Analytical Profiles of Drug Substances and Excipients Volume 13

Ana1yticaI Profiles of Drug Substances Volume 13 EDITORIAL BOARD Abdullah A. Al-Badr Steven A. Benezra Rafik Bishara...

Author: Klaus Florey

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Ana1yticaI Profiles of

Drug Substances Volume 13

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

Klaus Florey Salvatore A. Fusari Lee T. Grady Boen T. Kho Joseph A. Mollica James W. Muiison Milton D. Yudis

Academic Press Rapid Manuscript Reproduction

Analytical Profiles of Drug Substances Volume 13 Edited by

Klaus Florey The Squilh Institute for Medical Research New Rriiiiswick, N e w Jersey

Contrilmting Editors

Abdullah A. Al-Badr Norman W. Atwater Steven A. Benezra Rafik Bishara Gerald S.

Glenn A. Brewer, Jr. Salvatore A. Fusari Lee T. Grady James W. Munson Brenner

Compiled tinder the auspices of tlw Pliuriiiaceutical Analysis (ind Control Section APhA Ac(idcmy of P l w t-titcrcentical Scimces

ACADEMIC PRESS, INC.

1984

(Harcourt Brace Jovanovich, Publishers)

Orlando San Diego New York London Toronto Montreal Sydney Tokyo

COPYRIGHT 0 1984, B Y THE AMERICAN PHARMACEUTICAL ASSOCIATION ALL RIOHTS RESERVED. NO PART OF THIS PUBLICATION M A Y BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC

OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAQE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

Orlando, Florida 52887

United Kingdom Edition published by ACADEMIC PRESS, INC. LONDON) LTD.

24/28 Oval Road, London

NWI 76X

Library of Congress Cataloging in Publication Data

Analytical profiles of drug substances. Compiled under the auspices of the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences. Includes bibliographical references and indexes. 1. Drugs--Analysis. I. Florey, Klaus. 11. Brewer, Glenn A. 111. Title: Academy o f Pharmaceutical Sciences. Pharmaceutical Analysis and Control Section. [DNLM: 1. Drugs--Analysis--Yearbooks. QV740 AAl A551 RS189.A58 615l.1 70- 187259 ISBN 0-12-260813-5 ( V . 13)

PRINTED IN THE UNITED STATES OF AMERICA 84 85 86 81

9 8 7 6 5 4 3 2 1

CONTENTS

Affiliations of Editors, Contributors, and Reviewers Preface

vii ix

1

Atenolo1

Vesna caplar, Zooniinira MokotiC-Milaun, Hrvoje Hofinan, Josip Kuftinec, Franjo KajfeZ, Antun Nagl, and Nikola BlaZeviC 27

Camphor

Jaber S . Mossa and M u h o u d M . A . Hassan 95

Chloroy uine

Molaammad Tariq and Abdullalz A. Al-Badr Cimetidine P. M . G. Bazjin, Alex Post, arid John E . Zarenibo

127

Disopyramide Phosphate

183

Alan Wickinan and Patricia Finnegan 211

Indomethacin

Matthew O’Brien, laines McCauley, and Edward Cohen 239

Ketotifen

Zvonirnira MikotiC-Mihun, Josip Kuftinec, Hrcoje Hofman, Mladen ZiniC, Franjo Kajfei, and Zlatko MeiC 265

Melphalan

L . Valentin Feyns 305

Moxalactam Disodium

Leslie J . Lorenz and Patricici N . Thomas V

vi

CONTENTS

Oxyphenbutazone Abdullah A . Al-Badr and Hunieidn A . El-Obeid

333

Pentazocine Terry D . Wilson

361

Yhen ytoin Jose Philip, Ira J. Hokoinh, and Snhatare A. Fusnri

417

Pyridoxine Hydrochloride Hassun Y. Aboul-Eneiii and Mohaninied A . Lout$/

447

Saccharin Muhammad Uppnl Zubnir and Mahinoud M. A. Hnssan

487

Salicylamide Salini A. Babhair, Abdullnh A . Al-Buds. and Hassan Y. Aboul-Enein

52 1

Silver S ulfadiazine Auke Bult and Cees h4. Plug

553

Sulindac Fotios M . Plakogiannis and James A. McCnuley

573

Tetracycline Hydrochloride Syed Laik Ali

597

Vitamin D3 (Cholecalciferol) K . Thonias Koshy and Willianc F . Beyer

655

PROFILE SUPPLEMENTS Tolbu tamide Said E . lbrahiin and Abdullah A. Al-Badr

719

Reserpine Farid j . Muhtadi

737

ERRATUM Phenylpropanolamine Hydrochloride

767

Cumulative Index

769

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

H . Y. Abod-Enein, King Saud University, Riyadh, Saudi Arabia A . A . Al-Badr, King Saud University, Riyadh, Saudi Arabia S , L. Ali, Zentrallaboratoriuin Deutscher Apotheker e. V . , Eschborn, Federal Republic of Germany

N . W. Atwater, E. R. Squibb & Sons, Princeton, New Jersey S. A . Babhair, King Saud University, Riyadh, Saudi Arabia P. M . G. Bauin, Smith Kline & French Research, Ltd., The Frythe, Welwy11, Hertfordshire, United Kingdom S . A . Benezm, Wellcoine Kesearch Laboratories, Research Triangle Park, North Carolina W. F . Beyer, The Upjohn Company, Kalamazoo, Michigan R. Bishara, Lilly Research Laboratories, Indianapolis, Indiana N . BlaZeuiC, Institute for the Control of Drugs, Zagreb, Yugoslavia G. S. Brenner, Merck Sharp & Dolime Research Laboratories, West Point, Pennsylvania G . A. Brewer, J r . , The Squibl) Institute for Medical Research, New Brunswick, New Jersey A . Bult, State University of Leiden, Leiden, The NetherIands V. Cuplnr, Podravka-Institute, Zagreb, Yugoslavia E . Colzen, bferck Sharp & Dohme Rcscwdi Laboratories, West Point, Pennsylvania N . DeAngelis, Wyeth Laboratories, Philadelphia, Pennsylvania H . A . El-Obeid, King Saud University, Riyadh, Saudi Arabia J. E. Fairbrother, Stiefel Lahratories Ltd., Sligo, Ireland L. V. Feyns, The United States Pharmacopeia, Rockville, Maryland P. M . Finnegan, G . D. Searle and Co., Skokie, Illinois K . Florey, The Squibb Institute for Medical Kesearch, New Brunswick, New Jersey

vii

...

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

Vlll

S. A . Fusari', Warner-Lambert Company, Morris Plains, New Jersey L. T. Grady, The United States Pharinacopeia, Rockville, Maryland M . M . A. Hnssan, King S a d University, Riyadh, Saudi Arabia H . Hofinan, Podravka-Institute, Zagreb, Yugoslavia I . J . Holcomb, Warner-Lambert Research Institute, Morris Plains, New Jersey S. E . Ibrahim, King Saud University, Riyadh, Saudi Arabia F . Kajfei , Podravka- Inst it u tc , Zagreb , Yugoslavia B . T. Kho, Ayerst Laboratories, Rouses Point, New York K . Thomas Koshy, The Upjohn Company, Kalamazoo, Michigan J . Kuftinec, Podravka-Institute, Zagreb, Yugoslavia L. J . Lorens, Lilly Research Laboratories, Indianapolis, Indiana M . A . Loutfy, King Saud University, Kiyadh, Saudi Arabia J . A . McCauley, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey

Z. MeiC, Institute Ruder Boskovii., Zagreb, Yugoslavia Z. MikotiC-Mihun, Podravka-Institute, Zagreb, Yugoslavia J . A . Mollica, CIBA-GEIGY Corporation, Summit, New Jersev J . S. Mossa, King Saud University, Riyadh, Saudi Arabia F . J . Muhtadi, King Saud University, Riyadh, Saudi Arabia J . W. Munson, The Upjohn Company, Kalainazoo, Michigan A . Nagl, University of Zagreb, Zagreb, Yugoslavia M . O'Brien, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania J . Philip, Warner-Lambert Company, Morris Plains, New Jersey F . M . Plakogiannis, Arnold and Marie Schwartz College of Pharmacy and Health Sciences, Brooklyn, New York C. M . Plug, State University of Leiden, Leiden, The Netherlands A . Post, Smith Kline & French Laboratories, Philadelphia, Pennsylvania M. Tariq, King Saud University, Riyadh, Saudi Arabia P. N . Thomas, Lilly Research Laboratories, Indianapolis, Indiana A. Wickman, Searle Laboratories, Skokie, Illinois T.D. Wilson, Sterling-Winthrop Research Institute, Rensselaer, New York M . D.Yudis, Shering-Plough Inc., Bloomfield, New Jersey J . E . Zarembo', Smith Kline & French Laboratories, Philadelphia, Pennsylvania M . &nit, Podravka-Institute, Zagreb, Yugoslavia M . U . Zubair, King Saud University, Riyadh, Saudi Arabia

'Present address: 22625 St. Joan, St. Clair Shores, Michigan 48080. 'Present address: Revlon Health Care Group, Tuckahoe, New York 10707

PREFACE

The compilation ofdnnlyticcil Profiles of Drug Szibstunces to supplement the information contained in the official compendia is now a well-established activity. That we are able to publish one voluine per year is a trilxite to the diligence of the editors to solicit articles and even more so to the enthusiastic response of our authors, an international group associated with pharmaceutical firms, academic institutions, and coinpendial authorities. I would like to express my sincere gratitude to them for making this venture possible. Over the years, we have had queries concerning our publication policy. Our goal is to cover all drug sulxtaiices of medical valiie, and therefire, we have welcomed any papers of interest to an individual contributor. We also have endeavored to solicit profiles of the most useful and used medicines, hut inany in this category still need to be profiled. In the preface to the eleventh voliime, I announced that we would try to supplement previously published profiles with new data. Unfortunately, most of the original contributors are no longer available to undertake this task, and it has proven difficult to find other volunteers. We shall continue to pursue the updating program, but it will not be as comprehensive as originally envisioned. Again, I would like to request those who have found these profiles useful to contribute papers of their own. We, the editors, stand ready to receive such contributions.

ix

This Page Intentionally Left Blank

ATENOLOL Vesna Caplar, ' Zvonimira Mikotid-Mihun, Hrvoje Hofman, Josip Kuftinec, Franjo Kajfei Podravka-I nstitute Zagreb, Yugoslavia

Antun Nag1 IJnioerszt!l of Zagreb Zagreb, Yzigoslacia

Nikola Blaievid lnstitiite for the Control of Drugs

Zagreb, Ytigoslnoin 1. 2.

3. 4.

5.

6. 7. 8. 9.

Foreword, History, Therapeutic Category Description 2. I Name, Formula, Molecular Weight 2.2 Appearance, Color, Odor, Taste Synthesis Physical Properties 4.1 Spectra 4.2 Solid Properties 4.3 Solution Properties Methods of Analysis 5.1 Elemental Analysis 5.2 Titrimetric Determination-Electrochemical 5.3 Chromatographic Methods 5.4 Determination of Impurities Stability-Degradation Drug Metabolism. Pharrnacokinetics, Bioavailability Identification and Determination in Body Fluids and Tissues Determination in Pharmaceuticals References

2 2 2 2 2 5 5 15 19

20 20 20 20 22 22 22 23 23 24

'Correspondence,

ANALYTrCAL PROFILES OF DRUG SUBSTANCES VOLUME 13

1

Copyright 01984 by the i\rnencan Pharmaceutical A\\ociation ISBS 0-1?-?60813-5

2

VESNA

CAPLAR ET AL.

1. Foreword, History, Therapeutic Category Atenolol is a &adrenolytic, cardioselective drug, having no intrinsic sympathomimetic activity. J3-Receptor blocking drugs were introduced in 1966, t o treat cardiovascular disorders, These drugs are especially efficient in cases of coronary failure (angina pectoris), arterial hypertension and cardiac arrhythmia. The s thesis o f atenolol was first reported in 1970, (lyand the first pharmacological and clinical studies were made in 1973. and 1974. (2-4). 2. Description Hame, Formula, Molecular Weight

2.1

b

Atevlolol is 4-/2-hydroxy-3-/(1-methylethyl)amino/-propoxy/-benzenacetamide and is also known as 2-/p-/2-hydroxy-3- (isopropylamino)propoxy/phenyl/acetamide or l-p-carbamoylmethylpheno~-3-isopropylamino-2-propanol,

CH2CO NH2 I

OCH2CHCH2 I NHCH(CH3)z OH Mole ‘1 4H22”203 2.2. Appearance, Color, Odor, Taste

Wt;.

266.33

Atenolol is a white powder. It is odorless and h a s a slightly bitter taste.

3 . Synthesis Atenolol is synthetized starting from comercially available 4-hydroxyphenylacetaide (&) and epichlorohydrine (2) in 15-fold molar excess (1) with piperidine as a catalyst (see Scheme 1.j. zhe reaction mixture is stirred 4-6 hours at 95-100 C, then allowed to cool to room temperature and left

0

I" z 0

t

0

+

G

I

0

t (Y

n

i?

0

Y

I 0 I

I"

0

2 0

2 0

I" z

g o 0

N -r

2 0

0--0

i?

m

(Y n

0

4

I

Y

0

0-0 I

I 0 X I

hl

I I

-0

' 0 I \I

0

0 4

I"

0-0

+

0

I" z

0

0

5

ATENOLOL

standing overnight. The precipitate is collected by filtration and thoroughly trashed with methanol to remove unreacted epichlorohydrine. The product, 1-(4-carbarnoylmethylphenoxy)2,3-epoxy-propane ( ) may be recrystallized from methanol, water or 2i:xane. The recrystallized compound 2 is reacted with isopropylamine (24-fold molar exoess), in methanol as solvent, at the boiling point, f o r 5-30 minutes. The solvent and excess of isopropylamine are distilled o f f , and the crude atenolol recrystallized from water. The use of isopropylamine in the equimolar amount, or in a small excess, resulted in formation of an impurity, namely the product of a reaction of atenolol 2 and its precursor, 2, which had the structure of di-/3-(4*-carbamoylmethylpheno )-2-hydroxy-propyl/-isopropylamine (3) (3cheme 2.

7.

The infrared spectrum o f atenolol, presented in Fig. l., w a s recorded with a R3r pellet, The-l bands a r e assigned as f o l l o w s : 3340 and 3160 cm -CO-YH), 2940 (=CH), 1625 (-C=O, amide I), 1500 -N-'J=O, amide 11), 1400 (H N-CO-) 1385 (i-APr), 1390, 1235 (arylether) 117g (i-Prj, 1105, 1080, 1030, 910, 880, 810, 740, 700, 660. (5) The ir spectrum of the "dimeric" compound 5 exhibits the same bands wit5 the same intensities in the region 4000-1100 cm- as does coypound2. However, t h e bands at 1085, 910 and 880 cm (weak t o medium) present in the spectrum of a t e n o l o l are lacking in the spectrn of compound 24.1.2.

Ultraviolet

The ultraviolet spectrum of atenolol is presented in Fig, 2. It was recorded2with a methanolic solution at the concentration lo- g/L. The values of bands of atenolol and compound-5 lie at the same wavelenths (i.e. 225, 275 and 283 nm), but their intensities differ slightly.

.

4.1 3 . Proton Nagnetic R e s o n a E The proton magnetic resonance spectrum of atenolol in GD OD is presented. in Fig. 3. The spec-

3

10 0

80 CI

\

L 60

w

0

z

U

LO I

v)

z

U

=

c 20

0'

1

I

LOO0 3000

I I

I

r

2000 1800 1600

I

I

I

1200 cm-l

a

800

F i g . 1. I n f r a r e d s p e c t r u m of a t e n o l o l ( K B r p e l l e t ) . I n s t r u m e n t : Pye Unicam SP3-200.

ATENOLOL

A 0.4

0.3

0.2

0.1

nm 200

300

Fig. 2. Ultraviolet spectrum of' atenolol. Instrument: Pye Unicam SP8-loo UV-VIS

I

!

L

% I

8

I

7

I

6

I

5

I

4

1

3

I

2

Fig. 3. 80 MHz proton MR spectrum of atenolol. Bruker WP 80 DS at 80 MHz (8 K points).

’ I

6 (pprn,d Instrument:

9

ATENOLOL

Table 1. Assignment of proton MR s i g n a l s in t h e spectrum of a t e n o l o l

s h i f t Type Inten- Coupling const. F r o t o m Chemical d cppm) s i tg J (Hz) 8 '(95 d(A9) ? H Ha Ja,b 7-05 d(AB) 2 H J 8.755 % b ,a 4. 99 S 2+1+1 H 4.28-4.iO m 1 H Hd 4-12 S 2 H He 3.59 S 2 H Hf H 3-03 dd JgA,gB 12.5 gA JgA, d 5.00 HgB 2.81 dd J g ~ , g12.5 ~ I

.

.

%

Hh i'

-

3 14-2.65 1.25

m d

.1 B 6 R

JgB, d

5.00

Ji t

6.254

-

t r a l assignments are shown i n !Table 1. (5) Charact e r i s t i c peaks of t h e proton PYX s p e c t r u a of compound 2 (which may be p r e s e n t as an impurity i n crude a t e n o l o l ) i s presented i n Table 2. 13C-hT!R s p e c t r a o f a t e n o l o l and compound 5 (presented i n Fig. 4.) were run in p u l s e FT mode on a J e o l PX-loo spectrometer o p e r a t i n g a t 25-05 FlHz. The samples were measured i n 5 mm t u b e s with TIMS as an i n t e r n a l standard. An 8 R computer memory and i n t e r n a l deuterim lock ygre used., (5) The broad-band C' Nl'JR s p e c t r a o f DMSO-d sol u t i o n s o f atenolol and i t s " d i n e r " 2 a r e ShOiA i n

a

0

Ea

I

rg 0 N

0 *

0 u)

0 OD

0

c

0

s!

3

0

0

0

0

s

0

-nk

10 0 (v

0 -l

0 (0

0

co

0 0

c

0 (v c

*

0 c

0

P

0 W

c

VESNA CAPLAR ET AL.

12

Table 2. Assignment of p r o t o n MR s i g n a l s i n the spectrum o f compound 2

(H* Nc 0c

NCH(CH3) h

\c

eotons

H

a Hb HC

hemical s h i f t

6 (ppm)

'? 36 7,02

Type d

n en;&

4 Y

2 13

4.13

s

4

rj

9f 9

3.59 2 875

s

H@

2.98 2.8-3. o

4 2 2 1 6

B R H H H

He &.A

Hh

H,

Couplin

Ja.b

4.98 m

Id '

4.2-4. o

1.20

d

d

m d

i

-

-

JgA, gB JgB, $4

4.0 5.0

J

6.504

-

Fig. 4. S i n c e t h e s o l v e n t peaks c o v e r one carbon s i g n a l , t h e complementary CD OD s o l u t i o n s were a l s o used i n making t h e complete assignment. I n t h i s way an unambiguous assignment was achieved, a p p l y i n g off-resonance and g a t e d decoupling (?JOY) s p e c t r a , as well as t a b u l a t e d data of chemical s h i f t s f o r r e l a t e d c o n s t i t u t i o n a l p a r t s c o n t a i n e d i n t h e atenolol molf3ule. ( 6 ) The C ITIIR s p e c t r a of t h e S D OD s o l u t i o n s r e v e a l two signals belonging t o noneauivalent methyl carbons o f t h e i s o p r o p y l group. I n t h e Y313 spectrum o n l y one complex q u a r t e t was produced correspond i n g l y , s o t h a t o n l y one f i r s t o r d e r 3-€1 c o u p l i n g c o n s t a n t was determined, t h e o t h e r one was o n l y est i a a t e d , as being h i g h e r by a3out 4 Hz (see Table 3 . ) . The o t h e r m u l t i p l e t s were a l s o v e r y complex, due t o s e v e r a l long-range c o u p l i n g s and o v e r l a p s , with both t h e s o l v e n t s used. l e v e r , a r a t h e r comp l e t e a n a l y s i s of t h e coupled9E'C MT4R spectrum w a s made w i t h numerous f i r s t and h i g h e r C-H c o u p l i n g

ATENOLOL

13

T a b l e 3.13C

Chemical s h i f t s a and I3C-'H coupling constantsb f o r a t e n o l o l dissolved i n CD,OD

C-atom C-lC

c-2

c-3

c-1'

C-2' '6'

c-3' '5' c-4' C-1' ' (3-2''

d (PPd 50.75 (t)

69.59 71.78 22.61 22.42 49.68

(d) (t)

2

lJCH ~~

~

.

132.6 1+2 6 144.o

(a_) (9)

129d

(d)

131e

125.5

-

115.36 (d 130.84 ( d )

160.1

128.83 ( s ) 176.80 ( s ) 42.41 ( t )

-

158.7 128.4

JcR

2.0;1.5 2.4;2.4 2.4

JCH

-

-

-

5.4

2.0

2 4.9 2.4

9.7

7.1;4.9

5.7

7.8

-

3.9

6.6;4.4

-

downfield from i n t e r n a l TKS; accuracy +d i n pprn ppm; off-resonance m u l t i p l e t s a r e given i n

-0.02

parentheses + n ~ C Hi n HZ; accuracy -0.5 HZ nJCHv a l u e s determined from t h e DMSO-d6 s o l u t i o n estimated t o 21 Hz 4estimated t o -2 Hz; t h e long range coupling cons t a n t s can not be determined due t o very complex multiplets c o n s t a n t s which may be u s e f u l f o r a f u r t h e r s t r u c t u r a l study. The complete data a r e l i s t e d i n Table

3.

I n t h e DMSO-d

s o l u t i o n only one CH - s i g n a l

was found i n t h e spgctrum o f a t e n o l o l , an8 t h e s o l -

vent caused a s l i g h t downfield e f f e c t upon he chemical s h i f t . On t h e o t h e r hand, a l l o t h e r I3C chemical s h i f t s were moved s u b s t a n t i a l l y u p f i e l d , t h e changes rangin f r o m 0.60 f o r C-4' t o 4.27 ppm f o r t h e C=O group ?Table 4.). The NOIT spectrum shows r a t h e r smeared m u l t i p l e t s which were not suitable for p r e c i s e determination of coupling constants, except i n very few cFges. The broad band C NPlR spectrum of t h e compound 5 dissolved i n DFE0-d (Fig. 4 B ) shows t h e doubling o f t h e s i g n a l s a r o h d t h e c h i r a l c e n t r e (Tab l e Yo), which demonstrates t h e presence of d i a s t e reomers. I n comparison with t h e a t e n o l o l carbon

VESNA CAPLAR ET AL.

14

Table 4. 1 3 C Chemical s h i f t s a f o r a t e n o l o l and compound 5 d i s s o l v e d i n DMSO-d,

C-atom

'me

Atenolol

t

70 71

CH3

9

22 83

.

CH3

22.83

CH c-1 '

48.12

157.15 114.08 123.87

C-3' '5'

c-4'

C-1' c-2'

128.23

'

172.57

'

2

b

c-3

C-2'' 6'

Compound

~c.1.27

70 32b 70.32 18. 66c

17 77 17 77c 16.76 51.41 50.78

157.25

.

114.08

129 91 128.16 172.90 41.27

a +& i n ppm downfield from i n t e r n a l T X 3 ; accrJracy -0.02 ppm; off-resonance r n u l t i p l e t s a r e given b 9 c broadened s i g n a l i n d i c a t e s t o chemical s h i f t s separated by l e s s t h a n 0.02 ppm chemical s h i f t s , t h e methyl s i g n a l s a r e s i g n i f i c a n t l y moved u p f i e l d , and those belonging t o isopropyl CH and 2-1 a r e moved downfield. A l l o t h e r s i g n a l s a r e c l o s e t o those of a t e n o l o l .

4.1.5.

Nass

The mass s p e c t r a of a t e n o l o l and compound 2 were obtained by d i r e c t i n s e r t i o n of t h e sample iat o CEC 21-110 I3 mass spectrometer. C h a r a c t e r i s t i c s of t h e s e mass s p e c t r a a r e summarized i n Tables 5. and06, and Fig. 5, The i o n source temperature was 150 C and 200-250 C , r e s p e c t i v e l y , and t h e i o n i z i n g e l e c t r o n beam ener,gy was 70 eV. Atenolol gave a molecular i o n , with very l o w i n t e n s i t y , a t m/e 266. Other s i g n i f i c a n t peaks are due t o t h e following fra ments: isopropyl group (PI-43), carboxamide group $M-44) and carboxamidomethylene group (M-56). The base peak i n t h e a t e n o l o l

ATENOLOL

15

Table 5. Peak assignments in the mass spectrum of atenolo1

,* .J

m/e

1.2 24.5

266 222

3.9

223

12.9

116

43.1

107

fragment

01'

14.3 3.7 lo 0

23.5

107- C HO m

151 - C H 2 N HC H(C H3)2

72

73

- HC H H C H2N HOH

spectrum is due to isopropylaminomethylene group (m/e 7 2 ) . The base peak in the spectrum of compound 2 corresponds to radical-ion of protonated p-methylenequinone (m/e 107).

4.2. Solid Properties 4.2.1. Melting Range Atenolol melts begween 150:

und

5 melts between 154 and 156

4.2.2.

.

and 1 5 2 O C ; compo-

Crystal Properties

X-Ray diffraction data of atenolol are presented in Table 7.(5) The diffraction spectrum w a s produced by onochromatic radiation from the CuK line (1.542 ) which was obtained by excitation at 35 kV and 20 mA, Recording condhtions were as foll g w s . Optics: detector slit 0.2 ; I"I.R6 soller slit, 3 ; beam slit, 0.0007"; N& filter, 3 take o f f angle. Goniometer: scan at 2 , 2o/min. Detector: amplifier gain 16 coarse, 3.1 fine. Scintillation co-

!!

E

0) \

0 In N

0

0 h(

5

0

5

0

0 In

17

VESNA CAPLAR ET AL

18

Table 6. Peak assignements in the mass spectrum of compound 2 /??

Table 20 9.62 12.84 16.04 17.74 18.22 19.22 20.60 22.24 23 80 24.36 26.40

.

29. lo

m/e

7. X-Ray

fragment

diffraction data of atenolol crystal

Ia

.

43

9 1936

54

5 5254 4.9996

6 20 61 74

loo 91

81

72 92

31.84

11 42

34.58 35 94

14 17

40.46

db(interplanar distance)

15

.

6.8943

4.8689 4.6178 4.3115

. 3 .3759

3 9971 3 7385

3.6538

3.0686

2.8105 2 5938 2.49~7 2.2296

a based on the highest intensity which is selected as unity d = nk/2 sin@

ATENOLOL

19

unter tube and DC voltage at plateau. Fulse light selection 9V, En out. Rate meter: T.C. 1.0, 5000 CPS.

(5)

q . 3 . Solution Properties Solubility

4.3.1.

All solubilities were determined at room temperature and are presented in Table-8. The lities are reported in accordance with U.S.P.(XX ed.) definitions.(5)

solub4.

Table 8. Solubilities of atenolol Solvent methano 1 acetic acid dirnethylsulfoxyde 96$ ethanol water isopropanol acetone dioxane aceto n i t r i l e ethylacetate chloroform

Solubility

freelg soluble soluble so 1ub1e sparingly soluble slightly soluble slightly soluble very slightly soluble very slightly soluble insoluble insoluble insoluble

The negative logarithm of the proton dissociation constant for atenolol was determined as pRa= =8.6. (7)

4.3.3.

Partition Coefficient

Atenolol has a l o w partition coefficient f o r n-octanol-phosphate buffer (0.16 M). Its values were 0.008 at pH 7.0 and 0.052 at pH 8.0.(7) 4.3.4.

Dipole Moment

The dipole moment of atenolo& was determined in propionic acid solutions, at 20 C, using a Dipolmeter DM 01 (Wiss. -Techn. Werks-$Btten, D 812 Weilheim) The value found was 5.71-0.20 D. (5)

.

VESNA CAPLAR ET AL.

20

4.3.5.

Optical Rotation

The atenolol molecule contains an asymmetric carbon atom. The commercial product, however, is a racemic mixture and its resolution has not been reported so f a r .

. Methods of Analysis Elemental Ana1.y l’s s

.lo

Atenolol C14K22Nz03

(266.33) calc.: C 63.13% H 8.33% N 10.52% 0 18.02$

3.2.

Titrimetric Determination

-

loo 00% Electrochemical 0

Atenolol may be assayed in glacial acetic acid/acetic anhydride 15:2 by titration with 0.1 PI perchloric acid. The endpoint may be determined potentiometrically using a glass/calomel electrode pair. (5)

2.3. Chromatographic Methods 2.3.1.

Thin Layer

Thin-layer chromatography was performed using silica gel 60 F plates (Nerck); the solvent system was dioxane228etonitrile-methanol-conc ammonia (25f;) (60:36:5:4). One hundred mg of the sample was dissolved in lo ml of methanol, the solution spotted on the plate and subjected to ascending chromatography. After a front migration f o r at least 16 cm the plate was air-dried and sprayed with Dragendorff’s reagent. Atenolol and compound 2 appear as orange-brown spots, on a yellow background, at Rf 0 . 3 and 0.6, respectively.(5) High performance thin-layer chromatography (HPTLC) on silica %el plates was used for the separation A-adrenoceptor blocking drugs. The detection limit for atenolol was 25.0 ng, and the absorption wavelength selected f o r its determination was

.

205 nm.(8)

3.3.2.

Gas-Liquid

For gas-liquid chromatographic determinations a 1 m x 4 m (I.D.) g l a s s column packed with 3$ OVlo1 on 80-loo mesh Gas Shrom Q was used. (5) The in-

ATENOLOL

21

jector tempBrature was set at

30Ooc,

oven tem era-

ture at 190 C, Bad detector (flame ionization7 temperature at 300 C. The sample was pretreatred with trifluoroacetic acid anhydride as f o l l o w s : to 20 mg of atenolol in lo-ml volumetric flask was added 5 ml of dioxane and 0.5 r n l of trifluoroacgtic acid anhydride; the mixture was heated to 40 C and kept 5 min. at this temperature. The flask was then coo l e d to room temperature and the contents made up to lo r n l with dioxane. One ml of this solution was mixed with 0.2 r n l of internal standard solution (2 mg/ml of methyl ester of margarinic acid in dioxane) and 1 ,AL~ of the mixture forced into the injection block. The relative weight response of the detector was calculated according to equation (1): A* x RWR=

A

s

cs

x CA

(1)

in which symbols have the following meanings: AA = atenolol peak area of standard AS = internal standard peak area C, = concentration of atenolol (mg/ml) Cs = concentration of methyl margarinate ( m g / m l ) In actual analyses the content of atenolol is calculated from the areas of atenolol and internal standard according to eq. (2): AA x CS x 1000 mg of atenolol = AS Rm

in which the meanings of symbols are following: AA = atenolol peak area of sample

AS = internal standard peak a r e a

concentration of methyl margarinate (mg/ml) RWR= relative weight response W = weight of the sample (ng)

Cs

=

5 . 3 . 3 . Hiah Pressure Liquid The apparatus used for high pressure liquid chromatography was by Fye-Unicam (Cambridge), LPU-4011 equipped with a PU-4020 UV detector. The co-

VESNA CAPLAR ET A L

22

lumn was prepacked with Partisil l o p m ODS (25 cm; 4.6 mm ID, 6 mm OD). The mobile phase was acetonitrile-dist. water-orthophosphoric acid (35:loo:o.l) having a pH of 3.5. In some analyses benzimidazole was used as an inOernal standard. The flow rate was 1.0 ml/min. and the detector monochromator was adjusted at 222 nm; the amplifier gain was set at 0.08 A.U.F.S. The retention time of atenolol was 5.48.(5) In another case, 8 LiChrosorb 3 (RD-2, ethylsilaniaed) column and a mobile phase consisting of 351.;' MeOH, and 65,: of an aqueous solution 0.0005 M in HCl and 0.05 M in NaC1 were used. The analyte was detected at its peak absorption 220 nm, using a variable-wavelength detector.(9)

5.4.

Determination of Impurities

s

The "dimeric" com3ound , which may be present i n atenolol can be detec ed by all chromatographic methods and determined by Sas-liquid and I3PL chromatography.

6. Stability

- Degradation

Atenolol, as the powder and in the form of tableljs was kept at 50-60;L relative hunidity and 45-50 C for seven days. No changes were observed i n HPL chromatogram, neither did the color and appearance of the material show change.

7.

Drug Metabolism, Pharmacokinetics, Bioavailabi; litg

Owing to l o w hydrophilicity only a small of atenolol is metabolised (about lo$ of a dose The d r u g is also poorly bound to plasma protein (less 5% of the amount in blood). (lo) Plostly of the drug is eliminated, in its unchanged form, by several routes, but prevailing via the kidney.(ll) After oral administration atenolol is excreted with mine to the extent of about 4073; (12-14), after intravenous administration to the total urinary excretion encompasses 75-1007 of the dose (about l o -14% appeared in t h e form of catabolites).(l2,15, 16) Urinary pH variations do not change the extent ob excretion with the wine.(17) M d e n c e s was put forward to show that atenolol is not eliminated exclusively by glomerular filtration.(ll) Atenolol is also excreted with the faeces. After intravenous

7Y

ATENOLOL

-. 77

administration, about lo$ of the dose excreted unchanged; after oral administration, 40-50;: of the dose appears in faeces, of which 6-8;; accounts for catabolites.(E) Atenolol is characterized by a low hepatic clearance; for the most part o f body clearance is extrahepatic.(l8) The hepatic biotransformation of atemlo1 gives rise t o the pharmaceutically inactive catabolites, such as compound hydroxylated at the methylene group attached to the benzene ring and compouEds conjugated with glucuronic acid. ( 7 j Only a s m a l l fraction of a dose of atenolol reaches the brain and there is ratio of 2:lo between the brain and plasma concentration. Atenolol does not accumulate in tissues, such as lungs, heart and others.(7) The pharrnacokinetics of atenolol was studied in young and o l d subjects: no age-related differences were found with respect to volume of distribution, and bioavailability.(ll) The absolute bioavailability (E), defined as the fraction of an oral dose which reaches the systemic circulation in unchanged form, was 0.55-0.56.(11) Other authors report bioavailabilities of 50-63,;for oral dosage forrns.(15,16,19-22) However, a given oral preparation possesses unifhrm bioavailability, and exhibits but little variation in neak plasma concentration.(l8) Postprandial intake reduces the bioavailability of atenolol.(l9) Several investigators reported less than loo;: bioavailabilities of injectable preparations: with intravenously administered dosage forms, bioavailabilities between 85-98;s were obtained.(15,16,21,22)

8. Identification and Determination in Body Fluids and Tissues The concentrations of atenolol in various biological material was determined by gas chromatography.(23-25) Another method, EELC, w a s used f o r determination in whole blood,(26) plasma, (27,281 and urine.(28)

9. Determination in Pharmaceuticals In tablets atenolol is determined titrimetrically as follows: A sufficient number of tablets are powdered in a mortar, and a powder portions corresponding to one-half of average tablet mass

VESNA CAPLAR ET AL

24

a r e t r m s f e r e d t o t h e t i t r a t i o n v e s s e l containing 14 ml of g l a c i a l a c e t i c acid. Two m l o f a c e t i c a c i d anhydride and 2 m l o f a s a t u r a t e d s o l u t i o n of mercury a c e t a t e i n a c e t i c a c i d a r e added per v e s s e l , and t h e r e s u l t i n g suspension i s t i t r a t e d with 0.1 M HC104 using a g l a s s electrode as endpoint indicat o r (VS. a satd. calomel reference electrode). The a t e n o l o l content i n one t a b l e t i s calculated by using equation ( 3 ) . One ml of 0.1 PI HC104 corresponds t o 26.63 m g o f atenolol. V x f x 26.63 x WA mg of a t e n o l o l i n one t a b l e t =

W

where V

volume of 0.1 M IIC10,: consumed, normality f a c t o r , WA= .average mass of a t a b l e t , W = mass of t h e powder sample.

= f =

(3)

Acknowledgement. 'The authors a r e thankful t o dr. Z. ,lei&, "Ruder Ro%ovib " I n s t i t u t e , Zagreg Yugosla;a, f o r h i s h e l p i n i n t e r p r e t a t i o n o f 3C PR4R spectra.

l o . References 1. A.M. B a r r e t t , R. Hull, 3.J. LeCount, C.J. Squire, J. C a r t e r ( I C I L t d . ) , Ger. Offen. 2,007.751 (1370); Z.A. 22, 1 2 0 3 1 8 ~(1970). 2. J.F. GiudicelB, J.3. 3 o i s s i e r , Y. Dunas, C. Advenier, Compt. Rend. SOC. B i o l . , 232 (1973)3. L. Hansson, H. Aberg, S. Jameson, Karlberg 549 (1973). R. Malmcrona, Acta Pled. Scand. 4. A. Amery, L. B i l l i e t , R. k g l . J. Ned, 2 o 284 (1974). 5. &8aplar, 2. Mikoti6-P?5.hunC H. Eofman, J. Kuftinec, PI. Bkreblin, F. Kajfez, A. ITa I, IT. Blazev i 6 , Acta Pharm. Jugoslav., 2 ( 2 ) , 1983) i n press, 6. EfidBreitmaier, 'iJ. Voelter, l 3 C "IR Spectroscopy, %I.Verlag ., Chemie, !.Jeinheim, 1978. 2 7. E. F'Jarmo, Drugs Bcptl. Clin. Ses. 6 , 639 (1980). 8. %. Zhou, C.F. Poole, J. Triska, A.-Zlatkis, J. Chromatogr., Chromatogr. Commun.

%,

7

Kirschbaum, X.E. Poet, J. Pharm, Sci., 2, 336 (1981). lo.iJ. Kirch, K.G. G B r g , W r o J. Drug Metab. Pharmacokinetics, 81 (19827. 11.P.S. Rubin, P. .1W. S c o t t , K. McLean, A. Pearson,

5,

.

ATENOLOL

.

25

D. Ross, J.L. 235 (1982)-

Reid, Br. J. c l i n . Pharmac.

..

12 J. McAinsh, Postgrad. Med. J. 74 (1977).

z,(suppl.

3, 3),

13. J. 6Ieffih, S.R. Harapat, D.C. Harrison, Res. Commun. Chem. Pathol. Pharmacol. I + 31 (1976). 14. Ha. Ochs, D.J. Greeblatt, T.11. Smi h, "Farmac o l o g i a c l i n i c a e t e r a p i a " , 'a.Medico-ScientiTorino, 1978. f i c h e s.r.l.,

Brown, 3.G. Carruthers, G.D. Johnston, J. K e l l y , J. McAinsh, D.G. NcDevitt, R.G. Shan Shanks, Clin. Pharmacol. Ther. 20 524 (1976). 16. J.D. F i t z g e r a l d , R. R u f f i n , I

COOH

CAMPHOR

Scheme I1 ~-

$-yo

HN03 lo]+

kCO COOH

Camphoric acid

Camphor

0

Na--Hg

t-

0

G

Camphoric \ O

a-Campholide

anhydride

eCN &

( i )KCN

( i i )H+

-

N i t r i l e of hornocamphoric a c i d

;

~

Hornocamphoric COOH acid

JABER S . MOSSA AND MAHMOUD M . A . HASSAN

58

Scheme I I a .

--

., 0

Ill

[31

121

0

Scheme I11

MeC(=CH2)0AC lsopropenyl acetate ____Ic

I

[31

[ll

[41

CAMPHOR

59

Money e t a1 (1969) (65)IlaYe now c a r r i e d o u t a two s t e p s y n t h e s i s of (*)-camphor from dihydrocarvone. (Scheme 111). Dihydrocarvone [l] w a s t r e a t e d w i t h i s o p r o p e n y l a c e t a t e i n t h e presence o f p - t o l u e n e s u l p h o n i c a c i d and convert e d i n t o a m i x t u r e of e n o l a c e t a t e s [ 2 ] and [ 3 ] , sepaTreatment o f [ 2 ] w i t h boron t r i f l u o r i d e r a t e d by GLC i n methylene c h l o r i d e a t room t e m p e r a t u r e f o r 1 0 minut e s gave ( + ) Camphor [4]. This s y n t h e s i s i s p a r t i c u l a r l y i n t e r e s t i n g i n t h a t it is a chemical analogy f o r t h e b i o s y n t h e s i s conversion o f a monocyclic i n t o a b i c y c l i c monoterpenoid.

4.3 Commercial S y n t h e s i s Camphor i s o f considerab1Le importance t e c h n i c a l l y , bei n g used i n t h e manufacture of c e l l u l o i d and m e d i c i n a l p r o d u c t s . It i s manufactured i n d u s t r i a l l y from a-pinene, o b t a i n e d from t u r p e n t i n e , , by s e v e r a l p r o c e s s e s (66-107) which d i f f e r mainly i n d e t a i l . S y n t h e t i c camphor i s u s u a l l y o b t a i n e d as t h e racemic m o d i f i c a t i o n . The f o r m a t i o n of camphor i n v o l v e s t h e Wagner-Meerwein r e a r r a n g e ments, e . g . : Camphene ( i )a-Pinenc HC1 gas* B o r n y l c h l o r i d e AcoNar 10% ( -HC1)

Ac OH

Isobornylacetate

PhNo2 Nas Isoborneol

-

pp

Camphor

H2S04

( i i )a-Pinene

HCooH, ( i i i )a-Pinene

acetate

HC1 gas10°C

Bornylchloride

Isobornylformate

’*

(-HC1)

Camphene

O2

IsoborneolNi ,2000c* Camphor

AcOH ______) Isobornyl(rearrangement. ) Catalytic Camphor. > Isoborneol dehydrogenation

Isomerization>g a p h e n e

NaoH

-

One such process i s d e s c r i b e d i n d e t a i l i n Scheme I V .

60

JABER S. MOSSA AND MAHMOUD M . A . HASSAN

Scheme I V

Saturated at

Meerwein rearrangement

Base-catalysed elimination

131

Il51

+ 1

Sod. A c e t a t e - g l a c i a l

a c e t i c acid.

CAMPHOR

61

The most s t r a i g h t f o r w a r d method i n v o l v e s t h e convers i o n of p i n e n e [ I ] i n t o 13inene h y d r o c h l o r i d e [ 2 ] , b o r n y l c h l o r i d e [ 3 ] and hence i n t o b o r n e o l [6] (or i s o b o r n e o l ) from which cam3hor [7] i s o b t a i n e d by o x i d a t i o n . Also b o r n y l c h l o r i d e [ 3 ] i s c o n v e r t e d t o i s o b r o n y l a c e t a t e [ 5 ] upon h e a t i n g w i t h sodium a c e t a t e and g l a c i a l a c e t i c a c i d . A l L e r n a t i v e l y , camphene [ 4 ] i s o x i d a t i v e l y h y d r a t e d t o camphor [ T I . By s t a r t i n g w i t h o p t i c a l l y a c t i v e a - p i n e n e , o p t i c a l l y a c t i v e camphor can be s y n t h e s i s e d . Commercial camphor is b e s t p u r i f i e d by s u - b l i m a t i o n .

CH 1 3

WOH I I

I

I

I

i

I

H Borneo1

ICH3

[OI

H Isoborneol

5. BIOSYNTHESIS OF CAMPHOR The b i o s y n t h e s i s of m o n o t e r p m o i d s and camphor h a s been d e s c r i b e d by s e v e r a l a u t h o r s (108-114). Ruzicka (115,116) proposed a u n i f i e d b i o g e n e t i c scheme for t e r p e n e s . The biosynthetic building blocks f o r t h e s e terpenes a r e isop r e n e u n i t s . The b i o s y n t h e 1 ; i c a l l y a c t i v e i s o p r e n e u n i t s a r e i s o p e n t e n y l pyrophosphate [ 1 1 and d i m e t h y l a l l y l pyrophosphate [ 2 ] t h e compounds t h a t are d e r i v e d from a c e t a t e v i a mevalonic a c i d (Scheme V ) , Geranyl pyrophosphate [ 3 ] i s t h e C-10 p r e c u r s o r for t h e t e r p e n e s (117). Banthorpe and Baxendale (118) confirmed t h e b i o s y n t h e t i c pathway o f Camphor v i a a c e t a t e mevalonate by c o n d u c t i n g d e g r a d a t i o n s t u d y o f camphor, biosynthesfized from 14c l a b e l l e d mevalon i c a c i d . The b i o s y n t h e s i s of camphor i s summarised i n Scheme V I . The i s o p e n t e n y l pyrophosphate [ 1 3 d e r i v e d from mevalonic a c i d i s o m e r i z e d i n t o dimethy:L a l l y l pyrophosphate [ 2 ] and

(R-mevdlonk

aid1

/ \ /

CH3

-OPP=OP,O,H3

Scheme V

c I

CHzOPP

CAMPHOR

63

Scheine VI -

POH

JABER S. MOSSA A N D M A H M O U D M . A . HASSAN

64

t h e subsequent condensation y i e l d g e r a n y l pyrophosphate [ 3 ] . The g e r a n y l pyrophosphate a g a i n isomerized i n t o n e r y l pyrophosphate [4]. The n e r y l pyrophosphate w i l l be converted i n t o a common enzyme bound c y c l i c i n t e r m e d i a t e , a - t e r p i n e y l c a t i o n [ 5 ] which would undergo proton l o s s from C-5 and 1-2 h y b r i d e s h i f t w i t h t h e expulsion o f enzyme t o y i e l d borneyl c a t i o n [6]. T h i s on f u r t h e r c y c l i z a t i o n g i v e s borneol [7] and t h e n f i n a l l y camphor [8].

6. METABOLISM OF CAMPHOR ( - ) and ( + ) Camphor a r e m e t a b o l i s e d by h y d r o x y l a t i o n of a methylene group acd subsequent g l u c u r o n i d e c o n j u g a t i o n . Hydroxylation produced t h e 3 s n d o d e r i v a t i v e [ 2 ] and t h e 5-endo-derivative [ 31, t h e l a t t e r predominating. These r e s u l t s a r e c o n s i s t e n t w i t h t h e more s t r a i n e d n a t u r e of t h e 5-versus t h e 3 - p o s i t i o n o f camphor. The two hydroxyl d e r i v a t i v e s c a n j u g a t e w i t h g l u c u r o n i c a c i d t o g i v e t h e 3and 5-glucuronides r e s p e c t i v e l y and e x c r e t e d as such. Also it i s hydroxylated i n t h e 2 - p o s i t i o n t o g i v e ( + ) b o r n e o l [11 ( 1 1 9 , 1 2 0 ) Metabolism o f Camphor i s p r e s e n t e d i n Scheme V I I .

7 . PHARMACOLOGY 7.1 Action and Use Taken i n t e r n a l l y camphor i s i r r i t a n t and c a r m i n a t i v e . It has been used as a mild e x p e c t o r a n t and r e l i e v e s g r i p p i n g . Applied e x t e r n a l l y , camphor a c t s as a r u b e f a c i e n t and m i l d a n a l g e s i c and i s employed i n l i n i ments as a c o u n t e r - i r r i t a n t i n f i b r o s i t i s , n e u r a l g i a and s i m i l a r c o n d i t i o n s . Camphor h a s been a d m i n i s t e r e d a s a s o l u t i o n i n o i l by subcutaneous o r i n t r a m u s c u l a r i n j e c t i o n as a c i r c u l a t o r y and r e s p i r a t o r y s t i m u l a n t , but t h e r e i s l i t t l e evidence o f i t s v a l u e f o r t h i s purpose (121-125).

7.2 Toxicity Poisoning has occured through t h e a c c i d e n t a l administ r a t i o n of camphor l i n i m e n t to young c h i l d r e n by m i s t a k e f o r c a s t o r o i l . The symptoms are nausea, vomiti n g , c o l i c d i s t u r b e d v i s i o n , d e l i r i u m , mental confusion and e p i l e p t i c c o n v u l s i o n s . Recovery i s t h e r u l e . But i n rare c a s e s d e a t h may occur f r o m r e s p i r a t o r y f a i l u r e ( 126-128) .

CAMPHOR

65

H OOC

Glucuronic Acid

Glucuronic Acid

OH

S c h e m e .EZ

H


66

7.3 Dosage 120 t o 300 mg ( 2 t o by i n j e c t i o n s ( 1 2 ) .

5

g r a i n s ) ; 60 t o 200 mg (1-3 g r a i n s )

7.4 Preparations The f o l l o w i n g are t h e p r e p a r a t i o n s of camphor o f f i c i a l i n Pharmacopoeas: Camphor Liniment ( 4 ) Camphorated opium T i n c t u r e (126) W r p e n t i n e Liniment ( 4 ) Camphor Ointment ( 1 2 6 ) Camphor S p i r i t ( 1 2 6 ) f ) Camphor Water ( 1 2 6 ) g ) Camphor I n j e c t i o n ( 1 2 6 ) h ) Camphorated Parachlorophenol ( 1 5 2 ) .

a) b) c) d) e)

8. METHODS OF ANALYSIS 8.1 I d e n t i f i c a t i o n

8.11 Pharmacopeial T e s t The following t e s t s have been d e s c r i b e d i n B r i t i s h Pharmacopoeia for t h e i d e n t i f i c a t i o n o f clamphor ( 4 ) :

a ) Burns r e a d i l y w i t h a b r i g h t smoky flame, and v o l a t i l i s e s a t ordinary temperature. b ) The l i g h t a b s o r p t i o n i n t h e range 230 t o 350 nm, of a 2-cm l a y e r of a 0.25 p e r c e n t w/v s o l u t i o n i n a l c o h o l ( 9 5 % ) e x h i b i t s a maximum o n l y a t 289 nm; e x t i n c t i o n a t 289 nm, about 1 . 0 6 .

8.12 Colour Tests The following c o l o u r t e s t s have been d e s c r i b e d for t h e i d e n t i f i c a t i o n of camphor: a ) Bohrisch camphor r e a c t i o n : When 0.5g of natural camphor i s t r e a t e d w i t h 1 c c . of v a n j - l l i n h y d r o c h l o r i d e s o l u t i o n (1 p a r t o f v a n i l l i n d i s s o l v e d i n 1 0 0 p a r t s of 25% h y d r o c h l o r i c a c i d ) and h e a t e d on a water-bath t o 75-100°, a b l u e c o l o u r p a s s i n g t o green i s o b t a i n e d . S y n t h e t i c camphor g i v e s no colour (129-131).

67

CAMPHOR

Bohrisch ( 1 3 2 ) i n reexamining t h e o f f i c i a l t e s t s f o r t r u e camphor, s u b s t i t u t e s t h e following : To 0 . 1 g. o f powliered camphor i n t e s t t u b e i s added 2 c c . of 1% vanillin-hydrochloride solut i o n and t h e m i x t u r e p u t i n t o a beaker w i t h water which i s warmed g r a d u a l l y . A t 30° t h e c o l o u r i s yellow, a t 60° b l u i s h g r e e n , a t 7580' indigo-blue. The l a t t e r c o l o u r p e r s i s t s f o r s e v e r a l h o u r s , even a f t e r c o o l i n g . Synthet i c camphor by t h e same t r e a t m e n t g i v e s only a yellow colour. b ) When 1 g o f n a t u r a l camphor i s t r e a t e d w i t h 1 0 drops of Rosentk.al's Reagent ( e q u a l volume of v a n i l l i n - h y d r o c h l o r i d e and 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 ) a & u l l g r e e n c o l o u r changing in 7-8 hours t o indigo-blue i s o b t a i n e d ( 1 2 9 ) . c ) With barium peroxide-sulphuric a c i d r e a g e n t , camphor g i v e s a yellow c o l o u r ( 1 3 3 ) .

d) When 2 m l of a 0.5% o f a l c o h o l i c s o l u t i o n s o f camphor i s trealied w i t h 5 ml o f 5% phosphomol y b d i c a c i d s o l u t i o n and 5 m l of s u l p h r i c a c i d , a greenish-yellow p r e c i p i t a t e i s o b t a i n e d which r a p i d l y t u r n s g r e e n and d i s a p p e a r s on shaking ( 1 3 4 ) .

e ) With f u r f u r a l and s u l p h u r i c a c i d camphor g i v e s r e d d i s h brown l i q u i d which slowly t u r n s purplish-violet f o r s y n t h e t i c and p u r p l e f o r natural (134). f ) Fluorescence t e s t :

When camphor i s i r r a d i a t e d by x-rays it e x h i b i t s a b l u i s h v i o l e t f l u o r e scence i n t h e v i s i b l e r e g i o n ( 1 3 5 ) .

8.13 D e t e c t i o n of S y n t h e t i c Camphor i n Natural Camphor:

a ) About 1 g of camphor i s thoroughly mixed w i t h 2 g of l i m e , t h e mixture heated u n t i l t h e camphor i s completely v o l a t i l i z e d , t h e r e s i d u e i s e x t r a c t e d wi.th water, and t h e s o l u t i o n f i l t e r e d . The f i l t r a t e , a c i d i f i e d w i t h n i t r i c achd BhOuld give no p r e c i p i t a t e w i t h AgNO i f 3 s y n t h e t i c camphor i s a b s e n t from t h e o r i g i n a l s u b s t a n c e (136, 137).

JABER S. MOSSA AND MAHMOUD M . A. HASSAN

68

b)

5 g o f t h e camphor t o be t e s t e d i s d i s s o l v e d i n 50 c c 90% a l c o h o l , an aqueous s o l u t i o n o f 5 g hydroxylamine h y d r o c h l o r i d e and 8 g . sodium hydroxide are added, and t h e n enough a l c o h o l t o produce a c l e a r s o l u t i o n . The s o l u t i o n i s h e a t e d for 90 min. on a water-bath, water i s t h e n added, and i f t u r b i d i t y a p p e a r s , t h e presence o f i s o b o r n e o l o r camphene i s proved. I f t h e t u r b i d s o l u t i o n i s now n e u t r a l i z e d w i t h h y d r o c h l o r i c a c i d , t h e p r e c i p i t a t e which forms should be s o l u b l e i n excess of h y d r o c h l o r i c a c i d and a l s o i n sodium hydroxide (136).

8.2 Gravimetric Methods I n o r d e r t o determine t h e camphor c o n t e n t of e s s e n t i a l o i l s (which c o n t a i n no o t h e r carbonyl compounds) t h e g r a v i m e t r i c d e t e r m i n a t i o n proposed by Aschan (138) may be employed: Procedure: I n t r o d u c e about 1 g . o f t h e o i l , a c c u r a t e l y weighed, i n t o a t e s t t u b e and d i s s o l v e t h e o i l i n 2 g. o f g l a c i a l a c e t i c a c i d . Add 1 g. of semicarbaz i d e h y d r o c h l o r i d e and 1 . 5 g . o f f r e s h l y f u s e d anhydrous potassium a c e t a t e . T r i t u r a t e thoroughly with a g l a s s r o d , s t o p p e r t h e t u b e w i t h a plug of absorbent c o t t o n and h e a t t h r e e hours i n a water b a t h a t 70'. Cool t h e m i x t u r e , add 1 0 t o 15 c c . of water, s t i r t h o r o u g h l y and t r a n s f e r t h e p r e c i p i t a t e q u a n t i t a t i v e l y t o a t a r e d 4 t o 5 em. f i l t e r . Wash w i t h water u n t i l a l l water s o l u b l e m a t t e r i s removed, a i r d r y , wash w i t h petroleum e t h e r and dry i n a i r t o c o n s t a n t weight. Determine t h e weight of semicarbazone from t h e i n c r e a s e i n t h e weight of t h e f i l t e r . C a l c u l a t e t h e c o n t e n t of camphor i n t h e o r i g i n a l o i l by means of t h e f o l l o w i n g formula: P e r c e n t a g e of camphor =

V.7P S

where: p = weight o f semicarbazone i n grams; s = weight o f o i l i n grams. Other methods f o r d e t e r m i n a t i o n of camphor v i a s e m i c a r b a z o n e , i n pharmaceutical p r e p a r a t i o n s were a l s o r e p o r t e d (139, 1 4 0 ) . Estimation of camphor i n camphor s p i r i t ( 1 4 1 ) : The method isbased on t h e a b i l i t y of camphor t o enter

CAMPHOR

i n t o molecular combination w i t h s a l o l , t o y i e l d salolcamphor, C ~ O H ~ ~ O . C ~ ~ H ~ O O ~ . I n t o a 1 0 0 cc .weighed Erlenmeyer f l a s k introduce about

log. ( a c c u r a t e l y weighed) camphor s p i r i t and 70 c c of water. Add e x a c t l y 8 g. d e s i c c a t e d chemically pure s a l o l , shake t h e f l a s k , f i n a l l y allowing t h e camphor and s a l o l t o c o l l e c t on t h e bottom of t h e c o n t a i n e r . Pass t h e c l e a r superr.atant l i q u i d through a weighed Gooch c r u c i b l e ( t h e bottom of which c a r r i e s 2 s m a l l f i l t e r s covered by a p r o c e l a i n n e t ) . Wash t h e cruc i b l e with a few drops of c o n c e n t r a t e d s a l o l s p i r i t . P l a c e t h e Erlenmeyer f l a s k i n a water b a t h a t 50-60' whereby t h e e n t i r e c o n t e n t o f t h e f l a s k m e l t s t o a uniformly o i l y l i q u i d c o n s i s t i n g o f salol-camphor and s a l o l . Remove t h e c o n t a i n e r from t h e b a t h , s e t a s i d e 1 h r . i n a cold room i n o r d e r t h a t t h e s a l o l excess may c r y s t a l l i z e . Should t h i s not t a k e p l a c e , shake t h e f l a s k , whereupon r a p i d c r y s t a l l i s a t i o n u s u a l l y r e s u l t s . T r a n s f e r t h e e n t i r e c o n t e n t of t h e f l a s k t o t h e c r u c i b l e s e p a r a t i n g t h e o i l y salolcamphor from t h e c r y s t a l s s a l o l by s u c t i o n . Wash t h e l a t t e r by s u c t i o n w i t h 5 c c . c o n c e n t r a t e d s a l o l s p i r i t . Remove t h e combined p o r t i o n s o f s a l o l t o a s h e e t of smooth paper. Wash t h e i n s i d e o f t h e f l a s k w i t h a few cc o f e t h e r , r e c e i v i n g and e v a p o r a t i n g t h e l a t t e r on a watch g l a s s . P r e s s t h e s a l o l r e s i d u e on t h e watch g l a s s 'c,etween 2 s h e e t s o f w h i t e paper u n t i l no f u r t h e r g r e a s e s p o t forms. Add t h i s s a l o l t o t h e main p o r t i o n , t h e n d r y a t f i r s t i n t h e a i r u n t i l t h e odor of csmphor i s no l o n g e r p e r c e p t i b l e , t h e n i n a d e s i c c a t o r t o c o n s t a n t weight. The percentage of camphor j.s t h e n determined according t o t h e following formula:

% camphor = ( a 7 1 . 0 6 ) / b a = i s t h e amount of camphor combined w i t h salol. b = t h e amount of sample t a k e n for a n a l y s i s and t h e v a l u e 71.06 = t h e molecular weight of s a l o l i n t o t h a t of zamphor m u l t i p l i e d by 1 0 0 .

Good r e s u l t s a r e r e p o r t e d . Another method u t i l i z i n g hydroxylamine h y d r o c h l o r i d e ( 1 4 2 , 143) i s as f o l l o w s :

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1 0 g . of s p i r i t of camphor, 1 g . o f hydroxylamine h y d r o c h l o r i d e and 2 g. o f sodium hydroxide i n 20 c c d i s t i l l e d water are h e a t e d i n a f l a s k provided w i t h a r e f l u x condenser on a w a t e r b a t h f o r t h r e e hours. After c o o l i n g d i l u t e s u l f u r i c a c i d i s added, phen o l p h t h a l e i n being used as i n d i c a t o r . The l i q u i d i s t h e n e x t r a c t e d w i t h e t h e r t h r e e t i m e s . The e t h e r e a l s o l u t i o n o f camphor oxime i s d r i e d w i t h anhydrous sodium s u l p h a t e and poured i n t o a weighed f l a s k . After d i s t i l l i n g t h e g r e a t e r p a r t o f e t h e r , t h e remaining e t h e r i s removed w i t h a i r a t room t e m p e r a t u r e , u n t i l a c r y s t a l mass i s formed which i s d r i e d i n a d e s s i c a t o r and weighed. The r e s u l t s o b t a i n e d by t h i s method a r e s a t i s f a c t o r y .

Hampshire and Page ( 1 4 4 ) evolved a u s e f u l p r a c t i c a l method f o r t h e d e t e r m i n a t i o n o f camphor i n g a l e n i c a l s u s i n g 2,h-dinitrophenylhydrazine as a p r e c i p i t a n t . The procedure used i s as f o l l o w s : D i s s o l v e about 0 . 2 g of camphor, a c c u r a t e l y weighed by d i f f e r e n c e , i n 25 m l aldehyde-free e t h a n o l i n a 300 m l c o n i c a l f l a s k and slowly add w i t h c o n s t a n t shaking, 75 m l of t h e r e a g e n t ( p r e p a r e d by d i s s o l v i n g 1 . 5 g of 2,4-dinitrophenylhydrazine i n a m i x t u r e of 1 0 m l o f c o n c n e t r a t e d s u l p h u r i c a c i d and 1 0 m l o f water, d i l u t i n g w i t h w a t e r t o 100 m l and f i l t e r i n g ; t h e r e a g e n t decomposes on s t a n d i n g and must be made up j u s t b e f o r e u s e ) . Then h e a t t h e m i x t u r e on a water-bath under a r e f l u x condenser f o r f o u r h o u r s , a l l o w it t o c o o l , d i l u t e w i t h 2 p e r c e n t v/v sulphur i c a c i d t o 200 m l (weaker a c i d allows c r y s t a l l i s a t i o n of t h e excess o f base from t h e s o l u t i o n ) and a l l o w t o s t a n d f o r twenty-four hours. C o l l e c t t h e p r e c i p i t a t e on a weighed gooch c r u c i b l e w i t h paper m a t , o r on a s i n t e r e d - g l a s s f i l t e r , wash it w i t h s u c c e s s i v e q u a n t i t i e s of 10 ml of c o l d d i s t i l l e d w a t e r u n t i l t h e washings are no l o n g e r a c i d , and d r y at 80'; 1 g o f camphor 2,h-dinitrophenylhydrazone corresponds t o 0.458 g o f camphor. A comparison o f v a r i o u s methods for d e t e r m i n a t i o n o f camphor and o t h e r compounds u t i l i z i n g 2,k-din i t r o p h e n y l h y d r a z i n e were a l s o r e p o r t e d (145-149).

A vacuum oven method and a comparison w i t h t h e U . S . P , X . method as w e l l as a m o d i f i c a t i o n , u t i l i z i n g a n t i - o x i d a n t s such a s p y r o g a l l o l , a-naphthol,

71

CAMPHOR

hydroquinone and ParaFhenylenediamine f o r t h e d e t e r m i n a t i o n of camphor i n camphor l i n i m e n t were desc r i b e d by Poe ( 1 5 0 , 1 5 1 ) . The U.S.P. X I X ( 1 5 2 ) d e s c r i b e d a method f o r t h e d e t e r m i n a t i o n of camphor i n camphorated p a r a c h l o r e phenol. Procedure: T r a n s f e r about 300 mg of Camphorated Parachlorophenol, a c c u r a t e l y weighed, t o a 200 m l p r e s s u r e b o t t l e c o n t a i n i n g 50 m l o f f r e s h l y prepared TS. Close t h e p r e s s u r e dinitrophenyl-hydrazire b o t t l e , immerse it i n a w a t e r b a t h , and m a i n t a i n it a t about 75' f o r 4 hours. Cool t o room temperat u r e , t h e n t r a n s f e r t h e c o n t e n t s t o a beaker w i t h t h e a i d o f 1 0 0 m l of d - i l u t e s u l f u r i c a c i d (1 i n 12), and a l l o w it t o s t a n d o v e r n i g h t . C o l l e c t t h e prec i p i t a t e on a t a r e d f i l t e r c r u c i b l e , wash w i t h 1 0 0 ml o f d i l u t e s u l f u r i c a c i d (1 i n 1 2 ) and t h e n w i t h 75 ml of c o l d w a t e r , i n d i v i d e d p o r t i o n s , t o remove t h e a c i d . Dry a t 80' f o r 2 h o u r s , c o o l and weigh. The weight of t h e p r e c i p i t a t e so o b t a i n e d , m u l t i p l i e d by 0.4581, r e p r e s e n t s t h e weight of C H 0 i n t h e l o 16 sample t a k e n . Another o f f i c i a l method d e s c r i b e d by t h e pharmacop e i a of Japan I X (153:i f o r t h e a s s a y o f camphor i s as f o l l o w s : T r a n s f e r about 0.2 g o f d-camphor, a c c u r a t e l y weighe d ¶ t o a 300-ml g l a s s s t o p p e r e d f l a s k , and d i s s o l v e i n 25 m l o f aldehyde-j7ree e t h a n o l . Add slowly 75 m l o f 2,4-dinitrophenylhydrazine TS w h i l e shaking, and h e a t f o r 4 hours on a water b a t h under a r e f l u x cond e n s e r . Cool, add 1 0 0 m l of d i l u t e d s u l f u r i c a c i d (l+ 5 0 ) , and a l l o w t o s t a n d f o r 24 hours. C o l l e c t t h e p r e c i p i t a t e so obliained on a g l a s s f i l t e r ( G 3 ) , wash t h e r e s i d u e on t h e f i l t e r w i t h c o l d water unt i l t h e washings become n e u t r a l , d r y f o r 2 hours a t 105' and weigh as t h e 2,h-dinitrophenylhydrazone o f 332.36) d-camphor ( C 1 6 ~ 2 0 ~ 4 0: 4

.

Amount (mg) of d-camphor

(C10H160) = amount (mg) of

2,4-dinitrophenylh:~drazoneof d-camphor ( C16H20N404 ) X 0.,4580. The o f f i c i a l method O F NF X I V (154) f o r t h e a s s a y of camphor i n camphor s p i r i t i s g i v e n below:

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

Assay: T r a n s f e r 2 . 0 ml of camphor s p i r i t t o a s u i t a b l e p r e s s u r e b o t t l e c o n t a i n i n g 50 m l of f r e s h l y prepared d i n i t r o p h e n y l h y d r a z i n e TS. Close t h e p r e s s u r e b o t t l e , immerse it i n a water b a t h , and m a i n t a i n a t about 75' f o r 1 6 hours. Cool t o room t e m p e r a t u r e , and t r a n s f e r t h e c o n t e n t s t o a beaker w i t h t h e a i d o f 1 0 0 ml o f d i l u t e s u l f u r i c a c i d (1 i n 1 2 ) . Allow t o s t a n d not l e s s t h a n 1 2 hours a t room t e m p e r a t u r e , t r a n s f e r t h e p r e c i p i t a t e t o a t a r e d f i l t e r c r u c i b l e , and wash w i t h 100 m l of d i l u t e s u l f u r i c a c i d (1 i n 1 2 ) followed by 75 ml of c o l d water i n d i v i d e d p o r t i o n s . Continue t h e s u c t i o n u n t i l t h e excess water i s removed,dry t h e c r u c i b l e and p r e c i p i t a t e a t 80' f o r 2 h o u r s , c o o l , and weigh. The weight o f t h e p r e c i p i t a t e so o b t a i n ed, m u l t i p l i e d by 0.4581, r e p r e s e n t s t h e weight o f C H 0 i n t h e sample t a k e n .

10 16

8.3 T i t r i m e t r i c Methods 8.31 Aqueous Mital and Gaind (155) claim good r e c o v e r i e s f o r camphor i n pharmaceutical p r e p a r a t i o n s w i t h a volumetric method i n which hydroxylamine hydroc h l o r i d e i s used. For s p i r i t , l i n i m e n t and solut i o n of camphor i n t u r p e n t i n e , t h e following procedure i s a p p l i c a b l e : Reflux t h e sample ( c o n t a i n i n g 0 . 2 g of camphor) f o r f o u r hours w i t h 20 m l of aldehyde-free 95 p e r c e n t e t h a n o l , 1 0 ml of a 4 p e r c e n t s o l u t i o n of hydroxylamine h y d r o c h l o r i d e i n aldehyde-free 90 per c e n t e t h a n o l and 0.3 g o f sodium b i c a r b o n a t e . Cool, r i n s e t h e condenser w i t h 20 m l o f l i g h t petroleum ( b . p . 50' t o 60°), c o l l e c t i n g t h e r i n s i n g s i n t h e f l a s k and t i t r a t e t h e m i x t u r e f i r s t w i t h 0.2N hydroc h l o r i c a c i d t o dimethyl yellow and t h e n with 0.2N potassium hydroxide t o p h e n o l p h t h a l e i n . Carry o u t a c o n t r o l o m i t t i n g t h e camphor and addicg 5 m l of s t e a m - d i s t i l l e d t u r p e n t i n e o i l b e f o r e r e f l u x i n g f o r t h e s p i r i t and s o l u t i o n and 1 g of a r a c h i s o i l before refluxing f o r t h e liniment. Calculate t h e amount o f camphor p r e s e n t from t h e d i f f e r e n c e i n t h e volume of 0.2R potassium hydroxide used i n t h e two t i t r a t i o n s . 1 m l of 0.2N KoH = 0.0304 g camphor

.

13

CAMPHOR

Other r e p o r t e d procedures w i t h minor m o d i f i c a t i o n s and u s e of d i f f e r e n t i n d i c a t o r s have been a l s o r e p o r t e d (156-165). A method w a s a l s o d e s c r i b e d f o r d e t e r m i n a t i o n of camphor i n d i l u t e s o l u t i o n s by photometric t i t r a t i o n u s i n g bromophenol b l u e as t h e i n d i c a t o r (166). 8.32 Vanadometry

An i n d i r e c t VasladOmetriC method of a s s a y f o r camphor w a s developed. The method i n v o l v e s t h e formation o f 2,4-dinitrophenylhydrazone of camp$or. The n i t r o groups i n t h e hydrazone a r e t h e n reduced t o m i n e s by t r e a t m e n t w i t h vanadium s u l f a t e (VSO4) and t h e excess o f t h e r e a g e n t i s back t i t r a t e d w i t h sodium dichromate (Na2C1-207). The e n t i r e procedure i s c a r r i e d o u t i n a modified s e p a r a t o r y f u n n e l i n t o which s t a n d a r d v a n a d a t e , 6N H2SO4, and z i n c amalgam a r e i n t r o d u c e d . A carbondioxide c u r r e n t i s t h e n passed and t h e m i x t u r e i s shaken t o o b t a i n a b l u e c o l o u r a f t e r which t h e z i n c amalgam i s drawn o f f . Then s u l f u r i c a c i d i s added t o g e t h e r w i t h s e v e r a l drops o f phenosafranine and excess vandate i s t i t r a t e d with standazd sodium dichromate s o l u t i o n . 8.33 Polarographic Schwabe and Berg ( 1 6 7 ) have s t u d i e d t h e polarography of t e r p e n e d e r i v a t i v e s . The s o l v e n t s were 80% e t h a n o l , 80% dioxane, 80% acetone o r water and t h e s u p p o r t i n g e l e c t r o l y t e s were 0 . 1 M L i C 1 , L i O H , EtkNI, o r Eti+rlOH. Camphor was found t o d e p r e s s t h e polarogi-aphic maxima and t h i s could be used f o r i t s d e t e r m i n a t i o n .

8.4 R e f r a c t o m e t r i c Method Rapaport and Solyanik (168) have developed a r a p i d method of r e f r a c t o m e t r i c d e t e r m i n a t i o n of 23 m i x t u r e s c o n t a i n i n g a n e s t h e s i n , barbamyl , bromcanphor, bromisoval, camphor , a n t i p y - r i n e, amidopyrine , a c e t a l s a l i c y l i c a c i d , b a r b i t a l , codeine, s a l o l , t e r p i n h y d r a t e , hexamethylenetetramine, znd p h e n o b a r b i t a l . The met hod d e s c r i b e d i s s u i t a b l e f o r a n a l y s i s o f pharmaceutical m i x t u r e s c o n t a i n i n g cornpxnds which a r e i n s o l u b l e i n water and s o l u b l e i n e t h a n o l . A m i x t u r e of 2 pharmac e u t i c a l s ( 0 . 1 g ) i s d i s s o l v e d i n 1 m l . e t h a n o l and nD of t h i s s o l u t i o n i s determined. One component i s

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

chemically determined i n a n o t h e r 0 . 1 g of t h e sample. The amount o f such component i s c a l c u l a t e d from formulas

A = [(n-no) - (CxF)] P/100F1P1 C = VT 1 0 0 , B = VTP/P1 where A i s wt. o f t h e 1st component i n grams, V i s t h e volume o f 0.1N s o l u t i o n used for t i t r a t i o n o f t h e 2nd component, T i s t h e g . e q u i v a l e n t o f t h e 1 s t compound, B is t h e w t . of t h e 2nd component ( g . ) , n i s t h e r e f r a c t i v e index o f t h e s o l u t i o n , no i s t h a t o f e t h a n o l , C i s t h e amount of t h e 2nd component i n 1 0 0 m l . s o l u t i o n ( c h e m i c a l l y d e t e r m i n e d ) , F and F1 a r e c o n s t a n t s determined e x p e r i m e n t a l l y , P i s t o t a l wt. of t h e m i x t u r e and P1 i s wt. of sample (0.1 g . ) .

Other r e p o r t e d procedures f o r t h e d e t e r m i n a t i o n o f camphor, by u s i n g r e f r a c t i v e index and o t h e r p h y s i c a l c o n s t a n t s have a l s o been r e p o r t e d (169,170).

8.5 Nephlometric Method Nephlometric methods f o r d e t e r m i n a t i o n o f camphor vapors i n a i r (171) and camphor i n s p i r i t s were desc r i b e d ( 1 7 2 ) . Optimum r e s u l t s w e r e o b t a i n e d w i t h a m i x t u r e c o n s i s t i n g o f e t h e r , 9676, e t h a n o l and w a t e r i n t h e p r o p o r t i o n 0 . 1 : 0.9 : 3. The t u r b i d i t y of t h e m i x t u r e i s i n c r e a s e d by t h e presence of camphor, i n p r o p o r t i o n t o t h e c o n c e n t r a t i o n o f camphor i n s o l u t i o n . Approximately, 0.2 mg of camphor can be determined i n 2 ml o f t h e m i x t u r e ( 0 . 0 1 mg /L can be determined i f 100 L of a i r and 1 0 m l of t h e s o l u t i o n a r e u s e d ) .

8.6

Chromatographic Methods

8.61 Thin

Layer Chromatography

Miller and Kirchner (173) have developed a n analyt i c a l method f o r v o l a t i l e c o n s t i t u e n t s i n c l u d i n g camphor. They used s i l i c i c a c i d c o a t e d - g l a s s c h r o m a t o s t r i p s . E t h y l a c e t a t e hexane, chloroform, benzene, i s o p r o p y l e t h e r and t h e i r m i x t u r e s were used as d e v e l o p e r s and t h e s t r i p s were examined w i t h v a r i o u s r e a g e n t s l i k e bromine, f l u o r e s c e n c e , 0 - a n i s i d i n e , Bulphuric a c i d - n i t r i c a c i d , and 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 . The R f v a l u e s were calculated.

CAMPHOR

75

Table 7:

S. No.

R f v a l u e of Camphor on S i l i c i c a c i d u s i n g different solvents.

Colour of s p o t Df w i t h H2SO)++For- Value. I\L Solvent System.

maldehyde Reagent.

1.

n-Hexane.

Light green.

2.

Carbon t e t r a c h l o r i d e

0.31

3.

C yc l o hexane

0.33

4.

Benzene

0.10

5.

Chloroform

0.61

6.

Ethyl acetate

7.

Benzene : Carbon t e t r a c h l o r i d e

0.00

Spot a p p e a r s a t t h e solvent f r o n t .

(50 : 50)

0.62

8.

E t h y l a c e t a t e : n-Hexane (1.O:gO)

0.38

9.

E t h y l a c e t a t e : n-Hexane

10.

E t h y l a c e t a t e : Carbon t e t r a c h l o r i d e (1j:8j)

(15:85)

0.39 0.42

(15:85) ,, Benzene (1j:85)

11. E t h y l a c e t a t e : Cyclohexane

12. E t h y l a c e t a t e :

9 9

3 3

0.42

3 3

0.37

Conditions Glass p l a t e s 10.5 x 25 cm. (coated with s i l i c i c a c i d (Mesh No.100). Temperature 2 5 O C R e l a t i v e humidity 25 5%. Normal Chromatographic Chamber, s a t u r a t e d f o r 15 minutes b e f o r e u s e .

.

+_

76

JABER S. MOSSA AND MAHMOUD M. A . HASSAN

Egon S t a h l (174) h a s invented a s p e c i a l chromatog r a p h i c d e v i c e for t h e s e p a r a t i o n of v o l a t i l e o i l s u s i n g uniform t h i c k n e s s of adsorbent on p l a t e . El-Deeb, e t a1 (175) have r e p o r t e d t h e s e p a r a t i o n and e v a l u a t i o n o f camphor by TLC t e c h n i q u e on s i l i c i c a c i d p l a t e . T h e behaviour of camphor on chromatoplates w a s studied using d iffe re n t solvents of i n c r e a s i n g p o l a r i t y and t h e i r m i x t u r e s as devel o p e r s . The s p o t s were l o c a t e d by s p r a y i n g t h e p l a t e s f i r s t w i t h s u l p h u r i c a c i d and t h e n w i t h 40% s o l u t i o n of Formaldehyde and h e a t i n g t h e p l a t e t o 100°C f o r 3 t o 5 minutes. The R f v a l u e s were c a l c u l a t e d . It i s given i n Table 7.

8.62 Column Chromatography The column t e c h n i q u e has been used for t h e separat i o n and d e t e r m i n a t i o n of t h e p u r i t y of camphor. Montes (176) analysed t h e e s s e n t i a l o i l c o n s t i t u e n t s u s i n g t h e p r i n c i p l e o f chromatographic a d s o r p t i o n w i t h t h e a i d of columns of s i l i c i c a c i d and b e n t o n i t e . The compounds were t h e n c r y s t a l i z e d and t h e i r m e l t i n g p o i n t s were d e t e r determined. Gustave Vavon and Bernard Gastambide (177) and Bernard Gastambide (178),s t u d i e d t h e e f f e c t s o f s t e r i c h i n d r a n c e of camphor and fenchone on chromatographic alumina column. Eero Sjostrom (179) h a s d e s c r i b e d a method of t h e s e p a r a t i o n of camphor and o t h e r ketones u s i n g a n i o n exchange r e s i n s column. A s u c c e s s f u l attempt of s e p a r a t i o n and i d e n t i f i c a t i o n of camphor from m i x t u r e s w a s achieved by Karawya (180) and by Karawya and El-Deeb (181) u s i n g alumina column.

El-Deeb, e t a1 (175) have r e p o r t e d t h e comparative s t u d y of d i f f e r e n t i a l a d s o r p t i o n o f camphor on both aluminium oxide and s i l i c i c a c i d . The r e t e n t i o n volumes were determined by u s i n g Petroleum100 mg of m a t e r i a l e t h e r and e t h e r as s o l v e n t s w a s chromatographed on column o f 1 em diameter and 1 0 g of e i t h e r Alumina or s i l i c i c a c i d s ( m e s h No. 3 0 ) . The r e s u l t s are p r e s e n t e d i n Table 8.

.

17

CAMPHOR

Table

S.

No.

8:

R e t e n t i o n of Volume of Camphor on d i f f e r e n t columns.

Adsorbent.

1. S i l i c i c a c i d 2.

Alumina

-

R e t e n t i o n volume

Petroleum E t h e r

Ether

More t h a n 25 m l .

20 m l .

100 m l .

15 m l .

8.63 G a s Liquid Chromatography A GLC method f o r t h e d e t e r m i n a t i o n of camphor ( s y n t h e t i c and n a t u r a l ) has been c a r r i e d out i n

o u r l a b o r a t o r y ( 1 8 2 ) , u s i n g a Varian GC-3700 g a s chromatograph equipped w i t h Varian CDS 111 integrator. Column c o n d i t i o n s : 3 % OV1 on chromosorb W-Hp (801 0 0 mesh s i z e ) ; g l a s s column (2% x h m ) . The column r u n i s o t h e r m a l l y a t 100' + 2OO0C f o r 2 minu t e s ( t h e t e m p e r a t u r e i s i n c r e a s e d at t h e r a t e of 10' p e r m i n u t e ) , C a r r i e r g a s : Nitrogen, flow r a t e w a s a d j u s t e d t o 40 ml/minute. D e t e c t o r : FID, hydrogen and a i r , flow r a t e s were a d j u s t e d t o 30 ml/minute and 300 ml/minute r e s p e c t i v e l y . E t h a n o l w a s used as s o l v e n t . The i n j e c t i o n t e m p e r a t u r e was 150OC and t h e c h a r t speed w a s a d j u s t e d t o g i v e 1 cm/minute. The r e t e n t i o n t i m e = 1 . 4 5 minutes. Camphx c o n t e n t of t h e sample i s c a l c u l a t e d from t h e 2hromatogram by u s i n g peak a r e a r a t i o method. The GLC of camphor i s p r e s e n t e d i n F i g . 11. Camphor has been determined by Baines and P r o c t o r ( 1 8 3 ) i n a number of e s s e n t i a l o i l s and pharmaceut i c a l p r e p a r a t i o n s u s i n g g a s chromatographic t e c h nique. The a p p a r a t u s employed has a t h e r m a l cond u c t i v i t y d e t e c t o r w i t h 4 i n . platinum w i r e 0 . 0 0 1 i n . d i a m e t e r of nominal r e s i s t a n c e 250 Ohms. The w i r e s i n t h e channel:; being matched t o w i t h i n 0 . 1 Ohm. The b r i d g e c u r r e n t w a s 200 mA and t h e o u t p u t r e c o r d e d on a 2 . 5 mV r e c o r d e r . 2% ethylbenzene w a s added t o t h e s t a n d a r d s and samples as an i n t e r n a l s t a n d a r d . The o p e r a t i n g c o n d i t i o n s w e r e as

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

78

v

Fig. 11:

GLC of Camphor.

( a ) : Natural Camphor. ( b ) : S y n t h e t i c Camphor.

.

follows: Column l e n g t h 7 f t Column diameter 4 t o 5 mm. Column temperature 13OoC. S t a t i o n a r y phase 20% of squalane on 100 t o 1 2 0 mesh c e l i t e . Sample s i z e 30 pl, C a r r i e r gas 4:1 hydrogen and n i t r o g e n mixture a t 100 ml/min. The r e t e n t i o n volumes were camphor1200 m l , ethylbenzene 270 m. camphor content of t h e sample i s c a l c u l a t e d from t h e chromatogram by using peak a r e a r a t i o method. Yoshio Hanada and Masayoshi Kitajima (184) have r e p o r t e d t h e gas chromatographic determination of camphor using naphthalene as t h e i n t e r n a l standard

.

GLC procedure f o r t h e s e p a r a t i o n of camphor and o t h e r t e r p e n e s on polypropyleneglycol-impregnated

79

CAMP H 0R

column w a s a l s o d e s c r i b e d by Egon S t a h l and et a 1 (1.75) Trennheuser ( 1 8 5 ) . El-Deeb, h a v e r e p o r t e d G L C t e c h n i q u e f o r determini n g t h e c o n s t i t u e n t s of v o l a t i l e o i l u s i n g s i l i con o i l DC 550 as a s t a t i o n a r y phase.

8.64 High-Performance Liquid Chromatography Q u a n t i t a t i v e ana1ysj.s of b o t h camphor and parachlorophenol i n chaniphorated parachlorophenol by h i g h - p r e s s u r e l i q u i d . chromatography i s d e s c r i b e d

(186). The HPLC chromatograph (Waters A s s o c i a t e Model A . I . C . 202) was used w i t h s o l v e n t system o f 60% hexane and 40% chloroform. A UV d e t e c t o r a t 254 nm was used a t a n a t e n u a t i o n of x8 f o r camphor and x 64 f o r pa.rachlorophenol and phenol. The flow r a t e was 2 . 5 ml/minute a t an i n l e t p r e s The c h a r t speed w a s 0 . 5 cm/ s u r e of 0.131 Nrn-2. min. The column (Hi--Eff M i c r o p a r t ) w a s 0.64 cm 0.d X 1 5 em l o n g , packed w i t h 5 Y m s i l i c a g e l (Applied S c i e n c e L a b o r a t o r i e s ) . The i n j e c t i o n volume w a s 7-15 1 arid t h e e l u t i o n over was camphor ( 2.76 min. parachlorophenol ( 10.64 min) and phenol (12.61 min) .

!

The r e s u l t s i n d i c a t e d t h a t camphor and parachlorophenol can be s i m u l t a n e o u s l y assayed by simple d i l u t i o n s of t h e sample and i n j e c t i o n i n t o a h i g h p r e s s u r e l i q u i d chromatograph. The l i n e a r i t y c u r v e i n d i c a t e d t h a t t h e components have been anal y s e d g i v e l i n e a r response. R e s u l t s a r e shown i n Table 9. Table 9:

Assay R e s u l t s on USP Camphorated Parac h:Lorophenol

Assay

Camphor, 7;

1 2 3

64.3 65.6

34.7

65.0

35.4 35.6 0.42

4 SD

cv

Average deviation.

64.6 0.56 0.86 0.43

Parachlorophenol,%

34.9

1.19 0.35

JARER S MOSSA A N D MAHMOIJD M . A . HASSAN

80

8 . 7 S p e c t r o s c o p i c Methods 8.71 C o l o r i m e t r i c Camphor can be determined c o l o r i m e t r i c a l l y w i t h s u l f u r i c a c i d and f u r f u r a l (187). To l c c of camphor s o l u t i o n , add 3 cc of e t h a n o l ( 9 5 % ) and 1 0 drops of 1% s o l u t i o n o f f u r f u r a l i n 95% e t h a n o l . S t i r t h e m i x t u r e , add two drops of c o n c e n t r a t e d s u l f u r i c a c i d and h e a t i n a water b a t h . Then c o o l and add 5cc. of e t h a n o l and compare t h e r e d t o v i o l e t c o l o u r produced w i t h a s t a n d a r d . The amount p r e s e n t can be determined w i t h i n 10%. The c o l o u r r e a c t i o n s o f camphor w i t h aldehydes have been a p p l i e d w i t h p -dimethylaminobenzaldehyde i n s u l f u r i c a c i d (188). The yellow c o l o u r formed conforms t o B e e r ' s law and checks c l o s e l y with t h e o f f i c i a l N F method ( 1 5 4 ) . The a s s a y is c a r r i e d out a s follows : Dissolve 0.125 g of pdimethylaminobenzaldehyde i n 65 ml o f 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 and 35 m l of water. Add 7 ml of t h i s r e a g e n t by b u r e t t e t o an a l i q u o t o f a d i l u t i o n of t h e sample i n chloroform t o c o n t a i n between 0 . 6 and 7 . 5 mg o f camphor. S t o p p e r , shake and allow t o s t a n d two hours f o r c o l o u r development and measure t h e e x t i n c t i o n a t 460 mp. P r e p a r e a s t a n d a r d curve i n a s i m i l a r manner from a l i q u o t s o f a prepared s o l u t i o n of camphor i n chloroform i n t h e r a n g e 0 . 6 t o 7.5 mg of camphor. A method has been d e s c r i b e d f o r d e t e r m i n a t i o n o f camphor i n d i l u t e s o l u t i o n s and i n p l a n t s (189). The a s s a y i s based on t h e formation o f a yellow c o l o u r w i t h phenylhydrazine (Camphor phenylhydraz o n e ) . The c o l o u r produced a f t e r a b s o r p t i o n on Whatman No.120 paper and t h e i n t e n s i t y o f t h e yellow c o l o u r produced was compared with t h a t produced from a sample of known c o n c e n t r a t i o n . A m i c r o a n a l y t i c a l procedure w a s r e p o r t e d f o r d e t e r m i n a t i o n o f camphor ( 1 9 0 ) . The method i s based on t h e formation o f camphor oxime. The oxime i s s u b j e c t e d t o a c i d h y d r o l y s i s and t h e r e s u l t i n g hydroxylamine r e a c t e d w i t h f o r m a l i n and

81

CAMPHOR

f e r r i c ammonium. The r e s u l t i n g f e r r i c formohydroxamate i s t r e a t e d w i t h ammonium p e r s u l p h a t e and t h e c o l o u r produced i s t h e n determined.

8.72 Nuclear Magnetic Resonance Hassan et a 1 (29) r e c e n t l y have d e s c r i b e d a proton magnetic resonance a n a l y t i c a l procedure f o r t h e d e t e r m i n a t i o n of camphor a s b u l k drug and t h e simultaneous a s s a y o f camphor and p-chlorophenol. The method i s based on t h e i n t e g r a t i o n of t h e n i n e p r o t o n s of t h e t h r e e methyl groups of camphor c e n t r e d a t 0.88 ppm and t h e t e n aromatic p r o t o n s of benzophenone c e n t r e d a t 7.55 ppm employed as an i n t e r n a l r e f e r e n c e s t a n d a r d . The method i s s p e c i f i c and a c c u r a t e w i t h s t a n d a r d dev i a t i o n o f 2 0.86 and an average r e c o v e r y of 100.28%. I n c a s e of camphor parachlorophenol a s s a y , i n t e g r a t i o n of t h e n i n e protons o f t h e t h r e e methyls o f camphor c e n t r e d a t 0.83 ppm, t h e f o u r aromatic p r o t o n s of parachlorophenol c e n t r e d at 6.97 ppm, and t h e two o l e f i n i c prot o n s s i n g l e t of maleic a c i d appearing a t 6.37 ppm were c a r r i e d o u t . The method w a s s p e c i f i c and a c c u r a t e with s t a n d a r 5 d e v i a t i o n s of t 1.13, 2 1.01 f o r camphor and t 0.62, t 0.89 f o r parachlorophenol, i n s t a n d a r d mixtures and i n camphorated parachlorophenol r e s p e c t i v e l y . The PMR spectrum i n a d d i t i o n , provides a very s p e c i f i c means f o r i d e n t i f i c a t i o n of camphor and parachlorophenol. ACKNOWLEDGEMENT

The a u t h o r s g r a t e f u l l y thank M r . K.U. Abdul Hameed, L e c t u r e r , Department of Pharmacognosy, College of Pharmacy, King Saud U n i v e r s i t y , Riyadh, Saudi Arabia f o r doing t h e l i t e r a t u r e survey and f o r h i s ef:eort and hard work which have been undertaken d u r i n g t h e prepa:-ation of t h e manuscript and a l s o acknowledge Mr. A l t a f Hussain Naqvi, f o r t y p i n g t h e manus c r ip t .

JABER S. MOSSA AND MAHMOUD M . A. HASSAN

82

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Anales. Assoc. Quim.


CHLOROQUINE Mohammad Tariq and Abdullah A. Al-Badr King Satad Utrirersity Riycidh, Saudi Arabia I . History 2. Description 2.1 Nomenclature 2.2 Formulae 2.3 Molecular Weight 2.4 Elemental Composition 2.5 Appearance, Color, Odor, and Taste 3. Physical Properties 3.1 Melting Point 3.2 Boiling Point 3.3 Solubility 3.4 Acidity 3.5 Moisture Content and Hygroscopicity 3.6 Dissociation Constants 3.7 Stability 3.8 Storage 3.9 Sterilization 3.10 Spectral Properties 4. Synthesis 5. Pharmacokinetics 5.1 Absorption. Distribution, Metabolism, and Excretion 5.2 Distribution and Metabolism in Neonate:; 6. Therapeutic Uses 7. Toxicity 8. Methods of Analysis 8.1 Identification 8.2 Microbiological Assay 8.3 Spectrophotometric Methods 8.4 Chromatographic Methods References

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 13

95

96 96 96 97 91 97 97 91 97 98 98 98 98 98 98 98 98 98 Ill I I3 I I3 114 115 1 I5 115 1 I5

116 1 I6 1 I8 123

Copyright 0 1984 by the American Pharmaceutical Association ISBN 0-12-260813 5

M O H A M M A D TARIQ A N D ABDULLAH A . AL-BADR

96

1.

History Chloroquine i s one of a l a r g e series of 4-aminoquinolines investigated i n connection with t h e extensive cooperative program of a n t i m a l a r i a l r e s e a r c h i n t h e United S t a t e s durinE World War 11. The o b j e c t i v e w a s t o d i s c o v e r more e f f e c t i v e and l e s s t o x i c s u p p r e s s i v e a g e n t s t h a n quinac r i n e . Although t h e 4-aminoquinolines had p r e v i o u s l y been d e s c r i b e d a s p o t e n t i a l a n t i m a l a r i a l s by Russian i n v e s t i g a t o r s , s e r i o u s a t t e n t i o n w a s n o t p a i d t o t h i s chemical c l a s s u n t i l t h e French r e p o r t e d t h a t 3-methyl-7-chloro-4(4-diethylamino-1-methylbutylamino) q u i n o l i n e (SN-6911; S o n t i c h i n , Sonotoquin) w a s w e l l t o l e r a t e d and had h i g h a c t i v i t y i n human m a l a r i a s . Beginning i n 1943, t h o u s a n d s of t h e s e compounds were s y n t h e s i z e d and t e s t e d f o r a c t i v i t y i n a v i a n malaria and f o r t o x i c i t y i n m a m m a l s ; t e n of t h e s e r i e s were t h e n examined i n human v o l u n t e e r s w i t h e x p e r i O f t h e s e , c h l o r o q u i n e proved m e n t a l l y induced m a l a r i a s . most promising and w a s r e l e a s e d f o r f i e l d t r i a l . When h o s t i l i t i e s c e a s e d , i t w a s d i s c o v e r e d t h a t t h e chemical had been s y n t h e s i z e d and s t u d i e d under t h e name of Resochin by t h e Germans a s e a r l y as 1934. (1)

2.

Description

2.1

Nomenclature

2. I. 1

Chemical N a m e s 4 N -(7-chloro-4-quinolinyl~-N’, 4-pentanediamine.

MI-diethyl-1

,

7-Chloro-4-(4-diethylamino-l-methylbutylamino) q u i n o l i n e . The CAS R e g i s t r y No. i s [54-05-71.

2.1.2

& 3)

Generic Name Ch l o r o q u i n e RP 3377, SN 7 , 618.

2.1.3

(2

(2)

Trade N a m e s

Base:

Aralen, Nivaquine B , Sanquine, A r t r i c h i n , B i p i q u i n e , Reumachlor, Bemaphate, Tanakan, Resoquine. (3)

97

CHLOROQUIN E

Diphosphate: Valaquine, Resochin, S i l b e s a n , T r e s o c h i n , Arechin, A v l o c l o r , Imagon. ( 2 , 3) Sulphate:

2.2

Nivaquine and Remasulph. ( 2 , 3 & 8)

Formulae

2.2.1

Empirical

1gH2 6'IN3

2.2.2

Structural CH / C2H5 1 3 NH-CH-CH2CH 2CH2-N

,

JQJ@

c1

8

C2H5

N 1

Chlor oqu i n e

2.2.3

ldiswesser L i n e N o t a t i o n

T66 R N J EM & 3 N2 & 2 1GSQP QQ0 2 DL (4) 2.3

Molecular Weight 319.89

2.4

Elemental Composition C 6 7 . 5 9 % , H 8 . 1 9 % , C 1 11.08% N 13.14%.

2.5

(2)

Appearance, C o l o r , Odor and T a s t e White o r s l i g h t l y y e l l o w c r y s t a l l i n e powder, o d o r l e s s and b i t t e r i n t a s t e . ( 5 & 6 ) .

3.

Physical Properties

3.1

Melting Point M e l t s between 87'

and 97'.

(6)

M O H A M M A D TARIQ A N D ABDULLAH A . AL-BADR

98

3.2

Boiling Point 212'

3.3

t o 214'

a t 0.2 mm Hg.

(7)

Solubility Very s l i g h t l y s o l u b l e i n water; s o l u b l e i n d i l u t e (7) a c i d s , chloroform and e t h e r .

3.4

Acidity Chloroquine phosphate h a s a pH of 3.5 t o 4.5 of 10% s o l u t i o n . While t h e 10% s o l u t i o n of c h l o r o q u i n e s u l p h a t e h a s a pH of 4 t o 5 . (7)

3.5

M o i s t u r e Content and H y g r o s c o p i c i t y Not more t h a n 1.5% determined by d r y i n g a t 105'. It a b s o r b s i n s i g n i f i c a n t amounts of m o i s t u r e a t temperat u r e s up t o 37O a t r e l a t i v e h u m i d i t i e s up t o about 80%. (7)

3.6

Dissociation Constants pKa Values a r e 8 . 4 ,

3.7

10.8 (20').

(7)

Stability S o l u t i o n s of pH 4 t o 6 a r e s t a b l e when h e a t e d b u t sensitive to light. (7)

3.8

Storage I t should be p r o t e c t e d from l i g h t .

3.9

(7)

Sterilization S o l u t i o n s f o r i n j e c t i o n a r e s t e r i l i z e d by h e a t i n g i n a n a u t o c l a v e o r by f i l t r a t i o n . ( 7 ) .

3.10 S p e c t r a l P r o p e r t i e s 3.10.1

U l t r a v i o l e t Spectrum The u l t r a v i o l e t spectrum of c h l o r o q u i n e i n n e u t r a l methanol s o l u t i o n i n t h e r e g i o n of 200 t o 400 nm e x h i b i t s m a x i m a a t 218 nm, 253 nm and 328 nm and minima a t 243 and

99

CHLOROQUINE

275 nm. The spectrum i s shown i n Fig. I . The spectrum wasobtained on Varian AG U V - V i s Spectrophotoineter model DMS 90. 3.10.2

I n f r a r e d Spectrum The i n f r a r e d spectrum of c h l o r o q u i n e i s pres e n t e d i n F i g . 2. The spectrum w a s o b t a i n e d from K B r d i s c on a Perkin-Elmer I n f r a r e d Spectrophotometeir model 580 B , i n t h e r a n g e of 4000 - 400 CIn-1. It shows t h e f o l l o w i n g : Frequency (cm-l) 3260

Assignments

NH group

1540 1580

quinoli n e

1610 I n a d d i t i o n t o o t h e r bending and s t r e t c h i n g v i b r a t i o n bands € o r t h e a l i p h a t i c moiety of t h e molecule. C l a r k e (8) r e p o r t e d t h a t t h e p r i n c i p a l peaks f o r c h l o r o q u i n e b a s e i n K B r d i s c a r e 1573, 1538, 1372 o r 1 4 4 8 . 3.10.3

Mass Spectrum

The chemical i o n i s a t i o n (CI) m a s s spectrum F i g . 3 , w a s r e c o r d e d on a Finnigan 4000 Mass Spectrometer w i t h i o n s o u r c e p r e s s u r e 0.3 T o r r , i o n s o u r c e t e m p e r a t u r e 15OoC, e m i s s i o n c u r r e n t 300 VA, e l e c t r o n energy 100 e V u s i n g methane a s a r e a g e n t g a s . The e l e c t r o n impact ( E I ) m a s s spectrum F i g . 4 , w a s r e c o r d e d on Varian MAT 311 S p e c t r o m e t e r , w i t h a n i o n s o u r c e p r e s s u r e 10-6 T o r r , i o n s o u r c e t e m p e r a t u r e 180, e m i s s i o n c u r r e n t 300 UA and e l e c t r o n energy of 7 0 e V . Table 1 and t h e f o l l o w i n g f i g u r e i l l u s t r a t e t h e prominent f r a g m e n t s i n t h e E I spectrum and t h e i r proposed a s s i g n m e n t s .

L

i

100.0-

50.0

r

-

-

Bc

211

so

-

320

100.0-

207’

-

50.0-

284

Fig. 3.

I

348

Mass spectrum of chloroquine, CI.

L

I03

m a

r,

MOHAMMAD TARQ AND ABDULLAH A . AL-BADR

104

Table

1

E I Mass Spectrum Assignments of Chloroquine.

mfe

R e l a t i v e Abundance

319

13

304

3

I o n o r Fragment M+'.

M-CHT t

290

M - C ~ H ~

247

M - N E ~

231

M-CH2-NEg+

+

+

2-Nhq+

219

5

205

5

126

5

C ~ H ~ N E ~ J

112

13

C ~ H , N E ~

99

13

c 2~ 3 ~ ~ t 2 )

86

100

58

40

M-( (XI2)

M-(CFT2) 3NEt2 --I+

+

+

+

CH

2~~;-;1

H-N -Et

+

I05

CHLOROQUINE

3.10.4

'H-NMR

Spectrum

1 The H-NMR s p e c t r a of c h l o r o q u i n e b a s e and p h o s p h a t e a r e shown i n F i g . 5 and 6 and were o b t a i n e d o n V a r i a n T6-A NMR S p e c t r o m e t e r w i t h CDC13 and D:,O r e s p e c t i v e l y as s o l v e n t s and t e t r a m e t h y l s i l a n e a s a n i n t e r n a l r e f e r ence. The s i g n a h a r e a s s i g n e d i n T a b l e 2 . Table

2

.

'H-NMR

S p e c t r a l Assignments of C h l o r o q u i n e .

Chemical S h i f t (ppm) and M u l t i p l i c i t y [

Proton

Base

- CH3CH2;

0.83 -. 1.33 [m]

CH3-CH

--

-CH2-N

\

1.2

2.33

-'

- 1.5 [m] 1.82 [ b s ]

1.66 [ t ]

-CH CH2CH2 CH2-

-

Phosphate

2.67 [m]

3.2 [ 4 ]

CH2-

F 3 -CH -

3.66 [41

NH

5.64 [ d ]

I

Aromatic C H2

4.00 [ b s ] Exchanged

8.2 [ d ]

8.43 [ d ]

I _

Aromatic C H3 -

6.4

Other Aromatic P r o t o n s

s = singlet

d

=

b s = broad s i n g l e t

7.2

doublet

[d]

- 7.96 [m]

t = triplet

b m = broad m u l t i p l e t .

6.65 [ d ] 7.15

q =

- 7.9 [m] quartet

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

3.10.5

C-13 NMR Spectrum The noise-decoupled and o f f - r e s o n a n c e C-13

NMR s p e c t r a of c h l o r o q u i n e a r e p r e s e n t e d i n F i g u r e s 7 and 8 r e s p e c t i v e l y . The s p e c t r a were o b t a i n e d on Varian FT-80A S p e c t r o m e t e r . S p e c t r a l a s s i g n m e n t s are l i s t e d i n T a b l e 3. 9 2 1 CH3 7 6 5 /CH2-CH3 NH -CH- CH -CH -CH2 -N

I

2

\

m2 -(=H3 4 3 12

14

Table

3

. Carbon-13

Carbon No.

NMR S p e c t r a l Assignment of Chloroquine. Chemical S h i f t (ppm) r e l a r i v e t o 'INS

Mu1t i p l i c i t y

1, 4 2, 3

11.7

quartet

46.27

triplet

5

52.25

triplet

6

23.60

triplet

7

33.46

triplet

8

47.72

doublet

9

19.83

quartet

10

149.60

singlet

11

98.79

doublet

12

151.85

doublet

13

149.47

singlet

14

127.55

doublet

15

133.36

singlet

16

123.71

doublet

17

124.40

d oub 1e t

18

117.67

singlet

Fig. 7 .

I3C-NMR spectrum of chloroquine in DMSO-D6, noise-decoupled.

.

l

Fig. 8.

~

l

.

l

~

~

l

l

~

l

l

l

~

l

~

l

l

l

I3C-NMR spectrum of chloroquine in DMSO-D

~

6'

l

~

l

~

l

~

o f f -resonance.

l

l

~

'

l

~

i

~

Ill

CHLOROQUINE

4.

Synthesis a) One of the earliest and most effective antimalarials is "chloroquine", in whose synthesis 4,7-dichloroquinoline, prepared from '7-chloro-4-quinolone, is key intermediate. (9, 10).

250'

> &

oo+

COOEt

(1) NaOH

Cl

NHCH=C(C02C2H5)2

HO@'& c1

250'

Cl

~

( 2 ) HC1

d , 00

'OC13

~

c1

fH3

Chloroquine

Et


112

b) In another synthesis methyl acrylate is reacted with

m-chloroaniline,the resulting methyl m-chloroanilinopropionate is tosylated and then hydrolyzed to the acid. The acid is converted to acid chloride and cyclized to give 4-keto-7-chloro-1,2,3,4-tetrahydroquinoline reaction of this compound with 4-diethylamino-1-methylbutylamine under the dehydrogenating influence of nitrobenzene gives chloroquine in about 252 overall yield (11 6 12).

COOCH3

COOH

I

I

H

Tos

-m n

C

$3 B t N H ~ ~ ( c H3~;j3~ ~) 5 -. nitrobenzene

Chloroquine

I

H

c) Chloroquine is obtained by coupling 4,7-dichloroquinoline with 5-diethylamino-2-aminopentane. (13)

c1

YH3 /Et NH2-CH-(CH 2) 3N,Et t

Chloroquine

CHLOROQUINE

5.

I13

Pharmacokinetics 5.1

A b s o r p t i o n , D i s t r i b u t i c i n , Metabolism and E x c r e t i o n Chloroquine i s r e a d i l y absorbed from t h e g a s t r o i n t e s t i n a l t r a c t and about 50% i n t h e c i r c u l a t i o n i s bound t o plasma p r o t e i n . I t d e l a y s g a s t r i c emptying. I t a c c u m u l a t e s i n h i g h c o n c e n t r a t i o n s i n some tiss u e s , such as t h e k i d n e y s , liver l u n g s and s p l e e n and i s s t r o n g l y bound i n m e l a n i n - c o n t a i n i n g c e l l s such a s t h o s e i n t h e e y c s and t h e s k i n ; i t i s a l s o bound t o double s t r a n d e d DNA, p r e s e n t i n r e d blood c e l l s c o n t a i n i n g s c h i z o i i t s ( 1 ) . A t a d o s e of 310 mg, plasma c o n c e n t r a t i o n s r e a c h a p l a t e a u a t 10 d a y s w i t h a c o n c e n t r a t i o n of about: 120 ng/ml; a f t e r a 500 mg d o s e , c o n c e n t r a t i o n s of about 700 ng!ml are a t t a i n e d i n haemolysed blood i n 4. h o u r s and a f t e r s i n g l e o r a l d o s e s of 400 t o 500 mg, peak plasma c o n c e n t r a t i o n s of 35 t o 220 ng/ml a r e a t t a i n e d i n 2 t o 6 h o u r s ( 6 ) . About 7 0 4 5 % of t h e t o t a l whole blood c o n t e n t of c h l o r o q u i n e and i t s m e t a b o l i t e d e s e t h y l c h l o r o q u i n e were r e c o v e r e d i n blood c e l l s i s o l a t e d from whole b l o o d , i n d i c a t i n g t h a t t h e s e compounds have a c e l l / It h a s been plasma c o n c e n t r a t i o n r a t i o ( 1 4 ) . r e p o r t e d t h a t small amounts of c h l o r o q u i n e may b e found i n t h e blood and u-rine of p a t i e n t s f o r as l o n g a s 5 y e a r s a f t e r t h e lasir known a d m i n i s t r a t i o n , and t h e e x i s t e n c e of l a r g e t i s s u e r e s e r v o i r s h a s been p o s t u l a t e d (15 ti 1 6 ) . Other s t u d i e s are p u b l i s h e d on u n i d e n t i f i e d metabo1it:es p r e s e n t i n plasma and u r i n e of t h e t r e a t e d s u b j e c t s . The p o l a r and nonp o l a r m e t a b o l i t e s of chlclroquine are shown below: NHR

Fig. 9

c1 CH(CH3)-(CH2)3-N-(C2H5)2

I

R

=

I1

R

= CH(CH3)-(CH2)3-NH-C2H5

I11

R

= CH(CH3)-(CH2)3-NH2

IV

R

=

CH(CH~)-(C:H~)~-~JC~H~)~

V same a s I V , b u t r i n g N i s a l s o N@

--+

Oe VI R

=

H

MOHAMMAD TARlQ AND ABDULLAH A. AL-BADR

I14

F i g . 9 C h l o r o q u i n e ( I ) , d e s e t h y l - ( 1 1 ) and d i d e s e t h y l c h l o r o q u i n e (111). S i d e - c h a i n N-oxide ( I V ) , d i N-oxide (V) , and 7-chloro-4-aminoquinoline (VI) IV and V , p o l a r m e t a b o l i t e s ; I1 and 111, r e l a t i v e l y nonpolar metabolites. (17).

.

Chloroquine and i t s m e t a b o l i t e d e s e t h y l c h l o r o q u i n e found i n thrombocytes and g r a n u l o c y t e s i n v i v o and i n v i t r o . C h l o r o q u i n e i s c h a r a c t e r i z e d by i t s e x t e n s i v e t i s s u e d i s t r i b u t i o n which c a u s e s a slow e l i m i n a t i o n from t h e body. Plasma h a l f - l i f e i s v a r i a b l e 21/,-7 d a y s . B r o h u l t e t a 1 (18) o b t a i n e d a v a l u e f o r plasma h a l f - l i f e of c i i l o r o q u i n e i s 4 d a y s , w h i l s t Frisk-Holmberg e t a1 (19) found h a l f - l i v e s of 3.1 h o u r s t o 13 d a y s depending upon t h e dose. M e t a b o l i c r e a c t i o n s i n v o l v e o x i d a t i v e N - d e a l k y l a t i o n and o x i d a t i v e d e a m i n a t i o n and a l s o c o n j u g a t i o n , p o s s i b l y w i t h g l u c u r o n i c a c i d , of t h e c a r b o x y l i c a c i d m e t a b o l i t e s d e r i v e d from d e a l k y l a t i o n and d e a m i n a t i o n ; t h e metab o l i t e s i n c l u d e mono- and b i s d e s e t h y l c h l o r o q u i n e , 4 (7-chloroquinol-4-ylamino) pentan-1-01, and 4-(7chloroquinol-4-ylamino) p e n t a n o i c a c i d and i t s conChloroquine i s e l i m i n a t e d v e r y s l o w l y j u g a t e (7). from t h e body and i t may p e r s i s t i n t i s s u e f o r a prolonged p e r i o d . About 55% i s e x c r e t e d i n t h e u r i n e and a b o u t 10% i n t h e f a e c e s by 90 d a y s f o l l o w i n g t h e r a p y w i t h 310 mg f o r 1 4 d a y s ; t h e u r i n a r y excret i o n o f t h e unchanged d r u g i s dependent upon u r i n a r y pH and l a r g e r amounts are e x c r e t e d i n a c i d u r i n e t h a n i n a l k a l i n e u r i n e ; of t h e m a t e r i a l e x c r e t e d i n t h e u r i n e , a b o u t 70% i s unchanged, 23% i s d e s e t h y l c h l o r o q u i n e , 1 t o 2% i s b i s d e s e t h y l c h l o r o q u i n e and an u n i d e n t i f i e d m e t a b o l i t e , and 1 t o 2% i s e x c r e t e d a s c a r b o x y l i c a c i d m e t a b o l i t e s i n c o n j u g a t e d form. (21 &

--

22).

5.2

D i s t r i b u t i o n and Metabolism i n Neonates C h l o r o q u i n e , i t s N-dealkylated m e t a b o l i t e s , and c h l o r o q u i n e N-oxides were d e t e c t e d i n t h e u r i n e of p r e g n a n t women who were r e c e i v i n g c h l o r o q u i n e medic a t i o n whereas c h l o r o q u i n e and i t s n o n p o l a r m e t a b o l i t e s , d e s e t h y l and d i d e s e t h y l c h l o r o q u i n e and 7chloro-4-aminoquinoline have been found i n t h e neon a t e s ’ u r i n e , b l o o d and cord blood. T h a t c h l o r o q u i n e and i t s r e l a t i v e l y n o n p o l a r m e t a b o l i t e s ( i n c l u d i n g one w i t h o u t t h e a l k y l s i d e - c h a i n , 7-chloro-4-aminoq u i n o l i n e ) c r o s s t h e p l a c e n t a i s d e m o n s t r a t e d by t h e

CHLOROQUINE

115

p r e s e n c e of t h e s e compounds i n t h e c o r d blood, neon a t a l s y s t e m i c b l o o d , and n e o n a t a l u r i n e . The s e l e c t i v e t r a n s f e r of t h e compounds a c r o s s t h e p l a c e n t a h a s a l s o been r e p o r t e d ( 1 7 ) . Both i n v i t r o and i n -v i v o m e t a b o l i c s t u d i e s have shown t h a t some drug metabolism o c c u r s o r can be induced i n p l a c e n t a l t i . s s u e s ( 2 0 ) . F u r t h e r degradat i o n of p a r e n t c h l o r o q u i n e t h a t r e a c h e s t h e p l a c e n t a would r e s u l t i n t h e appearance of less c h l o r o q u i n e and more of t h e n o n p o l a r m e t a b o l i t e s i n t h e f e t a l o r n e o n a t a l c i r c u l a t i o n . (117). I

6.

Therapeutic

Uses

I t i s h i g h l y e f f e c t i v e i n t e r m i n a t i n g a c u t e v i v a x and f a l c i p a r u m malaria. I t i s a l s o v a l u a b l e i n t h e t r e a t m e n t of i n t r a i n t e s t i m l a m e b i a s i s and some cases of g i a r d i a s i s (1). Chloroquine a l s o produce symptomatic improvement i n B a r b e s i a i n f e c t i o n s . It can b e used a s an a n t i r h e u m a t i c agent (23).

7.

Toxicity I n t h e r a p e u t i c d o s e s c h l o r o q u i n e may c a u s e headache, v i s u a l d i s t u r b a n c e s , g a s t r o i n t e s t i n a l u p s e t and p r u r i t i s . I n some c a s e s i t may c a u s e d i s c o l o r a t i o n of n a i l b e d s and mucous membranes. Prolonged t r e a t m e n t may c a u s e l i c h e n o i d s k i n e r u p t i o n , v i s u a l b l u r r i n g and b l e a c h i n g of h a i r . Prolonged l a r g e d o s e s (250 t o 750 mg d a i l y ) may c a u s e l o s s of c e n t r a l v i s u a l a c u i t y , g r a n u l a r p i g m e n t a t i o n of macula and r e t i n a l a r t e r y c o n s t r i c t i o n . The v i s u a l l o s s a p p e a r s t o be i r r e v e r s i b l e ( 2 4 ) . O t o t o x i c i t y h a s been r e p o r t e d i n few c a s e s . Hart and Naunton, (25) have i m p l i c a t e d c h l o r o q u i n e i n f e t a l a b n o r m a l i t y . Blood d y s c r a s i a s have been r e p o r t e d r a r e l y . They i n c l u d e r e v e r s i b l e agranul o c y t o s i s , thrombocytopenia, and n e u t r o p e n i a ( 6 ) .

8.

Methods of A n a l y s i s

8.1

Identification a ) D i s s o l v e a b o u t 25 mg i n 20 m l of water and add 8 m l of t r i n i t r o p h e n o l s o l u t i o n ; a p r e c i p i t a t e i s produced which, a f t e r washing s u c c e s s i v e l y w i t h w a t e r , a l c o h o l ( 9 5 % ) , and e t h e r , m e l t s a t about 207O. (8).

1I6

MOHAMMAD TARlQ AND ABDULLAH A. AL-BADR

b) Micro: P i c r i c a c i d - r o s e t t e s of p l a t e s ( s e n s i r o s e t t e s of t i v i t y : 1 i n 1000); s t y p h n i c a c i d p l a t e s ( S e n s i t i v i t y 1 i n 1000). (8). M i c r o b i o l o g i c a l Assay

-

8.2

A r a p i d semiautomated m i c r o d i l u t i o n method f o r t h e m i c r o b i o l o g i c a l a s s a y of t h e c h l o r o q u i n e h a s been developed by Desgardins ( 2 6 ) . A n t i m a l a r i a l a c t i v i t y of c h l o r o q u i n e may be s t u d i e d a g a i n s t c u l t u r e d Plasmodium f a l c i p a r u m , m i c r o p l a t e s a r e used t o prep a r e s e r i a l d i l u t i o n of t h e drug. P a r a s i t e s o b t a i n e d fromcontinuous s t o c k c u l t u r e s a r e s u b c u l t u r e d i n t h e m i c r o - p l a t e s f o r 4 2 h. I n h i b i t i o n of u p t a k e of a r a d i o l a b e l e d n u c l e i c a c i d p r e c u r s o r by p a r a s i t e s serves a s t h e i n d i c a t o r o f a n t i m i c r o b i a l a c t i v i t y .

8.3

Spectrophotometric Methods

8.3.1

U l t r a v i o l e t Spectrophotometric A n a l y s i s

8.3.1.1

Ap, Method J

Chloroquine h a s been assayed s p e c t r o p h o t o m e t r i c a l l y ( 2 7 ) u s i n g t h e Apj method (28) t h a t depends on d i f f e r e n c e s i n orthogonal function c o e f f i c i e n t s . The l a t t e r method i s based on t h e f a c t t h a t t h e peaks of c e r t a i n compounds may s p l i t i n t o s u b s i d i a r y peaks without any a p p r e c i a b l e change i n i n t e n s i t y by changing t h e pH i n a s u i t a b l e i n t e r v a l . The b e h w i o u r of t h e s e compounds r e s t r i c t s t h e a p p l i c a t i o n of t h e AA method ( 2 9 & 30) because AA may n o t f u l f i l t h e requirements specified f o r its successful application. I n these c i r c u m s t a n c e s , t h e n p j method o f f e r s a s o l u t i o n f o r t h e d e t e r m i n a t i o n of such compounds i n t h e presence of i r r e l e v a n t absorption. Thus, t h e c o n t r i b u t i o n of a pH-insensitive i r r e l e v a n t absorption may be c a n c e l l e d by means of

+ P..(Z)I - [ajih 11

AP. .=[ajia Cx J1

and C

x

Cx

+ pji ( Z > I

=Ap../Aa.. 31

11

where p j i s t h e c o e f f i c i e n t o f t h e polynomial, p . ( 4 ) a i i s p j ( I X , 1 cm), t h e c o e f f i c i e i t f o r A (151, 1 cm) o f the

I17

CHLOROQUINE

p u r e compound, x ; C, i s t h e c o n c e n t r a t i o n ; pji(Z) d e n o t e s t h e c o n t r i b u t i o n from i r r e l e v a n t a b s o r p t i o n ; t h e subs c r i p t s i , a , and b d e n o t e t h e wavel e n g t h r a n g e and t h e two d i f f e r e n t s o l u t i o n s , r e s p e c t i v e l y . Thus, by choosing P . and a l s o t h e s e t of wavel e n g t h s , i: s o t h a t p j i i s optimum i n one s o l v e n t and n e g l i g i b l y small i n t h e o t h e r s o l v e n t , Apji can be used t o e v a l u a t e compound x. The a s s a y p a r a m e t e r s u s i n g t h e Ap. J method f o r c h l o r o q u i n e : Solvents

:

0.1 N H2S04 & 0.1 N NaOH

Concentration

:

1 . 6 mg/100 m l s o l vent

P

:

P

Number of Points

:

12-point o r t h o g o n a l polynomials.

Wave 1en g t h range

:

324-346

:

- 0.00170

:

8008

:

0.160

j

*p4 N;

. .

(the quartic polynomial).

nm each 2 nm.

1,

(Ap,.

N')"*

4

fc

N4 = normalizing f a c t o r f o r P

f 5 ~

should exceed 0.14 f o r t h e c o e f f i c i e n t of v a r i a t i o n t o be l e s s t h a n

4'

17.

MOHAMMAD TARIQ AND ABDULLAH A . AL-BADR

I18

8.3.2

F l u o r i m e t r i c Method

A h i g h l y s e n s i t i v e , a c c u r a t e and improved f l u o r i m e t r i c method h a s been developed f o r a n a l y s i s of c h l o r o q u i n e i n b i o l o g i c a l samples (31). The c h l o r o q u i n e i s e x t r a c t e d w i t h h e p t a n e o r methylene d i c h l o r i d e from a n a l k a l i medium b u f f e r e d w i t h b o r a t e , pH 9.5. Chloroquine i s r e e x t r a c t e d i n t o 0.1 N H C 1 which i s t h e n mixed w i t h an e q u a l volume of 0.2 M a l c o h o l i c NaOH and f l u o r e s c e n c e i s r e a d a t a c t i v a t i o n and emission wavelengths of 335 and 400 nm r e s p e c t i v e l y . 8.4

Chromatographic Methods 8.4.1

Paper Chromatography Several s o l v e n t systems have been d e s c r i b e d , which are used f o r t h e i d e n t i f i c a t i o n of c h l o r o q u i n e ( 8 ) a s shown i n t h e f o l l o w i n g table:

S o l v e n t Systems

V i s u a l i z i n g Agents (Rf)

C i t r i c a c i d : water: nb u t a n o l 4.8 g : 103 m l : 870 m l .

Ultraviolet, iodoplatinate s p r a y ( s t r o n g react i o n ) , bromocresol g r e e n s p r a y ( s t r o n g r e a c t i o n ) (0.19).

A c e t a t e b u f f e r (pH 4 . 5 8 )

Ultraviolet, iodoplatinate spray ( p o s i t i v e r e a c t i o n ) . (0.70).

Phosphate b u f f e r (pH 7 . 4 )

Ultraviolet, iodoplatinate spray. (0.14).

8.4.2

Thin-Layer

Chromatography

Clarke ( 8 ) d e s c r i b e d t h e f o l l o w i n g system f o r t h e i d e n t i f i c a t i o n of c h l o r o q u i n e . S o l v e n t System: S t r o n g ammonia s o l u t i o n : methanol ( 1 . 5 : 100) should be changed a f t e r two r u n s . Absorbent:

Silica G e l G

I I0

CHLOROQU INE

V i s u a l i z i n g Agent : A c i d i f i e d i o d o p l a t i n a t e spray ( p o s i t i v c r e a c t i o n )

.

Rf

8.4.3

: 0.31.

Gas Chromatography

a)

C l a r k e (8) h a s d e s c r i b e d t h e f o l l o w i n g g a s chrom5,tographic method € o r t h e i d e n t i f i c a t i o n of c h l o r o q u i n e .

1Z SE-30 on 100-120 mesh Arkrom 6 f t X 4 m i n t e r v a l diameter b o r o s i l i c a t e g l a s s column. Column:

ABS.

Col.umn Temperature .: 2500 Carrier Gass G a s Flow:

.

Argon.

80 m l p e r min.

Argon D e t e c t i o n and R e t e n t i o n Time: i o n i s a t i o n d e t e c t o r or flame i o n i s a t i o n d e t e c t o r . R t 1.44 r e l a t i v e t o c o d e i n e . b)

C h u r c h i l l e t a 1 1 9 8 3 (23) h a s developed two gas chromatographic methods u s i n g Tandem f u s e d - s i l i c a c a p i l l a r y f o r t h e d e t e r m i n a t i o n of c h l o r o q u i n e and i t s m a j o r m e t a b o l i t e d e s e t h y l c h Loroquine. The method I employed a s i n g l e e x t r a c t i o n s t e p and i n t e r n a l s t a n d a r d i z a t i o n to permit r a p i d p r e c i s e a n a l y s i s f o r c h l o r o q u i n e i n whole blood. Whereas method I1 employes d e r i v a t i z a t i o n with pent:afluoropropionic a n h y d r i d e b e f o r e e x t r a c t i o n and e s t i m a t i o n of c h l o r oqu i n e and d e se t hy l c h l o r o q u i n e t h e d e t e c t i o n l i m i t f o r chloroquine i n blood by b o t h methods are 5 ng/ml and f o r d e s e t h y l c h l o r o q u i n e i s 1 5 ng/ ml. Following s p e c i f i c a t i o n column;

MOHAMMAD TARlQ AND ABDULLAH A . AL-BADR

I20

1.83 m X 2 mm I D g l a s s column packed w i t h OV-101 on 100-120 macsh g a s chrome Q and a helium f l o w r a t e 15 ml/min. Temperature : 250°C. E l e c t r o n impact i o n i s a t i o n performed a t 70 eV. 8.4-3.1

L i q u i d Chromatography The l i q u i d chromatographic system c o n s i s t e d of an M45 pump a U6K i n j e c t o r . The e x c i t a t i o n wavelength w a s 335 nm and a 370 nm f i l t e r w a s used. The column (0.15 m X 4.6 mm I . D . ) w a s s l u r r y - packed w i t h L i c h r o s o r b S i 6 0 , 5 pm and e l u t e d . The flow r a t e of t h e e l u e n t w a s 1 m l / min. A f t e r a d d i t i o n of water and e x t r a c t i o n with d i e t h y l e t h e r , t h e o r g a n i c phase w a s d r i e d (sodium su1phate)and t h e s o l v e n t evaporated ,the p r o d u c t could be used d i r e c t l y . The l i m i t of d e t e c t i o n of t h i s method w a s 1 ng/ml of plasma f o r c h l o r o q u i n e and 0.5 ng/ml f o r d e s e t h y l c h l o r o q u i n e . The p r e c i s i o n of t h e method w a s 3.5 of (n=lO) a t 50 ng/ml plasma ( u r i n e ) f o r c h l o r o q u i n e and 5% (n=10) a t t h e 25 ng/ml f o r d e s e t h y l c h l o r o q u i n e . (32).

8.4.4

High-pressure L i q u i d Chromatography 8.4.4.1

Reversed-Phase HPLC A n a l y s i s B e r g q v i s t and Frisk-Holmberg, (33) r e p o r t e d a r a p i d and s e n s i t i v e method f o r t h e a n a l y s i s of c h l o r o q u i n e and i t s metabolite desethyl chloroquine i n plasma, blood and u r i n e u s i n g high-performance l i q u i d chromatography. Chloroquine i s e x t r a c t e d from the alkalinized biological f l u i d with e t h y l e n e d i c h l o r i d e and r e - e x t r a c t e d w i t h d i l u t e a c i d . It i s t h e n chromato-

121

CHLOROQUINE

graphed on a r e v e r s e d - p h a s e column. U l t r a v i o l e t a b s o r p t i o n ( 2 5 4 o r 340 nm) o r f l u o r e s c e n c e d e t e c t o r s are used. C h l o r o q u i n e and d e s e t h y l chloroquine concentrations i n t h e r a n g e of 10 n m o l / l (UV-detection) and of 0.5 n mol/ 1 ( f l u o r e s c e n c e d e t e c t i o n ) c o u l d be a c c u r a t e l y measured w i t h a r e l a t i v e s t a n d a r d d e v i a t i o n of 12%. 8.4.4.2

S t r a i g h t - P h a s e HPLC A n a l y s i s B e r g q v i s t and O l i n ( 3 4 ) h a v e d e s c r i b e d a s t r a i g h t - p h a s e HPLC method f o r t h e a n a l y s i s of c h l o r o q u i n e a s follows : LiChrosorb S i 100 ( 7 um) w a s u s e d a s support:. The m o b i l e and s t a t i o n a r y p h a s e s were e q u i l i b r a t e d b e f o r e u s e . A t u b e ( l e n g t h 1.5 m and volume 4 m l ) w a s i n s e r t e d i n f r o n t of t h e i n j e c t o r and p l a c e d i n a t h e r m o s t a t i n o r d e r t o e n s u r e good t e m p e r a t u r e c o n t r o l 25 m l of a n of t h e m o b i l e p h a s e . aqueous p e r c h l o r a t e s o l u t i o n was p r e s e n t as a s e p a r a t e l a y e r i n t h e s o l v e n t r e s e r v o i r i n o r d e r t o keep t h e mobile phase s a t u r a t e d . The column c o a t i n g w a s a c h i e v e d by t h e pumping t e c h n i q u e . Columns w e r e f i r s t t e s t e d i n t h e a d s o r p t i o n mode 1 - b u t a n o l (199 1) w i t h n-hexane as t h e m o b i l e p h a s e . The v o i d volume V,, w a s 2.86 m l . It w a s d e t e r m i n e d w i t h t o l u e n e , which i s n o t r e t a i n e d . The c o n c e n t r a t i o n of 1 - p e n t a n o l i n t h e m o b i l e phase w a s c o n t r o l l e d by g a s chromatography on a Chromosorb 102 cclumn w i t h 1 - b u t a n o l a s i n t e r n a l standard.

+

8.4.4.3

+

I o n - P a i r HPLC A n a l y s i s R e c e n t l y a method f o r d e t e r m i n a t i o n of chl.oroquine and i t s m e t a b o l i t e s h a s been d e s c r i b e d by Brown ( 3 5 ) .

MOHAMMAD TARlQ A N D ABDULLAH A . AL-BADR

I22

T h i s method i s s i m p l e s p e c i f i c and s e n s i t i v e f o r s e p a r a t i o n and q u a n t i f i c a t i o n of c h l o r o q u i n e d e s e t h y l c h l o r o q u i n e and b i d e s e t h y l c h l o r o q u i n e i n biological f l u i d s using ion p a i r I t can r e v e r s e phase HPLC procedure. d e t e c t amount as low as 5 ng and a n a l y s i s t i m e i s 12 minutes. The f o l l o w i n g c o n d i t i o n s have been u s e d . Column

30 mm X 3.9 mm I . D . packed w i t h 10 Um U Bondspak CI8.

Mobile Phase :

0.02 m l I-heptane s u l p h o n i c a c i d and acet o n i t r i l e

.

8.5

PH

3.4

Pumping r a t i o :

6 6 ~ 3 4of P I C B-7 acetonitrile.

Column P r e s - : sure

62-76 b a r .

to

Photolysis Analysis Aron and Fidanza 1982 h a s d e s c r i b e d a photochemical method f o r d e t e r m i n a t i o n and s e p a r a t i o n of c h l o r o q u i n e a t nanogram level c o v e r i n g a r a n g e between 4 and l o 4 ng. They showed t h a t r e v e r s i b l e photo i s o m e r i z a t i o n of c h l o r o q u i n e t a k e s p l a c e and y i e l d s a f l u o r e s c e n t photoproduct. The c h l o r o q u i n e s o l u t i o n a r e s p o t e d on s i l i c a g e l p l a t e s and developed i n a l c o h o l ammonia m i x t u r e . The d r i e d p l a t e s a r e i r r a d i a t e d w i t h t h e image of mercury a r e focused on c h l o r o q u i n e s p o t s . The f l u o r e s c e n c e i n t e n s i t y w a s r e c o r d e d . For p h o t o l y s i s s t u d i e s c h l o r o q u i n e zone w e r e s c r a p e d from t h e p l a t e s and mixed w e l l w i t h t h e 4 m l of water. The c l e a r s u p e r n a t a n t decanted and s t u d i e d by a b s o r p t i o n photometry and s p e c t r o f l u o r o metry ( 3 6 ) .

Acknowledgements The a u t h o r s are t h a n k f u l t o M r . Syed R a f a t u l l a h f o r h i s s k i l l f u l a s s i s t a n c e and M r . Tanvir A. B u t t f o r t y p i n g t h e manuscript.

123

CHLOROQUINE

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2.

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c,

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I24

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J. B r o h u l t , L. Rombo, V. Trop. Med. P a r a s i t . ,

Rubin, H.M. B e r n s t e i n and N . J . Opthalmol. 7 , 474 (1963).

Arch.

Zvaifler,

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Z v a i f l e r and M. Rubin, A r t h e r i t i s Rheum, (1962)

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Afamefuna, Clin. Chem.,

330

285,

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S i r l e a f and E. Bengtsson: (1979).

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B. Testa and P. J e n n e r , I n Drug Metabolism: Chemical and Biochemical A s p e c t s , J.J. S w a r d r i k , Ed., Marcel Dekker I n c . N . Y . , 397-404 (1976).

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S . A . Adeluse and L.A. (1982).

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S a l a k o , Gen. pharmac.

McChesney, M . J . F a s c o and W.F. Therap. 323 (1967).

E.W.

158,

Exp. -

2, 433 2. Pharm.

Banks, Jr.,

Schwartz, J.

23.

F.C. C h r u c h i l l , D.L. Mount and I . K . Chromatogr., 111 (1983).

24.

H.N. B e r n s t e i n , N . J . S v a i f l e r , F. Robin and A.M. 2 , 384 ( 1 9 6 3 ) . I n v e s t . Ophthalmol V i s u a l S c i . , -

274,

Mansour,

--

80,

25.

C.W.

26.

R.E. D e s g a r d i n s , J. C a n f i e l d , J . D . Haynes and J . D . Chulay, A n t i m i c r o b i a l Agents and ~- Chromotherapy, 710 (1979).

27.

H a r t and R.F. 407 (1964).

Naunton, Arch. O t o l a r y n g o l ,

16,

A.M. Wahbi, H. Abdine and S.M. R l a i l , 33, 96 (1978).

Die Pharmazie,

I

28.

H . Abdine, A.M.

24,

518 (1972).

Wahbi and M.A.

Korany, J. Pharm. Pharmac.

I25

CHLOROQUINE

74,

29.

G . Erdtman, Chem. I n d . ,

581 ( 1 9 5 5 ) .

30.

R.A. F i s h e r and F. Yates. S t a t i s t i c a l Tables f o r B i o l o g i c a l , A g r i c u l t u r a l and Medical Research, 6 t h Ed., 9 8 , O l i v e r b Boyd, E d i n b u r g h , ( 1 9 7 4 ) .

31.

S . A . Adeluse and L.A.

Salako,

Gen. Pharmac., 13,433

(1982). 32.

G. Alvan, L. Ekman and B. L i n d s t r o m , J. Chromatogr.,

2 2 9 , 241 ( 1 9 8 2 ) . 33.

Y.

B e r g q v i s t and M.

Frisk-Holmberg,

J.

Chromatogr.,

221, 119 ( 1 9 8 0 ) . 34.

Y . B e r g q v i s t and A . O l i n ,

Acts

Pharmaceutica S u ecica,

1 9 , 161 (1982).

I

35.

N.D.

Brown, B.T.

Poon and J . D .

Chulay,

2. Chromatogr.,

2 2 9 , 248 ( 1 9 8 2 ) . 36.

J . J . Aaron and J . F i d a n z a , T a l a n t a , 2 9 , 383 ( 1 9 8 2 ) .


CIMETIDINE P. M . G. Bavin Smith Kline cind French Rewnrch, Ltd The Frytlze, Welwyn Hertfordy h ire, United Kingdom

Alex Post and John E. Zarembo' Smith Kline cind French Laboratories Pliilacielphici, Peniisyltjnnin Introduction 1. Description 1.1 Nomenclature 1.2 Formula, Molecular Weight, Structure 1.3 Appearance, Color, Odor 2. Physical Properties 2.1 Spectral Properties 2.2 Physical Properties of the Solid 2.3 Solubility of Cimetidine 2.4 Physical Properties of Cimetidine Solutions 3. Synthesis 3.1 Synthesis of Cimetidine 3.2 Synthesis of Cimetidine Hydrochloride 4. Stability 4.1 Cimetidine 4.2 Cimetidine Hydrochloride 5. Analytical Chemistry 5.1 Identity Tests 5.2 Non-Aqueous Titration 5.3 Spectrophotometric Procedure 5.4 Thin Layer Chromatography 5.5 High Pressure Liquid Chromatographic Procedure 5.6 Determination of Cimetidine Sulfoxide Content 6. Metabolism and Pharmacokinetics 6.1 Determination of Cimetidine and Its Metabolites in Biological Fluids References

i28 i28 129

129 129 129 129 152 158 161 161 161 164

164 164 166 166

166 167

168 169 171 176 176 177 181

'Present address. Revlon Health Cure Group, Tuckuhoe, Neu' York 17070.


127

Copyright C 1984 by the American Pharmaceutical Asaocidtion ISBN 0-12-260813-5

P. M . G . BAVIN Er AL.

i 28

INTRC)DUCTION

Cimetidine i s a histamine H2-receptor antagonist which inhibits the secretion of basal and gastric hydrochloric acid secretion and also reduces the output of pepsin. The drug has been widely used i n the treatment o f ducdenal and gastric ulceration. It has also been used i n the t r e a h e n t of reflux esophagitis and f o r the reduction of gastric acid secretion and for the managanent of Zollinger-Ellison syndrcxne. The ccBnpound is given orally a s a tablet, syrup, i.v., and as an injection fluid.

1. Description 1.1 Ncpnenclature

1.11 Ch&cal

Names

(a) N”-cyano-N-methyl-N’- [ 2- [ (5-methyl-1~imidazol-4-yl)mthyll thiol ethyll guanidine (c.a. name)

1.12

(b) N”-cyanc-N-mthyl-N’- [ 2- [ (5-methylimidazol-4-yl)methylthiolethyll guanidine Trade Name Marketed by SnithKline Corporation under the trade nanie, Tagatnet.

129

CIMETIDINE

1.2

Formula, Molecular Weight Structure 1.21

mirical F o d a , Pblecular Weight

ci oHi 6 N 6 S 1.22

252.352

Structure dN-CSN H3C)

(CHZSCH~MZNHC b m 3

v 1.23

Hydrochloride S a l t C1 I ) H ~ ~ NHC1 ~S

1.3 Ap-

288.816

ance, Color, odor

The f r e e base and hydrochloridesalt are w h i t e c r y s t a l l i n e s o l i d s with l i t t l e or no odor. A slight sulfur-mercaptan odor may be present.

2.

Physical Properties

2.1

Spectral Properties

2.11

Infrared Spectra Figure 1 i s the infrared spectrum of cimetidine f r e e base. Figure 2 i s the infrared spectrum of cimetidine hydrochloride. The spectra were obtained as mineral o i l dispersions from 400-625 an-’ on a PerkinElmer Model 457A infrared spectrometer. The s i g n i f i c a n t absorption bands in t h e spectra are :

Cimetidine wavelength (an-’) 3220-3150

Assignment

NH s t r e t c h

2180

- E N stretch

1620

>C=N-,

9~10-

guanidine 1590

C-C, C-N,

armtic

I

4000

1

I

I

I

I

I

t

2500 WVENUMBER (CW)

Fig. 1.

1

Boo WVENUMBER (CM')

Infrared spectrum of cimetidine.

1

I

I 1

1 II

400

250

I

01

w 0

5 02 m

g

3

03 04 0-5

14J 0

)o

35m

3ooo

2500

WVENUMBER (CM')

Fig. 2.

2mo

leoo

1600

1-

1200

loo0

800

WVENUMBER (CW)

Infrared spectrum of cimetidine hydrochloride.

io

P. M . G . BAVIN E T A L

132

C h t i d i n e €iydrochloride: wavelength (un-l) Assignment

3280

NH stretch NH stretch

3130

NH stretch

3418

2780-2600 2175

NH2+

1705-1595

X - N stretch

>N-C!%

stretch

of the i n t r a m o l e c u l a r hydrogen bonding i n cimetidine and several related H2-receptor antagonists has been H i s study reported by R. C. Mitchell.' shaws that c d t i d i n e can form intramolecular bonds in solution and that it can adopt a tenmembered ring conformation i n which the basic imidazole nitrogen atan is believed to be intrmlecular hydrogen bonded t o the NH group furthest f m the imidazole ring. 2.12 Ultraviolet Spectrum & I infrared study

The ultraviolet absorption spectrum of cimetidine i n 0.lN aqueous sulfuric acid is sham in Figurz 3. A sumnary of the ultraviolet absorbance characteristics obtained i n several solvents is shown in Table 1. Table 1

S-

x max

Solvent 0.1N- aqueous

of Cimetidine Ultraviolet Absorption Data

(m)

E

max-&260

log

E

218

20,530

4.31

95% ethanol

220

22,600

4.35

0.1N- aqueous HC1

216

19,360

4.29

H2m4

0.6-

0.5-

0.4 -

w 0.3-

18

0.2-

0.1-

0.0-

I 220

1

5

230

240

1

250

270

1 280

1

290

NANOMETERS

Fig. 3.

Ultraviolet absorption spectrum of cimetidine in 0.1 N aqueous sulfuric acid.

1

300

P. M. G. BAVIN E T A L

I34

The absorption maxirmnn is due primarily to the T T* transition of t h e cyanoimino ( ~ N - C Z N ) grow with a p a r t i a l contribution frcan the conjugated double bond system of the imidazole ring. The pH absorption dependency of c k t i d i n e frm pH 1.36 t o 10.35 has been described by Kajfez, e t al. -f

2.13

Nuclear Magnetic Resonance Spectra 2.131 Proton Magnetic Resonance of Spectnm Cimetidine

The proton magnetic resonance spectrum (Figure 4 ) of cimetidine was recorded for a deuterated dimethyl sulfoxide solution containing approximately 100 mglrril of the ccmpclund with tetramethylsilane as t h e internal standard. The spectrum i l l u s t r a t e d w a s obtained using a P e r k i n - E m R32 proton magnetic resonance (CW) spectraneter The resonances are:

.

h

I

H h Protons Chtsnical Shift

At ca

cc ‘d ce

Nf“9 ch

(HE) 2 -50-2.58

ZJUtlkr

Multiplicity

lnteqral

multiplet

2

2

2.6C

singlet

3

3

2.65

doublet

3

multiplet

2

3 2

singlet

2

2

multiplet

2

2

singlet

1

1

3.09-3.44 3.60 6.45-7.34* 7.43

11.7

singlet (broad) *quartet and t r i p l e t overlapped

‘i

of Protons

0

5 I

6

7

I

I

8

9

1

2

I

I

I PPM 9

I

I

I

I

1

1

I

7

I

0

6

5

4

3

2

1

3 I

4

1

10

w

Fig. 4. Proton magnetic resonance spectrum of cimetidine in deuterodimethylsulfoxide.

P. M. G . BAVIN E T A L .

I36

2.132

Proton Spectrum-cimetidine Hydrcchloride

3'4

The proton magnetic resonance spectrum of cimetidine hydrochloride was obtained by preparing a solution of approximately 100 mg/ml of the chemical in deuterated dimethyl sulfoxide containing tetramethylsilane as the internal standard. The chemical s h i f t values of the hydrochloride s a l t d i f f e r f r m those of the free base and are: &N-C-sN

H g

Proton Position

Chem. Shift

~

No. of Protons

Structure

(ppn)

Multiplicity

a b

ringG3

2.29

singlet

3

s a 2

2.60

2-

c

-ma33

2.70

t r i p l e t overlapped by mso m l t i p l e t at 2.5 p p & a doublet a t 2.7 PPn doublet overlapped bY -SCH2 t r i p l e t

d

-2CH2NH

3.32

multiplet

e

ring-CH2S

3.87

singlet

3.

. 2

2 2

$7

N-CH=N

8.99

singlet

1

9.0-10 .o broad singlet 1 -NH The chemical p f t s at positions d and fwere verified by E. S. Pepper using spin decoupling and spin-decouplhg

h

CIMETIDINE

137

with D20 exchange, respectively. These shifts were later cmfby mjfez, et a l . 2.133 Double Resonance Spin Decouplinq and Deuterium Exchange Studies of Cimetidine Spin Decoupled at: 7.12 p p Results: 2.69 ppn doublet collapses to a singlet. Shms that a coupling exists between NH-cH3.

7.1 ppn Results: 2.09-3 -44 mltiplet (2H) is reduced to a less ccanplex pattern. This is assigned to 4X2NH< (=NCN) protons. Deuterium (D20)exchange causes the chmical shifts at 7.18 and 9.0-10.0 to disappar. These resonances are assigned to three exchangeable hy&ogens attached to nitrogen on the imidazole ring and in the side-chain. 2.134 Carbon-13 NMR Spectrum The broad band decoupled carbon-13 NMR spectrum of cimetidine hydrochloride (Figure 3) was obtained by using a solution of approximately 100 mg/ml in deuterated dimethylsulfoxide. The deuterium signal of dimethylsulfoxide was used as the internal reference and the spctrum was obtained on a Varian Associates Model ET-80 fourier transform NMR spectrcmeter. The chemical s h i f t assignments are: Spin Decoupled at:

4

1

.N

H-N 91

H

2

E

P. M . G . BAVIN E T A L .

Postition of carbon

Chemical Shift, p p

1

9.68

2

26.01

28.17

2.14

4

30.05

5

40.81

6

118.18

7

124.91

8

130.00

9

133.34

10

159.96

Mass Spectrum 2.141

Electron-Ionization Impact specrtrum

3,4

The mass spectrum of cimetidine using a Hitachi Perkin-Elmer W - 7 E medium resolution mass spectrmeter and a direct insertion probe of the sample is shown in Figure 6. m e mass spectrum was recorded on magnetic tape and relative abundances were calculated with a PDP-8/Idigital ccrmputer coupled to the instrument. The results are presmted in tabulated fonn in Table 2. Table 2

Cimetidine Mass Spectral Fragment Ions By 5 Electron-Ionization Irnpact

m -

ion

252

M+

+ 158

157 L

(continued)

CIMETIDINE

Table 2

139

Continued

ion -

m -

128

127

126

125

116 115 NCN

111

cH&kCH3

95

NCN 82

II

c==fiHcH3 (continued)

P. M. G. BAVIN E T A L .

140

Table 2

Continued

m -

ion -

69

CH2=&-C-NCH3

30

CH2-h2

The fragmentation of cimetidine base appears t o follcm t h e three major pathways. Sham in Schmes 1, 2, and 3. The mlecular ion is observed a t m/e = 252. 2.142

Mass Spctrum-Field Desorption The f i e l d desorption mass spectrum of cimetidine base and a table of the fragmntation peaks are presented i n Figure 7 and tabulated in Table 3. The spectrum was obtained using a V a r i a n MAT M-5 D F mass spectrometer. The &tter w a s loaded by t h e dipping techniques fran an acetone solution. An e m i t t e r m e n t of 20 m i l l i a n p r e s was used t o obtain the spectrum. The spectrum shcms a m l e c u l a r ion,,'M at m / e = 252 and a MH+ a t m / e = 253. The peak a t m / e = 258 is due t o acetone used for instrument tuning purposes. Several low intensity f r a q e n t ions are found and are related t o the electron-ionization fragmentation mechanisn described above.

2.143

Mass Spectrum42hmical Ionization (CI) The chemical ionization mass spectrum of cimetidine and the major fragmentat i o n ions are presented in Figure 8 and Table 4. The spectrum was obtained using a Finnigan H e 1 3200 quadruple mass spectrmter f i t t e d with a chemical ionization source. The sample, applied t o the probe f r m an acetone solution, was introduced v i a t h e d i r e c t inlet system. Methane w a s used as the reactant gas.

The spectrum shcrws an (M+1)+ peak f o r t h e m l e c u l a r ion and characteristic

(200)ppm

'H-

3

I

Fig. 5.

Carbon-13 NMR spectrum of cimetidine hydrochloride.

1

I

125

7 kwlralion Wlage. 7OeV Source Temperalure. 200°C Direct Sample Inlet Temperalure. 300°C Inslrwnenl: Hilachi-Perkln Elmer RMU 6E

I

C

c)

130

170

210

250

m/e Fig. 6.

Electron-ionization mass spectrum of cimetidine.

Fig. 7.

Field desorption mass spectrum of cimetidine.

r

I

5.W

2:

I:

1

1

117.6

28.4 I

Fig. 8.

Mass spectrum cimetidine- chemical ionization- methane reactant gas.

I45

ClMETlDINE

Table 3

Field &sorption Mass Spectrm of C h e t i d i n e . Peak Intensity D a t a . 04116/811SPEC#

185lKMIFD 20 MA

BASE 11306 PEAK 1

2 3 4

5 6 7 8 9 10 11

12 13

14 15 16 17 18 19 20 21 22 23 24 25

ItBASE 0.30'10

MASS 43.8

12.48% 4.49 '10 0.26% 0.09% 3.02% 0.44 '10 0.14% 2.98% 0.09% 0.12O/O 0.62% 0.64%

58.0

0.38% 0.92% 1.78% 1.27% 0.37% 0.35% 0.13% 1.71% 0.78%

29

1.4O '10 0.84% 1.63/o' 6.O9% 69.95 /'o 100.00% 16.45%

30 31

0.51O/o

26 27 28

SUM 27545

5.98%

59.0 77.9 81.O 95.1 96.1 97.0 105.9 106.3 117.0 125.9 127.0 141.9 157.0 158.0 158.9 188.0 188.1 197.0 21 1.1 226.9 228.0 249.0 250.0 251.O 252.1 253.1 254.0 255.0 256.1


146

peaks a t (M+29)+ and (M+41)+ when using methane as the reactant gas a t 1 1-1 t o r r pressure. The major fragwntation ions are given in Table 4. An INCOS ccknputer system coupled w i t h the mass spectrometer w a s used t o obtain, normalize, and plot the spectral data. Table 4

Chemical Ionization Mass Spectral Fragrtlent Ions of Cimetidine (CHs-Reaction Gas)

m

ion

293.2 (m + 41) 281.2

(m + 29)

253.1

159.1

117.0 [Em2a2-v*Nm3 +H

I+

95.1

68.9

CH2=h-C-NCH3

2.144

Negative Ion Mass Spectrum

The negative ion mass spectrum of cimetidine was obtained using a Finnigan We1 3200 quadrupole mass SpeCtKHneter quipped with an INCOS data system. The mass spectrum is presented in Figure 9 and the major fragnents are tabulated in Table 5. A caparison of the mlecular ion peak intensity obtained in the negative ion mde w i t h that obtained in the chemical ionization mode shows the former t o be 11.4 times mre intense. As the fragwntation i n the negative ion mode is less, identification of snaller s i z e samples of c k t i d i n e can be made.

2:

I:

9

53.9

I

I50

'! E

Fig. 9.

-.s ; . & l

-:no

Negative ion-mass spectrum of cimetidine.

Illdl28

mle 126

Scheme 1.

mle 127

Fragmentation of cimetidine.

[C.H.N-J

1: mle 158

We 68

/I

NCN

t II

HSCHaCH = NHCNHCH, m/e 157

CHa

*+ YN I NHCNHCH,

s "'

NCN

rn'e

II t

C = NHCH,

mle 82

Scheme 2.


+

r

CH~SCHaCHaNHCNHCHa d

I

m/e 120

' mle 158

-

+

Scheme 3.

I'

= C = NCH, -HSCHaCHaN mla 116


*

CIMETIDINE

151

Table 5

Negative Ion Mass S p c t r a l Fracpent Ions of Cimetidine

m

ion

251.0

[M-HI-

N-CN

II

156.9

-

M3wm

93.9

Nk9N

2.15

Photoelectron Spectrum of C i m e t i d i n e The photoelectron spectrum of cimetidine has ken reported2. The folbwing bands w e r e found and their assignrents have been described: Assignmen t

Band

Ionization potential of both imidazole TT electrons and the lone electron p a i r of imidazole

8.41

N (3) atcsn.

9.03

I o n i z a t i o n potential of unshared s u l f u r e l e c t r o n pair.

9.4

IT^ electrons of imidazole r i n g and TT electron pairs of three nitrogen atcans in guanidinium

unit. n electrons -CW

11.57, 12.08 13.03, 13.36, 14.20, 14.95, 15.88

1

N o t assigned

The photoelectron spectrum w a s obtained4 with a Vacuum Generator W 63 instrumnt a t l a v r e s o l u t i o n (35 eV) using He e x c i t a t i o n a t 140' and xenon internal c a l i b r a t i o n . 2.16

X-Ray D i f f r a c t i o n S i n g l e c r y s t a l x-ray diffraction s t u d i e s of

P. M . G . BAVlN E T A L .

I52

cimetidine w e r e carried out by Hadicke, " C i m e t i d i n e was found Frickel, and F'ranke.6 to form mnoclinic crystals (space group P21/C). The molecule is internally hydrcqen bnded by the N.. .N-N b n d between the imidazole and guanidine residue t o form a tene r -red ring system."2 The X-ray m diffraction spectnrm of cimetidine is sham in Figure 10. The spectnrm was obtained using a General E l e c t r i c XRD-5 @er diffractmeter using CuKci irradiation. Table 6 lists the d-spacings (interplanar distances), the diffraction angle, 28, and the relative peak intensities. Crystals w e r e found t o be prisnatic and monoclinic. Prodic-Kojic , et a17 have reported the cell constants t o be a = 6.82 ( l ) ,b = 18.813 (3), and c = 10.374 (2) A , ' B = 106.42 (1)'.

2.2

Physical P r o p e r t i e s of the Solid

2.21

Identity T e s t s

2.211

Thin Layer Chrmtography/Color T e s t s This test may be used with the chemical, tablet, or liquid dosage form containing cimetidine o r cimetidine hydrochloride. A methanol extract of the sample is prepared and chram-aphed on a Silica G e l GF c h r m t o p l a t e with ethyl acetate, mthanol, m n i m hydroxide (100, 1 0 , 1 0 , v/v) m b i l e phase. The Rf comparison t o a reference similarily chrcanatqraphed, the r e s p n s e to diazotized p-nitroaniline (imidazole nucleus detection), U.V. (254 nm), and I p vapors serves to identify cimetidine.

2.212

Infrared Identification The infrared spectrum of a mineral o i l dispersion of the sample, previously dried a t 105OC for four hours, corresponds t o the reference sample. Refer t o Section 2.11.

Fig. 10.

X-ray powder diffraction pattern of cimetidine.

P. M . G . BAVIN ET AL

IS4

Table 6

X-Ray - - Diffraction Pattern o f Cimetidine

20

d,

i*

Relative Intensity ~~

7 -20 9.40 10.10 13.00 14.75 16.58 16.80 17.80 18.46 19.10 19.72 20.58 21.26 23.58 26.06 26 -90 27.30 28.10 28.50 29.10 29.96 30.50 31.62 32.60 33.92 34.60 35 .OO 36.06 37.64 38.80 40.50 41.40 42.64 43.40 44.80 46.40 47 -30

* d

12.27 9.40 8.75 6.80 6 .OO 5.34 5.27 4.98 4.80 4.64 4.50 4.31 4.18 3.77 3.42 3.37 3.26 3.17 3.13 3.07 2.98. 2.93 2.83 2.74 2.64 2.59 2.56 2.49 2.39 2.32 2.22 2.18 2.12 2.08 2.02 1.96 1 .92 = interplanar distance =

5.7 7.4 2.8 14.5 31.9 38.3 58.2 24.1 32.3 4.6 39.0 4.3 4.3 100 .o 24.1 6.4 14.2 5.7. 19.1 4.3 0.7 7.1 1.4 7.8 14.9 9.2 2.8 2.1 1.4 2.1 2.1 2.1 5.3 4.3 2.1 2.1 1.4 nX

ClMETlDlNE

IS5

2.213

Ultraviolet Absorption Identification The ultraviolet absorption spectrum of a solution of cimetidine in 0.1N sulfuric acid o r 0.1N HC1 exhib% a maximum between 217-319 and a minimum between 210 m and 212 m, (Figure 4 ) . Refer t o Section 2.12.

2.214

Color Identification Test I 'Ib 0.1 m l of solution of the sample prepared by dissolving 1 mg of cimetidine i n 1 m l of ethanol add 5 ml of a solution of 1 g citric acid in acetic anhydride to make 50 ml, Heat the mixture (freshly prepared) on a waterbath f o r ten to fifteen minutes. A red violet color develops.

.

2.215

Color Identification T e s t I1 'Ib 0.1 g of sample add 5 ml of 1 N

hyckochloric acid, and heat gentiy. Add 5 m l of sodium hydroxide solution (3 g in 1 0 ml H20) t o the solution and heat additionally. The reaction mixture evolves an amnonia odor. When a piece of mistened red l i t m u s paper is exposed t o the evolved gas, it tums blue.

2.22

Them1 Properties 2.221

Melting Range Cimetidine m e l t s between 140 t o 143.5OC procedure for class 1 using the USP substances.

2.222

Differential Scanning Calorimetry (DSC) The DSC thesmogram for cimetidine is shown h Figure 11. A single endothem i s found a t 142OC. As it does not recrystallize f r m its m e l t a f t e r prolonged coolincj a t room temperature, seeding, scratching, or annealing, it is considered an mrphous glass. The hydrochloride s a l t of cimetidine shows a single endothem a t 193O C (Figure 12)

.

156

P. M. G.BAVIN E T A L .

Fig. 11. Differential scanning calorimetric thermogram of cimetidine.

167

177

187

"C

197

207

217

207

Fig. 12. D i - f f e r e n t i a l scanning calorimetric thermogram o f cimetidine hydrochloride.

P. M . G. BAVIN E T A L .

158

2.23

Densitv/Specific Surface Area' Density measurments were carried out by the use of an air canparison pycnoweter and t h e specific surface area was determined on milled and unmilled sample using a Stroklein areameter. The data are sumnarized in Table 7. Table 7 Property Units Unmilled Milled Mean Particle Microns 19.0 7.8 Size 12.1 Specific m2/g 0.666 1.304 Surface Area Absolute q/cc 1.2815 Density Tappea Density

2.24

g/cc

0.40

0.51

Themcgravimetric Analysis (TGA)

The themqravimetric curves for cimetidine

and cimetidine hydrochloride are sham in Figure 13 and 14, respectively. The curves shaw that the compounds are stable up to a tenperatwe of approximately 175-185OC and then decompse. The therrmgrams were obtained at a heating rate of lO"C/minute. 2.3 Solubility of Cktidine 2.31 Solubility in Various Solvents The solubility of cimetidineg * was determined in a variety of solvents using essentially the mthod of Schefter and Higuchi. Solvent Solubility (mg/ml) 37.0"C 37.5OC -2OoC - - _ 24OC _ _ _25OC Acetonitrile 2.7 Chlorofo m 1 .o Cyclohexane 250 - HC1

161

ClMETlDlNE

A. Prdic-Kojec , e t a17 reported a high dependence of equilibrium solubilities of cinetidine which ranged f r m 0.75% a t pH 8.92 t o 2.72% a t pH = 2.26 when determined a t Similar data was obtained by 35.0 f 0.1OC. Mitchell.

2.311

Aqueous Solubility of C i m e t i d i n e

HydrocNoride I n a study of apparent equilibrium solubility cimetidine hydrochloride is approximately 1.0 g/ml a t 25OC. The equilibrium solubility i s approximately 160 mg/ml. 2.4

Physical Properties of Cimetidine Solutions1 Aqueous solutions of cimetidine and cimetidine hydrochloride are usually colorless t o a very pale s t r a w color. A saturated solution of the free base has a pH of approximately 9.0 and a 15%solution of the hydrochloride salt, a pH of approximately 3.7. 2.41

Dissociation Constants The apparent pKa of cimetidine is 7.11 t 0.04 at 25OC masured potentimetrically in 0.1Naqueous KCll * In water, the pKa of the imidazole ring nitrogen (-NH-) is 6-80?' At pH 7.4, 20.7% of cimetidine is present as cations. The nitrogen a t m on the side chain t o which the cyan0 (-EN) group is attached is essentially neutral and has a pKa = -0.4?3 It is essentially non-ionized i n a broad pH range ( ~ 2 - 1 2 ) .

.

2.42

Partition Coefficient The "Apparent Partition Coefficient" for cimtidine in an cctanol/pH 7.4 phosphate buffer system a t 25OC and in an octanol/water system a t 37OC w e r e found t o be 2.0 and 2.5 respectively.

3.

synthesis 3.1

Synthesis of C h t i d i n e Synthesis of cimetidine has been described by Durant, G. J., e t a1.13r14

I62

P. M G . BAVIN E T A L .

ClMETlDlNE

163

Route 2

CIMETIDINE


164

3.2

sy"thesis of C i m e t i d i n e Hydrochloride N"-cyano-N-Inethyl-N ' - [2- [ (5-~thyl-l.~-imidaZ01-4-yl) mthyll thiol ethyl] guanidine, hydrochloride To an ethanolic suspension of cimetidine,

concentrated hydrocholoric acid and ethyl acetate are added, the product is collected, washed w i t h ethyl acetate, and dried. The schematic is illustrated k l w :

II

CHzSCH,CHzNH-C-NHCH3

4.

-HC1

Stability

4.1

Cimetidine 4.11

D r y State

C i n e t i d i n e in the dry s t a t e , stored i n a closed container a t temperature shwed no d e c m p s i t i o n a f t e r five years when examined by high pressure liquid c h r m t o graphy, thin layer chrmtography, infrared spectrophotmetry, and mass spectranetry. It is stable for a t least 48 hours a t 100OC.

4.12

Acid Hydrolysis Acid hydrolysis of cimetidine13 follows the folloWing scheme:

A

I65

ClMETlDLNE

C The formation of the amide is pH and t-ature depmdmt. Solutions of cimetidine in hydrochloric acid a t p H %5.4 showed no d e c a p s i t i o n a t 50' over a period of t h i r t y days. Hydrolysis t o the guanylurea (B) occurred when the caarq?ound w a s heated a t 45' for 36 hours w i t h an excess of 1N hydrochloric acid a t p H 4.The guanidine was formed by heating cimetidine for tm hours a t l0O'C with concentrated hydrochloric acid. 4.13

Effect of Ultraviolet Light N o damnpsition of cimetidine was noted 1s a f t e r several mnths exposure t o U.V. light.

4.14

Effect of Hydrogen Peroxide and Oxyqen

When cimetidine i s treated w i t h 3% H202 a t room t m p r a t u r e or exposed t o oxygen a t 50'C for short time periods, formation of sulfoxide is observed. Refer t o the following:

P. M . G . BAVlN E T A L .

I66

4.2

C i m e t i d i n e Hydrochloride The s t a b i l i t y of cimetidine hydrcchloride w a s

reported by Rosenberg, e t al. 4.21

Aq~musSolutions

pqueous solutions (vials for injection) shmed a shelf l i f e of a t least two years when examined by high pressure liquid and e x e e d by high pressure liquid and thin layer chrmtography . 4.22

Infusion Fluids Solutions i n comnon infusion fluids a t concentrations of 120 o r 500 mg/lOO m l w e r e found t o be stable for a t l e a s t one week a t rocan temperature when examined by high pressure liquid and thin layer chranatography?6

5.

Analytical Chemistry 5.1

Identity T e s t s 5.11

Identification T e s t s for C h e t i d i n e 2 m l of Dissolve about 20 mg of sample distilled w a t e r with gentle warming. Add 1 m l of 5% aqueous sodium n i t r i t e and allm t o stand for approximately three minutes. Add 1 m l of sulfanilic acid test solution (250 mg of sulfanilic acid dissolved in 25 ml d l 0 8 v/v aqueous HC1) and allow t o stand f o r a b u t five minutes. The formation of a yellaw-orange t o reddish brown color indicates the presence of cimetidine.17

A.

Place about 20 mg of sample in a s m a l l test tube. Add one drop of concentrated hydrochloric acid, 5 m l of glacial acetic acid, stir t o dissolve, and thm add 1 ml of mrcuric acetate test solution (USP). formation of a white precipitate indicates the presence of cimetidine.

B.

C.

A solution of the sample of the hydrochloride s a l t responds positively t o the test for chloride.

5.12

Identity T e s t s by I R , U V , TLC and HPLC Cimetidine and cimetidine hydrochloride have characteristic spectra (Figures 1 and 2 ) .

A.

167

CIMETIDINE

The ultraviolet absorption spectrum of a solution of cimetidine or cimetidhe hydrochloride in 0.1g H2%4 exhibits a maximum between 217 and 219 nm and a minimum a t 210212 nm (Figure 3 ) .

B.

C.

Characteristic TLC and HPLC retention times are described for cimetidine in the text and may be used a s confirmatory tests for c h e t i d i n e (Section 5.4, 5.5).

D.

Single crystals or trace amounts of cimetidine respond t o color tests and w i l l give a positive mass spectral peak for the mlecular ion. These have been described above.

5.2

Non Aqyeous Titration 5.21

Chemical Dissolve about 240 mg of sample, accurately weighed, in 75 ml of glacial acetic acid. T i t r a t e with standardized 0.1 N perchloric acid in acetic acid t o a potenFianetric endpint using glass-calm1 electrodes. Each ml of 0.1N perchloric acid is equivalent t o 25.234 mgof cimetidhe.

5.22

Tablet Determine the average tablet weight of twenty tablets. Powder and weigh accurately a sample equivalent t o about one gram of cimetidine into a 200 ml volumetric flask. Add about 150 m l of methanol, and shake mechanically for t h i r t y minutes. Dilute a v o l m with methanol, mix and f i l t e r through a Whatxan #1 f i l t e r paper, discarding the f i r s t 15 m l of the f i l t r a t e . To 50 ml add 25 ml of glacial acetic acid and t i t r a t e with 0.1N acetous perchloric acid t o a potenticmetric endpoint using glasscalmel electrodes. Calculation: mg Cimetidine tablet = ml x N

x 252.34 x 200 x Average Tablet Weiqht Sample Weight (mg) x 50

P. M . G . BAVIN ET AL.

168

5.3

spectrophanetric Procedure 5.31 Chemical Transfer about 65 mg of sample, accurately weighed, t o a 200 m l v o l m t r i c flask. Dissolve in and d i l u t e t o volume w i t h 0.1Ns u l f u r i c acid. Transfer 5.0 ml of t h i s solution t o a 200 ml volurnetric flask, and d i l u t e t o volume w i t h the same solvent. Record the ultraviolet absorption spectrum of t h i s solution i n 1 cm cells on a suitable spectrophotawter fran 320 t o 210 nm using 0.1N sulfuric acid as a blank. Measure the abs&ance difference ( PA) between the absorbance a t 218 nm (maximum) and that a t 260 nm. Calculation: % Cimetidine =

A??, x 0.01212* x Dilution Factor x 100 Weight of Sample (mg) *0.01212 = mg cirrretidine/ml/unit AA This absorptivity factor was experimentally determined using accurately weighed samples of cimetidine reference standard carried through the sarne procedure. 5.32

Tablet Weigh and finely p d e r twenty tablets. Weigh accurately a portion of the p d e r , equivalent to a b u t 100 mg of cimetidine and transfer t o a 200 m l voluretric flask. Add 75 ml of 0.1N sulfuric acid, and shake mechanically for-thbrty minutes. Dilute t o volm w i t h the same solvent, mix, and f i l t e r through Whatman #1 f i l t e r paper, discardjng the f i r s t 15 m l of f i l t r a t e . P i p e t 5.0 m l of f i l t r a t e into a 200 volurnetric flask, add 0.1N sulfuric acid t o v o l w , and mix. G o r d the ultraviolet absorption spectrum of t h i s solution frm 300 t o 215 nm in a 1 cm cell

v e r s u s 0.1N s u l f u r i c a c i d on a s u i t a b l e spectrophotometer. S u b t r a c t the a b s o r b a n c e a t 260 nm from the a b s o r b a n c e o f the maximum a t a b o u t 218 nrn *

CIMETIDINE

I69

Calculation: mg c h t i d i n e / t a b l e t = AA

x 0.01212" x Average Tablet Weight (mg) x Dilution Factor Sample Weight (mg)

5.4

Thin Layer Chrmtqraphy Adsorbent:

Silica G e l GF, 250 microns

Eluting Solvent:

ethyl acetate, methanol, concentrated m n i u m hydroxide ( l O : l : l / v/v)

Ethyl acetate must be freshly distilled. Equilibration:

15 minutes in a closed paper lined tank

Concentration:

50 mg/ml mthanol

Detection: spotting:

iodine, W (254 m) 100 ?Jg

The solvent i s allowed t o rise t o the 15 cm line, the plate i s remved frcnn the chrmtographic chamber and dried in a wrent of a i r u n t i l no solvent odor is detected. The plate is examined under W light a t 254 nm. To obtain maximum sensitivity the plate is placed i n a chamber containing iodine crystals for about t h i r t y t o forty minutes o r u n t i l maximum contrast of the spots i s obtained. The R f of c k t i d i n e is approximately 0.35. Several additional thin layer chrmtography systems have been reported by Kajfez, e t a l . These systems separate the sulfoxide of cimetidine f r m the free base. A l l work was carried out using Merck-Kieselgel 60F254 plates. Visualization of the spots W 254 and iodine absorption.

0

d: 0

0

'9 0

..

N

h

W Y

! A..

B

u

n

171

CIMETIDINE

5.5

20 High PressiJre Liquid Chrmatoqraphic Procedure Adsorption and reverse phase systems have been used t o evaluate cimetidine. The procedures are rapid, have high s p e c i f i c i t y and are amenable t o the analysis of cimetidine and c h t i d i n e formulations. Table 9 contains the parameters of several c i t e d investigations.

5.51 Analysis of Cimetidine Mobile Solvent: Mix 975 m l of a c e t o n i t r i l e , 20 ml of d i s t i l l e d water, and 5 m l of concentrated m n i u m hydroxide.

Assay Preparation: Accurately weigh about 50 q of sample i n t o a 50 ml volumetric flask. Dissolve i n and d i l u t e t o volume with mobile solvent. Standard Preparation: Accurately weigh about 50 nq of cimetidine reference standard i n t o a 50 ml volumetric flask. Add 25 m l of mobile solvent, shake f o r one-half hour, d i l u t e to volume with mobile solvent, and mix.

Procedure : I n j e c t 20 p 1 of Assay and Standard Preparations i n t o a l i q u i d chromatograph adjusted t o t h e f o l l w i n g operating conditions: Instrment: C h r m t r o n i x 3100

Column: Zorbax-Sil ( W o n t ) Column D i a m e t e r : 2.1 mn i . d . and 0.25 inch 0.d.

Column Length: 25 cm Column Temperature: A m b i e n t

Column Pressure: 500 psig Attenuation: 16 F l w Rate: 0.2 ml/minute

Detector: W (254 nm) NOTE: Under the above operating conditions,

cimetidine has a retention time of about

P. M . G. BAVIN E T A L .

172

1 0 minutes.

Calculation :

%Cimetidine= Where:

C x

AU x dilution factor x 100 As x Sample Weight (q)

A u = area under the peak relating t o

the Assay Preparation

A s = area under the peak relating t o the Standard Preparation C = concentration of c h t i d i n e

reference standard in q/ml 5.52

Analysis of Tablets: Wbile Solvent:

Mix 975 ml of acetonitrile, 20 m l of d i s t i l l e d water, and 5 m l of concentrated

ammonium hydroxiae. Assay Preparation : Weigh and finely M e r twenty tablets. Weigh accurately a portion of powder (equivalent t o 80 mg of c k t i d i n e ) into a 50 ml volumetric flask. Add 25 m l of m b i l e solvent, shake for one-half hour, d i l u t e t o volume with m b i l e solvent, and m i x . Standard Preparation: Transfer about 80 mg of c k t i d i n e reference standard accurately weighed, into a 50 m l v o l m t r i c flask. Add 25 ml of m b i l e solvent, shake for one-half hour, and dilute to volume with m b i l e solvent, and m i x .

Procedure: Inject 20 1.11 of Assay and Standard Preparations into a l i q i d c h r m t q r a p h adjusted t o the following operating conditions: Instrument: Column:

Qrrcanatranix Liquid Chranatcqaph o r equivalent

Zorbax-Sil (DuPont), o r equivalent

Column Diameter:

2.1 mn i.d. and 0.25 inch

0.d. ColumnLength:

2 5 m

Column Temper ature:

Ambient

CIMETIDINE

I73

Colurrm Pressure:

1200 p s i

x 16 0.54 ml/minute

Attentuation:

Flow Fate: Detector:

W (254 nm)

- Under the operating conditions, NOTE: cimetidine has a retention t i m e of about 10 minutes. Calculation:

q cimetidine/tablet = C

x Au x dilution factor x Av Tablet W t (q) As x S q l e Weight (mg)

Where:

Au = area under the peak relating t o Assay Preparation As = a-rea under the peak relating t o

the Standard Preparation C = concentration of cimetidine

reference standard in mg/ml 5.53

Analysis of Capsules Pbbile Phase (0.05M borax) :

Dissolve 39.1 g of Na2B,+07-10 H 2 0 in d i s t i l l e d water and d i l u t e t o 2000 ml. the pH t o 7.5 w i t h formic acid.

Adjust

Internal Standard Solution:

Dissolve about 120 mg of benzoic acid (ACS grade) in 100 m l of mobile phase. Assay Preparation: Transfer as ccanpletely as possible the contents of not less than twenty capsules t o a tared container, determine the average content weight per capsule, and mix the cmbined contents thoroughly. Transfer an accurately weigh& portion of the pwder, equivalent t o about 50 mg of c h t i d i n e , t o a 25 ml volumetric flask. Add about 8 m l of methanol, and shake mechanically for 10 minutes. Add 15.0 ml of Internal Standard Solution, d i l u t e t o volurne w i t h methanol and mix.

HPIC Parameters For C i m e t i d i n e

Table 9

Mobile Phase

COlUmn

Lichrosorb Si 60

CH 3CN ,H 2 0 , CH 30H,

F l w Rate W/min. 1

W Detector

'LRT (min.)

Reference

Inm)

0.8

228

10.0

40

conc. NH40H (1000, 20,60,5)

Lichrosorb Si 60

0.1% ~ M L + O H / C H ~ ~

0.8

228

40.0

40

Zorbax S i l

m3at

3.0

228

3.4

38

0.5

228

10.0

37

a30H,H20,

coric. NH40H (1000, 500,20,2)

Zorbax S i l

CH3CN,H20,conc. NH4OH (975,20,5)

p Bondapak C18

CH3C", lorrfil potassium phospnate buffer (pH 3.0) (90,910)

2.0

220

4.0

39

p Bondapak C18

0.3% 2 m 4 CH3OH (450,550)

1.0

254

16.5

4 1

1.0

228

3.5

40

0.75

254

2.8

42

2.5

228

-

43

Partisil

sex

P a r t i s i l SAX

0.9%

,

( M 4 ) 2 m4,

a30H (200,800) 0.M Borax Buffer (pH-8 .5 )

P a r t i s i l 1O-ODS

CH3CN,H2O, NH40H (1000,50,1)

CIMETIDINE

175

Standard Preparation: Transfer %50 mg of cimetidine reference standard, accurately weighed, into a 25 m l volumetric flask. Dissolve i n 8 ml of methanol, add 15.0 ml of Internal Standard Solution, dilute t o voluw with methanol, and mix. Procedure:

Using a loop injector, inject 20 $ of Sample and Standard Preparation.

Instrumental Conditions: The follming instrumental conditions are typically employed when a Chranatronix Liquid Chrmtograph is employed: Instrument:

Chrmatronix,

Column Packing:

Column D i m t e r :

p/lodel

3100

Strong Anion Exchange Resin 2.1 m (i.d.) 1 mter

ColmLength:

Column T a p e rature: Ambient Colm Pressure:

%500 p s i

d.40 ml/minute Attentuation: x 64 Flaw Rate: Qlart

Sped:

Detector:

4 minutes/cm

W (254 nm)

Using the ahwe conditions, the peaks are recorded in the order: cimetidine (approximately 4 minutes) and benzoic acid (approximately 8 minutes). Calculation : Calculate the ration 'R' for each chranatcqam where : h t i d i n e Peak Height R=C Benzoic Acid Peak Height

q cimetidine/capsule =

% x Wt

of Std (9) x 1000 x Av N e t Contents (9) % x W t of Sample (9)

P. M. G . BAVlN ET AL.

176

where:

5 = Ratio of % = Ratio

5.6

Assay Preparation

of Standard Preparation

Determination of C i m e t i d i n e Sulfoxide Content White21 used the previously cited procedure (Section 5.5) to separate and estimate the m u n t of the sulfoxide of cimetidine in the new drug substance. With sample concentration of 1 m g / d and an injection of 20 1.11,as l i t t l e as 1 ppn of the 'sulfoxide' could be detected and estimated a t the F t T of about 13 minutes. 0

N-CTJ + 11 CH2SCH2CH2NHCNHCH3

m*N

'Sulfoxide ' white4 also detennined the presence of the 'sulfoxide' using a P a r t i s i l SCX column (25 cm x 4.6 m i.d.); Pbbile Phase: 80% CH30H and 20% H z 0 containing 1.8 g ( N H 4 ) 2 = 4 / 1 , a flaw r a t e of about 1.5 rnl/minute; Detection: W a t 228 m a d an injection of 25 plof a 25 mg/ml solutian of sample. 6.

Metabolism and Phamxokinetics

The m e t a b o l i s m and pharmacokinetics of cinetidine has been studied and reviewed by several investigators. The CcBnpOund is absorbed rapidly whether given orally or i.v.. About 15-20% of cimetidine appears t o be plasna protein bound and this is r e p r t e d t o be of no pharmarological significance. In r a t s and dcgs, c k t i d i n e is readily absorbed and has a plasna halfl i f e of about one-half t o t m hours. In human studies, cinetidine was Shawn to have a half-life of 123 f 1 2 minutes in the blood.23 Cimetidine has been found f r m radioactive trace studies w i t h 2-1 4 C - c i m e t i d i n e in healthy humans as w e l l as dogs and r a t s t o be mainly excreted in the urine. It is widely distributed throughout a l l the tissues and is rapidly eliminated w i t h the exceptim of the liver, kidney and adrenal cortex. TLC, HPLC , and radioautographic studies indicated 56-85% of cimetidine (I) was unchanged, up to 30% was excreted as the sulfoxide (11), 5-8% as the &droxymz?thyl (111) ccmpund, approximately 2% as the

CIMETIDINE

171

guSnylurea (IV) and 7417% a s unidentified material.

'-"

The r e p r t e d metabolism products are i l l u s t r a t e d in Figure 15.

6.1

Determination of Cimetidine and its Metabolites i n Biological Fluids C h t i d i n e may be assayed i n blood, urine, plasna, 30,31,32,33,38

serum, b i l e , pancreatic fluid, gastric fluid, plueral fluid, ceribrospinal fluid, a s c i t i c fluid and body tissues by HPLC procedures? I The major identified metabolite, cirnetidine sulfoxide' which is eliminated f r m the M y mainly by renal excretion has been determined in blood and urine by Lee and O s b r n e using a high pressure liquid chrcmtcqraphic procedure. Their procedure allows the operator to cleanly separate creatinine, which i s frequently present in c l i n i c a l samples of patients suffering f r m renal failure and t o determine both the c h t i d i n e a s w e l l as the cimetidine sulfoxide. The separation of cimetidine and its metabolites i s usually carried out by extraction of the biological medium with 1-octanol fran an aqueous alkaline pH %9 solution followed by mixing, addition of an internal standard and centrifugation. The extraction with octanol i s repeated and the combined extracts are re-extracted with dilute hydrochloric acid. The aqueous acid solution is then separated, ethanol is added and mixed. This is then followed by saturating the mixture with a large munt of potassium o r sodium c a r b n a t e to " s a l t out" the ethanol layer which contains the cimetidine and its metabolite, the sulfoxide. Several different internal standards have been used: Metiamide, l) 1-nrtthyl-3- [ 2- [ [ ( 5 - ~ t h y l - ~ d a ~ o l e - 4 - y-rraethyl] thio] ethyl] -2-thiourea, (~-cyan~-~'-~~thylN"- (3- (4-imidazolyl) -yopyl) guanidine and Bhydroxy-theophylline. After extraction the samples are either evaporated t o dryness and reconstituted w i t h a hm m u n t of ethanol, injected directly o r dissolved in the m b i l e phase for the HPLC analysis. 29,31,32,34 and The c o l m s used for HPIC w e r a reverse phase C18 B E?~ndapa@'? The separation of cimetidine sulfoxidell and the guanidine derivat i v e of cimetidine , N-methyl-N ' - [ 2- ( (4-methyl-5imidazolyl)methyl] thio) ethyl]guanidine , (VI) plus the polar decmposition and metabolic products of

CIMETIDINE

I79

cimetidine may be carried out using a P a r t i s i l SC@ (whatman Inc. ) strong ion exchanqe column using the fcdlowing set of conditions :

'

Column:

P a r t i s i l 5cx @ 1 0 p (25 cm x 4.6 rm i.d.) Whabnan Inc.

While Phase:

%l ml/minute

Flow Rate: Detector:

weigh 1.8 grams of ammnium sulfate into a one liter volumetric flask. Add 200 m l of d i s t i l l e d water t o dissolve, then make up t o volume w i t h methanol.

W (228 nm)

Sensitivity:

Variable

Sample Diluent:

Mobile Phase

Retention Times:

Minutes

man01 (carboxamide) Derivative (IV) SK&F 92422

-10

Guanidine Derivative (VI) SK&F92408

$15

'Guanidine' Derivative of C h t i d i n e

VI

In the recent rwthcd of Z i M a k , e t a133 , a liquid chrmatoqraphic procedure has been developed which separates and permits quantitative analysis of cimetidine ( I ) ,c k t i d i n e sulfoxide (11), its h y d r o p t h y l 013 and guano1 urea derivatives (IV) Metimide, SK&F 92058, i s used as the internal standard. Their procedure involves the precipitation of protein with acetonitrile, addition of anhydrous K2J3E04, extraction of the separated aqueous phase w i t h mthylene chloride and KH2Po4 t o saturate and salt out the solution. The methylene chloride is evaporated t o dryness, the sample is reconstituted with mobile phase (CE3CN:CH30H:H20:NH,+OH,

.

P. M. G . BAVIN ET AL..

Detection is by W a t 228 nm and a Zorbax S i l Column, 4.6 mn x 25 cm (DuPont Instrum t s , Wilmington, DE) equipped w i t h a Whatman HC Pellosil p r e c o l m (maInc., Clifton, N J ) is used. An apparent pH of 10.5 has been used, and according t o the authors column performance was satisfactory for more than a five mnths period in spite of the high alkalinity. 1000:50:50:2).

Identification of d e a m p s i t i o n products and

mtablic products is mst conveniently done by their isolation using thin layer chramtoqraphy and identification by use of f i e l d desorptim mss spectrcanetry.

The authors muld like to achowledge and thank minkers of SnithKline Corporation’s Analytical & Physical C h b s t r y staff and other scientific staff a t Philadelphia, Guayama , Welwyn, Tonbridge,

Ireland, and France for t h e i r assistance, advice, recarmendations and their personal efforts. The authors would further like t o thank SmithKline Corporation for s u p p r t h g them t o carry out t h i s mrk

.

181

CIMETIDINE

7.

References

1

Mitchell, R. C., J.C.S.,

2

Kajfez, F., e t a l , Acta. Pharm. Yugoslav.,

3

Warren, R. J . , smith Kline & French Labs., Phila., PA.

4

Pepper, E. S., Smith K l h e & French Labs., Ltd., Welwyn Garden C i t y , England.

5

Darkin, D., Smith K l h e & French Labs., Ltd., Welwyn Garden City, England.

6

111, (9), 3222 (1978). Hadicki, E., e t a l , Chem. Ber. -

7

Prodic-Kojiz, B., (1979).

8

Leonard, G., smith Kline & French Labs., Ltd., Welwyn Garden C i t y , England.

9

Mitchell, R. C., Smith K l h e & French Lab., Ltd., Welwyn Garden City, England

Perkin 11, 915 (1980).

27,

199 (1977).

et a l , Gaz. Chbn. I t a l . , 109, 535

10

Baldinus, J., Smith Kline & French Labs., Phila., PA.

11

Schefter, E. and Higuchi, T., J. Pharm. S c i . , 52, 781 (1963)

.

12

Graham, M. J., Smith K l h e

&

French Ltd., Unpublished

results. 13

Durant, G. J., e t a l , J. W. Chem.,

14

Smith K l h e c.a.

&

20,

901 (1977).

French Labs., German Patent 2,344,799

80,1461681.

15

Z a r a t b , J. E., Smith K l i n e & French Labs., Phila., PA, unpublished data.

16

Rosenberg, H. A., (1980).

17

Eqosh, P. and smith, M., Smith K l k e Phila., PA, unpublished data.

18

United States Pharmacopeia, XX, p. 727.

e t a l , Am. J. Hosp. Pharm.,

&

37, 390

French Labs.,

P. M. C. BAVIN ET AL

182 19

l3eqosh, P., Smith K l i n e and French Labs., Phila., PA, m d l i s h e d data.

20

aid.

21

White, E. R., Smith K l i n e unpublished data.

22

Pedersen, P. V. arid Miller, R. J., Pharm. Sci., (1980).

23

Kinetics G r i f f i t h s , R.; Lee, R. M., and Tavlor, D. C., of Cimetidine'in M k and &p=r&tal'Animals in Proc. Sec. Int. Symp. of H i s t a m i n e H2-Receptor Antagonists FCxerpta M i c a , Amsterdam4xford, p. 38 (1977).

24

Brimblecombe, R. W.; Duncan, W. A. M.; Durant, G. J.; Bnnett, J. C.; Ganellin, G. R., and Parsons, M. E., J. Int. M e d . Res., 3, 86 (1975).

25

&

French Labs., Phila., PA,

Taylor, D. C. and C r e s s w e l l , P. R.,

69,

394

Biochem. Soc. T r a n s .

3, 884 (1975). 26

Jacobs, R. S. and Catania, H., Drug. I n t e l l . C l h . Pharmac., 11, 723 (1977).

27

Taylor, D. C.; Cresswell, P. R., and Bartlett, D. C . , D r u g . %tab. D i s p . , 6, 21 (1978).

28

Burland, W. L.; Duncan, W. A. M.; HesselbO, T . ; Mills, J. G., and Sharpe, P. C., Br. J. C l b . Pharmac,, 2, 481 (1975).

29

Soldin, S. J., et a l , Therap. D r u g Monitoring, (1979)

30

Larsen, N. E., et a l , J. Chromatog. ( B i d . Applic.), 163, 57 (1979).

31

m d o l p h , W. C.; O s b o m e , V. L.; Walkensteb, S. S., and Intoccia, J., Pharm. Sci., 66, 1148 (1977).

32

Ice, R. M. and Osborne, P. M.,

J. QvQMtog.,

I,371

146,

354 (1978). 33

Ziermnak, J. A.;

Chiannmte, D. A., and Schentog, J. J.,

Clin. Chem. 27, 272 (1981). 34

Chianmnte, D. A. and Schentaq, J. J., Therap. Drug 545 (1979). plbnitoring,

I,

DISOPYRAMIDE PHOSPHATE Alan Wickman and Patricia Finnegan G. D. Seurle and Co. Skokie, Illinois I . Description 1. I Name. Formula, Molecular weight 1.2 Color, Odor, Appearance 2. Synthesis 3. Physical Characteristics 3.1 Infrared Spectrum 3.2 Proton Magnetic Resonance Spectrum 3.3 Carbon Magnetic Resonance Spectrum 3.4 Ultraviolet Spectrum 3.5 Thermal Analysis 3.6 Mass Spectrum 3.7 Solubility 4. Metabolism and Pharmacokinetics 5 . Methods of Chemical and Dosage Form Analysis 5.1 Titrimetric Analysis 5.2 Spectrophotometric Analysis 5.3 Chromatographic Analyses 6. Methods of Analyses in Biological Fluids 6.1 Spectrophotometric Analysis 6.2 Spectrofluorometric Analysis 6.3 Chromatographic Analyses References


183

184 i84

184 184 186 186 188 190 193 194 194 194 198 199 199 199 199 204 204 204 205 208

Copyright 0 1984 by the American Pharmaceutical Association ISBN 0-12-260813-5

184

1.

ALAN WICKMAN AND PATRICIA FINNEGAN

Description 1.1.

Disopyrami.de Phosphate C21H29N30'H3P04 Mol, Wt. 437.5

0I t

a-[2-(Diisopyropylamino~ethyll-a-phenyl2-pyridineacetamide phosphate (1:1)

1.2.

Color, Odor, Appearance Disopyramide phosphate is an odorless white, or slightly off-white free flowing powder.

2.

Synthesis Nitrilopyramine is heated with concentrated sulfuric acid for 4 hours on a steam bath, the mixture is poured into ice after which it is made alkaline with 10 normal sodium hydroxide. The pH of the solution is then adjusted to 6 with acetic acid and the solution is washed with toluene. The mixture is again made alkaline with 10 normal sodium hydroxide and extracted with toluene, The toluene is evaporated and the residue dissolved in ethanol and treated with activated carbon. The ethanol is then evaporated and the residue recrystallized from hexane to give disopyramide. The phosphoric acid salt of disopyramide is prepared by reacting disopyramide with a phosphoric acid solution.' The synthesis pathway is illustrated in Figure 1.

I85

DISOPYRAMIDE PHOSPHATE

Nitrilopyramine

oFNH 1 CH,N CH{CH,} 2

2

D i sopy r am ide

Disopyramide Phosphate

Figure 1

-

Synthesis of Disopyramide Phosphate


I86

3.

Physical Characteristics 3.1.

Infrared Spectrum The infrared spectrum of disopyramide phosphate in a Nujol and Fluorolube split mull is shown in Figure 2.' Band assignments are summarized below. (cm-l)

Assisnment

3480, 3290

amide N-H stretch

23 00, broad

N+-H stretch

1678, 1640

amide C=O stretch and NH2 deformation

1590, 1560 1480, 1460

benzene and pyridine ring vibrations

13 95

CH -N+ methylene dezormation

1263

C-N+ stretch

1065, 940

H2POa stretches

760

4 adjacent H wag (a - substituted pyr idine 1

7 4 0 , 695

5 adjacent H wag (mono-substituted benzene1

u)

E W

Y C

n Y n C

U

C

c (I

C

E U

P

; C

c

c

3 3

0

m

LD D

f /.*?

*

0

NOISSIWSNML

N 0

0

c N

c 9 c

c c

c c D:

0 c 0 e

0 c e m

0 c

d m

c c c N

n Y

c c

c c e c

ALAN WICKMAN AND PATRlClA FINNEGAN

188

3.2.

Proton Magnetic Resonance Spectrum The PMR spectrum of disopyramide phosphate in deuterated water is shown in Figure 3.* Shifts are reported in pprn downfield from the methyl signal of 3trimethylsilyl propanoic acid-d sodium salt which was used as an interial reference. Band assignments are summarized below.

0 I I

d

2 "CHC

Assiunment a 2.93 singlet

b

3.67 septet

C

-

d

7.10

7.55 multiplet

7.88 triplet of

doublets

e

8.57 doublet of

doublets

f

0

a

0 N

a D

3 9

3

.5

0,

"p D 0

a c

D ID

3 m

3

3

aJ

(d

c, m

a

.c 0

c a,

pc

a

-4

5k

h

a

m

0

a

.d

0

w

si

5 a,

u

u

a a

ZI]

u d

(d

0

c

(I)

a,

u

p:

v i

tr

c, a, I:

c

0

0 k

u I

PI

a,

m $4

3 D

L4

.4


I90

3.3.

Carbon Magnetic Resonance Spectrum Noise and single frequency off-resonance decoupled CMR spectra of disopyramide phosphate in deuterated water solution are shown in Figures 4 and 5.2 Shifts are reported in ppm downfield from the shift of tetramethyl silane as referenced to a shift of 67.4 ppm for dioxane which was used as an internal reference. Band assignments are given below.

0 H

H,PO, 2

18.7, 17.1

a

35.4

b

45.2

C

55.8

d

62.7

e

125.4, 124.5

f

130.0, 129.2

9

139.4

h

141.8

1

149.6

j

160.6

k

178.2

1

3

3

n

3 3 4

3

n

-I

3 3

v

3

n u

E PI

3

3

n

3

3 -4

5

n -4

3

3

u

3

n

u

E P4

193


3.4

Ultraviolet Spectrum

210

230

1.0 0.9 0.8 0.7

w

0.6

U

2

m

0.5

a

$ m

0.4

a: 0.3 0.2

0.1

250

270

290

310

330

350

370

WAVELENGTH (nm)

Fig. 6. phosphate.

Ultraviolet spectrum of disopyramide


I94

3.5.

Thermal A n a l y s i s T h e DSC thermogram of disopyramide phosp h a t e o b t a i n e d u s i n g a h e a t i n g r a t e of 10°C/minute i s shown i n F i g u r e 7.2 The endothermic change of 1 3 8 J / g a t a b o u t 213OC i s due t o m e l t i n g and, a s can be s e e n by t h e TG i n F i g u r e 8 , 2 i s accompan i e d by weight l o s s . T h e TG was o b t a i n e d u s i n g a h e a t i n g r a t e

of 20°C/minute. The maximum r a t e of w e i g h t l o s s i s a t 236OC. The t o t a l weight l o s s i s 82.4% of t h e sample weight. 3.6.

Mass Spectrum A chemical i o n i z a t i o n mass spectrum of

disopyramide phosphate c o n t a i n s a b a s e peak a t M/z = 340 which i s due t o t h e p r o t o n a t e d f r e e base of disopyramide No peaks g r e a t e r t h a n 1 0 % of phosphate. t h e base peak a r e o b s e r v e d below M/z = 340. No peak due t o t h e s a l t i s p r e sent. * 3.7.

Solubility The s o l u b i l i t y of disopyramide phosphate i n v a r i o u s o r g a n i c s o l v e n t s i s summar i z e d i n Table 1 belowO3 Table 1 S o l u b i l i t y i n Organic S o l v e n t s * Solvent

*

s, 01 u b j J i . Q (a/J.L

Ethanol

1.089

Isopropanol

0.103

Chloroform

1.413 x 1 0 -2

Cyclohexane

6.524 x lo-)

S o l u b i l i t y d e t e r m i n e d a t 24

-

26°C


197

The solubility of disopyramide phosphate free base in various aqueous media is summarized in Table 2 below.3 Table 2 Solubility in Various Aqueous Media** Aqueous Medig

Molar i ty

. o

pH***

Solubility

0

Acetate

0.1

3 -93

62.0

Phosphate

0.1

5.85

32.0

Phosphate

0.16

7.45

8.71

Phosphate

0.1

7.98

4.32

Glyce r ine

0.1

9.86

3.78

0.1 N NaOH

0.1

** ***

12.7

Solubility at 27OC Represents pH of Aqueous Media prior to Addition of Disopyramide

1.29

I98

4.


Metabolism and Pharmacokinetics After oral administration of 100 mg of the drug to healthy men and w ~ m e n ~ ,peak ~ , plasma levels of 2.4 ug/ml were obtained 2 hours after administration. The drug had an absorption rate of 1.35 hours-’. The drug was eliminated with a biological half life of 7 hours. Twenty four hours after administration, plasma levels were. low but urinary excretion was still appreciable. An average of 60% of the oral dose was recovered unchanged in the urine within 48 hours, with 50% being recovered within 24 hours of administration. After oral administration of the drug to dogs and rats, the biological half life, after peak plasma levels were obtained, is 1.5 hours and 1.4 hours respectively. Urinary excretion after 24 hours was much lower for dogs (20%) and rats (35%) than for man. It is assumed that the rapid disappearance of disopyramide from the blood of both dog and rat is due to both urinary excretion and formation of metabolites which undergo biliary excretion. Karim et a16 determined that the main pathway of disopyramide metabolism involved N-dealkylation of the isopropyl group and arylhydroxylation, with a marked species difference in biotransformation between dog, rat, and man. Oral administration of I b C labeled disopyramide to dogs yielded 79% of radioactive compound in the urine after 72 hours. Of this 17% was the unchanged compound, 12% the monoN-dealkylated species 2, 29% the pyrrolidone species 4, and 18% a water soluble conjugate that gave the pyrrolidone upon acid hydrolysis. Figure 9 is the proposed metabolic pathway for disopyramide in dogs. (See Figure 9) Intraperitineal administration of “C labeled disopyramide to rats yielded 44% of the radioactive compound in the urine after 72 hours. Of this, 80% was the unchanged disopyramide 1. The minor metabolic products recovered were two phenolic compounds 5 and 6 arising from arylhydroxylation. (See Figure 10.1


199

Oral administration of disopyramide 1 to man yielded 56% of disopyramide and 4 % of the mono-N-dealkylated species 2 in the urine after 24 hours (refer to Figure 9). 5.

Methods of Chemical and Dosage Form Analysis 5.1.

Titrimetric Analysis Accurately weigh a portion of the disopyramide phosphate and add it to 50 mL of glacial acetic acid in a 250 mL Erlenmeyer flask. Titrate with 0.1 N perchloric acid to a potentiometric endpoint. A blank determination is performed and any necessary corrections made. Each mL of 0.1 N perchloric acid is equivalent to 21.87 mg of disopyramide phosphate.’

5.2.

Spectrophotometric Analysis The analysis of disopyramide phosphate may be performed by W spectrophotometric analysis employing 0.1 N sulfuric acid in absolute methanol as solvent. The ultraviolet absorption maximum is at about 268 nm.O

5.3. 5.3.1.

Chromatographic Analyses Thin Layer Chromatography Several TLC systems are available for the analysis of disopyramide phosphate.s Solvent, adsorbent, and detection parameters are summarized in Table 3.


200

0

0

1

c

0 I -1

Of

0

IH

Q L

N

Figure 9

-

Proposed Metabolic Pathway of Disopyramide in Dogs

1

20 1

DlSOPYRAMIDE PHOSPHATE

HO

Figure 10

-

Proposed Metabolic Pathway of Disopyramide in Rats

ALAN WICKMAN A N D PATRICIA FINNEGAN

202

Table 3 ~~

~

Solvent System ~~~

Adsorbent

Detection

Benzene:ethanol 2B:ammonium hydroxide 170: 28:2 (v/v/vi

Silica Gel 0.25 mm

1,2

To1uene:ethanol 2B :ammonium hydroxide 170: 28:2 (v/v/v)

Silica Gel 0.25 mm

1

.34

Methylene chloride:ethanol 2B:ammonium hydroxide 70: 28:2 (v/v/v)

Silica Gel 0.25 mm

2

0.65

~

Rf 0.39

Detection Systems 1.

Spray with Dragendorff Reagent

2.

Color test the chromatographic plate by exposure to excess t-butyl hypochlorite. Evaporate the excess reagent (20 minutes in fume hood) and then spray the plate with 0.5% (w/v) potassium iodide and 0.5% (w/v) starch in water.

5.3.2.

High Performance Liquid Chromatography Disopyramide phosphate may be chromatographed under the following HPLC conditions.10 System 1 Column:

u-Bondapak C18 (30 cm x 4 . 0 mm i.d.1

Mobile Phase:

70% triethyl ammonium phosphate buffer/30% methanol (v/v)

Flow Rate:

2 mL/min

203


Detection:

254 nm

Temperature:

Ambient

Retention Time:

Approximately 6 minutes

System 2 Column: Mobile Phase:

DuPont Zorbax C8 (15 cm x 4.6 mm i.d.1 65% TEAP Buffer pH 3.7/35% MeOH

5.3.3.

Flow Rate:

2 mL/min

Temperatur e :

Ambient

Detection:

254 nm

Retention Time:

Approximately 5 minutes

Gas Liquid Chromatography Disopyramide phosphate can be chromatographed as the free base. The free base is obtained by dissolving disopyramide phosphate chemical or capsule in an aqueous base medium and extracting into an organic solvent such as chloroform, ethyl ether, methylene chloride, etc.. Disopyramide is chromatographed under the following conditionsl l. System 1 Co1umn :

3% OV-1 on Gas Chrom Q, 80/100 mesh (1.8 m x 2 mm i.d., glass column)

Oven Temp.:

21 o o c

Carrier:

Helium

Flow:

50 mL/min


204

Detection:

Flame Ionization

System 2

6.

Column:

3% Silar 1 O C on Gas Chrom Q 1 0 0 / 1 2 0 (1.8 m x 2 mm i.d., glass column)

Oven Temp.:

25OOC

Carrier:

Helium

Flow Rate:

50 mL/min

Detection:

Flame Ionization

Methods of Analyses in Biological Fluids

6.1.

Spectrophotometric Analysis A portion of the plasma/serum is added to 0.5 M phosphate buffer pH 7.5 and extracted with methylene chloride. The organic portion is washed with potassium hydroxide and then extracted with 0.1 N sulfuric acid. The ultraviolet absorption of the aqueous extract is measured at about 2 6 0 nm to quantitate the disopyramide12.

6.2

Spectrofluorometric Analysis

A portion of the sample fluid is added to sodium hydroxide solution and extracted with methylene chloride. The methylene chloride solution is then extracted with 50% sulfuric acid. To quantitate disopyramide, the fluorescence of the sulfuric acid extract is determined at hemission = 410 nm, 3c excitation = 275 nm5.


6.3. 6.3.1.

20s

Chromatographic Analyses Gas Liquid Chromatography Many investigators have used gas chromatography to analyze disopyramide l 3 - 2 0 , and to a lesser extent disopyramide and its mono-N-dealkylated metabolite * l - 2 3 , in biological fluids. The majority of the methods involve basifying the sample fluid and extracting the free base into an organic medium. This organic medium may then be evaporated and the sample reconstituted in ethanol or another solvent, or the sample can be injected directly into the chromatograph. Some of the methods described use a double extraction where the organic medium containing disopyramide is extracted with acid solution, which is then made basic and again extracted with organic solvent. Many of the methods also employ an internal standard for quantitation. Internal standards commonly used are p-chlorodisopyramide, and aminopentamide. Table 4 gives a summary of the gas chromatographic conditions employed.


206

Table 4 Column Temp.

Column

Detector

Carrier Plow

Ref.

~

1.58 BE30 on Gas Chrom Q (1.8 m x 2 mm, g l a s s )

230.C

FID

30 mL/mln N2

13

38 OV-17 on Gas c h r w Q 100/200 mesh (1 m x 2 mm, g l a s s )

255.C

Nitrogen

20 mL/min N2

14

38 OV-101 on Gas Chrom Q 80/100 mesh (1.8 I x 6 m e g l a s s )

23 O*C

PID

30 mL/min 2'

15

38 OV-1 on Supelc o p o r t 80/100 mesh

240.C

PI0

4 0 mL/min N2

16

230.C

Nitrogen

30 mL/min 2'

17

38 ocw 98 on as Chrom Q 100/120 mesh (1.7 m x 2.6 mm, g l a s s )

210-C

PID

100 mL/min 2'

18

38 OV-1 Gas Chrom Q 100/100 mesh ( 2 m x 2 mm, g l a s e )

200.C

Nitrogen

40

a h i n N2

19

38 OV-17 on Gas Chrol Q 100/120 mesh ( 6 0 cm x 2 m, g l a s s )

260.C

PID

30 W m l n 2 '

20

38 OV-17 on Chrooos o r b W 100/120 mesh (1 x 2 mm, g l a s s )

250.C

PID

2 1 pLL/min 2 '

21

38 OV-17 on Gas Chrom Q 100/120 mesh (60 cm x 2 mm, g l a s s )

245.C

Nitrogen

25 a h i n

22

2.68 OV-17 on Chromo-

21 0-3 230.C

PI0

60 W m f n Be

23

(1.5 D

X

4

silanised glass) 28 SP2250, 2) OV-101

on Ehromosorb w-BP

100/120 mesh (1.2 D x 2 mm, g l a s s )

sorb W-BP 80/100 mesh (.6 D x 2 me g l a s s )

6.3.2.

High Performance Liquid Chromatography Several HPLC methods have been reported that can be used to analyze disopyramide phosphate a l o n e Z b - 2 8 or in combination with its mono-N-dealkylated metabolitez9 - 3 2 . Sample preparation is similar to that for gas chromatography. For most methods the sam-

DISOPY RAM1 DE PHOSPHATE

207 Table 5

5 oicron ODs-18

ketonitri1e:potasrium phosphate (monobasic) buffer

254

---

24

10 b ODS-18 (25 cm x 3 nun i.d.1

1t Acetic acid:methanol: triethylamine (34.5145:

254

1

25

pBondapak Cl8 (30 cm x 4 QIP i.d.1

Ilethano1:heranczsulfonic acid (pE 4-51 (60r401

254

1

26

Lichrosorb S160 (15 cm x 4 mm i.d.1

Dich1orocthane:methanolr percbloric acid (95.7:41

26 5

1

21

pBondapak C18 (30 cm a 4 mm i.d.1

- 0 5 I4 Potassium phosphate

258

2

28

pBondapak CN (30 cm x 4 mm i.d.1

Acetonitriler.01 H sodium acetate tpH 4 ) (50:50)

254

1.2

29

PBondapa k CN 125 cm x 5 mm i.d.1

.06 H Sodium acetate, 4.78 acetic acid CpR 3.5): methanol (85:15)

254

1.9

30

ODs C18

Hethanolrwater with .005 H heptanesulfonic acid ( 5 3 ~ 4 7 1

254

1

31

0.5 H sodium phosphate% acetonitrile (73x27)

254

1.8

32

(25 cm x 5 am i.d.l

Lichrosorb RP-8 10

.5)

.3)

(debasic1:acetonitrile (65:35)

micron (25 cm x 4.6 nm i.d.1

ple fluid is basified and extracted into an organic medium. The medium is then evaporated and the sample reconstituted in mobile phase solution or other organic medium. Table 5 gives a summary of different HPLC Systems.

Acknowledgments The authors wish to thank Dr. J. Witt, Dr. R. Bible, Dr. A. Karim, Dr. J. Hribar, C. Seul, and E. Hajdu for their contributions and suggestions and to A. Fenton for her secretarial assistance in preparing this manuscript.

208


References 1.

U.S. Patent 3,225,054 C.A. 58, 12522 (1963)

2.

Physical Methodology Group - G. D. Searle Co. - Internal Communication

3.

Y. W. Chien - G . D. Searle Communication

4.

Norpace/Investigational Brochure Searle & Co.

5.

Ranney, R. E., Dean, R. R., Karim, A,, Radzialowski, F. M., Arch Int. Pharmacodyn 191 (1971) 162-188

6.

Karim, A., RanneyI R. E., Kraychy, S., Pharm Sci 61 No. 6 (1972) pp. 888-893

7.

USP

8.

G. D. Searle

9.

C. Wohlt, J. Drury, C. Murphy - G. D. Searle & Co. - Internal Communication

&

Co.

&

-

Internal

-

G. D.

J

xx &

Co.

-

Internal Communication

10.

G. D. Searle

b

Co.


11.

G. D. Searle

&

Co.

12.

Martin, D., Burke, L., Norden, W., Chen, S., Clinical Chemistry 24 No. 6 (19781 991

13.

Daniel, J. W., Sobramanian, G., J Inst. Med. Res. 4 , Supp (1) (1976) 2

14.

Duchateau, A.* Merkus, F., J Chromatog 109 (1975) 432-5

15.

Johnson, A., McHaffie, D., J. Chromatog 152 (19781 501-506

16.

Hayler, A . * Flanagan, R.? J Chromatog 153 (1978) 461-471

17.

Vasiliades, J., Owens, C., Pirkle, D., Clin Chem 25 (1979) 311-313



, Reid,

209

P. , J Chromatog 178 (1979)

18.

Foster, E. 571-574

19.

Gai, J., Brady, J., 4 (1980) 15-19

20.

Doedens, D., Forney, R., J Chromatog 161 (1978) 337-339

21.

Aitio, M. L., J Chromatog 164 (1979) 515-520

22.

Bredesen, J., Clin Chem 26 (1980) 638-640

23.

Hutsell, T., Stachelski, S., J Chromatog 106 (1975) 151-158

24.

Clark, C., Lankford, G., Am Chem SOC Abstr 176 Anal 57 (1978)

25.

Broussard, L., Frings, S., Clin Chem 24 (1978) 1007

26.

Draper, P., Shapcott, D., Leonieux, B., Clin Biochem 12 11979) 52-55

27.

Lagerstrom, P. O., Perpson, B. A., J Chromatog 149 (1978) 331-40

28.

Ilett, K., Hackett, L., Dusci, L., Jokrosetio, R., J Chromatog 154 (1978) 325-29

29.

Nygard, G., Shelver, W., Wahba Khalil, S., J Pharm Sci 68 (1979) 1318-20

30.

Lima, J., Clin Chem 25 (1979) 405-8

31.

Meffin, P., Harapet, S., Harrison, D., J Chromatog 132 (1977) 503-11

32.

Ahokas, J., Davies, C., Ravenscroft, P. J., J Chromatog 183 (1980) 65-71

Kett, J., J Anal Toxic01


INDOMETHACIN Matthew O’Brien Merck Sharp 6 Dolime Research Laboratories West Point, Pennsylcania

James McCauley Merck Sliarp 6 D o h e Research Laboratories Rahway, New Jersey

Edward Cohen Merck Sharp 6 Dolinie Research Lahoratories West Point, Pennsylcania 1.

2.

3. 4. 5.

6.

212 212 212 213 213 213 213 213 214 220 222 222 226 221 221 227 228 229 229 229 229 230 230 234 234 235

Introduction 1.1 Historical I .2 Name, Formula, Molecular Weight 1.3 Definition I .4 Appearance, Color, Odor Physical Properties 2. I Ultraviolet Absorbance 2.2 Mass Spectrum 2.3 Nuclear Magnetic Resonance 2.4 Crystal Properties 2.5 Infrared Absorbance 2.6 Thermal Behavior 2.7 Solubility 2.8 Dissociation Constant 2.9 Partition Behavior Synthesis Stability Methods of Analysis 5.1 Elemental Analysis 5.2 Spectrophotometric Analysis 5.3 Fluorescence Analysis 5.4 Polarographic Analysis 5.5 Mass-Transport Techniques 5.6 Titrimetric Analysis Metabolism and Pharmacokinetics References


21 1


212

MATTHEW O'BRIEN ET AL

1. Introduction 1.1 Historical Indomethacin is a non-steroidal, anti-inflamatory agent with anti-pyretic and analgesic properties discovered and developed by the Merck Sharp and Dohme Research Laboratories(1). Indomethacin has been used effectively in the management of patients with moderate to severe rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, acute painful shoulder (bursitis and/or tendinitis) and acute gouty arthritis(2-6). Recently, indomethacin has been found effective in the treatment of neonates with patent ductus arteriosus and in patients with acute cystoid macular edema follcwing cataract surgery(7-9). Worldwide, indomethacin has been formulated into many dosage forms, including formulations designed for long duration of activity. The discovery of this compound continues to provide new insights into medical treatment of disabling diseases(l0). 1.2 Name, Formla, Molecular Weight Indomethacin (I) is l-(p-chlsrobenzoyl)-5-methoxy-2mthylindole-3-acetic acid. Other names for the compound include (l-p-chlorobenzoyl-5-methoxy-2-methylindol-3-yl) acetic acid(l0). Proprietary names for indomethacin include AMUNO INDACID, INDACIN, INDOCIN, INDOCIN-SR, INCKIN-IV, INEOMEE, INDOPTIC, METINDOL, and MEZOLIN. Additional chemical and proprietary names are listed in the monograph for indomethacin in the Merck Index(l1).

CH,O

CHzCOOH

Official monographs for indomethacin are given in U.S.P. B.P. 1980 , European Ph.1980 ,and the Nord. Ph.1969 , (12-15)

XX

213

INDOMETHACIN

1.3

Definition

Indomethacin is defined as the Form I crystalline, nonsolvated free acid moiety of the compound unless otherwise noted. Indomethacin as described below can exist as several crystalline forms but has been used in pharmaceutical preparations principally as Form I and less frequently as the crystalline sodium trihydrate. 1.4 Appearance, Color, Odor Pale yellcw to yellow-tan, crystalline powder that is odorless or almost odorless. 2. Physical Properties 2.1

Ultraviolet Absorbance et al. as Indomethacin was first characterized by Shen -having ultraviolet absorbance maxima at 319 and 230 nm with an inflection at 260 nm in ethanol. The corresponding values reported were 6290, 20800 and 16200, respectively(1). U.S.P.XX lists a W absorbance maximum at 318 nm in methanolic 0.1N hydrochloric acid. Figure 1 shows an ultraviolet absorption spectrum of indomethacin (Merck Standard 6375-66-1) in methanolic 0.1N HC1 at a concentration of 1.429mg/100 ml. Absorbance maximum is at 318 with E l % l c m value of 182 ( =6510).

2.2

Mass Spectrum The mass spectrum of indomethacin obtained on an LKB5000 at 70 eV ionization energy is found in Figure 2. Important Features of the spectrum include:

(1) Molecular ion peak at 357 (m/e). (2) Peak at m/e 312 ( M . W . 4 5 )

attributed to loss of C02H.

(3) The most intense peak of the spectrum occurs at m/e 139 which corresponds to p-chlorobenzoyl(16).

2 I4

MATTHEW O’BRlEN ET AL.

2.3 Nuclear Magnetic Resonance 2.3.1

The proton NMR spectrum of indomethacin in deuterated dimethylsulfoxide (d6 DMSO) at a concentration of 20% (w/v) is reproduced in Figure 3. A tabulation of the assignments and chemical shifts is found in Table I( 17). The carboxylic proton is not visible in the above spectrum due to exchange broadening involving the water in the solvent.

2.3.2

Carbon 13 Spectrum The I3C magnetic resonance spectrum is given in Figure 4 and was obtained using a Varian CFT-20 (FT mode) spectrometer with d DMSO as the solvent at a concentration of 10% w/v. $he spectrum is consistent with the structure and the assignments are in Table I1 (17).

CH

215

INDOMETHACIN

Wavelength (nm 1 F i g u r e 1. U l t r a v i o l e t A b s o r p t i o n Spectrum of Indomethacin i n M e t h a n o l i c H y d r o c h l o r i c Acid a t 0.00143% C o n c e n t r a t i o n ; max @ 318 nm; E l % l c m = 182. Merck S t a n d a r d 6375-66-1

A

u

M

h a,

C

8 0 .rl N

rd

u

C 0

-4 *rl

3 a, I

b

0

0 0 0 In

INDOMETH ACIN

217

Table I. Solvent:

Proton NMR Spectrum of Indomethacin Merck Standard 6375-66-1 d6 DMSO; Conc: 20% w/v; Inst.: JEOL C-60 Hz

Chemical. Shift

Integral

(PIa

(m)

Rel. No. Proton

Assigmnt

w-

6.55-7.10

(m)

44 1/2

[2*97I

H q r H6 and H7

7.45-7.80

(m)

61 1/2

[4.11I

c1

H H a

-

TMS used as internal reference.

Table 11.

Carbon 1 3 Chemical Shifts for Indomethacin Shift d 3 C

(PP) 172.0 167.8 155.5 137.6 135.1 134.1 1.31.1 130.7 130.2 129.0 114.5 113.4 111.2 101.7 55.3 29.5 13.1

Assignment*

I

I

I

I

I

1

I

I

5ppm Offset

f

1

AMPL. 2.5 x I 0

4

I

8.0

I

I

I

7.0 6.0 5.0

I

4.0

L

I 3.0 2.0 I

I

i.0

Chemical Shift (ppm) F i g u r e 3. P r o t o n N4R Spectrum of I n d o m e t h a c i n i n d -DMSO. 6 Merck Standard 6375-66-1

0

200

100

Chemical Shift 6 13C (ppm)

Figure 4 . 13C NMR Spectrum of Indomethacin. Merck Standard 6375-66-1.

0


220

2.4

Crystal Properties

Tndomethacin is known to exist in more than one nonsolvated crystalline modification. Yamamto described three polynorphs oc,\B and 7with melting point, TR and x-ray powder diffraction data(l8). Monkhouse and Lach reported two modifications characterized by melting point and IR(19). Borka found four and reported hot-stage microscopic melting point, IR and solubility data (solubility on only three)(20). Others have also observed four crystalline polymrphs(21). While there is disagreement as to the existence of some of the polymrphs, all agree on two of the crystalline modifications. There are consistent melting point, IR and x-ray powder diffraction data for these two modifications. Most authors refer to these polymorphs as Form I and Form 11. Yamamoto, however, uses 7-type and a-type, respectively. Form I is the highest melting (see 2.6 k l w ) and lowest- solubility (see 2.7 below) polymorph and is, therefore, the thermodynamically stable crystalline modification of indomthacin. However, from a practical view, both Form I and I1 are equally biologically available and active (22). The x-ray powder diffraction data for Forms I and I1 of indomethacin are found in Table 11. The d-spacings and relative intensities for Form I are consistent with those cited in the National Formulary X I V (1975) (13). Kistenmacher and Marsh determined the crystal and molecular structure of indomethacin by single crystal x-ray diffraction methods(23). Although not explicitly stated by Kistemcher and Marsh, the crystals grown from anhydrous acetonitrile are most likely Form I. They reported that the crystals are triclinic, space group P1 with cell constants a = 9.295(2)A, b = 10.979(1)A, C = 9.742(1)A, a=69.38(1)', g3 = 110.79(1)', V = 9 .78(1 and 2 = 2. The calculated density i3 1.37 gm/cm ; the observed density is 1.38(1) gm/cm

.

3

In addition to the polymorphism described above, indomethacin is known to form solvates with benzene and tbutyl alcohol(21). Borka has also observed solvates with a number of different solvents(20).

22 I

INDOMETHACIN

Table I11 X-Ray Powder Diffraction of Indomethacin Forms I and I1 Sample Merck Standard Cu & Radiation Form I 2

Rel. Intensity

('1

10.18 11.60 12.75 16.65 17 .OO 17.30 18.55 19.30 19.60 20.30 20.90 21.80 22.90 23.15 24.00 25.50 26.62 27.50 28.3 28.9 29.35 30.40 33.60 34.15 34.75 37.50

8.69 7.63 6.94 5.32 5.21 5.13 4.78 4.60 4.52 4.37 4.25 4.08 3.88 3.84 3.71 3.49 3.35 3.24 3.15 3.09 3.04 2.94 2.67 2.63 2.58 2.40

22 100 15 54 89 25 19 36 60 10 15 89 17 15 22 13 42 15 11 23 31 14 10 11 8 13

2

('1

4.85 6.89 8.80 10.20 11.35 11.95 13.85 14.19 14.45 14.70 14.90 16.05 17.50 18.00 18.90 19.00 19.70 20.15 20.60 21.10 22.02 22.60 23.35 24.01 24 .50 24.90 25.28 26.25 27.20 28.35 3 1 .O

Form I1 Re1 . d(g) Intens ity 18.22 12.83 10.05 8.67 7.80 7.46 6.39 6.24 6.13 6.03 5.95 5.52 5.07 4.93 4.70 4.67 4.51 4.41 4.31 4.21 4.04 3.93 3.81 3.71 3.63 3.58 3.52 3.40 3.28 3.15 2.89

14 43 100 36 32 77 34 80 79 30 30 31 45 50 55 16 54 39 46 29 77 68 52 39 57 23 38 36 14 39 25

MATTHEW O'BRIEN ET A L

222

2.5

Infrared Absorbance

The infrared absorption spectrum for Form et al. indomethacin in KEr is given by Hayden -Sadtler(24,25). Monkhouse and Lach, and Borka, to IR spectrum for Form I, give IR spectra for crystalline modifications of indomethacin that observed(l9,20).

I of and in addition the other they

The IR spectrum which is consistent with the literature for Form I of indomethacin is found in Figure 5. Some band assignments for the solid state infrared absorption spectrum are given belw(26). Wavelength (an-') *3400-2500 1715, 1695 1600 1450 1230 925 900-600 750

2.6

Aromtic C-H stretch Carboxylic acid 0-H stretch C=O stretch Aromatic C=C stretch 0-CH3 deformation stretch plus 0-H deformation Carboxylic 0-H out of plane deformation Various C-H out of plane deformation for substituted aromatic c-c1 ? ? (C-0)

Thermal Behavior

Shen et al. initially reported a melting point of 153154O for indomethacin(1). The N.F. XIV states that the melting point of indomethacin is 162°C while N.F. XI11 gives 1550 and 1620 for the melting points of two polymrphs of indomethacin. The British Pharmacopoeia 1980 specifies a ntelting point of 158u to 162uC(14). Tabulated in Table IV are the melting points observed for the various polymorphs previously reported in the literature.

INDOMETH AClN

223

Table N Melting Points of Indomethacin Polymorphs Form Form I (type y )

Melting Point (OC) 160-161.5( 18) 160(20,18) 158( 19)

Form I1 (typeo ( )

154.5-155.5(28) 154(20,28) 152( 19)

Form I11

148( 21)

Form IV

134(21)

Type

4

158-160.5(18)

A LYTA curve for indomethacin (Form I - Merck Standard) obtained on a du Pont 990 thermal analyzer is given in Figure 5. Observed DTA peak temperature is 162OC.

0

0

%

1

0 a0

I 0 a0

0 W

0 W

U

0

d

0

224

cu

0

N

c,

0

0

u

3

22s

3 LD N 3 N N

0 00 v

*0 Y

0 0 7

(D

0

N

0

226

2.7

MATTHEW O'BRIEN ETAAL.

Solubility ~

The following s o l u b i l i t y d a t a have been reported:

Sol vent

Temp ( OC )

Sol ubi li ty

Reference

Water

25

0.40 mg/100 m l a

20

Water

25

0.52 mg/lOO mlb

20

Water

25

0.88 mg/lOO m l c

20

Water

RT

P r a c t i c a l l y Insoluble

13

Phosphate Buffer pH 5.6

25

3 mg/100 m l a

28


25

5 mg/100 m l b

28


25

11 mg/IOO m l a

28

Phosphate Buffer p U 6.2

25

16 mg/lOO rnlb

28


25

54 mg/100 m l a

28


25

80 mg/lOO nilb

28

Ethyl alcohol (95%)

RT

1 i n 50

13

Chloroform

RT

1 i n 30

13

Ether

RT

1 i n 45

13

Metl-ranol.

25

32 mg/gm

29

Benzene

25

4 mg/W

29

n-butanol

25

19 W/gm

29

sec-butanol

25

27 mg/gm

29

aForm I,

bForrn 11,

'Form 111

INDOMETHACIN

2.8

221

Dissociation Constant

A pKa of 4.5 for the carboxyl group of indomethacin was calculated from aqueous solubility data(30). Potentiometric titration data for indomethacin in 50% methanol-water yielded pKa of 4.5 using a correction factor for the solvent(31).

2.9

Partition Behavior

Apparent distribution coefficients for indomethacin are tabulated below(31). Additional information is given in the references. Solvent Pai.rs

K*

Methylene Chloride/pH 7.1 Phosphate Buffer Ether/pH 7.1 Phosphate Buffer

16.3 8.2

K* - conc. in organic phase conc. in aqueous phase

3.Synthesis 3.1 Indomethacin(1) was originally prepared as shown in the scheme below by conversion of 5-methoxy2-methylindole-3-acetic acid to its anhydride using dicyclohexylcarbodiimide in THF(1). Treatmnt of the anhydride in the presence of zinc chloride and butanol produced the butyl ester, which was then acylated with p-chlorobenzoic acid to yield indomethacin butyl ester. The ester was then pyrolyzed and purified to produce indomthacin. Indomethacin ethyl ester is synthesized by the reaction of N-p-chlorobenzoyl-N-p-methoxyphenylhydrazine with l-hydroxy2-propanone and 2-bromoethylacetate after the method of Yamamto et.al. .(32) Indomethacin has also been synthesized from sodium p-methoxyphenylhydrazine sulfonate and from saffrole(33,34).

. ~.DCHC/THF 2. ZnC12/BuOH

3


228

4. Stability

In general, the integrity of indomethacin powder and formulate( products exists for at least five years at room temperature(35,36). Exposure to strong direct sunlight induces an increase in the color of indomethacin(37);however,degradation is slight, but the precaution of employing light resistant containers should be taken to minimize discoloration of solid. Indomethacin undergoes alkalinc hydrolysis to p-chlorobenzoate and 2-methyl-5-methoxy-indole-3acetate. These transformation products are also primary metabolic products(section on metabolism and pharmacokinetics).

-

co2

6-0

I

c1

The half-life at room temperature is about 200 hours in pH 8.0 buffer and about 90 minutes in pH 10.0 solutions(36). In a patent specification, Sumitom Chemical Co. Ltd. reports stability conditions for seven different injectable formulations of indomethacin(38). Several of these formulations were stable after four months storage at 5OoC. Formulations of indomethacin as the free-acid and as the sodim salt are available in various parts of the world. They include: Capsules 25mg and 50mg Time Release Capsules 75mg Injection l.Omg/ml Ophthalmic Suspension lOn!q/ml Oral Suspension lOmg/ml Suppositories 5Omg and lOOmg

INDOMETHACIN

229

5. Methods of Analysis 5.1

Elemental Analysis

Analysis of Merck Sharp & Dohme reference lot 590,226-0A38 for C, H, N and 0 was reported as follows:

C H N 0

Theory

Observed

63.78 4.51 3.92 9.91

63.87 4.44 4.09 10.01

Reference material is obtainable from both the Pharmacopeia and the British Pharmacopeia (13-14).

U.S.

5.2 Spectrophotometric Analysis Analysis of capsules containing indomethacin is described in the USP/NF and the British Pharmacopeia (13,141. After an extraction into Ethylene chloride from a methanolic pH 7.2 phosphate buffer, the ultraviolet alxorbance is measured near 318 nm. This analysis measures intact indomethacin in the presence of its hydrolytic degradation products. If esterification has occurred, an ether wash prior to extraction from phosphate buffer has been used to prevent interference caused by esters(39). This procedure is a l s o applicable to injections, suppositories, suspensions and tablets. Allesandro and co-workers have also described analysis of indomethacin by formation of a nitroso derivative which has an ultraviolet absorption maximum at 317 nm(40). 5.3

Fhorescence Analysis

The indomethacin hydrolysis product 2-methyl-5-methoxy indole acetic acid fluoresces at 385 nm after excitation at 300 nm in 0.1N NaOH,(41) and at 387 nm after excitation at 312 nm in pH 11.6 buffer(42). The latter procedure claimed a threefold increase in detectability. Neither method distinguishes indomethacin from salicylates. Clinical studies employing subjects administered aspirin must use a separation prior to fluorescence analysis. Without adequate separation the indole metabolites as well as salicylate, produce a positive assay bias.

MATTHEW O'BRIEN ET AL.

230

5.4 Pol.arographic Analysis In aqueous methanol, indomethacin exhibits a half-wave potential (El 2) at the dropping mercury electrode which is dependent up& pH. In 0.1M methanolic lithium chloride, indomthacin has two waves between -1.4~and -1.6~ (vs. S.C.E.). The first step height is diffusion controlled and corresponds to a two electron reduction of the amide carbonyl. The second wave is believed to be a kinetic wave. The method as described is specific for nonhydrolyzed indomethacin and is suitable for analysis of capsules, suppositories and +0.7% and +1.2% for suspensions with precision of +1.2%, the respective formulations(. ) 3 4 5.5

Mass-transport Techniques 5.5.1

Liquid-liquid Extraction

Distribution ratios of indomethacin and its metabolites, N-deschlorobenzoylindomethacin and 0desmethylindomethacin, have been described for heptane and aqueous solutions of buffers between pH 5.0 and 7.0(44). In addition, a comparison was made on samples extracted from sera of man, dog and rat in the above pH range. 5.5.2

Paper Chromatography

Harman and co-workers described a series of solvent systems for ascending chromatography on Whatman 3MM paper(45). The following table compares the R values of indomethacin ( T) , N-deschlorobenzoyl j ndomet6acin (I1) and 0-desmethylindomethacin (111).

System A B C

I1 -

0.75 0.95 0.95

0.40 0.88 0.95

I11 0.66 0-92 0.95

isopropyl alcohol - 15N arnmonium hydroxidewater (8:l:l) B - methanol-water-n-butyl alcohol-benzene (2:1:l: 1) C - acetic acid - isopropyl alcohol (5:95)

System A

-

I -

INDOMETHAClN

23 I

Other workers achieved separation of I, 11, and I11 using as a developing system Xylene-toluene-dioxaneisopropanol-20% morpholine (1:1:3:3:2)(46). 5.5.3

Thin-Layer Chromatography

Sondergaard and Steines measured indomethacin in plasma and urine by direct quantitative TLC using chloroform-methanol (30:6) on silica gel.(47). Reliable quantitation was achieved at the 30 ng/ml level(+l2&). In addition to the systems described above, Harman and co-workers developed TLC systems on silica gel (45).

System D E

D E

-Rf I1 -

I 0.50 0.57

0.55

0.37

I11 0.65 0.15

ethyl acetate - isapropyl alcohol-10% m n i u m hydroxide (5:4:3) acetic acid - chloroform (5:95)

Allessandro and co-workers using silica gel G were able to separate indomethacin from drugs of similar pharmacological properties(40). Likewise Thompson and Johnson reported Rf values for a variety of analgesics, antipyretics and anti-inflmatory drugs which were separable from indomethacin(48). Polyamide et al. to resolve a series gels were also used by Hsiu of antipyretics including indomethacin(49). Curran,et.al. , used TLC to resolve and identify impurities in formulations(50). The systems employed had detection limits on the order of 0.1 to 0.2 ug. Rf System A R

I 0.80 0.40

I1 0.75 0.38

PCB 0.90 0.50

W

V

0.24 0.33

0.35

0.28

PCB = parachlorobenzoic acid

IV = alpha monoglyceride of I from suppositories V = alpha monoglyceride of PCB from suppositories

MATTHEW O’BRIEN ET Af

232

5.5.4

Gas-Liquid Chromatography

GC-MS characterization of indomethacin has been reported using trimethylsilyl esters separated on SE-52 liquid phase(51). The same paper also reports precision and accuracy using an electron capture detector, which for plasma was 92219%( 5ng/ml) and 9651.5% ( 1000ng/ml). For aqueous humors , the values were 9755.6% and 99+2.2%,respectively.

Indomethacin has been determined without any preparatory work by Saito and Hara on 2% (w/w) CN-17 supported on Diasolid L and on 1.5% (w/w) SE-52 supported on high purity Chromsorb W(52). Indomethacin has also been determined as its ethyl ester on a lm x 2.5 mm column packed with 100-120 mesh ChromosorbW (AW-DMCS) coated with 2% (w/w) OV-1(53). Under optimum conditions utilizing an electron capture detector, a recovery from spiked serum of 96 2 3% was attained. Formation of the ethyl ester prevents assay inclusion of O-desmethylindomethacin, a metabolite which after methyl.ation would be identical with the methyl ester of indomethacin. Measurement with an electron capture detector has been made using the pentafluoropropyl derivative in the plasma of neonates with comparable precision and linearity over the range 10 to 1000 ng(54). 5.5.5

Liquid-Sol id Chromtography

Separation of salicylic acid and indomethacin was accomplished by packing 6 gms. of kieslguhr ground with citrate buffer on top of 2 gms. of kieslguhr mixed with pH 7.0 phosphate buffer. Material was eluted with heptane(42). 5.5.6

Hiqh Performance Liquid Chromatography

Table V contains references for chromatographic systems used to measure indomethacin in various matrices.

Table V SEPARATION SYSTEMS FOR INDOMETHACIN BY HPLC Sample

Colurrm

Mobile Phase

Detection

REF -

Biological

Hypersil ODs

76% methanol in 0.025M pH4 Phosphate Buffer

Fluorometric exc @ 295nm emm @ 340nm 1.5ng/ml

55

Dosage Form

Ultrasphere ODs

Methanol-water-acetonitrile-

Ultraviolet@254

56

acetic acid(55:35:10:1) Biological

Zorbax ODs

Mobile phase optimizatiom

Ultraviolet@235

57

Dosage Form

Silica,lOum

Gradient: A:8% acetic acid in heptane B:8% acetic acid and 20% ethanol in heptane

Ultraviolet@254

58

Dosage Form

C18uBondapak

27%acetonitrile in 1% aqueous formic acid

Ultraviolet@254

59

Dosage Form

C18uBondapak

60% methanol in 2.5% phosphoric acid

Ultraviolet@240

60

Bulk Drug

C18uBondapak

Gradient: Ultraviolet@254 A:250ml acetonitrile in 750 phosphate buffer ( PH 7, 0 . 0 2 ~ ) B:700ml acetonitrile in 300

61

234

MATTHEW O’BRIEN ET A L

5.6

Titrimetric Analysis

Assessment of indomethacin p d e r had been specified in the U.S.P XIX and the British Pharmacopeia,l980 as a back-tTtyazn with hydrochloric acid after alkaline hydrolysis(l3,14). This method can attain a precision of 2 0.8%(62). A direct titration for tablets and capsules is described using sodium hydroxide. If performed rapidly with phenolphtha in indicator, a precision of 50.3% is The latter procedure serves to differentiate attainable. ester formation as well as hydrolysis products from intact material. The presence of parachlorobenzoic acid and 5-methoxy2-methylindole-3-acetic acid are cause for positive error.

&

6.

Metabolism ant3 Pharmacokinetics

The only known routes of indomethacin elimination are renal., biliary and metabolic(46,63). Unchanged indomethacin (I), 0desmethyl-indomethacin (DMI), N-deschlorobenzoylindomethacin (DBI) and 0-desmethyl-N-deschlorobenzoylindomethacin (DMBI) and their respective acyl glucuronides account for all drug-related moieties in plasma urine and feces. I, DMI and DBI are the major components in plasm and urine: they appear predominantly as conjugates in urine but totally unconjugated in plasma. DMBI is the major chemical species in the feces. In a l l animal species studied, all four chemical moieties undergo enterohepatic circulation to varying degrees. The same can be inferred from the time courses of each in man. Quantitatively, it has been estimted that approximately 50% of an intravenous dose undergoes enterohepatic circulation as I. Because of enterohepatic circulation the amount of I available to the systemic circulation can exceed the administered dose. On the average, the bioavailability of orally and rectally adminstered I is 100 and 80% relative to an intravenous reference dose. Within the therapeutic range, there is no evidence that the disposition of I is route- or dose-dependent. The mean plasma, renal, biliary and metabolic clearances of I are 107, 28, 60, 68 ml/min., respectively. Because of the sporadic and variable nature of gall bladder discharge and consequently the ensuing reabsorptior attempts to determine plasma half-life directly tend to be futile. Thus, values ranging from 90 minutes to 16 hours have been reportec in the literature. An effective half-life of about 4.5 hours has been estimated from the time course of I accumulation during repeated drug administration and is the mean value which must have prevailed for the observed accumulation to obtain.

INDOMETHACIN

235

References 1. T.Y.Shen, T.B.Windholz, A.Rosegay, B.E.Witze1, A.N.Wilson, J.D.Willet, W.J.Ho'ltz, R.L.Ellis, A.R.Matzuk, S.Lucas, C.H.Stamner, F.W.Aolly, L.H.Sarett, E.A.Risley, G.W.Nuss, and C.A.Winter, J.Am.Chem.Soc.,% ,488(1963 - ,300(1964). 2. G.M.Clark,Arthritis and Rheumatism,7

3.

C.J.Smyth, and R.Godfrey, Arthritis and Rheumatism,l ,345(1964).

4. C.J.Smyth, E.E.Velayos, and C.Amoroso, Arthritis and Rheumatism, 6, 229 (1963). 5. C.J.Smyth, E.E.Velayos, and C.Amoroso, Arthritis and Rheumatism, 9, 306 (1963). 6.

A.Sunshine, E.Laska, M.Meisner, and S.Morgan, Clin. 5, 609 (1964). Phannacol. & Therap., -

7. W.Anderson, and J.Kissane,(eds), Pathology ,C.V.Mosley St.Louis,pp2021-2024(1977).

Co.,

8. J.Hollander, and D.McCarty,(eds), Arthritis and Allied Lea and Fabiger ,Phila,,pp1057-1058(1972). Conditions 9. Sanders and Yannuzzi, Merck Sharp and Dohme Research Laboratories Study C m i c a t i o n s .

10. J.H.Talbot,ed.,Seminars in Arthritis and Rheumatism,l2 - (1982).

11. E.G.C.Clarke , "Isolation and Identification of Drugs," Pharmaceutical Press, London, (1969). 12. "Merck Index," Ninth Ed.Merck

&

Co., Inc., Rahway, N.J. (1976).

13. "U.S.Pharmacopeiafiationa1 Formulary XX", Mack Publishing Co., Easton, Pa. (1980). [N.F. X I V , 19701. 14. British Pharmacopoeia, Pharmaceutical Press, London, (1980). 15. Pharmacopea Nordica, AAS & WAHLSI Oslo, 1969.

16. J.Smith, Merck Sharp & Dohme Research Laboratories, Rahway, N .J ., Personal Camminication. 17. A.W.Douglas, Merck Sharp & Dohme Research Laboratories, Rahway,N.J., Personal Comunication.

MATTHEW O'BRIEN ET Af

236

18. H.Yamamoto,Chem. Pharm. Bull.(Tokyo) 16, 17-19 (1968). 19. D.C.Monkhouse, and J.L.Lach, J. Pharm. , Sci., 61, 1430 (1972). 20. L.Borka, Acta Pharm Suecica,

11,295

(1974).

21. J.Wittick, Merck Sharp & Dohme Research Laboratories, Rahway, N.J., Personal Communication. 22. G.V.Dming, Merck Sharp & Dohme Research Laboratories, Rahway,

N.J., Personal Comnication. 23. T.J.Kistenmacher,and (1972).

24. A.L.Hayden,

R.E.Marsh, J. Amer. Chem. SOC., 94 ,1340

-et al. , Ass.

O f f . Anal. Chem. J.,

2, 1109

(1966).

25. "Sadtler Pharmaceutical Spectra," Sadtler Research Laboratorie: Philadelphia, Pa. 26. A.Rein, Merck Sharp & D o h Research Laboratories, Rahway,

N. J., Personal Caminmication.

Pharm. Sci., 58, 1190 (1969).

27. D.J.Allen, and K.C.Kwan,J.

28. J.McCauley, Merck Sharp & Dohme Research Laboratories, Rahway,

N.J.

Unpublished Data.

29. G.Smith, Merck Sharp & Dohme Research Laboratories, Rahway,

N.J., Personal Cormnunication. 30. D.H.Rodgers, Merck Sharp & Dohme Research Laboratories, West

Point, Pa., Unpublished Data. 31. R.F.Mulligan,Merck Cmunication.

Sharp

&

Dohme Res. Lab., Personal

32. H.Yamamto, N.Yasushi and K.Tsuyoshi

(Sumitom) Japan 70 36,74!

33. D.Bertozzoni. B.Bortoletti and T.Pqrlotto, Boll.Chim.Farm., 60 ( 1970).

101

INDOMETHACIN

137

et.al. - ,J.Chem.Res.Synop., 34. E.J.Barreiro, -

4

,102(1980).

35. Indomethacin Product Information SumTlary, Merck Sharp & Dohme, May (1973 1

.

36. Indomethacin Resume of Essential Information, Merck Sharp & Dohme,June( 1965)

.

Ibid 37. 38. B. R. 1, 221, 506 39. H.C.Van Darrre, Merck Sharp & Dohme Res. Lab., Personal

Comnication. 40. A.Allessandro, F.Mari, and S.Settcase,Boll. Chim. Farm., 761 ( 1966).

105 ,

41. L.P.Holt, and C.F.Hawkins,Brit. Med. J., 1,1354(1965). 42. E.Hvidberg, H.H.Lausen, and J.A.Jansen,Eur. 4 ,119( 1974t. 43. G.Razemifard and L.Holleck,Arch. Pharm.,

J. Clin.Pharmacol.,

306 9,664(1973).

44. H.B.Hucker, A.G.Zacchei, S.V.Cox, D.A.Brodie and N.H.R.Cantwel1, ,237S1966t. J. Pharmcol. Exp. Ther.,

153

45. R.E.Harman,

et al. ,J. Pharm. Exp. Ther., 143 ,215(1964).

46. D.E.Duggan, A. F. Hogans,

Pharmcol. Exp. Ther.,

K. C. Kwan and F. G. McManon,J.

181 ,563(1972).

47. 1.Sondergaard and E.Steines,J.Chromtog.,x 48. R.D.Thompson

,485(1976).

and G.L.Johnson,J. Chromatogr., 88 ,361(1974).

88 ,491(1969). 49. H.C.Hsiu, T.B.Shib, and k.T.Wang,J. Chromtogr., 50. N.M.Curran, E.G.Lovering, K.M.McErlane and J.R.Watson, J.Pharm.Sci., 69 ,187( 1980). 51. B.Plazonnet, and W.J.A.Vandenheuve1, 587(1977).

J.Chromtog.,B

,

20 ,72 52. K.Saito and H.Hara,Bull. Nippon Vet. Zootech. Coll., S1971t.

MATTHEW O'BRIEN ET A L

238

53 D.G.Ferry, D.M.Ferry, P.W.Moller and E.G.McQueen,J.

Chromatogr.,

89

of

,110(1974).

54. M.A.Evans, J.Pharm.Sci.,E

,219,(1980).

55. W.F.Bayne, T.East, D.Dye, J.Pharm.Sci.,z ,458(1981). 5 6 . E.Kwong, G.K.Pillai, K.M.McErlane, J.Pharm.Sci.

,c

828(1982). 57. C.P.Terweij-Groen, S.Heemtra, and J.C.Kraak, J.Chromatog.,

181 ,385(1980).

58. M.L.Cotton, Merck Sharp and Doh= Research Labs.,Personal Cmunication.

59. M.J.O'Brien,Merck Sharp and Dohme Research Laboratories. 60. R.Roman,Merck Sharp and Dohme Research Laboratories,Personal C m nication. 61. C.Y.Wu, Merck Sharp and D o h Research Laboratories,Personal C m u nication. 62. G.V.Dming, Merck Sharp Cmnication.

&

Dohme Research Laboratories, Person

6 3 . K. C. Kwan, G. 0. Breault, E. R. Umbenhauer, F. G. McMahon and

E. Duggan,J. Pharmacokin. Biopharm.,A ,255(1976).

KETOTIFEN Zvoniinira Mikotib-Mihun', Josip Kuftinec, Hrvoje Hofman, Mladen Zinik, Franjo Kajfei Podrackn-lnstitute Zagreh, Iligorlatki

Zlatko Meie ' Institute Rtider BoikoLiC Zagreb, k'rigoslaaici I. 2.

3. 4.

5.

6. 7. 8. 9.

Foreword, History, Therapeutic Category Description 2.1 Nomenclature 2.2 Formula 2.3 Molecular Weight 2.4 Appearance, Color, Odor Synthesis Physical Properties 4.1 Spectra 4.2 Solid Properties 4.3 Solution Properties Methods of Analysis 5.1 Elemental Analysis 5.2 Chromatographic Methods 5.3 Titration Stability and Degradation Drug Metabolism, Pharmacokinetics, Bioavailability Identification and Determination in Body Fluids and Tissues Determination in Pharmaceuticals References

240 240 240 240 24 I 24 1 24 1 243 243 256 251 258 258 258 260 260 24 I 261 262 262

'Correspondence.


239


ZVONIMIRA MIKOTIC-MIHUN ET AL

240

1. Foreword, History, Therapeutic Category

Ketotifen is a nonspecific oral, mast cell stabilizer introduced in 1972 (1j. The prominent biochemical-pharmacological activities of ketotifen are H -receptor antagonism, phosphodiesterase inhibitioh, inhibition of the formation SRS-A, and inhibition of calcium f l u x in smooth muscle preparations: a l l these actions are suited to prevent a development of asthmatic conditions. Promising results vere obtained in early clinical trials: ketotifen was equipotent with disodimn cromoglycate in prevention of asthma induced by spontaneous excercise or by antigens. Also, ketotifen is more specific than clemastine as a H -receptor antagonist. Rowever, more recent, contholed trials failed to substantiate the early therapeutic optimism. The beneficial effect of ketotifen in the treatment of asthma is only small; it was noted that this effect is associated with pronounced sedation (2). 2. Description 2.1. Nomenclature 2.1.1. Chemical T?ame

-

4-(1-Methyl- iperidylidene)-4R-benzo- [4,5] -cyclohepta- [l, 2-bj)-thiophene-lo (9H)-one hydrogenf umarate 2.1.2.

Generic Name Ketotifen

2.1.3. Trade Name

Zaditen 2.2. 2.2.1.

Formula Zmpirical C19H19NOS.C4CF-ILF04

KETOTIFEN

24 1

2.2.2. Structural

0

HOOC x

1cCOOH

CH, J

2 . 3 . Molecular Weight

425.504

2.4. Appearance. Color. Odor The drug is marketed as the salt, either containing 2.5 molecule of water, or anhydrous. In either form it is a white, odorless crystalline powder. Ketotifen base: a yellow, odorless crystalline powder.

3 . Synthesis The reaction sequence leading to ketotifen hydrogen fumarate is shown in sheme I. 2-Thenylchloride (11), prepared by chloromethylation of thiophene (Ia) or 2-hydroxymethylthiophene (Ib), was subjected to Arbuzov’s rearrangement to give 2-thenyl-diethylphosphonate (111). This product was reacted with 2-carbolrybenzaldeh de whereupon 2-/2- (thieny1)-vinyl/-benzoic acid (IVY was obtained; IV was h drogenated t o 2-/2-(thienyl)ethyl/-benzoic acid (VT. Cyclisation under the influence of polyphosphoric acid, (PPAJ resulted in 9,lo-dihydro-4H-benzo[4,~]-cyclohepta~l, 2-bl-thiophene-4-one (VI) Alternatively (V) may be prepared by reduction of 2-thenylidene-phthalide obtained from 2-thi-

.

CH

OCH3

A

Scheme 1.

KETOTIFEN

’743

engl acetic-acid (Ic) and sodium ethylate ( 7 ) . Bromination of (VI) gives the dibromo derivative which, on refluxing in methanol, followed by dehydrobromination with-KOH in refluking methanol, gave lo-methoxy-413-benzo- [ 4,5]-cyclohepta- [l, 2-51 -thiophene-4-one (IX). This compound was condensed with t h e magnesium derivative of 4-chloro-1-methylpiperidine in tetrahydrofurane, and the resultin lo-methoxy-4-(l-methyl-4-piperidyl)-49-benzo[4,~ -cyclohepta- [1,2-b]-thbhene-%-one (X) was fin 11 dehydrated and demethylated with HC1 at 95-100Bflw , to give the base of ketotifen ( q ) .

7-

4. Physical Properties

The IR absorption spectrum of ketotifen base, and that of ketotifen hydrogen fumarate with 2.5 molecules of water, a r e shown in Fig. 1, These spectra were recorded with K3r-pelleted samples usin a m e Unicam SP-200 Infrared Spectrophotrometer (57. Some of the major frequencies and band assignements are given in Tables 1, and ,2. Table 1. Characteristic IR bands of ketotifen base frequency (crn-l) -

~

~

-

3100 3000-2840

1640 1620 1400 and 1280 1450 1120

lo60

935-700 4.1.2,

assignment -~

~

~

~

thiophene C-Ii stretching aromatic and CH stretchings C=O stretching 3 C=C (ring), stretching C-C-H, interaction C-11, bending C-C(=O)-C, stretching and bending C-R, in-plane bending thiophene

Ultraviolet

Fig. 2. shows the UV spectra (me-Unicam SP-8-100 Spectrophotoaeter) of ketotifen base (E) and ketotifen hydro en fumarate with 2.5 molecule of crystal water (27 both compounds being in methanolic solution 75).


244

I

B

Fig. 1. I n f r a r e d absorption spectra o f k e t o t i f e n base ( A ) and k e t o t i f e n hydrogen fumarate w i t h 2.5 H20 (B). Instrummt: Pye-Unicam

SP-200.

KETOTIFEN

24s

200

300

nm

400

2. U l t r a v i o l e t s p e c t r a o f k e t o t i f e n base (L), and k e t o t i f e n hydrogen fumarate with 2.5 c r y s t a l H20 (2). Instrument: Pye-Unicam SP8-100.

-

t 309(H' I

100-

>

c

5

I-

E W

> -

2

60-

_I

W

58

e

70

2%

98 165

108

237

1 II 50

100

150

2 00

2 50

mle

11111

1

300

Fig. 3. Mass spectrum of ketotifen hydrogen fumarate. The electron energy was 70 eV and the temperature of the ion source was 25OoC. Instrument: mass spectrometer CEC-21-110 B.

KETOTIFEN

247

Table 2. C h a r a c t e r i s t i c I R bands of k e t o t i f e n hydrogen Pumarate containing 2.5 molecules of c r y s t a l water frequency (crn-l)

3550-3450 3080-3030 3095-3075 3100 2800-2200

1710 1640

1390 1280

4.1.3.

assignment OH, broad band aromatic CH, s t r e t c i n g =CB s t r e t c h i n g

th&hene C9, s t r e t c h i n g ZOOH and YH COOH (fumaric a c i d ) C=C ( r i n g ) , s t r e t c h i n g C-11, s t r e t c h i n g -C-0-H, interaction

Mass

The mass spectrum (CEC-21-110 B Mass spectrometer) of k e t o t i f e n hydrogen fumarate i s shown i n Fig. 3 . The s t r o n g e s t peak corresponds t o t h e molec u l a r i o n o f k e t o t i f e n hydrogen fumarate (m/e 309). Fragmentation a p p m e n t l y s t a r t s by a s e p a r a t i o n of t h e methyl group (m/e 15) from t h e p i p e r i d y l i d e n e moiety, and t h e remainder of t h e o r i g i n a l molecule (m/e 294) does not fragmentate f u r t h e r , a t l e a s t not markedly. The h e t e r o c y c l i c p a r t s o f t h e nolecul e , thiophene and p i p e r i d i n e , should g i v e t h e f r a g ments (m/e 58) and CH2-;-CH3 (m/e 5 7 ) , respect i v e l y S (5).

CH2

4.1.4.

Proton Magnetic Resonance

.The proton MR s p e c t r a ( J e o l PX-loo Spectromet e r ) were recorded i n d i f f e r e n t s o l v e n t s : t h a t of k e t o t i f e n base i n d e u t e r a t e d chloroform, and t h a t o f k e t o t i f e n hydrogen fumarate i n d e u t e r a t e d dimet h y 1 sulphoxide ( i n e i t h e r case a g a i n s t TPMS as i n t e r n a l s t a n d a r d ) (5). These s p e c t r a a r e presented i n Pigs. 4. and 5. C h a r a c t e r i s t i c f e a t u r e s of t h e s p e c t r a a r e given s e p a r a t e l y i n Tables 3 . and 4. The proton ME spectrum of k e t o t i f e n base was p a r t l y described by Waldvogel e t a l . (6). Vhen k e t o t i f e n hydrogen f u n a r a t e was shaken with I) 0 , t h e qarboyylic group protons of fumaric a c i d &d t h e N H protons were exchanged f o r deuter o n s from D 0, a s shown by t h e absence of t h e broad peak a t 9.12 ppm (see Table 4.) (5).


248

Table 3 . 'H-NMR spectrum of ketotifen base; characteristics and assignments

I CH3

h

assignchem. shift intens. multipli- coupling d' (ppm) city const. J @z) ment doublet J = 4.9 7.50 1 H *a a,c 7.36-7.10 4H nuliplet Hb 7-05 1 H doublet Jc,a= 4.9 HC 1R doublet Jd,e=13.18 d' 4.24 1 3 doublet Je,d=13.18 He 3-74 2.94-2.58 2H multiplet *f 2 49-2-39 2H multiplet H 63 2.28-1.99 7H multiplet Hh, N-CH

4.1.5. 13C-Huclear Magnetic Resonance 13C-TTb7R spectra were recorded with a Jeol FX-loo spectrometer at 25.05 NHz. The sample of ketotifen base was dissolved in both CDCl and DMSO-d6, and hydrogen fumarate only in DWO-d 3 (5). The samples were measured in 5 mm tubes h t h TMS as internal standard and uy3ng internal deuterium lock. C-NNR spectra of >?GO-d soThe broad band lutions of base and the hydrogen fumarate are ghown in Figs. 6. and 7. In both cases all the signals were clearly resolved, The assignments of saturated carbon a t o m can be made on the basis of their well

1

a,

0

A 0

KETOTIFEN

15 I

T a b l e 4. 'H-NMR

spectrum of ketotifen hydrogen fu-

marate n

h chem. shift intens, multipli- coupling (ppm) city const. J (Hz) 9.14 2 H broad singlet 7.98 1 H doublet Ja,c= 4.9 7.43-7.26 1H multiplet 7.26-7.21 4H singlet 6.60

2 H

doublet

4.37

1 H 1H 2H 2H 8H

doublet multiplet multiplet multiplet multiplet

3.72

3 17-3 03 2.80-2.66 2.54-2.51

ass 1gnment COOH Ha HC

Hb Jd,,=13.17 i' (fummic ac.) J, ,d=13.i 7 d' He H

g

Hf ;"Ih

defined absorption regions ( 7 ) , but due to very closely spaced chemical shifts of numerous aromatic and two quaternary olefinic carbons, the assignment was possible only by means of gated decoupled (NOE) spectra. Eecause of the solute-solvent interaction in DMSO-d solutions these spectra were not quite suitable $or an unambiguous interpretation. Therefore we have recorded the 130E spectrum of the base in CDCl 3From the multiplets of the aliphatic part of the completely coupled spectrum, it was possible to assign the signals belonging to C - 2 ' , C-3' and

.


252

Table 4. I 3 C Chemical shiftsa and I3C-'H coupling constants" for ketotifen base dissolved in C D G l '1-

C-atom

(ppm)

c-2

c-3 c-4

c-5

137.73 ( s )

JCH

6.3 3.9

161.1

i1.0

-

-

159.0 < l o o 157.6 =2.0

C-6

c-7

C-8

158.0

134.9

(3-9

125.9

c-11

c-12

189.05 ( s ) 147.95 ( s ) 132.39 ($1

c-13

141.02 (s)'

(2-10

lJCH

185,l 169.9

--

-

*loo

8.0 -

'-

JCH

4JCH

-

7.8

7.5 6,8 4.0

5.0

3.9 3.9

-

9.3 overlaped with C-7 multiplet

-

-

(2-14 c-2'

c-3 '

c-4' GH,

a $in

ppm downfield from internal TMS; precision ppm; off-resonance multiplets a r e given in parentheses in the second column. + J in Hz; the precision is -0.5 Elz. The assignment can be interchanged. The long-range couplings give complicated multiplets. -0.05

C

methyl group of the N-methyl-piperidine part of the molecule, as well as the methylene carbon ((3-9) of the cyclic ketone part. The latter carbon atom displays a doublet of doublets indicating a pronounced departu&e of the CH group from the planarity. Two different first ord& C-H coupling constants were deteymined (Table 5.1, which is in agreement with the H-NMR spectrum (Fig. 4, Table 3 . ) which shows the typical pattern for geminal protons. The assignment of the aromatic-olefinic part o f the spectrum was not possible using only known substitution rules (8) due to simultaneous interaction of few different moieties. However, the NOE spectrum enabled a sound interpretation based on the long-range carbon-hydrogen couplings. The corn-

a, 11)

rd

Q

c

.d

..

KETOTIFEN

25.5

Table 6. I3C Chemical s h i f t s a f o r k e t o t i f e n base and hydrogen fumarate i n IlMSO-d, s o l u t i o n C-atom

base

f umarat e

Adb

c-2

0

G-3

+O

c-5

+0.07

c-4

.54

-0.84

C-6

+O

c-7

.4 1

+o. 23 +o. 23 +o 02 +o. 16 -0 62

.

C-8

C-9 c-lo 5-11 ’5-12

+o. 21

(2-13’

140.89 ( s ) 138.94 ( s ) 56.21 ( t ) 30.92 (t)

C-14’ c-2

’

c-3 ’

c-4’

-0 46

-1.96

-2.07 -2.10

+0.9 -2.15

C%/

c-1’ ’ (C=C

c-2’

’(c=o

a +d i n ppm downfield f r o m i n t e r n a l TWS; p r e c i s i o n -0.05 ppm; o f f resonance m u f t i p l e t s a r e given i n parentheses. Changes i n chemical s h i f t s are going from base t o fumarate; + denotes a downfield, and - an u p f i e l d shift The assignments can be interchanged.

.

p l e t e assignment i s given i n Table 5. i n c l u d i n g a nimber of long-range coupling c o n s t a n t s up t o t h e f o u r t h order. Figs. 6. and 7. show Che broad-band decoupled s p e c t r a o f k e t o t i f e n base an9 i t s fumarate i n DT4SO-d and Table 6. l i s t s t h e 3C chemical s h i f t s . Thg assignment i s made on t h e b a s i s of a comparison with t h e values from Table 5. The most remarkable changes (an u p f i e l d s h i f t o f about 2.1 ppm) a r e observed f o r t h e s a t u r a t e d carbons of t h e heterocycl i c moiety, The t h i r d column i n Table 6. i n d i c a t e s t h e changes i n chemical s h i f t s f o r a l l carbon atoms common t o b o t h species. It should be also mentioned t h a t t h e a d d i t i o n

,

ZVONlMlRA MIKOTIC-MIHUN ET AL

256

of D 0 to the C D C l solution cau es not only the vanishing of the 08 peak in the fH-IWR spectrum, but the diminishing of the signal at 49.67 ppm belonging to C-9 as well. This indicates the exchange of protons by deuterons causing the splitting of t h e signal into a multiplet.

4.2. Solid Properties 4.2.1. Melting Characteristics The melting range of ketotifen base is 156-158 that ofoketotifen hydrogen fumarate with 2.5 H20 is 124-130 G, whereas the anhydrousohydrogen fumarate decomposes between 184 and 200 C. OC,

4.2.2.

X-Ray Diffraction

X-Ray diffraction patterns were determined with a Nod. XRD-6 Spectrogoniometer (General Electric, Schenectady) (5). Pertinent data are presented in Table 7.

Table 7. X-Ray diffraction data of ketotifen hydrogen fumarate

@0.9

sec-Butanol--AcOH--water

Phenol--wat er n-But ano 1--(water-sat -

4:l

urated) n-But anol--AcOH--wat er 5:2:3 n-Butanol--AcOH--water 12:3:5 i-Propanol--ammonia--water 20:1:2

-

>0.9

0.43 0.87 0.80 0.53

67 68

68 43 43

48 48

Visualization of the spots was done by examination under Wlight or spraying with ninhydrin. The color-forming reaction with nitrobenzylpyridine (NBP) (see 5.2.1) has been used by dipping the chromatograms in solutions of NBP followed by heating at 90” and treating with triethylamine [681. 5.3.2

Thin-layer Chromatography

The TLC systems reported in the literature for the analysis of melphalan are reported in Table IV. The spots were located by examination under W light or spraying with one of following solutions (detection limits in pg): fluorescamine (0.5), ninhydrin (l), NBP followed by heating and treatment with a base (5) [51,63a] , and 0.5% iodine in chloroform. Visualization using 4-pyridine-carboxaldehyde 2-benzothiazolyl hydrazone has been described under 5.2.1. Quantitative determinations were reported by densitometry [63,72] or, in the case of I4Clabelled compounds, by scraping or cutting the chromatograms into strips and measuring the radioactivity in a liquid scintillation spectrometer [44,70].

L. VALENTIN FEYNS

286

Table IV Thin-layer Chromatography of Melphalan Plate Silica gel

Solvent (v/v) acid--water -n-Butanol--acetic (7:2:1) or (4:l:l) or (13:3:5)

Re f erence 47,51,69 70,71

or (4:1:5, the upper phase)

Silica gel Silica gel Silica gel Silica gel Silica gel Silica gel Silica gel

n-Butanol--pyridine--acet ic acid-water (8:1 :2:9, the upper phase) sec-Butanol--3% armnonia (100:44) Chloroform--methanol (9:1) n-Butanol--0.1 N ammonium hydroxide (1:l) n-Butanol--Acetic acid (2:l) Methanol Chloroform-methanol--acet ic acid

(75 :20:5) Ethyl acetate--acetone--tetrahydrofuran--water (4:2:2:2, upper phase) n-But anol water saturated Silica gel Silica gel i-Propanol--water (1:L) Benzene--methanol--acetone--acetic Silica gel acid (7:2:6:5) Kieselguhr n-Butanol--water 86:14 HPTLC Silica Ch loroform--me thano1--ace t ic acid (35:17:3) ge1 Cellulose n-Butanol--Ethanol--Propionic acid--water (10 :5 :2 :5) Silica gel

51 51 51 47 47 47 63a 63a 63a 63a 63a

-

54 72

-

73

287

MELPHALAN

5.3.3

Gas Chromatography

Melphalan has been converted to its trimethylsilyl derivative with bis(trimethylsily1)acetamide and has been analyzed by GC on a 1.8 m x 3 nun column packed with 2.5% (w/w) SE-54 on acid-washed, silanized Chromosorb W (80-100 mesh) at 210" (injector temperature, 250"; flame ionization detector temperature, 215") using nitrogen as the carrier at 30 ml/min. The order of elution from a partly hydrolyzed mixture was: melphalan, mono-hydroxy-derivative VI and di-hydroxy-derivative VII (Scheme 111). The same elution order was obtained on a SE-30 column; it was reversed on a more polar liquid phase (OV-17). Identification of the peaks was done by mass spectrometry [ 5 2 ] . In another procedure, melphalan was derivatized by successive acylation with trifluoroacetic acid and eserification with diazomethane. The samples were analyzed on a 3% OV-101 Supelcoport (80--100 mesh) 5 foot x 2 nun glass column with temperature programming from 210" to 280" at 8"/min with a helium flow of 20 ml/min. Analysis of samples extracted from plasma was performed by mass spectrometry using chemical ionization with isobutane gas and me1~halan-d~ as internal standard. Selected-ion monitoring at the M + H peak of derivatized melphalan gave linear calibration curves over a range of 2 to 200 ng of drug per ml of plasma. The detection limit was less than 500 pg 1411. 5.3.4

High-pressure Liquid Chromatography

The HPLC procedures reported in the literature (see Table V) were developed mainly for biological and stability studies , and they do separate Melphalan from its hydrolytic degradation products. Detect ion was performed by W absorbance measurements at 254--263 nm [40,53,74,75,76] or by fluorescence with excitation at 256 or 260 nm and emission at 350 or 360 nm [ 7 7 , 7 8 ] . For the W measurements a linear standard curve was reported from 10 to 500 ng per injection [ 7 5 ] . With the fluorescence monitoring the detection limit was about 500 pg of drug injected 1781.

L. VALENTIN FEYNS

288

Table V HPLC Methods

Column lO-prn

Mobile phase ( v / v )

p-Bondapak c18

10- pm S p h e r i s o r b ODS

R e f er enc e

2-methoxyethanol--O.l% 75 AcOH g r a d i e n t from 33.7:66.3 t o 51.3:48.7 Methanol--0.01 M NaH2P04 53 (pH 3) ( 1 : l ) Acetonitrile--0.0175 M AcOH 76 concave g r a d i e n t from 12:88 t o 80:20 Water-methanol ( 1 :1) , 40,74,79 c o n t a i n i n g 1%a c e t i c a c i d 2% a c e t i c a c i d - - a c e t o n i t r i l e 54 (78:22) Methanol--water (40 :60) 78 Methanol--0.01 M NaH2P04 77 (pH 3 ) ( 7 : 3 ) L

10- um HCODS/SIL

10- pm p-Bondapak

x-c18 c18

10-pm p-Bondapak c18 5-pm S p h e r i s o r b ODS 5-~RIS p h e r i s o r b ODS

5.4

-

Titration

T i t r a t i o n with s i l v e r n i t r a t e i s used both t o confirm t h e l a c k of c h l o r i d e ions i n t h e sample and, a f t e r a l k a l i n e h y d r o l y s i s , as an a s s a y f o r t h e drug. The compendia1 a s s a y s a r e performed p o t e n t i o m e t r i c a l l y u s i n g s i l v e r and calomel e l e c t r o d e s , t h e l a t t e r modified t o c o n t a i n s a t u r a t e d potassium s u l f a t e s o l u t i o n [25--271. V i s u a l t i t r a t i o n was a l s o r e p o r t e d , t h e excess of s i l v e r being t i t r a t e d with potassium t h i o c y a n a t e i n t h e presence of f e r r i c ammonium s u l f a t e i n d i c a t o r [51]. Mercuric n i t r a t e t i t r a t i o n s were a l s o r e p o r t e d [ 6 2 ] . P o t e n t i o m e t r i c and thymol b l u e i n d i c a t o r t i t r a t ions of s a r c o l y s i n i n dimethylformamide s o l u t i o n s were performed using a 0.1 N sodium methoxide s o l u t i o n 180). 5.5

I d e n t i f i c a t i o n and P u r i t v Tests i n O f f i c i a l Compendia

S e v e r a l of t h e f o l l o w i n g t e s t s a r e included i n melphalan monographs: ( a ) t h e W spectrum of a methanolic s o l u t i o n should have maxima at 260 and 301 nm [25,27] o r be i n agreement with t h e spectrum of a r e f e r e n c e s t a n d a r d [26] ; ( b ) t h e IR spectrum i n K B r d i s p e r s i o n should e x h i b i t maxima only a t t h e same wavelengths as a s i m i l a r p r e p a r a t i o n of a

MELPHALAN

289

r e f e r e n c e standard [26,79]; ( c ) a p o s i t i v e color r e a c t i o n w i t h 4-(p-nitrobenzyl)pyridine [ 25--27,791; ( d ) a 0.5% (w/v) s o l u t i o n i n methanol should be l e v o r o t a t o r y ; ( e ) a m e l t i n g p o i n t of about 177" [ 2 7 ] or about 180" [ 2 6 ] with decomposit i o n ; ( f ) t h e loss on d r y i n g i n vacuum a t 105" t o c o n s t a n t weight should not be more t h a n 7.0%, and t h e r e s i d u e on i g n i t i o n should n o t be more t h a n 0.3% [251 or 0.1% 1261; ( g ) a l i m i t t e s t f o r " i o n i s a b l e c h l o r i n e , " performed by d i r e c t p o t e n t i o m e t r i c t i t r a t i o n of t h e sample 125,271; and (h) a p o s i t i v e test f o r c h l o r i d e s a f t e r a l k a l i n e h y d r o l y s i s of t h e sample [25--27,79].

6.

S t a b i l i t y , Degradation, Mechanism of Action

I n d i c a t i v e of t h e l a c k of s e l e c t i v i t y of n i t r o g e n m u s t a r d s , t h e i r d e g r a d a t i o n and b i o l o g i c a l a c t i o n proceed through a common mechanism, an o u t l i n e of which i s p r e s e n t e d i n Scheme 111. According t o t h e n a t u r e of X, Scheme I11 i l l u s t r a t e s a wide range of r e a c t i o n s , such as t h e h y d r o l y t i c d e g r a d a t i o n of t h e compound o r b i o l o g i c a l l y s i g n i f i c a n t a l k y l a t i o n s of r e a c t i v e groups i n p r o t e i n s ( c a r b o x y l , s u l f h y d r y l ) and i n n u c l e i c a c i d s ( p h o s p h o r y l , h e t e r o a r o m a t i c amino, h e t e r o a r o m a t i c h y d r o x y l ) . The o v e r a l l k i n e t i c s of t h e a l k y l a t i o n depend on t h e r e l a t i v e r a t e s of t h e r e a c t i o n s involved. For t h e a l i p h a t i c n i t r o g e n m u s t a r d s , t h e f a s t f o r m a t i o n of t h e c y c l i c immonium i o n V I I I and t h e o v e r a l l second-order k i n e t i c s f o r t h e r e a c t i o n have been supported by s e v e r a l a n a l y t i c a l t e c h n i q u e s i n c l u d i n g N M R [ 8 1 ] . There i s l e s s agreement on t h e r e a c t i o n mechanism f o r t h e aromatic n i t r o gen mustards [ 4 ] . The reduced b a s i c i t y of t h e a r o m a t i c n i t r o g e n mustards r e t a r d s t h e i o n i z a t i o n s t e p and makes i t rate-determining. It w a s i n i t i a l l y p o s t u l a t e d t h a t t h i s i o n i z a t i o n i n v o l v e s t h e format i o n of t h e c a r b o c a t i o n I X [ 9 , 8 2 , 8 3 ] , but t h i s view has been c h a l l e n g e d by a u t h o r s who c l a i m t h a t t h e c y c l i c immonium i o n is common t o a l l n i t r o g e n mustards [ 4 0 , 6 2 , 8 1 ] . The h y d r o l y s i s of melphalan (and of most aromatic n i t r o g e n m u s t a r d s ) r e p r e s e n t s t h e most important d e g r a d a t i o n mechanism and has been e x t e n s i v e l y s t u d i e d . The f i r s t r e p o r t s showed a 22% h y d r o l y s i s under t h e s t a n d a r d c o n d i t i o n s designed by Ross f o r t h e assessment of aromatic n i t r o g e n mustard r e a c t i v i t y (30 min r e f l u x i n acetone--water 1:1, v / v ) [ 1 0 , 8 4 ] . Rate c o n s t a n t s f o r t h e d i s a p p e a r a n c e of melphalan i n v a r i o u s media a r e l i s t e d i n Table V I .

Scheme I11

VI

Helphalan

VII

A. Hydrolysis of melphalan. N 0 \o

VIII

CHZCHzC1 R-N'

X-H

] : :;i!-R[

'CH~CH~CL

B . P o s s i b l e mechanism of a l k y l a t i o n .

c

R-N-CHzCH2X I CHZCHZCl

Table V I Rate Constants For t h e Disappearance of Melphalan i n Various Media Solvent

N

2

TemDerature. " C

k. h-l

Reference

0 23 37 25 25 25 41 25

0.04 0.13 0.83 0.132 0.140 0.146 0.994 0.176

40 40 40,90 53 53 53 53 53

25 25 37 37 37 25

0.103 0.043 0.44 0.78 0.28 0.0057

53 53 a7 87 87 53

37

0.31--0.45

40

Water Water Water 0.01 M C i t r a t e b u f f e r (pH 3) 0.01 3 Phosphate b u f f e r (pH 5) 0.01 Z P h o s p h a t e b u f f e r (pH 7 ) 0.01 Phosphate b u f f e r (pH 7 ) 0.01 tris(hydroxymethy1)amiFomethane (pH 9) pH 7 Buffer + 0.019 M NaCl pH 7 Buffer + 0.15 M-NaC1 0.013 M HC1 (pH 2.4T 0.013 KH3P04 (pH 2.6) 0.156 NaCl (pH 5.4) Burroughs-We1 lcome i n j e c t a b l e kita Human plasma

a Three component k i t . Melphalan (100 mg) i s d i s s o l v e d i n 1 m l of 92% e t h a n o l c o n t a i n i n g 2% (w/v> HC1. This s o l u t i o n i s d i l u t e d with 9 m l of a s o l u t i o n c o n s i s t i n g of 1.2% (w/v) K2HP04 and 60% (w/v> propylene g l y c o l i n water. The f i n a l s o l u t i o n has a pH of about 7.

L. VALENTIN FEYNS

292

The d i s a p p e a r a n c e of melphalan from aqueous s o l u t i o n s h a s been shown t o f o l l o w f i r s t - o r d e r k i n e t i c s [62,82,85] and t h e c o n c e n t r a t i o n p r o f i l e d u r i n g h y d r o l y s i s is c o n s i s t e n t w i t h a mechanism c o n s i s t i n g of two c o n s e c u t i v e pseudo f i r s t o r d e r r e a c t i o n s (Scheme I11 A) 1531. Of t h e systems l i s t e d i n Table VI, melphalan i s most s t a b l e i n t h e Burroughs-Wellcome i n j e c t a b l e k i t . The i n s t r u c t i o n s recommend t h e use of t h e s o l u t i o n w i t h i n 15--30 minutes. According t o t h e m a n u f a c t u r e r , 8.5% h y d r o l y s i s t a k e s p l a c e i n 24 h o u r s a f t e r mixing 1861. The s t a b i l i t y o f melphalan i n c r e a s e s i n a c i d i c s o l u t i o n s and h i g h e r temperat u r e s a c c e l e r a t e t h e h y d r o l y s i s . The t y p e and c o n c e n t r a t i o n of a n i o n i c s p e c i e s p r e s e n t i n t h e s o l u t i o n a l t e r t h e h y d r o l y s i s r a t e . The formation of t h e i n t e r m e d i a t e i o n i z e d s p e c i e s i s r e t a r d e d a n d / o r r e v e r s e d by t h e presence of c h l o r i d e ions [9,82,87] , r e s u l t i n g i n a s i g n i f i c a n t l y increased s t a b i l i t y [53,87,88]. The s t a b i l i t y of melphalan is i n c r e a s e d i n t h e p r e s e n c e of bovine serum albumin, human plasma, b i l e a c i d s , and b i l e , probably by hydrophobic i n t e r a c t i o n s t h a t make t h e c h l o r o e t h y l moiety less a c c e s s i b l e t o n u c l e o p h i l i c a t t a c k [40,62,74,87,89--911

.

S o l u t i o n s i n methanol were found t o r e t a i n f u l l i n t e g r i t y f o r more t h a n 1 2 hours a t room t e m p e r a t u r e and f o r a t l e a s t 4 weeks at -20" [ 7 8 ] . Melphalan is s t a b l e i n s o l u t i o n s of d i m e t h y l s u l f o x i d e a t -20" f o r over a month even w i t h f r e q u e n t thawing [ 511. Melphalan i s l i g h t - s e n s i t i v e and t h e packaging and storing instructions call for tight, light-resistant containers. 7.

T o x i c i t y , D i s t r i b u t i o n , Metabolism, E x c r e t i o n 7.1

Toxicity

D e t a i l e d d a t a on t h e t o x i c o l o g y and pharmacology of melphalan (and o t h e r a n t i t u m o r a g e n t s ) were r e p o r t e d i n 1965 by Schmidt and c o l l a b o r a t o r s [ 2 1 ] . The LDIO and LD50 v a l u e s f o r mice and r a t s show c o n s i d e r a b l e v a r i a t i o n s among s t r a i n s ; i n t r a v e n o u s and i n t r a p e r i t o n e a l a d m i n i s t r a t i o n s were twice as t o x i c as t h e o r a l d o s i n g - The L D l o v a l u e s ranged from 4 t o 15 mg/kg f o r mice and from 1 . 3 t o 3 mg/kg f o r r a t s 13,211. LD values of 2 t o 16 mg/kg were r e p o r t e d f o r r a t s [ 2 1 , 7 7 , 9 2 j . It w a s a l s o r e p o r t e d t h a t t h e LD50 v a l u e s i n mice v a r i e d with t h e time of dosing [ 9 3 ] .

MELPHALAN

293

The c l i n i c a l t o x i c o l o g y is m o s t l y h e m a t o l o g i c a l [21. 7.2

Carcinogenicity

Melphalan induced cancer at t h e s i t e of i n j e c t i o n ( p e r i t o n e a l c a v i t y ) i n mice and r a t s given t h r e e i n j e c t i o n s per week a t t h e maximally t o l e r a t e d dose [ 9 4 ] . It was a l s o r e p o r t e d t o i n c r e a s e t h e r i s k f o r development of leukemias [ 3 ] .

7.3

Distribution

Following o r a l a d m i n i s t r a t i o n t o human s u b j e c t s , peak blood l e v e l s for melphalan were seen a t 2 hour [ 4 4 ] , much slower than i n animals [ 3 , 9 1 , 9 5 ] . The drug i s w e l l absorbed when given t o animals by t h e o r a l r o u t e [ 3 , 9 1 ] , but c o n s i d e r a b l e v a r i a b i l i t y i n a b s o r p t i o n by humans was reported [41,96,97]. A f t e r i n t r a v e n o u s a d m i n i s t r a t i o n , melphalan d i s a p p e a r s from t h e plasma with h a l f - l i v e s of 67 min and 160 hour [ 4 4 ] . I n a p a t i e n t administered a 5-minute i n t r a v e n o u s bolus of 140 mg of melphalan p e r m 2 , t h e a l p h a and b e t a h a l f - l i v e s i n plasma were found t o be 6.2 and 53.5 minutes, r e s p e c t i v e l y [ 5 4 ] . Binding of t h e drug t o plasma p r o t e i n s i s e x t e n s i v e [ 4 4 ] , confirming t h e i n - v i t r o s t u d i e s [ 4 0 , 4 8 , 7 4 , 8 9 , 9 8 ] ; t h i s might have a profound e f f e c t on t h e in-vivo s t a b i l i t y of t h e drug [ 9 7 ] . The dihydroxy d e r i v a t i v e VII is d e t e c t a b l e i n plasma more than t h r e e weeks a f t e r a d m i n i s t r a t i o n [ 7 6 ] . It was suggested t h a t t h e prolonged decay phase [541 i s due t o h y d r o l y s i s products and m e t a b o l i t e s r a t h e r than t h e p a r e n t compound [ 4 1 ] . The a b s o r p t i o n from t h e p e r i t o n e a l c a v i t y one hour a f t e r a d m i n i s t r a t i o n was 25% [ 5 7 ] . The kidney, l i v e r , and s p l e e n had t h e h i g h e s t s p e c i f i c a c t i v i t y a f t e r a d m i n i s t r a t i o n of l a b e l e d melphalan t o r a t s and mice [ 6 8 , 9 8 ] , while i n dogs and monkeys t h e h i g h e s t l e v e l s of drug were i n t h e b i l e [ 3 , 7 5 , 9 9 ] . No s i g n i f i c a n t c o n c e n t r a t i o n s i n t h e tumor were n o t e d , and t h e i n h i b i t i o n of t h e tumor growth could not be a t t r i b u t e d t o i n t e r a c t i o n s with p r o t e i n s , DNA, o r RNA w i t h i n t h e tumor c e l l [ 6 8 ] . C e l l u l a r t r a n s p o r t of melphalan by amino a c i d c a r r i e r systems has been r e p o r t e d [ 100--1051.

7.4

Metabolism Only t h e mono- and d i h y d r o x y - d e r i v a t i v e s V I and

L. VALENTIN FEYNS

294

VII were identified in plasma and urine [ 8 5 , 9 9 ] , and it is difficult to distinguish between m tabolic changes and chemical degradation. Up to ten “C-labelled products of metabolism or hydrolysis of melphalan were detected without identification in the serum, urine, and bile of experimental animals. Metabolic pathways similar to that of phenylalanine were suggested [ 3 1 .

7.5

Excretion

In man, urinary excretion accounts for most drug elimination after intravenous administration ( 5 0 % within 24 hr, 7 5 % over 7 days), and only small amounts are recoverable from the stool [ 4 4 , 8 6 ] . Following oral administration of labelled melphalan, 30% of the label was recovered in urine over 9 days and 20--50% in feces over 6 days [ 4 4 ] . In animals also, urinary excretion appears to be the major means of elimination of the drug from the body [3,68,91,99]. 8.

Determination in Body Fluids and Tissues

The separation and measurement of melphalan in biological samples are hampered by its instability and by the amphoteric nature of the molecule. Some of the earlier publications did not make corrections for reversible protein binding and decomposition during the work-up of the sample. They also lacked sensitivity and specificity. The following techniques were reported: administration of labelled melphalan, followed by radioactivity measurements [ 4 4 , 9 1 , 9 8 ] ; formation of a colored complex by oxidation with FeCl and H202 [ 1 0 6 ] ; colorimetric assay with 4(p-nitrobenzyljpyridine (detection limit--5 pg/ml of plasma) [60,61,107,108]; fluorescence measurements (interference by tissue constituents) [ 4 8 ] ; thin-layer chromatography and densitometry (detection limit--20 ng/ml of plasma) [ 7 2 ] ; thin-layer chromatography followed by radioactivity measurements or derivatization and mass spectrometry [ 4 4 ] ; high-pressure liquid chromatography (detection limit with fluorescence detection--less than 5 ng/ml of plasma) [3,40,54,74,76--78,91,99]; and derivatization--gas chromatography--chemical ionization mass spectrometry (detection limit--less than 2 ng/ml of plasma) [ 4 1 ] .

MELPH ALAN

9.

295

I d e n t i f i c a t i o n and Determination i n Pharmaceutical Preparations

9.1

Tablets Identification

The I d e n t i f i c a t i o n T e s t s i n t h e o f f i c i a l compendia a r e s i m i l a r t o t h o s e used f o r t h e drug s u b s t a n c e : ( a ) e x t r a c t i o n of t h e drug with methanol, t r e a t m e n t with 4( p - n i t r o b e n z y 1 ) p y r i d i n e a t 80" and a l k a l i n i z a t i o n w i t h potassium hydroxide o r ammonia a f t e r c o o l i n g , producing a v i o l e t t o r e d - - v i o l e t c o l o r [ 2 5 , 4 6 , 7 9 ] ; ( b ) t h e W spectrum of a methanolic e x t r a c t e x h i b i t s a maximum a t 260 nm and a l e s s w e l l d e f i n e d maximum a t 301 mn [ 2 5 , 4 6 ] ; and ( c ) t h e r e t e n t i o n t i m e of t h e major peak i n t h e HPLC chromatogram of t h e Assay P r e p a r a t i o n is t h e same a s t h a t of a Standard P r e p a r a t i o n of Melphalan Reference Standard [ 7 9 ] . 4 ssay

The following procedures were r e p o r t e d f o r t h e a s s a y of Melphalan T a b l e t s : ( a ) h y d r o l y s i s w i t h aqueous potassium hydroxide under r e f l u x and p o t e n t i o m e t r i c t i t r a t i o n of the l i b e r a t e d c h l o r i d e i o n s w i t h s i l v e r n i t r a t e i n t h e presence of n i t r i c a c i d . (Corrections for "ionisable c h l o r i n e " a r e made by t i t r a t i n g under t h e same c o n d i t i o n s a sample t h a t i s not s u b j e c t e d t o h y d r o l y s i s [ 2 5 , 4 6 ] . 1; ( b ) HPLC Assay of an e t h y l a l c o h o l - a c e t i c a c i d e x t r a c t a g a i n s t a Reference Standard m a t e r i a l (chromatographic c o n d i t i o n s : reversed-phase mode; c18 column; methanol--water--acetic a c i d , 50:49:1 ( v / v ) , mobile phase; d e t e c t i o n a t 254 nm [ 7 9 ] ) ; and ( c ) e x t r a c t i o n of t a b l e t s w i t h methanol or e t h a n o l and UV measurements a t 260 o r 262 nm. L i n e a r i t y i n t h e 3--15 pg/ml and s t a n d a r d e r r o r s of l e s s t h a n 2% were r e p o r t e d [65,109,110]. A s i m i l a r procedure i s used f o r t h e d e t e r m i n a t i o n of t h e Content Uniformity of t h e t a b l e t s [ 7 9 ] . .

The c o n c e n t r a t i o n p r o f i l e d u r i n g t h e d i s s o l u t i o n t e s t i n g of melphalan t a b l e t s i n water and simulated g a s t r i c f l u i d was determined by WLC. A method of d a t a a n a l y s i s was developed t o t a k e i n t o account t h e spontaneous drug degradat i o n [111]. 9.2

Melphalan I n j e c t i o n [46]

Melphalan I n j e c t i o n i s d e f i n e d a s a s t e r i l e s o l u t i o n of melphalan h y d r o c h l o r i d e . It i s a v a i l a b l e commercially as a k i t comprising melphalan i n a s e a l e d

296

L. VALENTlN FEYNS

c o n t a i n e r , a d i s s o l v i n g s o l v e n t , and a d i l u t i n g s o l v e n t ( s e e f o o t n o t e i n Table VI). The i d e n t i f i c a t i o n r e q u i r e m e n t s i n c l u d e examinat i o n of t h e W spectrum of a methanolic s o l u t i o n , p o s i t i v e r e a c t i o n with 4-(p-nit robenzyl ) pyr i d i n e , p o s i t i v e r e a c t i o n for chlorides a f t e r alkaline hydrolysis, levorotatory o p t i c a l a c t i v i t y f o r a s o l u t i o n i n methanol, and a m e l t i n g p o i n t of about 177" w i t h decomposition. The a s s a y i s performed by p o t e n t i o m e t r i c t i t r a t i o n of t h e c h l o r i d e i o n s l i b e r a t e d by a l k a l i n e h y d r o l y s i s , a s d e s c r i b e d under 9.1 Assay ( a ) . Other p u r i t y r e q u i r e m e n t s i n c l u d e a c l a r i t y and a c i d i t y of s o l u t i o n t e s t , and l i m i t s f o r i o n i z a t i o n c h l o r i n e , loss on d r y i n g , and s u l p h a t e d ash. Acknowledgments The a u t h o r wishes t o thank D r . Richard Lindauer of t h e United S t a t e s Pharrnacopeia, R o c k v i l l e , Maryland f o r h i s h e l p f u l comments on t h e manuscript and f o r p e r m i s s i o n t o use d a t a o b t a i n e d i n t h e Drug Research and T e s t i n g Laboratory of USP. The h e l p of D r . James Shoolery of Varian A s s o c i a t e s , P a l o A l t o , C a l i f o r n i a and of D r . J i m K e l l e y of t h e N a t i o n a l Cancer I n s t i t u t e , Bethesda, Maryland i n t h e i n t e r p r e t a t i o n of t h e NMR and mass s p e c t r a , and t h e t e c h n i c a l a s s i s t a n c e of M r . Anthony J . Manna and M r . G. S c o t t Kobler a r e g r a t e f u l l y acknowledged. C r e d i t f o r p r o c e s s i n g t h e manuscript belongs t o Mrs. P a t r i c i a Perando and Mrs. Barbara Bowman.

297

MELPHALAN

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F. Bergel and J. A. Stock, B r i t . 783,292, Sept. 18, 1957; C.A. 52 5464b (1958).

29.

F. Bergel and J. A. Stock, U.S. C.A. 58 7871d (1963).

30.

F. Bergel and J. A. Stock, B r i t . 1956; C.A. 51 1274g (1957).

-

-

-

3,032,584, May 1, 1962;

750,155, June 13,

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299

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L. V. Feyns and E. G. Dan, Rom. 57,195, 20 J u l . 1974, C.A. 83, 97932 (1975).

32.

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104,781, Feb. 25,

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33.

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G . F e r r a r i , Farmaco ( P a v i a ) Ed. S c i . 11, 524 (1956); C.A. 53 10056 (1959).

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35.

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Ger. 1,292,660;

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V . N. Konyukhov, e t al., B r i t . 1979; C.A. 67 73325 (1967).

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39.

R. Kliukene, e t a l . , 83 10858 (1975).

40.

S. Y. Chang, D. S. A l b e r t s , D. Farquhar, L. R. Melnick, P. D. Walson and S. E . Salmon, J. Pharm. S c i . 67 ( 5 ) 682 (1978).

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Brit.

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J . Blazek and J. Kracmar, Cesk. Farm. 11 ( 4 ) 210 ( 1 9 6 2 ) ; C.A. 62 10294c ( 1 9 6 5 ) .

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USP XX--NF XV, Third Supplement, The United S t a t e s Pharmacopeial Convention, R o c k v i l l e , Maryland, 1982, p. 181.

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-

For t h i s p r o f i l e , t h e l i t e r a t u r e h a s been searched through Chemical A b s t r a c t s Vol. 96 (1982).

MOXALACTAM DISODIUM Leslie J. Lorenz and Patricia N. Thomas Lilly Research Laborntvries Indititiapvlic, Indiana

1.

2.

3. 4.

5.

6.

7.

8.

Description 1.1 Name 1.2 Structure 1.3 Molecular Formula 1.4 Molecular Weight I .5 Appearance Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Mass Spectrum 2.4 Ultraviolet Spectrum 2.5 Optical Rotation 2.6 Differential Thermal Analysis 2.7 Thermogravimetric Analysis 2.8 Dissociation Constants 2.9 Solubility Properties 2.10 Crystal Properties Chemical Synthesis Stability 4.1 Bulk Stability 4.2 Solution Stability Bacteriology and Pharmacokinetics 5.I Bacteriology 5 . 2 Pharmacokinetics Methods of Analysis 6.1 ldentification Tests 6.2 Quantitative Tests 6.3 Related Substance Assays Analysis of Biological Samples 7.1 Microbiological Assay 7.2 Chromatography Analysis of Pharmaceutical Formulations References


305

306 306 306 306 307 307 307 307 307 312 313 315 315 315 315 315 316 316 318 318 318 318 319 320 322 322 323 326 327 321 327 327 329

Copyright C 1984 hy the American Pharmaceutical Association ISBN 0-12-260813-5

LESLIE J. LOREN2 AND PATRICIA N. THOMAS

306

1. Description 1.1.

Name

Moxalactam disodium is a drug entity which was discovered at Shionogi and Company, Limited, Osaka, Japan, and codeveloped with Eli Lilly and Company. The drug is marketed under the trade names of MOXAM" and LAMOXAMTN. The chemical entity is the disodium salt of (6R,7R)-7-[[carboxy(4-hydroxy-phenyl)acetyl]amino]-7-methoxy-3-[ [ l-methyl-1H-tetrazole-5-yl )thiolmethyl ]-8-oxo-5-oxa-l-azabicycl o[ 4.2.O]oct-2-ene-2-carboxyl i c acid. 1.2.

HO

Structure

H OCH3H .NI-~++JH E O 0

N1 2/

H

1 TET

S k C6* 3'

N

'=YN CH3

As seen in the structure, moxalactam disodium has three asymmetric centers. Two centers in the ring system, C6 and C7, are stereospecifically defined during the biosynthesis of the penicillin used to produce the compound. A third asymmetric center exists adjacent to the amide carbon on the side chain of the antibiotic. The configuration o f this center is free to equilibrate and thus a pair of diasterioisomers is possible for moxalactam disodium. The rate of interconversion between the isomers increases with increasing acidity, with the maximum rate occurring at a pH of about 2.5. For solutions with pH lower than 2.5, the rate of interconversion is decreased only slightly from the maximum rate. 1.3. c2

Molecular Formula

The molecular formula for moxalactam disodium i s oH2 0N609SNa2.

307

MOXALACTAM DISODIUM

1.4.

Molecular Weight

The molecular weight f o r moxalactam disodium i s 564.44. 1.5.

Appearance

Moxalactam disodium i s a white t o s l i g h t l y cream-colored amorphous powder. 2.

Physical Properties 2.1.

Infrared Spectrum

The infrared spectrum of moxalactam disodium i n a potassium bromide p e l l e t i s presented i n Figure 1. The assignments for the spectral peaks a r e given i n Table 1. Table 1 ~~~

~~

IR Absorption Band (cm-l) 3600-2500 (broad multiple bands) 1770 1680 1610

1515 1410 1380, 1350

1250 (broad) 820 2.2.

Assignment g-OH, H20, OH stretching, s t r o n g hydrogen bonding 8-lactam, C=O stretching Amide I , C=O stretching C O T , asymmetrical stretching Amide I1 CO;, symmetrical stretching Tetrazol e , N-CH3 , CH2 (C-H bending) @-OH C-0 stretching p - d i s u b s t i tuted phenyl (C-H bending )

Nuclear Magnetic Resonance Spectrum

Figure 2 shows the proton magnetic resonance spectrum f o r moxalactam disodium. The spectrum was recorded on a 60 MHz instrument. An interpretation o f the spectrum i s presented i n Table 2. The spectrum i s complex, f o r the configuration a t s i t e 01 undergoes rapid equilibrium and t h e proton a t cc i s exchanged w i t h deuterium. A t h i g h resolution such a s a t 360 MHz every resonance i s s p l i t except f o r that of Ho.

1

\I \ I 1

1.282

" V

.ooo -

I

1

I

I

I

1

I

I

I

I

I

I

I

4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400 I/CM Figure 1.

The Infrared Spectrum of Moxlactam Disodium i n a KBr Pellet

cv

c3

E

.-3

ir 0

V

a,

Q

Ln

S

0

L

0

42

a

LESLIE J . LORENZ AND PATRICIA N . THOMAS

310

Table 2 Proton Magnetic Resonance Spectrum

H 0

H oCH3 I I H

r - 0 Na+

I

Na+ -0 €!E 3.5 4.0 4.1 4.5 5.1 6.83

H

U

I

2=*

Multiplicity two singlets two singlets complex AB patterns complex AB patterns two singlets doublet j-8Hz not vi sibl e fully deuterium exchanged

Figure 3 shows the carbon-13 nuclear magnetic resonance spectrum obtained for moxalactam disodium. The spectrum was run in deuterium oxide with dioxane present as a spectral marker. Moxalactam again has a complex spectrum, for the configuration at the C(11) carbon undergoes inversion. This tends to cause doublipg of peaks; therefore, assignments for the aromatic resonances are difficult. Table 3 provides the assignments for the carbon-13 spectrum.

!

L

200

I

I

I

I

I

I

150

F i sure 3. -

I

I

I

I

I

I

I

1

100 PPM (6)

The Carbon-13 Nucl ear Magnetic Resonance S ectrum for Moxalactam Disodium in D20 Paus Dioxane

I

50

I

I

I

I

1

0

1

LESLIE J . LORENZ AND PATRICIA N. THOMAS

312

HO 4"\

/1"

1 TET

7

k

1

3'

N

'VN CHS

Table 3 p pm

Assignment

32.4 34.8 53.8 61.5 65.9 67.5 83.5 94.9 116.n 124.4 124.6 129.5

N-CH3

154.8 155.8 163.2 167.2 174.6 176.7 2.3.

C(3' 1 OCH 3 C(11) C(4)

Dioxane marker

C(6) C(7)

aromatic carbons C(2") ,C(3"), C(5") ,C(6"), C(2), and C(3) C (1-TET) C(4") C(8)

2c01

C(12) C(10)

Mass Spectrum

Moxalactam disodium can y i e l d a mass spectrum when r u n i n the f i e l d desorption mode. The major ions f o r moxalactam disodium are a t m/z o f 116 and 130. These are a t t r i b u t e d t o

MOXALACTAM DISODIUM

313

1+'

1 +'

N-N

I

I

CH,

derived from t h e t e t r a z i n e moiety a r i s i n g from p y r o l t i c processes. The f i e l d desorption mass spectrum o f moxalactam d i a c i d gives i o n s a t m/z o f 543, 521, 477, and 361. The majortion A i s a t m/z o f 521 which i s t h e quasi-molecular i o n [ M t H ] small i o n i s observed a t m/z o f 543 which i s t h e c a t i o n i z e d molecular i o n o f moxalactam [MtNa]'. The i o n a t m/z o f 477 i s t h e r e s u l t o f t h e decarboxylation o f t h e parent compound. The i o n observed a t m/z o f 361 i s l i k e l y caused by t h e e l i m i n a t i o n o f C o n and

.

N-N N\N " A

s t i

I

CH,

from t h e parent compound. 2.4.

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

F i g u r e 4 shows t h e u l t r a v i o l e t spectrum f o r moxalactam disodium. The major chromophore c o n t r i b u t i n g t o t h e u l t r a v i o l e t absorbance spectrum i s t h e 3-oxacephem nucleus. This e n t i t y leads t o peaks a t 271 nm (~=12,100) which i s assigned t o a r-m* t r a n s i t i o n and a t 229 nm (~=18,000) which i s a n-m* t r a n s i t i o n .

253-

.ooo I

210.0

I

235.0 260.0

I

I

285.0 310.0

I

335.0

I

360.0 385.0

NM Figure 4.

I

The U l t r a v i o l e t Spectrum of Moxalactam Disodium i n Water

I

410.0

MOXALACTAM DISODIUM

2.5.

315

Optical Rotation

In section 1.2. moxalactam disodium i s described as consisting of a pair of diasterioisomers which can interconvert in solution. The observed value for optical rotation is therefore a function of the isomer distribution in solution at the time of measurement. No experimental data areavailable for the rotation of the pure isomers. 2.6.

Differential Thermal Analysis

The thermogram for moxalactam disodium shows an exotherm at about 170°C. This exotherm indicates the decomposition of the substance. 2.7.

Thermogravimetric Analysis

The thermogram for moxalactam disodium shows a weight loss throughout the curve beginning at about 30°C. A major loss in mass occurs near 170°C which corresponds to the exotherm in the DTA curve for moxalactam disodium. Moxalactam disodium is thermally labile and undergoes increased decomposition with increasing temperature. 2.8.

Dissociation Constants

The following dissociation constants have been determined for moxalactam disodium: Solvent H 20 66% DMF 2.9.

Carboxyl 1

pKa

Carboxyl 2

2.4 4.9

3.5 6.1

Phenol 9.95 12.9

Solubility Properties

The solubility properties of rnoxalactam disodium are described in Table 4.

316

LESLIE J . LOREN2 AND PATRICIA N . THOMAS

Table 4 Sol vent

Sol ubi 1i t y (mg/ml )

>loo. >loo. goo. >loo.

Water pH 1.2 (USP XIX) pH 4.5 (USP XIX) pH 7.0 (USP XIX) Methanol Octanol Isopropanol Diethyl e t h e r Ethyl a c e t a t e Chloroform Benzene Cycl ohexane

2.10.

-

>50. b u t .

2.2 S o l u b i l i t y P r a c t i c a l l y i n s o l u b l e i n water, s o l u b l e i n 3 p a r t s of a l c o h o l ( 9 5 % ) , i n 20 p a r t s of chloroform, i n 20 p a r t s of s o l v e n t e t h e r and i n 6 p a r t s of a c e t o n e ; a l s o s o l u b l e i n s o l u t i o n s of a l k a l i h y d r o x i d e s ( 3 ) . 2.3 I d e n t i f i c a t i o n 2.3.1 I n f r a r e d S p e c t r o s c o p i c Test The i n f r a r e d a b s o r p t i o n c h a r a c t e r i s t i c s of oxyphenbutazonehave been used by t h e B.P. (3) and U.S.P. ( 4 ) as a meansfor i d e n t i f y i n g t h e d r u g . The i n f r a r e d a b s o r p t i o n spectrum e x h i b i t s m a x i m a which a r e only a t t h e same wavelengths a s , and have s i m i l a r r e l a t i v e i n t e n s i t i e s t o , t h o s e i n t h e spectrum of a s i m i l a r p r e p a r a t i o n of a s t a n d a r d oxyphenbutazone. The i n f r a r e d characterist'ics of oxyphenbutazone s h a l l be d i s c u s s e d l a t e r i n t h e s p e c t r a l p r o p e r t i e s of t h e drug.

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2.3.2 U l t r a v i o l e t S p e c t r o s c o p i c T e s t The B.P. and U.S.P. a l s o recommend t h e u s e of u l t r a v i o l e t c h a r a c t e r i s t i c s of oxyphenbutazone as an i d e n t i f i c a t i o n t e s t . A s o l u t i o n of t h e d-.,- I n sodium hydroxide e x h i b i t s maxima and minima a t t h e same wavelengths a s t h a t of a similar s o l u t i o n of a s t a n d a r d d r u g . The u l t r a v i o l e t a b s o r p t i o n c h a r a c t e r i s t i c s of oxyphenbutazone s h a l l b e d i s c u s s e d l a t e r i n t h e s p e c t r a l p r o p e r t i e s of t h e d r u g . LUh

2.3.3 Color Tests Table 1 l i s t s some of t h e c o l o r t e s t s r e p o r t e d f o r t h e i d e n t i f i c a t i o n of oxyphenbutazone. Table 1.

Color T e s t s f o r Oxyphenbutazone.

Reagents

Color

Ref.

i) Hot s o l u t i o n of t h e drug i n g l a c i a l a c e t i c

(3)

and h y d r o c h l o r i c a c i d s . Cool and f i l t e r . F i l t r a t e p l u s sodium nitrite A d d i t i o n of 2-naphthol solution P p t + 95% a l c o h o l i i ) Drug i n a m i x t u r e of sodium hydroxide and aminoacet i c a c i d .

i i i ) S o l u t i o n of d r u g i n methanol p l u s M i l l o n ' s Reagent, h e a t . iv) Sulfuric acid dehyde

-

formal-

v) Ammonium molybdate vi) Vitali's Test

Yellow c o l o r B r i g h t orange p p t . Orange s o l u t i o n Solution c l e a r with a n e x t i n c t i o n of 4cm l a y e r a t 420 nm i s n o t more t h a n 0.40.

(3)

Cherry r e d p p t .

(4)

P a l e yellow (sensit i v i t y : 1.0 pg).

(2)

Pale orange ( s e n s i t i v i t y : 1.0 ug).

(2)

Pale yellow o r red ( s e n s i t i v i t y : 0.25 pg)

.

(2)

337

OXYPHENBUTAZONE

2.3.4 C r y s t a l T e s t s Crystal tests reported (2) include: a ) Treatment w i t h p l a t i n i c i o d i d e s o l u t i o n g i v e s r o s e t t e s of p r i s m s , forming o v e r n i g h t . The s e n s i t i v i t y of t h e t e s t i s 1 i n 100. b ) Treatment w i t h p o t a s s i u m i o d i d e s o l u t i o n g i v e s s h e l l - l i k e c r y s t a l s , forming o v e r n i g h t . The s e n s i t i v i t y of t h e t e s t i s 1 i n 100. 2.3.5

Chromatographic T e s t s

a) C l a r k e ( 2 ) d e s c r i b e d a p a p e r c h r o m a t o g r a p h i c method f o r t h e i d e n t i f i c a t i o n of t h e d r u g u s i n g t h r e e d i f f e r e n t s o l v e n t systems. System I. C o n s i s t s of c i t r i c a c i d : water: n - b u t a n o l ( 4 . 8 Em: 130 ml : 870 m l ) . Detect i o n c a r r i e d out using u l t r a v i o l e t l i g h t a b s o r p t i o n . p-Dimethylaminobenzaldehyde o r p o t a s s i u m permanganate can b e used a s s p r a y t o l o c a t e t h e d r u g . Rf 0.96. DetecSystem 11. A c e t a t e b u f f e r (pH 4.58). t i o n i s e f f e c t e d by u l t r a v i o l e t l i g h t a b s o r p t i o n . Rf 0.04, System 111. p h o s p h a t e b u f f e r (pH 7 . 4 ) . Detect i o n i s e f f e c t e d by u l t r a v i o l e t l i g h t a b s o r p t i o n . Rf 0.58. b) An i d e n t i f i c a t i o n t e s t f o r t h e d r u g by t h i n l a y e r chromatography w a s a l s o r e p o r t e d by The s o l v e n t system c o n s i s t s o f : Clarke (2). s t r o n g ammonia s o l u t i o n : m e t h a n o l (1.5 : 100). D e t e c t i o n i s e f f e c t e d by p o t a s s i u m permangan a t e s p r a y . Rf 0 . 7 7 .

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2.4 S p e c t r a l P r o p e r t i e s 2.4.1 U l t r a v i o l e t Spectrum The u l t r a v i o l e t spectrum of oxyphenbutazone i n n e u t r a l methanol u s i n g Cary, 219 Spectrophotometer i s shown i n F i g u r e 1. The spectrum of t h e d r u g e x h i b i t s two maxima a t 243 and 265 nm and a minimum a t 221 nm. The u l t r a v i o l e t a b s o r p t i o n c h a r a c t e r i s t i c s of t h e drug a r e used by t h e B.P. ( 3 ) and U.S.P. ( 4 ) , as an i d e n t i f i c a t i o n t e s t . A 0.001 % w/v s o l u t i o n of oxyphenbutazone i n 0.01 N sodium hydroxide s o l u t i o n e x h i b i t s a maximum o n l y a t 254 nm w i t h an e x t i n c t i o n of about 1.5. Oxyphenbutazone i n e t h a n o l w a s a l s o r e p o r t e d ( 2 ) t o have a maximum a t 242 nm ( E l % 1 cm 564) and an i n f l e x i o n a t 275 nm. 2.4.2 I n f r a r e d Spectrum The i n f r a r e d spectrum of oxyphenbutazone i n n u j o l m u l l i s p r e s e n t e d i n F i g u r e 2. An i n t e r p r e t a t i o n of t h e spectrum i s g i v e n below. The i n f r a r e d a b s o r p t i o n spectrum of oxyphenbutazone i s used a s an i d e n t i f i c a t i o n t e s t by t h e B.P. and U.S.P. Frequency (cm

-1

)

Assignment

1288

C-N s t r e t c h

1702 1748

C=O s t r e t c h

3220

0-H s t r e t c h

1180

C-0

s t r e t c h of phenol

Reported (2) p r i n c i p a l i n f r a r e d a b s o r p t i o n peaks of oxyphenbutazone i n potassium bromide d i s c s are 1683, 1725, 1275 and 1518 cm-1. 2.4.3

1

1

H Nuclear Magnetic Resonance ( H NMR) Spectrum

1 The H NMR Spectrum of oxyphenbutazone i s pres e n t e d i n F i g u r e 3 . The drug was d i s s o l v e d i n acetone-d6 and i t s spectrum determined on a Varian-TGOA NMR Spectrometer u s i n g TMS as t h e

OXYPHENBUTAZONE

339

T C

c

Fig. 1. Ultraviolet spectrum of oxyphenbutazone in neutral methanol.

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

342

i n t e r n a l standard. a r e shown below. P r o t o n Assignments*

The s t r u c t u r a l assignments

Chemical S h i f t s (6)

Mu 1t i p 1i c it y

a

7.33

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I3C Nuclear Mangetic Resonance (I3C NMR) Spectrum The I 3 C NMR spectrum of oxyphenbutazone i n acetone-dg u s i n g TMS as an i n t e r n a l r e f e r e n c e w a s o b t a i n e d u s i n g a J e o l FX 100 MHz Spectrometer a t an ambient t e m p e r a t u r e u s i n g 1 0 mm sample and i s p r e s e n t e d i n F i g u r e 4 . The carbon chemical s h i f t v a l u e s are d e r i v e d from t h e o f f - r e s o n a n c e spectrum and are l i s t e d below. The r e s u l t s a r e consistent with those reported earlier ( 5 ) .

Carbon No.*

Chemical S h i f t s (PPm)

Carbon No.

Chemical S h i f t s (PPd

1'

137.2

6

28.7

3

170.9

6'

126.4

3'

129.3

7

28.0

4

46.4

7'

116.1

4'

124.3

8

20.0

5

171.5

8'

157.1

5'

127.2

9

13.9

*See s t r u c t u r e i n F i g u r e 4 f o r carbon assignment.

I

~

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,

I

-

I

I

I

13NMR spectrum of oxyphenbutazone in acetone -d Fig. 4. 6 with TMS internal reference.

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

344

2.4.5

Mass Spectrum and Fragmentometry

The f r a g m e n t a t i o n p a t t e r n s of oxyphenbutazone and o t h e r p y r a z o l i d i n e d i o n e s were d e s c r i b e d ( 6 ) b u t t h e v e r i f i c a t i o n by e v i d e n c e of m e t a s t a b l e i o n s , a c c u r a t e mass d e t e r m i n a t i o n s o r u s e of l a b e l e d d e r i v a t i v e s w a s p r e s e n t e d by Locock et a1 ( 7 ) . The mass s p e c t r a of oxyphenbutazone i s r e p r o duced i n F i g u r e 5. According t o Locock e t a1 ( 7 ) t h e drug undergoes McLafferty rearrangement t o g i v e a r a d i c a l i o n a t m / e 268. A m e t a s t a b l e i o n is p r e s e n t i n t h e spectrum t o i n d i c a t e t h a t t h i s i o n i s a d i r e c t f r a g m e n t a t i o n from t h e m o l e c u l a r i o n w i t h t h e l o s s of t h e e l e m e n t s of b u t e n e (Scheme I ) . L-

The l o s s of a p r o p y l r a d i c a l from t h e b u t y l s i d e c h a i n of t h e m o l e c u l a r i o n r e p r e s e n t s a minor bathway t o g i v e an i o n a t m / e 281. The most c h a r a c t e r i s t i c f r a g m e n t s i n t h e m a s s spectrum of oxyphenbutazone w e r e a s e r i e s of p e a k s a t m / e 198, 199 and 200. The peak a t m / e 198 i s d u e t o t h e f o r m a t i o n of t h e azobenzene r a d i c a l i o n ( C ~ H ~ N ~ C G H , ) ~The r e m a i n i n g p e a k s a t m / e 199 and 200 t o g e t h e r w i t h o t h e r p e a k s r e s u l t i n g from t h e f r a g m e n t a t i o n o f oxyphenbutazone are e x p l a i n e d by Scheme I.

.

Other p e a k s i n t h e low m a s s r a n g e , below m / e 100, a r e common t o s u b s t i t u t e d a r o m a t i c s y s t e m s (Fig. 5 ) .

3. Synthesis Oxyphenbutazone w a s s y n t h e s i z e d (8) b y c o n d e n s a t i o n of t h e p r o t e c t e d aminophenol ( I , Scheme 2 ) w i t h n i t r o b e n z e n e (11). Reduction of t h e r e s u l t i n g azo-compound (111) gave t h e hydrazobenzene ( I V ) which when condensed w i t h d i e t h y l b u t y l -

Fig. 5 .

Mass spectrum of oxyphenbutazone (7).

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

348

malonate (V) gave t h e h e t e r o c y c l e ( V I ) . Removal of t h e b e n z y l group was a c h i e v e d by h y d r o g e n o l y s i s .

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4 . Absorption, Metabolism and E x c r e t i o n Oxyphenbutazone i s one of t h e main m e t a b o l i t e s of phenylIt is rapidly butazone and i s c o n s i d e r e d t o be more t o x i c . absorbed from t h e g a s t r o i n t e s t i n a l t r a c t and slowly metab o l i z e d and e x c r e t e d mainly i n u r i n e . The r a t e of i t s metabolism v a r i e s i n d i f f e r e n t s p e c i e s a s r e f l e c t e d by t h e great variance i n half-lives. I n h i s s t u d y on the metabolism of b u t a z o l i d i n e , Herrmann (9) measured t h e h a l f - l i f e of oxyphenbutazone i n r a t s and r a b b i t s a f t e r i n t r a v e n o u s a d m i n i s t r a t i o n of t h e d r u g . They w e r e 8 and 4-5 h r s r e s p e c t i v e l y . Weiner e t a1 (10) s t u d i e d t h e d r u g d i s p o s i t i o n p a t t e r n s i n subhuman p r i m a t e s a s compared t o humans and o t h e r s p e c i e s . I n t h e babooqoxyphen-

OXY PHENBUTAZONE

349

b u t a z o n e h a s a s h o r t h a l f - l i f e , w h i l e i n man it h a s a l o n g half-life. The plasma b i n d i n g and a p p a r a n t volumes o f d i s t r i b u t i o n of t h e d r u g are s i m i l a r i n t h e baboon and man. T h e r e i s s u g g e s t i v e e v i d e n c e t h a t a b s o r p t i o n may n o t Studies b e a s complete i n t h e baboon a s i t i s i n man. i n dogs i n d i c a t e d t h a t t h e d r u g is n o t absorbed i n t h e l y m p h a t i c s . Burns (11) compared t h e r a t e s o f metabolism of oxyphenbutazone i n man, r h e s u s monkey and dogs. The h a l f l i v e s showed g r e a t v a r i a t i o n s . P r e v i o u s s t u d i e s i n d i c a t e d t h a t metabolism of t h i s and o t h e r compounds may b e accomp a l i s h e d by d i f f e r e n t mechanisms s o t h a t s p e c i e s d i f f e r e n ces become b o t h q u a n t i t a t i v e and q u a l i t a t i v e . P e r e l -e t a1 ( 1 2 ) c o r r e l a t e d t h e p h y s i c o c h e m i c a l p r o p e r t i e s of phenylbutazone a n a l o g s w i t h t h e i r p h y s i o l o g i c a l d i s p o s i t i o n , i n p a r t i c u l a r w i t h r e f e r e n c e t o s p e c i e s d i f f e r e n c e s . While i n man t h e r e e x i s t s a d i r e c t r e l a t i o n between pKa and h a l f l i f e , no such c o r r e l a t i o n w a s o b s e r v e d i n dogs. The h a l f l i f e i n d o g s a p p e a r s t o depend on f a c t o G such as f a t l b u f f e r p a r t i t i o n c o e f f i c i e n t , plasma p r o t e i n b i n d i n g , t i s s u e d i s t r i b u t i o n and d r u g m e t a b o l i z i n g enzyme a c t i v i t y . The r a t e of metabolism of t h e a n a l o g s , based on plasma l e v e l d e c l i n e , v a r i e d e x t e n s i v e l y i n b o t h s p e c i e s . Oxyphenbutazone h a s a h a l f - l i f e of 0.5 h r i n t h e dog, whereas i n man it i s 7 2 h r s . The r e s u l t s of b i n d i n g of oxyphenbutazone t o human and dog plasma i n d i c a t e t h a t t h e d r u g i s h i g h l y bound ( 9 8 % ) . T h i s h i g h e r b i n d i n g o f t h e d r u g t o human plasma compared t o dog plasma s h o u l d be a s i g n i f i c a n t f a c t o r i n a c c o u n t i n g f o r some of t h e s p e c i e s d i f f e r e n c e s . In t h e i r s t u d y of t h e mechanism of h e p a t i c d r u g o x i d a t i o n and i t s r e l a t i o n t o i n d i v i d u a l d i f f e r e n c e s i n r a t e s of o x i d a t i o n i n man, Davies and T h o r g e i r s s o n (13) s u g g e s t e d t h a t plasma h a l f - l i v e s of a n t i p y r i n e d e t e r m i n e d f o l l o w i n g a s i n g l e d o s e w e r e a good measure of a n i n d i v i d u a l ' s a b i l i t y t o m e t a b o l i z e p h e n y l b u t a z o n e and oxyphenbutazone, i f t h e h a l f - l i v e s of t h e s e two d r u g s were d e t e r m i n e d u n d e r c o n d i t i o n s which d i d n o t change michrosomal enzyme a c t i v i t y . Human l i v e r microsomal p r o t e i n and cytochrome P-450 conc e n t r a t i o n s d i d n o t show l a r g e i n d i v i d u a l d i f f e r e n c e s and were s i m i l a r t o v a l u e s f o r a d u l t m a l e r a t s . Hoffmann et a1 (14) c a r r i e d o u t s t u d i e s on t h e o r g a n d i s t r i b u t i o n and s i d e e f f e c t s of oxyphenbutazone and p h e n y l b u t a z o n e u s i n g r a d i o a c t i v e i s o t o p e s . No i n f l u e n c e on t h e h e m o p o i e t i c s y s t e m w a s d e m o n s t r a t e d by oxyphenbutazone o r by phenylA m i l d e f f e c t on oxyphenbutazone metabolism w a s butazone. seen. I n d i r e c t r e l a t i o n of t h e c o n c e n t r a t i o n of oxyphenb u t a z o n e and phenylbutazone a d e c r e a s e of oxyphenbutazone u p t a k e b y t h e t h y r o i d was n o t e d , w i t h an i n c r e a s e i n i t s p r o t e i n binding. I n t h e presence of f e v e r , organs w i t h a I

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OXYPHENBUTAZONE

35 1

r e t i c u l a r e n d o t h e l i a l c e l l system, e s p e c i a l l y t h e l i v e r , had a h i g h e r u p t a k e of oxyphenbutazone w h i l e muscle t i s s u e S t i e r l i n and Saubermann showed a d e c r e a s e a t t h a t t i m e . (15) s t u d i e d t h e t r a n s p o r t of l o c a l l y a d m i n i s t e r e d r a d i o a c t i v e oxyphenbutazone i n t o t h e eye t i s s u e s of t h e r a b b i t . The r e s u l t s showed t h a t t h e c o r n e a l - s c l e r a l b a r r i e r can be passed by t h e drug. The c o n c e n t r a t i o n of oxyphenbutazone l o c a l l y a d m i n i s t e r e d i n t h e t i s s u e and f l u i d s of t h e eye w a s h i g h e r t h a n w i t h a s i n g l e 10 mg/kg i.v. dose. The d i r e c t a p p l i c a t i o n of oxyphenbutazone on t h e eye d o e s n o t r e s u l t i n a n accumulation of t h e d r u g w a s demonstrated by t h e f a c t t h a t no oxyphenbutazone could be d e t e c t e d i n t h e e y e a f t e r 24 h r s . The o x i d a t i o n of oxyphenbutazone by sheep v e s i c u l a r gland microsomes and l i p o x y g e n a s e w a s s t u d i e d by P o r t o g h e s e et a 1 ( 1 6 ) . Oxyphenbutazone w a s o x i d i z e d when i n c u b a t e d w i t h a c e t o n e powder p r e p a r e d from sheep v e s i c u l a r g l a n d microsomes o r w i t h l i p o x y g e n a s e a t pH 4 o r 5. Oxidation a l s o occured a t pH 8 o r 9 , i f a r a c h i d o n a t e o r l i n o l e a t e w a s added t o e i t h e r of i n c u b a t i o n m i x t u r e s . The oxygenated p r o d u c t w a s found t o b e i d e n t i c a l w i t h 4-hydroxyoxyphenbutazone (Scheme 3 ) which w a s s y n t h e s i z e d and a n a l y s e d by g a s - l i q u i d chromatography and mass s p e c t r o m e t r y . The oxygenated compound w a s n o t an i n h i b i t o r of p r o s t a g l a n d i n biosynthesis. P e r e l e t a1 (12) found t h a t oxyphenbutazone w a s s l o w l y m e t a b o l i z e d i n man and r a p i d l y m e t a b o l i z e d i n dogs. When t h e d r u g i s a d m i n i s t e r e d i n t r a v e n o u s l y t o d o g s , 10-15% of unchanged d r u g and 18-28% of i t s g l u m r o n i d e were e x c r e t e d i n 24 h r s . When e i g h t t i m e s t h i s d o s e of t h e drug w a s given o r a l l y t o human s u b j e c t s , t h e y e x c r e t e d 1-5% of t h e d o s e as g l u c u r o n i d e s i n 24 h r s .

I

The s u l f a t e of oxyphenbutazone i s p r o b a b l y n o t a major It h y d r o l y s e s i n water a t room t e m p e r a t u r e metabolite. and s i n c e less t h a n 2% of oxyphenbutazone i s e x c r e t e d i n 24 h r s (17) t h i s would r e p r e s e n t t h e maximal c o n v e r s i o n of oxyphenbutazone t o s u l f a t e .

5 . Methods of A n a l y s i s 5.1 T i t r i m e t r i c Methods a ) The BP 1980 ( 3 ) d e s c r i b e s a t i t r i m e t r i c method i n which oxyphenbutazone solution i n acetone is t i t r a t e d w i t h s t a n d a r d sodium hydroxide s o l u t i o n u s i n g bromothymol b l u e a s i n d i c a t o r u n t i l a b l u e c o l o r p e r s i s t s € o r a t l e a s t t h i r t y seconds. The

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operation is repeated without t h e drug; t h e d i f f e r e n c e between t h e t i t r a t i o n s r e p r e s e n t s t h e amount of a l k a l i r e q u i r e d by oxyphenbutazone. b) The U S P 1980 ( 4 ) a l s o recommends a s t a n d a r d sodium hydroxide t i t r i m e t r i c method b u t t h e d r u g s o l u t i o n i s made i n methanol and t h e end p o i n t i s determined p o t e n t i o m e t r i c a l l y u s i n g a calomel-glass e l e c t r o d e system w i t h a s a t u r a t e d s a l t b r i d g e of potassium A blank determination is c h l o r i d e i n methanol. necessary. c ) Walash and Rizk (18) determined oxyphenbutazone i n c e r t a i n dosage forms by a nonaqueous t i t r a t i o n proc e d u r e i n t e t r a m e t h y l u r e a u s i n g s t a n d a r d sodium methoxide a s a t i t r a n t . Recovery from compound p r e p a r a t i o n s of t h e a n a l g e s i c w a s q u a n t i t a t i v e and t h e end p o i n t could be l o c a t e d e i t h e r p o t e n t i o m e t r i c a l l y u s i n g glass-colomel e l e c t r o d e system o r v i s u a l l y u s i n g thymol b l u e as i n d i c a t o r .

5.2 E l e c t r o c h e m i c a l Methods a ) Fogg and Ahmed (19) adopted a B.P. 1980 ( 3 ) t e s t f o r t h e i d e n t i f i c a t i o n of oxyphenbutazone t o t h e q u a n t i t a t i o n of t h e drug by d i f f e r e n t i a l p u l s e polarography. The drug s o l u t i o n i n a c e t i c and hydroc h l o r i c a c i d s w a s t r e a t e d w i t h sodium n i t r i t e and coupled w i t h 1-naphthol. The azo-dye formed w a s determined by d i f f e r e n t i a l p u l s e polarography. Polarograms were reproduced f o r t h e d e t e r m i n a t i o n of t h e drug a t c o n c e n t r a t i o n s i n t h e r a n g e s 0 t o 0.9 UM and 0 t o 70 uM. The c a l i b r a t i o n g r a p h s w e r e rectilinear. The p r o c e d u r e , however, does n o t d i s t i n g u i s h between oxyphenbutazone and phenylbutazone.

--

b) P e l i n a r d e t a 1 (20) s t u d i e d t h e e l e c t r o c h e m i c a l b e h a v i o r of a n t i i n f l a m m a t o r y d e r i v a t i v e s of pyrazol o n e and t h e a p p l i c a t i o n t o d e t e r m i n a t i o n i n medicaments. Oxyphenbutazone and o t h e r 3,5-pyrazolined i o n e s w i t h mobile hydrogen atoms i n p o s i t i o n 4 were d i r e c t l y reduced e l e c t r o c h e m i c a l l y i n n e u t r a l e t h a n o l . Monoketones, phenazone and aminophenazone, were n o t reduced under s i m i l a r c o n d i t i o n s . The a p p l i c a t i o n of t h e method t o t h e d e t e r m i n a t i o n of 3 , 5 - p y r a z o l i d i n e d i o n e s , w i t h l a b i l e hydrogen i n t h e 4 - p o s i t i q g a v e a p r e c i s i o n of about 3% when 2 X 10-6 mole of a s u b s t a n c e were used.

OXYPHEN BUTAZON E

353

5.3 S p e c t r o p h o t o m e t r i c Methods 5.3.1 U l t r a v i o l e t S p e c t r o p h o t o m e t r i c Methods a ) Herrmann ( 9 ) r e p o r t e d a n u l t r a v i o l e t method f o r t h e d e t e r m i n a t i o n of p h e n y l b u t a z o l i d i n e together with i t s metabolic products, chiefly oxyphenbutazone, i n serum. The serum i s shaken w i t h 3N h y d r o c h l o r i c a c i d i n 3% isoamyl alcohol i n heptane. After c e n t r i f u g a t i o n an a l i q u o t of t h e o r g a n i c p h a s e i s shaken w i t h 2.5 N sodium h y d r o x i d e and t h e e x t i n c t i o n of t h e a l k a l i n e e x t r a c t i s d e t e r m i n e d a t 265 nm. A s i m i l a r method h a s a l s o been r e p o r t e d (21). b ) Fuchs ( 2 2 ) d e s c r i b e d a method f o r t h e blood l e v e l d e t e r m i n a t i o n of t a n d e r i l [ oxyphenbutazone] u s i n g a s i n g l e d r o p of c a p i l l a r y b l o o d . The sample of t h e serum i s mixed w i t h water and 3N h y d r o c h l o r i c a c i d . The m i x t u r e i s shaken with p u r e 1,2-dichloroethylene. After c e n t r i f u g a t i o n , a n a l i q u o t of t h e o r g a n i c p h a s e i s added t o a s e p a r a t i n g f u n n e l c o n t a i n i n g 1 m l of 2.5 N sodium h y d r o x i d e and shaken. The aqueous e x t r a c t of T a n d e r i l i s s e p a r a t e d by c e n t r i f u g a t i o n and d e t e r m i n e d by measuring a t 240, 245, 250, 252.5, 255, 257.5, 260, 265 and 270 nm. The e r r o r w a s < 4%.

5.3.2 C o l o r i m e t r i c Methods a ) S v a t e k and Hradkova (23) d e t e r m i n e d oxyphenb u t a z o n e and r e l a t e d k e t o d e r i v a t i v e s i n serum a c o l o r i m e t r i c method i n v o l v i n g t h e using r e a c t i o n of t h e d r u g s w i t h d i a z o t i z e d s u l f a n i l i c a c i d . According t o t h e procedure, t h e d i l u t e d serum is shaken w i t h N h y d r o c h l o r i c a c i d , h e p t a n e and isoamyl a l c o h o l . The h e p t a n e p h a s e i s shaken w i t h 0.1 N sodium h y d r o x i d e . The a l k a l i n e e x t r a c t i s shaken with a m i x t u r e of 1%s u l f a n i l i c a c i d , N h y d r o c h l o r i c a c i d , 1% sodium n i t r i t e and methanol. The a b s o r b a n c e of t h i s s o l u t i o n i s measured a t 525 nm a g a i n s t sodium h y d r o x i d e b l a n k . The e r r o r was 3-5%. Oxyphenbutazone combines w i t h d i a z o t i z e d s u l f a n i l i c a c i d i n a l k a l i n e medium w i t h o u t need f o r preliminary hydrolysis. The m e t h y l e n e group of t h e s i d e c h a i n i s a c t i v a t e d by t h e

354


y k e t o group w i t h t h e f o r m a t i o n

o f a formazan derivative. The formazan d e r i v a t i v e of s u l f a n i l i c a c i d i s v e r y s o l u b l e i n w a t e r and h a s a maximum a t 5 2 5 nm. The c o l o r i s s t a b i l i z e d by t h e presence of 25% methanol. The method i s s e n s i t i v e t o 0.5 y/ml and t h e l i n e a r r a n g e i s 0-15 y/ml.

b ) Wahbi -e t a 1 (24) used sodium c o b a l t i n i t r i t e as a r e a g e n t f o r d e t e r m i n i n g some p h e n o l i c d r u g s . When oxyphenbutazone is r e a c t e d w i t h t h e r e a g e n t i n aqueous a c e t i c a c i d a yellow c o l o r measurable a t 3 2 0 nm i s produced. Subs t i t u t i n g sodium hydroxide f o r a c e t i c a c i d g i v e s a c o l o r measurable a t 3 8 0 nm. The c o l o r e d product i s e x t r a c t a b l e w i t h c h l o r o form w i t h a maximum a b s o r p t i o n a t 3 2 5 nm. The method i s a p p l i e d t o t h e d e t e r m i n a t i o n i n t a b l e t form. The r e s u l t s o b t a i n e d are reasonably reproducible with a coefficient of v a r i a t i o n < 2%.

c ) Sanghavi e t a 1 (25) t r e a t e d oxyphenbutazone w i t h a m i x t u r e of anhydrous a c e t i c a c i d and h y d r o c h l o r i c a c i d . The product of t h e h e a t e d mixture i s reacted with v a n i l l i n or 4dimethylaminobenzaldehyde i n a c e t i c a c i d . The r e s u l t i n g c o l o r i s measured a b s o r p t i o m e t r i c a l l y o r a t 4 2 4 nm w i t h v a n i l l i n o r a t 406 nm with 4-dimethylaminobenzaldehyde. Beer’s Law i s obeyed f o r 8 t o 12 y/ml when measured s p e c t r o p h o t o m e t r i c a l l y o r 5 0 t o 2 5 0 y/ml when measured a b s o r p t i o m e t r i c a l l y .

5. 3.3 S p e c t r o f l u o r o m e t r i c Method Miller e t a 1 (26) s t u d i e d t h e luminescence prop e r t i e s of oxyphenbutazone and some o t h e r a n t i inflammatory and a n t i p y r e t i c d r u g s . Although oxyphenbutazone showed no s i g n i f i c a n t f l u o r e s cence a t room t e m p e r a t u r e i t w a s s t r o n g l y f l u o r e s c e n t a t 77K. Submicrogram q u a n t i t i e s of t h e drug could be r e a d i l y d e t e c t e d . The wavelength of e x c i t a t i o n maximum i n e t h a n o l was 2 8 0 nm and t h a t of t h e f l u o r e s c e n c e maximum w a s 4 3 0 nm.

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5.4 Chromatographic Methods 5.4.1 Thin-Layer Chromatography (TLC) S e v e r a l TLC methods a r e a v a i l a b l e f o r t h e s e p a r a t i o n , i d e n t i f i c a t i o n and d e t e r m i n a t i o n of oxyphenbutazone and i t s m e t a b o l i t e s . Schmidt (27) a n a l y s e d oxyphenbutazone t o g e t h e r w i t h o t h e r p h a r m a c e u t i c k l s by s t a i n i n g on s i l i c a g e l GF254 u s i n g W l i g h t v i s u a l i z a t i o n . An A v i c e l l a y e r chromatography w a s developed by L e e (28) f o r t h e s e p a r a t i o n and i d e n t i f i c a t i o n of s e v e r a l k i n d s of d r u g s . A c i d i c m a t e r i a l s w e r e s e p a r a t e d by a c i d i c s o l v e n t s and b a s i c materials w e r e s e p a r a t e d s a t i s f a c t o r i l y by b a s i c s o l v e n t s u s i n g mixed A v i c e l k i e s e l g e l p l a t e s . The proc e d u r e w a s a p p l i e d t o t h e d i f f e r e n t pharmaceutic a l f o r m u l a t i o n s . MoYe TLC methods are i n c l u d e d i n T a b l e 2. Table 2. TLC Methods f o r t h e D e t e r m i n a t i o n of Oxyphenbutazone. Adsorbent

S o l v e n t System

Visualizing agent

R

f

value

Ref.

S i l i c a g e l MethanollCHC131 G-0.06 M Aq. NH3 KH2PO4 (100:300:1) b u f f e r (1:2) dry p l a t e and a c t i v a t e at llOo. Silica gel

HexaneIAcetone (1:l)

UV r a d i a t i o n

0.76

(30)

CHC13/Ethylacetate

UV r a d i a t i o n

0.28

(30)

Silica gel

S t r o n g NH3 solution/Methano1 (1.5:lOO)

KMn04

0.77

(2)

Silica gel sheets

CyclohexaneIAcetone/Acetic acid (40: 50:l)

W radiation a t 254 nm

0.58

(31)

HF254

+

366

Silica gel HF254

+

366


356

5.4.2


A paper chromatographic method w a s d e s c r i b e d by C l a r k e ( 2 ) and i s o u t l i n e d above under Chromatographic T e s t s .

5.4.3

Gas Chromatography A g a s chromatographic method w a s r e p o r t e d by Hasegawa e t a 1 (32) f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n of p y r a z o l i d i n e d i o n e a n a l g e s i c s p r e s e n t i n blood plasma u s i n g a n e l e c t r o n c a p t u r e t e c h nique. The method i s claimed t o b e s i m p l e and a b l e t o d e t e c t low c o n c e n t r a t i o n s . The g a s chromatographic r e t e n t i o n i d e x e s of several compounds of t o x i c o l o g i c a l i n t e r e s t i n c l u d i n g oxyphenbutazone were determined ( 3 3 , 3 4 ) i s o t h e r m a l l y a t 180° on SE30, O V 1 , O V 1 7 and QF4. The d a t a f o r many of t h e s e s u b s t a n c e s w e r e compared s t a t i s t i c a l l y among themselves when a p p r o p r i a t e (SE30 and O V l ) and w i t h l i t e r a t u r e d a t a . I n g e n e r a l , no s t a t i s t i c a l l y s i g n i f i c a n t d e v i a t i o n s were found i n t h e series compared. The v a l u e s of t h e r e t e n t i o n i d e x e s w e r e r e p r o d u c i b l e t o a good d e g r e e which means t h a t t h e s e i n d e x e s a r e of c o n s i d e r a b l e d i a g n o s t i c v a l u e s i n chemical toxicological investigations.

Bruce -e t a1 ( 3 5 ) determined oxyphenbutazone and phenylbutazone i n plasma and u r i n e by g a s - l i q u i d chromatography. While phenylbutazone w a s d e t e r mined d i r e c t l y a f t e r e x t r a c t i o n from blood plasma o r u r i n e , oxyphenbutazone w a s r e a c t e d with heptafluorobutyric anhydride p r i o r t o gas e t a 1 (36) d e s c r i b e d chromatography. Tanimura -a g a s - l i q u i d chromatographic method f o r t h e d e t e r m i n a t i o n of phenylbutazone and i t s metabol i t e s , oxyphenbutazone and y-hydroxyphenylbutazone i n human and r a b b i t f o l l o w i n g a d m i n i s t r a t i o n of phenylbutazone. A modified Herrmann’s extract i o n method h a s been used and coupled w i t h t h e g a s - l i q u i d chromatographic p r o c e d u r e w i t h o u t d e r i v a t i v e f o r m a t i o n f o r phenylbutazone and using trimethylsilylation f o r the metabolites. The procedure w a s d e s c r i b e d as a c c u r a t e and s u f f i c i e n t l y s e n s i t i v e f o r use i n routine c l i n i c a l a s s a y and t h e e s t i m a t i o n of t h e pharmacokin e t i c p a r a m e t e r s of phenylbutazone and i t s metabolites.

OXYPHENBUTAZONE

3.57

5 . 4 . 4 High Performance L i q u i d Chromatography (HPLC) Pound and S e a r s (37) d e s c r i b e d a s e n s i t i v e , s p e c i f i c , high-speed l i q u i d c h r o m a t o g r a p h i c proc e d u r e f o r t h e s i m u l t a n e o u s d e t e r m i n a t i o n of phenylbutazone and i t s m e t a b o l i t e , oxyphenbutazone i n blood plasma. According t o t h i s proced u r e , a c i d i f i e d plasma i s p a r t i t i o n e d w i t h c y c l o h e x a n e : e t h e r (1:l) c o n t a i n i n g t h e 2,4d i n i t r o p h e n y l h y d r a z o n e o f 3,4-dimethoxybenzaldehyde as an i n t e r n a l s t a n d a r d . The o r g a n i c e x t r a c t i s reduced t o d r y n e s s , t h e r e s u l t i n g r e s i d u e i s r e d i s s o l v e d i n c h l o r o f o r m and a l i q u o t s of t h i s s o l u t i o n a r e chromatographed on an a d s o r p t i o n column, u s i n g a m o b i l e p h a s e of 0.002% a c e t i c a c i d and 23.0% t e t r a h y d r o f u r a n i n n-hexane a t 35OC. Use of a UV d e t e c t o r p e r m i t s q u a n t i t a t i v e a n a l y s i s of samples c o n t a i n i n g less t h a n 0.25 ug/ml of p h e n y l b u t a z o n e o r oxyphenbutazone. H a r z e r a n d B a r c h e t (38) s e p a r a t e d v a r i o u s combin a t i o n s of a n a l g e s i c s by HPLC, u s i n g b o t h r e v e r s e - p h a s e and a d s o r p t i o n columns and UV d e t e c t i o n a t 254 nm. For t h e r e v e r s e - p h a s e t e c h n i q u e , v a r i o u s p r o p o r t i o n s of methanol-water were used a s e l u a n t , whereas t h e a d s o r p t i o n system r e q u i r e s a ) c h l o r o f o r m - m e t h a n o l - a c e t i c The a c i d and b) chloroform-C7H16 - a c e t i c a c i d . a d v a n t a g e of t h e r e v e r s e - p h a s e column w a s t h a t i t r e q u i r e d changes o n l y i n column t e m p e r a t u r e and s o l v e n t p r o p o r t i o n s i n o r d e r t o e f f e c t a l l s e p a r a t i o n s . U s e of a v a r i a b l e wavelength d e t e c t o r enhanced t h e s e n s i t i v i t y by p e r m i t t i n g a d j u s t m e n t t o t h e a b s o r p t i o n maximum of e a c h drug. Oxyphenbutazone, and o t h e r n o n - n a r c o t i c a n a l g e s i c s are s c r e e n e d by r e v e r s e - p h a s e h i g h - p r e s s u r e l i q u i d chromatography ( 3 9 ) . The column i s L i C h r o s o r b RP8 and t h e m o b i l e p h a s e i s a a c e t o Blood and o r g a n s a r e n i t r i l e - w a t e r mixture. homogenized i n 4% p e r c h l o r i c a c i d b e f o r e e x t r a c t i o n w i t h t h e s o l v e n t s . U r i n e i s e x t r a c t e d without primary treatment. D i e t e r l e e t a1 ( 4 0 ) used a p r e p a r a t i v e r e v e r s e p h a s e chromatography f o r s e p a r a t i o n of p o l a r and n o n p o l a r m e t a b o l i t e s , i n c l u d i n g t h o s e of phenyl-

ABDULLAH A . AL-BADR A N D HUMEIDA A . EL-OBEID

358

b u t a z o n e , on column packed w i t h m i c r o n i z e d XAD2 r e s i n . By e q u i l i b r a t i o n w i t h s i n g l e - p h a s e m i x t u r e s of water, a lower a l i p h a t i c a l c o h o l and a hydrophobic s o l v e n t a r e v e r s e - p h a s e p a r t i t i o n system i s formed i n s i t u . The c h r o m a t o g r a p h i c technique is claimed t o b e c h a r a c t e r i z e d by a l a r g e c a p a c i t y , a h i g h power of r e s o l u t i o n and a h i g h d e g r e e of s e l e c t i v i t y and r e p r o d u c i b i l i t y and v e r s a t i l e a p p l i c a t i o n i n t h e i s o l a t i o n of p o l a r and n o n p o l a r d r u g m e t a b o l i t e s from complex mixtures. Acknowledgement

A sample of oxyphenbutazone w a s k i n d l y d o n a t e d by CibaGeigy L i m i t e d , Basle, S w i t z e r l a n d . The a u t h o r s would l i k e t o t h a n k M r . T a n v i r A. B u t t f o r t y p i n g t h i s m a n u s c r i p t .

Reference

1. "The Merck Index", 9 t h Ed., Merck and Co. Inc., Rahway, N.J.,

U.S.A.,

p . 903 (1976).

2. E.G.C. Clarke, " I s o l a t i o n and I d e n t i f i c a t i o n of Drugs", The P h a r m a c e u t i c a l P r e s s , London, p . 462 (1969). 3. " B r i t i s h Pharmacopoeia", V o l . 1 , H e r M a j e s t y ' s S t a t i o n a r y p. 321 (1980). O f f i c e , London, U.K.,

4 . "The U n i t e d S t a t e s Pharmacopeia", 2 0 t h Ed., Mack P u b l i s h i n g Co., E a s t o n , P e n n s y l v a n i a , p. 575-6 (1980). 5. S.P. Singh, S.S. Parmar, V . I . S t e n b e r g and S.A. J. H e t e r o c y c l . Chem., 1 5 ( 1 ) , 1 3 (1978). 6. B. U n t e r h a l t , -Arch. Pharm., ___

305,

Farnum,

334 (1972).

7 . R . A . Locock, R.E. Moskalyk, L.G. C h a t t e n and L.M. J. -Pharm. S c i . , 6 3 ( 1 2 ) , 1896 (1974). 8. R. P f i s t e r and F. H a f l i g e r , Helv. Chim. A c t a , ( 1 957)

.

9. B. Herrmann, Med. E x p t l . ,

1,170

(1959).

40,

Lundy,

395

OXYPHENBUTAZONE

250

10. M. Weiner, P.G. Dayton and A.G. Hendrickx i n " U s e Nonhuman P r i m a t e s Drug Eval., Symp." 1967 (pub. 1968), E d i t e d by Vagtborg, H a r o l d . Univ. of Texas P r e s s : 3-22. A u s t i n , Tex.

11. J.J. Burns, Ann. N.Y.

151 ( A r t .

Acad. S c i . ,

21,

959

(1968). 12. J . M . P e r e l , M.M. S n e l l , W. Chen and P.G. Pharmacol, , 13(9) , 1305 (1964).

Dayton, Biochem.

13. D.S. Davies and S.S. T h o r g e i r s s o n , Ann. N.Y. Acad. S c i . , 179, 411 (1971). 14. G . Hoffmann, H. Heimpel and P. P f a n n e n s t i e l i n "Nucl. Med., Suppl.," 1968 (Pub. 1970), No. 8, 187.

-

15. H. S t i e r l i n and G. Saubermann i n "Nucl. Med., 1968 (Pub. ,1970) , No. 8, 183. 16. P.S.

Suppl.",

P o r t o g h e s e , S. K e r s t i n and B. Samuelsson, Biochem. Commun., 63(3), 748 (1975).

Biophys.

x.

17. A.B. Gutman, P.G. Dayton, T.F. L.E. Sicam and J.J. Burns, (1960).

&.

Yu, L . B e r g e r , W. Chen, J. K . , 1017

2,

I n d i a n J. Pharm., 18. M . I . Walash and M. R i z k , -(1977).

39(4),

82

__I

19. A.G. Fogg and Y.Z. (1977).

Ahmed, Anal. Chim. A c t a , 94, 453

20. C. P e l i n a r d , E. Nivaud and P. Boucly, T a l a n t a , (1978). 21. F. F r e y t a g , A r z n e i m i t t e l - F o r s c h .

--

,20,

22. W. Fuchs, Muench. Med. Wochschr., 23. E. S v a t e k and A. Hradkova, Cesk.

25,

119

1273 (1970).

107,1267 (1965). Farm., 15,76 (1966).

24. A.M. Wahbi, H. Abdine, M. Korany and M.H. Abdel Hay, J. Assoc. O f f . Anal. Chem 61, 1113 (1978). -

- -.'-

25. N.M. S a n g h a v i , N.G. J i v a n i and P.D. -'Pharm * 39, 87 (1977).

Mulye, -I n d i a n J.

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

360

26. J . N .

Miller, D.L. P h i l l i p p s , D.T. T a l a n t a , 25, 46 (1978).

Burns and J . W .

Bridges,

27. F. Schmidt, Dtsch. Apoth. Z t g . , 114, 1593 (1974). 28. S.C. Lee, -Hua Hsuch, 4, 117 (1973).

E.,

29. F. Gouezo, J . P . Roman and B. C r i s t a u , Annls. Pharm. 26, 301 (1968).

30. T. Pomazanska-Kolodziejska, -Acta P o l . Pharm., 29, 389 (1972). 31. R.D. Thompson and G.L. Johnson, J. Chromatogr., (1974).

88,

361

32. M. Hasegawa, T. Maeda, H. Takenaka, T. Noguchi and Y. Hirayama, J a p a n Kokai, 76 25, 194 ( C l , GOlN31/08). 01 Mar. 1976. Appl. 74/97, 698. 26 Aug. 1974. 33. E . Marozzi, V. Gambaro, F. L o d i and A. P a r i a l i , Farmaco. Ed. P r a t. 31, 180 (1976). 34. Idem., Ibid.,

32,

330 (1977).

35. R.B. Bruce, W.R. Maynard and L.K. Dunning, 63, 446 (1974).

2.

Pharm.

g.,

I

36. Y. Tanimura, Y. S a i t o h , F. Nakagawa and T. S u z u k i , Chem. Pharm. B u l l . , 23, 651 (1975). 37. N.J.

Pound and R.W.

S e a r s , J. Pharm. S c i . ,

38. K. H a r z e r and R. B a r c h e t , D1. Apoth-Ztg., (1976).

64,248

(1975).

116,1226

Med., 39. H. K a e f e r s t e i n and G. S t i c h t , Beitr. G e r i c h t l . 36, 457 (1978).

40. W. Dieterle, J . W . 168, 27 (1979).

F a i g l e and H. Mory, J. Chromatogr.,

PENTAZOCINE Terry D. Wilsoii

I. 2.

3.

4.

5.

6.

7.

8.

Foreword Description 2.1 Nomenclature 2.2 Formula 2.3 Molecular Weight 2.4 Structure 2.5 CA Registry Number 2.6 Appearance, c o k x , Odor 2.7 Recognized Dosage Forms Synthesis and Resolution 3.1 Synthesis 3.2 Isomers and Resolution Physical Properties 4. I Polymorphism 4.2 Melting Point 4.3 Differential Scanning Calorimetry 4.4 Optical Rotation 4.5 Elemental Analysis 4.6 Ionization Constant 4.7 Partition Coefficient 4.8 Solubility 4.9 Spectral 4.10 X-Ray Diffraction 4.1 I Dissolution 4.12 Identification Stability 5. I Hydrolysis 5.2 Dosage Form Stability Methods of Analysis 6. I Titrimetric 6.2 Ultraviolet Spectrophotometry 6.3 Fluorescence Spectrophotometry 6.4 Radioimmunoassay 6.5 Chromatography Biological Disposition and Pharmacokinetics 7.1 Disposition 7.2 Pharmacokinetics Determination in Body Tissues and Fluids References

ANALYTICAL PROFILES O F DRUG SUBSTANCE VOLUME 13

36 I

362 362 362 362 362 362 362 362 365 365 365 365 365 365 366 366 366 366 366 372 372 37 5 382 382 388 388 3x8 389 389 389 389 389 390 39 I 40 I 40 I 40 I 406 41 1

Copyright I C 1984 by the American Pharmaceutical Association ISBN 0- 12-260813-5

362

TERRY D. WILSON

Foreward Pentazocine i s a p o t e n t a n a l g e s i c o f t h e benzomorphan s e r i e s w i t h a 50 m g o r a l dose e q u i v a l e n t t o 60 m g o f codeine. I t i s a l s o a weak n a r c o t i c a n t a g o n i s t w i t h about o n e - f i f t i e t h t h e a c t i v i t y o f n a l o r p h i n e . Pentazocine i s i n d i c a t e d i n t h e t r e a t I t i s a l s o used ment o f moderate t o s e v e r e p a i n . a s a p r e a n e s t h e t i c and a n e s t h e t i c supplement (1,2).

1.

Description 2.1 Nomenclature Pentaz c i n e Talwin 8 Fortral ++ (2d P 6 0 1 , l l R ) - ( !> -1,2,3 ,4,5,6-hexahydro6,11-dimethyl-3-(3-methyl-2-bu ?nyl)-2,6-methano3-benzazocin-8-01

2.

2.3 2.4

K o l e c u l a r Weight

2a5*43

Structure

H

CA R e g i s t r y Number base 7 3-83 -11 h y d r o c h l o r i d e [68964-90-9] 2.6 Appearance, Color, Odor Pentazocine i s w h i t e t o p a l e t a n , odorl e s s , c r y s t a l l i n e powder. 2.5

363

PENTAZOCINE

- WoMe - @z$

Me

Me

NaBH4

I

-

HBr

Me

Me

HO

CN

/

-

Br C H2CH = C (C H3)2

1) HCI

--Me

Me

HO

Me HO

NORPENTAZOCINE

Scheme 1.

DMF

PENTA ZO C1NE

Synthesis of pentazocine.

Ap

I

\

z

a

PENTAZOCINE

365

Recognized Dosage Forms The forms o f p e n t a z o c i n e r e c o g n i z e d by t h e U.S. P . i n c l u d e p e n t a z o c i n e b a s e , h y d r o c h l o r i d e - and l a c t a t e s a l t s i n t h e f o l l o w i n g f o r m u l a t i o n s (1 ,3 , & I . p e n t a z o c i n e h y d r o c h l o r i d e t a b l e t s 50 mgbase p e n t a z o c i n e l a c t a t e i n j e c t i o n 30 m g base/mL p e n t a z o c i n e h y d r o c h l o r i d e and a s p i r i n 12.5 mg base tablets 325 mg a s p i r i n

2.7

S y n t h e s i s and R e s o l u t i o n Synthesis The s y n t h e s i s o f p e n t a z o c i n e was o r i g i n a l l y accomplished by Archer and coworkers (5,6,7)and h a s been reviewed p r e v i o u s l y ( 8 , 9 , 1 0 , 1 1 ) . The i n t e r m e d i a t e : 1,2,3,4,5,6-hexahydro-8-hydroxy6,11-dimethyl-2,6-methano-3-benzazocine ( n o r p e n t a z o c i n e ) w a s s y n t h e s i z e d e a r l i e r by Nay and Eddy (12) as shown i n Scheme 1. Treatment o f n o r p e n t a z o c i n e with l-bromo-3-methyl-2-butene i n DNF gave p e n t azocine. A v a r i e t y o f a l t e r n a t e r o u t e s t o penta z o c i n e have been demonstrated. Included a r e methods employing: a 3-benzyl i n t e r m e d i a t e (13,141, an i n i t i a l r e a c t i o n o f 2-lithio-2-butene with f o r maldehyde (151, a thermal rearrangement o f aminomethylcyclopropyl ketone (16), a n abnormal Hofmann d e g r a d a t i o n o f N-(&-hydroxybenzyl)-3-benzazociniurn h a l i d e s ( 1 7 ) and a B i s c h l e r - N a p i e r a l ski reaction(l8).

3.

3.1

Isomers and R e s o l u t i o n Geometrical ( c i s - t r a n s ) i s o m e r s o f benzomorphans r e s u l t from t h e c y c l i z a t i o n s t e p i n t h e i r s y n t h e s i s when t h e bond t o C-6 i s formed (Scheme 2). When t h i s i s c o n s i d e r e d a t r a n s a d d i t i o n t o t h e double bond t h e preponderance o f t h e c i s o r d form i s e a s i l y explained ( 1 0 ) . R e s o l u t i o n o f o p t i c a l i s o m e r s f r o m b o t h c i s and t r a n s f o r m s has been r e p o r t e d (7,19,20). The n o r p e n t a z o c i n e p r e c u r s o r i n t h e c i s form c r y s t a l l i z e d f i r s t as t h e ( - ) base w i t h ( + ) t a r t a r i c a c i d which would g i v e t h e t w e n t y - f o l d more a c t i v e ( - 1 p e n t a z o c i n e . The ( + ) base ( - ) t a r t a r a t e was r e c overed f r o m r e s o l u t i o n l i q u o r s . Q u i n i c a c i d s a l t s were formed f o r r e s o l u t i o n o f t h e t r a n s isomers. 3.2

4.

Ph s i c a l P r o p e r t i e s h y m o m h i s r n

TERRY D. WILSON

366

Two d i f f e r e n t i n t e r c o n v e r t i b l e c r y s t a l polymorphs o f p e n t a z o c i n e h y d r o c h l o r i d e a r e known w i t h m e l t i n g p o i n t s o f 2180 and 254' ( 2 , 2 1 ) .

4.2

Melting P o i n t The r e p o r t e d m e l t i n g p o i n t s o f p e n t a z o c i n e base and h y d r o c h l o r i d e salts a l o n g w i t h t h o s e o f r e s o l v e d o p t i c a l isomers a r e shown i n Table 1. D i f f e r e n t i a l Scanning C a l o r i m e t r y curve f o r p e n t a z o c i n e i s shown i n F i g u r e 1. It w a s c a r r i e d o u t on a P e r k i n Elmer D.S.C. 1B a t a s c a n r a t e o f 2.5O,per minute, a range s e t t i n g o f 4 and a n i t r o g e n \ e r g e r a t e o f --20 mL p e r minute ( 2 3 ) .

4.3

A D.S.C.

4.4

Optical Rotation Table 2 i n c l u d e s r e s u l t s for o p t i c a l r o t a t i o n o f r e s o l v e d o p t i c a l isomers o f Pentazocine c a r r i e d o u t by T u l l a r et g ( 1 9 ) . A l s o i n c l u d e d i s t h e v a l u e o b t a i n e d by Nambara et a1 for ( - ) pentazocine (22). 4.5

Elemental_ A n a l y s i s R e s u l t s o f e l e m e n t a l a n a l y s i s f o r pentazoc i n e and p e n t a z o c i n e h y d r o c h l o r i d e a r e shown i n Table 3 .

4.6

Ionization Constant The PKa v a l u e for p e n t a z o c i n e l i s t e d i n g e n e r a l t e x t s as 8.76 ( 2 4 ) or 8.95 ( 2 ) i s t h e t e r t i a r y amine p r o t o n a t i o n c o n s t a n t . Borg and Nlikaelsson however have shown t h a t t w o i o n i z a t i o n s f o r pentazocine s h o u l d be cDnsidered. F i r s t t h e pentazocinium i o n l o s e s t h e p r o t o n f r o m t h e n i t rogen and t h e n t h e p h e n o l i c p r o t o n i s l o s t a t h i g h e r pH. T h i s i s p i c t u r e d i n Scheme 3 where two s e p a r a t e i n t e r m e d i a t e s a r e p o s s i b l e ; t h e n e u t r a l p e n t a z o c i n e b a s e (111) and a z w i t t e r i o n i c form (11). The f o u r microscopic d i s s o c i a t i o n c o n s t a n t s were determined u s i n g 0.1 i o n i c s t r e n g t h c a r b o n a t e / b i c a r b o n a t e b u f f e r s with t h e v a l u e s : p k = 9.74, pk2= 10.56, pk12 = 11.17 and pk21 = 10.3). From t h e s e t h e macroscopic d i s s o c i a t i o n c o n s t a n t s were determined from t h e r e l a t i o n s :

H+

+

Me

>

H+

+

H+ HO

I

HO

Iu Scheme 3.

Me

Pentazocine ionization.

Me

TABLE 1 MELTING POINTS OF PENTAZOCINE

Isomer

Form base base

Optical

(3 (+I

Geometrical

Melting Point

OC

Reference

cis

145.4-148.6

5

cis

179

-182

7

cis

176

-179

7

base

(-1 (-1

cis

164

-170

22

base

(+>

cis

180 .4-182.0

19

base

(-1

cis

180.6-182.2

19

hydrochloride

(+)

trans

254.5-255.0

19

hydrochloride

(-1

trans

246.0-254.0

19

base

PENTAZOCINE

369

1

1

1

1

1

1

1

1

1

1

n

n

n

0

0 0

0

0

(D

0

m

-

d-

Y

-

I

0

-

0

In

Y

Y

Fig. 1. Differential scanning calorimetry curve f o r pentazocine.

I

TABLE 2 OPTICAL R O T A T I O N OF PENTAZOCINE ISOMERS

W

d

Fo m

Isomer

[d]o;

base

cis (+)

base

Solvent

Reference

+I35* 5

CHC13

19

c i s (-)

-138.0

CHC13

19

base

cis (-)

-131

CHC13

22

hydrochloride

trans(+)

+115.4

2% HOAc

19

hydrochloride

trans(-)

-116 3

2% HOAc

19

TABLE 3 PENTAZOC INE ELEMENTAL ANALYSIS

$I Carbon

$I Hydrogen

%' N i t r o g e n

% Chlorine"

C a l c ' d Found

C a l c ' d Found

Form

Isomer

C a l c ' d Found

base

(T) c i s

79.95

79.84

9.34

9.28

4.91

5.23

(+)trans

70.89

70.62

8.77

8.46

4.35

4.25

HC1

*

C a l c ' d Found

( - ) t r a n s isomer

-

-

Ref.

5

11-01 11.25 1 9

TERRY D.WILSON

312

aH+

[Iv]

[II]

+ LII]

K2= Thus pKI was f o u n d t o e q u a l 9.68 and pK2 e q u a l s 11.23 (25).

4.7

Partition Coefficient The p a r t i t i o n c o e f f i c i e n t f o r p e n t a z o c i n e h a s been measured between v a r i o u s o r g a n i c and aqueous phases. The r e s u l t s o f f o u r s t u d i e s a r e shown i n Table 4. The f i r s t v a l u e was o b t a i n e d by a s i n g l e e x t r a c t i o n u s i n g 50 mL o f each phase while t h e second value i s from a c o u n t e r c u r r e n t d i s t r i b u t i o n s t u d y with pentazocine added t o t h e aqueous phase and e x t r a c t e d e i g h t t i m e s i n t o benzene. The t h i r d value was o b t a i n e d by t h e s i n g l e e x t r a c t i o n method with t h e a d d i t i o n a l d e f i n i t i o n o f a d i s t r i b u t i o n r a t i o , DNOH, f o r p e n t a z o c i n e u s i n g t h e p a r t i t i o n c o e f f i c i e n t kd(NOH) i n t h e r e l a t i o n :

1 DNOH

-

1

+-

kd ( N O H )

Here kl, k2 and k2 are t h e microscopic ioni z a t i o n c o n s t a n t s d e f i n k d i n s e c t i o n 4.6. From t h i s it was p o s s i b l e t o c a l c u l a t e and p l o t v a r i a t i o n i n DNOH with pH f o r pentazocine a s shown i n F i g u r e 2. The f o u r t h p a r t i t i o n c o e f f i c i e n t w a s o b t a i n e d by s t r u c t u r a l group c o n t r i b u t i o n t o t h e measured p a r t i t i o n c o e f f i c i e n t o f a r e f e r e n c e compound (morphine). T h i s w a s done f o l l o w i n g t h e Hansch approach.

4.8

Solubility The s o l u b i l i t y o f pentazocine base i s

TABLE 4 PARTITION COEFFICIENTS FOR PENTAZOCINE -~

0r g a n ic.

Aqueous

CHC13

0 . 1 M pH 7.35 phosphate b u f f e r

benzene

0.2 M pH 6.7 phosphate b u f f e r

benzene

0.1 i o n i c s t r e n g t h pH 6.9-8.2 phosphate b u f f e r

octanol

water

Coefficient

26

25.4

1.17

27 25

630

1.07

Reference

104

28

TERRY D. WILSON

3 74

I

2

n

s

1.0 -

2.0

Fig. 2. Relationship between log (benzene/water) distribution ratio and p H for pentazocine (after Borg and Mikaelsson)

.

-- 40 cI

0

c

E

\

- 30

0 2

4

6

8

10

12

PH

Fig. 3. Relationship between aqueous solubility and pH for pentazocine.

315

PENTAZOCINE

shown for v a r i o u s s o l v e n t s i n Table 5. I n a d d i t i o n a pH/ e q u i l i b r i u m s o l u b i l i t y s t u d y h a s been c a r r i e d o u t i n which s a t u r a t e d s o l u t i o n s o f p e n t a z o c i n e were prepared. u s i n g H C 1 and NaOH t o a d j u s t pH. A p l o t o f t h e pH/ s o l u b i l i t y r e l a t i o n s h i p i s shown i n F i g u r e 3(30).

IC*’

m

s

s Spectrum A d i r e c t i n l e t e l e c t r o n impact mass spectrum o f p e n t a z o c i n e ( S t e r l i n g - W i n t h r o p ) o b t a i n e d on a HP 5980 i s shown i n F i g u r e 4(31). The e l e c t r o n energy, emission c u r r e n t and s o u r c e t e m p e r a t u r e were 150 e . v , 0.42 mA and 185.6O r e s p e c t i v e l y . The m o l e c u l a r i o n i s found a t m/e o f 285.1, w i t h t h e b a s e peak a t 217.1 ( 1 0 0 % ) . G.C./N.S. r e s u l t s f o r p e n t a z o c i n e p r e v i o u s l y r e p o r t e d i n c l u d e b o t h d e r i v a t i z a t i o n and nonderi v a t i z a t i o n procedures, Underivatized pentazocine gave a molecular i o n a t m/e 285 and t h e nor-ion a t m/e 217 ( 3 2 ) while t h e 217 base Peak and a 202 peak were observed by a m u l t i p l e i o n d e t e c t o r (33). Chemical i o n i z a t i o n w i t h methane gave a molecular i o n o f m/e 286 and fragments shown below (34).

m/e 230

(M+

217

(M+ (M+

270

-

55)

68) 15)

30%

155 10%

(M+ - CH-CH-( CH3)z) (M+ - CH2CH-C(CH3)2) (M* - CH3)

The most f r e q u e n t l y encountered d e r i v a t i v e of p e n t a z o c i n e i n GC/NS w o r k h a s been t h e t r i f l u o r o a c e t y l with a molecular i o n a t m/e 381 (35,36,37). A p e n t a f l u o r o b e n z y l bromide was r u n g i v i n g a mole c u l a r i o n o f m/e 4-65 (38). A t r i m e t h y l s i l y l d e r i v a t i v e gave a M+ o f 357 w i t h a base peak o f 45 m/e f o r p e n t a z o c i n e (39), while a permethylated p e n t a z o c i n e g l u c u r o n i d e gave a m o l e c u l a r i o n a t 517, a n aglycone a t 217 and a b a s e peak a t m/e 201. H y d r o l y s i s o f t h e g l u c u r o n i d e f o l l o w e d by s i l a t i o n gave t h e 357 monoTMS d e r i v a t i v e ( 4 0 ) .

4.92 Nuclear Ma n e t i c Resonan= The p r o t o__&--r n and 3C nmr s p e c t r a o f p e n t a z o c i n e ( S t e r l i n g - W i n t h r o p ) a r e shown i n F i g u r e s 5 and 6 r e s p e c t i v e l y . Interpretations are g i v e n i n Tables 6 and 7 . The pmr spectrum was r u n o n a Varian HA-100 on a 10% s o l u t i o n i n

TERRY D.WILSON

316

TABLE 3 PENTAZOCINE SOLUBILITY Solvent

S o l u b i l i t y (%w/v) Reference:

29

2

CHC13

95% e t h a n o l benzene

1.25

-

ac e t one

3.3

-

ethyl acetate

ether

water 1 N HC1 1 N lactic acid

2.94

3 03-5

0

2.4

0.005

0.1

3

15

-

lnt

378

/

/

I

379

t

c

a,

u

4

0, a

a,

c

4J

a 0

w

5 k a,

u

4J

a [O

a,

u

c rd c

0 [O

u

a, k 4J

-ti

a, 5l

c

2

k

rd a,

u

rl

3

c

u m

d

a;

' 5

w -ti k

c

- 0 5l-l

- 0

h u

-4

h

E k 25

:.c E

380

TERRY D. WILSON

TABLE 6 PENTAZOCINE 1 H NMR ASSIGNMENTS No. H

Assignment

1.07

3H

CH3-CH

1 m53

3H

CH3-C -

1.92, 1.85

6 H

(CH3)2C=

1.4-3.6

5H

CH2x2

3.8-4.0

4H

NCH2x2

5.45

1 H

N-CH

6.86-7.25

3H

aromat ic s

7.5

1 H

OH, p a r t i a l l y exch * d

11.36

2 H

exch'd H

Chemical S h i f t (ppm)

, CH

38 1

PENTAZOCINE

TABLE 7 PENTAZOCINE I 3 C NMR ASSIGNMENTS CHZ-CHZCH-CH~ 1

d

*

HC1

CH3

HO 9,

Carbon

h

P PPdTMS

Multiplicity from partial off resonance dec oupling

a b

156.1 140.9

C

139.6

S

d e

127.9 122.5 113.7 111.7

d

f g h

i

57.3 50.8 44.8

S

S

S

d, d d d

k

38.1

t t t

1

37.2

d

m

34.6

S

n

25.1 23.7 21 .a 17.7 12.3

9"

j

0

P

sr

9" t 9 9

"values may be interchanged

TERRY D. WILSON

382

CF3COOD. The l 3 C nmr w a s o b t a i n e d on a J e o l FX 60 f o r a 10% DMSO dg s o l u t i o n (41). P r e v i o u s p r o t o n nmr data h a s been p u b l i s h e d f o r p e n t a z o c i n e ( 3 2 ) , N-benzylpentazocinium bromide (42) and p e n t a z o c i n e c h l o r o a c e t i c e t h e r ( 2 2 ) . These a r e summarized i n Table 8.

4.93

I n f r a r e d Spectrum The i n f r a r e d suectrum o f Dentazocine ( S t e r l i n g - W i n t h r o p ) , 1/2 7% i n - KBr, taken- on a P e r k i n E l m e r 467 i s shown i n F i g u r e 7 (43). The previousl y noted a l l y l i c (C = C ) s t r e t c h i n g a t 1670 cm-1 i s a l s o seen (32).

4.94

U l t r a v i o l e t Spectrum An u l t r a v i o l e t spectrum o f pentazoc i n e ( S t e r l i n g - W i n t h r o p ) was o b t a i n e d on a Cary 15 and i s shown i n F i g u r e 8 . The r e l a t i v e f o r t h i s 0.0318 g/L s o l u t i o n i n 95% a l c o h o l i s s e e n a t 283 nm. The c a l c u l a t e d a b s o r p t i v i t y t h e r e i s 2052 ( 4 4 ) .

4.10 X-Ray D i f f r a c t i o n A normalized powder x-ray d i f f r a c t i o n p a t t e r n f o r p e n t a z o c i n e ( S t e r l i n g - W i n t h r o p ) i s shown i n Figure 9 . I t w a s r e c o r d e d on a Rigaku X-ray D i f f r a c t o m e t e r M i n i f l e x w i t h Cu K a , Ni f i l t e r e d r a d i a t i o n a t 4000 c p s , TC/1, ss/s, 0 . 5 2 e/ min. The s c a n w a s done from 400 t o 3.50 2 8 with 1/1 peak h e i g h t r a t i o s r e l a t i v e t o t h e 1 2 . 4 O 2 0 100% peak. The c a l c u l a t e d d-spacings a s s o c i a t e d w i t h t h i s p a t t e r n a r e l i s t e d i n Table 9 ( 4 5 ) .

4.11 D i s s o l u t i o n The d i s s o l u t i o n method f o r p e n t a z o c i n e h y d r o c h l o r i d e t a b l e t s u t i l i z e s a UV d e t e r m i n a t i o n o f pentazocine i n f i l t e r e d t e s t samples a t 278 nm. The a p p a r a t u s used i s USP number two a t 50 rprn with water a s t h e d i s s o l u t i o n medium. The s o l u t i o n s are measured i n h y d r o c h l o r i c a c i d d i l u t e d with d i s s o l u t i o n medium t o g i v e 0.01 N HC1 and q u a n t i t a t i v e l y compared t o a s u i t a b l e s t a n d a r d ( 4 6 ) . A d i s s o l u t i o n procedure f o r p e n t a z o c i n e hydroc h l o r i d e and a s p i r i n t a b l e t s i s s e t f o r t h i n Addendum a , Second Supplement t o t h e USP XX. The procedure i s c a r r i e d o u t w i t h a p p a r a t u s number one a t 80 rpm u s i n g w a t e r as t h e d i s s o l u t i o n medium.

w 2

H

0

u N

E m

nw

0

.. a, 5 W

k a,

4-(

p:

I

A

%

\o II -

?? 5 v

0

a

..

coo

c2

(v n

I2 u v u II u

I

I

N

I

%

O & a 3 1

E h

-

k \ o

P

w oc

7 0 c k

.rl

a

u \ o .r(

*

b

E

. . . m m u l

a % a 3

p

a;

a

k 0

-d

rl

c 0 0 k

h

a a,

,G

c

0

u

-4 N

c

rd 4

a

a,

0

w

5

a,

u

k c,

a In

aa, k rd k

c,

w

H

r-

m

-4 E

385

PENTAZOCINE

I.o

0.9 0.8 0.7

8

0.6

z a 0.5 0

cn

m

a 0.4 0.3 283 nm 0.2 0.I 0.0

210

240

270

300

330

WAVELENGTH (nm) Fig. 8. Ultraviolet absorption spectrum of pentazocine.

001 x

O1/1

I

a

u k

w w

a,

k

a

50a

PENTAZOCINE

387

TABLE 9 PENTAZOCINE d SPACINGS d(i) =

2

eo

x 2

0

sin 8

d(1)

11

8.03

11.5

7.68 7.12 6.72 6.34 5-54 5.25

12.4

13.15 13 95 15 95 16.85 17.1 17.75 18 35 20.0

20.45 21 *55 22.85 25 75 25.85 28.5 29.9 34.6 36.05

= 1.54

1/10

69 69 13.13 100 18.18

60.6 34 3 4 81.81

5.17

71.71

4.99 4.82 4.43 4.33 4.11 3.88 3.45 3.44 3.12 2.98

20.2

2.58 2.48

A

12.12

31 - 3 1 50.5 33 33 17 1 7 19 1 9 15.15 12.12

6.06 9.09 6.06

TERRY D.WILSON

388

Twenty-five mL of t e s t s o l u t i o n i s f i r s t shaken with a s t r o n g l y basic a n i o n exchange r e s i n . Five mL o f t h e s u p e r n a t a n t i s t h e n e x t r a c t e d with a mixture o f 20 mL chloroform and 1 0 mL o f 1 i n 4000 bromocresol purple i n g l a c i a l acetic acid. A spectrophotometric d e t e r m i n a t i o n o f t h e i o n p a i r formed between pent a z o c i n e and t h e i n d i c a t o r i n t h e chloroform l a y e r i s t h e n made a t 408 nm and compared t o a s i m i l a r l y e x t r a c t e d pentazocine standard (4).

4.12

Identification Pentazocine i s i d e n t i f i e d a c c o r d i n g t o USP XX by u s e o f i n f r a r e d o r u l t r a v i o l e t s p e c t r o photometric methods. I n t h e f i r s t a d i s p e r s i o n i n KBr of t h e d r i e d d r u g e x h i b i t s maxima o n l y a t t h e same wavelengths as a similar p r e p a r a t i o n o f USP Pentazocine Refemnce Standard. I n t h e u l t r a v i o l e t method a 1 i n 12,500 s o l u t i o n o f p e n t a z o c i n e i n 0 . 0 1 N H C 1 e x h i b i t s maxima and minima a t t h e same wavelengths as r e f e r e n c e standard. C a l c u l a t e d abs o r b a n c e s a t 278 nm o f d r i e d drug and s t a n d a r d do n o t d i f f e r by more t h a n 3.0 % (3). Pentazocine i s i d e n t i f i e d i n t h e combination p r o d u c t : p e n t a z o c i n e h y d r o c h l o r i d e and a s p i r i n t a b l e t s by a method g i v e n i n Addendum a , 2nd Supplement o f USP XX. A s i l i c a g e l TLC system i s used w i t h t h e s o l v e n t system: e t h y l a c e t a t e : methanol : formic acid ( 9 0 : 5 : 5 ) . D e t e c t i o n i s by UV, i o d i n e vapor and i o d o p l a t i n a t e s p r a y ( 4 ) .

5.

Stability 5.1 H y d r o l y s i s The h y d r o l y s i s r e a c t i o n of p e n t a z o c i n e i s t h e o n l y c h e m i c a l l y d e g r a d a t i v e pathway which h a s been observed. An e a r l y r e p o r t d e s c r i b e d t h e r e a c t i o n and c h a r a c t e r i z e d t h e p r o d u c t : 1,2,3,4, 5,6-hexahydro-6,11-dimethyl-3-(3-hydroxy-~-~ethylbutyl)-2,6-methano-3-benzazocin-8-ol which w a s o b t a i n e d when p e n t a z o c i n e w a s h e a t e d a t 90° f o r two h o u r s i n 1 N HC1. A c i d i c h y d r o l y s i s o f pent a z o c i n e g1ucuroni.de a l s o y i e l d e d t h i s p r o d u c t while p e n t a z o c i n e i n b i o l o g i c a l f l u i d s was found t o be s t a b l e ( 3 2 ) . T h i s h y d r o l y s i s r e a c t i o n was i n f a c t used f o r t h e d e t e r m i n a t i o n o f p e n t a z o c i n e i n human u r i n e (34). The r e a c t i o n was e l u c i d a t e d by a k i n e t i c s s t u d y which showed t h a t a carbonium i o n p s e u d o - f i r s t o r d e r mechani m w a s involved. The kobs a t 80° a t a. 3.9 x lo-’ M H C 1 c o n c e n t r a t i o n

PENTAZOCINE

389

w a s 3.412 x hr w i t h an a c t i v a t i o n energy o f 35.9 k c a l mol-1 and a n a c t i v a t i o n e n t r o p y o f 27.3 c a l ( m o l deg1-l ( 4 7 ) . 5.2

Dosage Form S t a b i l i t y The s t a b i l i t y o f repackaged p e n t a z o c i n e h y d r o c h l o r i d e i n j e c t i o n w a s s t u d i e d t o determine t h e e x p i r a t i o n d a t e . An e x t r a p o l a t i o n o f t h e Arr h e n i u s p l o t o b t a i n e d u s i n g 40: 50'an 600 r a t e c o n s t a n t s gave a k o f 5.448 x 10-3 day-l a t 250. T h i s meant t h a t a 10% l o s s o f p o t e n c y occurr e d i n 1909 days a t 25' ( 4 8 ) .

6.

Methods o f A n a l y s i s 6.1 T i t x m e t r i c Pentazocine can b e determined by a t i t r i m e t r i c procedure i n which t h e compound i s d i s s o l v e d i n g l a c i a l a c e t i c a c i d . C r y s t a l v i o l e t i s used as i n d i c a t o r and t h e s o l u t i o n i s t i t r a t e d t o a g r e e n end-point w i t h 0 . 1 N p e r c h l o r i c a c i d . Each mL of 0.1 N perchloric acid a t t h i s point i s equivalent t o 28.54 m g o f p e n t a z o c i n e base ( 3 ) . 6.2

E r a v i o l e t Spectrophotometry Pentazocine can be determined i n p e n t a z o c i n e h y d r o c h l o r i d e t a b l e t s by a UV s p e c t r a l method. The sample i s e x t r a c t e d w i t h s u l f u r i c a c i d , b a s i f i e d , e x t r a c t e d w i t h e t h e r and backe x t r a c t e d i n t o 1 i n 7 0 d i l u t e s u l f u r i c a c i d . The absorbance o f t h e s o l u t i o n i n 0 . 5 N H2SO i s d e t ermined a t 278 nm and compared t o t h a t o f t h e s t a n d a r d p r e p a r a t i o n . A c a t i o n exchange column i s o l a t i o n method can b e u t i l i z e d t o determine pentazocine i n pentazocine l a c t a t e i n j e c t i o n . P e n t a z o c i n e i s e l u t e d from t h e column with methanol: 6 N HC1 ( 1 : l ) . Sample absorbance i s r e a d a t 278 nm and compared t o a p e n t a z o c i n e s t a n d a r d a t approxi m a t e l y 1 2 0 a g / mL ( 3 ) . 6.3

Fluorescence Spectrophotometry Highly s e n s i t i v e s p e c t r o f l u o r o m e t r i c measurements have been used e x t e n s i v e l y i n p e n t a z o c i n e a n a l y s i s e s p e c i a l l y i n measurements from b i o l o g i c a l s o u r c e s . Themethod developed e a r l y by Berkowitz, Way and coworkers ( 2 7 , 4 9 , 5 0 , 5 1 , 5 2 ) h a s been modif i e d and extended by o t h e r i n v e s t i g a t o r s (53,511, 55,56). Pentazocine l e v e l s i n plasma, u r i n e , b r a i n , and i n t e s t i n e have been determined by t h i s method. Briefly the t i s s u e o r f l u i d is b a s i f i e d with a

TERRY D. WILSON

390

carbonate/bicarbonate buffer at pH 8-9 and the pentazocine is extracted into benzene. An aliquot is washed with 0.2 M sodium phosphate buffer, pH 6.7 and is then back-extracted into 0.2 N H C 1 . The fluorescence excitation wavelength used most commonly has been 278 nm with emission measured at 310 nm.

An extraction, ion-pair partition chromatography separation method was developed by Borg and Nikaelsson for pentazocine using fluorescence detection ( 2 5 ) and has been used by others (57.58). The tissue, plasma, brain or serum was extracted with benzene following basification with a carbonate/bicarbonate buffer at pH 10.5. An ethanolized cellulose column was used with a stationary phase of 0.1 PI HC1 and a mobile phase: cyclohexane: 1-pentanol (614) followed by extraction into 0.1 M phosphoric acid for fluorescence measurements. Other fluorescence methods for pentazocine have been used on plasma ( 5 9 ) and for TLC quantitation (47). A fluorescent 2-p-chlorosulfophenyl-3-phenylindone ( D I S - C 1 ) derivative of pentazocine has been measured by TLC (60) while another extraction method using heptane with 0.5:’. isoamyl alcohol on basified plasma has been developed. In the latter, fluorescence was measured after back-extraction into 0.1 N HC1 (61).

6.4 Radioimmunoassay Radioimmunoassay methods have been developed for pentazocine which were shown to be both sensitive and specific. An early method utilized tritium labeled pentazocine at 1.5 Ci/mrnol. Antibodies were produced in rabbits to both an azobenzoic acid and a carboxymethyl derivative of pentazocine coupled by carbodiimide condensation to poly-L-lysine. No cross reactivity was found for metabolites and benzomorphan analogs to the former antiserum with only conjugated pentazocine crossreacting with the latter antiserum (62). In a second method rabbit antibodies were produced against a butanoic acid derivative of pentazocine which was conjugated to bovine se albumin via a carbodiimide condensation. An € 8 1 labeled methyltyrosine pentazocine giving 25,000-

PENTAZOCINE

39 1

cpm/ 1 0 0 d was used in the assay. An approximate detection limit of 1 ng/mL was measured

30,000

(63) Antiserum specific for the biologically more active 1-pentazocine has been produced in rabbits using an&pentazocine-2’-carboxymethyl ether-bovine serum albumin conjugate. A low 0.084% cross-reactivity was found for d-pentazocine while 51.2% was measured for dP -pentazocine (22).

6.5 Chromatography 6.51 Thin L?yer Chromatography Extensive use has been made of TLC separation for pentazocine on both qualitative and quantitative levels. Table 10 indicates mobile and stationary phases used and Rf values obtained. 6.52

Gas Chromatography

Gas chromatography has been the most frequently used method for pentazocine analysis as indicated by published accounts. It has found especially wide spread application in the field of biological disposition of pentazocine. Table 11 summarizes column packing, detection, derivatization, percent recoveries and detection limits where applicable.

6.53 High-Performance LiquidEhromatoPraphy Several reports of high-performance liquid chromatographic analysis of pentazocine have appeared. The separations have been made using both normal phase and reversed-phase systems. Detection was by ultraviolet, fluorescence and electrochemical means. W measurements were generally made at 278-280 nm on underivatized pentazocine although a greater sensitivity was found using a 2-p-chlorosulfophenyl-3-phenylindonederivative (91). Recoveries found for particular processes described include 78% by extraction from blood (91) and 98.6 and 98.9% on pentazocine tablet extractions by normal and reversed-phase methods respectively ( 9 2 ) . Fluorescence measurements were made on a dimer of pentazocine produced by on-column oxidation with 0.04 M K3Fe(CN)6. This product was separated from a dihydromorphine fluorescent dimer and a pentazocine-dihydromorphine product (93).

TABLE 10 THIN LAYER CHROMATOGRAPHY OF PENTAZOCINE Stationary Phase

Mobile Phase

Visualization

Rf

Re ference

.35-.42

64

nBuOH:KOAc:H20 Folin-Ciocal( 4 11:2) teau and 20% benzene:MeOH:. Na26O3 28%NH3 (79:21:1)

.60

50

EtOAcrtriethyl- Dragendorff and iodine amine (911) HOActEtOAcracet- vapor one (3:5:5)

.62

EtOAcretherrEtz- fluorescence NH (10110:1) (280/380nm) iodine vapor

.4

Silica gel glass Et0Ac:cyclo- iodoplatinate microfiber sheets hexanerMe0H: and iodine/ MI OH KI ( 52 :40r 0.8 t

0.4)

Silica gel W \o N

Silica gel G

Kieselgel 60 F254

35

9

32

33

47

Silica gel G

Silica gel

benzenetMeOH:28% NH4OH (79:20:1) nBuOH :HOAc I H20 ((hlr2) Me0H:HzO ( 9 5 : 5)

Folin-Ciocalteau 20% Na2C03

30

49

.62 2-p-chlorosulfo-

-

ph e nyl -3- ph enyl

27

60

indone derivative Na in MeOH:DMSO (8g:100:8) a w '&

ninhydrin Silica gel G- EtOAc:MeOH:NH4254 OH (85t10:5) iodoplatinate EtOAc:MeOHi H20 fluo Ee scamine (80:15:5) hexanerEt0Ac:EtOH: NHbOH (45:50:5:2)

.68

34

- .

Silica gel

EtOAc :MeOH:formic acid ( 9 0 : 5 15)

iodine vapor iodoplatinate

57

-

4 (Continued)

T A B L E 1 0 (Continued)

THIN LAYER CHRONATOGRAPHY OF P E N T A Z O C I N E Stationary Phase Silica gel G

\o W

Silica gel F-254

P

Kobile Phase

Visualization

Rf

MeOH:NH3 (100: radiochromatog. 67 1.5) Folin-Ciocalteau benzeneiMe0H: diazotized p-nitro- .53 28%NH3 ( 7 9 : 20I 1 )aniline, naphthareso rc ino1 benzene:MeOH:iso- liquid scintilpropylamine ( 9 5 : lation

Reference

65

-

66

-

67

513) Preparative Silica gel G

dioxane :MeOH: formic acid

radiochromatog.

(100:5: 5 )

benzene iMeOH : isopropylamine (95I 5 :3 1 Silica gel F-254

benzene SlKeOH I isopropylamine ( 9 5 : 5: 3)

liquid scintillation

68

Silica gel

benzene:MeOH:NHbOH (80:20:1) EtOAc :st3N (90: 10)

Silica gel glass EtOAcrcyclohexmicrofiber sheets aneiNH4OH:MeOH: H20 (70:1512: 8:0.5) Silica gel G and GF

w

u \D l

benzene:MeOH:isopropylamine(95:5:

3) d i o xane :H20 I HO Ac

radiochromatog.

52

26

-

-

ninhydrin 0 . 5 % H2SO4 io do plat inate ammoniacal AgN03

-

69

UV iodine vapor Dragendorff

65

70

-45

(100: 2 I5)

dioxane:H20:NH3 (100:15:15) dioxanerMe0H: formic acid (100:5: 5) EtOAc:Et3N (9O:lO) ~~

87 70

57

~

(Continued)

TABLE 10 (Continued) THIN LAYER CHROMATOGRAPHY OF PENTAZOCINE Stationary Phase

Mobile Phase

Silica gel glass micro fiber

Kieselgel 60/ Kieselgur FZ54

Rf

Referenc e

.60

71

73

39

Visualization

EtOAcrcyclohexninhydrin ane :MeOH :NH iodoplatinate i o d i ne/KI ( 7 0 : 15: 10: 57 develop to 9 cm then: EtOAcrcyclohexane:NH (50:40:0.1) to 14.3 c m

benzenerMe0H:isopro-UV pylamine (95:5:3) iodoplatinate dioxanerMe0H:formic Dragendorff acid (100:5:5) Folin-CiocalEtOActEt N (90110) teau benzene I J e O H :NH3 ( 7 9 :20: 1

9

79 9

65 72

TABLE 11

Column

1.5% OV-I

Detection

3% O V - 1 7

mass fragmentography

3% O V - 1 7

nitrogen

1.5% OV-101

F.I.D. mass s p e c t -

GAS CHROIViATOGRAPHY OF PENTAZOCINE Col. Derivatization Temp.oC D e t e c t i o n L i m . %Recovery Ref.

t r i f l u o roac e t yl -

220

t r i m e t h y l s i l y l 100-320 permethyl

0 . 5 -ug/m~

-

81

72

-

40

89

36

-

73

96-100

74

ral w

5

3% O V - 1 7

mass s p e c t ral

trifluoroacetYl

ov-1

6 3 N i E.C.

pentafluoropro pionyl

180-260 220

1 0 ng/mL

-

3% ov-1

F.I.D.

-

210

2,S!%,SE-30

F.I.D.

-

200

-

93-98

75

2,5%SE-30

F.I.D.

-

220

5 M g/mL

97

76

-

206

2.4 ng

5% QF-1

nitrogen

25 ng

100.3

57

(Continued)

TABLE l l ( C o n t i n u e d )

Column

Detection

5% O V - 1 7

63Ni E.C

3PDexs i 1 300

6 3 N i E.C.

10% OV-1

3 H E.C.

P \D w

F.I.D.

.

F.I.D.

GAS CHRONATOGRAPHY OF PENTAZOCINE Col. Derivatization Temp.OC D e t e c t i o n L i m . $Recovery pentafluorobenzyl

220

235

3 Pg

-

38

p e n t a f 11-10robenzyl

265

5 ng

-

77

h e p t a f l u o r o b u t y r y l 215 245 trimethylsilyl

2.5%SE-30

F.I.D.

4%Apiezon L

F. I .D.

-

nitrogen

-

5% QF-1

ace t y l

3% O V - 1 7

F.I.D.

trimethylsilyl

3% ov-1

F.I.D.

trimethylsilyl

3% SE-30

mass fragmentography

Ref.

-

5 ng 0.5~g/mL

66.4-95

78

200

-

-

32

206

2.4 ng

100.3

65

ng

79.9

80

-

81

92-94

33

90-220 225 21 0

10

2 ng/mL

a3

c7

a3

I

cu

I

.c

hl)

u\

b

0

cl

u\

aJ

0

a3

I

I

a3

=t

I

I

I

co m I n m 4

s

I

I

0 u\

N I 0

cu

c?

rn

rl

4

0 (v

cl

u\

d

F

H

n

I

cu

0

0 0

(\I

Lu

4

u\

0 0

h

rl

rl .A

a, &

h c P

n

k 4

H

I

n

k

H

k P

.d

L

H

1

Lu

0 u\

0

v3

c?

z

.ri

w

V

k

0

P

a,

2

I

cu

cl

4

0 4

N

u\

u'\

Lu

I

B4

H

r=l

I

N

V

w v3

399

TABLE l l ( C o n t i n u e d )

GAS CHROMATOGRAPHY OF PENTAZOCINE Column

3% SE-30 3% OV-7

Detection

Derivatization

F.I.D.

5% SE-30 ov-101

3% Dexsil

Ref.

150-290

F.I.D.

trimethylsilyl

mass s p e c t ral

F.I.D. nitrogen 63Ni

E.C.

300

3% SE-30

D e t e c t i o n L i m . $Recovery

130-290

3% OV-17 3% ov-1

Col. Temp.oC

230

39

260

pentafluoropropionyl

220

pentafluorobenz-

265

89

130-290

90

Yl

F.I.D.

-

PENTAZOCINE

40 I

Glassy carbon working e l e c t r o d e s were used i n electrochemical d e t e c t i o n o f pentazocine while no response w a s measured i n one s t u d y a t an o p e r a t i n g p o t e n t i a l o f 0.8 V (94). Table 12 summarizes t h e HPLC column and mobile phase c o n d i t i o n s u t i l i z e d i n reported investigations.

7. B i o l o g i c a l D i s p o s i t i o n and Pharmacokinetics 7.1

Disposition The a b s o r p t i o n . d i s t r i b u t i o n . metabolism and e x c r e t i o n of pkntazocine have been widely s t u d i e d i n both man and animal s p e c i e s . Early work w a s c a r r i e d out by t h r e e major groups: Pfttman and coworkers, Beckett and coworkers and Berkowitz, Way and coworkers, Reviews o f metabolism (51) and pharmacokinetics (97) o f pentazocine have been published. The e f f e c t o f r o u t e of a d m i n i s t r a t i o n on t h e d i s p o s i t i o n of pentazocine h a s been explored (27,49,75,85,86). The metabolic pathways a v a i l a b l e t o pentazocine a r e o u t l i n e d i n Figure 10. Also shown a r e u r i n a r y e x c r e t i o n data f o r t h e metabo l i t e s expressed as p e r c e n t o f administered dose. A r h e s u s monkey study i s a l s o included (66) which s p e c i e s shows t h e t r a n s - a l c o h o l m e t a b o l i t e excreted as well. Urinary e x c r e t i o n c o n s i s t s of unchanged pentazocine and m e t a b o l i t e s p l u s phenolic glucur o n i d e s o f each. Fecal e x c r e t i o n c o n s i s t s of l e s s t h a n 1%administered dose ( 8 6 ) . D i f f e r e n c e s i n pentazocine d i s p o s i t i o n i n smokers and nonsmokers h a s been observed with t h e former metabolizing 40% more rapidly(98).The e f f e c t o f c i r r h o s i s on pentazocine b i o a v a i l a b i l i t y h a s also been s t u d i e d . A 46% decrease i n c l e a r a n c e and a 233-2’785 i n c r e a s e i n b i o a v a i l a b i l i t y o f pentazocine for t h e s e p a t i e n t s was found (84,89). Other d i s p o s i t i o n s t u d i e s have been done on p o s t - o p e r a t i v e p a t i e n t s (591, mother and i n f a n t a t time o f b i r t h (99) and normal s u b j e c t s with c i r c a d i a n v a r i a t i o n

(36) 7.2

Pharmacokinetics The uharmacokinetics of uentazocine have been i n v e s t i g a t e d i n humans (11,76,86). Table 13 shows values f o r h a l f - l i f e , volume of d i s t r i b u t i o n , b i o a v a i l a b i l i t y and c l e a r a n c e c a l c u l a t e d by vari o u s a u t h o r s . The plasma h a l f - l i f e values obtained range from 2 t o 5.7 hours. The p o s s i b i l i t y t h a t

TABLE 12 HPLC ANALYSIS OF PENTAZOCINE Detection

Column

Mobile Phase

Mobile Phase pH

Reference

UV-278 nm

ABondapak C18

methanoltwater (66: 34) PIC B7

3.5

48

W-280 nm

ABondapak c18

acetonitrile:0.7% NH4Cl (80:20)

8.0

91

UV-254 nm

/lh

Bondapak C18

methanolrwater (28: 12) (0.0048 M K2HPO4, 0.0077M KH2PO4)

7.0

28

UV-254 nm

&

Bondapak c18

methano1:water (20: SO), 0.05 M tetrabutylammonium hydroxide, H3PO4 pH adjustment

6.1

94

Partisil 5& Silica

CHC13 :methanol:isopropylamine ( 960:40:

-

92

2.4

92

0 P N

W-280 nm

2)

UV-280 nm

JU

Bondapak c18

sodium octanesulfonate ( 0 . 0 0 5 M) : methanol: H3PO4 ( 6 0 0 : 400:1

fluore sc enc e 320/436 nm

Partisil porous silica

methanol: 2N NH4OH: 1 N NH4C1 (3O:zO: 10)

9.5

93

electrochem. 0.6 V, 50 nA/V

Syloid 74 silica

methano1:ammonium nitrate buffer ( 9 : 1)

10.2

95

M Bondapak C18

methano1:water (20: 6.1 8 0 ) , 0 . 0 5 M tetrabutylammoni um hydrox ide, H3P04 pH adjustment

94

LiChrosorb ODS

methanol :0.01 M KH2PO4 (85:15)

4.6

96

P

W 0

electrochem. 0.8 v, 5-20 n

v

elec trochem. 1.0

v

~/

TERRY D.WILSON

404

URINARY EXCRETION ( % DOSE 1 C" 3

\

UNC HA NG ED

62) dpCH3

-N

CH3

GLUCURONIDE

3.0

'I

)-

9.5

(5-20)

13

6.9 20.0

UNIDENTIFIED METABOLITES

no PENTAZOCINE CH20H 2.3

UNCHANGED

>-)cH~

11.6

-

II 19.0

GLUCURONIDE HO

-

CIS ALCOHO L

1 2 fpG

CH20H

-

UNCHANGED GLUCURONIDE

CH3

HO

TRANS-ALCOHOL

UNCHANGED 38.9

-

39

24

81

70

51

66*

GLUCURONIDE HO

TRANS-ACID REFERENCE )c

Rhesus Monkey

Fig. 10. Pentazocine metabolic pathways and urinary excretion products in man and monkey.

TABLE 13

HUMAN P E N T A Z O C I N E F'HARMACOKINETICS PARAMETERS

Half -1if e

0-4

Volume o f Distribution (L)

Oral B i o a v a i l a b i l i t y ($>

Clearance

Reference

(L/rnin)

3 04

396

18.4

1.38

100

3.8

342

18

1.25

84

2.41 2.20

2 58 26 5

1.741 day 1.672 n i g h t

36

2.2

208

-

33

-

-

59

2

TERRY D. WILSON

406

t h e i n t e s t i n e m e t a b o l i z e s p e n t a z o c i n e t o account f o r t h e l o w b i o a v a i l a b i l i t y was shown i n a r a b b i t

jejunum s t u d y . Here s u l f a t i o n and g l u c u r o n i d a t i o n o c c u r r e d by s a t u r a b l e p r o c e s s e s ( 1 0 1 ) . Data i n r e f e r e n c e 76 was f i t t o a t h r e e comDartment open model w i t h a d e t e r m i n a t i o n o f m e t a b o l i c , a b s o r p t i o n and e l i m i n a t i o n r a t e c o n s t a n t s from u r i n a r y Data c a l c u l a t e d t o f i t excretion rates. t h e equation: + B e- 8 t Cp=Ae e

a

t

f o r a two compartment open model f o r p e n t a z o c i n e i s shown i n Table 1 4 . I t w a s shown t h a t c i r c u l a t i n g p e n t a z o c i n e is d i s t r i b u t e d w i t h 48% i n blood c e l l s , 33% bound t o plasma p r o t e i n and 19% i n plasma w a t e r ( 6 7 ) . S t u d i e s o f pentazocine b i o l o g i c a l d i s p o s i t i o n i n a n i m a l s c o n s i s t of t i s s u e uptake ( 2 6 , 5 2 , 5 8 , 6 5 , 6 8 , 7 9 ) , blood l e v e l ( j 0 , 6 1 , 7 4 ) and u r i n e l e v e l d e t e r m i n a t i o n s ( 8 8 ) . While animal m e t a b o l i c pathways i d e n t i f i e d ( 3 9 , 4 0 , 7 O ) a r e similar t o t h o s e found i n man, a d d i t i o n a l p r o d u c t s have been demons t r a t e d . I n t h e r a t 9-methoxypentazocine ( o r i t s 8-methoxy-9-hydroxy i s o m e r ) , 8 , 9 ( o r 9,8)-methoxyhydroxy m e t a b o l i t e o f t h e c i s - a l c o h o l p r o d u c t and 8 , 9 ( o r 9,8)-methoxyhydroxy m e t a b o l i t e o f t h e t r a n s a l c o h o l p r o d u c t were found. Pharmacokinetic s t u d i e s have been done i n r h e s u s monkey ( 6 6 ) and i n t h e d o g ( 3 5 , 5 4 , 6 3 ) . T y p i c a l plasma h a l f - l i v e s measured i n dog plasma a r e 1.2-1.6 h o u r s . A day-night v a r i a t i o n w a s a l s o observed i n t h e c a l c u l a t e d p a r a meters ( 3 5 ) . Determination i n Body T i s s u e s and F l u i d s Pentazocine a n d i t s m e t a b o l i t e s have been d e t ermined i n human and animal t i s s u e s and f l u i d s by a v a r i e t y o f a n a l y t i c a l methods. References f o r t h e s e t e c h n i q u e s a r e shown i n Table l 5 A f o r humans and Table l 5 B f o r animal s t u d i e s ,

8,

Acknowledgement The a u t h o r wishes t o e x p r e s s h i s t h a n k s t o members of t h e P h y s i c a l Chemistry S e c t i o n , Analy t i c a l c h e m i s t r y Department, Sterling-Wjnthrop Research I n s t i t u t e e s p e c i a l l y A l l a n G. Hlavac; a l s o t o E . L . P r a t t , L.J.Dombrowski, A.V.R.Crain, R.Lorenz and J . Edelson f o r reviewing t h e manuscript.

TABLE 14

TWO COMPARTNENT OPEN NODEL PARAMETERS FOR PENTAZOCINE

553

102

8.82

0.0942

5.81

1.638

1.668

33

495

108

11.39

0.357

7 357

2.74

1.653

36*

324

98

10.58

0.349

6.65

1.32

36**

2.98 ~~

* **

day

night

~~~

TABLE

15

REFERENCES F O R PENTAZOCINE DETERMINATION I N BODY T I S S U E S AND F L U I D S A)

HUMAN S T U D I F S

GC

Method I

GC/MS

TLC

HPLC

f 1u o r e sc enc e

RIA

Tissue/Flui d

62

Cerebrospinal Fluid

-

33

-

-

-

B)

ANIMAL S T U D I E S

M et h o d I Species

GC

GC/NS

brain plasma

79 79

-

cat

bile plasma urine

-

-

56

66 66

-

C SF

plasma

monkey

f 1u o r e sc e n c e

RIA

Tissue/Fluid

baboon

P \o 0

TLC

brain plasma urine

-

-

-

-

26

-

26

61

-

54 54

68 68 66

23

-

(Continued )

TABLE 1 5 (Continued ) REFERENCES FOR PENTAZOCINE DETERPINATION I N BODY TISSUES AND FLUIDS

B)

ANIP!AL STUDIES

Method:

Species mouse

Pony

f 1uo r e sc e nc e

GC/M

TLC

-

-

61

40

50

-

50

-

53

80 103

39 -

-

-

-

GC

RIA

Tissue/Fluid

65

bile brain gut plasma serum

65

plasma

-

bile brain gut

4;

-

-

rabbit

rat

plasma

urine swine

wbc plasma

4;

52

52

39 I

61

-

41 1

PENTAZOCINE

References 1. P h y s i c i a n ' s Desk R e f e r e n c e , C h a r l e s E. Bake r , J r . , I e d i c a l Economics C o . , O r a d e l l , N . J . ,

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TERRY D.WILSON

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413

PENTAZOCINE

36.

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J . E'harmacol.

TERRY D. WILSON

414

53.

Brown, N . ,

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2217. (.1 9. .7.9.) . 55. Kigasawa, K . , S h i m i z u , H . , O h t a n i , H . , H a y a s h i d a , 3 . , and I s h i o d o r i , T . , Yaku a k u Z a s s h i , 99, 4-02 ( 1 9 7 9 ) .

%Kigasawa, K . , S h i m i z u , H . , S a i t o , I
57.. James, Sl,

a n d '"daring R . ,

2.

Chromatog.,

78, 4-17 ( 1 9 7 3 ) . 58. Paalzow, L, a n d Arbin, A , , J. Pharm. Pharmac., 24, 552 ( 1 9 7 2 ) . 59. G o t o h , Y . , Arakawa, Y . , T a n a k a , H . , a n d Furukawa, K . , I n t l . C o n g r e s s S e r i e s , 533,

66 ( 1 9 8 0 ) . 60. V i n s o n , J . , P a t e l , D. a n d P a t e l , A . , Anal. Chem., 49, 163 ( 1 9 7 7 ) . 61.Davis, L . , a n d Sturm, B . , 5. J . vet. Res., 31, 1631 ( 1 9 7 0 ) . 62. Xiilliams, T . a n d P i t t m a n , K . , Res. Corn. Chem. P a t h . P h a r m a c . , 7, 119 (1974). 63.P z s o n , J . , Graham, 1.':. , Banks, d . , B e n z i g e r , D . , Rowe, E . , Clemans, S . , a n d Z d e l s o n , J . , J . Pharm. S c i . , 68, 626 ( 1 9 7 9 ) . 64. K a i s t h a , K . , a n d T a d r u s , R . , J. Chroma., 155, 214 ( 1 9 7 8 ) . J a m e s , S., ?ugh, G., a n d d a r i n g , R. , Xenobiot., 4, 521 ( 1 9 7 4 ) . 66. P i t t m a n , K . , a n d P a r t m a n n , G . , ?harm. 63, 8 4 ( 1 9 7 4 ) . F E h r n e b o , f , , , A g u r e l l , S. , Bo r g u s , L . , Gordon, E . , a n d L o n r o t h , U., C l i n . Pharmac. T h e r . , 16, 424 ( 1 9 7 4 ) . 68.P i t t m a n , K . , L i f e S c i . , 1 2 , 131 (1973). 69. K a i s t h a , K . , a n d J a f f e e , J . , J. Pharm. -*S c i ' 61, 305 ( 1 9 7 2 ) .

a,, .

70.

J 0

P i t t m a n . K . . R o s i . 9.. C h e r n i a k , R . , A . , a n d Conway; d . , Biochem. Pharmac.

i erola,

18, 1673 ( 1 9 6 9 ) K a i s t h a , K . , T a d r u s , R., a n d J a n d a , R . , J . Chromatog., 1 0 7 , 359 ( 1 9 7 5 ) .

71.

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R a c k e l l , la., a n d P o k l i s , A . , J. Chromatog., 235, 4-45 (1982). 73. E d l u n d , P . , J. C h r o m a t o g . , 206, 109 ( 1 9 8 1 ) . 74. I e d z i h r a d s h y , F . , a n d Ahmad, K . , L i f e -*, Sci 1 0 , 711 (1971). 75. B e c k e t t , A . , T a y l o r , J . , a n d Kour o u n a k i s , P . , 2. Pharm. rharmac., 22,

123 ( 1 9 7 0 ) 76. Vaughan, D . ,

a n d B e c k e t t , A , , J. Pharm. Pharmac., 26, 789 ( 1 9 7 4 ) . 77. Swezey, S., B l a s c h k e , T . , a n d K e f f i n , P . , 2. Chromatog., 154, 256 (1978). 78. Isittman, K . , a n d D a v i s o n , C . , J. Pharm. ---" Sci 62, 765 (1973). 79. C o r o n e o s , N . , Keaney, N . , b c D o w a l l , D., P i c k e r o d t , V . , Glynn, J . , a n d R o b e r t son, A . , J. Pharmac., 50, 393

(1974)

&.

80. r a r u y a m a , Y . , Sawa, A , , a n d Hosoya, E . , B u l l . - N a r c . , 27, 37 (1975). 81. P i + t m a n , K.,Biochem. Pharmac., 19,

1833 (12701.

Brotell, H., J . Chromatog., 196, 489 ( 1 9 8 0 ) . 83. Ahmad, K . , a n d E l e d z i h r a d s k y , F., L i f e ScL., 1 0 , 707 (1971). 82.

Neal, E . , P r e f f i n , P . , G r e g o r y , P. a n d B l a s c h k e , T . , G a s t r o e n t e r o l . , 77,

,

96 ( 1 9 7 9 ) 85. B e c k e t t , A , , K o u r o u n a k i s , P . , Vaughan,

.,

a n d 1 8 . i t c h a r d , lv J . Pharm. Pharmac., 22 Supp., 169s (1970). - _ 85. B u r t , R . , a n d B e c k e t t , A,, J. Anaesth., 43, 427 (1971). 87. P e r r i g o , B . , a n d P e e l , H . , J. Chrom19, 219 ( 1 9 8 1 ) . atog. 88. I i i l l e r , J . , R o b e r t s , B . , B l a k e , J . , V a l e n t i n e , R . , a n d T o b i n , T . , Res. Corn. Chem. P a t h . Pharmac., 17, 447 (1977). 89. Pond, S , , Tong, T . , B e n o w i t z , N . , and Jacob, P., Aust. N -. Z-. J . 10,

D.,

z.

=.,

515 (1980).

90. Peel,H., Forens.

m.,

and Perrigo, B., a. J., 9 , 69 (1976).

Can.Soc.

TERRY D. WILSON

416

9 1 . Anderson, R . , and I l e t t , K . , J. Chromatog., 227, 239 ( 1 9 8 2 ) . Wilson, T . , J . Chromatog., 243, 92 99 ( 1 9 8 2 ) . 9 3 . J a n e , I . , and T a y l e r , J . , J . Chroma t 0 ., 1 0 9 , 37 ( 1 9 7 5 ) .

fJ . ,land P e t e r s o n , R . , Rumack, B . , S u l l i v a n , blakowski, A , , J . Chromatog., 1 8 8 , 420 ( 1 9 8 0 ) . 9 5 . White, K., J. Chromatog., 1 7 8 , 229 (1979) 9 6 . Wallace, J . , Harris, S . , and Peek, by., Anal. Chem., 52, 1328 ( 1 9 8 0 ) . 9 7 . M i t c h a r d , K., Acta Pharmac. T o x i c , , 2 9 , S U P P ~ .3 , 172 ( 1 9 7 1 ) 9 8 . Vaughan, D . , B e c k e t t , A . , and Robbie, D . , E. J . C l i n . Pharmac., 3 , 279 ( 1 9 7 6 ) . 9 9 . B e c k e t t , A . , and T a y l o r , J . , J. Pharm. Pharrnac., 1 9 , _Suppl, 5 0 s ( 1 9 6 7 ) .

-

100. Ehrnebo, M . , Boreus, L . , and Lonr o t h , U . , C l i n . Pharmacol. T h e r . , 2 2 ,

888 (1977)101. Anderson, R. and I l e t t , K . , C l i n . Exp. Pharmac. P h y s i o l . , 8 , 622 (1981). 102. S t o n e r , R . , and S u l l i v a n , P!. , C l i n . Chem., 2 5 , 1097 ( 1 9 7 9 ) . 1 0 3 . Medzihradsky, F . , Karks, F’!., and Carr, E . , Biochem. Pharmacol., 2 1 ,

--

1625 ( 1 9 7 2 ) . The p r e s e n t l i t e r a t u r e review i n cludes m a t e r i a l published through J u l y ,

1982.

PHENYTOIN Jose Philip, Ira J. Holcomb, and Salvatore A. Fusari' Wurrier-Lumbert Compuny Morris Plains, New Jersey 418 418 41 8 418 418 419 419 425 425 426 426 427 429 430 43 1 43 1 43 1 43 I 43 I 433 433 435 435 436 439 440

I. General Information

2.

3. 4. 5. 6. 7.

1.1 Nomenclature 1.2 Formulas and Molecular Weight 1.3 Description 1.4 Forms in Official Compendia Physical Properties 2.1 Spectra 2.2 Crystal Properties 2.3 Solubility 2.4 Dissociation Constants 2.5 Partition Coefficients Synthesis Stability and Degradation Metabolism and Pharmacokinetics Identification Methods of Analysis 7.1 Differential Thermal Analysis 7.2 Thermo-Gravimetric Analysis 7.3 Elemental Analysis 7.4 Colorimetric 7.5 Spectrophotometric 7.6 Polarographic 7.7 Titrimetric Assay 7.8 Chromatographic 7.9 Immunoassay References

'Present address: 22625 St. Joan, St. Clair Shores, Michigan 48080.


417

Copyright 0 1984 hy the American Pharmaceutical Association ISBN 0-12-260813-5

418

1.

JOSE PHILIP ET AL.

General I n f o r m a t i o n 1.1

Nomenclature 1.1 1

Chemical N a m e s

5,5-Diphenyl-2,4-imidazolidinedione 5,5-Diphenylhydantoin 1 .12

Generic Names Phenytoin, diphenylhydantoin

1 .13

Trade Names

-

Dantoin, Diphentyn,

D i v u l s a n , Novod i p h e n y l ( a l l Canad. ) :

D i f h y d a n , F e n a n t o i n ( b o t h Swed.); Di-Hydan, P y o r e d o l , S o l a n t y l ( a l l F r . ) ; D i l a b i d , Ekko, K e s s o d a n t e n ( a l l USA); D i l a n t i n ( A u s t r a l . , C a n a d . , USA); D i t o i n , P h e n t o i n ( b o t h A u s t r a l . ) ; Phenhydan, Z e n t r o p i l ( b o t h Ger.); T o i n U n i c e l l e s (S. A f r . ) 1.2

Formulas and M o l e c u l a r Weight C15H12N202

Molecular W e i g h t = 2 5 2 . 2 6

NH

1.3

Description W h i t e o r almost w h i t e c r y s t a l l i n e p o w d e r .

1.4

Forms i n O f f i c i a l Compendia T h e USP c o n t a i n s P h e n y t o i n , P h e n y t o i n Sodium and t h e f o l l o w i n g f o r m u l a t i o n s : P h e n y t o i n Oral S u s p e n s i o n Phenytoin Tablets E x t e n d e d P h e n y t o i n Sodium C a p s u l e s P r o m p t P h e n y t o i n Sodium C a p s u l e s . USP r e f e r e n c e s t a n d a r d s f o r P h e n y t o i n and P h e n y t o i n Sodium are a v a i l a b l e ,

PHENYTOIN

2.

419

P h v s i c a l P r o D e r t ies 2.1

Spectra 2.11

Infrared: T h e i n f r a r e d spectrum of phenytoin is presented i n Figure 1. The band a s s i g n m e n t s 1 are summarized i n T a b l e 1. T a b l e 1:

I n f r a r e d Band A s s i g n m e n t s f o r Phenytoin.

Wavenumber

Assignment

(cm-1) 3275, 3205

N-H

st r e t c h i n g 3064

a r o m a t i c C-H stretch

1774, 1740, 1719

carbonyl stretching vibrations o f hyd a n t o i n ring phenyl r i n g breathing vibrations

1403

C-N s t r e t c h i n g v ibrat ion

7 4 7 , 690

C-H

o u t of plane vibrations o f mo nosubs t i t u t e d phenyl

I

S8

I

1

I

1

0

PHENYTOIN

42 1

2.1 2 Nuclear Maqnet ic Resonance

The Proton NMR spectrum of phenytoin is shown in Figure 21 and t h e spectral assignments are i n Table 2. Table 2:

NMR spectral assignments for Phenytoin

Resonance Mult ipl i c i t y I n t q r a t ion Assiqnment (PM)

7.3 9.2 10.9

2.13

singlet

10

singlet very broad singlet

1 1

phenyl protons amide

protons

Mass Spectrum

"he mass spectrwn is shown i n f i g u r e 31. These are t h e p r i n c i p a l ions observed. Table 3:

m/e 252 223 209 180 175 165 147 104 77

2.14

Fragmentat ion molecular ion M-CHO M-HNCO M-HNCDCHO 6H5 C13H9 HgHg-Co C6H5a

C6H5

Ultraviolet Spectrun

The w spectrun o f phenytoin i n methanol is given i n Figure 4'. 'Ihe a b s o r p t i v i t y , a, a t 258 nm is 2.93.

--

--==D

422

I

L

\

In

R

N

423

U

JOSE PHILIP ET AL

424

220

260

300

340

WAVELENGTH (nm)

Fig. 4. Ultraviolet absorbance spectrum of phenytoin in methanol.

PHENYTOIN

2.2

425

Crystal Properties 2.2 1

C r y s t a l Morphology M i c r o c r y s t a l l o g r a p h i c c h a r a c t e r i s t i c s of P h e n y t o i n were d e t e r m i n e d and r e p o r t e d b y G . L. Keenan*. R o d l i k e p l a t e s and f i n e n e e d l e s of P h e n y t o i n were examined w i t h a p o l a r i z i n g microscope. I n para l l e l p o l a r i z e d l i g h t t h e e x t i n c t i o n is p a r a l l e l and t h e s i g n o r e l o n g a t i o n n e g a t i v e . In convergent p o l a r i z e d l i g h t no i n t e r f e r e n c e figures are observed. R e f r a c t i v e i n d i c e s : a = 1.600 2 002; y = 1.635

2.22

-+

0.002.

M e 1 t i n g Range

P h e n y t o i n melts a t 2 9 5 - 2 9 8 ' . 2.3

-

Solubility Phenytoin is p r a c t i c a l l y i n s o l u b l e i n water, 1 g. d i s s o l v e s i n about 6 0 ml a l c o h o l , a b o u t 30 m l a c e t o n e . The compound is s o l u b l e i n a l k a l i h y d r o x i d e s . Hong e t al3 s t u d i e d t h e s o l u b i l i t y o f p h e n y t o i n i n b u f f e r s of d i f f e r e n t pH v a l u e s .

pH 1.6 4.4 5 .O 5.9 6.9

8.0 9.0 10.0 1 1 .o 12.0

S o l u b i l i t y (mq/ml) 0.02 0.02 0.01 0.02 0.02 0.01 0.10 0.96 9.6 96.0

426

2.4

JOSE PHILIP ET AL.

D i s s o c i a t i o n Constant The i o n i z a t i o n c o n s t a n t , pKa, of p h e n y t o i n was d e t e r m i n e d t o b e 8.31 by u l t r a v i o l e t s p e c t r o p h o t o m e t r y and 8.33 by p o t e n t i o m e t r i c t i t r a t i o n 4 . A v a l u e of 8.06 was r e p o r t e d by S c h w a r t z e t a 1 . 5

2.5

Partition Coefficients Riedel et a16a determined p a r t i t i o n c o e f f i c i e n t s ( K ) f o r p h e n y t o i n b e t w e e n a few s e l e c t e d o r g a n i c s o l v e n t s and aqueous s o l u t i o n s a t t h r e e d i f f e r e n t p H v a l u e s . The r e s u l t s a r e t a b u l a t e d i n T a b l e 4. T a b l e 4. P a r t i t i o n c o e f f i c i e n t s ( K ) o f p h e n y t o i n ( D P H ) , b e t w e e n aqueous l a y e r s p H 4-10 and o r g a n i c s o l v e n t s ; DCE = d i c h l o r o e t h a n e , K = C o r g a n i c solventfaqueous layer.

pH

4 5 6 7

a

9 10

K (DPH) i n : CH7C17 CHCi-(

26.0 26.0 15.7 4.6 3.0 1 .o 0.37

DCE

10.1 9.9 3.2 4.3 2.0 1 .o 0.56

13.5 13.5 10.1 9.0 9.0 3.2 0.70

K v a l u e s o b t a i n e d by P h i l ip6b between c h l o r o f o r m and a q u e o u s b u f f e r s o f p H 4, 5

and 6 a r e g i v e n below: Solvent

Chloroform

K

pH = 4.0

p H = 5.0

pH = 6.0

28.9

24.1

18.9

PHENYTOIN

427

I n Table 5, t h e p a r t i t i o n r a t i o s o f p h e n y t o i n w i t h s i x s o l v e n t s and a q u e o u s b u f f e r s a t t h r e e d i f f e r e n t pH v a l u e s d e t e r m i n e d b y G o d h a r t e t a 1 6 c a r e g i v e n . T a b l e 5. P a r t i t i o n r a t i o s K ( c o n c e n t r a t i o n i n s o l v e n t / c o n c e n t r a t i o n i n aqueous p h a s e ) .

Solvent p H 3.4

Hexane Toluene Chloroform Dichloroethane Ether Ethylacetate

0.008 4.1 1 28.8 28.7 40.8 197

pH 7 . 4 0.004 2.59 25.4 26.0 30.3 178

pH 1 2 . 4

0.0001 0.001 0.007 0.02 0.009

-

S i m i l a r r e s u l t s were o b t a i n e d b y G o l d b e r g and T o d o r o f f 6 d i n 0.1 M p h o s p h a t e b u f f e r of p H 7 . 0 , (Table 6). T a b l e 6.

P a r t i t i o n c o e f f i c i e n t s ( K ) i n p H 7.0. Chloroform Dichloroethane Toluene Hexane

25.5 24.1 4.3 0.02

3. Synthesis

P h e n y t o i n c a n b e s y n t h e s i z e d i n s e v e r a l ways: U.S. P a t e n t 2 , 4 0 9 , 7 5 4 ( 1 9 4 6 ) t o H . R. Henze, P a r k e - D a v i s and C o . i n v o l v e s r e a c t i n g b e n z o p h e n o n e , p o t a s s i u m c y a n i d e and ammonium c a r b o n a t e i n 6 0 % e t h a n o l . I t c a n - a l s o be prepared b y r e a c t i n g b e n z o p h e n o n e , s o d i u m c y a n i d e and ammonium b i c a r b o n a t e a c c o r d i n g t o Scheme 1 .

428

JOSE PHILIP ET AL.

SCHEME 1 - Synthesis of Phenytoin (Henze)

SCHEME 2

-

Synthesis of Phenytoin (Biltz)

I1

J SCHEME 3

-

Hydrolysis of Phenytoin

PHENYTOIN

429

B i l t z 7 p r e p a r e d p h e n y t o i n by h e a t i n g urea with a previously prepared a l k a l i n e a l c o h o l i c s o l u t i o n of b e n z i l S i k d a r a n d Ghosh8 s t u d i e d t h e m e c h a n i s m of t h e B i l t z t e c h n i q u e a n d h a s shown t h e r e a c t i o n t o t a k e place a s shown i n Scheme 2.

.

4. S t a b i l i t y a n d D e g r a d a t i o n P h e n y t o i n is v e r y s t a b l e e v e n u n d e r c o n d i t i o n s of e x t r e m e stress. A f t e r r e f l u x i n g p h e n y t o i n i n 2.5 N h y d r o c h l o r i c a c i d f o r s e v e n h o u r s , e s s e n t i a l l y complete r e c o v e r y o f s t a r t i n g m a t e r i a l was o b t a i n e d b y F u ~ a r i . ~D u s c h i n s k i l reported t h a t p h e n y t o i n , h e a t e d 24 h o u r s a t 170-180" i n 2 0 % s o d i u m h y d r o x i d e (5N) g a v e a n Hong3 d e t e r m i n e d 8 2 % y i e l d of d i p h e n y l g l y c i n e . a 75% c o n v e r s i o n t o d i p h e n y l y c i n e for a s o l u t i o n o f p h e n y t o i n s o d i u m h e a t e d a t r e f l u x i n 2N s o d i u m He f o u n d d e t e c t a b l e hydroxide for f i v e days. amounts o f benzhydrol i n phenytoin s o l u t i o n s h e a t e d i n 0.2 N s o d i u m h y d r o x i d e i n a m p u l e s i n a 105" o v e n a f t e r 7 5 d a y s . S t a b i l i t y s t u d i e s on Dilantinm Infusion concentrate, a s o l u t i o n of p h e n y t o i n s o d i u m i n t e t r a g l y c o l t r o m e t h a n a m i n e - w a t e r , h a v e shown t h e p r e s e n c e o f d i p h e n y l hydantoic a c i d as a decomposition product along with d iphenylglycine. Hydrolysis s t u d i e s of p h e n y t o i n i n 0.1M p h o s p h a t e b u f f e r of pH 1 1 a n d 1 2 a t 7 0 , 8 0 a n d 9 0 " showed d e c o m p o s i t i o n t o f o l l o w p s e u d o - f i r s t o r d e r k i n e t i c s . Extrapol a t i o n of t h e A r r h e n i u s p l o t s a t 25°C i n d i c a t e d t h a t t h e times f o r d e c o m p o s i t i o n o f 1 0 % of t h e d r u g a t room temperature, t 0 . g would b e a b o u t 61 y e a r s a t pH 11 a n d 35 y e a r s a t p H 12.11. A l k a l i n e d e g r a d a t i o n of p h e n y t o i n was i n f e r r e d t o proceed v i a h y d r o l y s i s t o d iphenylhydantoic a c i d , a r e v e r s i b l e r e a c t i o n , and i r r e v e r s i b l e h y d r o l y s i s of t h e l a t t e r t o d i p h e n y l g l y c i n e (Scheme 3 ) . A l k a l i n e p e r m a n g a n a t e o x i d a t i o n of p h e n y t o i n w i l l r e s u l t i n q u a n t i t a t i v e y i e l d o f benzophenone.

430

5.

JOSE PHILIP ET AL

Metabolism and Pharmamkinetics Phenytoin is p a r t i a l l y metabolized and p a r t i a l l y excreted unchanged i n t h e urine. About 4% of t h e dose is excreted i n t h e urine unchanged; t h e excretion of unchanged drug is increased when t h e urine is alkaline.12 The principal metabolic products of phenytoin are phydroxy-phenyl d e r i v a t i v e (HPPH) and a conjugate of HPPH with glucuronic acid ( 1 3-21). The most l i k e l y pathways involved i n t h e metabolic d i s p o s i t i o n of DPH are sumnarized belaw:

--- >Diphenylhydantoic acid (minor canponent) DPH-->meta or para-HPPH----> conjyates)

I

I----- > I

(glucuronic acid

(Epox ide? )-->3,4 ,-Dihydrod iol-->catecho1

I

(Con] ugates)

1

3+Methylcatechol

Effective serum concentrations are i n t h e range 10 to 20 ug/ml; a t concentrations i n t h e range 20 t o 40 ug/ml, s i d e e f f e c t s may occur. Plasma h a l f l i f e v a r i e s considerably and is i n t h e approximate range of 7 to 40 hours; a t doses above 100 mg, t h e h a l f l i f e .is dose dependent. Fhenytoin is widely and r a p i d l y d i s t r i b u t e d throughout t h e body; it is secreted i n t h e milk, accunulates i n red blood cells and amounts secreted i n s a l i v a show a l i n e a r r e l a t i o n s h i p t o nowprote i n bound concentrations i n serum.

PHENYTOIN

6.

43 1

Identification 1.

T h e i n f r a r e d a b s o r p t i o n spectrum o f a pot a s s ium bromide d ispers i o n of p h e n y t o i n i s i d e n t i c a l t o t h a t of a r e f e r e n c e s t a n d a r d , s i m i l a r l y prepared ( F i g u r e 1)

.

2.

D i s s o l v e a b o u t 100 mg i n a m i x t u r e of 0 . 5 m l of 1N s o d i u m h y d r o x i d e a n d 2 m l of a 1 0 % s o l u t i o n of p y r i d i n e , a d d a f u r t h e r 8 m l s o l u t i o n of p y r i d i n e , s h a k e , a d d 1 m l of copper s u l f a t e w i t h p y r i d i n e s o l u t i o n and allow t o s t a n d f o r 1 0 m i n u t e s ; a b l u e p r e c i p i t a t e is producedl2. Pennington, e t a1 h a v e d e s c r i b e d i d e n t i f i c a t i o n d a t a , u s i n g m i c r o c r y s t a l 1i n e , c h r o m a t o g r a p h i c , s p e c t r o p h o t o m e t r i c methods and t e c h n i q u e s f o r color t e s t s . 2 2 .

7.

M e t h o d s of A n a l y s i s

7.1

D i f f e r e n t i a l Thermal A n a l y s i s D i f f e r e n t i a l t h e r m a l a n a l y s i s was c a r r i e d o u t on a Perkin-Elmer d i f f e r e n t i a l scanning colorimeter, m o d e l DSC-2C, a t a h e a t i n g r a t e o f 1 0 " / m i n , i n a n a t m o s p h e r e of n i t r o gen. I t g a v e a s i n g l e s h a r p m e l t i n g endot h e r m i c p e a k a t 298.2"C, f o l l o w e d b y decomp o s i t i o n ( F i g u r e 5 ) .23

7.2

Th e r m o - G r av i m e t r i c A n a l y s is TGA a n a l y s i s 2 3 was c a r r i e d o u t o n a P e r k i n - E l mer T h e r m o g r a v i m e t r i c a n a l y z e r , model TGS-2 a t a h e a t i n g r a t e o f 1 0 " m i n . I t showed n o w e i g h t l o s s u p t o 2OOoC, i n d i c a t i n g n o n - v o l a t il i t y a n d a b s e n c e of s o l v e n t of c r y s t a l l i z a t i o n .

7.3

Elemental Analvsis Element C H N

% Calculated

71.42 4.80 11.10

NORMALIZED PHENYTOIN USP YTr 4.75 m g SCAN RATL 10.00 drg/min

FIGURE 5

PEAK FROMc 288.01 TOt 381 ONSETi 295.98 CAL/CRAMi 31.8

FXLEI PMNZ DATES

83/02/06

TEMPERATURE CC)

M TIMES

03138

DSC PERKIMLMER [Thermal Analysis

PHENYTOIN

7.4

433

Colorimetric

colorimetric p r o c e d u r e f o r p h e n y t o i n d e v e l oped by D i l l e t a124, p r o vi d e d t h e f i r s t r e l i a b l e means f o r d e t e r m i n i n g b l o o d a n d t i s s u e l e v e l s of t h i s d r u g . The p r o c e d u r e involves quantitative n i t r a t i o n , reduction o f t h e n i t r o group t o a primary amine, d i a z o t i z a t i o n and c o u p 1 i n g w i t h t h e B r a t t o n Marshall reagent.

A

7.5

Spectrophotometric 7.51

Ultraviolet U l t r a v i o l e t absorption i n methyl alcohol: maximum a t 2 5 8 nm; a = 2.93. T h e l a c k of a w e l l d e f i n e d UV a b s o r p t i o n s p e c t r u m f o r p h e n y t o i n makes i t s d e t e r m i n a t i o n s b y d i r e c t UV s p e c t r o p h o t o m e t r y d i f f i c u l t : nevertheless, t h e literature d e s c r i b e s s e v e r a l m e t h o d s which d e p e n d upon t h e s p e c t r u m o f t h e unchanged d r u g 2 5 . Wallace e t a126 and F e l l e n b e r g e t a127 reviewed t h e u s e of UV s p e c t r o p h o t o m e t r i c m e t h o d s f o r t h e A spectrophotoa n a l y s i s o f phenytoin. metric method f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n of phenytoin h a s been d e s c r i b e d b y Bgorge e t a12* b a s e d on t h e d i f f e r e n c e i n absorbance of a n a l c o h o l i c s o l u t i o n a t 233 nm b e f o r e a n d a f t e r conversion of phenytoin t o t h e s o d i u m s a l t . Methods t h a t r e q u i r e c o n v e r s i o n o f t h e d r u t o benzophenone h a v e b e e n r e p o r t e d 2 9 I 3 0 I 31 I 3 2. A m e r e t a133 have d e s c r i b e d a s i n g l e u l t r a v i o l e t s p e c t r o p h o t o m e t r i c method f o r t h e d e t e r m i n a t i o n of p h e n y t o i n i n pharmaceutical preparations. The mean w a s 100.9% ( s i x d e t e r m i n a t i o n s ) and t h e r e l a t i v e s t a n d a r d d e v i a t i o n was 1.2%. T h e method was a p p l i e d s u c c e s s f u l l y t o t h e d e t e r m i n a t i o n of p h e n y t o i n i n c a p s u l e s and s u s p e n s i o n s .

434

JOSE PHILIP ET A t .

Cunningham and c o w o r k e r s 3 4 h a v e d e s c r i b e d a column c h r o m a t o g r a p h i c method f o r t h e d e t e r m i n a t i o n of p h e n y t o i n i n c a p s u l e s . The method is b a s e d o n t h e a c i d i c n a t u r e of p h e n y t o i n and p e r m i t s t h e s e p a r a t i o n and d i r e c t UV q u a n t i t a t i o n o f t h e d r u g . The sample i s d i s s o l v e d i n d i m e t h y l s u l f o x i d e and h y d r o c h l o r i c a c i d , and t h e n d i l u t e d t o volume w i t h s o d i u m c a r b o n a t e . An a l i q u o t o f t h i s is mixed w i t h C e l i t e and a d d e d t o a c h r o m a t o g r a p h i c column c o n s i s t i n g o f 0.5 M s o d i u m c a r b o n a t e a b s o r b e d o n C e l i t e . A p r e w a s h of 30 p e r c e n t c h l o r o f o r m - i s o o c t a n e removes i n t e r f e r i n g s u b s t a n c e s and 75 m l c h l o r o f o r m c o m p l e t e l y removes t h e d r u g from t h e column. The e l u a t e i s e v a p o r a t e d and t h e r e s i d u e is d i s s o l v e d i n e t h a n o l i c HC1 a n d d e t e r m i n e d s p e c t r o p h o t o m e t r ically. F u s a r i and c o w o r k e r s 3 5 h a v e d e v e l o p e d a similar method f o r t h e a s s a y o f p h e n y t o i n a n d p h e n o b a r b i t a l i n d o s a g e f o r m s . They h a v e u s e d S i l a n i z e d C e l i t e 545 a s t h e s u p p o r t m a t e r i a l , 25% amyl a l c o h o l i n c h l o r o f o r m as t h e s t a t i o n a r y p h a s e a n d 0.05M b o r a t e b u f f e r o f pH 9.5 a s t h e m o b i l e p h a s e . P h e n o b a r b i t a l was e l u t e d w i t h t h e m o b i l e p h a s e and p h e n y t o i n e l u t e d with absolute ethanol. U l t r a v i o l e t a b s o r p t i o n w a s u s e d t o q u a n t i t a t e t h e substances

.

7.52

Fluorometric A simple f l u o r o m e t r i c a s s a y f o r p h e n y t o i n i n plasma was r e p o r t e d b y D i l l e t a136.

PHENYTOIN

435

The assay w a s done d i r e c t l y on 0.2 m l o f plasma by t r e a t i n g with a l k a l i n e potassiun permanganate to form benmphenone, e x t r a c t i n g with heptane and shaking t h e heptane layer with s u l f u r i c acid. me fluorescence of t h e r e a c t i o n product was measured with e x c i t a t i o n a t 360 nm and emission a t 440 nm. Detection of 1 ug phenytoin per m l o f plasma was reported. Abrahamsson e t a137 described a f l u o r i metric microdetermination o f phenytoin i n plasma. The phenytoin is e x t r a c t e d from a c i d i f i e d plasma and oxidized to benzomenone which is then condensed with N ' ~ e t h y 1 n i m t i t ~ amide. The r e s u l t i n g product is s t r o n g l y f l u o r e s c e n t with e x c i t a t i o n a t 440 nm and emission a t 500 nm. 7.53

phosphor imetric The method38 is based on Wallace method f o r phenytoin, conversion to benzophenone i n an a l k a l i n e permanganate medium, e x c i t i n g a t 260 nm and emitting a t 446 nm. ?he limit of d e t e c t i o n is 0.05 ug/ml.

7.6

Polarograph ic Brooken e t a139 described a s e n s i t i v e d i f f e r e n t i a l pulse polarographic assay for phenytoin i n blood. It involves e x t r a c t i o n of phenytoin i n t o chloroform followed by n i t r a t i o n . The a n a l y s i s of t h e n i t r o d e r i v a t i v e is then acccmpl ished

.

7.7

T i t r i m e t r i c Assay weigh a c c u r a t e l y about 500 mg o f phenytoin and transfer t o a 125-1111c o n i c a l f l a s k . Dissolve i n 50 ml of dimethylformamide, add 3 drops of a s a t u r a t e d s o l u t i o n o f a z o v i o l e t i n benzene, and titrate with 0.1N sodim methoxide to a blue end-pint. Ferform a blank determinat ion , and make any necessary correction. Each m l of 0.1 N sodium methoxide is equivalent to 25.23 mg of C15H12~202.40

436

JOSE PHILIP ET AL.

Numerous assay procedures for phenytoin i n pharmac e u t i c a l preparations have been devised, based on t h e s e p a r a t i o n of phenytoin fran phenobarbital by ion exchange chranatography or s o l v e n t e x t r a c t i o n procedure, followed by nowaqueous t i t r a t i o n . 4 1 7.8

Chrmatographic 7.81

T h i w l a y e r (TLC) The TIC system f o r i d e n t i f i c a t i o n of phenytoin i n capsules and t a b l e t s 4 2 is ccnnpsed o f s i l i c a g e l G plate (Analtech) and c h l o r o f o r m isopropyl alcohol-ammonia (45-45-10). Rf = 0.6. Mas0nd43 h a s given t h e Rf values for Phenytoin i n t h e following systems on silica g e l GF plate: Chlorofomether-methanol-NH40H( 75-25-5-1 Ethylacetatel-propanol-NH40H (40-30-3) MethanOl-NH4OH ( 100-1.5) Ethanol-Acetic acid-H20 (60-20-1 0 ) Benzene

)

0.48 0.83 0.86 0.95 0

Detection of phenytoin on t h e plate can be achieved by W l i g h t , iodine exposure and Ehrlich's s p r a y followd by Iodoplatinate spray. Wad e t a144 described a rapid q u a n t i t a t i v e method for t h e determination of phenytoin i n serum by TLC. Perhaps t h e most exacting TLC s t u d i e s were c a r r i e d o u t by Simon e t a145. Fhenytoin w a s e x t r a c t e d f r m plasma, concentrated by evaporation, and subjected t o TLC on s i l i c a g e l plates. After t h e phenytoin spot was scraped off t h e colorimetric procedure of D i l l et a146 was applied d i r e c t l y t o t h e scrapings. Phenytoin l e v e l s of 5 ug/ml or more could be recognized by v i s u a l inspection of t h e plates under u l t r a v i o l e t l i g h t , and serum l e v e l s of 1 ug/ml were d e t e c t a b l e with t h e c o l o r i m e t r i c procedure.

PHENYTOIN

7.82

431

Gas Chranatography ( G K ) Glazko47 reviewed sane e a r l y s t u d i e s on t h e g a s chranatographic assay of phenytoin. These included d e r i v a t i z a t ion with d iazanethane48, t h e use o f t r i m e t h y l s i l y l derivative49r50, on colunn methylation using tetramethyl anmnium hydroxide51 and trimethylanil inium hydroxide52. A large number o f GKC procedures have been reported i n which phenytoin is chrcnratographed d i r e c t l y a t high temperatures without d e r i v a t i v e formation.53~54955 I n general t h e direct GLC methods without d e r i v a t i v e formation tend t o produce asymmetric peaks which increase t h e d i f f i c u l t y of q u a n t i t a t i o n by measurement o f peak heights. me on-colm methylation techniques appear t o be convenient to run and sharper peaks are obtained a t lower temperatures. Cramers e t a156 described high resolution g a s chranatography f o r t h e q u a n t i t a t i v e determination of underivatized anti-convulsant drugs w i t h support coated open tubular c o l m s . A s i l i c e o u s support material (-Sil) is deactivated with benzyltriphenyl phosphcnium c h l o r i d e and deposited on t h e inside w a l l of a glass c a p i l l a r y colm. After additional coating with OV-225, a nmber of anticonvuls a n t drugs were run.

Vandemark e t a157 described a procedure for determining phenytoin i n 50 u l volurne of serum by u s e of GLC and a detector with heightened s e n s i t i v i t y and s e l e c t i v i t y for nitrogenous compounds. mtal a n a l y s i s time for a s i n g l e s q l e is 15 minutes. 7.83

High Performance Liquid Chranatography (HPLC) Holccsnb e t a1 58 developed a s t a b i l i t y indicating HPLC procedure f o r t h e simul-

taneous determination of phenytoin and phenobarbital i n formulations. ?hey used a Waters Associates, 2 f t x 1/8 in. O.D. s t a i n l e s s steel column packed with u Bondapak C18 on Corasil 11. ?he mobile phase was a mixture

438

JOSE PHILIP ET AL.

of m e t h a n o l - w a t e r - a c e t i c a c i d i n t h e r a t i o 25-75-0.1. W i t h t h e d e v e l o p m e n t o f new c o l u m n s , t h i s p r o c e d u r e was l a t e r r e v i s e d b y 59 T h e c u r r e n t met h o d uses Fusari et al. a Waters Associates, 1 0 u , 3.9 mm x 30 c m u Bondapak C18 c o l u m n ; UV d e t e c t i o n a t 254 nm, t h e mobile p h a s e i s a m i x t u r e o f methanol-water-acetic acid i n t h e ratio 40-59.5-0.5. A t a f l o w r a t e of 1.5 m l p e r m i n u t e , p h e n o b a r b i t a l e l u t e d a t 6.6 m i n u t e s and p h e n y t o i n a t 13.8 m i n u ts

.

A t w e l l e t a 1 6 0 d e s c r i b e d a n HPLC p r o c e d u r e

f o r t h e s i m u l t a n e o u s a n a l y s i s of p h e n y t o i n a n d p h e n o b a r b i t a l i n plasma. S l o n e k e t a16I d e v e l o p e d a rapid and s i m p l e HPLC m i c r o a n a l y t i c a l m et h o d f o r t h e d e t e r m i n a t i o n of c l i n i c a l l y e n c o u n t e r e d plasma p h e n y t o i n l e v e l s . T h i s method is a c c u r a t e down t o a b o u t 1 ug of p h e n y t o i n / m l of plasma a n d requires a s l i t t l e a s 1 0 u l o f sample. C h a f e t z e t a l l 1 u s e d a n HPLC met h o d t o s t u d y t h e h y d r o l y s i s of p h e n y t o i n s o d i u m i n a q u e o u s s o l u t i o n s a t pHs 1 1 a n d 12. T h ey used a n A 1 t e x U 1 t r a s p h e r e m r e v e r s e - p h a s e bonded o c t a d e c y l s i l a n e column, 4.6 mm x 25 c m ; t h e mo bile p h a s e was a m i x t u r e of 550 m l of m e t h a n o l w i t h 450 m l of a n a q u e o u s s o l u t i o n a t p H 3.2 c o n t a i n i n g 30 m l o f 0.067 M p o t a s s i u m p h o s p h a t e a n d 1 g of h e x a n e s u l f o n i c a c i d s o d i u m s a l t ; t h e UV detector was s e t a t 220 nm. Under t h e s e conditions, phenytoin e l u t e d a t 10.3 m i n , d i p h e n y l g l y c i n e a t 4.0 m i n , d i p h e n y l d y d a n t o i c a c i d a t 5.6 m i n , b e n z h y d r o l a t 2 1 .6 m i n a n d b e n z o p h e n o n e a t 37.0 m i n .

PHENYTOIN

7.9

439

Immunosassay I m m u n o l o g i c m e t h o d s f o r q u a n t i t a t i n g t h e plasma c o n c e n t r a t i o n s of many h o r m o n e s a n d a f e w d r u g s 4. Tigelaar have been d e s c r ibed62 e t a165 h a v e developed a r a d i o i m m u n o s a s s a y for phenytoin. Cook e t a 1 6 6 s t u d i e d t h e s a l i v a a n d plasma l e v e l s o f p h e n y t o i n i n a s e r i e s of e p i l e p t i c p a t i e n t s b y m e a n s o f r a d i o immunoassay t h a t r e q u i r e d o n l y 1 0 u l of s a l i v a o r plasma. The r a d i o immunoassay i s v e r y s e n s i t i v e , q u a n t i t a t i o n of s u b s t a n c e s p r e s e n t i n c o n c e n t r a t i o n s a s l o w as a few p i c o g r a m s per m i l l i l i t e r of a b i o l o g i c a l f l u i d c a n be achieved. The d e v e l o p m e n t of s imple r a d ioimmunosassy is r e v i e w e d b y Cook e t d7. P i p p e n g e r e t a 1 6 8 r e p o r t e d a homogenous immunoassay s y s t e m w h i c h h a s t h e a d v a n t a g e s o f r a d i o i m m u n o a s s a y w i t h o u t t h e u s e of r a d i o a c t i v e compounds. Quantitative correlation to GLC w a s o b t a i n e d . P h e n y t o i n i n human s e r u m w a s determined by spinimmunoassay t e c h n i q u e by A s p e c i f i c radioimmuneMontgomery e t a l 6 9 . a s s a y f o r p h e n y t o i n , t h a t w i l l measure t h e r a p e u t i c l e v e l s d i r e c t l y i n 1 u l of p l a s m a is d e s c r i b e d by C h r i s t e n s e n 7 0 . C a s t r o e t a17’ made c o m p a r a t i v e d e t e r m i n a t i o n s of p h e n y t o i n b y v a r i o u s m e t h o d s . Serum from p a t i e n t s b e i n g t r e a t e d w i t h p h e n y t o i n were analyzed f o r t h e drug by spectrophotometry, g a s c h r o m a t o g r a p h y , r a d i o i m m u n o a s s a y , enzyme immunoassay a n d l i q u i d c h r o m a t o g r a p h y . The a s s a y v a l u e s o b t a i n e d were c o m p a r e d s t a t i s t icall y En zyme immunoassay a n d 1i q u i d c h r o m a t o g r a p h y appear t o b e a t t r a c t i v e a l t e r n a t i v e s t o t h e more t r a d i t i o n a l m e t h o d s of s p e c t r o p h o t o m e t r y a n d g a s c h r o m a t o g r a p h y .

.

440

JOSE PHILIP ET AL.

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34. C. G. Cunningham, C. A. Brunner and J. &vine Journal of t h e ADAC 55, 170 (1972).

-

35. S. A. Fusari, I. J. tIoLccanb, and C. A. Emus, Warner-lambert Co., unpublished data. 36. W. A. D i l l , A. Leung, A. W. Kinkel and A. J. Glazko; C l h . Chem. 22, 908-911 (1976).

-

37. Abrahamsson, L and Wassen, A.M., Lab. Invest. 29 1313 (1972).

Scand. J. Clin.,

38. Morrison, L.D., (1974).

Anal Chem. 46, 1119-20

-

O'Donnell, C.M.,

-

-

39. Brooken e t a1 Anal Chim. Acta 64, 165 (1973). 40. USP XX, pp. 620.

PHENYTOIN

443

and Blake, M.I., J. A s s o c . Offic. Agric. Chem. 51:1013-1016 (1968).

41. Agarwal, S.P.,

-

42. S. A. F u s a r i , Warner-Lambert Co., Personal Comunication. 43. Masond, A. N., J. Pharm. Sci. 65 1585-89 (1976).

-- 167-8.

44. Wad, N. Rosenmund H., J. Chrmatogr. 1978, 146 45. Simon, G.E.,

Jatlow, P.I.

Seligson, H.T.

and Seligson,

D. Measurement of 5,511iphenylhydantoin i n blood u s i n g t h i n layer chrmatograpy. An. J. C 1 i Pathol. 55: 145-151,

1971. 46. D i l l , W. A . , Kazenko, A., Wolf, L.M., and Glazko, A . J . S t u d i e s on 5,Sdiphenylhydantoin ( D i l a n t i f l ) i n animals and man. J. Pharmacol. Exp. 'Iher 118:270-279, 1956. 47. Glazko, A n t i e p i l e p t i c Drugs e d i t e d by D. M. Woodbury, J. K. Penry and R. P. S c h i d t Raven prep. New York, 1972. 48. Sandberg, D.H.,

Resnick, G.L, and Bacallao, C.Z. Measurement of serum d iphenylhydantoin by gas-1 iquid chrmatcqraphy. Anal. Chem. 40:736, 1968.

49. Chang, T., and Glazko, A. J. Q u a n t i t a t i v e assay of 5, 5 4 iphenylhydanto i n (DPH) and 5- (p-Hydroxyphenyl ) -5phenylhydantoin (HPPH) i n plasma and u r i n e o f human subjects. Clin. Res.16:339, 1968. 50. Chang, T. and Glazko, A. J. Q u a n t i t a t i v e assay of 5, 5 4 iphenylhydantoin ( D i l a n t in@) and 5- (p-hydroxyphenyl ) 5-phenylhydantoin by gas-1 iquid chranatography. J. Lab, C l h . M e d . 75:145-155, 1970.

51. MacGee, J. Rapid determination o f diphenylhydantoin i n blood plasma by g a s l i q u i d chromatography. Anal. Chem. 42:421-422, 1970. 52. Kupferberg, H. J. Quantitative estimation of d iphenyldantoin, primidone, phenobarbital i n plasma by gas-liquid chrmatograpy. Qin. Chim. Acta 29: 283-288, 1970.

444

JOSE PHILIP ET AL.

and Gillen, H.W. Gas chromatographic a n a l y s i s f o r anticonvulsant drugs i n b i o l o g i c f l u i d s . Clin. Chem. 15:582-590, 1969.

53. Pippenger, C.E.

54. Van Meter, J. C. Buckmaster, H.S. and Shelley, L.L. Concurrent assay of phenobarbital and d iphenylhydantoin i n plasma by vapor phase chranatography. Clh. Chem. 16:135-138, 1970. 55. Sampson, D. Harasymiv, I., and Hensley, W.J. Gas chromatographic assay of underivatized 5,5-d iphenylhydantoin ( D i l a n t i n @ ) i n plasma extracts. Clh. Chem. 17:382-385, 1971. 56. C. A. Crmers, E. A. V e n n e e r , L. G. Vankuik, J. A. Hulsnan and C. A. Meigers, C l h . Chem. Acta 73:97-107 ( 1 976).

-

57. Vandemark, F. L. and Adas, R.F., 1062 (1976).

-

C l i n . Chem. 22,

58. I. J. Holccanb, S. A. Fusari and C. Remus, Warner-Lambert Co , unpubl i s h d d a t a .

.

59. S. A. Fusari and J. Macri, Warner Lambert OO., unpublished d a t a .

60. S. H. A t w e l l , V. A. Green, and W. G. Haney, J. Pharm. Sci. 64, 806 (1975). 61. J. E. Slonek, G. W. Penz, and W. L. Chion, J. Pharm. S c i , 67, 1462 (1978). 62. S. Spector and E. J. Flynn, Science, (1971) 1036.

174

63. E. J. Flynn and S. Spector, J. Pharmacol. Exp. Therap. 181, (1972) 547.

-

64. H. VanVunakis, E. Wassennan and L. &vine, J. Phannacol, Exp. Therap., 180 (1972) 515.

PHENYTOIN

445

65. R.E.

Tigelaar, R. L. Rapport, J. K. Inman and H. J. Kupferbeq. Clin. Chimica Acta, 4 3 (1973) 231-241.

-

66. C. E. Cbok, E. Pmerson, W.K, mole, P. Lesser, and L. O'Tuama, C l i n i c a l Pharmacology and 'Iherapeutics, 18 742 (1975).

-

Cook, H.C. Christensen, E. W. Anerson, J. A. Kepler, C. R. T a l l e n t and G. F. Taylor i n W a n t i t a t i v e Analytic S t u d i e s i n Epilepsy, e d i t e d by P. Kellaway and I. Petersen, Raven Pres. New York, 1976.

67. C.E.

68. C. H. Pippenger, R. J. B a s t i a n i and R. S. Schneider i n C l i n i c a l Pharmacology of A n t i e p i l e p t i c Drugs Ed. H. Schneider, Springen Verlag 1975. 69. M. R. Montgomery, J. L. Holtzrnan, R. K. Leute, G. Eiolz and J. Dewees, Pharmacologist, 16, Abst. 178 ( 1 974). 70. Christensen, H.D., J. A. Kepler, and C.E. Pharmacologist 15, Abst. 228 (1973).

-

Cook,

71. Castro, A. Ibanez J., DiCesare J. L., M a n s , R. F. Malkins H., C l i n Chem. 24, 710 (1978).

-


PYRIDOXINE HYDROCHLORIDE Hassan Y. Aboul-Enein and Mohammed A. Loutfy K i n g Soud University Riyndh, Saudi Arabia 1,

2.

3. 4. 5. 6.

448 448 448 449 449 449 449 449 449 450 450 450 45 I 45 I 462 463 466 468 468 469 413 417 41I 478

Description 1.1 Nomenclature 1.2 Formulae I .3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Odor, and Taste 1.6 Dissociation Constants Physical Properties 2.1 Melting Point 2.2 Solubility 2.3 X-Ray Diffraction 2.4 Stability 2.5 Identification 2.6 Spectral Properties Biosynthesis Synthesis Metabolism Methods of Analysis 6.1 Titrirnetric 6.2 Chromatography 6.3 Spectrophotometry 6.4 Microbiological 6.5 Enzymatic References


441

Copyright 0 1984 by the American Pharmaceutical Association lSBN 0-12-260813-5

HASSAN Y. ABOUL-ENEIN AND MOHAMMED A. LOUTFY

448

1. D e s c r i p t i o n 1 . 1 Nomenclature

1.1.1 Chemical Names

5-Hydroxy-6-rnethyl-3,4-pvr i d ined irnethanol hydrochloride ; 2-Methyl-3-hydroxy-4,5-bis (hydroxymethyl) p r y i dine hydrochloride; 5-Hvdroxy-6-methyl-3,4-pvridinedicarbinol hydrochloride; 3-Hydroxy-4,5-dirnethyl-ol-ci-picoline hydrochlor i d e (1); 3-Hydroxy-4,5-d i (hydroxymethyl) -2-methylpyr i d i n e h y d r o c h l o r i d e (2) ; 4,5-Bis (hydroxymethy1)-3-hydroxy-Z-methylpyridinium c h l o r i d e (3) ; 3,4-Pyridinedimethanol, 5-hydroxy-6-methyl-, hydrochloride ( 4 ) ; (hpdroxymethyl)-2-methylpyri3-Hydroxy-4,5-bis dinium c h l o r i d e ( 5 ) . 1 . 1 . 2 Generic N a m e s P y r i d o x i n e h y d r o c h l o r i d e ( 5 , 6) ; P y r i d o x i n i chloridum; P y r i d o x i n i hydrochloridum; Pyridoxinium c h l o r i d e ; P y r i d o x o l h y d r o c h l o r i d e ; Vitamin $6; kdermine h y d r o c h l o r i d e ; p i r i d o s s i n a c h l o r i d r a t o ( 2 ) ; Vitamin 8 6 h y d r o c h l o r i d e (1); Hexabione hydrochloride. 1.1.3 Trade N a m e s Beadox; Benadon; Hexa-Betalin ( 7 ) ; Hexavibox, Hexabion; Pyr i v e l ; Pyr i d i p c a ; B e c i l a n , Hexermin; Campoviton 6 (1); Comploment; Ancoloxin (with meclozine hydrochloride) ( 3 )

.

1 . 2 Formulae

1 . 2 . 1 Empirical C8H1 1N03,

HC1.

1.2.2 S t r u c t u r a l P y r i d o x i n e i s o n l y one of t h r e e s i m i l a r compounds

PYRIDOXINE HYDROCHLORIDE

449

t h a t may b e r e f e r r e d t o as v i t a m i n B 6 , t h e o t h e r two a r e p y r i d o x a l and pyridoxamine ( 8 ) . Only p y r i d o x i n e h y d r o c h l o r i d e , however, i s used i n pharmaceutical preparations (9).

c1H3cQ HO

CH20H

1 . 2 . 3 Wiswesser L i n e N o t a t i o n T6 N J B C Q D I OEIQ and GH (10) 1 . 2 . 4 Chemical A b s t r a c t R e g i s t r y Number 58-56-0

(5

,

10)

1 . 3 M o l e c u l a r Weight

205.64. 1 . 4 E l e m e n t a l Composition C, C1,

46.72%; 17.24%;

H , 5.88%; N , 6.81%, 0 , 23.34%.

1 . 5 Appearance, C o l o r , Odor and Taste A w h i t e o r a l m o s t w h i t e , c r y s t a l l i n e powder, p l a t e l e t s acetone); o r t h i c k b i r e f r i n g e n t r o d s (from a l c o h o l odorless, with a s l i g h t l y b i t t e r s a l i n e taste, acid.

+

1 . 6 Dissociation Constants pKa

5.0,

9 . 0 (25O)

(3).

2. P h y s i c a l P r o p e r t i e s 2.1 M e l t i n g P o i n t About 205O ( 3 ) ; 202-206O

( 5 ) ; 205-212'

(I), w i t h decom-


450

position. (1).

I t sublimes.

The f r e e b a s e m e l t s a t 160°

2.2 S o l u b i l i t y F r e e l y s o l u b l e i n w a t e r (1 gm : 5 m l ) , s l i g h t l y s o l u b l e i n a l c o h o l (1 gm : 100 ml) , s o l u b l e i n propylene glycol; sparingly soluble i n acetone; insoluble i n e t h e r and i n chloroform.

2 . 3 X-Ray D i f f r a c t i o n X-ray d i f f r a c t i o n p a t t e r n s of v i t a m i n B 6 and o t h e r v i t a m i n s by a p r e c i s i o n x-ray camera, have been d e t e r mined. Elementary cell-dimension s t u d i e s have been r e p o r t e d (11). 2.4 S t a b i l i t y P y r i d o x i n e , H C 1 i s more s t a b l e t h a n ppridoxamine and p y r i d o x a l . Acidic aqueous s o l u t i o n s of v i t a m i n B6 a r e s t a b l e d e f i n i t e l y a t room t e m p e r a t u r e and may be heated a t 120° f o r 30 m i n u t e s w i t h o u t decomposition ( 1 3 ) . P y r i d o x i n e i s d e s t r o y e d by W r a d i a t i o n i n n e u t r a l o r a l k a l i n e solutions, but not i n a c i d i c solution (9). P y r i d o x i n e i s u n s t a b l e toward v a r i o u s food, d r u g , and cosmetic c o l o r s , because of t h e p o s s i b i l i t y of t h e formation of complex a d d i t i o n p r o d u c t s between t h e c o l o u r s and p y r i d o x i n e (14). S e v e r a l s t a b i l i t y s t u d i e s of p y r i d o x i n e , when added t o b r e a d , f l o u r ( 1 5 ) , cereals (16) and d u r i n g canning and f r e e z i n g of sweet c o r n (17), have been p u b l i s h e d . S h i r o i s h i and Hayakawa (18) have d e s c r i b e d t h e e f f e c t of s u n l i g h t i r r a d i a t i o n on p y r i d o x i n e and r e l a t e d compounds i n aqueous s o l u t i o n a t v a r i o u s pH's. Pyridoxine and pyridoxamine a r e r e l a t i v e l y s t a b l e i n a c i d i c medium, w h i l e p y r i d o x a l decomposes independently of pH. The a u t h o r s suggested t h a t t h e a l d e h y d i c 0 p a r t i c i p a t e s i n t h e p h o t o l y s i s of p y r i d o x a l . The p r e s e n c e of Cu* had no e f f e c t on t h e s t a b i l i t y of t h e s e s u b s t a n c e s . P. b o r a t e complex of p y r i d o x i n e i s s t a b l e t o s u n l i g h t i r r a d i a t i o n , h e a t i n g , o r a u t o c l a v i n g . Acetic a c i d and a r e d d i s h brown s u b s t a n c e were s e p a r a t e d as t h e decomp o s i t i o n p r o d u c t s from an i r r a d i a t e d s o l u t i o n of p y r i d o x i n e (18).


45 1

The e f f e c t of s h e l f - l i f e on t h e s t a b i l i t y of vitamin B6, i n o r a l pharmaceutical p r e p a r a t i o n s , h a s been r e p o r t e d (19). 2.5 I d e n t i f i c a t i o n The f o l l o w i n g t e s t s have been d e s c r i b e d f o r t h e i d e n t i f i c a t i o n of p y r i d o x i n e hydrochloride:

a. To 0.1 m l of a 5% w/v s o l u t i o n add 10 ml of a 5% w / v s o l u t i o n of sodium acetate, 1 ml of w a t e r and 1 m l of a 0.5% w/v s o l u t i o n of 2,6-dichloroquinone-4chloroimine i n e t h a n o l (96%) and shake; a b l u e c o l o r is produced which r a p i d l y f a d e s and t u r n s t o brown. R e p e a t t h e o p e r a t i o n adding 1 m l of a 3X w/v s o l u t i o n of b o r i c a c i d i n p l a c e of t h e 1 m l of w a t e r ; no b l u e c o l o r i s produced (3-5, 1 2 ) . b. To 2 ml of a s o l u t i o n (1 i n 200) add 0.5 m l of phosphotungstic a c i d , a w h i t e p r e c i p i t a t e i s formed

(4)* c . A 5% w/v s o l u t i o n a c i d i f i e d w i t h 2H n i t r i c a c i d y i e l d s r e a c t i o n s c h a r a c t e r i s t i c of c h l o r i d e s . d. pH of a 1% s o l u t i o n , 2.5-3.2 s o l u t i o n s , 2.3-3.5 ( 7 ) . 2.5.1

Micro-Color

(12); pH. of a 5% w/v

Tests

a. Ammonium molybdate t e s t g i v e s f a i n t b l u e c o l o r ( s e n s i t i v i t y : 0.25 yg) ( 7 ) . b . Ammonium v a n a d a t e t e s t produces b l u e then grey-green c o l o r ( s e n s i t i v i t y : 0.25 yg). 2.5.2 Micro-Crystal Tests

a. P l a t i n i c i o d i d e s o l u t i o n forms r e c t a n g u l a r c r y s t a l s ( s e n s i t i v i t y : 1 i n 1000) ( 7 ) . b. Potassium bismuth i o d i d e s o l u t i o n g i v e s bunches of p l a t e s o r b l a d e s ( s e n s i t i v i t y : 1 i n 200) ( 7 ) .

2.6 S p e c t r a l P r o p e r t i e s 2.6.1

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


452

The UV s p e c t r u m of p y r i d o x i n e , HC1 i n n e u t r a l e t h a n o l i s shown i n F i g . 1. I t e x h i b i t s a maximum a t a b o u t 291 nm ( E l % , 1 cm = 523 nm). I n a b a s i c s o l u t i o n , p y r i d o x i n e g i v e s two maxima a t 245 nm ( E l % , 1 cm = 219), and a t 307 nm a s shown i n F i g . 2 . These are i n agreement w i t h I n phosp r e v i o u s l y p u b l i s h e d d a t a ( 7 , 20-21). p h a t e b u f f e r of pH = 7 , two maxima are observed a t 324 and 254 nm ( E l X , 1 c m = 345 and 183, r e s p e c t i v e l y ) (9). M e t z l e r and S n e l l (22) have r e p o r t e d t h e UV a b s o r p t i o n s p e c t r a and i o n i s a t i o n c o n s t a n t s of v i t a m i n B 6 group. The a u t h o r s have c a l c u l a t e d t h e e q u i l i b r i u m c o n s t a n t s between d i p o l a r i o n i c and uncharged n e u t r a l forms of t h e 3-hydroxypyridines investigated. 2.6.2

I n f r a r e d Spectrum The I R spectrum of p y r i d o x i n e , H C 1 i n KBr-disc i s shown i n F i g . 3. Major band a s s i g n m e n t s a r e g i v e n i n T a b l e 1.

T a b l e 1.

I R S p e c t r a l Assignments of P y r i d o x i n e , HC1. Frequency, cm-l

Assignment

3340

OH p h e n o l i c

3250

OH a l c o h o l i c

1550 1630

1

C=C and C=N aromatic r i n g

Other f i n g e r - p r i n t bands c h a r a c t e r i s t i c of p y r i d o x i n e , H C 1 are: 1275, 1220, 1100, and 1570 cm-1. These d a t a are i n agreement w i t h p r e v i o u s l y r e p o r t e d o n e s ( 7 , 20, 2 3 ) . 2.6.3 N u c l e a r Magnetic Resonance Spectrum The PMR spectrum of p y r i d o x i n e , H C I i n d e u t e r a t e d water w a s r e c o r d e d on a V a r i a n T-60AY 60-m~ NMR s p e c t r o m e t e r . The spectrum i s shown i n F i g . 4. The f o l l o w i n g s t r u c t u r a l a s s i g n m e n t s have been e l i c i t e d from F i g . 4 .

PYRIDOXINE HYDROCHLORlDE

453

wave fength

Fig. 1. Ultraviolet spectrum of pyridoxine hydrochloride in ethanol solution.

Fig. 2. Ultraviolet spectrum of pyridoxine in basic ethanol solution.

454

c, a,

rl

a,

rl

P

k

m x

Y

a,

a .d

k 0 rl

V 0

c k

a,

a h c c

-d

x 0 a h

k

-4

a v-4 0

s k

4

u a, a z n

a

a, k rd k

H

G

Icl

m

l h

bl -4

LA P

1 Fig. 4.

'H-NMR

(6OMHz) spectrum of pyridoxine hydrochloride in D 0 2 -


456

Chemical S h i f t (6) -

P r o t o n Assignments

2.73

singlet

CH3 a t C 2

5.06

singlet

CH2 a t C4 and C

8.13

singlet

aromatic proton a t

5'

'6' When t h e spectrum i s d e t e r m i n e d i n DMSO-d6, t h e hydroxymethyl groups a r e r e s o l v e d i n t o two s i n g l e t s a t 4.7 6 ( f o r CH20H a t C5) a t 4.8 6 ( f o r CH20H a t C4). These s p e c t r a are i n agreement w i t h p r e v i o u s l y published d a t a (24). The e l e c t r o n i c p r o p e r t i e s of v a r i o u s i o n i c forms of v i t a m i n B6 group have been s t u d i e d and r a t i o n a l i z e d u s i n g PMR ( 2 5 ) . F u r t h e r m o r e , t h e PMR s p e c t r o s c o p y of v i t a m i n B6 compounds h a s been s t u d i e d by Korytnyk and Paul (26). 2.6.4 Mass Spectrum The mass spectrum of p y r d o x i n e i s shown i n F i g . 5. The most prominent i o n s a r e shown in T a b l e 2. T a b l e 2.

Mass S p e c t r a l F r a g m e n t a t i o n of p y r i d o x i n e .

m le 169

Fragment

M o l e c u l a r i o n M+.

151

122

H3 106

94

+

H

HB I!

H3

Y

A

L

L

A

a,

c

0

x

-4

a

k

-4

h

a

5

k

u

+,

a,

m

a m m

c

2

0

a,

0 m k

3

0 I4


458

+

r

1'

Ho\

&:

H*o 0

H3CO f i 2 0 H

H3C

1

J

m / e 151

m / e 169

l o s s of

l o s s of CO and HO'

H3c

possible

co

gcH 'OH

i

m / e 106 Azatropylium ion

m / e 106 t

CHZOH

Y

c,

H -H'

*I

H m / e 123

H

H

m / e 122

Scheme 1.

N

H m / e 94 ( b a s e peak)

Mass S p e c t r a l F r a g m e n t a t i o n of P y r i d o x i n e .

PYRIDOXINE HYDROCHLORlDE

459

The mechanism by which t h e major fragments a r e formed, i s shown i n Scheme 1. The d e t a i l e d mechanism i s r e p o r t e d by D e Jongh e t a l . (27) w i t h t h e a i d of m e t a s t a b l e peaks, deutrium l a b e l i n g , and a n a l y s i s w i t h s i m i l a r system. 2.6.5

13C-NMR The 13C-nuclear map,netic resonance s p e c t r a of p y r i d o x i n e h y d r o c h l o r i d e , o b t a i n e d i n D20 a t ambient temperature u s i n g dioxane a s r e f e r e n c e l i n e , i s shown i n F i g . 6 (off-resonance) and F i g . 7 (lH-decoupled?. The s p e c t r a have been recorded a t 20 MHz f o r The carbon-13 on a Varian FT80A spectrometer. chemical s h i f t s and s p e c t r a l assignments a r e given i n Table 3 . Table 3.

13C-NMR

of P y r i d o x i n e , H C 1 i n D20. H

c1CH20H 7

HO


CH20H 8

Carbon N o .

153.52

S

3

143.42

S

2

141.32

S

4

137.62

S

5

130.57

d

6

58.92 57.88

t

15.23

Q

s = singlet t = triplet

7 and 8. 9

d = doublet 4 = quartet

P 01 0

1

Fig. 6.

1

t

1

"

1

1

1

1

"

1

1

'

1

I

I

'

I

'

I

I

I

, I

l

~

l

~

l

'

l

13C-NMR Off -Resonance Spectrum of Pyridoxine Hydrochloride in D20.

'

l

r'

I 1

46 I

HASSAN Y. ABOUL-ENEIN AND MOHAMMED A . LOUTFY

462

These d a t a w e r e r e f e r e n c e d from dioxane which a p p e a r s a t 67.39 ppm. The assignments were based on p u b l i s h e d d a t a of s e v e r a l p y r i d i n e d e r i v a t i v e s (28).

3 . Biosynthesis Yoshiki (29) h a s s t u d i e d t h e b i o s y n t h e t i c pathways of v i t a m i n B6, i n c l u d i n g t h e discGvery of t h e p r e c u r s o r glyc o l a l d e h y d e , g l y c o l a l d e h y d e dehydrogenase, and i s o l a t i o n of microorganisms d e f i c i e n t i n r e g u l a t i o n mechanism of Bf, biosynthesis. The s t a t u s of g l y c o l a l d e h y d e i n t h e b i o s y n t h e s i s of v i t a min Bg h a s been a l s o d e s c r i b e d by Vella, e t a l . (30). Comp e t i t i o n e x p e r i m e n t s , employing 14C-labeled samples of g l y c e r o l and glycoaldehyde, i n d i c a t e t h a t i n E. C o l i B ware 2 independent pathways l e a d i n g t o p y r i d o x a l . I n mutant WG2 t h e major pathway u t i l i z e s g l y c e r o l and r e l a t e d t r i o s e s as t h e s o l e C s o u r c e i n t h e c o n s t r u c t i o n of t h e Cg N s k e l e t o n of p y r i d o x o l . I n t h e minor pathway glycoaldehyde, and n o t g l y c e r o l , s u p p l i e s C-5 and C-5’ of p y r i d o x o l , w h i l e g l y c e r o l i s t h e s o u r c e of t h e o t h e r 6 carbon atoms. Pope and Smith (31) have r e p o r t e d t h e s y n t h e s i s of p y r i doxine by t u b e r c l e b a c i l l i when grown on s y n t h e t i c media. P y r i d o x i n e h a s been s y n t h e s i z e d , t o g e t h e r w i t h o t h e r Bcomplex v i t a m i n s , by H37 and Ravenel s t r a i n s of t h e t u b e r cle bacillus. Tani, e t a l . (32) have d e s c r i b e d a paper d i s c a s s a y method t o s c r e e n t h e b i o s y n t h e t i c i n t e r m e d i a t e s of v i t a m i n B6. Dempsey ( 3 3 ) h a s p u b l i s h e d t h e b i o s y n t h e s i s and c o n t r o l of v i t a m i n B6 i n E s c h e r i c h i a c o l i . Yamada, e t a l . ( 3 4 ) have s t u d i e d t h e v i t a m i n I36 b i o s y n t h e sis of a pdx B m u t a n t , E s c h e r i c h i a c o l i R WG3, i n amino a c i d - supplemented medium. An amino a c i d m i x t u r e and g l y c o l a l e d h y d e , which s a t i s f y t h e v i t a m i n B 6 requirement of E . C o l i B WC, 3, a pdx B m u t a n t , have been examined f o r t h e i r e f f e c t on growth of t h e mutant i n l i q u i d c u l t u r e . The amount of B6 s y n t h e s i z e d by t h e mutant i n t h e amino a c i d - supplemented medium i s lower t h a n t h e amount synt h e s i z e d i n t h e g l y c o l a l d e h y d e - supplemented medium.


463

4. S y n t h e s i s S e v e r a l r o u t e s have been d e s c r i b e d f o r t h e s y n t h e s i s of pyridoxine hydrochloride. Route 1: By t h e condensation of cyanoacetamide and e t h o x y a c e t y l a c e t o n e (35-38). The r e s u l t i n g 3-cyano-4-ethoxymethyl-6methyl-2-pyridone ( I ) w a s n i t r a t e d t o g i v e n i t o r p y r i d o n e (11). The sequence of r e a c t i o n s of t h i s r o u t e may be r e p r e s e n t e d g r a p h i c a l l y (Route 1 ) . Some v a r i a t i o n s and improvements i n t h i s r o u t e have been r e p o r t e d (37). Route 2: A l k y l - s u b s t i t u t e d o x a z o l e s have been found t o react w i t h maleic a c i d o r i t s anhydride i n a d i e n e s y n t h e s i s t o y i e l d s u b s t i t u t e d p y r i d i n e r e a d i l y converted t o p y r i d o x i n e ( 3 9 ) . I n t h i s r o u t e , e t h y l d , 1 - a l a n i n a t e h y d r o c h l o r i d e i s treated w i t h f o r m i c - a c e t i c anhydride t o y i e l d e t h y l N-formyl d , l - a l a n i n a t e (78X). T h i s compound i s r e f l u x e d i n c h l o r o form w i t h phosphorous p e n t o x i d e ( 4 0 ) , quenched w i t h aqueous potassium hydroxide, and t h e o r g a n i c l a y e r d i s t i l l e d t o g i v e 4-methyl-5-ethoxyoxazole ( I ) ( 6 0 % ) . The r e s u l t i n g oxazole ( I ) i s condensedreadily w i t h a number of a p p r o p r i a t e d i e n o p h i l e s t o form 2-methyl-3-hydroxy-4,5d i s u b s t i t u t e d - p y r i d i n e s c o n t a i n i n g s u b s t i t u e n t s (111, a , b , c ) which could be converted t o p y r i d o x i n e as f o l l o w s :

1.

A m i x t u r e of the oxazole ( I ) w i t h two moles of d i e t h y l m a l e a t e ( I I a ) i s heated and t h e adduct cleaved with e t h a n o l i c hydrogen c h l o r i d e t o form t h e d i e t h y l ester of 2-methyl-3-hydroxy-pyridine-4,5-dicarboxylic acid h y d r o c h l o r i d e ( I I I a ) ( 8 5 X ) . T h i s d i e s t e r i s reduced w i t h l i t h i u m aluminum h y d r i d e t o p y r i d o x i n e (41-42) i s o l a t e d a s i t s hydrochloride.

2.

A m i x t u r e of the oxazole ( I ) w i t h one mole of fumaron i t r i l e ( I I b ) i s r e f l u x e d i n methanol and t h e adduct cleaved w i t h 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 t o g i v e c r y s t a l l i n e 2-methyl-3-hydroxy-4,5-dicyanopyridine ( I I I b ) (75X). Hydrogenation of I I I b i n m e t h a n o l i c hydrogen c h l o r i d e over 5% palladium on c h a r c o a l g i v e s 2-me t h y 1-3 -hydroxy-4,5 -d lam inomethyl pyr i d i n e tr ihydr oc h l o r i d e (111, Y=CH2NH2) ( 7 0 % ) , which on d i a z o t i s a t i o n

HASSAN Y.ABOUL-ENEIN A N D MOHAMMED A. LOUTFY

464

0

0

0

CH3-C-CH2-C-CH II II

0C H 2 2 5

+ N.CCH2-C-NH2 It

Ethano 1 P i p e r i d i n e as' Catalyst

CH OC H 2 2 5

*

0: 0 02N.

H N O /~A C ~ O

H3C

>

H3C

I

H

' \-N

HI

CH20Et

I

I1

I

CN H 2 / P t and \

PhCl

c1

Pd/Charcoal

HONO F

CH20Et

CH2Br

Ho'@cH20HE:i

____1_3 28120/AgC1

H3C

H3

HBr Pyridoxine, HC1

R o u t e 1.

S y n t h e s i s of P y r i d o x i n e H y d r o c h l o r i d e .


CH -CHC00C2H5, 3 1 NH2

+

HC1

46.5

CH3 CO,

/x3 H

- CO

CH CH-COOC2H5 31 NH-CHO

00

+

YCH=CHY

\

I1

I

Y

V

CH20H

I11

I

CH20H

Pyridoxine

a.

Y =

-

COOC2H5

b.

Y

-

CN

C.

R o u t e 2.

=

Y-Y= - CH -0-CH 2

2

-

S y n t h e s i s of P y r i d o x i n e .

>--


466

i n w a t e r a t 85'

3.

y i e l d s pyridoxine hydrochloride (797).

A s o l u t i o n of t h e oxazole ( I ) i n a 20-mole e x c e s s of 2,5-dihydrofuran ( I I c ) c o n t a i n i n g 1%of t r i c h l o r o a c e t i c a c i d is h e a t e d t o g i v e 2-methyl-3-hydroxy-4,5-epoxyd i m e t h y l p y r i d i n e ( I I I c ) of 95% p u r i t y (58Z). The c y c l i c e t h e r ( I I I c ) i s cleaved w i t h 48% hydrobromic a c i d t o t h e dibromide hydrobromide (111, Y=CHZBr) and hydrolysed t o p y r i d o x i n e (35-36).

S e v e r a l r e p o r t s and p a t e n t s a r e l i s t e d i n t h e l i t e r a t u r e f o r t h e s y n t h e s i s of t h e drug (43-54).

5. Metabolism Pyridoxine, p y r i d o x a l , and pyridoxamine, which occur i n f o o d s t u f f s , a r e c o l l e c t i v e l y known a s v i t a m i n B6. I n t h e body, a l l t h r e e are converted t o p y r i d o x a l phosphate which i s t h e coenzyme f o r amino-acid d e c a r b o x y l a s e and f o r transaminase. The s t r u c t u r e s of t h e t h r e e a c t i v e forms of v i t a m i n B6 and t h e p y r i d o x a l phosphate, a r e shown below (55). R Pyridoxine

CH20H

Pyridoxal

CHO

Pyr idoxamine CH2NH2

R

0

II

CHO

P y r i d o x a l phosphate

4-Pyridoxic a c i d

COOH


461

Although, owing t o t h e wide d i s t r i b u t i o n of v i t a m i n B6 i n n a t u r e , c l i n i c a l d e f i c i e n c y symptoms are seldom o b s e r v e d , t h e r e i s l i t t l e doubt t h a t p y r i d o x i n e i s e s s e n t i a l i n human n u t r i t i o n . P y r i d o x i n e i s absorbed from t h e g a s t r o i n t e s t i n a l t r a c t and i s c o n v e r t e d t o t h e a c t i v e form p y r i d o x a l phosphate. Absorption i s d e c r e a s e d i n g a s t r o i n t e s t i n a l d i s e a s e s and a l s o i n s u b j e c t s t a k i n g i s o n i a z i d ( 3 ) . It i s e x c r e t e d i n t h e u r i n e a s 4-pyridoxic a c i d ( 2 ) . The metabolism of v i t a m i n B6 i n human b e i n g s h a s been i n v e s t i g a t e d (56). Moller (57) h a s r e c o g n i z e d p y r i d o x i n e , p y r i d o x a l , pyridoxamine, and 4-pyridoxic a c i d a s t h e e x c r e t i o n p r o d u c t s of v i t a m i n Bg. Complete b a l a n c e s t u d i e s have been made i n p i g s and i n b a b i e s on a l l t h e known v i t a m i n B6 m e t a b o l i c compounds. I n b a b i e s , t h e t o t a l o u t p u t exceeded t h e i n t a k e . The assumption t h a t a l i m i t e d s v n t h e s i s of v i t a m i n R6 o c c u r s seems j u s t i f i a b l e . Lumeng e t a l . (58) have r e p o r t e d t h e plasma c o n t e n t of B6 v i t a m e r s and i t s r e l a t i o n s h i p t o h e p a t i c v i t a m i n B6 metabolism. O r a l l y i n g e s t e d p y r i d o x i n e i s r a p i d l y m e t a b o l i s e d i n l i v e r and i t s p r o d u c t s are r e l e a s e d i n t o t h e c i r c u l a t i o n i n t h e form of p y r i d o x a l phosphate, p y r i d o x a l , and pyridoxic acid. Johansson e t a l . (59) have s t u d i e d t h e metabolism of l a b e l e d p y r i d o x i n e i n man. 15-20% of t h e l a b e l e d adminstered pyridoxine i s excreted i n t h e u r i n e during t h e f i r s t day. The r a t e of e l i m i n a t i o n of v i t a m i n B6 from t h e body r e s e r v o i r i s 2.3% /day. Wozenski e t a l . (60) have r e p o r t e d t h e metabolism of s m a l l d o s e s of v i t a m i n B(j i n men. It h a s been found t h a t responses t o pyridoxamine are g e n e r a l l y slower t h a n f o r p y r i d o x i n e o r p y r i d o x a l , s u g g e s t i n q t h a t pyridoxamine i s absorbed more s l o w l y o r m e t a b o l i z e d d i f f e r e n t l y , o r b o t h , t h a n p y r i d o x a l o r p y r i d o x i n e . A d o s e of a t least 1 mg of B6 i s n e c e s s a r y t o o b t a i n measurable changes i n v i t a m i n B6 metabolism. Cox, e t a l . (61) have d e s c r i b e d t h e metabolism of tritiuml a b e l e d p y r i d o x i n e i n rats. U r i n e , blood, and f e c a l samples from r a t s i n j e c t e d i n t r a v e n o u s l y w i t h l a b e l e d p y r i d o x i n e are c o l l e c t e d f o r e x c r e t i o n and chromatographic s t u d i e s . Approximately 70% of t h e l a b e l i s e x c r e t e d i n t h e u r i n e i n 5 hours. F e c a l e x c r e t i o n i s less t h a n 0.03 X i n

HASSAN Y. ABOUL-ENElN AND MOHAMMED A . LOUTFY

468

96 h o u r s . Chromatographic s t u d i e s of u r i n e have showed t h a t p y r i d o x i n e i s e x c r e t e d p r i m a r i l y unchanged w i t h a small amount of o n l y one m e t a b o l i t e , probably 4-pyridoxic acid. Johansson, e t a l . (62) have r e p o r t e d t h e metabolism of t r i t i u m - l a b e l e d p y r i d o x i n e i n r a t s and humans. 4-pyrid o x i c a c i d accounted f o r 25% of t h e t o t a l i s o t o p e i n t h e u r i n e of r a t s and humans. Ham (63) h a s d e s c r i b e d t h e t r a n s p o r t and i n t e r c o n v e r s i o n of Bg v i t a m e r s i n t h e p e r f u s e d i n t e s t i n e and kidney of t h e

rat. It h a s been r e p o r t e d t h a t v i t a m i n B6 metabolism becomes a l t e r e d w i t h age ( 6 4 ) .

Some a b n o r m a l i t i e s of v i t a m i n R 6 metabolism i n human b e i n g s have been d e s c r i b e d (65-66). The m e t a b o l i c s i g n i f i c a n c e of v i t a m i n B 6 and i t s r o l e t o t h e growth and f u n c t i o n a l development have been published (67-78). S e v e r a l o t h e r i n v i t r o and i n v i v o m e t a b o l i c s t u d i e s of v i t a m i n B6 have been p u b l i s h e d (79-86). 6. Methods of A n a l y s i s 6.1 T i t r i m e t r i c 6.1.1 Non-Aqueous P y r i d o x i n e , H C 1 has been assayed by non-aqueous t i t r a t i o n s , u s i n g 0.1 N p e r c h l o r i c a c i d i n t h e p r e s e n c e of m e r c u r i c a c e t a t e and c r y s t a l v i o l e t i n d i c a t o r ( 4 , 6 , 10, 8 7 ) . 6.1.2

Complexometric The drug h a s been determined by complexometric t i t r a t i o n of t h e Cuu l i b e r a t e d by p a s s i n g t h e s o l u t i o n through a column of c a t i o n exchange r e s i n i n t h e cupper form. A s o l u t i o n of 0.005 o r 0.0025 M of disodium EDTA i s t h e t i t r a n t and u s i n g murexide a s an i n d i c a t o r (88).


469

6 . 1 . 3 Amperometric Lemahieu, e t a l . (89) have r e p o r t e d an amperom e t r i c method f o r t h e d e t e r m i n a t i o n of hydrochlor i d e s of several o r g a n i c b a s e s , i n c l u d i n g pyridoxine. T i t r a t i o n , w i t h AgN03 s o l u t i o n i n d i m e t h y l s u l p h o x i d e , of s u c h h y d r o c h l o r i d e s i n d i m e t h y l s u l p h o x i d e g i v e s a n end-point c o r r e s ponding t o t h e f o r m a t i o n of AgC12- and a l e s s s h a r p end-point f o r t h e p r e c i p i t a t i o n of AgC1. U s e of t h e f i r s t end-point and two p l a t i n u m indicator electrodes with a potential difference of 100 mV a l l o w s t i t r a t i o n down t o a 0 . 2 mM c o n c e n t r a t i o n of p y r i d o x i n e , H C 1 . 6.1.4 Polarographic Manousek and Zuman (90) have d e s c r i b e d a d i r e c t 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 of p y r i d o x a l i n t h e p r e s e n c e of p y r i d o x a l 5-phosphate. The s i m u l t a n e o u s d e t e r m i n a t i o n of b o t h compounds i s p o s s i b l e o n l y when t h e c o n c e n t r a t i o n s a r e comparable. The same a u t h o r s a l s o have r e p o r t e d t h e p o l a r o g r a p h y of p y r i d o x i n e and several a n a l o g u e s i n b u f f e r e d s o l u t i o n s . A 2 - e l e c t r o n wave w a s observed which corresponds t o a diffusion-controlled reduction o f t h e a c t i v a t e d C-OH bond i n p o s i t i o n 4 . Nakaya (91) h a s d e s c r i b e d p o l a r o g r a p h i c s t u d i e s o f p y r i d o x i n e , FC1 and several p y r i d i n e d e r i v a t i v e s a t v a r i o u s pH's. Another p o l a r o g r a p h i c method f o r t h e e v a l u a t i o n of v i t a m i n B6 and i t s m e t a b o l i t e s h a s been r e p o r t e d (92). 6.2 Chromatography 6 . 2 . 1 P a p e r Chromatography S e v e r a l s o l v e n t s s y s t e m s (93-97) have been u s e d f o r t h e i d e n t i f i c a t i o n , s e p a r a t i o n , and q u a n t i t a t i o n of p y r i d o x i n e i n p h a r m a c e u t i c a l s and b i o l o g i c a l f l u i d s . These a r e p r e s e n t e d i n T a b l e 4 .


470

Table 4. Solvent System

P a p e r Chromatowaphv of Pyridoxine. Localising F-f agent

1. Citric a c i d : water: n-butano 1 (4.8 gm : 130 m l : 870 m l )

.

UV l i g h t ( b l u e

fluorescence) , iodo p l a t i n a t e spray (white), bromocresol g r e e n spray (strong r e a c t ion)

Ref.

0.18

(7)

.

2. Acetate b u f f e r (pH=4.58)

UV l i g h t ( b l u e fluorescence)

0.90

(7)

3 . Phosphate buffer(pH=7.4)

UV l i g h t ( b l u e

0.86

(7)

a. Collidine: l u t i n e (1:l) saturated with water.

0.88

(98)

b. Butanol:

0.63

(98)

-

(99)

fluorescence).

4 . Two dimensional chromatography consists of:

a c e t i c acid: w a t e r (40:10:50)

5. Amy1 a l c o h o l a c e t o n e : water (2:l:Z). Isoamyl a l c o h o l : p y r i d i n e : water (2:1:2).

6. Two dimensional chromatography consists of: a. Propanol: 10% formic a c i d (80:20).

b. Butanol saturated with 1%ammonia.

UV l i g h t

-


47 1

The s e p a r a t i o n of w a t e r - s o l u b l e v i t a m i n s , i n c l u d i n g v i t a m i n B 6 , on ion-exchange paper h a s been d e s c r i b e d by K l o t z and H u e t t e n r a u c h , (101) u s i n g Amberlite SB-2, Amberlite WB-2, and Amberlite WA-2. For t h e s e p a r a t o r y d e t e r m i n a t i o n of p y r i d o x i n e i n multi-vitamin preparations, o t h e r vitamins are s e p a r a t e d by paper chromatography i n which t h e upper l a y e r of butanol-ammonium hydroxidewater (4:1:5) are used a s t h e d e v e l o p e r , and by t h e one-dimensional a s c e n d i n g method (102).

6.2.2

Thin-Layer Chromatography C l a r k e (7) h a s d e s c r i b e d a s o l v e n t system cons i s t i n g of s t r o n g ammonia solution-methanol (1.5: 100) and potassium permanganate s p r a y a s a detecting agent. Lyle and T e h r a n i (103) have s e p a r a t e d s e v e r a l vitamins, i n c l u d i n g v i t a m i n Bf,, by TLC, u s i n g anhydrous a c e t i c a c i d - a c e t o n e - methanol benzene (1:1:4:14). A r a p i d TLC method f o r t h e r o u t i n e i d e n t i f i c a t i o n of v i t a m i n B 6 , and o t h e r d r u g s , i n b i o l o g i c a l f l u i d s , h a s been d e s c r i b e d (104).

6.2.3

Electrophoresis A r a p i d e l e c t r o p h o r e t i c method f o r t h e s i m u l t a n eous s e p a r a t i o n and e s t i m a t i o n o f v i t a m i n B6 and d e r i v a t i v e s from v a r i o u s t i s s u e s h a s been desThe method i s based on h i g h c r i b e d (105). v o l t a g e e l e c t r o p h o r e s i s of s m a l l a l i q u o t s of t i s s u e e x t r a c t s , a p p l i e d on c e l l u l o s e - c o a t e d p l a t e s . The method i s s e n s i t i v e u p t o 1 pg o f v i t a m i n B6.

6.2.4

Column Chromatography S e v e r a l methods have been r e p o r t e d f o r t h e s e p a r a t i o n of water s o l u b l e v i t a m i n s i n c l u d i n g vitamin B6. Some of t h e s e systems a r e d e s c r i b e d as f o l l o w s :


472

T a b l e 5.

Column

Gas-Chromatography of P y r i d o x i n e , H C 1 .

Cond it i o n s

Comment

1. 3% SE-30

100-270° temperat u r e programming.

Derivatized using NO-bis(trimethy1s i l y l ) acetamide.

2. 152 DON -

Column t e m p e r a t u r e D e r i v a t i z e d 175O, w i t h t h e r m a l above. conductivity d e t e c t i o n and H e as carrier g a s .

as

( 1 09)

Column t e m p e r a t u r e D e r i v a t i z e d silanized 165O, N2 a s above. Gas Chrom P c a r r i e r gas. (60-80 mesh).

as

(110)

Corning h i g h vacuum s i l i cone g r e a s e on Gas-Chrom Q (60-80 mesh).

3 . 3% SE-52 on

4 . 3% OV-101 on Gas Chrom Q (100-200 mesh).

Ref.

(108)

Column t e m p e r a t u r e D e r i v a t i z e d w i t h (111) 160'. diazomethane and butaneboronic a c i d a s p y r i d o x i n e 0methyl e t h e r c y c l i c butaneboronate.

r e a c t i o n h a s been u t i l i z e d f o r t h e q u a n t i t a t i o n of t h e d r u g (122-126). R e c e n t l y , Moussa ( 1 2 2 ) h a s d e s c r i b e d a m o d i f i e d p r o c e d u r e t o improve t h e s t a b i l i t y of the c o l o r , t h e s p e e d , and t h e s e n s i t i v i t y of a s s a y . I n t h i s p r o c e d u r e , t h e r e a c t i o n i s c a r r i e d o u t i n 2p r o p a n o l medium ( i n s t e a d of w a t e r ) , t r i e t h a n o l amine i s used as a b u f f e r , and i o d i n e i s used as an oxidant. The d e t e c t i o n l i m i t of t h i s method i s 10 pg of v i t a m i n B 6 .


473

a . Amberlite WA-2, c a r b o x y l i c a c i d r e s i n , u s i n g a c e t a t e b u f f e r (pH=4.62) a s a n e l u e n t (106). b. SD c a t i o n i t e , u s i n g c o n c e n t r a t e d aqueous ammonia a s a n e l u e n t ( 1 0 7 ) . Another system c o n s i s t i n g of Amberlite IRA-400, u s i n g 80% a c e t i c a c i d a s a n e l u e n t , h a s been a l s o described (107). 6.2.5 Gas-Chromatography S e v e r a l gas-chromatographic procedures have been r e p o r t e d f o r t h e d e t e r m i n a t i o n of v i t a m i n R6 i n pharmaceutical f o r m u l a t i o n s and i n b i o l o g i c a l fluids. Table 5 summarises some of t h e s e methods. A p p l i c a t i o n of p y r o l y s i s - g a s chromatography f o r t h e d e t e r m i n a t i o n of pyridoxone, H C 1 and o t h e r w a t e r - s o l u b l e v i t a m i n s , h a s been r e p o r t e d (103). Id en t i f i c a t i o n and s e m i -quan t i t a t i v e e s t imat i o n of v i t a m i n B 6 , u s i n g microphase e x t r a c t i o n and gas-chromatography, h a s been d e s c r i b e d ( 1 0 4 ) . 6.2.6 High-Performance Liquid Chromatography S e v e r a l HPLC procedures have been published f o r t h e d e t e r m i n a t i o n of p y r i d o x i n e , H C 1 and o t h e r water-soluble vitamins, i n b i o l o g i c a l f l u i d s and i n f e e d s (112-1.16). Table 6 summarises, some of t h e HPLC systems used f o r t h e a n a l y s i s of v i t a m i n B6. 6.3 Spectrophotometry 6.3.1 C o l o r i m e t r i c S e v e r a l c o l o r i m e t r i c a s s a y s have been developed f o r p y r i d o x i n e . The U.S.P. method ( 4 ) f o r t h e d e t e r m i n a t i o n of p y r i d o x i n e H C 1 , i s based on c o u p l i n g of p y r i d o x i n e w i t h 2,6-dichloroquinone chloroimide t o give a blue c o l o r , according t o t h e method d e s c r i b e d by Kuhn and Low (121). The absorbance of t h e b l u e c o l o r i s measured a t 650 nm and compared w i t h s t a n d a r d s o l u t i o n s . The


414

Table 6.

HPLC systems of p y r i d o x i n e .

Column

Mobile Phase

Detection

1. LiChrosorb

Methanol-phosphate buffer (1 :1) c o n t a i n i n g 0.002 M C TAB.

uv

The method i s applicable i n a n a l y s i s of v i t a m i n B6 i n u r i n e but not i n blood.

Sodium p e r c h l o rate - potassium dihydrogen phosphate

mr

A p p l i c a b l e i n (117) u r i n e and blood analysis. Detection l i m i t 30 ng.

RP-8

2. TSK-Gel LS-160

-

3. Octadecyl silica

4. LiChrosorb NH2

Comment

Ref.

(116)

Sodium chloride.

- Acidic potass i u m phosphate

Acetonitrile - phosphate buffer

5. U l t r a s p h e r e 0.033 M p o t a s IP sium phosphate b u f f e r (pH=2.2) c o n t a i n i n g 2.5% acetonitrile.

Fluorescence

Recovery 93.9 ? . 7 . 6 %

(118)

uv

A p p l i c a b l e f o r ( 119) the analysis of v i t a m i n B6 i n i.v. solut i o n s , and beverages. Standard d e v i t i o n s l-2X.

Fluorescence

Recovery 82.4- (120) 105.1X. Applicable f o r the analysis of v i t a m i n B6 i n animal t i s s u e s and i n milk.


475

Among other colorimetric methods for the determination of vitamin B6, are the following: a. Pyridoxine and other derivatives give a stable, alcohol-soluble, red-violet color with zinc chloride-stabilized diazotised sulphathiazole at pH 6.5-7.0 (127). b. Murai (102) has determined vitamin B6 by its reaction with dimethyl-p-phenylenediamine, HC1 and sodium hypochlorite in a buffered solution (pH=8). The resulting color is extracted with isobutyl alcohol and the absorbancy of the solution is measured at 625 nm. c. A colorimetric method similar to that described in b, using N,N-diethyl-p-phenylenediamine, HC1 and potassium ferricyanide at pH 7 (128). Other colorimetric methods for the analysis vitamin B6 have been reported (9, 129).

of

6.3.2 Ultraviolet Bosch (130) has published a spectrophotometric determination of vitamin R6, and other vitamins, in pharmaceutica1,preparations with N-bromosuccinimide, in methanol at 370-380 nm. The method allows determination of 100-1000 pg of vitamin B6. Vitamin B 1 , BIZ, A , and menadione bisulphite interfere with this method. Vitamin B6 has been spectrophotometrically determined at 290 run, after chromatographic separation from other related analogues(131). A survey of normal and derivative spectra of numerous vitamins, including vitamin B6, has been reported. The second derivative spectra of these vitamins are especially suitable for their quantitative determinations (132). Quantitative determination by derivative spectroscopy is superior to direct spectrophotometric measurement in many problem situations.

HASSAN Y. ABOUL-ENEIN A N D MOHAMMED A . LOUTFY

476

Barrosa (133) h a s r e p o r t e d t h e a n a l y s i s of a m i x t u r e of p y r i d o x i n e and i s o n i a z i d i n t a b l e t f o r m u l a t i o n . P y r i d o x i n e i s determined by d i f f e r e n t i a l spectrophotometry a t 324 nm. 6.3.3 I n f r a r e d P y r i d o x i n e , H C 1 has been determined by I R spectrophotometry, u s i n g t h e r e g i o n 6-11 u , and water a s a s o l v e n t ( 1 3 4 ) . 6.3.4 Nuclear Magnetic Resonance A PMR s p e c t r o m e t r i c method h a s been d e s c r i b e d (135) f o r s i m u l t a n e o u s d e t e r m i n a t i o n of v i t a m i n B1 and Bg. The r e l a t i o n s h i p of t h e peak a r e a s of t h e s i g n a l s a t 6 2.55 ppm ( v i t a m i n B1) and a t 6 2.65 ppm ( v i t a m i n B1 + B6) t o t h a t a t 6 6.6 ppm (maleic a c i d ) a r e d e r i v e d , whereby t h e compounds of t h e m i x t u r e s can be r e a d i l y quantitated.

Recently, a n o t h e r PMR method f o r t h e s i m u l t a n eous a n a l y s i s of v i t a m i n B 6 and B 1 , u t i l i s i n g s i m p l e r e q u a t i o n f o r c a l c u l a t i o n , h a s been developed (136). The mean p e r c e n t r e c o v e r y of v i t a m i n B6 and B1 i n t h e i r dosape forms i s 97.42 f 2.99 and 97.57 t 1.52, r e s p e c t i v e l y . T h i s method a v o i d s o v e r l a p i n t h e peak a r e a s used i n t h e former method. 6.3.5 Absorption-Luminescence The t e c h n i q u e of absorption-luminescence s p e c t r o s c o p y h a s been r e c e n t l y a p p l i e d f o r t h e analys i s of v i t a m i n B 6 and r e l a t e d compounds (137138). Razhulina and Morozov (137) have i n v e s t i a g e d t h e e q u i l i b r i u m c o n s t a n t s between v a r i o u s ions and t h e t a u t o m e r i c forms of v i t a m i n B6 t o g e t h e r w i t h t h e e f f e c t of t e m p e r a t u r e , s u b s t i t u t i o n , and medium on t h e a b s o r p t i o n p r o p e r t i e s of t h e s e forms. The e x p e r i m e n t a l s p e c t r o s c o p i c d a t a a r e i n good agreement w i t h quantum c a l c u l a t i o n .


6.3.6

477

Spectrofluorimetric A l e K s e i c h i k , -e t a 1 (139) have p u b l i s h e d q u a l i t a t i v e a n a l y s i s of v i t a m i n B6 and o t h e r d r u g preparations. The s p e c t r a are d e t e r m i n e d i n e t h a n o l , h y d r o c h l o r i c a c i d , and sodium h y d r o x i d e , u s i n g v i t a m i n B6 c o n c e n t r a t i o n s from 0.01-r).001 mg/ml. F u j i t a , 5 a l . (140) have r e p o r t e d f l u o r o m e t r i c d e t e r m i n a t i o n o f v i t a m i n B6. The a u t h o r s have made e x t e n s i v e s t u d y i n t h i s r e s p e c t ( 1 4 1 ) . F u j i n o (142) h a s improved a method f o r t h e d e t e r m i n a t i o n of v i t a m i n B6, b y u t i l i s i n g t h e f l u o r e s c e n c e of 4 - p y r i d o x i c a c i d which i s produced by o x i d a t i o n of v i t a m i n €56 w i t h permanganate. T h i s method i s a p p l i e d f o r t h e d e t e r m i n a t i o n of v i t a m i n B6 i n b i o l o g i c a l f l u i d s (85, 143-144).

6.4 Microbiological S e v e r a l m i c r o b i o l o g i c a l methods f o r t h e a n a l y s i s of v i t a m i n B6 i n b i o l o g i c a l materials and o t h e r p r o d u c t s have been r e p o r t e d (145-147). The growth r e s p o n s e of some m i c r o o r g a n i s m s , e . g . ,

Streptococcus faecal& ( 1 48) and Saccharomyces carzsbergensis (99, 1 4 9 ) , p r o v i d e a r e l i a b l e estimate of v i t a m i n B6 c o n t e n t i n n a t u r a l p r o d u c t s . The v i t a m i n B6 c o n t e n t i s measured n e p h e l o m e t r i c a l l y a f t e r t h e p r o p e r t r e a t m e n t of t h e sample. Gregory (118) h a s d e t e r m i n e d v i t a m i n B6, u s i n g The method i s used f o r t h e e s t i m a t i o n of v i t a m i n B6 i n f o r t i f i e d b r e a k f a s t cereals.

Saccharomyces uvarum. 6.5 Enzymatic

Wada and S n e l l (150) have developed a method f o r t h e a s s a y of p y r i d o x i n e and pyridoxamine p h o s p h a t e , based on e n z y m a t i c o x i d a t i o n t o p y r i d o x a l which i s allowed t o r e a c t with phenylhydrazine.

HASSAN Y. ABOUL-ENEIN A N D MOHAMMED A . LOUTFY

478

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81, 1731 (1960); t h r o u g h 91. J . Nakaya, Nippon Kagoku Z a s s h i , Chem. A b s t r . , 2, 2275 (1962).

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99. J.Q. Snyder and S.H. Wender, Arch. Biochem. and Biophys., 46, 465 (1953) ; t h r o u g h Chem. A b s t r . , 49, 8364 (1955). I

100. G.C. D e R i t i s , G. C a t t a n e o , and U. F a n t o l i , Ann. 1st. !!Carlo F o r l a n i n i " , 2 3 , 168 (1963) ; t h r o u g h Chem. A b s t r . , 61, 2332 (1964). I


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Food Chem., 28, 486 ( 1 9 8 0 ) ; 213571 (1980).

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617 ( 1 9 8 0 ) .

120. J.F. Grepory,D.B. Manley and J . R . K i r k , J. Agri. Food Chem., 921 ( 1 9 8 1 ) ; through Anal. A b s t r . , 43, 79 (1982).

2,

1 2 1 . R. Kuhn and I. Low, Ber. d t s c h . (1939). 122. A.A.

Moussa,

123. J . V .

S c u d i , J. B i o l . Chem.,

Mikrochim.Acta,

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1,1 6 9 2, 707

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K e r e s z t e s y , Am. J .

1 2 5 . M. Hochberp,, D. Melnick, and B.L. 1 5 5 , 1 0 9 (1944).

Oser, J. B i o l . Chem.,

_ I

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126. J . P . Sweeney and W.L. H a l l , J . Assoc. O f f i c . Agric. Chemists, 38, 697 (1955). 127. A.M. Aliev, Aptechn. Delo, 1 3 , 31 ( 1 9 6 4 ) ; through Chem. A b s t r . , 62, 6343 ( 1 9 6 5 ) . I

1 2 8 . B.E. A l b r i g h t and E.F. Degner, Automat. Anal. Chem., Technicon Symp., 3 r d . , 1, 461 (1967); through Chem. A b s t r . , 2, 33460 (19697. 129. Y. S a k u r a i , T. Akao, and H. H o r i , B u l l . I n s t . Phys. Chem. Research, 307 (1944); t h r o u g h Chem. A b s t r . , 42, 5488 (1948)

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S n e l l , J. B i o l . Chem.,

236,

2089 (1961).

SACCHARIN Muhammad Uppal Zubair and Mahmoud M. A. Hassan King Snud Unimrsity Riyadh, Saudi Arabia 1.

2.

3. 4. 5.

488 488 488 489 489 489 489 489 490 490 49 1 50 1 504 506 506 506 508 508 509 5 I4

Description I,1 Nomenclature 1.2 Formula 1.3 Molecular Weight 1.4 Elemental Composition I.5 Appearance. Color, Taste, Odor Physical Properties 2.1 Crystal Properties 2.2 Solubility 2.3 pK,, Dissociation Constant 2.4 Spectral Properties Synthesis Metabolism Methods of Analysis 5.1 Detection and Identification 5.2 Titrimetric Methods 5.3 Gravimetric 5.4 Spectrophotometric 5.5 Chromatographic Methods References


487

Copyright 0 1984 by the American Pharmaceutical Association lSBN 0-12.260813-5

MUHAMMAD UPPAL ZUBAIR AND MAHMOUD M. A. HASSAN

488

1. Description 1.1 Nomenclature

1.1.1 Chemical Names

1.1.2

a.

1,2-Benzisothiazole-3 (2H)-one 1, 1dioxide.

b.

1,2-Benzisothiazol-3-one 1, 1-dioxide.

c.

2,3-Dihydro-2-ketobenzisosulfonazole.

d.

l,2-Dihydro-2-ketobenzisosulfonazole.

e.

Benzosulphimide.

f.

o-Benzoicsulphimide.

g.

o-Sulfobenzimide.

Generic Names Benzoic sulfimide, benzosulfimide, benzoic sulfimide, o-sulfobenzimide , o -sulfobenzoic acid imide , saccharin, saccharin insoluble.

1.1.3

Trade Names Garantose, Glucid, Gluside, Hermesetas, Sacarina, Saccarina, Saccharinol, Saccharinose, Saccharol, Saxin, Sykose, Zaharina.

1.2

Formula

1.2.1 Empirical C H N O

75

1.2.2

3

S

Structural

489

SACCHARIN

1.2.3

CAS' No.

81-07-2 1.2.4 Wiswesser Line Notation

T56 BSWMVJ 1.3 Molecular Weight

183.18

1.4 Elementa1 Composition C, 45.89%; H, 2.75%; N, 7.65%; 0, 26.20%; S, 17.50%.

1.5

Appearance, Colour, Taste, Odor White odorless or faintly aromatic crystals or crystalline powder (l).In dilute aqueous solution it is 500 times as sweet as sugar, the sweet taste is detectable even at 1:100,000 dilution. (2)

2. Physical Properties

2.1

Crystal Properties

2.1.1

Crystallinity The crystal structure of saccharin was determined (3) by 3-dimensional integrated intensity data collected on a computercontrolled diffractometer operated by an IBM 1620, in a closed-loop manner. The crystals were found to be monocligic with a 9.55*53, b 6.91923, c 11.80324 A, and f3l03.g0, and the space group is 'p21/c. The H atoms were also located and are included in the refinement. The centrosymmetric dimer (C6H4C0 NHS02)* mols. are formed by hydrogen bonds between the N of imide and 0 of the carbonyl atoms, (NH.. .OC), of the five membered ring system of saccharin. The six-sided ring formed by the hydrogen bonds around the center of symmetry is completely planar. The location of the H atom eliminates the possibility of the lactim structure for

.

490

MUHAMMAD UPPAL ZUBAIR A N D MAHMOUD M . A . HASSAN

t h e molecule. The c o n t a c t mode of phenyl r i n g s i s normal. A c h a r a c t e r i s t i c feature of t h e molecular c o n f i g u r a t i o n i s t h e narrow C-S-N a n g l e of 92.2' i n t h e five-membered ring. The narrow a n g l e diminishes s t r a i n from t h e r i n g and t h u s makes it p o s s i b l e f o r t h e e n t i r e molecule t o assume a p l a n a r shape. Other bond a n g l e s and bond d i s t a n c e s were found t o be normal. Luigi e t a1 ( 4 ) have a l s o s t u d i e d t h e c r y s t a l and molecular s t r u c t u r e s of s a c c h a r i n , and observed some n d e l o c a l i z a t i o n i n t h e C-0 and C-N bonds [ t r i c l i n i c (Pi), k 8 2 O 4 5 ' ( 2 0 ' ) , k82O 1 7 1 ( 1 0 1 ) , ~ = 6 5 O4 0 ' ( 1 2 ' ) , Z=4]. The C-C, C-S and S-N bonds i n t h e i s o t h i a z o l e r i n g are s i n g l e , and s t r o n g hydrogen bonding (NH 0 ) g i v e r i s e t o centrosymmetrical dimers, as was observed w h i l e determining t h e c r y s t a . 1 s t r u c t u r e of s s c c h a r i n .

...

2.1.2

Melting Range The m e l t i n g ranges of s a c c h a r i n are ( a ) 228.8-229.7 ( 2 , 5 ) and ( b ) 226-230(1,6,7) dec. 228.8-229.7 (8).

2.2

Solubility Saccharin i s s o l u b l e a t 20°, i n 290 p a r t s of water, i n 30 p a r t s o f a l c o h o l , i n 1 2 p a r t s o f a c e t o n e , i n 50 p a r t s of g l y c e r o l , i n about 25 p a r t s of b o i l i n g water. It i s v e r y s l i g h t l y s o l u b l e i n e t h e r and chloroform; s o l u b l e i n d i l u t e ammonia s o l u t i o n , a n d i n s o l u t i o n s of a l k a l i b i c a r b o n a t e s w i t h e v o l u t i o n of carbon dioxide ( 9 ) . I t s aqueous s o l u t i o n i s a c i d t o l i t m u s ( 2 ) . On a l k a l i n e and a c i d h y d r o l y s i s s a c c h a r i n g i v e s r i s e t o 0-sulphamoyl-benzoic a c i d and ammonium 0-sulphobenzoic a c i d r e s p e c t i v e l y ( 2 ) .

2.3

pKa , D i s s o c i a t i o n Constant

The d i s s o c i a t i o n c o n s t a n t of s a c c h a r i n a t 25' has been r e p o r t e d t o be pKa 1.6.

SACCHARIN

49 I

2.4 Spectral Properties 2.4.1

Infrared Spectrum The IR spectrum of saccharin as KBr disc was recorded on a Perkin Elmer 580-~Spectrophotometer and shown in Fig 1 (10). The structural assignments have been correlated with band frequencies and are given in Table 1. Table 1. IR Characteristics of Saccharin

-1 Frequency Cm

Assignment

3410, 3100

-co-x-

2970, 2700

-C-H

1722

-8-NH-

1595

aromatic- C=C-

1340 1165, 1180, 1188

-so2 -

0

frequencies

N

-

700-900

C-H out of plane deformation showing 1,2-disubstituted benzene ring.

760

Four adjacent hydrogens, ortho-disubstituted benzene. Other characteristic bands are: 2010, 1980, 1870, 1462, 1300, 1260, 1200, 1140, 1122, 1055, 1020, 1010, 975, 902, 688, 632. Other IR studies were also reported (11,121.

2.4.2

Ultraviolet Spectrum The W spectrum of saccharin in ethanol was scanned from 200 to 400 nm using Varian DMS 90 spectrophotometer. It exhibits a characteristic UV spectrum Fig 2 with maxima at 283, 275, 225 and 208 nm (13). Other UV spectral data of saccharin have also been reported (14).

492

493

MUHAMMAD UPPAL ZUBAIR AND MAHMOUD M . A. HASSAN

494

Xmax

&

775 951 8570 in methanol.

285 276 226

1% Elcm

hax -

234.5

351 in 0.01 N NaOH (5,9)

268 284

Inflexion

89

69

-

1% 1cm

Amax

267.5

244.5 2.4.3

-

minima saccharin sodium in water (5)

Nuclear Magnetic Resonance Spectra 2.4.3.1

Proton Spectrum

The PMR spectrum of saccharin in DMSO-d6 was recorded on a Varian FT80 NMR spectrometer using tetramethylsilane as a reference standard, Fig 3 (15). The following structural assignments have been made. Table 2. PMR Characteristics of Saccharin Assignments Aromatic protons

- NH

Chemical Shifts

8.11 (m) 12.45 (s)

m = multiplet; s = singlet

Natural abundance l5N NMFt data has been reported (16). N-H chemical shift is 215.4 ppm.


496

2.4.3.2

l 3 C NMR S p e c t r a The 13C-NMR n o i s e decoupled and off-resonance s p e c t r a a r e shown i n F i g 4 and F i g 5 r e s p e c t i v e l y (17). Both w e r e recorded over 5000 Hz range i n DMSO-d6 on FT 8 0 , 80 MHz NMR s p e c t r o m e t e r , u s i n g 1 0 m l sample t u b e , and t e t r a m e t h y l S 1 a n e as r e f e r e n c e The s t a n d a r d a t ambient temperature. carbon chemical s h i f t s a s s i g n e d on t h e b a s i s of a d d i t i v i t y p r i n c i p l e and o f f resonance s p l i t t i n g p a t t e r n a r e given i n Table 3.

3

Table 3. Carbon Chemical S h i f t s of Saccharin Carbon no.


c-1

127.57 ( s )

c-2

139.75 ( s )

c-3

c-4 c-5

124.53(d) 134.72(d) 135.55( d)

C-6

121.18(d)

c-7

160.79 ( s ) d = doublet; s = s i n g l e t

2.4.4

Mass Spectrum The m a s s electron ded on a equipped

spectrum of s a c c h a r i n o b t a i n e d by impact i o n i z a t i o n and which w a s r e c o r Ribermag R-10-10 m a s s spectrometer w i t h d i r e c t i n l e t probe and a d a t a

a

a a

d

? 0

u a, a

a, (I)

d

0

-d

0

2

n

W

a 0

m

c

2

c

-d

-4 k rd

.c rd m

u u

0

w

5 0 a,

k +r

a m

a,

u

c rd c

m

0 a, I

k

0

w w

i2

499

SACCHARIN

system ( 1 8 ) . The spectrum ( F i g 6 ) shows a molecular i o n peak a t m / e 183 and a base peak m/e 184 (M+l). The most prominent fragment, t h e i r r e l a t i v e i n t e n s i t i e s and p o s s i b l e s t r u c t u r e s a r e l i s t e d i n Table 4 . Table

m/e

184

Mass Fragments of S a c c h a r i n

Relative Intensity

Fragment

M+1

100

183

30.7

168

6.9

120

4.

M+

54

GY

CONH2

119

59

[ 104

25.6

+ 9

( g C O N H

1 I (Continued)

I

SACCHARIN

SO I

Table 4 (continued) m le

Relative Intensity

Fragment

11.8

103

+'

46

92

+.

76

51

H

+'

Other mass s p e c t r a l d a t a a r e a l s o r e p o r t e d ( 1 9 ) .

3.

Synthesis Route 1 Saccharin w a s f i r s t s y n t h e s i s e d i n 1879 from o-sulphanoylbenzoic a c i d ( 2 0 ) . The s t a r t i n g m a t e r i a l i s o b t a i n e d from t h e m i x t u r e o f o-and p - s u l f o n i c a c i d s , r e s u l t i n g from s u l f o n a t i o n of t o l u e n e . The s u l f o n i c a c i d s are t h e n conv e r t e d t o t h e s u l f o n y l c h l o r i d e s by t h e a c t i o n o f phosphorous p e n t a c h l o r i d e . The p - t o l u e n e s u l f o n y l c h l o r i d e i s l a r g e l y removed by f r e e z i n g , and t h e l i q u i d r e s i d u e c o n t a i n i n g t h e compound i s t r e a t e d w i t h ammonia t o g i v e t h e o-toluenesulfonamide which on o x i d a t i o n w i t h a l k a l i n e potassium permanganate s o l u t i o n g i v e o-sulfonamidobenzoic a c i d which undergoes spontaneous l o s s of water t o y i e l d s a c c h a r i n which can be o b t a i n e d on a c i d i f i c a t i o n of t h e r e a c t i o n mixture.


502

Bo2c1

Sac c h a r i n

Route I1

It may be prepared ( 2 1 ) by t r e a t i n g t o l u e n e w i t h c h l o r o s u l f o n i c a c i d and s e p a r a t i n g t h e c- and p-toluenesulfowlchloride. The o -compound i s t h e n t r e a t e d with ammonia and t h e r e s u l t i n g sulfonamide i s o x i d i z e d w i t h potassium permanganate i n a l k a l i n e s o l u t i o n y i e l d s a s a l t of S s u l f a m o y l b e n z o i c a c i d . The f r e e a c i d which i s l i b e r a t e d on a c i d i f i c a t i o n l o s e s w a t e r spontaneously t o give saccharin.

503

SACCHARIN

aCH3 acH NH3

s02c1

~

S02NH2

COOH

S02NH2

Saccharin

Route I11 Saccharin i n h i g h y i e l d may be o b t a i n e d by o x i d i s i n g o-toluenesulfonamide with dichromate and s u l f u r i c a c i d .

0

Saccharin Route IV, Many o t h e r i n d u s t r i a l methods are now employed e.g. one s t a r t s ( 2 2 ) w i t h a n t h r a n i l i c a c i d which i s d i a z o t i s e d and t r e a t e d w i t h l i q u i d s u l f u r d i o x i d e i n presence of copper as c a t a l y s t . The s u l p h i n i c a c i d d e r i v a t i v e t h e r e b y obtained i s t r e a t e d with chlorine i n a lk a lin e solution and sulfonyl c h l o r i d e so o b t a i n e d i s t r e a t e d w i t h m o n i a and heated.


504

COOH

N2HS04

NH2

NaOH

4.

Metabolism Early s t u d i e s r e p o r t e d (23) t h a t 90-95% of an i . v . dose of saccharin could be recovered from t h e u r i n e of a c a t h e t e r i z e d dog. Staub ( 2 4 ) r e p o r t e d t h a t o r a l l y admini s t e r e d saccharin w a s excreted unchanged i n t h e u r i n e . Herter ( 2 5 ) demonstrated t h e presence of s a c c h a r i n i n various organs of a r a b b i t a f t e r an o r a l dose. I n recent s t u d i e s u t i l i z i n g r a d i o - l a b e l l e d saccharin ( 26-28 ) t h e compound w a s shown t o be metabolised t o a s l i g h t e x t e n t i n r a t s and o t h e r animals. Lethio and Wallace ( 2 9 ) have used ( 3 - 1 4 C ) saccharine ( I ) i n r a t , dog, r a b b i t , guinea p i g and hamster, i n d i c a t e d t h a t t h e r e w a s l i t t l e d i f f e r ence i n p a t t e r n of metabolic p r o f i l e s due t o animal speci e s or dose l e v e l . They a l s o showed t h a t l a b e l l e d C 0 2 i n t h e expired a i r i n d i c a t e d t h a t s a c c h a r i n w a s decarbox y l a t e d t o a s l i g h t degrec. (11), and GEAE chromatographic s e p a r a t i o n and i s o l a t i o n of l a b e l l e d compounds i n d i c a t e d t h a t more t h a n 99% of t h e u r i n a r y 1 4 C w a s unchanged

SACCHARIN

505

saccharin and up to 1% of 1 4 C was a metabolite identified as o-sulphamoylebenzoic acid (111) (Fig 1, Scheme ). Scheme

*

0

I11

a

2

99% I Saccharin

S02NH2

I1

Fraction Number Fig. 1. Elution diagram of the chromatography on DEAE cellulose of urine from a rat which received [ 3 1 4 C ] saccharin at 50 mg/kg. Each fraction contained 10 m l . Peak I: carbonate; Peak 11: sulfamoylbenzoic acid; Peak 111: saccharin.

MUHAMMAD UPPAL ZUBAIR AND MAHMOUD M. A . HASSAN

506

I n man s a c c h a r i n i s r a p i d l y absorbed from t h e g a s t r o i n t e s t i n a l t r a c t and e x c r e t e d unchanged mostly i n t h e u r i n e w i t h i n 24 h o u r s ( 5 ) .

5.

Methods of A n a l y s i s

5.1

D e t e c t i o n and I d e n t i f i c a t i o n

a. Saccharin i s h e a t e d w i t h r e s o r c i n o l and s u l p h u r i c a c i d over a low flame; on d i l u t i o n and a d d i t i o n of sodium hydroxide s o l u t i o n , a f l u o r e s c e n t green l i q u i d i s o b t a i n e d (5,30,31). b. Saccharin i s f u s e d w i t h sodium hydroxide t i l l t h e e v o l u t i o n of ammonia c e a s e s . N e u t r a l i s e d s o l u t i o n on a d d i t i o n o f f e r r i c c h l o r i d e g i v e s r i s e t o v i o l e t c o l o u r (30 J1). C.

Thermal a n a l y s e s ( 3 2 ) and microchemical r e a c t i o n s of s a c c h a r i n w i t h Mn, Co, N i and Cu have been employed f o r t h e c h a r a c t e r i s a t i o n of s a c c h a r i n ( 3 3 ) .

d. Saccharin can a l s o b e i d e n t i f i e d by i t s r e a c t i o n

w i t h H202 and NaNO2 and subsequent b a s i f i c a t i o n w i t h a l k a l i a f f o r d s dark brown-red c o l o u r ( 3 4 ) . e. I d e n t i t y t e s t s f o r s a c c h a r i n p r e s e n t i n food have a l s o been based on column and t h i n l a y e r chromatography ( 3 5 ), m a s s s p e c t r o m e t r y (36) , spectrophotometry ( 3 7 ) and m i c r o a n a l y t i c a l methods ( 3 8 ) .

5.2

T i t r i m e t r i c Methods 5.2.1

Aqueous The USP XIX(7) and B.P 1980 ( 6 ) methods c o n s i s t of d i s s o l v i n g an a c c u r a t e l y weighed sample of s a c c h a r i n i n h o t water and t i t r a t i n g w i t h 0 . 1 N sodium hydroxide, u s i n g phenolphthalein as indicator. Another o f f i c i a l procedure ( 3 0 ) i n v o l v e s d i g e s t i o n of an a c c u r a t e l y weighed sample o f s a c c h a r i n i n h y d r o c h l o r i c a c i d . The r e s u l t i n g ammonium s a l t i s r e a c t e d w i t h sodium hydroxide and t h e l i b e r a t e d ammonia i s d i s t i l l e d i n t o 0 . 1 N s u l p h u r i c a c i d . The excess of t h e a c i d i s determined by t i t r a t i n g w i t h . sodium hydroxide u s i n g methyl r e d solut i o n as i n d i c a t o r .

507

SACCHARIN

5.2.2

Non-aqueous

A non-aqueous differentiating titration procedure (39) involves passage of the artificial sweeteners mixture through a cationic exchange resin , Dowex 50-x8, to separate and convert saccharin to its acid form. The solution obtained is titrated potentiometrically in methyl isobutyl ketone with 0.1 N sodium methoxide. The method has been applied in presence of cyclamate and benzyl alcohol. 5.2.3

Ion Selective Electrode Hazemoto et a1 (40) developed an ionselective electrode sensitive to saccharin, by establishing an ion association between Fe2+-bathophenanthroline chelate and saccharin in nitrobenzene. The electrode developed could measure saccharin ion in presence of other sweetening agents e.g., sucrose, glucose, sodium cyclamate and sorbitol in the Concentration range of 10-1to 10-5 M.

5.2.4

Polarography Polarographic methods of analysis have been applied to samples of foods containing saccharin (41-44). In a procedure (44) saccharin is extracted into organic solvents in an acidic medium. Further purification is achieved by column chromatography. The residue obtained is dissolved in 0.1 N NaOH and an aliquot is polarographed in a supporting electrolyte of 0.1 N HC1, 0.1 N KC1 and 0.1% Bu4 N Br. Nasierowska (45) has applied alternatecurrent oscillopolarographic method to pharmaceuticals containing saccharin and sodium saccharin. Both forms a well developed oscillopolarographic curve in MH2SO4 in air with mercury anode and dropping-mercury cathode, and the results are comparable to those obtained by UV spectrometry.

MUHAMMAD UPPAL ZUBAIR AND MAHMOUD M . A . HASSAN

508

5.3

Gravimetric Saccharin has been determined gravimetrically by the oxidation of its sulfur to sulfate ion (46). An accurately weighed amount of the sample is boiled with concentrated nitric acid and the vapours evolved are reacted with sodium hydroxide in a tube. The sulfate content is then determined gravimetrically

.

5.4

Spectrophotometric

5.4.1 Colorimetric A method involving salt formation between saccharin and methylene blue has been developed (47)and its concentration as low as 10 p M can be determined by this method. Taneka et a1 (48) developed another procedure in which saccharin is reacted with phenothiazine and copper acetate in 50% acpeous ethanol. The resulting complex is extracted into xylene and its absorbance is measured at 510 nm. Belagy (49) analysed saccharin by forming a complex with safranine and measuring its absorbance at 510 nm.

5.4.2 Ultraviolet Several procedures have been developed for the extraction and isolation of saccharin when present in beverages (50-53), and food ( 54-57) before its estimation spectrophotometrically. The absorbance was measured in chloroform at 278 nm ( S O ) , in ethanol at 271 nm (55) and in 1% aqueous sodium carbonate at 235 and 244 nm (52 1. 5.4.3

Infrared

A procedure has been developed (58) for the determination of saccharin by IR spectrophotometric method. Saccharin is separated from cyclamate by extracting it into a mixture of benzene and ether from an acid medium.

509

SACCHARIN

The IR absorption at 1721 cm-l is then measured. Saccharin up to 0.2 mg can be determined by this method.

5.4.4 Spectrofluorometric Nakamura (59) had developed a procedure for the isolation and determination of saccharin in foods by measuring fluorescence at 410 nm (excitation at 277 nm) However recovery of saccharin added to 23 types of food ranged from 82 to 98%.

.

5.4.5

PMR Spectrometry

A PMR procedure was described (60) involving the integration of the aromatic protons singlet of benzisothiazoline ring occurring at 8.00 ppm and that singlet due to protons of the internal standard maleic acid occurring at 6.25 ppm. The method was employed for saccharin sodium tablets resulted in an average of 97.1% ? 3.15 and for saccharinlactose mixture with an average of 98.92 2 3.5 for saccharin.

5.5 Chromatographic Methods 5.5.1

Paper Chromatography Saccharin has been detected and estimated after extraction from food samples on Whatman No. 1 filter paper (61,62). The sample in solution form, such as carbonated water can be used for paper chromatography. The spotted paper is developed in BuOH-ACOH-H20 (40:10:22) for 18 hours; and sprayed with a solution of phthalic acid and aniline for spot development. Saccharin with an Rf 0.17 can be estimated colorimetrically after elution of the spot with 60% AcOH (62).

5.5.2

Thin Layer Chromatography Thin layer chromatography has been used in qualitative and quantitative analysis of saccharin, when present in artificial sweetening agents , beverages , food and pharmaceuticals. Several systems have been used and are listed in table 5.

MUHAMMAD UPPAL ZUBAIR AND MAHMOUD M . A. HASSAN

510

Table

5

Thin Layer Chromatography Systems f o r Saccharin Analysis ~~

~

~

~~

~

S t a t i o n a r y Phase

Developing Phase

Detection

Reference

10%-Acetylated CellulosePolyamide ( 3 :2 )

S h e l l s o l Apropanola c e t i c acidformic a c i d

U.V. and m u l t ip l e colorimetric methods

63

(45:6:7: 2 ) S i l i c a gel Kieselgel G

chloroformacetic acid

acetone-ammonia, ethanol-ammonia and benzene

Wakogel B-5 plates Acetyl cellulosepolymi.de powder (3:2)

64

U.V.

(9:l) silver n i t r a t e , peroxide-ferric c h l o r i d e and 2,6-dichloroquinone chloroimide spray reagent

65

66

multiple colour react ion xylene-propanola c e t i c acidformic a c i d

67

(45: 6 : 7 : 2 ) Silica gel ( Adsorbosil-1 )

Avicel SF

but anol-et hanol (95%)-ammonia (28%)-water,

254 m

(40:4:1:9 )

irradiation

ethanolammonia-acetone,

spray with 0 .l% pina c r y s t a l yellow i n 95% e t h a n o l and i r r a d i a t e

(1:1:8) dime t h y l formamideethanol-water

,

(5:4:1)

68 69

a t 365OAO.

dioxanepyrimidinewater. ( 7 : 2 : 1 ) Polyamide

benzeneethyl acetateformic a c i d ( 5 :10:2)

methyl r e d bromocresol green

70

(Continued)

SACCHARIN

51 1

Table 5 (continued) S t a t i o n a r y Phase .Developing Phase

A1 0 /Polyamide 2 3

toluenepropionic acidformic a c i d

Detection

Reference

multiple colour r e a c t ions

71

U.V.

72

(5:4:2) Silica gel G

5.5.3

but anolethanol25% aqueous ammonia (40 :4 :5 ) G a s Chromatography

Saccharin has been d e t e c t e d and analysed by gas chromatography, when p r e s e n t i n pharmac e u t i c a l s and food. It i s u s u a l l y , f i r s t e x t r a c t e d from t h e sample w i t h an o r g a n i c s o l v e n t and t h e n converted t o i t s e s t e r , such as methyl or s i l a l y l t o make it more v o l a t i l e . shows v a r i o u s systems t h a t have Table 6 been used.

5.5.4

High-Performance L i q u i d Chromatography High-Performance Liquid Chromatography h a s been used e x t e n s i v e l y f o r a n a l y s i n g s a c c h a r i n , p r e s e n t i n pharmaceutical s , beverages and food. The d i f f e r e n t HPLC systems used for t h e a n a l y s e s are given i n Table 7.

l n w t - t -

cu

2

0

I

(u

0

I

0

ri

0

co ffl

X k a,

c,

2

d;J.

a 3 I

U P k 0

0 0

ffl

cu

z

0

V

!2

k .c V

a,

u

.rl

4

m

-rl

ri

0

W

0

co

. .

?3?

rlmN 0 0

rl I 0

co

a c cd

W R ri

m m

cu

a3

cu

2

cu

ri

0

I

I

E

0

k

e m Ld t7a

F

&

d

0

m

%

SACCHARIN

__

513

Table 7. HPLC of Saccharin Analyte or Parameters and Comments Matrix

Pharmaceuticals

Beverages

Reference

Micronized silica gel treated with 3-aminopropyltriethoxysilane; 0.04 M phosphate buffer; 254 nm.

83

p Bondapak/C18(S/S 30 cm x

84

4 m m ) ; 5% acetic acid, -1 at 2000 p.s.i, (2 ml min ) ; 254 nm. Alcoholic products

Partisil-10 ODS ( S / S , 25 cm x 4 mm); 0.01 M ' KH2P04 at pH 2.2 (1.2 ml min-l); 254 nm.

85

Food

Permaphase AA X or Zipax SAX; 10 nm-KClO4 in 10 nm-Na2B407 buffer of pH 8.0;

86

Pharmaceuticals and non-alcoholic drinks

ODS-Sil-X -1, 400500 p.s.i.; aqueous 45% methanol or phosphate buffer solution (pH 3.0) and methanol ( 3 :2 ) ; U.V. detection.

87

Pharmaceuticals

1-1 Bondapak cl8; acetic acid-methanolH2O (1:60:139); 280 nm

88

Plasma and urine

Partisil PXS ODS-2 (10 m) (30 cm x 4 m m ) , 1.2 ml min-l ; H20-methanolanhyd. acetic acid (399:lOO:l); fluorimetric detection (excitation at 230 nm; emission at 350 nm)

89

Beverages

HC ODS Sil-X (25~0.26cm); 1M CH3COOH

90 91 9


514

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SALICYLAMIDE Salim A. Babhair, Abdullah A. Al-Badr, Hassan Y. Aboul-Enein King Sotid Unizjer,sity

Riyadh, Saudi Arabia 1.

2.

3. 4. 5. 6.

522 522 522 522 523 523 523 523 523 524 524 524 53 1 534 534 535 535 536 536 537 538 538 542 542 547 547 548

Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, and Odor Physical Properties 2.1 Crystal Properties 2.2 Solubility 2.3 pK, 2.4 Identification 2.5 Spectral Properties Synthesis Stability Metabolism and Pharmacokinetics Methods of Analysis 6.1 Non-Aqueous Titration 6.2 Colorimetric Analysis 6.3 Spectrophotometric Analysis 6.4 Spectrophotofluorimetric Analysis 6.5 Infrared Spectrophotometric Analysis 6.6 Chromatographic Analysis 6.7 Radiochemical Analysis 6.8 Flame Emission Spectrometry 6.9 Thermomicroscopic Method 6.10 The Ring-Oven Technique References


521

Copyright 0 1984 by the Amencan Pharmaceutical Association ISBN 0-12-260813-5

522

SALIM A . BABHAIR ET AL

SALICYLAMIDE 1. Description

1.1 Nomenclature

1.U Chemical Names o-Hydroxybenzamide, 2-Hydroxybenzamide. 1 . 1 2 Generic Name

Salicylamide

.

1.13 Trade Names Afko-sal, Amid-sal, Doldram, Drosprin, L i q u i p r i n , Raspberin, S a l r i n , Uromide, Salamide, Samid, Acket, Algiamida, Cidal, Oramid, P a n i t h a l , S a l i z e l l , Novecyl, Dolomide, S a l i p u r , Salymid, Saliamin, u r t o s a l , Algamon, Benesal (1,2).

1.14 Chemical A b s t r a c t R e g i s t r y Number [ 65-45-21. 1 . 2 Formulae

1 . 2 1 Emperical C H NO

7 7 2

1.22 S t r u c t u r a l

4

1.3 Molecular Weight

137.13

523

SALICYLAMIDE

1.4 Elemental

Composition

C , 61.27%; H, 5.11%;

N, 10.28%; 0, 23.34%.

1 . 5 Appearance, Color, and Odor White o r yellowish white c r y s t a l l i n e powder or c r y s t a l s darkens on exposure t o a i r , o d o r l e s s .

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

2.1 Cryst a1 P r o p e r t i e s 2 . 1 1 C r y s t a l System

Monoclinic, Rods from s o l v e n t s o r sublimation.

2.12 X-Ray D i f f r a c t i o n C e l l Dimensions a = 12.93 A., c = 24.80 0.2A.

b = 5.02 A.,

Formula Weight per c e l l . 8 (8.05 c a l c u l a t e d from X-ray d a t a ) . Density. 1.320 ( f l o t a t i o n and pycnometer) ; 1.308 (X-ray). Optical Properties. R e f r a c t i v e i n d i c e s (5893 A , , 25OC) a = 1.595 ?r 0.002. b = 1.637 0.001, q = 1.727 2 0.005. Optic Axil Angles (5893 A., 25'C) 2H = (+) 82.5' 2 V = ( + ) 76' ( c a l c u l a t e d from b and 2H), 2V = 75' ( c a l c u l a t e d from a , b and q) (3).

2.13 Melting Point and Fusion Data Salicylamide sublimes b e f o r e melting (142OC; h o t s t a g e ) . A s t h e m e l t c o o l s , broad and uns t a b l e form appears. This form i s m e t a s t a b l e below t h e melting point. When seeded o r on standing t h e s t a b l e form grows slowly through t h e u n s t a b l e form. The r a t e of transformation i s very slow a t room temperature (3).

2.2 S o l u b i l i t y One gram i s s o l u b l e i n about 500 m l o f water, about

1 5 ml o f a l c o h o l , about 35 m l o f e t h e r , about 100

ml

524

SALIM A. BABHAIR ET AL.

of

chloroform and about 20 m l of propylene g l y c o l .

I n s o l u b l e i n benzene, carbon t e t r a c h l o r i d e and xylene. Soluble i n s o l u t i o n of a l k a l i e s . A s a t u r a t e d s o l u t i o n of salicylamide i n water has a pH 5.2-6(1,4,5). 2 . 3 pKa

8.1 ( 6 ) . 2.4 Identification Salicylamide g i v e s a b l u e v i o l e t c o l o r w i t h f e r r i c chlor i d e solution, a color reaction indicative t o s a l i c y l a t e derivatives. T r i n d e r ' s reagent g i v e purple c o l o r w i t h salicylamide

(5). Matta and Nunes ( 7 ) i d e n t i f i e d salicylamide from chemic a l l y r e l a t e d analogues by t h e determination of i t s r e f r a c t i v e index at t h e melting p o i n t which w a s found t o be 1.55. Furthermore, salicylamide could be c h a r a c t e r i z e d through t h e formation of diphenylmethyl d e r i v a t i v e prepared by r e f l u x i n g with diphenylmethanol and p-toluene sulphonic a c i d i n a c e t i c a c i d ( 8 )

A f u l l r e p o r t f o r t h e i d e n t i f i c a t i o n of salicylamide through melting p o i n t , r e a c t i o n s of t h e a i d e group, t h e phenolic group, t h e benzene r i n g , and t h e format i o n of c h e l a t e compounds has been published ( 9 ) . Salicylamide can be d e t e c t e d microchemically by means of t h e formation of t h e dibromo-derivative ( 9 ) . Furthermore, t h e i d e n t i f i c a t i o n of salicylamide by chromatographic a n a l y s i s a r e discussed f u r t h e r i n t h e monograph.

2.5 S p e c t r a l P r o p e r t i e s 2.51 U l t r a v i o l e t spectrum The u l t r a v i o l e t spectrum of salicylamide i n methan o l and water shows maxima a t 235 nrn and 302 nm. The u l t r a v i o l e t spectrum i n e t h a n o l i s shown i n Figure [l] and i s recorded on Varian AG UV-VIS spectrophotometer model DMS 90 with maxima a t

S ALICYLAMIDE

5 25

F i g . 1. The UV spectrum of s a l i c y l a mide i n e t h a n o l .

I

I

f’

’,

Q,

2

cd P

k 0 (0

5

2

Fig. 2 . The UV spectrum of salicylami.de i n 0 . 1 N NaOH s o l u t i o n .

SALIM A . BABHAIR ETAL.

526

235 nm (El%, 1 cm 543) and 302 nm (E;$, 1 cm 295). I n a l k a l i n e medium, t h e spectrum e x h i b i t s two maxima a t 242 nm ( E l % , 1 cm 536) and 328 nm ( E l % , 1 cm 435) as shown i n f i g u r e [ 2 ] . 2.52 I n f r a r e d spectrum The i n f r a r e d of salicylamide i n KBr d i s c i s presented i n f i g u r e [ 3 ] and i s recorded i n PerkinE l m e r spectrophotometer model 580 B. The frequenc i e s and t h e i r s t r u c t u r a l assignment a r e shown below: Frequency( cm-1) 3400 3200 1680

:760 ;:I 750

Assignments OH s t r e t c h JTH s t r e t c h CO (amide) C=C aromatic CH out-of -plane bending aromatic

.

Other c h a r a c t e r i s t i c f i n g e r p r i n t bands a r e 1500, 1450, 1430, 1360, 1310, 1250 cm-1. 2.53 Mass Spectrum The m a s s spectrum of salicylamide i s shown i n f i g u r e [ 4 ] . The spectrum w a s c a r r i e d out on Varian MAT 311 spectrometer with an i o n i s i n g energy of 70 e V . The sample w a s introduced by d i r e c t probe a t 12OoC. A molecular ion peak w a s observed a t m/e 137. Some c h a r a c t e r i s t i c peak observed a r e l i s t e d below: Mass (m/e) 137

120

Species or Fragment M"

+=

d-O

v)

2

'k

4

-1

100 0

.

500

Fig. 4. The mass spectrum of salicylamide determined by electron impact.

529

SALICY LAMIDE

92

65 39

2.54 Nuclear 2.541

I

aromatic fragmentations.

Magnetic resonance spectrum

1 H-NMR Spectrum 1 The 60 MHz H-NMR spectrum o f s a l i c y l a m i d e i n DMSO-Db i s shown i n f i g u r e [ 5 ] . The spect r u m w a s recorded on Varian T ~ O - ANMR spect r o m e t e r u s i n g TMS as t h e i n t e r n a l s t a n d a r d . The f o l l o w i n g s t r u c t u r a l assignments have been e l i c e t e d from f i g u r e [51. Chemical s h i f t (ppm1 Multiplet centered at

6.83 Multiplet centered at

7.36 Mult i p l e t c e n t e r e d a t

7-90

Assignments Aromatic p r o t o n s a t C3 and C 5 Aromatic p r o t o n at C 4 Aromatic p r o t o n at Cg

Broad s i n g l e t a t 8.33

NH2 amide( exchanga b l e w i t h D~o).

S i n g l e t a t 13.00

OH phenolic (exchangable w i t h D20, Fig. [ 6 1 ) .

Long range coupling between p r o t o n s a t C 3 C5, C 4 and C 6 i s observed.

2.542 l3C-NMR Spectrum The Cwbn-13 NMR spectrum o f Salicylamide have been determined on Varian FT 80 Spectrometer. The

530


.... .,,. ,... , . . . ..,.

,.. .... I

I

'

I

I

.

I " " I '

0

f / r A

A

, . a0

1

1

.

l . . . . l . . . . l . . . . l . . . . I . . . . I ZO 6.0 $U PPw(I)+R 3.0

Fig. 5. DMSO-D6.

'H-NMR

....

I

1 1 . . . 1 . . . ,

20

I

1.0

spectrum of salicylamide in

6

1

r

I I I . 1 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 ,

8.0

TO

5.0 w(r)*.O

6.0

35

2.0

t.0

Fig. 6. IH-NMR spectrum of salicylamide in DMSO-D6, showing D 0 exchange. 2

, 0

SALICYLAMIDE

53 1

sample w a s d i s s o l v e d i n DMSO-DG i n a tube with a diameter of 1 0 mm. S p e c t r a l width: 4000 Hz, a c q u i s i t i o n time: 1.023 seconds, seconds, and number of d a t a p u l s e width 6 p o i n t s 8192. The noise-decoupled spectrum i s shown i n f i g u r e [ 7 ] shows seven s i n g l e t s . The complete o f f - r e s o n w e e spectrum i s shown i n f i g u r e [ 8 ] and s p e c t r a l assignments a r e l i s t e d below:

XY0* 6

5

Chemical s h i f t ( i n ppm r e l a t i v e t o TMS)

114.60

Assignment

(s)

c2

161.31 ( s )

c3

117.39 ( d )

c4

(a)

c5

118.49 ( a )

c6

128.31 ( d )

c7

172.39 ( s )

c=o

134.16

s = singlet. d = doublet.

The assignments a r e i n agreement w i t h r e c e n t l y published d a t a (11). 3. Synthesis Although s e v e r a l methods were published f o r t h e s y n t h e s i s of salicylamide, y e t t h e main r o u t e i s through t h e aminol y s i s of methyl or e t h y l s a l i c y l a t e ( 1 2 , 13, 1 4 , 15, 16, 17) as shown i n t h e following scheme:

532

533

SALIM A . BABHAIR ET AL.

534

0 =C-OR

0c ,

-N H,

4. Stability Salicylamide i s s t a b l e i n s o l i d s t a t e , however, it i s recommended t o be s t o r e d a t room temperature i n a drydark container. The hydrolysis of salicylamide under experimental conditions designed t o simulate t h e g a s t r o i n t e s t i n a l environment proved t h a t t h e amide bond i s r e s i s t a n t t o a c i d cleavage (18). The k i n e t i c s and mechanism of a l k a l i n e hydrolysis was reported and found t o follow pseudo f i r s t order k i n e t i c s ( 1 9 ) .

5. Metabolism and pharmacokinetics Salicylamide i s r e a d i l y absorbed from t h e g a s t r o i n t e s t i n a l t r a c t and d i s t r i b u t e d t o a l l body t i s s u e s but t h e drug does not bind appreciably t o plasma p r o t e i n s . Several s t u d i e s had been r e p o r t e d i n l i t e r a t u r e s d e a l i n g with t h e absorption of salicylamide and i t s plasma concentration ( 2 0 ) , ( 2 1 ) , (22), ( 2 3 ) . It i s r a p i d l y excreted i n t h e u r i n e mainly as t h e glucuronide and sulphate conjugates. S a l i cylamide i s metabolized i n human e n t i r e l y as shown i n Scheme 1. The percentage o f t h e u r i n a r y conjugated m e t a b o l i t e s v a r i e s according t o animal s p e c i e s ( 2 4 ) , ( 2 5 ) , age ( 2 6 ) and r o u t e of administration ( 2 7 ) and ( 2 8 ) .

535

SALICYLAMIDE

4% oc, 9'

6'

I Hr Sulphate

Glucuronide Gen tisamide

Schemei, Metabolic pathways OFsalicylmnide in humans. Tne proportion of a dose of salicylamide excreted as sulphate decreased with i n c r e a s e d doses ( 2 9 ) . Peak serum l e v e l s are reached between 30 minutes and 1 hour a f t e r o r a l a d m i n i s t r a t i o n ( 5 ) . I n v e s t i g a t i o n has revealed t h a t s a l i c y l a m i d e i n h i b i t s t h e a c y l a t i o n of sulphonamides leading t o i n c r e a s e i n t h e i r c o n c e n t r a t i o n ( 3 0 ) .

6. Methods

of Analysis

6.1 Non-aqueous T i t r a t i o n Rhodes e t al (31) determined a mixture containing acet y l s a l i c y l i c a c i d , acetaminophen and salicylamide, p o t e n t i o m e t r i c a l l y by non-aqueous t i t r a t i o n . The titr a n t w a s 0 . 1 N tetrabutylammonium hydroxide i n benzene/ methanol mixture and t h e t i t r a t i o n solvent w a s dimethylformamide. The d i f f e r e n c e i n t h e pKa values f o r t h e s e weak a c i d i c drugs ( a s p i r i n pKa 3.5, acetaminophen pKa 8.31) w a s s u f f i c i e n t t o permit u s e f u l d i f f e r e n t i a t i o n . Walash and R i z k ( 3 2 ) r e p o r t e d a non-aqueous t i t r a t i o n o f several a n a l g e s i c s i n dosage form including s a l i c y l a mide i n t e t r a m e t h y l u r e a using 0.1 N sodium methoxide as t h e t i t r a n t , t h e end point w a s measured e i t h e r p o t e n t i o m e t r i c a l l y or with thymol b l u e as i n d i c a t o r . The r e sults were comparable with t h o s e obtained using dimethylformamide as a s o l v e n t .

SALIM A. BABHAIR ET A t .

536

6.2 Colorimetric Analysis Rutkowski ( 3 3 ) r e p o r t e d t h e simultaneous c o l o r i m e t r i c assay of salicylamide, s a l i c y l i c a c i d and g e n e t i s i c a c i d i n pharmaceutical p r e p a r a t i o n s . These compounds g i v e b l u e c o l o r complexes with f e r r i c s a l t ( n i t r a t e i n ethanol s o l u t i o n ) . The b l u e c o l o r complex of s a l i c y l amide i s e x t r a c t e d with e t h e r while t h e o t h e r s a r e n o t . Another method r e p o r t e d by Souza (34) for t h e determin a t i o n of s e v e r a l phenols i n c l u d i n g salicylamide and aminopyrazoles i n pharmaceutical products, where a solut i o n of 2% potassium f e r r i c y a n i d e g i v e s a colored indophenol d e r i v a t i v e which i s measured a t 500 nm. 6.3 Spectrophotometric Analysis Salicylamide had been estimated spectrophotometrically as a s i n g l e component, i n complex pharmaceutical mixt u r e and a n a l g e s i c t a b l e t s by s e v e r a l procedures among them a r e t h e following: a ) The treatment of salicylamide with chloramine a t pH 8.00 i n t h e presence of thiobromine sodium s a l i c y l a t e , c a f f e i n e , s a l i c y l i c a c i d , acetaminophen, and methanamine. The method could be used t o determine as low as 0 . 1 mg o f salicylamide i n t h e mixture ( 3 5 ) . b ) The mixture of salicylamide, papaverine H C 1 , phenob a r b i t a l and g l y c e r y l g u a i a c o l a t e i n glycol-glycerol aqueous s o l u t i o n w a s determined i n 0 . 1 M H2S04 at 250, 273 and 299 nm ( 3 6 )

-

c ) Soliman and Salaheldin ( 3 7 ) r e p o r t e d a simple r a p i d method f o r r o u t i n e a n a l y s i s of salicylamide i n analg e s i c t a b l e t containing acetaminophen, phenobarbital, c a f f e i n e , codeine phosphate, ascorbic a c i d , chloroamine phosphate and prednisone. The method does not r e q u i r e preliminary s e p a r a t i o n of salicylamide from o t h e r c o n s t i t u e n t s p r i o r t o determination. The absorbance w a s l i n e a r f o r concentration of s a l i cylami.de up t o 4 mg/lOO r r i i s o l u t i o n a t 308 nm. d ) Salicylamide may be estimated as s a l i c y l i c a c i d i n

b i o l o g i c a l f l u i d s following t h e a d d i t i o n of T r i n d e r ' s reagent ( 3 8 ) o r a f t e r e x t r a c t i o n i n t o 0.4% malonic acid i n butyl ether (39).

SALICYLAMIDE

537

6.4 Spectrophotofluorimetric Analysis The spectrophotoflourimetric determination of s a l i c y l amide i n b i o l o g i c a l f l u i d s (blood, serum and u r i n e ) w a s r e p o r t e d by Vercsh e t a1 ( 4 0 ) . The procedure involves t h e simultaneous determination of salicylamide and s a l i c y l i c a c i d a t pH 11. The method i s s e n s i t i v e and capa b l e of estimating a c o n c e n t r a t i o n of salicylamide and/ o r i t s m e t a b o l i t e s as low as 0 . 1 pg/ml blood, serum o r o t h e r b i o l o g i c a l fluids. The method may a l s o be used i n monitoring t h e b i o a v a i l a b i l i t y o f salicylamide provided i n d i f f e r e n t dosage forms. The fluorescence of salicylamide w a s measured d i r e c t l y a t maximum a c t i v a t i o n and e m i s s i o n wavelengths of 340 and 430 nm resp e c t i v e l y while f o r s a l i c y l i c a c i d a t 310 and 435 nm r e s p e c t i v e l y . Lange e t a1 ( 4 1 ) employed t h e f l o u r e s cent p r o p e r t y of salicylamide i n b a s i c s o l u t i o n t o determine t h e drug and o t h e r s a l i c y l a t e s following t h e g e l f i l t r a t i o n separation. Barr and Riegelman ( 4 2 ) d e t a i l e d a s p e c t r o f l o u r i m e t r i c procedure f o r t h e determination of salicylamide and i t s m e t a b o l i t e s i n b i o l o g i c a l f l u i d s following h y d r o l y s i s as described by Levy and Matsuzawa ( 2 9 ) . The method involves t h e h y d r o l y s i s of a l l m e t a b o l i t e s t o s a l i c y l a t e and determining them at emission t o e x c i t a t i o n wavel e n g t h of 350 and 430 nm r e s p e c t i v e l y . T o t a l s a l i c y l a mide w a s determined by hydrolysing a l l m e t a b o l i t e s t o s a l i c y l i c a c i d i n aqueous b a s i c s o l u t i o n a t a maximum a c t i v a t i o n and e m i s s i o n wavelengths of 350 and 430 nm r e s p e c t i v e l y . Recently, S t r e e t and Schenk ( 4 3 ) reported a s p e c t r o f l u o r i m e t r i c a n a l y s i s of salicylamide i n t h e presence of a c e t y l s a l i c y l i c a c i d and s a l i c y l i c a c i d as i m p u r i t i e s by d i r e c t and i n d i r e c t methods. S a l i c y l i c a c i d i s determined i n t h e range of 10-7M concentration a f t e r s e p a r a t i o n from salicylamide. The instrumental condit i o n s f o r t h e s p e c t r o f l u o r i m e t r i c determination of salicylamide, a c e t y l s a l i c y l i c a c i d and s a l i c y l i c a c i d i s shown i n Table 1.

538

SALIM A. BABHAIR ET AL

Table 1 Ingredient determined

Wavelength nm

S l i t Width nm

Excita- -istion sion

Excita- -istion sion

Salicylamide i n ethanol. i n c,hloroform Acetylsalicylic acid Salicylic acid

300 315

42 5

445

10 10

10 20

280

335

10

20

315

445

10

10

The presence of o t h e r medicinals such as phenacetin, ascorbic a c i d , methapyreline hydrochloride among o t h e r a n a l g e s i c s does not a f f e c t t h e determinations of salicylamide, a c e t y l s a l i c y l i c a c i d and s a l i c y l i c a c i d .

6.5 I n f r a r e d Spectrophotometric Analysis Salicylamide i n mixed pharmaceutical p r e p a r a t i o n s w a s determined by i n f r a r e d spectrophotometry using t h e amide absorption a t 3425 cm-l as t h e key band ( 4 4 ) . The absorbance measured with a base l i n e at 3300 em-l o r 3470 cm-l. The c a l i b r a t i o n curves were l i n e a r i n t h e c o n c e n t r a t i o n range between 0.5 t o 2 mg/ml and t h e r e s u l t s were i n good agreement with t h o s e determined c o l o r i m e t r i c a l l y . The determination w a s not i n t e r f e r r e d w i t h t h e coexistance of a l i m i t e d amount of 40 p o s s i b l e drug c o n s t i t u e n t s e.g., phenacetin and p y r a b i t a l ( 4 4 ) . Another I R spectrophotometric method for t h e determinat i o n of salicylamide among o t h e r medicinally used amides w a s reported ( 4 5 ) . The amide carbonyl absorpt i o n band a t 1732 cm-I w a s used as t h e b a s i s of quant i t a t i v e assay.

6.6 Chromatographic Analysis

6.61 Paper Chromatography ( 5 ) described a method f o r t h e separat i o n of s a l i c y l a t e s using t h e following t.wo systems :

1) Clarke

SALICYLAMIDE

539

a ) Strong ammonia: n- butanol: water 33:100:66 by descending technique. The l o c a l i z i n g agent used w a s f e r r i c c h l o r i d e spray o r ult r a v i o l e t l i g h t a t 254 mu.. b ) Phosphate b u f f e r (pH 7.4) and d e t e c t i n g agent bromine-starch potassium i o d i d e . 2 ) Other method was described by Wagner ( 4 6 ) f o r t h e a n a l y s i s and d e t e c t i o n of s a l i c y l i c a c i d and d e r i v a t i v e s including salicylamide. Development w a s e f f e c t e d by using c o l o r r e a c t i o n s , w i t h f e r r i c ammonium s u l p h a t e , ammoniacal s i l ver n i t r a t e s o l u t i o n , potassium n i t r a t e , HC1 and 5% KOH through d i a z o t i z a t i o n and coupling with, 2-naphthol and coupling with d i a z o t i z e d sulphanilic acid.

3 ) It w a s r e p o r t e d ( 4 7 ) t h a t salicylamide and o t h e r aromatic hydroxy carboxylic a c i d amides undergo autooxidation, when chromatographed i n a l k a l i n e media. This autooxidation products hinder t h e development of t h e complex colorat i o n with f e r r i c ion. However, paper chromatographic s e p a r a t i o n of salicylamide and analogs i n a l k a l i n e b u f f e r media can be accomplished by ionophoresis ( 4 8 ) . 6.62 Column Chromatography Column chromatography has been a p p l i e d t o s e p a r a t e and analyse a mixture of salicylamide and a c e t minophen i n t a b l e t s and capsules ( 4 9 ) . Strongly a c i d i c and b a s i c c e l i t e columns a r e used t o separ a t e acetaminophen and salicylamide r e s p e c t i v e l y . Q u a n t i t a t i o n i s performed using u l t r a v i o l e t spectrophotometry . Cataionic and anionic ion-exchange r e s i n s ( 5 0 ) were used as s t a t i o n a r y phases f o r t h e s e p a r a t i o n of s e v e r a l analgesic drugs including salicylamide using aqueous ethanol as t h e mobile phase. 6.63 Thin Layer Chromatography S e v e r a l r e p o r t s have been published on t h e i d e n t i f i c a t i o n and s e p a r a t i o n o f salicylamide which a r e summarized i n Table 2.

Table 2: S t a t i o n a r y Phase

Thin Layer Chromatography of Salicylamide.

Solvent System

Detecting Agent

References

'A

0 P

1) S i l i c a g e l

Butyl a c e t a t e : Chloroform : 85% formic a c i d (6:4:2)

2 ) Polyamide resin

a ) Isopropanol : Water : 90% f o r mic acid. ( 1 . 5 : 6 : 0.1) b ) Chloroform : benzene : 90% Formic a c i d . ( 5 : 1 : 01). c ) Chloroform : 90% Formic a c i d . (20 : 0.1). d) Cyclohexane : Chloroform : a c e t i c a c i d . ( 4 : 5 : 1).

3 ) SGF p l a t e s

Benzene : Acetic a c i d : Methanol Chloroform : Petroleum e t h e r ( 3 0 - 7 0 ' ) (10:5:5:10:70).

5% s o l u t i o n o f f e r r i c c h l o r i d e i n acetone followed 1% a l c o h o l i c o-phenanthroline.

(51)

-

U l t r a v i o l e t l i g h t 254 nm.

(53)

4) Silica

Methanol

10% Copper s u l p h a t e , 2% ammonium hydroxide, d a r k yellow c o l o r .

(54)

a ) Chloroform : E t h e r : Acetone ( 6 0 : 20 : 2 0 ) .

F e r r i c c h l o r i d e and 2,6-dibromoquinone-4-chloroimide.

(55)

g e l G.

5) S i l i c a gel

b ) Chloroform : Benzene : Acetone ( 6 0 : 20 : 2 0 ) .

6) S i l i c a

A c e t i c a c i d : Carbon t e t r a c h l o r i d e : Chloroform : Water (100:60:90:50)

g e l G. Kieselguhr G impregnated with f ormamide

a ) Benzene : Chloroform (30:120) s a t u r a t e d w i t h formamide.

.

'A

c. -

b ) Carbon t e t r a c h l o r i d e s a t u r a t e d w i t h f ormamide

.


542

6.64 Gas Liquid Chromatography Various gas l i q u i d chromatographic methods have been developed f o r t h e i d e n t i f i c a t i o n and determin a t i o n of salicylamide i n pharmaceuticals and biol o g i c a l f l u i d s . It i s of i n t e r e s t t o mention t h a t salicylamide could be gas chromatographed without d e r i v a t i z a t i o n . Some of t h e d a t a b t a i n e d from t h e l i t e r a t u r e a r e summarized i n Table 3. The a n a l y s i s of s e v e r a l drugs including s a l i c y l a i d e using semiatoumated gas l i q u i d chromatography has been r e p o r t e d ( 5 7 ) . Nieminen (58) described a gas chromatographic method f o r simultaneous s e p a r a t i o n and q u a n t i t a t i o n of some a n t i p y r e t i c s including salicylamide containing phenobarbital, c a f f e i n e and codeine phosphate. Codeine phosphate w a s f i r s t separated from t h e samples as t h e base. Although t h e separat i o n w a s e x c e l l e n t f o r a l l components , y e t a c e t y l s a l i c y l i c a c i d i n t e r f e r e s with t h e s e p a r a t i o n of sa1icylami.de.

6.65 High-pressure Liquid Chromatography High-pressure l i q u i d chromatography has been u t i l i z e d f o r t h e d e t e m i n a t i o n of salicylamide i n blood, serum and o t h e r b i o l o g i c a l f l u i d s as w e l l as pharmaceutical formulations. Some of r e c e n t l y reported methods are summarized i n Table 4.

6.7 Radiochemical Analysis The radiochemical a n a l y s i s of salicylamide i n plasma using t h e r i n g - l a b e l l e d t r i t i a t e d compound was r e p o r t e d by S t e l l a e t al ( 5 9 ) . The t r i t i a t e d salicylamide w a s synthesized by r e a c t i n g c o l d salicylamide with t r i t u i m oxide i n t h e presence o f h e p t a f l u o r o b u t y r i c a c i d . This procedure i s e f f e c t i v e f o r t h e a n a l y s i s of salicylamide and i t s m e t a b o l i t e s ( t h e s u l p h a t e and glucuronide) i n t h e presence of s i m i l a r phenolic compounds.

6.8 Flame m i s s i o n Spectrometry Dialonzo and S i g g i a r e p o r t e d a method f o r determining several compounds including salicylamide based on t h e use of Hofmann r e a c t i o n f o r t h e s y n t h e s i s of m i n e s

Table 3: Gas Chromatography o f Salicylamide Column

Carrier Gas

Detector

Remarks

Reference

27% OV-225 +1% OV-17 Gas Chrom-Q

Helium

Flame ionization

1% Silicon Gum SE-30 on Chromosorb-G

Helium

Thermal conducting

Applied for simultaneous determination of dextromethorphan, chlorpheniramine norephedrine and salicylamide.

(61)

-

Flame ionization

Simultaneous analysis of salicylamide, phenylpropanolamine HC1, caffeine, chlorpheniramine maleate phenylephrine HC1 and pyrilamine maleate, the sample should be treated with b-(dimethylamino)-pyridine in pyridine-acetic anhydride 1:l.

(62)

Flame ionization.

Detection limits 5 mg/l.

(63)

8% OV-101

2% SP-2501 DA

Helium

Quantitation was carried out by combined GC/MS. Sensitivity ranges from 1 to 25 u g / d (whole blood plasma and saliva) and 5-100 ug/50 ul urine.

(60)

(continued)

Table 3 (continued)

Column

2 P

C a r r i e r Gas

Mixture of Helium g:1 2% methyl/phenyl s i l i c o n (SP 2110) and 1% SP 2510 on 100-120 mesh a c i d washed dimet h y l d i ch l o r o s i l a n e t r e a t e d diat o m i t e support.

Detector Flane ioniza-

t ion.

Remarks. Detection l i m i t s 5 mg/l.

Reference (63)

Table Column

Mobile Phase

4:

HPLC for Salicylamide

Detect i o n

Reference

Remarks

RPz

Methanol : Water (60: 40)

w

L i m i t of d e t e c t a b i l i t y i s 0 . 2 Vg/ml. Caffeine and paracetamol do not i n t e r f e r e i n t h e determination of salicylamide.

(64)

RP-5c18

Methano1:Buffer pH 3 (10: 50)

w

Method i s s u i t a b l e f o r c l i n i c a l determinan a t i o n and t o x i c o l o g i c a l use.

(65

0 . 0 1 M potassium a c i d phosphate i n w a t e r with 19% v/v methanol, pH w a s a d j u s t e d t o 2 . 3 with 80% aq. phosphonic a c i d s o l u t ion.

uv

Simultaneous q u a n t i t a t i o n of acetaminophen, a s p i r i n , c a f f e i n e , codeine phosphate, phenacetin and salicylamide i s a f f e c t e d .

(66

Solvent system with pH 2 . 3 a f f e c t s b e t t e r separation.

Same as above except pH w a s a d j u s t e d t o 4.85

Reverse-HPLC technique w a s used.

(66)

1% a c e t i c a c i d i n 25% v/v methanol / w a t e r solution.

uv

LOW concentration of unconjugated s a l i c y l a mide i n human serum and s a l i v a were assayed.

(67)

3 mM t e t r a b u t y l ammonium hydroxide i n a mixture of 8 p a r t s methanol and 92 p a r t s v/v a c e t i c a c i d 7%.

w

Reverse phase ion-pair chromatography i s used.

(67)

Salicylamide and i t s conjugated m e t a b o l i t e s were assayed.

(continued)

Table

4 (continued)

Column

p-Bondapak

'18

Mobile Phase

Detect ion

1% Acetic a c i d i n

W

15% v/v methanol/Water/

Remarks

Reference

G e n t i s a i d e and i t s conjugates i s assayed.

(67)

Can d e t e c t salicylamide as l o w as 5 m g / d i n plasma.

(68)

Applied f o r r o u t i n e s e p a r a t i o n of common components o f a n a l g e s i c tablets.

(69)

solution, f-

-Bondapak oDS-cl8

0.15 M Sodium d i b a s i c phosphate pH 8 : Methanol ( 7 0 : 30).

LFS P e l l i cuar anionexchange resin.

One molar T r i s (pH 9 ) a t 60'

Flourometric detector

w

SALICYLAMIDE

541

w i t h one l e s s of carbon atom t h a n t h e s t a r t i n g amide. B a r i u m n i t r a t e i s used as reagent. B a r i u m carbonate formed i n t h e r e a c t i o n i s d i s s o l v e d i n n i t r i c a c i d and i s determined by flame atomic emission spectroscopy; t h e p r e c i s i o n w a s t 1 . 5 t o 6.5% i n t h e range 1 t o 4 mM (4 t o 9 r e p l i c a t e r e s u l t s f o r each compound). The mean r e c o v e r i e s were g e n e r a l l y between 2 95 and 105% (70).

6.9 Thermornicroscopic Method Analysis o f t e r n a r y mixtures of c e r t a i n drugs including salicylamide were s t u d i e d by p l o t t i n g t h e r e f r a c t i v e index isotherms of t h e fused m a s s and t h e isotherms of t h e primary c r y s t a l s on t r i a n g u l a r conc e n t r a t i o n diagrams. The i n f l u e n c e of each f a c t o r upon t h e q u a n t i t a t i v e determination could be found. I n each case t h e r e w a s an a r e a u n s u i t a b l e f o r measurement where t h e isotherms were p r a c t i c a l l y p a r a l l e l ; when t h i s w a s not t h e c a s e , q u a n t i t a t i v e determination w a s p o s s i b l e . Precaution should be taken t o prevent l o s s of highly v o l a t i l e components ( 7 1 ) .

6.10 The ring-oven Technique. Salicylamide w a s determined a t concentrations of 10400 ng w i t h a m a x i m u m e r r o r o f 7.656 by t h e ring-oven technique. Drugs such as paracetamol and c a f f e i n e d i d not i n t e r f e r e i n t h e determination ( 7 2 ) .

Acknowledgement The a u t h o r s uould l i k e t o thank Mr. Altar H . Naqvi f o r t y p i n g t h e manuscript.

SALIM A . BABHAIR ET AL.

548

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J. Chem. -


SILVER SULFADIAZINE Auke Bult and Cees M. Plug

1. History 2. Description 2.1 Name, Formula, Molecular Weight 2.2 Appearance, Colour, Odour 3 . Synthesis 4. Physical Properties 4.1 Infrared Spectrum 4.2 Nuclear Magnetic Resonance (NMR) Spectrum 4.3 Ultraviolet Spectrum 4.4 Mass Spectrum 4.5 Melting Range 4.6 Differential Scanning Calorimetry 4.7 Crystal Structure and X-Ray Powder Diffraction 4.8 Stability Constant 4.9 Conductivity 4.10 Solubility 5 . Methods of Analysis 5.1 Elemental Analysis 5.2 Identification Tests 5.3 Purity Tests 5.4 Volumetric Methods 5.5 Spectroscopic Methods 5.6 Thin Layer Chromatography 5.7 High Performance Liquid chromatography 6 . Stability 7. Pharmacology 8. Identification and Determination in Body Fluids and Pharmaceuticals 8.1 Determination in Body Fluids 8.2 Identification and Determination in Pharmaceuticals References


553

554 554 554 554 554 555 555 557 559 560 560 560 562 563 564 565 566 566 566 567 561 568 568 568 569 569 569 569 570 570

Copyright 0 1984 by the American Pharmaceutical Association ISBN 0-12-2608 13-5

AUKE BULT AND CEES M . PLUG

554

1. HISTORY Silver sulfadiazine was first described in 1943 by Wruble (1) and was found to be mildly antiseptic. Fox (2)rediscovered the compound in 1968 in his search for a suitable substance for topical treatment of burns. His philosophy was to combine the oligodynamic effect of the silver ion with the antibacterial activity of sulfadiazine. The compound has been in clinical use in the USA since 1973 and appears to be active against a wide range of micro-organisms (3). The usual application form is a 1% w/w oil-water cream.

2.

DESCRIPTION 2.1

Name, Formula, Molecular Weight

Nomenclature -

Generic name

Silver sulfadiazine

The following nomenclature is used in Chemical Abstracts: monosilver(1) salt of 4-amino-N-2-pyrimidinylbenzenesulfonamide 122199-08-21 Synonyms sulfanilamide

-

Silver compound (salt) of N1-2-pyrimidinyl-

Structure

Molecular Weight: 357.14

2.2

Appearance, Colour, Odour

White or creamy-white micro-crystalline powder which is odourless or almost odourless. It is stable in air but slowly becomes yellow on exposure to light.

3. SYNTHESIS The synthesis of silver sulfadiazine is based on the following reaction scheme (1,4): AgN03 + NaSD

-

AgSD+ + NaN03

Mixing of equimolar amounts of silver nitrate and sodium sulfadiazine (NaSD), both dissolved in water, results in the almost

SILVER SULFADIAZINE

555

q u a n t i t a t i v e p r e c i p i t a t i o n of s i l v e r s u l f a d i a z i n e . An a l t e r n a t i v e procedure makes u s e of ammoniacal s o l u t i o n s of s i l v e r n i t r a t e and s u l f a d i a z i n e ( 5 ) . R e c r y s t a l l i z a t i o n o f t h e compound can t a k e p l a c e from 25% ammonia s o l u t i o n ( 4 ) . D e r i v a t i v e s of s i l v e r s u l f a d i a z i n e can be prepared according to: AgSD + nY-

AgSD.Yn

Examples of such d e r i v a t i v e s a r e : Y = m o r f o l i n e , n = 1 (6) and Y = imidazole, n = 2 ( 7 ) .

4.

PHYSICAL PROPERTIES 4.1

Infrared Spectrum

The i n f r a r e d spectrum g i v e n i n F i g u r e 1 w a s determined f o r a KBr p e l l e t p r e p a r a t i o n of s i l v e r s u l f a d i a z i n e w i t h t h e u s e o f a Beckman I R 1 0 s p e c t r o m e t e r . The assignments f o r i m p o r t a n t abs o r p t i o n bands are p r e s e n t e d - as f a r as p o s s i b l e - i n Table I. The assignments a r e based on v a r i o u s l i t e r a t u r e r e f e r e n c e s (4, 8,s). I n p r a c t i c e i t i s found t h a t d i f f e r e n t b a t c h e s of s i l v e r s u l f a d i a z i n e do produce s l i g h t l y d i f f e r e n t I R s p e c t r a , w h i l e t h e batches assay closely t o the t h e o r e t i c a l values (4,5,9). The d i f f e r e n c e s o c c u r i n t h e r e g i o n s above 3000 c m - l , between 1630 and 1660 c m - l , n e a r 1600 cm-l and 1360 c m - l , and between 1200 and 1300 cm-I and 650 and 750 cm-I ( 9 ) . The SO2 s t r e t c h i n g v i b r a t i o n s a r e known t o show a m u l t i p l e s t r u c t u r e w i t h subbands on t h e s i d e s of t h e main peaks u s u a l l y ( 1 0 ) . The o r i g i n of t h e s e sub-bands i s n o t clear. Wysor and S c o v i l l (5) d i s t i n g u i s h e d two forms of s i l v e r s u l f a d i a z i n e based u p o n d i f f e r e n c e s i n t h e I R s p e c t r a . Both forms of t h e compound were a c t i v e

Table 1. Infrared Assignments for Silver Sulfadiazine ~~

Wavenumber (cm-I)

~

Assignment

3390 3340 1655

6 (NH2)

1595 1500

phenyl s k e l e t a l v i b r a t ' i o n s phenyl s k e l e t a l v i b r a t i o n s

1560

pyrimidine s k e l e t a l vibration

1230 1125

V a s y m (SO2) vsym (332)

1070

aromatic v i b r a t i o n

m

0 0 0 0

U

0 0 u)

I

0 0 03-

Z L0,

0 5

0

E

n

8 2

hl

;

>

Pa2
9 9 % ) of silver remains located in the wound environment (31). The sulfadiazine is absorbed to about 10% into the general circulation ( 3 2 ) . Depending on the burnt surface, blood levels up to about 3 mg/100 ml and renal excretion to 2 g / 2 4 hours have been reported ( 3 2 ) . Details about the metabolism of sulfadiazine have been reported before (12).

8. IDENTIFICATION AND DETERMINATION IN BODY FLUIDS AND PHARMA-

CEUT I CALS 8.1

Determination in Body Fluids

A U K E BULT AND CEES M. PLUG

570

From the topically applied silver sulfadiazine only sulfadiazine is absorbed to some extent (about l o x ) . The analysis of sulfadiazine in biological fluids can be based on the methods described by Stober and De Witte ( 1 2 ) . Delaveau and FriedrichNoue (33) employed the Bratton-Marshall reaction for the measurement of sulfadiazine levels in plasma, serum and urine after topical application of silver sulfadiazine cream.

8.2 Identification and Determination in Pharmaceuticals The common application form of silver sulfadiazine is a I % w/w oil-water cream. Prior to the identification and determination of silver sulfadiazine the cream basis can be removedby dissolution into a suitable organic solvent (or mixture of solvents). The following extraction media are described: a) a mixture of ethanol 94% and chloroform ( I : ] ) and b) the successive extraction with n-butanol and diethyl ether ( 9 , 2 8 ) . The identification and/or determination of the isolated silver sulfadiazine can be based on the methods of analysis described in sections 5.2 - 5.7. Direct treatment of the whole cream with 2 M hydrochloric acid by refluxing for 30 minutes followed by filtration results in isolation of sulfadiazine. The sulfonamide present in the filtrate is determined by biamperometric titration ( 2 4 ) . Treatment of the cream with concentrated ammonia followed by acidification with 1% acetic acid is suitable as a sample preparation method for the high performance liquid chromatographic determination ( 9 ) . The clear supernatant is used for the analysis. ACKNOWLEDGEMENT

The authors are indebted to Dr J.M. Gillingham, Dr A. van der Hoek, Dr M. Horlington and Prof. A.W.M. Indemans for contributing relevant analytical information, to Mr C . Erkelens, Drs H. Jousma, Mr B.E. Pareraand DrJ. vanThuij1 for performing the physical measurements, to Mr A.G. Huigen for drawing the figures and to Miss S. Oosterwijk for preparing the manuscript. REFERENCES (the literature is cited through 1983) 1 . M. Wruble, J. Am. Pharm. Ass. 32, 80 (1943). 2. Ch.L. Fox, Arch. Surg. 96, 1 8 4 7 1 9 6 8 ) .

3. J.J.M. van Saene, A. B u z and C.F. Lerk, Pharm. Weekblad, Sci. Ed. 5, 61 (1983). 4. A. Bult azd H.B. Klasen, J. Pharm. Sci. 67, 284 (1978). 5. M.S. Wysor and J.P. Scovill, Drugs ExptlyClin. Res. VIII, 155 (1982). 6 . B.J. Sandmann, R.U. Nesbitt and R . A . Sandmann, J. Pharm.

SILVER SULFADIAZINE

S c i . 63, 948 (1974). K l a s e n , Arch. Pharm. (Weinheim) 313, 1016 (1980). K.K. Narang and J . K . Gupta, C u r r . S c i . 45, 744 (1976). M. H o r l i n g t o n , Smith & Nephew R e s e a r c h E d . , Harlow, p e r s o n a l communication. L. J. Bellamy, Advances in Infrared Group Frequencies, Chapman and H a l l , London (1968), p . 219. P.P. d2 W i t , H. van Doorne and A. B u l t , Pharm. Weekblad, S c i . Ed., i n p r e s s . H. S t o b e r and W. DeWitte, Analytical Profiles of D m g Substances, Vol. 1 1 , Ed. K. F l o r e y , Academic P r e s s , New York ( 1 9 8 2 ) , p . 523. C. Chang, H.G. F l o s s and G.E. Peck, J . Med. Chem. 18, 505 (1975). A.W.M. Indemans, L a b o r a t o r y of t h e Dutch P h a r m a c i s t s , The Hague, p e r s o n a l communication. J . van T h u i j l , S u b f a c u l t y o f C h e m i s t r y , L e i d e n , p e r s o n a l communication. P. Gundu Rao, P. Ranganatham, K.K. S r i n i v a s a n and N.R. S h a r a d a , C u r r e n t S c i . I n d i a 48, 99 (1979). H. Jousma, S u b f a c u l t y of Pharmacy, L e i d e n , p e r s o n a l communication. D.A. Cook and M.F. T u r n e r , J. Chem. SOC., P e r k i n T r a n s 11, 1021 (1975). N . C . B a e n z i g e r and A.W. S t r u s s , I n o r g . Chem. 15, 1807 (1976). G.J. Boelema, A . B u l t , H . J . M e t t i n g , B.L. Bajema and D . A . Doornbos, Pharm. Weekblad, S c i . Ed. 4 , 38 (1982). W. J . Geary, Coord. Chem. Rev. 7, 81 T1971). R.U. N e s b i t t and B . J . Sandmanny J. Pharm. S c i . 66, 519 (1977). Ch.L. Fox, S. Modak, J . W . S t a n f o r d and P.L. Fox, Scand. J . P l a s t . R e c o n s t r . Surg. 13, 189 (1979). A. v a n den Hoek, ACF C h z i e f a r m a NV, Maarssen, p e r s o n a l communi c a t i o n . European Pharmacopoeia, 2nd E d i t i o n , P a r t I, C o u n c i l of Europe, V . 3 . 1 . 1 . (1980). European Pharmacopoeia, Ed. I , P a r t 111, C o u n c i l of Europe, p. 347 (1975). European Pharmacopoeia, 2nd E d i t i o n , P a r t I , C o u n c i l of Europe, V.3.5.1. (1980). Anonymous, Pharm. Weekblad, 109, 884 (1974). A.C. B r a t t o n andF.K. M a r s h a l r J . B i o l . Chem. 128, 537 (1939). A . B u l t , Pharmacy I n t e r n a t i o n a l 2, 400 (1982). H.N. H a r r i s o n , Arch. Surg. (Chicago) 114, 281 (1979). A.R. Grossman, Am. Fam. Phys. 1 , 69 (1970). P. Delaveau and Ph. Friedrich-Goue, T h g r a p i e 32, 563 (1977).

7. A. B u C and H.B. 8. 9. 10. 11.

12.

13. 14. 15. 16.

17.

18. 19. 20. 21. 22. 23. 24. 25. 26.

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

57 I


SULINDAC Fotios M. Plakogiannis Arnold and Marie Schwarts College of Pharmucy and Health Sciences Brooklyn, New York

James A. McCauley Merck Sharp G Dokine Research Laboratories Rnhtoay, New Jersey 1, Foreword, History, Therapeutic Category 2. Description 2.1 Name, Formula, Molecular Weight 2.2 Appearance, Color, Odor 3 . Synthesis 4. Physical Properties 4.1 Infrared Spectrum 4.2 Mass Spectrum 4.3 Nuclear Magnetic Resonance Spectra 4.4 Ultraviolet Absorption Spectrum 4.5 Solubility 4.6 Dissociation Constant 4.7 Partition Behavior 4.8 Thermal Behavior 4.9 Polarographic Behavior 4.10 Polymorphism 5. Methods of Analysis 5.1 Identification 5.2 Elemental Analysis 5.3 Chromatographic Analysis 5.4 Titrimetry 6. Drug Metabolic Products, Pharmacokinetics, Bioavailability 7. Identification and Determination in Body Fluids and Tissues References


573

574 574 574 574 575 575 575 577 577 582 582 584 584 584 586 586 59 1 59 1 591 59 1 592 593 594 595


FOTIOS M . PLAKOGIANNIS AND JAMES A. McCAULEY

514

1. Foreword, History, Therapeutic Category. Sulindac is a new nonsteroid anti-inflammatory drug with analgesic and antipyretic activity1,2. Sulindac and indomethacin had similar pharmacologic profiles in laboratory animals but sulind c showed less gastrointestinal bleeding and ulcers’, 3. 2. Description 2.1 Name, Formula, Molecular Weight 1H-Indene-3-acetic acid, 5-fluor0-2-methyl-lI4(methyl-sulfinyl) phenyllmethylene -, (2)-;CIS-5fluoro-2-methyl-1- (p-methyl-sulfiny1)benzyl-idenel indene-3-acetic acid. Empilrical formula C20H17F03S Molecular weight 356.41 The CAS Registry number is CAS-38194-50-2 0

2.2 Appearance, Color, Odor Sulindac is an odorless, yellow, crystalline powder.

SULINDAC

S75

3. Synthesis A

synthesis4r5 of sulindac is summarized as. follows:

SULINDAC

4. Physical Properties 4.1 Infrared Spectrum The infrared spectrum of sulindac, run as a nujol mull, is reproduced in Figure 1 and is consistent with the structure. Band assignments which may be made with confidence are tabulated below: TABLE I Band Assiqnment Infrared Spectrum of Sciliiidac v (crn-l)

Assignment

2 500-2 650

-CO H dimer v c=o

1700 1600 1580 1270 1155 1 0 0 0-10 20

Aromatic ring n d e s -CO H dimer v C-F v s-0

In addition to these, sharp bands between 8 0 0

0 CD

0 d

0

cu

0 0

cu 0 0

ro 0

0 aD 0

0 w

0 0 d

-

0 0 Ic

0 0 0

cu

0

0 rT)

cu

M

0 0 0 0 0 v)

M

0 0 0 0-J-

rl

7

rl

E rl

0 7

z

-n

c

-4

c

u a a 7

rl

-4

0

cn w

5 k

a,

u

4J

a c

v)

0

-4 4J

0

e i k

a,

a

a k

H

c

Icl

4

a,

k

b,

F

-4

517

SULINDAC

aqd 900 c m ' l are expected from the ArH out-of-plane modes in both rings. The nujol mulling agent, of course, accounts for the very strong band from 2 8 2 5 to 2 9 7 5 cm , as well as for all or part of the bands at 1 4 6 0 and 1 3 7 5 cm-l.f6) 4 . 2 Mass Spectrum

The electron impact ionization spectrum is given in Figure 2. A LKB model 9 0 0 0 mass spectrometer was used with ionization potential of 70eV and accelerating voltage of 3.5kv. The mass spectrum shows a molecular ion at 3 5 6 (m/e) with two relatively intense peaks at 3 4 1 and 2 3 3 (m/e). Loss of a methyl group from the parent molecule will account for the 3 4 1 m/e fragment. Loss of CH COOH, CH SO n hydrogen will account for the other fragment. 7 7 7 4.3

Nuclear Magnetic Resonance Spectra

4.3.1

Proton Spectrum

The proton NMR spectrum is found in Figure 3 and assignments to the characteristic protons are recorded below. The complex pattern located at 6.4 to 7 . 2 ppm is consistent with aromatic 1,2,4 substitution with fluorine splitting. The singlet with weak satelites ( ' ~ 7 . 7 4 ppm) is consistent with an aromatic AA'BB' pattern which is nearly collapsed into a singlet. A solvent (DMSO-ds) resonance appears at approximately 2.5 ppm ( 6 )

.

578

u KJ a

. (v

519

f

FOTIOS M. PLAKOGIANNIS A N D JAMES A . MCCAULEY

580

TABLE I1 H '

NMR Sulindac Assignments

Chemical Shift ppm ( 6 1

Pattern

2.17

singlet

2.83

singlet

3.60

singlet

~6.4 to 7.2

complex

7.36

singlet

7.74

singlet with weak satelites

Proton Assignment

4.3.2 Carbon 13 Spectrum The I3C magnetic resonance spectrum is given in Figure 4 and was obtained using a Varian CFT-20 (FT mode) spectrometer with CDC13/CK30D (9:l) as The specthe solvent at a concentration of 0.5M. trum is consistent with t e structure and the assignments are as follows:?8)

I

I

I I I I I I I

I

I I I I I I I I I

1

I I I I I I I I I

1

I I I

r

c2;6 c33 CH,DH (Solvent 1

CO,H

(Solvent 1

Chemical Shift Si3C ( ppm) Figure 4.

1 3 C NMR of S u l i n d a c

I I 1 . 1 7

FOTIOS M. PLAKOGlANNlS AND JAMES A. McCAULEY

582

TABLE I11 Carbon-13 Chemical Shifts for Sulindac Shift, 613C PPm) 141.9 138.2 132.4 106.2 163.4 110.7 123.7 147.1 129.7 128.0 140.1 130.5 124.1 144.2 10.4 31.7 172.9 43.2

Assignment C C C C C C C C C

c

(Benzal) C1 ' CZ', c6' C3', C5' c4 1 2 - c ~ ~ 3-CH2 Carboxyl SCH3

4.4 Ultraviolet Absorption Spectrum The ultraviolet absorption spectrum of suindac in methanolic 0.1N hydrochloric acid is found in Figure 5 and is characterized by maxima at 327, 284, 258 and 228 nm with El%lcm values of approximately 370, 420, 405 and 540 respectively. 4.5 Solubility Sulindac is sparingly soluble in methanol and 95% ethyl alcohol, slightly soluble in ethyl acetate, and practically insoluble in water. The water solubility increases with increasing pH as indicated in the following table. (9)

5x3

SULINDAC

0.9

1

I

1

I

250

300

350

400

0.8

0.7

0.6 0)

0

C 0

n L

0.5

a

n

a

0.4 0.3

0.2

0.1

Wavelength ( n m )

Figure 5.

U l t r a v i o l e t A b s o r p t i o n S p e c t r u m of S u l i n d a c i n M e t h a n o l i c 0.1N H C 1 1 . 6 2 5 mg/100 m l

FOTIOS M. PLAKOGIANNIS AND JAMES A . McCAULEY

584

TABLE IV Solubilitv of Sulindac Solvent

Temperature ("C)

0.1N IlCl Water p H 7.0 (Phosphate) pH 7 . 4 (Phosphate) p H 3.9 (Citrate) pH 4.8 (Citrate) p H 5.9 (Citrate) pH 6.4 (Citrate) Acetone Chloroform Ethanol Ethyl Acetate Hexane Isopropanol Methanol

Solubility (mg/ml)

25 25 25 25

0.003 0.003 2.7 3.4

37 37 37 37

0.01 0.05 0.35 1.0 18 25

25 25 25 25 25 25 25

15

4 1

7 30

4.6 Dissociation Constant The pKa of sulindac is 4.7

at 25°C.

4.7 Partition Behavior The distribution coefficient for sulindac between phosphate buffer pH 7 . 0 and octanol at 25°C is 0.28. Sulindac can be extracted quantitatively from aqueous acidic media into the following organic solvents: methylene chloride, chloroform, ethyl acetate and 3 volume % isoamyl alcohol in heptane. Sulindac aan be back extracted into dilute alkali from these solvents. 4.8 Thermal Behavior Sulindac melts with decomposition and therefore the observed temperature is dependent upon the conditions of the observation. Under USP Class la con it'ons, the temperature ranges from 182185°C. The DTA curve (Fig. 6) f o r s u l i n d a c at 20°C/minis characterized b y a single endotherm with

c

rn


586

peak temperature at approximately a186 C. 4.9 Polarographic Behavior

In 0.1M tetraethylammonium perchlorate in acetonitrile, half-wave potentials of -1.38 and -1.52 were observed for sulindac. The reductions involve the C=C bonds and not the sulfur functionalities by analogy to the work of Hawson et a1 (I1 and 1 2 ) * 4.10 Polymorphism

Sulindac is a polymorphic substance and can exist in two nonsolvated enantiotropic crystal forms designated Forms I (191°C) and I1 (186°C). Form I1 is the more stable polymorph at room temperature and temperatures below the transition point which is estimated to be 157°C from heats of fusion and melting points of the polymorphs. The transition temperature has a l s o been estimated to be 165°C by extrapolations of solubility measurements of the two forms over the temperature range of approximately 20 to 80°C. The average of the two determinations yields a value fo 161 f 7°C for the transition point. Form I is the more stable form above the transition point up to its melting point of 191°C which is about five degrees ab0v.e the metastable melting point of Form 11. Although Form I is metastable with respect to Form I1 at room temperature, the rate of conversion of Form I to Form I1 is dependent upon the conditions. With solid Form I alone, the rate is so slow that it cannot be conveniently measured. In contact with water or aqueous buffered systems, the conversion requires weeks or months. In contact with ethanol Form I converts to Form I1 within 16 hours or less. Form 11, the more stable form at room temperature, is the preferred form. X-ray powder diffraction (Figs. 7 and 8), infrared absorption (Figs. 1 and 9) and differential thermal analysis (Figs. 6 and 10) are capable of distinguishing the two forms

.

0

c

U (d

c

a

k

rd

b

w w a,

0

3

a

PI

I

x

c

3-

h

IZ 0

4J

-4

E

0

d

0

4J

-4

fd k

.. Ill

3

a

0

PI

h

x

2I

. 03

7

k

a, b -4 I%

Iv

0 0

Iv

0

Iv N

03 0

A

0

P

A

0 0

&

0 0

0 Endotherm

Exot her m,

Wavelength Microns

2.5 100

3

4

5

6

7 8 9 10 12 14 18 22 3.550

80 60 40 20 0 4000 3500 3000 2500 2000 1700 1400 I100

800

500

200

Frequency (cm-'1 Figure 1 0 .

I n f r a r e d A b s o r p t i o n Spectrum of S u l i n d a c Form I in Nujol Mull

59 1

SULINDAC

5 . 0 Methods of Analysis

5 . 1 Identification

Comparison of infrared and absorption spectra with those of reference standard are me hods of identifications specified by the USP-NF413)

.

5.2 Elemental Analysis ( 1 0 ) The following elemental composition was observed for sulindac when approximatley 2 mg of sample was analyzed with a Perkin-Elmer, Model 2 4 0 CHN Analyzer. Element C H

Theory % 67.40 4.81

Funds % 67.63 4.72

5 . 3 Chromatoqraphic Analysis 5.3.1

dac.

Thin Layer Chromatography Several TLC systems have been used for sulinTable V lists the solvents and the sulindac

RF’s.

TABLE V TLC Solvents and Rf for Sulindac Solvent 1. Chloroform/Ethyl Acetate/Acetic Acid 1 6 : 5 : 1 2 . Ethyl Acetate/Acetic Acid 9 7 : 3 and 9 4 : 6 3. Chloroform/Acetic Acid 9 5 : 5 4 . Ethyl Acetate/Chloroform 1:1 5 . Carbon Tetrachloride/Acetic Acid 9 : l 6 . Chloroform/Dioxane/Acetic Acid 2 5 : l : l 7 . Benzene/Methanol/Ammonia 50:20:1

Rf -0.4 -0.4 ~ 0 . 3 QO. 1

s0.2 -0.7 s0.2

Silica gel GF absorbents are used with all the solvent systems and detection is with short wave UV light ( ~ 2 5 4nm) or with exposure to iodine vapor. Solvent system #1 has been used for quantitative determination of impurities in sulindac. Quantum Q-7G plates are used and the plate is

592

FOTIOS M. PLAKOGIANNIS A N D JAMES A. McCAULEY

.

s c a n n e d a t 280 nm w i t h a s u i t a b l e d e n s i t o m e t e r (13)

Approximate R f ' s f o r s u l i n d a c r e l a t e d compounds f o r s o l v e n t s y s t e m s #1 and # 2 a r e l i s t e d below. Compound Sulfone analoq S u l f i d e analog E t h y l ester s u l i n d a c T r a n s isomer

#1

Rf -

#2 -

%0.6

%O. 7

-0.7

-0.8 %0.5 %O. 3

%Lo. 5 'LO. 3

5 . 3 . 2 G a s ChromatouraDhv S u l i n d a c h a s been c h r o m a t o g r a p h e d a s the m e t h y l ester ( d i a z o m e t h a n e ) o n a 6 ' x 1 0 . 2 5 " 1% SE-30 on S u p e l c o n 80/100 column u n d e r i s o t h e r m a l a t 2 6 2 ° C w i t h n i t r o g e n as a c a r r i e r gas a t + l o 0 ml/min. Det e c t i o n w a s w i t h e l e c t r o n c a p t u r e and s a m p l e s i z e s r a n g e s from 450 t o % l o 0 ng. The r e t e n t i o n t i m e of s u l i n d a c w a s a p p r o x i m a t e l y 7 m i n u t e s . The s u l f i d e and s u l f o n e a n a l o g s h a v e r e t e n t i o n t i m e s o f 1 . 5 and 5 m i < y i f 3 s r e s p e c t i v e l y u n d e r t h e same c o n d i tion 5.3.3 L i q u i d Chromatography S e v e r a l HPLC s y s t e m s h a v e b e e n u s e d f o r s u l i n d a c i n c l u d i n g o n e employing c h l o r o f o r m / e t h y l acet a t e / a c e t i c a c i d ( 8 0 0 : 2 0 0 : 2 ) as t h e mobile phase w i t h a LI P o r a s e l , 30 c m , 1 0 p p a r t i c l e s i z e column; UV d e t e c t i o n a t 280 nm and a f l o w r a t e of 2.0 m l / min (500-3000 p s i ) . Under t h e s e c o n d i t i o n s s u l i n d a c c h r o m a t o g r a p h e d w i t h a r e t e n t i o n t i m e o f approxi m a t e l y 6 . 3 m i n u t e s . The s u l f i d e and s u l f o n e anal o g s chromatographed w i t t i m e s of %1.8 2 . 5 minutes r e s p e c t i v e l y O t h e r HPLC s y s t e m s u s e Zorbax ( s i l i c a g e l ) column 0 . 5 m x 8 mm o r 0.25 m x 2 . 1 mm w i t h m o b i l e phases of chloroform/ethyl acetate/acetic a c i d 38 :5 :1( 8 ) o r m e t h y l e n e chloride/hexane/isobutanol/ a c e t i c a c i d / w a t e r 50:50:10:4:.046. 5.4 T i t r i m e t r y S u l i n d a c c a n be q u a n t i t a t i v e l y a s s a y e d by pot e n t i o m e t r i c t i t r a t i o n . Methanol i s u s e d a s a

SULINDAC

593

s o l v e n t f o r s u l i n d a c and t h e s o l u t i o n i s t i t r a t e d w i t h s t a n d a r d i z e d sodium h y d r o x i d e ( 0 . 1 N ) t o a p o t e n t i o m e t r i c end p o i n t using a qlass-calomel electrode system(l5). 6.

Drug M e t a b o l i c P r o d u c t s , P h a r m a c o k i n e t i c s , B i o availability

The major u r i n a r y m e t a b o l i t e s are s u l i n d a c and i t s g l u c u r o n i d e , s u l f o n e and i t s g l u c u r o n i d e and i n t r a c e amounts t h e i n s o l u b l e s u l f i d e m e t a b o l i t e and i t s g l u c u r o n i d e . The major b i o t r a n s f o r m a t i o n i n v o l v e s i r r e v e r s i b l e o x i d a t i o n of t h e s u l f o x i d e g r o u p o f s u l i n d a c t o s u l f o n e and a r e v e r s i b l e r e d u c tion t o the sulfide. Studies i n healthy subjects revealed t h a t a f t e r oral administration of sulindac a t l e a s t 88% i s a b s o r b e d . A f t e r a s i n g l e 2 0 0 mg o r a l dose t h e peak plasma c o n c e n t r a t i o n s o f s u l i n d a c and i t s s u l f o n e and s u l f i d e m e t a b o l i t e s were 4 , 2 , and 3 mg/ml r e s p e c t i v e l y , and w e r e a t t a i n e d a f t e r 1 hour f o r t h e p a r e n t d r u g and a f t e r 2 h o u r s f o r each of the metabolites. The s u l f o n e and i t s c o n j u g a t e s are t h e major p r o d u c t s e x c r e t e d i n u r i n e , and a c c o u n t f o r 27.6 + 2 . 8 % o f t h e a d m i n i s t e r e d dose. S u l i n d a c and i t s g i u c u r o n i d e accounted f o r 2 0 . 2 + 0 , 2 % o f t h e dose e x c r e Mean reFal c l e a r a n c e o f s u l i n d a c ted i n the urine. and i t s s u l f o n e m e t a b o l i t e w a s 4 5 . 1 + 1 6 . 2 and 33.2 + 1 2 . 2 ml/min r e s p e c t i v e l y ( l 6 ) . The e f f e c t i v e h a l f - i i f e f o r accumulation i s about 7 hours f o r s u l i n d a c ( l 7 ) . The l o n g h a l f - l i f e i s d u e t o e x t e n s i v e e n t e r o h e p a t i c r e c i r c u l a t i o n (18). There a r e no r e p o r t e d b i o a v a i l a b i l i t y p r o b l e m s w i t h sulindac tablets. No s i g n i f i c a n t d i f f e r e n c e i n plasma c o n c e n t r a t i o n s o r u r i n a r y e x c r e t i o n o f s u l i n d a c w a s found f o l l o w i n g a d m i n i s t r a t i o n of t a b l e t s or s o l u t i o n ( l 7 ) .


594

7. Identification and determination in body fluids and tissues. The following methods have been used for the assay of sulindac in body fluids and tissues. Methods Radioactivity Isotope dilution-radioimmunoassay Ultraviolet Thin-layer chromatography Gas-liquid chromatography Mass spectrometry HPLC

References

(17) (191 (171 (16) (161 (16) (20)

595

SULINDAC

References

1. T . Y . Shen, B . E . W i t z e l , H. J o n e s , B.O. L i n n , J. McPherson, R. G r e e n w a l d , M . F o r d i c e and A. Jacobs: Fed. Proc. 31, 5 7 7 ( 1 9 7 2 ) . 2.

C.G. V a n A r m a n , E.A. R i s l e y , and G.W. Proc. 31, 5 7 7 ( 1 9 7 2 ) .

T.Y. Shen and C.A. Winger i n " A d v a n c e s i n Drug R e s e a r c h " , vol. 1 2 , N . J . Harper and A.B. S h m o n s , E d . , A c a d e m i c Press, New Y o r k , N . Y . 1 9 7 7 . 89-228.

5 . S h u m a n , R.F.

et-al., JOC, 42, 1914 (1977).

6. D o u g l a s , A.W., Merck Sharp L a b o r a t o r i e s , Rahway, N . J . , tion. 7.

Fed.

S p e i g h t and G . S .

3 . R.N. B r o g d e n , R.C. H e e l , T.M. Avery: D r_ u g_ s -1 6 , 9 7 ( 1 9 7 8 ) . _ 4.

Nuss:

Smith, J.L.,

Dohme R e s e a r c h

&

Personal Communica-

Merck Sharp & Dohme R e s e a r c h L a b o r Personal C o m m u n i c a t i o n .

atories, Rahway, N . J . , 8 . D o u g l a s , A.W.,

C a n . J. C h e m . ,

56,

2129(1978).

9 . B r e n n e r , G . , Merck Sharp & D o h m e R e s e a r c h L a b o r atories, Rahway, N . J . , Personal C o m m u n i c a t i o n .

10. S h u m a n ,

R.F.

et.al.,

JOC, 42, 1 9 1 4 ( 1 9 7 7 ) . -

11. T a i t , W.E., Merck Sharp & D o h m e R e s e a r c h L a b o r a t o r i e s , Rahway, N . J . , Personal C o m m u n i c a t i o n . 1 2 . N e w t o n , P . , Merck Sharp & D o h m e R e s e a r c h L a b o r a tories, Rahway, N . J . , Personal C o m m u n i c a t i o n .

13. M a n c i n e l l i , R . J . , Merck Sharp & D o h m e R e s e a r c h Laboratories, R a h w a y , N . J . , Personal Communication. 1 4 . T h e U n i t e d S t a t e s P h a r m a c o p e i a , 2 0 t h rev., A d d e n d u m a t o S u p p l e m e n t 2 , t h e U.S. P h a r m a c o p e i a l C o n v e n t i o n I n c . , 1 9 8 0 , page 2 9 5 .

596

FOTIOS M . PLAKOCIANNIS AND JAMES A. McCAULEY

15. The United States Pharmacopeia, 20th rev., Addendum a to Supplement 1, the U.S. Pharmacopeial Convention Inc., 1980, page 151. 16. H.B. Hucker; S.C. Stauffer; S.D. White et.al., Drug Metab. and Disp. 1:721(1972). 17. D.E. Dugqan, L.E. Hare, C.A. Ditzler, B.W. Lei, and K.CI-Kwan: Clinical Pharmacoloqy and Therapeutics. 21, 326 (1977a). 18. R.W. Walker et.al., Anal. Biochem. 95, 579 (1979).

19. L.H. Hare, C.A. Dirtzler, M. Hichens, A . Rosejay, and E.E. Duggan: J. Pharm. Sci. 66, 414 (1977). 20. W.O.A. Thomas, T.M. Jeffenes, and R.T. Parfitt. J. Pharmacy and Pharmacol. 31, 91P(1979).

TETRACYCLINE HYDROCHLORIDE Syed Laik Ali Zentrallaborntoriul,1 Deutscher Apotlzeker e . V Eschborn, Feder(11 Republic of Germiny 1. History 2. Nomenclature 3 . Description 3.1 Name, Formula. Molecular Weight 3.2 Appearance, Colour, Odour 4. Synthesis 5. Physical Properties 5. I Solubility 5.2 Loss on Drying 5.3 Dissociation Constant 5.4 Specific Optical Rotation 5.5 pH Value 5.6 Ultraviolet Spectrum 5.7 Infrared Spectrum 5.8 Nuclear Magnetic Resonance Spectrum 5.9 I3C NucIear Magnetic Resonance Spectrum 5. I0 Mass Spectrometry 5.1 I Circular Dichroism Spectra 5.12 Differential Thermal Analysis 6. Colour Reactions 7. Degradation and Stability 8. Related Impurities 9. Toxicity 10. Dissolution 1 I . Microbiological Methods 11. I Agar-Diffusion Method I 1.2 Turbidimetric Method 12. Methods of Analysis 12.1 Titrimetry 12.2 Visible and UV Spectrophotometry 12.3 Fluorimetry 12.4 Chromatographic Methods 12.5 Polarographic 13. Drug Metabolism and Pharmacokinetics References


591

598 598 598 598 600 600 60 I 60 1 602 602 602 602 603 603 606 606 608 610 613 613 613 620 620 62 1 622 623 623 623 623 623 625 628 642 642 645

Copyright 0 1984 by the American Phanndccuticai Associdtlon ISBN O-L2-ZM)X13-5

598

SYED LAlK ALI

Te t racycl ine Hydrochloride Tetsacycline and tetracycline hydrochloride have been mostly used synonymously in this monography. Literature cites quite often tetracycline but it is presumed to be valid als o for tetracycline hydrochloride. Generally solutions of tetracycline are made in dilute acids. Physical properties, spectra etc are given f o r tetracycline hydrochloride. 1.

History During the course of experiments for the elucidation o f the structure of the two earlier discovered compounds chlortetracycline (CTC) and oxytetracycline (OTC) it was found that hydrogenation of chlortetracycline resulted in halogenolysis and the product tetracycline (TC) retained the useful activity spectrum of the first two members of the family. TC appears to represent the first clinically successful antibiotic produced by shere chemical manipulation of preexisting antibiotic. TC was found to be present in fermentations of both cultures streptomyces aureofaciens and streptomyces rimosus as well as in streptomyces viridofaciens (1).

2.

Nomenclature The name tetracycline derives from the naphthacene nucleus that it possesses. The rings are lettered A through D from right to left and the numbers start at the bottom o f ring A . The enolized form as illustrated on next page has been selected arbitrarily and is used most exclusively in the literature. It is further defined as hydrochloride of 4F-dimethylamino-1,4,4a,5,5a,6,ll,l2a-octahydro-3,6 o(,lO ,12,12a -pentahydroxy-6-methyl-l,11 dioxonapht acene-2-carboxamide.

R

3. Description 3.1. Name, Formula, Molecular Weight Tetracycline hydrochloride 480.90

0 1

e

SYED LAIK ALI

600

3 . 2 . Appearance, Colour, Odour

A yellow crystalline powder, odourless, with a bitter taste. 4.

m

S nthesis h c a l total synthesis of tetracycline hydrochloride is economically not feasible. The original process involved catalytic hydrogenolysis of CTC to TC and then a directed biosynthesis using chlorine-free-culture-mediums ( 2 , 3 ) . In fact the removal of chlorine from the medium o r decreasing chlorine incorporation with a wide variety of chlorination inhibitors is technically quite important in order to facilitate processing without the need of expensive chromatographic steps as otherwise undesirable quantities of CTC are coproduced along with TC ( 4 ) . Fermentation and Isolation ( 5 ) : Various te] fermentation of different streptomyces species isolated in soil screening programms and then nutritionally and genetically manipulated to optimize yields. The fermentations are done on a very large scale of about 20,000 to 50,000 gallons. The organism and strain chosen not only determine maximal yields but als o the identity of the antibiotic formed. Chloretracycline fermentations frequently have up to 10% of tetracycline present. A wide variety of media have been employed for TC production. Fermentation starts with a tube of lyophilized culture which must be opened aseptically and transferred to agar slants for incubation. The cell population is then built up in stages. The pH of the fermentation is generally closely controlled between 6 and 7 by the periodic addition of the base, usually solid CaC03 and the temperature is maintained between 2 5 0 and 30 OC. Many energy and carbon sources such as starch, dextrin, maltose, glucose and nitrogen sources including amino acids, casein, meat extracts, nitrates and ammonium salts have been employed. A variety of inorganic ions must be present and calcium

TETRACYCLINE HYDROCHLORIDE

60 I

carbonate is specially noteworthy as it stabilizes the tetracyclines formed and may help increase yields by precipitating the antibiotics as they are formed and thus decreases their autotoxic effect on the producing o r ganism. Several patents dealing with the fermentative production of tetracyclines are reported in literature. The yields of tetracyclines are reported to lie about in the range of 10 g/liter ( 6 ) . There are a variety of procedures which have been employed for isolating the various tetracyclines from high-yielding fermentations. The processes in use are governed by the physical and chemical properties of the antibiotics and by economic factors. Tetracycline is an amphoteric substance and has an isoelectric point at 4.8 and its water solubility near neutral pH is about 1 mg/ml. The solubility in water is about ten times greater at pH 1 - 3 than at pH 5 - 6 . The isolation of TC can be affected through the use of a liquid ion exchange process, extraction from filtered beer at pH 8 . 5 with n-butanol or M I B K and precipitation with a non-polar solvent as the free base ( 7 1 , fractional precipitation of the less soluble TC-HC1 from CTC-HC1 (coproduced in the fermentation) (8) and precipitation as the calcium-complex ( 9 ) . The tendency of TC fermentations to produce sufficient quantities of CTC also renders refining difficult on a commercial scale (10). TC-oxalate is less soluble than CTC-oxalate s o that fractional crystallization is quite effective (11). Precipitation at about pH 4 in the presence of citrate (121, urea (13) and phosphate (14) has also been reported. Various cultures known to produce major fermentation-derived TC are also mentioned in literature ( 5 ) . 5.

Physical Properties 5.1. Solubility: It is freely soluble in water giving a clear solution' which becomes turbid on standing, soluble in aqueous solutions of alkali hydroxides and carbonates, slightly soluble in alcohol and practically insoluble in various organic solvents (15,16).

602

SYED LAIK ALI

Following solubility parameters are given in Table 1. Table 1 Solubility of TC-HC1 and TC in various solvents in mg/ml at 28 OC I17). TC TC-HC1 Water Methanol Ethanol Benzene Petroleum ether Carbon tetrachloride Ethyl acetate Acetone Ether Ethylene chloride Dioxane Chloroform Pyrid ine Benzyl alcohol

10.9 2o+ 7.9 0.3 0.0

0.1 0.8 0.8 0.6 0.8 7.7 2.9 20+ 10.8

1.7 20+ 20+ 1.0 0.005 0.3 17.3 17.4 3.7 11.3 14.6 13.8 20+ 14.4

5.2. Loss on Drying (15) Not more than 2.08, determined with 1.00 g by drying for 3 hours at 60 0 over phosphorus pentoxide at a pressure not exceeding 5 Torr.

5.3. Dissociation Constant The macroscopic PKa values of tetracycline hydrochloride in water at 25 OC are reported to be 3.30, 7.68 and 9.69 (18, 19). The most acidic value is attributable to the unusual 8-tricarbonyl system in ring A , the middle PKa is associated with the vinylogous acidic 8-dicarbonyl system o f rings B and C and highest PKa is then assigned to the protonated dimethylamino function at C4. A fourth PKa value of 10.7 has also been reported and is also attributed to the phenolic OH group at C 10 (20). 5 . 4 . Specific Optical Rotation (15)

- 239 0 to - 258 0 , determined in a 1.00% w/v solution in 0.01N HC1 and calculated with reference to the dried substance.

5.5. pH Value (15) The pH of a 1.0% w/v solution is between 1.8 and 2.8.


603

5.6. Ultraviolet Spectrum The quaternary carbon at C 12a separates the chromophores of the tetracyclines into two relatively distinct regions. The 13-tricarbonyl system of ring A contributes a strong absorption band at about 260 nm while the aromatic ring D conjugates with the 13-diketone moiety in rings B and C to produce a visible band at about 360 nm (21). tjte molecular exTC-HC1 in tinction coefficients and %(,,of different solvents are rep0 ed to be (22): Methanol 0.1 N HC1 0.1N NaOH 363 nm 356 nm 380 nm Absorption 270 nm 269 nm 268 nm maximum 366 301 A1 % 331 345 382 299 14480 17580 15940 18380 14360 16590 The UV spectra are given in Fig. 1.

1m

€

5.7. Infrared Spectrum The infrared spectrum of TC-HC1 is given in Fig. 2. The spectrum was obtained with a Perkin-Elmer Spectrophotometer 257 from a KBr pellet. The several carbonyl groups of the TC are either amide in character o r conjugated to double bond system o r strongly hydrogen bonded. Thus the amide band at about 1670 cm-1 and the large carbonyl envelope near 1620 cm-1 are usually identical for almost all the tetracyclines whose spectra then differ mostly in the finger print region (21). The structural assignments may be correlated with the foll wing band frequencies: Frequenc.(cm-?) Assignment 3350 3100 2600-2800 1670 1620 1450

streching vibrations of NHz stretching vibrations of OH Amine halide salt bands stretching vibrations of amide group stretching vibrations of carbony1 group characteristic vibrations of the aromatic ring

604

SYED LAlK ALI

El

220

240 260Ainnm#300

320 340

- Methanol ---- -

OlN-HCI 0.1N- NaOH

Fig. 1 UV Spectrum Of Tetracycline Hydrochloride

In Different Solvents

SYED LAlK ALI

606

Nuclear Magnetic Resonance Spectrum The nuclear magnetic resonance spectrum of TC-HC1 as shown in Fig. 3 was obtained on a Varian T-60 NMR Spectrometer in deuterated methanol containing 1% tetramethyl silane as the internal standard. The following spectral assignments are made f o r Fig. 3. Chemical Shift 1.72 singlet 3.10 singlet 7.20 doublet 7.58 doublet 6 . 9 5 doublet

Ass i gnment CH3 at C 6 N(CH3)2 at C 4 H a t C 7 H a t C 8 H a t C 9

They are approximately in agreement with values reported in literature, but the spectrum there was obtained with a HA-100 (23). The nmr shifts appear to be general regardless of solvent. Similar shifts are obtained in pyridine, trifluoroacetic acid and dimethyl sulfoxide. Particularly notable is the substantial downfield shift in the resoncance due to the C 4 proton when the tetracyclines epimerize at C 4 . This is especially convenient as both isomers are usually available and can be distinguished easily in this way. NMR was used to monitor the epimerization o f TC since the dimethylamino resonance of TC and its C 4 epimer differ by 0.1 ppm ( 2 4 ) . Formation of anhydrotetracycline could also be readily detected through NMR (23). 5 . 9. 13C Nuclear Magnetic Resonance Spectrum

carbon magnetic resonance measurements have been performed using the natural 13C abundance. Many more resonances are present in these spectra compared with the proton nmr. The spectrum of TC-HC1, dissolved in D 2 0 , i s presented in Table 2 . The signals are reported to be slightly different in DMSO ( 2 5 ) .

500

I ,

400

. . .. . .

...

I . . . . 1 . . . . 1 . . . . l . . . . l . . . . l . . . . , . . . . 1 . . . . 1 . . . . ,

I , . I ,

80

I . . 70

. .

I . 60

I . . . . I . . . . I . . . . I . . . . l . . . . I 5 0 FPM(0 40 30 20 10 0

Fig. 3 N M R Spectrum Of Tetracycline Hydrochloride, Varian T 6 0 Spectrometer

608

SYED LAIK ALI

Table 2 13C NMR Spectrum o f TC-HC1 in D 2 0 (25) Carbon Assignment Chemical Shift c 1 193.9 c 2 97.1 CONH 2 173.0 187.3 c 3 c 4 70.7 43.1 (quartet) HN+(CH3)2 35.0 (doublet) C 4a c 5 27.3 (triplet) 42.3 (doublet) C 5a 70.4 C 6 22.1 C - CH3 146.7 C 6a c 7 118.6 138.4 C 8 c 9 116.8 161.9 c 10 115.1 C 10a 194.3 c 11 107.3 C lla 172.3 c 12 74.3 C 12a 5.10. Mass Spectrometry Due to its non-volatility and thermal instability the electron-impact mass spectra o f tetracyclines is rich in fragment ions and weak in molecular ion intensity (26). In the highest mass region one sees m/e 426 for dehydration followed by ions 409 and 391 resulting from loss of NH3 and water from the carboxamide function as well as 381 for l o s s of the whole group a s CONH3. Most of the fragments seen involve rings A and B because rings C and D become s o quickly naphthalenoid. The mass spectrum of TC-HC1 is given in Fig. 4. Instrument: Varian Mat 44 Sample temperature: (direct inlet) 50OC - 250OC in 5 minutes Source temperature: 2 0 0 OC Electron energy: 80 eV The prominent ions o f this spectrum can be correlated to the structure as following: M-HC1 H20=426; M-HC1 H20 NH3=409; M-HC1 2H20 NH3=391; M-HC1 H20 CONH3 = 381;

-

2.

3

.

6

E

50-

lAi1 300

400

,i,

,

, , ,

Fig. 4

Mass Spectrum Of Tetracycline Hydrochloride

,

, 500

610

SYED LAIK ALL

5.11. Circular Dichroism Spectra TC-HC1 gives useful ORD-CD spectra. The intensity of the A ring band and its relative absence in 4-epitetracycline provides a sensitive means of assay for epimeric mixtures. Mixtures of tetracycline and 4-epitetracycline were assayed utilizing the large difference in their circular dichroism spectra at 2 6 2 nm. The method is very rapid and requires only the experimental measurements of ellipticity at 2 6 2 nm and absorbance at 3 5 6 nm ( 2 7 ) . The disadvantages of the assay are its inability to determine the content of anhydrotetracycline if it is present in large amounts. In Fig 5 - 6 molar ellipticity is illustrated graphically ( 2 7 ) . The ability of this technique to reflect subtle conformational shifts in dilute hydroxylic solvents under a wide variety of conditions is a particular strong point. Interactions with complexing ions gave dramatic changes in the spectral bands from both chromophoric regions providing evidence for chelation in both areas. Quantitative studies of ion-binding led to the conclusion that little if any binding takes place below pH 3 , that the binding of B C D rings takes place at pH 5 and the A ring binds at physiological pH's ( 2 8 ) . The study of C a z L and M g 2 L complexes of tetracycline in buffered solution was undertaken to determine their stoichiometry and the chelation sites. Circular dichroism was used to follow complex formation. Circular dichroism spectra of calcium and magnesium complexes of tetracycline in aqueous and methanol-water (9O:lO) systems are reported. Fig. 7-9 show these spectra as published by Newman and Frank (29). Calcium formed a 2:l metal ion to ligand complex at C 10 and C 12 sites, while magnesium formed a 1:l ratio complex, the chelating site being at C 11 ( 2 9 ) . Circular dichroism spectra of TC-calcium complexes in different organic solvents and the effect o f barbital sodium and L-tryptophan on it in alkaline and other buffer s o lutions are also known ( 3 0 ) .

61 I


250

300

350

Wavelength, nm. Fig. 5 Molar ellioticity ( 8 ) of tetracycline & 4-erpitetracycline ammonium hydrochloride (-) as a function of wavelength in 0.03 N salt (---+ HC 1

250 300 350 400 Wavelength, nm.

450

Fig. 6 Molar ellipticity ( 8 ) of anhydrotetracycline hydrochloride (----) and 4-epianhydrotetracycline (-1 as a function of wavelength in 0.03 N H C 1

SYED LAlK ALI

612

-T -5

Fig. 7 Circular dichroism spectrum of tetracycline in methanol-water ( 9 O : l O )

lot

Fig. 8 Circular dichroism spectrun of the calcium complex of tetracycline in methanol-water ( 5 0 : 5 0 )


613

5.12. Differential Thermal Analysis Stability studies of TC have been done with differential thermal analysis (DTA) (31, 32). DTA was employed to study the effect of certain additives, magnesium stearate, talc and lactose on the stability of tetracycline. The incorporation of magnesium stearate has a profound influcence upon the characteristic thermogram features of TC (Fig 10). The analysis was performed with a thermal analyzer with a heating rate of 50/min (33). DTA was further used to identify some vitamins a s the inactivating components in formulations containing TC-HC1 and lactose with uncoated and coated vitamins' granules. The thermal analysis was done employing a Mettler T.A. 2000 differential thermal analyser with a constant heating rate of lOO/min (34). 6.

Colour Reactions To about 0 . 5 mg TC-HC1 when conc. sulfuric acid is added a purplish-red colour is produced. On addition of 1 ml water the colour changes to deep yellow (15). When a solution of 2 mg TC-HC1 in 2 m l water is reacted with 5 drops of iodine solution, a brown voluminous precipitate of periodide is obtained. 2 mg TC-HC1 dissolved in 1 ml dilute sodium hydroxide are treated with 1 m l sulfanilic acid and few drops of sodium nitrite solution. A brown-orange coloured solution is obtained ( 3 5 ) .

7.

Degradation and Stability The dimethyl amino function at C 4 of TC-HC1 stands 1,3-diaxially eclipsed with respect to C 12a OH group in acidic solutions. Epimerization takes place and the product 4-epitetracycline has no significant antibacterial activity. The reaction takes place most rapidly between pH 2 - 6 in aqueous solutions and is a reversible first-order reaction (36-42). In alkaline solutions using precise conditions epitetracyclines are converted almost comletely back to the bioactive isomer in the presence o f chelating metals (43). This is due to the complexing metals tying together the C 4 -

SYED LAlK ALI

614

I

-tk--\

i7L

300 A(nm) 50

\ VI

-41 .5

1

Fig. 9 Circular dichroism spectrum of the magnesium complex of tetracycline in aqueous solution

I At

100 200 300 400°C Fig. 10 Thermograms of tetracycline 8, mixtures with magnesium stearate; l.tetracycline, 2. tetracycline + Mq. stearate (freshly prepared), 3. tetracycline + Mg.stearate (stored for 1 year)


dimethyl amino and C 12a - hydroxy functions thus overcoming their steric repulsion (44). Other groups which influence the epimerization include phosphate (41), citrate (411, urea (45) and some pharmaceutical ingredients (46). Additionally the reaction is catalysed by light. The equilibrium is reached, depending on the reaction-medium, at the level of about 40-50 % 4-epitetracycline following the 1st order reaction kinetics. Further epimerization is strongly dependent on pH, temperature and the buffer system used (47). In a solution o f 0 . 2 mg TC-HC1 in 10 ml water at pH 3.5 about 7% epitetracycline is formed in 48 hours (48). The C 6 - OH group is axial and antiperiplanar trans to the H at C 5a s o that it is sterically ideal for an elimination reaction o f second order. The anhydrotetracycline is produced due to dehydration at C 5a, 6. It is progressively formed at pH's lower than 2 , specially upon heating. Anhydrotetracycline possesses in vitro bioactivity but has not found clinical applications (49-55). The degradation of tetracycline itself is superimposed by the concurring degradation reactions of the primary tetracycline degradation products. Epimerization of anhydrotetracycline (ATC) is faster and the dehydration of 4-epitetracycline (ETC) is slower than the corresponding degradation reactions of the intact tetracycline. Kinetics of dehydration of epitetracycline in solution to form epianhydrotetracycline (EATC) were studied using UV and visible spectrophotometry. The reaction was found to be first order with respect to epitetracycline and hydronium-ion concentrations (56). Degradation pathway of TC to ETC, ATC and EATC is presented in Fig. 11 as reported by Leeson (77). I n alkaline medium tetracycline decomposes at the C 6 - OH site and isoderivatives with phthalide structure are obtained. The tetracyclines are stable in neutral o r mildly acidic solutions. At pH extremes, specially at elevated temperatures bioactivity i s progressively lost (57). Table 3 gives relevant stability data for

615

-N

I

z

r o

11

I ___,

11

a,

a

617


the fermentation derived tetracycline. Table 3 Stability Data for the Fermentation-derived Tetracycline in Solution ~~

Half-life 2 min 1 min 15.5 hr 6.8 min 11 hr 101 min 12 hr 12 hr

Conditions

Reference

0.2 N H2SO4/lOOO 1.0 N H2S04/1OOO 1.0 N H2S04/250 0.1 N NaOH/1000 pH 10 buffer/220 0.1 N NaOH/600 pH 8.8 buffer/240 pH 8 . 8 5 phosphate buffer

58 59 40 59

58 40 60 61

The stability of TC-HC1 injection solutions can be enhanced through the addition of stabilising agents such as magnesium gluconate (62). Generally bivalent and trivalent cations such as magnesium, calcium, mangnese, copper, nickel, iron and aluminium form chelates with TC which are mostly microbiologically inactive. This sort o f complex-formation can be eliminated through the addition of citric acid, polyphosphates and glucoseamine hydrochloride (48). TC does not decompose in glucose solutions in the pH range 2-7.5 during a period of 6 hours. Propylene glycol, pyridin derivatives, patothenic, lactic and tartaric acids may be used as stabilising agents and the TC solutions should be stored cool (47). Differential thermal analysis was empolyed to study the effect of certain additives such as magnesium stearate. It was found that magnesium stearate affects the stability o f TC even in freshly prepared formulations (33). Photodecomposition o f TC at oil-water interfaces has been studied. When the oils were irradiated in presence of TC, much larger amounts of peroxide were formed. This increase in the peroxide content was presumably due t o the formation of the oil-soluble anhydrotetracycline derivatives (63). The stability of TC and some TC-complexes in suspensions has been tested.

618

SYED LAIK ALI

Maximum stability of TC and its complexes with CaC12, urea and sodium hexametaphosphate has been reported in weakly acidic medium (pH 4-5). Ascorbate anion influences the stability negatively. In non-aqueous vehicles the TC-urea-complex shows better stability than TC itself. Sodium metabisulfite and sodium hydrogensulfite influence the stability positively. Further it can be increased by treating the TC-suspensions with an inert gas (64). Studies of TC stability in aqueous solutions and the influence of pyridin derivatives, magnesium salts and aliphatic and aromatic acids has been extensively investigated by Kassem and coworkers (62, 65). Commercial tetracycline samples and formulations have been the subject of stability studies by various authors (66, 67, 68). It was found that newly manufactured TC preparations contain only small amounts of anhydrotetracycline and 4-epitetracycline. Storage under adverse conditions markedly increase the amount of degradation products, but storage under normal conditions results in only a slow increase in anhydrotetracycline and 4-epitetracycline. In syrups this can be correlated with loss in tetracycline potency. Citric acid was found to increase greately the tendency of tetracycline degradation. TC-HC1 is more stable than TC-phosphate (68). Epianhydrotetracycline could not be found over 1% in any of the commercial formulations tested ( 6 6 ) . Problems of TC stability in eye-drops, injections and ways and means to increase this has been reported (69, 70, 71, 72). Powder o r crystaline TC and TC-HC1 samples darken in strong sunlight in a moist atmosphere. In aqueous solutions TC and TC-HC1 degrade by epimerisation which leads to further decomposition products. It is rapidly inactivated at a pH less than 2 and is slowly destroyed at pH 7 and above. The rate of degradation of TC to epianhydrotetracycline is increased in the presence of citric acid (16). Influence of temperature on the stability of solid TC-HC1


619

was investigated by Dihuidi and coworkers ( 7 3 ) . At 3 7 0 and 70 OC the stability was studied over a period of 2 7 months and at 5 0 OC over a period of 16 months. At 37 OC and 5 0 OC no decomposition is observed for TC nor for its related substances. At 7 0 OC a distinct decrease in TC is observed as well as a small increase in anhydrotetracycline. It is inferred from these values that at 2 0 OC and under conditions of low humidity TC-HC1 is a very stable product having a tgo of at least 50 years but probably of about 3 5 0 years ( 7 3 ) . The stability of solid samples of TC-HC1, TC and TC-phosphate aged at room temperature for up to 8 years has been followed. No appreciable decomposition was observed except for TC-phosphate which showed a significant increase of 4-epitetracycline and anhydrotetracycline after a period of 4 years ( 7 4 , 7 5 ) . Dony-Crotteux ( 7 6 ) reported that aqueous TC solutions containing riboflavin degrade in the presence of air and light. In addition suppression of degradation by the antioxidant ascorbic acid was observed. Some of the potency loss encountered results from epimerization catalysed by the buffer system. Parenteral TC-formulations containing ascorbic acid may be mixed with riboflavine for intravenous administration without concern for antibiotic stability. Although ascorbic acid itself appears to induce another degradation pathway, the loss is adequately covered by normal product overage ( 7 6 , 7 7 ) . The dehydration of TC at the C 5a-6 position as a function of acidity was investigated at various temperatures ( 7 8 ) . TC epimerization kinetics has also been investigated using NMR spectrometry ( 2 3 ) . The concentration of TC, ETC (epitetracycline), ATC (anhydrotetracycline) and EATC ( e p i a n h y d r o t e t r a c y c l i n e ) were studied in pH 1.5 phosphate solution at four temperatures in a degradation-pattern investigation. The study showed that epimerization of TC and ATC can take place at a low pH ( 7 9 ) . Stability of TC and TC-HC1 in methanol at 26 OC has also been performed (130).

620

SYED LAlK ALI

Siewert and coworkers made a thorough study of TC stability in large number of formulations such as ointments, creams and hydrogels. All formulation showed a relatively quick TC decomposition at temperatures above 50 OC. Preparations containing non-aqueous ointment bases demonstrated a higher degree of stability than aqueous ones like creams and hydrogels. TC-HC1 decomposed quicker than TC-base in aqueous systems (79a). In another study of the same authors 14 suspensions and 8 solutions were subjected to an accelerated stability test. 1 2 out of 2 2 preparations were calculated to be stable for a period of over three years under normal storage conditions. Two formulations decomposed to an extent of more than 10% at 61 OC within one day. Suspensions did not show a better stability than TC-containing solutions (79b). 8.

Related Impurities Impurities of 4-epitetracycline, anhydrotetracycline and 4 - e p i a n h y d r o t e t r a c y c l i n e have been limited in tetracycline hydrochloride by european pharmacopoeia (15), british pharmacopoeia ( 8 0 ) to 4 % (ETC) and 0.5% each for ATC and EATC. USP XX (81) allows the content of EATC up to 2 % . The permissable content o f EATC in various TC formulations such as capsules and injections has been even increased to 3 . 0 % by USP X X .

’*

w a c y c l i n e s have a safety margin which is quite satisfactory under most circumstances. Acute toxicity data f o r mice is given for TC as following: Route of Administration Acute Toxicity LD50 (mg/kg) oral 6300 IV 170 IP 2 80 Overaged TC preparations degrading t o EATC show a definite toxicity. Fatal reactions have been reported, although withdrawal of


62 I

the drug usually causes a slow reversal of the damage. Expiration dates should then be respected and TC should not be stored under conditions of excess heat and humidity. The LD50 of EATC is 4.8 times higher than that of unchanged TC. The immunosupressive effect is even reported to be 40-100 times higher (83). A comparative toxicity of ATC has not been reported. But the further degradation of ATC to EATC takes place much faster than the epimerisation of tetracycline. 10.

Dissolution The dissolution of TC was investigated using in vitro dissolution system of Stricker. Simulated gastric and intestinal juices were used as dissolution media separately. A good in vitro - in vivo correlation for TC was obtained (84). The aminomethylation of TC, a known process to make tetracyclines watersoluble and useful for parenteral administration has no significant influence on the bioavailability of the orally administered antibiotic. The in vitro adsorption of TC-HC1 was studied on various antacids, namely magnesium trisilicate, magnesium oxide, calcium carbonate, bismuth oxycarbonate, aluminium hydroxide and kaolin. The reversibility of the adsorption process was studied in different media and at pH values similar to those of the gastrointestinal tract. In general 0.0143 n NaHC03 was found to possess higher eluting properties than 0.01 N HC1 (85). The effect of various additives, PEG 6000, PVP, CMC on the adsorption of TC-HC1 on magnesium trisilicate and milk was also studied. Conclusions were made that the incorporation of some of these additives may possibly reduce TC-antacid interactions and consequently improve the antibiotic availability when coadministered with antacids ( 8 6 ) . The influence of additives on the in vitro release of drugs from hard gelatin capsules has been studied. A factorially designed experiment was used to assess the total percentage of TC-HC1 released from hard gelatine capsules as the function of a) diluent - the diluents being

622

SYED LAlK ALI

lactose, primojel and dry-flow starch - b) the quantity of diluent c) the presence and absence of magnesium stearate and d) the presence and absence of sodium lauryl sulphate (87). In a further study in vitro release of TC-HC1 from hard-gelatine, softgelatine capsules and TC-HC1 tablets in a number of commercial formulations was investigated. Majority of the commercial preparations conformed to the FDA regulations. The paddle and the flow-through-cell systems were used for the in vitro release of the drug (88). BP 80 prescribes a dissolution test for TC-tablets using a basket method and 0.1 M HC1 as medium (88a). According to FDA regulations (89) tablets and hard-gelatine capsules containing 250 and 500 mg TC-HC1 should release in first 30 minutes more than 60 and 50% of the drug repectively. 11.

Microbiological Methods 'fn vitro antimicrobial spectrum of TC as minimum inhibitory concentration in pg/ml can be given a s following (89a): Ps.aeruginosa 10

A.aerogenes 1.66

Shigella Sonnei 2.5

S . typhosa

Vibrio comma 0.58

Kl.pneumoniae 1.56

1.56 S.aureus 5 0.21

6 0loo+

S.aureus TC resist.

S.pyogenes 0.06

Ps.Morganii 2

Ps.vulgaris E.coli 0.73

The antibacterial activity of TC expressed in MIC-values (minimum inhibitory concentration) for a large number of gram-positive and gram-negative coccis, gram-negative bacteria and other microorganisms have been given in detail (89b). Microbiological assay methods are used due to their sensitivity but they are correspondingly nonspecific. The method is based upon a comparison of the inhibition of the growth of bacteria by


623

measured concentrations of the antibiotic to be examined with that produced by known concentrations of a reference standard of known activity. The assay for TC-HC1 may be carried either by diffusion method or by turbidimetric method. 11.1. Agar-Diffusion Method The micro-organism used for TC is Bacillus pumilus NCTC-8241 and Bacillus cereus ATCC 11778. Buffer solutions of pH 4.5 in both cases and the incubation temperatures of 37 - 390 and 35 - 3 7 OC were used respectively (90). 11.2. Turbidimetric Method The growth of the test organism is measured by means of a suitable optical apparatus. The microorganism used for tetracycline are Staphylococcus aureus NCTC 6751 and Staphylococcus aureus ATCC 6538 P. The buffer s o lution with a pH of 4.5 and the incubation temperature between 35 - 37 OC are used (90, 91). Additives in TC formulations can influence the results obtained by microbiological methods (92). Comparison of the microbiological results has been made with those obtained through other methods (93, 94). Effects of certain tablet-formulation-additives such as starch, bentonite, veegum F , talc, liquid paraffin, stearic acid etc. on the antimicrobial activity of TC-HC1 has been investigated (95). 12. 12.1

12 2

Methods of Analvsis Titrimetry Titrations of TC-HC1 in non-aqueous medium are known. TC-HC1 is dissolved in glacial acetic acid, 6% mercuric acetate solution added and then titrated potentiometrically with 0.1 N perchloric acid in dioxane (96, 97, 9 8 ) . Indirect volumetric determination of TC-HC1 after formation of a tetracycline-thiocyano-chrom (111)-complex through oxidation with KMn04, KBr03 and KIO3 has also been reported (99). Visible and UV-Spectrophotometry TC-HC1 can be determined after dissolving i t in 0.25 N NaOH. The yellow colour of the s o lution has an absorption maximum at

624

SYED LAlK ALI

3 8 0 nm. Another method is the addition of ferric chloride solution to a dilute HC1 s o lution of TC-HC1 which gives an orange-brown colour with an absorption maximum at 4 9 0 nm (100, 101). Another procedure, originally developed f o r chlortetracycline, in which TC is dehydrated by heating in acid and the resulting solution is examined at 440 nm. This is preferred to direct spectrophotometric determination at 3 6 0 nm because there are fewer interferences at longer wavelengths (102, 103). The simplest method o f all is to measure absorbance directly at 355 nm in dilute acid solutions (0.01N HC1) when relatively "clean" pharmaceutical formulations are involved. If dimethylformamide is used as solvent, the method can also be applied for TC determination in dragees (104). A spectrophotometric method for determining the amount of 4-epitetracycline is also known ( 4 0 ) . The content of TC in presence of ATC can be determined through the application of absorbancy ratios, but the results are higher compared to those obtained in other studies (105). TC forms a rose colour with diazobenzene sulfonic acid in acid s o lutions which can be measured spectrophotometrically without interference from CTC and OTC (106). Another dye reaction used for analysis is the butanol soluble blue colour developed by TC with p-aminodimethyl aniline and sodium hypochlorite in neutral solutions (107). A spectrophotometrically screening method was used to screen powder, tablet and capsule samples for the contents of ATC and EATC in TC samples ( 6 8 , 108). TC and its degradation products ATC and EATC have been determined spectrophotometrically as the metal complexes of titanium (1111, zirconium (IV) and thorium ( I V ) salts at different wavelengths (109). The method has some advantages in respect to sensitivity and rapidity of measurements. Colorimetric determinations of TC after chemical reactions such as treatment with boric acid in sulfuric acid (110) o r heating it with a mixture o f sodium tungstate,


12.3

625

sodium carbonate and hydrogen peroxide (111) or reacting it with zirconium oxychloride in methanol and diazobenzoic sulphonic acid (106) are known. Other colorimetric methods for the determination of TC-HC1 involve the use o f ferric thiocyanate reagent, nitrosation method and an iodobismuthate reagent. TC-HC1 was determined in various formulations such as powder, capsules and tablets (1121, utilising these reagents. Spectrophotometric determination of TC and its degradation products ETC, ATC and EATC in synthetic mixtures and some solid-dosage forms on account of the differences in molar absorptivities at the wavelengths 254, 260, 267, 357 and 390 nm has been reported (113). A sensitive colorimetric method of analysis for TC-HC1 and other antibiotics involves the formation of a coloured complex between TC and thorium (IV). The influence of pH and time upon the stability of the complexes was investigated. The method was applied to 29 different pharmaceutical dosage forms (114). Complex formation of uranyl acetate with TC has been utilized for its microdetermination. The mean stability constant of the 1:l complex with absorption maxima at 404, 350 and 270 nm, as determined spectrophotometrically, amounted to 1.2 x lo5, thus permitting the use of this procedure for TC microdetermination ( 1 1 5 ) . Fluorimetr quantum corrected fluorescence spectrum of TC HC1 (11 mg/50 ml) in 0.05 N NaOH taken with a Perkin-Elmer spectrofluorometer LS 5 is given in Fig. 12. Various fluorimetric methods of determination of tetracycline antibiotics are reported in literature (116, 117, 118, 119, 120). TC can be dehydrated to ATC by heating in acid solutions and taking the advantage of the greater lipophilicity of ATC i t is extracted at about pH 4 - 5 with chloroform and then the fluorescence of aluminium-chelate is measured (116, 121). The content of TC has been determined fluorimetrically in partially decomposed aqueous solutions of different pH values. Calcium and diethyl barbituric acid complexes of TC were extracted into an

+

SYED LAIK ALI

626

E

3

EXCITATION

SPECTRUM

Z I

60 NANOMETERS

Fig.

12

Fluorescence Spectrum Of Tetracycline Hydrochloride in 0.05 N NaOH, Perkin-Elmer Spectrofluorometer LS 5


621

organic solvent and the fluorescence intensity was determined by using an excitation wave length of 365 nm mercury line and an emission wave length of 530-550 nm (glass filter). Strongly fluorescent extractable complexes of calcium and barbituric acid are formed only with undecomposed biologically active TC. Decomposition products of TC do not form such complexes o r they are not extractable with an organic solvent (122). Spectrofluorimetric determination of TC in serum and urine is performed after isolation through adsorption on A1203 at an excitation wavelength of 335 nm and emission wavelength of 414 nm. The assay is based on the strong fluorescence of a degradation product o f TC formed on heating in alkaline solution. 0.2 - 5 g TC/ml in serum and 5 5 0 ug/ml in urine can be determined (123). Fluorimetric assay of TC in mixtures of antibiotics is performed by formation of strongly fluorescent aluminium-TC complex without prior extraction o r separation of individual antibiotics. Estimation was done by measuring fluorescence at 465 nm excitation and 555 nm emission wavelengths (124). TC-HC1 is determined fluorimetrically after addition of perchloric acid t o its solution in acetic anhydride as well as after extraction of ATC at pH 4 . 5 with chloroform. The excitation wavelength of 365 nm and emission wavelength 5 3 0 - 5 5 0 nm (glass filter) were used for measurements (125). TC formed ternary calcium complexes with barbital sodium and tryptophan in aqueous s o lutions. The fluorescence of TC and its complexes was measured at the emission maximum of 5 1 2 nm with the excitation wavelength of 390 nm (30). Fluorimetric determination o f TC in biological materials is based on solvent extraction with ethyl acetate of mixed tetracycline-calcium-trichloroacetate-ion-pairs from aqueous solution. The excitation and emission maxima are reported to be 400 nm and 500 nm respectively. Detection limits of 0.05 and 0.125 g/ml were obtained (126, 127).

P

I"

SYED LAIK ALI

628

12.4

Chromatographic Methods Paper Chromatography Paper partition chromatographic methods have been widely applied to the analysis of tetracyclines (128, 129). Pharmaceutical aqueous suspensions for oral use are acidified with HC1 and diluted with methanol. Crystalline formulations are dissolved only in methanol. A paper chromatographic method for TC determination in pharmaceutical preparations is based on the complexation of the antibiotic with a mixture of urea and disodium edetate on paper at pH 7.4. Urea helped in the separation of degradation products and led to the formation of well defined spots (130). Samples from fermentations must be acidified with oxalic acid to liberate TC from the mycelium. TC in filtrates may be precipitated in saturated s o lution of sodium tetraphenyl borate, precipitate dissolved in ethyl or butyl acetate and applied for paper chromatography. Various solvent systems and hRF values for paper chromatography are given in Table 4 . Table 4 Solvent System hRF Reference

n-butanol-acetic acidwater (4:1:5)

68 TC

131

n-butanol-conc. NH4OHwater (4:1:5)

24

TC

132

28

TC

133

chloroform-n-butanol (4:l) saturated with Mcllvaine's buffer pH 4.5

49

TC

134

n-butanol-acetic acid water (4:1:5) on paper impregnated with EDTA and dried

65 65

TC ETC

135

chloroform-nitromethanepyridine (10:20:3) on paper impregnated with Mcllvaine's citrate-phosphate buffer pH 3.5

87 ATC 87

EATC

(continued)


Table 4 (continued) n-butanol-ammonia-water 39 TC (4:1:5) on paper impreg 15 ETC nated with EDTA and dried 62 ATC 40 EATC n-butanol-chloroform (9:l) saturated with O.15M 34 buffer pH 3.0, Whatman no. 4 paper impregnated with a 3.7% solution of chelaton I11 and wetted with aqueous phase ethyl acetate-acetone (4:l) saturated with disodium edetate and urea; What 37 man no. 1 paper impregnated with a mixture 45 (1:3) of 10% urea in Mcllvaine's buffer pH 98 7.4 and 5% disodium ede2 tate in the same buffer

- 42 TC

135

136

TC TC-!-ICl

130

ATC EATC

Detection is made in UV light. The yellow fluorescence of tetracyclines and their epimers in UV light is greatly enhanced by exposing the paper to ammonia vapours. They may also be detected by Ehrlich's reagent or by starch-iodine following N-chlorination (137). Column Chromatography A partition chromatographic procedure was developed for the assay of tetracycline mixtures. Washed diatmaceous earth impregnated with buffered stationary phases containing EDTA have been used to separate and quantify TC mixtures in presence of degradation products. The compounds are eluted from a column of Celite 545 coated with a pH 7 buffer containing EDTA, glycerine and PEG 400 and then determined spectrophotometrically (138, 139, 140). ATC and ETC were determined in commercial and aged TC formulations through column chromatography using Celite 545 o r Kieselgur moistened with EDTA in buffer solution as stationary phase and chloroform, methanol and n-butanol etc as eluents (68, 141, 142, 143).

630

SYED LAIK ALI

Sephadex G-25 buffered at alkaline pH's has been found to possess excellent resolving power in some cases (144, 145, 146). A column liquid chromatographic method f o r determining TC in various dosage forms was automated. Each chromatogram consisted of a continuous flow separation of components followed by a spectral determination of the column eluate at a rate o f 12 samples per hour (147). Gel chromatography has also been applied to the analysis of TC antibiotics using a Bio-Gel P - 2 column with 0.1M acetic acid as an eluent (148). Thin Layer Chromatography TLC h a s been applied in european pharmacopoeia for the identification of TC-HC1 as well as for detection of impurities and related products. TLC plates were prepared with a slurry of kieselguhr and sodium edetate solution adjusted to pH 7 o r 7.5 and mobile phases of chloroform + ethyl acetate + acetone (4:4:2), previously saturated with 0.1M sodium edetate,and water + ethyl acetate + acetone (1:3:23) were used for identification and detection of impurities respectively (15). TLC has been widely used for the analysis of TC and related products (ETC, ATC, EATC) with various stationary phases and solvent systems. Stationary phases impregnated with EDTA solutions have been chosen often to avoid the complexation of TC with metal cations. Lloyd and Cornford (149) described an improved TLC limit test for the detection of ATC and EATC in TC using cellulose layer as stationary phase, buffered before development by spraying with a buffer solution (0.1 M disodium EDTA - 0.1 NH4C1), and chloroform as mobile phase, previously saturated with the same buffer solution. The Rf-values for TC, ATC, EATC were found to be 0.25, 0.93 and 0.4% respectively (149). A method for quantitative analysis of TC-HC1 on microcrystalline cellulose thin layer plates is described (150). Determination of ATC and EATC in microgram range in degraded TC tablets through TLC is also reported (151).


63 I

Table 5 gives a number of stationary phases, solvent systems and where available hRf-values for TC and related products for TLC. TLC has been used for qualitative and quantitative assay of degradation products of TC in TC-HC1, commercial formulations of various dosage forms such as powder, tablets and capsules etc. Determination has been performed after scratching the zone from TLC plates, elution of the drug and spectrophotometric measurement at 430 nm (155). Direct fluorimetry of the spots on TLC plates applying a 360 nm excitation and 540 nm emission wavelengths has been also performed. A linear relationship was found between the concentration of TC and related compounds in the range of 0.05-1 g and fluorescence intensity (157, 59, 160, 161, 162, 163). Chromatograms may be detected by bioautography with Bacillus subtilis using microbiological techniques (137, 169, 170, 171). Spraying with methanolic solution of ferric chloride (dark greyish spots), with antimony trichloride solution, MgC12 in ethanol or methanolic triethanolamine solutions have been reported (137). Aqueous fast blue solution, diazotized p-nitroaniline, modified Sakaguchi reagent (boric acid in conc. sulfuric acid) and diphenyl picrylhydrazyl spray reagents have also been used for detection of TC and degradation products on TLC plates. Coloured spots on white background or yellow spots on a bluish-white background were obtained (156). The tetracyclines have been most commonly detected by their fluorescence under long wavelength UV light at 365 nm either with or without exposure of the chromatograms to ammonia vapours ( 8 0 , 162, 164, 165, 168). TC-HC1 was allowed to epimerize in phosphate and acetate solutions at room temperature and the reaction products were studied at suitable time intervals by TLC (156). High Performance. Liquid Chromatography HPLC can seDarate and resolve TC-HC1 from its degradaiion products~ETC,ATC, EATC sensitively and reproducibly. The technique

P

Stationary phase

Table 5 Mobile Phase hRF-values

Kieselguhr, impregnated with citrate-phosphate buffer pH 5 . 5 containing 10% glycerine

dichloromethane - ethanol 95 % (9:l); TC:55-65; ETC:10; ATC:100; EATC:85;

152

Silica Gel G

n-butanol-oxalic acid-water (100:6:100); TC:38;

153

Silica Gel G

n-butanol-tartaric acidwater (100:6:100); TC:26;

153

Silica Gel impregnated with EDTA-water and activated

n-butanol saturated with water; TC:23

153

Kieselguhr G, impregnated with glycerol-phosphatecitrate buffer, pH 3.7

Chloroform-acetone(1:l) saturated with the same buffer

154

Kieselguhr impregnated with 5% aqueous EDTA solution adjusted to pH 9 with NaOH

Acetone-water (1O:l) TC:69; ETC:29 ATC:84; EATC:55;

155

The same sorbent as above, but adjusted to pH 7 . 5

acetone-ethyl acetatewater (20:10:3); TC:71 ETC:38; ATC:88; EATC:46;

155

References

?

'd

Kieselguhr G impregnated with EDTA+PEG 400+glycerine

methyl ethyl ketone saturated with Mcllvaine's buffer pH 4.7 TC:53; ETC:20; ATC:93; EATC:47;

156

Same as above

dichloromethane-ethyl formateethanol (9:9:2) saturated with buffer pH 4 . 7 ; TC:36; ETC:12 ATC:83; EATC:50;

156

Kieselguhr Impregnated with mobile phase

ethylene glycol-water-acetoneethyl acetate (2:2:15:15) TC:50; ETC:28; ATC:95; EATC:30;

157

Cellulose TLC plates impregnated with EDTA, pH adjusted to 9.0

n-butanol saturated with water TC:18; ETC:10; ATC:23; EATC:23;

158

Kieselguhr G impregnated with 0.1M EDTA

MIBK-ether-water-methanolethylene glycol (50:60:3:7.5:2.5) TC:16; ETC:4; ATC:83; EATC:22;

159

Microcrystalline cellulose plates, Merck

aqueous 0.25M MgCl2 TC:72; ETC:68; ATC:29; EATC:16;

160

Same a s above

aqueous 0.20M CaC12 TC:67; ETC:63; ATC:25; EATC:25;

160

Table 5 continued ~

Same as above

aqueous 0.25M BaC12 TC:63; ETC:60; ATC:24; EATC:17;

160

Same as above

aqueous 0.15M ZnCl2 TC:65; ETC:65; ATC:22; EATC:18;

160

Microcrystalline cellulose impregnated with phosphatecitrate buffer and subsequently submerged in methanolic ethylene glycol solution

twice in ethyl acetate saturated with water TC :35

161

w P

Powdered cellulose m TLC plates chloroform saturated with 0.1M EDTA, after developimpregnated with 0.1M rnent exposed t o ammonia EDTA TC:6-23(band); ETC:3.5; ATC:92; EATC:52;

162

m

Kieselguhr plates impregnated with EDTA, adjusted to pH 7 . 5

acetone-ethyl acetate-water (20:10:3); TC:10; ETC:40; ATC:95; EATC:50;

163

Kieselguhr G impregnated with 0.1M EDTA

ethyl methyl ketone saturated with Mcllvaine buffer pH 4 . 7 or alcohol; later plates exposed to ammonia; TC:56; ETC:30;

164, 165

Microcrystalline cellulose on TLC plates

0.1M EDTA with 0.1% NH4C1; chloroform saturated with EDTA-NH4Cl solution; TC:72; ATC:36; EATC:52;

166, 167

Kieselguhr G treated with a buffer containing 0.1M EDTA at pH 7.0, glycerine and PEG 400

ethyl acetate saturated with 0.1M EDTA at pH 7.0

168

SYED LAIK ALI

636

has been successfully applied for the quantitative assay of TC and related compounds in all sorts of commercial solid and liquid dosage forms, stability studies, in body fluids, pharmacokinetics and drug metabolism investigations. Quite often tests for identity, potency and assay determination are performed with this method. Micro to submicrogram amounts could be determined with ease. Different modes of HPLC, adsorption, reversed phase, ion-exchange and paired-ion chromatography have been applied. Butterfield separated various tetracyclines using an ion-exchange system (172). Later workers included EDTA in the system to prevent binding with the metal columns (75). Advantages of reversed phase HPLC over ion-exchange techniques have been given (173). Serum levels of as little as 0 . 3 g/ml TC have been determined through HPLC 174). A preliminary study of the effectiveness of ion-exchange, adsorption, liquid-liquid partition and reversed phase ion-pair chromatography indicated that only the last method showed promise. A 18 m partisil column gave plate heights in the range of 0.15 - 0.3 mm. The method was used to quantify the impurities in TC at round the 1% level (175). T C s may be separated with high efficiency (plate heights around 0.05mm) using SASand ODs-Hypersils in the pH range 3-5. Within this pH range the presence of EDTA at a concentration of about M is essential for s a tisfactory chromatography. EDTA is thought to act as a zwitterion pairing agent with the zwitterionic form of te TCs (176). Table 6 gives various columns, mobile phases etc. reported in literature for the analysis of TC-HC1 and TC. Concentrations as low as 4 g/mg TC in urine were quantitated accuratelK TC was extracted from urine as calcium comlex ( 1 8 3 ) . Calcium complex of TC was extracted from urine and plasma with ethyl acetate and then reextracted into HC1. Concentrations of less than l p g / m l in urine and 1.5 pg/ml in plasma were determined with a relative standard deviation of less than 5 % (184).

P

P

rETRACYCLINE HYDROCHLORIDE

637

The addition of phenylbutazone to urine o r plasma enhances the extraction of these compounds by ethyl acetate. The formation of p h e n y l b u t a z o n e - t e t r a c y c l i n e ion pairs and their role in extraction process is discussed (185). C 18 reversed phase column was used with the mobile phase water-acetonitrile-perchloric acid in two steps with varying amounts of acetonitrile. Six different TC preparations analysed contained ETC, ATC and EATC from undetectable to 7.78% (186). Limits of detection for ATC and EATC in TC were found to be 12 and 7 ng respectively through HPLC analysis (187). TCs can be extracted from serum o r organ homogenates by means of acetonitrile containing buffers. The lowest concentrations of the intact molecule detectable are 0.2 pg/ml serum o r blood and 0.4 g/g organ. HPLC analysis yielded the s me results as microbiological method (191). Reversed phase ion-pair chromatography with the use of tertiary amines as counter ions has been applied for the separation of TC analogs and their potential impurities (192). Retention of TC can be regulated by the addition of organic ammonium compounds to the mobile phase in reversed phase ion-pair chromatography (194). The addition of inorganic anions C1-, Br-, ClOT as their sodium salts to the mobile phase and their influence on the retention of TC has been studied (194). TC has been extracted from plasma as an ion-pair with tetrabutyl ammonium hydrogen phosphate into chloroform-1-heptanol and after reextraction into an acidic aqueous phase the separation was performed through reversed phase HPLC. Analysis of urine was done by direct injection (193).

zr

Table 6 Column Pellicular cation-exchange resin

Mobile Phase 0.003M EDTA in waterethanol (60:40)

Detection Application

References

TC in formulat ions

177

nm

TC im pharm. preparations

178

280 nm

separation

179

254

6 ;ygaPa;k P

0.02M phosphate buffers, pH 2 - 5 in wateracetonitrile gradient

TMS bonded WLCU Si gel

0.1M HClO4 in wateracetonitrile gradient 85:15 to 70:30

280 nm

ATC, EATC

water-methanol-0.02M phosphate buffer (pH 7.6) and 1mM EDTA, gradient 25-50% v / v methanol

267 nm

TC from penimocycline

180

350 nm

separation of TC from related compounds

181

w QI m

Micropak CH

Vydac RP 18 and Lichrosorb 0,15M (NHq12C03RP 8 acetonitrile (96:4) pH adjusted t o 8 . 4 with ammonia; citrate-phosphate buffer (pH 2.20)acetonitrile ( 6 5 : 3 5 )

o f TC, ETC,

rm

Vydac RP 18 lo

isopropanol-diethanolamine-phosphate ammonium EDTA-water

254,280,365 TC from degradation 405 nm products

182

TMS-bonded partisil 18

0.1M HC104 o r O.lM H2SO4 or 0.05M HNO3 - acetonitrile (85:15 t o 70:30)

2 8 0 nm

separation of TC, ETC ATC, EATC

175

Zipax HCP polymer

0.001M EDTA, 0.01M H3PO in water-methalon 487313, v / v >

2 8 0 nm

separation of TC, ETC, ATC, EATC

ODs-Sil-X-1

0.005M EDTA, 0.05M (NH4)2C03 in watermethanol ( 9 2 : 8 )

2 8 0 nm

TC

P"

Pellionex CP 172

0.1M Na+, 0.002M EDTA in water-ethanol (70:30)

ODs-Hypersil, water-acetonitrile-dime- 272 nm thyl-formamide-0.001M 2 8 0 nm Na2EDTA-citric acid and acetic acid, pH a d justed to 3.0 or 5.0

75

173

TC, ETC, ATC, EATC TC, ETC, ATC, EATC

176

P

continued

Table 6 continued 0.005M EDTA, 0.05M NaCl in water-methanol, 95:s and 70:30

375 nm

TC in urine extracts as calcium cornplex

0.01M NaH2P04 in water-acetonitrile 60:40 pH 2.4

355 nm

TC in urine plasma, tissues as calcium complex

Water-acetonitrile-perchloric acid 76:22:1.8; 61:37:1.8

254 nm

TC, ETC, ATC, EATC

186

429 nm

ATC, EATC in TC

187

lo Lichrosorb NHz, RP 8, 5 and 10 im

0.3M EDTA buffer, pH adjusted to 7.0 10% acetonitrile in 0.1M phosphoric acid with 1 0 - 3 ~HIBS

357 nm

TC in mixtures

188

Lichrosorb RP 8, 5 m

acetonitrile-0.1M citric acid 25:75

3 5 0 nm

Re tent i on mechanism of TCs

189

Ion-X SA, conditioned with EDTA

183

184+185

a. P O

ODs-Sil-X-1 lo

r""

Zipax SCX

P"

t

r"

Bondapack {henyl C 18,

a step gradient of 12-22% acetonitrile in 0.2M phosphate buffer at pH 2.2

Nucleosil C 8, 10 m

25% acetonitrile-0.01M NaHzP04 , p1-i adjusted to 2.4

L ichrosorb RP 8 , 5 m

phosphate buffer, tri280 nm propylamine o r N,N-dimethyloctylamine, pH 8.0, with 20-35% acetonitrile

Lichrosorb

0.01M phosphoric acid in 357 nm water-35% acetonotrile organic ammonium compounds in mobile phase

plasma and urine

methanol-water-1 M phosphoric acid, gradient elution

influence o f temperature on solid TC

I"

r

RP 2, 8, 18,

ETC, ATC, EATC in TC powder and capsules 270, 357 nm TC in blood and organs o f animals

280 nm

separation of TC and analogs and their impurities ion-pair LC

190

191

192

193,194

of TC, TC in

195

642

SYED LAIK ALI

Gas Chromatography The GLC determination of TC, ETC, ATC and EATC in aged TC samples has been reported. Trimethylsilyl derivatives of TCs were formed and gas chromatographed on six different columns, 3% OV-1, 3% JXR, 3 8 SE 52, 3% OV-17, 10% OV-25 and 10% OV-210. A 6 feet 3% JXR column at 260 OC isotherm separated most of the compounds. Different TC-HC1 powder samples were analysed through GLC for their degradation products and the results compared with those obtained from microbiological and UV assay methods (74). 12.5 Polarogra h T Z T Z d & $ antibiotica have been determined qualitatively and quantitatively through polarographic methods (196, 197). Polarographic behaviour of tetracycline and some of its degradation products has been investigated in phosphate-acetate buffer solutions. The first reduction wave is attributed to the carbonyl group at C 1 and the second reduction step corresponds to the conjugated system at C 11 - C 12 (198). Oscillopolarographic methods have been also applied for the analysis of tetracycline and its degradation products (199, 200). Optimum conditions for the dc polarographic reduction of TC were studied. A boric acid-sodium borate buffer provided the best conditions for the elctro-reduction o f TC. Quantitative determination in various dosage forms were carried on (201). TC can be analysed through polarography and different i a l - c a t h o d e - r a y p o l a r o g r a p h y in a supporting electrolyte o f 0.1M diethyl barbituric acid in 40% methanol and 0.1M lithium chloride at pH 4.7 (202). TC-HC1 gives a polarographic step in a phosphate buffer solution, pH 6.2, with a half-wave potential of -1.45 to -1.51 V (203). A method involving ac polarography is also reported in literature (204). Electrochemical methods for the determination of TC have been reviewed by Unterman and Hughes (205, 206). 13,

Drug Metabolism and Pharmacokinetics TC-HC1 is incompletely and irregulary absorbed after oral administration. The


643

presence of metal ions such as calcium, magnesium, aluminium, zinc and iron reduce its absorption due to formation of chelates and coordination compounds. Alkalis also lower the serum concentration. Rectal absorption from suppositories is unsatisfactory. Absorption from stomach is minimal. Absorption from the intestines is incomplete and subject to considerable variation between individuals and is affected substantially by the contents of GI tract. Milk and food reduce TC absorption, phosphates may enhance it. Some practical improvement in absorption appears to have resulted from the exclusion of "inert" magnesium stearate or calcium carbonate from TC capsules (207, 208, 209, 210). Therapeutic blood concentrations are achieved more readily by increasing the frequency of administration than by increasing the dose. Therapeutic serum concentrations lie in the range g/ml whilst the toxic concentration Of is abo t 12 g/ml o r over. Serum concentrations of l-tyg/ml are maintained when oral doses of 250 500 mg are administered daily. An intramuscular dose of 100-250 mg brings about serum concentration of 2 - 4 g/ml during 1-2 hours. A serum concentration of 5-30 g/ml is reached 30 minutes after an intravenous dose of 250-300 mg TC. Serum half-life is about 9 hours when given orally in a single dose (16). A mean serum half-life is about 11 hours when administered orally in divided doses (211). TC is secreted in milk and saliva and crosses the placents. It is readily taken up by newly formed bones and teeth in which it forms a TC-calcium orthophosphate complex. It remains bound to malignant cells for a longer time than to non-malignant cells. TC is excreted both in urine and bile. The concentration in bile may reach 10 to 20 times that of blood (212, 213). About 208 is excreted in urine in 24 hours, mainly as unchanged drug, after oral administration and 50% after intravenous doses. The average concentration of TC in urine after oral and im + i v administration is in the range of 100-300 rg/ml. Urinary excretion is increased when

"-"9

P

P

634

SYED LAIK ALI

urine is alkaline (16). TC appears to be excreted in the urine by glomerular filtration as its half-life is strongly dependent upon the extent of protein-binding. TC bounds to plasma proteins in the range of 20-70% (16). It is also bound reversibly to serum proteins. In serum 53% TC is in the form of complexes with protein. Among the bound TC, 54% is with albumin, 1 3 and 19% with low and high density lipoproteins respectively, 6% with very high density lipoprotein and 8% is bound with other serum proteins. TC appears to dissolve in the lipophilic portion of the lipoprotein molecule, rather than t o be associated with specific binding sites (214). Acknowledgement The author thanks Mr. Martin Siewert, Pharmacist, f o r providing some useful literature and Mrs. Monica Hediger f o r typing the manuscript.


645

References Mitscher, L.A., The Chemistry of the Tetracycline Antibiotics, p. 1-2, Marcel Dekker, (1978) New York. 57 Boothe, J.H. et al, J. Amer. Chem. SOC., 3263 (1953). U.S. Patent 2,699,054 (1955). 3) U.S. Patent 2,734,018 (1956). 4) Mitscher, L.A., The Chemistry of the 5) Tetracycline Antibiotics, p. 46-49, Marcel Dekker (1978) New York. U.S. Patent 2,923,688 (1960). U.S. Patent 2,739,924 (1956). U.S. Patent 2,804,476 (1957). U.S. Patent 2,847,471 (1958). U.S. Patent 2,905,714 (1959). U.S. Patent 2,929,837 (1960). U.S. Patent 3,013,074 (1961). 13) U.S. Patent 3,037,973 (1962). 14) U.S. Patent 3,230,254 (1966). European Pharmacopoeia, amendment to Volume 11, p . 39 (1971) Maisonneuve S . A . , France The Pharmaceutical Codex, 11. edition, page 924, The Pharmaceutical Press, London (1979). Weiss, P.J. et a l , Antibiotics chemother., 7 376 (1957). 26 18) Parke, T.V. and W . W . Davis, Anal. Chem., 642 (1954). 19) Stephens, C. R . et al., J . Amer. Chem. SOC., 7a 4155 (1956). K g l e r , N.E. et al., Anal. Chem., 37 872 (1965). Mitscher, L.A., The Chemistry of the Tetracycline Antibiotics, p. 92-93, Marcel Dekker (1978) New York. Dibbern, H.W., UV and I R spectra of some important drugs, Editio contor, Aulendorf (1979). Schlecht, K.D. and C.W. Frank, J. Pharm. Sci., 62 2 5 8 (1973) Von Wittenau, M.S. and R.K. Blackwood, J. Org. Chem., 31 613 (1966). 21 162 2 5 ) Barbatschi, F. et al., Experientia (1965). Hoffman, D.R., J. Org. Chem., 31 792 (1966). Miller, R.F. et al., J. Pharm. Sci., 62 1143 (1973). 1)

646

29) 30

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Mitscher, L.A. et al., Antimicrob. Agts. Chemother., 111 (1970). Newman, E.C. and C.W. Frank, J . Pharm. Sci., 65 1728 (1976). Day, S.T. et al., J. Pharm. Sci., 67 1518 (1978). Navolotskaya, T.I., Antibiotika 18 1101 (1973). Navolotskaya, T.I. and G.I. Lose=, Antibiotika 19 47 (1974). Geneidi, A . S E and L. El-Sayed, Pharm. Ind., 40 1074 (1978). 49 172 Geneidi, G.S. et al., Sci. Pharm., (1981). Gsterreichisches Arzneibuch, 9. Ausgabe, page 1507, Vieanna (1960). Doerschuk, C.R. et al., J. Amer. Chem. S O C . , 77 4687 (1955). Stephens, C.R. et al., J. Amer. Chem. SOC., 78 1515 (1956). McCormick, J.R.D. et al., J. Amer. Chem. SOC., 78 3547 (1956). 7 Kaplan, M.A. et al., Antibiot. Chemother., 569 (1957). McCormick, J.R.D. et al., J. Amer. Chem. SOC., 79 2849 (1957). Remmers, E.G. et al., J. Pharm. Sci., 52 752 (1963). Hussar, D.A. et al., J. Pharm. Pharmacol., 20 539 (1968). 90 Korst, J.J. et al., J. Amer. Chem. SOC., 439 (1968). Mitscher, L.A. et al., Antimicrob. Agts. Chemother., 78 (1968). Agostini, C.T., Pharm. Acta Helv., 47 560 (1972). Naggar, V. et al, Die Pharmazie 29 129 (1976). Kreilgard, B., Arch. Pharm. Ogchxi, Copenhagen 80 1083 (1973). EuropaischesArzneibuch, Kommentar, page 1206, Govi Verlag, Frankfurt (1976). Conover, L.H. et al., J . Amer. Chem. SOC., 75 4622 (1953). Boothe, J.H. et al., Ibid 75 4621 (1953) Miller, M.W. and F.A. Hochzein, J. Org. Chem., 27 2525 (1962). Waller,C.W. et al., J. Amer. Chem. SOC., 74 4981 (1952). Blackwood, R.K. and C.R. Stephens, Can. J. Chem., 43 1382 (1965).


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Hlavka, J.J. and H.M. Krazinski, J. Org. Chem. , 28 1422 (1963). 82 3946 Green, A. et al., J. Amer. Chem. SOC., (1960). 63 1901 Hoener, B.A. et al., J. Pharm. Sci., (1974). Mitscher, L.A., The Chemistry of the Tetracycline Antibiotics, p . 54, Marcel Dekker (1978) New York. Doerschuk, A.P. et al., J . Amer. Chem. SOC., 81 3069 (1959). KCormick, J.R.D. et al., J. Amer. Chem. Soc., 79 4561 (1957). K n i e r i , P.P. et al., Antibiotics Annual 81 (1953-54). De Smit,-C.P., Pharmac. Weekbl. 99. 49, 73 (1964). 111 1932 Kassem, B.M.A. et al., Pharmaz.Ztg. (1966). Sanniez, W.H.K. and N. Pilpel, J. Pharm. Sci., 69 5 (1980). Kassem, M.A. et al., Pharmaz. Ztg., 115 1036 (1970). Kassem, M.A. et al., Pharmaz. Ztg., 112 221 (1967). Steinbach, D. and Th. Strittmatter, Pharmaz. Ztg. 123 1083 (1978). Griffiths, B.W. et al., Can. J. Pharm. Sci., 5 101 (1970). Walton, V.C. et al., J. Pharm. Sci., 59 1160 (1970). 116 196 Dessing, R.P. et al., Pharm. Weekl., (1981). Wassiak, H., Acta Polon. Pharm., 22 141 (1965). 9 44 Sina, A. et al., Can. J. Pharm. Sci., (1974). Petrick, R.J. et al., Amer., J. Hosp. Pharm., 35 1386 (1978). Dihuidi, K. et al., J. Chromatogr., 246 350 (1982). 45 Tsuji, K . und J.H. Roberson, Anal. Chem., 2136 (1973). Tsuji, K . et al., Anal. Chem., 46 539 (1974). 12 179 Dony-Crotteux, J., J. Pharm. Be%., (1957). Leeson, L.J. and J.F. Weidenheimer, J. Pharm. Sci., 58 355 (1969). Schlecht, K.D. and C.W. Frank, J. Pharm. Sci., 64 352 (1975).

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Yuen, P.H. and T.D. Sokoloski, J. Pharm. Sci., 66 1159, 1648 (1977). Erittmatter, Th. and M. Siewert, Pharmaz. Ztg., 127 1300 (1982). S i e w e r C M . and Th. Strittmatter, Pharmaz. Ztg., 128 (1983) in print. British Pharmacopoeia, p. 468 (19731, London's Her Majesty's Stationary Office. United States Pharmacopia X X , p . 781 (1980), Rockville, Md. 20852, USA. Redin, G.S., Antimicrobiol. Agts. Chemother., 371 (1966). Klimova, N.E. and O.B. Ermolova, Antibiotiki 21 1018 (1976). Fleischmann, L . , Pharm. Ind., 41 1218 (1979). 31 105 Khalil, S.A. et al, Die Pharmazie (1976). 31 125 Daabis, N.A. et al., Die Pharmazie (1976). Newton, J.M. and F.N. Razzo, J. Pharm. 26 suppl. 30 P (1974). Pharmac., MGller, H., Pharmaz. Ztg., 127 2045, 2223 (1982). British Pharmacopoeia, p. 826, (19801, London's Her Majesty's Stationary Office. Federal Register 44 48186 (1979). English, A.R., P r z . SOC. Exptl. Biol. Med., 122 1107 (1966). Antibiotika-Fibel, published by H. Offen, M. Plempel and W. Siegenthaler, page 321, George Thieme Verlag, Stuttgart (1975). European Pharmacopoeia, Volume 11, pages 49, 433, 436, (1971), Maisonneuve S.A., France. Kavanagh, F., Analytical Microbiology, Vol. I1 Acad. Press, New York (1972). Csillaghy-Rutschmann, Th. and A . Lindner, Pharm. Acta Helv., 36 617 (1961). Bose, B.K. et al., Indian J. Pharm., 26 221 (1964). Amer., M.M. et al., Bull. Fac. Pharm. Cairo 8 87 (1970). Univ., Asker, A. et al., Die Pharmazie 28 474, 476 (1973). Sideri, C.N. and A . Osol, J. Amer. Pharm. ASSOC., Sci. Ed. 42 688 (1953). Yokoyama, F. and K L . Chattere, J. Amer. Pharm. Assoc.. 47 548 (1958). Safarik, L. and-V. Spinkova, Cesk. Farm., 7 76 (1958).


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Ganescu, I. et al., Archiv der Pharmazie 310 544 (1977). Woolford, M.H. Jr. and F.S. Chiccarelli, J. Amer. Pharm. ASSOC., Sci., Ed., 45 400 (1956). Monastero, F. et al., Ibid 40 241(1951). Levine, J. et al., Ibid 38 473 (1949). Chiccarelli, F . S . et al.,Ibid 48 263 (1959). Cruceanu, I. et al., Die Pharmaze 24 733 (1969). 58 470 Pernarowski, M. et al., J. Pharm. Sci., (1969). Vogth, H., Arch. Pharm., 289 502 (1956). Murai, K . , Yakugakku Z a s s K 8 0 544 (1960). Dijkhuis, I.C., Pharm. Weekbc, 102 1308 (1967). Pradeau, D. et al., Ann. Pharm. Francaises 37 369 (1979). Sekiguchi, K., J. Pharmac. SOC. Japan (Yakugakuzasshi) 78 965, 970 (1958). 75 973 Kakemi, K . , J. Phzmac. SOC. Japan (1955). Roushdi, I.M. et al., Die Pharmazie 28 236 (1973). Regosz, A. and G. Zuk, Die Pharmazie 35 24 (1980). Chatten, L.G. and S.I. Krause, J. Pharm. Sci., 60 107 (1971). Mahgoub El-Said, A . et al., J. Pharm. Sci., 63 1451 (19741. Hayes, E.Jr. and H . G . Du Buy, Anal. Biochem., 7 322 (1964). Tbsen, K.H. et al., Anal. Biochem., 5 505 (1963). Kohn, K.W., Anal.Chem., 33 862 (1961). Hajdu, P. and A. HauslerTArzneim. Forsch., 12 206 (1962). Winkelmann, J. and J. Grossman, Anal. Chem., 39 1007 (1967). 28 222 Kellly, R . G . et al., Anal. Biochem., (1969). Regosz, A . , Die Pharmazie 32 681 (1977). Schatz, F., Arzneim. Forscx, 23 426 (1973). Hall, D., J. Pharm. Pharmac., 'ZB 420 (1976). Regosz, A. e t al., Sci. Pharm., 46 249 (1978). Poiger, H. and Ch. Schlatter, Analyst 101 808 (1976). Van den Bogert, C. and A.M. Kroon, J. Pharm.Sci., 70 186 (1981). Hughes, D.W.and W.L. Wilson, Can. J. Pharm.

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Sci., 8 67 (1973). 129) Wagman, G.H. and M.J. Weinstein, Tetracyclines in "Chromatography of Antibiotics", Elsevier (1973) New York. 62 1998 130) Youssef, M.K. et al., J. Pharm. Sci., (1973). 131) Regna, P.P. and I.A. Solomans, Ann. N.Y. Acad. Sci., 53 229 (1950). 53 132) CaffauTS. and S. Jaccobacci, Minerva Med., 3747 (1962). 133) Selzer, G.B. and W.W. Wright, Antibiot. Chemother., 7 292 (1957). 134) Urx, M. et ai., J. Chromatogr., 11 62 (1963). 135) Kelly, R.G. and D.A. Buyske, Antziot. Chemother., 10 604 (1960). 136) Vondrackova,T. and 0. Strauchova, J. Chromatogr., 32 780 (1968). 137) Macek, K., PhZEnaceutical Applications to TLC and PC, p. 522-523 Elsevier (1972) New York. 61 138) Fike, W.W. and N.W. Brake, J. Pharm. Sci., 615 (1972). 139) Kelly, R.G., J. Pharm. Sci., 53 1551 (1964). 121 405 140) Bailey, F., J. Pharm. Pharmacx., (1969). 141) Ascione, P.P. et al., J. Pharm. Sci., 56 1396 (1967). 142) Ascione, P.P. and G.P. Chrekian, J . Pharm. Sci., 59 1480 (1970). 143) Regoasc A., Sci., Pharm. 47 225 (1979). 5 144) Griffiths, B.W. et al., Can- J. Pharm. Sci., 101 (1970). 145) Griffiths, B.W., J. Pharm. Sci., 55 353 (1966). 146) Griffiths, B.W., J . Chromatogr., 38 41 (1968). 147) Ascione, P.P. et al., J. Chromatos., 65 377 (1972). 148) Ragazzi, E. and G. Veronese, J. Chromatogr., 134 223 (1977). 149) Lloyd, P.B. and C.C. Cornford, J. Chromatogr., 53 403 (1970). 150) R m m o n s , D.L. et al., J. Chromatogr., 43 141 (1969). 55 1313 151) Simmons, D.K. et al., J. Pharm. Sci., (1966). 152) Dijkhuis, I . C . and M.E. Brommet, J.Pharm. Sci., 59 559 (1970). 153) Kapadic G.J. and G.S. Rao, J. Pharm. Sci., 53 223 (1964). 154) Sonanini, D. and L. Anker, Pharm. Acta Helv., 39 518 (1964).


65 I

155) Fernandez, A.A. et al., J. Pharm. Sci., 58 443 (1969). 59 156) Gyanchandani, N.D. et al., J. Pharm. Sci., 224 (1970). 64 1681 157) Willekens, G.J., J. Pharm. Sci., (1975). 158) Langner, H.J. and U. Teufel, J. Chromatogr., 78 445 (1973). 159) E n Hoeck, G. et al., Pharm. Acta Helv., 47 316 (1972). 160) Ragazzi, E. and G. Veronese, J. Chromatogr., 132 105 (1977). 151 256 (1978). 161) Szabo, A., J. Chromatogr., 205 486 162) Omer, A.I.H. et al., J. Chromatogr., (1981). 163) Massa, V. et al., Trav. SOC. Pharm., Montpellier 35 373 (1975). 164) Keiner, J. etal., Pharm. Zentralhalle 105 705 (1966). 165) Keiner, J. et al., Pharm. Zentralhalle 108 525 (1969). 166) Simmons, D.L. et al., J. Pharm. Sci., 55 219 (1966). 167) Simmons, D.L. et al., J. Pharm. Sci., 55 1313 (1966). 56 1393 168) Ascione, P.P. et al., J. Pharm. Sci., (1967). 169) Nicolaus, B.J.R. et al., Farmaco Ed. Prat. 16 349 (1961). 170) Nicolaus B.J.R. et al., Experientia 17 473 (1961). 42 552 171) Zuidweg, M.H.J. et al., J. Chromatogr., (1969). 172) Butterfield, A.G. et al., Antimicrob. Agts. chromatogr., 4 11 (1973). 173) White, E.R. et al., J. Antibiot., 28 205 (1975). 174) Nilsson-Ehle, I. et al., Antimicrob. Agts. Chemother., 9 754 (1975). 175) Knox, J.H. and J. Jurand, J. Chromatogr., 110 103 (1975). 176) Knox, J.H. and J. Jurand, J. Chromatogr., 186 763 (1979). 64 177) Butterfield, A.G. et al., J. Pharm. Sci., 316 (19751. 178) Tusij, K.-and J.H. Robertson, J. Pharm. Sci. , 6 5 400 (1976). 179) f i o x , J.H. and A. Pryde, J. Chromatogr., 112 171 (1975).

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180) Chevalier, G. et al., J . Chromatogr., 124 343 (1976). 181) De Leenheer, A.P. and H.J.C.F. Nelis, J. Chromatogr., 140 293 (1977). 182) Mack, G.D. a n m . B . Ashworth, J. Chromatogr., Sci., 16 93 (1978). 183) Sharma7J.P. et al., J. Pharm. Sci., 66 1319 (1977). 184) Sharma, J.P. et al., J. Chromatogr., __ 134 441 (1977). 185) Sharma, J.P. and E.F. Bevill, J. Chromatogr., 166 213 (1978). 186) S.L. and Th. Strittmatter, Int. J. Pharm., 1 145 (1978). 187) Lindauer; R . F . et al., Anal. Chem., 48 1731 (19761. 188) Eksborg, S . , J. Chromatogr., 208 78 (1981). 189) Nelis, H.J.C.F. and A.P. De Leenheer, J. Chromatogr., 195 35 (1980). 190) Muhammad, N. and J.A. Bodnar, J . Pharm. Sci., 69 928 (1980). 191) m c k e r , R . and C.J. Estler, Arzneim.-Forsch., 29 1690 (1979). 192) Hermansson, J. and M. Andersson, J. Pharm. Sci., 71 222 (1982). 193) Eksborc S. et al., J. Chromatogr., 185 583 (1979). 194) Eksborg, S . and B. Ekqvist, J. Chromatogr., 209 161 (1981). 195) Dihuidi, K. et al., J. Chromatogr., 246 350 (1982). 196) Krestynova-Telupilova, 0 . et al., Chem. Listy, 47 636 (1953). 197) Eechoslovak Pharmacopoeia 11, page 185 (19541, Prag. 198) Doskocilova, D., Die Pharmazie 13 548 (1958). 199) Faith, L . , Farmac. Obzor 3 2 52 n963). 2 0 0 ) Parrak, V . et al., Die PhEmazie 21 418 (1966) 201) Chatten, L.G. et al., J. Pharm. S S . , 65 1315 (1976). 202) Hetman, J.S., Lab. Pract., 12 727, 740 (1963). 2 0 3 ) Doan, L. and B.E. Riedel, Canad. Pharmac. J . , Sci. Sect..* - 96 109 (1963). 204) Caplis, M.E. et al.; J . Pharm. Sci., 54 694 (1965). 205) Unterman, H.W. and S . Weissbuch, Die Pharmazie 29 752 (1974). -

m,

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653


VITAMIN D3 (CHOLECALCIFEROL) K. Thoinas Koshy and William F. Beyer The Upjohn Coinpaizy

Kalnmazoo, Michigan I.

2.

3.

4.

5.

6.

Description 1.1 Name 1.2 Formula and Molecular Weight 1.3 Storage and Handling I .4 Brief History 1.5 Biosynthesis 1.6 Potency Definition Physical Properties 2.1 Spectra 2.2 Crystal Properties 2.3 Solubility 2.4 Optical Rotation Chemical Properties 3. I Chemical Synthesis 3.2 Previtamin D3-Vitamin D3 Equilibrium 3.3 Cis-Trans Equilibrium 3.4 Other Chemical Reactions Methods of Analysis 4. I Identification Tests 4.2 Quantitative Methods Metabolites of Vitamin D3 5.1 Primary Metabolites 5.2 Secondary Metabolites 5.3 Metabolites of Very Low or Unknown Biological Activity Vitamin D3 as Prohormone References

ANALYTICAL PROFILES OF DRUG SUBSTANCE5 VOLUME 13

655

656 656 656 656 656 658 658 660 660 668 672 672 672 672 677 677 679 679 679 680 70 1 70 1 704 705 705 707

Copyright 0 1984 by the Americae Phannaceutlcal Association ISBN 0-12-260813-5

K . THOMAS KOSHY AND WILLIAM F. BEYER

656

1.

Description

1.1

Name: V i t a m i n D Generic Name: C2olecal c i f e r o l Chemical Name: 9,lO-Secochol esta-5,7 , l o ( 1 9 ) - t r i en38-01 ,(38,52,7E)-,

1.2

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

The chemical s t r u c t u r e o f v i t a m i n 03 i s c l o s e l y r e 1ated t o i t s p r e c u r s o r , 7-dehydrochol ester01 , from which i t i s produced by a photochemical r e a c t i o n . Therefore, vitamin D i s closely related s t r u c t u r a l l y t o the four-ring nucleus o! s t e r o i d s d e r i ved from t h e c y c l opentanoperhydrophenanthrene r i n g system. No v i t a m i n D a c t i v i t y i s n o t i c e d u n t i l t h e B r i n g o f 7-dehydrocholesterol i s opened between C-9 and C-10. Thus, v i t a m i n D3 i s a 9,lO-seco s t e r o i d and i t s carbon s k e l e t o n i s numbered a c c o r d i n g l y (Scheme I ) . The i m p o r t a n t aspects o f i t s c h e m i s t r y c e n t e r about t h e 5,6,7c i s - t r i e n e s t r u c t u r e . The formula f o r v i t a m i n D3 i s C27H44O and i t s formula weight i s 384.64. 1.3

Storage and Handling

C r y s t a l l i n e v i t a m i n D3 i s s t o r e d i n h e r m e t i c a l l y sealed c o n t a i n e r s under n i t r o g e n i n a cool p l a c e and prot e c t e d from l i g h t . Under such c o n d i t i o n s , d e y r a d a t i o n i s n e g l i g i b l e f o r one year. V i t a m i n D 3 e x i s t s i n thermal e q u i l i b r i u m w i t h p r e v i t a m i n D3 and t h e r a t e o f e q u i l i b r a t i o n markedly i n c r e a s e s w i t h temperature and t i m e ( 1,Z). 1.4

Brief History

The symptoms o f v i t a m i n D d e f i c i e n c y disease has been documented i n 1 6 t h c e n t u r y l i t e r a t u r e . A c l e a r p i c t u r e o f t h e b a s i s f o r t h e disease and methods o f t r e a t m e n t were u n c l e a r u n t i l t h e experiments by S i r Edward Mellanby (3,4). I n t h e e a r l y 1920's, cod l i v e r o i l was known t o c u r e r i c k e t s and xerophthalmia. The name v i t a m i n D was g i v e n t o

VITAMIN D3 (CHOLECALCIFEROL)

657

H

SCHEME I:

Vitamin D3 structure with hydrocarbon number assignments.

t h e a n t i r a c h i t i c substance i n cod l i v e r o i l ( 5 - 7 ) and d i f f e r e n t i a t e d from v i t a m i n A by t h e f a c t t h a t v i t a m i n A a c t i v i t y i n t h e o i l was l o s t by h e a t i n g w h i l e t h a t o f D was not . Hess and Steenbock d i s c o v e re d , i n d e p e n d e n t l y ( 8 - l l ) , t h a t t h e a n t i r a c h i t i c a c t i v i t y o f t h i s v i t a m i n c o u l d be induced by UV i r r a d i a t i o n o f r a c h i t i c animals o r t h e i r food. Steenbock's d i s c o v e r y t h a t a n t i r a c h i t i c a c t i v i t y c o u l d be induced by i r r a d i a t i o n o f foods, p a r t i c u l a r l y o f the sterol fraction, ultimately led t o the i d e n t i f i c a t i o n o f t h e s t r u c t u r e o f v i t a m i n D and t o t h e e r a d i c a t i o n o f r i c k e t s as a major medical problem (9,10,12).

K . THOMAS KOSHY A N D WlLLIAM F. BEYER

658

It was l a t e r d i s c o v e r e d t h a t v i t a m i n D occurs i n two a c t i v e forms, e r g o c a l c i f e r o l and c h o l e c a l c i f e r o l . Ergocalc i f e r o l i s t h e s y n t h e t i c form d e r i v e d by t h e i r r a d i a t i o n o f e r g o s t e r o l and i s designated as v i t a m i n D2 (13,14). Cholec a l c i f e r o l , t h e n a t u r a l form, was i d e n t i f i e d (15,16) and designated as v i t a m i n D3. Vitamins D and D3 a r e e q u a l l y a c t i v e i n humans and o t h e r mammals, a u t D2 i s v i r t u a l l y inactive i n poultry.

1.5

B i osynthesis

It has been known f o r a l o n g t i m e t h a t v i t a m i n D3 i s synthesized i n t h e s k i n when exposed t o s u n l i g h t from 7d e h y d r o c h o l e s t e r o l (7-DHC, P r o v i t a m i n D ). The sequence o f steps l e a d i n g t o t h e cutaneous photosyn?hesis o f v i t a m i n D3 from 7-DHC was r e c e n t l y demonstrated (17). I n Caucasian s k i n , a l l of t h e epidermal s t r a t a c o n t a i n 7-DHC. When ir r a d i ated, 7-DHC i s photochemical l y converted by a f a s t r e a c t i o n t o p r e v i t a m i n D throughout t h e epidermis and t h e dermis. Once formed i n ?he s k i n , p r e v i t a m i n D3 i s isomeri z e d t o D by a slow nonphotochemical rearrangement a t a r a t e d i c t a z e d by s k i n temperature. V i t a m i n D3 i s then bound t o a s p e c i f i c b i n d i n g p r o t e i n (DBP) and t r a n s p o r t e d from t h e s k i n i n t o t h e c i r c u l a t i n g blood. Thus t h e s k i n serves as t h e source o f 7-DHC, a r e s e r v o i r f o r t h e s t o r a g e o f t h e p r i m a r y photoproduct, p r e v i t a m i n D3, and t h e organ where t h e slow thermal conversion o f p r e v i t a m i n D3 t o D3 occurs. The t h i r d process p e r m i t s t h e s k i n t o c o n t i n u o u s l y s y n t h e s i z e D3 and r e l e a s e i t i n t o t h e c i r c u l a t i o n f o r up t o 3 days a f t e r a s i n g l e exposure t o s u n l i g h t . Scheme I 1 (17) i l l u s t r a t e s t h i s sequence o f events.

1.6

Potency D e f i n i t i o n

The o r i g i n a l r e f e r e n c e s t a n d a r d o f v i t a m i n D was a s o l u t i o n of i r r a d i a t e d e r g o s t e r o l i n o l i v e o i l . The present i n t e r n a t i o n a l standard i s a s o l u t i o n o f p u r e c r y s t a l l i n e c h o l e c a l c i f e r o l i n o l i v e o i l c o n t a i n i n g 0.025 pg/mg o f solution. One i n t e r n a t i o n a l u n i t ( I U ) i s e q u i v a l e n t t o 0.025 pg o f c r y s t a l l i n e D3 o r 1 pg o f D3 i s e q u i v a l e n t t o 40 IU. The USP r e f e r e n c e standards a r e c r y s t a l 1ine e r g o c a l c i f e r o l o r c h o l e c a l c i f e r o l packaged i n sealed ampules. The USP standards and t h e i n t e r n a t i o n a l standards a r e e q u i v a l e n t i n t h a t 1 pg o f each i s e q u i v a l e n t t o 40 IU.

HjC 7-Dehydrocholesterol

Previtamin D3

A,c

H?

Vitamin D3

Blood

DBP

SCHEME 11:

Diagram of the formation of previtamin D3 in the skin, its thermal conversion to vitamin D 3 , and transport by binding proteins ( D B P ) in plasma into the general circulation (Reference 17). From ref. 17, copyright 1980 by the AAAS.


660

2.

Physical Properties 2.1

Spectra

2.11 U l t r a v i o l e t Spectrum F i g u r e 1 i s t h e u l t r a v i o l e t spectrum o f a 10 mcg/ml s o l u t i o n o f v i t a m i n D3 i n methanol. The spectrum was o b t a i ned u s i n g a Cary Model 219 r e c o r d i ng spectrophotometer Palo A l t o , CA). Vitamin D3 and ( V a r i a n Instrument Co., r e l a t e d compounds have a c h a r a c t e r i s t i c UV a b s o r p t i o n maximum a t 265 nm and a minimum a t 228 nm. The e x t i n c t i o n c o e f f i c i e n t a t 265 nm i s about 17,500 and 15,000 a t 254 nm. An i n d e x o f p u r i t y o f v i t a m i n D3 i s a value o f 1.8 f o r t h e r a t i o o f t h e absorbance a t 265 t o t h a t a t 228 nm. The h i g h absorbance a t 254 nm enables one t o use t h e most common and s e n s i t i v e s p e c t r o p h o t o m e t r i c d e t e c t o r used i n h i g h performance 1 i q u i d chromatography (HPLC) f o r t h e a n a l y s i s o f v i t a m i n D3 i n m u l t i v i t a m i n p r e p a r a t i o n s , f o r t i f i e d m i l k , o t h e r food products, animal feed a d d i t i v e s e t c . 2.12

I n f r a r e d Spectrum

The i n f r a r e d spectrum o f a m i n e r a l o i l m u l l o f v i t a m i n D3 recorded on a Model FTS 15E i n s t r u m e n t ( D i g i l a b , Cambridge, Mass.) i s shown i n F i g u r e 2 (18). Table I shows a s s i ynments f o r t h e s i g n i f ic a n t in f r a r e d a b s o r p t i on bands (18) 2.13 Nuclear Magnetic Resonance Spectram P r o t o n and C-13 NMK s p e c t r a o f v i t a m i n D3 r e s p e c t i v e l y a r e shown i n F i g u r e s 3 and 4 (19). Spectra were o b t a i n e d w i t h a Model XL-200 NMR spectrophotometer (Varian, Palo A l t o , CA). Tables I1 and I11 a r e t h e corresponding chemical s h i f t s i n t h e p r o t o n and C-13 NMR s p e c t r a which are based on t h e assignments made by Wing e t a1 , (20) and W i l l i a m s e t a1 (21) , r e s p e c t i v e l y .

.

'9

0

0

0

100

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

1

I

I

I

I

I

I

I

I

I

I

I

I

I

1

I

I

I

1

I

n

s W

w

0

z

a

-B

k 50 I-

to z a Pe

I-

0 3800

I

I I I I I

3000

I

2000 1800 1600 1400

1200 1000

FREQUENCY (CM-') FIGURE 2:

I n f r a r e d spectrum o f a m i n e r a l o i l m u l l o f v i t a m i n

D3.

800

600

663

0

0

cv

t

0

0 (0

0 Qo

~

0 0 n m

' 5

o a ' cv 0

'

t

0

'

(0

Qo

0 r

cv

0 0

0

cv cv

VITAMIN Di (CHOLECALCIFEROL)

665

TABLE I

I n f r a r e d Band Assignments f o r V i t a m i n D? (Ref. 18)

Wave Numbers cm-’

Structural Feature

Assignment

3289

a1 cohol

0-H s t r e t c h

3055, 3031

a1 kenes

C-H s t r e t c h

1648, 1634, 1604

Conjugated a1 kenes

C=C s t r e t c h

1053

secondary a1 cohol (equitorial)

C-0 s t r e t c h

895

a1 kene R 1 R 2 C=CH2 t y p e

C-H Out O f plane bending

968, 942, 888 and/or 860

a1 kene

C-H Out O f p l a n e bending

3077,

K.THOMAS KOSHY AND WILLIAM F. BEYER

666

TABLE I 1 Proton NMR S p e c t r a l Assignment f o r V i t a m i n D,

(Ref. 20)

Assignment

Chemical Shift

18 methyl

0.54

Singlet

27 methyl

0.85

Doublet

26 methyl

0.88

Doublet

21 methyl

0.92

Doublet

4 a H

2.55

Dou b l e t Doublet

9 B H

2.80

Doublet Doublet

3 a H

3.92

Mu1t i p l e t

19 E H

4.83

Doublet

19 Z H

5.05

Doublet of Triplet

7 H

6.03

Doublet

6 H

6.24

lloubl e t

Shape

VITAMIN D? (CHOLECALCIFEROL)

667

TABLE I 1 1

13C Chemical S h i f t s o f V i t a m i n D2 a t 200 MHz i n 070 ( R e f . 21) Carbon

Chemi c a l Shifts

Carbon

No.

No.

Chemical Shifts

1

31.95

14

56.33

2

35.15

15

23.58

3

69.17

16

27.66

4

45.90

17

56.56

5

145.05

18

12.00

6

122.41

19

112.41

7

117.47

20

36.14

8

142.26

21

18.84

9

29.02

22

36.14

10

135 '04

23

23.86

11

22.25

24

39.49

12

40.52

25

28.01

13

45.83

26

22.83

27

22.56

K. T H O M A S K O S H Y AND WILLIAM F. BEYER

668

2.14 Mass Spectrum The e l e c t r o n impact mass spectrum o f v i t a m i n D3 o b t a i n e d w i t h a CEC Model 21-llOB i n s t r u m e n t ( C o n s o l i dated Electrodynamics Corporat ion , Pasadena, CA) by d i r e c t probe sample i n t r o d u c t i o n i s shown i n F i g u r e 5 (22). It shows a s t r o n g m o l e c u l a r i o n a t m/z 384 and a f r a g m e n t a t i o n p a t t e r n c h a r a c t e r i s t i c o f v i t a m i n s D2 and D3, t h e i r p r i m a r y metabol it e s and o t h e r chemical l y re1 a t e d compounds. The fragment i o n assignment i s shown i n Scheme I11 (22). 2.2

Crystal Properties 2.21 M e l t i n g Range

C r y s t a l l i n e v i t a m i n D3 o b t a i n e d as f i n e needl e s from d i l u t e acetone m e l t s i n t h e range o f 84-88". 2.22 C r y s t a l Conformation Trinh-Toan e t a l . ( 2 3 ) have shown by x-ray d i f f r a c t i o n a n a l y s i s t h a t v i t a m i n D3 c r y s t a l l i z e s i n an equimolar r a t i o of t h e a c h a i r f o r m i n which t h e 19-CH2 group i s s i t u a t e d below t h e mean A r i n g p l a n e and t h e p c h a i r form i n which t h e 19-CH2 group i s s i t u a t e d above t h e mean A r i n g plane. The 3-OH s u b s t i t u e n t occupies an equit o r i a l p o s i t i o n i n t h e a form and an a x i a l p o s i t i o n i n t h e p form. F i g u r e 6 shows t h e two independent molecules o f v i t a m i n D3 d i s p l a y i n g d i f f e r e n t s o l i d - s t a t e conformations o f the A ring. These s o l i d - s t a t e conformations i n t h e A r i n g correspond t o t h o s e i n f e r r e d from t h e ' H NMR i n v e s t i g a t i o n s on t h e s o l u t i o n conformations o f v i t a m i n s D2 and D3 (24) and several metabol i t e s i n c l u d i n g 1 ~ , 2 5 - ( 0 H ) ~ - v i t a r n i n D ~(2527). The o b s e r v a t i o n t h a t b o t h t h e mean t o r s i o n a l angles o f 53.8 and 50.1 i n r i n g A o f molecules a and @, r e s p e c t i v e l y , a r e small e r t h a n t h e correspondi ny experimental v a l u e o f 55.9" found i n cyclohexane i s a t t r i b u t e d t o t h e f l a t t e n i n y e f f e c t caused by t h e two e x o c y c l i c double bonds (23). Trinh-Toan e t a l . (23) have a l s o r e p o r t e d t h a t t h e r e i s s t r o n g hydrogen bonding between t h e a and p c o n f o r m a t i o n a l forms. The molecules are w e l l separated i n t h e u n i t c e l l w i t h a l l i n t e r m o l e c u l a r nonhydrogen c o n t a c t s b e i n g g r e a t e r t h a n 3.4 A except f o r t h e r e l a t i v e l y s h o r t O-H--0 d i s t a n c e s o f 2.71 t o 2.73 A which i n d i c a t e reasonably s t r o n g OH--0 bonds. The a u t h o r s conclude t h a t t h e c r y s t a l packing o f v i t a m i n D3 i n c l u d i n g t h e presence o f b o t h conformers i s m a i n l y d i c t a t e d by s t e r i c c o n s t r a i n t s imposed by t h e i n f i n it e he1 ic a l hydrogen-bonded oxygen chain.

0

5:

0

m

0

Im

0 3

IlISN31NI 3 h I l M 3 U

N

0

I

0 In

m

0

0

-

S 0 .C

d l n

Low

0

0

sce L L

OaJ LI

V E

L

aJ0 7

wce

670


- - tl- - - - 1 I

I 1 I I

I

i ,136

I I 1 I I

Scheme 111: Mass s p e c t r a l fragmentation p a t t e r n of vitamin D (from r e f e r e n c e 22. Copyright 0 1972. Reprinted by per3 mission of John Wiley & Sons, Inc.)

U

Q t s 4-u

cr)

n . m E 54-

C N *r

E

4aJ L >

-7

4-

O L

- 0

v)

.-

E S L O 0


672

2.23 Thermal A n a l y s i s D i f f e r e n t i a l scanning c a l o r i m e t r i c (DSC) and t h e t h e r m o g r a v i m e t r i c (TGA) curves f o r v i t a m i n D3 a r e shown i n F i g u r e s 7 and 8 ( 2 8 ) . The curves were generated on a DuPont thermal a n a l y z e r (Model No. 1090, DuPont DeNemours and Co., Wilmington, Del .). The sample was c o n t a i n e d i n aluminum pans and t h e a n a l y s i s was conducted under N2 atmosphere. The h e a t i n g r a t e f o r b o t h was 5"Imin. There i s a p r e c i s e m e l t i n g endotherm i n t h e DSC c u r v e which peaks a t 86.3'. The exotherm a t 206' may be due t o decomposition. The TGA curve shows a l i t t l e w e i g h t g a i n i n t h e m e l t i n g range o f 84-88' when t h e sample volume i s reduced and t h e buoyancy e f f e c t o f N i s a l t e r e d , The sharp drop i n w e i g h t a t 128' may be due f o b o i l i n g and p o s s i b l e s p l a t t e r i n g of t h e sample.

2.24 X-Ray D i f f r a c t i o n F i g u r e 9 i s t h e x-ray powder d i f f r a c t i o n p a t t e r n o f c r y s t a l 1i n e v i t a m i n D3 (USP r e f e r e n c e standard) ( 2 9 ) . The values f o r t h e 2 8 angles and t h e corresponding "d" spacings a r e shown i n Table I V .

2.3

Solubility

The s o l u b i l i t y o f c r y s t a l l i n e v i t a m i n D (99 + % pure, A l d r i c h Chemical Company, I r ~ c . , Milwaukee, d i s c o n s i n ) was determined i n our l a b o r a t o r i e s i n commonly used l a b o r atory solvents. It had o v e r 10% s o l u b i l i t y i n methanol, ethanol, acetone, e t h y l a c e t a t e , hexane, chloroform, methyl ene c h l o r i de, d i met hyl f ormami de, c y c l ohexane, t e t rahydrof u r a n , m e t h y l e t h y l ketone, i s o p r o p a n o l and cyclohexanone. The o n l y s o l v e n t w i t h low s o l u b i l i t y was a c e t o n i t r i l e i n which t h e s o l u b i l i t y was about 0.18%.

2.4

Optical Rotation

The s p e c i f i c r o t a t i o n o f a f r e s h l y prepared 5 mg/ml ethanol s o l u t i o n o f v i t a m i n D3 i s between +100.5' and +112"

(30) 3.

Chemical P r o p e r t i e s Chemical Synthesis

3.1 ation

of

V i t a m i n D3 i s c h e m i c a l l y synthesized by t h e i r r a d i 7-DHC and subsequent c o n t r o l l e d h e a t i n g o f t h e

1

2

-t

0 A

3

E -1

Y

%

0 I I&

-2

l-

a

I -3 -4 -5

40

80

120

160

200

240

280

320 360

TEMPERATURE ( " C ) FIGURE 7:

D i f f e r e n t i a l scanniny c a l o r i m e t r i c curve f o r v i t a m i n D3.

400 440

480

614

0

d

r

0 0

0 0 0

0

r -

0 (D

v)

0

0

e 0 0

> L 0

Ic

P L

= I

v

.. W

w

Y 3 W U

AlISN31NI 3 A I l V l 3 t l 675

0 d

8

N

mi W W K

>

.r

0

L

c

rc

L

a, c,

5 L

c, V


676

TABLE I V X-Ray Powder D i f f r a c t i o n P a t t e r n o f V i t a m i n

28 4.89 5.05 6.60 8.60 8.85 9.20 10.15 13.00 13.59 14.25 14.95 15.15 15.75 16.15 16.65 18.05 18.65 19.35 21.79 22.85 23.35 23.65 25.10 25.60 27.05 27.75 29.80 31.30 31.90 33.65 38.45

d-spaci ng ( A )

18.06 17.48 13.38 10.27 9.98 9.60 8.71 6.80 6.51 6.21 5.92 5.84 5.62 5.48 5.32 4.91 4.75 4.58 4.08 3.89 3.81 3.76 3.54 3.48 3.29 3.21 3.00 2.86

D2 (Ref. 29) Re1a t i ve Intensity 4 3

VITAMIN D j (CHOLECALCIFEROL)

677

previtamin. A number o f by-products a r e formed, most o f which a r e removed d u r i n g t h e clean-up steps. Scheme I V shows t h e s t r u c t u r e s o f t h e known i m p u r i t i e s and d e g r a d a t i o n p r o d u c t s (31-33) t h a t may be p r e s e n t i n t h e s y n t h e t i c v i t a min D3 concentrates. Some o f t h e by-products have b i o l o g i c a l a c t i v i t y b u t o t h e r s such as l u m i s t e r o l , t h e s u p r a s t e r o l s , and t h e pyro- and isopyrocal c i f e r o l s have no anti rachitic activity. T a c h y s t e r o l and t r a n s - v i t a m i n D3 have o n l y s l i g h t a n t i r a c h i t i c a c t i v i t y . Thus, the b i o l o g i c a l a c t i v i t y i s p r i m a r i l y due t o v i t a m i n D and p r e v i t a m i n 0. The b i o l o g i c a l a c t i v i t y o f p r e v i t a m i n 3I? i s a t t r i b u t e d t o i t s i n v i v o c o n v e r s i o n t o v i t a m i n D3. It may be mentioned t h a t analogous compounds are formed d u r i n g t h e The two forms s y n t h e s i s o f v i t a m i n U2 from e r g o s t e r o l . d i f f e r o n l y i n t h e s i d e chain. V i t a m i n D2 has an e x t r a methyl group on C-24 and a double bond between C-22 and 23. 3.2

P r e v i t a m i n D-,-

-

-.

V i t a m i n D-, E q u i l i b r i u m

The thermodynamics and k i n e t i c s o f t h e thermal e q u i l i b r i u m between p r e v i t a m i n D3 and v i t a m i n D3 have been s t u d i e d (34,35). The i s o m e r i z a t i o n o f p r e v i t a m i n D3 t o v i t a m i n D3 i s an exothermic f i r s t o r d e r r e a c t i o n . The v i t a m i n D 3 / p r e v i t a m i n D3 e q u i l i b r i u m r a t i o depends on t h e t e m p e r a t u r e and can be c a l c u l a t e d from t h e a p p r o p r i a t e e q u i l i b r i u m and k i n e t i c c o n s t a n t s r e p o r t e d by Hanewald e t a l . (36). The r a t e c o n s t a n t s f o r t h e e q u i l i b r i u m have been shown t o be independent o f t h e n a t u r e o f t h e s o l v e n t , o f a c i d i c o r b a s i c c a t a l y s i s and o f f a c t o r s known t o a f f e c t f r e e r a d i c a l process (37,38). The percentages o f v i t a m i n D3 i n e q u i l i b r i u m w i t h p r e v i t a r n i n D3 ranges from 98% a t -20" t o 78% a t 80'. Thus, when v i t a m i n D3 i s s t o r e d i n t h e c o l d , t h e e q u i l i b r i u m c o n s t a n t h i n d e r s t h e conversion t o p r e v i t a min D3. 3.3

Cis-Trans E q u i l i b r i u m

The 5,6,7-cis-triene configuration of vitamin D i s i m p o r t a n t f o r i t s b i o l o g i c a l a c t i v i t y as t h e 5,6-trans has v e r y low a c t i v i t y . Exposure t o i o d i n e i n non-polar s o l v e n t s under d i f f u s e l i g h t (39,40) or t o mild a c i d i c c o n d i t i o n s (41) a f f o r d s f o r m a t i o n o f t h e 5,6-trans isomer. The r e v e r s e t r a n s f o r m a t i o n occurs p h o t o c h e m i c a l l y (42). The 5,6-trans isomer can undergo f u r t h e r i s o m e r i t a t i o n s upon exposure t o heat (43) o r a c i d s o r t r e a t m e n t w i t h antimony trichloride. The c i s and t h e t r a n s forms o f v i t a m i n D3 d i s p l a y c h a r a c t e r i s t i c u l t r a v i o l e t p r o p e r t i e s . The c i s form as shown e a r l i e r has a UV maxima a t 265 nm. The t r a n s form has a UV maxima a t 273 nm.

?arm

lsotachysterol D 3

Transvitamin D3

lsovitamin D3

R

no Provitamin D3

no Prewitomin D 3

Pholoisopyro D3

t

Pholopyro D3

Vitamin D3

no Tachysterol D3

no Lumisterol D 3

Pyro D3

HO

lsopyro D3

Suprasterol D3

Scheme I V . P h o t o c h e m i c a l t h e r m a l , and c h e m i c a l r e a c t i o n pathways i n t h e s y n t h e s i s o f v i t a m i n D3 ( r e f e r e n c e s 31,33). From J . Pharm. S c i . 71, 137 (1982). Reproduced w i t h p e r m i s s i o n o f t h e c o p y r i g h t owner.

VlTAMlN Dz (CHOLECALCIFEROL)

3.4

679

Other Chemical Reactions

V i t a m i n D3 can undergo v e r y complex chemical react i o n s upon exposure t o heat, l i g h t and chemical reagents. It i s beyond t h e scope o f t h i s a r t i c l e t o r e v i e w t h e s e r e a c t i o n s . The reader i s r e f e r r e d t o r e c e n t reviews on t h e s u b j e c t (99,100).

4.

Methods o f A n a l y s i s

The a n a l y s i s o f v i t a m i n O3 i s d i f f i c u l t f o r t h e f o l l o w i n g reasons: a. As shown i n Scheme I V a number o f by-products a r e formed d u r i n g t h e chemical s y n t h e s i s o f v i t a m i n D3, some o f which may be p r e s e n t i n t h e f i n i s h e d b u l k form. V i t a m i n D3 c o n c e n t r a t e s are commerci a1 l y a v a i 1a b l e b. i n a r e s i n form i n peanut o i l and as a d r y g e l a t i n beadlet. The v i t a m i n D3 c o n c e n t r a t e s u s u a l l y c o n t a i n o t h e r o i l s o l u b l e v i t a m i n s , e s p e c i a l l y v i t a m i n A. The v i t a m i n D3, t h e r e f o r e , has t o be r e l e a s e d from i t s m a t r i x which i s accomplished by s a p o n i f i c a t i o n and e x t r a c t i o n . It t h e n has t o be analyzed by a method t h a t would d i s t i n g u i s h i t from a1 1 i n t e r f e r i n g isomers and o t h e r i n g r e d i e n t s . e x i s t s i n thermal e q u i l i b r i u m w i t h c. Vitamin D p r e v i t a m i n D3 (See s e c t i o n 3.2). There i s always u n c e r t a i n t y o f t h e e x t e n t o f t h i s r e a c t i o n even when a v i t a m i n D3 s t a n d a r d i s t r e a t e d under t h e same c o n d i t i o n s as t h e sample. d. The c o n c e n t r a t i o n s o f v i t a m i n D3 i s low i n food p r o d u c t s such as f o r t i f i e d m i l k , m i l k formulae, b r e a k f a s t c e r e a l s , animal feeds e t c . These samples, t h e r e f o r e , need e x t e n s i ve c l ean-up p r i o r t o a n a l y s i s .

4.1

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

The USP XX (30) d e s c r i b e s t h e f o l l o w i n g i d e n t i f i c a t i o n t e s t s f o r v i t a m i n D3, which a r e reproduced verbatum: a. The i n f r a r e d a b s o r p t i o n spectrum o f a potassium bromide d i s p e r s i o n o f it, i n t h e range o f 2 p t o 12 pm, e x h i b i t s maxima o n l y a t t h e same wavelengths as t h a t o f a s i m i l a r p r e p a r a t i o n o f USP C h o l e c a l c i f e r o i RS.


680

b. The u l t r a v i o l e t a b s o r p t i o n spectrum o f a 1 i n 100,000 s o l u t i o n i n a l c o h o l e x h i b i t s maxima and minima a t t h e same wavelengths as t h a t o f a s i m i l a r s o l u t i o n o f USP Cholecal c i f e r o l RS, c o n c o m i t a n t l y measured, and t h e respect i v e a b s o r p t i v i t i e s a t t h e wavelength o f maximum absorbance a t about 265 nm do n o t d i f f e r by more t h a n 3.0%. c. To a s o l u t i o n o f about 0.5 my i n 5 m l c h l o r o f o r m add 0.3 m l o f a c e t i c anhydride and 0.1 m l s u l f u r i c acid, and shake v i g o r o u s l y ; a b r i g h t r e d c o l o r produced, and i t r a p i d l y changes through v i o l e t and b l u e green.

of of is to

d. Prepare w i t h o u t h e a t i n g , and handle w i t h o u t delay, a 1 i n 100 s o l u t i o n o f squalane i n c h l o r o f o r m cont a i n i n g 50 mg o f c h o l e c a l c i f e r o l p e r m l , and p r e p a r e a Standard s o l u t i o n o f USP C h o l e c a l c i f e r o l RS i n t h e same s o l v e n t and having t h e same c o n c e n t r a t i o n . Spot 10 p1 o f t h e t e s t s o l u t i o n and 10 p l o f t h e Standard s o l u t i o n on a l i n e p a r a l l e l t o and about 2.5 cm from t h e bottom edge o f a t h i n - 1 ayer chromatographic p l a t e (See ) coated w i t h a 0.25 mn l a y e r o f chromatographic s i l i c a gel m i x t u r e . Place t h e p l a t e i n a developing chamber c o n t a i n i n g and e q u i l i b r a t e d w i t h a m i x t u r e o f equal volumes o f cyclohexane and d i e t h y l ether. Develop t h e chromatogram u n t i l t h e s o l v e n t f r o n t has moved about 15 cm above t h e l i n e o f a p p l i c a t i o n . Perform t h e development and subsequent o p e r a t i o n s i n t h e dark. Remove t h e p l a t e , a l l o w t h e s o l v e n t t o evaporate, and spray w i t h a 1 i n 50 s o l u t i o n o f a c e t y l c h l o r i d e i n antimony t r i c h l o r i d e TS: t h e chromatogram o b t a i n e d w i t h t h e t e s t s o l u t i o n shows a y e l l o w i s h orange area ( c h o l e c a l c i f e r o l ) h a v i n g t h e same Rf v a l u e as t h e area o f t h e Standard s o l u t i o n , and may show below t h e c h o l e c a l c i f e r o l area a v i o l e t area a t t r i b u t e d t o 7-dehydrocholesterol .

4.2

Q u a n t i t a t i v e Methods

Since v i t a m i n D3 i s i n c o r p o r a t e d i n t o a number o f food a d d i t i v e s f o r human and animal consumption, t h e r e i s no one method t h a t may be b e s t s u i t a b l e f o r a l l such products. Therefore, several a n a l y t i c a l systems are d e s c r i b e d here t o f i t t h e need f o r a s p e c i f i c s i t u a t i o n .

4.2.1

B i o l o g i c a l Methods

B i o l o g i c a l methods a r e t h e o l d e s t f o r t h e a n a l y s i s o f v i t a m i n D3 and t h e one t h a t i s a p p l i c a b l e f o r a l l t y p e s o f samples. The b i o l o g i c a l methods can be d i v i d e d

VITAMIN

D3

68 I

(CHOLECALCIFEROL)

i n t o t h r e e groups: c u r a t i v e , p r o p h y l a c t i c , and t h o s e based on c a l c i u m a b s o r p t i o n i n t o t h e b l o o d stream. A l l are based on t h e a d m i n i s t r a t i o n o f measured doses o f a standard v i t a min D3 p r e p a r a t i o n t o a group o f t e s t animals and comparison o f t h e b i o l o g i c a l responses w i t h a s i m i l a r group g i v e n t h e substance under t e s t . A t h i r d group o f t e s t animals i s used as c o n t r o l s . The b i o l o g i c a l methods are p o p u l a r f o r t h r e e reasons: 1) i n most b i o l o g i c a l samples t h e c o n c e n t r a t i o n of v i t a m i n D3 i s v e r y low and i s n o t s u i t a b l e f o r more s p e c i f i c methods because o f i n t e r f e r i n g m a t e r i a l s even a f t e r e x t e n s i v e c l e a n up; 2) v i t a m i n D3 i s e f f e c t i v e b i o l o g i c a l l y i n t r a c e amounts making i t more s e n s i t i v e than t h e b e s t chemical methods; and 3) t h e b i o l o g i c a l methods a r e s p e c i f i c f o r v i t a m i n D3 and its biologically active metabolites. The chief disadvantages o f t h e b i o l o g i c a l methods a r e i n t h e h i y h i n d i v i d u a ? v a r i a t i o n s i n t h e responses o f t e s t animals, t h e high cost, t h e t i m e f a c t o r and t h a t t h e y do n o t d i f f e r e n t i a t e v i t a m i n D3 f r o m i t s b i o l o g i c a l l y a c t i v e impu r it ie s o r met a bo 1it es

.

The USP XX (30) and t h e O f f i c i a l Methods o f A n a l y s i s o f t h e A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l Chemists (AOAC) (44) d e s c r i b e i n d e t a i l t h e c u r a t i v e method more commonly known as t h e r a t l i n e method. This method i s n o t a p p l i c a b l e t o p r o d u c t s o f f e r e d f o r p o u l t r y feeding. For p o u l t r y feeds, and f i s h l i v e r o i l s and t h e i r e x t r a c t s , t h e AOAC (44) a l s o d e s c r i b e s i n d e t a i l t h e c h i c k bone ash method. I n addition t o t h e above two methods, t h e r e i s a method based on t h e comparative measurement o f serum c a l c i u m i n r a t s r e f e r r e d t o as I n t e s t i n a l Calcium T r a n s p o r t Assay and another method based on t h e comparative amounts o f c a l c i u m absorbed by c o n t r o l and t e s t c h i c k e n r e f e r r e d t o as Calcium A b s o r p t i o n Test. These methods are d e s c r i b e d by DeLuca and B l u n t (45). 4.2.2

Chemical Methods

The most w i d e l y used chemical method i s t h e antimony t r i c h l o r i d e c o l o r i m e t r i c method. The method i s a p p l i c a b l e t o v i t a m i n s D2 and D3 f o r t h e i r a n a l y s i s i n pharmacopeial p r e p a r a t i o n s . The r e a c t i o n product o f v i t a m i n D w i t h antimony t r i c h l o r i d e i s b e l i e v e d t o be i s o v i t a m i n D ( i 6 , See Scheme I V ) . Antimony t r i c h l o r i d e r e a c t s w i t 8 v i t a m i n A also. V i t a m i n A occurs a l o n g w i t h D3 i n many b i o l o g i c a l samples and i s a l s o an i n g r e d i e n t i n many commerci a1 products. Therefore, i t i s necessary t o remove i t and o t h e r i n t e r f e r i n g substances p r i o r t o r e a c t i o n w i t h


682

t h e reagent. This i s g e n e r a l l y achieved by s a p o n i f i c a t i o n w i t h a l c o h o l i c potassium hydroxide, e x t r a c t i o n o f t h e u n s a p o n i f i a b l e f r a c t i o n w i t h petroleum ether, and clean-up on chromatographic columns. The procedure f o r t h e d e t e r m i n a t i o n o f v i t a m i n D i n pharmaceutical p r e p a r a t i o n s i s d e t a i l e d i n t h e USP ( 3 0 ) . This method i s a p p l i c a b l e w i t h s u i t a b l e m o d i f i c a t i o n s t o b i o l o g i c a l samples l i k e f i s h l i v e r o i l s , f o r t i f i e d m i l k , etc., b u t t h e c o l o r i m e t r i c procedures are being replaced when p o s s i b l e by more s p e c i f i c i n s t r u m e n t a l methods, u s u a l l y HPLC.

4.2.3


Paper chromatographic s e p a r a t i o n o f some s t e r o l s , provitamins and v i t a m i n s D and D has been r e p o r t ed by Peereboom e t a l . (47). ?he b e 4 chromatographic s e p a r a t i o n was achieved by reverse phase chromatography on paper impregnated w i t h reagents shown i n Table V, which a l s o gives t h e R f values obtained. D e t a i l s r e g a r d i n g t h e preparA number o f a t i o n and development o f t h e paper are given. d e t e c t i o n systems are described which i n c l u d e d exposure t o U V l i g h t and r e a c t i o n s w i t h phosphomolybdic acid, antimony t r i c h l o r i d e , bismuth c h l o r i d e , phosphotungstic acid, s i l i c o t u n y s t i c acid, a m i x t u r e o f dimethyl-p-phenylenediamine and m-to1 uenedi ami ne, M i 11ion's reagent and sodi um iodate. Paper chromatographic systems are a1 so d e s c r i bed (48) by manufacturers o f r a d i o l a b e l e d v i t a m i n D3 f o r m o n i t o r i n g purity The systems r e p o r t e d a r e reverse phase chromatography w i t h 95% aqueous methanol on paper t r e a t e d w i t h 10% minera o i l i n benzene and w i t h 1 O : l a c e t i c acid-water on p a r a f f n coated paper.

4.2.4.

Thin Layer Chromatography

Thin Layer Chromatography i s used by major manufacturers o f r a d i o l a b e l e d v i t a m i n D3 (48) f o r m o n i t o r i n g p u r i t y . The systems described are; 1) s i l i c a gel G w i t h 10% acetone i n hexane; 2) s i l i c a gel G w i t h acetone-hexane 1:l and acetone-chloroform 1:l; 3) s i l v e r n i trate-impregnated s i l i c a gel G w i t h c h l o r o f o r m o r chloroform-acetone 9 : l ; and 4) s i l i c a gel s a t u r a t e d w i t h s i l i c o n e o i l u s i n g 4:l acetoneIt may be mentioned t h a t these operations art? carwater. r i e d o u t under very c a r e f u l l y c o n t r o l l e d c o n d i t i o n s t o prevent o r minimize decomposition. TLC i s an e x c e l l e n t technique f o r s e p a r a t i n g v i t a m i n D from i n t e r f e r i n g materi a l s , b u t i t has t o be used w i t h due care i n t h e q u a n t i t a -


683

TABLE V

3 - (Ref. 47) S t a t iona ry Phase

Mobi 1e Phase

Rf x 100

Q u i1on

MeOH-H20-ethyl ene g l y c o l monomethyl e t h e r (65:20: 20)

76

Qui1on

MeOH-H20 (95: 5 )

91

Paraf f in

A c e t i c Acid-H20 (84: 16)

36

Paraf f i n

Ethylene g l y c o l monoethyl ether-n-propanol-MeOHH20 (35:10:30:25)

46

Para f f i n

N-propanol -MeOH-H20 (15:82:3)

68

Paraffin

MeOH-H20 (85: 1 5 )

68

K . THOMAS KOSHY AND WlLLlAM F. BEYER

684

t i v e a n a l y s i s o f v i t a m i n D , e s p e c i a l l y i n b i o l o g i c a l samp l e s c o n t a i n i n g small q u a n j i t i e s o f t h e v i t a m i n . The R f values f o r v i t a m i n D3 on S i l i c a Gel GF i n common o r g a n i c s o l v e n t s as determined i n t h e authors'*j!boratory a r e shown i n Table V I (49). These values are r e p o r t e d t o enable one t o develop s u i t a b l e systems i n unknown m a t r i c e s . Hashmi e t a l . (50) have r e p o r t e d R f values f o r b o t h water and o i l s o l u b l e v i t a m i n s on s i l i c a gel and alumina p l a t e s by c i r c u l a r t h i n l a y e r chromatography. These systems a r e f a s t ( < 2 minutes development t i m e ) and convenient f o r d e t e c t i n g and e s t i m a t i n g t h e amounts o f v i t a m i n D3 i n d i f f e r e n t matrices. The authors have a l s o l i s t e d d e t e c t i o n systems f o r a l l t h e vitamins. The chromatographic systems d e s c r i b e d i n t h i s r e p o r t a r e shown i n Tables V I I and V I I I .

4.2.5.

Gas L i q u i d Chromatography (GLC)

Although GLC procedures have been used e x t e n s i v e l y f o r t h e determi n a t i on o f numerous p h y s i o l o g i c a l l y i m p o r t a n t s t e r o i d s and hormones, o n l y a few i n v e s t i y a t o r s have attempted t o develop s i m i l a r techniques f o r v i t a m i n D3. The major problem w i t h t h e GLC a n a l y s i s o f v i t a m i n D3 i s due t o i t s open r i n g s t r u c t u r e i n c o r p o r a t i n g t h r e e Conjugated double bonds i n a 5,6-cis configuration. The 5,6-cis double bond causes l e s s c o n t r o l l a b l e thermal c y c l i z a t i o n i n t o t h e pyro- and i s o p y r o c a l c i f e r o l s a t operat i n g GLC temperatures, r e s u l t i n g i n two peaks. Another reason f o r t h e l a c k of i n t e r e s t i n GLC a n a l y s i s i s t h a t v i t a m i n D3 c o n c e n t r a t i o n i n b i o l o g i c a l systems i s low and t h e samples g e n e r a l l y c o n t a i n s t r u c t u r a l l y s i m i l a r compounds r e q u i r i ny e x t e n s i v e sample c l ean-up. Murray e t a l . ( 4 6 ) overcame t h e d i f f i c u l t y o f t h e thermal i s o m e r i z a t i o n o f v i t a m i n D3 i n t o two peaks by conv e r t i n g i t i n t o i s o v i t a m i n D3 by t r e a t m e n t w i t h antimony t r i c h l o r i d e which gave a s i n g l e peak by GLC. They r e p o r t e d s u c c e s s f u l a p p l i c a t i o n o f t h e method f o r t h e d e t e r m i n a t i o n o f v i t a m i n D3 i n b i o l o g i c a l samples. Sheppard e t a l . ( 5 1 ) t r e a t e d v i t a m i n D3 w i t h a c e t y l c h l o r i d e t o c o n v e r t i t quant i t a t i v e l y t o i s o t a c h y s t e r o l D3 (See Scheme I V ) which gave a s i n g l e peak by GLC. N a i r and deLeon ( 5 2 ) prepared t h e 5,6t r a n s v i t a m i n D3 by t r e a t i n g v i t a m i n D3 w i t h i o d i n e f o l l o w e d by exposure t o UV l i g h t . They then prepared t h e pentaf 1u o r o p r o p i onyl o r t h e h e p t a f l u o r o b u t y r y l e s t e r s o f t h e trans-D3 which chromatographed as t h e i s o t a c h y s t e r o l D3 e s t e r s as a s i n g l e peak. Using 63Ni e l e c t r o n c a p t u r e detec-

VITAMIN

D3

(CHOLECALCIFEROL)

685

TABLE V I R f Values o f Vitamin D on S i l i c a Gel GFZs4 TLC Plates' f o r Pommon s o l v e n t s

Sol vent

Rf

Hexane (1.9)

0

1,4-Di oxane (2.2)

0.67

Benzene (2.3)

0.10

Toluene (2.4)

0.07

E t h y l Ether (4.3)

0.55

Chl o r o f o n (4.8)

0.23

E t h y l Acetate (6.0)

0.70

Methylene C h l o r i d e (9.1)

0.23

Cyclohexanone (18.3)

0.90

Acetone (21)

0.78

Ethanol , Absol Ute (24.3)

0.73

Methanol ( 32.6)

0.67

Acet o n i t r i 1e ( 38.8)

0.78

Hexane-Acetone, 9 : l

0.27

Hexane-Acetone,

0.75

1: 1

Acetone-Chloroform,

'Anal t e c h Inc.,

1:l

0.70

Newark, DE.

2The numbers i n parentheses are t h e d i e l e c t r i c constants o f t h e s o l v e n t s a t 2OOC.

K . THOMAS KOSHY AND WILLIAM E BEYER

686

TABLE V I I C i r c u l a r Thin La.yer Chromatographic Systems f o r Vitamin D3 on S i l i c a Gel D-0. Camag Without Binder (Ref. 50) Mobile Phase

Rf x 100

Benzene-Petrol eum Ether ( 4 : l )

33

D e t e c t i n g Reagents and Colors A B C Grey

(0.7)’

Cycl ohexane-Methyl E t h y l Ketone (21:4)

35

II

To1 uene-Methyl E t h y l Ketone ( 9 9 : l )

40

II

Cycl ohexane-Cyclopentanone (24: 1)

48

11

35

II

-

Cy c 1oh exa ne Et hy 1 Ether ( 4 : l )

Cyclohexane-Methyl Iso- 50 p r o p y l Ketone ( 9 : l )

II

To1 uene-Chl oroform (9: 1)

32

11

Cycl ohexane-Acet ic Acid (22:3)

66

II

Benzene-Cycl ohexane (9: 1)

34

II

Toluene-Cyclohexane

23

11

32

II

Bluish Grey

Greyish Green

(0.7)l

(0.8)l

11

II

II

II

(9:l) Cycl ohexane-Methanol (43: 7 )

(Continued)

687


TABLE V I I Mobile Phase

Rf x 100

-

Continued D e t e c t i n g Reagents and Colors A B C

Cycl ohexane-Methyl E t h y l Ketone (21:4)

50

Grey (0.7)l

Cycl ohexane-Ethyl Ether ( 4 : l )

52

I1

Bluish Grey (0.7)' I1

Greyish Green (0.8)' I1

A

70% P e r c h l o r i c A c i d

B

Concentrated S u l f u r i c A c i d

C

S a t u r a t e d S o l u t i o n o f antimony p e n t a c h l o r i d e i n carbon tetrachloride. The numbers i n parentheses r e p r e s e n t t h e minimum amounts (11.9)o f v i t a m i n D3 t h a t can be d e t e c t e d and i d e n t i f i e d on the plates.

68 8

K . THOMAS KOSHY A N D WILLIAM F. BEYER

TABLE

VIII

C i r c u l a r T h i n Layer Chromatographic Systems f o r V i t a m i n D3 on Aluminum Oxide G (E. Merck) (Ref. 5 0 ) Mobile Phase

R f x 100

D e t e c t i n g Reagents and C o l o r s A B C

Benzene-Pet r o l eum Ether ( 4 : l )

53

Cycl ohexane-Met h y l E t h y l Ketone (21:4)

53

II

11

To1 uene-Methyl E t h y l Ketone ( 9 9 : l )

52

11

11

Cycl ohexane-Cyclopentanone (24: 1)

47

II

II

Cyc 1 o hexa ne E t hy 1 Ether (4:l)

30

11

II

Cy cl o hexane -Met hy 1 I s o p r o p y l Ketone (9: 1)

31

II

11

II

To1 uene-Chl o r o f o r m (9:l)

45

II

II

II

Cyclohexane-Acetic Acid (22:3)

63

II

II

Benzene-Cycl ohexane (9:l)

59

II

II

To1 uene-Cycl ohexane

38

II

47

II

-

Brown

Grey

(0.6)’

(0.3)’

L iyht Brown (0.4)’ 11

II

(9:l) Cyclohexane-Methanol

II

II

(43: 7 ) (Continued )

VITAMIN Di (CHOLECALCIFEROL)

689

TABLE V I I I Mobile Phase

-

Continued

Rf x 100

Methanol - g l a c i a l A c e t i c Acid (49:l)

82

A

S a t u r a t e d S o l u t i o n o f antimony tetrachloride.

B

70% p e r c h l o r i c acid.

C

Concentrated s u l f u r i c a c i d .

D e t e c t i n g Reagents and C o l o r s A B C Brown

Grey

(0.6)l

(0.3)l

Light Brown (0.4)'

p e n t a c h l o r i d e i n carbon

The numbers i n parentheses r e p r e s e n t t h e minimum amounts (pg) o f v i t a m i n D3 t h a t can be d e t e c t e d and i d e n t i f i e d on the plates.

K. THOMAS KOSHY AND WILLIAM F. BEYER

690

t o r , t h e y were a b l e t o d e t e c t nanogram q u a n t i t i e s o f v i t a m i n D3I n r e c e n t y e a r s GLC methods have been superceded by HPLC methods. There has n o t been much i n t e r e s t i n c a p i l l a r y GLC a n a l y s i s o f v i t a m i n D3. C a p i l l a r y column technology, however, has improved so much i n r e c e n t y e a r s t h a t t h i s t e c h n i q u e may have a p p l i c a b i l i t y i n t h e d e t e r m i n a t i o n o f t r a c e amounts o f v i t a m i n D3 i n b i o l o g i c a l m a t r i c e s e s p e c i a l l y i n c o n j u n c t i o n w i t h an e l e c t r o n c a p t u r e d e t e c t o r . 4.2.6.

High Performance L i q u i d Chromatography (HPLC)

I n r e c e n t y e a r s HPLC procedures have evolved as t h e methods o f c h o i c e f o r t h e a n a l y s i s o f v i t a m i n D i n b u l k d r u g (32,33,53-61), pharmaceutical p r e p a r a t i o n s (22-73), f o r t i f i e d m i l k (74-82), animal feed supplements (83-89), margarine (79), i n f a n t formulae (79), cod l i v e r o i l It may be p o i n t e d o u t t h a t (90-93), and chicken egg (96). these reports represent t h e l a t e s t i n t h e state-of-the-art i n t h e HPLC analyses o f v i t a m i n s D2 and 03.. In s p i t e of t h e i n t e n s i v e e f f o r t by a number o f i n v e s t i g a t o r s and a few c o l l a b o r a t i v e s t u d i e s , t h e r e i s no consensus on w i d e l y acceptable HPLC methods f o r v i t a m i n D a n a l y s i s i n t h e b u l k drug, pharmaceutical p r e p a r a t i o n s , and f o r t i f i e d m i l k . 4.2.6.1

HPLC A n a l y s i s o f Bulk V i t a m i n D3

B u l k v i t a m i n D may c o n t a i n some o f s y n t h e t i c by-products shown i n Scheme Tartivita et al. (33) have r e p o r t e d an excel 1e n t chromatographi c system which showed r e s o l u t i o n of most o f t h e photochemical isomers and r e a c t i o n by-products. The chromatograms o b t a i n e d on a 30-cm x 4-mn i.d. commercial m i c r o p a r t i c u l a t e s i l i c a column u s i n g a 70:30:1 m i x t u r e o f c h l o r o f o r m ( f r e e f r o m ethanol and water), n-hexane, and t e t r a h y d r o f u r a n a t a f l o w r a t e of 1 ml/min i s shown i n F i g u r e 10. The d e t e c t i o n was by a 254 nm UV d e t e c t o r . Using t h i s system, v i t a i n D3 was q u a n t i t a t e d i n a r e s i n sample c o n t a i n i n g 20 x 10' IU/g w i t h a r e l a t i v e standard d e v i a t i o n o f 1.37%. T h i s procedure i s e s s e n t i a l l y t h e b a s i s f o r t h e USP XX (30) procedure f o r t h e a n a l y s i s of b u l k v i t a m i n D3 which i s reproduced below, i n i t s e n t i r e t y .

1".

Standard P r e p a r a t i o n - T r a n s f e r about 30 my o f USP C h o l e c a l c i f e r o l RS, a c c u r a t e l y weighed, t o a l o w - a c t i n i c , 25-ml v o l u m e t r i c f l a s k , add i s o o c t a n e t o volume, mix, and s o n i c a t e f o r 5 minutes. S t o r e a t 0 f 5O, and use t h i s s t o c k


69 1

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6 8 1 0 12 14 1 6 18 20 2 2 2 4 2 6 RETENTION TIME (minutes)

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10 12 14 RETENTION TIME (minutes) 6

8

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FIGURE 10: High Performance Liquid Chromatograms: ( A ) A synthetic mixture of photochemical isomers and reaction products of vitamin D3. Key: ( 1 ) unknowns,; ( 2 ) transvitamin D3; (3) previtamin D3; (4) lumisterol3; (5) isotachysterol3; ( 6 ) p-dimethylaminobenzaldehyde (internal standard); ( 7 ) tachysterol3; (8)vitamin D3; (9) 7-dehydrocholesterol. ( B ) A typical vitamin D3 resin. Key: (1) resin impurities; ( 2 ) p-dimethylaminobenzaldehyde (internal standard); (3) tachysterolj; ( 4 ) vitamin D3. (Both from Reference 33; reproduced with permission of the copyright owner. 1

692


s o l u t i o n w i t h i n 3 days. Pipet 5 ml o f i n t o a l o w - a c t i n i c , 50-1111 v o l u m e t r i c i s o o c t a n e t o volume, and mix t o o b t a i n known c o n c e n t r a t i o n o f about 120 pg per

t h i s stock s o l u t i o n flask, d i l u t e with a s o l u t i o n having a ml.

Assay P r e p a r a t i o n - T r a n s f e r about 30 my o f Cholecalc i f e r o l , a c c u r a t e l y weighed, t o a l o w - a c t i n i c , 25-1111 v o l u m e t r i c f l a s k , and proceed as d i r e c t e d f o r Standard Prepara t i o n , b e g i n n i n g w i t h "add i s o o c t a n e t o volume," t o o b t a i n a s o l u t i o n having a c o n c e n t r a t i o n o f about 120 11.4 per m l . A l c o h o l -Free C h l o r o f o r m - Prepare 2 Chromatographic columns by packing 2 chromatographic tubes w i t h an amount o f dry, a c t i v a t e d , 80- t o 200-mesh alumina s u f f i c i e n t t o h a l f f i l l each t u b e (See Column A d s o r p t i o n Chromatography under Chromatoyraphy ), and mount one column above t h e o t h er. E x t r a c t 500 m l o f c h l o r o f o r m w i t h t h r e e 75-ml p o r t i o n s o f water, and d i s c a r d t h e aqueous e x t r a c t s . Pass t h e c h l o r o f o r m l a y e r through t h e Chromatographic columns, c o l l e c t t h e e l u a t e i n a glass-stoppered f l a s k , i n s e r t t h e stopper, and mix. Prepare t h i s s o l v e n t on t h e day o f use. M o b i l e Phase

-

Prepare a f i l t e r e d and de-gassed m i x t u r e n-hexane, and t e t r a h y d r o f u r a n (about 70:30:1 by volume). The r a t i o o f components and t h e f l o w r a t e may be v a r i e d t o meet system s u i t a b i l i t y r e q u i r e ments.

of a l c o h o l - f r e e c h l o r o f o r m ,

-

Chromatographic System Typically, a high-pressure l i q u i d chromatograph, operated a t room temperature, i s f i t t e d w i t h a 30-cm x 4-mm s t a i n l e s s s t e e l column packed An u l t r a v i o l e t w i t h chromatographic column p a c k i n g L3*. d e t e c t o r t h a t m o n i t o r s a b s o r p t i o n a t t h e 254-nm wavelength i s used. System S u i t a b i l i t y P r e p a r a t i o n - P i p e t 5 m l o f t h e stock s o l u t i o n , prepared as d i r e c t e d under Standard p r e p a r a t i o n , i n t o a 25-11-11 v o l u m e t r i c f l a s k , d i l u t e w i t h i s o o c t a n e t o volume, and mix. Heat t h i s s o l u t i o n a t r e f l u x f o r 2 hours, cool , and i r r a d i a t e f o r 1 hour under lony-wavelength and short-wavelength u l t r a v i o l e t l i g h t . This s o l u t i o n contains c h o l e c a l c i f e r o l , p r e - c h o l e c a l c i f e r o l , and t a c h y s t e r o l

.

System S u i t a b i l i t y l e s t

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Chromatograph f i v e i n j e c t i o n s

o f t h e h e a t - e q u i l ib r a t e d Standard P r e p a r a t i o n , and measure *Porous s i l i c a m i c r o p a r t i c l e s , 5 t o 10 pm i n diameter.


693

t h e peak response as d i r e c t e d under Procedure. The r e l a t i v e s t a n d a r d d e v i a t i o n f o r t h e peak response does n o t exceed 2.0%. The r e t e n t i o n t i m e s observed f o r t h e System S u i t a b i 1 ity P r e p a r a t i o n , chromatographed as d i r e c t e d f o r Procedure, a r e between 10 and 11 minutes f o r p r e - c h o l e c a l c i f e r o l , between 14 and 16 minutes f o r t a c h y s t e r o l , and between 16 and 18 minutes f o r c h o l e c a l c i f e r o l . The r e s o l u t i o n between t a c h y s t e r o l and c h o l e c a l c i f e r o l i s n o t l e s s t h a n 1.0.

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Procedure Equi 1ib r a t e t h e Standard P r e p a r a t i o n and t h e Assay P r e p a r a t i o n i n t h e dark a t 80' f o r 2.5 hours, accura t e l y timed. Cool, and i n t r o d u c e equal volumes ( 5 t o 10 k l ) o f t h e h e a t - e q u i l ib r a t e d Standard P r e p a r a t i o n and Assay P r e p a r a t i o n i n t o t h e high-pressure l i q u i d chromatograph (See chromatography ) by means o f a s u i t a b l e sampling valve. Measure t h e peak responses o b t a i n e d f o r t h e Assay P r e p a r a t i o n and t h e Standard P r e p a r a t i o n , and c a l c u l a t e t h e q u a n t i t y , i n mg, o f C27H440 i n t h e p o r t i o n o f C h o l e c a l c i f e r o l t a k e n by t h e f o r m u l a 0.25C(A /As), i n which C i s t h e c o n c e n t r a t i o n , i n p g per m l , o f UP! C h o l e c a l c i f e r o l RS i n t h e Standard Preparation, and A, and As are t h e peak r e s ponses f o r c h o l e c a l c i f e r o l o b t a i n e d f o r t h e Assay Prepara t i o n and t h e Standard P r e p a r a t i o n , r e s p e c t i v e l y . 4.2.6.2.

HPLC A n a l y s i s o f v i t a m i n D? i n Pharmaceutical P r e p a r a t i o n s

HPLC methodology p e r m i t s r a p i d and g e n e r a l l y q u i t e simple a n a l y s i s o f v i t a m i n D3 i n pharmaceut i c a l b u l k drugs, o c c u r i n g as o i l s o l u t i o n s , beadlets, o r resins. However, f o r many m u l t i v i t a m i n products, e x c i p i e n t components w i t h d i f f e r i n g degrees o f p u r i t y , o t h e r v i t a m i n s and t h e i r d e g r a d a t i o n products, and whether v i t a m i n D i s p r e s e n t as beadlets, o i l , o r r e s i n , can make HPLC methoqogy a c h a l l e n g i n g procedure. Since t h e e x t i n c t i o n c o e f f i c i e n t f o r D3 a t 254 nm i s very h i g h (-15,000) standard, s i n g l e wavelength d e t e c t o r s a t 254 nm a r e g e n e r a l l y used. Reverse-phase HPLC procedures f o r v i t a m i n Dg i n b u l k d r u g were r e p o r t e d i n t h e e a r l y 70's by W i l l i a m e t a l . ( 5 4 ) u s i n g a DuPont Permaphasee ODs column (DuPont , W i l m i ngton, Del.) and 78% methanol i n water as t h e m o b i l e phase. A s i m i l a r column and m o b i l e phase (DuPont's Zorbax" ODS Column and 95% methanol i n w a t e r ) were r e p o r t e d t o g i v e i n c r e a s e d r e s o l u t i o n o f t h e two forms o f v i t a m i n D (62) i n m u l t i v i t a min f o r m u l a t i o n s . Complete s e p a r a t i o n o f v i t a m i n s D2 and D3 by reverse-phase chromatography i n model m u l t i v i t a m i n prepar a t i o n s were r e p o r t e d by Osadca and A r a u j o (63). In this

694

K. THOMAS KOSHY A N D WILLIAM E BEYER

a n a l y s i s t h e y used a Vydac column ( S e p a r a t i o n s Group, Hespera, CA.) and a m o b i l e phase o f 90% methanol i n water. Tscherne and Capi t a n 0 (64) a l s o r e p o r t e d complete s e p a r a t i o n column (Waters, M i l f o r d , o f D2 and D3 w i t h a 12 Bondapake C Mass.) and a m o b i l e phase o f 86!!!?% methanol i n water w i t h added s i l v e r n i t r a t e . S e p a r a t i o n o f t h e two v i t a m i n s was s a t i s f a c t o r y , however, t h e presence o f s i l v e r n i t r a t e i n i t s e l f can g i v e r i s e t o a m u l t i t u d e o f problems.

A reverse-phase HPLC assay, as p a r t of t h e A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l Chemists r e p o r t on a n a l y s i s o f f a t s o l u b l e v i t a m i n s , was d e s c r i b e d by DeVries e t . a l . (65). A n a l y s i s were made w i t h a Merck LiChrosorb RP-18 column OH) and a ( M a n u f a c t u r i n g Chemists, Inc., Cincinnati, a c e t o n i t r i 1 e : p r o p i o n i t r i 1e:water (79:15:6) m o b i l e phase. A1 though adequate chromatography was r e a l i z e d , t h e a u t h o r s were concerned t h a t problems arose concerning i n f l u e n c e o f temperature, d i s s o l u t i o n of sample and p u r i f i c a t i o n of s o l vents i n t h e m o b i l e phase. F o r these reasons they recommended normal -phase chromatography. Separation o f v i tami ns D2 and D3 w i t h t h e i r systems was n o t discussed. Normal-phase HPLC on s i l i c a columns a r e a l s o used e x t e n s i v e l y i n D3 a n a l y s i s o f v i t a m i n products w i t h nonp o l a r m o b i l e phase c o n t a i n i n g p o l a r m o d i f i e r s . Krol e t al. (66) separated D3 from pre-D3 and from a m i x t u r e o f o t h e r v i t a m i n s u s i n g an a d s o r p t i v e s i l i c a support i n t r o d u c e d i n 1972 (Vydaco, s u p p l i e d a t t h a t t i m e by A p p l i e d Science The hand-packed l a b o r a t o r i e s , Inc. S t a t e Col 1ege , Penn. ) column was used i n c o n j u n c t i o n w i t h a m o b i l e phase o f pent a n e : t e t r a h y d r o f u r a n (97.5:2.5). S t e r u l e (32) used aluminum o x i d e as column support w i t h c h l o r o f o r m as t h e m o b i l e phase. Separation o f O3 and i t s isomers and from v i t a m i n A a c e t a t e was achieved.

.

DeVries e t a l . (67) r e p o r t e d t h e sumnary o f s t u d i e s by a number o f c o l l a b o r a t i n g l a b o r a t i e s f o r t h e HPLC assay o f v i t a m i n D i n m u l t i v i t a m i n p r e p a r a t i o n s . S a p o n i f i c a t i o n and a reverse-phase Merck LiChrosorb RP-8 column were used f o r sample cleanup. The a n a l y t i c a l column was a P a r t i s i l a , 5 pm column (Whatman, C l i f t o n , N.J.) w i t h hexane-amyl a l c o h o l (99.65:0.35%) as t h e m o b i l e phase. The cleanup procedure a l t h o u g h a deparature from t h e usual a n a l y t i c a l methods, was incorporated to ensure predictable, interference-free v i t a m i n D assays (D2 and D3 c o - e l u t e ) . Normal-phase columns, e i t h e r Water's p P o r a s i l o o r DuPont's Zorbax-sil@, were used f o r t h e data o f Sheridan's

VITAMIN DI(CHOLECALCIFEROL)

695

(68) f i n a l r e p o r t o f t h e Pharmaceutical M a n u f a c t u r e r ' s Associ a t i on-Qua1 it y C o n t r o l S e c t i o n (PMA-QC) The d a t a covered c o l l a b o r a t i v e s t u d i e s on v i t a m i n D u s i n g HPLC. A m o b i l e phase o f ch1oroform:hexane:tetrahydrofuran (70:30:1) was used w i t h d e t e c t i o n a t 254 nm. The HPLC method separated v i t a m i n D from i n t e r f e r i n g compounds t h a t c o u l d be present, namely 7-dehydrocholesterol , t a c h y s t e r o l , i s o t a c h y s t e r o l p r e - v i t a m i n D, t r a n s - v i t a m i n D, l u m i s t e r o l , and e r g o s t e r o l , from o t h e r v i t a m i n s ( A a c e t a t e , A p a l m i t a t e , t o c o p h e r o l , t o c o p h e r y l acetate, and phytonadione), and from antioxidants. Samples and standards were t r e a t e d i d e n t i c a l l y as t o t i m e and temperature so t h a t o n l y v i t a m i n D need be measured, s i n c e f o r p r a c t i c a l purposes p r e v i t a m i n D would be t h e same f o r sample and standard. Vitamins D3 i n r e s i n and v i t a m i n D2 i n b e a d l e t s were t r e a t e d d i f f e r e n t l y f o r sample p r e p a r a t i o n s . The r e s i n was d i s p e r s e d i n i s o octane, t r e a t e d w i t h u l t r a s o n i c a t i o n , and d i l u t e d W i t h isooctane. Beadlets were added t o a m i x t u r e o f DMS0:water (3:1), shaken v i g o r o u s l y , and heated a t 5OoC f o r 20 minutes. A f t e r c o o l i n g t o room temperature, hexane was added, t h e m i x t u r e was t h e n shaken and c e n t r i f u g e d . The hexane l a y e r was removed and t h e e x t r a c t i o n procedure w i t h hexane was c a r r i e d o u t t h r e e more t i m e s . The combined hexane e x t r a c t s were t h e n d r i e d w i t h anhydrous sodium s u l f a t e and reduced t o dryness w i t h a r o t o - e v a p o r a t o r . The d r y r e s i d u e was t h e n d i s s o l v e d i n i s o o c t a n e f o r i n j e c t i o n on t o t h e column. The HPLC procedure was considered advantageous because m i c r o - p a r t i c u l a t e columns were r e p r o d u c i b l e and a v a i l a b l e commercially, ambient temperature requirements reduced thermal breakdown o f 0, and time-consuming s a p o n i f i c a t i o n was n o t necessary. The method was considered s u p e r i o r t o methods o f t h e USP (30) and AOAC (44).

.

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Based on t h e r e p o r t o f Sheridan (68), Walker e t a1 (69) were a b l e t o assay a v a r i e t y o f pharmaceutical p r o d u c t s f o r D (and r e p o r t e d l y D3, a l t h o u g h no d a t a were given). A S i 5g s i l i c a column (Brownlee Labs., Santa Clara, C a l i f . ) was used i n c o n j u n c t i o n w i t h a m o b i l e phase comprised o f a m i x t u r e o f ch1oroform:water-saturated hexane: hexane: t e t r a h y d r 0 f u r a n : a c e t i c a c i d (60:15:25:1.5:0.4). As w i t h t h e HPLC assay o f t h e PMA-QC r e p o r t (68), D2 and D3 co-eluted. Comments were made concerning t h e a d d i t i o n o f p o l a r modif i e r s o r changes i n t h e r a t i o of m o b i l e phase t o enhance chromatography. B u i l d i n g on p u b l i s h e d l i t e r a t u r e and based on e m p i r i c a l t r i a l s , an e f f i c i e n t reverse-phase HPLC system and sample p r e p a r a t i o n scheme has been developed f o r v i t a m i n D3 i n

696


m u l t i v i t a m i n products i n t h e author's laboratory f o r i n house use (70). Although t h e method does n o t appear i n open l i t e r a t u r e , i t has been used e x t e n s i v e l y i n our l a b o r a t o r i e s and w i l l be d e s c r i b e d i n some d e t a i l . It i s f e l t t h a t t h e sample p r e p a r a t i o n and t r e a t m e n t scheme, t h e use o f two i n t e r n a l standards, and t h e c a p a b i l i t y t o s w i t c h m o b i l e phases t o r i d t h e column o f v e r y slow e l u t e r s a r e h e l p f u l i n r o u t i n e q u a l i t y c o n t r o l assays and f o r s t a b i l i t y e v a l u a t i o n o f v i t a m i n D3 i n a v a r i e t y of pharmaceutical products. I n t h i s procedure, t h e HPLC a n a l y s i s i s c a r r i e d o u t on a DuPont Zorbax ODs@ column u s i n g methanol , acetoni t r i l e and water 10:2:1 a t t h e m o b i l e phase. T h i s system r e s o l v e s v i t a m i n s D3 from D2 and from d e g r a d a t i o n products and o t h e r f a t s o l u b l e vitamins. D i d e c y l and d i n o n y l p h t h a l a t e were used as t h e i n t e r n a l standards. The l a t t e r was i n c l u d e d f o r use i n t h e event t h a t extraneous peaks i n t e r f e r e d w i t h t h e d i d e c y l p h t h a l a t e peak. F i g u r e 11 shows a chromatogram o f an e x t r a c t o f a m u l t i v i t a m i n f o r m u l a t i o n w i t h t h e two added i n t e r n a l standards. M u l t i v i t a m i n l i q u i d suspensions were prepared f o r assay u s i n g a hexane e x t r a c t i o n o f t h e sample from d i m e t h y l s u l foxide:lO% g l a c i a l a c e t i c a c i d ( 9 : l ) . A small volume o f hexane c o n t a i n i n y t h e i n t e r n a l standards was added quant i t a t i v e l y t o t h e e x t r a c t i n g s o l u t i o n . P r o v i s i o n s were made t o a u t o m a t i c a l l y r i n s e t h e column between i n j e c t i o n s w i t h methanol : t e t r a h y d r o f u r a n : d i m e t h y l s u l f o x i d e (25:25:1) to remove any slow e l u t i n g m a t e r i a l s . Column r i n s i n g was i n i t i a t e d and c o n t r o l l e d ( D i g i t a l Valve Sequence Programmer, Valco, Houston, Texas), by an automatic sampler. Mobile phase and column r i n s e s w i t c h i n g was made w i t h a v a l v e (Model 5302 Rheodyne, C o t a t i , C a l i f . ) , f i t t e d w i t h an a i r a c t i v a t o r (Model 5300, Rheodyne). A i r t o t h e v a l v e was c o n t r o l l e d by a s o l e n o i d v a l v e (No. 062E1-3-10-20-35, HumWith t h i s chromatophrey Products, Kalamazoo, Mich.). graph c system d i n o n y l p h t h a l a t e e l u t e d i n about 15 minutes, v i tam n D i n 25 minutes, and d i d e c y l p h t h a l a t e i n 30 minCo3umn r i n s e was complete i n approximately 10 minUtes. Utes, p e r m i t t i n g an a n a l y s i s t i m e o f about 40 minutes. 0 1 f o r m u l a t i o n s o f m u l t i v i t a m i n Droducts were d i l u t e d w i t h hexane, passed t h r o u g h a SEP-PAK@ s i l i c a c a r t r i d g e (Waters, M i l f o r d , Mass.) where t h e o i l was r e t a i n e d . The c a r t r i d g e was t h e n f l u s h e d w i t h methanol t o e l u t e D3 i n t o a c o n t a i n e r h a v i n g t h e a p p r o p r i a t e amount o f i n t e r n a l standard, p r e v i o u s l y evaporated t o dryness. The c a r t r i d g e ,use was based on t h e r e p o r t s f r o m Water's ( 7 1 ) and from R.A.

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Dinonyl Phthalate

RESPONSE, 0.0032 AUFS


698

Pask-Hughs and D.H.Ca1 am (72). HPLC procedures f o r v i t a m i n D i n most i n s t a n c e s have s o l v e d problems o f i n t e r f e r e n c e f r o m t h e m a t r i x and d e g r a d a t i o n products, presence of isomers and homo1ogues, o t h e r f a t - s o l u b l e v i t a m i n s and t h e i r companion compounds. S t a b l e and h i g h l y e f f i c i e n t columns, an e a s i l y o b t a i n a b l e and s t a b l e d e t e c t o r operated a t 254 nm ( n e a r absorbance maximum of 265 nm) , and r e 1ia b l e sample p r e p a r a t i v e techniques and apparatuses have made HPLC a most appeal ing technique. A choice of techniques w i t h o r w i t h o u t i n t e r n a l standards i s available, depending on t h e amount o f development t i m e t h a t can be committed and t h e presence o r absence o f s u i t a b l e areas on t h e chromatogram f o r t h e i n s e r t i o n of an i n t e r n a l standard peak. The a d d i t i o n o f an i n t e r n a l standard a t t h e e a r l i e s t t i m e p o s s i b l e i n t h e sample p r e p a r a t i o n scheme can add c o n s i d e r a b l y t o t h e accuracy o f t h e HPLC method. It can a i d i n v a r i o u s ways, namely; c o r r e c t f o r recovery, negate b i a s f r o m l o s s e s i n i s o l a t i o n steps, improve p r e c i s i o n a s s o c i a t e d w i t h a l i q u o t removal and d i l u t i o n , and i m p o r t a n t l y , compensate f o r changes i n i n s t r u m e n t a l performance, p a r t i c u l a r l y , long chromatographic runs. Valve s w i t c h i n g f o r columns and m o b i l e phases can decrease chromatographic t i m e by d i v e r t i n g unwanted peaks o r f l u s h i n g t h e column a f t e r d e s i r e d peaks have been obtained. I n essence, HPLC has been u t i l i z e d as one o f t h e most powerful a n a l y t i c a l t o o l s a v a i l a b l e f o r v i t a m i n D a n a l y s i s i n pharmaceutical products. Even g r e a t e r u t i l i t y o f HPLC can be expected as h i g h e r e f f i c i e n c y columns and e x t r a c t i o n procedures, b e t t e r column hardware, and in c r e a s i n y l y e f f ic i e n t in s t r u m e n t a t i on a r e developed and made a v a i l a b l e .

4.2.6.3

A n a l y s i s V i t a m i n D? i n M i l k and M i l k Powder

M i l k s o l d i n t h e USA and Canada i s r e q u i r e d by law t o be f o r t i f i e d w i t h e i t h e r v i t a m i n D o r D a t t h e l e v e l o f 400 I U / q u a r t which i s e q u i v a l e n t t o 16 ng/m!? o r 10 ppb. It i s i m p o r t a n t f o r n u t r i t i o n a l reasons t h a t compliance w i t h t h e r e g u l a t i o n be checked by a n a l y z i n g t h e f o r t i f i e d milk. Moreover, i t i s q u i t e p o s s i b l e t h a t a harmful excess c o u l d be added by mistake, and t h e r e f o r e i t i s i m p e r a t i v e t h a t e r r o r s i n f o r t i f i c a t i o n be d e t e c t a b l e . O f a l l t h e methods a v a i l a b l e HPLC appears t o be t h e most r e l i a b l e one even though i t r e q u i r e s c a r e f u l h a n d l i n g o f t h e sample. A number o f methods have been r e p o r t e d f o r t h e a n a l y s i s o f v i t a m i n D3 i n whole m i l k (74-79), f o r t i f i e d


699

d r i e d m i l k s (76,77,80), skim and c h o c o l a t e m i l k (81), i n s t a n t n o n f a t d r i e d m i l k (82) and i n f a n t formula (79). There i s s t i l l no w i d e l y accepted method b u t t h e general approach ( 7 6 ) i n v o l v e s s a p o n i f i c a t i o n and e x t r a c t i o n o f t h e unsaponif i a b l e r e s i d u e f o l l o w e d by an HPLC cleanup on a n i t r i l e bonded s i l i c a column t o separate v i t a m i n D3 and i t s isomers from i n t e r f e r i n g substances. The f r a c t i o n corresponding t o v i t a m i n D3 i s c o l l e c t e d and q u a n t i t a t e d on a m i c r o p a r t i c u l a t e s i l i c a column u s i n g 0.35-1% amyl a l c o h o l i n n-hexane as t h e m o b i l e phase. The HPLC method i s 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 e i t h e r v i t a m i n D or D3 i n m i l k . It i s remarkable t h a t i t i s p o s s i b l e t o teetermine f a i r l y accuratel y t h e amounts o f v i t a m i n D p r e s e n t i n whole m i l k and i n d i f f e r e n t forms o f r e c o n s t i t u t e d m i l k . 4.2.6.4

D e t e r m i n a t i o n o f V i t a m i n D2 i n Animal Feeds

The b u l k use o f commercially produced v i t a m i n s D2 and D a r e f o r t h e supplementation o f p o u l t r y , c a t t l e , swine an$ p e t feeds. The c o n c e n t r a t i o n o f v i t a m i n D i n animal feed premixes ranges from 30,000 t o 150,000 I i / k g . It i s i n t h e range o f 500 t o 4000 IU/kg i n t h e f i n a l feed. The animal f e e d composition v a r i e s depending on t h e a v a i l a b i l i t y and p r i c e s o f t h e feed ingredients. Therefore i t i s a very c h a l l e n g i n g problem t o develop an acceptable method f o r t h e a n a l y s i s o f v i t a m i n D i n animal feeds. However, t h e r e a r e a l r e a d y r e p o r t s (83-897 o f p a r t i a l l y successful e f f o r t s i n t h i s regard. A c o l l a b o r a t i v e study (88) was conducted under t h e auspices o f Associ a t ion o f O f f ic i a1 Ana 1y t ica 1 Chemi s t s , Wash in g t on, D.C. The procedure based on t h e one developed by Knapstein e t a1 (89) i n v o l v e s s a p o n i f i c a t i o n o f 25 g o f t h e powdered sample, e x t r a c t i o n o f t h e u n s a p o n i f i a b l e s , cleanup on an a1 umi na column u s i ng ether-hexane m i x t u r e s , a d d i t i o n a l cleanup on a c18 bonded m i c r o p a r t i c u l a t e s i l i c a column u s i n g CH$N-MeOH-H20 (50:50:5) as t h e m o b i l e phase and f i n a l q u a n t i t a t i o n on a m i c r o p a r t i c u l a t e s i l i c a column (250 x 4.6 ( i d ) mn) u s i n g 0.35% amyl a l c o h o l i n n-hexane as t h e m o b i l e phase. R e s u l t s are very encouraging and i t can be expected t h a t p r a c t i c a l methods w i l l be a v a i l a b l e i n t h e near f u t u r e .

.

4.2.6.5.

D e t e r m i n a t i o n o f V i t a m i n D2 i n Cod Liver O i l

P r i o r t o t h e a v a i l a b i l i t y o f synthet i c v i t a m i n s D2 and U3, f i s h l i v e r o i l s were t h e p r i m a r y sources of v i t a m i n D3. The commonly used o i l i s t h a t from

700


t h e cod l i v e r which c o n t a i n s about 100-150 I U o f v i t a m i n D per gram. F i s h l i v e r o i l s a l s o c o n t a i n s l a r g e amounts o v i t a m i n A and c h o l e s t e r o l . I n cod l i v e r o i l t h e w e i g h t r a t i o may be 1:100:3000 f o r v i t a m i n D3, v i t a m i n A and c h o l e s t e r o l . The main d i f f i c u l t y i n a n a l y z i n g cod l i v e r o i l f o r v i t a m i n D3 i s i n s e p a r a t i n g i t from t h e l a r g e excess o f cholesterol. Even though p r e c i p i t a t i o n of c h o l e s t e r o l w i t h d i g i t o n i n or by f r e e z i n g a r e s t a n d a r d procedures, t h e s e methods a r e p h y s i c a l l y cumbersome and a l s o r e s u l t i n l o s s o f v i t a m i n D by o c c l u s i o n . Therefore, t h e a n a l y s i s o f v i t a m i n D3 i n c o d f i v e r o i l i s a d i f f i c u l t problem. A l i (90) has r e p o r t e d a method based on a column chromatographic clean-up f o l l o w e d by a t h i n l a y e r chromatography and spectrophotometry. HPLC procedures have been r e p o r t e d by A l i (91), Egaas and Lambersten (92) and Stancher and Sonta (93). 3f these t h e procedure by Stancher and Zonta (93) appears t o be t h e simplest. The procedure i s r e p o r t e d l y a p p l i c a b l e f o r t h e simultaneous d e t e r m i n a t i o n o f v i t a m i n s D3 and E, b u t t h e recoveries o f t h e p r e l i m i n a r y e x t r a c t i o n step using e i t h e r hexane o r e t h y l e t h e r i s i n t h e range 60-75% only. The a u t h o r s do n o t r e p o r t t h e o v e r a l l recovery f o r t h e procedure.

9

From t h e above d i s c u s s i o n i t i s c l e a r t h a t t h e r e i s scope f o r more work i n d e v e l o p i n g a b e t t e r method f o r v i t a min D3 i n cod l i v e r o i l . 4.2.6.6.

D e t e r m i n a t i o n o f Vitamin D2 i n Chicken Eaa Yolk

The c h i c k e n egg y o l k , as deduced from i t s a n t i r a c h i t i c a c t i v i t y , c o n t a i n s 1-2 pg o f c h o l e c a l c i f e r o l and t h i s can be r a i s e d f u r t h e r by i n c r e a s i n g t h e i n t a k e o f v i t a m i n D3 o f t h e l a y i n g hen (94). The concent r a t i o n i n y o l k i s 5-10 t i m e s h i g h e r than t h a t o f t o t a l v i t a m i n D3 p l u s i t s m e t a b o l i t e s i n b l o o d plasma o f normal chickens and 50-100 t i m e s h i g h e r than t h e c o n c e n t r a t i o n i n any o t h e r t i s s u e (95). Consequently, t h e chicken egg y o l k i s a p o t e n t source o f v i t a m i n U3. Because several o f t h e m e t a b o l i t e s o f v i t a m i n D3 a r e b i o l o g i c a l l y a c t i v e , t h e i n o l e c u l a r species o f v i t a m i n D3 which passes i n t o t h e y o l k cannot be determined j u s t from measurement o f a n t i r a c h i t i c a c t i v i t y . Consequently, a r e l i a b l e and s e n s i t i v e method f o r d e t e r m i n i n g t h e amount o f t h e unchanged form o f v i t a m i n D3 would be e x t r e m e l y benef ic i a1 t o those i n t e r e s t e d i n t h e metabol ism and o t h e r f a c t o r s t h a t i n f l u e n c e t h e c h i c k e n t o d e p o s i t v i t a m i n D3 i n

70 I

VITAMIN Dj (CHOLECALCIFEROL)

t h e egg y o l k . Packson e t a l . ( 9 6 ) have r e c e n t l y r e p o r t e d an HPLC method f o r t h e a n a l y s i s o f v i t a m i n D3 i n egg y o l k . They use v i t a m i n D2 as an i n t e r n a l s ta n d a rd which i s added t o 50 g of t h e f r e e z e - d r i e d egg a t t h e b e g i n n i n g o f t h e a n a l y s i s t o compensate f o r l o s s e s d u r i n g t h e s e v e r a l m a n i p u l a t i v e s t e p s i n t h e procedure. The sample i s s a p o n i f i e d f o r 30 m i n u t e s i n a N2 atmosphere. The u n s a p o n i f i a b l e s a r e e x t r a c t e d w i t h 1:l m i x t u r e o f p e tro l e u m e t h e r and d i e t h y l e t h e r . The ext r a c t a f t e r washing f r e e o f base i s evaporated. The excess s t e r o i d s a r e removed by p r e c i p i t a t i o n by c h i l l i n g from 9O:lO methanol-water s o l u t i o n and removed by f i l t r a t i o n . The e x t r a c t i s evaporated t o dryness, d i s s o l v e d i n a s m a l l volume of l i g h t p e tro l e u m e t h e r and s u b j e c t e d t o TLC on s i l i c a gel p l a t e s . The band r e p r e s e n t i n g v i t a m i n D i s scraped, e l u t e d w i t h HPLC grade methanol , c o n c e n t r a t e d and stainless steel s u b j e c t e d t o HPLC on a 250 x 4 mm i.d. column packed w i t h a C reversed-phase p a c k i n g (Magnusil C 5x) u s i n g e i t h e r 9$:5 o r 9O:lO methanol-water as t h e m % i l e phase. Under t h e HPLC c o n d i t i o n s v i t a m i n D2 e l u t e d b e f o r e v i t a m i n D3 i n about 14-17 minutes. The two peaks were n o t c o m p l e t e l y re s o l v e d . Replicate analysis o f 5 samples showed v i t a m i n D3 c o n t e n t i n t h e range 1.3-1.9 pg/lOO g (mean 1.6 f 0.2 pg/lOO g) o f whole f r e e z e - d r i e d sample. T h i s i s e q u i v a l e n t t o 0.32 pg/20 g o f whole egg. T h i s v a l u e i s c o n s i d e r a b l y l o w e r t h a n t h e c u r r e n t l y accepted v a l u e of 1-2 p g p e r egg y o l k w e i g h i n g 15-20 g (94).

5.

Metabolites o f Vitamin

5.1

D3

Pri m a ry Metabol it e s

F o r n e a r l y 30 y e a r s f o l l o w i n g t h e d i s c o v e r y o f v i t a m i n D3, r e l a t i v e l y l i t t l e was known about i t s m e t a b o l i c It was th o u g h t t h a t v i t a m i n D3 a c t e d d i r e c t l y f a t e i n man. on t h e t a r g e t t i s s u e s o f i n t e s t i n e and bone. However, t h e time lag reported by Carlsson (97) between the admi n i s t r a t ion o f v i ta m i n D3 and i t s p h y s i o l o g i c a l response D3 was led investigators to suspect that vitamin m e t a b o l i c a l l y a l t e r e d b e f o r e i t became b i o l o g i c a l l y a c t i v e . Major e v e l o p y g n ts i n t h e m e t a b o l i c s t u d y came a f t e r s e l e c t i v e H' and C labeled vitamin D w i t h high specific a c t i v i t i e s were s y n t h e s i z e d i n t h e mid-?960's. S i n c e then, massive e f f o r t s by a number o f i n v e s t i g a t o r s have l e d t o t h e d i s c o v e r y o f a l a r g e number o f m e t a b o l i t i e s , t h e hormonal n a t u r e of t h e p r i m a r y m e t a b o l i t i e s i n t h e maintenance o f

702


calcium homeostasis, t h e n a t u r e o f t h e s p e c i f i c p r o t e i n s t h a t c a r r y t h e m e t a b o l i t e s t o t a r g e t t i s s u e s , t h e enzymes i n v o l v e d i n t h e t r a n s f o r m a t i o n s and t h e i n t r i c a t e r o l e played by o t h e r endocrine organs i n t h e c o n t r o l o f t h e metabolism o f v i t a m i n D These f i n d i n g s have been reviewed e x t e n s i v e l y (98-100). ?he c u r r e n t l y known metabol i c pathway o f v i t a m i n D3 i s shown i n Scheme V. Vitamin D from t h e d i e t o r t h a t generated i n t h e s k i n i s absorbed i n t o t h e blood. The c o n c e n t r a t i o n o f v i t a m i n 03 i n t h e blood i s very low, but excess v i t a m i n i s s t o r e d i n t h e f a t depots. Deplet i o n o f v i t a m i n from f a t t y t i s s u e s depends on t h e t u r n o v e r o f f a t which i s g e n e r a l l y a slow process.

.

Lund and DeLuca (101) administered C3H] v i t a m i n 03 t o r a t s and found b i o l o g i c a l l y a c t i v e m e t a b o l i t e s i n t h e bone, 1iver and serum. The aqueous-sol u b l e metabol it e s f rom t h e t i s s u e s and t h e feces d i d not have v i t a m i n D a c t i v i t y . A t l e a s t t h r e e b i o l o g i c a l l y a c t i v e m e t a b o l i t e s were i s o l a t e d from t h e c h l o r o f o r m - s o l u b l e p o r t i o n o f t h e e x t r a c t . One o f these was found i n l a r g e amounts i n t h e l i v e r , blood and bone. I n 1968, B l u n t et. a l . (102) proved c o n v i n c i n g l y t h a t t h i s major m e t a b o l i t e i s 25-hydroxyvitamin D3. (25-OH-0 ). Two o t h e r groups o f i n v e s t i g a t o r s (103,104) independenhy found c l u e s t o t h e metabolic h y d r o x y l a t i o n o f v i t a m i n D It was soon e s t a b l i s h e d t h a t 25-hydroxylation o f v i t a m i n takes p l a c e p r i m a r i l y i n t h e l i v e r (105,106) and t h a t 25-OHD i s t h e major form o f c i r c u l a t i n g v i t a m i n D3 i n human p?asma (107).

a;

I n i t i a l l y , 25-OH-D3 was considered t o be t h e main b i o l o g i c a l l y a c t i v e m e t a b o l i t e o f v i t a m i n 0. But soon i t was discovered t h a t p h y s i o l o g i c a l c o n c e n t r a t i o n s o f Z5-OH-D3, l i k e v i t a m i n D , are incapable o f s t i m u l a t i n g e i t h e r i n t e s t i nal c a l c i um %ransport or bone c a l c i urn y o b i 1 iz a t i on (108110). E a r l i e r work (111,112) w i t h [ l a - H I v i t a m i n D3 had shown t h a t one o f t h e unknown m e t a b o l i t e s had l o s t i t s t r i t i u m from t h e C - 1 p o s i t i o n . Fraser and Kodicek (113) e s t a b l i s h e d t h a t t h i s a c t i v e m e t a b o l i t e i s 1-oxygenated 25OH-D3 and t h a t it was produced i n t h e kidney. A s h o r t t i m e l a t e r Lawson e t a l . (114) i d e n t i f i e d t h i s m e t a b o l i t e t o be l a , 25-dihydroxyvitamin D3 (1,25-(OH)2D3) which was confirmed by o t h e r i n v e s t i g a t o r s (115,116). The endogenous l e v e l o f 25-OH-03, t h e primary m e t a b o l i t e o f v i t a m i n 03, has been determined by a number o f i n v e s t i gators and found t o be i n t h e 15-30 ng/ml i n normal Caucas i a n plasma. The c o n c e n t r a t i o n o f 25-OH-03 increases w i t h increased v i t a m i n D3 i n t a k e and t o extended exposure t o

703


lo-OH-03

1,25-~OH1203- 2 6 . 2 3 - l o c l o n e

(Ref 130,1311

Tn

\ U"k"O.".i

no" 25-(OH1-24-ora D3

( R e f 133)

\\

t

25,26(OH1203

25-01-03-26,23- loctone

( R e 1 1251

( R e f 128.1291

(Ref.1321

25-OH-D3

Unknowns

( R e f . 1021

c."nnn

&on

Unknowns 8n the b t l i

( R e f . 127)

AZ5 -Ia-OH-D3 ( R e f . 1271

Scheme V .

M e t a b o l i c pathway f o r v i t a m i n D3. From J . Pharm. Reproduced w i t h p e r m i s s i o n o f t h e copy-

S c i . 71, 137 (1982). r i g h t owner.

A Z 4 - .1 - O H - 0 3 ( R e f 127)


704

sunlight. As p o i n t e d o u t e a r l i e r , i t i s t h e major c i r c u l a t i n g and r e a d i l y a v a i l a b l e form o f v i t a m i n D The endogenous l e v e l o f 1,25-(OH) D , on t h e o t h e r %nd, i s very low, between 20 and 40 pgfm? o f plasma. The f o r m a t i o n o f 1,25-(OH)2D3 i s feedback r e g u l a t e d depending on t h e need f o r c a l c i u m i n t h e blood. A t t h e present time, 1,25-(OH)2D3 i s c o n s i d e r e d t o be t h e most p o t e n t o f a l l t h e known metabo1it e s o f v i t a m i n D3 f o r c o n t r o l 1ing c a l c i u m and phosphorus a b s o r p t i o n f r o m t h e in t e s t i ne and mobi 1iz a t ion o f these elements from t h e bone when necessary. 5.2

Secondary Metabol it e s

It became known d u r i n g t h e work on t h e i d e n t i f i c a t i o n o f 25-OH-D3 and 1,25-(0H)2D3 t h a t t h e r e were o t h e r m e t a b o l i t e s some o f which were i d e n t i f i e d i n q u i c k succession. Of these t h e i m p o r t a n t ones from a b i o l o g i c a l standp o i n t a r e 24R,25-dihydroxyvitamin D3 [24,25-(OH)3D ] and 1,24,25-t r i h y d r o x y v i tami n D3 [ 1,24,25-( OH) 3D3]. Omd&l and DeLuca r e p o r t e d (117) t h a t when t h e s y n t h e s i s o f 1,25-(OH) D3 was suppressed by a b l o c k o f t h e kidney hydroxylase whic produces 1,25-(OH)2D3 from 25-OH-D , a new m e t a b o l i t e appeared. T h i s m e t a b o l i t e i s made e x c j u s i v e l y i n t h e kidney (118,119) and was i d e n t i f i e d as 24,25-(OH)?D3 (120). The c o n c e n t r a t i o n o f t h i s m e t a b o l i t e i n human i s low b u t much T a y l o r e t a l . (121) h i g h e r t h a n t h a t o f 1,25-(OH) D3. r e p o r t e d a value o f 1.68 f 0.8 ng/ml among several normal volunteers. It was demonstrated (122,123) t h a t under normal o r hypercalcemic c o n d i t i o n s , 24,25-(OH) D-3 i s t h e major c i r c u l a t i n g m e t a b o l i t e o f 25-OH-D3. L i k e $b-OH-D3 and 1,25(OH)2D3, i t i s capable o f e l e v a t i n g serum c a l c i u m and supp o r t i n g bone growth on a normal calcium, normal phosphorus diet. However, u n l i k e 1,25-(OH) D i t has l i t t l e a b i l i t y t o m o b i l i z e c a l c i u m f r o m t h e bon$.3’Boyle e t a l . (122,123) had a l s o shown t h a t 24,25-(OH)2D3 has t o be metabolized t o a more p o l a r m e t a b o l i t e i n t h e k l d n e y b e f o r e i t became b i o l o y i c a l l y active. This m e t a b o l i t e was i s o l a t e d by H o l i c k e t a l . (124) i n pure form from c h i c k e n kidney homogenates and i d e n t i f i e d as 1,24,25-(OH)3D3. The b i o l o g i c a l a c t i v i t y o f D3 p a r a l l e l s t h e b i o l o g i c a l a c t i v i t y o f 24,251,24,25-(OH) (OH)2D3 (1233. There i s no c o n c l u s i v e d a t a on t h e concent r a t i o n o f 1,24,25-(OH)3D3, It i s known t o be lower than t h a t o f 1,25-(OH) D3 and, t h e r e f o r e , i s i n t h e very low picogram per m i l l i ? i t e r range i n t h e plasma.

i

8

705


5.3

M e t a b o l i t e s o f Very Low o r Unknown B i o l o g i c a l Activity

A number o f m e t a b o l i t e s o f v i t a m i n D3 w i t h extremel y low o r o f unknown b i o l o g i c a l a c t i v i t y have been d i s c o v e r ed. Scheme V shows t h e s t r u c t u r e s o f these m e t a b o l i t e s , t h e i r o r i g i n and a1 so a p p r o p r i a t e 1 it e r a t u r e c i t a t i o n s o f t h e i r discovery. The m e t a b o l i t e s a r e 25,26-di h y d r o x y v i t a m i n D3 d i s c o v e r e d by Suda e t a l . (125), la-OH-24,25,26,$it e t ranor-D3-23-carboxyl ic a c i d c a l c i t r o i c a c i d ) ( 126), A l a - h y d r o x y v i t a m i n D (127), b2'-la-hydroxyvitamin D (127), -26,23-lactone (128,129) , la-25-dihy25-hydroxyvitamin d r o x y v i t a m i n D3-26,2\-lactone (130,131), 25,26,27-tris-norv i t a m i n D -24-carboxyl ic a c i d ( 132), 25-hydroxy-24-oxocho1 ec a l c i f e r o ? (133), and p o s s i b l y 25-hydroxyvi tami n D3-25g l u c u r o n i d e (134,135). I n i t i a l experiments w i t h r a d i o l a b e l ed v i t a m i n D3 showed very l i t t l e r a d i o a c t i v i t y i n t h e aqueous p o r t i o n s a f t e r t h e t i s s u e s were e x t r a c t e d w i t h a mixt u r e o f c h l o r o f o r m and methanol. However, t h e presence o f w a t e r s o l u b l e m e t a b o l i t e s has always been suspected. The presence o f v i t a m i n D s u l f a t e i n human m i l k was r e p o r t e d by a few i n v e s t i g a t o r s ( h 6 - 1 3 8 ) R e c e n t l y H o l l i s e t a1 (139) used a more s p e c i f i c HPLC method f o r v i t a m i n D s u l f a t e i n human m i l k whey and d i d n o t f i n d any ( d e t e c t i o n l i m i t 1 ng/ml). However, i t i s e s t a b l i s h e d t h a t human m i l k has antirachitic activity. Lakdawala and Widdowson (137) have r e p o r t e d t h a t even i n w i n t e r i n t h e U n i t e d Kingdom, b r e a s t m i l k p r o t e c t s i n f a n t s from r i c k e t s . Therefore, t h e metabo1 it e t h a t p r o t e c t s in f a n t s f rom r i c k e t s whi l e on b r e a s t m i 1k remains t o be c l e a r l y i d e n t i f i e d .

d

.

.

Leading i n v e s t i g a t o r s a r e c e r t a i n t h a t t h e r e a r e many u n i d e n t i f i e d m e t a b o l i t e s o f v i t a m i n 03. For example, acc o r d i n g t o Onisko e t a l . (127), t h e r e a r e s u b s t a n t i a l amounts o f unknown m e t a b o l i t e s o f 1,25-(OH)2D3 i n b i l e . These a r e water-sol u b l e, n e g a t i v e l y charyed compounds which a r e rendered c h l o r o f o r m - s o l u b l e a f t e r m e t h y l a t i o n suggesting t h a t t h e s e are unknown c a r b o x y l ic a c i d metabol it e s . Admi ni s t r a t i o n o f r a d i o a c t i v e v i t a m i n D3 d a i l y f o r 1-2 weeks (140) have l e d t o t h e d i s c o v e r y o f a few unknown metabolites. I t i s n o t known i f any o f these have i m p o r t a n t b i 01o y i c a l a c t iv i t y 6.

V i t a m i n D1 As

A Prohormone

V i t a m i n 03 i s unique i n t h a t under normal circumstances i s formed i n t h e s k i n in s u f f i c i e n t q u a n t i t i e s when humans and animals a r e exposed t o sunshine, and e x t r a supit

K. THOMAS KOSHY AND WILLIAM E BEYER

706

plementation i s unnecessary. The p r e v i t a m i n D3 t h a t i s formed i n t h e s k i n i s s l o w l y released i n t o t h e body as v i t a m i n D3. Vitamin D3 i s a l s o unique i n t h a t i t has t o be metabolized i n sequence t o 25-OH-D3 and t o 1,25-(OH) D3 o r t o some as y e t unknown m e t a b o l i t e b e f o r e i t becomes pEysiol o g i c a l l y a c t i v e . Since 1,25-(OH)*D3 a c t s on t i s s u e s remote from i t s p r o d u c t i o n s i t e , it meets t h e c r i t e r i a and d e f i n i t i o n o f a hormone. I n t r u e hormonal form, i t s biogenesis i s r e g u l a t e d by hypocalcemia o r hypophosphatemia. Then v i t a m i n D3 can be considered a prohormone and 25-OH-D3 as a prehormone. Acknowledgement The authors acknowledge t h e excel l e n t s e c r e t a r i a l support o f Mrs. Becky B. Hughson throughout t h e p r e p a r a t i o n o f t h i s manuscript

.

VITAMIN Dz (CHOLECALCIFEROL)

701

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Proc. N a t l . Acad. and H.F.DeLuca,

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Biophys.,


PROFILE SUPPLEMENTS


TOLBUTAMIDE Said E. Ibrahiin and Abdullah A. Al-Badr King Saud University Riyadh, Saudi Arabia Physical Properties 1. I Crystal Shape I .2 Crystal Structure 1.3 Mass Spectrum 1.4 Carbon-I3 Nuclear Magnetic Resonance Spectrum 2. Methods of Analysis 2.1 Titrimetric Methods 2.2 Spectrophotometric Methods 2.3 Chromatographic Methods 3. Pharmacokinetics 4. Identification References 1.

ANALYTICAL PROFILES OF D R U G S U B S T A N C E S VOLUME 13

719

720 720 720 721 72 1 726 726 727 729 73 1 733 734

Copytghr 0 19x4 by [he American Phannaceutical Asrociation ISBN 0-I?-260813-5

SAID E. IBRAHIM AND ABDULLAH A . AL-BADR

720

TOLBUTAMIDE

Tolbutamide, N- [ (butylamino) c a r b o n y l ] -4-methylbenzenesulphonamide; N-(butylamino) carbonyl-p-toluene sulphonamide is a s u l p h o n l y l u r e a t h a t i s o r a l l y a c t i v e a s a hypoglycemic a g e n t . The drug s t i m u l a t e s t h e p o n c r e a t i c i s l e t b e t a c e l l s t o release e x t r a i n s u l i n . I t a l s o i n h i b i t s p h o s p h o d i e s t e r a s e , which p r e s e r v e s c y c l i c AMP and t h u s f a v o r g l y c o g e n o l y s i s i n a number of t i s s u e s . 0

-NH

CH3

II

- C - NH-

CH -CH2-CH2-CH 2

3

Tolbutamide

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

1.1 C r y s t a l Shape Tolbutamide e x i s t s i n two polymorphs, one w a s o b t a i n e d e i t h e r by c r y s t a l l i z a t i o n of t o l b u t a m i d e from benzene s o l u t i o n a f t e r a d d i t i o n of hexane, o r by p r e c i p i t a t i o n from s o l u t i o n i n aqueous ammonia by a d d i t i o n of a c e t i c a c i d , and t h e o t h e r , m e t a s t a b l e form w a s o b t a i n e d from e t h a n o l i c s o l u t i o n a f t e r a d d i t i o n of water. The two forms w e r e c h a r a c t e r i z e d by i n f r a r e d s p e c t r o s c o p y , xr a y d i f f r a c t i o n and d . t . a . ( l ) 1.2 C r y s t a l S t r u c t u r e Nirmala and Gowda ( 2 ) have r e p o r t e d t h a t t o l b u t a m i d e c r y s t a l l i z e s i n t h e orthorhombic s p a c e group P n 2 l a , w i t h a=20.16 (2), b=9.05 ( l ) , C=7.85 (1) Ao, 2=4, D c = l .25, h=l. 24 Mgm-3 and The (CuKa) = l .968 mm-1. s t r u c t u r e w a s s o l v e d by t h e P a t t e r s o n s e a r c h method and r e f i n e d t o a n R f a c t o r of 0.083 f o r 615 v i s u a l l y measured r e f l e c t i o n s . The s t r u c t u r e i s s t a b i l i z e d by hydrogen bonding between t h e p o l a r group and Van d e r waals i n t e r a c t i o n s between t h e non p o l a r groups. The

12 I

T'OLBUTAMIDE

hydrogen-bond network and t h e dynamics of t h e p r o t o n s a r e of g r e a t importance i n e x p l a i n i n g t h e p r o p e r t i e s of t h e c r y s t a l s and t h e i r phase t r a n s i o n s . The f o l l o w i n g f i g u r e r e p r e s e n t s a p r o j e c t i o n of t o l butamide molecule showing t h e atom numbering and bond The a u t h o r s excluded t h e hydrogen atoms lengths (Ao). f o r c l a r i t y (2).

1.3 Mass Spectrum The e l e c t r o n impact ( E I ) m a s s spectrum a t 70 eV r e c o r d ed on Varian Mat 311 m a s s s p e c t r o m e t e r and t h e methane d e r i v e d chemical i o n i z a t i o n (CI) m a s s spectrum o b t a i n e d w i t h F i n n i g a n 4000 m a s s s p e c t r o m e t e r are shown i n F i g u r e s 1 and 2 r e s p e c t i v e l y . The ( E I ) spectrum F i g . 1 shows a b a s e peak a t m / e 91. Other major fragment a t m / e ( r e l a t i v e abundance) 108(71) , 1 5 5 ( 4 8 ) , 65(27) , and 1 9 7 ( 1 5 ) . The ( C I ) spectrum F i g . 2 i s s u r p r i s i n g l y s i m p l e , t h e base peak m/e 271 c o r r e s p o n d s t o E.t' i o n . Other major f r a g m e n t s a r e a t m / e 117, 172, 200, 61 and

m f e 74.

1 . 4 Carbon-13 Nuclear Magnetic Resonance Spectrum The carbon-13 NMR c o m p l e t e l y decoupled and off-resonance 3 and 4 r e s p e c t i v e l y . s p e c t r a are shown i n Fig. Both were r e c o r d e d o v e r 5000 Hz r a n g e i n d e u t e r a t e d d i m e t h y l s u l p h o x i d e on FT-SO A-80 MHz NMR s p e c t r o m e t e r u s i n g t e t r a m e t h y l s i l a n e a s r e f e r e n c e s t a n d a r d . The carbon chemical s h i f t v a l u e a r e a s s i g n e d on t h e b a s i s of s i g n a l m u l t i p l i c i t y , chemical s h i t s and t h e comp a r i s o n w i t h t h e chemical s h i t s of model compounds. Table 1 summarizes t h e carbon chemical s h i f t s of t o 1b u t a m i d e

.

122

s

4

at

a a, c .;i

E

a,

a, 4

a rl

h

-w

a,

a

5

-4

7

u

Q

0

rl

0

c, w

IOO.0-

50.0 -

100.0-

-

271

4

w N

-

50.0-

'

325

344 353

351 849

378 337 395 l.'**'"''I

Fig. 2. Mass spectrum of tolbutamide (C1) determined by direct probe insertion.

124

7

1

Fig. 4. C-13 nuclear magnetic resonance spectrum ( o f f resonance) of Tolbutamide.

SAID E. IBRAHIM A N D ABDULLAH A . AL-BADR

726

Table 1.

Carbon Chemical S h i f t s of Tolbutamide.

"0; 0

!I

-NH

H3C

8'

7'

4

3

2

- 5C - IW-CH 2-CH 2-CH2-CH

1

3

0

Carbon

Multiplicity


(9)

13.26

(t)

19.30

(t>

20.90

(t1

38.98

(s)

151.45

(s)

148.36

(d)

127.09

c7 '

(d)

127.09

'8

(d)

129.23

(d)

129.23

(s)

137.62

(9)

31.28

c2 c3 c4

c5 '6

c7

'

'8 c9

clo

2 . Methods of A n a l y s i s 2.1 T i t r i m e t r i c Methods

2.1.1 Aqueous T i t r a t i o n e t a1 (3) i n v o l v e s The methods of E l - F a t a t r y -measurements of t h e pKa v a l u e s of t o l b u t a m i d e i n d i f f e r e n t s o l v e n t s and t i t r a t i o n of t h e d r u g i n a s u i t a b l e medium w i t h s t a n d a r d a l k a l i t o t h e

TOLBUTAMIDE

121

pH v a l u e c o r r e s p o n d i n g t o t h e pKa of i t i n t h e medium. Accurate and p r e c i s e r e s u l t s were o b t a i n e d i n t h e c o n c e n t r a t i o n r a n g e 1 t o 10 mg m l - 1 i n aqueous 60% a c e t o n e . 2.1.2 Non-Aqueous T i t r a t i o n Tolbutamide i n t a b l e t s and p u r e forms w a s d i s s o l v e d i n t e t r a m e t h y l u r e a and t i t r a t e d w i t h 0.1 N l i t h i u m methoxide i n benzene-methanol medium, t h e end p o i n t w a s determined v i s u a l l y w i t h 0 . 2 % a z o - v i o l e t i n t o l u e n e as i n d i c a t o r ( 4 ) . 2.2 SDectroDhotometric Methods 2.2.1 U l t r a v i o l e t a. Abdel Hady -et a l ( 5 ) r e p o r t e d two s p e c t r o p h o t o m e t r i c methods f o r t h e q u a n t i t a t i o n of t o l butamide w i t h o u t i n t e r f e r e n c e from t h e t a b l e t e x c e p i e n t s . I n t h e f i r s t (Glenn's) method, t h e absorbance of t o l b u t a m i d e i n 952 e t h a n o l w a s measured a t 250-270 nm a t 4 nm i n t e r v a l s The coand the p2 c o e f f i c i e n t c a l c u l a t e d . e f f i c i e n t w a s l i n e a r l y r e l a t e d t o concentraThe t i o n w i t h i n a r a n g e of 0.1-0.4 mg/ml. second method i s based on t h e f o r m a t i o n of a complex of a r a t i o 1:l w i t h b a s i c dye b r i l l i a n t c r e s y l b l u e o r s a f r a n i n e T , t h e complex w a s e a s i l y e x t r a c t e d w i t h chloroform and t h e absorbance of t h e chloroform e x t r a c t w a s measured a g a i n s t e i t h e r a b l a n k o r r e f e r e n c e experiment a t 615 o r 510 nm f o r b r i l l i a n t c r e s y l b l u e o r s a f r a n i n e T methods r e s p e c t i v e l y . The r e s u l t s of t h e s e methods a r e more a c c u r a t e t h a n t h o s e of t h e t r a d i t i o n a l u l t r a v i o l e t s p e c t r o p h o t o m e t r i c methods.

b. Tolbutamide w a s determined i n p r e s e n c e of t h i a m i n e h y d r o c h l o r i d e and p y r i d o x i n e hydroc h l o r i d e , by measuring t h e d i f f e r e n c e i n absorbance a t 274 and 276 nm i n 95% e t h a n o l medium, t h e i n t e r f e r e n c e by t h i a m i n e hydrochloride o r pyridoxine hydrochloride i s n e g l i g a b l e . Mean r e c o v e r i e s (10 determinat i o n s ) w e r e 100.0 t o 100.8 and t h e s t a n d a r d d e v i a t i o n = 0.5% ( 6 ) .

SAID E. IBRAHIM A N D ABDULLAH A . AL-BADR

728

c. Tolbutamide w a s determined i n c i t r a t e d blood contaminated w i t h i t s m e t a b o l i t e s l-butyl-3(4-carboxybenzenesulphonyl) u r e a and l - b u t y l 3-(hydroxymethylbenzenesulphonyl) u r e a ( 7 ) . The haemolyzed blood i n aqueous phosphate b u f f e r of pH 5 was shaken w i t h heptane-chloroform ( 4 : l ) f o r 20 m i n u t e s , t h e o r g a n i c phase was shaken w i t h 0.1 M sodium hydroxide s o l u t i o n f o r 20 minutes t h e n t h e aqueous a l k a l i phase w a s mixed ( 2 0 : l ) w i t h 3M h y d r o c h l o r i c a c i d s o l u t i o n and t h e e x t i n c t i o n w a s measured a t 230 nm. Blank s o l u t i o n c o n t a i n i n g uncontaminated drug w e r e used i n a l l i n s t a n c e . 2.2.2

Spectrofluorimetry Among o t h e r a n t i d i a b e t i c d r u g s , t o l b u t a m i d e w a s determined i n f o r m u l a t i o n s and i n b i o l o g i c a l f l u i d s by a c i d h y d r o l y s i s , r e a c t i o n of t h e r e s u l t i n g amine w i t h a c e t y l a c e t o n e and fonnaldehyde, and f l u o r i m e t r i c d e t e r m i n a t i o n of t h e r e s u l t i n g s u b s t i t u t e d d i h y d r o p y r i d i n e a t 600 nm, w i t h e x c i t a t i o n a t 480 nm (8).

2.2.3

P r o t o n Magnetic Resonance Spectrometry Al-Badr and Ibrahim (9) r e p o r t e d a n a c c u r a t e , r a p i d and p r e c i s e method f o r t h e d e t e r m i n a t i o n of t o l b u t a m i d e and some o t h e r hypoglycemic a g e n t s i n p u r e and t a b l e t forms u s i n g p r o t o n magnetic resonance spectroscopy. The method i n v o l v e s comparing t h e i n t e g r a t i o n of t h e a r o m a t i c doubl e t s of t o l b u t a m i d e a t 7.33-7.8 ppm w i t h t h a t of t h e s i n g l e t of hexamethylenetetramine ( i n t e r n a l s t a n d a r d ) a t 4.61 ppm u s i n g d i m e t h y l s u l f o x i d e as s o l v e n t . Recovery i s 1 0 0 % f 1 . 5 and 9 9 . 6 % ? 1 . 4 f o r p u r e t o l b u t a m i d e and i t s t a b l e t dosage form respectively

.

2.2.4

Mass Spectrometry

a. Tolbutamide and i t s m e t a b o l i t e s i n plasma and u r i n e were determined u s i n g chemical i o n i z a t i o n mass spectrometry. The d r u g s and i t s m e t a b o l i t e s were e x t r a c t e d w i t h e t h y l e t h e r from samples of plasma and u r i n e t o which 2Hl a b e l e d i n t e r n a l s t a n d a r d s have been added. The extract w a s t h e n t r e a t e d w i t h e x c e s s

129

TOLBUTAMIDE

diazomethane. Quantilative measurements were made on protonated molecular ion (which is free from interference), with use of isobutane as reagent gas, by single ion monitoring for determination of single component or in scan mode for multi-component mixtures. Levels down to 0.5 ug ml in plasma can be determined by this method (10).

b. A high resolution mass spectroscopic analysis of methylated tolbutamide derivatives was reported by Sabih (11). The methyl derivative of tolbutamide obtained by treatment with dimethyl sulphate was subjected to g.1.c. on a stainless steel column ( 6 ft. X 0.125 in) packed with 1% OV-1 on Gas-Chrom Q , temperature programmed from 150 to 1900 at 20 per minute and operated with helium as carrier gas (30 ml per minute). A mass spectrum of the column effluent was obtained at 70 eV with a double-focussing instrument linked to a digital data system. c. Braselton et a1 (12) determined tolbutamide and its metabolites in human serum by the formation of thermally stable derivatives (Nmethyl-N--trifluoroacetyl) and analyzing this derivatives by g.1.c.-m.s. method. The g.1.c. was performed on a glass column (1.5 m X 2 nun) operated at 170° with helium as carrier gas. system was coupled with PDP-8 The g.1.c.-m.s. computer for data processing, a detection ion limit of 1 p mol is claimed. 2.3 Chromatographic Methods 2.3.1 Gas Chromatographic Gas chromatography has frequently been used for the analysis to tolbutamide. The type of column, carrier gas and other experimental conditions are listed in Table 11. 2.3.2 Liquid Chromatography Tolbutamide, among other sulphonylureas, was determined in pharmaceutical products by highspeed liquid chromatography. The column used

Table I1 G a s Liquid Chromatography of Tolbutamide.

Drug Source

Phase and Column

Carrier g a s

1%SP-2100 on s u p e l c o p o r t n i t r o g e n

Tempera- D e r i v a t i v e Detector t u r e (OC) used

Internal Standard

Ref.

165

N-MethylN--trif luoroacetyl.

FID*

Chlorpropamidie.

(12)

nitrogen

220

methyl

EC**

Hexadocosane

(13)

nitrogen ( 50 m l /min-l)

190

methyl

FID

4-chloro-Npropylbenzene sulphonamide

(14)

Plasma 10%W-98 on Chromosorb W- helium 6 u r i n e HP(80-100 mesh) , s t a i n l e s s (30 ml/mi

Analytical Profiles of Drug Substances and Excipients Volume 13

Analytical profiles of drug substances and excipients

Analytical profiles of drug substances and excipients