Analytical Profiles of Drug Substances
and Excipients
EDITORIAL BOARD
Abdullah A. Al-Badr
Lee T. Grady
Gerald S. ...
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Analytical Profiles of Drug Substances
and Excipients
EDITORIAL BOARD
Abdullah A. Al-Badr
Lee T. Grady
Gerald S. Brenner
Larry D. Kissinger
Glenn A. Brewer
David J. Mazzo
Harry G. Brittain
John N. Staniforth
Klaus Florey
Timothy J. Wozniak
George A. Forcier
Analytical Profiles of Drug Substances and Volume 22 Edited by
Harry G. Brittain Bristol-Myers Squibb Pharmaceutical Research Institute New Brunswick, New Jersey
Founding Editor
Klaus Florey
ACADEMIC PRESS, INC. A Division of Harcourt Brace & Company
San Diego New York Boston London Sydney Tokyo Toronto
Academic Press Rapid Manuscript Reproduction
This book is printed on acid-free paper. @
Copyright 0 1993 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.
Academic Press, Inc. 1250 Sixth Avenue, San Diego. California 92101-431I United Kingdom Edition puhlished hy
Academic Press Limited 24-28 Oval Road. London NW1 7DX International Standard Serial Number: 0099-5428 International Standard Book Number: 0-12-260822-4 PRINTED IN THE UNITED STATES OF AMERICA 9 3 9 4 9 5 9 6 9 7 9 8
BC
9 8 7 6 5 4 3 2 1
CONTENTS
vii ix
Afiliations of Editors and Contributors Preface
1
Ace tazolamide Jagdish Parasrampuria Aminobenzoic Acid Humeida A. El-Obeid and Abdullah A. Al-Badr
33
Bumetanide Prasad N. V. Tata, Raman Venkataramanan, and Swroop K. Sahota
107
Clozapine Michael J. McLeish, Benny Capuano, and Edward J. Lloyd
145
Didanosine Munir N. Nassar, Tracy Chen, Michael J. Re8 and Shreeram N. Agharkar
I85
Dipivefrin Hydrochloride G . Michael Wall and Tony Y. Fan
229
Lactic Acid Fahad J. Al-Shammary, Neelofur Abdul Aziz Mian, and Mohammad Saleem Mian
263
Methixene Hydrochloride E u a t M . Abdel-Moety, Nashaat A . Khattab, and Mohammad Saleem Mian
317
Simvastatin Dean K . Ellison, William D. Moore, and Catherine R . Petts
359
V
vi
CONTENTS
Sulfathiazole Vijay K. Kapoor
389
Tenoxicam Abdulrahman Mohammad Al-Obaid and Mohammad Saleem Mian
43 1
Thiamphenicol Gunawan Indrayanto, Dian L. Trisna, Mulja H. Santosa, Ratna Handajani, Tekad Agustono, and Purnomo Sucipto
46 1
Tolazamide John K. Lee, Kazimierz Chrzan, and Robert H. Witt
489
Profile Supplement
Vincristine Sulfate Farid J. Muhtadi and Abdul Fattah A. A. AJifr
517
Excipient Profile
Povidone Christianah M . Adeyeye and Eugene Barabas
555
Cumulative Index
687
AFFILIATIONS OF EDITORS AND CONTRIBUTORS
Ezzur M . Abdel-Moery, Pharmaceutical Chemistry Department, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia Chrisrianah M . Adeyeye, School of Pharmacy, Duquesne University, Pittsburgh, Pennsylvania 15282 Abdul Fattah A . A . A$fy, Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia Shreeram N. Aghurkar, Bristol-Myers Squibb Pharmaceutical Research Institute, Syracuse, New York 13221 Tekad Agusrono, PT. New Interbat Pharmaceutical Laboratories, Buduran, Sidoarjo, Indonesia Abdulluh A . Al-Budr, Pharmaceutical Chemistry Department, College of Pharmacy, King Saud University, Riyadh 1 1451, Saudi Arabia Abdulrahman Mohummud Al-Obaid, Pharmaceutical Chemistry Department, College of Pharmacy, King Saud University, Riyadh 1 1451, Saudi Arabia Fuhud J. Al-Shammury, Clinical Laboratory Sciences Department, College of Applied Medical Sciences, King Saud University, Riyadh 1 1433, Saudi Arabia Eugene Burubas, ISP Corporation, Wayne, New Jersey 07470 Benny Cupuuno, Victorian College of Pharmacy, Monash University, Parkville, Victoria, Australia Tracy Chen, Bristol-Myers Squibb Pharmaceutical Research Institute, Syracuse, New York 13221 Kuzimierz Chrzun, RhBne-Poulenc Central Research, Collegeville, Pennsylvania 19426 Hurneidu A . El-Obeid, Pharmaceutical Chemistry Department, College of Pharmacy, King Saud University, Riyadh 1 1451, Saudi Arabia Dean K . Ellison, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486 Tony I:Fun, Alcon Laboratories, Inc., Fort Worth, Texas 76134 Rurna Hundujani, PT. New Interbat Pharmaceutical Laboratories, Buduran, Sidoarjo, Indonesia Gunuwun Indrayunto, Faculty of Pharmacy, Airlangga University, Surabaya, Indonesia
vii
viii
AFFILIATIONS OF EDITORS AND CONTRIBUTORS
Vijay K . Kapoor, Department of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India Nashaat A. Khattab, Pharmaceutical Chemistry Department, College of Pharmacy, King Saud University, Riyadh 1145 1, Saudi Arabia John K . Lee, RhBne-Poulenc Central Research, Collegeville, Pennsylvania 19426 Edward J. Lloyd, Victorian College of Pharmacy, Monash University, Parkville, Victoria, Australia Michael J. McLeish, Victorian College of Pharmacy, Monash University, Parkville, Victoria, Australia Mohammad Saleem Mian, Pharmaceutical Chemistry Department, College of Pharmacy, King Saud University, Riyadh 1145 1, Saudi Arabia Neelofur Abdul Aziz Mian, Clinical Laboratory Sciences Department, College of Applied Medical Sciences, King Saud University, Riyadh 11433, Saudi Arabia William D. Moore, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486 Farid J. Muhtadi, Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 1145 1, Saudi Arabia Munir N. Nassar, Bristol-Myers Squibb Pharmaceutical Research Institute, Syracuse, New York 13221 Jagdish Parasrampuria, Abbott Laboratories, Abbott Park, Illinois 60064 Catherine R. Petts, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486 Michael J. Reg Bristol-Myers Squibb Pharmaceutical Research Institute, Syracuse, New York 13221 Swroop K . Sahota, Hoffmann-La Roche, Inc., Nutley, New Jersey 071 10 Mulja H. Santosa, Faculty of Pharmacy, Airlangga University, Surabaya, Indonesia Purnomo Sucipto, FT. New Interbat Pharmaceutical Laboratories, Buduran, Sidoarjo, Indonesia Prasad N. V: Tutu, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Dian L. Trisna, Faculty of Pharmacy, Airlangga University, Surabaya, Indonesia Raman Venkataramanan, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 G. Michael Wall, Alcon Laboratories, Inc., Fort Worth, Texas 76134 Robert H. Witt, RhBne-Poulenc Central Research, Collegeville, Pennsylvania 19426
PREFACE
The profiling of the physical and analytical characteristics of drug compounds remains as important today as it was when the Analytical Profiles series was first initiated. The compilation of concise summaries of physical and chemical data, analytical methods, routes of compound preparation, degradation pathways, and the like, is a vital function to both academia and industry. The expansion of the series mission to include profiles of excipient materials reflects the realization that all aspects of a drug formulation need to be fully specified. It is no longer sufficient to consider excipients as merely representing the inactive portion of a dosage form. In the future, it is likely that more complete chemical and physical characterization work will have to be performed, which will in turn increase the existing body of knowledge. For the sake of the pharmaceutical community, this accrued information will be summarized in a series of excipient profiles. The extensive chapter on Povidone contained in this volume is an indication of how detailed these excipient profiles can be. The success of the Analytical Profiles series will continue to be based on the contributions of the chapter authors, and on the quality of their work. We seek to profile new drug compounds as they become marketed, but also wish to profile important older compounds which have somehow escaped attention. Updates on compounds profiled earlier in the series are always welcome, especially once new and significant information has become available as a result of the continuing advances in the field. A complete list of available drug and excipient candidates is available from the editor. We look forward to hearing from new and established authors, and to working with the pharmaceutical community on Analytical Profiles of Drug Substances and Excipients.
Harry G. Brittain
ix
This Page Intentionally Left Blank
ACETAZOLAMIDE
Jagdish Parasrampuria
Abbott Laboratories
Pharmaceutical R&D Abbott Park, Illinois 60064
ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 22
1
Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any form reS0Ned.
2
1.
JAGDISH PARASRAMPURIA
I NTRODUCTION 1.1 1.2 1.3
2.
DESCRIPTION Name, Formula, and Molecular Weight Appearance, Color, Odor, and Taste Pharmaceutical Dosage Forms PHYSICAL PROPERTIES 3.1 Dissociation Constant (pKa) 3.2 U1 traviolet Spectra 3.3 Solubility 3.3.1 pH-Sol ubil i ty Profi 1 e 3.3.2 Solubility in Various Commonly Used Pharmaceutical Sol vents 3 -3-3 Sol ubi 1 i zation through Cosol vency 3.4 Polymorphism 3.5 Melting Point 3.6 Differential Scanning Calorimetry 3.7 Infrared Spectra 3.8 X-Ray Diffraction 3.9 Nuclear Magnetic Resonance Spectra METHODS OF ANALYSIS 4.1 Identification Tests 4.2 U1 traviolet Spectroscopy 4.3 Polarography 4.4 Nuclear Magnetic Resonance Spectroscopy 4.5 Chromatography 4.5.1 High-Performance Liquid Chromatography 4.5.2 Gas Liquid Chromatography STABILITY 5.1 pH-Rate Profile 5.2 Stability in Intravenous Admixtures 5.3 Effect of Temperature 5.4 Effect of Buffer Species, Buffer Concentration, and Ionic Strength 5.5 Stability in Various Solvents BIOPHARMACEUTICS 6.1 Pharmacokinetics and Metabolism 6.2 Bi oavai 1 abi 1 i ty REFERENCES 2.1 2.2 2.3
3.
4.
5.
6. 7.
History Therapeutic Category Chemistry and Structure-Activity Re1 ati onshi p
3
ACETAZOLAMIDE
1.
INTRODUCTION
1.1
History
I n t h e e a r l y 1930s, Roughton (1) discovered t h e existence i n erythrocytes of an enzyme which promotes t h e hydration o f carbon d i o x i d e and dehydration o f carbonic acid. The enzyme was found t o be carbonic anhydrase. Since then, t h e presence of carbonic anhydrase has been demonstrated i n p r a c t i c a l l y every physiological b a r r i e r where i o n exchange occurs, e.g., t h e kidney, sweat glands, s a l i v a r y glands, b r a i n and spinal cord t i s s u e , choroid plexus, p a r i e t a l t i s s u e o f g a s t r i c mucosa, pancreatic tissue, and t h e eye. I n the eye, carbonic anhydrase has been found i n t h e lens, r e t i n a , c i l i a r y body, i r i s , and v i t r e o u s body. According t o the c l assf cal concept developed between 1930 and 1950, carbonic anhydrase was thought t o have a s i n g l e chemical r o l e , t h a t i s , c a t a l y s i s of t h e r a t e o f a t t a i n i n g e q u i l i b r i u m i n t h e primary r e v e r s i b l e r e a c t i o n : H20 + CO2
d CARBONIC ANHYDRASE
A secondary, p r a c t c a l l y instantaneous e q u i l i b r i u m
L H2CO3
H+ + ~ ~ 0 3 -
7
i s n o t c a t a l wed. Thus t h e enzyme-sensitive r e a c t i o n i s t h e r a t e - c o n t r o l i i n g step i n t h e o v e r a l l process (2). I n 1950, a series o f unsubstituted h e t e r o c y c l i c sulfonamides were synthesized. Among these was acetazol ami de (3 *4), a compound found t o possess speci f ic carbonic anhydrase-inhibiting a c t i v i t y and a low incidence o f acute and chronic t o x i c i t y . Thus, acetazolamide was f i r s t used by physicians i n 1953 as a d i u r e t i c . 1.2
Therapeutic Category
Acetazolamide i s a potent and r e v e r s i b l e carbonic anhydrase i n h i b i t o r , e f f e c t i v e i n t h e c o n t r o l o f f l u i d secretion, e.g., glaucomas ( 5 ) , i n t h e treatment of c e r t a i n convulsive disorders, e.g., epilepsies (6), and i n t h e promotion o f d i u r e s i s i n instances o f abnormal f l u i d r e t e n t i o n (7).
4
JAGDISH PARASRAMPURIA
Acetazolamide i s indicated f o r centrencephal i c e p i l e p s i e s ( p e t i t malt unlocalized seizures), chronic simple (open angl e) glaucoma, secondary glaucoma, and preoperatively i n acute angle closure glaucoma where delay o f surgery i s desired i n order t o lower i n t r a o c u l a r pressure ( 8 ) . Acetazolamide i s used as an adjuvant i n t h e treatment o f c e r t a i n dysfunctions of t h e central nervous system i n which cerebral spinal f l u i d pressure i s increased, e.g., hydrocephalus (9-13). Acetazolamide i s a l s o used f o r t h e f o l l o w i n g i n d i c a t i o n s n o t included i n USA product l a b e l i n g : i n t h e treatment o f hypokalemic and hyperkalemic forms o f f a m i l i a l p e r i o d i c p a r a l y s i s (14,15); as prophylaxis f o r and treatment o f a1t i t u d e (mountain) sickness (16-19); t o f o r c e a l k a l i n e d i u r e s i s t o increase e l i m i n a t i o n o f weakly a c i d i c medications (20); and as a a n t i u r o l i t h i c t o a l k a l i n i z e t h e u r i n e t o prevent c y s t i n e and u r i c a c i d renal stone formation (21). Acetazolamide has a l s o been used t o prevent metabolic a l k a l o s i s (22) and as a d i u r e t i c i n t h e treatment o f edema due t o congestive heart f a i l u r e o r due t o drugs. However, i t has been replaced by newer d i u r e t i c s f o r these indications. 1.3
Chemistry and Structure-Activity Relationship
Acetazolamide i s n o t a mercurial d i u r e t i c . Rather, i t i s a nonbacteriostatic sulfonamide possessing a chemical s t r u c t u r e and pharmacol og ical a c t i v i t y d ist in c t l y d if f erent from t h e b a c t e r i o s t a t i c sulfonamides. Among t h e enormous number o f sulfonamides t h a t have been synthesized and tested, acetazolamide has been studied most extensively as an i n h i b i t o r o f carboni c anhydrase. The most s t r i k i n g s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p i s t h a t aromatic sulfonamides, unsubstituted on -S02NH2 nitrogen, are speci f ic carbonic anhyqrase i n h i b i t o r s . Thi s i n h i b i t o r y e f f e c t i s l o s t when t h e N -(sulfonamide) n i t r o g e n i s s u b s t i t u t e d (23).
2.
DESCRIPTION
2.1
Name, Formula, and Molecular Weight
Acetazolamide i s 2-acetylamido-1,3,4-thiadiazole-5sul fonamide; N-( 5-Sul famoyl-l,3,4-thiadiazol e-Z-yl )
ACETAZOLAMIDE
acetamide with a molecular formula of CqH6Nq03S2 and molecular weight o f 222.24. The structure of acetazolamide is shown in Figure 1.
Figure 1: Structure o f Acetazolamide
Appearance, Color, Odor, and Taste
2.2
A fine, white-to-faintly-yellowish-white, odorless, bitter crystalline powder. A 3.85% solution of acetazolamide is iso-osmotic with serum (24). Pharmaceutical Dosage Forms
2.3
Acetazolamide Tablets, USP: 125 and 250 mg; Diamox by Lederle Laboratory, Division of American Cyanamid Co.; Generic version by Bolar Pharmaceuticals, Danbury Pharmaceuticals, Lannett Co., and Mutual Pharmaceutical Co. (25)
-
Acetazol ami de Extended-Re1ease Capsul es : 500 mg ; D i amox Sequel s by Lederl e Laboratory, Division of Ameri can Cyanamid co. Sterile Acetazolamide Sodium, USP: 500 mg; Diamox by Lederle Laboratory, Division of American Cyanamid Co., Generic version by Quad Pharmaceuticals (25).
S
JAGDISH PARASRAMF'URIA
6
3.
PHYSICAL PROPERTIES
3.1
D i s s o c i a t i o n Constant
Acetazolamide i s a weak acid w i t h a d i s s o c i a t i o n constant (pKa) value o f 7.2. 3.2
U l t r a v i o l e t Spectroscopy
The u l t r a v i o l e t absorbance o f acetazolamide scanned from 200 t o 400 nm i s presented i n F i g u r e 2. The wavelength o f maximum absorbance i s a t 265 nm (26). 3.3
Solubility
Acetazolamide i s very s l i g h t l y s o l u b l e i n water: s l i g h t l y s o l u b l e i n alcohol and acetone; p r a c t i c a l l y i n s o l u b l e i n carbon t e t r a c h l o r i d e , chloroform, and ether. Dissolves i n s o l u t i o n o f a l k a l i hydroxides; s p a r i n g l y s o l u b l e i n p r a c t i c a l l y b o i l i n g water (27). 3.3.1
pH-Sol ubi 1it y prof ile
The concentration o f acetazolamide i n saturated s o l u t i o n s o f various pH values i s shown i n F i g u r e 3 (28). The s o l u b i l i t y ranges from 0.8 t o 2.8 mg/mL between pH values o f 1.7 and 8.2. The p H - s o l u b i l i t y p r o f i l e i n d i c a t e s t h e s o l u b i l i t y between pH 4 and 7 i s approximately t h e same (0.8-1 mg/mL). S o l u b i l i t y i s higher on t h e basic s i d e because o f sodium s a l t formation. However, degradation increases manyfold on t h e basic side, which precludes measurement o f e q u i l i b r i u m s o l u b i l i t y above pH 8.2. 3.3.2
S o l u b i l i t y i n Various Commonly Used Pharmaceutical
Sol vents: Table 1 shows t h e s o l u b i l i t y o f acetazolamide i n various common1y used pharmaceutical solvents. The maximum s o l u b i l i t y i s i n polyethylene g l y c o l 400 (PEG 400), w i t h a value o f about 87.8 mg/mL. The s o l u b i l i t y i n propylene g l y c o l , alcohol, g l y c e r i n , and water i s 7.4, 3.9, 3.6, and 0.7 mg/ml, r e s p e c t i v e l y (28).
ACETAZOLAMIDE
Wavelength (nrn)
Figure 2: UV Spectra o f Acetazolamide
JAGDISH PARASRAMPURIA
I
I
1
2
I
I
3
4
I
i
I
I
1
5
6
7
8
9
PH
Figure 3: pH-Sol ubi 1 ity Prof i1 e of Acetazolamide
ACETAZOLAMIDE
Table 1:
9
S o l u b i l i t y o f Acetazolamide i n Various Solvents a t 25'C
Sol vent
Sol ubi 1 it y (mg/mL)
Pol y e t h y l ene G1y c o l
87.81 (3.45)*
Propyl ene G1ycol
7.44 (1.20)
Ethanol
3.93 (0.11)
Glycerin
3.65 (0.09)
Water
0.72 (0.05) ~
*Mean 3.3.3
( + std. dev.)
S o l u b i l i z a t i o n through Cosolvency
F i g u r e 4 ( A and 6) i l l u s t r a t e s t h e e f f e c t o f g l y c e r i n , 1,Z-propylene g l y c o l , PEG 300, PEG 400, polypropylene g l y c o l 420, e t h y l alcohol, dimethylacetamide, and dimethylsulpoxide a l l mixed w i t h water a t d i f f e r e n t r a t i o s . There i s an increase i n aqueous s o l ubi 1it y o f acetazol ami de upon t h e a d d i t i o n o f cosolvents. A s o l u b i l i t y - e n h a n c i n g e f f e c t i s dependent on t h e t y p e and volume f r a c t i o n o f cosolvent used. G l y c e r i n showed l e a s t e f f e c t , w h i l e dimethylsulphoxide i s t h e most e f f i c i e n t cosolvent f o r i n c r e a s i n g t h e s o l u b i l i t y
(29). According t o t h e equation derived by Yalkowski e t al., (30) l o g a r i t h m s o l u b i l i t y i s p l o t t e d against volume f r a c t i o n o f t h e cosolvent. As depicted i n F i g u r e 5, t h e r e i s a 1 inear r e 1a t i onshi p between 1og sol ubi 1 it y o f acetazol amide and volume f r a c t i o n o f g l y c e r i n , 1,2-propylene g l y c o l , methyl alcohol, e t h y l alcohol, PEG 300, PEG 400, and dioxane. The r e l a t i v e s o l u b i l i z i n g power o f these s o l v e n t s can be obtained from t h e slope o f t h e l i n e as l i s t e d i n Table 2 (29).
10
JAGDISH PARASRAMPURIA
0'
Figure 4A:
I
lo
20 30 40 Concentration (% v/v)
50
Sol ubi 1 i t y o f Acetazolamide in Some Mixed Solvent Systems. a--17 Polypropylene Glycol 420; A-A PEG 400; m.PEG 300; 04 1,2 G1 ycerol ; Propyl ene G1 ycol ; O-.
ACETAZOLAMIDE
Figure 46:
Solubility o f Acetazolamide in Some Mixed Solvent Systems. 0---0Dimethyl Sulfoxide; A---A Dimethyl Acetamide; 0---o Dioxane; Methanol ; A---A Ethanol
.---.
JAGDISH PARASRAMPURIA
12
0
2.4
2.0 -
2.2
1.8
.5 .-
-
1.4-
a
3
*
0.0
F i g u r e 5:
0.1
0.2
0.3
0.4
0.5
Log S o l u b i l i t y versus Volume F r a c t i o n o f Cosolvent f o r Acetazolamide. w Polypropylene Glycol 420; A-A PEG 400; PEG 300; 04 1,2 Propylene Glycol ; Glycerol ; (7---o Oimethyl Sulfoxide; A---A Oimethyl Acetamide; 0---o Oioxane; Methanol ; A---A Ethanol
Fc
.-. .---. m-
.m
ACETAZOLAMIDE
Table 2 Solubilizing Power of Some Binary Solvents Toward Acetazolamide
Cosol vent
Solubilizing Power
G1 ycerol
5.294 x
1,P-Propylene Glyco?
8.437 x
Methyl A1 coho1
10.00
x 10-3
Ethyl Alcohol
12.00
x 10-3
PEG 300
16.50
x
PEG 400
20.60
x
Oioxane
26.70
x
JAGDISH PARASRAMPURIA
14
3.4
Polymorphism
Acetazolamide has two polymorphic forms (Forms A and The solubility and dissolution rate of Form B is about 1.1 times higher those of Form A (31). The transition temperature obtained by solubility measurement is 78"C, and the heats of transition (AHtrans) calculated by solubility measurement and by differential scanning calorimetry are 2.6 and 1.7 kJ.mol-l, respectively. The free energy change ( ~ G 2 5 0 ~between ) the two polymorphic forms is 357 J. mol-l, which is a relatively small value. It is therefore presumed, following Aguiar and Zelmer, that acetazolamide polymorphic forms do not significantly affect bioavailability. The kinetics of isothermal transition from Form A to Form B at high temperature follows the mechanism of random nucleation with first-order kinetics. The activation energy for this tran ition as derived from Arrhenius plots is 246 kJ. mol-I. The results from the scanning electron microscope indicate that the crystal shape of acetazolamide during isothermal transition from Form A to Form B does not change significantly (31).
B).
3.4
Melting Point
The me1 ting point range of acetazol amide is 258-263OC, accompanied by decomposition. 3.5
Differential Scanning Calorimetry
Differential Scanning Calorimetry (DSC) curves of acetazolamide's two polymorphic forms are shown in Figure 6 (31). The DSC curve of Form A exhibits two endothermic peaks: one at 205OC corresponding to the transition from Form A to Form 3, and the other at 263OC attributable to the melting point accompanied by decomposition. Form B gives only one endothermic peak at 263OC corresponding to the melting point which is accompanied by decomposition as i ndi cated by the exothermic peak. 3.6
Infrared Spectra
Infrared spectra of acetazol amide's two polymorphic forms are shown in Figure 7 (31). The spectrum of Form A is different from that o f Form B. In particular, Form A shows
ACETAZOLAMIDE
3.4
IS
Melting Point
The me1t i ng p o i n t range o f acetazol ami de is 258-263OC, accompanied by decomposition. 3.5
Differential Scanning Calorimetry
D i f f e r e n t i a l Scanning Calorimetry (DSC) curves o f acetazolamide's two polymorphic forms a r e shown i n F i g u r e 6 (31). The DSC curve o f Form A e x h i b i t s two endothermic peaks: one a t 205OC corresponding t o t h e t r a n s i t i o n from Form A t o Form B, and t h e o t h e r a t 263OC a t t r i b u t a b l e t o t h e m e l t i n g p o i n t accompanied by decomposition. Form B g i v e s o n l y one endothermic peak a t 263OC corresponding t o t h e m e l t i n g p o i n t which i s accompanied by decomposition as i n d i c a t e d by t h e exothermic peak.
form A
100
150
200
Temperature
F i g u r e 6:
250
("c
OSC-TG Curves o f Acetazolamide Polymorphic DSC Curves; --- TG Curves; Forms. -, S e n s i t i v ' t y Range 41.8 mJ.s-l; Heating Rate, 5"C.min
-1
16
JAGDISH PARASRAMPURIA
3.6
Infrared Spectra
Infrared spectra of acetazolamide's two polymorphic forms are shown i n Figure 7 (31). The spectrum of Form A is different from that of Form 6. In particular, characteristic absorption peaks in the 1100-900 and Form B gives a specific peak at about 940 cm-
n
Form A
1
1
I
I
I
I
4000
3000
2000
1500
1000
650
Wave number (cm-l)
Figure 7:
Infrared spectra of Acetazolamide Polymorphic Forms (in N u j o l )
17
ACETAZOLAMIDE
3.7
X-Ray Diffraction
The X-ray powder diffraction patterns of the two polymorphic forms a r e shown i n Figure 8 (31). The d i f f r a c t i o n pattern of Form A is different from t h a t of Form B. Very h i g h peaks a t 9.9', 24.8' and 29.4" were observed i n the diffraction pattern o f Form A ; these were not found i n the diffraction pattern of Form B. On the other hand, Form B gave the h i g h e s t diffraction pattern a t 13.7" w i t h c h a r a c t e r i s t i c peaks a t 19.6" 22.3" 26.0' and 26.9".
Form A
5
10
'0
Form B
5
10
28 degrees
Figure 8:
X-Ray Diffraction Pattern of Two Polymorphic Forms o f Acetazol ami de
18
3.8
JAGDISH PARASRAMPURIA
Nuclear Uagnetic Resonance Spectra
The NMR spectra of acetazolamide contains broad peaks centered a t 790 cps ( 6 13.17, 1 H) and 514 cps ( 6 8.57, 2H) a r i s i n g from the N-protons of the carboxamide and sulfonamides, respectively (32).
The 15N NMR spectra of acetazolamide was measured in hexadeuteriodimethyl sufoxide. I n i t i a l l y protondecoupled ad protoncoupled spectra of the nitrogens bearing hydrogens
were measured. Then, tris-
(acetylacetonate)Cr(111) [Cr(acac) was added t o sol ution because the Cr(acac)3 considerab y shortens the relaxation tim and thus enables f a s t e r pulse r e p e t i t i o n . The changes of f S N chemical s h i f t s induced by the addition of t h i s able 3 l i s t s laxation reagent i s small (usually 4 ppm “N chemical s h i f t s and coupling constants ;(’ 15NH) of
1
acetazol ami de. Table 3:
1 5 N hemical s h i f t s and coupling constants J (“NH) in acetazol amide ~
~~~
6 15N Chemical S h i f t
‘J(15NH) Coupling Constant
-241.2 (CONH) [-241.4b (CONH)] -282.4 (S02NH2) -282.6b (SOzNHz)
-15. 7b [ N=C ( S ) -SO21 -58.3b [N=C(S)-NH] aCoupl ing constants were observed e i t h e r f o r reasons of small intermolecular proton exchange or small sol ubi 1 i t y of sampl es bmeasured a f t e r the addition o f 10 rng/mL Cr(acac)3, as re1 axati on agent
ACETAZOLAMIDE
4.
19
UEMODS OF ANALYSIS
Several assay methods f o r acetazolamide have been reported, such as u l t r a v i o l e t absorption spectroscopy (3335), p o l oragraphy (34), e l e c t r o n capture gas-1 iq u i d chromatography (36), high-performance 1i q u i d chromatography (37-44), amperometric determination using a s e s s i l e mercurydrop d e t e c t o r (45), and nuclear magnetic resonance (32). 4.1
Identification Tests
A) The i n f r a r e d absorption spectrum of a potassium bromide d i s p e r s i o n e x h i b i t s maxima o n l y a t t h e same wavelength 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 acetazolamide reference standard. I f a d i f f e r e n c e appears, p o r t i o n s o f both t h e t e s t specimen and t h e Reference Standard should be dissolved i n methanol, t h e s o l u t i o n s evaporated t o dryness, and t h e t e s t on residues repeated.
B ) When d i ssol ved acetazol ami de is m i xed w i t h hydroxylarnine hydrochloride and c u p r i c s u l f a t e and heated i n a steam bath f o r 5 minutes, a c l e a r b r i g h t y e l l o w s o l u t i o n i s produced. No heavy p r e c i p i t a t e o r dark brown c o l o r r e s u l t s a f t e r t h e mixing o r heating. 4.2
Ultraviolet Spectroscopy
The USP-NF methods f o r t h e q u a n t i t a t i o n o f acetazolamide sodium i n i n j e c t i o n i s based on UV spectroscopy i n HC1 solution (34). Since t h e h y d r o l y s i s product 5-arnino-1,3,4-thiadiazole-2-sulfonamide a l s o absorbs l i g h t a t same wavelength (265 nrn), t h e USP-NF method t h e r e f o r e cannot be s t a b i l i t y - i n d i c a t i n g . I n t e r f e r i n g absorbance due t o e x c i p i e n t s i n pharmaceutical formulations a f f e c t s t h e accuracy o f t h e spectrophotometric method. A p p l i c a t i o n o f pH-induced s p e c t r a l changes f o r acetazol ami de, however, nu1 1 i f ies t h e i r r e l e v a n t absorbance i f we assume t h a t t h e e x c i p i e n t i s n o t a f f e c t e d by these pH changes. As seen i n F i g u r e 9, i n 0.1 N NaOH acetazol ami de e x h i b i t s a bathochromi c s h i f t together w i t h a hyperchromic e f f e c t . The bathochromic s h i f t i s a t t r i b u t e d t o t h e formation o f SQ.NH-, which i s conjugated with the t h i a d i a z o l e r i n g (33).
20
JAGDISH PARASRAMPURIA
fi''\\,, I,t-\ I \.
0 5 . Ob
I
.
0 3 .
.-
a2 0Ob a 13 '. a2 '
a1
t
'.
; 0 ' L'
u \\
\ 1
b
Figure 9:
4.3
'\\
211
250
270
290
30
330
A
UV Spectrum of Acetazolamide i n 0.1 N H2SO4 -, and 0.1 N NaOH ---
Poloragraphy
Acetazolamide can be assayed i n a polarographic c e l l t h a t i s immersed i n a water bath regulated a t 25 k 0.5"C, and de-aerated by b u b b l i n g nitrogen through the solution f o r 10 minutes. A mercury-drop electrode should b e immersed i n a s u i t a b l e polarograph, and the polarogram recorded from 0.20 volts t o -0.75 volts, using a saturated calomel electrode (SCE) as the reference electrode i n 0.1 M HC1. Diffusion current i s recorded a t -0.70 volts. A method of determining acetazolamide by reductive amperometry w i t h flow injection using a s e s s i l e mercury-drop electrode i s reported ( 4 5 4 Acetazolamide was determined i n the range of 10-70 mcg m l a t -0.85 V vs. SCE i n 0.1 M HC1. 4.4
Nuclear Magnetic Resonance Spectroscopy
Acetazolamide can be assayed using an NMR spectrometer equipped w i t h a variable temperature probe having a six-turn insert. Spectra were scanned a t a probe temperature of 42'C. A 25% ammonia solution i s selected as the solvent and t-butanol is used as the internal standard (32).
?I
ACETAZOLAMLDE
4.5
Chromatography
4.5.1 High-performance Liquid Chromatography Several systems have been developed for acetazolamide. Table 4 describes assays published in the literature.
Table 4:
HPLC Conditions for Acetazolamide Assay
Internal Std.
Col umn
Conc.
Mobile Phase
Ref.
( PLghL)
Sul famerazine
C18
15
Chl orothi azide
C18
1-20 6% ACN, 0.05 M Acetate, pH 4.5
39
Sul fadiazine
C 18
1-50 3% ACN, 2% MeOH, pH 4.0
40
1-50 9.7% Ethanol , 79.4% Di chloromethane, 1%HAC
41
Chl oroth azide
Silica Gel 1-30 65% Hexane, 25% Chloroform, 10% MeOH, 0.25% HAC
42
Chl oroth azide
U1 trasphere 0.05 10% ACN, 0.05 M -20 Acetate, pH 4.5
43
Propazolamide
Porasil
cia 2-Acetamido-4methyl-5-thiadiazole sul fonamide
12% MeoH, 2% ACN, 0.02 M Phosphate
1-25 23.8% MeOH, 0.02 M Ammonium Phosphate
37 3a
44
t
JAGDISH PARASRAMPURIA
22
5,
STABILITY
5.1
pH-Rate Profile
The decomposition o f acetazolamide follows a firstorder kinetics (Figure 10). The pH-rate profile curve (Figure 11) is V-shaped, which indicates specific acid-base catalysis. The slopes o f the pH-rate profile curve for the acidic and alkaline solutions is -1.72 and 1.15, respectively. The pH of maximum stability i s 4 (46, 47).
Time (days)
Figure 10: First-order plots o f acetazolamide at different pH values. (0)pH 5.46; ( 0 ) pH 6.06; (A) , pH 6.86; (*) pH 1.68; (A) pH 8.17
ACETAZOLAMIDE
-3
23
-
-4
-
-5
-
-6
-
-7
-
-8
-
Y
C
-
d -1-g/ 0
1
2
3
4
5
6
7
8
9
PH
Figure 11:
5.2
The pH-rate p r o f i l e curve of acetazolamide
Stabi 1 ity in Intravenous Admixtures
Acetazolamide sodium s o l u t i o n s i n 5% dextrose and 0.9% sodium c h l o r i d e i n j e c t i o n s a r e s t a b l e f o r 5 days a t 25°C w i t h a l o s s o f potency of l e s s than 7.2%. A t 5 ' C t h e l o s s i n potency i n both t h e s o l u t i o n s i s 6% a f t e r 44 days o f storage. A t -10°C t h e l o s s i n potency i s l e s s than 3% i n both s o l u t i o n s ( 4 8 ) . The s o l u t i o n s remain c l e a r throughout but a s l i g h t change i n pH occurs. Results of frozen samples thawed i n a microwave oven a r e s i m i l a r t o those samples thawed using tap water ( 4 8 ) . Thawing i n a microwave can be completed i n l e s s than two minutes.
5.3
Effect of Temperature
The decomposi t i on of acetazol ami de f o l 1ows f ir s t - o r d e r k i n e t i c s a t higher temperature. The higher-temperature data
24
JAGDISH PARASRAMPURIA
f o l l o w t h e Arrhenius values o f 3.1, 5.85, values, are d i r e c t l y equation i s reported
equation a t a l l three d i f f e r e n t pH and 6.64. The energy o f a c t i v a t i o n , Ea r e l a t e d t o pH values and t h e f o l l o w i n g (49) using t h e experimental data
I n Ea = I n K + 0.083 pH
Eq. 1
where k i s a constant w i t h a value o f 11.94 kcal/mole and 0.083 i s t h e slope o f t h e s t r a i g h t l i n e . Using Equation 1, t h e Ea value a t pH 4 (pH value o f maximum s t a b i l i t y ) i s calculated t o be 16.6 kcal/mole. The other a c t i v a t i o n parameters a r e l i s t e d i n Table 5. The p o s i t i v e enthalpy value determined using Equation 1 i n d i c a t e s endothermic r e a c t i o n . The increase i n enthalpy as pH values a r e increased i n d i c a t e s higher heat contents o f t h e s o l u t i o n a t higher pH values. The change i n t h e entropy values from -46.77 cal/mole/deg a t pH 3.10 t o -20.75 cal/mole/deg a t pH 6.64 i n d i c a t e s t h a t l e s s energy i s a v a i l a b l e f o r work due t o random motion and t h a t t h e r e i s greater d i s o r d e r l i n e s s o f t h e molecules a t pH 3.10. Table 5:
PH
A c t i v a t i o n Parameters f o r the Degradation o f Acetazolamide i n Aqueous Solutions a t D i f f e r e n t pH Values
AH kcal /mol e
Ea kcal /mol e
In A
3.10
15.43
29.24
28.77
14.84
-46.77
5.85
19.35
26.04
28.03
18.76
-31.11
6.64 20.69
18.27
26.28
20.10
-20.75
5.4
AG kcal /mol e
As
cal /mol /deg
Effect of Buffer Species, Buffer Concentration, and Ionic Strength
The phosphate b u f f e r has very l i t t l e e f f e c t on t h e kobs values o f acetazolamide a t pH 7.5 w i t h b u f f e r concentrations between 0.05 M and 0.15 M. S i m i l a r r e s u l t s
ACETAZOLAMIDE
25
a r e reported w i t h a c i t r a t e buffer a t pH 6.25. The kobs values a r e very similar w i t h different ionic strengths. T h i s indicates t h a t it is the unionized acetazolamide which reacts w i t h e i t h e r H or OH-. Furthermore, the pH-rate p r o f i l e i s a l s o a typical plot of a s p e c i f i c acid-base c a t a l y s i s (47). The hydrolysis of acetazolamide may b e represented as f 01 1ows :
kobs
=
ko + kH (H+) + k0H (OH-)
Eq. 2
where kobs i s the overall observed r a t e constant, and ko, kH, k0H a r e r a t e constants f o r hydrolysis due t o solvent, hydrogen ion concentration ( H ), and hydroxyl ion concentration (OH-), respectively. Assuming the k o t o be 0.0001 per day (k value a t pH 4 where hydrolysis is a t i t s m i n i m u m ) , and neglecting the effect of OH- a t pH 1.68, the kH value i s estimated t o be 0.23 per day. Using the kobs valuf of 0.0495 per day a t pH 12.5 and neglecting the e f f e c t of H , the k0H value i s estimated t o be 1.56 per day. 5.5
Stability in Various Solvents
The stabi 1 i t y of acetazolamide i n pure unbuffered sol vents 1 i ke propyl ene glycol , pol yethyl ene glycol 400, and water i s not optimum, probably because of the higher pH values of these solutions a t which acetazolamide i s not stab1e. Progressive rep1 acement of water w i t h propyl ene glycol improves the s t a b i l i t y of acetazolamide because of solution pH values approaching 4 , which i s the pH value for maximum s t a b i l i t y (26, 50). T h e s t a b i l i t y of acetazolamide i n an extemporaneous suspension compounded from t a b l e t s w i t h a predicated shelfl i f e of 371 days i s also reported (51).
6
BIOPHARIUICEUTICS
6.1
Phariacokinetics and Metabolism
Acetazolamide i s rapidly and almost absorbed from the gastrointestinal t r a c t . not appear t o influence absorption (52). concentrations i n plasma occur w i t h i n two
completely Food intake does Peak hours (53). Usual
JAGDISH PARASRAMPURIA
26
therapeutic serum acetazol amide concentration range is 15-20 mcg/mL, with variations in response from patient to patient (54). Acetazol ami de is 70-90% protei n-bound (55). The apparent volume of distribution i s about 0.2 L/kg (56). Acetazolamide is not metabolized (56) and 90% of the administered dose is excreted unchanged in the urine within the first 24 hours. This process involves both active tubular secretion and passive reabsorption. Plasma concentrations of acetazolamide are proportional to dose, fall in the therapeutic range, and can be detected for six to 12 hours after administration (57). The saliva concentration is about 1% o f the plasma levels, elimination half-life about 4-8 hours, and the therapeutic index 2.7. Renal clearance is approximately two-thirds of simultaneously administered creatinine. Acetazolamide is widely distributed throughout the body, including the CNS (58). No tissue, with the exception of red blood cells, has special affinity for acetazolamide. In red blood cells the drug remains for several days after a single dose and appears to reach a fixed range of concentration, independent of dose or duration of administration, due to the apparent saturation and delay in elimination. Only a small proportion of the carbonic anhydrase in circulating red cells is apparently inhibited by acetazolamide (59). Peak acetazolamide levels in erythrocyte are 45% higher in the elderly group (60). It is unknown if acetazolamide is excreted into human breast milk; however, no harmful effects have been reported in breast-fed infants whose mothers were taking acetazolamide (61). 6.2
Bioavailability
Bi oequi Val ent comparisons of two sustained re1 eases and an immediate-release acetazolamide dosage form performed in normal human volunteers (n = 18) demonstrates a large statistical difference between the preparations. The sustained-re1 ease dosage forms are 40-70% 1 ess avai lab1 e than the immediate-release dosage form, based on the AUC data (62). Comparisons were made between the ocular hypotensi ve effects and blood levels achieved with the single-dose administration of either generic acetazolamide or brand-name acetazolamide (Diamox). The generic and brand-name acetazolamide were equivalent in their effects on intraocul ar pressure. Comparable bl ood 1 eve1 s of acetazolamide were obtained with the two products.
21
ACETAZOLAMIDE
7.
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ACETAZOLAMIDE
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49.
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Alexander, K . S . , Haribhakti, R. P., and Parker, G. A., Stability of Acetazolamide in Suspension Compounded From Tablets. Am. J . Hosp. Pharm. a, 1241 (1991).
52.
Ellis, P. P., Price, P. K., Kelmenson, R., and Rendi, M. A . , Effectiveness of Generic Acetazolamide. Arch. Opthalmol . lQQ, 1920 (1982).
53.
Straughn, A. B., Gollamudi, R., and Meyer, M. C., Relative Bioavailability of Acetazolamide Tablets. Biopharm. Drug Dispos. 3, 75 (1982).
JAGDISH PARASRAMPURIA
32
54.
Yakatan, G. J . , Frome, E. L., Leonard, R. G., Shah, A. C., and Doluisio, J . T., B i o a v a i l a b l l i t y o f 252 Acetazol amide Tab1ets. J . Pharm. Sci
(1978).
. u,
55.
K e l l y , R., B i o a v a i l a b i l i t y o f Sustained Release Acetazolamide. J . Pharm. Pharmacol . 38, 863 (1986).
56.
Diamox (Acetazol amide) Package I n s e r t , Lederl e Laboratories, Inc., American Cyanamid Company, New York.
57.
Kunka, R. L., and Mattocks, A. L., Relationship o f Pharmacokinetics t o Pharmacological Response f o r Acetazolamide. J . Pharm. Sci. 68, 347 (1979).
58.
Kunka, R. L., and Mattocks, A. Acetazolamide. J . Pharm. Sci.
59.
Sweeny, K. R., Chapron, D. J . , and Kramer, P. A., E f f e c t o f S a l i c y l a t e on Serum P r o t e i n Binding and Red Blood C e l l Uptake o f Acetazolamide i n v i t r o . J . Pharm. Sci. Lz, 751 (1988).
60.
Chapron, 0. J . , Sweeny, K. R., Feig, P.. U., and Kramer, P. A., I n f l u e n c e o f Advanced Aye on t h e D i s p o s i t i o n o f Acetazol amide. Br. J . C l in. Pharmacol .
L., Nonlinear Model f o r
a,342 (1979).
l9, 363 (1985). 61.
White, G. J . , and White, M. K., Breast-feeding and Drugs i n Human M i l k . Vet. Human Toxicol. 2,1
(1980). 62.
Schoenwald, R. O., Garabedian, J . N., and Yakatan, G. J ., Decreased B i oaval abi 1it y o f Sustained Re1ease Acetazol ami de Dosage Forms. Drug Oevel op. Indus Pharm. 4, 599 (1978).
.
AMINOBENZOIC ACID
Humeida A. El-Obeid and Abdullah A, Al-Badr
Pharmaceutical Chemistry Department College of Pharmacy King Saud University Riyadh 11451, Saudi Arabia
ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 22
33
Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR
34
Contents 1
Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Appearance, Color and Odor
2
Physical Properties 2.1 Melting Range 2.2 Acidity 2.3 Solubility 2.4 Incompatibility 2.5 Stability 2.6 Detection 2.7 Thermal Analysis 2.8 X-Ray Powder Diffraction 2.9 Crystal Structure 2.10 Spectral Properties 2.10.1 Ultraviolet Spectrum 2.10.2 Infrared Spectrum 2.10.3 Proton NMR Spectrum 2.10.4 Carbon-13 NMR Spectrum 2.10.5 Nitrogen-15 NMR Spectrum 2.10.6 Mass Spectrum
3
Synthesis
4
Biosynthesis
5
Method of Analysis 5.1 Titrimetric Methods 5.2 Polarography 5.3 Enthalpimetry 5.4 Spectrophotometric Methods 5.5 Chromatographic Methods 5.6 Electrophoresis
6
Medicinal Use
AMINOBENZOIC ACID
7
Adverse Effects
8
Precautions
9
Yharmacokinetics 9.1 Absorption 9.2 Distribution 9.3 Metabolism 9.4 Elimination
Acknowledgements References
35
HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR
36
1
Description 1.1
Nomenclature 1.1.1 Chemical Names
p-Aminobenzoic acid 4-Aminobenzoic acid 1.1.2 Generic Names Amben, Anticanitic vitamin, Antichromotrichia factor, Chromotrichia factor, PAB, PABA, Pabacidum, Paraaminobenzoic acid, Trichochromogenic factor, Vitamin Bx, Vitamin H' (1-3). 1.1.3 Propietary Names Amben, Hachemina, Hill-Shade, Pabacyd, Pabafilm, Pabagel, Pabanol, Pabasin, Paraminan, Paraminol, Presun-8, RVPaba, Sunbrella. As potassium p-aminobenzoate: Fibroderm, Pabak, Potaba. As sodium p-aminobenzoate: Epitelplast (1-4). 1.2
Formulae 1.2.1 Empirical
1.2.2 Structural
AMINOBENZOIC ACID
1.2.3 CAS ReEistrv No,
1.3
Molecular Weight 137.14 (5)
1.4
ADDearance. Color and Odor
White or slightly yellow odorless or almost odorless crystals or crystalline powder (3). It forms monoclinic prisms from dilute alcohol (1). 2
Physical Properties 2.1
Meltinn Range 187.0 '-187.5 O C (1) 186.0 O - 189.0O C (2)
2.2
Acidity pKa: 4.65, 4.80 (1) pKa: 2.40, 4.90 (25°C) (2) pH : (0.5% solution): 3.5 (1)
2.3
Solubility
One gram of p-aminobenzoic acid dissolves in 170 ml water, in 90 ml boiling water, in 8 ml alcohol, in 50 ml ether. Soluble in ethyl acetate, glacial acetic acid; slightly soluble in benzene, practically insoluble in petroleum ether (1); slightly soluble in chloroform, freely soluble in solutions of alkali hydroxides and carbonates (2). 2.4
Incompatibility Incompatible with ferric salts and oxidizing agents (43).
37
HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR
38
2.5
Stability
p-Aminobenzoic acid gradually darkens on exposure to air or light (3). The Merck Index (1) states that it may turn slightly yellow on prolonged exposure to light and air. The drug, therefore, should be stored in airtight containers and protected from light.
2.6
Detection
A solution of ethchlorvynol in chloroform (2%) in the presence of phosphoric acid reacts with p-aminobenzoic acid and other primary amines to give colored products (6). A 2% solution of the reagent in CHC1,-isopropyl alcohol-H,PO, (10:89:1) was also used as spray reagent for drugs separated by TLC on silica gel. R, values and colors are reported. Detection limits in TLC ranges from 3 to 100 pg and in solution 0.5 mg of a primary arylamine can be detected. p-Aminobenzoic acid can be detected by the yellow color produced on oxidation with aqueous 1% KClO, in the presence of H,SO, (7). The limit of detection was 0.048 pg
mr1.
p-Aminobenzoic acid was identified by IR spectroscopy (8) using KBr disc in the absorption region of 600-4000 cm-'. The USP (5) requires the IR absorption spectrum of a KBr dispersion of the drug exhibit maxima only at the same wavelengths as that of a similar preparation of USP Aminobenzoic acid RS. The ultraviolet absorption spectrum of a solution of the drug in NaOH should exhibit maxima and minima at the same wavelengths as that of a similar solution of USP Aminobenzoic acid RS (5).
2.7
Thermal Analysis
Thermal analysis of p-aminobenzoic acid was done on Dupont differential scanning calorimetry (Dupont TA 9900 computerlthermal analyzer). The analysis was done after the calibration of the instrument using 3-5 mg of the sample under constant flow of N, gas (150 ml/min) betweem 100°C and
39
AMINOBENZOlC ACID
25O0C/min heating rate (Figure 1) and using purity program. Purity of the sample was found to be 100.72%. Heat of fusion of the sample was found to be 24.1 KJ/mole (5.76 Kcal/mole). X-Rav Powder Diffraction
2.8
The X-ray powder diffraction pattern of para aminobenzoic acid was determined using Philips full automated X-ray diffraction spectrogoniometer equipped with PW 1730/10 generator. Radiation was provided by a copper target (CU anode 2000 w, y = 1.5480 A), high intensity X-ray tube operated at 40 Kv and 35 d. The monochromator was a curved single crystal one (PW 1752/00). Divergence and receiving slits were 10 and 0.10, respectively. The scanning speed of the goniometer (PW 1050/81) used was 0.2-20 per second. The instrument is combined with philips PM 8210 printing recorder with both analogue recorder and digital printer. The goniometer was aligned using silica sample before use. The X-ray pattern of para aminobenzoic acid is presented in Figure 2. The interplanner distance d(A) and the relative intensity 1/10 are shown in Table 1. I
108 5
188 0
1 A A & A a
A
' A
187.5
A
A
87 0
-f u
6,.
I86 5
2 186 0
/
-7-
Correciion : Mol.Weigh( : Cell Cons11 ; Onsel Slobc : I
-8 -9
60
0 80
20 O+/. 137.1 glMole 1.244 -4.52 mW/*C
10
100
I85 5
85 0 Tolal A t M I P a r t ; a l A i e a 20 3,O 40
120
140
160
50 180
200
6.0 220
2
PUF TcmperalurQ(C')
Figure 1. Thermal curve of p-aminobenzoic acid.
ac s V I 1A
f
40
HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR
nEGREES(20)
Figure 2. X-ray powder diffraction pattern of p-aniinobenzoic acid.
AMINOBENZOlC ACID
41
Table 1. X-ray Powder Diffraction Pattern of para aminobenzoic acid. dA 16.48 9.42 6.43 5.81 4.66 4.07 3.70 3.57 3.27 3.11 2.97 2.89 2.66 2.5 1 2.4 1 2.26
1/10
dA
11.31 6.22 22.35 100.00 3.54 22.04 10.82 34.98 15.72 4.13 20.35 7.16 6.28 9.20 3.92 3.95
2.23 2.18 2.13 2.10 2.03 1.99 1.92 1.86 1.80 1.76 1.70 1.67 1.63 1.60 1.55
1/10
4.18 3.73 3.55 3.11 2.69 2.88 2.92 3.22 2.5 1 2.97 3.15 1.82 2.89 1.92 1.90
dA = Interplanner distance. 1/10 = Relative intensity (based on highest intensity as 100). 2.9
Crystal Structure
Lai and Marsh (9) determined the crystal structure of a monoclinic modification of p-aminobenzoic acid from threedimensional X-ray diffraction data. The unit-cell dimensions are: a = 18.551, b = 3.860, c = 18.642 A, D = 93.56O; the space group is P2,/n. The unit cell incorporate 8 molecules and hence two in the asymmetric unit. The structure was refined by least-squares methods to an R index, for 1916 observed reflections, of 0.073 and a goodness of fit of 1.29; the resulting standard deviations in the bond distances are 0.006 A. The dimensions of the two structurally distinct molecules are closely similar, and suggestive of a small amount of
42
HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR
quinoid character. The amino and carboxyl groups are displaced slightly from the planes of the benzene rings, and the nitrogen atoms are nonplanar. Dimers formation takes place by linking together pairs of molecules through two 0-Ha.-0hydrogen bonds arranged about a center of symmetry; an additional N-H-0 hydrogen bond is formed by one of the two kinds of molecule. Twinning and disorder are common for these crystals. In the disordered structure, which is based on an orthorhombic unit cell half as large as the monoclinic cell, the hydrogen-bonded dimers apparently remain intact but the arrangement of N-H...O bonds becomes random (See Tables 2-6 and Figures 4-6). Killean et. al. (10) reported similar crystallographic findings for p-aminobenzoic acid. Apparently the same sort of twinning and disorder was observed; and after suitable axes transformation, the two dimensional structure they reported is in satisfactory agreement with that reported by Lai and Marsh (9) above. Anulewicz et. al. (11) reported on the refinement of the crystal and molecular structure of p-(dimethy1amino)benzoic acid (DABA) and on the full ab inito STO-3G optimization of its molecular geometry and empirical analysis of substituent effects on the geometry of benzene rings in p-substituted benzoic acids. The crystal and molecular structure of DABA was examined by X-ray diffraction. The DABA molecules are almost planar and form cyclic dimers exhibiting no orientational disorder with separation of oxygen atoms RO-0 = 2.627 (2) A. The pattern of molecular geometry in DABA suggests a relatively strong through-resonance effect between NMe, and COOH groups and shows nonadditivity of substituent effects on valence angles and bond lengths in the ring. Mesomeric equalization of CO bond lengths in the carboxylic groups of 10 well solved p-substituted benzoic acids depends linearly on the R O - 0 distance in the H-bridged dimers. Full ab initio STO-3G optimization of molecular geometries for monomers of DABA, p-aminobenzoic acid and benzoic acid show reasonable agreement with experimental data.
AMINOBENZOIC ACID
43
Table 2. Crystal data (9).
F.W. 137.14 F(000) = 576
p-Aminobenzoic acid C7H7N02
Monoclinic; space roup P2,/n a = 18.551+0.002 b = 3.860_+0.010 c = 18.642+0.003 R = 93.56+0.02O
1
D, = 1.360 g . ~ m - ~ D, = 1.367 g.cm”
V = 1332A3 Z = 8
Table 4. The parameters, and their standard deviations, of the hydrogen atoms (9). Values for the coordinates have been multiplied by lo3. The temperature factors are in the form exp (-B sin2 O/ X2).
y
Molecule B H(1) H(2) W3) H(4)
H(5) H(6) H(7)
U”
2
uz
B(uB)
-009 (2) 224 (2) 330 (2) 390 (2) 346 (2) 215 (2) 106 (1)
135 (10) 065 (8) 238 (8) 484 (9) 678 (10) 666 (9) 490 (7)
-053 (2) 011 (2) -044 (2) -139 (2) -199 (2) -217 (2) -163 (1)
5.1 (0.9) 2.7 (0.7) 3.2 (0.7) 4.6 (0.8) 6.9 (1.0) 5.2 (0.9) 2.2 (0.7)
450 (2) 508 (2) 457 (2) 359 (2) 301 (2) 282 (1) 337 (1)
-111 (11) -067 (8) -250 (8) -517 (9) -711 (11) -671 (7) -506 (7)
-013 (2) 220 (2) 330 (2) 383 (2) 341 (2) 206 (1) 100 (2)
7.7 (1.1) 3.1 (0.7) 3.8 (0.8) 4.2 (0.8) 6.3 (1.0) 2.2 (0.6) 2.9 (0.7)
Table 3. The heavy-atom parameters and their standard deviations (in parentheses). All values have been . multiplied bv lo4. T h e anisotroDic temDerature factor is expressed in the form exp[-(b,,h2+ b2i2+ b3,I2+ b,,hl + b,,hl+ b23ki)l. (9). Y
2
b,
b33
b,,
Molecule B 3304 (1) 4286 (1) 4624 (2) 4309 (2) 3642 (2) 3300 (2) 3622 (2) 4615 (2) 5213 (1) 4248 (1)
-5445 (7) -2821 (7) -2073 (8) -2967 (8) -4648 (8) -5452 (8) -4530 (8) -1727 (8) -0236 (6) -2343 (6)
3401 (1) 1519 (2) 2194 (2) 2817 (2) 2783 (2) 2111 (2) 1489 (2) 0862 (2) 0883 (1) 0262 (1)
1154 (28) 594 (23) 708 (25) 716 (25) 748 (25) 762 (25) 708 (25) 706 (24) 1198 (24) 1218 (23)
27 (1) 28 (1) 30 (1) 29 (1) 27 (1) 29 (1) 27 (1) 29 (1) 30 (1) 25 (1)
-57 (8) 27 (7) -8 (8) -2 (8) 37 (8) -33 (8) 16 (8) 21 (7) -61 (7) -81 (7)
Molecule A 3461 (1) 1542 (1) 2214 (2) 2842 (2) 2829 (2) 2160 (2) 1531 (2) 0879 (2) 0866 (1) 0276 (11
5079 (8) 2733 (7) 1898 (8) 2738 (8) 4379 (8) 5258 (8) 4422 (8) 1681 (8) 0089 (6) 2586 (6)
-1666 (2) -0711 (1) -0372 (2) -0675 (2) -1343 (2) -1686 (2) -1374 (2) -0394 (2) 0180 (1) -0754 (1)
1388 (34) 649 (24) 846 (27) 832 (28) 821 (27) 756 (27) 722 (26) 708 (24) 1083 (23) 1192 (22)
42 (1) 25 (1) 28 (1) 31 (1) 31 (1) 31 (1) 29 (1) 24 (1) 28 (1) 35 (1)
-37 (9) -6 (7) -4 (8) 4 (8) -28 (8) 0 (8) 16 (8) 8 (7) -22 (6) -2 (6)
49 (10) -12 (7) -11 ( 8 ) -23 (8) -56 (8) 21 (8) -4 (7) -7 (7) 57 (6) 115 (6)
AMINOBENZOIC ACID
45
Table 5. The best planes of the benzene rings, atoms C( l)-C(6), and the deviations of the individual atoms from these planes (9). The direction consines q are relative to a, b, and c*, respectively; D is the origin-to-plane distance. Molecule A q, = -0.0094 q, = 0.8878 q3 = 0.4599 D = 0.287 A
Molecule B q, = 0.4645 92 = -0.8855 e = 0.0031 D = 4.570 A Deviation -0.001 -0.004 -0.004 0.003 0.009 0.002 -0.009 -0.005 0.005 0.003 0.001 0.001 -0.061 -0.057 -0.073 -0.058 -0.1 18 -0.046 -0.065 -0.137 -0.29 -0.22 -0.01 -0.08 0.08 0.05 0.09 0.07 0.23 0.25 0.05 0.02 -0.05 -0.01
46
HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-AADR
Table 6. Description of thermal ellipsoids (9). qil, q:, qi3are the direction cosines of the principal axes relative to the unit cell axes. Axis i Molecule A
Bi
(A2)
1
2
3
8.51 5.78 3.64
-0.099 0.234 -0.967
0.965 -0.213 -0.15 1
0.248 0.932 0.265
3.96 3.89 3.21
-0.493 -0.640 0.589
-0.630 0.730 0.265
0.629 0.279 0.726
5.06 4.32 3.47
-0.004 -0.716 0.698
-0.991 0.097 0.093
0.135 0.734 0.665
5.16 4.46 3.40
-0.260 -0.547 0.796
-0.848 0.524 0.082
0.477 0.686 0.550
5.59 3.78 3.55
0.246 0.328 -0.912
-0.785 0.620 0.011
0.552 0.69 1 0.466
4.71 4.40 4.03
0.093 0.970 -0.225
0.824 -0.201 -0.530
0.552 0.076 0.830
4.44 4.02 3.73
-0.445 -0.275 0.853
-0.854 0.417 -0.311
0.297 0.882 0.366
4.29 3.89 3.21
-0.379 -0.876 0.299
-0.909 0.413 0.060
0.199 0.303 0.932
AMINOBENZOIC ACID
41
Table 6 (cont.)
O(1) 1 2 3
6.76 4.04 3.52
-0.157 -0.890 0.429
0.946 -0.260 -0.193
0.292 0.430 0.854
O(2) 1 2 3
8.00 4.06 3.14
-0.061 -0.305 0.950
0.88 1 -0.465 -0.092
0.473 0.849 0.237
7.29 4.86 3.68
-0.369 0.925 0.950
0.920 0.348 -0.182
0.159 0.094 0.983
4.26 3.65 3.15
-0.602 -0.363 0.712
-0.433 0.601 0.672
0.707 0.689 0. I60
4.68 4.22 3.54
-0.678 0.007 0.735
0.146 -0.979 0.144
0.76 1 0.204 0.615
4.67 4.27 3.43
-0.69 1 -0.111 0.7 14
-0.083 0.994 0.074
0.759 0.016 0.65 1
4.75 3.82 3.43
-0.49 1 -0.452 -0.745
-0.871 0.229 0.436
0.004 0.889 0.459
4.78 4.11 3.52
-0.465 -0.383 0.798
0.885 -0.223 0.409
0.050 0.9 18 0.393
4.60 3.88 3.40
-0.547 -0.6 11 0.572
-0.690 0.716 0.105
0.507 0.375 0.776
48
HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR
Table 6 (Cont.)
C(7)
1 2 3
4.47 3.95 3.37
-0.355 0.044 0.934
-0.779 0.537 -0.322
0.537 0.838 0.098
O(1) 1 2 3
7.39 4.10 3.78
-0.255 0.233 -0.938
0.966 0.013 -0.259
0.063 0.956 0.287
O(2) 1 2 3
7.82 4.60 3.28
-0.399 -0.856 0.330
0.910 -0.414 -0.027
0.138 0.363 0.921
Figure 4. A composite representation of the final 3-dimentional difference map of p-aminobenzoic acid, the hydrogen contributions were omitted from the Fc’s contours are at interval of 0.1 e.A-3beginning with 0.1 e...4-3 (9).
Figwe 5. Bond distances and angles of p-aminobenzoic acid (9).
51
P
HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR
52
2.10
Spectral Properties 2.10.1
The UV absorption spectrum of PABA in water was obtained on a Cary 219 spectrophotometer and is shown in Figure 7. The spectrum is characterized by an absorption maxima at 275 nm. The literature report a maxima at 270 nm in aqueous acid and at 265 nm in aqueous alkali (2). 2.10.2 Infrared (IR) Spectrum The IR absorption spectrum we presented in Figure 8 is obtained for PABA from a potassium bromide dispersion and is recorded on a Pye Unicam SP IR spectrophotometer. The characteristic bands and their assignment are presented in Table 7. Many reports appeared in the literature on the IR characteristic of PABA. The infrared absorption spectroscopy was used for the determination of the structure of p-aminobenzoic acid in solid, solution and gas states, for study of the properties and structures of its metal and nonmetal complexes as well as for identification purposes. Inomata and Moriwaki (12) studied the IR absorption spectra of p-aminobenzoic acid and its oand m-isomers in solid and solution states. The study indicated that the p- and o-isomers assume a nonpolar structure (dimer) in the solid state and in solutions, whereas the m-isomers has a polar structure in the solid state but a nonpolar structure in solutions. The absorptions were assigned to various vibrational modes with the aid of the spectra of sodium aminobenzoate, aminobenzoic acid hydrochloride, N-deuterated and compounds with similar structure. The intensity of the C-N stretching vibration of compounds containing a COOH group was greater than that for compounds containing a COOgroup. These results were explained on the basis of electron delocalization in the benzene ring and the double bond character of the C-N bond.
AMINOBENZOIC ACID
53
Figure 7. Ultraviolet spectrum of p-aminobcnzoic acid in watcr.
Wavelength pn
01
4000
3500
3000
25G0
2000
1800
1600
lLOO
I200
1000
Figure 8. Infrared spectrum of p-aminobenzoic acid, KBr disc.
Boo
600
55
AMINOBENZOIC ACID
Table 7: Assignment of the IR characteristic bands
Frequency (cm-')
Assignment
3443,3343
N-H stretch
3200-2500
Hydrogen bonded 0-H stretch (dimeric assocation)
3040
Aromatic C-H stretch 0
II
1670
Conjugated -C- stretch
1620
N-H deformation
1594-1500
C - C stretch
1320-1280
C - 0 and C-N stretch and 0 - H deformation
837
Out of plane bending C-H (p-substituted benzene)
The molecular and zwitterion structures of the three isomeric aminobenzoic acids were also studied (13) in different crystalline forms using IR spectroscopy. In one of the crystalline forms, form 1, m-aminobenzoic acid existed as the zwitterion structure: +NH,-C,H,COO-, whereas in crystal form 11, which is reported for the first time by these authors, the misomer assumed the molecular structure: NH,C,H,-COOH. This new crystal form was obtained by direct sublimation of the isomer on a KBr window precooled to -1900C. The analysis of IR spectra of the three isomers of aminobenzoic
56
HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR
acid confirmed that the molecular assocation in the solid state takes place by either formation of acid dimers or chains In the case of formation by strong hydrogen bonds 0-H-N. the m-isomer in form 1 a "proton transfer" gives an O-...HN+ hydrogen bond. The structure of some aromatic carboxylic acids in the vabor phase was also examined using the IR spectra (14). The results showed that the acids, including paminobenzoic acid, were intermolecularly hydrogen bonded in the crystal but exist as monomers in the gas phase. A comparative infrared spectroscopic study of complexes of ammonia and aliphatic or aromatic primary amines with iodine and chloroform was reported by Lauransan and Corset (15). The aromatic amines form weaker complexes than aliphatic amines. However, complexation with aromatic amines caused a large shift in the NH, stretching vibration to lower frequency. These large shifts are explained by a relocalization of the lone-pair electrons on the nitrogen atom through complexation. As a result intermolecular H-bonds involving the amine function become readily detectable in the infrared spectrum. The analysis of these shifts indicated that the NH, group is not involved in complexation in the y-form of p-aminobenzoic acid due to dimer formation. In the or-form, chains are formed by O-H...NH bonds. The IR technique was also used to study the properties and structure of divalent manganese, cobalt and nickel complexes with p-derivatives of benzoic acid (16). The complexes were prepared, characterized and their stability constants determined. The acid dissociation constants, the dependence of the thermal stability on the nature of the metal and the p-substituent and the lattice parameter were determined for the complexes. The 0 - H stretching frequencies of p-aminobenzoic acid together with many p- and msubstituted benzoic acids were measured in dil CCl, solution (17). Their values were correlated by the Hammett equation with normal u constants and slope p = -11.7 cm-' on the one on the other hand and by the equation V,-V" = 1.14 (Vm-Vo) hand, where the frequency V" refers to the unsubstituted compound. The validity of the latter for substituents without
AMINOBENZOIC ACID
51
an a lone electron pair was confirmed even in IR spectroscopy. 2.10.3 Proton Nuclear Magnetic Resonance ('H-NMR) Spectra The 'H-NMR spectra of PABA in DMSO-d, is recorded on a Varian XL 200, 200 MHz spectrometer with TMS as the internal standard. The simple proton (Figure 9) and HOMCOR spectra (Figure 10) are used to determine the exact chemical shifts and coupling of protons. The HETCOR spectrum (Figure 11) is used to assign the protons to their respective carbons in the molecule. The assignment of the chemical shifts to the different protons presented in Table 8. Table 8: Assignment of protons chemical shifts. Chemical shift ( 6 )
Multiplicity
Assignment
No. of protons
5.9
Broad singlet
Exchangeable NH2
2
6.59-6.63
Doublet
C3-H
2
7.67-7.71
Doublet
C,-H
2
11.85 (centre)
Broad singlet
Exchangeable -COOH 1
2.10.4 Carbon-13 Nuclear Magnetic Resonance (13C NMR) Spectra The 13C NMR spectra of PABA in DMSO-d, using TMS as internal standard are obtained on a Varian XL 200, 200 MHz spectrometer. The S2PUL pulse sequence 13CNMR spectrum is shown in Figure 12. The APT and HETCOR spectra are presented in Figure 13 and 11, respectively. The carbon chemical shifts assigned on the basis of the theories of
I
Dmso
f
Figure 9. Proton N M R spectrum of p-aminobenzoic acid in DMSO-d,, internal reference TMS.
'1
2
3A -
5-
G-
1-
PDM
Figure 10. HOMCOR spectrum of p-aminobenzoic acid.
bo
+
I ii
1;
16
s
i
6 S i
j
i
I’
6
P?M
Figure 11. HETCOR spectrum of p-aminobenzoic acid
I
1
Figure 12. Carbon-13 NMR spectrum of p-arninobenzoic acid in DMSO-d,, internal reference ThlS.
~
I
Dmso
Y
I
~
Figure 13. APT spectrum of p-aminobenzoic acid.
~
-
AMINOBENZOIC ACID
63
chemical shifts and the 13C NMR spectra are shown in Table 9. Table 9: Assignment carbon chemical shifts.
Carbon No.
Chemical shift (6) 112.60 116.93 131.26 153.07 167.57
In a study on the importance of .rr-polarization in determining the 13C substituent chemical shifts in the sidechain carbons of aromatic system, Bromilow et d. (18) discussed the concepts of "extended" and "localized" 71polarization. Thus for a series of the general formula below, the p-or m-substituent X induces changes in the 13Cchemical shifts at the a-C atom which correlate with substituent parameters via the d.s.p. equation indicating that they are electronic in origin. The inductive effect of X is largely
x
HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR
64
determined by localized n-polarization of the C = 0 Relectrons, and is independent of the adjacent Z group. Removal of the n-electrons of the carbonyl by complexation or protonation removes the possibility of a n-polarization mechanism and results in a change in the sign of PI values. The resonance effect of X varies considerably from one series to another, and is determined by both the inductive and resonance effects of the Z group. 2.10.5 Nitrogen-15 Nuclear Magnetic Resonance (15N NMR) Spectrum The N-15 chemical shifts of p-substituted anilines and related compounds were determined (19) at the naturalabundance level. All compounds that are not highly hindered show systematic changes in chemical shifts that can be correlated with inductive and resonance parameters by using a dual-substituent-parameter analysis. In these cases, resonance effects play a slightly more important role than do inductive effects. Highly hindered compounds show no systematically significant correlations although qualitative trends are discernible. The absence of correlation reflects the different extents to which steric constraints allow or inhibit lone-pair delocalization as the deman changes with substituent. 2.10.6 Mass Spectrum A mass spectrum of PABA is shown in Figure 14 (20). The literature (2) also reports principal peaks at m/e 137, 120, 92,85,39, 138, 121, 63. The spectrum shows a large molecular ion peak at m/e of 137 characteristic of aromatic acids. Other prominent peaks are formed by loss of OH (M-17) and of COOH (M-45). Scheme 1 shows a proposed fragmentation pattern of PABA. 3
Svnthesis A. Mallonee (21) synthesized p-aminobenzoic acid by agitating a mixture of water, sodium hydroxide, aqueous ammonia and p-nitrobenzoic acid charged into a steel make-
80
60 40 20
0
20
40
60
80
100
120
140
160
m/e
Figure 14. Electron impact mass spectrum of p-aminobenzoic acid (20).
66
HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADK
up tank and heated to 550C to give a clear light-amber solution (pH 7.3) containing a mixture of ammonium and sodium p-nitrobenzoates. The mixture was then hydrogenated over Pd-C in an autoclave, previously flushed with nitrogen at 9O0C/4O0 psig. The charge was pumped through a catalyst recovery filter press at > 6OoC, acidified with 10% HC1 to p H 4, and cooled to room temperature to give slurry of paminobenzoic acids which was filtered, washed with water and dried at 60°C in a current of nitrogen to give 95% product with 99.5% purity (Scheme 2). B. p-Aminobenzoic acid was also obtained (22) by irradiation of a mixture of p-nitrobenzoic acid, conc. HCl and anthraquinone-2,5-bis(sodiumsulfonate) (ABSS) in isopropyl alcohol. The irradiation was carried for 6 hr at 8 2 O using Hg lamp (Scheme 2). C. p-Aminobenzoic acid was prepared by amination of benzoic acid in the y-radiolysis of liquid ammonia. Thus ammonia solution of benzoic acid (14COOH labeled) was irradiated with 6oCo y-rays at OoC. It was deduced that the aminating agent was the amino radical (23) (Scheme 2).
4
Biosvnthesis p-Aminobenzoic acid is a growth factor for certain microorganisms. This moiety is incorporated in foIate conenzymes in bacterial biosynthesis (Scheme 3). pAminobenzoate itself derives from chorismate by enzymatic steps that are still poorly characterized because of the difficulty in detecting the enzyme activity in wild-type Escherichiu coli crude extracts and the problem of difficult purification (24-27). Genetic studies have led to the characterization of two genes, pabA and pabB (28-30). The pabA gene encodes PabA, a 21-kDa protein with high sequence homology to the TrpG component in oaminobenzoate (required for anthranilate synthesis) biosynthesis (29). Each of PabA and TrpG is capable of encoding a glutaminase activity, providing nacent ammonia for the two regiospecific chorismate aminations. The pabB gene product, SlkDa (30) is substantially homologous to the trpE
f
0
I
;1
I/
\$ -0
+
+=I
u
0
I
__f
I
I
$1 I
I
=
I
-
I
2
q / \ ; ; 0 ;
I
+
a
U
00 0
u
0
r 0
L
0
( =, I 0
a
68
0
I 0
6 V
S
0
Z
0
0 ON
m
i zI
I
6 z
I
0 z
5
U
.-U
.-Dc
i I
z
r 0 V
0
v)
N
B T
a
a ii (d
.d
.. Q)
M
I
vl
E 0
L
u
0
c u
69
C
5
1 0 0
0
1 0 0
aJ
c
E
m
.-ILn 0
h
V
L X
0 aJ -0 0
t
'E
4
aJ
-4-
V
w
C
N
x
-
'0
h
L
a
Q
500 76.5
0.4 21.6 50.2
soluble
Table I1 : U V Spectral Characteristics of Bumetanide Solvent
Absorution Maxima (nm)
M
Water 0.05M SDS Aqueous Acid Aqueous Alkali 0.1N NaOH Methanol Acetonitrile
260 (18.9). 220 (17.1) 260 (25.9), 220 (27.0)
15
340 (80)
9
317 (87) 326 270, 345 270, 345
9 14 13
15
13
1I4
PRASAD N. V.TATA ET AL.
3.6.2 Infrared Soectrum The principal peaks reported [ 1 1 1 in the IR spectrum of bumetanide (KBr disc) are found at energies of 1695. 1215 , 1199, 1153 and 1587 cm-l. The IR spectrum shown in Figure 1 was recorded on a Perkin Elmer model 1760X infrared spectrometer. The major observed bands have been correlated with the following functional groups:
Table 111 : IR Spectral Assignments of Bumetanide Functional Group
13406, 3273 .3076 :2959, 2932, 2872 1694 1604, lS88, 1509, 1490 1339, 1162
NH stretch Unsaturated CH stretch Saturated CH stretch C = O stretch aromatic ring vibrations SO, stretch
3.6.3 Nuclear Magnetic Resonance 3.6.3.I 'HNMR Spectra The proton nmr spectrum of bumetanide in DMSO-d6 was obtained on a Varian XL-200 NMR, using tetramethylsilane an internal reference. The spectrum is shown in Figure 2 and the proton chemical shifts are assigned in Table 1V.
3.6.3.2 I3C NMR Spectra
1131
The I3C-NMR spectra of bumetanide was recorded in DMSO-,d6,using tetramethylsilane as the internal reference, on a Varian XL-200 NMR spectrophotometer. The spectrum is shown in Figure 3, and the chemical shifts were assigned as listed in Table V.
100
a0
60
t$?
40
20
0 4000
I
I
I
I
I
I
3500
3000
2500
2000
1750
1500
I
1250
cm-’
Figure 1. Infrared Spectrum of B w t a n i d e
I
I
I
1000
750
400
0.77 PPM *H2C!!,
-
7.35 PPM
J ~ ~ 2 . 7.3 ~ ~~t3
-NH
*6!2 *ll2CIl,
--J 3.05 PPH 5.07 PPM -NII
-
,
Figure 2.
1H
WR Spectrum of Bunetanide
-NHCH2
' 1
1 . 3 7 PPM -CI12CII*CH,
li .I
.r
.s-
I I7
=c
=
~
u M
PRASAD N. V. TATA ET AL.
118
Table IV : 'H NMR Characteristics of Bumetanide A ssimment
Chemical Shift (ppm) 0.77 1.18 3.05 5.07 6.82 - 7.31 7.35 7.43 - 7.70
Table V : I3C NMR Chemical Shifts of Bumetanide
&& Chemical Shift D D ~Assimment a
13.48
-CH,
bl
19.20 30.10
~x-CH,
C
d
41.96
-NHCH2
e f
114.75 115.14 115.48 122.15 129.02
protonated aromatic carbons
k I rn n
128.00 137.58 139.52 142.29 156.23
non protonated aromatic carbons
01
166.45
C=O
?!. h
j 1
BUMETANIDE
3.6.4 Mass Snectrum
I I9
I131
The electron impact (El) mass spectrum of bumetanide is presented in Figure 4, with the spectrum being obtained on a HP5989 MS system operating at an ionization potential of 70 eV. The spectrum shows a base peak at the masslcharge (m/z) ratio of 304, and the most prominent ions and their relative intensities are listed in Table VI. The loss of -C3H,, -NH3, -OH, -SO, and -CO, fragments were noted.
Table VI : The Mass Fragments of Bumetanide
mL.z
Relative Intensity
364 32 1 304 240 196 168 91 77
3.7
Metal Complexinv Abilitv
74.0 91.8 100.0 63.0 19.2 30.8 17.0 10.4
1651
Bumetanide forms metal complexes with Cu(lI), Mg(l1). and Zn(I1) in an HCI-Dioxane-Water medium. The site of complexation is believed to be between the carbonyl and the imino groups, with the complex being formed as a 1 : 1 metal-ligand species.
L
P
~
L
I
55
B
$
I
?
d 9
s
t
t
BUMETANIDE
3.8
X-Ray Crystallographic Data
121
I131
X-ray crystallographic information was obtained on a Scintag model XTS-2000. Powder patterns displaying d-spacings under the operating conditions listed below are given in Figure 5 , and the full data is collected in Table VII. Instrument Target Generator Detector
HV
Filter Divergent Beam Scatter Slit Receiving Beam Slit Receiving Beam Scatter Slit 20 Scanning period Goniometer Radius Goniometer Type Wavelength Full Scale Step Size Scan Time Preset Time* Target Size Fine Gain Coarse Gain Ratemeter Lower level potentiometer Window SCA Amp. Window
*
Scintag X D S - 2 0 Copper 45 KV. 40 mA LN - cooled solid state with intrinsic high purity germanium crystal lo00 volts negative (control setting - 5 . 6 4 ) None 4 0.3 0.5 I . 13 "/min. 250 mm. Theta/Theta I .S406"A
2-70' 20 0.03" 60 mins. 1 .S seconds 1.0 x 10 I .o 0.20 IOk cps. 0.08 0.84
: Time spent to collect each data point.
PRASAD N. V. TATA ET AL.
122
8.?38
:Ps
2.976
4 436
2856.0
--A-
2570.4-
UO 104331 L O T #031021
2.252
1.541
1.823
1.343%
100 BWETANIDelUSm AS A REFFRENCEl.
F
U PLOT
- so
- a0
2284,s1999.2-
70
1713.8-
- 60
1421.0'
- !lo
I14Z.4-
.. 40
858. 8-
- 30
57i.a-
- 20
28s.8-b
-
LO
r n 7 . m
0
0.01
I
I
1
1
,
'
I
I
1
1
1
9
1
1
1
7
.6 -8 . 8 3 8
1
I
8
I
t
I-r- ~ T T
40
10
5.901
4.436
I
3.559
70
60
2.978
2.582
2.252 -1-.
2.
RO 1 0 4 3 3 8 L O T #031021
Figure 5 . X-Fay Diffraction Pattern of Bmetanide
.
BUMETANIDE
123
Table VII : X-Ray Diffraction Data of Bumetanide
4.2200
~~
I
20.92176
I
21
I
20.7638
I
4.27451
I
29
~
19.4000
4.57180
5
3 I .4103
2.84572
4
20.7638
4.2745 I
18
35.2034
2.54730
6
PRASAD N. V. TATA ET AL.
124
4. Stability
~ 3 1
Bumetanide may react in an acidic environment to yield the following products:
+
n-butylchloride
COOH
This process is analogous to the Hofmann-Martius reaction, but is conducted under less stringent conditions. This results in the formation of the debutylated amine and butyl chloride rather than in the rearrangement product commonly associated with this reaction when performed under pyrolysis conditions. No other butylated compounds have been detected in samples stored under accelerated conditions for as long as 1 month at 55°C.
5. Synthesis
[ 10.16-191
The synthetic route for bumetanide is shown in Figure 6. 4-chloro5-(chlorosulfony1)benzoic acid (I) is nitrated in the 3'd position using a mixture of concentrated nitric and sulfuric acids to yield 4-chloro3-nitro-5-(chlarosulfamyl)benzoicacid (11). This is treated with ammonia to give 4-chloro-3-nitro-S-(sulfamyl) benzoic acid (111). This is in turn is treated with a mixture of phenol and sodium bicarbonate to give 4-phenoxy-3-nitro-5-sulfamyl benzoic acid in its sodium salt form (IV),which treated with hydrochloric acid gives 4-Phenuxy-3-mitro-5-sulfamyl benzoic acid (V). The nitro group in V is reduced to an amino group by either treating with sodium acid
BUMETANIDE
I25
\
COOH
COOH
II
utyraldehydc or -BuOH/H$O.
t
NH(WW
-Ac,-
Sa_W
MII
IN NaOH
for 45 min H2NW
w
COOBu
J W C I
Bumetanide
Figure 6. Route of Synthesis of Bumetanide
PRASAD N. V. TATA ET AL.
126
bisulfite or with lithium hydroxide in the presence of palladium catalyst, to yield 3-amino-4 phenoxy-5-sulfamyl benzoic acid (VI). This compound is treated with either butyraldehyde or n-butanol and sulfuric acid to yield VII. This product is then saponified by sodium hydroxide to yield the sodium salt of bumetanide (VIII) which is treated with hydrochloric acid to yield bumetanide (BMT).
6. Methodsof Analvsis
6.1
Elemental Analysis
(71
C: 56.03%. H: 5.53%. N: 7.69%. 0:21.95%, S: 8.80%.
6.2
I dentif ication
[8,111
USP specifies either the IR spectrum in mineral oil and a U V spectrum in isopropyl alcohol, or a TLC test for positive identification.
Bumetanide responds to the following color tests: Koppanyi-Zwikker test Liebermann’s test Mercurous Nitrate 6.3
Loss on Drying
Violet Brown Orange Black
r 81
USP specifies that when bumetanide is dried at 105°C for 4 hours, the LOD cannot be more than 0.5% of the sample weight .
BUMETANIDE
6.4
Residue on Ignition -
I27
181
USP specifies not more than 0.1% residue by weight when one gram of bumetanide powder is used.
6.5
Related Impurities
PI
USP specifies tests for the following related impurities of bumetanide: 3-Nitro-4-phenoxy-S-sulfamoyl benzoic acid. a. b. 3-Amino-4-phenoxy-S-sulfamoyl benzoic acid. Butyl 3-butylamino-4-phenoxy-5-sulfarnoyl c. benzoate.
6.6 An acid-base titrimetric method has been used for the determination of bumetanide. Bumetanide in alcohol is titrated with 0.1N NaOH. using phenol red as the indicator. 6.7 A coulometric method for the determination of bumetanide and furosemide has been described. The method uses electrogenerated C1- in a supporting electrolyte of 0.5M H,SO, and 0.2M NaCI, with methyl orange being used as an indicator.
6.8.
SPectroohotometrv
[ 14.27.281
All the reported spectrophotometric methods are based on the UV absorption of bumetanide in 0. I N NaOH at 326 nm.
I28
PRASAD N. V. TATA ET AL.
6.9
Colorimetric Methods
[27.33.34]
Patel et al. [27] estimated bunietanide by classical diazotization, followed by coupling with the Bratton-Marshall reagent. The method makes use of the absorption maximum a1 550 nm. The method linearity was reported to be 33-333 pg/mL. The low sensitivity can be attributed to the fact that the drug is diazotized without prior hydrolysis to liberate the primary amino group. Sastry et. al. reported two methods. The first method 1331 utilizef the formation of molybdenum blue when bumetanide was treated with Na,CO, and the Folin-Ciocalteu reagent. The derivative has an absorption maximum at 760 nm, and the method exhibits a linear range of 2-24 pg/mL. The second method [34] involves the reaction of bumetanide with MBTH and Ce(IV), which produces a green derivative. This derivative exhibits an absorption maximum at 660 nm, and the method is linear over the range of 1-10 &mL. 6.10. Fluorimctry
[21,28,31,32]
Bumetanide exhibits strong fluorescence in both alkaline and acidic media over the pH range of 3.2-3.5. It has an excitation maximum at 355 nm and an emission maximum at 415 nm. In a 1M solution of glycine buffer at pH 11.2, the compound exhibits an excitation maximum at 355 nm and an emission maximum of 415 nm. Bumetanide in 0.02N AcOH was measured at an excitation maximum of 350 nm and an emission maximum of 570 nm. The detection limit is 0.7 pg/mL.
129
BUMETANIDE
6.1 1. Ion Selective Electrode Method
1301
Chao et al. described a method for the estimation of bumetanide, in the range of 0.01M to 0.0001M at physiological pH, through its ability to interfere with the response of a chloride ion selective microelectrode. 6.12
Radioimmuno Assav (.RIA)
[14,351
Bumetanide was estimated either by quantifying precipitated antibody bound fractions [35], or by unbound fractions [ 141. The RIA method uses 3H labelled bumetanide. An immunogen consisting of 40 moles of N-(3-N-butyl amino-4phenoxy-5-sulfamoyl benzoyl) glycine and one mole of bovine serum albumin was prepared and introduced into a rabbit. After sufficient time, the drawn serum was suitably harvested to obtain the antiserum. To generate the standard curve, 0.1-20 ng/O. 1 mL of bumetanide was added to 0.1 mL of plasma or urine. To each tube, 0.05 mL of 1M sodium acetate buffer (pH 5 . 5 ) and 2 mL of ether were added and vortexed for 5 seconds. The ether phase was separated and evaporated to dryness. To the residue, 0.6 mL of phosphate buffer and 0.2 mL of 3 H-labelled bumetanide were added. Then to each tube, 0.2 mL of antiserum was added, the solution stored overnight, and on the following day 1 mL of saturated ammonium sulfate was added to each tube to precipitate the globulin. The tubes were centrifuged at 3000 rpm at 2°C for 30 minutes and either supernatant or bound portion were analyzed for radioactivity in a scintillator after adding one drop of concentrated H,SO, and 10 mL of toluene.
PRASAD N . V. TATA ET AL
130
6.13
Radiometric Method
[ 14,25,38]
A radiometric method for the determination of plasma levels of intact bumetanide in dogs (when given both by oral and intravenous routes) has been reported. This method makes use of '*C-lahelled bumetanide and the subsequent measurement of radioactivity.
6.14
Electrophoresis
1251
Halladay et al. [25] used paper electrophoresis to separate bumetanide from its metabolites in urine, bile, and feces. A system comprising of Whatman filter paper no. I , borate buffer at pH 9.0 as the electrolyte solution, and a potential of 950 V (applied for 45 minutes), was found to resolve bumetanide and its metabolites.
6. IS
Extraction from Biolopical Fluids
(371
For estimation of bumetanide in biological fluids, various workers extracted the drug with either ether or ethyl acetate after the samples were acidified with various agents. A more specific solid phase extraction procedure was reported by Ameer et al. [37]. The procedure uses a SepPak disposable column containing ODS bonded phase, and the loaded sample containing 4-Benzyl bumetanide (the internal standard) was eluted with methanol. The eluent was analyzed by HPLC.
131
BUMETANIDE
6.16
Chromatographic Techniques 6.16. I
Paper ChromatograDhv
1211
A Schleicher and Schill paper (No.2043b) containing Mgl was used as the stationary phase, and n-BuOH saturated with 25% aqueous NH,OH was used as the solvent system. Bumetanide was detected by its intrinsic fluorescence at 360 nm, at a R, of 0.48-0.58, over the range of 5-10 pg.
6.16.2
Thin Laver Chromatom-aphy
6.16.3
High Performance Thin Layer C h r o m a t o m
[8,11,20,21, 25,26,60] Kolis et al. [20] used multi-dimensional TLC for the quantitative separation of bumetanide and its metabolites in urine, which were detected either by UV, fluorescence, or radioactivity means. Similarly Pentikainen et al. [26] used two-dimensional TLC for the identification of bumetanide metabolites in human volunteers. In this method, detection was performed either by UV or radioactivity. A summary of the reported methods is given in Table VIII.
1391 Zivanovic et al. [39] reported a HPTLC method for the quantification of bumetanide and other diuretics in bulk samples and in their dosage forms. The method uses a plate coated with silica gel GF2C4.and chloroform-diisopropyl ether-Methanol-AcOH (5 :3: 1 :1) as the solvent system. The plates were scanned by a HPTLC scanner operating at 254 nm. The method allows the determination of diuretics in the range of I - 10 gg.
132
PRASAD N. V. TATA ET AL.
Table Vlll : TLC Methods For Analysis Of Bumetanide Stationary Phase
Sample Type
Solvent System
Silica Gel
Bumetanide and related substances
CHC1,CyclohexaneGI.AcOH-H~O (80:10:10:2.5)
Silica Gel
TV
8
--
EtOAC,EtOACMeOHStr.NH,OH (85:105)
AX0
CHC1,-Acetone (4:11
B.M. Reagent
11
Bumetanide metab
n-BuOH sat. with 25 % NH40H
Flu. 360.nm
21
Kiesel gel Q4F
Bumetanide metab
Bz:BuOAc:AcOH (85:35:3.25)
Kiesel gel HF254
Bumetanide/U.Pl n-BuOHNH40H(3: 1)
Cellulose
Bumetanide and Other Diuretics
6.16.4 High Performance Liquid Chro-
coupling with
-Flu.
-UV,FIu. 3351415 nm.
I 25 160
r
HPLC methods play an important role in the determination of bumetanide due to the low therapeutic dose levels used. The reported HPLC methods are summarized in Table IX. In most cases, the samples are subjected to one of the preliminary clean up procedures discussed in 6.15.
BUMETANIDE
I33
-
Table IX: HPLC Methods for Analysis of Bumetanide SL
Compounds
No.
Seppnted
01
BMT.4-Ethylbennl&byde*/ Tab, hj.
02
Column
Mobile Phase
ODS(MOX3.9
MeOH-HzO-THFAcOH (5&455:2)
W254 nm
8
ACNPH 23 . Phaphate Buffer (156344)
UVm
40
nm
mm) RP-18
BWalong with srn
(250X4mm)
compounds.
Detector
Referena
5 MPPH' 03
04
BMT, CLT, SPL 557 olhen
C18Sil-X-10
' BMT, B-HET', Hypersil ODs (200X4.6 mm, mu,CRN T, 5 um) HCT, HFT,I cr,PT,Az MCT, BZT, CLT, CyT,
M?Z% DCP, BMTZ SPL, TRI,T a QT, AML, PCD ia urine
0.05M NaH Polbuffer ofPH23.0 Contg.0.016M propyl amine Hcl (A) and ACN (B) 15% - 80% in u) minuta
UV detn
41
Wwf&
42
275 nm
20mhiBDS in
44
50 mM PH 3.0
phosphate buffer MeOH - H$Conc AcOH ( 7 0 Q X 1)
BBASBA'PISJ LG3-DB
BAPMSBA'IPI,
-
0.03M Phosphate buffer pH3.0-ACN
RU
45
W418 RU
46
w 4 4 0
-
RP-18
UV 231
47
Radial Pak qs MeOH-Hp AcOH (68:34:1) (1OOx 8mm)
Flu 2uV418
48
SGE100GL-4 C18P (1oo~o.4mm,
W 270 nm or Plasma spray
49
forms BMT in urine,
I
10
BMT,PCD,
EA, SPL in urine
I
H$-ACN-MeOH TFA (70: 15:150.5)
-
PRASAD N. V. TATA ET AL.
I34
Table I X (continued)
sl.
Refenna
3emor
No. flu Dclq WMS
50
BMT. AML
LIv,GGEI
51
BMT, FRU/
LIV
32
uv
35
Rev P
11
12
W
others in urlns
13
14
-
nu*
h-PrOHNH,OH (l1:l)
BMT, FRU, TCM
ACN in 0.01M
13
Phaspbre Buffer of pH3.0 10% at 1.5 minutes to 35% 81 3 s minula till
16
13 minutes
BMT, Meubs, FRU'
ODs Radial compressed
17
-
ACN-THF. PO, Buffer 3.5 (151k7S)
MtoH-HP
Fluwz
AcOH (M30.1)
am
I
am
20
PmiSU 10 ODs-3
-
57
Ppnisil 10 ODS-3
I
M6H.W AcOH(7&3& 10) for BMT
ACNaOlN H$O, (1:l)
21
l
nlL3wvua
18
19
I 5 6
am for BMT
w 254 lor
AP UPOnSil ODs M(h3.9 mm
izzx==-
CHC13
Flu. W 4 1 8
MeOHAcOH (%3:1)
nJn
-
59
BUMETANIDE
6.16.5 Micellar Chromato&
135
~5,441
Berthod et al. [IS] described a method by which micellar chromatography, followed by UV and fluorescence detection, was used to determine bumetanide. This method utilized Nucleosil C,8 columns, and a n-BuOH sodium dodecyl sulfate mixture as the mobile phase. In a similar way, Sentell et al. [44] reported the estimation of bumetanide in serum and urine. This methods is rapid since it allows the direct injection of physiological fluids, avoiding details of sample preparation. 6.16.6 Gas ChromatograDhv
[60-651
Bumetanide can be determined by gas chromatography in physiological fluids or in dosage forms. In most cases, the samples were derivitized and extracted from the biological matrix. Fagurlund et al. [64]estimated bumetanide along with various other drugs in plasma after extractive alkylation. GC was performed using a column packed with 1 % SE 30 on Gas Chrom Q (80-100 mesh), a Ni EC detector, and nitrogen as the carrier gas. The injector and detector temperatures were 280" and 270", respectively. 2(2-chlorophenyl)-5-sulphamyl-I ,3,4-thiadiazole was used as an internal standard. Lisi et al. [61] used a selected ion monitoring GC-MS technique for the detection of bumetanide with various other components in urine samples. The samples were alkylated and analyzed on a column packed with a fused silica coated with HP Ultra. Hydrogen was used as the carrier gas, and the injector and detector temperatures were maintained at 280°C and 320°C respectively. Bumetanide was monitored at m/z values of 254, 363, and 406.
PRASAD N. V. TATA ET AL.
136
Feit et al. [62] determined bumetanide in urine after converting it into its methyl derivative by a flash methylation technique. GLC was performed on a column packed with 1.5% OV-17 silicone on 100-120 mesh diatomaceous earth, using nitrogen as the carrier gas, and a flame ionization detector. The injection port, column, and detector were maintained at 370", 270," and 300°C. respectively. 4benzyl bumetanide was used as an internal standard. Davies et al. [60] determined bumetanide after flash methylation, using a column packed with 1.5% OV-17 silicone on chromosorb W HP, flame ionization detection, and nitrogen as the carrier gas. The temperatures of the injection port, detector, and column were 350"C, 350"C, and 270°C respectively. Hioki et a]. [63] reported a method for the determination of bumetanide in urine after converting it to a methyl derivathe.
A method was reported by Yoon et al. 1661, in which electron impact mass spectrometry was used to quantify bumetanide and nine other diuretics after preparation of their deuterated methyl derivatives.
7 . Metaboli sm
[20-24,26,36]
In dogs, bumetanide is excreted unchanged, whereas in humans and rats, almost complete biotransformation is observed to either urinary or fecal metabolites. The structures of bumetanide and its metabolites are given below. Desbutyl bumetanide (V)is common to all species, and the metabolites are relatively inactive.
BUMETANIDE
Bumetanide I I1 111 IV V VI VII Vlll
8. Pharmacakinetics
CH,CH,CH,CH, CH,CH,CH,COOH CH,CH,CH,CH,OH CH,CH,CHOHCH, CH,CH,CHOHCH,OH H COCH, CH,(CH2)2CH, H
I37
COOH COOH COOH COOH COOH COOH COOH CONHCH2COOH CONHCH2COOH
[9.I1,12,24-26,35,36,60]
The various pharmacokinetics parameters for bumetanide in humans have been found to be:
Dosage information; Pediatric dose ( >6 months) 0.015 mg/kg/day usual adult oral dose 0.5 to 2 mg/day usual adult iv dose 0.5 to 1 ing/day over I -2mins. Maximum daily dose
10 inglday
PRASAD N. V. TATA ET AL.
I38
Absorption:
Bioavailability C,,, after 2 nig/dos Time to peak
9s96 80 nglmL about 30 minutes
Distribution:
Plasma protein binding Apparent distribution vol.
94-96% 12-35 L or 0.2 LlKG
Elimination:
1-1.5 hour Half-life Total body clearance 125-250 mL/min. Fraction excreted unchanged about 50%
80%of the dose is excreted in urine in 48 hours; 4560%as unchanged drug and 20-35% as metabolites. lO-lS% of the dose is excreted in feces. Pharmacodynamicq: Onset of action Peak effect Duration of effect
iv within mins. P.O. 30-60 mins. iv 15-30 mins. P.O. 30-60 mins. iv 3.5-5 hours oral 4 hours with 2 mg dose 4-6 hours with higher doses.
Practical considerations: No dosing adjustments are needed in renal failure, although the compound should be used with caution in patients with renal dysfunction to minimize alteration in electrolyte balance. No dosing changes are needed for patients with congestive heart failure. Bumetanide should be used with caution in combination with other Otto-toxic agents.
BUMETANIDE
I39
9. Referenca 1.
Asbury M.J., Gatenby P.B.B., O’Sullivan S. Bourke E.. Brit. Med. J . 1972, 1, 211-3.
2.
Drug facts and comparisons (Ed. Bernie R . Olin) 1992, 535-42. Facts and Comparisons Inc., MI 63146-3098.
3.
Davies D.L., Lant A.F., Millard N.R., Smith A.J., Ward J. W., Wilson G.M., Br. J. Pharmacol. 1973, 47, 61 8-19.
4.
Olesen K.H., Sigurd B., Steiness E., Leth h . , Acta Med. Scand. 1973, 193, 119-31.
5.
Chemical Abstacls Index Guide, 1990, Part I, Chem. Abstr. Service, Columbus. OH 43210.
6.
International Nonproprietary Names for Pharmaceutical Substances, 1988, p. 66, W.H.O. Publications. Geneva.
7.
The Merck Index 1989, 11th edition, p. 1473, Merck & Co. Inc., New Jersey.
8.
IJSP 1988, pp. 191-2.
9.
The Pharmaceutical Codex XI edn. 1979, pp. 113-4, p. 1038, The Pharmaceutical Press, London.
10.
Pharmaceutical Manufacturers Encyclopedia (I 988), 2nd ed., Vol I., p. 200-2, Ed. Marshall S . , Noyes publications.
11.
Clarke’s-Isolation and identification of drugs (1986). p. 408-9, 2nd Edn.. Ed. A.C. Moffat.
12.
Martindale - The Extra Pharmacopeia, 29th Edition, (Ed. Reynolds J.E.F.) 1990, p. 980, The Pharmaceutical Press London.
140
PRASAD N. V . TATA ET AL.
13.
Sahota, S.K., personal communication, Hoffmann-la Roche, Nutley, New Jersey.
14.
Dixon W.R., Young R.L., Holazo A., Jack M.L., Weinfeld R.E., Liebman A., Kaplan S.A., J. Pharm. Sci, 1976, 65(5), 701-4.
IS.
Berthod A . , Asensio J.M., Laserna J.J., J. Liq. Chromatogr., 1989, 12( 13), 262 1-34.
16.
Feit P.W., Brunn H., Nielsen C.K., J. Med. Chem., 1971, 13(6), 1071-5.
17.
Feit, P.W. J. Med. Chem.. 1971. 14(5), 432-9
18.
Organic Chemistry of Drug Synthesis (1980), 2. p. 87. (Ed. L . A . Mitscher).
19.
Swinyard E . A., Remingtons Pharmaceutical Sciences, 18th edn., 1990, p- 939, (Ed. Gennaro A.R.) Mack Publishing Co. Easton, PA. 18042.
20.
Kolis. S.J., Williams T.H., Schwartz, M.A., Drug Metab. Disp., 1976, 4(2), 169-76.
21.
Ostergaard E. H.,Magnussen M.P., Nielsen C.K., Eilertsen, E., Prey H.H., Arzneim-Forsch., 1973, 22, 66-72.
22.
Magnussen M .P., Eilersten E., Naunyn-Schmiedberg’s Archives of Pharmacology, 1974, 282, p. 61.
23.
Halladay S.C., Carter D.E., Sipes I.G., Brodie B.B., Bressler R., Life Sci. 1975. 17, 1003-10.
24.
Ward A., Heel R.C., Drugs, 1984, 28, 426-64.
25.
Halladay S.C., Sipes, I.G., Carter D.E., Clin. Pharmacol. Ther. 1977, 22(2), 179-87.
BUMETANIDE
26.
Pentikainen P.J., Pentilla A., Neuvonen P.J., Gothoni G., Br. J. Clin. Pharmacol., 1977, 4, 39-44.
27.
Patel R.B., Patel A.A., Gandhi T.P, Patel M.R., Patel S.K., Ind. J. Pharm. Sci., 1979, 41, 124-5.
28.
Ramaswami P.S., Kurani S.P., Desai D.K., personal communication, through Ind. J. Pharm. Sci. 1982, 44, 5.
29.
Nikolic K.I., Velasevic K., J., Pharm. Belg., 1989 44(6), 387-90.
30.
Chao A.C., Armstrong W.M., Am. J. Physiol., 1987 253(2. I ) , C343-C347.
31.
Davies D.L., Lant A.F., Millard N.R., Smith A.J., Ward J. W., Wilson G.M., Clin. Pharmacol. Ther., 1973, 15(2), 141-55.
32.
Patel R . B . , Patel A.A., Patel M.R., Patel S.K., Manikwala S.C., Ind. J. Pharm. Sci., 1987. 49(4), 142-3.
33.
Sastry C.S.P., Prasad T.N.V., Roma Mohana Rao A, Venkata Rao E., Indian Drugs, 1988, 25(5), 206-8.
34.
Sastry C.S.P., Prasad T.N.V., Sastry B.S., Venkata Rao E . , Analyst, 1988, 113(2), 255-8.
35.
Halazo A.A., Colburn W.A., Gustafson, Young R.L., Parsonnet M.,J. Pharm. Sci., 1984. 73(8), 1108-13.
36.
Therapeutic Drugs, 1991, Vol 1 , 8122-6 (Ed. C. Dollary) Churchill Livingston.
37.
Ameer B., Mendoza S.M., LC-GC, 1989, 7(7), 590-2.
38.
Pentikainen P.J., Neuvonen P.J., Kekki M., Pentilla A . , J. Pharmacokinet, Biopharm., 1980, 8, 219.
141
PRASAD N. V. TATA ET AL.
142
39.
Zivanovic L., Agatonovic S., Radulovic D., Pharmazie, 1989,
44(12), 864. 40.
Daldrup T., Susanto F., Michalke P., Fr. Z., Anal. Chem., 1981, 308, 413-427.
41.
Daldrup T., Michalke P., Boehme W., Chromatogr. Newsl., 1982, 10(1), 1-7.
42.
Cooper S.F., Masse R . , Dugal R.J., J. Chromatogr., 1989, 489(1), 65-88.
44.
Sentell K.B., Clos J.F., Dorsey J.G., Biochromatography, 1989, 4(1), 35-40.
45
Ameer B., Burlingame M.B., Anal. Lett., 1988, 21B, 15891601.
1
46.
Boekens H., Bourscheidt C., Mueller R. F., J. Chromatogr. , 1988, 434, 327-9.
47.
Zivanov S.C.D., Solomun L.J., Zivanovic L.J., J. Pharm. Biomed. Anal. 1989, 7(12), 1889-92.
48.
Wells T.G., Hendry I.R., Kearns G.L., J. Chromatogr. , 1991, 570(1), 235-42.
49.
Ventura R . , Fraisse D., Becchi M., Paisse O . , Segura J., J. Chromatagr., 1991, 562(1-2), 723-36.
50.
Gradeen C'.Y,, Billay D.M., Chan S.C., J. Anal. Toxicol., 1990, 14(2), 123-6.
51.
Park S.J.,Pyo H.S.,Kim Y.J., Kim, M.S., Park J., J. Anal. Toxicol., 1990, 14(2), 84-90.
52.
Singh A.M., McArdle C., Gordon B., Ashraf M. Granley K., Biomed Chromatogr., 1989, 3(6), 262-5.
BUMETANIDE
143
53.
Fullinfaw R.O., Bury R.W., Moulds R.F.W., J. Chromatogr., 1987, 415(2), 347-56.
54.
Howlett M.R., Auld W.H.R., Skellern G.G., Method]. Surv. Biochem. Anal., 1984, 14, 337-42.
55.
l k h i n o K., Yamamura Y., Saitoh Y., lsozaki S., Tamura Z.. Nakagawa F., Sekine K.,Kozima I., Yakugaku Zasshi, 1984, 104(10), 1101-7.
56.
Marcantonio L.A., Auld W.H.R., Skellern G.G., J. Chromatogr., 1980, 183( I), 1 18-23.
57.
Walmsley L.M., Chasseaud L.F., Miller J . N . , J. Chromatogr., 1981, 226(2), 441-9.
58.
Smith D.E., J. Pharm. Sci., 1982. 71(5), 520-3.
59.
Berkersky I . , Popick A., Drug Metab. Disp., 1983, 1 1 , 5 12-
3. 60.
Davies D.L., Lant A.F., Millard N.R., Smith A.J., Ward J.W., Wilson J. W., Clin. Pharmac. Ther., 1974, 15(2), 14155.
61.
Lisi A.M., Trout G . J . , Kazalauskas R.,J. Chromatogr., 1991, 563(2), 257-70.
62.
Feit P.W., Roholt K., Soernsen H., J. Pharm. Sci., 1973, 62, 375-9.
63.
Hioki M., Ariza T., Shindo H., Sankyo. Kenkyusho Nempo., 1973, 26, 85-93.
64.
Fagurlund C., Hartvig P., Lindstrom B., J. Chromatogr., 1979. 168(1), 107-16.
144
PRASAD N. V. TATA ET AL.
65.
Orita Y., Ando A . , IJrakabe S., Abe H., Arzneim.-Porsch., 1976, 26( I), 11-33.
66.
Yoon C.N., Lee T.H., Park J., J. Anal. Toxicol., 1990, 14(2), 96- 101.
Note: The following abbreviations have been used in the paper for the purposes of brevity: AZ: acetazolamide, AML: amiloride Hcl, BMT: Bumetanide, 4BzBMT: 4-benzyl derivative of Bumetanide, CT: Chlorthiazide, CLT: Chlorthalidone, AcOH: acetic acid, AcN or MeCN: acetonitrile, Bz: benzene, sds: Sodium Dodecyl Sulfate, -HET: Hydroxyethyl theophylline, SPL: Spironolactone. QT: Quithazone, CRN: Carneone, HCT: Hydrochlorthiazide, HFT: Hydroflurnethiazide, FT: Flumethiazide, PT: Polythiazide, DCP: Dichlorophenamide, PCD: Probenacide, RZT: Benzthiazide, MCT: Methyclothiazide, CyT: Cyclothiazide, CyPT: Cyclopenthiazide, BfMTZ: Bendrufluomethiazide, TRI : Triamterene, IDP: indepamide, MTZ: Metalazone, EA: Ethacrynic acid, TCM: Trichloromethiazide, BfZ: Bendrufluazide, MFS: Mefruside, SA: Salycilic acid, CXN: clorexolone, BBASBA: 4-benzyl-3-butylamino-5sulfamoylhenzoic acid, BAPMSBA: 3-n Butylamino-4-phenoxy5-methyl sulfamoyl benzoic acid, AP: Acetophenone, Metabs: Metabolites, Grad.Elu. : Gradient elution, Flu.Detn. : Fluorescent Detection, xxx/xxx nm: Excitation/Emission wavelengths, IJ: IJrine, PI: Plasma, S: serum B1: blood, 5MPPH:5-(p-methyl pheny1)-5 phenyl hydantoin; PRT :pi ratanide.
CLOZAPINE
Michael J . McLeish, Benny Capuano, and Edward J. Lloyd
School of Pharmaceutical Chemistry Victorian College of Pharmacy Monash University Parkville, Victoria, Australia
ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 22
145
Copyright 0 1993 by Academic Press, Inc All rights of reproduction in any form reserved
MICHAEL J. MCLElSH ET AL.
I46
CONTENTS 1.
DESCRIPTION
1.1
Nomenclature 1.1.1 Chemical Names 1.1.2 Proprietary Names
1.2
Formulae 1.2.1 Empirical 1.2.2 Structural
1.2.3 CAS Registry Number
1.2.4 Drug Code Number 1.3
Molecular Weight
1.4
Efemental Composition
1.5
Appearance, Color and Odor
2.
PHYSICAL PROPERTIES
2.1
Melting Range
2.2
Solubility Data
2.3
Dissociation Constant
2.4
Partition Coefficients
2.5
Spectral Properties 2.5.1 Ultraviolet Absorption Spectrum 2.5.2 Infrared Absorption Spectrum 2.5.3 Nuclear Magnetic Resonance Spectrum 2.5.4 Mass Spectrum 2.5.5 X-ray Crystal Structure
CLOZAPINE
3.
SY NTHESlS
4.
METHODS OF ANALYSIS
4.1
Extraction
4.2
Spectrophotometric Analysis
4.3
Chromatography 4.3.1 Thin Layer Chromatography 4.3.2 Gas Chromatography 4.3.3 High Performance Liquid Chromatography
4.4
Radioimmunoassay
5.
METABOLISM
6.
PHARMACOLOGY
7.
USES, ADMINISTRATION and CONTRA-
INDICATIONS
8.
PHARMACOKINETICS
9.
REFERENCES
I47
MICHAEL I. MCLEISH ET AL.
I48
1.
DESCRIPTION
1.1
Nomenclature
1.1.1 Chemical Names 8-Chloro-ll-(4-rnethyl-l-piperizinyl)-5Hdibenzo[b,el [1,4ldiazepine [1,2]
1.1.2 Proprietary Names Clorazil, Clozaril, Leponex, Lepotex [l, 21 .
1.2
Formulae
1.2.1 Empirical C,,H,,N,Cl
(Clozapine
1.2.2 Structural
1.2.3 CAS Registw Number 5786-21-0 [ Z ]
1.2.4 Drua Code Number HF-1854 [ 1,2]
-
free base)
CLOZAPINE
1.3
149
Molecular Weight 326.83
1.4
1.5
Elemental Composition C:
66.15%
H:
5.86%
C1:
10.85%
N:
17.14%
Appearance, Color and Odor Clozapine is a yellow crystalline
or
finely
crystalline powder which is odorless or has a weak characteristic odor.
2.
PHYSICAL PROPERTIES
2.1
Melting Range Clozapine,
recrystallized
from
acetone-
petroleum ether, melts at 183-184 "C [l].
Solubility Data Solubility at 25 "C (w/w, vibration 24 hours, gravimetry) :
> 5%
Acetone Acetonitrile Chloroform Ethyl Acetate Ethanol (Absolute) Water (pH 7.4)
2.3
1.9%
> 20% > 5% 4.0%
< 0.01%
Dissociation Constant pK,(1)
= 3.70
[3]
pK,(2) = 7.60
[4]
MICHAEL I. MCLEISH ET AL.
150
mure 1
W epectrum of clozaplne in methanol.
200
250
350
300
400
Wavelength (nm)
Wavelenghth mox (nm)
Molar absorptivity (mo?L cm-’)
215
24300
230
23200
259
16200
296
10000
CLOZAPINE
2.4
151
Partition Coefficients The partition coefficient of clozapine in octanol/water is 0.4 at pH 2, 600 at pH 7, 1000 at pH 7.4 and 1500 at pH 8 [4].
2.5
Spectral Properties
2.5.1 Ultraviolet Absorption Spectrum The ultraviolet absorption spectrum of clozapine (E.lpg/mL) in methanol was obtained on a Shimadzu W-160A recording W-Vis spectrophotometer. The spectrum (Figure 1) shows the presence of four distinct absorption maxima. The wavelengths of maximal absorption (L) and corresponding molar absorptivities ( & ) are provided in Table 1. Kracmar et al. [5] have recorded the W spectra of clozapine in methanol, 95% ethanol and 0.1M HC1. The wavelengths of the maxima and minima, as well as optimal concentrations f o r W analysis were also reported [5]. Absorption maxima in ethanol were 215, 230, 261 and 297 nm [l].
2.5.2 Infrared Absorption Spectrum The infrared absorption spectrum of clozapine as a KBr disc is shown in Figure 2. The spectrum was obtained on a Hitachi 270-30 infrared spectrophotometer. Table 2 details the wavenumber and assignment of the principal absorption bands.
i
Q rl
d k
al ti: (d
m Id Q)
c
rl
4
N
0 rl U
0
44
3! U
1
i
0
'E
?-
A
I
0 '0
0
W
.o
0
W
.o
d
0 0 '0
0 0
' N .-I
0 0 V d
0
% -
E3
13
. - I %
0
3
"z gc 0 0 0 (Y
0 0 Y) (Y
0
*
0
0
m
y1
0
0
0 0
0
k
0
m
0
VI
&
0 0 d
a,
a
m pt
H
a
152
*I
CI
U
d
G
U
Q1
4
G
a
4
id. N 0
3k!
0)
U U
4 I
8 X
0
0
m
153
Q Q
E
MICHAEL J. MCLEISH ET AL.
1 54
able 3
IH-NMR
characteristics of clotapine
Chemical Shift 6 (ppm)
Multiplicity (Number of Hydrogens)
2.35
singlet (3H)
2 -52
broad singlet (4H)
3.49
broad singlet (4H)
4.93
singlet (1 H)
6.61
doublet (1H) multiplet (2H)
6.82 I
multiplet (1 H)
7.01 I
7.07
I
7.25
I
7.28
1
doublet (1H) multiplet (1 H) multiplet (1H)
'H Assignment
CLOZAPINE
Table 2
155
Infrared charactersistics of c 1o zapine
Wavenumber (cm-')
Infrared assignment
3320
N-H stretch
3016
Aromatic C-H stretch
2980 - 2800
Aliphatic C-H stretch
1595- 1550
Amidine C=N stretch
1430- 1460
Aromatic C=C stretch
1042
Aromatic C-CI stretch
2.5.3 Nuclear Magnetic Resonance Spectrum The aromatic region of the 'H NMR spectrum of clozapine,
recorded
spectrometer,
has
on
a
been
Varian
XL-100
described
[ 61
.
However, no detailed analysis was undertaken. The 'H-NMR spectrum of clozapine in CDC1, was recorded
on
a
Bruker
AMX-300
spectrometer
at
ambient
temperature
portrayed and
in Figure 3.
spectral
clozapine
assignments
are
of
internal reference.
(TMS)
was
MHz)
and
The chemical
presented
Tetramethylsilane
(300
is
shift
the protons
of
in
3.
Table
used
as
the
7 5 MHz
13C-NMR
spectrum of clozaplne in CDC1,.
157
CLOZAPINE
Table 4
13C-NMR characteristics of clozapine
CH, 16
i3c
Chemical Shift 6 (ppm)
Assignment
46.04
C-16
47.20
C-14, C-18
54.96
C-15, C-17
119.96
c-8
120.05
c-5
123.02
c-3
123.09
c-9
123.49
c-1
126.80
c-11
129.06
c-10
130.26
c-2
131.87
C-4
140.40
c-7
141.84
c-12
152.77
C-6
162.76
C-13
MICHAEL J. MCLEISH ET AL.
158
The 13C-NMR spectrum of clozapine in CDC1, was also recorded on a Bruker AMX-300 spectrometer,
again
at
ambient
(75 MHz)
temperature,
and is presented in Figure 4.
The chemical
shift and spectral assignments of the carbon atoms
are
displayed
in
Table
4.
TMS
was
again used as the internal reference.
2.5.4 Mass Spectrum The
electron
impact
(EI) mass
spectrum
of
clozapine was recorded using a JEOL JMS-DX3OO mass spectrometer and is shown in Figure 5. The
following
spectrometer
conditions
were
used: Acceleration voltage
3 kV
Ionization voltage
70 eV
Ionization current
0.3 mA
Ionization chamber temp.
170 "C
The
spectrum
compares
favourably
reported by Stock et al.
[6].
shows
m/z
prominent
intensity (32%)
22%),
and
ions 256
192
at
that
The spectrum 326
(77%), 243
(relative
(loo%),
227
A
possible
is provided
in Scheme
(34%).
fragmentation pathway
to
1. The fast atom bombardment was
(FAB) mass spectrum
also recorded on the same instrument and
6. The sample was analyzed in a 3-nitrobenzylalcohol matrix and bombarded with xenon atoms at an acceleration is presented
in Figure
0
W
k c, Q
u
2
n
F4
0 0
P N u i 3 NA"
ay-JD mle 326
J /=-
N
mle 268
mle 256
mle 243
Fragmentation pathway for Clozapine
Fast Atom Bombardment (FAB) ma88 spectrum o f clozapine
Fiuure 6
~~
327
192
243
1
256
164
u 150
200
250
m/z
300
350
MICHAEL J. MCLEISH ET AL
I62
voltage
of
expected
6 keV.
The
(M+H)+ peak
spectrum
at
m/z
shows the
327
with
a
relative intensity of 100%.
2.5.5 X-ray Crystal Structure The crystal structure of clozapine has been reported
[7] and is presented,
ball
stick diagram,
and
as a stereo
in Figure
7.
The
crystals were obtained by slow evaporation of a
saturated
clozapine. data
were
1 :1 methanol-water
solution
The three dimensional collected
on
a Hilger
of
intensity and
Watts
linear diffractorneter by use of graphitemonochromatized Mo-K, radiation (A = 71.07 Pm)
-
Crystals of clozapine are orthorhombic, space group P212121, with a = 1804(3), b = 957(1), c
lo6 pm3, D, = 1.32, 2 = 4. In addition to molecular geometry data, crystallographic x-ray co-ordinates were also = 950(1) pm, U = 1640 x
reported. that
the
From the data, central
it was
seven-membered
concluded
heterocycle
a boat conformation with an almost exact mirror plane passing through the benzenamine nitrogen atom and the centre of the C-N double bond. The dihedral angle between the planes of the benzene rings was found to be 115". Intermolecular hydrogen bonds were not evident in the molecular packing of clozapine molecules in the C-N bond connecting the crystal. The is
in
Computer-generated stereo view of clozapine showing the molecular conformation as determined by X-ray analysis.
,,
..
MICHAEL J. MCLEISH ET AL.
164
tricyclic system to the N-methylpiperazine moiety displayed considerable double bond character [7].
3.
SYNTHESIS Methods for the synthesis of clozapine have been described [3,8,91 and are shown in Scheme 2. The initial step in the synthesis involves the formation of the aminocarboxylic acid 9 (R=H). Condensation of anthranilic acid 2 with 2-bromo-5-chloronitrobenzene 1 in the presence of potassium carbonate and powdered copper, followed by the reduction of the nitro group (preferably with sodium dithionite in aqueous alkali) readily affords 3 [lo]. Subsequent thermal cyclization of 3 in boiling toluene produces the cyclic amide 4, which is the key intermediate in the synthesis of clozapine [3,8,10] . As an alternative to the thermal cyclization, the methyl ester of 3 (R=CH,) will also provide a via a base-catalyzed cyclization (dioxane as solvent) [lo].
The cyclic amide 4 is readily converted to clozapine, &, in a one-pot reaction with Nmethylpiperazine and titanium tetrachloride (TiC1,) 19,111. N-Methylpiperazine was
CLOZAPINE
a
165
+
..
H'
I
a Thermal cyclizatbn (R = H) Base catatyzed cyclizatbn (R = %)
r R
N=C'
5
R = a
Sdmms-22:
Synthesis of Cloeapine
MICHAEL J. MCLEISH ET AL.
I66
treated, at 50 "C,
with TiC1, in a mixture of
toluene and anisole.
The amide,
A,
was added
and the mixture heated at reflux to give 8 in 90% yield [9]. The preparation
3
chloride However,
has
of
clozapine
also
via
been
the
imido
described
[3].
yields obtained in this method
3
[3].
synthesized
by
low, presumably due to the lability of Clozapine
may
also
be
are
of the thioether 5, but the reaction is reported to proceed slowly [3].
aminolysis
Finally, reaction of the thiolactam methylpiperazine also affords
1
with N-
8 in good yield
131.
4.
METHODS OF ANALYSIS
4.1
Extraction Clozapine is a basic compound and has a pK, of 7.6
[4].
Its
partition
coefficients,
in
octanol/arater, are 0.4 at pH 2 and 1500 at pH 8
[4].
As
a
consequence
most
methods
of
extraction require that any tissue, urine or plasma be made alkaline prior to extraction with some organic solvent. Generally this is accomplished using sodium hydroxide concentrated ammonia solution carbonate
[4] and
phosphate
have also been employed.
although
[6,12], sodium buffer
[13,14]
CLOZAPINE
167
Diethyl ether is the solvent most commonly used for extraction of clozapine [4,15-181 . Others employed include chloroform [ 12,191 , n-hexane [20,21], benzene [22], toluene [22], ethyl acetate [22,23], methylene chloride [6] and methyl t-butyl ether [24]. Some success has also been achieved using mixed solvent systems such as hexane/isoamyl alcohol [25], chloroform/2-propano1 [6,12,15], toluene/2butanol [26] and methylene chloride/2propanol [13]. The extraction of clozapine with diethyl ether is efficient with recoveries of greater than 90% being the norm Toluene and benzene are similarly [4,17]. efficient [ 221 , however n-hexane is less effective [20,21]. In some cases a backextraction has also been used for sample clean-up [23,251 . The choice of solvent for extraction may be based on whether the extraction of clozapine metabolites is also required. The single solvent systems are suitable for clozapine and N-desmethyl clozapine but chloroform or a mixed solvent system seems to be required for the extraction of clozapine N-oxide. Recently, methods have been developed for the solid phase extraction of clozapine and its metabolites. Columns used have included Chem-Elut [ 131 and a silica-RP,, matrix [ 141 .
MICHAEL J. MCLEISH ET AL.
168
4.2
Spectrophotometric Analysis In methanol W
maxima
[l,51
.
used,
and ethanol,
around
215,
clozapine exhibits
230,
260
and
295 nm
Although W methods are not generally clozapine
determined
by
in
tablets
dissolution
in
has
been
methanol
and
measuring the absorbance at 214.5 and 294 nm ~ 7 1 .
4.3
Chromatography
4.3.1 Thin-Layer Chromatoaraphy One of the earliest methods used to measure clozapine levels in plasma was W reflectance photometry of thin layer chromatograms In
this
process,
metaboli.tes were subjected
to
clozapine
extracted
two
from
dimensional
chromatography on silica gel. d i m e n s i o n
t h e
and the
and
its
plasma
and
thin
layer
In the first
e l u a n t
isopropanol/chloroform/25% (16:8:1:1)
[22].
w a s
ammonia/water
Rf of clozapine was 0.68.
In the second dimension ethyl acetate/ethanol/acetic acid/water/l,2(26: 12 :8 :7.5 :15)
dichloroethane
eluant and the R, was 0.35. visualized under W these
conditions
desmethyl
clozapine
was
The plates were
light at 295 nm. the
major
and
the Under
metabolites,
clozapine
N-oxide
could also be measured.
The R, values in the
first/second
were
dimension
0.23/0.35, respectively [22].
0.37/0.45
and
A densitometer
Table 5
GC methods for the determination of Clozapine ~~
COLW/SUPPORT
DETECTOR
DERIVATIZATION
SENSITIVITY
REFERENCE
DB5
Nitrogen
none
1-2 ng/mL
4
SIM
none
n.s.
13
Nitrogen
none
1.2 ng/mL
18
ECD/Nitrogen
TFAA
1 ng/mL
23
S 1M
PFPA
1.0 ng/mL
26
5% SE-54 on 100/120
Supelcoport 1% SP 1000 on 100/120
Supelcoport 3% OV-1 on 100/200
Supelcoport OV- 17 0 1
MICHAEL J. MCLEISH ET AL.
I70
was
used
clozapine limits
to
quantitate
and
being
the
metabolites, in
the
levels
with
region
of
detection
of
5-10
ng/g
plasma 1221. TLC
A
method
has
detection
of
including
clozapine
clozapirie columns
is
and
R,
The
a
also
broad
been
spectrum
[13].
extracted
In
when
on
eluted
for
of
drugs,
this
method
using
chromatographed
values
described
solid-phase silica
gel.
with
ethyl
acetate/methanol/ammonia/water (86:10:1 :3) (1OO:l) are 0.46 and and methano1Iammonia 0.60,
respectively.
identifed
by
its
Clozapine colour
may
reactions
reagents including Fast Black K Salt and Dragendorff spray nm
(black).
be with
(blue)
(brown), or W at 254
In addition, two metabolites of
clozapine appeared as red (R, 0.3) and orange ( Rf
0.4) spots when eluted with methanol/ammonia and sprayed with Fast Black K
Salt.
The detection
limit was 250 ng/mL
1131.
4.3.2 Gas Chramatoaraphv Table that
5 provides have been
chromatographic
a
summary
developed analysis
of
the methods
for of
the
gas
clozapine
[4,13,18,23,26]. These methods have proven to be relatively selective and provide sensitivity of approximately 1 ng/mL.
Table 6 COLUMN
Conditions employed for the HPLC determination of Clozapine MOBILE PIIASE
DETECTOR
INTERNAL STANDARD
SENSITIVITY
REF
C18 5 P
KPO, (0.05M pH 2.9)
W, 2 2 6 nm
C1 one zapam
450 Pg
14
C18 10 Cun
MeOH/Acetate (pH 5.5) ( 6 7 : 3 3 ) MeOH/H,O (80:20)
w,
2
~
W, 2 3 0 nm
MeOH (0.O5%ET3N)/H,O
254 nm
(78:22)
4
~ Diazepam
D iazepan, D iazepam
15
n.s.
16
5.0 ng
17
500 Pg
20
EC XL-Octyl 3 P
MeCN/NH,Ac (0.25 mM) (9O:lO)
C8 5 P
MeCN/NH,Ac (0.25 mM) (9O:lO)
C18 10 Cun
C18 5 P
=04 (pH 4,
(64:36)
MeOH/NH,CL (2M)/ NH,Ac (2M) ( 6 0 : 40:2)
+ o . 7 vs
Dibenzepine
Ag/AgCl
w,
254
21
Fluphenazine
W, 230 nm
Protryptyline
15 ng
25
W
Diazepam
n.s.
28
MICHAEL 1. MCLEISH ET AL.
172
The extraction of clozapine was carried out using
extraction
section did
4.1.
not
procedures
In most
prove
to
trifluoroacetic used.
In
sometimes
necessary,
anhydride
anhydride
addition,
have
been
maprotiline
[23]
used
to
although [ 261
and
have
been
internal
accuracy of the assay. acetyl
in
cases derivatisation
be
pentaf luoropropionic
described
standards ensure
the
These have included
[4],
propyl-norclozapine
[26], arnoxapine [23] and dibenzepine [18].
4.3.3 High Performance Liquid Chromatoaraphv The
first
HPLC
assay
for
clozapine
was
developed so as to detect and differentiate between clozapine and its N-oxide metabolite [19].
The assay was performed using normal
phase chromatography, with a stationary phase of
10
pn
silica
and
a
gradient
chloroform/methanol/water
serving
mobile
these
phase.
Under
as
of the
conditions
clozapine and its N-oxide could be determined to
a
minimum
concentration
blood or urine [19]. silica
of
10 ng/mL
of
One other assay using a
column has been
described,
having
a
sensitivity limit of 3 ng/mL [24].
More
recently
developed
assays
[14-17,19-
21,25,28] have employed reverse phase columns and, in general, W detection. are summarised in Table 6.
These methods
In all cases an
173
CLOZAPINE
internal standard has been used with diazepam being the most popular [15-17,271. The
differences
attributed
to
in
the
assays
whether
the
can
be
simultaneous
determination of clozapine and any or all of its
metabolites
methods
was
merely
required.
Thus
some
measured
clozapine
[16,17,20,21], others clozapine and desmethyl clozapine
[25] or clozapine
metabolites clozapine
[151.
Still
in the presence
and both
major
others
measured
of drugs
such as
neuroleptics and benzodiazepines [14]. As with gas chromatography, the sensitivity
of most HPLC methods was of the order of 1-5 ng/mL which is sufficient for monitoring the therapeutic
range
of
100-800
ng/mL
of [29
and references therein].
A
radioimmunoassay
has
been
developed
that
permits the quantitation of clozapine in the presence of its major metabolites
[30].
described,
to
the
assay
ng/mL human plasma. that
is
limited
However,
it is possible to detect
100 pg/mL plasma [30].
METABOLISM
As
15-480
it is claimed as little as
MICHAEL J. MCLEISH ET AL.
I74
I / 3:-
h
\
Metabolism of Cloeapine
CLOZAPINE
I75
metabolites which have limited or no activity [31,32]. These metabolites are primarily unconjugated [6,12,31] with the ratio of unconjugated to conjugated metabolites being 30:l [12]. The main route of elimination of clozapine appears to be urinary excretion: 50% of an oral does of 'H-clozapine was recovered in the urine and 35% in the faeces [291.
Scheme 3 shows the major routes of clozapine metabolism [6]. In urine, the principal metabolites of clozapine were found to be the N-oxide (a), the N-desmethyl compound (b) and a phenolic derivative of the N-desmethyl compound
(presumably c), in a 2:l:l ratio [12]. The other metabolites are present at much lower levels (61. In plasma the ratio of the N-oxide to the desmethyl metabolite seems to be reversed, with one study showing N-desmethyl clozapine occurring in quantities equal to 64-82% and clozapine N-oxide equal to
10-25%
of
the
clozapine
concentration
[221*
PHARMACOLOGY Clozapine is an antipsychotic drug used in the treatment of schizophrenia [28,33-351. Its pharmacology, first described in 1961, [3,36] showed that it produces very few of
the extra-pyramidal side effects (EPS) usually associated with classical
MICHAEL J. MCLEISH ET AL.
I76
antipsychotics such as chlorpromazine and haloperidol. For this reason, it has become the prototype of the 'atypical' class of antipsychotic drugs [ 371 . However, clozapine was found to induce the blood disorder agranulocytosis [3,29,32,38,39], which in some cases can be fatal, and this led to its withdrawal from the market [3]. Recently it has been reintroduced by Sandoz, with close monitoring of patients' blood [40] for treatment-resistant schizophrenic patients [341 *
Clozapine differs from classical antipsychotics in having a relatively low affinity for dopamine receptors [29,33], both D, (PIC,, 7.3) and D, (PIC,, 7.01, but somewhat higher affinity (PIC,, 8 . 0 ) at the recently cloned D, receptor [41]. Experiments using positron emission tomography [42] have established that the highest D,-dopamine receptor occupancy of 41% is found using clozapine treatment, and that the effects of clozapine may be a combined effect on both D, and D, receptors. Clozapine acts at a multiplicity of receptors [29,33] (muscarinic, q-noradrenergic, serotonergic (5HT,) and histaminergic), but it is not clear which specific combination of these determines its unique properties. Earlier
explanations,
which
have
recently
CLOZAPINE
177
been revived [43], presume that clozapine' s potent anticholinergic affinity and activity produce a combined antipsychotic/ anticholinergic effect, but this alone does not account for its atypical activity. Clozapine selectively binds to limbic brain regions (where the antipsychotic effect is mediated), as distinct from striatal brain regions (at which EPS develop) [37]. BY contrast, classical antipsychotics show either no preference or else striatal selectivity [37]. Clozapine is designated 'atypical' because it shows a large separation between measures of antipsychotic activity and acute EPS [37], as determined by selectivity for limbic dopamine receptors using behavioural, biochemical and electrophysiological techniques. Thus, at pharmacological dose levels, clozapine does not produce catalepsy (a striatal effect) yet it inhibits stimulation of locomotion (a limbic effect), but not the stereotypy (a striatal effect) induced by the dopamine agonist, apomorphine [ 371 . Biochemically, clozapine produces a greater increase in dopamine turnover in limbic than in striatal brain regions [37]. Using electrophysiological techniques, it has been shown that clozapine predominantly alters firing rates of neurons projecting to the
MICHAEL J. MCLEISH ET AL.
178
limbic region [37]. Preclinical and clinical data [44] for clozapine support the hypotheses that (i) D, receptor antagonism is necessary and sufficient for an atypical profile, but that interaction with subtypes of the D, receptor differentiates classical from atypical antipsyc:hotics; and (ii) a high ratio of 5HT,:D, :receptor antagonism is required for an atypical profile .
7.
USES, ADMINISTRATION and CONTRAINDlCAT1ONS Clozapirie is an atypical antipsychotic whose use is indicated only in the management of severely ill schizophrenic patients who have failed to respond to other neuroleptic agents or who cannot tolerate the adverse side effects produced by those agents [32]. It is effective in a substantial proportion (3050%) of such cases [3,29]. Clozapine is administered as an oral dose, initially 25 mg one to two times a day and, if tolerated, increasing to 300-400 mg a day at the end of two weeks. The usual adult prescribing limit is 900 mg/day [29,32]. Clozapine should not be used in cases of severe CNS depression and myeloproliferation
CLOZAPINE
179
disorders, specifically blood dyscrasias or a [32]. history of bone marrow depression Further, depending on the amount present, some interaction may combined with alcohol, depression, bone lithium [32].
be observed when agents causing CNS
marrow
depressants
or
As clozapine has been implicated in cases of [3,29,32,38,39] clozapine agranulocytosis therapy must be carried out in conjunction with close haematological monitoring. If the white blood cell count falls below 3000 per mm3 or the granulocyte count exceed 1500 per mm3, clozapine should be discontinued [29,32] . Patients who have recovered from agranulocytosis should never be restarted on clozapine [29].
8.
PHARMACOKINETlCS Although the pharmacokinetics of clozapine have not been examined in great detail, there are some studies following the administration of single and multiple doses in psychiatric patients [24,45-471 . Clozapine is moderately well absorbed [ 481 with, in general, plasma concentrations reaching a peak at 1-4 hours [24,45,47]. Maximum physiological effect is observed after 4 hours [48]. The plasma levels could be described with a two compartment model of
MICHAEL J . MCLEISH ET AL.
I80
elimination with first order absorption [24]. The terminal half life of clozapine shows considerable individual variation with values Mean ranging from 5.8 to 33 hours [24]. values for terminal half life that have been reported include 10.3 f 2.9 hours [45], 6.0 -+ 1.5 hours [47] and 17.4 f 7.7 hours [24]. The estimated bioavailability of clozapine, when administered orally, ranges from 27-50% [29]. Approximately 95% of the drug is bound to plasma proteins [29,48J . Consistent with observations of individual variability in clozapine half-life, there is considerable variation in steady state plasma concentrations at a given dose [49]. Plasma levels of clozapine are higher in women than men, and in older (45-54) rather than younger (18-35) patients [49]. Smoking also seems to lower c1ozapine concentrations [491. However, provided the plasma concentrations were maintained within a therapeutic range of 100-800 ng/mL, the antipsychotic effect does not appear to be directly related to the clozapine concentration [18,29,47].
9.
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CLOZAPINE
181
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Choc MG, Lehr RG, Hsuan F, Honi.gfeld G, Smith Pharm. Res. HT, Borison R and Volavka J, (1987) 4 , 402-405.
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A,
HI
DIDANOSINE
Munir N. Nassar, Tracy Chen, Michael J . Reff, and Shreeram N. Agharkar
Bristol-Myers Squibb Pharmaceutical Research Institute Syracuse, N Y 13221
ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 22
185
Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
MUNlR N. NASSAR ET AL.
I86
CONTENTS 1.
Introduction
2.
Description 2.1
2.2 2.3 2.4 2.5 2.6 3.
Nomenclature 2.1.1 Chemical Names 2.1.2 Generic Names 2.1.3 Proprietary Name 2.1.4 Laboratory Code 2.1.5 Chemical Abstract Registry Number (CAS) Formulae 2.2.1 Empirical 2.2.2 Structural Molecular Weight Elemental Composition Appearance and Color Dosage Forms
Physical Properties 3.1 3.2 3.3
3.4 3.5 3.6 3.7 3.8
Melting Point and Melting Range X-Ray Powder Diffraction Differential Scanning Calorimetry (DSC) Dissociation Constant Solubility Hygroscopicity Partition Coefficient Spectral Properties 3.8.1 Ultraviolet Spectrum 3.8.2 Infrared Spectrum 3.8.3 Optical Rotation
DIDANOSINE
3.8.4 Circular Dichroism 3.8.5 Nuclear Magnetic Resonance Spectra 3.8.5.1 *H-NMRSpectrum 3.8.5.2 13C-NMRSpectrum 3.8.6 Mass Spectrum 4.
Synthesis
5.
Methods of Analysis 5.1 5.2
6.
Determination in Biological Fluids 5.1 5.2 5.3
7.
Plasma Samples Urine Samples Cerebro Spinal Fluid Samples
Stability - Degradation 7.1 7.2 7.3
8.
Elemental Analysis High-performance Liquid Chromatography
Solid State Stability Solution Stability Dosage Form Stability
Pharmacokinetics and Metabolism 8.1 8.2 8.3
Absorption and Bioavailability Metabolism Pharmacokinetics
9.
Drug Interactions
10.
Toxicity
11.
Acknowledgements
12.
References
I87
MUNIR N. NASSAR ET AL.
188
1.
Introduction
Didanosine (ddI, dideoxyinosine), a synthetic purine nucleoside analog, is an inhibitor of the in vitro replication of the Human Immunodeficiency Virus HIV (also known as HTLV I11 or LAV) in human primary cell cultures and in established cell lines. It is the second antiretroviral agent, after zidovudine, approved by the Food and Drug Administration (FDA) for the treatment of HIV infection. After didanosine enters the cell, it is converted by cellular enzymes to the active antiviral metabolite dideoxyadenosine triphosphate (dd-ATP). The intracellular half-life of dd-ATP varies from 8 to 24 hours (1). A common feature of dideoxynucleosides is the lack of a free 3I-hydroxyl group. In nucleic acid replication, the 3I-hydroxyl of a naturally occurring nucleoside is the acceptor for covalent attachment of subsequent nucleoside 5'-monophosphates; its presence is therefore requisite for continued DNA chain expansion. Because dd-ATP lacks a 3'-hydroxyl group, incorporation of dd-ATP into viral DNA leads to chain termination and thus, inhibition of viral replication. In addition, dd-ATP continues the inhibition of viral replication through interference with the HIVRNA dependent DNA polymerase (reverse transcriptase) by competing with the natural nucleoside triphosphate, d-ATP, for binding to the active site of the enzyme (1,2). Didanosine is indicated for the treatment of adult and pediatric patients (over 6 months of age) with advanced HIV infection who are intolerant to zidovudine therapy or who have demonstrated significant clinical or immunologic deterioration during zidovudine therapy (1).
DIDANOSINE
2.
Description 2.1
Nomenclature 2.1.1 Chemical Names a. b.
Inosine, 2',3'-dideoxy (3) 2',3'- Dideoxyinosine (43)
2.1.2 Generic Names
didanosine (3) 2.1.3 Prourietarv Name
VidexB (Bristol-Myers Squibb Company) 2.1.4 Lsboratorv Code
BMY-40900 (3) (Bristol-Myers Squibb Company) 2.1.5
Chemical Abstract Registrv Number (CAS) 69655-05-6 (3)
2.2
Formulae 2.2.1 Emuirical ClO H12 N4 0 3
2.2.2 Structural
Figure 1 depicts the structure of didanosine (4).
I89
MUNIR N. NASSAR ET AL
190
H 3’
2‘
Figure 1. The chemical structure of didanosine. 2.3
Molecular Weight 236.23
2.4
Elemental ComDosition
C 50.84% 2.5
H 5.12%
N 23.72% 0 20.32%
Amearance and Color Didanosine is a white crystalline powder.
2.6
Dosage Forms
Videx is formulated for oral administration and is available as a chewable/dispersible buffered tablet, buffered powder for oral solution and pediatric powder for oral solution. The chewable/dispersible buffered tablet is available in strengths of 25, 50, 100 or 150 mg, has a mint flavor and contains aspartame. The buffered powder for oral solution, packaged in single-dose packets, is available in 100, 167, 250 or 375 mg strengths. The pediatric powder for oral solution is unbuffered and is packaged in either a four ounce bottle containing two grams of drug or an eight ounce bottle containing four grams of drug (6).
191
DIDANOSINE
Physical Properties Data presented in this section were obtained from the Pharmaceutical Research Institute of Bristol-Myers Squibb Co. unless otherwise indicated. 3.1
Melting Point and Melting Range
The melting point of didanosine is reported to be 160" - 163" C (5). 3.2
X-Ray Powder Diffraction
The X-ray diffraction pattern of didanosine was determined using a 114.6 mm. diameter Debye-Scherrer powder camera with a copper target X-ray tube and nickel filter (1.54505 A). The sample was irradiated for one hour at 25 Kv/40 mA and the film was developed and hand measured. The values of interplanar distance, D(A), and relative intensity, VI,, are shown in Table I. The diffraction pattern is shown in Figure 2. Table I. X-ray powder diffraction pattern of didanosine.
8.20 7.21 6.43 6.02 5.55 5.18 4.80
70 10 10 10
20 30 30
3.50
100
3.32
60
3.18 3.02 2.96 2.37
40 20 20 10
D(A) = interplanar distance I/I, = relative intensity (based on the highest intensity of 100)
c
?
E. 3
E
Q W
P
%
t 4
5
00
l
-
0
i
o
w
Z ! O
- 20.5
>
-- 17.7
- 27.0
0
0
0
c
--
--6.1
I
10.7
-c
- 25.7
I93
DIDANOSINE
3.3
Differential Scanning Calorimetrv CDSC)
The DSC curve for didanosine was obtained using a Perkin-Elmer DSC-7. A-weight of 1.308 mg was sealed into an aluminum sample pan. The temperature was scanned from 40" to 250" C at a rate of 10" C h i n under a nitrogen purge at 30 cc/min. The thermogram of didanosine is presented in Figure 3 and shows that the compound melts with decomposition at a peak onset of 176" C. 3.4
Dissociation Constant
The apparent pKa value of didanosine, uncorrected for activity coefficients, was obtained by titration of a 0.01 M solution of didanosine in water with standardized solution of 0.1 N NaOH at room temperature. The apparent pKa of didanosine was found to be 9.12 0.02 (* S.D., n =2) (7).
*
3.5
Solubilitv
The aqueous solubility values of didanosine at 25" C as a fbnction of pH are listed in Table I1 (7). Table 11. Aqueous solubility of didanosine at 25" C as a hnction of pH.
I 1 I
pH 6.21 8.06
10.01 10.08
I I I
Solubility (mg/mL) 27.3 31.1
460
1I I
7.0
TI
162.800 .C
T2
183.600 'C
Peak
181.364 'C
Aroa
i4e.9sa nJ
Ealto H
I I3.876 J/g
6.0
5.0
F
Oneat
E
v
6
4.400 n Y
Halght
175.648 'C
4.0
c
m a3 I
3. a
2. a
1.
c
0. c
I
50. 0
1 75. 0
I
100.0
1 125.0
I 150.0
I 175.0
Temperature ("C)
Figure 3 .
DSC therniogram of didanosine.
1 200.0
I
225.0
25,
DIDANOSINE
I95
The solubility values of didanosine in various organic solvents at ambient temperature (23' C) are reported in Table 111. Table 111. Approximate solubility of didanosine at ambient temperature (23" C) in various organic solvents.
Solvent Acetone Acetonitrile t-Butanol Chloroform Dimethyl Acetamide Dimethyl Sulfoxide Ethanol Ethyl Acetate Hexane Methanol Methylene Chloride Polyethylene Glycol 300 1-Propano1 2-pro pano
Solubility (mg/mL) 95% potency following storage for 8 weeks at 50" C. The drug substance should be stored in a tightly closed container with a desiccant. 7.2.
Solution Stability
The aqueous solution stability of didanosine was studied as a fimction of temperature, pH, and drug concentration (7). Degradation followed apparent first-order kinetics with optimal stability in the pH range of 12 to 13. The pH-rate profile for didanosine at 25" C is shown Figure 17 (7).
100 10 1
0.1 0.01 0.001 0.0001 0.00001 1e-06
10-07 0
2
4
6
6
10
12
PH
Figure 17. The pH-rate profile of didanosine.
14
218
MUNIR N. NASSAR ET AL.
Didanosine is highly acid labile but is quite stable in alkaline environment. It has at,, at 37" C of less than 2 minutes at pH 3 and 509 days at pH 9.5. In aqueous solution, didanosine undergoes hydrolysis to its corresponding purine, hypoxanthine and 2'3'dideoxyribose as shown Figure 18 (7).
dihsine
hypoxanthine
2',3'dideOxyribose
Figure 18. Structures of didanosine and its major degradation products. An aqueous solution of didanosine (0.18 M, phosphate buffer pH 6.9) containing 3% hydrogen peroxide showed about 40%
potency remaining after 6 hours at 37" C. Aqueous didanosine exposed to high intensity light (minimum 1000 foot-candles) for 8 weeks, pH 9.0 at 30" C, maintained a potency greater than 96%. 7.3
Dosage Form Stabilitv
Didanosine Chewable Buffered Tablets are stable for 36 months at Controlled Room Temperature (15"-30' C). When the tablets are dispersed in water to form a suspension, the dispersion is stable for at least one hour at room temperature (20" to 22" C) and lighting conditions (0.45 to 0.55 kilolux). Didanosine Buffered Powder for Oral Solution and Didanosine Pediatric Powder for Oral Solution have an expiry period of 24 months and 12 months at Controlled Room Temperature (15"30" C), respectively.. Following constitution with water, Didanosine Buffered Powder for
DIDANOSINE
219
Oral Solution can be stored up to 4 hours at room temperature (20" to 22" C) and lighting conditions (0.45 to 0.55 kilolux). When Didanosine Pediatric Powder for Oral Solution is constituted with water as directed and mixed with the appropriate antacid, it is stable for 30 days under refiigeration (2" to 8" C) (1).
Pharmacokinetics and Metabolism 8.1
AbsorDtion and Bioavailabilitv
Didanosine is a highly acid labile compound which is quite stable in alkaline environment. The oral bioavailability of didanosine administered 2 minutes after ingesting 30 mL of antacids (e. g., magnesium hydroxide) averaged 35 % (2 1). Similar bioavailability data with pre- or co-administered antacid were also reported as follows: 43 % by Rozencweig et a1 (22); 40% for oral doses of 0.8-10.2 rng/kg and 23% for oral doses of 15.2-33.0 mg/kg by Dollin et a1 (23); 35%-40% by Yarchoan et a1 (24); 43% for oral doses of 0.8-10.2 mg/kg by Knupp et a1 (25); and 38% by Hartman et a1 (26). For pediatric administration, a lower bioavailability of 21 % (range .-a
‘D
IW.4
-
Y. 4
T t
'P
6
sz
Figure 7. EI Mass spectrum of dipivefrin hydrochloride.
118144.
DlPIVEFRlN HYDROCHLORIDE
24.5
Table IV.Mass spectral data of dipivefrin hydrochloride Chemical Ionization (CI)
Electron Impact (EI) m/z
7% re1 abundance
Assignment
m/z
% re1 abundance
100 35 1
11
250 223 85 57 44
2 11 20 20 100
18
Mass spectrometry has also been used to idenhfy dipivefrin and hydrolysis products in aqueous humor samples from rabbit eyes.5 3.2
Thermal Properties
3-2.1 Melting Range The melting range of dipivefrin hydrochloride is 158-159OC;17the melting range of the base is 146-7OC.17 3.2.2 Differential Scanning Calorimetry A 2-mg sample of dipivefrin hydrochloride drug substance was heated from 25OC to 200OC at a linear rate of 200C/min using a Perkin-Elmer DSC-4.45 One major endothenn was observed with an onset of 158.6OC and a maximum of 163.7oC. corresponding to the melting range of dipivefiin
hydrochloride (Figure 8).
u v)
0
n
t!
99.5%typically) and an overall yield of 86%. (1)
SlMVASTATlN
365
I
1Y
B
I
a) 2NNaOH b) HCI
c) NH,OH
TBSCl a iinidazole
R=H R = CH,
R=H R = CH,
Scheme I Synthesis of Simvastatin from Lovastatin
DEAN K. ELLISON ET AL.
366
4.
Physical Properties 4.1
Infrared Spectrum The infrared absorption spectrum of simvastatin is shown in Figure 1 (4). The spectrum was obtained as a potassium bromide pellet using a Mattson Polaris Model NU-10000 FT-IR spectrometer. Assignments of characteristic absorption bands are shown below. Frequency, (cm-') 3546 cm-' 3422 301 1 2969 2929 287 1 1718 1701 1459 1389 1369
Assignments Free O-H stretch Associated O-H stretch Olefinic C-H stretch Methyl C-H asymmetric stretch Methyl C-H symmetric stretch; methylene C-H asymmetric stretch Methylene C-H symmetric stretch Lactone C=O stretch, associated Ester C=O stretch, associated Methylene C-H symmetric bend; methyl C-H asymmetric bend Gem-dimethyl C-H bend
1267
Lactone -C-0-C stretch
1225 1166 1056 870 670
Ester -C-0-C stretch Secondary alcohol C - 0 stretch Trisubstituted olefinic C-H wag Cis olefinic C-H wag
d
0 0
a3
0 0
0 7
cu
0
0 7
co
0
cu
0 0 0
0
d
0
cu 0
0 a3
cu
0
cu
0 (3
0 0
a
m
DEAN K. ELLISON ET AL.
368
4.2
Proton Nuclear Magnetic Resonance Spectrum The proton magnetic resonance spectrum of simvastatin is shown in Figure 2 (5). This spectrum was obtained using a Bruker Instruments Model WM250 NMR spectrometer and an approximately 6.6% w/v solution of sinwastatin in deuterated chloroform. The reference compound was CHCl, ( aH = 7.27 ppm). Signal assignments are tabulated following the numbered structural formula for simvastatin shown below.
Proton Nuclear Magnetic Resonance Assignments Multiplicity (‘)/J
Relative No. of Protons 1 1 1 1 1 1 1 2
5.98 5.11 5.50 5.35 4.62 4.36 2.92 2.71-2.57
d, J=9.6 Hz dd,J=9.6,6.0 Hz t (broad), J=3.0 Hz m m m (broad) in (broad) m (AB) 2J=17.6
2.50-2.23 2.01-1.2
overlapping in overlapping in’s
3 11
1.11. 1.1:. 1.08 0.88 0.82
s
6 3 3 3
Assignments (’)
C5H C6H
C4H ClH
c, ,H c4 +H
c4#OH c, 8, C3H. C7H C&. c, .H,. C&
c8%
Notes:
d. J=1.4 Hz d, J=7.0 Hz t, J=6.8 Hz
(1) (2)
‘$2,
clO%
C2”(C!!33)* C,-CH3 C7-% CVH3
Multiplicity: s=singlet; d=doublet; m=multiplet; t=triplet. Assignments made in reference to numbered structure of simvastatin shown above.
- 0
.. r4
DEAN K.ELLISON ET AL.
370
4.3
Carbon-13 Nuclear Magnetic Resonance Spectrum The carbon-13 magnetic resonance spectrum of simvastatin (4) shown in Figure 3 was obtained using a Bruker Instruments Model WM250 NMR spectrometer and an approximately 6.6% w/v solution of simvastatin in deurerated chloroform. The reference compound was deuterated chloroform. Signal assignments are tabulated following the numbered structure shown in Section 4.2. Chemical Shift
-
MX?!! 177.9 170.4 132.8 131.5 129.6 128.3 77.0 76.4 68.1 62.5 43.0 38.6 37.6 36.6 36.1 33.0) 32.9) 30.6 21.2 24.7 24.3 23.0 13.9 9.3
Assignment (l) C,. c61 c6
c7 c3
- 0
c\I
0 - 0
.. m
DEAN K. ELLISON ET AL.
312
4.4.
Ultraviolet Spectrum The ultraviolet (UV) absorption spectrum of simvastatin is characterized by absorption maxima at 23 1,238 and 247 nm with Al%lcm values of 516,604 and 408 respectively. The absorption maximum at 238 nm is typical for a substituted diene chromophore (6). A UV spectrum of simvastatin (c = 0.02 mg/mL in acetonitrile) is shown in Figure 4.
4.5
Mass Spectrum The mass spectrum of simvastatin is shown in Figure 5 . This spectrum was obtained by the direct probe electron impact (90 eV) method using a Finnigan MAT 212 mass spectrometer (7). The spectrum (molecular ion M+ = 418) is consistent with that expected for simvastatin. Other pertinent fragment ions can be rationalized by the fragmentation pattern shown in Figure 6.
4.6
Optical Rotation Simvastatin has seven chiral centers and is optically active. The specific rotation rag5is +292' for a 0.5% solution in acetonitrile (8).
4.7
m i a l Behavior The differential scanning calorimetry (DSC) curve for simvastatin at a heating rate of 2'C/min under a nitrogen atmosphere is shown in Figure 7. The thennogram is characterized by a single melting endotherm with an extrapolated onset temperature for melting of -141 " C which is independent of heating rate from 2-20' C/min. In contrast, the DSC thermogram for simvastatin obtained at a heating rate of 2' C/min in air (Figure 8) exhibits an exotherm at -128 C which is attributed to oxidative reactions occumng in air. Thermogravimetricanalysis (TGA) of simvastatin, heated from 25 C to 160"C at 5 a C per minute in an inert atmosphere (nitrogen) shows no significant weight loss until melting occurs at about 140a C. The weight loss seen thereafter is due to sublimation (10). O
7
N 7-
9
0
cq
(9 0
a3ueqJosqv
0
7-
f\! 0
1
8
(D (u
0
(u
3 0 (u
m
d
v) (u
0
d (\I
In
m
N
0
m
N
In N N
0 N N
,-
0 0
aJ ,-I d
(D
0
l ' " " ' " ' l ' "'""I
a2
0
.' .
d
"I""'"
0 0.
I '
d
0
N
0
m
gE
0
d
0 0
0
!n m
i2
B
a
375
SIMVASTATIN
/
M+ = 418 -H20
'
m/z 400
/ J
mlz284
'-120
-'C3H5
mlz 199
mlz 240 I
\ m/z 159
t m/z 173 Figure 6:
Proposed Fragnentation Pattern to Explain the Mass Spectrum of Simvastatin.
DEAN K . ELLISON ET AL.
376
I
I
I
50.
100.
150.
Figure 7:
DSC Thermogram for Simvastatin under Nitrogen.
m
\
I. 0
a 0 4
In
4-
d7
Figure 8:
I
DSC Thermogram for Simvastatin in Air.
I
'C
377
SIMVASTATIN
At elevated temperatures in an atmosphere of air, a small gradual weight gain due to oxidation may be observed. The thermal properties of sinivastatin, in particular those derived from DSC experiments, have been used to assess the oxidative stability of the compound (9, 10). 4.8
Solubility Simvastatin is insoluble in water, but is soluble in polar organic solvents. Solubility data obtained at room temperature are tabulated below (10). Solvent Chlorofomi Diniethyl sulfoxide Methanol Ethanol n-Hexane Hydrochloric acid, 0.1M Polyethylene glycol 400 Propylene glycol Sodium hydroxide, 0.1M Water
Solubility (mg/mL) 610 540 200
160 0.15
0.06* 70 30 70 ** 0.03
NB: Hydrolysis of the lactone moiety of the nlolecule occurs in acid and in alkaline media. Solubility data are therefore for the free hydroxy acid* and the sodium salt of the hydroxy acid** of simvastatin. 4.9
Crystal Properties Simvastatin is a white, crystalline, non-hygroscopic solid. The X-ray powder diffraction pattern for simvastatin is shown in Figure 9 (9). This spectrum was obtained on a Phillips APD 3720 X-ray diffractonieter. No polyniorphs other than that represented by the X-ray pattern in Figure 9 have been observed.
1.oo
0.90
0.80 0.70 0.60 0
0
0.50 0.40
0.30 0.20 0.1 0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
Degrees 20
Figure 9: Powder X-Ray Diffraction Pattern of Simvastatin.
35.0
40.0
379
SIMVASTATIN
X-Ray Powder Diffraction Pattern Data Of Simvastatin
Peak
Angle
w
@@
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 26
2.4825 7.7600 9.3000 10.7875 12.6800 15.5350 16.4800 17.0850 17.5425 18.6000 19.2275 20.1950 21.8750 22.4375 23.5050 24.1450 24.9850 25.8175 26.3300 28.2275 29.5625 3 1.8000 34.7 100 36.4675 38.1750 39.0875
D spac
w
35.5588 11.3834 9.5016 8.1945 6.9754 5.6993 5.3746 5.1856 5.0514 4.7665 4.6133 4.3935 4.0597 3.9592 3.7817 3.6829 3.5610 3.4480 3.3820 3.1589 3.0192 2.81 17 2.5823 2.46 18 2.3555 2.3026
VImax (%)
1.33 36.78 89.46 15.77 5.01 49.56 100.00 27.40 6.20 99.28 19.72 2.19 14.64 10.32 4.23 5.85 5.85 24.46 5.68 29.72 1.33 13.83 7.93 2.52 8.13 7.14
DEAN K. ELLISON ET AL.
380
4.10 Dissociation Constants
Simvastatin exhibits no acidbase dissociation constants. Potentiometric titration of a sample in 50% aqueous methanol revealed no observable buffering action in the pH range of 2-10 (4). 4.11 Partition Behavior
At room temperature, the partition coefficient of simvastatin (K, o/w) between octan-1-01 and either pH 4 acetate or pH 7.2 acetate is > 1995 (4). 5.
Methods of Analysis 5.1
Elemental Analysis Analysis of Merck Sharp & Dohme reference lot L-644.128-00OU066for carbon and hydrogen gives values compared to calculated values as given below (1 1):
Carbon Hydrogen
5.2
Calculatcd
Found
71.74 9.15
71.79 8.88
Chromatography 5.2.1
Thin Layer Chromatography. Simvastatin may be chromatographed using E. Merck Silica Gel 60 F254 high performance thin layer chroniatographic plates with ;1 mobile phase of 5:2:1 cyclohexane: chloroform: isopropanol solution (0.05% w/v butylated hydroxytoluene). Visualization is either by viewing the developed plate under ultraviolet light or by spraying the developed plate with a dilute methanolic sulfuric acid solution and application of heat. Sulfuric acid spray detection is the most useful system because non-UV absorbing impurities are detectable. The Rf of simvastatin is approximately 0.4 (4).
38 I
SlMVASTATlN
5.2.2
High Performance Liquid Chromatography (HPLC). A variety of gradient and isocratic reverse phase HPLC
systems has k e n used to chromatograph simvastatin (see Table 1).
Table 1: High Performance Liquid Chromatographic Systems
Application
System No. Column
Mobile Phase
nin Detection
Drug Substance Purity
I
Spherisorb ODS
Isocratic and Gradient A = acelonitrile R = 0.025M NaH2 PO,
238
Drug Substance Potency
I1
Perkin-Elmer C-18CR
Isocratic 50:50 A = acelonitrile
238
B = 0.1% (V/V %) H3P0, aqueous
Measurement of Simvastatin and metabolites in plasma
111
GIadie n t Jones Chromatography A = 0.025M sodium acetate Apex 1. C I l 8 B = ncetonitrile
Measurement of low levels in fermentation broth
IV
PRP
Gradient A = aqueous ammonium phosphate pH = 6.1 B = acetonitrile
260
Hyprsil ODS
A = acetonitrile
238
Hypersil 5 micron ODS
A = 0.025M NaH2P0,
Measurement in tablets
V
Measurement in tablets
VI
238
B = water (0.025M NaH2P0, pH = 4.5) 60:40 A:B
pH=4 B = CH3CN C = MeOH 33:55:12 A:R:C
230
Ref
DEAN K. ELLISON ET AL.
382
5.3
Gas Chromatography/Mass Spectrometry A sensitive and selective method for monitoring simvastatin in human plasma using derivatization and GCMS with selected ion monitoring was described by Takano et al. (15).
5.4
Mid-infrared Spectrometry A mid-infrared spectrometry method has been described by Ryan
et al. which provides rapid verification of content and identity of simvastatin formulations (16). 5.5
Identifcation Tests Three methods are routinely used to identity simvastatin: 1. the infrared spectrum; 2. the ultraviolet spectrum; and 3. the chromatographic retention time (4).
6.
Stability and Degradation 6.1
Solid State Stability Solid simvastatin stored under ambient conditions undergoes very slow oxidation of the naphthalenyl diene bond system to give trace amounts of several polar oxidation products. These degradates are difficult to detect by HPLC with UV detection as they are present at low levels and have very little UV absorption. The oxidative degradation process has been studied by chromatography, isolation and identification of degradates, differential scanning calorimetry, thermogravimetric analysis and heat conduction calorimetry (4,10). No degradation products other than those generated by oxidative processes have been found. Degradation is prevented by storage in an inert atmosphere.
SIMVASTATIN
6.2
Solution Stability In aqueous solutions, the lactone ring of simvastatin readily hydrolyses to fonn the P-hydroxy acid. The hydrolysis is very slow in buffered aqueous/acetonitrile solutions and in buffered aqueous surfactant solutions, provided that the apparent pH of the system is approximately 7 (10). In acidic solutions, an equilibrium exists between the hydroxy acid and the lactone. Conversion to the hydroxy acid is rapid in alkaline solutions and is irreversible. The equilibrium constants and the rtlte of the acid-catalysed hydrolysis of simvastatin in pH 2.0 buffer at 37 C have been studied (17). The results indicate that the hydrolysis is reversible. In aqueous surfactant solution in the presence of an initiator. simvastatin was shown to be susceptible to oxidation at the diene functional group (17). In addition, oxidation of simvastatin in chloroform solution has been demonstrated, and the effects of oxidation inhibitors investigated (18).
7.
Pharmacokinetics and Metabolisni Simvastatin is a pharmacologically inactive prodrug for several active metabolites which are HMG-CoA reductase inhibitors. The metabolites, of which the most potent with respect to HMG-CoA reductase inhibition is simvastatin 0-hydroxyacid, are formed by hydrolysis of the lactone ring (19). The inhibitors may be referred to as active (ie: total active p-hydroxyacid metabolites) or total inhibitors (ie: active inhibitors plus lactones and conjugates).
383
384
DEAN K. ELLISON ET AL.
7.1
Absorption and Distribution After oral administration in humans, simvastatin is rapidly extracted by the liver where it is metabolised. The systemic bioavailability of the p-hydroxy acid after administrationof simvastatin is therefore low (less than 5% compared with an intravenous reference dose of the 0-hydroxy acid). Using inhibition of HMG-CoA reductase as a basis for assay, studies have shown that the maximum concentration of inhibitors occurred between 1.3 and 2.4 hours after dosing. The areas under the plasma concentration - time curves (AUC) indicated that the relationship between circulating levels of inhibitors and dose is linear. The plasma profile is essentially unaffected by concomitant administration of food (20). Simvastatin and the 0-hydroxy acid are extensively protein bound (95%) (20).
7.2
Metabolism and Excretion Simvastatin undergoes extensive first-pass metabolism in the liver. Studies with radiolabelled drug have shown that the levels of circulating total inhibitors accounted for 42% of the AUC, indicating that most of the metabolites were inactive or weak inhibitors. Recoveries from urine were 13% and from feces were 60%, the latter including both unabsorbed drug and drug excreted in bile. Less than 0.5% of the administered dose of drug was present as active inhibitors in urine (20). In humans, the main metabolite is the 0-hydroxy acid. Other active metabolites are the 3-hydroxy-, 3-hydroxy-3-methyl-, and 3-exomethylene derivatives. Biliary metabolites include (as hydroxy acids and lactones) the 6-hydroxymethyl- and 6-carboxylic acid analogues, in both of which the chiral centre at position 6 has been inverted (21).
SIMVASTATIN
Qualitatively, the metabolism of simvastatin closely resembles that of lovastatin (2 1). The formation of one metabolite:
has been demonstrated only in rodents (21). Using rat liver microsonies. two male-specific metabolites were identified ( 3 ’ ’-hydroxy and 3 ’ , 3 ’ ‘-dihydroxy 64 ‘ 5 ’ derivatives of simvasta t in) ( 2 2 ) . 8.
Determination in Biological Fluids A procedure for the determination of total HMG-CoA reductase inhibition in biological fluids has been described (2) and applied to simvastatin. This procedure is non-specific, and is based on the inhibition in vitro of the HMG-CoA reductase catalyzed conversion of 14C-HMG CoA to ‘‘C-mevalonic acid. The levels of total inhibitors and active inhibitors can be determined. The detection limit is about 5 ng/mL. Another non-specific procedure applied to simvastatin ( 2 3 ) involves measurenieni of plasma mevalonic acid by gas chromatography-electron capture mass spectrometry. The detection limit in plasma is 0.1 ng/niL.
385
DEAN K.ELLISON ET AL.
386
Simvastatin and its metabolites have been determined selectively in dog plasma by MPLC (13), and the profile compared with that of lovastatin and pravastatin. A sensitive procedure involving gas chromatography-mass spectrometry-selected ion monitoring has been reported (15). This method involves derivatization of the simvastatin in plasma with ferroceneboronic acid. The quantification limit is 0.1 ng/mL. Acknowledgemen& The authors wish to thank Mrs. Barbara Cresswell and Ms. K. Dirmeitis for typing the manuscript.
SIMVASTATIN
9.
References 1.
D. Askin, T.R. Verhoeven, T. M.-H. Liu and I. Shinkai, J . 0rg.Cheni. Xj,4929 (1991).
2.
A.W. Alberts, J. Chen. G. Kuron, V. Hunt, J. Huff, C. Hoffman, J. Rothrock, M. Lopez, H. Joshua, E. Harris, A. Patchett, R. Monaghan, S . Currie, E. Stapley, G. Albers-Schonberg, 0. Hensens. J. Hirshficld, K. Hoogsteen, J. Liesch and J. Springer, Proc. Notl. Acad. Sci. USA 77,3957 (1980).
3.
W.F. Hoffman, A.W. Alberts, P.S. Anderson, J.S. Chen, R.L. Smith and A.K. Willard, J . Med. Chem. 29, 849 (1986).
4.
Merck Sharp & Dohme Research Laboratories, Rahway, NJ., unpublished data.
5.
R. Reamer, Merck Sharp & Dolune Research Laboratories. personal communication.
6.
A.I. Scott Interpretation of the Ultrcr\)iolet Spectra of Natural Products, Pergamon Press, Oxford, 1964.
7.
D. Zink, Merck Sharp & Dohme Research Laboratories. personal communication.
8. S. Thomas. Merck Sharp & Dohme Research Laboratories. personal communication. 9.
J. McCauley, Merck Sharp & Dohme Research Laboratories, personal communication.
10. W.D. Moore, Merck Sharp & Dotulle Research Laboratories, personal communication. 11.
J. Wu, Merck Sharp & Dohme Research Laboratories, personal communication.
3x7
388
DEAN K.ELLISON ET AL.
12. A. Halfpenny, Merck Sharp & Dolme Research Laboratories, personal communication. 13. R.J. Stuhbs, M.D. Schwartz, R.J. Gerson,T.J. Thornton, and W.F. Bayne, Drug. Invest. 2 (supplement 2), 18 (1990).
14. K. Gkwonoyo, B.C. Buckland, and M.D. Lilly, Biotechnol. Bioeng. 2 ( 1 1). 1101 (1991).
15. T. Takano, S. Abe, and S. Hata, Bionied. Environ. Mass Spectrom. 19 (9), 577 (1990). 16. J.A. Ryan, S.V. Compton, M.A. Brooks, and D.C. Compton, J. Pharm. Biomed. A n d . 9 (4), 303 (1991). 17. M.J. Kaufman, Pharni. Res. 7,289 (1990). 18. C.R. Petts, Merck Sharp & Dohme Research Laboratories, personal communication.
19. E.E. Slafer and J.S. MacDonald. Drugs 36 (supplement 3) 72 (1988). 20. International Physicians’ Circular, Zocor (Siniwsfurin),Rahway, NJ. Merck Sharp & Dohme International, Sept. 8. 1989. 21. S. Vickers, C.A. Duncan, K.P. Vyas, P.H. Kari, B. Arison, S.R. Prakash, H.G. Ranjit, S.M. Pitzenberger, G. Stokker, D.E. Duggan. Drug Merob. Dispos. lJ, (4) 476-483 (1990). 22. N. Uchiyama, Y. Kagami, Y. Saitoh. M. Ohtawa. Chent. Pharm. Bull. 3, ( 1 ) 236-8 (1991). 23. A. Scoppla. V.M.G. Maher, G.R. Thompson, N.B. Rendell, G.W. Taylor, J. Lipid. Res. 2,( 6 ) 1057-1060 (1991).
SULFATHIAZOLE
Vijay K. Kapoor
Department of Pharmaceutical Sciences Panjab University Chandigarh - 160014 INDIA
ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 22
389
Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
VIJAY K. KAPOOR
390
1.
D e sc ription 1.1 1.2
2.
Name, Formula and Molecular Weight Appearance, Color and Odor
Physical P rop erties 2. 1 2.2 203
204 2^;5 2a 6 2 07 2.8 2.9
2.10 2.11
Solubility D i s s o c i a t i o n Const ant Partition Coefficient Optical A c t i v i t y Polymorphism and Crystal Pr ope r t ie s Melting Range lnfrared Spectrum U l t r a v i o l e t Spectrum 13C-Nuclear Magnetic Resonance Spectrum Raman Spectra Mass Spectrum
3.
Synthesis
4.
S t a b i l i t y and Degradation
5.
Pharmacokinetics and Metabolism
60
P r o t e i n Binding
7
.
SULFATHIAZOLE
Bloavailabll i t y
8.
Toxicity
9.
Methods of A n a l y s i s 9.1 9.2 9.3 9.4 9.5 9.6 9.7
9.8 9.9
9.10 9.11 9.12 9.13
10.
Elemental Composition I d e n t i f l c a t i o n Color T e s t s Titrirnetric Analysis Bioassay P o l a r o g r a p h i c Analysis A d s o r p t i v e S t r i p p i n g Voltamaretry Thermal A n a l y s i s Atomic Absorption Spectroscopy Spectrophotometric Analysis 9.9.1 Colorimetric 9.9.2 Fluorometric 9.9.3 Ultravlolet 9.9.4 Wrared lH-Nuclear Magnetic Resenance Spectroscopy Raman SpeCtrOmetry Mass Spectrometry Chromatographic A n a l y s i s 9.13.1 Ion-exchange Chromatography 9.13.2 Paper Chromatography 9.13.3 Thin-Layer Chromatography 9.13.4 Gas Chromatography 9.13.5 High Performance Liquid Chromatography 9.13.6 Supercritical Fluid Chromatography
References
39 I
VIJAY K. KAPOOR
392
1.
Description 1.1
Name, Formula and Molecular Weiqht
S u l f a t h i a z o l e is a s h o r t - a c t i r g sulfonamide. I t is a l s o c a l l e d as norsulfazolum, s u l f anilamidothiazolum, s u l p h a t h i a t o l e , sulfonazolum, and as s o l f a t i a z o l o . Chemically, it i s N~-thiazol-2-ylsulphanilamide O t h e r chemical names are: N1-2=thiazolylsulfanilamide, 4-amino-N-2=th i a z o l y l benzenesul f onamide , 2-sulf a n ~ l a m i d o t h i a z o l e 2 4 s u l f anilylam1no)t h i a zole,2-( paminobenzenesulf onamido ) th i a z o l 8 I t a l s o o c c u r s as s u l f a t h l a z o l e sodium ( s o l u b l e sulf athiazole)
.
-
,
.
.
The CAS r e g i s t r y number f o r s u l f a t h i a z o l e is 72-14-0, and f o r s u l f a t h i a m l e sodium is 14674-1.
CgH9N302S
Molecular Weight: 255.31
The coamon p r o p r i e t a r y names are C i b a z o l , Sulfamul, and Thiazamlde. 1.2
Appearance, Color and Odor
S u l f a t h i a z o l e is a w h i t e or almost w h i t e , c r y s t a l l i n e powder; o d o r l e s s o r almost o d o r l e s s . The sodium salt i s a white o r y e l l o w i s h w h i t e m i c r o c r y s t a l l i n e powder. 2.
Physical P r o p e r t i e s 20 1
SOlUbill
ty
S u l f a t h l a t o l e is very s l i g h t l y s o l u b l e
SULFATHIAZOLE
3Y 3
( 1 i n 2 5 0 0 ) i n w a t e r , s p a r i n g l y s o l u b l e ( 1 i n 120) i n e t h a n o l ,and p r a c t i c a l l y i n s o l u b l e i n chloroform and ether.192 A t pH 6 i t s s o l u b i l i t y 3 i n water ( m S / l O O m l ) is 60 a t 26 100 a t 37'; and a t pH 7.5 i s 2 3 5 - a t 37'. It d i s s o l v e s i n a c e t o n e , d i l u t e mineral a c i d s l and i n aqueous s o l u t l o n s of a1 k a l i hydroxides and c a r b o n a t e s . S o l u b i l i t y of s u l f a t h i a z o l e i n human blood serum3 is 330 mg/lOO ml a t 37'. S u l f a t h i a z o l e i s r e p o r t e d 4 t o be more s o l u b l e I n 0.3M phosphate b u f f e r a t pH 7.2 t h a n a t pH 6.3. The s o l u b i l i t y d e c r e a s e s by a d d i t i o n of in: r e a s l n g c o n c e n t r a t i o n s of sodium t a u r o g l y c o c h o l a t e or sodium t a u r o c h o l a t e up t o c o n c e n t r a t i o n s approximating t h e c r i t i c a l miceller concentrations ( 5 &).
-
The s o l u b i l i t y of s u l f a t h i a z o l e h a s been determined i n mixtures of dimethylacetamide g l y c e r o l and water, and t h e s o l u b i l i t y p r o f i l e s were w e l l reproduced by t h e u s e of extended Hildebrand s o l u b i l i t y approach.5 The s o l u t i o n r a t e and s o l u b i l i t y of an aqueous s u l f a t h i a z o l e s u s p e n s i o n have a l s o been studied.6 D i s s o c l a t i o n Constant
2.2
pka
7.1
(25O)l
Partition Coefficient
2.3
-
Log P (octanol/pH 7.5), Optical A c t i v i t y
2 04
a c ti v i t y 2.5
- 0.4'
.
S u l f a t h i a z o l e e x h i b i t s no optical Polymorphism and Crystal Properties
The polymorphism of s u l f a t h i a z o l e has The been i n v e s t i g a t e d by numerous norkers.7-21 e a r l i e r confusion a b o u t t h e s e p a r a t e e x i s t e n c e of two polymorphs, forms 111 and IV of s u l f a t h i a z o l e h a s now been resolved21; b o t h forms exist. Single c r y s t a l s were u s e d t o generate X-ray d i f f r a c t i o n p a t t e r n s , e q u i v a l e n t t o t h o s e normally o b t a i n e d from p o l y c r y s t a l l i n e samples, u s i n g t h e G a n d o l f i camera. The p a t t e r n s o b t a i n e d were d i s t i n c t and i n agreement w i t h t h e t h e o r e t i c a l
VIJAY K. KAPOOR
394
powder p a t t e r n s c a l c u l a t e d from t h e r e p o r t e d s t r u c t u r e s of t h e two forms, t h u s confirrnlrg t h e s e p a r a t e e x i s t e n c e . The e x i s t e n c e of four polymorphs, form I , f I , l I I and IV of s u l f a t h z o l e has been confirmed by I n f r a r e d , Raman, and n u c l e a r magnetic resonance s p e c t r a and powder X-ray d i f f r a c t i o n (PXRD) patterns.21 The powder X-ray d i f f r a c t i o n p a t t e r n s f o r t h e forms have been o b t a i n e d u s i n g a Siemens D500 d i f f r a c t o m e t e r f i t t e d w i t h a s c l n t f l l a t i o n c o u n t e r and a C u K a r a d i a t i o n source. T h e divergence and t h e d e t e c t o r s l i t s were 0.3 and 0.05O a p e r t u r e , r e s p e c t i v e l y . Data were c o l l e c t e d I n a step-scan mode using a s t e p size of 0 . 0 5 O 2 0 and a c o l l e c t i q time of 5 s per step. A post-sample n i c k e l f l l t e r was used The f o r t h e monochromation of t h e X-rays. observed PXRD p a t t e r n s of t h e forms are shown i n F i g u r e 121. The e s t i m a t e d d-spacings and r e l a t i v e i n t e g r a t e d intensifies are g i v e n I n Table I.
1%
c
*
-A
Y
c
I
10
I
15
2
I
I
20
25
I
30
I
35
W")
F i g u r e 1- Powder X-ray d i f f r a c t i o n p a t t e r n s of olymorphs1 , I J , I I I , and IV of sulf a t h i a z o l e A = C U K )~
P
SULFATHIAZOLE
39.5
Table I- Observed _d-Spacings ( A o ) and R e l a t i v e Integrated I n t e n s i t i e s (1)for Polymorphs I , I f , 111, and IV of S u l f a t h i a z o l e
Form
I
I I1
IV d
10.03 8.11 7 e56 6.91 6.61 6 056 6. 12 5.52 5.10 5 002 4.98 4.69 4.24 4 005 4.00 3 088 3.8 1 3.78 3.68 3.63 3 .57 3.46 3.35 3.31 3.28 3.14 3.09 3.03 2.98 2.87 2 084 2.80 2.78 2.76 2.74 2 070 2.67 2.65 2.61
12 50 7
a
10 10 7 72 15 58 22 84 64 100 35 14 7 12 12 27 14 7 1 5 14 23 3 4 5 5 4 2 10 3 1 1
.rl
k
cd
c, A
3 N
M L4
.d
/-I
i', rransinittance.
uE 0 d
0
0 0 d
0
0
m
0 d
0 0 0 N
0
0 0 M
TENOXICAM
3.7.3.
44 1
Nuclear Magnetic Resonance Spectra 3.7.3.1.
PMR Spectrum
The PMR spectrum o f t e n o x i c a m i n DMSO-da was The r e c o r d e d on V a r i a n XL-200 NMR s p e c t r o m e t e r . s p e c t r u m o f t h e d r u g was c o n s i s t e n t w i t h t h e s t r u c t u r e , with c h a r a c t e r i s t i c N-methyl p r o t o n s which appeared as a sharp s i n g l e t a t 2.90 ppm ( F i g . 4 ) . The spectrum a l s o d i s p l a y e d s i x s i g n a l s i n t h e aromatic r e g i o n due t o C s - H , C l o - H , C 1 1 - H and C 1 2 - H of t h e p y r i d y l r i n g , i n a d d i t i o n t o C4-H and C5-H o f t h e fused thiophene r i n g . The chemical s h i f t values a r e presented i n t a b l e V. Both amide and h y d r o x y l p r o t o n s disappeared i n t h i s spectrum.
Table V. PMR c h a r a c t e r i s t i c s o f tenoxicam. Proton assignment
Chemical s h i f t 6 (ppm)
N-CH3
2.90
(s)
C4 -H
8.04
(d)
C5-H
7.46
(d)
CS-H
7.33
(t)
CIO-H
8.16
(t)
CI 1-H
7.74
(d)
C i 2-H
8.35
(d)
s
singlet,
d = doublet,
t
= triplet.
I
12
I
I
10
Fig.
L
4
8 90.6
I
1
I
6
4
PMR s p e c t r a o f tenoxicam i n DPISO-d6.
I
-2
1
0 63.5
PPM
443
TENOXICAM
3.7.3.2.
3C-NMR SDectrum
' 3 C - N M R spectrum o f tenoxicam i n DMSO-ds F i g . (5-8 1 was recorded on V a r i an XL-200 NMR spectrometer. The m u l t i p l i c i t y o f t h e resonances was obtained from DEPT p r o g r a m ( D i s t o r t i o n l e s s Enhancement b y P o l a r i z a t i o n t r a n s f e r ) and APT program ( A t t a c h e d Proton T e s t ) . The 13C-NMR spectrum d i s p l a y e d a l l t h e t h i r t e e n carbon resonances, t h e narrow resonance range o f some o f t h e s e carbons makes t h e s p e c t r a r a t h e r complex and d i f f i c u l t f o r proper assignments. However, a l l resonances have been assigned by t h e a i d o f t h e DEPT and APT programs. The carbon chemical s h i f t assignments are presented i n t a b l e V I .
3.7.4.
Mass S w c t r u m
The mass spectrum o f tenoxicam o b t a i n e d by e l e c t r o n impact i o n i z a t i o n (Fig. 9) was recorded on a Finnigan MAT 90 spectrometer. The spectrum was scanned E l e c t r o n energy was 70 ev. f r o m 10 t o 350 a.m.a. Emission c u r r e n t 1 mA and i o n source p r e s s u r e t o r r . The most prominent fragments and t h e i r r e l a t i v e i n t e n s i t i e s are presented i n t a b l e V I I . Table V I . Carbon-I3 chemical s h i f t s o f tenox icam
Carbon assignment C-13 c-12 c-11 c-10 c- 9 C-8 c-7
Chemical s h i f t
6
(wm)
39.22 143.60 122.80 140.10 116.24 149.30 165.60
Carbon assignment C- 6 c- 5 c-4 c- 3 c- 2 c- 1
Chemical s h i f t i 3 (PPm) 137.56 118.40 132.70 108.23 163.00 40.70
444
ABDULRAHMAN MOHAMMAD AL-OBAID AND MOHAMMAD SALEEM MIAN
Table VII. Mass fragments of tenoxicam.
m/z
Relative intensity X
18
62
83
25
94
44.2
Ions
-
o-" 0
i-co
ON" I
121
50.2
153
42
bH+ 179
100
co 256
213
337
12.2
I
200
180 Fig.
160 5
1
I3C-NMR
PPM
s p e c t r a o f tenoxicam i n DMSO-d
6'
I
l
l
~
~
l
l
l
l
l
I
l
I l1
1 1 1 1 1 " l ~ l 1 1 1 1 1 1 1
LA
46 Fig.
6
42 "C-NMR
I
1 1 1 1 1 1 1 1 '
40
1
30
1
'
1
1
1
1
1
1
1
'
36
s p e c t r a of tenoxicam (Peak Expansion).
1
1
~
'
l
I
l
' 1 1 1 1 1 1 1
3 4 PPM
447
I
! L
0
a -
I
-cJ
-0
0 -cJ
0
-U
0
-W
0
'a3
.-
0 '0
cJ
0
0 7
.U
0 W
-
0
s 0 cJ
.O
c Ii3
c ti x I
I
180
200 Fig.
8
I
160
I
140
I
120
I
100
1
80
I
60
I3C-NMR spectra o f tenoxicam (DEPT program).
I
40
I
20
I
0
I
20 I
PM
I
8 W
I 0
I
0
N
0
I
U
1 0
449
\
'
N
E-
l
0
-
0 In
m
m
0 0
In
0 N
0 0
cw
C
LI: c
C
v-
*C
LT
.c
9 u
x
.?I
0 E a, c, 0
w
k
(d
u
c,
m
a, C, v)
m (d
P
m M Lr,
.?I
ABDULRAHMXN MOHAMMAD AL-OBAID AND MOHAMMAD SALEEM MIAN
450
4
I
Phannacokin e t 4 cs 4.1
AbsorDtion and D i s t r i b u t i o n
Tenoxicam i s completely ( 7 ) absorbed a f t e r o r a l administration. I t has h i g h p r o t e i n b i n d i n g capacity and low volume o f d i s t r i b u t i o n (0.12 t o 0.152 L/Kg). I t e f f i c i e n t l y p e n e t r a t e s i n t o synov-ial f l u i d and systemic clearance i s a l s o low (0.1 t/h). Tenoxicam has got a long e l i m i n a t i o n h a l f l i f e (60-75 hours). The average time t o achieve peak plasma concentrations i s 1 t o 2 . 6 h o u r s f a s t i n g and 4 t o 6 h o u r s postprandial. Following o r a l administration o f s i n g l e o r m u l t i p l e doses o f tenoxicam t o healthy subjects, peak plasma concentrations occur between 0.5 and 2 hours a f t e r a d m i n i s t r a t i o n (8,9) compared w i t h intravenous administration. Tenoxicam i s nearly 100% b i o a v a i l a b l e a f t e r o r a l a d m i n i s t r a t i o n and about 8% a f t e r r e t a l dose (10,ll). A f t e r a d m i n i s t r a t i o n o f a s i n g l e o r a l dose o f 20 mg o f drug t o h e a l t h y s u b j e c t s t h e mean plasma concentration was about 2.3 mg/L and area under the plasma concentration-time curve (AUC) v a r i e d from 86-243 my/L.h w i t h a mean value o f 151 mg/L.h. ( 1 1 ) . A b s o r p t i o n o f tenoxicam i s delayed by t h e presence o f food, as pre- and post-prandial mean times t o maximum plasma concentration o f 1.3 and 4.1 hours r e s p e c t i v e l y were a t t a i n e d i n 6 healthy subjects given s i n g l e o r a l doses o f 40 mgs (8). Following intravenous administration o f tenoxicam, the time course o f t h e plasma concentrat i o n s was representative o f a 2-compartment model (9). Tissue d i s t r i b u t i o n studies i n r a t s using r a d i o l a b e l l e d tenoxicam have shown h i g h l e v e l s o f r a d i o a c t i v i t y p r e s e n t i n l i v e r and kidney. Blood concentrations i n other t i s s u e s were lower than those i n the blood (12). S t u d i e s suggest t h a t t h e pharmacokinetics o f t e n o x i c a m a r e u n e f f e c t e d b y age, s e x , r e n a l impairment, hepatic disease o r rheumatic disorders, b u t these p r e l i m i n a r y conclusions r e q u i r e c o n f i r m a t i o n i n l a r g e groups o f p a t i e n t s t r e a t e d f o r longer periods (13).
TENOXICAM
4.2
45 I
Uetabolism
Tenoxicam i s a l m o s t c o m p l e t e l y m e t a b o l i z e d before e x c r e t i o n from t h e body. Up t o two t h i r d s o f t h e a c t i v e substance i s e l i m i n a t e d v i a t h e kidney and t h e remainder v i a t h e b i l e . The m e t a b o l i t e e l i m i n a t e d m a i n l y by t h e r e n a l r o u t e i s I
F.
Therrnoanalytical Behavior
1. Melting Range A fairly wide melting range has been reported for tolazamide (possibly due to polymorphism). These include 170-173oc (9.20), 165-17OOC (21). 168-171OC (22) and 163.5-166.5OC (23). Decomposition has been noted at these elevated temperatures.
2.
Loss on Drying
Weight loss is not more than 0 . 5 X for an accurately weighed sample of tolazamide heated at 60OC under vacuum at a pressure not exceeding 5 m of mercury for 3 hours. 3.
Differential Thermal Analysis
The differential thermal analysis (DSC) behavior of tolazamide is shown in Figure 8 . The thermogram was obtained using a Perkin Elmer Series 7 DSC scanning from 30OC to 22OOC at SOC/minute. A primary endotherm corresponding to melting is observed at a peak onset 169OC. A 3.33 mg sample of tolazamide, USP temperature of reference standard, lot G was used.
.
Peak-
P.sk
f-
to:
170.70
1Bl.M 174.70
Onsat- 160.01 J/g l22.tl.Y
-
I I
4.w
h.bo
$.a
lm.mx
14.66
I-----
Idb.a6-id6.M
Terriperalure ( C )
Pjgure
8.
Differential thermoanalytical behavior of tolazamide.
A
k
4.
Thermogravimetric Analysis
Thermogravimetric analysis of tolazamide indicates no weight loss until a temperature of 170QC is reached. Significant weight loss is observed above 212% due to decompositionlvaporization. A typical TGA curve is shown in Figure 9. The curve was obtained using a Perkin Elmer Series 7 TGA scanning from 30QC to 280QC at SoC/minute. A 3.24 mg sample of tolazamide, USP reference standard, lot G was used. -
I----
ilo.00
Figure
9.
llb.00
190.00
_______
&.w
Temperature ( C ) Thermogravimetric behavior of tolazamide.
&.w
i
IV.
SYNTHESIS Tolazamide has been synthesized by Wright and Willette
( 9 ) according to the general method of Marshall and Sigal ( 2 4 ) . The p-toluenesulfonyl derivative of methyl urethane
is reacted with the appropriate disubstituted hydrazine (N-aminohexamethyleneimine) to yield tolazamide ( 5 4 % yield) as illustrated in Figure 10.
Figure 10.
Synthesis of tolazamide.
V.
PHARMACOKINETICS
A.
Absorption
Several reports indicate tolazamide as being rapidly allsorbed (3.25) whereas others indicate a somewhat slower absorption (1,2,11,13,26). The drug is probably best characterized as well absorbed from the gastrointestinal tract after oral administration with peak blood levels occurring between 4 - 8 hr. It has an elhfnatfon half-life of about 7 hr and a duration of action of between 10-18 hr. After four to s i x doses, an equilibrium state is reached in most patients and the drug does not accumulate in the blood even during long term therapy (1.25). A recent study by Wright and Antal ( 2 7 ) demonstrated that the pharmacokinetics of tolazamide are independent of age.
JOHN K.LEE ET AL.
506
B.
Metabolism
A n extensive investigation of the metabolism of tolazamide in both man and the rat has been reported by Thomas and co-workers (28). Using tritium labeled tolazamide (labeled predominantly in the aromatic ring), they found the drug to be extensively metabolized in both species with 85% of the radioactivity found in the urine after five days (normal male subjects) and 79% found in the urine after the same period (female Sprague-Dawley rats). Both studies utilized a single oral dose of tritium labeled drug. In each case most of the radioactivity was recovered in the first 24 hr (urine). Figure 11 shows the major pathways of metabolism found for both species. Based on a 24 hr urine collection, metabolite 4 was the most abundant metabolite in the rat (80%) but one of the least abundant in man (10%). Whereas metabolites 2 and 3 were the least abundant metabolites in the rat ( - 5% each), they were the most abundant in man ( " 25% each). Unidentified metabolite 5 was detected in man ( - 15%), but was not detected in the rat. Studies indicated that it may be labile and transformed to metabolite 6. Metabolites 2 and 4 were tested for hypoglycemic activity and found to be, respectively, 70% and 20% as potent as tolazamide in the rat. Metabolite 6 was 5 % as potent as tolazamide. Based on these studies. both metabolites 2 and 4 have greater hypoglycemic activity than tolbutamide.
507
TOLAZAMIDE
0 CH3-54
--NH
--C
II -NH
-N
TOLAZAMIDE 14hexahydroazepi n-1-yl)-3-p-to1ylsulfon ylurea
\ UNKNOWN 3
3
Metabolite 2
3
4 5 6
Figure 11.
t
Identity
dl-l-(4-hydroxyhexahydroazepin-l-yl)-3-ptolylsulfonylurea p-toluenesulfonamide l-(hexahydroazepin-l-yl)-3-p-(hydroxymethylpheny1)sulfonylurea Unknown l-(hexahydroazepin-l-yl)-3-p-(carboxyphenyl)sulfonylurea Metabolism of tolazamide in man and the rat [modified from ref. (28)J : a previously isolated and identified (29).
JOHN K. LEE ET AL.
SO8
VI. A.
DETERMINATION IN PHARMACEUTICALS Dissolution Testing
Tablet dissolution testing has been reported by Welling and co-workers (30) in their study of tolazamide bioavailability. The method used is identical t o the procedure described in USP XXII (31). The dissolution rate was determined in 900 mL of 0.05 M tris(hydroxymethy1)aminomethane aqueous buffer (pH 7.6) using a 75 rpm paddle stirring rate. W measurement of the dissolution medium was performed at 224 nm by continuously pumping the medium through a 0 . 5 m path length flow cell. (The USP method specifies filtration of samples).
B.
High Performance Liquid Chromatography
The official monograph in the United States Pharmacopeia XXII (31) describes an HPLC method for both tolazamide bulk drug and tolazamide in tablets. This procedure employs a stainless steel column (300 rmn x 4 mm I.D.) that contains 10-um particle size silica and a mobile phase consisting of hexane, water-saturated hexane, tetrahydrofuran, alcohol, and glacial acetic acid (475:475:20:15:9). Detection is achieved at ambient temperature using W absorbance at 254 nm. The tolazamide standard and sample solutions having a known concentration of about 3 mg per mL are prepared in an internal standard solution of Tolbutamide (” 1.5 mg per mL) in alchol-free chloroform. This procedure may not be suitable in all cases depending on the specific tablet formulation. Therefore, other HPLC procedures map need to be employed and have been reported in the literature. A relatively recent microbore column HPLC method using pre-packed microbore columns packed with 10-um particle size silica ( 5 0 0 nun x 1 m I.D., microsphere silica) and RP-8 ( 2 5 0 rn x 1 mm I.D., Partisil 10 CCS/CS) (32) claimed a 16-fold increase in sensitivity over conventional HPLC systems with high column efficiency, good peak resolution, and very little, if any, mobile phase modifications. In addition to sulfonylureas such as tolazamide, steroids and antibiotics were also successfully analyzed.
TOLAZAMIDE
509
A prior HPLC method for tablets ( 3 3 ) utilized a mobile phase consisting of 0.01M monobasic sodium citrate containing either 10% or 15% methanol (v/v), at an apparent pH of 4.4. The stainless steel column (1000 mm x 2.1 mm I.D.) was dry-packed with hydrocarbon polymer support (1% ethylene propylene copolymer on DuPont Zipax). The method consists of 1 pL injections with detection at a fixed wavelength of 254 nm. In the method, the average weight of ten tablets is determined followed by grinding to a fine powder. The equivalent of - 8 0 mg of drug is transferred into a vial containing 20 mL of internal standard (acetohexamide) followed by shaking, centrifugation, and injection of a portion of the supernatant. Linearity was demonstrated over the concentration range of 1.8-9.0 p g [unpublished data ( 3 4 ) J as well as good resolution and quantitative recovery. In another recent investigation, Severin (35) developed an HPLC method for the determination of N-nitrosohexamethyleneimine at the low ppb level in both bulk drug and tablets without interference from impurities and degradates of tolazamide. This compaound is a potential carcinogen and is an intermediate in the synthesis of tolazamide. Following extraction in diethyl ether and on-line cleanup and enrichment, the nitrosamine is detected by W at 228 run.
C.
Thin-Layer Chromatography
Several TLC systems have been reported for the identification of tolazamide or other oral hypoglycemic agents in biological extracts, standards, bulk drug or tablets. A partial listing is presented in Table 111.
B
P
0
rl
0 0
d
@
0 W 0 1.1 0 rl
0
m
H
B 3 rl
A
V
00 N
6
a,
6 i4
0
8
m m
m
r-
...
crl d
00
Y)
0)
ld
4J
0,
u
9
@ 0
l-l
0
r-l
A
1.1
9 w
W 0
e
0
V
N
e 0
ld U *4
d
rl
t3
In
N
v)
8
rl
m
v)
rl .rl
V
v)
.rl rl .rl
0
d
a
'A 3 8"
" N rl
TOLAZAMIDE
D.
511
Spectroscopic
Recently, Al-Badr reported a proton NMR spectrometric analysis of tolazamide in both bulk drug and tablets using the CH - group signal of the drug (2.42 ppm) and comparing it to $he -CH signal of the benzyl benzoate internal standard (5.38 ppm) . Quantitative recoveries were obtained without interferences from excipients, and the method was shown to be specific (38).
-
E.
Spectrophotometric
Saleh and Askal ( 3 9 ) described a spectrophotometric method for the assay of tolazamide and other similar compounds based on the formation of charge-transfer complexes formed between the drug as electron donor and iodine as electron acceptor. Measurements were made at 295 tun with a sensitivity of 1 pglmL. Hussein and co-workers (40) described a similar method based on the reaction between tolazamide and p-chloranil (as electron acceptor). An intensely colored complex was formed and measured at 445 tun. In addition, p-bromanil and 7,7,8,8-tetracyanoquinonedimetharie were used. F.
Non-Aqueous Titration
A non-aqueous titration method has been reported (41) which involves titration with lithium methoxide in methanol-benzene to either a visual endpoint with azoviolet (yellow to blue) or to a potentiometric endpoint. Tolazamide is dissolved in tetramethylurea prior to titration. Quantitative recoveries were claimed. G.
Complexometric Titration
A complexornetric titration method, applicable to tablet assay, has been reported by Guerello and Dobrecky (42). The method involves hydrolysis of the drug with alkali, neutralization with acid, and addition of excess cupric sulfate. Following adjustment to a pH of 6 and filtration, excess cupric ion is titrated with disodium EDTA using 1-(2-pyridylazo)-2-napthol as an indicator.
JOHN K.LEE ET AL.
512
H.
Voltammetric Method
In the synthesis of tolazamide (Figure 10) the disubstituted hydrazine, N-aminohexamethyleneimine, is reacted with the appropriate urethane. The hydrazine, in turn, is synthesized from the lithium aluminum hydride reduction of N-nitrosohexamethyleneimine. This N-nitroso compound is produced by the reaction between hexamethyleneimine and nitrous acid ( 9 ) . N-nitrosamines have long been suspected of being carcinogenic. Recently, Buldini and co-workers (43) recommended monitoring the level of this N-nitrosamine residue in bulk drug and also developed a sensitive voltammetric method for its determination.
VII. A.
DETERMINATION IN BIOLOGICAL MATRICES High Performance Liquid Chromatography
In their investigation of the bioavailability of tolazamide from various tablet formulations, Welling and co-workers (30) described an HPLC method which they developed for the determination of the drug in serum. In this procedure, 0.5 mL of serum is added to 0.5 mL of a chloroform solution of the internal standard, 5-(p-methylphenyl)-5-phenylhydantoin. After adjustment to a pH of 4.5, samples are extracted with methylene chloride and the organic layer is evaporated to dryness. Reconstitution in methanol is followed by HPLC on a Lichrosorb C-18 column, 10 pm particle size (250 nun x 4.6 mm I.D.) using a mobile phase consisting of 52.3% methanol in acetate buffer (pH 5.6). Detection is at a fixed wavelength of 254 nm. The procedure was reproducible and specific for tolazamide with a sensitivity of 1 pg/mL serum.
TOLAZAMIDE
513
Starkey and co-workers described a method for determination of tolazamide and several other sulfonylureas in serum which involves extraction into ethyl ether, followed by pyrolysis with dinitroflourobenzene to form a derivative detectable by HPLC at 360 run (44). They used a Spherisorb ODS-2 column (250 nun x 4.5 mm I.D.), glibonuride as an internal standard, and a phosphoric acid-acetonitrile mobile phase. Separation of tolazamide from the other drugs was achieved with a recovery of at least 93% for all components and a detection limit of 0.04 pg/mL. An advantage of this method is that serum components usually do not interfere at 360 run.
-
B.
Gas-Liquid Chromatography
A GLC method for assaying tolazamide in guinea pig plasma has been reported (45). In this method, plasma specimens from dosed guinea pigs are first adjusted to an acidic pH and then extracted with chloroform. After evaporation of an accurately measured volume, the residue is subjected to a TLC cleanup procedure (Silica Gel F-254, 250 pm) to separate tolazamide from metabolites and plasma residues prior to GLC analysis. Levels are calculated from a standard curve obtained using spiked control plasma as well as chloroform standards. Using an internal standard of l-(n-butyl)-3-p-chlorobenzenesulfonylurea and an 0 . 5 % Carbowax 20M column on 80-100 mesh Chromosorb G (column, 19OOC [isothermal]: flash heater, 236OC; detector, 220OC), a sensitivity of 0 . 7 p g / m L plasma was obtained. Under these conditions, tolazamide is fragmented to p-toluenesulfonamide and the internal standard is fragmented to p-chlorobenzenesulfonamide.
REFERENCES 1.
AHFS Drug Information@, American Society of Hospital Pharmacists, Inc., 1903-1905 (1992).
2.
R.H. Travis and G. Sayers, in L.S. Goodman and A . Gilman, The Pharmacological Basis of Therapeutics, 4th Edition, The Macmillan Co., 1581-1603 (1970).
JOHN K.LEE ET AL.
514
3.
Drug Evaluations Annual 1992, American Medical Association, 939-940, 953-959 (1992).
4.
Diabetes, 19 (supp. 2). 747-830 (1970).
5.
M. Janbon, J. Chaptal, A. Vedel and J. Schaap, Montpell. med., 21-22, 441-444 (1942).
6.
A. Loubatieres, Ann. N.Y. Acad. Sci., 71, 4-11 (1957).
7.
S.L. Mukherjee and A. David Ltd., Indian pat. 59,097 (1958)
8.
H. Ruschig, W. Aumueller. G. Korger, H. Wagner, J.
.
Scholz and A. Baender, U.S. pat. 2,968,158 (1961 to Upjohn). 9.
J.B. Wright and R.E. Willette, J. Med. Pharm. Chem., 5, 815-822 (1962).
10.
W.E. Dulin, H.L. Oster and F.G. McMahon, Proc. SOC. Exptl. Biol. Med., 107, 245-248 (1961).
11.
J.E. Jackson and R. Bresisler, Drugs, 22, 211-245 (1981).
12.
Merck Index, 11th Edition, Merck h Co., Inc. (1989).
13.
Clarke’s Isolation and Identification of Drugs, 2nd Edition, The Pharmaceutical Press, London, 1029 (1986).
14.
Material Safety Data Sheet, United States Pharmacopeial Convention, Inc., No. 66800 (1985).
15.
Remington’s Pharmaceutical Sciences, 17th Edition, Mack Publishing Co., 977 (1985).
16.
T. Mills 111, W.N. Price and J.C. Roberson, Instrumental Data For Drug Analysis, Volume 2, Elsevier Sclence Publishing Co., Inc., 1228-1229 (1984).
17.
J. McMurry, Organic Chemistry, BrookslCole Publishing
Co., Monterey, CA, 470-473 (1984). 18.
J.D. Roberts and M.C. Caserio, Basic Principles of Organic Chemistry, W.A. Benjamin, Inc., New York, 101, 773 (1965).
TOLAZAMIDE
19.
R . T . Conley, Infrared Spectroscopy, Allyn and Bacon, Inc., Boston, 179-181 (1966).
20.
Ger. pat. 1,186,865 (1965 to Upjohn).
21.
H. Ueda and T. Nagai, Chem. Pharm. Bull. (Japan),
515
26(5), 1353-1356 (1978). 22.
I. Butula, V. Vela and M.V. Prostenik, Croat. Chem. Acta, 52(1), 47-49 (1979).
23.
Fr. pat. 1,458,907 (1961 to Upjohn).
24.
F.J. Marshall and M.V. Sigal, Jr., J. Org. Chem.. 23, 927-929 (1958).
25.
Physicians’ Desk Reference@ , 45th Edition, Medical Economics Co. Inc., 2259 (1991).
26.
M.C. Balodimos and A. Marble, Curr. Ther. Res., 13(1), 6-12 (1971).
27.
C.E. Wright I11 and E.J. Antal, (Abstract 66), DICP, 19, 458 (1985).
28.
R.C. Thomas, D.J. Duchamp, R.W. Judy and G.J. Ikeda, J. Med. Chem., 21(8), 725-732 (1978).
29.
A.A. Forist and R.W. Judy, J. Pharm. Phamacol., 26(7), 565 (1974).
30.
P.G. Welling, R.B. Patel, U.R. Patel, W.R. Gillespie, W.A. Craig and K.S. Albert, J. Pharm. Sci., 71(11), 1259-1263 (1982).
31.
United States Pharmacopeia, 22nd Revision, 1384 (1990).
32.
K. T s u j i and R.B. Binns, J. Chromatogr., 253(2), 227-236 (1982).
33.
W.F. Beyer, Anal. Chem., 44, 1312-1314 (1972).
34.
K. Tsuji (Editor), GLC and HPLC Determination of Therapeutic Agents, Chromatographic Science Series, 9(3), Marcel Dekker, Inc., 1080, (1979).
JOHN K. LEE ET AL.
516
35.
G. Severin, J. Chromatogr., 386, 57-63 (1987).
36.
United States Pharmacopeia, 22nd Revision, 6th Supplement, 2916-2917 (1992).
37.
P.G. Takla and S.R. Joshi, J. Pharm. Biomed. Anal., 1(2), 189-193 (1983).
38.
A.A. Al-Badr, Spectrosc. Lett., 16(9), 673-682 (1983).
39.
G.A. Saleh and H.F. Askal, J . Pharm. Biomed. Anal., 9(3), 219-224 (1991)
9
40.
S.A. Hussein, A.M.I. Mohamed and A.A.M. Abdel-Alim, Analyst (London), 114(9), 1129-1131 (1989).
41.
S.P. Agarwal and M.I. Walash, Indian J. Pharm., 34(5), 109-111 (1972).
42.
L.O. Guerello and J. Dobrecky, Rev. Asoc. Bioquim. Argent., 33(178-179). 185-188 (1968).
43.
P.L. Buldini, V. Rossetti and A. Toponi, Freaenius Z. Anal. Chem., 328, 265-267 (1987).
44.
B.J. Starkey, G.P. Mould and J.D. Teale. J. Liq. Chromatogr., 12(10), 1889-1896 (1989).
45.
J.A.F. Wickramasinghe and S.R. Shaw, J. Pharm. Sci., 60, 1669-1672 (1971).
VINCRISTINE SULFATE (SUPPLEMENT)
Farid J . Muhtadi and Abdul Fattah A. A. Afify
Department of Pharmacognosy College of Pharmacy King Saud University Riyadh, Saudi Arabia
ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 22
517
Copyright 0 1993 by Academic Press, Inc All rights of reproduction in any form reserved.
FARID J. MUHTADI AND ABDUL FATTAH A. A. AFIFY
518
VI.NCRISJINE SULFATE Contents Foreword
1. Description. 1.1 1.2 1.3 1.4 1.5 1.6 1.7
Nomenclature Empirical Formulae Molecular weight Structure Elemental Composition CAS Registry Number Appearance, Color and Odor
2. Physical Properties 2.1 Melting Range 2.2 Solubilit 2.3 S ecific ptical Activity 2.4 D Range 2.5 Loss on-Drying 2.6 Dissociatiori Constant 2.7 X-Ray Crystal Structure 2.8 Spectral Properties
L
2.8.1 2.8.2 2.8.3 2.8.4
L
Ultraviolet Spectrum Infrared Spectrum 1 H-NMR Spectrum Mass Spectrum
3. Isolation of Vincristine
4. Total Synthesis of Vincristine 4.1 Total Synthesis of Vinblastine 4.2 Total Synthesis of Vincristine
5. Biosynthesis of Vincristine
6. Pharmacokinetic 6.1 Drug Absorption 6.2 Drug Distribution 6.3 Metabolism 6.4 Drug Excretion 6.5 Half-Life 7. Preparation and Preservation 8. Uses of Vincristine Sulfate
9. Methods of Analysis
VINCRlSTlNE SULFATE (SUPPLEMENT)
9.1 9.2 9.3 9.4
Identification Tests Titrimetric Determinations Voltametric Determination Spectrophotometric Determinations 9.4.1 U V Spectrophotometry 9.4.2 Colorimetric Determination
9.5 Chromatographic Methods 9.5.1 Thin Layer Chromatography 9.5.2 Gas Liquid Chromatography 9.5.3 High Performance Liquid Chromatography 9.6 Radioimmunoassay Methods 9.7 Enzyme-linked lmmunosorbent Assay Acknowledgement References
519
FARID J. MUHTADI AND ABDUL FATTAH A. A. AFIFY
520
Foreword Vincristine or leurocristine is an indole alkaloid obtained along with vinblastine from the common periwinkle, Catharanthus roseus G. Don., F a m i l y Apocynaceae. This plant was previously known as rrinca roseae L . Vincristine is one of the cytotoxic drugs and used in the treatment of acute leukemias particularly in children and in other cases such as lymphosarcoma, reticulum sarcoma, neuroblastoma, Wilms tumor and tumors of the brain, breast and lung (1). It is used as the sulfate salt which is also formulated as IV vials. 1. DescriDtion 1.1 Nomenclature Vincristine; Leurocristine; 22-0x0-vincaleukoblastine; VCR; LCR. (The Base). - Vincristine sulfate; Leurocristine sulfate (1 :1) (salt); Vincaleukoblastine, 22-0x0-sulfate (1:l) (salt) ; Oncovin; Kyocristine (The sulfate salt).
-
1.2 Empirical Formulae C46H56N40 10 (Vincristine), C46H56N4010, H2S04 (Vincristine sulfate) . 1.3 Molecular Weight 824.97 923.05
(Vincristine) . (Vincristine sulfate).
1.4 Structure The following is the absolute configuration of vincristine ( 2 ) . The absolute stereochemistry of vincristine has been deduced by X-ray crystallographic analysis of the methiodide salt ( 3 ) . The absolute stereochemistry of vincristine was in complete agreement with that deduced forvindoline (4,s) and velbanamine half [X-ray crystallography of cleavamine methiodide (6)]. However, the confirmations of the nine member rings are considerably different in vincristine and cleavamine methi.odides owing to the attachment of the bond (10-16') joining the two parts of the dimeric molecule ( 7 ) .
VINCRISTINE SULFATE (SUPPLEMENT)
W f i N > o ;
\\\
HFOOC
\
52 1
\
H
\
\
\ \
\ \
\ \
H,CO
1.5
Elemental Composition
C, 66.97%; H, 6.84%; N, 6.79%; 0, 19.40% (vincristine). 59.86%; H, 6.33%; N , 6.07%; S, 3.47%; 0, 24.27% (vincristine sulfate.
C,
1.6 CAS Registry Number [57-22-71 [2068-78-21
vincristine. vincristine sulfate.
1.7 Appearance, Color and Odor The base occurs as blades from methanol (8); odorless. A white to slightly yellow crystalline or amorphous powder; odorless, very hygroscopic (1,9) or crystals from ethanol (8) (The sulfate salt). 2.
Physical Properties 2.1
Melting Range
Vincristine melts at 218-220' (8). Vincristine sulfate melts at 273-281' ( 9 ) .
FARID I. MUHTADI AND ABDUL FATTAH A. A. AFIFY
522
2.2
Solubility
V i n c r i s t i n e p r a c t i c a l l y insoluble i n water; soluble i n a l c o h o l s and chloroform. One p a r t o f v i n c r i s t i n e s u l f a t e i s s o l u b l e i n 2 p a r t s o f water; i n 30 p a r t s of chloroform; s l i g h t l y s o l u b l e i n e t h a n o l (96%); p r a c t i c a l l y i n s o l u b l e i n e t h e r ( 9 ) . 2.3
Specific Optical Rotation
[,ID2' + 26.2' ( e t h y l e n e c h l o r i d e ) ; Both f o r v i n c r i s t i n e ( 8 ) .
+ 8.5' 2.4
+ 17"
(c=0.8) f o r t h e s u l f a t e ( 9 ) .
pH Range - (The s u l f a t e s a l t )
pH of a 0.1% w/v s o l u t i o n i s 3.5 t o 5.0 ( 9 ) . Between 3 . 5 and 4 . 5 i n a s o l u t i o n 1 i n 1000 ( 1 0 ) . 2.5
Loss on Drying
When v i n c r i s t i n e s u l f a t e i s d r i e d a t 40' a t a p r e s s u r e not exceeding 0 . 7 kPa f o r 16 h o u r s , l o s e s n o t more t h a n 12.0% of i t s weight ( 9 ) . 2.6
D i s s o c i a t i o n Constant
pKa 5.0, 7.4- ( i n 33% DMF) ( 8 ) . 2.7
X-Ray C r y s t a l S t r u c t u r e
X-Ray d i f f r a c t i o n s t u d y of s i n g l e c r y s t a l s of v i n c r i s t i n e methiodide has been determined by t h e combination of two c r y s t a l l o g r a p h i c methods based on t h e anomalous s c a t t e r i n g of X-rays ( 3 ) : C r y s t a l s of v i n c r i s t i n e methiodide d i h y d r a t e , (C47H59010N4) + I - . 2H20, a r e monoclinic i n s p a c e group P2, w i t h two molec u l e s i n t h e u n i t c e l l , which h a s p a r a m e t e r s a=10.96+0.05, b=21.89+0.05, c=12.68?0.01 A' and !3=124' 53'+10'. This s t u d y has e s t a b l i s h e d t h e a b s o l u t e s t e r e o c h e m i s t r y and t h e complete molecular s t r u c t u r e of v i n c r i s t i n e and therefore of vinblastine.
s23
VINCRISTINE SULFATE (SUPPLEMENT)
2.8
Spectral Properties U l t r a v i o l e t SDectrum fUV1
2.8.1
The UV absorbance spectrum of v i n c r i s t i n e s u l f a t e i n methanol was scanned from 200 t o 400 nm u s i n g a PyeUnicum SP 8-100 Spectrophotometer. The spectrum i s shown i n F i g u r e 1. V i n c r i s t i n e s u l f a t e e x h i b i t e d t h e f o l l o w i n g a b s o r p t i v i t y v a l u e s (Table 1 ) . T a b l e 1 : UV A b s o r p t i v i t y Values
X max. nm
log
218
4.72
568.75
252
4.24
188.75
285
4.18
165.60
293
4.23
185.6
E
A(1%, lcm)
The UV d a t a of v i n c r i s t i n e s u l f a t e have a l s o been r e p o r t e d ( 11- 1 4 )
.
2.8.2
I n f r a r e d Spectrum (IR)
The IR a b s o r p t i o n spectrum of v i n c r i s t i n e s u l f a t e a s a K B r d i s c (1%) was recorded o v e r a Pye-Unicum SP 3 300 I n f r a r e d Spectrophotometer. The spectrum i s p r e sented i n Figure 2. Assignment of t h e f u n c t i o n a l groups have been c o r r e l a t e d with t h e f o l l o w i n g f r e q u e n c i e s (Table 2 ) . Table 2 : I R C h a r a c t e r i s t i c s o f v i n c r i s t i n e Frequency Cm-I 3450 2920 1725 168.0 1225
F u n c t i o n a l Group F r e e OH -CH s t r e t c h E s t e r C=O (acetoxy) Lactam C=O
c-0-c
524
FARID J. MUHTADI AND ABDUL FA'ITAH A. A. AFIFY
-
200 '250 300 350
400
FIGURE 1 : UV SPECTRUM OF VINCRISTINE.
k
i
3 3
W
-
0 0 0
0 0 -T c
0 0
2
m
0 0
N
0 Lo
0
m
FARID J. MUHTADI AND ABDUL FATTAH A. A. AFIFY
526
The I R o f v i n c r i s t i n e e x h i b i t e d t h e f o l l o w i n g o t h e r peaks : 1365, 1280, 1150, 1065, 1035, 885, 780, 745, 1230 , 1445 , 1500 cm-l The I R d a t a of v i n c r i s t i n e s u l f a t e have a l s o been r e p o r t e d (12-14)
.
.
2.8.3
Spectrum
'H-NMR
The p r o t o n magnetic resonance spectrum of v i n c r i s t i n e s u l f a t e i s shown i n F i g u r e 3. I t was o b t a i n e d on a Varian XL 200 NMR spectrophotometer €or a s o l u t i o n i n D2O. The p r o t o n chemical s h i f t s a r e p r e s e n t e d i n Table 3 . 1
T a b l e 3 : H-NMR Assignment t o V i n c r i s t i n e Chemical S h i f t 6 (ppm) 7.18 - 7.49 (m)
Assignment
4 H , aromatic p r o t o n s of catharanthine ~ c 9 f , 1 0 f , l l &f 1 2 1 ) H , aromatic proton of
6.66 ( s )
v i n d o l i n e (C,) 6.42 ( s )
.
H, a r o m a t i c p r o t o n of
v i n d o l i n e (C12). 3.913 (s)
3H, methoxy p r o t o n s ( C l l ) .
3.757 ( s )
3H, e s t e r p r o t o n s of c a t h a r a n t h i n e (CI6
3.650 ( s )
3H, e s t e r p r o t o n s of v i n d o l i n e (C16).
2.068 (s)
3H, a c e t a t e p r o t o n s o f v i n d o l i n e (C17).
m = multiplet. s
=
.
singlet.
1 Other H-NMR s p e c t r a f o r v i n c r i s t i n e have been r e p o r t e d ( 1 2 , 15-17).
t+
1
FIGURE 3 :
'H
- NMR SPECTRUM OF
VINCRISTINE.
FARID J. MUHTADI AND ABDUL FATTAH A. A. A F I N
528
2.8.4
Mass Spectrum
High resolution mass spectra of vinblastine and vinblastine hydrazide have been reported (18). This technique has established the correct elemental composition of vinblastine and provided completely independent additional information regarding the point of attachment of the two halves (vindolinevelbanamine). This could be applied to vincristine in view of the known relationship between vinblastine and vincristine. 3.
Isolation of Vincristine Initial methods for the isolation and separation of vincristine from the periwinkle plants ( C a t h a r a n t h u s r o s e u s ) had been described (19, 20, 21) and well documented in several texts including the previous profile of vincristine sulfate (13). Isolation of vinblastine and vincristine from C. r o s e u s continues to receive attention, and several procedure have been reported (mainly in the patent literature) for the isolation and separation of these alkaloids (22-27). Extracts of c. r o s e u s have been found to contain Ndemethylvinblastine and this can be used to prepare vincristine by formylating the alkaloid mixture before separation and purification (28) High performance liquid chromatography is recommended as a rapid, reliable, reproducible and sensitive method for the quantit(3tiveseparation and determination of vinblastine, vincristine and the other dimeric alkaloids. Reten tion times o f these alkaloids differ widely, therefore, good separation are possible (29).
.
Vincristine Sulfate This salt is prepared by addition of aqueous o r ethanolic sulfuric acid to solution of vincristine in ethanol or acetone. The resulting mixture is evaporated under vacuum and the residue is crystallized from absolute ethanol to give crystals of vincristine sulfate (20).
VINCRISTINE SULFATE (SUPPLEMENT)
529
4. Total Synthesis of Vincristine Since vincristine chemically is 22-oxo-vinblastine, it can therefore be prepared from vinblastine by specific N-oxidation. Vinblastine is first synthesized from catharanthine and vindoline followed by conversion into vincristine. 4.1 Total Synthesis of Vinblastine (30-36).
Catharanthine [ 11 underwent N-oxidation with mchloroperbenzoic acid to give catharanthine N-oxide [ 2 ] . This was treated with trifluoroacetic anhydride to afford the trifluoroacetate derivative [ 3 ] . Coupling of [3] with vindoline in methylene chloride at - 50°C gave. the immonium ion [ 4 ] . This was reduced with sodium borohydride to furnish anhydrovinblastine 20 -deoxyvinblastine [ S ] . Substance [ S ] was treated with thallium triacetate followed by borohydride reduction to produce vinblastine [61* This s y n t h e s i s of v i n b l a s t i n e i s presented i n schemeI.
4.2
Synthesis of Vincristine Two methods are available:
- The preferred method is to oxidize the N-methyl group of vinblastine with chromium trioxide in acetone at
low temperature 1-60') to give vincristine directly[7] (scheme I); [3] (scheme 11) (37-38). - The other method involved microbial N-demethylation followed by N-formylation procedure as follows: Vinblastine [ l ] was incubated with the microorganism streptomyces alboqriseolus to afford N-demethylvinblastine [2] ( 39 ) . Substance [ 2 ] was N-formylated to produce vincristine [3]. These syntheses are shown i n scheme I I . A highly efficient and commercially important synth-
esis of vinblastine from catharanthine and vindoline has also been reported (40).
FARID J. MUHTADI AND ABDUL F A l T A H A . A. A F I N
530
Scheme I : Synthesis of Vinblastine -
4!
vindol ine +
coupling
red.
VlNCRlSTINE SULFATE (SUPPLEMENT)
53 I
i) TL(OAC)~ -
i i ) NaBH4
[51 R = vindoline
A
Oxid.
/
/
--
CO 2 C H ~
OH H3C0 H 3co
OHC OHC
II
C02CH3
[71
-,
FARID J. MUHTADI AND ABDUL FATTAH A. A. AFIFY
532
Scheme 11. Synthesis of V i n c r i s t i n e
It
II
I
Microbial
u'
Oxidation cr20,/acetone~ ~
Vinblast'.le
[I1
Vincristine [31
VINCRISTINE SULFATE (SUPPLEMENT)
533
5. Biosynthesis of Vincristine
It is now established that the monomeric alkhloids vindoline and zatharanthine are precursors of the dimeric alkaloids, both vinblastine and vincristine (41,42). Thus when radioactive vindoline [l] and labeled cathananthine [2] were fed into 6-week old differentiated C a t h a r a n t h u s r o s e u s plants, labeled anhydrovinblastine [3] has been isolated (42).
/ /
vindoline
[31
Administration of labelled [ 3 ] to cell-free extracts of C. r o s e u s afforded radioactive vinblastine [4] (43,44). It has also been shown that when anhydro [Ar-jH] vinblastine ( [Ar-3H]- 3 ) was incubated at room temperature in solutions of the cell-free extracts from C. r o s e u s leaves at pH 6.3, both radioactive vinblastine [4] ( [Ar-3H]-4) and vincristine [5] ([AI--~H]-S)were obtained after 3 and 50 hours respectively (44). Later it was found that anhydrovinblastine [3] can be converted into vinblastine [4] by cell-free homogenates of C. r o s e u s cell suspension cultures (45).
534
FARID J. MUHTADI AND ABDUL FATTAH A. A. AFIFY
[31 Cell-free extracts leaves (7. roseus
I
or Cell-free homogenates of C. roseus cell suspension cultures
COOCH3
H3C0 COOCH3
535
VINCRISTINE SULFATE (SUPPLEMENT)
Further studies with cell-free systems from
Catharanthus
roseus plants have shown that the enzyme catalyzed synthesis of vindoline [l] from tryptamine [ 6 ] and secologanin [7l (44,461.
m $ $ & YToGlu ’ 4 I
CHO
,4
Enzymes from
r
C . roseus
OCH3
CH3
:-i COOC H’
! -OH
C02CH3
3
In another study, catharanthine [ 2] and vindoline [l] are utilized by these enzymes systems and coupled into anhydrovinblastine [ 3 ] which is, in turn transformed into vinblastine [ 4 ] , vincristine [S] as well as into the dimeric alkaloids leurosine and Catharine ( 4 6 ) .
T
%]
OCH3
“3
[ 11
’ OH C02CH3
1L
-
-free extract
J
C . roseus
leurosine Catharine
~4114 vindoline [ c ] /
[31
[51
FARID J. MUHTADI AND ABDUL FATTAH A. A. AFIFY
536
6. Pharmacokinetics
6.1 Drug Absorption Vincristine is not reliably absorbed from the gastrointestinal tract thereby requiring intravenous (IV) administration (47). Vincristine should not be administered intrathecally Intrathecal administration with vincristine that is intended for IV use is uniformly fetal ( 48 ) . It is readily absorbed after fV administration.
.
6.2
Drug Distribution Vincristine is rapidly distributed within 15 to 30 minutes after IV administration., greater than 90% distributes from the blood to the tissues where it is tightly bound (49). Vincristine presumably penetrates the blood brain barrier poorly and does not appear in the cerebrospinal fluid (CSF) in therapeutic concentrations (49). Volume of distribution (VD) : vincristine shows considerable VD variation values, little difference between VD values among patients with impaired hepatic function and normals : 1635131 and 165+105 liters/ square meter respectively ( 5 0 ) . Another study reported a mean steady state VD for vincristine of 167.6 liters/1.73 square meter of body surface area ( 51 ) . A third study indicated a VD of 8.4 L/Kg for vincristine ( 5 2 ) . Utilizing a 3 compartment model, and a radioimmunoassay t o determine pharmacokinetic paranmeters following the administration of vincristine to children ages 5 to 16 years of age, a mean steady state VD of 215.9 Kiters/l.73 square meter of body surface area was reported (53)
6.3 Metabolism Vincristine is extensively metabolized in the liver. Blood concentration : after an IV dose of 2 mg, plasma concentrations of about 100 to 400 ng/ml are obtained after 5 minutes, falling to less than 40 ng/ml by 10 minutes (1). 6.4
Drug Excretion Vincristine is primarily excreted via the bile and
VINCRISTINE SULFATE (SUPPLEMENT)
531
f e c e s , about 10 t o 20% of a dose i s e x c r e t e d i n t h e urine (49,52). About 1 2 2 of a dose i s e x c r e t e d i n t h e u r i n e i n 72 h o u r s and 70% i s e l i m i n a t e d i n t h e f e c e s i n t h e same period ( 1 4 ) . Following t h e a d m i n i s t r a t i o n of v i n c r i s t i n e , 10% of t h e dose h a s been r e p o r t e d t o a p p e a r i n t h e u r i n e w i t h 24 h o u r s , w i t h a c u m u l a t i v e t o t a l of 13%a f t e r 72 h o u r s ( 5 2 ) . A f t e r a d m i n i s t r a t i o n of v i n c r i s t i n e t o c h i l d r e n 5 t o 16 y e a r s of age, u t i l i z i n g a radioimmunoassay, i t was found t h a t w i t h i n 90 h o u r s , 37% of t h e dose h a s been e x c r e t e d i n t h e u r i n e e i t h e r as v i n c r i s t i n e o r i t s metabolites (53). 6.5
Half-Life V i n c r i s t i n e showed c o n s i d e r a b l e v a r i a t i o n i n t h e l e n gth of h a l f - l i f e : A mean gamma t e r m i n a l h a l f - l i f e of 22.6 h o u r s h a s been o b t a i n e d by 3-compartment model ( 5 1 ) . Beta t e r m i n a l h a l f - l i v e s o f 5.1k3.5 and 13.0f10.8 h o u r s f o r normal and f o r p a t i e n t s w i t h compromised l i v e r f u n c t i o n were r e p o r t e d by u s i n g 2-compartment model ( 5 0 ) . A gamma t e r m i n a l h a l f - l i f e of 85 h o u r s h a s r e c e n t l y been r e p o r t e d ( 5 2 ) . Serum h a l f - l i f e , about 75 minutes ( 1 ) . Plasma h a l f - l i f e , about 3 h o u r s , and a t e r m i n a l e l i m i n a t i o n h a l f - l i f e o f about 23 h o u r s h a s a l s o been r e p o rted (14). Following t h e a d m i n i s t r a t i o n of v i n c r i s t i n e t o c h i l d r e n a g e s 5 t o 15 y e a r s of a g e , a mean gamma t e r m i n a l h a l f - l i f e o f 25.5 h o u r s was o b t a i n e d ( 5 3 ) .
7.
Preparation 6 Preservation V i n c r i s t i n e s u l f a t e should b e s t o r e d i n a i r t i g h t c o n t a i n e r s , a t a t e m p e r a t u r e between 2' and l o " , p r o t e c t e d from light (1). V i n c r i s t i n e s u l f a t e i s administered intravenously. V i n c r i s t i n e i n j e c t i o n i s a s t e r i l e s o l u t i o n of v i n c r i s t i n e s u l f a t e and l a c t o s e i n t h e p r o p o r t i o n of 1 p a r t and 10 p a r t s r e s p e c t i v e l y i n water f o r i n j e c t i o n . I t i s prepared by d i s s o l v i n g t h e c o n t e n t s of a s e a l e d c o n t a i n e r , which include the lactose i n water f o r i n j e c t i o n s h o r t l y before use ( 1 ) . A v a i l a b l e a s a powder i n ampoules c o n t a i n i n g 1 mg v i n c r i s t i n e s u l f a t e and 10 mg o f l a c t o s e and i n ampoules c o n t a i n -
538
FARID J. MUHTADI AND ABDUL F A T A H A. A. AFIFY
i n g 5 mg v i n c r i s t i n e s u l f a t e and 50 mg of l a c t o s e . The s e a l e d c o n t a i n e r s h o u l d be s t o r e d i n a r e f r i g e r a t o r a t a t e m p e r a t u r e between 2' and l o o , p r o t e c t e d from l i g h t ( 1 ) . The i n j e c t i o n c o n t a i n s no b a c t e r i o c i d e , should be used a s soon a s p o s s i b l e a f t e r p r e p a r a t i o n , and i n any c a s e w i t h i n 24 hours ( li ) . In t h e p r e s e n c e o f a s u i t a b l e b a c t e r i o c i d e such a s benzyl a l c o h o l 0.9%, i t may be used f o r up t o 14 days when s t o r e d a t 2' t o 10' ( 1 ) . 8.
Uses of V i n c r i s t i n e S u l f a t e V i n c r i s t i n e i s an a n t i n e o p l a s t i c drug which may a c t s i m i l a r l y t o v i r i b l a s t i n e by a r r e s t i n g m i t o s i s a t t h e metaphase. I t a l s o h a s some immunosuppressant a c t i v i t y . I t i s used p r i n c i p l y i n combination chemotherapy regiments f o r a c u t e leukemia and Hodgkin's d i s e a s e and o t h e r lymphomas, i n c l u d i n g B u r k i t t ' s lymphoma. I t i s a l s o used i n t h e t r e a t m e n t o f W i l m ' s tumor, neuroblastoma, sarcomas and i n tumors o f t h e b r e a s t , b r a i n and lung ( 4 7 ) . V i n c r i s t i n e used t o g e t h e r w i t h c o r t i c o s t e r o i d s i s p r e s e n t l y t h e t r e a t m e n t of c h o i c e t o induce r e m i s s i o n s i n childhood leukemia ( 5 4 ) . Remissions a r e i n c l u d e d i n a c u t e lymphoblastic leukemia with v i n c r i s t i n e i n a s s o c i a t i o n w i t h p r e d n i s o n e a l o n e o r with d a u n o r u b i c i n ( o r doxorubicin) o r c o l a s p a s e . In Hodgkin's d i s e a s e , v i n c r i s t i n e i s given w i t h mustine, p r o c o r b a z i n e and p r e d n i s o n e ( 4 7 ) . V i n c r i s t i n e s u l f a t e 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 a t weekly i n t e r v a l s . For c h i l d r e n with leukemia t h e commencing weekly dose i s 50 pg/kilogram body-weight w i t h weekly i n c r ements of 25 pg p e r Kg, u n t i l a maximum o f 150 micrograms p e r kilogram i s reached. A f t e r r e m i s s i o n has o c c u r r e d t h e dosage i s reduced t o a maintenance l e v e l of 50 t o 75 micrograms p e r kilogram weekly. Fo:r a d u l t leukemia t h e weekly dose i s from 25 t o 75 micrograms p e r kilogram body-weight. For o t h e r malignant c o n d i t i o n s , t h e dose i s s m a l l e r , o f t h e o r d e r of 25 microgram p e r kilogram body-weight weekly u n t i l a r e m i s s i o n o c c u r s . The dose i s t h e n reduced t o 5 t o 10 micrograms p e r kilogram f o r a s long a s any a n t i tumor e f f e c t b:an be o b t a i n e d ( 1 ) . Neuromuscular t o x i c i t y may o c c u r , t h e r e f o r e r e p u a r examination of deep tendon r e f l e x e s should b e c a r r i e d o u t and t h e dose reduced i f t h e y are impaired. White-cel coun t should a l s o be made. Dosage should be reduced o r empo r a r i l y omitted i f i n f e c t i o n i s present (1).
VINCRISTINE SULFATE (SUPPLEMENT)
9.
539
Methods of Analysis 9.1
Identification Tests
The following i d e n t i f i c a t i o n t e s t s are mentioned i n the BP (9).
To lmg Vincristine sulfate add 0.2 ml of a freshly prepared 1% w/v solution of vanillin in hydrochloric acid. An orange color is produced in about 1 minute (distinction from vinblastine sulfate). Mix 0.5 mg vincristine sulfate with 5 mg of 4-dimethylaminobenzaldehyde and 0.2 ml of glacial acetic acid and add 0.2 ml of sulfuric acid; a reddish-brown color is produced. Add 1 ml of glacial acetic acid; the color changes to pink. T h e following i d e n t i f i c a t i o n t e s t s are mentioned i n the
usp (10) The infrared absorption spectrum o f a potassium bromide dispersion of vincristine sulfate, previously dried in vacuum at 40' for 16 hours, exhibits maxima only at the same wavelengths as that of a similar preparation of USP vincristine sulfate RS. Other i d e n t i f i c a t i o n t e s t s (55 1 :
With 1% ceric ammonium sulfate in 85% phosphoric acid, vincristine gives a bluish violet color. With 1% ferric ammonium sulfate in 75% sulfuric acid, it produces a blue color which changes to gray-blue. With 1% ferric ammonium sulfate in 85% phosphoric acid, it gives a pink color after heating on a water bath for 10 minutes. 9.2 Titrimetric Determinations Non-Aqueous Titrations Quantitative determinations of the alkaloids including vincristine in drugs and extracts are reported as follows (56). Aerial parts of Vinca rosea were ground, treated with 25% NH40H for 30 minutes, and extracted with MeOH. The extract was evaporated and the residue was dissolved in 2% H2SO4 on a water bath. The H2SO4 extract was alkalinized with NH40H, reextracted with CHC13, the extract was dried, and evaporated. The residue was dissolved in HOAC and titrated with 0.1N HClO4. Aerial parts of vinca minor were treated with 25% NH40H € o r 30 minutes, extracted with CHC13, the concentrated extract was reextracted with 2% tartaric acid adjusted to
540
FARID J. MUHTADI AND ABDUL FATTAH A. A. AFIFY
pH 9.0 with 25% NHdOH, and alkaloids were extracted with CHC13, dissolved in HOAc and titrated with HClO4 by using crystal violet indicator. 9.3 Voltametric Determination Ultrasensitive voltametric measurements based on coupling hydrogen catalytic systems with controlled interfacial accumulation of the catalyst has been reported (57). By combining adsorptive stripping voltammetry with a catalytic hydrogen process, trace Pt can be measured with a detection limit of -1X10-12M. The supporting electrolyte (1 -08 M H2S04) contains 0.04% (wt ./vol .) formaldehyde and 0.001% (wt./vol.) hydrazine. The adsorption of the Ptformazone complex (the catalyst) results in a well-defined catalytic H peak at - 0.92 V, with a peak half-width of 60 mV. The peak height increases rapidly with increasing preconcentra.tion time, indicating a large enhancement of the complex on the surface of the hanging Hg drop electrode (SDS) and gelatin causes serious interference by competing with the catalyst on adsorption sites. By using 0.25 M ammoniacal buffer (pH 9.3), the vinca alkaloids vincristine and vinblastine can be* determined at subnanomol levels (57). 9.4 Spectrcphotometric Determination 9.4.1 UV Spectrophotometry The BP ( 9 ) recommends the following procedure for the assay of vincristine sulfate: Dissolve 10 mg of vincristine sulfate in sufficient methanol to produce 500 ml and measure the absorbance of the resulting solution at the maximum at 297 nm. Calculate the content of C46H56N4010. H2SO4 taking 177 as the value of A(l%, 1 cm) at the maximum at 297 nm. The former USP ( 58 ) described the following procedure to assay vincristine sulfate: Dissolve about 5 mg of vincristine sulfate, accurately weighed in methanol and dilute quantitatively and stepwise with methanol t o obtain a solution containing about 20 pg of anhydrous vincristine sulfate per ml. Dissolve an accurately weighed quantity of USP Vincristine sulfate Reference standard previously dried in vacuum at 40' for 16 hours in methanol and dilute quantitatively and stepwise with methanol to obtain a Standard solution having a known concentration of about 20 pg per ml. Concomitantly determine thie absorbances of both solutions in 1-cm cells
VlNCRlSTlNE SULFATE (SUPPLEMENT)
54 I
at the wavelength of maximum absorbance at about 297 nm, with a suitable spectrophotometer, using methanol as the blank. Calculate the quantity in mg of CqgH56N4010. H2SO4 in the portion of Vincristine sulfate taken by the formula 0.25C (Au/As), in which C is the concentration in pg per ml of USP vincristine sulfate Reference standard in the standard solution and Au and As are the absorbance5 of the solution of vincristine sulfate and the standard solution respecti vely. 9.4.2
Colorimetric Determination
The colorimetric method which was described to assay vinblastine sulfate can be also used to determine vincristine sulfate (55,59). The method depends on the formation of a deep rose color upon heating vincristine sulfate at 80' with a reagent consisting of pyridine (35 ml), concentrated sulfuric acid (1 ml) and acetic anhydride (35 ml) containing 0.05% acetyl chloride. The color so produced is measured at 574 nm. 9.5 Chromatographic Methods
9.5.1
Thin Layer Chromatography (TLC)
The following TLC systems were recommended for the identification and separation of vincristine. Chromatogram 1.
Silica gel HF 254
2.
0.5
Solvent System
Rf value
Toluene-chloroformdiethylamine (80:40:6)
N LiOH/alumina Absolute alcohol-
0.51
acetonit ri1e (5 :95) 3.
Silica gel G
Methanol
4.
Silica gel G
Chloroform-methanol (95 :5)
Silica gel G
Ethy 1acetate- ab solute alcohol ( 3 : l )
0.18
6. Silica gel G
n-Butanol-glacial acetic acid-water (4:1 :1)
0.16
5.
Ref.
(55)
FARID I. MUHTADI AND ABDUL FATTAH A. A. AFIFY
542
A double development TLC t e c h n i q u e on alumina l a y e r s was
recommended f o r t h e s e p a r a t i o n of v i n c r i s t i n e from t h e o t h e r d i m e r i c c a t h a r a n t h u s a l k a l o i d s (11). The loaded chromatograms were f i r s t developed i n e t h y l a c e t a t e , t h e n a f t e r d r y i n g , a second run was performed i n ethylacetate-absolute alcohol ( 3 : l ) . Vincristine e x h i b i t e d R f v a l u e of 0.54 i n t h i s t e c h n i q u e ( 1 1 ) . A two dimensional TLC t e c h n i q u e was 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 more complex m i x t u r e s of Catharanthus alkaloids (62). S p o t s on TLC can be d e t e c t e d by one o r more of t h e followings:1. Under s h o r t U V l i g h t (254 nm) 2 . Spraying t h e c h r o m a t o p l a t e s w i t h e i t h e r : a- Dragendorff’s reagent ( 6 3 ) . b- A c i d i f i e d i o d o p l a t i n a t e r e a g e n t ( 6 3 ) . c - 1%Ceric ammonium s u l f a t e i n 85% phosphoric a c i d (55) * The BP (9) adopted a TLC t e c h n i q u e t o t e s t f o r t h e p r e s ence of r e l a t e d a l k a l o i d s i n v i n c r i s t i n e s u l f a t e sample (checking t h e p u r i t y o f t h e sample) as f o l l o w s : TLC c h r o m a t o p l a t e s a r e c o a t e d w i t h s i l i c a g e l HF254. 5 ~ 1 of each of t h e f o l l o w i n g t h r e e s o l u t i o n s i n methanol a r e s e p a r a t e l y a p p l i e d t o one chromatoplate: 1- 1.0% w/v of t h e s u b s t a n c e b e i n g examined. 2- 0.02% W / V of s t a n d a r d v i n b l a s t i n e s u l f a t e BPCRS. 3- 1.0% w/v of s t a n d a r d v i n c r i s t i n e s u l f a t e BPCRS. The chromatogram i s t h e n developed i n t h e s o l v e n t t o l u e n e chloroform-diethylamine (80:40:6). A f t e r development, t h e p l a t e i s allowed t o d r y i n a i r and examined under UV l i g h t (254 nm) Any secondary s p o t i n t h e chromatogram o b t a i n e d with solut i o n (1) i s n o t more i n t e n s e t h a n t h e s p o t o b t a i n e d w i t h solution (2).
.
.
Two TLC methods € o r t h e d e t e r m i n a t i o n o f v i n c r i s t i n e were r e p o r t e d ( 6 4 ) . In t h e first method, two dimensional TLC was performed followed by d e n s i t o m e t r i c scanning o f t h e s p o t s a t 289 nm. The second method involved one dimensional TLC followed by e l u t i o n of t h e s p o t s , f u r t h e r TLC of t h e r e s u l t i n g s o l u t i o n and e l u t i o n of t h e s p o t s and f i n a l l y spectrophotomet r i c measurement a t 289 nm. A s t a n d a r d s o l u t i o n of v i n c r i s t i n e was run i n each i n s t a n c e . The c o e f f i c i e n t o f v a r i a t i o n o f t h e d e n s i t o m e t r i c and s p o t e l u t i o n p r o c e d u r e s were 7.5 and 8.2% r e s p e c t i v e l y ( 6 4 ) .
VINCRISTINE SULFATE (SUPPLEMENT)
9.5.2
543
Gas Liquid Chromatography
The following system has been reported for the identification of vincristine as well as other vinca alkaloids ( 65 1: Column condition: Glass column (lm x 3.2 mm) precoated with hexamethyldisilazane and packed with 3% OV-101 on Gas-Chrom Q (80-100 mesh), with temperature programming from 200' to 300' at 5' min.-l. Carrier gas:Nitrogen, at a flow rate 30 ml/min.-l Detector:
FID.
Condition:
Vinca alkaloids including vincristine were derivat ized before application by heating f o r 5 minutes at room temperature with trif luorob i s - (trimethylsilyl) acetamide-pyridine (1:l).
9.5.3
High Performance Liquid Chromatography (HPLC)
Several HPLC methods have been employed to determine vincristine and its metabolites in biological fluids and tissues. Some of these methods are as follows: The following system has been recommended €or quantitative determination of vincristine and other vinca alkaloids in plasma and urine (66). Conditions: The drugs are extracted from biological materials by an ion pair extraction with sodium octylsulfate as counter ion at pH 3.0. The extracts are injected onto a reversed-phase system with a cyano column as stationary phase. Mobile Me CN-phosphate buffer pH 3 . 0 (65:35). phase : The following system is a reversed-phase with System 2 : electrochemical detection. It is employed for quantitative determination of vinca alkaloids including vincristine in plasma and urine. Quantification of substances in human plasma and urine is possible down to lng/ml (67) * Column : Hypersil ODs. Mobile Methanol - 10m M phosphate buffer pH 7.0. phase: System 1:
FARID J. MUHTADI AND ABDUL FATTAH A. A. AFIFY
544
System 3 : Column: Mobi 1e phase :
T h i s system i s employed f o r t h e a n a l y s i s of Catharanthus a l k a l o i d s i n c l u d i n g v i n c r i s t i n e I: 29 ) . k s t a i n l e s s s t e e l (25cm x 4mm), packed w i t h Li-Chrosorb RP-8; o p e r a t e d a t ambient temperat:ure. 0.01M ammonium c a r b o n a t e - a c e t o n i t r i l e (53 :47)
.
Flow r a t e : l . 5 m l min. Retention time :
-1
7.22 minutes f o r v i n c r i s t i n e .
D e t e c t i o n : U V a t 298 nm. System 4:
T h i s system has been employed f o r t h e s e p a r a t i o n , d e t e c t i o n and c o r r e l a t i o n of p l a t e h e i g h t and m o l e c u l a r weight of v i n c r i s t i n e and o t h e r Vinca a l k a l o i d s ( 68 1. Column: 25cm x 4.6mm, packed w i t h R S i l C18 HL-D ectadecyl-silica gel. Mobile Gradient e l u t i o n with aqueous 50 t o 85%methanol phase : c o n t a i n i n g 0.1% ethanolamine. -1 Flow r a t e : 2 m l min. D e t e c t i o n : UV a t 290 nm. System 5 :
The f o l l o w i n g reversed-phase system h a s been used f o r t h e a n a l y s i s of C a t h a r a n t h u s a l k a l o i d s i n c l u d i n g v i n c r i s t i n e by thermospray l i q u i d chromatography-mass s p e c t r o m e t r y (69) Column: p Bondapak C18 (30cm x 3.9mm), r e v e r s e d - p h a s e column. Mobile I s o c r a t i c s o l v e n t , 0.1M ammonium a c e t a t e (pH 7.2) - MeCN (51:49). phase : -1 Flow r a t e : 1 m l min.
.
D e t e c t i o n : E l e c t r o c h e m i c a l and UV (The l i m i t o f d e t e c t i o n b e i n g 4 n g / i n j e c t i o n € o r each a l k a l o i d ) . System 6 :
Column:
The f o l l o w i n g r e v e r s e d - p h a s e system has 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 n c r i s t i n e and o t h e r a l k a l o i d s of Catharanthus r o s e u s leaves ( 7 0 ) . p Bondapak C18.
Mobile phase :
0.01M diammonium hydrogen orthophosphate - MeCN (25:75), pH 7.0.
D e t e c t i o n : UV a t 2 5 4 and 280 nm.
VINCRISTINE SULFATE (SUPPLEMENT)
System 7 :
Column: Mobile phase : I nt erna 1 standard :
545
T h i s r e v e r s e d - p h a s e system i s d e s c r i b e d f o r t h e d e t e r m i n a t i o n of v i n c r i s t i n e and o t h e r C a t h a r a n t h u s a l k a l o i d s . 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 , t h e l i m i t of d e t e c t i o n and t h e r e c o v e r y were 1.63-3.52%, 10 ug/ml and 96.6-102% r e s p e c t i v e l y ( 7 1 ) . A c a r t r i d g e column packed w i t h Spheri-SRP. 2 g r a d i e n t systems c o n t a i n i n g MeOH, Me C N , 0.025M ammonium a c e t a t e and t r i e t h y l a m i n e i n different ratios. 5 -Met hoxy t r y p t amin e .
D e t e c t i o n : UV a t 280 and 254 nm. System 8 :
Column: Mobile phase :
T h i s i s a l s o a r e v e r s e d - p h a s e system which h a s been a p p l i e d € o r s e p a r a t i o n and q u a n t i t a t i o n o f a l k a l o i d s from c e l l s u s p e n s i o n c u l t u r e s of Catharanthus roseus including v i n c r i s t i n e ( 7 2 ) u Bondapak c18. A m i x t u r e of methanol and (NH4)2HP04 i n w a t e r .
D e t e c t i o n : UV a t 298 nm. System 9 : Column:
The f o l l o w i n g system i s a r e v e r s e d - p h a s e system h a s been used t o i d e n t i f y v i n c r i s t i n e ( 7 3 ) . Reversed-phase C 1 8 (RP18), 10 pm (25cm x 4mm).
Mobile phase :
I s o c r a t i c composed of a c e t o n i t r i l e - p h o s p h a t e b u f f e r pH 2.3 (156g : 344g) i n 1 I t aqueous solution. Flow r a t e : 1 ml min.-l 1 . 1 4 (MPH=5 (p-Methylphenyl) -5-phenylhydantoin) Relative retention:
.
D e t e c t i o n : UV The USP ( 10 ) adopted HPLC p r o c e d u r e t o assay v i n c r i s t i n e s u l f a t e and v i n c r i s t i n e s u l f a r e f o r i n j e c t i o n as f o l l o w s : The l i q u i d chromatograph : T h i s i s equipped w i t h a 297 nm d e t e c t o r , a precolumn packed w i t h porous s i l i c a g e l i n s t a l l e d between t h e pump and t h e i n j e c t o r , a guard column (2 t o 5 cm) packed w i t h c h e m i c a l l y bonded o c t a d e c y l s i l a n e and i n s t a l l e d between t h e i n j e c t o r and t h e a n a l y t i c a l column. The a n a l y t i c a l column (25 cm x 4.6 mm) i s packed with chemi c a l l y bonded o c t y l s i l a n e (5 pm p a r t i c l e s ) .
FARID J. MUHTADI A N D ABDUL FATTAH A. A. AFIFY
546
T h e mobile p h a s e : Diethylamine (5 ml) is mixed with 295m1 water and adjusted with phosphoric acid to pH 7 . 5 , ethanol is then added to obtain a volume of 1 L, the solvent is filtered through 0.5 pm filter and degas under vacuum. The mobile phase i s maintained at a pressure and flow rate (about 2 mL per minute) capable of producing the required resolution and a suitable elution time. P r o c e d u r e : As described in USP XXI p. 1116. Other HPLC systems for vincristine have also been reported
(74-76). 9.6
Radioimmunoassay Methods
Radioimmunoassays developed for determining the neoplasm inhibitors ( 77 ) vinblastine ( I ) and vincristine (11) in blood involve use of antiserum raised in a rabbit immunized with ( I ) bovine serum albumin conjugate. Detection limits (ng ml-l) or 2 . 1 for ( I ) and 3 . 8 for (11) with use of tritiated (I) under non-equilibrium assay conditions. The antiserum showed no cross-reactivity with 25 other alkaloids and cytotoxic drugs used therapeutically in combination with (I) and ( 1 1 ) . Another method of Radioimmunoassay for vinca alkaloids vincristine and vinblastine was reported ( 78 ) as follows: Antisera were raised in rabbits by immunisation against compounds prepared by coupling carboxylic acid derivatives of vincristine and vinblastine to human serum albumin. For assay, antiserum was incubated with the sample, e.g. plant extract and the appropriate tritiated alkaloid for 1 h at 37O, and the mixture was allowed to react with a goat anti-rabbit serum or with polyoxyethylene glycol overnight at 4", and then centrifuged; the precipitate was dissolved in NaOH solution f o r scintillation counting. Either compound could be determined in amounts down to < 1 p mol. The specificity of the antisera is discussed; that raised against vincristine bound this alkaloid 200 times more effectively than it bound vinblastine. Several other compounds, including antineoplastic drugs, showed no crossreactivity. The assays were applied to the blood of rabbits injected with the drugs, and also to extracts of vinca rosea after preliminary fractionation by HPLC. A third sensitive radioimunoassay for vincristine and vinblastine was also reported ( 79 ) as follows: Antiserum for use in the method was raised in rabbits against a 4-deacetylvinblastine carboxazide-bovine serum albumin conjugate; [3H] -vincristine or [3H] -vinblastine was used as radio-ligand. Rates of binding of vincristine
VINCRISTINE SULFATE (SUPPLEMENT)
541
and vinblastine to the antiserum were similar. After incubation free and bound ligand were separated with use of a dextran-charcoal suspension; after centrifugation the activity of the supernatant solution was measured by liquid scintillation counting. Sensitivity was improved by a sequential saturation procedure (incubation with unlabelled drug, followed by incubation with radio-ligand). Of the drugs tested only bleomycin (> 0.1 unit) interfered. 9.7 Enzyme-linked Immunosorbent Assay Method An enzyme-linked immunosorbent assay for vincristine was recently developed ( 80 ) : The method is based on a new
procedure for synthesizing the hapten-protein conjugate. In both the immunogen and the enzyme tracer, a spacer group is introduced between the hapten and protein, the vincristine is coupled at a site far from its functional groups. The antibody produced, proved to be exceptionally specific as compared with previous immunoassays €or bis-indole alkaloids. Thousandfold antibody dilutions could be used and samples at femtomole range are assayable. Applications of the method to patient plasma samples and to plant materials are discussed.
ACKNOWLEDGEMENT The authors would like to thank both.Mr. Uday C. Sharma and Mr. Tanvir A. Butt, College of Pharmacy, Riyadh, Saudi Arabia for their valuable and sincere efforts in typing this manuscript.
FARlD J. MUHTADI AND ABDUL FATTAH A. A. AFIFY
548
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POVIDONE
'
Christianah M. Adeyeye and Eugene Barabas'
(1) School of Pharmacy
Duquesne University Pittsburgh, PA 15282 (2) ISP Corporation 1361 Alps Road Wayne, NJ 07470
ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 22
555
Copyright Q 1993 by Academic Press, Inc. All rights of reproducllon in any form reSeNed.
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
556
CONTENTS
1.
Introduction 1 .l Structure 1.2 Nomenclature
2.
The Monomer (N-Vinyl-2-Pyrrolidinone) 2.1 Physical Properties
3. Polymerization 4.
Description of the Polymer Appearance Molecular Weight MoleciJlar Weight Distribution Glass 'Transition Temperature Hygroscopicity Solubility Viscosity Chemi'cal Nature and Stability
4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8
5. Uses of PVP 5.1 Primary Uses 5.11 Pharmaceutical Applications 5.12 Medicinal Applications 5.13 Cosmetic Applications 5.2 Other Applications 5.21 Textile 5.22 Paper 5.23 Adhesive 5.24 Membrane 5.25 Suspending Aid 5.26 Elastomers, Plastics and Rubbers 5.27 Detergents and Soaps 5.28 Ceramics 5.29 Photochemistry 5.210 Radiation Curing 6. Health and Safety 6.1 Oral and Topical Application 6.2 Parenteral Administration 7. Compliance with Pharmacopeias and Food Regulations 7.1 Detection and Identification 7.11 Identification by Spot Tests 7.11 1 With Potassium Dichromate 7.112 With Cobalt Nitrate 7.113 With Iodine 7 1 14 With Dimethylamino Bentaldehyde
POVIDONE
7.12
7.2
7.3
Identification by Spectra 7.121 Infrared 7.122 Proton NMR 7.1 23 C-13 NMR Specifications 7.21 Water Content 7.21 1 Determination of Water 7.21 11 Karl Fischer Method 7.21 21 Toluene Distillation 7.22 pH 7.221 Determination of pH 7.221 1 By pH Meter 7.221 2 By Indicator Paper Test 7.23 Residual Monomer 7.23 1 Determination of Residual Monomer (VP) 7.24 Nitrogen Content 7.241 Determination of Nitrogen 7.25 Ash 7.251 Determination of Ash 7.251 1 Total Ash 7.251 2 Acid-Insoluble Ash 7.251 3 Sulfated Ash 7.26 Heavy Metals 7.261 Determination of Heavy Metals 7.27 Viscosity 7.271 Determination of Viscosity 7.28 Aldehydes 7.281 Determination of Acetaldehyde 7.281 1 By Enzymatic Reaction 7.281 2 With Hydroxylamine Hydrochloride 7.29 Hydrazine 7.291 Determination of Hydrazine Other Characteristics 7.31 Color and Clarity of Solution 7.31 1 Degree of Coloration of Liquids 7.31 11 European Color Test 7.31 12 APHA Color Test 7.31 2 Determination of Clarity 7.32 Peroxide Content 7.321 Determination of Peroxides 7.321 1 With Titanium Sulfate 7.321 2 By lodination 7.33 Arsenic 7.331 Determination of Arsenic 7.34 Loss on Drying 7.341 Determination of Loss on Drying 7.35 Surface Area 7.351 Determination of Surface Area
551
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
558
7.36
Particle Size Distribution 7.361 Determination of Particle Size Distribution 7.361 1 Determination by Mechanical Sieving Device 7.361 2 Determination by Air-Jet Sieve
7.37
Bulk Density 7.371 Determination of Bulk Density 7.38 Flow Properties 7.381 Determination of Flow Properties 7.39 Solubility and Rate of Dissolution 7.391 Determination of Solubility 7.392 Determination of the Rate of Dissolution 7.310 Sediment Content 7.3101 Determination of Sediment Content
8.
7.4
Instrumental Analysis 7.41 Chromatography 7.41 1 Gas Liquid Chromatography 7.41 2 High Performance Liquid Chromatography 7.41 3 Thin Layer Chromatography 7.42 Thermal Analysis 7.421 Differential Scanning Calorimetry and Differential Thermal Analysis 7.422 Thermogravimetric Analysis 7.43 X-Ray Diffraction 7.44 Methods of Instrumental Analysis
7.5
Microbial Limit Tests 7.51 Preparatory Testing 7.52 Preparation of Stock Solution and Media 7.53 Procedure (Total Aerobic Microbial Count) 7.531 Plate Method 7.532 Multiple Tube Method 7.533 Test for Staphylococcus Aureus and Pseudomonas Aeruginosa 7.534 Test for Salmonella and Escherichia Coli 7.535 Total Combined Molds and Yeasts Count 7.536 Retesting for Presence of Microorganisms
Pharmacokinetics 8.1 Absorption 8.1 1 Animal Studies 8.1 2 Human Studies 8.2 Distribution and Storage of PVP in the body 8.21 Distribution 8.22 Storage
POVIDONE
8.3 8.4 8.5
9.
Uptake of PVP into Isolated Tissues Metabolism Excretion 8.51 Animal Studies 8.52 Human Studies 8 . 5 3 Biliary Excretion
Toxicity 9.1 Acute Toxicity 9 . 2 Subchronic Toxicity 9 . 3 Chronic 9 . 4 Local Tissue Damage 9 . 5 Carcinogenicity
10.
Mutagenicity
11.
Cryoprotection
12.
Anticarcinogenicity
13.
Ackowledgements
14.
References
559
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
560
POLYVINYLPY RROLIDONE (POVIDONE)
1. Introduction Polyvinylpyrrolidone or PVP is a highly polar, amphoteric water soluble polymer which has been known since the early 1930s, when it's monomer was first synthesized. It is used in the pharmaceutical field as an excipient le.g. in tablet, ophthalmic, and sustained release dosage forms), in the medical field, cosmetic, food and textile industries. 1.1
Structure
1.2
Nomenclature
Chemical Abstracts Services Registration No.: 9003-39-8 Chemical Abstracts Name: 1-ethenyl-2-pyrrolidinone homopolymer PVP has been known under a variety of names. Some of those have been used as the "approved names" by the regulatory authorities of different countries. The commonly used names include: polyvinylpyrrolidone, povidone, polypovidone, polyvidon, polyvidonum, poly(N-vinyl-2-pyrrolidinone), poly(Nvinylbutyrolactam), polyt 1-vinyl-2-pyrrolidinone), 1-vinyl-2-pyrrolidinone homopolymer, polv[ 1-(2-0xo-l-pyrrolidinyl)ethylenel
POVIDONE
S6 I
PVP - beside being available in technical grades with different specifications is sold in pharmaceutical grade conforming to the requirements of various national and international Pharmacopeias, as well as to the demands of national and international drug and food regulatory authorities. The pharmaceutical grades are marketed under the trade name of Kollidon by BASF and Plasdone by GAF (recently changed to ISP - International Specialty Products) and referred to as Povidone by the United States Pharmacopoeia (USP).
2. Monomer (N-Vinvl-2-Pvrrolidinone1 N-vinyl-2-pyrrolidinone (N-vinylbutyrolactam) was first synthesized in Germany in the early 1930's in the laboratories of Badische Anilin und Soda Fabrik (BASF); commercial production started in the 1940's. The synthesis was part of a program headed by Dr. Walter Julius Reppe aimed at developing a new chemical industry based on the safe use of acetylene. The compound was prepared through a five step synthesis: acetylene was combined with formaldehyde in a catalytic vapor phase reaction to yield 1,4butynediol. This product was hydrogenated t o 1,4-butanedioI with 1,4butenediol as intermediate. The 1,4-butanedioI was oxidized with copper catalyst t o hydroxybutyric acid, which cyclized spontaneously to butyrolactone. The latter product was treated with ammonia under pressure t o yield 2pyrrolidinone. The final step was vinylation at the nitrogen atom of the pyrrolidinone ring with acetylene, using caustic catalyst. The synthesis is shown in scheme 1-5. H C S CH
+ 2HCHO
HOCH2Cz CCH20H
___t
(1)
HO. CH2CH2CH2CH20H ( 2 )
HO. CHZCH~CH~CH~OH
cu
(-& H
(3)
562
CI-IRISTIANAH M. ADEYEYE AND EUGENE BARABAS
In spite of the fact that the synthesis was discovered over 50 years ago, and it consists of as many as five steps, the Reppe method described above is still
the process of choice for the commercial production of N-vinyl-2-pyrrolidinone (2). Other methods (e.g. polymerization with cationic initiator or y-irradiation, polymerization with sodium borohydride or alkyl aluminum-transition metal combination catalysts, etc.) for the preparation of this monomer are not used commercially. 2.1
Phvsical ProDerties
Highly purified vinylpyrrolidinone is a water-clear, colorless liquid, though the commercial monomer normally has a light straw color. It is miscible with water and with organic solvents in all proportions. In water-organic systems vinylpyrrolidinone prefers the organic phase. The physical properties of the monomer are shown in Table 1.
Table 1
Phvsical Prooerties of N-Vinvl-2-Pvrrolidinone (3,4) Molecular Weight Assay Moisture Content Appearance Color (APHA) Vapor Pressure a t 17OC 24OC 45OC 54OC 64OC 77OC Boiling Point at 44 mm Hg Freezing Point Flash Point (open CUP) Fire Point Viscosity a t 25OC Specific Gravity (24/4OC) Refractive Index nZ6
111 98.5% min. 0.2% max. clear liquid 100 max. 0.05 0.10
0.50 1 .oo 2.00 5 .OO 193'C (209OF) 13.5'C 100.5°C (213OF) 98.4OC (213OF) 2.07 cps 1.04 1.51 1
POVIDONE
3.
563
Polvmerization
Polymerization of N-vinylpyrrolidinone may be carried out either by free radical or by ionic mechanism. The latter gives only low molecular weight products and is of no commercial importance (5). The pioneering work with the free radical polymerization of vinylpyrrolidinone was carried out in water with hydrogen peroxide as initiator and ammonia as activator (6). This method still is the most widely used for the polymerization of vinylpyrrolidinone. The interrelation of the respective concentrations of monomer, hydrogen peroxide and ammonia is expressed by the following equation: Rate of polymerization = K[HOOH]”* [NH,1’/4 [VPI3/’(7) pH does not significantly influence the rate in the range 7-12; above pH 12 the polymerization is slower and no polymerization takes place above pH 13 ( 8 ) . By using hydrogen peroxide-ammonia system, polymers with degrees of polymerization of 10 to 10,000 may be made, corresponding to molecular weights of about 1000 to about 1,000,000. Initiation occurs by the hydroxy radical, the concentration of which influences the molecular weight. Propagation proceeds through addition of the macroradical to the double bond of the monomer. Termination takes place through combination of the polymer radicals and the hydroxyl radicals present in the solution. The termination results in the formation of an unstable hydroxyl carrying endgroup which splits off a pyrrolidinone molecule with the formation of an aldehyde endgroup. See the scheme of free radical polymerization below. Polymerization with azo-catalysts also readily yields polymers. This polymerization takes place even a t room temperature in the case of purified monomer. The rate of polymerization is a function of the degree of purity (9). Isopropanol-water mixture is used as reaction medium for the polymerization in which t-butylperoxypivalate is used as initiator (10). In another method tbutylhydroperoxide is used as catalyst (1 1). When the initiator is an organic peroxide both the initiation and termination take place on the effect of solvent radicals formed through hydrogen abstraction. The mechanism is suggested by proton NMR spectra of the dry polymer and as illustrated (12). Several other methods described in the literature are only of laboratory interest.
564
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
The scheme of the free radical polymerization with hydrogen peroxide is as
follows:
Initiation H202
-*
00
H2C=CH
+HO.
-W
HO.+ HO.
HOHC-CH.
Propagation HOHC-CH.
+ n.
Termination
OHC-CH
POVIDONE
46.5
Free radical polymerization with organic radical.
Propagation
Termination: Chain Transfer
4. Descriotion of the Polvmer 4.1
ADDearance
Poly(vinylpyrro1idinone) is a white to off-white powder. All grades have a faint, characteristic odor, but are almost tasteless. The polymers of different molecular weights form clear, hard films.
4.2
Molecular Weiaht
Depending upon the conditions of polymerization PVP can be prepared in a variety of molecular weights. When the polymer is made by the hydrogen peroxide-ammonia process, the molecular weight range is from 2500 to about 1,100,000 amu. The molecular weights are conventionally expressed by the SO called "K-values", which are based on viscosity measurements and are calculated according t o a formula developed by Fikentscher (13).
where
c q,.,
K
concentration in g/100 ml solution, viscosity of the solution compared to that of the solvent = = 1000 KO =
566
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
The Fikentscher value is currently used mostly for the expression of the molecular weight of PVP and vinylpyrrolidinone copolymers. From the arrangement of the Fikentscher equation the K-value can be calculated directly K - v a l l l e = J 300 C l o g Z + ( C + 1 . 5 C l 0 g 2 ) ~+ l . S C ( l o g Z ) - C 0.15 C + 0.003 C2
where Z is the viscosity of PVP at concentration C, relative to water. Polymers can ba characterized by three types of molecular weights. Since the normal composition of a polymer is a mixture of chains of various lengths, the measurement provides an average, the value of which depends upon the definition of the average molecular weight and the method of determination. The most common expression of molecular weight are the number average (mn) and the weight average (mw). The former (hi) is the simple arithmetic mean of all the relative molecular weights of the molecules, while latter (mw) is the mean of the weighted relative molecular weights of the constituent molecules. The following calculations for a hypothetical polymer illustrate the differences between a n and mw. Number Averaae Molecular Weiaht Number of Molecules
Mol. Wt.
Relative Weight of Each Fract.
1
X
2,000,000
=
2,000,000
10
X
1,00,000
=
1,000,000
11
Total
Mn
3,000,000
=
11
3,000,000 = 273,000amu
Weiaht Averaae Molecular Weiaht Relative Weight 01 Each Fraction
Mol. Wt. of Molecules in Each Fraction
Weighted Mol. Wt. of Each Fract. ~~
2
X
2,000,000
-
1
X
100,000
-
Total
-
3
-
4,000,000 100,000 4,100,000
4,100,000
MW =
=
1,367,000 amu
3 Measurements of the colligative properties of solutions, which depend on the number of molecules present in a given amount of solution (e.g. osmometry or end-group analysis), lead to mn. Methods based on the properties of polymers influenced by the size of the molecules (e.g. ultracentrifugation or light
POVIDONE
567
scattering) give f l w . These measurements are absolute methods and give the respective values directly. Because the polymer solutions are non-ideal, the measurements have to be extrapolated t o infinite dilution so that the values are independent of the solvent. The correlation of weight average molecular weight, degree of polymerization and intrinsic viscosity is shown in Figure 1. Fiaure 1
0.225
0.547
1.01
1.61
[ql. d”g
Relation of Weight Average Molecular Weight, Degree of Polymerization, and Intrinsic Viscosity of Povidone A very frequently used value is the viscosity average molecular weight (flvl. Viscosimetry is not an absolute method, since the values are influenced by the solvent. The values must be calculated from equations or obtained from calibration curves. From the K-value the molecular weights may be calculated by the equations f l n = 24 x K 2 @ W = 15 x K’.’ MV = 22.22 (K + 0.0075 K21’‘*6
following ( 1 4)
1151 116)
The correlation between K-value and viscosity average molecular weight is illustrated in Figure 2.
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
568
Fiaure 2 100
90 80 70
-s t j60o 0,
3
?
4 40 30 20 10
0 lo3
~
10'
tos
'10'
Nl K-value vs viscosity-average molecular weight
4.3
Mu
Molecular Weiaht Distribution
The chain lengths of the polymers are never uniform. As it is typical for radical polymerizations, the molecular weight distribution of PVP follows the Schultz-Flory distribution curve. The distribution curve of PVP is generally broad due to chain transfer reactions during the polymerization. Those chain transfer reactions are more frequent during the preparation of higher molecular weight polymers because of higher degrees of branching (1 7). In general the lower the molecular weight grade, the narrower is the distribution curve. The absolute molecular weight distribution of PVP can be determined by size exclusion chromatography for most K-value grades ( 1 8 ) .
POVIDONE
Fiaure
569
9
Molecular Weiaht Distribution of Various PVP-s (Ref. 181
WP K - 3 0 0 01*
4.4
Glass Transition Temoerature
The glass transition temperature (Tg) of linear PVP increases with increasing molecular weight and is calculated according to the following equation:
where K is the Fikentscher K-value of the polymer ( 1 9). The glass transition temperature of various molecular weight PVP-s is given in Table 2.
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
570
Table 2 Glass Transition Temperatures of PVP Measured K-value
To. OC
PVP K-15
14.9
130
PVP K-25
22.5
160
PVP K-30
27.5
163
PVP K-60
55.5
170
PVP K-90
89.6
174
122.0
176
PVP K-120
The deviation ad DSC measured Tg from that calculated is 1.7OC while the standard deviation is 1. l0C,probably reflecting the different molecular weight distributions.
4.5
HvaroscoDi&
PVP is hygroscopic but the amount absorbed is nearly independent of the molecular weight of the polymer. This fact is quite important in many applications (20).The equilibrium water content varies with the relative humidity of the atmosphere and it is approximately 1 /3of the latter value (211. The correlation is shown in Figure 4.
Fiaure 4
60
y
40
,-
I
1
1
i
20
40
60
80
s .-$
P 5:
J3 CQ
s
20-
c.
0
0
Rcktive atmospheric humidity, %
POW DONE
4.6
57 I
Solubilitv of PVP
PVP is soluble in a variety of solvents, most important of which is water in which the polymer has remarkably high solubility. In the following table "Soluble" means that a saturated solution of the polymer contains a mass fraction of at least l o % , while "Insoluble" means that the mass fraction of PVP of a saturated solution is less than 1%.
PVP is soluble in: Water
Di(ethylene glycol)
Methanol
Poly(ethy1ene glycol) 400
Ethanol
Propylene glycol
Propanol
1,4-Butanediol
Butanol
Glycerol
Cyclohexanol
2-Vinylpyrrolidone
Chloroform
Triethanolamine
Dichloromethane
Formic acid
I ,2-Dichloroethane
Acetic acid
N-Methylpyrrolidone
Propionic acid
Ethylenediamine
2-Pyrrolidone N-Methylpyrrolidone y-Butyrolactone
PVP is insoluble in: Ethyl acetate
Carbon tetrachloride
Acetone
Light petroleum distillates
Dioxane
Toluene
Diethylether
Xylene
Pentane
Mineral oil
Cyclohexane
572
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
Five percent solutions in heptane, kerosene or toluene may be prepared from a 25% solution of PVP in butanol ( 2 2 ) .
4.7
Viscositv of PVP Because of the polar nature of the polymer, its viscosity can be determined
most conveniently in water or methanol. In these solvents the application of the Mark-Houwink equation (23,24) =
(TI)
m:
gives the following results methanol: 2.3 x 1 0 ' x MWo,''; water: 5.65 x 10.' x Mw0.66 where the larger exponent indicates that methanol is a better solvent for PVP than water (25). In aqueous solution the kinematic viscosity of the polymer solution is dependent on both the solid content and the molecular weight. The correlations are illustrated on Figure 5. Fiaure 5
----d
1 1 1 t I I I l l l l l l I l L 1
EITcct of
0
2
0 3 0 PVP. %
4
0
5
0
~
PVP conccnlralion on lhe viscosity of aqucous solution
513
POVIDONE
The chemical character of the solvent influences the viscosity of the polymer solution significantly (26). The values of the kinematic viscosities of PVP solution in various solvents are given in Table 3.
Table 3 Viscosities of PVP K-30 in Various Organic Solvents
Solvent Methylene Dichloride Nitroethane N-Methyl Pyrrolidinone Absolute Ethanol Butyrolactone Acetic Acid (glacial) Methyl Cyclohexanone Glyme lsopropanol Ethyl Lactate Diacetone Alcohol Ethylene Glycol Monoethanolamine Diethylene Glycol Propylene Glycol Cyclohexanol 1,4-ButanedioI Triethanolamine Glycerine Nonylphenol
Note:
Kinematic Viscosity (in centistokes)
2 % PVP 1 1 2 2 2 2 3 3 4 4 5 24 27 39 66 80 101 156 480 3,300
=
10% PVP 3 3 8 6 8 12 10 12 12 18 22 95 83 165 261 376 425
666 2,046 -
absolute viscosity (in centipoises) density
The viscosity of the polymer solution is inversely proportional to the temperature (27). The effect is a function of the concentration of the solution. The correlation is shown in Figure 6.
514
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
20
40
60
80
Terrperalwe. 'c
Effect of temperature on viscosity at various concentrations
4.8
Chemical Nature and Stability
PVP is quite inert chemically. The dry polymer can be stored under normal conditions without undergoing decomposition, degradation or other structural changes. The heat sensitivity of the polymer is low, and it is stable when kept at 13OoC for short intervals. However, reduced solubility and enhanced color is noted at 15OoCin air. If this treatment is carried out for an extended period of time, the polymer may become insoluble (81. PVP can be made insoluble by heating it with ammonium persulfate (28). Diazo-compounds (29) and oxidizing agents (30) (e.g. dichromates) in the presence of light can gel the polymer, too. Such gels when dried can retain their structure and then can be swollen with the absorption of large amounts of water (311. PVP precipitates from water solution permanently when heated with strong bases, trisodium phosphate, due to the formation of unsaturated compounds and subsequent crosslinking through the system (32).PVP acts as reducing agents towards ammoniacal silver nitrate, potassium permanganate or Fehling's solution (33). Due to its unique structure PVP forms complexes with a variety of compounds. With electronegative compounds carrying active hydrogens (e.g. carboxylic acids, phenols, etc.) PVP forms complexes through hydrogen
POVIDONE
575
bonding mechanism. With polybasic acids, such as poly(acrylic acid), poly(viny1ether-co-maleic acid), etc. PVP gives complexes in water solution. These complexes are insoluble in water or alcohol, but dissolve readily in caustic solutions, showing that the nature of the binding mechanism is through hydrogen-bonds. Phenols behave similarly and the strength of the binding is a function of the acid strength of the hydroxyl group (34). PVP exhibits strong affinity towards dyes and complexes then mostly through the different kinds of Van der Waals forces (35).Because of this affinity PVP is effective as dyestripping agent.
5. Uses of Polv(Vinv1 Pvrrolidinonel Because of it's unique physical and chemical properties, particularly its good solubility in water and a variety of organic solvents, its outstanding chemical stability, it's strong complexing ability towards both hydrophobic and hydrophilic substances, PVP is one of the most widely used specialty polymers. Particularly important is the use of PVP' in the pharmaceutical industry and medicine. It has found application also in the textile, paper, adhesive, membrane, suspending aid, plastics, detergents, ceramics, detergent and soap industries, as well as in the fields of photochemistry and radiation curing. Of particular importance is the use of PVP in the pharmaceutical industry and in medical and cosmetic applications.
5.1
Primarv Uses
5.1 1 Pharmaceutical ADDlications Because of its exceptionally low toxicity, PVP has become one of the most widely used polymers in the pharmaceutical industry. PVP becomes sticky with water, therefore, it is used extensively as a tablet binder. It holds the tablet together and prevents cracking and chipping at the edges (36). Because of good solubility and hydrophilicity, PVP facilitates the disintegration of the tablet and provides reliable rates of drug dissolution (37) particularly when used in conjunction with crosslinked PVP. The abbreviation "PVP" is used for the sake of convenience here, though the name "Povidone" is widely used in the pharmaceutical industry and publications. The proportion of PVP/K-30 relative t o the mass o f the tablet is usually 25%, however, PVP/K-90 - because of its higher binding power - may be usedin lower amounts. The polymer can be used also for direct tableting without previous granulation (38,39,40). Because of its good film forming and adhesive properties, and high dispersing power, PVP is widely used for tablet coating (38,411. Because of its hydrophilicity it prevents too rapid drying, which might create tension between the tablet body and the coating (27). Since PVP has excellent dispersing properties, it prevents the aggregation of the pigment in the coating. It
516
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
promotes the homogeneity of sugar crystallization and serves as color dispersant (27,42). PVP is used also in film coating where it serves several purposes: it helps film formation, promotes adhesion and ensures even dispersion of the pigment (43,441. The formulation of poorly water-soluble drugs has been a problem of the pharmaceutical industry for quite some time. PVP has been found useful in enhancing the water solubility and bioavailability of these drugs (45-49). PVP has been steadily gaining importance as controlled release matrix. The excellent compatibility, high complexing ability, unique solubility characteristics and low toxicity makes this polymer eminently suitable for this application. It is used in transdermal devices (50,51 I, hydrogels (52,531, membrane.s (541, osmotic devices (551, microcapsules (561, suppositories (57) and tablets. In some formulas PVP is used as thickener t o improve the appearance and texture of the formulation (81. PVP is widely used in a variety of ophthalmic applications it is used t o increase the viscosity of the eyedrop, thus increasing the contact time with the eye and improving the lubricating effect (601, to reduce irritation (61 and to promote availability of the medication (62). It is used also as part of cleaning and preserving preparations for contact lenses (631. 5.12 Medical ADDlications The first medical use of PVP was as a plasma volume expander during World War II (64). At that time, it was found that a 3.5% solution of PVP in isotonic salt solution was effective in the treatment of shock due to loss of blood. The 3.5% PVP solution has about the same colloidal osmotic pressure as plasma. Because of this and its prolonged residence in the bloodstream (it remains in effective concentration in the circulation for 2-3 days) (65.661, it could maintain circulating blood volume in the early reversible stage of shock (671. The application lof PVP in shock treatment was reviewed by the NAS/NRC Drug Efficacy Study Group, where it received an effective rating. PVP was found to be an effective cryoprotective agent for the preservation of whole blood and its various cellular components (platelets, It offers high recovery rate, lymphocytes, erythrocytes and leucocytes) (68). and the thawed product is suitable for clinical use (69,701. PVP has shown propensity to complex with a variety of toxic agents accidentally ingested and with certain bacterially generated toxins. These complexes are eliminated through the kidney (71-75). Recently it has been found that PVP in conjunction with certain drugs, e.g., cisplatin or thalidasine, enhances anti-neoplastic, immunosuppressant activity (76,77,78). Heavy metal salt PVP complexes have been used in computed tomographic diagnosis of tumors (79). PVP is known to prolong the effectiveness of certain drugs e.g., penicillin (801, insulin (811, vasodilators (821, local anaestetics (83), hormones (89) etc. by slowing down their availability. The mechanism of this process can be either the formation of complexes, from which the drug is slowly liberated, or slow diffusion due to the high local viscosity a t the site of administration caused by the polymer, or the partial blocking of the active sites. It is probable that these three mechanisms work simultaneously.
POVIDONE
577
PVP is used as a diagnostic tool in gastroenterology and hematology
(85). PVP has been used successfully as articular lubricant in the treatment of rheumatic arthritis and deforming osteoarthritis (86). Due t o its good film forming and adhesive properties, as well as its compatibility with animal tissues, PVP is used in various forms of bioadhesives 187). 5.13
Cosmetic ADDlications
PVP is probably the most widely used polymer in the cosmetic industry. It gained wide application in hair grooming products and in a variety of other cosmetic applications: it improves the physical properties and performance of skin and eye makeups, lipsticks, deodorants, suntan lotions. It is used as cream, lotion and foam stabilizer and pigment dispersant. It increases lubricity and acts as humectant in skin care products. It also reduces toxicity and sensitivity. PVP is a component of numerous face powders, facial rouges, wrinkle removers, acne preparations, creams, skin cleansers and cosmetic emulsions. PVP is used advantageously in personal and room deodorants, fragrance binders, bath preparations and disinfectants. It is used in preparations for mouth-hygiene, like toothpastes, mouthwashes, dental-floss coatings. Also in leg-care products, depilatory agents, nail lacquers, etc. (88). 5.2
Other ADDlications (891
5.21
Textile
PVP is used to improve dye receptivity and also to restore the color of faded textiles. It is used as ink acceptor and ink stabilizer in the textile printing process. PVP has wide application as a dye dispersant, a water holding aid for hydrophobic fabrics, and as a print paste thickener. 5.22
PaDer
PVP is used in a variety of paper coating applications: for printing paper, as a luster improver, as a whitener and sliding preventer. In addition it is useful also as a filler and white water flocculent. Moreover, PVP is used also in acrylic fiber substitute compositions and in other types of paper replacement. PVP is used extensively in the formulation of various paper adhesives. 5.23 Adhesives PVP has excellent adhesive properties. It adheres to glass, plastic and metal surfaces with high initial tack, high peel strength and hardness. Because of its physical properties, it is particularly suitable for hot-melt and remoistenable adhesive applications. The adhesive stick is a special application which uses a PVP more than any other polymers.
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
578
5.24 Membranes The good polymerizing ability and crosslinkability, coupled with desirable physical and chemical properties, such as complex formation, hydrophilicity, strongly polar character, etc. made PVP eminently suitable for the use in membrane technology. Examples are desalination, gas separating, liquid ultrafiltration, hemodialysis membranes. 5.25
SusDendina A&
The amphophilic character, complexing ability and protein-like chemical composition make PWP suitable for application as suspending aid. Some of the most extensive applications are: suspending aid for PVC, polystyrene, polyethylene, butadiene-styrene copolymers, acrylonitrile-styrene suspensions and emulsions. PVP is used also to suspend carbon black. 5.26
Elastomers, Plastics and Rubbers
PVP, through its physical and chemical properties, is important in the manufacture of poljrmeric structures, affording better processability and improved performance. It is used in the preparation of a variety of thermoplastic elastomers. PWP is employed as binder or carrier of various ingredients used in the production of plastics, rubbers, and elastomers. Because of its high hydrophilicity, PVP is important in hydrogel formation. 5.27
Deteraents and SoaDs
PVP is compatible in clear, liquid detergent formulations. It prevents soil deposition, particularly on synthetic fibers and resin treated fabrics. It also acts as a loose-color scavenger because of its dye-bonding power. Complete water solubility and effectiveness as tablet binder makes it eminently suitable for detergent briquette application. It also improves the detergent action of soaps and waterless hand cleanses. It is extensively used also as thickener in detergent systems. P'VP is used as additive in glass cleaner formulations. 5.28 Ceramics PVP is compatible with a wide variety of inorganic materials. It is specifically adhesive to glass and used advantageously as binder and hydrophilizing additive with various ceramic substances. The use of PVP as an additive to cement used in the oil industry, is of special significance. It finds application as a cement set-time retarder, as a fluidloss additive and well-casing sealant. PVP is used in glass fiber lubricant formulations and in glass fiber finishing compositions. It is used in coatings to prevent clouding of glass and in coatings for optical glass fibers.
POVIDONE
5.29
519
Photochemistry
PVP is used in diazo and silver halide emulsions, etch coatings and in the preparation of lithographic plates. It gives a water soluble, light hardenable colloid that can increase covering power, density, contrast and speed of emulsions. PVP can make the deep etching of plates unnecessary. It offers uniform viscosity and temperature stability. It is non-thixotropic and gives tight adhesion in non-image areas. The polymer also is an effective protective colloid in silver bromide emulsions. PVP is widely used in ink-jet printing receptor formulations and in a variety of other ink-jet recording applications. It finds application in photoimaging, in the preparation of photoresists, in photolithography and in the laser imaging process.
5.210 Radiation Curinq Vinylpyrrolidinone monomer finds wide utility as a reactive diluent in radiation curable systems. Its versatility makes it adaptable t o many different conditions and properties required for coating. It copolymerizes rapidly with acrylates, which are the most commonly used oligomers in radiation curing. Vinylpyrrolidinone is an excellent solvent and has l o w viscosity, 2.07 mPa.s (i.e. cp). It gives very good viscosity reduction. Due to its high polarity, vinylpyrrolidinone improves the wettability characteristics of the coating compositions and improves pigment dispersion. It accelerates curing rate and requires a lower dosage rate of irradiation than monoacrylates. Because of the high Tg of PVP, the inclusion of vinylpyrrolidinone into the radiation curable system gives films of increased hardness. The high boiling point of vinylpyrrolidinone is advantageous, since highly volatile and toxic ingredients are objectionable for environmental and health reasons.
6 . Health and Safety Poly(vinylpyrro1idinone) has been used in medical applications since the early
1940’s. I t was the first synthetic polymer used in such application. Because of the nature of these applications the health and safety aspects of Povidone are of primary importance.
6.1
Oral and Topical Amlication
The largest volume of use has been as an excipient in the manufacture of tablets containing antibiotics, cardial medications, analgesics, oral contraceptives and vitamins. Povidone is the most widely used binder in the wet granulation for tablet making. It is estimated that the total number of tablets which utilizes PVP as binder disintegrant, color coating aid and subcoating material, controlled release aid and dissolution rate improver is more than 100,000 million (10”) per year: about 15% of all oral dosage forms use PVP. The lack of adverse physiological reactions t o the use of Povidone in such
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
580
large number of cases as well as the extensive safety data available are good indications that Povidone is safe, in the amounts used in these applications. To determine the pharmacokinetic behavior of Povidone the correlation between molecular weight and molecular size has to be known. The size of the solvated molecule can be approximated by the unpenurbed radius of gyration (90). Using the equation
where ro = end to end distance of the unperturbed chain in Angstrom units and M = molecular weight. For PVP, 0.480A
Therefore, So
=
0.480
x
=
0.196
x
0
Based on that, the following molecular weight-molecular size correlations were calculated: Table 4 Correlation Between Molecular Weight ( a w l and Molecular Size K-Value
Molecular Weight (amu)
Molecular Size,
12
2,900
1,055
17
9,000
18,594
25
29,000
33,378
30
45,000
41,578
A
It has been found that the effective pore size of the human jejunum is between 6.7 and 8.8 8, and in the ileum between 3.0 and 3.8 A. Therefore, only minute quantities of very low molecular weight PVP-oligomers could penetrate through the gastrointestinal tract (911. On the other hand, the renal glomerulus is much more penetrable than the intestines. The size of pores of the renal glomerulus was found to be 17.8 8, to 66.0 A (92). Consequently, any low molecular weight oligomer that might pass through the intestinal pores, can easily be eliminated by the renal system. The Food and Drug Administration allows the use of PVP in a number of food uses (931, such as bodying agents, clarifiers, dispersants, tableting
POVIDONE
58 I
adjuvants and as indirect food additives in packaging for transporting and holding food (94). The Acceptable Daily Intake (ADI) permitted by the Joint Expert Committee on Food Additives of the United Nations is max. 50 mglkg bodyweight (95). PVP is safe for oral and topical applications. It is not absorbed through the skin and it is non-irritating and non-sensitizing. By oral route PVP is primarily excreted unchanged. The small molecular weight portions which may be absorbed are rapidly eliminated. LD, values range from 12-15 g/kg bodyweight (intraperitoneal, mouse) t o 100 g/kg bodyweight (oral, rat) (96).
6.2
Parenteral Administration
In the case of parenterally administered Povidone, urinary clearance is directly related t o the molecular size of the polymer used. For intravenous use, drugs may be used containing an unrestricted (but identified) amount of Povidone up t o K-18since lower molecular weight Povidone clears through the renal system readily. Large size molecules (mol. w t . > 1 10,000 amu) retained by the reticuloendothelial system and go through fluid-phase pinocytosis or phagocytized by the RES cells and deposited at the liver, spleen, kidneys, lungs, lymph nodes, bone marrow and other storage sites (97-1 00). For intramuscular use, PVP should be injected only into areas with good blood circulation, since the polymer is not biodegradable and removed mostly through the lymphatic system (101). It is advisable t o inject it at alternative sites and for periods not longer than 30 days. Subcutaneous application of Povidone is not recommended due t o the possibility of thesaurismosis (See section 8.22). There is no evidence, however, that the use of povidone would result in any histopathological changes, and it was not found t o be carcinogenic (1 021, mutagenic (1031or teratogenic (104).See Sections 9.5and 10.
7.
ComDliance with PharmacoDoeias and Food Reaulations
7.1
Detection and Identification of PVP
There are several methods for the identification of soluble grades of PVP. They are applicable t o all grades, but the sensitivity is not the same in every case. The polymers of lower molecular weight sometimes react less strongly than the polymers of higher molecular weight. Every pharmacopoeia and food grade regulations uses determination by potassium dichromate and by addition of iodine respectively. The European, British and Japanese Pharmacopoeias also recommend identification by dimethylaminobenzaldehyde and sulfuric acid. FCC and JECFA use the determination of cobalt nitrate hexahydrate. All the above methods are described in Section 7.11 . The various spectra (IR, proton and C13 NMR) obtained from International Specialty Products (1 05)are also suitable for the identification of the polymer, and the respective spectra are given in Section 7.12.
582
7.1 1
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
Identification bv Soot Tests
7.1 11 Detection with Potassium Dichromate To 10 mL OF a 2% aqueous solution of the sample add 20 mL 1N hydrochloric acid and 5 mL of a 10% aqueous solution of potassium dichromate. An orange-yellow precipitate shows the presence of Povidone.
7.1 12 Detection with Cobalt Nitrate Dissolve 75 mg of cobalt nitrate and 300 mg of ammonium thiocyanate in 2 mL of water. To the mix add 5 mL of a 2% aqueous solution of the sample, then make the resulting solution acidic with diluted hydrochloric acid TS. A pale, blue precipitate is formed if Povidone is present. 7.1 13 Detection with Iodine To 5 mL of a 2% aqueous solution of the sample add a few drops of iodine TS. In the presence of Povidone a deep red color is produced. 7.1 14 Detection with Dimethvlaminobenzaldehvde (DMABAL
To 1 mL of a 2% sample solution add 0.2 mL of dimethylaminobenzaldehyde reagent and 2 mL of concentrated sulfuric acid, then cool the mixture after 30 seconds. If the sample contains Povidone, a permanent pink color is formed. The dimethylaminobenzaldehydereagent is prepared by mixing 0.2 g of dimethylaminobenzaldehyde, 20 mL ethanol and 0.5 mL concentrated hydrochloric acid. The solution is decolorized by shaking it with activated charcoal followed by filtration of the solution. 7.1 2
Identification bv Infrared Snectra
7.1 21 Infrared Soectrum The presence of Povidone can be established by the examination of the infrared spectrum of the sample. The peaks of the spectrum are characteristic to Povidone, particularly the triplet a t 1495 crn-’, 1463 cm” and 1423 cm-’ wavelengths. The peaks are independent of the molecular weight of the polymer. The infrared spectrum of Povidone is given in Figure 7.
POVIDONE
Fiaure 7 Infrared Soectrum of the Povidone
(K-30, KBr pellet)
I on
.O
-
.o
LO
20
Significant absorptions occur at the following wavenumbers: 2954 2981 1706 1423 1387 1259 1267 1072 846
CH stretching Amide band CH,-bending (or deformation band) CH,- deformation and skeletal vibrations
583
584
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
7.122 Proton NMR SDectrum The spectrum of Povidone K-12 prepared in isopropanol by an organic initiator with t-butyl radical is shown in Figure 8. FiQure 8
3 Proton NMR
'cj
,
0-
a 5
,
. . , 1 3
,
.
. , ,
1 0
.
. , I
S
,
, ,
.
,
2 0
,
,
,
., I 5
,
.
.. , t
,
0 P?U
,
., 0
:
POVIDONE
585
7.123 C-13 NMR SDectrum The spectrum of Povidone obtained in deuterated water is given in Figure 9. Fiaure 9 C-13 NMR Soectrum of Povidone K-30
D
D
L
Note Methods 7.11 1 (Potassium Dichromate), 7.1 13 (Iodine) are used by all the Pharmacopoeias and food regulations covered in this monograph. Method 7.1 12 (Cobalt Nitrate) is used by the U.S. Pharmacopoeia as well as the FCC and JECFA food regulations. Method 7.1 1 4 (Dimethylaminobenzaldehyde) is used by the European, British and Japanese Pharmacopoeias. The European Pharmacopoeia prefers the IR spectrum as method for identification. Table 5 shows this arrangement.
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
586
Table 5
7.2
SDecificationS
7.21
Water
Polybinylpyrrolidinone) is quite hygroscopic, therefore, it usually contains some water. The amount of water allowed by the various Pharmacopoeias is max. 5.0%. The quantitative determination of water is important in demonstrating compliance with this standard. There are two methods allowed for the determination A. Karl Fischer Titrimetric Method B. Azeotropic Distillation Method Method A is described in Section 7.21 11. Method Section 7.21 12. Generally Method A is preferred.
B can be found in
7.21 1 Determination of Water (106) For the determination of water content all Pharmacopoeias and Food Regulations direct the use of the Karl Fischer titrimetric method. The determination is carried out as follows: 7.21 11 Karl F
i
s
c
h
e
w
d
Amaratus Any apparatus may be used that provides for adequate exclusion of atmospheric moisture and determination of the end-point. The end-point is determined electrometrically with an apparatus employing a simple electrical circuit that serves to impress about 200 mV of applied potential between a pair of platinum electrodes (about 5 square mm in area and about 2.5 cm apart)
POVIDONE
587
immersed in the solution t o be titrated. A t the end-point of the titration a slight excess of the reagent increases the flow of current t o between 5 0 and 1 5 0 microamperes for a minimum of 30 seconds. With some automatic titrators, the abrupt change in current or potential at the end-point serves t o close a solenoid-operated valve that controls the buret delivering the titrant. Commercially available apparatus generally comprises a closed system consisting of one or t w o automatic burets and a tightly covered titration vessel fitted with the necessary electrodes and a magnetic stirrer. The air in the system is kept dry with a suitable desiccant such as phosphorus pentoxide, and the titration vessel may be purged by means of a stream of dry nitrogen or current of dry air. Reaaent Prepare the Karl Fischer Reagent as follows: Add 1 2 5 g of iodine t o a solution containing 670 mL o f methanol and 170 mL o f pyridine, and cool. Place 1 0 0 mL of pyridine in a 2 5 0 mL graduated cylinder and, keeping the pyridine cold in an ice bath, pass in dry sulfur dioxide until the volume reaches 2 0 0 mL. Slowly add this solution, with shaking, t o the cooled iodine mixture. Shake well t o dissolve the iodine, transfer the solution t o the apparatus, and allow t o stand overnight before standardizing. One mL of this solution when freshly prepared is equivalent t o approximately 5 m g of water, but it deteriorates gradually; therefore, standardize it within 1 hour before use, or daily if in continuous use. Protect from light while in use. Store any bulk stock of the reagent in a suitably sealed, glass-stoppered container, fully protected from light, and under refrigeration.
A commercially available, stabilized solution of Karl Fischer type reagent may be used. Commercially available reagents containing solvents of bases other than pyridine andlor alcohols other than methanol may be used also. These may be single solutions or reagents formed in situ by combining the components of the reagents present in t w o discrete solutions. Test PreDaration Use an accurately weighed or measured amount of sample estimated t o contain 1 0 t o 2 5 0 mg of Povidone. Since Povidone is hygroscopic use a dry syringe t o inject an appropriate volume of methanol accurately measured, into the container, previously accurately weighed, and shake t o dissolve the specimen. Using the same syringe, remove the solution from the container and transfer it t o a titration vessel prepared as directed under Procedure. Repeat the procedure with a second portion of methanol, accurately measured, add this washing t o the titration vessel, and immediately titrate. Determine the water content, in mg,
588
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
of a portion of solvent of the same total volume as that used to dissolve the specimen and to wash the container and syringe, as directed under Standardization o f Water Solution for Residue1 Titrations, and subtract this value from the water content, in mg, obtained in the titration of the specimen under test. Dry the container and its closure a t 100' for 3 hours, allow to cool in a desiccator, and weigh. Determine the weight of specimen tested from the difference in weight from the initial weight of the container. Standardization of the Reagent Place enough methanol in the titration vessel to cover the electrodes, and add sufficient Reagent to give the characteristic end-point color, or 100 f 50 microamperes of direct current at about 200 mV of applied potential. For determination of trace amounts of water (less than 1 %) sodium tartrate may be used as a convenient water reference substance. Quickly add 150 to 350 mg of sodium tartrate (C,H,Na20,.2H20), accurately weighed by difference, and titrate to the end-point. The water equivalence factor F, in mg of water per mL of reagent, is given by the formula: 2(18.02/230.08)(W/V), in which 18.02 and 230.08 are the molecular weights of water and sodium tartrate dihydrate, respectively. W is the weight, in mg, of sodium tartrate dihydrate, and V is the volume, in mL, of the Reagent consumed in the second titration. For the precise determination of significant amounts of water (more than 1%), use purified water obtained by distillation as the reference substance. Quickly add betwee'n 25 mg and 250 mg of water, accurately weighed by difference. Titrate t o the end-point. Calculate the water equivalence factor, F, in mg of water per mL of reagent, by the formula:
F = W/V, in which W is the weight, in mg, of the water, and V is the volume, in mL, of the reagent required. Standardization of Water Solution for Residual Titration Prepare a Water Solution by diluting 2 mL of water with methanol t o 1000 mL. Standardize this solution by titrating 25.0 mL with the Reagent, previously standardized as directed under Standardization o f the Reagent. Calculate the water content, in mg per mL, of the Water Solution by the formula: V'F/2 5,
POVIDONE
5x9
in which V’ is the volume of the Reagent consumed, and F is the water equivalence factor of the Reagent. Determine the water content of the Water Solution weekly, and standardize the Reagent against it periodically as needed. Procedure Transfer 35 t o 40 mL of methanol or other suitable solvent t o the titration vessel, and titrate with the Reagent to the electrometric or visual endpoint. Quickly add the Test Prepafation, mix, and add an accurately measured excess of the Reagent. Allow sufficient time for the reaction to reach completion, and titrate the unconsumed Reagent with standardized Water Solution t o the electrometric or visual end-point. Calculate the water content of the specimen, in mg, by the formula: F(X’ - XRI,
in which F is the water equivalence factor of the Reagent, X‘ is the volume, in mL, of the Reagent added after introduction of the specimen, X is the volume, in mL, of standardized Water Solution required to neutralize the unconsumed Reagent, and R is the ratio, VY25 (mL ReagentlmL Water Solution), determined from the Standardization of Water Solution for Residual Titration. 7.21 21 Azeotrooic (Toluene Distillation) Method ADDaratus Use a 500 mL glass flask 1 connected by means of a trap 8 t o a reflux condenser C by ground glass joints (see Figure 10). Fiaure 1Q Aooaratus for Determination of Water
The figures are in mrn.
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
590
The critical dimensions of the parts of the apparatus are as follows: The connecting tube D is 9 t o 11 m m in internal diameter. The trap is 235 to 2 5 0 mm in length. The Condenser, if of the straight-tube type, is approximately 4 0 0 mm in length and not less than 8 mm in bore diameter. The receiving tube E has a 5 mL capacity and its cylindrical portion, 146 to 1 5 6 m m in length, is graduated in 0.1 mL subdivisions, so that the error of reading is not greater than 0.05 mL for any indicated volume. The source of heat is preferably an electric heater with rheostat control or an oil bath. The upper portion of the flask and the connecting tube may be insulated with asbestos. Clean the receiving tube and the condenser with chromic acid cleansing mixture, thoroughly rinse with water, and dry in an oven. Prepare the toluene t o be used by first shaking with a small quantity of water, separating the excess water, and distilling the toluene. Procedure Place in the dry flask a quantity of Povidone weighed accurately to the nearest centigram, which is expected t o yield 2 to 4 mL of water. Place about 2 0 0 mL of toluene in the flask, connect the apparatus, and fill the receiving tube E with toluene poured through the top of the condenser. Heat the flask gently for 15 minutes and, when the toluene begins t o boil, distill at a rate of about 2 drops per second until most of the water has passed over, then increase the rate of distillation t o about 4 drops per second. When the water has apparently all distilled over, rinse the inside of the condenser tube with toluene while brushing down the tube with a tube brush attached to a copper wire and saturated with toluene. Continue the distillation for 5 minutes, then remove the heat, and allow the receiving tube to cool t o room temperature. if any droplets of water adhere to the walls of the receiving tube, scrub them down with a brush consisting of a rubber band wrapped around a copper wire and wetted with toluene. When the water and toluene have separated completely, read the! volume of water, and calculate the percentage that was present in the subst#ance. 7.22
&!
Polyfvinylpyrrolidone) solutions are usually slightly acidic. The reason for this can be the presence of acidic groups, and/or some degree of enolization of the carbonyl group in the ring structure. Not all the Pharmacopoeias demand set pH values, but the ones that make such requirements - as well as the FCC and JECFA regulations set the pH values between 3.0 and 7.0. The determination of pH can be made potentiometrically using a suitable pH meter. The method is described in Section 7.221. Where approximate pH values suffice, indicator test papers may be used. The determination by test papers are described in Section 7.221 2.
POVIDONE
59 1
7.221 Determination of DH 7.221 1 Bv DH Meter ADDaratus 1. Magnetic stirrer and stirring bar, 2. pH meter, 3. Combination pH electrode, 4. pH indicator test paper. Procedure Prepare test solution by weighing approximately 2.00 g Povidone in a 4 oz jar. Place the jar on the magnetic stirrer, and from a burette add 1 8 fold amount of carbon dioxide-free distilled water. Place the stirring bar in the jar, start stirring and make clear solution. With the help of pH indicator test paper determine the approximate pH of the test solution (for method see B.). Use a suitable pH meter and follow the manufacturer's instructions. Each time the electrodes are used, rinse them with distilled or deionized water and carefully blot them dry with clean absorbent tissue. Form a fresh reference electrode liquid junction. Rinse the sample vessel three times with each new solution t o be introduced. Choose t w o standard buffers to bracket, the anticipated pH of the sample. Warm or cool these standards as necessary to match within 2OC the temperature of the unknown, and initially set the temperature compensator to that temperature. Immerse the electrodes in a portion of the first standard buffer, and following the manufacturer's instructions adjust the appropriate standardization control (knob, switch, or button) until the pH reading is that of the buffer. Repeat this procedure with fresh portions of the first standard buffer until t w o successive readings are within f 0.02 pH unit without an adjustment of the standardization control. Rinse the electrodes, blot dry, and immerse them in a portion of the second standard buffer of lower pH. Do not change the setting of the standardization control. Following the manufacturer's instructions, adjust the slope control (thumbwheel switch, knob, or temperature compensator) until the exact buffer pH is displayed. Repeat the sequence of standardization with both buffers until the pH readings are within f 0.02 pH unit for both buffers without any adjustment of either control. The pH of the sample solution may then be measured. 7.221 2 DH Indicator Test Papers There are presently available test papers that have impregnated acidbase indicators and are very convenient for the determination of the approximate pH of an aqueous solution. Indicator test papers that cover a wide
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
592
pH range or that cctver a narrow pH range may be obtained. The maximum accuracy attainable with these systems is It 0.25 pH unit. The accepted test procedure using a pH indicator test paper is to use a clean glass rod to remove a drop of the solution whose pH is being determined and place it on a test paper strip. When the indicator color is achieved, compare the color with the color comparison chart and determine by color similarity the pH of the solution. If the pH is alkaline, take care to minimize absorption of carbon dioxide by the solution on the pH paper, as this will result in pH change. Immersion of the test paper strip in the solution whose pH is to be determined is not recommended since some of the indicator may dissolve in the solution. 7.23
Residual Monomer (VinvlDvrrohdonel
Recent methods used for the production of PVP are designed to achieve close to quantitative conversion. Nevertheless, slight amounts of unreacted monomer may remain attached to the surface of the polymer, even after drying. The various Pharmacopoeias had permitted 0.2% residual monomer, with 1 % specified in food regulations (FCC, JECFA). However, lowering this value to 0.1 % has been recommended by the major producers. All the current monographs use iodometric method for the determination of unreacted monomer. The method is described in Section 7.231. 7.23 1
Determination of Residual Monomer (VinvlPvrrolidinone)
Procedure 1. Place 80 mL distilled water in an Erlenmeyer flask and with gentle stirring dissolve in it 10.0 g Povidone.
2. Add 1.O g sodium acetate to the solution and shake the flask gently until clear solution is obtained. 3. Titrate the solution with 0.1ON iodine until a faint yellow color appears, then add an additional 3.0 mL of 0.1ON iodine solution. 4. Set the flask aside for 10 minutes, preferentially in the dark.
5. Titrate the excess iodine with 0.10N sodium thiosulfate solution until the color is light yellow.
6. Add 3.0 mL of 0.5% starch solution. The color of the solution turns to bluish-green.
593
POVIDONE
7. Continue the titration with 0.10N sodium thiosulfate solution to a sharp decolorizing endpoint. Calculation
% VP =
(mLi
*
Ni
-
mL,,)
Weight of sample
where
7.24
(5.555)
*
%
100
mL,
=
total volume of iodine used in the sample,
N,
=
normality of iodine solution,
mLrs
=
volume of thiosulfate solution used for back titration
N,
=
normality for thiosulfate solution
Nitroaen
The vinylpyrrolidinone molecule has one nitrogen atom, corresponding to 12.6% of the total weight. The various Pharmacopoeias and the FCC specification allow a range of 11.5 - 12.8% (corresponding to 91.3 - 101.6%). The JECFA requirement is 12.2 - 13% (corresponding to 96.8 - 108.3%). The analysis is done by the Kjeldahl method. The determination may be carried out in full size (A) or semimicro (B) equipment. The decomposition is usually carried out with sulfuric acid using various catalysts in the procedure. During the treatment the nitrogen is transformed to ammonia and is determined as such. The British Pharmacopoeia also describes as an alternative (Dumas) method the oxidation of the nitrogen content to nitrous oxides. The Japanese Pharmacopoeia1 Forum in an effort to harmonize the various methods, recommends the semi-micro method described in the IV Supplement to USP XXII. The method can be found in Section 7.241. 7.241 Determination of Nitroaen (107) Aooaratus As shown in Figure 11. It is to be constructed of hard glass with ground glass stoppers and joints. All rubber parts should be boiled for 10 to 30 minutes in sodium hydroxide TS and for 30 to 60 minutes in distilled water, and washed again with distilled water before use.
594
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
Fiaure 11 Kieldahl ADDaratUS (From Pharmacopeia of Japan, Tenth Edition, 1981, p. 754)
A: Kjcldahl firsk. B: Sicam pencrator, cootzinin@uatcr, 10 which a few drops of sulfuric acid. 2nd I:zcmtou of boiliop tips 10 prcvcni bumpiog. bzvc k e n added.
C: Spray trap.
D: WZIC:supply funnel. E: Sieamtubc. F: FUMCI for zddition of alk21i solution (0 flzsk A. tubiog uitb I
G: Rubbcr
' The f ~ ~ *re finamm.
clamp.
H: A small
hole haviog diameter rpproximatelyequzl to :be delivery lube.
J : Condenser. the lourr end of which i s beveled. K: Absorptioa flask
A: Kjeldahl flask, 8: Steam generator, containing water, to which a few drops of sulfuric acid, and fragments of boiling tips to prevent bumping, have been added, C: Spray trap, D: Water supply funnel, E: Steam type, F: Funnel for addition of alkali solution to flask A., G: Rubber tubing with a clamp, H: A small hole having diameter approximately equal to the delivery tube, J: Condenser, the lower end of which is beveled, K: Absorption flask. Procedure
1. Weigh accurately a quantity of the sample corresponding to 2 t o 3 mg of nitrogen (N: 14.0071 and place in the Kjeldahl flask.
2. Add 1 g of a powdered mixture of 10 g of potassium sulfate and 1 g of cupric sulfate. Wash down any adhering sample from the neck of the flask with a small quantity of water.
POVIDONE
595
3. Add 7 mL of sulfuric acid, allowing it to flow down along the inside wall of the flask. 4. While shaking the flask, add cautiously 1 mL of strong hydrogen peroxide solution drop by drop along the inside wall of the flask. 5. Heat the flask, on a wire gauze, over a free flame until the solution has a clear blue color and the inside walls of the flask are free from carbonaceous material.Heat for an additional 45 minutes.
6. After cooling, add cautiously 20 mL of water, cool the solution, and connect the flask to distillation apparatus washed beforehand by passing steam through it. 7. To the absorption flask K, add 15 mL of boric acid solution (1-25). 3 drops of bromocresol green-methyl red TS and sufficient water to immerse the lower end of the condenser tube J. Add 30 mL of sodium hydroxide solution (2-5) through the funnel F, rinse cautiously the funnel with 10 mL of water, immediately close the clamp attached to the rubber tubing G .
8. Begin the distillation with steam and continue until the distillate measures 80 to 100 mL.
9. Remove the absorption flask from the lower end of the condenser tube J, rinsing the end part with a small quantity of water, and titrate the distillate with 0.01 N sulfuric acid until the color of the solution changes from green through pale grayish blue to pale grayish red-purple.
10. Perform a blank determination in the same manner, and make any necessary correction. Calculation Each ml of 0.01 N sulfuric acid = 0.14007 mg of N 7.25
&
The purpose of this test is to determine the amount of inorganic contaminants in the polymer. The amount of these contaminants are small, and result from the impurities of the ingredients used in the course of the preparation. The pharmacopoeias allow 0.1 % while the amount of ash in the polymer used for food purposes can be as much as 0.2%. The methods in the various pharmacopoeias determine the so called "sulfated" ash while the regulations of the food grade PVP describe the determination also of the total ash, as well as of the acid insoluble ash content. The respective methods can be found in Section 7.251.
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
596
7.251 Determination of Ash (108) ADDaratus 1. 2. 3. 4.
Porcelain or platinum crucible, Muffle furnace, Hot plate, Desiccator with suitable desiccant.
Procedure 7.251 1 Ash (Total) Weigh accurately about 3 g of the sample to the nearest 0.0002 g in a tared crucible, ignite at a low temperature (about 500°),not to exceed a very dull redness, until free from carbon, cool in a desiccator, and weigh. If a carbon-free ash is not obtained, wet the charred mass with hot water, collect the insoluble residue on an ashless filter paper, and ignite the residue and filter paper until the ash is white or nearly so. Finally, add the filtrate, evaporate it t o dryness, and heat the whole to a dull redness. If a carbon-free ash is still not obtained, cool the crucible, add 15 mL of alcohol, break up the ash with a glass rod, then burn off the alcohol, again heat the whole to dull redness, cool, and weigh. 7.251 2 Ash (Acid-insoluble) Boil the ash obtained as directed under Ash (Total) above, with 25 mL of dilute hydrochloric acid TS for 5 min, collect the insoluble matter on a tared Gooch crucible or other suitable ashless filter, wash with hot water, ignite at 800' f 25', cool, and weigh. Calculate the percentage of acid-insoluble ash from the weight of the sample taken.
Note:
For Sulfated Ash the European Pharmacopoeia describes a slightly modified method whi'ch follows Method II of the British Pharmacopoeia:
Heat a silica or platinum crucible to redness for 3 min, allow to cool in a desiccator and weigh. Place the substance to be examined in the crucible and add 2 mL of dilute sulphuric acid R . Heat at first on a waterbath, then cautiously over a flame, then progressively to about 60OoC. Continue the incineration until all black particles have disappeared and allow the crucible t o cool. Add a few drops of dilute sulphuric acid R, heat and incinerate as before and allow to cool. Add a few drops of ammonium carbonate solution R. Evaporate and incinerate carefully, allow t o cool, weigh, and repeat the ignition for periods of 15 min to constant mass.
POVlDONE
597
7.251 3 Sulfated Ash Weigh accurately approximately 2.000 g Povidone in a crucible that previously has been ignited, cooled and weighed. Heat gently at first until the substance is thoroughly charred. Cool, then moisten the residue with 2.0 ml (1:1) sulfuric acid. Heat gently until white fumes no longer are evolved. Ignite at 8OO0C f 25OC until the carbon is consumed. Cool in the desiccator, then weigh and calculate the ash content as follows: % ash
where Wo = W, = W, = 7.26
=
w -w * w, - wo
100
crucible weight sample and crucible before ashing sample and crucible after ashing
Heavv Metals
The heavy metal contaminants may be introduced in the polymer by ingredient impurities. These heavy metals are the metal inclusions which are darkened with the sulfide ion (Ag, As, Bi, Cd, Cu, K, Pg, Sb, Sn), and their total quantity is expressed in terms of the quantity of lead (Pb). The allowed limit is max. 1 0 ppm. The US Pharmacopoeia uses standardized dithizone solution as comparison, the European and British Pharmacopoeias require thiacetarnide solution, while the Japanese Pharmacopoeia, as well as the food regulatory processes use hydrogen sulfide (or sodium sulfide in acid solution) for comparing color intensity as test methods. The method (US Pharmacopoeia XXII) is given in Section 7.261. 7.261 Determination of Heavv Metals Amaratus 1 . 50 ml color comparison tubes (or Hunter colorimeter)
2. 100 m l volumetric flask 3. 100 ml capacity porcelain crucible 4. Hot plate (or Bunsen burner)
5. Muffle furnace
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
598
Reaaents 1. Stock Lead Nitrate Solution ( 1 00 pg/mL) - Dissolve exactly 0.1 598 g of Pb(NO,), in 100 mL distilled water. Add 1 mL nitric acid and dilute to 100 mL in a volumetric flask.
2. Standard Lead Solution (10 pg/mL) - Transfer 10 mL stock lead nitrate solution t o a 100 mL volumetric flask and make up t o volume with distilled water. Each mL of this standard is equivalent to 10 micrograms of lead. This solution must be prepared just before the test. 3. Ammonia Test Solution - To be prepared by diluting t o 40 mL of concentrated NH,OH solution t o 100 mL with distilled water.
4. 10% acetic acid solution in distilled water. 5. Hydrogen Sulfide Test Solution - Saturate 100 mL of distilled water with H,S by passing the H,S gas into cold water. The solution has t o be kept in a cold, dark place. Procedure 1 . Place the weighed quantity of Povidone (approx. 10 g) in a porcelain crucible. Add sulfuric acid to wet the substance and carefully ignite it at l o w
temperature until thoroughly charred.
2. Add to contents of the crucible 2 mL of concentrated HNO, and 5 drops of concentrated H,SO,
and heat cautiously until white fumes are evolved.
3. Place crucible in a muffle furnace for one hour at 500-600°C. 4. Cool, add 2 mL of concentrated HCI then 10 mL of distilled water and digest for 2 minutes on :steam bath. 5. Moisten residue with 1 drop of concentrated HCI, add 10 mL distilled water and digest for 2 minutes on steam bath.
6. Add dropwise ammonia test solution until solution is just alkaline. Dilute t o 25 mL with distilled water.
7. Add dropwise 10% acetic acid solution to neutralize, then add 2 mL of excess acetic acid solution.
8. Transfer the solution into a 50 mL graduated test tube by using as little as possible distilled water for rinsing. Bring the volume to 40 mL with distilled water. (Test Premration)
POVIDONE
599
9. In another test tube, pipette 2 mL of standard lead solution and add 2 mL of 10% acetic acid solution, then bring the volume to 40 mL with distilled water.
10. Add t o each tube 10 mL of saturated H,S solution. Mix well and allow to stand for 5 minutes. (Standard Preparation) 11. View the tubes downward over a white surface. The color of the solution from the Test Preparation should not be darker than that of the solution from the Standard Preparation, representing 2 0 ppm lead. Alternatively the tubes may be compared with a Hunter Colorimeter.
7.27
Viscosity
Viscosity is a property of liquids that is closely related to the resistance t o flow (109). It is defined in terms of the force required t o move one plane surface continuously past another under specified steady-state conditions when the space between is filled by the liquid in question. It is defined as the shearstress divided by the rate of shear strain (1 10). The basic unit is the poise, however, viscosities commonly encountered represent fractions of the poise, so that the centipoise (1 poise = 1 0 0 centipoises) proves to be the more convenient unit. The specifying of temperature is important because viscosity changes with temperature: in general viscosity is decreased as temperature is increased. While on the absolute scale viscosity is measured in poises or centipoises, for convenience the kinematic scale, in which the units are stokes and centistokes (1 stoke = 100 centistokes) commonly is used (1 11). To obtain the kinematic viscosity from the absolute viscosity, the latter is divided by the density of the liquid at the same temperature.
Kinematic viscosity
=
Absolute viscosity Densi t y
The sizes of the units are such that viscosities in the ordinary ranges are conveniently expressed in centistokes. Absolute viscosity can be measured directly if accurate dimensions of the measuring instrument are known, but it is more common practice to calibrate the instrument with a liquid of known viscosity and t o determine the viscosity of the unknown fluid by comparison with that of the known. The usual method for measurement of viscosity involves the determination of the time required for a given volume of liquid to flow through a capillary. Many capillary-tube viscometers have been devised, but the Ostwald type, Ubbelohde-type, and Cannon-Fenske type viscorneters are the most frequently used (1 12). The logarithm of the molecular weight of the polymer is directly proportional to the logarithm of its viscosity.
600
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
The viscosi1:y is expressed either as kinematic viscosity ( y ) in square millimeters per second ( m m z s ~ ’from ) the expression y
(q) in pascal seconds (Pa or as dynamic viscosity
= Kt
s) from the expression q
=
K{t
where t = time in seconds for the meniscus t o travel between the t w o marks, { = mass volume (g cm3) obtained by multiplying the relative density of the fluid being examined by 0.998203. The constant (K) of the instrument is determined using an appropriate reference liquid. The methods determining the flow viscosity of Povidone-solutions is described in Section 7.271. In polymer science it is not the absolute viscosity of a solvent or a solution that is of interest, but the increase in viscosity attributable to the dissolved polymer. Therefore, in the viscometry of it is the relative viscosity that is most often used. The relative viscosity is defined as the quotient of the viscosity of the solution, qe, and the viscosity of the solvent, q.
The molecular weight of Povidone is usually expressed in the so called K-values. The K-value can be calculated from the relative viscosity of the polymer solution. The formula used for the calculated is described in Section 7.271. The practical range of K-values lies between K-10 and K-120. From the K-values the various types of average molecular weights can be calculated using the formulas given in Section 7.27. The requirements of the food regulatory agencies are given in intrinsic viscosities. This vallue is derived from relative increase of the viscosity called specific viscosity and is denoted by q,,
When this quantity is divided by the concentration C, the viscosity number q& is obtained (1 13). The value of the viscosity number at infinite concentration is the intrinsic viscosity ([ql).The intrinsic viscosity of a polymer solution is a measure of the capacity of the polymer molecule to enhance the viscosity, which depends on the size and shape of the polymer molecule. Within a given series of polymer homologs, the intrinsic viscosity increases with the molecular weight, therefore, it is a measure of the molecular weight. It is
POVIDONE
60 I
obtained by extrapolating t o zero concentration the plotted measurements obtained at several concentrations and at the same temperature (1 14). For practical purpose the intrinsic viscosity may be calculated from the values of the relative viscosity using a so called "one-point" formula. A useful equation for a one-point approximation of the intrinsic viscosity is as follows:
All information in this equation is obtained from a single determination of q,sl at one concentration (1 15). The method of determining the intrinsic viscosity is described in Section 7.271e. A particularly convenient and rapid type of instrument is a rotational viscosimeter which utilizes a bob or spindle immersed in the test specimen and measures the resistance to movement of the rotating part. Different spindles are available for given viscosity ranges, and several rotating speeds are usually available. Other rotational instruments may have a stationary bob and a rotating cup. The Brookfield, Rotovisco and Stormer viscosimeters are examples of rotating-bob or spindte instruments, and the McMichael viscosimeter is an example of the rotating cup instrument. 7.271 Determination of Viscosity A.
K-value PrinciDle
The method is designed to measure the Fikentscher K-value of Povidone in an aqueous solution. K-value is an indirect measurement of molecular weight and increases with it. The K-value is not entirely independent of the concentration, therefore, an optimum concentration is specified for each grade of Povidone. If the nominal value is K-18 or less a 5 % w /v solution, if the nominal value is more than K-18 but less than or equal t o K-95, a 1% solution is t o be prepared. If the nominal K-value is higher than 95, 0.1% solution is tested. The K-value of Povidone in an aqueous solution is defined by the Fikentscher equation:
where K C
= =
q,
=
1000 KO concentration in g/lOO mL relative viscosity
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
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After determining the relative viscosity the K-value is calculated according t o the following equation:
K =
g-/
(Irel +
( C + 1.5C log 0.15C
+
(Ire1)2 +
1.5C l o g (qrel) -C
0.003C2
.
1 Volumetric flask, 100 mL glass stoppered,
2. Constant temperature bath which can be controlled at temperatures between about 25'C and 5OoC, constant t o rt O.O2'C,
3. Cannon-Fenske type or Ubbelohde type viscosimeter. The size of the viscosirneter should be such that solution flow time is about 100 seconds. C.
Procedure 1. Prepare sample by weighing the calculated amount to four decimal accuracy, add 50 mL distilled water and dissolve the solids by swirling the flask. After dissolution thermostat the flask in the constant temperature bath set to 25'C, then dilute to the mark with thermostated solvent.
2. Using gentle suction from a vacuum line, raise the solution meniscus t o the upper fiducial mark. Determine the time required to the meniscus to pass from the upper t o the lower mark. Record the time t o the nearest 0.1 second.
3. Repeat the measurement until t w o successive readings agree to within 0.1 seconds or 0.1 % of their mean, which ever is greater, then average these values. 4. Empty, clean and dry the viscosimeter, then repeat above steps using 10.0 mL of distilled water.
0.
Calculations a. Relative Viscosity
where t and to are the average flow times for the solution and solvent, respectively.
POVIDONE
603
b. K-value
To be calculated from the Relative Viscosity using the formula given in A. c. Reduced Viscosity Calculate the reduced viscosity q,,
-q r e-l
-
Orel
C
-
by the formula 1
C
where C is grams of polymer per 100 mL of solution. Since the reduced viscosity varies with concentration, with each value of q8, the concentration must be specified. d. Inherent Viscosity Calculate the inherent viscosity qlnh,by rlih
=
In
qrel
C
~
where In qra,is the natural logarithm of q,el (-2.303 x log,, too, the concentration must be specified.
q,=,)here,
e. Intrinsic Viscosity Estimate the intrinsic viscosity [ql,with the following equation using a single q,elvalue.
[ql
=
1 - 2(qrei
C
-
1
- I n qrei)
or determine [ql from q,elvalues taken at several concentration: qapversus C. Extrapolate to zero Concentration. The 1 ) PlotC intercept is [ql, or
21 Plot qlnhversus C and extrapolate to zero. This extrapolation should give the same intercept as 1). 7.28
Aldehvdes
Poly(vinylpyrrolidinone1 usually contains small amount of aldehyde as contaminant. The monomer itself is made by the reaction of formaldehyde with acetylene, though it was ascertained that no detectable amount of that l ow
604
CH RISTIANAH M. ADEYEYE AND EUGENE BARABAS
boiling material remained with the product after several steps of reaction and strenuous purification. A more probable source is the monomer itself, which has relatively low hydrolytic stability on the acid side. Under such conditions the monomers may decompose t o 2-pyrrolidone and acetaldehyde. The Pharmacopoeia and the JECFA regulations allow max. 0.2% aldehyde content (determined as acetaldehyde). The FCC regulation allows 0.5% aldehyde. The method of determination uses hydroxylamine hydrochloride as reagent. The Japanese Pharmacopoeia1 Forum as well as USP recommend an enzymatic method using aldehyde dehydrogenase enzyme as reagent and UV spectroscopy as test method. The respective methods are described in Section 7.281. 7.281
Determination of Acetaldehvde
7.281 1 Determination bv Enzvmatic Reaction and UV SDectrophotometrv
1. Aldehyde Test Kit, consisting of potassium pyrophosphate buffer solution (pH 91, nicotinamide adenine dinucleotide tablets and aldehyde dehydrogenase 2. Acetaldehyde, 99% 3. 0.3M potassium pyrophosphate buffer, pH 9. Dissolve 100 g K,P,O, (97%) in 800 mL distilled water. Adjust t o pH 9 with 1 M HCI, then dilute to 1 liter with distilled water.
1 . Double beam UV-visible spectrophotometer. 2. 1 cm quartz cuvettes with Teflon stoppers. If not available, standard cuvettes can be used. However, they must be covered with parafilm during analysis to prevent oxygen contamination. 3. Thermostated water bath set at 6OoC f 2OC. Procedure 1. Preparation of acetaldehyde standards
a) Pipet 'I30 pL acetaldehyde into a 100 mL volumetric flask half filled with distilled water. Mix and dilute to the mark with distilled water. Store at 4OC for 12 hours before use. This corresponds to 1000 ppm acetaldehyde. b) Prepare working standards of 1 ppm, 1 0 ppm and 100 ppm by serial dilution of the stock standard (0.1 mL, 1 mL, 10 mL diluted to 100 rnL). These standards should be prepared immediately before use.
POVIDONE
2. Preparation of the sample a) Weigh 1 .OOO g to 1.5000 g sample (W,) into a beaker. Add pH 9 buffer t o a known approximate weight of 50.0000 g (W,). Dissolve completely by shaking and cap or cover tightly. b) Add solution to a 50 mL centrifuge tube with screw cap (tightly capped) and heat at 6OoC for 1 hour. c) Cool t o room temperature. Do not unscrew cap until contents are cool.
3. Preparation of the reagents a) Dissolve 8 tablets of nicotinamide adenine dinucleotide [(NAD), bottle 21 in 2 4 mL of potassium pyrophosphate buffer (bottle 1 ) . For each replicate 1 tablet of NAD and 3 mL of buffer are required. b) Add 0.6 mL of deionized water to 4 units of aldehyde dehydrogenase (AI-DH, bottle 3). Dissolve completely. c) Allow the NAD and AI-DH solutions to reach 2OoC - 25OC before use. 4. Instrument set-up and analysis a) Pipet 3 mL NAD solution, 0.5 mL deionized water, and 5 0 p L AlDH solution into each quartz cuvette. Stopper tightly and mix (by inversion) thoroughly. b) Place cuvettes into instrument and set the absorbance to 3 4 0 nm. c) Analyze standards (1, 10, 100 ppm) by mixing 3 mL NAD solution, 0.5 mL standard and 50 p L AI-DH solution in cuvette. Stopper tightly. Record absorbance at 3 4 0 nm after 5 minutes. d) Analyze the sample in the same manner as the standards, using 0.5 mL as the sample size. Record absorbance at 3 4 0 nm after 5 minutes. e) Perform the sample analysis on duplicates. Calculations 1. Construct a calibration curve of absorbance at 3 4 0 nm versus acetaldehyde concentration.
2. Determine the acetaldehyde concentration in the sample solution by comparison of the absorbance to the calibration curve values.
605
CHIUSTIANAH M. ADEYEYE AND EUGENE BARABAS
606
3. p p m acetaldehyde =
where
C,,,,,,
=
W,, W,
=
calibration curve (ppm), weight {gt of sample solution and original sample respectively.
7.281 2 Determination with Hvdroxvlamine Hydrochloride
Distillation equipment with ice-cooled receiver. Procedure 1. Transfer 20.0 g of Povidone to a round-bottom flask containing 180 mL of 9N sulfuric acid.
2. Attach a suitable water-cooled condenser and reflux for 45 minutes. 3. Reassemble the apparatus, and distill until about 100 mL of distillate has been collected, receiving the distillate in a receiver emerged in an icebath. The receiver contains 20 mL of 1N hydroxylamine hydrochloride solution previously adjusted to a pH of 3.1. 4. Titrate the distillate solution with 0.1 ON sodium hydroxide to a pH of 3.1.
5. Perform a blank determination, and make the necessary corrections.
6. Not more than 9.1 mL of 0.10N sodium hydroxide is required corresponding to not more than 0.2% of aldehyde, calculated as acetaldehyde.
7.29
Hvdrazine
Hydrazine is a product of the reaction of ammonia and hydrogen peroxide which are parts of the initiator system. The Pharmacopoeias allow 1 ppm hydrazine in the product. The concentration is determined by thin-layer chromatographic method using salicylaldehyde as test solution. The method is given in Section 7.291.
607
POVIDON E
7.291
Determination of Hvdrazine Content ADDaratus 1. 2. 3. 4.
Bench top centrifuge, Constant temperature water bath, TLC developing tank (12" x 3 7/8" x l o " ) , UV lamp, 365 nm, 5. Pre-coated TLC plate coated with 0.25 m m dimethylsilanized chromatographic silica gel plate.
layer
of
Procedure
1. Transfer 2.5 g Povidone to a 50 mL centrifuge tube, add 25 ml distilled water and stir to dissolve.
2. Add 500 p L of a 1:20 solution of salicylaldehyde in methanol, swirl, and heat in a water bath at 6OoC for 15 minutes. 3. Allow solution to cool to room temperature, then add 2.0 mL toluene. Insert stopper in the tube and shake vigorously for 2 minutes. Centrifuge for 5 minutes. 4. Decant a portion of the upper layer and place it in a 5 mL capped vial. Discard lower laver.
5. On a pre-coated chromatographic plate spot 10 microliters of the clear upper toluene layer in the centrifuge tube and next to it 10 microliters of a standard solution of salicylaldazine in toluene containing 9.38 milligram per mL.
6. Allow the spots to dry, then develop the chromatogram with an eluent consisting of a 2: 1 mixture of methanol and water until the solvent front moved about three-fourth of the length of the plate.
7. Remove the plate from the developing chamber, mark the solvent front and allow the solvent to evaporate.
8. Locate the spots on the plate by examination under ultraviolet light at a wavelength of 365 nm. Salicylaldazine appears as a fluorescent spot having an RF value of about 0.3. The fluorescence of the salicylaldazine spot from the test specimen must not be stronger than that produced by the spot obtained from the standard solution corresponding to 1 ppm hydrazine.
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CHKISTIANAH M . ADEYEYE AND EUGENE BARABAS
7.3
Other Characteristics
7.31
Color and Clarity of Solution
Color may be defined as the subjective response of an observer to the objective stimulus of radiant energy in the visible spectrum extending over the range 400 nm to 700 nm in wavelength. The color is commonly identified by a) hue, that is the quality by which one color is distinguished from another (e.g. red, yellow, blue, green, etc.), b) value, that is the quality which distinguishes a light color from a dark one, c) chroma, that is a quality which distinguishes a strong color from a weak m e . These three attributes of color may be used to define a three dimensional color space in which any color is located by it's coordinates. Since the perception of color is dependent upon the conditions of viewing and illumination, the determination should be made with diffuse, uniform illumination and under conditions which reduces reflectance and shadows to a minimum. A solutions is termed clear when its appearance is the same as that of water or the solvent used, in the absence of the substance being examined. The divergence from this condition is called opalescence. Under ideal circumstances the solution of Povidone should be clear and colorless. However, in the course of production and/or handling slight amounts of side products and contaminants may get in the polymer, which are insoluble in the dissolving medium or form color when dissolved. Some of the pharmacopoeias are more stringent in this respect than the others. The U.S. Pharmacopoeia does not make any demands with respect of either clarity or color. Neither do the FCC and JECFA regulations. The Japanese Pharmacopoeia simply states that the 10% w/v solution should be colorless or pale yellow and clear. On the other hand the European Pharmacopoeia describes exactly what should be the color hue and intensity of the Povidone solution and uses an elaborate method for making the comparison. This method is given in Section 7.31 11. The British Pharmacopoeia uses the same method and demands the same comparative color value. Beside that, however, it regulates the requisites of clarity and gives a detailed method for its determination. This method is given in Section 7.312. 7.31 1
Deoree of Caloration of Liauids (1 16)
7.31 1 1 EuroDean Color Test The examination of the degree of coloration of liquids in the range brownyellow-red is carried out by one of the two methods below.
POVIDONE
609
A solution is colorless if it has the appearance of water or the solvent or is not more intensely colored than reference solution B,. Method I Using identical tubes of colorless, transparent, neutral glass of 12 mm external diameter, compare 2.0 mL of the liquid to be examined with 2.0 mL of water or of the solvent or of the reference solution (see Tables of reference solutions). Compare the colors in diffused daylight, viewing horizontally against a white background. Method II Using identical tubes of colorless, transparent, neutral glass with a flat base and an internal diameter of 15 mm to 25 mm, compare the liquid to be examined with water or the solvent or the reference solution (see Tables of reference solutions), the depth of the layer being 40 mm. Compare the colors in diffused daylight, viewing vertically against a white background. REAGENTS Primarv Solutions Ye//ow solution - Dissolve 4 6 g of ferric chloride R in about 900 mL of a mixture of 25 mL of hydrochloric acid R and 975 mL of water and dilute to 1000.0 mL with the same mixture. Titrate, and adjust the solution to contain 45.0 mg of FeCI,, 6H,O per milliliter by adding the same acid mixture. The solution should be protected from light. Titration - Place in a 250 mL conical flask fitted with a ground-glass stopper, 10.0 mL of the solution, 15 mL of water, 5 mL of hydrochloric acid R and 4 g of potassium iodide R, close the flask, allow to stand in the dark for 15 min. and add 10 mL of water. Titrate the liberated iodine with 0.1N sodium thiosulphate, using 0.5 mL of starch solution R, added towards the end of the titration, as indicator. 1 mL of 0.1N sodium thiosulphate is equivalent to 27.03 mg of FeCI,, 6H,O. Red solution - Dissolve 60 g of cobalt chloride R in about 900 mL of a mixture of 25 mL of hydrochloric acid R and 975 mL of water and dilute to 1000.0 mL with the same mixture. Titrate, and adjust the solution to contain 59.5 mg of CoCI,, 6H,O per milliliter by adding the same acid mixture. Titration - Place in a 2 5 0 mL conical flask fitted with a ground-glass stopper, 5.0 mL of the solution, 5 mL of dilute hydrogen peroxide solution R and 1 0 mL of a 30 per cent m/V solution of sodium hydroxide R. Boil gently for 1 min. allow t o cool and add 6 0 mL of dilute sulphuric acid R and 2 g of potassium iodide R. Close the flask and dissolve the precipitate by shaking gently. Titrate the liberated iodine with 0.1N sodium thiosulphate, using 0.5 mL of starch
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
610
solution R, added towards the end of the titration, as indicator. The end-point is reached when the solution turns pink. 1 mL of 0.1 N sodium thiosulphate is equivalent to 23.79 mg of CoCI,,
6H,O.
Blue primary solution . Dissolve 63 Q of copper sulphate R in about 900 mL of a mixture of 25 mL of hydrochloric acid R and 975 mL of water and dilute to 1000.0 mL with the same mixture. Titrate, and adjust the solution to contain 62.4 rng of CuSO,, 5H,O per milliliter by adding the same acid mixture. Titration - Place in a 2:50 mL conical flask fitted with a ground-glass stopper, 10.0 mL of the solution, 50 mL of water, 12 mL of dilute acetic acid R and 3 g of potassium iodide R. Titrate the liberated iodine with O.1N sodium thiosulphate, using 0.5 mL of starch solution R, added towards the end of the titration, as indicator. The end-point is reached when the solution shows a slight pale brown color. 1 mL of 0.1 N sodium thiosulphate is equivalent to 24.97 mg of CuSO,,
5H,O.
Standard Solutions Using the three primary solutions, prepare the five standard solutions as follows.
Standard Solution
Yellow solution
Red Solution
Blue Solution
Hydrochloric acid (1 per cent m/V HCI)
(brown) (brownishyellow) (yellow) (greenishyellow) (red)
3.0
3.0
2.4
1.6
2.4 2.4
1.o 0.6
0.4 0.0
6.2 7.0
9.6 1.o
0.2 2.0
0.2 0.0
0.0 7.0
B BY Y GY
R
POVIDONE
61 I
Reference solutions for Methods I and II
Using the five standard solutions, prepare the following reference solution. Reference solutions B II
Hydrochloric acid (1 per cent m/V HCI) ~~
75.0 50.0 37.5 25.0 12.5 5.0 2.5 1.5 1.o
25.0 50.0 62.5 75.0 87.5 95.0 97.5 98.5 99.0
Reference solutions BY
Reference solution
Standard solution BY
Hydrochloric acid (1 per cent m/V HCI)
BY, BY2 BY3 BY,
100.0 75.0 50.0 25.0 12.5 5.0 2.5
0.0 25.0 50.0 75.0 87.5 95.0 97.5
BY,
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
612
Reference solutions Y
Volume in milliliters ~
Reference solution
Standard solution Y
~~~
Hydrochloric acid (1 Der cent m/V HCI)
~~
y 2 y3
100.0 75.0 50.0 25.0 12.5 5.0
0.0 25.0 50.0 75.0 87.5 95.0
2.5
97.5
Reference solutions GY
II
II
Volume in milliliters Reference solution
Standard solution GY
Hydrochloric acid (1 per cent m/V HCI)
25.0 15.0 8.5 5.0 3.0 1.5 0.75
75.0 85.0 91.5 95.0 97.0 98.5 99.25
I I Reference solution
11
Standard solution R
Hydrochloric acid
(1 per cent m/V HCI) Rl R* R3 R4
R6
Re R,
100.0 75.0 50.0 37.5 25.0 12.5
0.0 25.0 50.0 62.5 75.0 87.5
5.0
95.0
613
POVIDONE
Storaae
For method I, the reference solutions may be stored in sealed tubes of colorless, transparent, neutral glass of 12 m m external diameter, protected from light. For method II, prepare the reference solutions immediately before use from the standard solutions.
Procedure 1.
25.0 g of Povidone are added t o 75 mL of distilled water in an 8 bottle and magnetically stirred until dissolved.
2.
Using the 3 primary solutions prepared under B, a "B" standard is made as follows:
02.
"B Standard" Yellow Standard: Red Standard: Blue Standard: 1 % (w/v) HCI: Total:
3.
4.
30 mL 30 mL 24 mL 16 mL 100 mL
From the "B" standard, the following 9 reference solutions are made:
mL Std "B"
mL 1 % ( w / v ) HCI c or gs to 100 mL
75.0 50.0 37.5 25.0 12.5 5.0 2.5 1.5 1 .o
25.0 50.0 62.5 75.0 87.5 95.0 97.5 98.5 99.0
Each Povidone solution is transferred t o a clear dry Vessler color tube filled t o the 20 mL mark (middle line) and compared t o the same depth of reference solutions against diffuse white background. Note and record the nearest color match for each sample. Note: The color intensities are inverted, that is, the darkest is B, and the lightest is Bg.
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
614
7.31 12 Determination of APHA Color Atmaratus Hellige Aquatester with APHA color disks. Reaaent Potassium Chloroplarinate
- Cobalt Chloride Stock - 500 units.
Procedure 1. Prepare solution of the material to be analyzed. If necessary remove bubbles by brief exposure to ultrasonic radiation. 2. Fill Vessler tube to the mark and insert optical stopper.
3. Fill reference Vessler tube to the mark and insert optical stopper. 4. Turn on light and match color intensity by rotating the wheel of the
apparatus. 5. Record indicated color. If color is over 100, make up reference standard by diluting the stock solution (1 part stock and 4 parts water). Use this as reference and add to the indicated reading. 7.31 2 Determination of Clarity (1 17) Procedure Into separate matched, flat-bottomed test tubes, 15 to 20 mm in diameter, and of colorless, transparent, neutral glass place sufficient of the solution being examined and of a suitable reference suspension, freshly prepared as specified below, such that the test tubes are filled to a depth of 40 mm, accurately measured. Five minutes after preparation of the reference suspension, compare the contents of the test tubes against a black background by viewing under diffused light down .the vertical axes of the tubes. The diffusion of the light must be such that Reference Suspension I can be readily distinguished from water and from Reference Suspension II. Standard of Opalescence - Dissolve 1.0 g of hydrazinium sulphate in sufficient water to produce 100.0 mL, and allow to stand for four to six hours. To 25.0 mL of this solution add a solution containing 2.5 g of hexamine in 25.0 mL of water, mix well and allow to stand for twenty-four hours. This suspension is stable for t w o months, provided that it is stored in a glass container free from surface defects.
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hi5
To prepare the standard of opalescence, dilute 15 mL of the suspension to 1000 mL with water. This suspension must be used within twenty-four hours of preparation. Reference Suspensions - Reference Suspension I to IV should be prepared as indicated in Table 6. Each suspension should be mixed well and shaken before use. TABLE 6 Reference suspension
Standard of opalescence (ml) Water (ml)
I
II
Ill
IV
5.0
10.0
30.0
50.0
95.0
90.0
70.0
50.0
Expression of clarity and degree of opalescence A solution is termed clear if its opalescence is not more pronounced than that of reference suspension I. A solution is termed slightly opalescent if its opalescence is more pronounced than that of reference suspension I, but not more pronounced than that of reference suspension II.
A solution is termed opalescent if its opalescence is more pronounced than that of reference suspension II, but not more pronounced than that of reference suspension Ill. A solution is termed very opalescent if its opalescence is more pronounced than that of reference suspension Ill, but not more pronounced than that of reference suspension IV.
7.32
Peroxides
Peroxides may be present in the system as residues of the initiator (hydrogen peroxide or organic peroxide) or through autoxidation of the polymer chain (1 18). This phenomenon may be produced during the polymerization by the reaction of the peroxy-radical with a tertiary methine-group of the polymer, according t o the following mechanism:
RO,
0
+
R,H
> ROOH + R,.
616
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
It may also be formed on the effect of atmospheric oxygen during the aging of the polymer (1 19). The decomposition of the hydroperoxide group may produce chain scission, branching or even crolsslinking. The various monographs - with the exception of the European Pharmacopoeia - make no requirements for allowable peroxide content. The European Pharmacopoeia uses titanium trichloride sulfuric acid reagents for the determination and allows a limit of max. 400 ppm representing H,O, and hydroperoxide content. Another method fa'r the determination is by iodometric titration which yields total peroxide content. Both methods are described in Section 7.321. 7.321 Determination of Peroxides 7.321 1 Determination with Titanium (IVI Sulfate ComDlexation ADDaratus 1. UV-Visible Spectrophotometer. 2. 1 cm cuvettes. Procedure 1. Prepare titanium (IV) sulfate reagent by dissolving 2.0 g titanium sulfate in 1L distilled water which contains 25 mL of concentrated H,SO,. (Note 1 ) 2. Prepare the stock standard by weighing 3.30 g of 30% H,O, into a 100 m l volumetric flask. Dilute to the mark with distilled water and mix thoroughly.
3. Compare the working standard by pipeting 1 mL of the diluted H,O, to 100 mL. This corresponds to 100 p g H,O, per mL solution (100 ppm). 4. Prepare H,O, calibration standards by pipetting 0, 2, 4, 10 and 20 mL of the stock standard into 100 mL volumetric flasks. Add 20 mL titanium sulfate reagent to each flask and dilute to the mark with distilled water. These solutions correspond to 0, 0.2, 0.4, 1.0 and 2.0 p g H,O, standards, respectively.
5. Accurately weigh 4.00 g Povidone into a 100 mL volumetric flask. Add 2 0 mL titanium sulfate reagent to the flask and dilute to the mark then mix thoroughly.
6. Determine the absorbance of the standard and sample solutions at 405 nrn versus the blank solution.
617
POVIDONE
Calculations 1. Construct a calibration curve of absorbance (y) versus p g of H,O, in the standard solutions (XI. Use linear regression to determine the slope and intercept of the straight line.
PPm H , 0 2
where
A b m W
= = = =
=
(A
- b) *
1000
m * W
absorbance of the sample solution versus blank solution y-intercept of calibration curve slope of calibration curve sample weight, g
Note 1 . The Japanese Pharmacopoeia describes titanium trichloride sulfuric acid reagent. The preparation of this reagent is carried out as follows: 1 . In a hood add t o 7 5 mL of a 20% aqueous TiCI, solution slowly with stirring, about 65 mL conc. H,SO,. Use a 2 5 0 mL wide-mouth Erlenmeyer flask and wear gloves and apron. Considerable heat evolves during this operation. 2. Start heating the stirred solution until dense fumes of SO, appear fabout 5 minutes).
3. A t this point, cautiously add dropwise from behind the hood glass shield, 30% hydrogen peroxide, until the solution clears and becomes colorless or pale yellow. (The originally milky purple suspension goes through various color changes, until clear solution is achieved. If too much hydrogen peroxide is added as evidenced by yellow to orange color of the solution, continue heating, until the color fades.) 4. Cool the solution to room temperature and dilute to 500 mL with distilled water. The solution - to be labelled as TiOSO, 2.9% - is stored stable for several months.
-
7.321 2 Determination with Iodine Titration Armaratus Magnetic stirrer with Teflon coated stirring bar. Procedure 1 . Dissolve in 500 mL Erlenmeyer flask 2 0 distilled water.
*
0.10 g Povidone in 200 mL
618
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
2. Titrate with 0.1 N iodine solution until a slight yellow color persists for 2 minutes. Cap the flask and swirl by hand. 3. If the yellow color fades before two minutes, add more 0.1 ON iodine solution and record the volume of iodine solution used.
4. Add 15 mL of 20% potassium iodide solution and 30 mL of a 6N H,SO, solution. 5. Allow to stand approximately 10 minutes with slow stirring with a Teflon coated magnetic bar. Keep the flask covered to exclude air. 6. Add 2 mL starch solution and titrate with 0.1 ON sodium thiosulfate to clear endpoint. Calculations ppm H,o, =
7.33
mL t h i o s u l f a t e * N * 1.7 g sample
* loa
1
( v/ w )
Arsenic
The customary methods of preparation and handling make the presence of arsenic in Povidone improbable. However, this polymer was declared suitable in direct secondary food applications and indirect food applications. Therefore, the respective regul,ations of the Food and Agricultural Organization of the United Nation (FA01 and the Food Chemicals Codex (FCC) - the specification of the Food and Nutrition Board of the U.S. National Research Council - specify the allowable limits of arsenic in Povidone. Some national pharmacopoeias (e.g. German, Italian) also set limits for the maximum allowable arsenic content. It is not more than 3 mg/kg in the FA0 regulations, not more than 1 ppm in the FCC specifications. The USP, British, European and Japanese Pharmacopoeias don't set limits on the allowable arsenic content. Methods to determine the amount of arsenic in general are given in all the major pharmacopoeias. 7.331 Determination of Arsenic (1 20) Atmaratus The apparatus (see Figure 12) consists of an arsine generator (a.) fitted with a scrubber unit (c.) and an absorber tube (e.) with standard-taper or ground glass ball-and-socket joints (b. and d.) between the units. However, any other suitable apparatus, embodying the principle of the assembly described and illustrated may be used.
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619
Fiaure 1 2
Arsenic Test Apparatus (Ref. 120)
Method Preoaration of Standard 1. Dissolve 132.0 mg of arsenic trioxide previously dried at 105OC for 1 hour and accurately weighed, in 5 mL of sodium hydroxide solution (1 in 5) in a 1000 mL volumetric flask.
2. Neutralize the solution with 2N sulfuric acid, add 1 0 mL more 2N sulfuric acid, then add recently boiled and cooled distilled water t o volume, and mix thoroughly. 3. Transfer 10.0 mL of this solution to a 1000 mL volumetric flask, add 1 0 mL of 2N sulfuric acid, then add recently boiled and cooled distilled water t o volume and mix thoroughly. Each mL of standard contains the equivalent of 1 p g of arsenic (As). Keep this solution in a glass container and use it within 3 days. Test Preoaration 1. Transfer to the generator flask the quantity, in g, of the test substance calculated by the formula 3.0/L, in which "L" is the arsenic limit, in ppm.
2. Add 5 m i of sulfuric acid and a f e w glass beads, and digest on a hot-plate, or other suitable heating unit in a fume hood until charring begins (additional sulfuric acid may be necessary t o meet the specimen completely, but the total volume added should not exceed 1 0 mL.1
620
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
3. After the test substance has been initially decomposed by the acid, cautiously add, dropwise, 30 percent hydrogen peroxide, allowing the reaction to subside than again heating between drops. Add the first few drops slowly t o prevent violent reaction and discontinue heating if foaming becomes excessive. 4. When the reaction has abated, heat cautiously,
rotating the flask occasionally t o prevent the specimen from caking on the glass exposed to the heating unit. Maintain oxidizing conditions all the time during the digestion by adding small quantities of hydrogen peroxide solution whenever the solution turns brown or darkens.
5. Continue the di'gestion until the organic matter is destroyed, fumes of sulfur trioxide are copiously evolved, and the solution becomes colorless or retains only a faint yellow color.
6. Cool, add cautiously 1 0 mL of distilled water, mix and again evaporate to strong fuming, repeating this procedure, if necessary, t o remove any trace of hydrogen peroxide. 7. Cool again, add cautiously 10 mL of distilled water, wash the sides of the flask with a f e w mL of water and dilute with water to 35 mL.
8. Add 20 mL of dilute sulfuric acid (1 in 51,2 mL of potassium iodide TS, and 0.5 mL of stronger acid stannous chloride TS and mix thoroughly. 9. Allow to stand at room temperature for 30 minutes.
10. Pack the scrublber tube (c) with t w o cotton rolls that have been soaked in saturated lead acetate solution, freed from excess solution by pressing, and dried in vacuum at room temperature, leaving a small space between the t w o rolls. Lubricate the joints (b and d) with a suitable stopcock grease (designed for use with organic solvents) and connect the scrubber unit to the absorber tube (el. 11. Transfer 3.0mL of silver diethyldithiocarbamate TS t o the absorber tube. 12. Add 3.0 g of granular zinc (No. 2 0 mesh) to the mixture in the flask, immediately connect the assembled scrubber unit, place the generator flask (a) in a water bath maintained at a temperature of 25 f 3OC, and allow the evolution of hydrogen and the color development to proceed for 45 minutes swirling the flask gently at 10-minute intervals. (If necessary t o obtain a more uniform gas evolution, 1 mL of isopropyl alcohol may be added t o the generator.) 13. Disconnect the absorber tube from the generator and scrubber unit, and transfer the absorbing solution t o a 1-cm absorption cell.
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62 I
14. Determine the absorbance at the wavelength of maximum absorbance up to 535 nm, with a suitable spectrophotometer or colorimeter, using silver diethyldithiocarbamate TS as the blank. The absorbance due to the red color of the test solution should not exceed that produced by 3.0 mL of Standard Preparation, corresponding t o 3.0 p g of As, when treated with the same quantities of the same reagents and in the same manner.
Note:
Metals or salts of metals such as chromium, cobalt, copper, mercury, molybdenum, nickel, palladium and silver may interfere with the reaction. Antimony, which forms stibine reacts similarly to arsine; when presence of antimony is suspected the readings should be made between 535 nm and 540 nm, since at this wavelength the interference is negligible.
7.34
Loss on Drving
The purpose of this analysis is to determine the amount of volatile matters of any kind that can be driven of at 100°C and atmospheric pressure. Loss on drying is a requirement only of the European Pharmacopoeia with a maximum 5.0% limit. There are several methods suitable for the determination of this value. The method as per the European Pharmacopoeia is described in Section 7.341. If it is presumed that water is the only volatile ingredient method described in Section 7.21 may also be used. For more accurate measurement TGA (Section 7.422) is suitable.
7.341 Determination of Loss on Drving
1. Electrically heated oven. 2. Glass stoppered, shallow weighing bottle. 3. Vacuum desiccator with diphosphorous pentoxide desiccant. Procedure 1. Place the glass stoppered weighing bottle in the oven. Remove the stopper and leave it in the oven. 2. Heat the oven to 100-105°C and hold the temperature for 30 minutes at atmospheric pressure.
3. Remove the bottle and cool it in the desiccator to room temperature. 4. Replace the stopper and tare the weighing bottle.
C'HRISTIANAH M. ADEYEYE AND EUGENE BARABAS
622
5. Weigh in the bottle 1 .OOOO to 2.0000 g Povidone. (W,)
6. By gentle, sidewise shaking, distribute the test specimen as evenly as practicable t o a depth of about 5 mrn. 7. Place the loaded bottle in the oven and removing the stopper, leave it also in the oven.
8. Heat the oven t o 100-105°C and hold it for 3 hours at atmospheric pressure. 9. Remove the weighing bottle and immediately replace the stopper. 10. Put the bottle in the desiccator and hold it there for 1 hour. 11. Weigh bottle accurately together with the tare of the bottle. (W,) % l o s s on drying
7.35
=
4 - w2 x -
100
WI
Surface Area
The spray dried or milled particle of PVP is not solid but contains t w o different types of air spaces of voids. a) b)
Open voids, which are open to the external environment. Closed voids, which are within the particle but separated environment by a continuous film of the polymer.
from the
The open voids of Type a) are important because they increase the liquid uptake of the particle, thus enhancing the disintegration of the tablet and the availability of the drug. Of course the surface area of the particle is dependent upon the size of the open intraparticulate pores (1 21 1. The calculation of the approximate surface area is relatively simple, if it is assumed that the particle is spherical, when the surface could be calculated from the average diameter of the particles. However, the particles are usually irregular, therefore, the accuracy of the calculation would depend upon the deviation of the particles of the spheric shape and that is usually considerable (122). The most widely used method for the determination of the surface area of the particles is the B.E.T. method (Brunauer, Emmett and Teller), which calculates the surface area by the determining volume of nitrogen absorbed as monomolecular layer on the surface of unit mass of powder ( 1 2 3 ) . The description of the BET method is given in Section 7.35. Instruments based on chemisorption are available which measure pressures, temperatures and the amount of aldsorbed gas very accurately in wide operating temperature
POVIDONE
623
range. With some of these instruments digital pressure readout is connected to a strip chart recorder, so that the amount of adsorbed gas as a function of time may be followed.
7.351 Determination of Surface Area (122) Aooaratus The determination may be carried out with an instrument built according to the BET principle. The schematic of such setup is shown below. Fiaure 13
TO McLEOD
CAUCC
1 C
Emmett-Tvoe Setuo for Surface Area Determination (Ref. 122) A. Sample Container B. Thermometer C. Water jacketed container D. Manometer mercury reservoir E. Drying container
F. Manometer G,H,J,K,L = Vacuum valves M. Acetone - Dry Ice trap N. Vacuum pump 0. Dewar flask with liquid nitrogen
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
624
Method The basic steps of the method are carried out as follows:
1. Determine the volume of each section of the apparatus by filling it with known volume of gas at known pressure. 2. Pretreat sample by degassing it. The rate of pressure decrease must be slow if sample is of low density or very fine. Such materials may have trapped within their volume sufficient amount of gas to cause fluidization if the pressure change is too fast. Fluidization may cause carryover of the particles into the filter or valves. By heating to 100-125'C, the removal of gas from the sample can be speeded.
3. The sample after degassing is immersed in a liquid nitrogen bath. (The l o w temperature is necessary during the procedure because the method of analysis is templerature dependent.) 4. Clean, dry nitrogen is introduced in small increments at constant temperature and the pressure is recorded at each equilibrium point. Calculation Since the volume of the sample and the container are known, the difference in pressure is a mealsure of the number of molecules adsorbed on the surface of the sample.
No. of Molecules
=
(Volume)( P r e s s u r e Change) (Avogadro No.) ( G a s C o n s t a n t ) (Absolute T e m p e r a t u r e )
that is
n = V
*
AP * 6 . 2 3 ~ 1 0 ~ 3 0.082 * T
where V is in liters A P is in atmospheres Gas Constant (0.82) is in liter-Atm/degree-mole The pressure adsorbed gas correlation can be followed on an adsorption isotherm illustrated in Figure 14. The linear portion of the curve is extrapolated to the mole-axis, anld the intercept expresses the number of molecules of gas adsorbed as a monolayer on the surface of the sample (1 24).
POVIDONE
625
Figure 14 (Ref. 122)
PRESSURE B. E.T.
absorption isotherm.
Various instruments measure above variables with good accuracy. The steps of the procedures are given in the individual instruction manuals of these instruments.
7.36
Particle Size Distribution
The particle size distribution of solid powdered PVP is important in pharmaceutical technology, particularly in direct tableting. The excipients used in pharmaceutical applications are usually milled, and this procedure gives irregularly shaped particles the size of which varies within the range of the largest and smallest particles. Since it is practically impossible t o define the dimensions of irregularly shaped objects in geometrical terms, statistical methods are used to define the size of the particles. There are several methods for the determination of particle size distribution which utilize different principles of measurement and used preferentially in various particle size ranges ( 1 25). A few of the most widely used methods are given in Table 7.
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
626
Table 7 Methods for the Determination of Panicle Sizes
Method
Preferred Size Ranae
Size Averam
Sieving
50 m or greater
weight
Light Scattering
1 - 200 p m
volume or weight
Electronic Sensing
1 - 300 pm
volume
Sedimentation
2 - 200 pm
weight
Optical Microscopy
0.5
- 100 p m
number or volume
The data obtained by such measurements may be presented graphically (126). This allows for easy visualization of the frequency of the various particle sizes. Depending upon the method used the frequency may be expressed by a cumulative distribution plot Figure 15, or a Gaussian-type size frequency distribution plot, as shown in the particle size distribution of commercial Povidone K-30 (Figure 16 ) Figure 15 (Ref. 126)
1.0
20
30
40
50
SIZE C u m u l a t i v e d i s t r i b u t i o n plot
POV IDON E
Figure
16
The most commonly used method is sieving. The method is not time consuming and it is independent of the individual skill of the operator. The procedure involves the mechanical shaking of the samples through sieves of successively smaller openings, followed by weighing of the portion of the sample retained o n each sieve. There are several methods b y which the powder can be swirled on the sieve. The most efficient method is vibration either mechanically or by ultrasound. Swirling by fast moving stream of air is also quite effective, though simple mechanical shaking may also be used with or without tapping. The time of sieving as well as the load of powder on the sieve have to be standardized. Since temperature and relative humidity influence the accuracy of PVP particle size classification by sieving, the condition of the sample has t o be kept within cenain limits. Temperatures between 70 and 85OF with relative humidity between 45 and 48% are acceptable ambient conditions.
7.361 Determination of Particle Size Distribution The test methods cover the measurement of the particle size of dry Povidone powder. The methods utilize dry sieving using various methods to move the particles over the surface of the sieves. The mass of the Povidone
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
628
powder is placed on a series of sieves arranged in order of increasing fineness and the mass is divnded into fractions corresponding to the sieve opening. Alternatively, single screen may be used in which case the weight percent of the "passing through" powder is determined. 7.361 1 Determinatioln with Mechanical Sievina Device
1. Balance - 500 g minimum with 1I1 0 g sensitivity.
2. Sieving Device - Mechanical sieve-shaking device equipped with a time switch. The device shall be capable of imparting uniform rotary motion and a tapping action at a rate of 1 5 0 1 0 taps per minute.
*
3. Wire Cloth Sieves - Woven wire cloth of the suitable fineness, mounted in 8-in. (203 mm) frames. The number of sieves is to be determined for the purpose of the test. 4. Screen Cleaning Accessories
- Brush, vacuum cleaner air hose.
SamDling
1. Plastic materials may segregate by particle size during handling. HOmOgeniZe the lot where possible before removing the test sample. PreDaration of ADDaratus 1 . Thorough cleaning and inspection of the sieve are required prior to initiating a test. Carefully clean the sieves with a brush and vacuum cleaner or compressed air, or both. Periodic washing with soap and water or suitable solvent may be required.
2. Tare each sieve and the pan. Record tare weights to the nearest 1/10 g. 3. Assemble sieves so that the sieve openings decrease in size in sequence from the top of the stack. Place the pan at the bottom. 4. Use full- or half-size screens to accommodate the holder in the shaker.
Conditioning 1 . The Povidone sample must be in a free flowing condition.
2. If possible, the sample should be conditioned to the laboratory temperature and humidity.
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Procedure
1. Select sieves in sufficient number t o cover the expected range of particle sizes, and nest them together in order of diminishing opening with the coarsest sieve on top and the pan on the bottom. Note 1: Select sieves in sufficient number t o have significant measurable quantities on four or more sieves.
2. Weigh 50 g of sample and transfer it t o the top of the stack. Note 2: This test may be made on a sample of any size. A larger sample size could cause screen blinding and skew the results t o the coarse particle size. A screen can be considered blinded if it is holding 20 or more g. For repeatable results, use a smaller sample size. Record the sample weight used. Note 3: The use of an antistat (or slip agent) is needed. Add 1 % of the antistat (or slip agent) t o the sample and mix in with a spatula. State in the report, the agent used.
3. Cover the stack and place it in the mechanical sieve shaker. Start the shaker and run for 10 min f 1 5 s. Longer times may be required depending on the efficiency of the shaker. 4. After shaking, carefully separate the stack of sieves, beginning at the top, and weigh the quantity of powder retained on each sieve and that contained in the pan t o the nearest 1/10 g. This may be accomplished either by transferring the fractions t o the balance or by weighing either the sieve or the pan and its contents and subtracting the tare weight from the total. If the powder is transferred t o the balance, carefully brush the sieve on both sides t o ensure that adhering particles are transferred.
Note 4: If the cumulative total of actual weight is less than 98%, carefully check the weights and operations and repeat the work if necessary. Calculation of Particle Distribution Obtain net weight of material retained on each sieve. Calculate percentage
by dividing net weight by total sample weight x 100. Repeat for each sieve. Calculation of Mean Particle Size Obtain net weight of material retained on each sieve. Determine an average particle size for each sieve. The average particle size is defined as the nominal opening size of that sieve plus the nominal opening size of the next larger sieve in the stack divided by two. Calculate the mean particle size as:
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
630
where: D, = mean particle diameter, in microns, P, = percent of material retained on sieve (or pan), and D, = average particle size in microns of material on sieve. Hazards The sieving operation and cteaning o f the sieves can introduce dust from the material and antistat agent into the atmosphere. Take precautions to avoid breathing these palnicles. 7.352 Determination with Air-Jet Sieve
1. Air-Jet Sieve Equipment
- Alpine type or equivalent.
2. Standard Testing Sieves - Five 200 mm U.S. Standard Testing Sieves (ASTM E-11 specification) or equivalent. The fineness of the sieves are 16, 30, 50, 80 and 200 mesh respectively. 3. Sieve cleaning brush - flat-ended, soft bristle brush, specifically made for cleaning sieves. 4. Ultrasonic cleaning bath - sized t o accommodate a 200 mm sieve. 5. Electronic Top-Loading Balance - readability to 0.01 g or equivalent.
6. Psychrometer - ,for measuring relative humidity. 7. Humidifier - ultri3sonically driven.
8. Dehumidifier. 9. Air blow gun, hose and filter - capable of removing oil, moisture and foreign matter from air supply. Procedure 1 . Evenly coat the plastic sieve cover with a coating of anti-static agent.
2. Adjust the temperature and relative humidity of the air supply t o the specifics mentioned previously.
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3. Run the sieving unit under the proper humidity/temperature requirements for about ten minutes with a clean sieve screen and cover (no sample) in place. Adjust the vacuum setting t o 10 t o 12 inches of water.
4. Turn off the unit, remove and accurately weigh together the sieve and cover t o f 0.01 g. Add 10 f 0.5 g of sample t o the center of the screen while it is still on the balance. In this manner any fine particles which pass through the screen will collect on the balance pan. Carefully replace the lid and record the weight t o the nearest 0.01 Q.
5. Place the covered sieve (with sample) on the unit and turn on the unit (timer set t o three minutes). After approximately t w o minutes gently tap the sieve and cover t o remove much of the powder held by static charge.
6 . After the three minute period is complete, weigh t o f 0.01 the sieve and the sample remaining on the screen, leaving the plastic cover in place.
7. Using the flat-end brush, brush the top and bottom of the screen, tapping the sieve frequently to remove the loose powder. Then clean both sides of the screen with the air gun. Clean the brush with the air gun and then repeat the above screen cleaning procedure.
CAUTION: The sieve should be cleaned in an area where breathing of the dust will be minimized, preferably in an exhaust hood. Calculations
I n i t i a l Wt of ( C + S c + S p ) - F i n a l W t of ( C + S c + Sp) Initial W t o f ( C + S c + S p ) - W t o f ( C + S c ) C
= Wt%
Cover Screen Sp = Sample
S
7.37
= =
Bulk Density
Bulk density of a powder is the ratio of the weight of the powder t o the volume it occupies. This value is expressed in g/cm3(g/l) or Ib/ft3. Bulk density differs from the so called "true" or absolute density of the powder in as much as latter accounts only for the solid portion of the particles, while the bulk density includes also the voids between the particles. Because of its dependence on particle packing, the packing condition of the sample should be reported. The "poured" bulk density is determined by slowly pouring the powder into a graduated cylinder. The "tapped" bulk density obtained by tapping the
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
632
cylinder in a predetermined fashion after pouring until the volume of the powder ceases to Change (127). The bulk density is important not only because of equipment size considerations in manufacturing, handling and storage, but also because of product uniformity due to possible differences between the densities of the drug and excipients. The method for the determination is given in 7.371. Typical values for PVP are listed in Table 8. Table 8 Bulk Densities of PVP
K-15 K-25 K-30 K-90
400 - 600 gll 400 - 600 gll 400 - 600 g/l 110 - 250 g/l
7.371 Determination of Bulk Density
Graduated cylinder, 50 milliliters Procedure Obtain the tare weight of a 50 mL graduated cylinder. Add Plasdone in approximately 5 mL increments. After each addition tap the base of the cylinder on a book until the measured volume is constant. Continue until the total volume between 45 and 50 mL is accumulated. Weigh the cylinder and contents to determine the sample weight, W, t o the nearest centigram. Read the volume, V, to the nearest 0.1 mL. Calculation
where D is bulk density V is volume of Povidone W is the weight of Povidone
7.38
Flow Properties
Pharmaceutical powders may be characterized as free flowing or cohesive, that is non-free flowing (128).These terms describe the ability of the particles t o move on the effect of gravity or other external forces. Good flow assures
POVl DON E
633
efficient mixing of the powder which is necessary for the production of uniform tablets. The f low of the powder is strongly influenced by its compressibility (1291, that is the ability of the powder to decrease its volume under pressure. This property can be calculated from the density characteristics. % Compressibility
=
v -v * v,
100
where V, is tapped bulk density and V, is the density before tapping. Table 9 shows the practical correlation between the t w o values. Table 9 Correlation Between ComDressibilitv and Flowability
% Commessibility 5-15 1 2 - 16 1 8 - 21 23 - 35 33 - 38 < 40
Flowability Excellent Good Fair Poor Very poor Extremely poor
The determination of flow properties may be done by placing the powder in a grounded metal tube and let it flow through an orifice onto a balance which is connected t o a suitable recorder. The flow rate (g/sec) is dependent upon the true particle density and the diameter of the orifice and can be calculated by the following equation (130).
where D, is the orifice diameter, W is the flow rate, p is the particle density and g is the gravity. A and n are constants dependent upon the material and the particle size. The flow rate may be influenced by factors which affect the free movement of the particles, their intrinsic adhesive properties, the electrostatic forces which develop as a consequence of the friction between the moving particles, and the adsorbed layer of moisture which can help to dissipate the electrostatic forces. On the other hand the moisture layer may form bridges among the particles thus holding them together through surface tension. The effect of these factors can be estimated by the so called "angle of repose", which is the maximum angle that can exist between the free-standing surface of the powder pile and the horizontal plane (1311. There are several ways to determine the "angle of repose". In Method A the powder is allowed to flow from a funnel onto a horizontal, flat surface. In
634
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
Method B the powder is rotated in a drum and after stopping the rotation the angle of the stationary powder is measured. The values obtained b y these methods are called “dynamic angle of repose’. In Method C the tabular container filled with the powder is resting on a horizontal, flat surface. The pile of powder is formed while the container is lifted away vertically from the surface. The value determined by Method C is called ”static angle of repose’ (1 32). The scheme of these methods are illustrated in Figure 17. Fiaure 17 Determination of the Angle of Repose
SLWE OF JIOE FORMEO OV W O E R C L O n l N I rOrVOER Fb
Q
ANGLE Of REPOSE tdvrumrl
mooin LEVEL SURFACE Method 1
fly.A
ROTATINQ O R W
Method 1
AWAY FROM SURFACE
\ W E N TUB€ FILLED WlTW COWOLR
.......
Method 3
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7.381 Determination of Flowability Figure 18 Apparatus for the Determination of Flowability
1
a) metal funnel stand b) funnel (glass or stainless steel) c) inside volume marking d) funnel holder ring, which sets the position of the funnel el stand (attached t o the funnel - stand) to hold the metal plate that closes the funnel bottom opening f) stainless steel plate g) chart holding clamps h) chart Any other apparatus that allows the formation of a pile of powder reproducibly, may also be used.
636
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
Procedure
1. The powder to ble tested is to be mixed with a stainless steel spatula until all the clumps are broken up.
2. Place the funnel on the stand. The bottom of the funnel should be exactly 1 inch from the platform of the stand.
3. Insert the stainlless steel plate which closes the bottom opening of the funnel.
4. Pour the powder into the funnel until it is filled exactly to the inside volume marking. Hit the top of the funnel a few times t o level the powder exactly at the marking.
5. With quick horizontal movement remove stainless steel plate (f) and let the powder flow.
6. With a pencil draw the contour of the powder on the chart paper. 7. Replace the stainless steel plate at the bottom of the funnel and collect the powder flown through the orifice. (It may be used to refill the funnel for test repetition.1
8. Determine
by averaging the diameter of the flown out pile of the
.XI
powder. Calculation
1
1
Angle of Repose ( a
I
= Arc Sin
-
1-
I
I
1
+
-
x2
I I
Arc tg X
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631
Since Povidone is quite hygroscopic it is possible that it Changes its flow properties during industrial manipulations with diminishing flow and increasing cohesiveness. In order t o obtain reliable flow measurements, the condition of the powder has t o be carefully controlled.7.39 7.39
Solubility
The solubility of polymers differs from that of nonpolymeric solids both qualitatively and quantitatively (133). Nonpolymeric solids have a well-defined limit of solubility which can be defined by the amount of solid which can be brought into solution by a certain volume of solvent. The amorphous polymers are usually soluble without limit. In this respect the solubilizing process is similar to the mixing of t w o liquids. Due t o the thermal motion the segments of the polymer make room for the more mobile solvent molecules, until the intimate mixing produces a solution. Therefore, the dissolving of the polymer is a diffusion controlled process. Usually there are several solvents capable to dissolve each polymer. The method of determining the suitability of a solvent is through its solubility parameter (61, which is the square root of the cohesive-energy density (134). This value is numerically equal to the potential energy of one cm3 of material. The energy is produced by the Van der Waals forces which exist between the molecules of the materials. In order t o form a solution it is necessary that polymer and the solvent have similar polarity, but - beyond that - the polymer will form solution only with those solvents whose cohesive-energy density is close t o its own. During the dissolution process intramolecular bonds of solvents and polymers are broken and new bonds between solvent molecules ( S ) and polymer molecules (P) are formed. If the S-S bonds, P-P bonds and P-S bonds are similar t o each other, the replacement of the former ones with newly formed P-Sbonds is energetically easy. On the other hand, if the S-S or P-P bonds are very strong as compared t o the S-P value, the entering of the polymer molecule into the solvent is not feasible (135.1 36). The only reliable way t o determine the cohesive-energy density of the solvent is by its molar heat of evaporation (AH,)(1371, using the following equation:
where V is the molar volume. Since the polymer does not evaporate without decomposition, its cohesiveenergy density can be best determined by measuring the intrinsic viscosity in a series of solvents. The 6, value is considered t o be equal to the 6, value of the solvent which gives the highest intrinsic viscosity (138).In another widely used method the crosslinked polymer is swelled in a series of liquids t o find the 6, of the solvent in which the polymer is swelled the most (139).
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
638
Small has published a table of "molar attraction constants" which allows the estimation of the solubility parameter without experimentation from the structural formula of the compound and its density (140). Solvents had been classified according to their proton donating or proton accepting character (141), as well as from the standpoint of the strength of their respective hydrogen bonding capacities (142). Povidone - itself a weak electron donor - dissolves best in strongly hydrogen bonding compounds of the type which can act simultaneously both as proton donors and acceptors, such as water, alcohols, carboxylic acids, primary and secondary amines. The determination of the solubility is given in Section 7.391 . In the pharmacological application an important factor is the "rate of dissolution" (1431, which is, while closely related to the solubility of the polymer, depends (also from its particle size, surface area and wetting properties. The rate of dissolution of the polymeric ingredients of tablets and capsules influences the bioavailability and delivery of the drug decisively. From the Noyes-Nernst equation a t constant temperature and constant surface area, the dissolution rate can be expressed by dC/dt, where "C" is the concentration of the polymer a t time It". The plot of "C" versus "t" gives the intrinsic dissolution rate constant the dimension of which is g/cm2 (144, 145, 146). The pellet used for the d!eterminationof the rate of dissolution contains an active ingredient (drug), the concentration of which in the solution is the measure of the rate. The rate of dissolution as expressed by the intrinsic rate constant is directly proportional to the solubility of the material tested. It has been found, however, that the solubility of acidic and basic drugs is pH dependent which may cause deviation from the correlation proposed by Noyes and Nernst (147,148). Therefore, in the determination of the intrinsic dissolution behavior the pH of the system has to be taken into consideration (149,150). The dissolution can be brought about either by agitating the solvent over the stationary pellet or by rotating the pellet in the solvent (151). The general concept of the procedure is shown in Figure 19. The method to determine the rate of dissolution is described in Section 7.392. 7.391 Determination of Solubility ADDaratUS 1. 2. 3. 4. 5.
Constant temperature bath (controllable at 25 k l0C) Thermometer 800 ml standard glass beaker Constant speed stirrer motor which can be adjusted to 465 rpm 3 blade stainless steel stirring propeller (blade diameter 4.5 cm)
rt
20 rpm.
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Fiaure
Synch-
639
19
Mom Sampling port
Stlrring shaft Ustcr Jxkttrd ylink
Rotating d t . Drug pellet
Constant surface area dissolution apparatus. Left: static disc dissolution apparatus (Paddle stirring), Right: rotating disc apparatus (Basket stirring). (Ref. 126). Procedure
1. Place an 800 ml beaker containing 465 g of distilled water in a constant temperature bath at 25 f l0C.
2. Set up a 3-blade stirring propeller calibrated at 465 rpm f 20 rpm over the beaker.
3. Submerge the stirrer blades in the water in the beaker such that the bottom of the stirrer rod is positioned at a distance of 3 cm from the surface of the water. 4. Weigh 3 5 g of PVP into a plastic tray.
5. Start the motor. 6. Simultaneously start stop watch and carefully pour the weighed amount of PVP uniformly over a period of 3 minutes into the vortex caused by the stirrer, taking care to minimize clumping. 7. Continue stirring for a total of 15 minutes. Then stop the stirrer and stop watch.
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
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8. If, during the 1 5 minute stirring, the polymer totally dissolves, record the time at which it dissolved and terminate the test. 9. Leave the stirrer off for 3 minutes, allowing air bubbles t o rise to the surface of the beaker.
10. Reposition the blades of the stirrer 1.5 cm deeper into the beaker (i.e. 4.5 c m from the surface of the liquid). 11. Restart simultaneously the stop watch and the stirrer.
12. In a rapid motion, carefully dislodge any polymer clumps or large air bubbles from the sides o f the beaker using a narrow spatula. 13. Continue stirring until no clumps or undissolved material are visible in the bulk of the liquid. 14. Stop stirrer and stop watch and record time taken for the complete solubilization of the polymer as indicated by the stop watch. 15. Run test in triplicate. 7.392 Rate of Dissolution For the determination of the rate of dissolution both basket stirring and paddle stirring methods may be used (150). The respective systems are illustrated in Figure 19. For following the rate of dissolution should contain an analyzable ingredient, which is released as the tablet disintegrates. Any active ingredient is suitable which offers fast and accurate analysis. ADDaratus (15 1) Basket stirring method - The assembly consists of the following: a covered vessel made of glass or other inert, transparent material; a motor; a metallic drive shaft; and a cylindrical basket. The vessel is partially immersed in a suitable water bath of any convenient size that permits holding the temperature inside the vessel at :37 -I- 0.5' during the test and keeping the bath fluid in constant smooth motion. Apparatus that permits observation of the specimen and stirring element during the test is preferable. The vessel is cylindrical, with a hemispherical bottom. It is 160 mm to 175 mm high, its inside diameter is 9 8 mm t o 106 mm, and its nominal capacity is 1000 mL. Its sides are flanged at the top. A fitted cciver may be used to retard evaporation. The shaft is positioned so that its axis is not more than 2 mm at any point from the vertical axis of the vessel and rotates smoothly and without significant wobble.
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A speed-regulating device is used that allows the shaft rotation speed to be selected and maintained within f 4%. Shaft and basket components of the stirring element are fabricated of stainless steel, type 31 6 or equivalent. Use 40-mesh cloth. A basket having a gold coating 0.0001 inch (2.5 pm) thick may be used. The dosage unit is placed in a dry basket at the beginning of each test. The distance between the inside bottom of the vessel and the basket is maintained at 25 t 2 m m during the test. Paddle stirring - Use the assembly from basket stirring, except that a paddle formed from a blade and a shaft is used as the stirring element. The shaft is positioned so that its axis is not more than 2 mm at any point from the vertical axis of the vessel, and rotates smoothly without significant wobble. The blade passes through the diameter of the shaft so that the bottom of the blade is flush with the bottom of the shaft. The distance of 25 t 2 mm between the blade and the inside bottom of the vessel is maintained during the test. The metallic blade and shaft comprise a single entity that may be coated with a suitable inert coating. The dosage unit is allowed to sink to the bottom of the vessel before rotation of the blade is started. A small, loose piece of nonreactive material such as not more than a few turns of wire helix may be attached t o dosage units that would otherwise float. The thickness of the test-tablet should be 0.175 inch and it should be compressed t o give a hardness of 6-8 kilopounds and friability not more than 2.0%. It should contain about 200 mg active ingredient. Procedure 1. Place 900 mL distilled water (or phosphate buffer solution, or 0.1 hydrochloric acid - depending upon the drug in the tablet) in the vessel of the apparatus and assemble the stirring unit.
2. Equilibrate the temperature of the bath to 37 t 0.5OC,then remove the thermometer.
3. Place one tablet in the apparatus and check that the surface of the tablet does not carry air bubbles. (Remove bubbles with a thin spatula, if necessary). 4. Immediately start operating the apparatus. The rate of agitation depends upon the nature of the drug of the tablet following the instructions of USP XXll (152).
5. The suitable time intervals withdraw a specimen from a zone midway between the surface of the water and the top of the rotating basket (or - if paddle stirring is used - the top of the blade), not less than 1 cm from the vessel wall.
642
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
6. Analyze the sample for the active ingredient.
7. Continue sampling until tablet is completely disintegrated and not less than 95% of the active ingredient is accounted for. 7.310 Sediment C o n m During the preparation and handling some insoluble inorganic or organic contaminants may get in the Povidone powder. The amount usually is small and typical products will contain no more than 100 ppm of such sediment. The determination of the sediment content may be carried out by a sediment testing apparatus developed for evaluating milk and similar dairy products. The particulate residue is retained on the filter element of the apparatus and it is visually compared to standards showing known amounts of particulate sediment. The equipment is shown in Section 7.3101. 7.31 01 Determination of Sediment Content Amaratus 1. Clark Dairy Supply Sediment Tester, model "J" or equivalent.
2. Clark Dairy SP-10 filters, or equivalent (Note 1I .
3. Water aspirator or' vacuum pump. 4. 4000 mL filtering flask, heavy wall.
5. Rubber stopper with hole bored to accommodate filter funnel pipe extension.
6. Magnetic stirrer-hot plate with 2 inch magnetic stirring bar. 7. APHA Official Milk Sediment Standard Photograph (Note 2) or equivalent. Fiaure 2Q Sediment Tester
643
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Procedure 1. Assemble filtration system with filter element as shown in Figure 20.
2. Pour approximately 2 5 0 0 mL distilled water into a clean 4000 mL beaker. Suspend the thermometer in such a way that it is suspended in the water with the bulb above the bottom of the beaker.
3. Place water filled beaker on the hot plate-stirrer and agitate water with a moderate motion. Adjust temperature control to heat the water just under the boiling point (approximately 98OC). Add water occasionally, t o maintain 2500 mL level. 4. Accurately weigh 100.0 g Povidone into a clean, dry beaker.
5. When the water reached the designated temperature, increase speed of agitation until a vortex developed which almost reaches the bottom of the beaker. Carefully pour some Povidone into the vortex and gently push Povidone farther with a small spatula. When the powder is dispersed in the water, ad additional load and continue until all the Povidone has been dispersed. Reduce agitation speed to gentle motion while maintaining the temperature at 98OC. 6. When Povidone is completely dissolved and the solution is clear, turn stirrer off. Turn filter apparatus vacuum source on and carefully pour the Povidone solution into the filter funnel. Rinse beaker and filter apparatus with distilled water. 7. Remove filter element from apparatus and place on white background. While wet, visually compare the retained sediment to the appropriate standards (course or fine) and determine the total mg content by matching the sample t o the closest standard photograph.
8. Alternatively (especially for lower molecular weight grades of Povidone) the sample can be shaken at room temperature for an extended time period to achieve complete dissolution. Calculations ppm sediment(dry b a s i s ) = (mg on filter)
Note 1: Note 2:
*
10
(Clark Dairy Supply Co., Inc., 430 East Main Street, Box 157, Greenwood, IN 461 42) (Standardization Branch, Dairy Division, Agricultural Marketing Service, U.S. Department of Agriculture, Washington, DC 20250)
CHRlSTlANAH M. ADEYEYE AND EUGENE BARABAS
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7.4 Instrumental A n a M 7.41
ChromatoaraDhy
The basic feature of chromatography is a system which consists of two immiscible phases, one of which is stationary and the other is mobile. The sample is introduced into the mobile phase and by the effect of an outside force (carrier gas or eluent11it is carried along through a container (column or plate) holding the stationary phase (153).In the course of this movement the sample undergoes repeated partitions between the mobile phase and the stationary phase. As a result of these repeated interactions the components of the sample separate into bands in the mobile phase. The separated components are received according to their increasing interaction with the stationary phase. The least strongly retained fraction appears first, followed by fractions of increasing retention (1 54). By the proper selection of the phases, the forces bringing about the separation, due to differences in adsorption (1551, molecular size (1561, polarity or ionic charye density (1571, can be maximized. 7.41 1 Gas Chromatoaraohv (GCL Gas chromatography is the most widely used method for the separation of volatile organic and inorganic compounds. It achieves the separation by partitioning the components of the sample between a mobile gas phase and a stationary liquid phase held by a solid support or by a porous solid support (158). The chromatograph consists of several elements: a) the mobile phase carrier gas that flows through the system with predetermined velocity. As carrier gas helium, nitrogen or other inert gas is used, b) a device to introduce the sample into the mobile phase (usually by injection), c) the chromatographic column of appropriate length holding the stationary phase on the proper carrier. (No carrier support is used in most capillary columns.) The stationary phase has to be a liquid or a solid with good thermal and chemical stability, d) an oven which maintains the column a t the appropriate programmable temperature,
e l a detector which can register the components of the sample. Detectors commonly used depend upon thermal conductivity, flame ionization or electron capture,
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f ) a device which provides a signal proportional to the amount of each component of the sample. The resulting record is a signal-time plot - the chromatogram - on which each component appears as a bell-shaped peak on a time axis (159). Ericsson and Ljunggren (160) recently published a study in which PVP was analyzed by pyrolysis-gas chromatography. 7.41 2 Hiah Performance Liauid ChromatoaraDhv (HPLC) HPLC - sometimes referred to also as "high pressure liquid chromatography" (161) - uses small-bore (2 to 5 mm) columns and small size (3 - 50 pm) packings, which allow fast equilibrium between mobile and stationary phases. Because of the small-bore, small-particle packing equipment, the delivery of the mobile phase requires pressure, which can be as much as 300 atmospheres (approximately 4400 PSI) to achieve flow rates of several mL per minute (162,163). The most often used types of HPLC are a) ion-exchange chromatography, for the separation of water soluble ionic or ionizable materials of molecular weights less than 1500. The stationary phases are usually synthetic organic resins with different types of active groups (cation- or anion-exchange resins) (162). b) partition chromatography in which the substances to be separated are partitioned between t w o differently polar, immiscible liquids one of which - the immobile phase - is adsorbed on a solid support. In this way a very large surface area is created for the mobile phase (the flowing solvent). The high number of successive liquid-liquid contacts allows a very efficient separation (164). c) adsorption chromatography, which utilizes the affinity of the ingredients of the sample towards the stationary phase as they move along the column at a characteristic rate resulting in a spatial separation (165,162). The advantage of HPLC is that it can separate quickly materials which could not be analyzed by GC, due to their high molecular weight or low thermal stability. Investigations involving PVP and HPLC analysis include the work of Koehler (166) and Kou, et a/ (167). 7.41 3 Thin Laver ChromatoaraDhy Chromatography is a procedure by which solutes are separated by a differential migration process in a stationary phase (168,169). In this process the individual substances of the mobil phase exhibit different mobilities due to differences in adsorption, molecular size, polarity or ionic charge density. The adsorbent is a relatively thin, uniform layer of dry, finely powdered material, such as silica gel or alumina, together with a binding agent. The adsorbent is spread on a glass plate and the latter functions as a physical support for the adsorbent layer which does not h w e sufficient rigidity by itself. The uniformity of the adsorbent is very critical with respect to the reproducibility of the method (170).
646
CHlRlSTlANAH M. ADEYEYE AND EUGENE BARABAS
The coated plate can be considered an open chromatographic column and the separation achieved is based upon adsorption, partition or the combination of these effects, depending upon the composition and physical properties of the adsorbent and the nature of the mobil phase. The recommended grain size of the adsorbent is 60 p or smaller, and the thickness of the layer is about 250 p . The detection usually requires the use of a color forming agent. Identification can be effected by the observation of spots obtained respectively with an unknown and a reference sample chromatographed on the same plate. Quantitative measurements are possible by densitometry, fluorescence or fluorescence quenching. The spots may be carefully removed from the plate followed by elution with a suitable solvent and spectrophotometric measurement ( 17 11. 7.42
Thermal Analvsis
Thermal analysis in general is a technique by which certain change in the physical properties of a material is measured as a function of temperature. The thermal analysis may give both qualitative and quantitative information in this respect (172). 7.421 Differential Scannina Calorimetry and Differential Thermal Analvsis Differential scanning calorimetry (DSC)determinesthe thermal energy which appears in the case of: a thermal transition, that is, when a chemical or physical change takes place, which results in the emission or absorption of heat. In DSC both the sample and the reference (a thermally inert material) heated with the same heat source. During the thermal transition both sample and reference are maintained at the same temperature, therefore, the thermal energy is added either to the sample or to the reference. Since the transferred energy is equivalent to the energy absorbed or produced during the transition, the temperature balancing energy is a direct calorimetric measurement of the energy of transition (173). Differential thermal1analysis (DTA) directly measures the heat-energy change that occurs in the substance as a function of temperature. The sample is heated side by side with the thermally inert reference material a t a uniform rate, and the temperature difference between them is measured as a function of temperature. The plot obtained that way shows whether the transition is exothermic or endothermic. DTA is used also to determine the glass transition temperature of the polymer, the point a t which the polymer changes from brittle, glass-like material to a tough, rubbery structure. It is an important feature, because the lower the glass transition temperature, the lower is the temperature a t which the polymer is useful in applications (174). Studies in which PVP alone, or with other polymer has been characterized using thermal analysis include publications of Cesteros, et a/ (175); and Thyagarajan and Janarthanan (176).
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7.422 Thermooravimetric Analvsis Thermogravimetric analysis (TGA) is a continuous process that involves the measurement of sample weight as the reaction temperature is changed by means of a programmed rate of heating. It provides quantitative data of any weight changes which occur as the consequence of thermally induced transitions, such as dehydration or decomposition. The chemical and molecular structure of the compounds determines the rates of thermally induced transitions. Therefore, the sequence of physical and chemical changes that take place over a definite temperature range will depend upon the chemical and physical properties of the compound in question. Consequently the thermogravimetric curves are characteristic of a given compound ( 1 77). On the effect of increasing temperature chemical bonds may break or new bonds may form, which can produce changes in the weight of the sample. TGA is a suitable method to characterize materials and to give data on the thermodynamic and kinetic descriptors of the reactions and transitions which take place on the effect of heat. It determines the thermal stability of the compound and produces useful data for it's compositional analysis ( 1 78). The thermogravimetric analysis of Povidone K-30 in both air and nitrogen is shown in Figure 21. Fiaure 21 (Ref. 105)
Solid line represents sample in air while dotted line represents sample in nitrogen.
648
7.43
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
X-Rav Diffraction
Polyvinylpyrrolidone does not have X-Ray diffraction properties because of its amorphous nature.
7.44
Methods of Instrumental Analvsis
In the instrumental analytical procedures follow the instructions supplied b y the manufacturers of the respective instruments. 7.5
Microbial Limit Tests (1 79)
Since Povidone is used in a variety of pharmaceutical applications, its sample has t o be entirely free of all kinds of microorganisms. The tests to prove that have t o show the absence o f bacteria of both Gram-positive and Gramnegative type, as well as the absence of mold and yeasts. Microbial limit t e s s provide information about the number of viable aerobic microorganisms present and for freedom from designated microbial species in Povidone used in pharmaceutical application. The validity of the results of the tests rests largely upon the adequacy of a demonstration that the test specimens t o which they are applied do not, of themselves, inhibit the multiplication, under the test conditions, of microorganisms that may be present. Therefore, preparatory t o conducting the tests it has ta be ascenained that the Povidone sample does not contain ingredients, which would prevent the growth of viable cultures of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Salmonella. The fact that Povidone is an excellent nutrient for bacterial growth, makes these tests particulatly important. The tests are carried out by inoculating sterilized growth media - containing the Povidone sample - with various bacteria or mold, then after incubation determine the number of microbial colonies of visual observation. A n automated method may be used for the analysis, provided it has been properly validated as giving equivalent or better results. Aseptic conditions have to be maintained in handling the specimens. Where the procedure specifies "incubate", the container has to be kept in air that is thermostatically controlled at a temperature between 30' and 35', for a period of 2 4 t o 48 hours. The term "growth" is used in a special sense herein, i.e., to designate the presence and presumed proliferation of viable microorganisms.
7.51
PreDaratorv Testinq
Preparatory testing is to be carried out with viable cultures of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella. The testing is to be done by adding 1 mL of not less than 10-3dilution of a 24-hour broth culture of the microorganism t o the first dilution (in pH 7.2 Phosphate Buffer, Fluid Soybean-Casein Digest Medium, or Fluid Lactose Medium) of the test material and following the test procedure. Failure of the organism(s) t o grow in the relevant medium invalidates that portion of the examination and necessitates a modification of the procedure by ( 1 ) an increase in the volume of diluent, the quantity of test material remaining the Same
orby
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(2) the incorporation of a sufficient quantity of suitable inactivating agent(s) in the diluents, or by (3) an appropriate combination of modifications (1 1 and (2) so as t o permit growth of the inocula. If the behavior of the test cultures indicate the presence of inhibitory substances in the Povidone sample, 0.5% soy lecithin or 4.0% polysorbate 2 0 may be added t o the culture medium to neutralize the effect of such substances. If in spite of the incorporation of suitable inactivating agents and a substantial increase in the volume of diluent it is still not possible to recover the viable cultures described above it can be assumed that the failure to isolate the inoculated organism is attributable to the bactericidal activity of the sample. This information serves to indicate that it is not likely t o be contaminated with the given species of microorganism. Monitoring should be continued in order t o establish the spectrum of inhibition and bactericidal activity of the sample. 7.52
PreDaration of Stock Solution and Media
7.521 PreDaration of QH 7.2 PhosDhate Buffer (Stock Solution) Stock Solution - Dissolve 3 4 g of monobasic potassium phosphate in about 500 mL of water contained in a 1000 mL volumetric flask. Adjust t o pH 7.2 f 0.1 by the addition of sodium hydroxide TS (about 175 mL), add water to volume, and mix. Dispense and sterilize. Store under refrigeration. For use, dilute the Stock Solution with water in the ratio of 1 t o 800, and sterilize. 7.522 Preoaration of Media For the formulas used in the preparation of media such as fluid Casein Digest-Soy Lecithin-Polysorbate 20, Mannitol-Salt Agar and Vogel-Johnson Agar - see Reference 179. 7.53
Procedure
Total Aerobic Microbial Count Dissolve 1O.Og of Povidone in pH 7.2 phosphate buffer t o make 100 mL. For viscous samples that cannot be pipeted at this original 1 :10 dilution until a solution is obtained, i.e. 1:50 or 1:100, etc., that can be pipeted. Ascertain that this solution does not have antimicrobial properties (See Section 7.5). Add the specimen to the medium not more than 1 hour after preparing the appropriate dilutions for inoculation. 7.531 Plate Method Dilute further, if necessary, the fluid so that 1 mL of the final dilution onto each of two sterile petri dishes. Promptly add to each dish 15 to 2 0 mL of Soybean-Casein Digest Agar Medium that previously has been melted and cooled t o approximately 45'. Cover the petri dishes, mix the sample with the agar by tilting or rotating the dishes, and allow the contents t o solidify at room temperature. Invert the petri dishes, and incubate for 4 8 to 7 2 hours. Following incubation, examine the plates for growth, count the number of colonies, and
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
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express the average for the two plates in terms of the number of microorganisms per g or per mL of specimen. If no microbial colonies are recovered from the dishes representing the initial 1:10dilution of the specimen, express the results as "less than 10 microorganisms per g or per mL of specimen".
7,532Multiole-Tube Method Into each of fourteen test tubes of similar size place 9.0 mL of sterile Fluid Soybean-Casein Digest Medium. Arrange twelve of the tubes in four sets of three tubes each. Put aside one set of three tubes t o serve as the controls. Into each of three tubes of one set ("1 00")and into a fourth tube (A) pipet 1 mL of the solution or suspension of the specimen, and mix. From tube A, pipet 1mL of its contents into the one remaining tube (B) not included in a set, and mix. These two tubes contain 100 mg (or 100 pL) and 10 rng (or 10 pL) or the specimen, respectively. Into each of the second set ("10") of three tubes pipet 1 mL from tube A, and into each tube of the third set ( " 1 " ) pipet 1 mL from tube B. Discard the unused contents of tubes A and B. Close well, and incubate all of the tubes. Following the incubation period, examine the tubes for growth: the three control tubes remain clear and the observations in the tubes containing the specimen, when interpreted by reference to Table 10, indicate the most probable number of microorganisms per g or per mL of specimen. Table 10 Most Probable Total Count bv Multiole-Tube Method Observed Combination of Numbers of Tubes Showing Growth in Each Set No. of mg (or mL) of Specimen per Tube
100 (1 00 PLI
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
10 (1 0 UL) 3 3 3 3 2 2 2 2 1 1 1 1 0 0 0
1 (1 pL) 3 2 1 0 3 2
0
0
1
0
3 2 1 0 3 2 1
Most Probable Number of Microorganisms per g or per mL
>1100 1100
500 200 290 21 0 150 90
160 120 70 40 95 60 40 23
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6.5 I
7.533 Test for StaDhvlococcus Aureus and Pseudomonas Aeruqinosa To the specimen add Fluid Soybean-Casein Digest Medium to make 100 mL, mix, and incubate. Examine the medium for growth, and if growth is present, use an inoculating loop to streak a portion of the medium on the surface of the Vogel-Johnson Agar Medium (or Baird-Perker Agar Medium, or Mannitol-Salt Agar Medium) and of Cetrimide Agar Medium, each plated on petri dishes. Cover and invert the dishes, and incubate. If, upon examination, none of the plates contains colonies having the characteristics listed in Tables 1I and 1 2 for the media used, the test specimen meets the requirements for freedom from Staphylococcus aureus and Pseudomonas aeruginosa. Coagulase Test (for Staphylococcus aureus) - With the aid of an inoculation loop, transfer representative suspect colonies from the agar surfaces of the Vogel-Johnson Agar Medium (or Baird-Perker Agar Medium, or MannitolSalt Agar Medium) t o individual tubes, each containing 0.5 mL of mammalian, preferably rabbit or horse, plasma with or without suitable additives. Incubate in a water bath at 37', examining the tubes at 3 hours and subsequently at suitable intervals up to 2 4 hours. Test positive and negative controls simultaneously with the unknown specimens. If no coagulation in any degree is observed, the specimen meets the requirements of the test for absence of Staphylococcus aureus. Oxidase and Pigment Tests (for Pseudomonas aeruginosa) - With the aid of an inoculating loop, streak representative suspect colonies from the agar surface of Cetrimide Agar Medium on the agar surfaces of Pseudomonas Agar Medium for Detection of Fluorescin and Pseudomonas Agar Medium for Detection of Pyocyanin contained in petri dishes. If numerous colonies are to be transferred, divide the surface of each plate into quadrants, each of which may be inoculated from a separate colony. Cover and invert the inoculated media, and incubate at 35 2' for not less than three days. Examine the streaked surfaces under ultraviolet light. Examine the plates to determine whether colonies having the characteristics listed in Table 1 2 are present. Confirm any suspect colonial growth on one or more of the media as Pseudomonas aeruginosa by means of the oxidase test. Upon the colonial growth place or transfer colonies to strips or disks of filter paper that previously has been impregnated with N,N-dimethyl-p-phenylenediamine dihydrochloride: if there is no development of a pink color, changing to purple, the specimen meets the requirements of the test for the absence of Pseudomonas aeruginosa. The presence of Pseudomona aeruginosa may be confirmed by other suitable cultural and biochemical tests, if necessary.
*
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
652
Table 11 MorDholoaic Characteristics of StaDhvlococcus aureus on Selective Aaar Media Selective Medium
Characteristic Colonial Morpholoav
I
I
Gram Stain
1
Mannitol-Salt Agar Medium
Baird-Parker Agar Medium
Black surrounded b y yellow zone
Yellow colonies with yellow zones
Black, shiny, surrounded b y clear zones 2 to 5 mm
Positive cocci (in clusters)
Positive cocci (in clusters)
Positive cocci (in c Iust e r s)
Vogel-Johnson Agar Medium
I
~
Table 12 Momholoaic Characteristics of Pseudomonas aeruainosa on Selective and Diaanostic Aaar Media
. Medium
Cetrimide Agar Medium
Pseudomones Agar Medium for Detection of Fluorescin
Characteristic Colonial
Generally greenish to yellowish
Fluorescence in Ultraviolet Light Oxidese Test Gram Stain
Medium for Detection of
Greenish
I
Positive Negative rods
Yellowish
I
Blue ~
Piitive
Positive
Negative rods
Negative rods
I
1 1
7.534 Test for Salmonella SDecies and Escherichia coli To the specimen, contained in a suitable vessel, add a volume of Fluid Lactose medium t o make 100 mL, and incubate. Examine the medium for growth, and if growth is present, mix by gently shaking. Pipet 1 mL portions into vessels containing, respectively, 10 mL of Fluid Selenite-Cystine Medium and Fluid Tetrathionate Medium, mix, and incubate for 12 to 24 hours. (Retain the remainder of the Fluid Lactose Medium.) Test for Salmonella species - By means of an inoculating loop, streak portions from both the selenite-cystine and tetrathionate media on the surface of Brilliant Green Agar Medium, Xylose-Lysine-Desoxycholate Agar Medium, and Bismuth Sulfite Agar Medium contain in petri dishes. Cover and invert the dishes, and incubate. Upon examination, if none of the colonies conforms t o the
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description given in Table 13, the specimen meets the requirements of the test for absence of the genus Salmonella. If colonies of Gram-negative rods matching the description in Table 1 3 are found, proceed with further identification by transferring representative suspect colonies individually, by means of an inoculating wire, to a butt-slant tube of Triple Sugar-Iron-Agar Medium by first streaking the surface of the slant and then stabbing the wire well beneath the surface. Incubate. If examination discloses no evidence of tubes having alkaline (red) slants and acid (yellow) butts (with or without concomitant blackening of the butt from hydrogen sulfide production), the specimen meets the requirements of the test for the absence of the genus Salmonella. Test for Escherichia coli - By means of an inoculating loop, streak a portion from the remaining Fluid Lactose Medium on the surface of MacConkey Agar Medium. Cover and invert the dishes, and incubate. Upon examination, if none of the colonies conforms to the description given in Table 1 4 for this medium, the specimen meets the requirements of the test for absence of Escherichia coli. If colonies matching the description in Table 1 4 are found, proceed with further identification by transferring the suspect colonies individually, by means of an inoculating loop, to the surface of Levine Eosin-Methylene Blue Agar Medium, plated on petri dishes. If numerous colonies are to be transferred, divide the surface of each plate into quadrants, each of which may be seeded from a separate colony. Cover and invert the plates, and incubate. Upon examination, if none of the colonies exhibits both a characteristic metallic sheen under reflected light and a blue-black appearance under transmitted light, the specimen meets the requirements of the test for the absence of Escherichia coli. The presence of Escherichia coli may be confirmed by further suitable cultural and biochemical tests. Table 1 3 MorDholoaic Characteristics of Salmonella SDecies on Selective Aaar Media Medium
Description of Colony
Brilliant Green Agar Medium
Small, transparent, colorless or pink to white opaque (frequently surrounded by pink or red zone)
Xylose-LysineDesoxycholate Agar Medium
Red, with or without back centers
Bismuth Sulfite Agar Medium
Black or green
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
654
Table 1 4 Moroholoaic Characteristics of Escherichia coli on MacConkev Aaar Medium
Characteristic Colonial Morphology
Brick-red; may have surrounding zone of precipitated bile
Gram Stain
Negative rods (cocco-bacilli)
7.535 Total Combined Molds and Yeasts Count Proceed as for the Plate Method under Total Aerobic Microbial Count, except for using the same amount of Sabouraud Dextrose Agar Medium or Potato Dextrose Agar Medium, instead of Soybean Casein Digest Medium, and except for incubating the inverted petri dishes for 5 to 7 days at 20' to 25'. 7.536 Retest For the purpose of confirming a doubtful result by any of the procedures outlined in the foregoing tests following their application to a 10.0 g specimen, a retest on a 25 g specimen of the product may be conducted. Proceed as directed under Procedure, but make allowance for the larger specimen size. 8.
Pharmacokinetics
8.1
AbsorDtion
Absorption of PVP has been determined using three main techniques, namely, the use of radiolabelled PVP, histological and histochemical methods. Since PVP is used most often in pharmaceutical products and foods, and are ingested orally, numerous reports about the oral absorption have been documented, Chronic and subchronic peroral route studies performed in the late 1950's and early 1960's and confirmed in the review of Burnette (180) indicated that only minimal absorption of PVP takes place. 8.1 1
Animal studies
Shelanski (181) studied the absorption of l4 C-PVP K-30 ( M W 40,000) in rats and reported that large dose of PVP caused diarrhea. 1 % of the PVP was collected in urine in first 24 hours, 0.25% in CO, and 0.5% in carcass; and the rest was assumed to be excreted via feces. Loehry e t a / (182) also studied the absorption of 1 3 ' I-PVP (MW 8,000 -80,000) and reported a linear log/log relationship between permeability and molecular weight. Plasma effusate,
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fractionated on G 200 Sephadex revealed the following ratio for PVP of different MWs: PVP of M W of 10,000 was 0.09, M W 22,000 = 0.01. Values for 8,000, 33,000 and 80,000 were 0.67%, 0.39% and 0.67% respectively. Studies suggested that PVP, M W of less than 2,000 would be freely absorbed. Haranaka (1 83), studied the absorption using small intestine of anesthetized rabbits perfused with 7 % PVP (MW 40,000) solution (14 9) and reported that maximal absorption was achieved after 10 minutes, while by 30 minutes, the blood level was at 10% of peak. Digenis eta/ (184) studied the absorption of l 4 C-PVP (K-30)in rats and reported that the plasma half-life was 1 1/2 hours, and that absorption was complete after 6 hours. They also reported that greater than 90% of the PVP was recovered from the feces at 48 hrs. Other animals such as sheep, calf and pig have been used respectively by Fell et a/ (185) and Hardy (186, 187). PVP uptake in rats into the columnar epithelium of the intestine by fluid phase pinocytosis has been reported by Beahon and Woodley (1 88). Single dose studies of intramuscular injection of PVP indicated rapid absorption from site of injection with majority of the PVP being excreted in urine and feces within t w o days. The rate of absorption was reported t o be dependent on molecular weight and the percent leaving the body was also dependent on molecular weight. Up t o 27% of PVP K-30 was retained within the animal carcass 30 days post administration while only 0.7% of PVP K-12 was retained when the polymer was similarly administered. A two-year feeding study in the dog showed slight swelling of the reticulo-endothelial cells in the mesenteric lymph nodes, an observation which may be due t o PVP absorption, though not confirmed. Study of intestinal permeability of PVP in pigs during rotavirus infection was conducted by Vallenga et a/ ( I 89) using '261-PVP ( M W40,000), orally administered They found that there was no increase in permeability of the PVP after the clinical signs of the infection had developed.
.
8.12
Human studies
PVP has been investigated for the effects of GIT disease on permeability ''' I-PVP was used t o measure blood t o lumen permeability changes in protein-losing gastroenteropathies and blood t o lumen permeability changes in ulcerative colitis. They all concluded that PVP was absorbed in l o w levels but could not be well quantified because of large degree of dissociation of the iodine label. In an attempt t o overcome the problem of low-level detection, Siber et a/ (193) administered l4 C-PVP orally t o patients along with 5- fluorouracil in an attempt t o assess the changes in GIT permeability and reported an increase in PVP absorption, a result of intestinal damage caused by 5FU. Chronic and subchronic studies of intramuscularly administered PVP ( M W > 2,500) - adjunct with drugs such as vasopressin, t o delay absorption from injection site, led t o accumulation of PVP in organs of the reticulo-endothelial cells - liver, kidney, lymph nodes and spleen. I t has been reported (194) that PVP is absorbed by inhalation from hair spray as indicated by granular phagocytosed material in the lymph nodes. However, these observations or findings are unclear because hair sprays contain other materials such as shellac, vinylpyrrolidone vinylacetate copolymer,
(190). (191) and (192).
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CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
any of which may also accumulate in the lymph nodes. It can be stated, based on the investigations discussed, that there is no evidence t o conclude the extent of absorption of PVP from aerosols or sprays.
8.2
Distribution and storaae of PVP in the body
8.21
Distribution
There have been many reports regarding the distribution of high M W PVP in the body after intravenous administration of large doses. The polymer storage was observed as foam cells or globular deposits in the liver of humans, when given as plasma expander, as early as 1940's and 1950's (195). Intravenous injection into animals also revealed similar results. The number of foam cells or globular deposits produced was found to be proportional to the M W and the amount of PVP administered. It is assumed that the foamy appearance of the cells is due to pinocytotic uptake of PVP into the cytoplasm. Although studies involving the use of radiolabelled PVP indicated storage, the identification by histochemical means has not been established. Seale e ta / (196) studied the distribution '261-PVPof different MWs (10, 4 0 and 360kDa) by intravenous administration in normal and adjuvant-induced arthritic rats. They found that half-life of PVP increased with increased molecular weight with mean half-lives of 2.2, 6.9 and 16.4 hours respectively. Accumulation of the polymer in inflamed tissues greatly exceed that of normal paws especially with 360kDa PVP. Single dose studies of tissue distribution and excretion of 14C-K30 (MW 40,000) done by Digenis et a/ (184) in male Sprague Dawley rats indicated that amounts of radioactivity in major tissues and blood were not significantly different from the untreated controls. Radioactivity equivalent to 0.04% of administered dose was detected in urine after 6 hours, leaving them to conclude that an oral dose of 14CPovidone is not significantly absorbed in the rat. Faiz-ur-Rehman et a/ ( 1 97) conducted studies of distribution of 99m Tc-tin phosphate PVP-stabilized colloid in bone marrow of rabbits and humans using scintigraphy technique. The PVP was found in high concentration in bone marrow collected from the femoral shaft and head. Some activity was detected in the spleen and liver, but the kidneys and compact bone showed no uptake. Horbach et a/ (1981, in their investigation of rates of fluid-phase endocytosis in several organs and tissues of WAG Rij rats, of different ages, using '261-PVP, reported that high amount of the polymer was found in liver, muscle and skin, 28 hours after injection. An age related increase in uptake was also observed for rats between 12 and 3 6 months old, in liver, kidneys and heart. The increase is said to be due to increase in wet weight of the organs and not by an increase in endocytic rate. Kingham e t a / (199) also reported that the distribution of '261-PVPin the mucosa of the small intestine of guinea pigs, is dependent on the molecular size and that PVP concentration was greatest in the vascular lamina, lower in the extravascular space and least in the epithelium. Hespe e t a / (200) also studied the distribution of 14C-PVP in rats and concluded that the distribution and retention of PVP in organs (reported as target organs for PVP toxicity) can be
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prevented by decreasing the fraction of molecules with a weight of more than 25,000. Earlier studies conducted by Ravin et a/ (201) using different grades of 14C-PVP, injected into rats and rabbits intravenously, indicated that largest fractions accumulated in skeletal muscle and skin, followed in order by liver, spleen, bone marrow and lymph nodes. They also reported that retention of PVP increased with molecular size, and that below a specific molecular size (equivalent t o K-26). uptake by the RES was minimal. Siber e t a / (1931,in their tissue distribution studies, using fractionated PVP K-17.8 ( intravenously given) in rabbits found that tissue uptake into the RES is a reversible process Studies involving induction of uptake into the RES have been unsuccessful in the reports of Cameron and Dunsire (202, 203), in which 14C-PVP (K-12, K-17 and K-30) were injected by IM route into the legs of rats. 8.22
Storaqe
Numerous reports exist in the literature regarding storage disease in humans after receiving large doses of PVP (greater than 70 g), intravenously. Storage of PVP, observed as foam cell or globular deposits, were found in the spleen, bone marrow, kidney and liver in earlier investigations (204) (205). Traenkner (206)reported persistent storage pattern in spleen, bone marrow, kidney and liver following intravenous administration of large amounts of PVP (MW 20,000 - 80,000) to 300 adult patients. Hizawa et a/ (207) reported formation of a pseudotumor in a patient that was given an antihypertensive drug containing PVP. Storage of PVP in the body after short term therapy has also been reported by Bubis et a/ (208). They found that after intravenous injection of a preparation containing PVP into the breast of a woman, foreign body granuloma, mimicking congenital mucolipid storage disease was observed. Storage of PVP after long term therapy has been investigated by several authors. Takahashi el a/ (2091 reported that following intravenous injection for more than 15 years in t w o cases, storage of PVP, indicated by foam and basophilic foam cells, were detected in the lymph nodes and bone marrow respectively. Tumor like swellings were also found by Dehlschlaegel e t a / (210 ) in the facial and shoulder areas of a woman who received parenteral preparation of a drug containing PVP (Depo-lmpetol) for many years. The tumor is result from excessive storage of high molecular fractions of PVP in the RES. Other workers that have documented formation of pseudotumors after long term therapy of drug formulations containing PVP include Bork and Hoede (21 1), Kossard er a/ (2121, Soumerai (2131, Edelmann et a/ (214) and Caulet et a/ (215). Earlier studies also showed that massive amount of PVP can result in storage related functional changes in target organs. Gall et a/ (216) administered 3.5% to 4.5% PVP solution (MW 40,000) to 25 patients and reported histological changes such as basophilic globular deposits within the Kupffer cells, or free in the sinusoids and sometimes accompanied by mild inflammatory exudates. However the authors questioned the nature of the deposits. Honda et a/ (217) also studied changes in organs as a result of storage of PVP. They concluded that complications due to PVP were limited
658
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
below a total dose 69 g of PVP, but were expected with total doses of 7 0 g or more. Kojima et a/ (218) conducted morphological study of PVP storage in tissues of humans who had previously received intravenous PVP and later died from neoplastic, cardiovascular diseases unrelated to PVP and reported similar results as Honda e t a/ (217). In addition, they observed that with PVP (MW 24,8001, there was little evidence of storage in any of the tissues, but with amount greater than 7 0 g, there was evidence of storage in the RES, heart tissues, GIT and urinary bladder. With lower M W PVP (MW 12,6001, there was minimal evidence of storage in the tissues up t o 500 g, an indication that storage is related as well to molecular weight as to the amount of PVP given. Honda et a/ (2171, using different set of patients, reported that PVP, in large doses, was observed t o be localized in the areas 01 neoplastic lesion, inflammatory loci and surgical wounds and concluded that the storage of the polymer may promote growth and dissemination of already present cancers. This assumption is questionable, because the evidence that large doses of PVP can promote metastasis is not supported by the data, and PVP might actually be a diagnostic aid in cancer chemotherapy or can be useful in focussing the delivery of antineoplastic agents (2011. Moinade e t a / (219)also reported that a minimum cumulative dose of 2 0 0 g is needed t o initiate PVP storage and even up t o 1 - 2 kg in some individuals. Storage is therefore dependent on molecular mass, amount injected, frequency of injection and extent of blood circulation at the site of injection. Cutaneous storage syndrome or cutaneous thesourismosis, observed after prolonged subcutaneous or intramuscular administration of large amounts of PVP has been observed and reported extensively. Workers who have documented incidence of this disease, later known as Dupont-Lachapelle disease include Dupont and LaChapelle (220), Bazex et a/ (2211, L'Epee e f a/ (222) and Rimaud e t a / (223). Storage of PVP has been reported following inhalation from hair sprays. Bergmann et a/ (2241, in earlier histological studies, reported pulmonary inflammation attributed to PVP, however, since then, investigators such as Lowsma et a/ (225) and Cambridge (226) Gowdy and Wagstaff (227); and Bergmann (228); all concluded that there was no proof of an etiological relationship between inhalation of PVP and storage syndrome resulting in pulmonary effects.
8.3
Uotake of PVP into isolated tissues
The relationship between intestinal absorption and rerial excretion t o the molecular weight of PVP has been established and the conclusion is that only small M W PVP can pass through the membrane pores (121). The mechanism of uptake has been reported t o be endocytic. Endocytosis can be distinguished into t w o types, namely phagocytosis (cell feeding) and pinocytosis (cell drinking) or fluid-phase endocytosis. Phagocytosis is a receptor mediated vesiculation process in which attachment of particles to the receptor sits plasma membrane triggers their incorporation into a vessicle or phagosome. In contrast, pincytosis is a continuous process in which very small vessicles are
POVIDONE
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pinched off from the cell membrane i. e. cellular uptake of extracellular fluid. Another mechanism is macropinocytosis which is similar to phagocytosis, but which does not involve enclosure of small vessicles. Many studies have been conducted in which uptake of PVP is reported t o be by pinocytosis. Horbach et a/ (198) demonstrated that '261-PVP was taken up by fluid-phase endocytosis into organs and tissues of WAG/Rij rats. England et a/ (229) studied intracellular transport of '261-PVP in rat liver parenchyma cells and reported that accumulation of the PVP was rapid initially and decreased t o a constant value. The diminished rate of accumulation was reported t o be due t o exocytosis of previously endocytosed PVP. Both uptake and release were temperature dependent because they ceased at 10°C. Michelakakis and Danpune (230) also studied uptake subcellular distribution of '261-PVP in isolated perfused rat liver and reported that PVP was taken up into the lysosomes. Pratten and Lloyd (2311 used suramin to study the pinocytotic uptake of 14C-Sucrose, 3H-Dextran and '261-PVP and reported that '261-PVP uptake was enhanced by suramin more than it does with the other t w o substances, indicating that the uptake is dependent of the substrate chosen to measure pinocytosis. In another study Pratten et a/ (232) studied the adsorptive pinocytosis of polycationic copolymers of '261-PVPusing rat yolk sac and peritoneal macrophage. They found that there was non-adsorptive pinocytotic capture of '261-PVPwhereas the cationic derivative was captured more efficiently probably because it adsorbs to the cell surface. Rowland e t a / (233) also demonstrated that whereas '261-PVPuptake into rat intestinal sacs in vitro was by fluid-phase endocytosis, liposomes entrapped '261-PVPuptake was by adsorptive endocytosis. A similar report was documented by Pratten et a/ (234). Other studies involving pinocytotic or endocytotic uptake of PVP into isolated tissues include reports by Praaning van Dalan e t a / (2351, Pratten el a/ (2361, Pratten e t a / (2371, Roberts el a/ (238), Leake and Bowyer (2391, Rudolph and Regoeczi (2401, Koostra eta/ (2411, Stevenson and Williams (242) and Williams e t a / (243). 8.4
Metabolism
Ravin e r a / (201 gave 14C-PVP(K-33) to humans and found that 0.15 - 2 % of the PVP was excreted as 14C carbondioxide in the first 12 hours, and 12 hours later, the excretion fell to 0.01 %. The radioactivity was detected after 3 6 hours.. Dialysis of the 14C-PVPK33 against running water indicated no loss of radioactivity, which shows that low M W material was responsible. Shelanski (244) also reported that using 14C-PVPK90 in rats via the oral route, 0.04% of the radioactivity was detected in the expired air within the first 6 hours and none thereafter up to 120 hours. McClanahan et a/ (245) in their earlier investigation, also reported that 3.5% of 14C carbondioxide was expired after a 45-hour period. Less than 0.2% of radioactivity was collected in the urine and 2 -3% of activity was found in bile due to unchanged monomer. However, the authors were not able t o identify the other metabolites. The detection of the monomers should not be misunderstood however for PVP metabolites, since PVP is assumed not t o be metabolized.
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
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8.5
Excretion
8.51
Animal studies
Earlier studies of PVP excretion into the urine, based on chemical estimate or viscosity measurement, indicated that PVP is excreted in the urine at a rate and in amount that depends on molecular size. After IV administration, extent of PVP clearance in the urine during the first 24-96 hours ranged from 90-95% for PVP with MW of 12,600 to 10-20% for PVP with MW in excess of 100,000 (195). The half-life for the elimination of PVP with an average MW of 40,000 ranges from 12 hours to 72 hours in experimental animals. Ravin et a/ (201) studied this using 14C-P\/P (of varied MW), administered intravenously into normovolenic dogs. They reported that blood levels fell rapidly during the first 2 hours, followed by a short inflection, and then a prolonged straight line decay. The prolonged decay was concluded to be due t o removal into lymph. Other workers (2461, (247) confirmed that the passage from plasma into lymph was dependent on molecular mass. Youlten (248) also related the molecular mass t o volume of distribution and concluded that the radii effect of molecular mass is critical between 2.5 and 3.1 nm (average MW 16,000 - 25,0001, an average M W most prevalent in PVP grades used for these experiments). The apparent volume of distribution ranged from 19.45 ml for PVP with average MW 6,5000 - 8,000 t o 3.66 ml for PVP with average M W 113,000 - 21 1,000. Carter el a/ (249) studied the I-PVP and reported that a biphasic clearance was observed. clearance of lZ6 There was an initial rapid fall for the first 4 hours, followed by a slower exponential fall corresponding to the phases of distributiori and renal clearance, and later uptake b y phagocytic cells of RES. Ravin et a/ (201) studied renal elimination in dogs using '3'1-labeled PVP and reported that 90% of PVP K-32, 65% of PVP K-35 and only 15% of PVP of PVP K-50 appeared in the urine within 72 hours after administration. Hespe eta/ (200) also investigated the urine excretion i n rats using PVP K-14 and PVP K-18 (50 mg/kg). They reported that 93% of the polymer was excreted in the urine 72 hours, but when the dose was increased to 2100 mg/kg, PVP was detected for extended period (22 days) in the urine. However, the cumulative amount recovered for PVP K-14 remained 92% while that of PVP K-18 decreased t o 86%. Schiller and Taugner (250) performed more precise study of renal excretion of PVP using Wistar rats and l 4 C-PVP, K-12 (150 mg/kg. Assessment of levels of radioactivity in urine and plasma, followed by measurement of radioactivity in excised kidneys revealed that clearance of PVP K-12 was identical to that of inulin and that distribution into tissues was independent of concentration (at concentration over the range of 5-10,OO nmol PVP/ml of plasma). The clearance was not affected by induction of diuresis with mannitol. This indicates that excretion is entirely by glomerular filtration and that active tubular reabsorption and secretion were not involved. Owen e r a / (251) studied the excretion or rate of elimination of PVR from circulation in sheep using '261-PVP. They found that the PVP elimination was in t w o phases. The first phase involves the equilibration of the polymer within the initial
POVIDONE
66 I
volume of distribution (half-life = 43 f 45 hours) and the rate was dependent on the molecular mass of PVP. In the second phase, the '261-PVP was eliminated considerably faster (half-life = 176 f 39 hours) principally via the kidneys. Characterization of sieving profile of the PVP in renal glomerulus in rabbit, dog, rat by fractionation of the urine and plasma have been reported by several authors, (252) and (253). They found that PVP can be fractionated to 20-30 different molecular MW fractions and that radii up to 2.4 nm are as readily cleared as inulin (i.e 100% GFR). The clearance decreased with increase in radius until there was no clearance a t 6 nm. Lambert et a/ confirmed this in their investigation, in which they reported that in the dog, the mean glomerular pore size range was 3.86 nm, and this would allow molecules of 104,000 or less to pass through into the urine (254). Unpublished data by GAF (ISP), reported by Stahl and Frauenfelder (255) correlated with these results. Gartner et a/ (256) also reported that molecules 25,000 or less are eliminated rapidly by glomerular filtration. Larger molecules passed into the renal interstitium to be removed by the lymph or passed back into the post glomerular capillaries. The interstitial movement of PVP within the kidney was further investigated by Vogel et a/ by measuring lymphatic flow of PVP (MW10,0001 in rabbit kidney (257). They concluded that macromolecules reach the interstitial space by diffusion along concentration gradient and returned into plasma by solvent drag. 8.52
Human studies
Few studies in humans have been conducted on urinary excretion of PVP despite the fact that it was once used as plasma expander. This is due to the difficulty of accurate quantitation without the use of radiolabelled PVP, which for ethical reasons (i. e. hazards to patients), cannot be justified, based on the research purposes alone. However, there have been reports on the use of the radiolabelled PVP given intravenously and the findings indicate that PVP of lower molecular weight is more quickly absorbed and excreted than higher molecular weight PVP ( 195). Heinrich e t a / (258) studied the urinary excretion of intravenously given '3'1-PVP (MW 30,000), and found that all the radioactivity detected in urine could not be attributed to PVP, and thus suspected that l3lI was liberated after PVP was taken up by the RES. Estimates of the maximal pore size of PVP that can be excreted via the glomerulus in humans have been investigated and reported by Hulme and Hardwicke (259); Hulme (260) and Lambert et a/ (254) to be as high as 6 nm, corresponding to MW of about 94,000. Slightly lower figures of molecular radii of maximal pore size of 4 nm, equivalent to 70,000, were reported by Blainey (261 and Robson et a/ (262). Campbell e t a / (263) also studied the excretion of PVP using chemical method to estimate the amount excreted in urine following IV administration of the polymer (MW 35,000). To patients who received PVP a plasma expander they reported that 60% was excreted in urine within 24 hours and 80% within 14 days, and that multicompartment model kinetics was involved due to the presence of different molecular weight PVP in the polymer used. Wilkinson and Storey (264) also
CHRISTIANAH M. ADEYEYE AND EUGENE BAR.4BAS
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studied the excretion of PVP using calorimetric assay in 20 patients who received PVP as plasma expander for treatment of shock following injury or surgery. They reported that in majority of the patients, 50 -70% of the PVP was excreted after 26 hours. Using chemical analysis, Brautigam and Gleiss (265) demonstrated that l o w M W PVP is more easily excreted than high MW. With M W 10,000, given t o children (patients), 95% was eliminated in urine within 6 hours and almost completely excreted wi,thin 1-2 weeks. In contrast, about 80 85% of the high M W polymer (MW 20,000and 25,000) and given t o adults, was excreted in 2 weeks.
-
8.53
Biliarv Excretion
Polyvinylpyrrolidone was found in feces after orall administration and since this reject have come from the bile, it is important to establish the extent of biliary excretion. Ravin et a/ (201) studied biliary excretion in dogs and humans by intravenously administering 14C-PVP K-33. They reported that 0.5% of the dose was found in stools within 24 hours and thereafter the concentration fell to about 0.01 %. Biliary excretion was confirmed by cannulation of the bile duct in dogs. Morgenthaler (266) administered 14C-PVP (K-12 and K-25) intraduodenally t o anesthetized rats, and reported that 1.2% of the dosage of K-12 and 0.09% of the dose of K-25 were excreted by biliary excretion over a 20 -25 hour period. McClanahan et a/ (2451, in their investigation using 14C N-vinylpyrrolidone (given intravenously) and on the assumption that monomers exist in commercial PVP, showed that 17-20% of the original radioactivity could be recovered in the bile and that amount excreted in feces (0.4%) was less than that found in the bile. This they concluded, is an indication that enterohepatic circulation took place.
9.
Toxicitv
Numerous investigations on the toxicity of PVP in animals have been carried out and exhaustively reported in literature. The LD50 values range from 12 g/kg t o 100 glkg, depending on the animal species, molecular weight of the polymer, amount of dose, frequency of administration and route of administration. High doses (about 100 glkg) have been known t o cause diarrhea in dogs, rats, and cats, a result of non-absorbable osmotic: load (195).
9.1
Acute toxicity
Studies have been carried out in rabbits, dogs and rhesus monkey, and no dose related histopathological changes were observed. Shelanski (2671, Scheffner (2681, in separate studies used PVP (MW 40,000; and 10,000 or 30,000) respectively to evaluate the LO50 of orally administered PVP. They reported LD50 of 40 glkg for both 10,000 and 30,000 M W and LD50 of 100 g/kg for M W 40,000. Mouse was also used by Scheffner (orally) (268) and b y Angerwall and Berntsson (intraperitoneally) (2691, and they reported that the
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LO50 were 4 0 glkg and 12-1 5 glkg respectively. Neumann et a/ (270) gave PVP by gastric gavage t o groups of 10 animals in doses of 300, 9 0 0 or 2 7 0 0 mg/kg, using water or hydroxypropyl cellulose as the controls. They found that there was no treatment related effects. Zendzian and Teeters (271, 272) administered PVP ( M W 40,000) intravenously t o beagle dogs and concluded that there were no adverse effects unless the dose exceeded 1 0 g/kg. A t this level, shock reactions (tumors, subconvulsive movements, defecation, salivation and ptosis) were observed. The animals recovered with occasional changes in transaminase and hematological values, but no changes in histopathology were observed at postmortem. Rhesus monkeys were also given PVP ( M W 15,000) intravenously and similar results were obtained. Animals survived at 5 glkg dose with only minor changes in blood chemistry. A t 1 0 g/kg dose, given as 20% solution, the animal died after receiving 5 3 % of the injection, an effect thought t o be due to viscous hypertonic nature of the solution. A t post mortem, no histological changes were observed. 9.2
Subchronic toxicity
Studies have been conducted in animals using M W ranging from 10,000 t o 1,500,000. Aside from diarrhea, loose bowel movement, the studies indicated overall lack of toxic effects. Shelanski (273) administered PVP K-90 as 5%, 1 0 % and 20% by weight of diet t o rats in groups of 5 0 ( 2 5 female and 25 male), and reported no significant weight or histological changes in both the treatment and controls, that can be attributed to PVP. In a different study, Shelanski (274) used dogs instead of rats and reported that the animals on 1 0 % PVP lost weight significantly, but there was no consistent pathology related t o PVP effects. Kirsch et a/ (275) reported similar results using Sprague-Dawley rats and beagle dogs. 9.3
Chronic Toxicity
PVP was orally administered through diet t o Wistar rats over a two-year period by Shelanksi (276). They reported no weight loss, normal hematological and urine chemistry. Albumin was found in the urine of the high dose group and they concluded that PVP was harmless orally. BASF (277) also conducted a two-year study in which PVP was incorporated into the diet of 7 5 Sprague-Dawley rats. Compared with the controls, no treatment related effects were observed following macroscopic and histological examination. Several other studies performed by Burnette (1801, Wolven and Levenstein (278) on dogs, using 5 or 10% PVP indicated no treatment related effects except for swollen reticulo-endothelial cells in lymph nodes in the group that received 1 0 % PVP. The swelling in the reticuloendothelial cells observed after 1 year was similar t o that observed at the end of 2 years, suggesting that there was no cumulative effect or progressive degenerative changes in the organs.
664
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CHRTSTIANAH M. ADEYEYE AND EUGENE BARAIiAS
Local tissue damaae
Rasmusses and Ladefoged (279) reported tissue damage at the site of injection after IM administration of oxytetracyline formulation containing PVP. In more recent studies conducted by BASF using PVP K-12, K-17, K-30 and K-90, local tissue tolerance of IM injections was investigated using female sprague-Dawley rats and 200 or 2000 mg doses per animal. After single injections, no evidence of specific tissue damage was noted at 3 days, but by 1 4 and 45 days, histological damage was observed. After repeated injections, granulomatous tissue changes were observed in many animals: 6 of 2 4 killed after 3 days, 5 of 2 4 animals killed after 1 4 days and 2 of 2 4 animals killed after 45 days. There was no MW related tissue damage and the tissue changes observed at the intervab may be due t o trauma of the injection procedures. Other investigators (203) and (103) also reported that there was no toxic effects or histopathological changes related to parenteral administration of PVP.
9.5
Carcinoaenicitv
Numerous reports on the carcinogenic effects of PVP have been reported in the literature and all the results indicate that PVP is not carcinogenic, although it may cause tissue reaction (if given parenterally, in large amount or over a prolonged period) that mimics certain benign tumors. Feeding (oral) studies done in different animals show no carcinogenic effects. Paluch eta/ (280) studied hydrogel-I containing 10% PVP dressings in humans and concluded that minimal tissue reaction was observed. Long-term therapy of an anesthetic agent containing PVP (for treatment: of facial neuralgia) resulted in a pseudotumor (PVP granuloma) which is not malignant (2811. In several long-term clinical studies of a drug formulation containing PVP (Depo-lmpletol) in different female patients (2821, (209) and (2831, PVP granuloma was also noted, however, the tumors were not malignant and were removed by surgery. Bode eta/ (284) and other workers (2851, (286) and (213) also reported that after several years of intracutaneous PVP-procaine injections, in humans, a foreign body allergic reaction of the granulomatous type was noted. Other studies in which non-carcinogenicity of PVP was reported include the investigation of Coulinaud e t a / (287) and Colomb e t a / (288).
10.
Mutaaenicity
Several mutagenic studies of PVP have reported in literature and all indicate that PVP has no mutagenic effect. Bruce (289) conducted an in vitro study (Ames test) using S. tr@hinuriumon 3.5% solution of PVP (MW 37,000) and reported no mutareversion of histidine auxotrophic mutatants to synthesize histidine freely (no mutagenic effect was observed). In a similar study, Clairol labs (290) used PVP K-30 in Ames test and reported identical findings. In another investigation by Kessler eta/ (2911. involving transformation of mouse cell using PVP K-30 (0.5 - lo%), non-mutagenicity of PVP was concluded. Zeller and Engelhardt (292) also studied mutagenicity of PVP K-30 using the
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dominant lethal test in male mice and reported that mutagenic index of the treatment was similar t o the control. Toxico-pharmacologic and genetic study of povidone solution on bone marrow cells, conducted by Laricheva etal (293) indicate that PVP is not mutagenic. 1 1.
CrvoDrotection
PVP solution is a good protective agent for living cells. Laricheva e t a l in their study of the toxico-pharmacology of PVP solution showed that the solution is harmless to myelokaryotic cells and stable during storage with no teratogenic effect (293). The mechanism of PVP cryoprotection has also been studied by Takahashi e t a / (294) and they concluded that polymers with glass transition temperature (Tg) of approximately -2OOC protect human monocytes the best. PVP (MW 10,000 and 40,000) has been reported t o have high cryoprotective efficiency on 10% sheep erythrocytes solution (irradiated up t o 13 kGy). The use of PVP at a level of 1 0 - 1 5 % reduced hemolysis from 90% to approximately 10%. Protective action of PVP on liver lysosomes was also reported by Korolenko (295) in their study of functional disorders of liver lysosomes in toxic hepatitis in rats. Clawson et a/ (2961 studied the modulation of RNA transport by PVP and concluded that the loss of nuclear protein during aqueous nuclear isolation procedure was counteracted by PVP, preventing nuclear swelling thus decreasing RNA transport. Other investigations in which the cryoprotection action of PVP was highlighted include the reports of Wrogmann et a/ (2971, Smille et a/ (2981, Neubert and Menger (2991, Perevozkina and Margolin (3001, Echlin e t a l (3011 and Meryman et a/ (302). 12.
Anticarcinonenicitv
PVP has been documented to have anticarcinogenic properties. Hueper et a/ (3031, Stern et a/ (3041, Chevallier et (305) conducted studies using a known carcinogen, 3,4 benzopyrene with 40% PVP. The activity of the carcinogen was reported to be reduced and Chevallier et a/ concluded that the polymer was binding competitively with the carcinogen for local protein in such a way that instead remaining at the protein site, the carcinogen was eliminated with the PVP. Another study was conducted with brickery fern (a GIT and bladder carcinogen), in which PVP (50 mg/g), when added to the diet, reduced the percentage of animals with bladder tumors, but not with gastrointestinal tumors. Stern et a/ (304) in their study, also reported that PVP reduced incidence of spontaneous mouse mammary gland cancer, using the carcinogen cited above and PVP. Takeuchi (3051 in their investigation, observed that 1 % PVP as subcutaneous injection reduced artificially formed Erhlich ascites tumor in mouse. The reduction in tumor occurrence (through immune lysis) was also attributed to PVP by Hartveit (306) in another study in which they use Erlich and Bergen a-4 ascites tumor cells.
666
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CHRISTIANAH M. ADEYEYE AND EUGENE BARAHAS
Acknowledgements The authors wish t o express their gratitude to Messrs. John F. Tancredi, Louis Blecher, Drs. Edward G. Malawer and Robert M. lanniello for their strong support and valuable advise. The authors would like t o also thank Suzanne Currie, Susan Thomas and Trudy King for their contributions in the typing of the manuscript. The authors are also grateful to the School of Pharmacy, Duquesne University, Pittsburgh, PA, for the use of the computer hardware and software in the preparation of the manuscript.
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257.
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682
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274. M. V. Shelanski, Unpublished report from the Industrial Biological Research and Testing Laboratory (USA) for GAF. Submitted t o WHO by BASF (1 959al
POVIDONE
683
275.
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CUMULATIVE INDEX
Bold numerals refer to volume numbers.
Acebutolol, 19, 1 Acetaminophen, 3, I ; 14,551 Acetazolamide, 22, 1 Acetohexamide, 1, 1; 2,573; 21, I Allopurinol, 7, 1 Alpha-tocopheryl acetate, 3, 1 I1 Amantadine, 12, 1 Amikacin sulfate, 12, 37 Amiloride hydrochloride, 15, 1 Aminobenzoic acid, 22, 33 Aminoglutethimide, 15, 35 Aminophylline, 11, 1 Aminosalicylic acid, 10, 1 Amiodarone, 20, 1 Amitriptyline hydrochloride, 3, 127 Amobarbital, 19,27 Amodiaquine hydrochloride, 21.43 Amoxicillin, 7 , 19 Amphotericin B, 6, 1; 7, 502 Ampicillin. 2, 1;4, 518 Apomorphine hydrochloride, 20, 12 1 Ascorbic acid, 1I , 45 Aspirin, 8, 1 Astemizole, 20, 173 Atenolol, 13, 1 Atropine, 14, 32 Azathioprine, 10, 29 Azintamide, 18, 1 Aztreonam, 17, I
Bacitracin, 9 , 1 Baclofen, 14,527 Bendroflumethiazide, 5, 1; 6, 597 Benperidol, 14, 245 Benzocaine, 1 2 ,7 3 Benzyl benzoate, 10.55 Betamethasone dipropionate, 6 .4 3 Bretylium tosylate, 9.71 Bromazeparn, 16, 1 Bromocriptine methanesulfonate, 8 , 4 7 Bumetanide, 22, 107 Bupivacaine, 19,59 Busulphan, 1 6 ,5 3 Caffeine, 15, 71 Calcitriol, 8, 83 Camphor, 1 3 ,2 7 Captopril, 1 1 ,7 9 Carbamazepine, 9, 87 Cefaclor, 9, 107 Cefamandole nafate, 9, 125; 10,729 Cefazolin. 4, 1 Cefotaxime, 11, 139 Cefoxitin, sodium, 11, 169 Ceftazidime, 19.95 Cefuroxime sodium, 20,209 Celiprolol hydrochloride, 20,237 Cephalexin, 4, 21 Cephalothin sodium, 1, 3 19 Cephradine, 5 , 21 687
Chloral hydrate, 2,85 Chlorambucil, 16, 85 Chloramphenicol, 4,47,518; 15,701 Chlordiazepoxide, 1, 15 Chlordiazepoxide hydrochloride, 1.39; 4 , 5 18 Chloroquine, 13,95 Chloroquine phosphate, 5,61 Chlorothiazide, 18, 33 Chloropheniramine maleate, 7 , 4 3 Chlorprothixene, 2.63 Chlortetracycline hydrochloride, 8, 101 Chlorthalidone, 14, 1 Chlorzoxazone, 16, 119 ChoIecalciferol, see Vitamin D, Cimetidine, 13, 127; 17,797 Cisplatin, 14,77; 15, 796 Clidinium bromide, 2, 145 Clindamycin hydrochloride, 10,75 Clioquinol, 18,57 Clofazamine, 18.91 Clofazimine, 21,75 Clofibrate, 11, 197 Clonazepam, 6 , 6 1 Clonidine hydrochloride, 21, 109 Clorazepate dipotassium, 4.91 Clotrimazole, 11,225 Cloxacillin sodium, 4, 113 Clozapine, 22, 145 Cocaine hydrochloride, IS, 151 Codeine phosphate, 10,93 Colchicine, 10, 139 Cyanocobalamin, 10, 183 Cyclandelate, 21, 149 Cyclizine, 6,83; 7, 502 Cyclobenzaprine hydrochloride, 17,41 Cycloserine, 1,53; 18,567 Cyclosporine, 16, 145 Cyclothiazide, 1,66 Cypropheptadine, 9, 155 Dapsone, 5.87 Dexamethasone, 2, 163; 4, 519 Diatrizoic acid, 4, 137; 5, 556 Diazepam, 1,79;4,518 Dibenzepin hydrochloride, 9, 181 Dibucaine and dibucaine hydrochloride, 12, 105 Diclofenac sodium, 19, 123 Didanosine, 22, 185
688
Diethylstilbestrol, 19, 145 Diflunisal, 14,491 Digitoxin, 3, 149 Digoxin, 9,207 Dihydroergotoxine methanesulfonate, 7,8 1 Dioctyl sodium sulfosuccinate, 2, 199; 12.7 13 Diperodon, 6,99 Diphenhydramine hydrochloride, 3, 173 Diphenoxylate hydrochloride, 7, 149 Dipivefrin hydrochloride, 22, 229 Disopyramide phosphate, 13, 183 Disulfiram, 4, 168 Dobutamine hydrochloride, 8, 139 Dopamine hydrochloride, 11,257 Doxorubicine, 9,245 Droperidol, 7, 171 Echothiophate iodide, 3,233 Emetine hydrochloride, 10, 289 Enalapril maleate, 16, 207 Ephedrine hydrochloride, 15,233 Epinephrine, 7, 193 Ergonovine maleate, 11, 273 Ergotamine tartrate, 6, I 13 Erythromycin, 8, 159 Erythromycin estolate, I, 101; 2, 573 Estradiol, 15,283 Estradiol valerate, 4, 192 Estrone, 12, 135 Ethambutol hydrochloride, 7.23 1 Ethynodiol diacetate, 3, 253 Etomidate, 12, 191 Etoposide, 18, 121 Fenoprofen calcium, 6, 161 Flecainide, 21, 169 Flucytosine, 5, 115 Fludrocortisone acetate, 3, 28 1 Flufenamic acid, 11,313 Fluorouracil, 2, 221; 18, 599 Fluoxetine, 19, 193 Fluoxymesterone, 7 , 2 5 I Fluphenazine decanote, 9,275; 10,730 Fluphenazine enanthate, 2,245; 4,524 Fluphenazine hydrochloride, 2,263; 4,519 Flurazepam hydrochloride, 3, 307 Folic acid, 19,221 Furosemide, 18, 153 Gentamicin sulfate, 9, 295; 10, 731 Glafenine, 21, 197
Glibenclamide, 10, 337 Gluthethimide, 5 . 139 Gramicidin, 8 , 179 Griseofulvin, 8 , 219; 9, 583 Guanabenz acetate, 15,319 Halcinonide, 8, 25 1 Haloperidol, 9,341 Halothane, 1, 119; 2,573; 14,597 Heparin sodium, 12, 215 Heroin, 10, 357 Hexestrol, 11,347 Hexetidine, 7,277 Homatropine hydrobromide, 16,245 Hydralazine hydrochloride, 8, 283 Hydrochlorothiazide, 10, 405 Hydrocortisone, 12,277 Hydroflumethiazide, 7, 297 Hydroxyprogesterone caproate, 4,209 Hydroxyzine dihydrochloride, 7, 3 19 Impenem, 17.73 Imipramine hydrochloride, 14, 37 Indomethacin, 13,21 1 Iodamide, 15, 337 Iodipamide, 2,333 Iodoxamic acid, 20,303 Iopamidol, 17, 115 Iopanoic acid, 14, 18 I Iproniazid phosphate, 20, 337 Isocarboxazid, 2,295 Isoniazide, 6, 183 Isopropamide, 2,315; 12,721 Isoproterenol, 14, 391 Isosorbide dinitrate, 4, 225; 5 , 556 Ivermectin, 17, 155 Kanamycin sulfate, 6, 259 Ketamine, 6, 297 Ketoprofen, 10,443 Ketotifen, 13,239 Khellin, 9, 371 Lactic acid, 22, 263 Lactose, anhydrous, 20, 369 Leucovorin calcium, 8,315 Levallorphan tartrate, 2,339 Levarterenol bitartrate, 1.49; 2, 573; 11, 555 Levodopa, 5 , 189 Levothyroxine sodium, 5 , 225 Lidocaine base and hydrochloride, 14, 207; 15, 76 1
Lisinopril, 21, 233 Lithium carbonate, 15, 367 Lobeline hydrochloride, 19,261 Lomustine, 19, 315 Loperamide hydrochloride, 19, 341 Lorazepam, 9,397 Lovastatin, 21, 277 Maprotiline hydrochloride, 15, 393 Mebendazole, 16,291 Mefloquine hydrochloride, 14, 157 Melphalan, 13, 265 Meperidine hydrochloride, 1, 175 Meprobamate, 1,209;4, 520; 11,587 6-Mercaptopurine, 7, 343 Mestranol, 11, 375 Methadone hydrochloride, 3,365; 4,520; 9, 601
Methaqualone, 4, 245, 520 Methimazole, 8, 35 1 Methixene hydrochloride, 2 2 ,3 17 Methotrexate, 5 , 283 Methoxamine hydrochloride, 20,399 Methoxsalen, 9,427 Methyclothiazide, 5, 307 Methylphenidate hydrochloride, 10,473 Methyprylon, 2, 363 Metipranolol, 19, 367 Metoclopramide hydrochloride, 16, 327 Metoprolol tartrate, 12, 325 Metronidazole, 5,327 Mexiletine hydrochloride, 20,433 Minocycline, 6, 323 Minoxidil, 17,185 Mitomycin C, 16,361 Mitoxantrone hydrochloride, 17,221 Morphine, 17,259 Moxalactam disodium, 13, 305 Nabilone, 10,499 Nadolol, 9,455; 10,732 Nalidixic acid, 8, 371 Naloxone hydrochloride, 14,453 Nalorphine hydrobromide, 18, 195 Naphazoline hydrochloride, 21, 307 Naproxen, 21,345 Natamycin, 10, 513 Neomycin, 8,399 Neostigmine, 16,403 Nicotinamide, 20,475
689
Nifedipine, 18,221 Nitrazepam, 9.487 Nitrofurantoin, 5, 345 Nitroglycerin, 9,519 Nizatidine, 19, 397 Norethindrone, 4,268 Norfloxacin, 20,557 Norgestrel, 4, 294 Nortriptyline hydrochloride, 1, 233; 2, 573 Noscapine, 11,407 Nystatin, 6, 341 Oxamniquine, 20,601 Oxazepam, 3,441 Oxyphenbutazone, 13,333 Oxytocin, 10,563 Papaverine hydrochloride, 17,367 Penicillamine, 10, 601 Penicillin-G, benzothine, 11,463 Penicillin-G, potassium, 15,427 Penicillin-V, 1,249; 17,677 Pentazocine, 13, 361 Pergolide mesylate, 21,375 Phenazopyridine hydrochloride, 3,465 Phenelzine sulfate, 2, 383 Phenformin hydrochloride, 4, 319; 5,429 Phenobarbital, 7. 359 Phenolphthalein, 20,627 Phenoxymethyl penicillin potassium, 1, 249 Phenylbutazone, 11,483 Phenylephrine hydrochloride, 3,483 Phenylpropanolamine hydrochloride, 12, 357; 13,771 Phenytoin, 13,417 Physostigmine salicylate, 18, 289 Phytonadione, 17,449 Pilocarpine, 12, 385 Piperazine estrone sulfate, 5, 375 Pirenzepine dihydrochloride, 16,445 Piroxicam, 15, 509 Polythiazide, 20, 665 Povidone, 22,555 Pralidoxine chloride, 17, 533 Prazosin hydrochloride, 18, 361 Prednisolone, 21,415 Primidone, 2,409; 17,749 Probenecid, 10,639 Procainamide hydrochloride, 4, 333 Procarbazine hydrochloride, 5,403
Promethazine hydrochloride, 5,429 Proparacaine hydrochlaride, 6.423 Propiomazine hydrochloride, 2,439 Propoxyphene hydrochloride, 1.30 I :4,520; 6, 598 Propylthiouracil, 6,457 Pseudoephedrine hydrochloride, 8,489 Pyrazinamide, 12,433 Pyridoxine hydrochloride, 13,447 Pyrimethamine, 12,463 Quinidine sulfate, 12, 483 Quinine hydrochloride, 12,547 Ranitidine, 15,533 Reserpine, 4, 384: 5,557; 13,737 Riboflavin, 19, 429 Rifampin, 5,467 Rutin, 12, 623 Saccharin, 13,487 Salbutamol, 10,665 Salicylarnide, 13.52 1 Scopolamine hydrobrornide, 19,477 Secobarbital sodium, 1, 343 Silver sulfadiazine, 13, 553 Simvastatin, 22,359 Sodium nitroprusside, 6,487; 15, 781 Sotalol, 21, 501 Spironolactone, 4,43 1 ; 18, 64 I Streptomycin, 16, 507 Strychnine, 15, 563 Succinycholine chloride, 10, 69 I Sulfadiazine, 11, 523 Sulfadoxine, 17,57 1 Sulfamethazine, 7, 40 I Sulfamethoxazole, 2,467; 4, 521 Sulfasalazine, 5, 5 15 Sulfathiazole, 22, 389 Sulfisoxazole, 2,487 Sulfoxone sodium, 19,553 Sulindac, 13, 573 Sulphamerazine, 6, 5 15 Sulpiride, 17,607 Teniposide, 19,575 Tenoxicam, 22,431 Terazosin, 20,693 Terbutaline sulfate, 19,1501 Terfenadine, 19,627 Terpin hydrate, 14, 273 Testolactone, 5, 533
690
Testosterone enanthate, 4,452 Tetracaine hydrochloride, 18, 379 Tetracycline hydrochloride, 13,597 Theophylline, 4,466 Thiabendazole, 16.61 1 Thiamine hydrochloride, 18,413 Thiamphenicol, 22, 461 Thiopental sodium, 21, 535 Thioridazine and Thioridazine hydrochloride, 18,459 Thiostrepton, 7, 423 Thiothixene, 18, 527 Ticlopidine hydrochloride, 21, 573 Timolol maleate, 16,641 Titanium dioxide, 21,659 Tolazamide, 22,489 Tolbutamide, 3,513; 5,557; 13,719 Trazodone hydrochloride, 16,693 Triamcinolone, 1, 367; 2, 571; 4, 521, 524; 11, 593 Triamcinolone acetonide, 1,397,416;2,571; 4,521;7,501: 11,615 Triamcinolone diacetate, 1,423; 11.65 1 Triamcinolone hexacetonide, 6, 579 Triclobisonium chloride, 2, 507
Trifluoperazine hydrochloride, 9,543 Triflupromazine hydrochloride, 2,523; 4,521; 5,557 Trimethaphan camsylate, 3, 545 Trimethobenzamide hydrochloride, 2 . 5 5 1 Trimethoprim, 7,445 Trimipramine maleate, 12, 683 Trioxsalen, 10,705 Tripelennamine hydrochloride, 14, 107 Ttiprolidine hydrochloride, 8, 509 Tropicamide, 3, 565 Tubocurarine chloride, 7,477 Tybamate, 4,494 Valproate sodium and valproic acid, 8, 529 Verapamil ,17,643 Vidarabine, 15, 647 Vinblastine sulfate, 1,443; 21, 61 1 Vincristine sulfate, 1,463; 22, 5 17 Vitamin D,, 13, 655 Warfarin, 14, 243 Xylometazoline hydrochloride, 14, 135 Yohimbine, 16, 731 Zidovudine, 20,729 Zomepirac sodium, 15,673
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