroin Hydrochloride 2. Physical Properties 2.1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectra 2.3 Ultraviolet Spectra 2.4 Mass Spectrum 2.5 Melting Range 2.6 Differential Scanning Calorimetry (DSC) 2.7 Solubility 2.8 Moisture Content 2.9 Specific Rotation 2.10 Crystal Properties 2.11 Polymorphism 3. Synthesis 4. Stability-Degradation 5. Metabolism 6. Pharmacokinetics 7. Methods of Analysis 7.1 Elemental Analysis 7.2 Color Tests 7.3 Microcrystalline Tests 7.4 Non-Aqueous Titrimetric Analysis 7.5 Chloride Titration 7.6 Phase Solubility Analysis 7.7 Thin-Layer Chromatography 7.8 Paper Chromatography 7.9 Gas Chromatography 7.10 High-Performance Liquid Chromatographic Analysis 8. Determination in Biological Fluids References

ANALYTICAL PROFILES OF DRUG SUBSTANCES, 10

357

358 358 358 359 359 359 366 370 370 370 370 373 373 374 374 374 377 377 377 379 379 379 380 380 381 38 1 38 1 381 388 388 388 397

358

1.

DOROTHY K. WYATT AND LEE T. GRADY

Description 1.1

Heroin

1.1.1

Name. Formula. Molecular Weieht Heroin is 3,6,-diacetoxy-7,8-dehydro-4,5 3,6,-diacetoxy-7,8-dehydr.0-4,5 epoxyThe CAS Iregistry :,egistry no. is 561-27-3 [l].

-. N-methylmorphinan t.hylmorphinan [4]. -.N-me

2' 1H23N05 molecular weight 369.4 OCCH3 1.1.2

ADDearance. Color. Odor

White crystals which turn pink and emit an acetic odor on prolonged exposure to air [ 2 ] . 1.1.3

Synonyms [Z] Acetomorphine Diacetylmorphine Diamorphine 7,8-Didehydro-4,5a-epoxy-l7-methylmorphinan-

3,6a-diol diacetate (ester)

1.2

Heroin Hydrochloride

1.2.1


Heroin hydrochloride is 3,6-diacetoxy-7,8dehydro-4,5 epoxy-N-methylmorphinan hydrochloride monohydrate [4]. The GAS registry no. i s 1502-95-0 [l].

&

cl-. N20 molecular C21H23N05-HC1 weight H20423.9

CH3CO0

OCCH3

HEROIN

1.2.2

359

Appearance. Color. Odor

The hydrochloride is an almost white, crystalline powder, odorless when freshly prepared but develops an odor characteristic of acetic acid on storage [50,51]. 1.2.3

Synonyms 3,6-di-O-acetylmorphine hydrochloride monohydrate [50] Diacetylmorphine hydrochloride [SO] Diamorphine hydrochloride [ 501 7,8-Didehydro-4,5a-epoxy-l7-methylmorphinan 3,6a-diol diacetate (ester) hydrochloride monohydrate 121

2.


Infrared Spectra

The infrared spectra are presented in Figure 1. The spectra were obtained from potassium bromide and potassium chloride dispersions of previously dried material (105', constant weight) using a Beckman 5260 grating infrared spectrophotometer. Principal bands are 1765, 1740, 1450, 1370, 1250, 1180 cm-I [61]. 2.2 2.2.1

Nuclear Magnetic Resonance Spectra Proton Spectrum The proton spectra are presented in Figure 2.

d

.rl

e,

0 k

c

a,

5

r;0

c .ri

a,

0 k

c 0

w


362

F i g . 2a.

-

P r o t o n NMR s p e c t r u m of heroin base.

.-_A,-

1200 Fig. 2b.

P r o t o n NMR s p e c t r u m of heroin h y d r o c h l o r i d e .

HEROIN

363

Spectral assignments are listed in Table I Table I H ' NMR Spectral Assignments for Heroin [ 4 8 ] Chemical Shift PPm ( 6 )

Multiplicity

Characteristic of proton acetyl N-methyl 10

2.27, 2.11 2.88 3.33 3.70

9

5.45

a

5.21

596 7 1 2

5.73 6.87 6.66 2.2.2

Carbon-13 Spectrum The carbon-13 spectra is presented in Figure 3.

Spectral assignments are listed in Table XI. Table I1 I3C NMR Spectral Assignments for Heroin 1711 Chemical Shift

Multiplicity -

Carbon Number

119.1

d

1

121.6

d

2

132.0

S

3

364

0 k

.ti Q,

c 0

k

7

E

k c, 0

a

d)

co

m

r:

d

k

0 P 0

(d

.. (d

m

365

's

1

i

P)

P

k 0

.rl

d

c 0

E

P

C

2 .A Q,

0 k

c 0

k

0

f

366

DOROTHY K. WYATT AND LEE T. CRADY

Table I1 -- Cont'd. Chemical Shift

Multiplicity

Carbon Number

S

4 5 6 7 8 9 10 11 12 13 14 15 16 NCH3

149.1 88.5 67.9 129.2 128.2 58.7 20.4 131.5 131.2 42.6 40.4 34.9 46.3 42.8 20.4 168.2 20.4 170.2 2.3

d d d d d t S S S

d t t 4 4

3CH3CO 3CH3CO

S

6CH3C0 6CH3CO

4 S

Ultraviolet spectra

The ultraviolet absorption spectra of heroin and heroin hydrochloride are shown in Figure 4 for the solvents listed in Table 111 (1 in 10,000 solutions used). Table 111 Solvent 0.1 N hydrochloric

X Max (nm)

278

4.8

cm

279

4.8

cm

281

5.3

4.3

[53]

4.3

[53]

4.9

[53]

39 [41

[53];

1% =

ethanol

Absorptivity Heroin HC1

[5];

1% =

acid 0.1 N sulfuric acid

Heroin

52 141

[53];

El% = 54 [68] 1 cm

HEROIN

Fig.

367

4a.

U l t r a v i o l e t spectrum i n 0.1 N hydrochloric a c i d ( r e p r e s e n t a t i v e of h e r o i n o r h e r o i n hydrochloride).

368

Fig.


4b. U l t r a v i o l e t s p e c t r u m i n 0 . 1 N s u l f u r i c a c i d ( representative o f heroin o r heroin hydrochloride 1 .

369

HEROIN

-

Fig.

4c.

--4

U l t r a v i o l e t spectrum i n 95% ethanol ( repr e s e n t a t i v e of h e r o i n o r h e r o i n hydrochloride).

370


2.4

Mass Spectrum

The electron impact ionization spectrum is given in Figure 5, and the fragmentation pattern is presented in Table IV [6]. A Finnigan 3000 Peak Identifier mass spectrometer was used. The masslcharge (m/e) range scanned was 40 to 400 atomic mass units. The ionization potential was 70 eV. Table IV Mass Spectrum Fragmentation Pattern of Heroin [47, 541

m/e 369 327 268 310

2.5

Species M+ COCH2 M+ - CH3CO0 and COCH2 M+ - CH3CO0 (c6 acetyl group) cleavage followed by of peripheral groups

Melting Range

The melting point of a heroin sample is about 170" 141. It is also given as 173°C [2]. Additional melting ranges of 170"-172"C, 171"-174"C, and 172"-173°C and a melting point of 173°C have also been reported [24]. (See 2.11.) The melting range of a heroin hydrochloride sample is between 229" and 233" [51,4]. It is also described as 243-244" [5J

.

2.6

Differential Scanning Calorimetry (DSC)

The DSC of heroin and heroin hydrochloride are shown in Figure 6 [53J

.

2.7

Solubility

The approximate solubilities obtained at room temperature are listed in Table V.

n

4 .h l

n

-....

k

b

L,

a,

a CO

3 E

372


I

--I-

I .

~

I

1 F i g . 6 a . 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 o f heroin base h e a t i n g r a t e : 5 / m i n . ; 25 t o 200 C.

I

I

j A,

I

f-

- 1

\ .

.

i

I

I I

F i g . 6 b . 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 of heroin h y d r o c h l o r i d e h e a t i n g r a t e : 5 / m i n . ; 2 5 t o 300 C .

HEROIN

373

Table V Solubility Data of Heroin at Room Temperature Approximate Solubility (gm) Heroin Heroin Hydrochloride

Solvent

1 in 1 in 1 in 1 in

water ethanol ether chloroform alkali 2.8

2.8.1

1700 [2,4] 3 1 [2,4] 100 [2,4] 1.5 [2,4] soluble [ 2 ]

1 in 1.6 [51,4]; 1 in 2 [ 5 2 ] 1 in 12 [51,4]; 1 in 11 [ 5 2 ] insoluble [51,4,52] 1 in 1.6 [51,4]

Moisture Content Karl Fischer Titration

An accurately weighed sample of heroin or heroin hydrochloride is dissolved in methanol which has been titrated to end-point and titrated with Karl Fischer reagent using the dead stop end-point technique and a 20-second delay (heroin hydrochloride exists as the monohydrate) [ 5 3 ] . 2.8.2

Loss on Drying

Heroin has been dried at 105" to constant weight [51.

Heroin hydrochloride has been dried at 105" to constant weight [ 5 1 ] . 2.9

Specific Rotation

The specific rotation of heroin determined in 0.015 N methanolic hydrochloric acid at a concentration o f 0.5% a t 25°C is given in Table VI [5,53]. Table VI Specific Rotation data Wavelength (nm) 589 578 546 436

[a]

25"

Specific Rotation ( " ) Heroin [ 5 ] Heroin Hydrochloride [ 5 3 ] -147 -154 -175 -303

-1 33 -139 -159 -27 5


374

The specific rotation of heroin in methanol [a]i5" is -166" (C = 1.49) [ 2 ] . The s e ific rotation of heroin hydrochloride in water at 24" [a]!56 is -156" (C = 1.044) [2]. 2.10

Crystal Properties

The crystal structure, configuration and bond distances are presented in Figures 7 , 8, and 9. Heroin crystals were formed after addition of heroin hydrochloride t o aqueous sodium acetate solution. Clear hexagonal crystals of diacetylmorphine free base were obtained. Reflections were measured with a Syntex P2 diffractometer with a 0-20 technique on a crystal 0.7 x 0.2 x 0.2 mm [49]. 2.11

PolvmorDhism Heroin can exist in two polymorphic forms.

Form

I, consisting of rods, oblique plates, and needles, has a

melting point of 172"-173°C. Form 11, consisting of spherulites, melts at about 168°C and is readily converted into Form I [24]. 3.

Svnthesis

1.

Synthesis from morphine [5].

HEROIN

Fig. 7. Arrangement of the molecules in the unit cell (0 oxygen). From an origin in the lower left front corner, c is to the right, b is vertical and a is into the page.

Fig. 8. Configuration drawing of diacetylmorphine with endocyclic torsion angles for rings A, B y C and D.

Fig. 9. Bond distances (A). The estimated average standard deviation in bond length is 0.014 A in diacetylmorphine.

DOROTHY K . WYATT AND LEE T. CRADY

376

2.

Synthesis from Sinomenine [ 2 9 ] .

,c1!3 Ti

catalytic S "

3

reduction

sinornenine

ec;;

3

HEROIN

4.

Stability

377

-

Degradation

it

Heroin is rapidly hydrolyzed in alkaline solutions. is rapidly hydrolyzed in vivo after mixing with blood to 0monoacetylmorphine and then at a slower rate t o morphine. Heroin also degrades to 06Inonoacetylmorphine in buffered aqueous solutions (pH 7.4) at 23°C. The hydrolysis is more rapid at higher pH value (pH 6.4). No evidence of further conversion to morphine at pH 7.4 is observed in 24 hours [7]. Heroin hydrolyzes to 06-acetylmorphine in 0.5 M sodium carbonate with a half-life Tf only 4.2 min. Subsequent hydrolysis to morphine has a half-life of 55.5 min. 191. Half-life for hydrolysis in human blood is 12.6 min.; in serum, 19.8 min. [9]. In pH 4 phosphate buffer, the halflife was 415 min.; in fresh dog plasma, 8 min. [9]. Heroin stability increases with increased alcohol content in Brompton mixtures [28]. Heroin reportedly is most stable at pH 4.0-4.5 [28] and at pH 4.3 [39]. 5.

Metabolism

Heroin is a short-acting (2 hours) narcotic analgesic. It is rapidly hydrolyzed in vivo by serum cholinesterase [38] to 06-monoacetylmorphine and then at a slower rate to morphine-[4]. Heroin rapidly passes out of the blood [32] after conversion to 06-monoacetylmorphine and appears in the brain as 06-monoacetylmorphine where it is sLowly hydrolyzed to morphine. Heroin and 06-monoacetylmorphine have a considerably greater ability to penetrate the blood brain barrier than does morphine which is the probable explanation for the higher potency of heroin [38]. 06monoacetylmorphine, morphine, and morphine 3-glucu~onideare the major metabolites of heroin excreted in the urine [9]. Minor or negligible amounts of normorphine and its glucuronide as well as morphine 6-glucuronide have been determined in urine [9]; dihydromorphinone [26], 6-acetylmorphine 3-glucuronide [26], and norcodeine [17] have also been detected in urine. Additional possible metabolic pathways of opiates in man are presented in Figure 10 [17]. 6.

Pharmacokinetics

Heroin is rapidly hydrolyzed to morphine and other metabolites and is rapidly excreted. Heroin, 6-acetylmorphine, morphine, the sum of morphine and 6-acetylmorphine and total normorphine, determined 24 hours after initial IV administration of a 10 mg/70 kg dose, was found to be 0.5, 1.5, 7.2, 54 and 4%, respectively, of administered dose.

Excretion products -free morphine morphine conjugates --free nomorphine normorphine conjugates ii

iv

16

H iii

I\ICH3

--free norcodeine norcodeine conjugates 4

--free codeine codeine conjugates

ii

CX30

OH

CH3O

OH

Figure 10: P o s s i b l e metabolic pathways of o p i a t e s i n man(ixP1-methylation; ii=EI-dimethylation; i i i = 0-methylation; i v = 0-demethylation)

HEROIN

379

Eighty-eight percent of free morphine and 84% of the total morphine (including morphine glucuronide) found in urine was excreted within the first eight hours [ 4 7 ] . Of the morphine found in the urine, 88% was bound as the glucuronide and 11% was free morphine [ 6 2 ] (50-60% bound and 7% free [ 2 6 ] ) . The amount of heroin detected in the urine as morphine after a single intramuscular injection of 5 mg is about one third that detected after a single intramuscular injection of 1 5 mg of morphine. Methods of Analysis

7.

7.1

Elemental Analysis

Heroin

Heroin Theoretical % [ 2 ]

carbon hydrogen nitrogen oxygen chlorine 7.2

Heroin HC1 (anhydrous) Theoretical % [ 2 ]

68.28 6.28 3.79 21.66

--__

62.14 5.96 3.45 19.71 8.74

Color tests

Agent

Color

Ref.

1.

sulfuric acidformaldehyde (Marquis)

purple (sensitivity 0.05 wg)

496

2.

ammonium molybdate

red-purple-bluelight green (sensitivity 0.05 Lig)

4

3.

ammonium vanadate

faint blue-gray (sensitivity 1.0 rig)

4

4.

Vitali's test

faint yellow & faint yellow/orange (sensitivity 1.0 $18)

4

5.

nitric acid base (heroin HC1)

yellow green

20,51

deep green

6

purple

42

6. Mecke test 7.

cobalt thiocyanate

380


Agent

Color

Ref.

8.

sulfuric aciddeep blue potassium hexacyanoferrate (111)iron (111) chloride heroin HC1)

51

9.

nitric acidphosphoric acid

57

7.3

yellow to red brown (depending on concentration)

Microcrystalline Tests

Agent

Crystal type

Ref.

mercuric iodide sodium acetate platinum chloride goId bromide mercuric chloridehydrochloric acid iodine-potassium iodide bromauric acid-phosphoric acid-hydrogen bromide

needles hexagon needles needles blades and needles

20 20 6,20 6,20 6,20

7.4

blades and needles amorphous precipitate followed by irregular dichroic plates or blades

70 70

Non-aqueous Titrimetric Analysis

An accurately weighed sample of heroin is dissolved in glacial acetic acid and titrated to the potentiometric end-point with 0.1 N acetous perchloric acid, using a glass indicating electrodeand a calomel reference electrode filled with 0.02 N lithium chloride in glacial acetic acid. The titrant i s standardized against dried potassium biphthalate. A blank titration is run [5]. An accurately weighed sample of heroin hydrochloride is dissolved in glacial acetic acid; mercuric acetate is added. The solution is titrated to the potentiometric end-point with 0.1 N acetous perchloric acid using a glass indicating electrodeand a calomel reference electrode filled with 0.02 N lithium chloride in glacial acetic acid. The titrant i s standardized against dried potassium biphthalate. A blank titration is run [53]. Crystal violet indicator may also be used [Sl].

38 1

HEROIN

7.5

Chloride Titration

An accurately weighed sample of heroin hydrochloride is dissolved in 1.5 N sulfuric acid and titrated to the potentiometric end-point with 0.1 N silver nitrate using a silver indicating electrode and a mercurous sulfate reference electrode. The titrant is standardized with dried sodium chloride and a blank titration is run [ 5 3 ] . 7.6

Phase Solubility Analysis

The United States Pharmacopeia procedure was followed [ 5 2 ] . The heroin solvent was (3:l) hexane:dioxane (solubility 13 mg/g) [ 5 ] . Dioxane was used for heroin hydrochloride (solubility 26 mg/g) [ 5 3 ] . The solvents were commercial distilled-in-glass solvents which had been degassed prior to mixing. The bath temperature was 25O, rotation was 28 rpm. 7.7

Thin-layer Chromatography

Thin-layer chromatography has frequently been used for the analysis of heroin. Methods of detection and solvent systems are listed in Table VII. 7.8

Paper Chromatography

Ascending paper chromatography was accomplished using Whatman #l paper which had been buffered by dipping into a 5% solution of sodium dihydrogen citrate, blotting, and drying at 25" for 1 hour. The solvent consisted of 4.8 g of citric acid in a mixture of 130 ml of water and 870 ml of 1-butanol. 2.5 p 1 of a 1% solution in 2 N acetic acid, 2 N hydrochloric acid, 2 N sodium hydroxide, o r ethanol were spotted on the paper. Visualization was accomplished using ultraviolet light or iodoplatinate spray (Rf = 0.33) [ 4 ] . Reversed phase ascending paper chromatography was conducted using Whatman 81 or 83 paper impregnated by dipping into a 10% solution of tributyrin in acetone and drying in air. Acetate buffer pH 4.58 was used as solvent. Samples were spotted from a 1 to 5% solution in ethanol or chloroform. Iodoplatinate spray was used for detection (Rf = 0.84) [ 4 ] . An additional reversed phase ascending paper chromatography system consisting of phosphate buffer (pH 7 . 4 ) solvent and Whatman "1 or #3 paper impregnated with a

Table VII Thin-layer Chromatography of Heroin

w

Rf x 100

Ref.

45, 38, 50

4 ,5 ,20 ,56

76, 60

4 , 20

A,B,C,D

35,44 ,50 , 35,35

4,5,20,21, 22

benzene-dioxane-ethanol25% aqueous ammonia (1O:g:l:l)

A,B,C,D

46,76

5,21

silica gel F-254

methanol

A,B,C,D

38

5

cellulose

2-propanol-water-glacial acetic acid (8:l:l)

A,B,C,D

72

5

silica gel

butyl ether-ethyl etherdiethylamine (45:45:10)

A

44

6

silica gel

chloroform-dioxane-ethyl acetate-aqueous ammonia (25:60:10:5)

A

85

6

silica gel

chloroform-methanol (9:l)

61,--

6,56

Plate

Solvent

silica gel

methano1:aqueous ammonia (100:1.5)

silica gel

aqueous ammonia-benzenedioxane-ethanol (5:50:40:5)

silica gel

acetic acid-ethanol-water (30:60:10)

silica gel F-254

Method of Detection A,B,C,D,E,J B

A,C,J

---

Table VII

--

Cont'd.

Plate

Solvent

silica gel

chloroform saturated with ammonia-methanol (18:l)

A

silica gel.

chloroform-methanol (8:2)

E

silica gel

chloroform-cyclohexanediethylamine (8:10:3)

HPTLC-silica gel F-254

toluene-methanol-aqueous ammonia (50:50:1)

60

19

HPTLC-silica gel F-254

2-propanol-n-heptaneaqueous ammTnia ( 5 0 : 5 0 : 1)

10

19

silica gel G+ 0.1 M KOH

cyclohexane-benzenediethylamine (75: 15:10)

22

21


39

21


20

21

Method of Detection

Rf x 100

Ref. -

70

6

9,67

18

silica gel+ 0.1 M NaHS04

methanol

-------

24

21

silica gel+ 0.1 M NaHS04

95% ethanol

-------

9

21

Table VII -- Cont'd.

kP?.

Rf x 100

Ref.

E

35,35

21,22

E

37,37

21,22

43

61

alcohol-n-butyl etherwater ( 8 0 : 7 : 1 3 )

15,15

21,22

silica gel

n-butanol-glacial acetic

61,61

21,22

silica gel

n-butanol-concentrated -

32,32

21,22

90,90

21,22

95,65

21,22

Plate

Solvent

Method of Detection

silica gel

methanol-n-butanol-benzenewater ( 6 0 y 1 5 : 1 0 : 1 5 )

silica gel

ethanol-pyridine-dioxanewater ( 5 0 : 2 0 : 2 5 : 5 )

cellulos e (previously dipped in 5% sodium dihydrogen citrate and dried one hour)

citric acid ( 4 . 8 g) in water-n-butanol (130:870) -

silica gel

A,E, K

acid-water ( 4 : 1 : 2 ) HC1

saturated with water ( 9 : l )

MN-cellulose powder 3 0 0 G

methanol-n-butanol-benzenewater ( 60y15 :10 :15)

MN-cellulose powder 3 0 0 G

-t-amyl

alcohol-n-butylether-water ( 8 0 : 7 : 1 3 )

Table V I I Plate

P

silica gel

Solvent chloroform-dioxane-ethyl-

--

Cont'd. Rf x 100

Ref.

E

67

21

E

73

21

Method of Detection

acetate-aqueous ammonia (25 :60: 10: 5)

silica gel

ethanol-chloroform-dioxanepetroleum ether-benzeneaqueous ammonia-ethyl acetate (5:10:50:15:10:5:5)

W 07 00

silica gel

ethyl acetate-benzene-aqueous ammonia (60:35:5)

19

21

silica gel

ethanol-dioxane-benzeneaqueous ammonia (5:40:50:5)

76

22

silica gel

acetone-methanol-aqueous ammonia (50:50:1)

58

23

silica gel

chloroform-acetone-aqueous ammonia (50:50:1)

64

23

silica gel

ethanol-dioxane-benzeneaqueous ammonia (5:40:50:5)

76

23

silica gel

ethanol-di-n- bu tyl etheraqueous ammonia (60:35:5)

11

23

Table V I I Plate

Solvent

silica gel

chloroform-acetone (9:l)

--

Cont'd.

Method of Detection A,C,J

Rf x 100

Ref.

--

56

Method of Detection A.

B. C. D. E. F. G. H.

I. J. K.

shortwave ultraviolet light longwave ultraviolet light 0.5% iodine in chloroform acidified iodoplatinate followed by exposure to ammonia vapor potassium iodoplatinate Dragendorff's reagent followed by heat (120OC) for 5 minutes and spraying with 10% sulfuric acid. potassium permanganate cobalt thiocyanate bromocresol green iodine in methanol + copper chloride (Ludy-Tenger) dilute hydrochloric acid

387

HEROIN

10% solution of tributyrin in acetone was used. The samples were spotted using a 1 to 5% solution in ethanol or chloroform. Iodoplatinate spray detection was used (Rf = 0.12)

[4].

Paper chromatography was also conducted using the systems in Table VIII. Table VIII

*

Paper Chromatography of Heroin Paper + Treatment

Solvent

Whatman #1 impreg- chloroform nated with formaldehyde and 1% acetic acid

Detection Rfxl00

Ref.

-----

76

21

-----

33

21

74

21

Whatman #l impregnated with 5% zirconium phosphate

5% acetic acid

Whatman #I.

l-butanolCYE,G,H,I glacial acetic acid-water (12: 3 :5)

Whatman #I.

1-butanol-1 N CyEyG,HyI sodium acetate1 N hydrochloric acih (7:120:60)

89

21

Whatman #l buffered with 5% sodium dihydrogen citrate

l-butanolCYEY GY H,1 glacial acetic acid-water

32

21

16

21

(12:3: 5)

Whatman #1 buffered with 5% sodium dihydrogen citrate

1-butanol-1 N C,E,G,H,I sodium acetate1 N hydrochloric acid (7:120:60)

*Spotting solvent was

not given.


388

7.9

Gas Chromatography

Gas chromatographic systems used for analysis are listed in Table IX. Flame ionization detection was used unless otherwise specified.

7.10

High-Performance Liquid Chromatographic Analysis

High-Performance Liquid Chromatography has been used extensively for the analysis of heroin. The various HPJX systems used for the analysis are given in Table X8. Determination in Biological Fluids

P1asma- Serum Organic solvent extracts were obtained with ethyl acetate-isopropanol (85:15), or benzene-butanol (85:15) from plasma mixed with pH 8.9 carbonate buffer. The extracts were dried and reconstituted in acetone and analyzed on a bonded phase column using a mobile phase consisting of methanol (0.1% (NH4)2C03 and 0.01 M (NH4)2HP04, pH 6.98) (55:45) and W detection (254 nm). Heroin, 6-0acetylmorphine, and morphine were separated [9]. Similarly, TLC was used for separation of heroin, morphine, 6-0acetylmorphine and morphine glucuronide [9] on silica gel plates using chloroform-methanol (80:20) and iodoplatinate for visualization. Blood Heroin has been analyzed by gas chromatography following the extraction scheme presented for urine below [62]. Silyl derivatives was analyzed on QF-1 or SE-30. Urine The following procedure was used for the quantitation of heroin and morphine by gas chromatography. Extraction from sodium bicarbonate solution into ethyl acetate is followed by extraction with 0.05 N hydrochloric acid. Ammonium hydroxide, sodium chloride, and sodium bicarbonate are added to the hydrochloric acid solution. Extraction with ethyl acetate, silylation and analysis on QF-1 and SE30 using flame ionization detection follows [62]. Metabolites in urine were also determined after acid hydrolysis and silylation followed by gas chromatography [26]. An alternative analysis using thin-layer

Table IX Gas ChromatoeraDhv of Heroin Flow (ml/min);

Column

SUDDOrt

Mesh

Length

Temp. ( " )

1% SE-30

Anakrom ABS

100-120

6ftx4mm glass

250

80; argon

4

2.5% SE-30

Chromosorb WAW HMDS

80-100

5ftx4mm glass

225

50; nitrogen

4

5% SE-30

Chrom0so rb WAW

60-80

5 ft x 118" 230 stainless steel

30; nitrogen

4,21

3% ov-1

Gas Chrom Q

100-120

1.2 m x 4 mm glass

210

5 0 ; helium

5

Carrier Gas

Ref.

3% OV-17

--------

----

6ftx4mm glass

250

6

3% ov-1

--------

----

6ftx4mm glass

250

6

----

0.6 m x 4 mm glass

220

40; 5% methane in argon

7

100-120

1.83 m x 4 mm glass

220-240

100-120; nitrogen

8,14

10% ov-1*

Chromosorb GHP

3% OV-17*

Gas Chrom Q

Table IX -- Cont'd. Flow (ml/min);

W CD 0

Mesh

Column

Support

0.04% SDBS 0.02% FFAP 0.06% SP-525

glass beads

70-90

3% OV-17

Gas Chrom Q

80-100

3% OV-17

Chromosorb WHP

3%

ov-1

Chromosorb W

Length

Temp. ( " )

1 . 6 m x 3 mm glass

240

0.5 kg/cm2; nitrogen

13

1 m x 6.35 mm 0.d. glass

235

55; nitrogen

16

Carrier Gas

Ref.

18

80-100

1.2 m x 6.35 glass

250

60; nitrogen

20

1.2 m x 6.35 mm 0.d. glass

280

60; nitrogen

20

mm 0.d.

3% OV-17

Chromosorb W

80-100

5% SP-2401-DB

Supelcoport

100-120

1.2 m x 2 mm i.d. glass

250

60; helium

55

5% SP-2401-DB

Supelcoport

100-120


255

50 ; he1ium

55

3% SP-2401-DB

Supelcoport

100-120


230, 245

55

Table IX -- Cont'd. Column

Support

3% OV-25

Gas Chrom Q

Length

Temp. ( " )

Flow (ml/min); Carrier Gas

Ref. -

1.8 m x 3.18 mm stainless

240

30; nitrogen

20

1.8 m x 3.18 mm stainless steel

240

30; nitrogen

20

1.83 m x 4 mm glass

210, 225, 250

(argon 6-ionization)

21

1.83 m x 3 mm glass

175, 200, 225

(argon B-ioniza-

21

t ion)

100-120

0.92 m x 3.2 glass

220, 250

HF I

21

80-100

5ftx4mm i.d.

200, 250

60; nitrogen

61

Mesh 80-1 00

steel

6% Dexsil 400 Gas Chrom Q

80-100

-----

1% SE-30

Chromosorb W

0.1% polyethylene glycol 9000 + 1.15% SE-30

----Chromosorb P washed with concentrated HC1 and methanolic potassium hydroxide and treated with hexamethyldisilazane

W

E

1%Hi EFF8B

Gas Chrom P

3% cyclohexChromosorb W ane dimethanol succinate

Table IX -- Cont'd. Flow (ml/min); Carrier Gas

Ref.

Column

Support

Mesh

Length

Temp. ( " )

3% SE-30**

Chromosorb WHP

100-120

5.3 ft x 2 mm i.d.

218

95% of controller; 62 nitrogen

2.7% QF-1

Chromosorb WHP

100-120

9 ft x 2 m i.d.

218

60% of controller; 62 nitrogen

3% OV-175% SE-30 (1: 1)

Varoport 30 (OV-17) Chromosorb WAW

80-100

6 ft x 2 i.d.

temperature 30 ml/min; helium program: 250 (12 min); 10/ min, 280 (12 min)

65

225

67

UUII

(SE-30)

0

co w

3.8% UCW-98

* **Derivatized Derivatized

Chromosorb WHP

80-100

6 f t x 4 mm

40 ml/min; helium

with heptafluorobutyric acid anhydride in acetonitrile, 5 min., with BSA.

60°C.

Table X High-Performance Liquid Chromatographic Systems for Heroin

Flow/ Temperature Pressure

Detector ( A nm)

Ref.

35"

1000 psi

UV ( 2 5 4 )

5

methanol-(0.1% ammonium carbonate 0.01 M ( N H 4 ) HP04) pH 6.% ( 6 : 4 3 ; ( 5 5 : 4 5 )

ambient

2 ml/min

uv

9

0.2 N aqueous ammonia

ambient

3 ml/min

-------

12

UBondapak c18 acetonitrile-(aqueous buffer containing 0.75 g ammonium acetate) (65:35)

ambient

1.5 ml/min

UV ( 2 8 0 )

15

LiChrosorb Li 6 0 ( 5 ~ )

ambient

1.5 ml/min UV ( 2 5 0 ) 175 bar pressure

18

Column

Mobile Phase

SCX (1.0 m)

0.4-1.4 M sodium perchloratein 0.01 M pH 6.8 aqueous phosphate buffer containing 10% ethanol

bonded phase

LiChrosorb Si60 (10 vm)

% w

diethyl ether-isooctane-methanoldiethylamine ( 5 2 . 8 : 35:12:0.2)

(--)

Table X Column

Mobile Phase

--

Cont’d.


Detector (A nm)

Ref.

acetonitrile-(0.015 potassium phosphate adjusted to pH 3.0 with 2 N phosphoric acix) (1 :3)

ambient

0.8 mljrnin (620 psi)

UV (235)

28

pBondapak c18 acetonitrile-(0.015 M (heroin KH2P04 adjusted to hydrochloride) pH 3.5 with 2 N phosphoric acid) (3:7)

ambient

1 ml/min

UV (235)

39

Whatman acetonitrile-water Partisil-10 with 0.1% (NH4)2C03 ODS (heroin (6:4) hydrochloride)

ambient

2 ml/min

UV (254)

40

UBondapak c18

M monobasic

(800 psi)

UBondapak C18

(50% methanol/0.05 M phosphate buffer pH6.2)-methanol; 0-100% methanol, l%/min linear gradient

ambient

1.2 ml/min

UV (254)

54

UBondapak c18

(50% methanol/0.05 M phosphate buffer pH7.4)-methanol; 0-100% methanol, l%/min linear gradient

ambient

1.2 rnljmin

UV (254)

54

Table X

~

--

Cont'd.


Detector (A nm)

Ref. -

(0.2 M H3BO3 adjusted to pH 9.7 with 40% sodium hydroxide)-(0.2 H3B03acetonitrile-z-propanol (86:12: 2) adjusted to pH 9.8 with 40% sodium hydroxide); 0-100% linear gradient

ambient

2 ml/min

UV (270)

60

Corasil I1

hexane-(chloroformmethanol-diethylamine (100:300: 1)) gradient

ambient

600 psi

UV (254)

64

Merckosorb Si-60

chloroform-methanol (9:1, 8:2, 7:3)

20"

50-250 kg/cm2

UV (254)

68

Merckosorb Si-60

diethylether-methanol (8:2, 7:3, 6:4)

20"

50-250 kg/cm2

UV (254)

68

Column

Mobile Phase

Zipax SCX

01 CD

396


chromatography with a 1-butanol-acetic acid-water (35:3:10) system on silica gel plates and ethyl acetate-methanolammonium hydroxide (17:2:1) on silica gel plates has also been reported [26]. Plates were visualized using iodoplatinate spray. Morphine metabolites in urine were also analyzed after incubation with acid followed by adjustment to basic pH and benzene extraction. Thin-layer chromatography was then done using ethanol-benzene-1,4dioxane-concentrated aqueous ammonia (50: 40: 5: 5) and 1,4dioxane-chloroform-ethyl acetate-concentrated aqueous ammonia (60: 25: 10: 5) systems, silica gel plates, and potassium iodoplatinate spray [17,66].

Acknowledeements The authors wish to thank the chemists of the USP Drug Research and Testing Laboratory for experimental data and Ann K. Ferguson for providing the computerized literature search, and William K. Wyatt and Barbara A. Bowman for their assistance.

HEROIN

397

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. l

~~~

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HEROIN

399

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29.

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33.

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36.

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39.

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40. Reuland, D.J., Trinler, W.A. ,"An Unequivocal Determination of Heroin in Simulated Street Drugs by a Combination of High Performance Liquid Chromatography and Infrared Spectrophotometry Using Microsampling Techniques," Forensic Sci. 11 195 (1978). 41.

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43.

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44. Garrett, E.R., Gurkan, T., "Pharmacokinetics of Morphine and Its Surrogates I: Comparisons of

Sensitive Assays of Morphine in Biological Fluids and Application to Morphine Pharmacokinetics in the Dog," J. Pharm. Sci. 67 1512 (1978).

HEROIN

45

a

40 1

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'I

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62.

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HEROIN

403

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HYDROCHLOROTHIAZIDE Hans Peter Deppeler 1. Description 1,1 General Information 1.2 Nomenclature 1.3 Formula and Molecular Weight 1.4 Appearance 1.5 Official Compendia 1.6 Other Compendia 2. Physical Properties 2.1 Spectra 2.2 Physical Properties of the Solid State 2.3 Solubility 2.4 Ionisation in Aqueous Solution 3. Synthesis 4. Stability and Degradation 4.1 Bulk Stability 4.2 Solid-Solid Interactions 4.3 Stability in Solution 5. Methods of Analysis 5.1 Elemental Analysis 5.2 Identification 5.3 Colorimetry 5.4 Ultraviolet Spectrophotometry 5.5 Phosphorimetry 5.6 Fluorimetry 5.7 Polarography 5.8 Titration 5.9 Chromatography 5.10 Electrophoresis 6. Interferences of Hydrochlorothiazide in Analytical Methods 7. Pharmacokinetic and Metabolic Studies 7.1 Analytical Methods Used for Biological Material 7.2 Absorption 7.3 Distribution 7.4 Basic Pharmacokinetics 7.5 Bioavailability 8. Acknowledgements 9. References

ANAI.Y?’Ii:AI. I’ROFII.ES OF DRUG SURSTANCES. 10

405

406 406 406 407 407 407 407 407 407 419 422 424 424 425 425 425 425 426 426 426 426 427 427 428 428 428 429 430 432 432 432 432 433 433 434 435 436

HANS PETER DEPPELER

406

1.

Description

1.1

General Information

Research in sulfonamide chemistry has brought a rich yield of valuable therapeutics. One of the great successes was the discovery of the benzothiadiazines as potent diuretics of low toxicity(1). In 1958 De Stevens et a1.(2) reported on the condensation product of 4-amino-6-chloro-3,5-disulfonamide and formaldehyde which was found to be identical with the hydrogenation product of chlorothiazide(3) and which soon became a widely used saluretic: Hydrochlorothiazide 1.2

Nomenclature

1.2.1 Chemical Names Hydrochlorothiazide is the recommended international nonproprietary name(4) of 6-Chloro-3,4-dihydro-7-sulfamoyl-2H-1,2,4-benzothiadiazine l,l-dioxide(5) or 6-Chloro-3,4-dihydro-2H-lf2,4-benzothiadiazine-7sulfonamide l,l-dioxide(5,6) or 6-Chloro-7-sulfamyl-3,4-dihydro-1,2,4-benzothiadiazine l,l-dioxide(5) or 2H-1,2,4-Benzothiadiazine-7-sulfonamide, 6-chloro-3,4-dihydro-,l,l-dioxide(6) CAS registry number: 58-93-5 1.2.2 Trade Names The Merck Index(5) quotes 28, and Index Nominum(7) 64 trade names not including the combinations with other active substances. Therefore, only a few examples can be listed here. Trade names including combinations in different countries: France: Adelphan-Esidrex, Esidrex, Esimil, Hydromet, Moduretic Germany(BRD1: Di-Chlotride, Diu 25, Esidrix Germany(DDR1: Disalunil, Urodiazin Great Britain: Direma, Esidrex, Hydrosaluric, Sa1upres Japan: Esidrix, Dichlotride Esidrix, Hydrodiuril, Oretic, USA : Serapes, Thiuretic.

IIYDROCHLOROTHIAZIDE

1.3

Formula and M o l e c u l a r Weight 0

0

0

0

H

c7H8C1N3O4S2

1.4

M o l e c u l a r Weight 297.73

Appearance

White, o r p r a c t i c a l l y w h i t e , p r a c t i c a l l y odourless, c r y s t a l l i n e powder(6). S l i g h t l y b i t t e r taste( 8 ) . 1.5

O f f i c i a l Compendia

Monographs o n h y d r o c h l o r o t h i a z i d e and hydroc h l o r o t h i a z i d e t a b l e t s are i n c l u d e d i n t h e f o l l o w i n g compendia: BP 73, DAB 7 ( D D R ) , Ph. I n t . 11, Ph. J a p . 1 9 7 1 , Ph. Nord. Add., USP X I X Monographs i n Ph. Eur. are p r o p o s e d . A USP Hydroc h l o r o t h i a z i d e Reference Standard i s a v a i l a b l e .

1.6

O t h e r Compendia

Summaries i n c l u d i n g a n a l y t i c a l and pharmaceut i c a l i n f o r m a t i o n s are g i v e n i n The P h a r m a c e u t i c a l Codex( 9 ) and i n Kirk-Othmer, E n c y c l o p e d i a of Chemical T e c h n o l o g y ( l 0 ) . 2.

P h y s i c a l Properties

2.1

Spectra

2.1.1

Infrared(l1)

The i n f r a r e d spectrum i s p r e s e n t e d i n F i g u r e 1. The s p e c t r u m was o b t a i n e d from a m i n e r a l o i l m u l l on a P e r k i n E l m e r Model 157 i n f a r e d s p e c t r o p h o t o m e t e r -f A s s i g n m e n t s f o r t h e i n t h e range of 4000-650 c m c h a r a c t e r i s t i c b a n d s i n t h e spectrum a r e l i s t e d i n Table I.

.

HANS PETER DEPPELEH

408

Table I

Infrared absorption Wavenumber cm’l

Assignments

3 3 7 0 , 3 2 7 0 , 3170 1600, 1550, 1520

NH + NH2

1335 1180

2.1.2

heterocyclic ring system

/ 1320 / 1 1 6 5 / 1150

s02

Raman( 11)

T h e Raman spectrum of h y d r o c h l o r o t h i a z i d e powder i s shown i n F i g u r e 2 a n d Table 11. I t w a s b b t a i n e d o n a C a r y Model 8 3 spectrometer u s i n g t h e a r g o n 488 nm e x c i t a t i o n of a Lexel Model 7 5 i o n laser

Table I1

Raman spectrum Frequency c m

-1

Assignments ~~

3380, 3080, 2960, 1600, 1335, 1165, 940, 710,

3 2 8 0 , 3180 3020 2900 1525, 1460 1320 1155 900 675

NH s t r e t c h i n g aromatic CH s t r e t c h i n g CH s t r e t c h i n g C=6 stretching

SO2 asym. s t r e t c h i n g S O sym. s t r e t c h i n g S-& s t r e t c h i n g + NH deformation r i n g deformations

F i g . 1.

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

HYDROCHLOROTHIAZIDE

2.1.3

411

Ultraviolet

T h e U V spectrum of h y d r o c h l o r o t h i a z i d e i n e t h a n o l i s shown i n F i g u r e 3 (11). I n f o r m a t i o n a b o u t t h e UV a b s o r p t i o n i n o t h e r s o l v e n t s i s g i v e n i n T a b l e 111.

T a b l e I11 U 1t r a v i o 1e t ab s o r p t i o n

So lve n t

e t h a n o l ( 11

methanol( 1 2 1

X max nm

1 min nm

225 269 316 226 241.5 271 294

water( 13 1 0.01 N H C l ( 1 3 ) 0 . 0 1 N NaOH( 1 3 )

0 . 1 N NaOH(14)

317 270 315 270 315 272 323 221

log

4.576 4.307 3.505 4.513 3.129 4.279 3.272 3.471 4.286 3.495 4.290 3.500 4.193 3.435 4.448

247 273

4.198 299

31 9 / 3 2 0

E

3.456

HANS PETER DEPPELEH

418

F i g u r e 3 . U l t r a v i o l e t s p e c t r u m of h y d r o c h l o r o thiazi.de i n ethanol

FO 6 2

Spectrum No 6 5 7 5 1 Sample 76-104 ?93 Prod.Std.76 M o l e c u l a r ),eight 297,75 S o l v e n t *ethanol

j

-

Concentra t 1 on 2 , 5.10-5H01 ./Lit C e l l p a t h 1,Ocm I n s t r u m e n t Cary 1 1 8 O p e r a t o r Dmo. D a t e 1 9 . 3 ~ 1 1 19.9

HYDROCHLOROTHIAZIDE

2.1.4

413

1H-Nuclear Maqnetic R e s o n a n c e ( l 1 )

The 'H-NMR s p e c t r u m shown i n F i g u r e 4 was o b t a i n e d from a s o l u t i o n i n a c e t o n e - d a t a m b i e n t t e m p e r a t u r e on a V a r i a n XL-100-12 s p e g t r o m e t e r a t 1 0 0 MHz. The a s s i g n m e n t s of t h e s i g n a l s are l i s t e d i n Table I V .

Table I V 'H-NMR I

Signal

Mu 1t i p 1i c i t y

Chem. shift ppm v s . TMS

Number

Species

Of

protons

I 7.16 7.04

6.75 6.65 4.93 3.0 2.05

I

1

arom. proton sulfonamide N H arom. proton arom. NH

2

Ar-S02NH2

2

me t h y l e n e protons solvent solvent

1 broad singlet singlet broad singlet broad singlet broad singlet singlet multiplet

1 1

-

S i g n a l f i s b r o a d e n e d due t o u n r e s o l v e d c o u p l i n g w i t h t h e NH p r o t o n s .

NMR spectrum no. 105523

I

1

C

j-

1 +

/

10

9

8

7

Figure 4. 'H-NMR

6

5

4

3

2

spectrum of hydrochlorothiazide

1

0 ppm

HYDROCHLOROTHIAZIDE

2.1.5

415

13C-Nuclear M a g n e t i c Resonance( 111

The 13C-NMR s p e c t r a shown i n t h e F i g u r e s 5 , 6 , 7 w e r e r e c o r d e d a t 25.2 MHz and a m b i e n t t e m p e r a t u r e w i t h a V a r i a n XL-100-15 spectrometer u s i n g a s o l u F i g u r e s 5 and 6 show d e c o u p l e d t i o n i n acetone-d spectra. The undegoupled s p e c t r u m i n F i g u r e 7 shows t h e m u l t i p l i c i t i e s of t h e s i g n a l s . The a s s i g n m e n t s of t h e s i g n a l s are l i s t e d i n Table V.

.

Table V 13C-NMR ~~~

I

M u l t i p l i c i t y (umber S p e c i e s S i g n a l Chem. If shift zarbons ppm vs. TMS I

/

1

55.9

1

5 9

118.9 120.8

1 1

-N - 2 \ arom. C-H arom C-S02 -NH-CH2

8 6

127.3 129.6 135.9

1 1 1

arom. C-H arom. C-C1 arom.

7

h-CH /

.

C-S02NH2 10

147.7

d,m ( b r o )

d : d o u b l e t ; t: t r i p l e t ;

( b r o ) : broadened

in:

1

multiplet;

arom. C-NH-R

416 0

Figure 5. 13C-NMR spectrum of hydrochlorothiazide

m

n m

2 l-.t

0

N

-I

417

a,

5

.rl N

Id .d 2 4J 0 k 0 d

c V 0 k

a

2 0

w E 3

k

V a,

4J

4 P;

B z I

u

m

d

W

br

a, k 3

E

4

m

r-

F i g u r e 7 . Undecoupled 13C-NMR chlorothiazide

spectrum of hydro-

HYDHOCHLOROTHIAZIDE

2.1.6

419

Mass( 11)

S p e c t r a were r e c o r d e d o n a V a r i a n CH7 mass s p e c t r o m e t e r u s i n g t h e d i r e c t i n l e t s y s t e m , 70eV e l e c t r o n e n e r g y a n d a n i o n s o u r c e t e m p e r a t u r e of 18OoC. A t a sample t e m p e r a t u r e o f a b o u t 24OoC t h e m o l e c u l a r i o n m/e 297 c o u l d be d e t e c t e d b u t t h e s p e c t r u m w a s c o m p l i c a t e d and d i f f i c u l t t o i n t e r p r e t d u e t o p y r o l y t i c d e g r a d a t i o n . R o u t i n e mass s p e c t r o s c o p y i s c o n s i d e r e d t o be i n a d e q u a t e f o r t h e c h a r a c t e r i z a t i o n of h y d r o c h l o r o t h i a z i d e . P h y s i c a l P r o p e r t i e s of t h e S o l i d S t a t e

2.2 2.2.1

Thermal A n a l y s i s ( 1 5 ) Meltinq p o i n t

Melting p o i n t s reported i n l i t e r a t u r e (5,14,16,17,18,19,20) vary within t h e temperature r a n g e o f 263 t o 275OC. The s t r o n g dependence o f t h e m e l t i n g p o i n t on h e a t i n g c o n d i t i o n s h a s b e e n c o n f i r m e d w i t h t h e Mettler FP-2 h o t s t a g e micros c o p e as w e l l as w i t h t h e P e r k i n - E l m e r DSC-2. The e f f e c t which c a u s e s t h e anomalous m e l t i n g behaviour(20) i s not c l e a r l y understood. D i f f e r e n t i a l Scanninq Calorimetry The m e l t i n g p o i n t of h y d r o c h l o r o t h i a z i d e a c c o r d i n g t o DSC-2 m e a s u r e m e n t s , O v a r i e s from 266.OoC f o r a s c a n s p e e d of 1125 C min-' t o 273.3OC f o r a s c a n s p e e d o f 8OoC min( F i g u r e 8 1 . A s any e f f e c t (e.g. decomposition or t r a n s i t i o n i n t o another c r y s t a l modification) i s suppressed a t a h i g h s c a n speed, one can r e g a r d t h e v a l u e f o r an i n f i n i t e l y f a s t s c a n s p e e d as t h e t r u e m e l t i n g p o i n t . E x t r a p o l a t i o n of t h e c u r v e shown i n F i g u r e 8 g i v e s a m e l t i n g p o i n t o f 274.5 + 0.3OC*. T h i s a g r e e s w e l l w i t h t h e m e l t i n g p o i n t r e p o r t e d i n The Merck I n d e x ( 5 ) . However, t h e p u r i t y v a l u e s o b t a i n e d from m e l t i n g c u r v e s measured w i t h t h e DSC-2 a r e i n d e p e n d e n t of t h e s c a n s p e e d as w e l l as of t h e s u r r o u n d i n g a t m o s p h e r e . When a sample of hydro-

*

E r r o r of t h e mean v a l u e i n terms of c o n f i d e n c e

i n t e r v a l s on a 95 % l e v e l

HANS PETER DEPPELER

420

c h l o r o t h i a z i d e w a s t s t e d a t scan speeds of 1.25, 2 . 5 , 5 and 2OoC min -7 t h e mean v a l u e o b t a i n e d f o r t h e p u r i t y w a s 99.3 2 1 . 0 mole per c e n t . A t y p i c a l DSC m e l t i n g c u r v e is shown i n F i g u r e 9. A s l i g h t exotherm, i n d i c a t i n g decomposition i n t h e l i q u i d p h a s e , i s s e e n above t h e m e l t i n g p o i n t . Thermogravimetry Measurements were p e r f o r m e d w i t h a P e r k i n The r e s u l t s were: less t h a n 0 . 1 % v o l a t i l e i m p u r i t i e s up t o a t e m p e r a t u r e of 28OoC. d e c o m p o s i t i o n s t a r t s a t 307OC.

E l m e r t h e r m o b a l a n c e TGS-1.

-

Density

2.2.2

-

1.68 + 0 . 0 1 g ~ m ’ ( 1~ 9 , 2 1 1 2.2.3

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

S t u d i e s by Dupont a n d D i d e b e r g ( l 9 ) o n a s i n g l e c r y s t a l m e a s u r i n g 0.2xO.4xO.l mm g a v e t h e f o l l o w i n g crystallographic data Monoclinic System : Space group: p2 1 Unit cell: Z = 2 molecules a = 7.419 + 0 . 0 0 6 8 b = 8.521 7 0 . 0 0 3 8 c = 10.003 7 0 . 0 0 2 8 6 = 111.720:v = 587.5 Calculated density: 1.672 g

a

L i n e a r a b s o r p t i o n c o e f f i c i e n t p = 6 . 7 1 cm” (Mo K : 0 , 7 1 0 7 ) . T h e s e r e s u l t s , found on a c r y s t a l c r y s t s l l i z e d from e t h a n o l , c o r r e s p o n d w e l l w i t h t h o s e f o u n d on a sample c r y s t a l l i z e d from m e t h a n o l i n a preliminary study(22 1 . A powder d i a g r a m , c a l c u l a t e d from t h e s i n g l e c r y s t a l d a t a , a g r e e s v e r y w e l l w i t h measurements o n i n d u s t r i a l p r o d u c t i o n l o t s . The powder d i f f r a c t i o n p a t t e r n , a s shown i n T a b l e V I , was o b t a i n e d w i t h a Guinier-DeWolf N o . 2 camera w i t h CuKa ( 1.54178 8) r a d i a t i o n ( 2 3 ) .

F i g u r e 8 . M e l t i n g p o i n t of h y d r o c h l o r o t h i a z i d e as a f u n c t i o n of scan s p e e d (DSC)

275 274 273 272 271 270 269 268 267

0

266

265 80 2 0

5

2.5

1.25

Scan s p e e d OC v i n - 1 4

F i g u r e 9. DSC m e l t i n g p o i n t c u r v e of h y d r o c h l o r o t h i a zi d e -

-

-

--

Range 2 mcal s-l Scan speed: 1-25

OC

Sample w e l g h t 4 . 1 5 0

rn1n-l

rns Heat of f u s i c - :

260

261

262

263

264

Temp. OC

265

266

267

266

269

422

HANS PETER DEPPELER

Table V I X-ray d i f f r a c t i o n p a t t e r n of h y d r o c h l o r o t h i a z i d e powder

I n t e n s it y 9.3 6.9 6.3 5.35 4.75 4.65 4.26 4.15 4.09 3.87 3.62 3.44 3.39 3.19 3.14 3.10 2.89 2.74

2.3 2.3.1

v e r y weak weak v e r y weak very strong strong strong very strong very strong v e r y weak moderate very strong moderate weak strong weak strong weak weak

Intensity 2.71 2.67 2.62 2.50 2.45 2.40 2.38 2.35 2.29 2.25 2.21 2.19 2.16 2.12 2.07 2.06 2.03

v e r y weak

strong medium weak moderate v e r y weak v e r y weak v e r y weak moderate v e r y weak v e r y weak v e r y weak weak weak v e r y weak v e r y weak moderate

Solubility S o l u b i l i t y i n Homogeneous Media

H y d r o c h l o r o t h i a z i d e i s s o l u b l e i n a q u e o u s sol u t i o n s of i n o r g a n i c b a s e s l i k e sodium h y d r o x i d e ( 6 ) o r ammonium h y d r o x i d e ( 5 ) and i n o r g a n i c b a s e s l i k e n - b u t y l a m i n e ( 6 ) . S o l u b i l i t i e s i n aqueous s o l u t i o n s a r e g i v e n i n T a b l e V I I , and i n some commonly u s e d organic solvents, i n Table V I I I . The s u r f a c e t e n s i o n of t h e s a t u r a t e d a q u e o u s s o l u t i o n a t 23OC w a s f o u n d t o b e 724 !.IN p e r c m by L e r k and L a g a s ( 2 1 ) . The i n c r e a s e of t h e s o l u b i l i t y upon a d d i t i o n of n o n - i o n i c s u r f a c t a n t s was s t u d i e d by A b o u t a l e b e t a l . ( 2 4 1 .

423

HYDROCHLOROTHIAZIDE

Table V I I S o l u b i l i t y i n aqueous s o l u t i o n s ( 13 1

Solvent

t°C

p H of t h e solution

water water 0.9 % N a C l 0.1 N HC1 0.1 N acetic acid 0.1 N acetic b u f f e r pH 4.4 0.067 M p h o s p h a t e b u f f e r p H 7.4 0.05 M borate b u f f e r p H 9.0 1 M ammonia( 2 5 ) 0 . 1 N NaOH simulated gastric f l u i d p H 1.1 simulated i n t e s t i n a l f l u i d p H 7.4

Solubility g i n 100 m l solution

25 37 25 25 25 25

6.2 7.2 6.1 1.0 2.9 4.5

60.9 108 59.4 60.8 63.6 62.3

25

7.4

61.6

25

8.9

25 25 37

11.6 10.2 1.1

37

7.5

... . -3 .. 1 0 .

103

.

2.2 1.79 108

.

109

.

T a b l e VIII S o l u b i l i t y i n non aqueous s o l v e n t s

r-Solvent

temp. 0C ca.

Solubility g i n 100 m l s o l u t i o n I acetone 25 13.7 (25) 25 acetic acid 0.15 (25) ace t o n i t r i l e 25 2.0 (25) ethylacetate 25 0.59 (25) chloroform 23 0.1 ( 131 ethanol (96 % 1 23 1.3-1.4 ( 131 methanol 23 3.9-4.1 ( 131 d i c h l o r o m et h a n e 23 < 0.02 ( 13 1

HANS PETER DEPPELER

424

2.3.2

Partition Coefficients

The p a r t i t i o n between n - o c t a n o l and a q u e o u s p h a s e s a t 25OC i s e x e m p l i f i e d by t h e f o l l o w i n g d a t a ( 13 1 0.1 N H C 1 (pH 1 . 0 6 ) : 1.94 pco r g l c a q 0.1 M glycine b u f f e r (pH 3 . 0 ) : pCorg/Caq = 0.866 0.067 M p h o s p h a t e pCorg/Caq = 0.855 b u f f e r (pH 7 . 4 ) : 2.4

I o n i s a t i o n i n Aqueous S o l u t i o n

The i o n i s a t i o n c o n s t a n t s q u o t e d i n t h e l i t e r a t u r e d i f f e r . The v a l u e s r e p o r t e d by Mollica e t a 1 . ( 2 6 ) and by S t a h l a g r e e b e s t w i t h t h e s o l u b i l i t y b e h a v i o u r ( l 3 ) . They a r e l i s t e d below i n Table I X .

Table I X

pK-Values

i n aqueous s o l u t i o n s Method

pK-Value pKa pKa pKa pKa PKa

3.

1 2 1 2

-

8.81 + 0 . 0 5 10.4 ,+ 0 . 1 8.6 9.9 8.7

photom. t i t r . photom. t i t r potent. titr. potent. titr. spectrophotom.

.

13 1 13 1 (26) (26) (26) ( (

Synthesis

A c c o r d i n g t o Kleemann(27) t w o ways of s y n t h e s i s are u s e d

5-Chloro-2.4-disulfamoylaniline and p a r a f o r m a l d e h y d e r e a c t i n non a q u e o u s media t o g i v e hydrochlorothiazide.

HYDROCHLOROTHIAZIDE

425

b) 0

0

0

0

0

0

(29)

0

0

H 6-chloro-7-sulfamoyl-2H-l.2.4-benzothiadiazine1.1-dioxide reacts with formaldehyde in aqueous alkaline solution to form hydrochlorothiazide.

4.

Stability and Degradation

4.1

Bulk Stability(25)

Hydrochlorothiazide stored at room temperature for five years shows no degradation and heat affectg it very slowly, e.g. treatment for 2 hours at 230 C gives a yellowish discoloration but no significant change of the physical properties. Although hydrochlorothiazide is fairly stable in normal daylight, it should not be exposed to intense light: 38 hours at 180 0 0 0 Lux (Xenotest) destroyed about 3 per cent of a sample spotted on glass fibre paper. 4.2

Solid-Solid Interactions

Bornstein and Lach(30) found that hydrochlorothiazide reacts under the influence of humidity with adjuvants containing metal compounds. The changes in UV-absorption spectra obtained by diffuse reflectance spectrometry were interpreted as the result of charge-transfer chelation. In a compatibility study with Aerosil 2000, calcium stearate and talc using diffuse reflectance spectroscopy, tlc and UV spectroscopy after extraction, no indications of degradation under usual manufacturing and storage conditions were found( 13 1. 4.3

Stability in Solution

In aqueous solutions, hydrochlorothiazide undergoes hydrolysis to give formaldehyde and

HANS PETER DEPPELER

126

6-Chloro-2.4-disulfamoylaniline. The dependence o f t h e r e a c t i o n r a t e on t e m p e r a t u r e and p H was s t u d i e d by Mollica e t a 1 . ( 2 6 , 3 1 ) and Yamana e t a l . ( 3 2 ) . Between pH 2.5 and pH 1 1 . 5 t h e r a t e f o l l o w s a b e l l s h a p e d c u r v e w i t h a maximum a t a b o u t pH 7 . 2 . B e l o w p H 2 and above g H 1 2 t h e r e a c t i o n r a t e increases rapidly. T h i s pH r a t e p r o f i l e and t h e l a c k of s i g n i f i c a n t b u f f e r c a t a l y s i s was e x p l a i n e d by M o l l i c a by t h e f o r m a t i o n of i n t e r m e d i a t e s of t h e i m i n e t y p e a n d , upon h y d r a t i o n , of t h e hydroxymethylamine type( 26 1.

-

H2°

h’

+

+

CH20

H

5.

Methods of A n a l y s i s

5.1

Elemental A n a l y s i s ( 3 3 ) Element

% calculated % found

5.2

C

H

c1

N

0

S

28.24 2 . 7 1 1 1 . 9 1 1 4 . 1 2 21.49 21.54 28.21 2 . 7 2 1 2 . 1 5 14.18 2 1 . 2 6 21.45

Identification

Chemical t e s t s were d e s c r i b e d by K e r t e s z ( 3 4 ) K a l a ( 3 5 ) and P e r e z ( l 8 ) . M i c r o c h e m i c a l i d e n t i f i c a t i o n methods were r e p o r t e d by d e Z o e t e n ( 3 6 1 , Groenewegen(371, K a l a ( 3 8 ) and A u e r b a c h ( 3 9 ) . Usuall y , h y d r o c h l o r o t h i a z i d e i s i d e n t i f i e d by spectros c o p i c means, e.g. by i t s I R ( 6 , 1 4 , 4 0 ) and UV ( 6 , 1 4 , 4 0 , 4 1 ) s p e c t r a o r by o n e of t h e 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 c i t e d i n s e c t i o n 5.9. 5.3

Colorimetry

Hydrochlorothiazide i s r a p i d l y hydrolysed i n a c i d o r a l k a l i n e s o l u t i o n s . By d i a z o t i s a t i o n of t h e h y d r o l y s i s p r o d u c t 5-chloro-2,4-disulfamoyla n i l i n e and s u b s e q u e n t c o u p l i n g w i t h a n aromatic amine o r a p h e n o l , s t a b l e a z o d y e s are p r o d u c e d .

HYDROCHLOROTHIAZIDE

427

N-(l-naphthyll-ethylenediamine( 4 2 , 4 3 , 4 4 ) , chromot r o p i c a c i d ( 451, g u a j a c o l s u l f o n i c a c i d ( 4 6 1 , and t h y m o l ( 3 5 ) h a v e b e e n r e p o r t e d as c o u p l i n g a g e n t s . T h e s e methods are c o n s i d e r e d t o b e s u i t a b l e f o r t h e a n a l y s i s of p h a r m a c e u t i c a l s and may a l s o s e r v e as l o w cost techniques i n biopharmaceutical s t u d i e s . O t h e r methods a r e b a s e d o n c o n d e n s a t i o n r e a c t i o n s of t h e h y d r o l y s i s p r o d u c t s ( 2 6 , 4 7 ) or o n d i r e c t c o l o r r e a c t i o n s of h y d r o c h l o r o t h i a z i d e w i t h d i f f e r e n t r e a g e n t s i n t h e p r e s e n c e of c o n c e n t r a t e d s u l f u r i c acid(18,42,48). R e c e n t l y , E l s a y e d and Nwakanma(49) r e p o r t e d o n i o n p a i r e x t r a c t i o n w i t h s a f r a n i n b a s i c dye as a s i m p l e , s e l e c t i v e and s e n s i t i v e new method f o r t a b l e t analysis. 5.4

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

Rehm and S m i t h ( 45 1 showed t h a t UV-spectrophotometry i s n o t s u i t a b l e f o r t h e d e t e r m i n a t i o n o f hydrochlo r othiazide i n t h e presence of i t s h y d r o l y s i s p r o d u c t 5-chloro-2,4-disulfamoyla n i l i n e . I n s p i t e of t h i s l i m i t a t i o n , t h e method w a s d e s c r i b e d f o r t a b l e t a n a l y s i s by S t e i n b a c h , M o e l l e r ( 5 0 ) and Ruiz Rodriguez e t a 1 . ( 5 1 ) . F a z z a r i combined UV-Spectrophotometry w i t h a column extract i o n t e c h n i q u e ( 5 2 ) . The s p e c i f i c i t y of t h e method may a l s o b e improved by c a l c u l a t i n g t h e c o n c e n t r a t i o n s from pH-induced s p e c t r a l c h a n g e s ( 5 3 , 5 4 , 5 5 ) . An a u t o m a t e d d e t e r m i n a t i o n of h y d r o c h l o r o t h i a z i d e i n s i n g l e m u l t i c o m p o n e n t t a b l e t s was d e s c r i b e d by U r b a n y i and O ' C o n n e l l ( 5 6 ) . The d e t e r m i n a t i o n of h y d r o c h l o r o t h i a z i d e i n ( b o v i n e ) serum i s p o s s i b l e a f t e r s e p a r a t i o n by d i a l y s i s ( 5 7 ) . Q u a l i t a t i v e r e s u l t s were o b t a i n e d by UV-spect r o m e t r y of p o l y e t h y l e n e c o n t a i n e r m a t e r i a l ( 5 8 ) a n d by means of d i f f u s e r e f l e c t a n c e s p e c t r o s c o p y o n powder m i x t u r e s ( 3 0 ) . U V - s p e c t r o p h o t o n e t r y i s also applied i n d i s s o l u t i o n rate s t u d i e s , e.g. i n USP X I X ( 6 ) . 5.5

Phosphorime t r y

B o w e r and W i n e f o r d n e r ( 5 9 ) r e p o r t e d on a t e c h n i q u e f o r room t e m p e r a t u r e p h o s p h o r e s c e n c e measurements o n h y d r o c h l o r o t h i a z i d e and found i t t o be a s i m p l e and s e l e c t i v e method s u i t e d t o c e r t a i n c l i n i c a l analyses.

HANS PETER DEPPELER

428

5.6

Fluorimetry

S c h a f e r , Geissler and M u t s c h l e r ( 6 0 ) developed two methods based on f l u o r i m e t r i c measurement on t l c p l a t e s . By c o u p l i n g t h e d i a z o t i s e d h y d r o l y s i s p r o d u c t of h y d r o c h l o r o t h i a z i d e t o a f l u o r e s c e n t compound, followed by chromatographic s e p a r a t i o n , 0 . 6 ng of 6-chloro-2,4-disulfamoyl-aniline could b e d e t e c t e d . Because t h e a u t h o r s found t h e hydrol y s i s s t e p d i f f i c u l t t o reproduce q u a n t i t a t i v e l y t h e y recommend measurement of t h e f l u o r e s c e n c e of u n d e r i v a t i s e d h y d r o c h l o r o t h i a z i d e . The s e n s i t i v i t y of t h e l a t t e r method i s lower b u t still s u f f i c i e n t f o r t h e a n a l y s i s of human plasma, u r i n e and s a l i v a a f t e r o r a l a d m i n i s t r a t i o n of 25 m g hydrochlorothiazide

.

5.7

Polaroqraphy

Cohen e t a1.(61) and Woodson and S m i t h ( 6 2 ) r e p o r t e d on t h e p o l a r o g r a p h i c r e s p o n s e of hydroc h l o r o t h i a z i d e and r e l a t e d compounds. P r a c t i c a l a p p l i c a t i o n s were d e s c r i b e d i n USP X V I I I ( 6 3 ) f o r t a b l e t s and i n a p a p e r of Kkolos and W a l k e r ( 6 4 ) f o r multicomponent t a b l e t s . The p o l a r o g r a p h i c d e t e r m i n a t i o n i s r e p o r t e d t o be s u i t a b l e f o r s i n g l e t a b l e t a n a l y s i s w i t h o u t s e p a r a t i o n of o t h e r components. 5.8

Titration

The t i t r a t i o n of t h e p u r e compound w i t h s t r o n g b a s e s i n non aqueous s o l v e n t s h a s found widespread application(35,65,66,67,68). USP X I X ( 6 ) t i t r a t e s h y d r o c h l o r o t h i a z i d e w i t h sodium methoxide i n n-butylamine w i t h azo v i o l e t a s i n d i c a t o r . BP 7 3 ( 8 ) d e s c r i b e s a potentiometric t i t r a t i o n with t e t r a butylammoniumhydroxide i n p y r i d i n e . Other t i t r a t i o n t e c h n i q u e s a p p l i e d t o hydrochlorothiazide a c t i v e substance o r formulations a r e l i s t e d below: Complexome$$ic a f t e r p r e c i p i t a t i o n w i t h Pb++(69 1 o r w i t h H g ( 7 0 , 7 1 1 . Amperometric w i t h n i t r i t e (72). Bromatometric(73). Thermometric w i t h sodium h y d r o x i d e ( 7 4 ) . S u l f a t e d e t e r m i n a t i o n a f t e r mineral i s a t i o n ( 7 5 ) . Argentometric a f t e r m i n e r a l i s a t i o n (76).

HYDROCHLOROTHIAZIDE

5.9

429

Chromatoqraphy

5 . 9 . 1 T h i n Layer Chromatography P a p e r c h r o m a t o g r a p h y w a s of some i m p o r t a n c e f o r t h e i d e n t i f i c a t i o n of h y d r o c h l o r o t h i a z i d e ( 1 7 , 4 0 , 7 7 , 7 8 , 7 9 , 8 0 ) b e f o r e i t w a s r e p l a c e d by t h i n l a y e r chromatography. I n t l c , s i l i c a g e l l a y e r s a r e t h e most o f t e n u s e d s o r b e n t s . They were shown t o be s u i t a b l e f o r q u a l i t a t i v e ( 7 9 - 9 1 ) and q u a n t i t a t i v e ( 5 0 , 6 0 ) a n a l y s i s of h y d r o c h l o r o t h i a z i d e i n pharmaceuticals(50,79,87) and i n b i o l o g i c a l mater i a 1 ( 6 0 , 8 8 , 9 0 , 9 1 ) . O t h e r s o r b e n t s l i k e aluminum o x i d e ( 8 0 , 8 3 , 8 7 ) and c e l l u l o s e ( 8 1 ) t h e r e f o r e h a v e n o t r e c e i v e d much a t t e n t i o n . The d e t e c t i o n o f h y d r o c h l o r o t h i a z i d e o n p a p e r ( 1 7 , 4 0 , 7 8 ) , c e l l u l o s e l a y e r s ( 8 1 ) and aluminum o x i d e ( 8 0 , 8 3 , 8 7 ) was n o t s t u d i e d i n t e n s e l y . On s i l i c a g e l , t h e q u e n c h i n g of f l u o r e s c e n c e on l a y e r s containing a fluorescence indicator ( d e t e c t i o n l i m i t 0 . 2 p g ) and c o l o u r r e a c t i o n by h y d r o l y s i s , d i a z o t i s a t i o n and c o u p l i n g w i t h sodium chromotrop a t e ( d e t e c t i o n l i m i t below 0 . 1 p g ) were found t o b e r e l i a b l e and s e n s i t i v e methods. O t h e r r e p o r t e d v i s u a l i s a t i o n techniques(81,85,87,88,92) w e r e n o t found t o be of comparable s e n s i t i v i t y . S u c c e s s f u l l y t e s t e d s y s t e m s ( e x a m p l e s ) ( 93 1 : E t h y l acetate+chloroform+methanol ( 1 1 + 8 + 1 ) , a t a b o u t 23OC, o n s i l i c a g e l 60 F-254 ( M e r c k ) , Rf o f h y d r o c h l o r o t h i a z i d e a b o u t 0.3. Used f o r semiq u a n t i t a t i v e s t a b i l i t y t e s t s on d o s a g e forms. Two s t e p d e v e l o p m e n t s y s t e m . a ) Diethylether+chloroform+etyhl acetate+ methanol ( 1 0 + 8 + 6 + 1 . 5 ) , b) Ethyl agetate+chloroform ( 2 2 + 3 ) , a t a b o u t 23 C , on s i l i c a g e l S i l - G 25 HR UV 254 ( M a c h e r e y - N a g e l ) , R f a +Rfb) o f h y d r o c h l o r o t h i a z i d e a b o u t 0.6. Used kor p u r i t y t e s t i n g o n a c t i v e substance.

-

The d i r e c t q u a n t i t a t i o n o f h y d r o c h l o r o t h i a z i d e on t l c p l a t e s was r e p o r t e d by S t e i n b a c h e t a 1 . ( 5 0 ) ( d e n s i t o m e t r y a t 2 7 2 nm) and S c h a f e r e t a l . ( 6 0 ) ( f l u o r i m e t r y ) . The l a t t e r method allows t h e d e t e r m i n a t i o n of 2 ng h y d r o c h l o r o t h i a z i d e on t h e p l a t e and w a s u s e d f o r t h e a n a l y s i s of human body f l u i d s a f t e r s i n g l e d o s e a p p l i c a t i o n .

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430

5.9.2

L i q u i d Chromatoqraphy

S e p a r a t i o n of h y d r o c h l o r o t h i a z i d e from t a b l e t i n g r e d i e n t s by chromatography o n a l k a l i n e c e l i t e c o l u m n s ( 5 2 , 9 4 ) and from b a s i c compounds i n m u l t i component p h a r m a c e u t i c a l s on i o n exchange columns ( 56,951 was shown t o be p o s s i b l e u n d e r l o w p r e s s u r e conditions (gravity). Later on, h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y r e p l a c e d t h e low p r e s s u r e methods c o m p l e t e l y . The d i v e r s i t y of r e p o r t e d HPLC methods i s i l l u s t r a t e d by t a b l e X . 5.9.3

G a s Chromatography

The d e t e r m i n a t i o n i n p l a s m a , b l o o d c o r p u s c l e s a n d u r i n e by g a s c h r o m a t o g r a p h y w a s r e p o r t e d by Lindstroem e t a1.(104,105). Hydrochlorothiazide w a s methylated with methyliodide, using t h e e x t r a c t i v e m e t h y l a t i o n p r o c e d u r e . For t h e q u a n t i t a t i v e evaluation, an i n t e r n a l standard, chlort h a l i d o n e , was u s e d . Chromatographic c o n d i t i o n s ( l 0 4 ) : 1 %oSE-30 o n Gas-Chrom Q (80-100 m e s h ) , Column : 225 C: C a r r i e r n i t r o g e n Injector: 23OoC Detectors: ECD, 3OO0C; F I D , 27OoC The method h a s been a p p l i e d e . g . by Beermann e t a 1 . ( 1 0 6 - 1 1 3 ) i n p h a r m a c o k i n e t i c and b i o a v a i l a b i l i t y s t u d i e s and by Wallace e t a 1 . ( 8 8 ) as a c o n f i r m a t o r y method t o t l c i d e n t i f i c a t i o n methods. Vandenheuvel e t a1.(114) d e v e l o p e d a method f o r t h e a n a l y s i s of b l o o d and plasma b a s e d on t h e 'on-column m e t h y l a t i o n t e c h n i q u e ' w i t h t e t r a m e t h y l a n i l i n i u m h y d r o x i d e and t h e u s e of 6-bromo-3,4dihydro-2H-1.2.4-benzothiadiazine-7-sulfonamide 1 , l - d i o x i d e as t h e i n t e r n a l s t a n d a r d . The same i n t e r n a l s t a n d a r d was u s e d by R e d a l i e u e t a 1 . ( 1 1 5 ) i n a m o d i f i c a t i o n of t h e L i n d s t r o e m method. 5.10 E l e c t r o p h o r e s i s R u g g i e r i ( 1 1 6 ) proposed e l e c t r o p h o r e t i c s e p a r a t i o n of h y d r o c h l o r o t h i a z i d e f o r q u a n t i t a t i v e analysis.

Table X

HPLC m e t h o d s

column

eluent:

CSP a n i o n e x c h . o n Zipax 30 pm 1 0 0 0 ~ 2 . 1mm

0.005 Na2S04 i n p H 9.2 borate b u f f e r : methanol :

volumes

sample H. + h y d r o l y s i s

35 5

product +hydralazine

ref. 96 97

artificial m i x t u r e s of antihypert.

98

serum/urine

99

85 15

serum (gel filtered)

00

n-hexane : 2-propanol: chloroform: d i e t h y 1a m in e :

77 18 5 0.01

tablets ( H. + r e s e r p i n e 1

101

Lichrosorb 5 pm, 5 0 0 ~ 4 . 4 mm

n-hexane : ethanol :

55 45

serum

102

N u c l e o s i l 10-CN 1 0 pm, 2 0 0 ~ 4 . 8 mm

0 . 0 1 M a q . C12H25Na04S: 2-propanol : 0 . 1 N a q . H2S04:

75 23 5

tablets

1 03

Corasil-C18

2 2 0 ~ 2 . 3 mm Corasil-phenyl 1 2 2 0 ~ 2 . 3mm

sc r e e n i ng

p Bondapak C18

300x4 mm

0.01 M aq. NaH2P04: methanol :

S p h e r i s o r b ODS 10 pm, 250x3 mm

water: methanol :

L i c h r o s o r b S160 5 pm, 2 5 0 ~ 2 . 1mm

H.

= hydrochlorothiazide

4 1

H. + r e s e r p i n e

+hydralazine

HANS PETER DEPPELER

432

6.

I n t e r f e r e n c e s of Hydrochlorothiazide i n A n a l y t i c a l Methods

Hydrochlorothiazide i n t e r f e r e s with t h e u r i n a r y e s t r i o l d e t e r m i n a t i o n s by g a s c h r o m a t o g r a p h y ( l l 7 ) and by c o l o r i m e t r y w i t h t h e Kober r e a c t i o n ( 118 1 . 7.

P h a r m a c o k i n e t i c and M e t a b o l i c S t u d i e s

7.1

A n a l y t i c a l Methods Used f o r B i o l o g i c a l Material

B e s i d e s r a d i o m e t r i c p r o c e d u r e s used i n a b s o r p t i o n and d i s t r i b u t i o n s t u d i e s ( 7 7 , 1 0 6 , 1 1 9 , 1 2 0 1 , s e v e r a l colorimetric methods were d e v e l o p e d f o r t h e d e t e r m i n a t i o n of h y d r o c h l o r o t h i a z i d e i n plasma and u r i n e ( 4 2 - 4 4 , 1 2 1 - 1 2 4 ) . The p e r f o r m a n c e o f b i o a v a i l a b i l i t y s t u d i e s by colorimetric methods may p r o d u c e errors as c o u l d be s e e n f o r c h l o r o t h i a z i d e ( 1 2 5 ) . T h e r e f o r e g a s c h r o m a t o g r a p h i c methods were p r e d o m i n a n t l y u s e d , i . e . t h e method of Lindstroem e t a 1 . ( 1 0 4 ) i n k i n e t i c s t u d i e s o f t h e g r o u p of Beermann ( 1 0 7 - 1 1 3 ) and by J o r d o e e t a 1 . ( 1 2 6 ) , t h e method of Vandenheuvel e t a 1 . ( 1 1 4 ) u s e d by S u n d q u i s t e t a 1 . ( 1 2 7 ) and t h e p r o c e d u r e of R e d a l i e u e t a 1 . ( 1 1 5 ) . H i g h - p r e s s u r e l i q u i d chromat o g r a p h y ( 1 0 0 ) and f l u o r i m e t r i c ( 6 0 ) or s p e c t r o p h o t o m e t r i c ( l 2 8 ) d e t e r m i n a t i o n a f t e r s e p a r a t i o n by t l c may be a l s o s u i t a b l e f o r t h e d e t e r m i n a t i o n of h y d r o c h l o r o t h i a z i d e i n b i o l o g i c a l material. 7.2

Absorption

Aft?$ s i n g l e i n t r a v e n o u s o r o r a l a d m i n i s t r a t i o n of C - l a b e l l e d h y d r o c h l o r o t h i a z i d e (Doses: i . v . 1, 3 5 , 65 mg, n = 3; 2.0. 5 , SO, 65 m g ; n = 4 , n = 6) t o v o l u n t e e r s and p a t i e n t s 90-93 % a n d 53-83 % o f d o s e , r e s p e c t i v e l y were e x c r e t e d i n u r i n e . T h e r e f o r e a b s o r p t i o n of a n o r a l d r u g d o s e w a s i n t h e r a n g e o f 60-80 %. I t was found t o b e reduced i n p a t i e n t s w i t h c o n g e s t i v e h e a r t f a i l u r e o r r e n a l and h e p a t i c d i s e a s e s s i n c e o n l y a b o u t 4 0 % o f d o s e or less were e l i m i n a t e d r e n a l l y ( l 0 6 ) . H y d r o c h l o r o t h i a z i d e w a s e x c r e t e d i n u r i n e of r a t o r man almost c o m p l e t e l y as t h e i n t a c t substance( 77,106).

HYDROCHLOROTHIAZIDE

433

The peak plasma c o n c e n t r a t i o n s of t o t a l r a d i o a c t i v i t y i n p a t i e n t s ( n = 3 ) were 260, 386 and 616 ng/ml r e a c h e d w i t h i n 3-4 h o u r s , t h e corresp o n d i n g v a l u e s i n b l o o d c e l l s were a b o u t 3 times h i g h e r ( l 0 6 ) . S i m i l a r blood/plasma c o n c e n t r a t i o n r a t i o s were a l s o r e p o r t e d f o r v o l u n t e e r s ( l 0 6 , 1 1 4 , 1 1 5 ) . The n a t u r e of t h e b i n d i n g of h y d r o c h l o r o t h i a z i d e i n t h e e r y t h r o c y t e s i s s t i l l unknown. I n v i t r o experiments with bovine carboanhydrase showed no b i n d i n g ( 1 0 4 , 1 0 6 ) . 7.3

Distribution

Organ d i s t r i b u t i o n p a t t e r n i n r a t s a f t e r s i n g l e o r a l a d m i n i s t r a t i o n of t r i t i u m l a b e l l e d h y d r o c h l o r o t h i a z i d e ( d o s e : 5 mg) r e v e a l e d h i g h e s t c o n c e n t r a t i o n s of t o t a l r a d i o a c t i v i t y i n l i v e r ( 2 7 . 8 pg/g) and g a s t r o - i n t e s t i n a l t r a c t ( 3 6 . 0 pg/g) w i t h i n 1 h o u r a f t e r d o s i n g . A t t h e same t i m e t h e c o n c e n t r a t i o n i n plasma was 1.53 pg/ml, t h a t i n s p l e e n , m u s c l e and b r a i n 0.36-0.46 p g / g ( 7 7 ) . A l o w d e g r e e of h y d r o c h l o r o t h i a z i d e b i n d i n g t o b o v i n e serum a l b u m i n was o b t a i n e d w i t h o n l y o n e binding site c l a s s ( 5 7 ) . 7.4

Basic P h a r m a c o k i n e t i c s

I n v o l u n t e e r s a f t e r s i n g l e o r a l d o s e admini s t r a t i o n of h y d r o c h l o r o t h i a z i d e ( n = 8; d o s e s : 1 2 . 5 , 25, 5 0 , 75 mg) t h e peak plasma c o n c e n t r a t i o n s o f i n t a c t d r u g r e a c h e d w i t h i n 1.5-5 h o u r s a n d t h e area u n d e r t h e c o n c e n t r a t i o n c u r v e s ( A U C , 0-9 h o u r s ) were l i n e a r l y c o r r e l a t e d w i t h t h e d o s e . Peak c o n c e n t r a t i o n s i n d e p e n d e n c e o f t h e i n c r e a s i n g d o s e s were 70 + 1 9 , 1 4 2 + 50, 2 6 0 + 88 and 376 + 70 ng/ml, r e s p e c t i v e l y - ( % ,+ s 1 . Hydrochlor o t h i a z i d e w a s e l i m i n a t e d from plasfia m o s t l y i n a b i p h a s i c way w i t h t e r m i n a l h a l f - l i v e s of 5.6-14.8 h o u r s ( l 0 7 ) . I n t h e same v o l u n t e e r s u s i n g t h e same e x p e r i m e n t a l d e s i g n t h e u r i n a r y e x c r e t i o n and t h e d o s e a d m i n i s t e r e d were s i g n i f i c a n t l y c o r r e l a t e d too. A t o r a l d o s e s of 12.5, 25, 50 and 75 mg t h e u r i n a r y e x c r e t i o n ( 0 - 4 8 h o u r s ) was 8.5 + 2 . 0 , 1 7 . 9 + 4 . 2 , 33.4 + 8.6 a n d 4 8 . 9 ,+ 7 . 6 respect i v e l y . The c u m u l a t i v e u r i n a r y r e c o v e r y of t h e d r u g was 65-72 % o f d o s e f o r a l l d o s e s a d m i n i s t e r e d . R e n a l c l e a r a n c e was a l s o i n d e p e n d e n t of d o s e w i t h

-

ms,

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345 + 1 2 3 t o 319 + 86 m l / m i n ( l 0 7 ) . I n s e v e n pat i e n & w i t h c o n g e s t i v e h e a r t f a i l u r e ( d o s e : 50 mg, n = 6 ; 75 mg, n = 1) h i g h e s t c o n c e n t r a t i o n s o f i n t a c t h y d r o c h l o r o t h i a z i d e i n plasma were f o u n d w i t h i n 1.5-8 h o u r s w i t h 282-672 ng/ml. The t e r m i n a l h a l f - l i v e i n plasma w a s 8.9-28.9 h o u r s ( n = 6 ) and 3 . 1 h o u r s i n one p a t i e n t w i t h t h e h i g h e s t h e a r t f a i l u r e . Urinary e x c r e t i o n of i n t a c t drug (0-7 d a y s ) i n t h e s e p a t i e n t s may be r e d u c e d (20.8-71.6 % of d o s e ) f 1111 . I n hypertensive p a t i e n t s during repeated treatment w i t h d i f f e r e n t d o s e s of h y d r o c h l o r o t h i a z i d e ( d o s e s : 12.5, 25, 5 0 , 75 mg/day f o r . 2 c o n s e c u t i v e weeks; 75 mg f o r a d d i t i o n a l 4 w e e k s ) p r e - d o s e p l a s m a l e v e l s of i n t a c t s u b s t a n c e showed a l i n e a r r e l a t i o n s h i p t o i n c r e a s i n g d o s e s w i t h 15 + 7 , 1 7 2 8 , 2 7 2 11 and 34 f. 1 7 ng/ml, r e s p e c f i v e l y . T h i s w a s a l s o o b t a i n e d f o r plasma c o n c e n t r a t i o n s 5 h o u r s a f t e r d o s i n g . S t e a d y s t a t e plasma concent r a t i o n o f i n t a c t d r u g a f t e r 6 weeks of d a i l y t r e a t m e n t w i t h 75 mg h y d r o c h l o r o t h i a z i d e w a s f o u n d t o be 111 ng/ml. U r i n a r y e x c r e t i o n of i n t a c t hydroc h l o r o t h i a z i d e w i t h i n t h e l a s t 2 4 h o u r s of e a c h t r e a t m e n t p e r i o d w a s a b o u t 6 0 % o f d o s e and r e n a l c l e a r a n c e a c c o u n t e d f o r 317 + 1 2 0 m l / m i n ( l l 3 ) . O t h e r a u t h o r s , u s i n g a i e s s s p e c i f i c method, h a v e o b s e r v e d s t e a d y s t a t e plasma levels o f 970 2 90 ng/ml and 2250 f. 2 0 ng/ml ( x + s ; ) d u r i n g a 1 2 week and 2 0 week t r e a t m e n t p e r i o d - i n h y p e r t e n s i v e p a t i e n t s a t d a i l y o r a l d o s e s o f 150 and 450 mg hydrochlorothiazide, respectively(44). 7.5

Bioavailability

S i n c e h y d r o c h l o r o t h i a z i d e i s e x c r e t e d almost c o m p l e t e l y as t h e i n t a c t s u b s t a n c e i n man, i t s c u m u l a t i v e u r i n a r y e x c r e t i o n i s t h e best m e a s u r e of bioavailability.

As c o u l d b e s e e n above, t h e u r i n a r y r e c o v e r y o f t h e d r u g a f t e r s i n g l e o r a l d o s e s of 12.5-75 mg h y d r o c h l o r o t h i a z i d e ( c o m m . 25 mg t a b l e t s ) i s i n d e p e n d e n t of t h e d o s e w i t h 65-72 % ( 1 0 7 ) . The u r i n a r y e x c r e t i o n was i n t h e same r a n g e i n a n e x p e r i m e n t where b i o e q u i v a l e n c e o f t w o d o s a g e f o r m s f c o m m . 25 m g t a b l e t s of d i f f e r e n t o r i g i n ) w a s o b s e r v e d w i t h 70.8 + 1 4 . 9 vs. 65.2 + 1 0 . 1 % o f d o s e ( 108 1 . T h i s was a l s o o b s e r v e d i n s t u d i e s comparing

HYDROCHLOROTHI AZIDE

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several dosage forms of hydrochlorothiazide, but less specific colorimetric methods were used(121, 123,124). Enhanced bioavailability of hydrochlorothiazide was obtained in volunteers ( n = 8; dose 75 mg) when the drug was administered with food (74.2 + 6.5 vs. 63.2 2 8.0 % of dose measured in urine)-after the pretreatment of volunteers with the anticholinergicum propantheline ( n = 6; dose 75 mg; 5 6 . 9 + 4.4 vs. 49.3 + 5.3 % of dose in urine)(109,1iO) or after concomitant administration of polyvinylpyrrolidone(l28~. No influence on the bioavailability of hydrochlorothiazide was observed when sotalol, metoprolo1 or hydralazine were administered in combined dosage forms or separately to volunteers(l22, 126,127,129). A significant malabsorption, measured in terms of urinary excretion of intact hydrochlorothiazide was found in patients with congestive heart failure ( n = 7; dose: 50-75 mg) or in patients after intestinal shunt surgery ( n = 5; dose 75 mg) with an average urinary recovery of 40.7 and 30.7 8 of dose, respectively(lll,ll2). The relationship between bioavailability data and in vitro dissolution test results has been investigated repeatedly(l21,123). 8.

Acknowledgements

The author gratefully acknowledges the assistance of K.O. Alt, K. Brugger, E. Felber, H. Fuhrer, 0. Heiber, H. Huerzeler, E. Marti, S . Moss, W. Padowetz, P.H. Stahl and R . Steiner who contributed much previously unpublished information about physical and chemical properties and especially of K.F. Feldmann who prepared the chapter about pharmacokinetic and metabolic studies.

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9.

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100. 101. 102. 103. 104. 105. 106. 107. 108. 109.

110. 111. 112.

Ghali G., Sohn D . , Simon J . , Hanna M.A., T o l b a R . ; J. C h r o m a t o g r . 8 7 , 570-575 ( 1 9 7 3 ) S c h m i d t F.; D e u t . Apoth. Ztg. 1593-1597 (1974) CIBA-GEIGY L t d . Basle; A n a l y t i c a l P r o c e d u r e s 1976-1979 F a z z a r i F.R.; J. Assoc. O f f . A n a l . Chem. 55, 161-162 ( 1 9 7 2 ) Chu R . ; J . Assoc. O f f . Anal. Chem. 54, 603-608 ( 1 9 7 1 ) S m i t h J . B . , Mollica J . A . , Govan H . K . , Nunes I . M . ; Amer. Lab. 4, 13-19 ( 1 9 7 2 ) K i r k l a n d J.J.; J . Chromatogr. S c i . 1, 361-365 ( 1 9 6 9 ) Honigberg I.L., Stewart J.T., Smith A.P., Hester D.W.; J . Pharm. S c i . 6 4 , 1201-1204 ( 1975) Cooper M . J . , S i n a i k o A.R., A n d e r s M.W., M i r k i n B.L.; A n a l . Chem. 4 8 , 1 1 1 0 - 1 1 1 1 ( 1 9 7 6 ) C h r i s t o p h e r s e n A.S., Rasmussen K . E . , S a l v e s e n B.; J . C h r o m a t o g r . 132, 91-97 ( 1 9 7 7 ) B u t t e r f i e l d A.G., Lovering E . G . , S e a r s R.W.; J . Pharm. S c i . 67, 650-653 ( 1 9 7 8 ) R o b i n s o n W.T., Cosyns L . ; C l i n . Biochem. -11 I 172-174 ( 1 9 7 8 ) F e l b e r E . , S t e i n e r R . ; CIBA-GEIGY L t d . B a s l e , i n t e r n a l report, O c t . 1 9 7 9 L i n d s t r o e m B . , M o l a n d e r M . , G r o s c h i n s k y M.; J . Chromatogr. 114,459-462 ( 1 9 7 5 ) F a g e r l u n d C . , H a r t v i g P., L i n d s t r o e m B . ; J . Chromatogr. 168,107-116 ( 1 9 7 9 ) Beermann B . , G r o s c h i n s k y - G r i n d M . , R o s e n A . ; C l i n . P h a r m a c o l . T h e r . l9, 531-537 ( 1 9 7 6 ) Beermann B., G r o s c h i n s k y - G r i n d M . ; E u r o p . J . C l i n . P h a r m a c o l . l.2, 297-303 ( 1 9 7 7 ) Beermann B . , G r o s c h i n s k y - G r i n d M . , L i n d s t r o e m B.; Europ. J. C l i n . Pharmacol. 203-205 ( 1 9 7 7 ) Beermann B . , G r o s c h i n s k y - G r i n d M . ; E u r o p . J . C l i n . P h a r m a c o l . 13, 385-387 ( 1 9 7 8 ) Beermann B . , G r o s c h i n s k y - G r i n d M . ; E u r o p . J . C l i n . P h a r m a c o l . 13, 125-128 ( 1 9 7 8 ) Beermann B., G r o s c h i n s k y - G r i n d M . ; B r . J . C l i n . Pharmac. 579-583 ( 1 9 7 9 ) Backman L . , Beermann B . , G r o s c h i n s k y - G r i n d M . , H a l l b e r g D . ; C l i n i c a l P h a r m a c o k i n e t i c s 4, 63-68 ( 1 9 7 9 )

114,

11,

z,

441

HYDROCHLOROTHIAZIDE

113. B e e r m a n n B., G r o s c h i n s k y - G r i n d M.; E u r o p . J . C l i n . P h a r m a c o l . l3, 1 9 5 - 2 0 1 ( 1 9 7 8 ) G r u b e r V.F., Walker R.W., 114. V a n d e n h e u v e l W.J.A., Wolf F . J . ; J. P h a r m . S c i . 6 4 , 1309-1312 ( 1 9 7 5 ) Wagner W.E. j r ; 115. R e d a l i e u E . , T i p n i s V.V., J . Pharm. Sci. 6 7 , 726-728 ( 1 9 7 8 ) 1 1 6 . R u g g i e r i R.; Ban. C h i m . F a r m . 99, 2 0 - 2 3 ( 1960) C l i n . Chem. 18, 1 1 7 . Rosenthal A.F., Tomson M.R.; 471-472 ( 1 9 7 2 ) 1 1 8 . Watanabe F . , N a k a h a r a M., T s u b o t a N . , T s u k i d a K . , S a i k i K . , I t o M.; C l i n . C h i m . A c t a 88, 2 1 - 2 5 ( 1 9 7 8 ) Brettell H.R., Aikawa J . K . ; 1 1 9 . A n d e r s o n K.V., Arch. I n t e r n a l Med. 107, 7 3 6 - 7 4 2 ( 1 9 6 1 ) 1 2 0 . C a l e s n i c k B., Sheppard H . , Bowen N . ; Fed. P r o c . 20, 409 ( 1 9 6 1 ) Needham T.E.; J. P h a r m . S c i . 68, 1 2 1 . Shah K . A . , 1486-1490 ( 1 9 7 9 ) 1 2 2 . C I B A P h a r m a c e u t i c a l Company, S u m m i t R N . J . , B i o a v a i l a b i l i t y Data of Apresazide , September 1 9 7 6 1 2 3 . Meyer M.C., M e l i k i a n A.P., W h y a t t P.L., S l y w k a G.W.A.; C u r r . T h e r . R e s . l7, 5 7 0 - 5 7 7 ( 1 9 7 51 1 2 4 . Cook D . ; P h a r m a c o l o g y 2 , 1 9 0 - 1 9 5 ( 1 9 7 2 ) 1 2 5 . Resetarits D.E, B a t e s T.R.; J. P h a r m . S c i . 6 8 , 1 2 6 - 1 2 7 ( 1 9 7 9 ) 1 2 6 . Jordoe L , J o h n G o n G. , L u n d b o r g F , P e r s s o n B.A., Regardh C.-G., R o e n n 0.; B r . J. C l i n . Phannac. 7 , 563-567 ( 1 9 7 9 ) 1 2 7 . S u n d q u i s t H . , A n t i l l a M., S i m o n A . , Reich J . W . ; J. C l i n . P h a r m a c o l . 2 , 557-564 ( 1 9 7 9 ) 128. Corrigan O.I., T i m o n e y R.F., Whelan M . J . ; J . P h a r m . P h a r m a c o l . 28, 7 0 3 - 7 0 6 ( 1 9 7 6 ) 1 2 9 . Wagner W.E., G i l l e r a n T., Zak S . ; C l i n . Pharmacol. Ther. 1 7 , 247 (1975)

.

L i t e r a t u r e (C.A.)

.

surveyed t h r o u g h 1 9 7 9

KETOPROFEN Gary G. Liversidge 1. Description 1.1 Nomenclature 1.2 Formula 1.3 Molecular Weight 1.4 Appearance, Colour, Odour, and Taste 2. Physical Properties 2.1 Melting Range 2.2 Solubility 2.3 pH 2.4 Dissociation Constant 2.5 Partition Coefficient 2.6 Thermal Analysis 2.7 Crystallinity 2.8 Ultraviolet Spectrum 2.9 Optical Rotation 2.10 Mass Spectrum 2.11 Photoelectronic Spectrum 2.12 Nuclear Magnetic Resonance Spectrum 2.13 Infrared Spectrum 3. Synthesis 4. Stability and Degradation 5. Absorption, Metabolism and Excretion 6. Methods of Analysis 6.1 Elemental Analysis 6.2 Thin Layer Chromatographic Analysis 6.3 Ultraviolet Spectroscopy 6.4 Potentiometric Titration 6.5 Gas Chromatography 6.6 Enantiomer Analysis 6.7 Colorimetric Analysis 7. Analysis of Biological Samples 7.1 Colorimetric Analysis 7.2 Polarographic Analysis 7.3 Gas Chromatographic Analysis 7.4 Thin Layer Chromatographic Analysis 7.5 Gas Chromatography-Mass Spectroscopy Analysis 7.6 High Pressure Liquid Chromatographic Analysis 8. Analysis of Pharmaceutical Formulations 8.1 Potentiometric Titration 8.2 Pyrolysis-Gas Chromatography-Mass Spectrometry 9. Acknowledgements 0. References

ANALYTICAL PROFI1,ES OF DRUG SUBSTANCES, 10

443

444 444 444 444 445 445 445 445 445 445 445 446 446 446 449 449 449 453 453 456 456 456 460 460 460 462 462 462 462 463 463 463 463 465 466 467 467 468 468 469 469 469

GARY G . LIVERSIDGE

441

1.

Description

1.1 Nomenclature 1.1.1. Chemical Names m-Benzoylhydratropic acid (1,2), a- (benzoylphenyl) propionic acid (3), a- (3-benzoylphenyl) propionic acid (4,5,6), 2-(3-benzoylphenyl) propionic acid (1,2,7-11), 2- (benzoyl-3-phenyl) propionic acid (12,131, Benzeneaceticacid, 3-benzoyl-a-methyl. The latter name is used by Chemical Abstacts. The Chemical Abstract's registry number for (2)ketoprofen is 22071-15-4 , for the ( + ) enantiomer it is 22161-81-5 and for the ( - ) enantiomer it is 56105-81-5

.

1.1.2. Nonproprietary Name Ketoprofen 1.1.3. Propietary Name

.

Orudis@,

Alrheumat@, Alrheumun6,

Profenid@

1.2 Formula 1.2.1.

Empirical C16H1403

1.2.2. Structural

1.3 Molecular Weight 254.29

KETOPROFEN

445

1.4 Appearance, Colour, Odour and Taste A slightly coloured, odourless, tasteless powder with an irritant dust. 2.

Physical Properties 2.1 Melting Range

93-95OC(5)I 94-95OC(14)I 94OC(1,2,15,16) 96OC, 92OC ( 1 7 ) , 91OC ( 1 8 ) .

,

2.2 Solubility soluble ether soluble ethano1 s1ightly water soluble octanol soluble disopropyl ether soluble acetone soluble chloroform dimethylformamide soluble soluble methanol soluble ethyl acetate 2.3 pH The pH of a 3.95 x is 6.5 (5).

M solution in water

2.4 Dissociation Constant The pKa in : dioxan : water (2:l) is 7.2 (20), acetonitrile : water (3:l) is 5.02 ( 5 1 , methanol : water (3:l) is 5.937 (14). 2.5 Partition Coefficient The partition coefficient of ketoprofen in an n-octanol/water (phosphate buffer pH 7.35 and initial ketoprofen concentration of 0.2542 mg/ml in this) is 0.105 ( 5 ) and at pH 7.4 (MacIlvaine's buffer and initial ketoprofen concentration of 0.0240 mg/ml in this) is 0.97 (20). At these pH's most of the ketoprofen is ionised (20) and thus an increase in the initial concentration of ketoprofen in the buffer will cause an alteration in the partition coefficient.

GARY G . LIVERSIDGE

446

2.6 Thermal Analysis 2.6.1. Differential Thermal Analysis A D.T.A. thermogram of ketoprofen at a heating rate of 5OC per minute and sample size of 4 mg in a static air atmosphere shows an endotherm at 96OC which indicates melting (Fig. 1). If the melted sample is cooled to O°C and then analysed again no peak corresponding to melting can be detected. The ketoprofen is in a glass like form. On storage the glass like form changes to the regular crystdllineform, conversion is complete in ten days at room temperature.

2.6.2. Thermogravimetric Analysis A TGA thermogram of ketoprofen at a heating rate of 20C per minute and sample size of 6 mg in a static air atmosphere shows no l o s s of weight until 223% when ketoprofen decomposes (Fig.2).

2.7 Crystallinity 2.7.1. Polymorphism Ketoprofen can exist in two Polymorphs as mentioned in section 2.6.1. on differential thermal analysis. Ketoprofen forms white crystal prisms when crystallised from di-isopropyl ether ( 5 ) . 2.8 Ultraviolet Spectrum The UV spectra of ketoprofen (3.95 x 10-4m) in the following solvents are given in Figure 3 (using Varian Techtron M 165) 1. 0.1N hydrochloric acid pH 1.2 (5) 2. distilled water pH 6.5 (5) 3. 0.1N sodium hydroxide pH 12.9 (5) appears at 261 nm and corresponds toa The A m K ban$. This maximum is independent of p H but the maximum absorbance is slightly decreased with increasing pH. The Xmax in methanol has been The reported as 255 nm and log E = 4.33 (14 2 1 ) Xmax in ethanol has been reported as 255nm and

.

% ’

cm

640 ( 7 ) .

447

KETOPROFEN

Figure 1.

Differential Thermogram of Ketoprofen

0

2

Figure 2.

g %

Weight

N"

Thermogravimetric Curve of Ketoprofen

GARY G. LIVERSIDGE

448

Of

0;

Of

0: u 0

9

p OL a 13

03 02 0.1

200

Figure 3

250

Mo

31

Ultraviolet Spectrum of Ketoprofen (see text for key)

'8D

.L

.a

.3

* 40

.2

2G

.I

-

0 A 0

'0

0; 2%

fi

-20

-1

-LO

-2

- 60

-3

-80

-L

200

Figure 4

nrn

250

I

CD Spectra of Ketoprofen's Enantiomers

KETOPROFEN

449

2.9 Optical Rotation Ketoprofen is a racemic mixture of

( 2 )a- (3-benzoylphenyl)-propionic acid (3,4,5 ) .

Both enantiomers show Cotton Effects at 223 nm (4) as demonstrated in Figure 4. The (+)-enantiomer shows a positive Cotton Effect indicating a S-absolute configuration and interacts more strongly with human serum albumin as well as with biotransformation enzymes than the (-)-enantiomer 23 (4) (+)-enantiomer [a], + 57.1° (C = 0.76 in CH2C12) 23 (415) (-)-enantiomer [a], - 57.4O (C = 0.88 in CH2C12) 23 (415) (+)-enantiomer [ a ] , + 49.6O ( C = 1.15 in CH2C12) 23 (3) . ( C = 1.05 in CH3C13) (-)-enantiomer [ a ] , - 52.4' (3) 2.10 Mass Spectrum

-

The mass spectrum of ketoprofen has not been published but the mass spectrum of the methyl ester has (8,10,13), see Figures 5 and 6 (using LKB900S gas chromatograph mass spectrometer) (13). The fragmentation pattern is reported in Figure 7. The fragmentation pattern for ketoprofen will be similar, the methyl of the ester being replaced by a hydrogen atom. 2.11 Photoelectronic Spectrum The photoelectronic spectrum on a Vacuum Generators UV G3 instrument (Figure 8 ) exhibits several b nds characteristic of the benzophenone group. Notably the partially overlapping bands at 9.07 and 9.45 eV, these bands are a result of the ionisation of the two degenerated phenyl orbitals. The energy at 10.62 eV corresponds to the ionisation of a free electron pair of the carboxylic carbonyl group. Bands have been assigned as:47r

0 0 N

.O

0

N 0

.O

N

z

1s

0

0

0

0

0

0

"2

GARY G . LIVERSID(;E

456

3.

Synthesis

Several methods for the synthesis of ketoprofen have been reported in the literature (15-20), 23-27, 43, 46-54). The synthesis starting from (3-benzoylphenyl)-acetonitrile is illustrated in Figure 11 (15,16,41). The synthesis starting from (3-carboxyl-phenyl)-2 propionitrile is illustrated in Figure 12 (17,20). The synthesis starting from 2- (4-Aminophenyl) propionic acid is illustrated in Figure 13 (15,17,19). 4.

Stabilitv and Dearadation

Ketoprofen must be protected from light and moisture. It is stable at room temperature. Ketoprofen has been dissolved in ethyl acetate and stored for several weeks at 4OC with no detectable decomposition (13). If ketoprofen is heated in an acid solution pH1 at 98OC for 30 min. no decomposition is detected (28). 5.

Absorption, Metabolism and Excretion

Ketoprofen is absorbed rapidly regardless of the route of administration. It reaches a peak maximum in the first hour of administration if taken by the oral, rectal and parental routes and six hours if taken by the subcutaneous route. Peak blood levels by the rectal route are observed after 45 mins. to 60 mins. (29,30,31). Peak blood levels by the oral route are observed after 60 mins. to 90 mins. (31,32,33) and by the intramuscular route after 30 mins. (32). The half life of ketoprofen has been reported from 1.5 hrs. to 2 hrs. (6,29,30, 32-36). From 60% to 90% of ketoprofen is bound to serum protein (29). The kinetics of elimination are first order and the rate constant is 0.350 hrs., 63% of the administered dose is excreted in the urine during the first 24 hrs. and 65. in the first 48 hrs. Minimal excretion occurs in the faeces, the rat being the exception (29). The metabolism of ketoprofen is due to two major processes, a hydroxylation process, predominant in the rat, although the prefered excretory form in the rat is unchanged ketoprofen and glucuronide conjugation in other species including man. The glucuronide conjugation pathway is predominant in the rabbit and man but in man the hydroxylation is not totallyabsent

KETOPROFEN

457

+

CH31

Figure 11 Synthesis of Ketoprofen Starting From (3-benzoylphenyl)-acetonitrile

GARY G. LIVERSIDGE

458

II

7

0

7

0

Ho’coC H3

It

cL’cocH H3

-cN

-

F RI C DEL C R A F 1

1)

HYDROLYSIS I

Figure 12 Synthesis of Ketoprofen Starting From (3-carboxy-phenyl)-2 propionitrile

459

KETOPROFEN

P O T A S S l U l l E THVLXANTHATE

7-IODOEENZOIC

ACID

POLYPHOSPHOAIC A C I D

RANEY N I C K L L

Figure 13 Synthesis of Ketoprofen Starting From 2- (4-Aminophenyl) propionic acid

GARY G . LIVERSIDGE

460

see Figure 14 ( 2 9 , 3 1 ) . As mentioned in section 2.9 Optical Rotation ketoprofen can exist in two enontiomeric formsl each having a different affinity for human serum albumin and different biotransformation pathways (Hydroxylation pathways) (4).

6.

Methods of Analvsis 6.1

Elemental Analysis C(75.58%) I H(5.55), 0(18.87%)

6.2

Thin Layer Chromatographic Analysis

Stationery phase and platecoating

Mobile phase

Cellulose 0.10 cm thick

sec-butanol: 1.35 absolute ethanol: water:ammonia 32% (50:30 19:l) v / v . ch1oroform:methanol: 1.42 ammonia 32% (120:60:0.5) v/v hexane:acetone:water 0.72 (12.15:3) v/v. iso-octano1:dioxan: 0.88 glacial acetic acid (20:20:1) v/v . butyl acetate:methyl 1.03 ethyl ketone: glacial acetic acid:water (4:4:2:1) v/v ch1oroform:methanol 0.42 (94:6) 3.05 n-hexane : dimethylketone: acetic acid (90:10:2) dichloromethane: 0.75 methapol. ammonia t98150: 3)

Silica gel 0.25 cm thick Silica gel 0.25 cm thick Silica gel 0.25 cm thick Silica gel 0.25 cm thick Silica Silica

Silica

Rf (References)

.

(21)

*

(21)

*

(21)

*

(21)

*

(21)

*

(5) (5)

(5)

P I C

751 c u o m

3

ucll 3

O n 3 c u 0u

r

0 0

u I

w

u--u -

1I I"

r P I C

o m

Yc u2 hell Y Z J

u n z c

u 0u

I 0

0

'i

-

u-u-0

w

I

0

3 04i 0

o=w O=U

6

GARY G . LIVEHSIDGE

462

6.3 Ultra Violet Spectroscopy Quantitative determinations of ketoprofen based on the peak maximum at 261 nm in distilled water (5) or 256 nm in methanol can be performed (21) see Section 2.8 and Figure 3. 6.4 Potentiometric titration If ketoprofen is accurately weighed and dissolved in acetonitri1e:water (3:l) and titrated with 0.1 NaOH the potentiometric curves are recorded between pH 3.45 - 12.0. This method is convenient for measuring the purity of ketoprofen in the crystalline preparation and also the content in tablets (5). 6.5 Gas Chromatography Gas chromatography is an inconvenient method for purity determination, as ketoprofen is partially decomposed by the procedure. This can be overcome by using the methylester or trimethylsilylester prepared quantitatively from ketoprofen. According to the method of Populaire et a1 (37) ketoprofen and the esters can be chromatographed on a 1 m x 3 mm column of OV-17, 5% GCQ 80-100 mesh at a temperature between 230 - 27OOC and carrier gases; argon:50 ml/min, hydrogen:79 ml/min, air:150 ml/min, and a Flame Ionisation Detector. The retention times Rt are for ketoprofen 6.35 min, for the methyl ester 4.05 min and for the trimethylsilyester 2.68 mins (5). 6.6 Enantiomer Analysis

The ratio of (+)-ketoprofen to (-)-ketoprofen in a racemic mixture can be determined by reaction with a stereospecific molecule and the product is then analysed by either gas chromatography (5) or high pressure liquid chromatography (38). The ratio of peak height or peak area respectively gives the ratio of the two enantiomers.

KETOPROFEN

463

6.7 Colorimetric Analysis

Ketoprofen can be complexed with safraniii and the absorption determined in chloroform at 5 2 0 nm ( 1 4 ) . Analysis of Biological Samples

7.

7.1

Colorimetric Analysis

This method is suitable for the analysis of ketoprofen in urine. The urine is made alkali by addition of NaOH then extracted with ether, the aqueous layer is then acidifiedandthe ketoprofen extracted with hexane and evaporated to dryness. The ketoprofen (via its carbonyl group) is reacted with p-nitrophenylhydrazine to give a p-nitrophenylhydrazone which gives a violet colour with trimethylbenzylammonium hydroxide. This violet complex is then assayed colorimetrically at 5 6 0 nm and 4 6 0 nm (see Figure 1 5 for spectrum and sample - ) . The full of blank experimental detail is reported by P. Populaire et a1 ( 3 7 ) . The p-nitrophenylhydrazone formed undergoes partial decomposition to give other hydrazones, but these also absorb at 5 6 0 nm and are directly proportional to the concentration of ketoprofen in total and this decomposition does not interfere with the assay. The absorption due to ketoprofen at 5 6 0 nm is determined by subtraction of the absorption by urine blanks, at 4 6 0 nm ( 4 6 0 nm Abs = 5 6 0 nm Abs for blank) from it, this is the case for humansand cats. But for rats and rabbits the absorbance at 480 nm is deducted from the absorbance at 5 6 0 nm i.e. interference is species dependent. The precision of this method 10% over the urinary concentration range of is 10-100 mg/L and the limits of detection of 2-5 mg/L in urine.

......

*

7.2

Polarographic Analysis

This method is suitable for the analysis of ketoprofen in urine. The same extraction procedure asin Section 7 . 1 is used as urinary substances will interfere with the polarogram. The carbonyl groupof ketoprofen is reduced at the dropping mercury electrode, in a 0 . 2 m solution of

0

0

0

0

Ln

0

0

0

r

0

0 .3

10

a,

a

E

d

rd

Id

CI]

Colourmetric analysis of Ketoprofan in Biological Samples d

Figure 15

KETOPROFEN

465

tetrabutylammonium hydroxide. The half wave potential Ef is -1.36 volts, see Figure 16 and the method employs a standard addition technique. For further experimental details see P. Populaire (37). The precision of this method is flO% of urinary concentrations in the range 10 to 100 mg/litre and the limit of detection is 5 mg/litre. 7.3 Gas Chromatographic Analysis If ketoprofen is analysed directly by gas chromatography partial decomposition results. This difficult can be overcome by working with the methyl ester (5,8,13,30,31,34,35,37,39), and using (Benzoyl-4-phenyl)-2 butyric acid as the internal standard as itsmethyl ester. Neither serum or urine samples of ketoprofen are gas chromatographed directly, an extraction procedure is employed. In the case of serum the sample is acidified then extracted by an organic solvent usually ether. In the case of urine the sample is made alkali and the unwanted products extracted with an organic solvent usually ether, the aqueous phase is then acidified and extracted with ether. The etherial extracts are washed with acid and then water, dried with magnesium sulphate and then evaporated to dryness. The samples are then methylated and chromatographed (5,29,30,31,34,35, 37). In some cases further purification prior to gas chromatography is performed using thin layer chromatography (37). Table IV gives the conditions of chromatography and retention times (Rt) of the methyl esters of ketoprofen (A) and the internal standard (Benzoyl-4-phenyl)-2 butyric acid ( B ) obtained from biological media. The accuracy of this technique has been claimed as f10% and a lower limit of detection of 0.03 - 0.04 mg/litre (37)

-

GARY G. LIVERSIDGE

466

TABLE IV

Support, s t a t i o n a r ] C a r r i e r phase and Temp. g a s and flow rate

Length and diameter internal (Reference)

R t (A)

R t (B)

6.29

8.85

I

I

6 f t x 2mm (35)

OV-17 3% GCQ 80/100 mesh 25OoC f o r - 1 0 mins t h e n 0 280 for 4 mins. I ov-1 3% GCQ 18O0C-25OOC a t z0c/min OV-1 1%GCP 100/120 mesh 25OoC OV-17 1% Chromosorb W AW DMCS 24OoC OV-17 3% GCQ 80/100 mesh 225'C OV-17 3% Chromasorb W AW DCMS 24OoC OV-17 5% GCQ 80/100 mesh H 2 0 230-270°C

N 2 60ml / min

I 1.5m x 2mm (8) 2m x 2mm (13) 2m x 3mm (29,391 1.5m x 2mm (10) 2m x 3mm (40) l m x 3mm

(5)

H e 2Oml /min

14

18

H e 3Oml /min

3

5

N2 3 0 m l /min

4.8

6.6

N2 3 0 m l /min N2 30ml /min

3

4.5

4.8

6.6

A r 50ml/mi H, 79m l / m i a i r 150ml/ min

4.05

7.4 Thin Layer Chromatographic Analysis A TLC method for the analysis of ketoprofen and its urinary metabolites has beendescribed (31) using a two dimensional development system. But the separation is incomplete and the system insufficiently sensitive. After extraction from biological samples as in Section 7.3 ketoprofen can be analysed by TLC, using 250 urn thick Merck 60 F254 plates, activated at 105OC for 1 hour, with a solvent system of ether-benzene-l-butanol-methanol (85:8:6:1), giving an Rf value of 0.75. The spot can be

KETOPROFEN

467

scraped off the plate, dissolved in ethanol and analysed under UV. Accuracy of + 6 . 0 1 % and a limit of detection of 11.19 is claimed by Ballerini et a1 (7). A further method involving methylation of ketoprofen has been described ( 3 7 ) . 7.5 Gas Chromatography Analysis

-

Mass Spectroscopy

Ketoprofen in biological extracts is converted to methyl ester with an internal standard (benzoyl-4-phenyl)-2-butyric acid before undergoing GC-MS, as ketoprofen undergoes rearrangement when subjected to gas chromatography, see Section 6.5 (8,10,13). The limits of detection by this method are 2.5 ng and an accuracy of 10% at plasma concentrations of 25 ng/ml (8). 7.6 High Pressure Liquid Chromatographic Analysis

Most methods for the analysis of ketoprofen in biological samples require the selective extraction of ketoprofen (42) similar to Section 7.3 (12,28,39-421, or by direct injection (44). Table V gives the chromatographic conditions retention times (Rt), limit of detection and accuracy for ketoprofen.

-

1.0

1.2

1-4

1.6

VOLTS

Figure 16

Polarogram of Ketoprofen

3 68

GARY G. LIVERSIDGE

TABLE V

Column, Packing, diameter intercal (Reference)

Mobile phase, flow rate pressure

Hewlard Packard RP8-79918 A 25Ox4mm (7)

Water/Methanol 85/15, 0.8 ml/min 32-38 atmos

8.3

0.1 pg/ml 25.1%

Spherisorb-5 ODS 50 x 5 mm (44)

35% aqueous methanol pH3 2 ml/min

2

2.5 ng f2.1%

Lichrosorb S 1 60 Spm 250 x 4.7 mm (39,401

dichloromethane/ hexane 60/40 1.3 ml/min 35 bars

:12.a

Lichrosorb RP 18 Sum 150 x 4.7 mm

methanol/ acetonitrile/ phosphate 15/35/45 gH3 0.83 ml/min 7 0 bars

8

Rt min

Detection limit and accuracy

0.1 pg/ml

24%

0.1 pg/ml

24%

0.5 phosphate pH 7.0 16 1 6 4 % acetonitrile 2.0 ml/min 1000 psi

10-23 ng i ml

methanol/water 45/44 1.1 ml/min 1800 p i

5.3

300 x 4 mm (12)

0.1 pg/ml -+5.4%

Lichrosorb RP 18 5 pm or Lichrosorb RP 8 5 pm. 100 x 4.6 mm (9)

acetonitrile/ phosphate 0.02 m pH3 45:55 1000 psi

5.2 4.2

40 x 4.6 mn (42)

*

methyl ester of ketoprofen

8. Analysis of Pharmaceutical Formulations 8.1 Potentiometric Titration 300 mg of ground sample are dissolved in 5 mls of acetonitrile or ethanol and 15 ml of water added. The titration is performed with 0.1 N N a O H

KETOPROFEN

469

and monitored using a glass electrode and a calomel reference electrode ( 5 ) . 8.2 Pyrolysis - Gas Chromatography Spectrometry

-

Mass

Ground samples are dissolved in a solvent and a known amount applied to a rotating wire. After evaporation of the solvent the material is pyrolysed in a Curie point pyrolyser at 77OoC for 5 seconds and the pyrolate purged onto a Carbowax 20 M - KOH column, that is temperature programmed from 100 to 24OoC. On pyrolysis ketoprofen is rearranged to (3-benzoylphenyl)-ethane and (3-benzoylphenyl)-ethylene which have retention indices of 2.27 and 2.52 respectively, analine having a retention of 1.00. Limits of reproducible detection range from 10 ng to 10 pg. This method can also be employed in the analysis of biological materials (45,221. 9.

Acknowledgements

I wish to express my thanks to Mrs. T. Bowler for typing this manuscript. References 1. 2. 3.

4. 5.

6. 7. 8. 9.

Drugs of Today, 1973, IX (II), 468-471. Merck Index, 9th E d . , Ed. M. Windholz, Pub. Merck & Co. Inc. Rahway, N.J., U.S.A., 1976 pp. 695-696. Rendic, S., Alebic-Kolbah, T., Kajfez, F., Sunjic, V. Farmaco, Ed. Sci. 1980, 35 (I), 51-9. Rendic, S., Sunjic, V., Kajfez, F., Blazevic, N., Alebic-Kolbah, T., Chimia 1975, 29 ( 4 ) , 170-2. Blazevic, N., Zinic, M., Kovac, T., Sunjic, V., Kajfez, F. Acta. Pharm. Jugosl. 1975, 25 ( 3 ) , 155-64. Lombardino, J.G., Otterness, I.G., Wiseman, E.H. Arzneim. Forsch. 1975, 25 ( l o ) , 1629-35. Ballerini, R., Cambi, A F D e l Soldato, P., Melani, F., Meli, A., J. Pharm. Sci 1979, 68 (3); 366-8. HeuSSe, D - , Raynaud, L., Ann. Pharm. Fr. 1978, 36 (11-121, 631-8. Bannier, A., Brazier, J.L., Quincy, C. Feuill. Biol. 1979, 106, 91-8.

4 $0

10

a

11. 12. 13. 14. 15. 16. 17.

18.

19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

GARY G. LIVERSIDGE

Stenberg, P., Joensson, T.E., Nilssen, B., wollheim, F. J. Chromatogr. 1979, 177 (1), 145-8. Ballerini, R., Cambi, A., Del Soldato, P. J. Pharm. Sci. 1977, 66 (2), 281-2. Farinotti, R., Mahuzier, G. J. Pharm. Sci. 1979, 68 (41, 484-5. De Grave, J., Frankinet, C., Gielen, J.E. Biomed. Mass. Spetrorn. 1979, 6 (61, 249-52. Unterhalt, B. Pharm. Ztg. 1978, 123 (41), 1801-3. Farge, D., U.S. Patent 3,641,127. Soci&t’e Des Usines Chimiques RhEne-Poulenc Fr. Patent6.444 M. Brunet, J.P., Cometti, A. Soci6t6 Des Usines Chimiques Rhane-Poulenc Fr. Patent 2,163,875. Allais, A., Rousseau, G., Meier, J., Deraedt, R., Benzoni, J., Chifflot, L. Eur. J. Med. Chem. - Chim. Ther. 1974, 9 (4), 381-9. Socigt’e Des Usines Chimiaues Rhhe-Poulenc Fr. Addn. 0296 to patentaM6,444. Brunet, J.P., Cometti, A. Ger. Patent 2,258,985. 114 (6) Lotti, B. Boll. Chim. Farm. 1975, 351-4. Irwin, W.J., Slack, J.A. Biomed. Mass Spectrom. 1978, 5 (12), 654-657. F6rge et a1 S - A , Patent 68,00,524. Farqe, D. et a1 Ger. Patent 1,668,648 Farge, D. et a1 Ger. Patent 2,024,389. Farge, D. et a1 Brit. Patent 1,164,585. Farge, D. et a1 Brit. Patent amended 1,164,585 Thomas, W.O.A., Parfitt, R.T., J. Chromatogr. 1979, 162 (1) 122-4. Courpron, P., Brazier, V.L., Meunier, P., Ribon, B., Bannier, A. Lyon Med. 1978, 239 (81, 477-82. Meunier, P., Courpron, P., Brazier, J.L., Ribon, B., Bannier, A. Rev. Rhum. Mal. OsteoArticulaires 1977, 44 (7-91, 519-24. Populaire, P., Terlain, B., Pascal, S., Decouvelaere, B., Renard, A., Thomas, J.P. 31 (121, 735-49. Ann. Pharm. Fr. 1973, Sala, G. Silvestri, N., CastegNarot E . Pollini, C., Farmaco, Ed. Prat. 1978, 33 (101, 455-60. I

KETOPROFEN

33* 34.

471

Brogden, R.N., Speight, T.M., Avery, G.S., Drugs, 1 9 7 4 , 8 ( 3 ) , 1 6 8 - 7 5 . Caill’e, G., Besner, J.G., Brodeur, J., 36, (5-6) , Vezina, M. Ann. Pharm. Fr. 1 9 7 8 , 243-52.

35. 36.

37.

Caill’e, G . , Besner, J.G., Lacasse, Y., Vezina, M. Biopharm. Drug. Disp. 1 9 8 0 , 1, 1 9 5 - 2 0 1 . Ishizaki, T., Suganuma, T., Sasaki, T., Watanabe, M., Horai, Y., Hoshi, H., Ashisuke, W. Rinsho Yakuri 1 9 7 9 , 10, ( 4 1 , 5 9 1 - 2 , Chem. Ahs. 1 9 8 0 , 9 2 : 2 0 8 7 n a . Populaire, P . , TGlain, B., Pascal, S., Decouvelaire, B., Lebreton, G., Renard, A., Thomas, J.P. Ann. Pharm. Fr. 1 9 7 3 , 3 1 , (ll), 679-90.

38.

McKay, S.W., Mallen, D.N.B., Shrubshall, P.R., Swann, B.P., Williamson, W.R.N. J. Chromatog.

39*

Bannier, A., Brazier, J.L., Quincy, C . Feuil. Biol. 1 9 7 9 , XX ( l O 6 ) , 9 1 - 9 8 . Bannier, A., Brazier, J.L., Ribon, B. J. Chromatogr. 1 9 7 8 , 1 5 5 ( 2 ) , 3 7 1 - 8 . Italfarmaco, S.p.A. Belg. Patent 8 3 3 , 2 6 6 . Upton, R.A., Buskin, J.N., Guentert, T.W., Williams, R.L., Riegelman, S. J. Chromatogr.

1979

40. 41. 42.

170,

(2)

482-5.

1 9 8 0 , 1 9 0 ( 1 1 , 119-28.

43* 44 * 45. 46. 47. 48. 49. 50.

Zupancie, B., Jenko, B., Aust. Patent 3 5 1 , 5 1 6 . Thomas, W.O.A., Jeffries, T.M., Parfitt, R.T. J. Pharm. Pharmacol. 1 9 7 8 , 30 (Suppl. British Pharm. Conf. 1 9 7 8 ) , 6 6 P . Slack, J.A., Irwin, W.J. Proc. Anal. Div. Chem. SOC. 1 9 7 7 , 14, ( 8 ) , 2 1 5 - 1 7 Aziende Chimiche Riunite Angelini Francesco S.p.A. Jpn. Kokai Tokyo Koho 7 9 0 9 , 2 5 1 . Chem. A b s . 1 9 7 9 , = : 1 6 8 3 0 8 p . LEK Tovarna Farmaceutskih in Kemicnih Izdelkov n.so1.0. Fr. Demande 2 , 3 6 7 , 7 2 8 . Ibid Fr. Demande 2 , 3 6 7 , 7 2 7 . Zupancic, B. Patent Ger. Offen 2,744,833. Zuparnic, B., Jenko, Patent Ger. Offen. 2,744,834.

51. 52. 53. 54.

Zoni, G. Spanish Patent 4 4 5 , 8 4 6 . Baiocchi, L. Patent Ger. Offen. 2 , 6 2 4 , 1 7 7 . Sigurta Farmaceutici S.p.A. Be1 Patent 8 3 7 , 6 2 4 . Zoni, G. Belg. Patent 8 3 9 , 6 3 4 .

METHYLPHENIDATE HYDROCHLORIDE Gandharva R. Padrnanabhan 1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance 2. Physical and Chemical Properties 2.1 Infrared Absorption Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Absorption Spectrum 2.4 Mass Spectrum 2.5 Optical Rotation 2.6 Melting Range 2.7 Differential Scanning Calorimetry 2.8 Thermogravirnetric Analysis 2.9 Solubility 2.10 X-Ray Diffraction 2.11 Polymorphism 2.12 Partition Coefficient 2.13 Dissociation Constant 3. Synthesis 4. Stability-Degradation 5. Drug Metabilism and Pharrnacokinetics 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 High Pressure Liquid Chromatography 7.7 Gas Chromatography 7.8 Gas Chromatography-Mass Spectrometry (GC-MS) 7.9 Colorimetric Methods 7.10 Infrared 7.11 Reineckate Salt 8. References 9. Acknowledgment

474 474 474 474 474 474 477 479 479 48 1 48 1 48 1 48 1 483 483 483 483 485 485 486 486 486 486 486 486 487 487 489 49 1 492 493 494

495 495 497

GANDHARVA R. PADMANABHAN

474

1.

Description 1.1. Name, Formula, Molecular Weight Methylphenidate hydrochloride is methyl a-phenyl2-piperidineacetate hydrochloride, (Rfc,Rfc)- (+) .

C14H19N02.HCl 1.2

Molecular Weight 269.71

Appearance Methylphenidate hydrochloride occurs as a white, odorless, fine, crystalline powder.

2.

Phvsical and Chemical ProDerties 2.1.

Infrared Absorption Spectrum The infrared spectrum of a mineral oil suspension o f methylphenidate hydrochloride is shown in Figure 1. The spectral assignments are listed in Table 1. TABLE 1

1

Wavenumber, cm-l 703, 737 1602 2300

2700

Assignment Monosubstituted benzene Aromatic Stretch Secondary Amine Salt

1739

C=O Stretch

-

C-0 Stretch

1150 2.2

-

I

1170

Nuclear Magnetic Resonance SDectrum (NMR) The NMR spectrum of methylphenidate hydrochloride i s shown in Figure 2. The spectrum was determined on a Perkin-Elmer R-24B 60 MHz spectrometer at ambient temperature. The sample was dissolved in a 1:l mixture o f deuterated chloroform and deuterated dimethylsulfoxide containing tetramethylsilane as an internal standard. The spectral assignments are shown in Table 2.

E

I 0 Q

N 0

I

>

a

?

0

W 0

0

8

8

: 8 2

2

;

a

3

2

3

‘D

8

f

8

7 8

I 1

0

m

U

I

-0 N

7

0 -N

N

0 .U

0

0

.W

0

.a,

0 0

W

METHYLPHENIDATE HYDROCHLORIDE

477

TABLE 2 Chemical Shift 6 (PPd

Multiplicity

7.1

-

7.6

Broad Singlet

Phenyl protons

4.0

-

4.4

Doublet

-CH-COOCH, -

3.6

-

3.8

Singlet

-0cg3

No. of

Assignment

Protons

I

I

2.8

- 3.6

Broad Mu1 tiplet

-N

Solvent

'CH2-

2.4

-

2.7

Broad Multiplet

1.0

-

1.9

Broad Multiplet

2.3.

CH/ -

Ultraviolet Absorption Spectrum The W spectrum of methylphenidate hyd,rochloride (1 mg/mL) in methanolic 0.1N HC1 exhibits maxima and minima as shown in Table 3 and Figure 3 . TABLE 3

A max, nm

A 1% 1cm

264

6.1

165

25 7

7.7

208

25 2

5 -9

159

247

4.5

122

A min. at

&

263 nm, 255 nm, 249 nm and 245 nm.

GANDHARVA R . PADMANABHAN

478

Figure 3: Ultraviolet Absorption Spectrum of Methylphenidate Hydrochloride

0.8

0.7

0.6

0.5 a, 0

c

m

g

0.4

0

n

Q

0.3

0.2

0.1

0.0

Wavelength , Nanometer

METHYLPHENIDATE HYDROCHLORIDE

2.4

479

Mass Spectrum The low resolution mass spectrum of methylphenidate hydrochloride obtained at 70 ev using a solid probe insertion is shown in Figure 4 . The spectrum was run on a Kratos MS25 spectrometer interfaced with a data handling system. Table 4 illustrates the prominent fragments and their masslcharge ratios. TABLE 4

';The spectrum is known to vary due toethermal decomposition (27).

2.5

Optical Rotation Although the methylphenidate hydrochloride molecule has two asymmetric carbon atoms, the drug exhibits no optical activity as it is a racemic mixture. The diastereoisomer of the drug, (R",Sf:) isomer, is also referred to as "erythro isomer". The conformations of methylphenidate hydrochloride and its (&:,Sf

20

(50,54)

u,12 (50,54) 1,5 (50,54)

0,01 0,11 0,17 0,Ol 0,01 14 (54); 20 (38 15 (50,54) 20 (38) 50 (38,50)

1Y5

,ee text ;ec text 1Y9 15

140 (38,54) 120 (38,5U) 185 (50,54)

250

0,21

>

20

0,0U3

*at L I O C , c a l c u l a t e d from evaporation r e s i d u e , corrected f o r sol vent bl ank. **mu1 t i pl c c r y s t a l 1i sed natainyci t i r e f e r e n c e standard; a t 20°C a f t e r e q u i l i b r a t i o n with a s o l v a t e , i f any; f i l t r a t e analyzed by d i f f e r e n t i a1 spectrophotoinetry ‘37. h - . , - h . , A - - & - . ......r e r . JJ. iieAarryuraLe;

+C+--C

.

-,C

iei.

‘>O

...-A

L A .

.>o ariu m.

,.-I,--.._

UIIK~IWWII.

541

NATAMYCIN

4.

P roduc t i on

4.1.

Discovery

I n 1955 S t r u y k e t a l . i s o l a t e d a new a n t i f u n g a l a n t i b i o t i c from a c u l t u r e o f Streptomyces n a t a l e n s i s nov. sp. (38). T h i s s t r a i n was i s o l a t e d from a s o i l sample which was taken near P i e t e r m a r i t z b u r g , a town i n t h e p r o v i n c e o f N a t a l , South A f r i c a . The t y p i c a l u l t r a v i o l e t spectrum o f t h e new a n t i m y c o t i c p o i n t e d t o a r e l a t i o n s h i p w i t h a l r e a d y known polyenes l i k e n y s t a t i n , t h e f i r s t i,iernber o f t h i s group which was discovered 5 years e a r l i e r .

.

I n 1959 Burns e t a1 ( 5 5 ) i s o l a t e d a compound from a c u l t u r e o f Streptonyces chattanoogensis, a s t r a i n from a s o i l sample of Chattanooga, Tennessee, which was c a l l e d t e n n e c e t i n . However, w i t h i n two y e a r s t h i s compound appeared t o be i d e n t i c a l w i t h natamycin ( 3 9 ) , so t h e name t e n n e c e t i n was del eted.

A nameless t e t r a e n e , d e s c r i b e d by Backus e t a l . i n 1959 ( 5 6 ) , i s most p r o b a b l y i d e n t i c a l w i t h natamycin. The substance was produced by Streptomyces g i 1vosporeus ATCC 13326. 4.2.

Biosynthesis

The b i o s y n t h e s i s o f t h e C-25 b u t y l homologue o f natamycin, 1ucensomyci n, has been s t u d i e d u s i n g 1%-1 abel ed p r o p i onate and a c e t a t e (57). These p r e c u r s o r s are i n c o r p o r a t e d i n t o t h e aglycone. 1 4 b l a b e l e d natamycin c o u l d be produced i n t h e same way (58). The carbon s k e l e t o n o f mycosamine i s p r o b a b l y d e r i v e d d i r e c t l y from glucose (59). 4.3.

Fermentation and I s o l a t i o n

Natamycin i s produced on an i n d u s t r i a l s c a l e by f e r m e n t a t i o n u s i n g Streptornyces n a t a l e n s i s ( 6 0 ) o r Streptomyces g i l v o s p o r e u s ( 2 5 ) . As most o f t h e a n t i m y c o t i c i s bound t o t h e mycelium i t i s i s o l a t e d e i t h e r by whole b r o t h e x t r a c t i o n o r by e x t r a c t i o n o f t h e mycelium, u s i n g l o w e r a l c o h o l s (25,bO). The crude compound i s p r e c i p i t a t e d by pH r e g u l a t i o n o r by e v a p o r a t i ve c o n c e n t r a t i o n .

HARRY RRIK

542

5.

S t a b i 1 it y

Natamycin i s a s t a b l e compound p r o v i d e d t h e powder i s p r o t e c t e d from l i g h t arid m o i s t u r e . Only a few percent l o s s o f a c t i v i t y i s observed a f t e r several y e a r s storage a t roointemperature. T h i s i s t r u e f o r t h e t r i h y d r a t e , t h e anhydrous form however i s n o t s t a b l e . T h i s form, prepared by h e a t i n g t h e tri h y d r a t e i n vacuo a t roomtemperature over phosphorus pentoxide (see a l s o S e c t i o n 3.10.3), l o s e s 15% o f a c t i v i t y when s t o r e d f o r 48 hours a t roomtemperature i n a c l o s e d b o t t l e i n t h e dark (49). Natamycin w i l l w i t h s t a n d h e a t i n g a t up t o 1 2 i ) O C ; f o r no more t h a n one hour. However, any anhydrous natarnycin produced d u r i n g h e a t i n g i s u n s t a b l P. The methanol s o l v a t e ( S e c t i o n 3.1.1) i s an u n s t a b l e substance as we1 1

.

N e u t r a l aqueous natamyci n suspensions a r e n e a r l y as stab1 e as t h e d r y powder. A n e u t r a l aqueous suspension can be b o i l e d f o r a s h o r t t i m e b e f o r e a r e d u c t i o n i n potency occurs. Aqueous s o l u t i o n s a r e q u i t e s t a b l e a t pH values between 5 and Y i f s t o r e d i n t h e dark ( 5 4 ) . A t extreme pH values natamycin i s r a p i d l y i n a c t i v a t e d w i t h formation o f various kinds o f decomposition products ( F i g u r e 1 7 ) . A t a low pH t h e mycosamine m o i e t y i s s p l i t o f f . The r e s u l t i n g i n s t a b l e aglycone r e a c t s w i t h e i t h e r a second molecule o f aglycone o r w i t h a s t i l l i n t a c t molecule o f natamycin. I n b o t h cases diiners w i t h a t r i e n e r a t h e r t h a n a t e t r a e n e group a r e formed. A t t h e same t i m e t h e epoxy group i s h y d r o l y s e d t o a d i o l . H e a t i n g a t l o w pH f a v o u r s d e c a r b o x y l a t i o n o f t h e aglycone (61). A t h i g h pH values, r a p i d l y a t pH 12, t h e l a c t o n e i s s a p o n i f i e d w i t h f o r m a t i o n o f t h e m i c r o b i o l o g i c a l l y i n a c t i v e natamycoic a c i d (33). Treatment w i t h s t r o n g a l k a l i r e s u l t s i n f u r t h e r d i s r u p t i o n o f t h e molecule owing t o a s e r i e s o f r e t r o a l d o l r e a c t i o n s . Among t h e r e a c t i o n products t h e f o l l o w i n g compounds c o u l d be detected: 13-hydroxy-2,4,6,8,1O-tetradecapentaene-l-a1 (1,62), acetone (4), acetaldehyde (3,4) and ammonia (1). Natamycin i s decomposed by u l t r a v i o l e t r a d i a t i o n w i t h l o s s o f Thoma ( 6 5 ) observed t h a t t h e t e t r a e n e s t r u c t u r e (33,b3,64). natamycin decomposed f a s t e r i n aqueous s o l u t i o n a t pH 4 than a t pH 8 upon r a d i a t i o n w i t h a xenon lamp. V i s i b l e l i g h t does n o t i n a c t i v a t e natamycin unless t r a n s f e r o f photo-energy by e.9. r i b o f l a v i n takes p l a c e (66). Gamma r a d i a t i o n decomposes natamycin as He1 1 , i t can t h e r e f o r e n o t be used t o s t e r i l i z e t h e substance.

543

NATAMYCIN

OH

0 HO

-IHO NHZ mycosamine

I Me

OH

: aponatarnycin

IR = R 1 )

11 : natarnycinolidediol dimer (“aglycone dirner”) (R E O H )

OH

t

\1

H+

pH >1/12-decarboxy-analogue

acetone,ethanal, NH,

natamycoic a c i d

13-hydroxy-2,4,6,8,10-tetradecapentaen-l-al

N”2

Figure 17. Decomposition o f natamyci n i n acid and al kal ine medium ( I and 11: t e n t a t i v e s t r u c t u r e )

HARRY BRIK

544

The i n a c t i v a t i o n by peroxides o r , e s p e c i a l l y a t h i g h e r temperatures, by oxygen can be prevented by a n t i o x i d a n t s l i k e c h l o r o p h y l l , a s c o r b i c a c i d (38,67,68) b u t y l a t e d hydroxyanisol e o r b u t y l ated h y d r o x y t o l uene ( 6 9 ) . O x i d a t i v e i n a c t i v a t i o n i s promoted by several metal i o n s , e s p e c i a l l y F e ( I I I ) , N i ( I 1 ) and C r ( I I 1 ) (33). This can be prevented by adding complexing agents l i k e EDTA o r polyphosphates (69). I n a c t i v a t i o n of natamycin by l i g h t , peroxides o r oxygen proceeds a t t h e f a s t e s t r a t e i n s o l u t i o n o r i n suspension, l e s s so i n t h e s o l i d form. O x i d a t i v e d e g r a d a t i o n u f natamycin p r o b a b l y l e a d s t o t h e f o r m a t i o n o f polymers o r coli1pounds formed by a d d i t i o n o f oxygen on t h e conjugated double bonds. The l a t t e r r e a c t i o n , which takes p l a c e a t one end o f t h e polyene chain, i s d e s c r i b e d f o r several polyenes. E i t h e r an epoxy-group ( f i1ip i n and 1agosi n, 70) o r a hydroperoxide ( n y s t a t i n , 71, o r l e v o r i n and mycoheptin, 72) i s formed. I n a c t i v a t i o n occurs a l s o i n t h e presence o f s u l p h i t e s o r sodi um formal dehyde sul phoxyl ate. 6.

B i opharmaceuti cs

6.1.

Pharmacokinetics

A b s o r p t i o n o f natamycin from t h e human i n t e s t 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 o f doses from 125 t o 500 mg per day d u r i n g a p e r i o d o f 1 up t o 7 days has n o t been observed. The serum c o l l e c t e d d i d n o t show any a n t i f u n g a l a c t i v i t y (73). I n animals t h e same r e s u l t s have been obtained. With r a t s and mice o r a l a d m i n i s t r a t i o n o f natamycin o n l y reduced t h e y e a s t count i n t h e faeces (38,74). 6

.%. T o x i c i t y

Uatamycin has a very l o w o r a l t o x i c i t y . The o r a l ills0 i n t h e male r a t i s 2,73 g/kg, i n t h e male r a b b i t 1,4Z y/kg (75). The c h r o n i c t o x i c i t y of natamycin was s t u d i e d by t h e a d m i n i s t r a t i o n o f natamycin i n t h e food o f r a t s and dogs. Only m i n o r e f f e c t s such as a s l i g h t decrease i n t h e i n t a k e o f food and a s l i g h t i n h i b i t i o n o f growth, were observed when 1 mg p e r kg p e r day was f e d t o r a t s f o r two years. Dogs t o l e r a t e d a dose o f 0,25 mg per kg per day f o r more tharl two years, a dose o f 0,s mg per kg per day r e s u l t e d i n a s l i g h t decrease i n body weight when administered f o r two y e a r s (75). A c i d degradation p r o d u c t s ( 1 ike aponatamycin, t h e aglycone dimer and mycosami ne) and products o b t a i n e d by a1 k a l ine d e g r a d a t i o n o r UV r a d i a t i o n o f natamycin a r e even l e s s t o x i c t h a n t h e p a r e n t compound (76,77).

NATAMYCIN

6.3.

545

Other

No s e n s i t i z i n g e f f e c t has been observed a f t e r continuous exposure t o h i g h c o n c e n t r a t i o n s o f natamycin (78,79). This i s p o s s i b l y due t o t h e low a f f i n i t y o f natarnycin f o r p r o t e i n s . Kesistance t o natamycin i s not observed (80), cross r e s i s t a n c e between nataniyci n and o t h e r polyenes has not been r e p o r t e d (81,82). The haemolytic a c t i v i t y o f natamycin i s l e s s than t h a t o f t h e more l y o p h i l i c polyenes n y s t a t i n, amphoterici n B and 1 ucensomyci n (83). tkrrcoiiler (84) r e p o r t e d nausea, v o m i t i n g and d i a r r h o e a when natamycin was given o r a l l y t o a d u l t s i n doses exceeding 1000 mg p e r day. 7.

Hnalysis

7 .l.

Identification

T y p i c a l c o l o u r s are formed when concentrated m i n e r a l a c i d s a r e added t o natamycin. T h i s r e a c t i o n i s based O H , x o t o n a t i o n of t h e polyene chrornophore (85). I n t h i s manner natamycin may be i d e n t i f i e d among o t h e r polyenes (33). See Table 9.

A s o l u t i o n o f antimony t r i c h l o r i d e i n c h l o r o f o r m ( C a r r - P r i c e reagent) y i ves d i f f e r e n t c o l ours as we1 1 ijatarnyci n and lucensomycin g i v e d r e d c o l o u r , n y s t a t i n a r e d - v i o l e t c o l o u r , a l l t h r e e s h i f t i n g r a p i d l y t o dark-brown. F i l i p i n g i v e s a b l u e c o l o u r , t h e heptaenes l i s t e d i n Table Y c o l o u r yreen, s h i f t i n g t o bl ue-green ( 3 3 ) .

.

Natamycin, 1 i k e o t h e r polyenes, r e a c t s w i t h F o l i n - D e n i s r e a g e n t (molybdotungstophosphoric a c i d ) w i t h f o r m a t i on o f a b l u e c o l o u r . T h i s t e s t i s d e s c r i b e d i n several pharmacopoeias f o r n y s t a t i n (86,87,88,89). The reagent i s h i g h l y a s p e c i f i c however s i n c e i t r e a c t s w i t h a l l r e a d i l y o x i d i z a b l e conpounds.

A somewhat more s p e c i f i c t e s t i s t h e r e a c t i o n w i t h d e c o l o u r i z e d magenta ( S c h i f f r e a g e n t ) . Upon h e a t i n y w i t h several polyenes in c l u d i ng natamyci n a r e d c o l o u r i s produced. The r e a c t i o n i s based on t h e f o r m a t i o n o f aldehydes, t h e heptaenes l i s t e d i n Table 9 do n o t r e a c t . T h i s t e s t i s a l s o d e s c r i b e d f o r n y s t a t i n i n a number o f pharmacopoei as (86,87,88).

546

HARRY BRIK

Table 9 Colour r e a c t i o n o f polyenes* polyene

group

conc. H C l :onc.

natamyci n

tetraene

brown

nystati n

tetraene

brown (gray 1 brown (gray) browngreen v i ole t

amphotcrici n A tetraene 1ucensomycin

tetraene

filipin

pentaene

amphoteric n B heptaene candi c i d i n

1e v o r i n partricin t r i chomyci n

aromati c heptaene a roma t ic h e p t aenc aromatic hept aene aroinat ic

v i 01 e t (gray1 green (brown) green (brown) green (brown) green (brown)

H2S04

brown v io le t brown v i 01e t brown viole t green brown v i 01e t brown blue b l ue blue blue

red (brown) rcd-brown (brown) red-brown (brown) green (brown green) v i 01 e t brown ilue (violet) blue (bluegreen) blue (bluegreen) blue (bluegreen) blue (yreyish-blue)

*in parentheses: c o l o u r a f t e r a few minutes Natamycin may be i d e n t i f i e d by means o f t h i n l a y e r chrornatoyraphy ( S e c t i o n 7.5.2), t h e d i f f e r e n t i a t i o n froiil o t h e r common polyenes i s n o t very c l ear, however. Natarnycin may be i d e n t i f i e d as a t e t r a e n e by r e c o r d i n g t h e u l t r a v i o l e t spectrum. By t h i s means i t can a l s o be d i f f e r e n t i a t e d from t h e t e t r a e n e s n y s t a t i n and a m p h o t e r i c i n A by r e c o r d i n g down t o 215 nrn (90). Natamycin shows an a b s o r p t i o n a t 220 nm (en-one), n y s t a t i n and a m p h o t e r i c i n i-i however show an a b s o r p t i o n a t 230 nm ( t r a n s , t r a n s - d i e n c ) . The b e s t methods f o r i d e n t i f i c a t i o n of natamycin a r e IK and UV spectrophotometry combined with t h e c o l o u r r e a c t i o n w i t h s t r o n g acids. By t h i s means i t can be d i f f e r e n t i a t e d from a l l o t h e r common polycncs. For p r e p a r a t i ons c o n t a i n i ng s i y n i f ic a n t q u a n t i t i e s o f e x c i p i e n t s a combi n a t i on o f t h i n-1 ayer chromatography and UV spectrophotometry i s t o be recommended.

NATAMYCIN

7.2.

547

Spectrophotometric a n a l y s i s

U l t r a v i o l e t spectrophotonietry, u s i n g methanol w i t h O , l % o f a c e t i c a c i d as t h e s o l v e n t , may be used f o r t h e assay o f natamycin and i t s dosage forms. The method i s u s e f u l f o r r o u t i n e c o n t r o l b u t n o t f o r s t a b i l i t y s t u d i e s because o f t h e n o n c o r r e l a t i o n o f m i c r o b i o l o g i c a l a c t i v i t y and t e t r a e n e c o n t e n t upon degradation. The h i g h e s t degree o f c o r r e l a t i o n i s o b t a i n e d w i t h d i f f e r e n t i a l spectrophotometry (33) , a p r i n c i p l e which i s a l s o used f o r t h e assay o f n y s t a t i n ( 9 1 ) and t h e heptaenes rnycoheptin and l e v o r i n (92). T h i s method i s based on t h e measurement o f absorbance a t t h e main maximurn a t 303 nrn and d t t h e minima on e i t h e r side, i.e. a t 295 and 311 nm. Froin t h e s e Val ucs t h e base-1 ine a b s o r p t i o n

A303

-

A295

' A311 2

i s calculated. As seen i n Tables 10 and 11 t h e b a s e - l i n e lilethod g i v e s more r e 1 i a b l e r e s u l t s as compared w i t h t h e m i c r o b i 01o y i c a l assay t h a n t h e 'lone p o i n t " s p e c t r o p h o t o m e t r i c riiethod u s i n g o n l y absorbance measurement a t t h e peak a t 303 nm. I n b o t h examples a 5% natarnycin suspension was degraded and analyzed a t s p e c i f i c t i m e s (33). Table 10 shows t h e r e s u l t s o f d e g r a d a t i o n a t pH 1,5. Under t h i s c o n d i t i o n m a i n l y dirners w i t h t r i e n e a b s o r p t i o n though w i t h remarkable ( f 1 a n k ) a b s o r p t i o n a t 303 nm a r e formed. A small amount o f i n a c t i v e t e t r a e n e (aglycone) i s r e s p o n s i b l e f o r t h e b a s e - l i n e method y i e l d i n g t o o h i g h r e s u l t s . Table 11 shows t h e r e s u l t s o f d e g r a d a t i o n by long-wave u l t r a v i o l e t r a d i a t i o n . I n t h i s way no i n a c t i v e t e t r a e n e i s formed so t h e r e s u l t s o f t h e b a s e - l i n e method are n e a r l y equal t o those o f t h e m i c r o b i o l o g i c a l assay. When natamycin i s degraded i n a l k a l i n e medium a compound i s forrned which s t r o n g l y i n t e r f e r e s w i t h t h e base-1 i n e method. F o r instance, a 5% aqueous s o l u t i o n o f natamycin a t pH 12 was t o t a l l y i n a c t i v a t e d w i t h i n a few hours, s p e c t r o p h o t o r n e t r i c a l l y however, u s i n g t h e base-1 i n e riiethod, no decrease i n t e t r a e n e c o n t e n t c o u l d be d e t e c t e d (33). T h i s can be r e a d i l y e x p l a i n e d by t h e f o r m a t i on o f t h e m i c r o b i o l o g i c a l l y i n a c t i v e t e t r a e n e natamycoic a c i d , forrned by simple s a p o n i f i c a t i o n o f natamyci n (see S e c t i o n 5).

HARRY BRIK

548

Table 10 Degradation o f natamycin a t pH 1 , 5

100 81 59 39 23 21

100 74 50 31 14

5

100 75 46 14 2 (

0,5

Table 11 Degradation o f natamycin by UV l i g h t *

100 83 67 46 32 8

100

78 62

40 22 6

100 79 60 39 20 5

Natamycin shows a t r a n s i e n t b l u e c o l o u r i n f a i r l y s t r o n g h y d r o c h l o r i c a c i d , owing t o t h e f o r m a t i o n o f a carbonium i o n (85). T h i s p r i n c i p l e was used by Dryon (93) t o perform a c o l o r i m e t r i c deterrni n a t i on o f natamyci n. To f o u r volumes o f a m e t h a n o l i c s o l u t i o n o f natamycin c o n t a i n i n g 30 t o 190 ug p e r m l a r e added t e n volumes o f c o n c e n t r a t e d h y d r o c h l o r i c a c i d c o n t a i n i n g 20% o f ethanol under c o o l i n g w i t h ice. A f t e r 13 - 15 minutes t h e absorbance i s measured a t 635 nm. The b l u e c o l o u r does n o t obey B e e r ' s law. A number o f a c i d and a l k a l i n e d e g r a d a t i o n products o f natamycin does n o t i n t e r f e r e i n t h i s method ( 3 3 ) .

549

NATAMYCIN

Sol vent system n-butanol /water, saturated n-butanol/ethanol/water (5:1:4) n-propanol /water (7:3 ) t r i e t h y l ami ne/formamide/water (10:3:10), upper layer

Kf value

Keference

0,33

55

*

38 38

*

u ,33**

*not reported, b u t separation from three other tetraenes possible **re1 a t i ve t o chromi n

25

HARRY BRIK

550

/

900

E

800

U

si=L A -4-

.-> 700 .--4U

3

73

c

0

sodium l a u r y l s u l p h a t e

U

u

.-

60C

ce .U

a,

a v)

50 0

40C b

I

0

I

2

I

I

4

1

I

6

I

1

8

I

1

10

ml of t i t r a n t Figure 18. Conductoinetri c t i t r a t i on o f natamyci n and sodi urn 1 auryl sul phate (each 0 , l mmol ) w i t h 0,02F1 c e t y l trirnethylarnmoni urn bromide.

NATAMYCIN

7.5.2

55 1

T h i n L a y e r Chromatography

T h i n l a y e r chromatography has been used i n q u a l i t a t i v e a n a l y s i s t o d i f f e r e n t i a t e t h e a n t i b i o t i c from o t h e r polyenes o r t o t e s t i t s p u r i t y . S e v e r a l systems a r e l i s t e d i n t h e T a b l e s 13, 14 and 15. I n t e s t i n g t h e s t a b i l i t y s o l v e n t systems 10 and 13 a r e u s e f u l t o e s t i r , i a t e mycosami ne i n n a t a m y c i n and i t s p r e p a r a t i o n s . N i n h y d r i n e i s used as t h e d e t e c t a n t . A f t e r w a r d s t h e same p l a t e can be s p r a y e d w i t h a u n i v e r s a l d e t e c t a n t l i k e s u l p h u r i c a c i d t o d e t e c t aglycone-1 i k e d e g r a d a t i o n p r o d u c t s (33,61). T a b l e 13 T h i n-1 a y e r chromatography systems f o r s t a t i o n a r y phase

Silicaqel G ( N C ckS ( P H 8 ) S i 1 c a g e l G (Flerck) S i 1 cayel G (Merck) S i 1 c a g e l G (Merck) S i 1 cagel G (Merck) S i 1 c a g e l C (Merck) S i l i c a g e l G (Merck) S i 1 i c a g e l C (Flerck) S i l i c a g e l G (Merck) Silicagel G (Flerck) (pH 3 ) S i 1 i c a g e l 60 F 254 (Merck) S i 1 i c a y e l 6 0 F 254 ( Mer c k ) S i l i c a g e l GF (Analtech) Polygram S i l G f o i l (M and 1.1) Sephadex G-15

sol vent system (Tabl e 14)

method of detection (Tabl e 15)

1 2 2 3 4 5

1 1 2,3

natamyci n "f Val ue

ttef.

u ,34

Y5

Y5 96

7

2,3 2,3

8

2,3

0,34 u,57 u,4u 0,54 U,18 u,55 0,75 0,GO

9

5

0,5Y

97

u,4

G1

G

4 4 2,3

93 93 YG 96 Y6

96

10

0,lO

11

7 ,8

u ,u

45

12

78

0,45

45

13

Y ,10 11

0Y7 0,7*

33 98

14

*relative t o benzylpenicillin

552

HARRY BRIK

Table 14 Thi n-1 a y e r chromatography systems f o r natamyci n Sol vent systems

1. ethanol/ammonia/water (8:l:l) 2. n-butanol / a c e t i c a c i d / w a t e r ( 3 : 1: 1) 3. methanol/isopropanol/acetic a c i d (90:10:1) 4. m e t h a n o l j a c e t o n e l a c e t i c a c i d (8: 1:1) 5. ethanol /ammoni a / d i oxane/water (8 :1:1:1) 6. n - b u t a n o l / p y r i d i ne/water ( 3 : 2 : 1) 7. n - b u t a n o l / p y r i d i n e / a c e t i c a c i d / w a t e r (15:10:3:i2) 8. n - b u t a n o l / a c e t i c a c i d / w a t e r / d i oxane (6:2:2: 1 ) 9. n - b u t a n o l / a c e t i c a c i d / w a t e r ( 2 : l : l ) 10. c h l oroforn/methanol / a c e t i c a c i d / w a t e r ( 6 : Z : Z : 1) 11. chloroform/methanol/~,05M b o r a t e b u f f e r pH 8 , 3 (2:2:l), lower l a y e r 12. n - b u t a n o l / a c e t i c a c i d / w a t e r (4:1:5), upper l a y e r 13. n - b u t a n o l / a c e t i c a c i d / w a t e r (4:1:2) 14. 0,025M phosphate b u f f e r pH 6,0 c o n t a i n i n g 0,5M NaCl

Table 15 Thi n-1 ayer chromatography systems f o r natamyci n Methods o f d e t e c t i o n

1. 10% p o t a s s i um permanganate/0 ,2% brornophenol b l ue 2. 5% potassium permanganate 3. c o n c e n t r a t e d phosphoric acid, 5 minutes a t 100°C 4. 0,2% p-dimethyl ami nobenzaldehyde i n concentraked sul p h u r i c acid containing a trace o f f e r r i c chloride 5. 1%p-dimethylami nobenzaldehyde + 20% antimony t r i c h l o r i d e i n ethanol w i t h 20 V / V % c o n c e n t r a t e d hydroc ti1 o r i c a c i d 6. c o n c e n t r a t e d s u l p h u r i c a c i d , 10 riiinutes a t 105OC 7. i o d i n e vapour 8. c o n c e n t r a t e d s u l p h u r i c a c i d / y l a c i a l a c e t i c a c i d (1:l) 9. concent r a t e d s u l p h u r i c acid/methanol (1:2) 10. n i n h y d r i ne 11. b i oautoyraphy

553

NATAMY CIN

7.5.3

High Pressure L i q u i d chromatography

HPLC has been used by Frede (99) f o r t h e i d e n t i f i c a t i o n o f natamycin i n cheese-extracts. The d e t e c t i o n l i m i t was 20 ny per i n j e c t i o n a t a d e t e c t i o n wavelength o f 303 nrn. As HPLC i s much more s e l e c t i v e t h a n t h e UV spectrophotornetric method i t i s a u s e f u l method t o assay p a r t i a l l y degraded samples, pharmaceutical dosaye forms o r b i o l o g i c a l m a t e r i a1 Several systems a r e l i s t e d i n Table 16. A chromatograrn o f t h e USP r e f e r e n c e standard i s shown i n F i g u r e 19.

.

Table 16 Systems f o r HPLC o f natamyci n Eluent

S t a t i o n a r y phase L i c h r o s o r b KP-8 25 cni (Merck) uBondapak C18 25 cm (Waters) VBondapak C18 25 cm (Waters)

7.6.

IleOH - H20 (65 : 35) MeOH-H20-HOAc (48 : 32 : 1) MeOH-H20-THF ( 4 4 : 47 : 2) c o n t a i n i n g 1 wlvX o f NH4OHc

II 10 1

E l ectrophoretic Analysis

Ochab (102) separated natamycin froin s e v e r a l o t h e r p o l yenes by means o f e l e c t r o p h o r e s i s on Whatman no. 4 and no. 34 paper, m o b i l i t i e s i n f o u r d i f f e r e n t e l e c t r o l y t e s a r e reported.

7.7.

Pol a r o g r a p h i c A n a l y s i s

Dornberger ( 103) determi ned natamyci n and i t s C25 b u t y l homo1ogue 1ucensomyci n p o l arographi c a l l y a t t h e d r o p p i n g mercury e l e c t r o d e i n 0,ZM hosphate b u f f e r pH 7 i n a c o n c e n t r a t i o n range o f 1 0 - t t o 10-5M. The epoxy group o f natamycin i s reduced a t a half-wave p o t e n t i a l o f -O,85 V versus t h e normal calomel e l e c t r o d e . Lucensomycin g i v e s a wave a t -1,O V. D e r i v a t i v e s o r polyenes which l a c k an epoxy group r e a c t negatively.

HARRY BRIK

554

Figure 19. Hi gh-pressure 1i quid chromatogram of 4 ug of natamycin USP r e f e r e n c e standard (104) I nstrurnent

: Spectra Physics SP 8000

Detection

chromatograph w i t h Schoeffel SF770 d e t e c t o r : WBondapak Cl8 3,9 x 300 mm : m e t h a n o l - d i s t i l l e d watert e t r a hyd rof u ran (440 : 470 : 20) c o n t a i n i ng 1%o f ammoni urn a c e t a t e . Kate o f flow: 2 ml/minute. : U1 t r a v i o l e t absorption a t

Sensi t i v i t y Ketention time

303 nm : 0,04 HUFS : 13,5 minutes

Col umn Mobile phase

NATAMYCIN

555

Elemental H n a l y s i s

7.8.

The presence o f ash, o r g a n i c i m p u r i t i e s ( e s p e c i a l l y i n e a r l i e r l o t s ) , s o l v e n t o f c r y s t a l l i z a t i o n ( i .e. 1,iethanol , w a t e r ) may b r i n y about s u b s t a n t i a l l a c k o f agreement between 01 d e r experimental data and t h e r e c e n t t h e o r e t i c a l composition. P o s s i b l y r a t h e r because o f t h e presence o f t h e above f o r e i g n c o n s t i t u e n t s t h e r e was sometimes a f a i r l y good agreement between experimental and -meanwhi 1e obsoletet h e o r e t i c a l d a t a ( 1 , 6 2 ) . Kecent experimental d a t a ( 4 9 ) , o b t a i ned w i t h natamyci n t r i h y d r a t e r e f e r e n c e standards , conform v e r y w e l l w i t h modern t h e o r e t i c a l data. Table 17 E l emental a n a l y s i s o f natamyci n t h e o r e t i c a l composi t i on i n %

H

c

0

Ii

59,54 55,06

natamycin anhydrous riatamyci n tri h y d r a t e

found i n L;

c

H

1.1

0

ref.

58,53

7,32

2,12

-

i

57,11

7,33

2,08

-

62

55,ll

7,41

1,99

34,98

49

remarks ibiean v a l u e o f seventeen analyses i n one sample (1958) mean v a l u e o f t h e a n a l y s i s o f seven r e c r y s t a l 1ized sampl cs ( 1964) mean v a l u e o f t h e analysis of three s p e c i a l l y prepared r e f e r e n c e standards

(1973-1976)

HARRY BRIK

556

7.9.

Microbiological Analysis

Natamycin i s assayed m i c r o b i o l o g i c a l l y w i t h Saccharomyces c e r e v i s i a e RTCC 9763 as t h e t e s t organism u s i n g t h e agar d i f f u s i o n method. The assay i s recommended f o r t h e d e t e r m i n a t i o n o f natamycin i n s o l u t i o n s o r e x t r a c t s o f t h e substance, i t s dosage forms o r i n b i o l o g i c a l m a t e r i a l . The s e n s i t i v i t y o f t h e agar d i f f u s i o n rnethod i s approxirnately 0,5 uy per m l o f s o l u t i o n (105). An i n t e r e s t i ng a1 t e r n a t i ve f o r t h e bioassay o f natamyci n i s based on measurement o f t h e decrease i n heat o u t p u t r a t e w i t h t i m e o f t h e r e s p i r a t i o n o f Saccharomyces c e r e v i s i a e (106). The determi n a t i on, which proceeds by f l ow m i c r o c a l oriinetry, was c a r r i e d o u t i n a c o n c e n t r a t i o n range of 1 t o 7 x 10-6PI. 8.

Acknowledgment

The a u t h o r thanks D r J . de F l i n e s , D r H.J. Kooreman, D r R.P. Morgenstern, D r 0.A.Smink and Ir J.A. van d e r S t r a a t e n f o r r e v i e w i n g t h e manuscript, D r s G.J.B. C o r t s and D r s C. van d e r V l i e s f o r t h e i r v a l u a b l e suggestions f o r improvements, t h e many c o n t r i b u t o r s c i t e d as "personal communication" and I r J.C. Monshouwer 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 i n p r e p a r i n g t h e manuscri p t

.

NATAMYCIN

9.

557

References

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L i t e r a t u r e surveyed through February 1981.

OXYTOCIN Friedrich Nachtmann, Kurt Krummen, Friedrich M a d , and Erich Riemer 1. Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Conformation 1.6 Appearance, Colour, Odour 1.7 Biological Activity 2. Physical Properties 2.1 Infrared Spectrum 2.2 Ultraviolet Absorption 2.3 Circular Dichroism 2.4 Raman Spectra 2.5 Proton NMR 2.6 W-NMR 2.7 Solubility 2.8 Optical Rotation 2.9 Isoelectric Point 3. Production 3.1 Extraction from Gland Material 3.2 Chemical Synthesis 4. Stability 5. Metabolism 6. Analysis 6.1 Identity Tests 6.2 Quantitative Physicochemical Methods 6.3 Biological methods 6.4 Determination in Biological Material 6.5 Determination in Dosage Forms 7. References

564 564 564 565 565 565 567 567 567 567 568 568 572 573 573 573 576 576 576 576 577 578 58 1 582 582 584 590 592 595 596

FRIEDRICH NACHTMANN et al.

564

1.

Description

Oxytocin i s t h e c y c l i c octapeptide') hormone released by t h e p o s t e r i o r p i t u i t a r y and having uterotonic and galactagenic activity i n mmmls and h y p t e n s i v e a c t i v i t y i n birds.

Its 20-memberd ring i s composed of f i v e amino acids cystine, tyrosine, isoleucine, glutamine and asparagine -, and t h e s i d e chain contains a f u r t h e r 3 amino acids - proline, leucine and glycinamide. A l l the o p t i c a l l y active amino acids belong t o t h e L-series. The s t r u c t u r e of oxytocin w a s elucidated by du Vigneaud e t al., and indepndently by Tuppy i n 1953 ( 1 , 2 ) . The structure was confirmed by du Vigneaud e t al . by synthesis shortly afterwards ( 3 ) . 1.1 Nomenclature 1.11 Chemical names

L-Cysteinyl-L-tyrosyl-L-isoleucyl-L-glutaminylL-asparqinyl-L-cysteinyl-L-prolyl-L-leucyl-glycinamide c y c l i c (16) disulphide L-H€tni-cystinyl-L-tyrosy~-L-isoleucy~-L-g~utaminylL-asparaginyl-L-hemi-cystinyl-L-prolyl-L-leucylg lyc inamide 1.12

Generic name oxytocin [50-56-61

1.13

Brand names

The following brand names are listed i n t h e Merck Index ( 4 ) : Alpha-hypphamine; Ocytocin; Endop i t u i t r i n a ; Pitocin; Syntocinon; Nobitocin S; Orasthin; Oxystin; Partocon; Synpitan; Piton+; U teracon

.

1.2

Formula 1.21

Amino acid sequence 1

2

3

4

5

6

7

8

9

Cys-~-Ile

Analytical Profiles of Drug Substances and Excipients Volume 10

Analytical profiles of drug substances and excipients

Analytical profiles of drug substances and excipients