Analytical Profiles of Drug Substances and Excipients Vol 22 1993 ISBN 0122608224 9780122608223

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.

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EDITORIAL BOARD
Abdullah A. Al-Badr Gerald S. Brenner Glenn A. Brewer Harry G. Brittain Klaus Florey George A. Forcier
Lee T. Grady Larry D. Kissinger David J. Mazzo John N. Staniforth Timothy J. Wozniak
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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 o Drug Substances and Excipients. f
Harry G. Brittain
ix
ACETAZOLAMIDE
Jagdish Parasrampuria
Abbott Laboratories
Pharmaceutical R&D
Abbott Park, Illinois 60064
ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 22
Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any form reS0Ned.
JAGDISH PARASRAMPURIA
1.
I NTRODUCTION
1.1 1.2 1.3
2.
3.
4.
5.
6. 7.
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 61 . Pharmacokinetics and Metabolism 6.2 Bi oavai 1 abi 1 i ty REFERENCES
2.1 2.2 2.3
DESCRIPTION
History Therapeutic Category Chemistry and Structure-Activity Re1 ati onshi p
ACETAZOLAMIDE
1 .
1.1
INTRODUCTION
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 -
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).
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 .
2.1
DESCRIPTION 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
2.2
Appearance, Color, Odor, and Taste
A fine, white-to-faintly-yellowish-white, odorless, bitter crystalline powder. A 3.85% solution of acetazolamide is iso-osmotic with serum (24).
2.3
Pharmaceutical Dosage Forms
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).
JAGDISH PARASRAMF'URIA
3.
3.1
PHYSICAL PROPERTIES
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 n (26). m 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 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 values o f 1.7 and 8.2. 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
PH
Figure 3: pH-Sol ubi 1 ity Prof i1 e of Acetazolamide
ACETAZOLAMIDE
Table 1:
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 G y c o l 1 Propyl ene G ycol 1 Ethanol Glycerin Water

~
87.81 (3.45)* 7.44 (1.20) 3.93 (0.11) 3.65 (0.09) 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
0'
lo
I
20 30 40 Concentration (% v/v)
50
Figure 4A:
Sol ubi 1 i t y o f Acetazolamide in Some Mixed Solvent Systems. a - 7 Polypropylene Glycol -1 420; A-A PEG 400; m.PEG 300; 0 1,2 4 Propyl ene G1 ycol ; O-. G1 ycerol ;
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
.---.
12
2.4 2.2
2.0 1.8
.5 .a
3
1.4-
0.0
0.1
0.2
0.3
0.4
0.5
Fc
F i g u r e 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; 0 1,2 Propylene Glycol ; 4 Glycerol ; (7---o Oimethyl Sulfoxide; A---A Oimethyl Acetamide; 0---o Oioxane; Methanol ; A---A Ethanol
.-. .---.
m -
.m
ACETAZOLAMIDE
Table 2 Solubilizing Power of Some Binary Solvents Toward Acetazolamide
Cosol vent
G1 ycerol
Solubilizing Power 5.294 x
1,P-Propylene Glyco?
Methyl A1 coho1 Ethyl Alcohol
8.437 x
10.00 12.00 16.50 20.60
x 10-3
x 10-3 x
PEG 300
PEG 400
x x
Oioxane
26.70
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 (~G250~) 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
IS
3.4
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
250
Temperature
("c
F i g u r e 6:
OSC-TG Curves o f Acetazolamide Polymorphic Forms. -, DSC Curves; --- TG Curves; S e n s i t i v ' t y Range 41.8 mJ.s-l; Heating Rate, 5"C.min
-1
16
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-
Form A
4000
3000
2000
1500
1000
650
Wave number (cm-l)
Figure 7:
Infrared spectra of Acetazolamide Polymorphic Forms (in N u j o l )
ACETAZOLAMIDE
17
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. O the other hand, n 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
10
'0
5
10
Form B
28 degrees
Figure 8:
X-Ray Diffraction Pattern of Two Polymorphic Forms o f Acetazol ami de
18
3.8
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).
were measured. Then, tris-
The 1 N NMR spectra of acetazolamide was measured in 5 hexadeuteriodimethyl sufoxide. I n i t i a l l y protondecoupled ad protoncoupled spectra of the nitrogens bearing hydrogens
(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 ; 15N ) of ( H
acetazol ami de. Table 3:
1 5 N hemical s h i f t s and coupling constants J (NH) in acetazol amide

~ ~~~
6 1 N Chemical S h i f t 5
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
19
4 .
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
0 5 .
Ob
. . -
0 3 .
a23 ' a 01 . Ob a2 '
; L' 0 '
a1
t
b
fi''\\,,
I,t-\ \ I .
'\\
'.
u \\
\
1
211
250
270
290
30
330
Figure 9:
UV Spectrum of Acetazolamide i n 0.1 N H2SO4 -,

and 0.1 N NO --aH
4.3
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).
ACETAZOLAMLDE
?I
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 Col umn C18 C18 Conc.
( PLghL)
Internal Std.
Mobile Phase
Ref.
Sul famerazine Chl orothi azide Sul fadiazine Propazolamide
15
12% MeoH, 2% ACN, 0.02 M Phosphate
37 3a 39
1-20 6% ACN, 0.05 M Acetate, pH 4.5 1-50 3% ACN, 2% MeOH, pH 4.0 1-50 9.7% Ethanol , 79.4% Di chloromethane, 1%HAC
C1 8
Porasil
40
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 1 % ACN, 0.05 M 0 -20 Acetate, pH 4.5

1-25 23.8% MeOH, 0.02 M Ammonium Phosphate
43
44
cia 2-Acetamido-4methyl-5-thiadiazole sul fonamide
22
5,
5.1
STABILITY
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 1 : First-order plots o f acetazolamide at different 0 pH values. (0)pH 5.46; ( 0 ) pH 6.06; (A) , pH 6.86; (*) pH 1.68; (A) pH 8.17
ACETAZOLAMIDE
23
-3
-4
-5
-6
Y
-7
-8
d
-10 -g/ 1
PH
Figure 11:
The pH-rate p r o f i l e curve of acetazolamide
5.2
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 25C 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 -10C 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
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 Eq. 1
I n Ea = I n K + 0.083 pH
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: 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
PH
Ea kcal /mol e
In A
AG kcal /mol e
AH kcal /mol e
As
cal /mol /deg
3.10
15.43 19.35
29.24 26.04 18.27
28.77 28.03 26.28
14.84 18.76 20.10
-46.77
5.85
-31.11
-20.75
6.64 20.69
5.4
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
6.1
BIOPHARIUICEUTICS Phariacokinetics and Metabolism

completely Food intake does Peak hours (53). Usual
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
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.
ACETAZOLAMIDE
21
7.
1. 2.
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AMINOBENZOIC ACID
Humeida A. El-Obeid and Abdullah A, Al-Badr
Pharmaceutical Chemistry Department College of Pharmacy King Saud University Riyadh 11451, Saudi Arabia
33
Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
34
HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR
Contents 1 Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Appearance, Color and Odor 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 Synthesis Biosynthesis Method of Analysis 5.1 Titrimetric Methods 5.2 Polarography 5.3 Enthalpimetry 5.4 Spectrophotometric Methods 5.5 Chromatographic Methods 5.6 Electrophoresis Medicinal Use
4
5
AMINOBENZOIC ACID
35
7
8 9
Adverse Effects Precautions Yharmacokinetics 9.1 Absorption 9.2 Distribution 9.3 Metabolism 9.4 Elimination
Acknowledgements References
36
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
37
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 C (1) 186.0 - 189.0 C (2)

O O O
2.2
Acidity pKa: 4.65, 4.80 (1) pKa: 2.40, 4.90 (25C) (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).
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 100C and
AMINOBENZOlC ACID
39
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).
2.8
X-Rav Powder Diffraction
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 The tube operated at 40 Kv and 35 d. 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
1
A A
& A a A
188 0
' A
187.5
87 0
6,.
-7Correciion : Mol.Weigh( : Cell Cons11 ; Onsel Slobc :
I
I86 5
f
u
2
186 0
20 O+/. 137.1 glMole 1.244 -4.52 mW/*C

Tolal A t M I P a r t ; a l A i e a 20 3,O 40
I85 5
-8
-9
85 0
60
0 80
10
50
180
100
120
140
160
200
6.0 220
ac s
V I 1A
PUF
TcmperalurQ(C')
Figure 1. Thermal curve of p-aminobenzoic acid.
40
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 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
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
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
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.-0 hydrogen 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). p-Aminobenzoic acid C7H7N02
F.W. 137.14 F(000) = 576
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
V = 1332A3 Z = 8
D, = 1.360 g . ~ m - ~ D, = 1.367 g.cm
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
-009 (2) 224 (2) 330 (2) 390 (2) 346 (2) 215 (2) 106 (1) Molecule B H(1) H(2) W3) H(4)
450 (2) 508 (2) 457 (2) 359 (2) 301 (2) 282 (1) 337 (1)
uz
B(uB) 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)
135 (10) 065 (8) 238 (8) 484 (9) 678 (10) 666 (9) 490 (7) -111 (11) -067 (8) -250 (8) -517 (9) -711 (11) -671 (7) -506 (7)
-053 (2) 011 (2) -044 (2) -139 (2) -199 (2) -217 (2) -163 (1) -013 (2) 220 (2) 330 (2) 383 (2) 341 (2) 206 (1) 100 (2)
H(5)
H(6) H(7)
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
Molecule B 3304 (1) 4286 (1) 4624 (2) 4309 (2) 3642 (2) 3300 (2) 3622 (2) 4615 (2) 5213 (1) 4248 (1)
2
b,
1154 (28) 594 (23) 708 (25) 716 (25) 748 (25) 762 (25) 708 (25) 706 (24) 1198 (24) 1218 (23) 1388 (34) 649 (24) 846 (27) 832 (28) 821 (27) 756 (27) 722 (26) 708 (24) 1083 (23) 1192 (22)
b33
b,,
-5445 (7) -2821 (7) -2073 (8) -2967 (8) -4648 (8) -5452 (8) -4530 (8) -1727 (8) -0236 (6) -2343 (6) 5079 (8) 2733 (7) 1898 (8) 2738 (8) 4379 (8) 5258 (8) 4422 (8) 1681 (8) 0089 (6) 2586 (6)
3401 (1) 1519 (2) 2194 (2) 2817 (2) 2783 (2) 2111 (2) 1489 (2) 0862 (2) 0883 (1) 0262 (1) -1666 (2) -0711 (1) -0372 (2) -0675 (2) -1343 (2) -1686 (2) -1374 (2) -0394 (2) 0180 (1) -0754 (1)
27 (1) 28 (1) 30 (1) 29 (1) 27 (1) 29 (1) 27 (1) 29 (1) 30 (1) 25 (1) 42 (1) 25 (1) 28 (1) 31 (1) 31 (1) 31 (1) 29 (1) 24 (1) 28 (1) 35 (1)
-57 (8) 27 (7) -8 (8) -2 (8) 37 (8) -33 (8) 16 (8) 21 (7) -61 (7) -81 (7) -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)
Molecule A 3461 (1) 1542 (1) 2214 (2) 2842 (2) 2829 (2) 2160 (2) 1531 (2) 0879 (2) 0866 (1) 0276 (11
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 0.002 0.009 -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
(A2)
8.51 5.78 3.64 3.96 3.89 3.21 5.06 4.32 3.47 5.16 4.46 3.40 5.59 3.78 3.55 4.71 4.40 4.03 4.44 4.02 3.73 4.29 3.89 3.21
Bi
-0.099 0.234 -0.967 -0.493 -0.640 0.589 -0.004 -0.716 0.698 -0.260 -0.547 0.796 0.246 0.328 -0.912 0.093 0.970 -0.225 -0.445 -0.275 0.853 -0.379 -0.876 0.299
0.965 -0.213 -0.15 1 -0.630 0.730 0.265 -0.991 0.097 0.093 -0.848 0.524 0.082 -0.785 0.620 0.011 0.824 -0.201 -0.530 -0.854 0.417 -0.311 -0.909 0.413 0.060
0.248 0.932 0.265 0.629 0.279 0.726 0.135 0.734 0.665 0.477 0.686 0.550 0.552 0.69 1 0.466 0.552 0.076 0.830 0.297 0.882 0.366 0.199 0.303 0.932
AMINOBENZOIC ACID
41
Table 6 (cont.)
O(1) 1 2 3
6.76 4.04 3.52 8.00 4.06 3.14 7.29 4.86 3.68 4.26 3.65 3.15 4.68 4.22 3.54 4.67 4.27 3.43 4.75 3.82 3.43 4.78 4.11 3.52 4.60 3.88 3.40
-0.157 -0.890 0.429 -0.061 -0.305 0.950 -0.369 0.925 0.950 -0.602 -0.363 0.712 -0.678 0.007 0.735 -0.69 1 -0.111 0.7 14 -0.49 1 -0.452 -0.745 -0.465 -0.383 0.798 -0.547 -0.6 11 0.572
0.946 -0.260 -0.193 0.88 1 -0.465 -0.092 0.920 0.348 -0.182 -0.433 0.601 0.672 0.146 -0.979 0.144 -0.083 0.994 0.074 -0.871 0.229 0.436 0.885 -0.223 0.409 -0.690 0.716 0.105
0.292 0.430 0.854 0.473 0.849 0.237

0.159 0.094 0.983
O(2) 1 2 3
0.707 0.689 0. I60 0.76 1 0.204 0.615 0.759 0.016 0.65 1

0.004 0.889 0.459 0.050 0.9 18 0.393
0.507 0.375 0.776
48
Table 6 (Cont.)
C(7)
1 2 3
4.47 3.95 3.37 7.39 4.10 3.78 7.82 4.60 3.28
-0.355 0.044 0.934 -0.255 0.233 -0.938 -0.399 -0.856 0.330
-0.779 0.537 -0.322 0.966 0.013 -0.259 0.910 -0.414 -0.027
0.537 0.838 0.098 0.063 0.956 0.287 0.138 0.363 0.921
O(1) 1 2 3
O(2) 1 2 3
Figure 4. A composite representation of the final 3-dimentional difference map of p-aminobenzoic acid, the hydrogen contributions were omitted from the Fcs 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
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 p n
01
4000
3500
3000
25G0
2000
1800
1600
lLOO
I0 20
1000
Boo
600
Figure 8. Infrared spectrum of p-aminobenzoic acid, KBr disc.
AMINOBENZOIC ACID
55
Table 7: Assignment of the IR characteristic bands
Frequency (cm-') 3443,3343 3200-2500 3040
Assignment N-H stretch Hydrogen bonded 0-H stretch (dimeric assocation) Aromatic C-H stretch
0
1670 1620
Conjugated -C- stretch N-H deformation C - C stretch C - 0 and C-N stretch and 0 - H deformation Out of plane bending C-H (p-substituted benzene)
II
1594-1500 1320-1280 837
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
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 hand and by the equation V,-V" = 1.14 (Vm-Vo) the other 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 ) 5.9 6.59-6.63 7.67-7.71 11.85 (centre) Multiplicity Broad singlet Doublet Doublet Broad singlet Assignment Exchangeable NH2 C3-H C,-H No. of protons 2 2
2
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
Dmso
Figure 9. Proton N M R spectrum of p-aminobenzoic acid in DMSO-d,, internal reference TMS.
'1
3A -
5-
G-
1-
PDM
Figure 10. HOMCOR spectrum of p-aminobenzoic acid.
bo
I
ii
1;
16
6 S i
P?M
Figure 11. HETCOR spectrum of p-aminobenzoic acid
I
Figure 12. Carbon-13 NMR spectrum of p-arninobenzoic acid in DMSO-d,, internal reference ThlS.
Dmso
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.
Chemical shift (6) 112.60 116.93 131.26 153.07 167.57
Carbon No.
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
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
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 60C 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).
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
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gene product, 60kDa, the larger subunit of the anthranilate synthase TrpG complex (31). The latter protein catalyses the ammonia-dependent chorismate amination to 2-amino-2deoxy-isochorismate and its subsequent aromatization by syn elimination of the elements of pyruvate (32,33). The studies carried on PabB anticipated that this protein catalyses similar regiospecific amination and then aromatization of 4-amino-4deoxychorismate, which is finally converted to PABA by crude bacterial extracts (34). Another protein has been recently reported (35,36) with substantial homoglogy to the TrpE and PabB. The enzyme, known as isochorismate synthase, catalyses the interconversion of chorismate and its dihydroaromatic isomer isochorismate without aromatization (37), at the start of the enterobactin biosynthesis pathway (38). Studies by Nichols et. al. (39) have demonstrated that in extracts overproducing PabA or PabB, still no PABA formation could be detected until soluble extract from a pabA pabB double mutant was added. This activity was proposed to act on a diffusible intermediate generated by PabB action and to convert it to the aromatic amino acid product PABA and is designed as enzyme X. This is confirmed by a recent report by Ye et. al. (40) who overexpressed the E. coli pabA and pabB genes separately and in tandem. Working on the purified PabB, they confirmed that PabB needs an additional protein, enzyme X, to convert chorismate and ammonia to paminobenzoate. The enzyme X was purified to near homogeneity from E. coli and showed no activity on chorismate but acts as an aminodeoxychorismate lyase, and quantitatively converts the preformed aminodeoxychorismate into PABA and pyruvate. Scheme 4 is proposed for the action of PabA, PabB and enzyme X in the biosynthetic pathway of PABA.
5
Methods of Analvsis
5.1
Titrimetric Methods
5.1.1 Aaueous Titration
British Pharmacopoeia 1980 (41) described the following method:
AMlNOBENZOlC ACID
73
Dissolve 0.3 g of p-aminobenzoic acid in 60 ml of ethanol (50 per cent) and titrate with 0.1M sodium hydroxide VS using phenolphthalein solution as indicator. Each ml of 0.1M sodium hydroxide VS is equivalent to 0.01371 g of C,H,NO,.
A method is performed for the assay of 4-aminobenzoic acid (42) by titration with 0.1M-NaOH using phenolphthalein as indicator. The product is described and its solubilities and m.p. are given. Identity tests and limit tests for insoluble matter, color of solution, heavy metal, CP SO:-? 2- and 3aminobenzoic acids, sulphated ash and loss on drying are included.
Kumar and Indrasenan (43) have reported a visual titrimetric method for the determination of p-aminobenzoic acid (used in pharmaceutical and cosmetics), using Nbromosaccharin in 10% aqueous acetic acid and amaranth as the indicator.
5.1.2
Non-Aaueous Titration
The pKb value of 4-aminobenzoic acid and other bases in acetic acid ('acetous' pKb) are reported (44), and differentiating titrations of five pairs of bases in acetic acid medium are considered; a glass-calomel (LiCIO, bridge) system is used and the titrant is HCIO, in acetic acid. It is concluded that differentiating titration of bases is possible if the difference between their pKb values exceeds 4. 5.1.3 Potentiornetry A potentiornetric titration with 2M- or 4M- NaNO, permits the determination of 4-aminobenzoic acid (1 to 1.5 g) in 2-3M- or 9M-HCI medium (45). A bright platinum indicator electrode was used. Satisfactory results (error < 3%) are obtained at 150 and 30" but not at 45". If more dilute reagent solution are used, there is some loss of accuracy.
5 O ,
Kumar and Indrasenan (43) have determined p-aminobenzoic acid (used in pharmaceuticals and cosmetics), by a
74
direct potentiometric method. The drug was titrated with Nbromosaccharin in 10% aqueous acetic acid. 5.1.4 Iodometry Kumar and Indrasenan (43) have reported an excessback titration method for the determination of p-aminobenzoic acid. In this method, p-aminobenzoic acid was treated with Nbromophthalimide or N-bromosaccharin and 10% KI solution and the released iodine was titrated against Na,S,O, solution.
An indirect volumetric method for the determination of 4-aminobenzoic acid and other amines is described (46). The method is based on the oxidizing action of NaC1O2, Under the reaction conditions, the action of NaCIO, on the amine is directly proportional to its concentration. The amine solution is prepared by dissolving of weighed amount in HC1, and aliquot is transferred into ground-glass stoppered Erlenmeyer flasks, with addition of a measured excess of 0.1N NaC10, into two flasks containing the sample and the standard sample (which is essential because of the impurity content of the intial NaCIO, and its variable water content). After addition of distilled water, 20% HCI was added at the ratio 1:2 with respect to the solution volume, and after shaking and leaving for 10 minutes, an excess of 10%. KI solution is added and the solution is agitated and titrated with 0.1NNa,S,O,; a little before the equivalent point 1% starch solution is added. The NaClO, solution must be prepared sometime before use, to allow establishment of a constant concentration and it is calibrated against 0.1N Na,S,O, after acidification with HCI and after addition of KI.
The use of chloramine-T for the estimation of 4aminobenzoic acid has been reported (47). The method is claimed to be simple and accurate and determines milligram amounts of the drug. Milligram amounts of the sample were allowed to react with a known excess of chloramine-T in acidic medium at room temperature for 20-30 minutes. After the completion of reaction, the unconsumed reagent was back titrated iodometrically. The accuracy of the method is k 0.1%.
AMINOBENZOIC ACID
75
Jayaram and Gowda (48) reported a method for the assay of 4-aminobenzoic acid with aromatic N-haloamines. The method involves the use of chloramine T, bromamine-T (the bromo-analogue of chloramine-T) or bromamine-B (the demethyl analogue of bromamine T) as oxidimetric reagents. An aliquot of the test solution containing 0.5 to 25 mg of 4aminobenzoic acid and 25 ml of a 0.05N solution of the reagent were adjusted to an appropriate volume, then, after thirty minutes, 20 ml of water, 30 ml of 2M-H2SO, and 10 ml of 10% KI solution were added, and the liberated iodine was titrated with S,O:- solution to a starch end-point.
A titrimetric method suitable for the determination of 50-2000 pg of p-aminobenzoic acid was developed (49). The method is based on iodination of the compound. The resulting iodide, after removal of excess iodine, is oxidized with Br to iodate which is determined by the Leipert amplification procedure.
5.1.5 Coulometrv
Delgado (50) have reported a coulometric determination of 4-aminobenzoic acid and other aromatic amines. The pH is a dominant factor in the titration of the amine with bromine. Displacement of certain substituent groups e.g., carboxyl, can be prevented at low pH, but may be total at higher pH; thus the titration can be carried out under either condition. Suitable pH, and the equivalent of bromine per molecule, for 4-aminobenzoic acid are 5 and 6, respectively. 5.2 Polarograuhy
The polarographic behavior of 4-aminobenzoic acid, and other substituted benzoic acids, in aprotic dipolar solvents are studied (51) by d.c. and a.c. polarography. The advantage of ax. polarography was observed in the determination of substituted benzoic acids in 0.0SM (C,H5), N + I- in dimethylsulfoxide. There is a linear relation between the reduction peaks of the acids and their concentration with a lower detection limit of (4-5) X 10JM.
76
5.3
Enthalpimetry
A direct-injection enthalpimetric method for the determination of 4-aminobenzoic and other aromatic amines has been reported (52). The method is based on diazotisation or nitrosation of the amine, the heat of the reaction being measured. The double injection method is used; the difference in the temperature jumps observed on making the two injections of reagent is correlated with the amine concentration. The procedure was verified by determination of 4-aminobenzoic acid and other amines. The reaction medium is usually OSM-HCI in H 2 0 or (for amine poorly soluble in H20), methanol: H,O (3:1), and the reagent is 1M-NaNO, in the same solvent as for the sample. 5.4 Spectrophotometric Methods 5.4.1 Raman Spectrometrv Laserna et al (53) have reported studies of sample preparation for surface-enhanced Raman spectrometry (SERS) on silver hydrosols of p-aminobenzoic acid, aniline and pnitrobenzoic acid. Several theories and practical aspects of the hydrosol preparation, protocols and sample preparation procedures, and their effects on the sensitivity and reproducibility of the Raman signals are discussed. The effect of acidity on SERS signal intensity is shown to depend on the time of the observation of the Raman spectra, illustrating the relevance of time to quantitative SERS data. The identification power of SERS at trace level of closely related compounds (p-nitrobenzoic acid, p-aminobenzoic acid and aniline) is illustrated. The determination of 4-aminobenzoic acid in PreSun-15 lotion using surface-enhanced Raman analysis has been published (54). Glass plates were spin-coated with an aqueous 5% (w/w) suspension of agglomerate-free alumina (0.1 pm). The plates were then vacuum-coated with a 100-nm layer of Ag. The lotion was diluted with ethanol to give two solutions expected to contain 6 and 14 ppm of p-aminobenzoic acid. Portions (1 pl) of these solutions and standards were applied
AMINOBENZOIC ACID
77
to the prepared plates, which were then kept in a dissicator overnight to complete the adsorption of p-aminobenzoic acid. For the measurement of the surface-enhanced Raman scattering, the plates were illuminated from the back with light from a Kr laser (647.1 nm), conducted to the surface of the plate with a optical fibre. The scattered light was transmitted to the photomultiplier tube with a second optical fiber and the p-aminobenzoic acid peak at 1132 cm-' was measured. In the range 4 to 16 ppm of p-aminobenzoic acid, the results were correct to within 3 ppm; no other constituents of the lotion interfered. 5.4.2 Ultraviolet Spectrometry p-Aminobenzoic acid, in the presence of the other ortho and meta isomers, can be selectively determined by measurement of absorbance of its aqueous solution at 265 nm because absorbance of the other two isomers under these conditions is nigligible (55). p-Aminobenzoic acid, which was obtained from the hydrolysis of procaine, was determined, at 268 nm (pH 6.8) (56). The other hydrolysis product (diethylaminoethanol) did not interfere with the determination of p-aminobenzoic acid.
1 Veinbergs et a (57) determined p-aminobenzoic acid as a hydrolysis product of procaine, by the Firordt method; thus a portion (5 ml) of an aqueous solution containing 100 mg/l of procaine is buffered at pH 6 with phosphate and diluted to 100 ml. A 5-ml portion of a Celnovocaine preparation is similarly diluted to contain 5 mg/l of procaine, the absorbance of each solution is measured at 291 and 266 nm.
Liu (58) determined p-aminobenzoic acid in procaine injection by UV spectrometry. Procaine injection were mixed with 60 ml potassium tartrate and water to 100 ml. Four ml of the solution was further diluted with water to 10 ml, which was extracted with ether. The organic phase was dried, dissolved in 10 ml water and analyzed at 269 nm for the determination of p-aminobenzoic acid.
The quantitative determination of p-aminobenzoic acid and other compounds present in a pharmaceutical preparation, (antiseborrhoeic shampoo) was reported (59). The drug was determined in a 5 g sample by dilution and measurement of the absorbance of the solution at 289 nm. p-Aminobenzoic acid in procaine hydrochloride injection was determined by dual-wavelength spectrophotometry (60). The injections were diluted with distilled water and the absorbance was measured at 276,264.5 and 291 nm. The p-aminobenzoic acid concentration was inversely related to (A276A,,.,)/A291. Wang (6 1) have applied secondary chemical equilibria in reversed-phase column partition chromatography, for the determination of procaine hydrochloride injections and quality control of 4-aminobenzoic acid. The injection solution containing 10 mg of procaine-HC1 was applied to an 8 g silanized siliceous earth support with 5 ml of hexanol as stationary phase previously percolated with 20 ml of 0.05MNa,CO, and eluted with 30 ml of 0.05M Na,CO,. The eluate was diluted with water to 50 ml and p-aminobenzoic acid was determined by absorbance measurement at 266 nm vs water. Procaine was then eluted from the column with 60 ml of 0.1M-HC1, the eluate was treated with 10 ml of acetic acidsodium acetate buffer of pH 6 and water to 100 ml and the absorbance was measured at 290 nm vs water. Equations for computation of procaine and p-aminobenzoic acid concentrations are presented. 5.4.3 Fluorimetry Taniguchi et a1 (62) described a procedure for the fluorimetric determination of 4-aminoebzoic acid and other aromatic amines with 4-methoxy-m-phenylenediamine as follows:
To an ice-cold solution (5 ml) of 4-aminobenzoic acid in 0.04M-HCI was added 1 ml of O.1M-KNO,. After ten minutes, 1 ml of 3% ammonium sulphamate solution (at room temperature) and, five minutes later, 1 ml of 0.06 mM-4-
AMINOBENZOIC ACID
79
methoxy-m-phenylenediamine dihydrochloride in 1M- acetate buffer (pH 5) were added. The mixture was kept for ten minutes at room temperature, then 1 ml of 10% ammonia solution and 1 ml of 0.06% CuS0,.5H20 solution were added, and the mixture was heated at 100" for ten minutes, cooled and diluted to 20 ml with ethanol. The fluorescence was measured at 462 nm (excitation at 358 nm).
5.4.4 Colorimetric Methods p-Aminobenzoic acid was determined colorimetrically (63). Samples and solutions containing 0.02-0.5 mg paminobenzoic acid were diazotized and coupled, in the presence of urea, with thyme camphor, to obtain color solution. Their extinctions were determined in a Pulfrich photometer using an S-47 filter. A curve of extinction values versus the content of the drug was given. The detection of a mixture of p-aminobenzoic acid and procainamide is also reported (63). The drug was photocolorimetrically determined (64) using its color reaction in acid media with glutaconaldehyde, the product of the alkaline decomposition of Npyridylpyridinuim chloride-HC1. Thus, to 1 ml of paminobenzoic acid solution (50-140 pg/ml) were added 1 ml N-pyridylpyridinium chloride-HC1(1% aqueous solution), 2 ml 2N NaOH, and 3 ml 2N HCl, successively and diluted to 25 ml. Thirty minutes later, the absorbance was measured at 530 nm. The drug was also determined (65) by diazotisation with 2N-HCl (0.2 ml) and O.1M-NaNO, (0.5 ml) at room temperature and after two minutes, 0.lM-sulphamic acid (0.5 ml) was added and the color was developed by the addition of mM-5-amino-4-hydroxy naphthalene-2,7-disulphonic acid monosodium salt (0.5 ml). The optimum pH for maximum color development is in the range of 7 to 11. The extinction of the solution was measured at 530 nm. The calibration graph was rectilinear in the range of 0.02 to 0.2 pmole.
80
p-Aminobenzoic acid and the other isomers were assayed by mixing with diazotizing solution (1 ml 1N-HC1 and 1 ml-1%NaNO,), 1 ml3% urea solution, and 1 ml2N-NaOH, and coupling with 1 ml 0.1% a-naphthol solution. The absorbance of the dye was measured at 490 nm (55).
A photometric determination of 4-aminobenzoic acid was reported (66). To 1 ml of solution containing 10 mg of the drug were added 5 ml of M-HCl and 1 ml of 0.1% NaNO, solution. After 2 minutes, 2 ml of 0.2% ethacridine lactate solution is added. After further 3 minutes, the solution is diluted to 50 ml with water and the extinction is measured at 505 nm.
An assay for the measurement of urinary 4-aminobenzoic acid in the oral pancreatic-function test was reported (67). The measurement of the acid in urine after oral acid administration of N-benzoyl-L-tyrosyl-4-aminobenzoic has been studied with 4-dimethylaminocinnamaldehyde as chromogenic reagent. The latter reacts with 4-aminobenzoic acid in acidic solution (pH 1.5) and the resulting red dye is measured at 550 nm. The calibration graph is rectilinear for up to 400 pg m1-I of p-aminobenzoic acid in the sample.
f Sastry et a (68) have reported a method for estimating p-aminobenzoic acid and other pharmaceutical primary aromatic amines by mixing with o-aminophenol and potassium iodate and pH 1.7 glycine-HC1 buffer and measuring the abosrbance at 520-530 nm.
Several colorimetric and fluorometric methods were described for the quantitation of primary arylamines (69). These include: formation of N-substituted derivatives of p(1) nitrophenylazobenzamide. reaction with succinic dialdehyde to give pyrrole (2) derivatives which can be developed with p-dimethylaminobenzaldehyde. Condensation with glutaconic dialdehyde to yield (3) colored Schiff's base. and (4) diazocoupling with p-nitrophenyldiazonium ion.
AMINOBENZOIC ACID
81
Bratton-Marshall method for urinary 4-aminobenzoic acid have been evaluated (70). Urine samples are hydrolysed with HC1 for .1 hour at 1000. Portion of the hydrolysate are diluted to 5 ml and treated with 0.5 ml each of 0.1% NaNO, solution in 1.3M-HC1,aqueous 0.5% NH,SO,NH, and aqueous 0.1% N-1-naphthylethylenediammonium chloride, and the absorbance is measured at 550 nm after 10 minutes. The calibration graph is rectilinear for 0.5 to 2.5 pg ml-' of paminobenzoic acid. The method was intended as a test for excocrine pancreatic function after administration of bentiromide. Bando et a1 (71) have reported an enzymic method for selective determination of 4-aminobenzoic acid in urine. Urine (1 ml) was heated at loo0 for 2 hours with 4M-KOH, then mixed with anhydrous acetic acid and incubated at 300 for 20 minutes with 4-aminobenzoate hydroxylase (50 miu). 0.1 pMpotassium phosphate buffer solution (pH 7), 0.17 mM-FAD, 0.75 mM-NADH and 0.3 g I-' bovine serum albumin. After addition of 200 g 1-' trichloroacetic acid, 0.83 M-NaOH, 0.2Mphenol and 1.67 M-Na,CO,, the solution was incubated for 20 minutes at 300 before determination at 630 nm.
A colorimetric determination of 4-aminobenzoic acid and other primary aromatic amines using N-alkyl aminophenol and iodine has also been reported (72).
The determination of urinary 4-aminobenzoic acid with fluorescamine in the pancreatic function test with bentiromide have been published (73). 5.4.5 Phosphorescence The use of internal standard (1-naphthoic acid) in the measurement of room-temperature phosphorescence of 4aminobenzoic acid, have improved the precision of detection of the compound (74).
An experimental procedure is described (75) whereby addition of poly(acry1ic acid) to solution of 4-aminobenzoic
Table 10: Thin layer chromatography of p-aminobenzoic acid

I
Solvent System Water: ethanol: 2M-HCl (225:240:13 Ethanolic 0.6% 4-dimethyl aminobenzaldehyde: anhydrous acetic acid (4: 1) Acetone, Acetone: cyclohexane (9:l) or Acetone: cyclohexane: Aq NH, (90: 10:1) Xylene, benzene, benzene: methanol (19:l) benzene 1.4 dioxan (195) or ethanol-aq. NH, : H,O (20:1:2)
Adsorbent
Detecting Agent
Ref.
(79)
Silica gel Silica gel
UV at 265 nm
NaNO, in HCI plus 2-naphthol in NaOH solution
~ ~ ~~
(80) (81)
N W
Silica gel G
Modified Ehrlich reagent
Silica gel Benzene: acetone (80:2) Silica gel C ,
2% Solution of ethclorvynol in CHCl, -isopropanol-H,PO, (10239:1) Rhodamine B solution, 4-dimethylaminobenzaldehyde-HC1 and diazotized sulphanilic acid.
(6)
(83)
Table 10 ( c o n t . )
Solvent System Cyclohexane: ethyl acetate: chloroform: acetone (2:4:3:4) Cyclohexane: ethylacetate: methanol (2:2:1) Cyclohexane: ethylacetate: chloroform (2:4:3) and (2:2:1) Alkaline or neutral solvent systems
w m
Adsorbent
~
Detecting Agent Solution of 4-dimethylaminobenzaldehyde (1 g) in 96% ethanol (loo), anhydrous acetic acid (10 ml) and water (90 ml)
Ref. (84)
Silufol sheets
lSilica gel Silica gel and CMcellulose
UV at 254 nm or spray with FeCl,, iodine, HgNO, or picryl chloride

Solution containing 1% solution of potato starch, 1% KI and 0.05% Triton X 100. Treatment with 3% dimethylaminobenzaldehyde: ethanol and analyzed with a dual wavelength thin-layer scanner.
Several solvents*
~
Chloroform: acetic acid: ethanol (10: 1:1)
Table 10 (cont.)
Adsorbent
~
Solvent System Benzene: acetone (90: 10) methanol: chloroform (90:10)
Detecting Agent Tosylchloramide sodium
Ref.
UV 254 silica layers and FND cellulose layers
*Followed by drying of the plates, N-chlorination of the amino group with chlorine vapor evolved from decomposition of calcium hypochlorite, selective reduction of excess chlorine with formaldehyde vapor, followed by treating with spraying agent.
AMINOBENZOIC ACID
RS
acid improved phosphorimetry.
its
detection
by
room-temperature
Karnes et a1 (76) reported a comparative evaluation of two substrates for urinary determination of 4-aminobenzoic acid by room-temperature phosphorimetry. The substrates considered were: (i) filter paper (S. and S. 903) impregnated with diethylenetriaminepentaaceticacid and (ii) ion-exchange paper (Whatman DE-81). In each instance, calibration graphs were rectilinear in the range 0 to 40 mg I-' of 4-aminobenzoic acid. Karnes et a1 (77) have also determined 4-aminobenzoic acid in urine by room-temperature phosphometry, with application to the bentiromide test for pancreatic function.
1 Long et a (78) have determined 4-aminobenzoic acid and other pharmaceuticals by derivatization-room-temperature phosphorescence. The drug was derivatized with fluorescamine. The optimum pH, buffer concentration and phosphorescence characteristics are discussed.
5.5
Chromatographic Methods
5.5.1 Thin Laver Chromatoeraphv
Table 10 summarizes the several thin-layer chromatographic methods reported on p-aminobenzoic acid (79-88). Two more TLC methods have also been reported (89,90).
5.5.2
Gas Liauid Chromatoeraphv
Cumpelik (9 1) described a gas liquid chromatographic system for analysis of p-aminobenzoic acid and other multiple absorber sunscreens. The gas chromatographic retention times of different UV absorbing agents used in sunscreen preparations are compiled as an aid to identification in cosmetic samples. The retention times were obtained using 10% SE-30 liquid phase on 80/100 Chromosorb W. The temperature was programmed from 160 to 300 at
Table 11: High performance liquid chromatography of p-aminobenzoic acid Support and column pBondapak C,, Mobile phase Flow rate
1.8 ml/min
Detector UV at 230 nm
Sample
Ref. (96)
Aqueous 10 mMNaH,PO,-methanol (24: 1)

5 mM-pentanesulphonic acid in 25 mMKH,PO, of pH 2.5.
pBondapak C,, (30 cm X 4.6 mm) washed with 0.1 M-oxalate (50 ml), H,O (150 ml) and 0.1% triethanolamine solution (50 ml) Diaion CDR-10 (25 cm X 3 mm) of LiChrosorb RP-18 (10 Pm)
UV at 254 nm
(97)
~ _ _ _ _ _ _ _
6 M-ammonium acetate (pH 4.4) Methanol-citrate buffer solution (1: 1)
0.8 to 1.6
ml/min
UV at 254 nm UV at 254 nm
Body fluid
(98) (99)
0.8 ml/min.
Table 11 (cont.)
Support and column Yanapak ODs-T, (10 pm), (25 cm X 4 mm), operated at 5 5 " .
Mobile phase
0.2 M-potassium phosphate buffer (pH 3.5): acetonitrile (7: 1)
Flow rate
0.7 ml/min.
Detector Electrochemical detection (Y anaco model VMD101 operated at + 1.1 V) UV at 254 nm
Sample Urine
Ref.
~~
Spherisorb 5 ODS
0.2 M-phosphate buffer (pH 3S)-acetonitrile (7:l)
1.5 ml/min
Urine
Spherisorb 5 ODS
0.2 M-phosphate buffer (pH 4)-acetonitrile (7: 1) A) Acetonitrile-water (15:85) containing M-HClO, and 5.10'* M-NaCIO, B) Methanokwater
(85:15)
1.5 ml/min
UV absorption
~
Blood
~~
LiChrosorb RP 18
UV at 280 t n u and 318 nm
Cosmetics
Support and column

(1) B-Cyclodextrin silica (CDS) (2) Macrophase MP-1 polymer (MP) (3) Macroporous polystyrene/divinyl benzene (MPD) (4) Octadecylsilica (ODS) (5) Propylphenylsilica (PPS)
Mobile phase
Flow rate
Detection
Sample
Ref. (1W
AMINOBENZOK AClD
89
8O/minutes increase. All the samples were silanized and centrifuged. Wurst et a (92) developed a gas-chromatographic 1 method for determining trimethylsilyl derivatives of 4aminobenzoic acid and other carboxylic acid in mixtures of biological materials. The separation was performed on 1,5-bis(m-phenoxypheny1)- 1,1,3,3,5,5-hexaphenyltrisiloxane as stationary phase. Its efficiency was compared with that of SE52. Nitrogen carrier gas, flame ionisation detection, and a temperature programming mode were used. Harahap et a1 (93) reported a gas-chromatographic method for the analysis of 4-aminobenzoic acid in the thermoplastic aromatic polyamides after alkali fusion. The sample of the thermoplastic aromatic polyamide, containing equimolar ratios of 4-aminobenzoic acid and other acid was subjected to alkali fusion at 300" for 2 hours with potassium hydroxide-sodium acetate (19: 1). The mixture was cooled and isophthalic acid was precipitated by adjusting the solution to pH 7. The precipitate was filtered off and isophthalic acid was derivatized to its dimethyl ester with methanolic BF, reagent. The filtrate was made slightly alkali with potassium hydroxide and the libaraled 4,4/-methylenedianiline was extracted into chloroform and derivatized to the TMS derivative. The remaining aqueous layer was adjusted to pH 6 and paminobenzoic and 3-aminobenzoic acid were extracted into chloroform and derivatized to their TMS derivatives. Separation of the three derivatives was carried out on a column (12 ft X 0.25 inch) of 5% of SE 30 on Celatom A W DCMS (72 to 85 mesh); the carrier gas (60 ml/rnin) was helium and flame ionization detector was used.
5.5.3 Column ChromatoPraDhy

The separation of the isomers of aminobenzoic acid has been reported (94). The 2-aminobenzoic acid is separated from the other two isomers by use of a column (390 mm X 14 mm) of Amberlite CG-120 resin (200 to 400 mesh; Cu2+ form), with aqueous NH, of pH 8.4 as eluent (0.3 ml per minute). The 3 and 4-aminobenzoic acids are separated on a
90
HCMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR
column (230 mm X 17 mm) of the resin equilibrated with aqueous NH, at pH 5.9, with water as eluent (0.3 mi per minute); 4-aminobenzoic acid is eluted first. Randau and Schnell (95) have separated 4aminobenzoic acid on columns (25 cm X 1.5 cm) of the basic resin M 5080 (gel) and MP 5080 (macroporous), with 0.1MHC1 in 50% ethanol as eluent. The MP 5080 was superior, optimal separation occuring at 380; on the M 5080 column, no separation was achieved at the lower temperature and only partial separation at 50 O . It is concluded that the macroporous resins permit separation only achieved with the gel resins at much smaller particle size and greater column pressure, 5.5.4 High Performance Liquid Chromatography Table 11 summarizes several high performance liquid chromatography method which have been reported (96-104). Another method has also been published (105). 5.6 Electrouhoresis
4-Aminobenzoic acid was determined (106) in procaine injection solution by electrophoresis on paper, A 5 111 portion of the injection solution was diluted to 2 mg/ml of procaine hydrochloride, and applied to No. 1 paper (25 cm X 24 cm) for electrophoresis in a JMDY-WI apparatus with the use of a buffer solution (9.76 g of citric acid and 1.03 g of sodium H citrate in 100 ml) of p 3 and a potential gradient of 20 V cm-' applied for 20 minutes. The paper is then dried and sprayed with solution containing 100 ml of ethanolic 2% pdimethylaminobenzaldehyde and 5 ml of anhydrous acetic acid. Standard solution containing 0.03 pg/pl of 4aminobenzoic acid are used for comparison. 6 Medicinal Use Aminobenzoic acid has sometimes been included as a member of the vitamin-B group, but deficiency of PABA in man or animals has not been demonstrated. PABA is used topically as a sunscreen agent usually in a concentration of
AMINOBENZOIC ACID
91
5%. The drug and its derivatives effectively absorb light throughout the UVB range but absorbs little or no UVA light (3). Its preparations are therefore effective in preventing sun burns but ineffecitve in preventing drug-relating or other photosensitive reactions associated with UVA light; combination with a benzophenone may give some added protection against such photosynthetic disorders. The drug appears to diffuse into the horney layer of the skin and significant protection remain for about three days, after a single application of a 5% alcoholic solution (108). Application of this solution once daily for 30 days did not give rise to cutaneous or systemic toxic symptoms. PABA has no protective effect when given by mouth.
The PABA or BTPABA test is used to assess pancreatic function by measuring concentrations of aminobenzoic acid and metabolites in urine following the administration of bentiromide, a synthetic peptide derivative of PABA. Some studies were reported on the improvement of the test specificity as well as comparison and combination with established or new tests (108-112). 7 Adverse Effects Contact and photocontact allergic dermatitis have been reported following the topical application of aminobenzoate sunscreen agents (1 13-116). Development of vitiligo in sunexposed areas following adminstration of aminobenzoic acid by mouth was reported (1 17). Toxic effects are infrequent and are usually associated with plasma concentrations greater than 600 pg/ml (2).
8
Precautions Aminobenzoate sunscreen agents should not be used by patients with previous experience of photosensitive or allergic reactions to chemically-related drugs such as sulfonamides, thiazide diuretics and certain local anesthetics, particularly benzocaine.
92
HlJMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR
Pharmacokinetics 9.1 Absorption
PABA is readily absorbed from the gastrointestinal tract after oral adminstration (2,3). The percutaneous absorption of PABA was determined in vitro through hairless guinea pig skin (109). The absorption of PABA was greater through nonviable skin. Illel et. al. (119) studied the role the follicles play in percutaneous absorption of a number of drugs including PABA. Using their skin model they compared the percutaneous absorption in appendage-free skin relative to normal skin. The results confirmed that appendageal diffusion is the major bathway in hairless rat skin. In the absence of follides, the steady state flux and the amounts diffusing in one or two days are 2-4 times lower than in normal skin. PABA serum and urine concentrations were measured in patients with normal, pathologic and pharmacologically inhibited pancreatic function (120). PABA serum concentration, in patients with normal pancreas function and volunteers, was characterized by a rapid increase during the first 1% h. A maximum increase of 32.42 k 10.04 mpmol/L was reached after 90 min. In patients with exocrine pancrease insufficiency or those with pharmacologically inhibited exocrine pancrease resulted in a significantly reduced PABAserum concentration. In correspondence to the delayed and smaller serum PABA increase, urine PABA concentrations were also diminished. Thyroid dysfunction was found to affect the small intestinal absorption of some drugs including p-aminobenzoic acid. Thus, examination of the effect in the i siru recirculating n perfusion and everted sac methods showed that the intestinal absorption of passively absorbed drugs were depressed in hyper- and hypothyroid rats (121). Studies using the same above methods were undertaken by Miyagi et. al. (122) to study the effect of concanavalin A(Con A) on the absorption and metabolism of p-aminobenzoic acid in the small intestine of rats. It %was found that the absorption of PABA in the small intestine of rats pretreated with Con A was not different
AMINOBENZOIC ACID
93
compared with control, but the formation of p-acetamidobenzoic acid increased. The transfer of p-acetamidobenzoic acid was not influenced by C o k When PABA was orally administered in the rat treated with Con A, the plasma concentrations of PABA and of acetamidobenzoic acid increased compared with control. No influence of Con A was observed in the I.V. administered PABA. The plasma concentrations of acetamidobenzoic acid was unchanged when this PABA metabolite was orally or I.V. administered in the rat treated with Con A.
9.2
Distribution
Branco and Torres (123) determined the levels of some water-soluble vitamins in Planorbidae. The determinations were carried out in total snail and in digestive tract extracts of Biomphalaria glubrata. While some vitamins like folk acid showed higher levels in the digestive tract extract than in the total snail extract, the concentration of other vitamins including PABA produced higher levels in the total snail extracts. Fendrich et. al. (124) studied the distribution of PABA in rats. Thus after whole-body irradiation with 600R, labeled PABA was administered I.V. to female rats. Greatest concentrations of PABA were noted in the kidneys, liver and intestines with almost none in the brain. Koren et al. (125) studied the disposition of PABA following ingestion of the free drug in 6 control and 18 cystic fibrosis (CF) patients. PABA distribution volume in CF patients was smaller, although not significantly so, than the controls. The value of 376+140 ml/kg was reported for controls as compared to the value of 268+107 ml/kg for CF patients. Good correlation was found between PABA distribution volume and T4 (r = 0.51, P = < 0.02).
9.3
Metabolism
PABA is mainly metabolized in the liver (4) and kidney (126). It is conjugated with glycine to form p-aminohippuric
94
HIJMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR
acid. Small amounts of p-aminobenzoyl glucuronide, pacetamidobenzoyl glucuronide and traces of p-acetamidohippuric, p-acetamidobenzoic acid and unchanged aminobenzoic acid are also detected in urine (2,127). Chan et al. (127) isolated all these metabolites and noted that at high doses saturation of glucuronidation of p-acetamidobenzoic acid appears to occur, which resulted in an increase in the formation of p-acetarnidohippuric acid. 4-Hydroxybenzoic acid was also reported to be generated as a metabolite of PABA treated with homogenate of Agan'cus hispurus (128). PABA may be detected in urine as a metabolite of amethocaine, benzocaine and procaine (2). The metabolism of PABA is reported to be influenced by many factors. Acetyltransferase activities in the small intestinal mucosa and the liver were increased in rats treated with cancanavalin A (122). These results suggest that concanavalin A will facilitate the metabolism of PABA in the small intestine and liver of rat. The effect of ethanolamine on the acetylation of PABA was studied in adult rats (129). The results showed that ethanolamine significantly increases the acetylation capacity of tissues. Griffeth et. al. (130) utilized a previously validated small mammal trauma model, (hind-limb ischemia secondary to infrarenal aortic ligation in the rat) to investigate the effects of traumatic injury on PABA acetylation. The N-acetyltransferase activity was depressed by 20-22%. Moreover, the i vivo reaction of acetylation was n found to be significantly decreased by model trauma. This effect on in vivu pharmacokinetics appeared to be correlated closely with trauma's influence on the conjugating enzymes and relatively independent of the post-traumatic response of the necessary co-substrates. It is thus suggested that traumatic injury appears to have wide-ranging effects on a variety of determinants of hepatic drug metabolism. In an overview on renal disease and drug metabolism, Gibson (126) reported that in a diseased kidney the metabolism of PABA, and other drugs known to be metabolized in the kidney, is reduced. Renal disease, therefore, has its potential to alter not only the renal clearance of unchanged drug but also may substantially modify the metabolic transformation of drugs in both the liver and the kidney.
AMINOBENZOIC ACID
95
The tissue distribution of acetyltransferase with PABA as a substrate in humans was investigated by Pacifici et. al. (131) in the cytosolic fraction of the placenta, liver, adrenals, lungs, kidneys and intestines. All tissue specimens catalyse the acetylation of PABA at a significant rate. High activity is also observed for the N-acetylation of PABA in skin cytosols of hamsters (132). These results together with the detection of Nacetylating activity in the skin of other experimental animals and humans (127), suggest that the skin may play an important role in the metabolism of the drug and other armatic amines. Relatively high levels of acetyl transferase activity was also found in urinary bladder cytosol of humans (132). A number of other studies on the metabolic acetylation of PABA appeared in the literature. These include reports on the genetic control (128-144), kinetics (134,145-147) and inhibition studies (148-150) of the acetyltransferase enzyme. Scheme 5 lists the major metabolites of PABA.
9.4
Elimination
PABA is mainly excreted in urine as its conjugate, paminohippuric acid together with small amounts of paminobenzoyl glucuronide, p-acetamidobenzoyl glucuronide. Traces of p-acetamidohippuric acid, p-acetamidobenzoic acid and unchanged benzoic acid are also detected in urine (2). The studies on the disposition of PABA in control and CF patients reported above (125) showed that the elimination half-life of PABA was significantly shorter in CF patients (58+21 min) compared to controls (93.5+28 min). The PABA clearance was similar in the control (2.99+ 1.21 ml/min/kg) and CF patients (3.27k1.02 ml/min/kg). Acknowledgements The authors would like to thank Mr. Tanvir A. Butt for typing this manuscript.
96
HLIMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR
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L.K. Griffeth, G.M. Rosen and E.J. Rauckman, Drug Metab. Dispos., 13, 398 (1985).
G.M. Pacifici, C. Bencini and A. Rane, Pharmacology, 32,283 (1986).
Y. Kawakubo, S . Manabe, Y. Yamazoe, T. Nishikawa and R. Kato, W h e m . Pharmacol., 37, 265 (1988).
AMINOBENZOIC ACID
I05
133. 134.
Y. Kawakubo, Y. Yamazoe, R. Kato and T. Nishikawa, Skin Pharmacol., 3, 180 (1990).
T. Yerokun, W.G. Kirlin, A. Trinidad, R.J. Ferguson, F. Ogolla, A.F. Andres, P.K. Brady and D.W. Hein, Drug Metab. Dispos., 17, 231 (1989).
I.B. Glowinski and W.W. Weber, J. Biol. Chem., 257, 1431 (1982).
D.W. Hein, T.N. Smolen, R.R. Fox and W.W. Weber, Pharm. Exp. Ther., 223, 40 (1982).
135. 136. 137. 138. 139.
ibid. 220, 8 (1982).
D.W. Hein, J.G. Omichinski, J.A. Brewer and W.W. Weber,
D.W. Hein, A. Trinidad, T. Yerokun, R.J. Ferguson, W.G. Kirlin and W.E. Weber, Drup Metab. DisDos., 16, 341 (1988).
A. Trinidad, W.G. Kirlin, F. Ogolla, A.F. Andrews, T. Yeroku, R.J. Ferguson, P.K. Brady and D.W. Hein, ibid. 67, 238 (1989).
140.
A. Trinidad, D.W. Hein, T.d. Rustan, R.J. Ferguson, L.S. Miller, K.D. Bucher, W.G. Kirlin, F. Ogolla and A.f. Andrews, Cancer Res, 50, 7942 (1990). W.G. Kirlin, F. Ogolla, A.F. Andrws, A. Trinidad, R.J. Ferguson, T. Yerokun, M. Mpezo and D.W. Hein, ibid. 51, 549 (1991). D.M. Grant, M. Blum, M. Beer and U.A. Meyer, Mol. Pharrnacol., 39, 184 (1991).
Y. Sasaki, S. Ohsako and T. Deguchi, J. Biol. Chem., 266, 13243 (1991).]
141.
142. 143. 144.
M.H. Heim, M. Blum, M. Beer and A.U. Meyer, J. Neurochem., 25, 309 (1980).
106
HLIMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR
145. 146. 147. 148. 149. 150.
R. Gollamudi, B. Muniraju and E.C. Schreiber, Enzyme, 25, 309 (1980).

S.S. Mattano and W.W. Weber, Carcinogenesis, 8, 133 (1987).
A.J. Kilbane, T. Petroff and W.W. Weber, D ~ U P Metab, Dispos., 19, 503 (1991). P.E. Hanria, R.B. Banks and V.C. Marhevka, Mol. Pharmacol, 21, 159 (1982).
T.J. Smith and P.E. Hanna, Biochem. Pharmacol., 37, 427 (1988).
M.J. Wick and P.E. Hanna, ibid.39, 991 (1990).
BUMETANIDE
Prasad N.V. Tata,' Raman Venkataramanan,' and Swroop K . Sahota2
(1) School of Pharmacy University of Pittsburgh Pittsburgh, PA I526 1 (2) Hoffmann - La Roche, Inc. 340 Kingsland Street Nutley, NJ 071 10
107
Copyright 0 1993 by Academic Press, Inc All rights of reproduction in any form reserved
I08
PRASAD N. V. TATA ET AL.
CONTENTS
I . Introduction
2. Description 2. I Nomenclature 2.2 Formulae 2.3 Compendia 2.4 Dosage Forms Available 3. Physical Properties 3.1 Appearance, Color and Odor 3.2 Melting Point 3.3 Ionization Constants 3.4 Storage 3.5 Solubility 3.6 Spectral Properties 3.7 Metal Complexing Ability 3.8 X-Ray Crystallographic Data
4. Stability 5. Synthesis
6. Methods of Analysis 6.1 Elemerita1 Analysis 6.2 Identification 6.3 Loss on Drying 6.4 Residue on Ignition 6.5 Related Impurities 6.6 Titrimetry 6.7 Coulometry 6.8. Spectrophotometry 6.9 Colorimetric Methods 6.10. Fluorirnetry 6.11. Ion Selective Electrode Method 6.12 Radioimmuno Assay
BU MET AN IDE
6.13 6.14 6.15 6.16
Radiometric Method Electrophoresis Extraction from Biological Fluids Chromatographic Techniques
7. Metabolism
8. Pharmacokinetics 9. References
1. Introduction
[ 1-41
Bumetanide fBMT) is a potent loop diuretic similar to furosemide (FRU) in its pharmacological action but equally effective at one fortieth the dose on a weight basis. It is indicated for the treatment o edema associated with congestive heart failure, hepatic and renal f disease, including the nephrotic syndrome.
2. Description
2.1
Nomenclature 2.1.1 Chemical Names
t 1-41
[S-71
Benzoic acid, 3-(aminosulfonyl)-S-(butylamino)-4phenoxy
3-(aminosulfonyl)-5-(butylamino)-4-phenoxybenzoic acid
3-(butylamino)-4-phenoxy-S-sulfamoyl bemoic acid
110
2.1.2 Other Names

RO 10-6338 PF 1593
171
2.13
Trade Names Aquazone (Spain) Bumex (USA)
[lo1
Diurama (Italy) Fordiuran (Germany, Spain) Butinet (Argentina. Spain) Farmadiuril (Spain) Bumet (India) Fontego(Ita1y) Bonures (Sweden) Lunetron (Japan) Cambiex (Argentina) Segurex (Argentina) Burinex (U.K.. Austria, Belgiurn,Denmark, France, Italy, Netherlands, Norway, South Africa, Sweden, Switzerland)
2.2
Formulae 2.2.1 (!hemica1

( 1,H,ON,OSS 1
2.22. Molecular Weight 364.62 2.2.3 CIAS Registry Number [28395-03- I]
BUMETANIDE
2.2.4 Structural
2.3
Compendia
Bumetanide and its dosage forms are official in the IJSP [8] and Italian Pharmacopeia. Other compendia which include analytical and pharmaceutical information are given in the Phurmuceurit*ul Cde.w [9], The Phurmuwurit~al Munufucruretx Encyclopediu [ 101, and Clarke's Isolution und Identijkurion cf Drug Substunc.es [ 1 11.
2.4
Dosage Forms Available

1.
1121
Scored tablets of bumetanide, at potencies of 0.5, 1.0. and 2.0 mgitab. Bumetanide for injection, at a potency of 0.25 mg/mL, in 2 ml, ampules, 2, 4. and 10 mL vials. Burnetanide liquid, potency of 1 mg/mL. Tablets of 0.5 mg Bumetanide + 573 mg of Potassium Chloride for sustained release of potassium.
2.
3.
4.
I12
3. Phvsical Properties
3.1 Apparance. Color and Odor ~3-91
Bumetanide is an odorless, white crystalline powder with a slightly bitter taste. 3.2 Meltin? Point The reported melting point range is 230-231"C. [8,131
3.3
Ionization Constants
1651
The pK, and pK2 values of bumetanide are reported to be 3.6 and 7.7, respectively.
3.4
Storage
PI
The lJSP recommends that bumetamide raw material be stored in air tight containers, and protected from light.
3.5
Solubility
[8,13]
The solubility of bumetanide in various solvents is listed in Table 1.
3.6
Spectral Properties
3.6.1 Ultraviolet Soectrum
[9.13-1s]
The principal IJV absorption peaks of bumetanide in various solvents are listed in Table 11, and the (E" values (where available) are given in parentheses.
BUMETANIDE
I13
Table I : Solubility of Bumetanide

Solvent Water Ethanol Propylene Glycol Dimethylacetamide Methanol Benzene Benzyl Alcohol Acetone Alkaline solutions mdlnL 0.1
30.6 18.7
> 500
76.5
0.4
21.6 50.2
soluble
Table I1 : U V Spectral Characteristics of Bumetanide

Solvent Water 0.05M SDS Aqueous Acid Aqueous Alkali 0.1N NaOH Methanol Acetonitrile Absorution Maxima (nm)
260 (18.9). 220 (17.1) 260 (25.9), 220 (27.0)
M
15
15
9
340 (80)
317 (87) 326 270, 345 270, 345
9 14 13
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 3.6.3 Nuc lear Magnetic Resonance 3.6.3.I 'HNMR Spectra
NH stretch Unsaturated CH stretch Saturated CH stretch C = O stretch aromatic ring vibrations SO, stretch
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
3500
3000
2500
2000
1750
1500
1250
1000
750
400
cm-
Figure 1. Infrared Spectrum of B w t a n i d e
0.77 P M P
*H2C!!,
7.35 PPM
J ~ ~ 2 . 7.3 ~~t3 ~
-NH
*6!2 *ll2CIl,
-J 3.05 PPH
5.07 PPM
-NII
-NHCH2
1 . 3 7 PPM
-CI12CII*CH,
' 1
Figure 2.
1 H
WR Spectrum of Bunetanide
=c
=
.r
~
.s
li
.I
I I7
118
Table IV : 'H NMR Characteristics of Bumetanide

Chemical Shift (ppm) 0.77 1.18 3.05 5.07 6.82 - 7.31 7.35 7.43 - 7.70
A ssimment
Table V : I3C NMR Chemical Shifts of Bumetanide

Assimment && Chemical Shift D D ~
a
bl
C
13.48 19.20 30.10 41.96 114.75 115.14 115.48 122.15 129.02 128.00 137.58 139.52 142.29 156.23 166.45
-CH,
~x-CH,
-NHCH2
d
e f
?. ! h
protonated aromatic carbons
j
1
k I rn n
01
non protonated aromatic carbons
C=O
BUMETANIDE
I I9
3.6.4 Mass Snectrum
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
m. L z
364 32 1 304 240 196 168 91 77
Relative Intensity
74.0 91.8 100.0 63.0 19.2 30.8 17.0 10.4
3.7
Metal Complexinv Abilitv
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.
55
I I
d
s
9
BUMETANIDE
121
3.8
X-Ray Crystallographic Data
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 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
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
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.
122
:s P
8.?38
-A --
4 436
2.976
2.252
1.823
1.541
1.343%
2856.0
100
2570.42284,s1999.21713.812.' 410 I14Z.4858. 8-
UO 104331 L O T #031021
BWETANIDelUSm AS A REFFRENCEl.
U PLOT
- so
- a0
70
- 60 - !lo
.. 40
- 30 - 20 I 1
57i.a28s.8-b
0.01
I
LO
0
'
I-r- ~ T T
r n 7 . m
60
10
40
70
6 8 .- . 8 3 8
5.901
4.436
3.559
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
20.92176
21
20.7638
4.27451
29
~~
19.4000 20.7638
4.57180 4.2745 I
3 I .4103
35.2034
2.84572 2.54730
4 6
18
124
4. Stability
~ 3 1
Bumetanide may react in an acidic environment to yield the following products:
+
COOH
n-butylchloride
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 55C.
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 benzoic acid in its bicarbonate to give 4-phenoxy-3-nitro-5-sulfamyl 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
II
COOH
utyraldehydc or -BuOH/H$O.
NH(WW
t
for 45 min
HN 2W IN NaOH
Sa_W
-Ac,MI I
COOBu
J W C I
Bumetanide
Figure 6. Route of Synthesis of Bumetanide
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 Liebermanns test Mercurous Nitrate 6.3 Violet Brown Orange Black
Loss on Drying
r 81
USP specifies that when bumetanide is dried at 105C for 4 hours, the LOD cannot be more than 0.5% of the sample weight .
BUMETANIDE
I27
6.4
Residue on Ignition -
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. c. Butyl 3-butylamino-4-phenoxy-5-sulfarnoyl 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
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.
BUMETANIDE
129
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 2C 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.
130
PRASAD N . V. TATA ET AL
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.
BUMETANIDE
131
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, the range of 5-10 pg. over
[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.
6.16.2
Thin Laver Chromatom-aphy
6.16.3
High Performance Thin Layer C h r o m a t o m
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
Table Vlll : TLC Methods For Analysis Of Bumetanide

Stationary Phase Silica Gel Sample Type Bumetanide and related substances Bumetanide and Other Diuretics Solvent System CHC1,CyclohexaneGI. AcOH-H~O (80:10:10:2.5) EtOAC,EtOACMeOHStr.NH,OH (85: 105) CHC1,-Acetone (4:11 Cellulose Kiesel gel Q4F Kiesel gel HF254 Bumetanide metab Bumetanide metab n-BuOH sat. with 25 % NH40H Bz:BuOAc:AcOH (85:35:3.25)
T V
AX0
Silica Gel
coupling with B.M. Reagent

11
Flu.
Flu. 360.nm
21
UV,FIu. 3351415 nm.
I 25
160
Bumetanide/U.Pl n-BuOHNH40H(3: 1)
6.16.4 High Performance Liquid Chro-
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
No. 01 Compounds Column ODS(MOX3.9 Mobile Phase MeOH-HzO-THFAcOH (5&455:2) ACNPH 2 3 . Phaphate Buffer (156344) Detector Referena
8
W5 24 nm
Seppnted
BMT.4-Ethylbennl&byde*/ Tab, h. j BWalong with srn
5 MPPH'
mm)
02
(250X4mm)
RP-18
nm
Um V
4 0
compounds.
03
BMT, CLT, SPL 557 olhen
C18Sil-X-10
UV detn
0.05M NaH Polbuffer ofPH23.0 Contg.0.016M propyl amine Hcl (A) and ACN (B) 15% - 80% in u) minuta
Wwf&
41 42
04
' BMT, B-HET', Hypersil ODs

(200X4.6 mm, mu, CRN HCT, HFT,I , 5 um) T cr,PT,Az MCT, BZT, CLT, CyT,
275 nm
M?Z%
DCP, BMTZ SPL, TRI,T a QT, AML, PCD ia urine
50 mM PH 3 0 .
20mhiBDS in
44
phosphate buffer
BBASBA'PISJ
LG3-DB
$ MeOH - H Conc AcOH ( 7 0 Q X 1)
RU
W418 RU
w0 4 4
45
BAPMSBA'IPI,
-
0.03M Phosphate buffer pH3.0-ACN
46
RP-18
UV 231
47
forms
BMT in urine,
Radial Pak qs MeOH-Hp AcOH (68:34:1) (1OOx 8mm)

SGE100GL-4 C18P (1oo~o.4mm,
Flu 2uV418
48
10
EA, SPL in
urine
BMT,PCD,
H$-ACN-MeOH 5.) TFA (70: 15:1 0 5
W 270 n or m Plasma spray
49
I34
No.
Table I X (continued)
3emor
s. l
11
Refenna
50
51
1 2
13
Rev P
flu Dclq WMS
others in urlns
BMT. AML
LIv,GGEI
BMT, FRU/
1 4
nu*
LIV
3 2
13
BMT, FRU, TCM
h-PrOHNH,OH (1l l:)

ACN in 0.01M
uv
35
Phaspbre Buffer of pH3.0 10% at 15 .

minutes to 35% 81 3 s minula till
13 minutes
16
BMT, Meubs, FRU'
ODs Radial compressed
17
ACN-THF. P , Buffer O 3.5 (151k7S)
MtoH-HP
AcOH (3.) M01
Fluwz
am
18 19
l
I
I 5 6
nL w u l 3 va
am
PmiSU 10 ODs-3 Ppnisil 10 ODS-3
57
I
59
M6H.W
AcOH(7&3& 10) for BMT
20
ACNaOlN H$O, (1:l)
am for BMT
AP
21
w 254 lor
UPOnSil ODs M(h3.9 mm
CHC13
izzx==-
MeOHAO c H (%3:1)
nn J
Flu. W 4 1 8
BUMETANIDE
135
6.16.5 Micellar Chromato&
~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- ,3,4-thiadiazole was used as I 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 280C and 320C respectively. Bumetanide was monitored at m/z values of 254, 363, and 406.
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 300C. 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 270C 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
I37
Bumetanide
I I1 111 IV V VI VII Vlll
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
COOH COOH COOH COOH COOH COOH COOH CONHCH2COOH CONHCH2COOH
8. Pharmacakinetics
[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
I38
Absorption:
Bioavailability C,,, after 2 nig/dos Time to peak Plasma protein binding Apparent distribution vol.
9s96 80 nglmL about 30 minutes

94-96% 12-35 L or 0.2 LlKG
Distribution:
Elimination:
Half-life 1-1.5 hour 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., OSullivan S. Bourke E.. Brit. Med. J . 1972, 1, 211-3. Drug facts and comparisons (Ed. Bernie R . Olin) 1992, 535-42. Facts and Comparisons Inc., MI 63146-3098. 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. Olesen K.H., Sigurd B., Steiness E., Leth h . , Acta Med. Scand. 1973, 193, 119-31. Chemical Abstacls Index Guide, 1990, Part I, Chem. Abstr. Service, Columbus. OH 43210. International Nonproprietary Names for Pharmaceutical Substances, 1988, p. 66, W.H.O. Publications. Geneva. The Merck Index 1989, 11th edition, p. 1473, Merck & Co. Inc., New Jersey. IJSP 1988, pp. 191-2. The Pharmaceutical Codex XI edn. 1979, pp. 113-4, p. 1038, The Pharmaceutical Press, London. Pharmaceutical Manufacturers Encyclopedia (I 988), 2nd ed., Vol I., p. 200-2, Ed. Marshall S . , Noyes publications. Clarkes-Isolation and identification of drugs (1986). p. 408-9, 2nd Edn.. Ed. A.C. Moffat. Martindale - The Extra Pharmacopeia, 29th Edition, (Ed. Reynolds J.E.F.) 1990, p. 980, The Pharmaceutical Press London.
2.
3.
4.
5.
6. 7.
8. 9. 10.
11.
12.
140
PRASAD N. V . TATA ET AL.
13.
Sahota, S.K., personal communication, Hoffmann-la Roche, Nutley, New Jersey. 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. Berthod A . , Asensio J.M., Laserna J.J., J. Liq. Chromatogr., 1989, 12( 13), 262 1-34. Feit P.W., Brunn H., Nielsen C.K., J. Med. Chem., 1971, 13(6), 1071-5. Feit, P.W. J. Med. Chem.. 1971. 14(5), 432-9 Organic Chemistry of Drug Synthesis (1980), 2. p. 87. (Ed. L . A . Mitscher). Swinyard E . A., Remingtons Pharmaceutical Sciences, 18th edn., 1990, p- 939, (Ed. Gennaro A.R.) Mack Publishing Co. Easton, PA. 18042. Kolis. S.J., Williams T.H., Schwartz, M.A., Drug Metab. Disp., 1976, 4(2), 169-76. Ostergaard E. H.,Magnussen M.P., Nielsen C.K., Eilertsen, E., Prey H.H., Arzneim-Forsch., 1973, 22, 66-72. Magnussen M .P., Eilersten E., Naunyn-Schmiedbergs Archives of Pharmacology, 1974, 282, p. 61. Halladay S.C., Carter D.E., Sipes I.G., Brodie B.B., Bressler R., Life Sci. 1975. 17, 1003-10. Ward A., Heel R.C., Drugs, 1984, 28, 426-64. Halladay S.C., Sipes, I.G., Carter D.E., Clin. Pharmacol. Ther. 1977, 22(2), 179-87.
14.
IS.
16. 17.
18.
19.
20. 21. 22. 23. 24. 25.
BUMETANIDE
141
26. 27. 28. 29.
Pentikainen P.J., Pentilla A., Neuvonen P.J., Gothoni G., Br. J. Clin. Pharmacol., 1977, 4, 39-44. Patel R.B., Patel A.A., Gandhi T.P, Patel M.R., Patel S.K., Ind. J. Pharm. Sci., 1979, 41, 124-5. Ramaswami P.S., Kurani S.P., Desai D.K., personal communication, through Ind. J. Pharm. Sci. 1982, 44, 5. Nikolic K.I., Velasevic K., J., Pharm. Belg., 1989 44(6), 387-90. Chao A.C., Armstrong W.M., Am. J. Physiol., 1987 253(2. I ) , C343-C347. 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. Patel R . B . , Patel A.A., Patel M.R., Patel S.K., Manikwala S.C., Ind. J. Pharm. Sci., 1987. 49(4), 142-3. Sastry C.S.P., Prasad T.N.V., Roma Mohana Rao A, Venkata Rao E., Indian Drugs, 1988, 25(5), 206-8. Sastry C.S.P., Prasad T.N.V., Sastry B.S., Venkata Rao E . , Analyst, 1988, 113(2), 255-8. Halazo A.A., Colburn W.A., Gustafson, Young R.L., Parsonnet M., Pharm. Sci., 1984. 73(8), 1108-13. J. Therapeutic Drugs, 1991, Vol 1 , 8122-6 (Ed. C. Dollary) Churchill Livingston. Ameer B., Mendoza S.M., LC-GC, 1989, 7(7), 590-2. Pentikainen P.J., Neuvonen P.J., Kekki M., Pentilla A . , J. Pharmacokinet, Biopharm., 1980, 8, 219.
30.
31.
32.
33.
34.
35.
36. 37. 38.
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. Daldrup T., Michalke P., Boehme W., Chromatogr. Newsl., 1982, 10(1), 1-7. Cooper S.F., Masse R . , Dugal R.J., J. Chromatogr., 1989, 489(1), 65-88. Sentell K.B., Clos J.F., Dorsey J.G., Biochromatography, 1989, 4(1), 35-40. Ameer B., Burlingame M.B., Anal. Lett., 1988, 21B, 15891601. Boekens H., Bourscheidt C., Mueller R. F., J. Chromatogr. , 1988, 434, 327-9. Zivanov S.C.D., Solomun L.J., Zivanovic L.J., J. Pharm. (2, Biomed. Anal. 1989, 7 1 ) 1889-92.
41.
42.
44.
45
46.
47. 48.
Wells T.G., Hendry I.R., Kearns G.L., J. Chromatogr. , 1991, 570(1), 235-42.
Ventura R . , Fraisse D., Becchi M., Paisse O . , Segura J., J. Chromatagr., 1991, 562(1-2), 723-36. Gradeen C'.Y,, Billay D.M., Chan S.C., J. Anal. Toxicol., 1990, 1 ( ) 123-6. 42, Park S.J.,Pyo H.S., Y.J., Kim, M.S., Park J., J. Anal. Kim 42, Toxicol., 1990, 1 ( ) 84-90. Singh A.M., McArdle C., Gordon B., Ashraf M. Granley K., Biomed Chromatogr., 1989, 3(6), 262-5.
49.
50.
51.
52.
BUMETANIDE
143
53. 54.
55.
Fullinfaw R.O., Bury R.W., Moulds R.F.W., J. Chromatogr., 1987, 415(2), 347-56. Howlett M.R., Auld W.H.R., Skellern G.G., Method]. Surv. Biochem. Anal., 1984, 14, 337-42. 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. Marcantonio L.A., Auld W.H.R., Skellern G.G., J. Chromatogr., 1980, 183( I), 1 18-23. Walmsley L.M., Chasseaud L.F., Miller J . N . , J. Chromatogr., 1981, 226(2), 441-9. Smith D.E., J. Pharm. Sci., 1982. 71(5), 520-3. Berkersky I . , Popick A., Drug Metab. Disp., 1983, 1 1 , 5 12-
56. 57. 58. 59. 60.
3.
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. Lisi A.M., Trout G . J . , Kazalauskas R.,J. Chromatogr., 1991, 563(2), 257-70. Feit P.W., Roholt K., Soernsen H., J. Pharm. Sci., 1973, 62, 375-9. Hioki M., Ariza T., Shindo H., Sankyo. Kenkyusho Nempo., 1973, 26, 85-93. Fagurlund C., Hartvig P., Lindstrom B., J. Chromatogr., 1979. 168(1), 107-16.
61.
62.
63.
64.
144
65.
Orita Y., Ando A . , IJrakabe S., Abe H., Arzneim.-Porsch., 1976, 26( I), 11-33. Yoon C.N., Lee T.H., Park J., J. Anal. Toxicol., 1990, 14(2), 96- 101.
66.
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
145
I46
MICHAEL J. MCLElSH ET AL.
CONTENTS 1.
1.1
DESCRIPTION
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
1.4
Molecular Weight Efemental Composition Appearance, Color and Odor
1.5
2.
2.1 2.2 2.3 2.4
PHYSICAL PROPERTIES
Melting Range Solubility Data Dissociation Constant Partition Coefficients 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
2.5
CLOZAPINE
I47
3.
4.
4.1 4.2
SY N HESlS T
METHODS OF ANALYSIS
Extraction Spectrophotometric Analysis Chromatography
4.3.1 Thin Layer Chromatography 4.3.2 Gas Chromatography 4.3.3 High Performance Liquid Chromatography
4.3
4.4
Radioimmunoassay
5.
6.
METABOLISM PHARMACOLOGY
USES, ADMINISTRATION and CONTRA-
7.
INDICATIONS
8.
9.
PHARMACOKINETICS RFRNE EE E CS
I48
MICHAEL I. MCLEISH ET AL.
1.
1.1
DESCRIPTION
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,,NC ,H,,l (Clozapine
free base)
1.2.2 Structural
1.2.3 CAS Registw Number

5786-21-0 [ Z ]
1.2.4 Drua Code Number

HF-1854 [ 1,2]
CLOZAPINE
149
1.3
Molecular Weight
326.83
1.4
Elemental Composition
C: C1: 66.15% 10.85% H: 5.86% 17.14%
N:
1.5
Appearance, Color and Odor

Clozapine is a yellow crystalline weak characteristic odor. or finely crystalline powder which is odorless or has a
2.
2.1
PHYSICAL PROPERTIES
Melting Range
Clozapine, recrystallized from acetonepetroleum ether, melts at 183-184 "C [l].
Solubility Data
Solubility at 25 "C (w/w, vibration 24 hours, gravimetry) : Acetone Acetonitrile Chloroform Ethyl Acetate Ethanol (Absolute) Water (pH 7.4)
> 5%
1.9%
> 20% > 5%

4.0%
< 0.01%
2.3
Dissociation Constant
pK,(1)
= 3.70
[3]
pK,(2) = 7.60
[4]
150
MICHAEL I. MCLEISH ET AL.
mure 1
W epectrum of clozaplne in methanol.
200
250
300
350
400
Wavelength (nm)
Wavelenghth mox (nm)
Molar absorptivity (mo?L cm-)
215
230
259
24300 23200 16200
296
10000
CLOZAPINE
151
2.4
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) corresponding and are provided in molar absorptivities ( & ) 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 The as a KBr disc is shown in Figure 2. spectrum was obtained on a Hitachi 270-30 infrared spectrophotometer. Table 2 details the wavenumber and assignment of the principal absorption bands.
0 '0
I
.o
W
.o
W
0 0 '0
d
0
0
' NI . -
d
?-
Q rl
d
A
i
0 V
al ti:
(d
k
0
'E
0
% -
. - I %
13
m
I d
Q)
rl
0 rl U
44
3 !
&
i
1
0 d
z c " g
0
0 0
(Y
E 3
0
0
Y)
(Y
0
0 0
a,
a
H
0
pt
y1
0 0
*
VI
0
152
* I
CI
U
4
Q1
i. d
0
E
Q Q
U U
0)
3 ! k
8
X
I
153
1 54
MICHAEL J. MCLEISH ET AL.
able 3
IH-NMR
characteristics of clotapine
Chemical Shift 6 (ppm) 2.35
Multiplicity (Number of Hydrogens) singlet (3H) broad singlet (4H) broad singlet (4H) singlet (1 H) doublet (1H) multiplet (2H)
I
'H
Assignment
2 -52 3.49
4.93
6.61
6.82 7.01
I
multiplet (1 H)
I
7.07 7.25 7.28
doublet (1H) multiplet (1 H) multiplet (1H)
CLOZAPINE
155
Table 2
Infrared charactersistics of c 1o zapine
Wavenumber (cm-') 3320 3016

2980 - 2800
1595- 1550
Infrared assignment N-H stretch Aromatic C-H stretch Aliphatic C-H stretch Amidine C=N stretch Aromatic C=C stretch Aromatic C-CI stretch
1430- 1460
1042
2.5.3 Nuclear Magnetic Resonance Spectrum

The aromatic region of the ' NMR spectrum of H clozapine, recorded has on been a Varian described
XL-100
[ 61
spectrometer,
However, no detailed analysis was undertaken. The 'H-NMR spectrum of clozapine in CDC1, was recorded portrayed and clozapine on a at Bruker ambient
AMX-300
(300
MHz)
spectrometer spectral
temperature
of
and
is
of
in Figure 3. assignments (TMS) are
The chemical in Table as
shift 3. the
the protons used
presented was
Tetramethylsilane internal reference.
7 5 MHz
13C-NMR
spectrum of clozaplne in CDC1,.
CLOZAPINE
157
Table 4
13C-NMR characteristics of clozapine
16
CH,
Chemical Shift 6 (ppm)

46.04
47.20 54.96 119.96 120.05 123.02 123.09 123.49 126.80 129.06 130.26 131.87 140.40 141.84 152.77 162.76
i3c
Assignment
C-16 C-14, C-18 C-15, C-17 c-8 c-5 c-3 c-9
c-1
c-11 c-10 c-2 C-4
c-7
c-12 C-6 C-13
158
The 13C-NMR spectrum of clozapine in CDC1, was also recorded on a Bruker AMX-300 spectrometer, again at ambient and is presented in Figure 4. atoms are displayed in Table (75 MHz) temperature, The chemical 4. TMS was
shift and spectral assignments of the carbon 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 used: Acceleration voltage Ionization voltage Ionization current Ionization chamber temp. The shows (32%) 1. The fast atom bombardment was is presented in Figure (FAB) mass spectrum spectrum compares ions 256 at 3 kV 70 eV 0.3 mA 170 "C to that following spectrometer conditions were
favourably [6]. m/z 326

A
reported by Stock et al. prominent 22%), and 192 intensity
The spectrum (relative 227 possible in Scheme

(loo%),
(77%), 243 (34%).
fragmentation pathway
is provided
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
0
n
k c,
0 0
F4
P N u i 3
ay-JD
mle 326
NA"
J
N
/=
mle 268
mle 256
mle 243
Fragmentation pathway for Clozapine
Fiuure 6
Fast Atom Bombardment (FAB) ma88 spectrum o f clozapine
~~
192
327
243
256
u
164
150
200
250
300
350
m/z
I62
MICHAEL J. MCLEISH ET AL
voltage expected
of
6 keV.
The at
spectrum m/z
shows the with a
(M+H)+ peak
327
relative intensity of 100%.
2.5.5 X-ray Crystal Structure

The crystal structure of clozapine has been reported ball a and [7] and is presented, stick diagram,
as a stereo
in Figure
7.
The of
crystals were obtained by slow evaporation of saturated were
1 :1 methanol-water
on
solution and
clozapine. data
The three dimensional collected a Hilger
intensity 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 is the in
From the data, central
it was
concluded
seven-membered
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 crystal. The C-N bond connecting the
Computer-generated stereo view of clozapine showing the molecular conformation as determined by X-ray analysis.
,,
..
164
tricyclic system to the N-methylpiperazine moiety displayed considerable double bond character [7].
3.
SYNTHESIS
Methods for the synthesis of clozapine have [3,8,91 and are shown in been described 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 19,111. N-Methylpiperazine was (TiC1,)
CLOZAPINE
165
I
N=C'
' H
..
a
Thermal cyclizatbn (R = H) Base catatyzed cyclizatbn (R = %)
r
R
R = a
Sdmms-22:
Synthesis of Cloeapine
I66
treated, at 50 "C,
with TiC1, in a mixture of The amide,
toluene and anisole. 90% yield [9]. The preparation chloride However, of
A,
was added
and the mixture heated at reflux to give 8 in
clozapine been
via
the
imido [3]. are [3]. by
has
also
described
yields obtained in this method
low, presumably due to the lability of Clozapine aminolysis may also be
synthesized
of the thioether 5, but the reaction is reported to proceed slowly [3].
Finally, reaction of the thiolactam methylpiperazine also affords 131.
with N-
8 in good yield
4.
4.1
METHODS OF ANALYSIS
Extraction
Clozapine is a basic compound and has a pK, of 7.6 8 [4]. [4].
As
Its a
partition consequence
coefficients, most methods
in of
octanol/arater, are 0.4 at pH 2 and 1500 at pH 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. buffer although [13,14] [6,12], sodium
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 [4,17]. Toluene and benzene are similarly 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 .
168
4.2
Spectrophotometric Analysis
In methanol
W
and ethanol, 215, in
clozapine exhibits 260 and has methanol 295 nm been and
maxima
around
230,
[l,51 used,
Although W methods are not generally clozapine by tablets in dissolution
determined ~ 7 1 .
measuring the absorbance at 214.5 and 294 nm
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, to two clozapine from dimensional t h e and plasma thin metaboli.tes were subjected extracted [22]. its and layer w a s
chromatography on silica gel. d i m e n s i o n

(16:8:1:1)
In the first ammonia/water
e l u a n t
isopropanol/chloroform/25% and the
Rf of clozapine was 0.68.
In the second dimension ethyl acetate/ethanol/acetic acid/water/l,2dichloroethane (26: 12 :8 :7.5 :15) was the Under N-oxide and
, eluant and the R was 0.35.
The plates were metabolites,
visualized under W these conditions clozapine desmethyl
light at 295 nm. the and major clozapine
could also be measured. first/second dimension
The R, values in the were 0.37/0.45 A densitometer
0.23/0.35, respectively [22].
Table 5
GC methods for the determination of Clozapine

~~
COLW/SUPPORT
DETECTOR
DERIVATIZATION
SENSITIVITY
1-2 ng/mL
REFERENCE
DB5 5% SE-54 on Supelcoport

1% SP 1000 on 100/120 100/120
Nitrogen
none
SIM
none
n.s.
13
Supelcoport
3% OV-1 on 100/200
Nitrogen
none
1.2 ng/mL
18
ECD/Nitrogen
TFAA
1 ng/mL
23
Supelcoport
OV- 17 0 1
S 1M
PFPA
1.0 ng/mL
26
I70
was
used
to and
quantitate metabolites, in the
the with of
levels 5-10
of
clozapine limits
detection ng/g
being
region
plasma 1221.
A
TLC
method of is
has a
also
been
described of this In
for
detection including clozapirie columns The

R,
broad
spectrum using on eluted
drugs, method gel. ethyl
clozapine
[13].
extracted when
solid-phase silica with
and
chromatographed
values
acetate/methanol/ammonia/water (86:10:1 :3) and methano1Iammonia (1OO:l) are 0.46 and

0.60,
respectively. by its Clozapine colour may be with (blue) identifed reactions
reagents including Fast Black K Salt and Dragendorff spray nm

(R f
(brown), or W at 254
(black).
In addition, two metabolites of
clozapine appeared as red (R, 0.3) and orange
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
summary
of of
the methods the gas clozapine
developed analysis
for
chromatographic
[4,13,18,23,26]. These methods have proven to be relatively selective and provide sensitivity of approximately 1 ng/mL.
Table 6 COLUMN
C18 5 P C18 10 Cun
Conditions employed for the HPLC determination of Clozapine

MOBILE PIIASE
KPO, (0.05M pH 2.9)
MeOH/Acetate (pH 5.5) ( 6 7 : 3 3 ) MeOH/H,O (80:20) MeOH (0.O5%ET3N) /H,O
(78:22)
DETECTOR
W, 2 2 6 nm
INTERNAL STANDARD
C1 one zapam 4 ~ Diazepam D iazepan, D iazepam
SENSITIVITY
450 Pg
REF
14 15
w,
W, 2 3 0 nm
n.s.
16
17
254 nm EC
5.0 ng
XL-Octyl 3 P C8 5 P C18 10 Cun C18 5 P
MeCN/NH,Ac (0.25 mM) (9O:lO) MeCN/NH,Ac (0.25 mM) (9O:lO)

=04 (pH 4,
Ag/AgCl
+ o . 7 vs
Dibenzepine Fluphenazine Protryptyline
500 Pg
20
w,
254
21 15 ng 25
(64:36)
W, 230 nm
MeOH/NH,CL (2M)/ NH,Ac (2M) ( 6 0 : 40:2)
Diazepam
n.s.
28
172
MICHAEL 1. MCLEISH ET AL.
The extraction of clozapine was carried out using section did not extraction 4.1. prove to procedures be described in In most cases derivatisation necessary, [23] to although
[ 261
pentaf luoropropionic trifluoroacetic used. sometimes acetyl In have
anhydride internal used
and been the
anhydride been
have ensure
addition,
standards
accuracy of the assay. maprotiline [4],
These have included 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]. of 10 The assay was performed using normal
pn
phase chromatography, with a stationary phase silica and a gradient as of the
chloroform/methanol/water
mobile to a phase. minimum Under concentration
serving these of
conditions
10 ng/mL
clozapine and its N-oxide could be determined of a blood or urine [19]. silica One other assay using a described, having
column has been
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
CLOZAPINE
173
internal standard has been used with diazepam being the most popular [15-17,271. The differences to in the assays the can be
attributed its
whether was
simultaneous Thus some
determination of clozapine and any or all of metabolites required. measured methods clozapine metabolites clozapine merely clozapine major
[16,17,20,21], others clozapine and desmethyl [25] or clozapine [151. Still in the presence and both others of drugs measured 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 and references therein].
of [29
radioimmunoassay
has
been
developed [30]. to
that
As
permits the quantitation of clozapine in the presence of its major metabolites described, that the assay is limited ng/mL human plasma. 100 pg/mL plasma [30]. However, 15-480
it is claimed as little as
it is possible to detect
METABOLISM
I74
I /
3:
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
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 [3,29,32,38,39], which in agranulocytosis some cases can be fatal, and this led to its withdrawal from the market [3]. Recently it has been reintroduced by Sandoz, with close blood [40] for monitoring of patients' 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 BY regions (at which EPS develop) [37]. 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
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 history of bone marrow depression [32]. Further, depending on the amount present, some interaction may combined with alcohol, depression, bone lithium [32]. be observed when agents causing CNS depressants or
marrow
As clozapine has been implicated in cases of agranulocytosis [3,29,32,38,39] clozapine 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
I80
MICHAEL J . MCLEISH ET AL.
elimination with first order absorption [24]. The terminal half life of clozapine shows considerable individual variation with values ranging from 5.8 to 33 hours [24]. Mean 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 [29,48 . Consistent with J 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.
1.
REFERENCES
The Merck Index, 11th Ed. (1989), Merck and Co. Inc., Rahway NJ, U.S.A., p 2421. Martindale The Extra Pharmacopeia, 29th Ed. (1989), p 727, The Pharmaceutical Press, London.
2.
CLOZAPINE
181
3.
Schmutz J and Eichenberger E, in "Chronicles of Drug Discovery", V o l . 1 (1982), pp 39-59, J.S. Bindra and D. Lednicer Eds., Wiley, New York . Richter K, 468.
J. Chromatogr.
(1988) 4 3 4 , 465-
4. 5.
Kracmar J, Kracmarova J, Remsova M, Bokovikova TN, Ciciro VE, Nesterova GA, Suranova AV and Trius NV, Pharmazie (1990) 4 5 , 411-413. Stock VB, Spiteller G and Heipertz R, Arzneim. Forsch./Drug Res. (1977) 27, 982990. Petcher TJ and Weber HP, J. Chem. Perkin Trans 11 (1976) 1415-1420.
SOC.,
6.
7.
8.
Hunziker F, Fischer E and Schmutz J, Helv. Chim. Acta (1967) 5 0 , 1588-1599. Schneider J, German Patent Wander Ltd. (CA 80: 1 4 9 6 9 ~ )
2,316,438
9. 10. 11.
to
Hunziker F, Lauener H and Schmutz J, Arzneim. Forsch/Drug Res. (1963) 13, 324-328. Fryer RI, Earley J V , Field GF, Zally W and Sterbach LH, J. O r g . Cbem. (1969) 3 4 , 11431145. Gauch R and Michaelis W, 26, 667-681. Lillsunde P and Korte, T (1991) 15, 71-81. Wilhelm D and Kemper (1990) 525, 218-224. A,
I1 Farmaco
(1971)
12. 13. 14. 15.
J. Anal. Toxicol.
J.
Chromatogr.
Zeren W, Minglian L, Peipei X I Yanlin 2 and Yanlin 2, Biomed. Chromatogr. (1986) 1, 5357. Chen D and Jia HI 113: 908232)
Sepu
(1990) 8 , 63-64.
16.
(CA
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MICHAEL J . MCLEISH ET AL
17. 18. 19. 20. 21.
Gu N, Duan J, Bo S, Wang Z, Yan H and Xia 2, Ziran Zazhi (1985) 8 , 846. (CA 1 0 4 : 1 2 2 4 8 4 ~ ) Heipertz R, Pilz H and Beckers W, Toxicol. (1977) 37, 313-318. Meier 440P.
J,
Brit.
Arch.
J. Pharmaco.2.
(1975) 53,
Humpel C, Haring C and Saria Chromatogr. (1989) 491, 235-239.
A,
J.
Haring C, Humpel C, Auer B, Saria A, Barnas J. C, Fleischhacker W and Hinterhuber H, Chromatogr. (1988) 428, 160-166. Breyer U and Villumsen K, Pharmacol. (1976) 9, 457-465. Simpson GM and Cooper MA, (1978) 135, 99-100.
Am.
22. 23. 24.
Eur. J. Clin.
J. Psychiatry
Choc MG, Lehr RG, Hsuan F, Honi.gfeld G, Smith Pharm. Res. HT, Borison R and Volavka J, (1987) 4 , 402-405. Lovdahl MJ, Perry PJ and Miller DD, Drug Monit. (1991) 13, 69-72. Bonde s son U and Lindst rom Psychopharmacology (1988) 95, 4'72-475. Stefyuk W and Turkevich OD, Farm. (Kiev) (1986) 2, 70-72. (CA 105:12244g) Eigendorf HG, Moeschwitzer G Pharmazie (1989) 44, 645-646. Fitton A and Heel RC, 747.
Ther.
25. 26. 27. 28. 29. 30.
LH 1
Zh.
and Budde R,
722-
Drugs (1990) 40,
Rosenthaler J, Nimmerfall F, Sigrist R and Munzer H, E u r . J. Biochem. (1977) 80, 603609. Pi EH and Simpson GM, (1985) 3, 129-138.
Drugs Psychiatry
31. 32.
USP DI, Drug Information for the Health Care
CLOZAPINE
I83
Professional, 12th Ed. (1992), pp 974-978. United States Pharmacopeial Convention Inc., Rockville MD, U.S.A.
33. 34. 35. 36. 37.
Baldessarini RJ and Frankenburg FR, New E n g l . J. Med. (1991) 324, 146-154. Stephens P, Comprehensive 31, 315-326.
Psychiatry
(1990) (1988) (1975)
Lindstrom LH, Acta Psychiatr. Scand. 77, 524-529. Schutz J, Arzneim. Forsch./Drug Res. 25, 712-720
Vinick FJ and Kozlowski MR, in Annual Reports in Medicinal Chemistry, Vol. 21 (1986) pp. 1-9, B. Hesp Ed., Academic Press, New York . Harrison I, Br. J. Pharm. Prac. (1990) 71-73. Claas FHJ, S113-117.
Psychopharmacology
(1989)
38. 39. 40. 41.
99,
Salzman C, New Eng. J. Med. 829.
(1990) 323, 827-
Van To1 HHM, Bunzow JR, Guan H-C, Sunahara RX, Seeman P, Niznik HB and Civelli 0, Nature, (1991) 350, 610-614. Farde L, Wiesel FA, Nordstrom A-L and Sedvall G, Psychopharmacology (1989) 99, S28-31. Tandon R and Greden JF, Psychiatr . (1989) 46, 745-753.
Arch.
Gen.
42. 43. 44.
Deutch AY, Moghaddam B, Innis RBI Krystal JH, Aghajanian GK, Bunney BS and Charney DS, Schizophrenia Res. (1991) 4, 121-156. Cheng YF, Lundberg T, Bondesson U, Lindstrom L and Gabrielsson J. Eur. J. Clin. Pharmacol. (1988) 34, 445-449. Choc MG, Hsuan F, Honigfeld G, Robinson WT, Ereshefsky L, Crismon ML, Saklas, SR,
45.
46.
I84
Hirschowitz
47.
48.
and
Wagner
R,
Pharm.
(1989)
Res.
99,
(1990) 7 , 347-351.
Ackenheil M,
S32-S3T.
Psychopharmacology
In Therapeutic Dollery Ed., Edinburgh, UK.
Drugs Vol. 1 (1991), c Churchill Livingstone,

A,
49.
Haring C, Meise U, Humpel C, Saria Fleischhacker WW and Hinterhuber Psychopharmacology ( 1 9 8 9 ) 99, S38-S40.
HI
DIDANOSINE
Munir N. Nassar, Tracy Chen, Michael J . Reff, and Shreeram N. Agharkar
Bristol-Myers Squibb Pharmaceutical Research Institute Syracuse, N Y 13221
185
I86
MUNlR N. NASSAR ET AL.
CONTENTS
1.
Introduction Description 2.1 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
2.
2.2 2.3 2.4 2.5 2.6

3.
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
I87
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. 5.
Synthesis Methods of Analysis

5.1 5.2
Elemental Analysis High-performance Liquid Chromatography
6.
Determination in Biological Fluids

5.1 5.2 5.3
Plasma Samples Urine Samples Cerebro Spinal Fluid Samples
7.
Stability - Degradation
7.1 7.2 7.3
Solid State Stability Solution Stability Dosage Form Stability
8.
Pharmacokinetics and Metabolism

8.1 8.2 8.3
Absorption and Bioavailability Metabolism Pharmacokinetics
9.
Drug Interactions Toxicity Acknowledgements References
10. 11.
12.
188
MUNIR N. NASSAR ET AL.
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
I89
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).
190
MUNIR N. NASSAR ET AL
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).
DIDANOSINE
191
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
3.50
3.32
100
60
20 30 30
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)
I
c
0
--6.1
c
00
Z ! O
5
-- 17.7
--
10.7
t 4
>
%
Q W
- 20.5
- 25.7 - 27.0
P
E
E. 3
?
DIDANOSINE
I93
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
I
I I
Solubility (mg/mL) 27.3 31.1
1 I I
10.01 10.08
460
7.0
TI
162.800 .C
183.600 ' C 181.364 'C
T2
6.0
Peak
Aroa
i4e.9sa nJ
Ealto H
I I3.876 J/g
4.400 n Y
5.0
Halght
Oneat
F
v
175.648 'C
m a 3 I
6
c
4.0
3 a .
2 a .
1.
0. c
5 .0 0
1 75. 0
100.0
1 125.0
I 150.0
I 175.0
1 200.0
225.0
25,
Temperature ("C)
Figure 3 .
DSC therniogram of didanosine.
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)
<1
<1 <1 <1 45
200 1
<1
<I 6
<1
1
<1 <1 8
3.6
HvProscoDicitv
Didanosine is slightly hygroscopic when exposed to high relative humidity. Samples of didanosine exposed to 87% relative humidity at 25" C for 8 weeks absorbed about 1.5% moisture. No evidence has been found for the formation of polymorphs, hydrates, or solvates.
3.7
Partition Coefficient
The partition coefficient of didanosine is reported to be 0.068 f 0.005 (f S.D., n=4) as determined by the traditional shake-flask method (8). The aqueous phase used was 0.05 M phosphate buffer pH 7.0 and the organic phase was I-octanol.
I96
3.8
Spectral Properties
3.8.1
Ultraviolet SDectrum
The ultraviolet absorbance spectrum of didanosine in water (3.18 mg/mL) is shown in Figure 4. Spectral acquisition was performed on a Hewlett-Packard 84SOA Spectrophotometer with 1 cm Quartz cells. The spectrum shows the characteristic x-m* transition and is essentially identical to that of the parent at 248 nm and a molar compound, inosine which has,,A absorptivity of 12,200. Didanosine exhibited an absorbance maximum at 248.8 nm with a molar absorptivity of 12,350 M-lcml.
3.8.2 Infrared SDectrum
The infrared spectrum of didanosine is shown in Figure 5 . The spectrum was obtained with a Bio-Rad Digilab FTS-45 infrared spectrometer operated at 4 c m l resolution. The sample was diluted in KBr and pressed into a clear pellet. The hydrogen stretching region exhibits a broad band due to the NH and OH stretching bands and small C-Hstretching bands. The intense carbonyl band at 1705 cm-1 is characteristic of conjugation with the purine base. Other strong bands are the C-N stretching band at 1194 cm-1 and the C-0-Cstretch of tetrahydrohrfiuyl alcohol. The band at 1062 cml is characteristic of the symmetrical C-0-C stretching mode. In the fingerprint region, there are numerous absorption bands, many of which are difficult to assign to specific vibrational modes. The infrared spectrum of didanosine shows most of the same major bands and intensities as the spectra of the two components, hypoxanthine and tetrahydrofurfury1 alcohol. The major fiequencies, intensities, and band assignments are listed in Table IV.
2.6
2.2
1.8
1.4
. n
2
a d
. n 4
. 6
u)
.2
- .2
D
240
7 1
280
320
360
400
Waveiengbth ( n m )
Figure 4. UVNisible spectrum of didanosine
I .G
I.2
.8
.4
0
I
3000
2000
1600
1200
800
400
Wavenumbers (cm- I )
Figure 5 .
Infrared spectrum of didanosine
DIDANOSINE
I99
Table IV. Infrared peak assignments of didanosine

Frequency (cm-1)
3 100-3400 2800-3000
Intensity
Assignment
M M
NH, OH stretches
CH stretches
1704
C=O stretch
3.8.3 ODtical Rotation
The optical rotation of didanosine at 589 nm was determined using a Perkin-Elmer 241-MC polarimeter at a concentration of 10 mg/mL in deionized water using a 1 dm tube. The specific rotation of didanosine is -26.3" at 25' under the conditions specified.
3.8.4
Circular Dichroism
The circular dichroism spectrum of didanosine was measured using a Jasco 5-600 spectropolarimeter scanning from 180 to 300 nm at 100 d m i n with sensitivity of 20 mdeg. Each sample was time averaged for 16 scans total. The sample concentration was 0.015 mg/mL,. The circular dichroism spectra of didanosine (ddI) and dideoxyadenosine (ddA) are compared in Figure 6. The didanosine band at 203 nm corresponds to the n + 6* transition of the terminal hydroxyl group. This functional group is directly adjacent to one of the two optical centers in the tetrahydrofufiryl alcohol portion of the molecule and thus exhibits circular dichroism. The molar ellipticity for didanosine at 203 nm was calculated to be 16583 deg.cm2.decimol-l. The similarity in the circular dichroism
'il
DIDANOSINE
20 1
spectra of didanosine (ddI) and ddA indicated that the optical centers of both molecules have identical configurations.
3.8.5
Nuclear Magnetic Resonance Spectra

3.8.5.1
1H-NMR Soectrum
The lH-NMR spectrum of didanosine was acquired on a Bruker AM-360 FTNMR spectrometer (Figure 7) using TMS as an external reference. Approximately 80 mg of the sample was dissolved in deuterated dimethyl sulfoxide (DMSO). The spectrum was obtained using a 30" pulse width with a 5-second relaxation delay in 32 scans. The proton NMR spectrum of didanosine is comprised of a complex 8-spin system from the tetrahydrofirftryl alcohol, the C-8H and the C-2H singlets from hypoxanthine and a broad singlet due to the exchangeable proton in hypoxanthine. In DMSO solution, the alcoholic hydroxyl proton is also observed. The small broad peak at 3.4 ppm is due to the small traces of residual moisture in this hygroscopic deuterated N M R solvent. The proton chemical shifts and assignments are listed in Table V and are consistent with the structure shown in Figure 1. Table V.
e
IH-NMR characteristics of didanosine

Coupling Constant Assignment
3c-
Chemical Shift (ppm) 12.36 8.34 8.05 6.19 4.98 4.10 3.56 3.40 2.49 2.39 2.00
Multiplicity
Singlet Singlet Singlet Doublet, Doublet Doublet, Doublet multiplet multiplet Singlet (broad)
---
N1H C8H C2H C1'H C5'OH C4'H C5'W H20
DMSO-D5
C2'H2 C3'H2
Doublet, ,multiplet Doublet, , multiplet
14
12
10
T-
- 1
Parts Per Million
Figure 7. Proton NMR spectrum of didanosine.
1
Chemical Multi-
DIDANOSINE
203
3.8.5.2 j3C-NMR Spectrum
The 1%-NMR spectrum of didanosine in d-DMSO using TMS as an external reference was recorded on a Bruker AM630 FTNMR spectrometer and is presented in Figure 8. The spectrum was acquired with composite pulse broad-band decoupling with 45" flip angle pulse and a I-second total recycle time for 4096 scans. Chemical shifts and assignments of the carbon-13 spectrum are listed in Table VI. Table VI. 13C-NMR characteristics of didanosine
Chemical Shift (6)
158.98 148.63 146.30 140.11 124.28 85.91 83.02 63.47 32.22 25.70
(PPm)B
156.7 147.6 145.7 138.3 124.3 84.5 82.1 62.6 39.5 32.2
a:
AEL Assignment
C6 c4 c2 C8 c5
D D
c 1'
C4'
C5'
T M T
C2' C3'
b: c: d:
Data from Bristol-Myers Squibb Co S=Singlet, D=Doublet, T=Triplet, M=Multiplet. Solvent used was deuterad dimethyl sulfoxide. Data f o Buchanan and Bourque, 1989 (9). rm
3.8.6
Mass SDectrum
The mass spectrum of didanosine was acquired using a HewlettPackard 5985B mass spectrometer equipped with a Vestec 701 thermospray interface and a Waters Associates 600MS high performance liquid chromatograph (HPLC). The sample was prepared by dissolving approximately 1 mg of material in 0.5 mL of
160
120
00
40
Parts Per Million
Figure 8. Carbon-13 NMR spectrum of didanosine.
DIDANOSINE
205
mobile phase. Sample introduction was by direct injection without separation. The experimental conditions are listed below:
HPLC Conditions: Flow Rate: Column: Detector: Injection Volume: Sample Concentration: Mobile Phase:
1.0 mL/min. None Thermospray LCMS 25 mL = 2 mg/mL mobile phase Acetonitrile and 0.1 M ammonium acetate (50:50)
Thermospray Interface: Source Temperature: 250' C Vaporizer: Standard fixed tip Vaporizer Temperature: 2 12" C Tip Heater Temperature: 255" C Filament: Of f Discharge Electrode: Off Repeller: Off Mass Spectrometer: Ionization (Thermospray): Scan Range: Scan Rate: Electron Multiplier: Signal Processor Sensitivity:
Positive ion C1 125 -300 amu 2.825 sechcan -1800 V
4
A typical thermospray mass spectrum of didanosine is shown in Figure 9. A strong protonated molecular ion is observed at 237 mass units. The base peak is found at 137 mass units and is due to the protonated hypoxanthine fragment. This mass spectrum is indicative of extensive adduct formation. The ion fragment at 273 is due to dimerization of the hypoxanthine in the highly ionized plasma of the thermospray source. The fragment at 154 is the ammonium adduct of oxanthine and the ion at 178 is the acetonitrile adduct.
l u
2
b 5
E
. I
LL
DlDANOSlNE
207
1.
Synthesis 2,3'-Dideoxynucleosides are typically synthesized from 2'-deoxy nucleosides & Barton-type deoxygenation reactions (1 0) or fiom I intact nucleosides by multistep routes involving deoxygenation reactions to 2',3'-unsaturated deoxynucleosides ( 11) which are then hydrogenated. Approaches through ketonucleosides (1 2) and a photoreductive process have also been described (1 3). A scheme depicting the synthesis of didanosine (ddI) is shown in Figure 10 (14). Selective benzoylation of the 5' hydroxyl group of 2'-deoxyinosine J was achieved by dropwise addition of a pyridine solution of benzoyl chloride to 2'-deoxyinosine suspended in pyridine. The 5'-O-benzoyl-2'-deoxyinosine formed was then treated in one portion with l,I'-thiocarbonyldiimidazoleto form the thioimidazolide III. Deoxygenation at the 3' position of the thioimidazolide 111 gave 5'-O-benzoyl-2',3'-dideoxyinosine E. Deprotection of & by treatment with anhydrous methanol J saturated with anhydrous ammonia at 0' C yielded didanosine V in 90% yield. Didanosine has also been prepared enzymatically by the deamination of 2',3'-dideoxyadenosine (ddA) using adenosine deaminase at room temperature (14). Recrystallization fiom methanol gave 85% yield.
Methods of Analysis
5.1
Elemental Analvsis
The elemental analysis of didanosine was done using a Control 1 Equipment Corporation Model 240/24 system. Data are presented in Table VII.
208
I
OH
OH
1, 1-thiocarbonyl
diimidazole
0
deoxygenation
4
1 ,Cdioxane
PhC(0)
PhC(0)
IV
I11
S
V
Figure 10, Synthesis of didanosine (dd1)
DIDANOSTNE
209
Table VII. Elemental analysis of didanosine.

Element
YOThe0
50.84
YOFound
50.47 23.60
5.2
High Performance Liquid Chromatography
Several stability-indicatingreverse-phase HPLC methods have been reported for the determination of didanosine and its impuritieddegradates in bulk drug substance or formulations. A summary of these methods is presented below. Method A (7): Column: Detector: Mobile Phase: Retention Volume: Method B (1 5): Column: Guard Column: Detector: Mobile Phase:
C1g pBondapak@, 10 pm, 4.6 mm X 250 mm (Waters Associate) UV at 254 nm acetonitri1e:phosphatebuffer pH 7 (5:95) hypoxanthine: 4.7 mL didanosine: 9.4 mL C1g Ultrasphere@ OD, 5 pm, 4.6 mm X 250 mm (Beckman) C18 pBondapak Guard-Pak@,4 mm X 6 mm, (Waters Associates) UV at 254 nm methanol : 0.2 M ammonium acetate buffer pH 7 (12:88) 1.OmL/min.
Flow Rate: Sample Concentration: 4 PdmL Injection Volume: 50 pL hypoxanthine: 3.8 mL Retention Volume: inosine: 5.3 mL 2'-deoxyinosine: 5.8 mL didanosine: 12.4 mL
210
Method B is capable of separating the possible synthesis impurities. A representative chromatogram of a spiked sample (all compounds are 4 pg/mL,) is shown in Figure 1 1.
1 0 MINUTES
IS
Figure 1 1 . Chromatographic separation of didanosine and its possible synthesis impurities.

6.
Several HPLC procedures have been developed for the analysis of didanosine and its major metabolite hypoxanthine in biological fluids. Laboratory specimens were handled according to the guidelines set forth by the Centers for Disease Control (CDC) (16, 17, 18). All sample manipulations are recommended to be performed in a biological safety cabinet using gloves and gown.
DIDANOSINE
21 I
Transfer of samples should be done with plastic transfer pipettes to reduce risk of puncture.
6.1
Plasma Samples
Method A (19):
All sampies were heated at 57" C for 45 minutes to inactivate HIV followed by centrifigation at 400 g for 10 minutes prior to extraction. C,, Sep-PakB (Waters) cartridges were primed with 6 mL of methanol followed by 12 mL of distilled, deionized water. The internal standard (2'-deoxyguanosine, 0.1 mg/mL) was added to the treated plasma to achieve a final concentration of 0.5 pg/mL. Samples (usually 1 mL) were then loaded onto the primed Sep-PakB cartridges and drawn through under vacuum at approximately 0.5 mL/min. After drawing through all liquid from the plasma, each cartridge was washed with 2 mL of water drawn through under vacuum at approximately 1 mL/min. Samples were then eluted with 2 mL of 100% methanol into test tubes (0.5 mL/min). Evaporation of the methanol was achieved under a stream of nitrogen while heating in a water bath at 37" C. Samples were reconstituted in a volume of 200 pL of water and votex-mixed for 30 seconds. After a final centrihgation for 3 minutes at 11,900 g, samples were analyzed by HPLC.
The recovery of didanosine (1.7 pM) and the internal standard, deoxyguanosine (1.9 pM)from spiked plasma was 80% f 15% and 85% f lo%, respectively. Spot checks of low (0.212 pM) and high (13 . 6 pM) concentrations demonstrated the same recovery throughout the range. The HPLC conditions for the assay of the extracts are as follows: Column: Detector: Mobile Phase: C,, NovaPakB, 4pm, 8mm X l o o m , Z-module mode, (Waters Associates) UV at 252 nm acetonitrile : 0.1% heptafluorobutyric acid in deionized, distilled water (5:95)
212
Flow Rate: Detection Limit: Injection Volume: Retention Time:
2.0 mL/min.
0.1 pM for didanosine 50 pL deoxyguanosine: 7.4 min 8.4 min. didanosine:
A wash cycle of 100% acetonitrile for 30 seconds after each run (beginning at 8.4 min) was programmed in to keep the interference to a minimum. A representative chromatogram of a plasma sample is shown in Figure 12. (3)
2.00
'*0
t
2
I1 I
6 Minutes
10
Figure 12. Chromatogram of an extracted plasma sample from a patient: didanosine (3), internal standard, deoxyguanosine, (2), and major metabolite, hypoxanthine (1). Method B (20): All clinical plasma samples were heated at 57" C for 3 hours to inactivate HIV prior to extraction. Samples not processed immediately were stored at -20" C. Samples were extracted using C18 SPE@ columns (500 mg, J. T. Baker Res.). SPE columns were activated with methanol, followed by 10 m M sodium phosphate buffer, pH 8.0. A 1.0 mL aliquot of internal standard solution (3',5'-anhydrothymidine, 3 pg/mL in sodium phosphate buffer) was placed on the cartridge, followed by 0.5 mL of plasma. The samples were aspirated slowly. Columns were washed with 3 mL of sodium phosphate buffer and 3 mL of water, dried, then
DIDANOSINE
213
eluted using two 0.5 mL aliquots of methanol. The eluates were evaporated under a stream of nitrogen at 40" C, followed by reconstitution in 0.20 mL HPLC mobile phase (15% methanol in 50 m M potassium phosphate buffer containing 0.05 % tetraethyleneamine, pH adjusted to 7.0). The linear response was in the range 0.025 to 10 pg/mL. The recovery of didanosine from extracted plasma samples was 85%. The HPLC conditions used in this assay method are listed below. A typical chromatogram is shown in Figure 13: Column: C , UltrasphereB, 5 pm, 4.6 mm X , 250 mm, (Beckman) Detector: W at 254 nm Mobile Phase: methanol:5OmM potassium phosphate buffer containing 0.05% tetraethyleneamine pH 4.0 (15:85) Flow Rate: 0.7 mL/min. Injection Volume: 50 pL
Patient
a
!I 1 :
100 ng/ml standard
Blank plasma
+L
8
*JL
12 16 20
Time (min)
Figure 13. Chromatograms of blank extracted human plasma, plasma spiked with didanosine, and a sample from a patient.
214
62 .
Urine Samples
Method. A (19): Urine samples (1 mL) were processed in the same manner as the plasma Method A samples except that the final reconstitution volume was 1 mL since didanosine concentration was usually high enough to not require concentrating the sample. In some cases, a hrther dilution was necessary before analysis. The amount of urine processed is dependent on the dose of didanosine the patient was given. The HPLC conditions used are the same as those in Method A for the plasma samples with the exception that 3% acetonitrile (5% in plasma sample analysis) was used due to the greater number of interfenng peaks in patient urine. This resulted in an elution time of 19.6 minutes for didanosine. A typical chromatogram is shown in Figure 14.
10 Minutes
15
20
Figure 14. Chromatogram of a patient's urine sample. Method B (21): Urine samples were buffered with 200 mM potassium phosphate buffer, pH 8.0 (1 part urine to 2 parts buffer) at the time of preparation to improve the stability of didanosine, which hydrolyzes rapidly under even mildly acidic conditions. Samples not processed immediately were stored at -20' C. All clinical samples were
DIDANOSINE
21s
heated at 57" C for 3 hours to inactivate HIV prior to extraction. Urine samples were extracted using phenylsilane columns (500 mg, J. T. Baker). Prior to extraction, the column was washed with methanol, followed by 20 mM potassium phosphate buffer, pH 8.0. Buffered sample (0.5 mL) was pipetted on the column, followed by 0.1 mL of a 400 pg/mL internal standard (2',3'-didehydro-3'deoxythymidine, d4T) in water. The columns were washed sequentially with 3 mL of each of the following: 20 mM monobasic potassium phosphate, 20 m M potassium phosphate buffer (pH 8.0) and water. Columns were dried under vacuum, then eluted with two 0.5 mL aliquots of aqueous 70% methanol containing 0.02% tetraethyleneamine. The eluate was mixed with 1.O mL potassium phosphate buffer (20 mM, pH 7.2) prior to HPLC assay. The linear range of urine assay was in the range 1.0 - 400 &mL. Extraction recovery average was 97%. Typical chromatogram are shown in Figure 15:
Patient
I!
7
11
11
rl
5 ug/ml standard
Blank urine
15
19
Time (min)
Figure 15. Chromatograms of blank extracted human urine, urine spiked with didanosine, and a sample from a patient.
216
The HPLC conditions used in this method to produce chromatograms in Figure 15 are described below. Column: C Zorbax@,5 pm,4.6mmX250mm (Dupont) , W at 254 nm Detector: methoxyethanol : 18.3 mM potassium Mobile Phase: phosphate buffer pH 7.2 (3.9:96.1) Flow Rate: 1.OmL/min. Injection Volume: 20 pL
6.3
Cerebro Spinal Fluid Samples
CSF samples were processed in the same manner described under plasma samples Method A (1 8) except that only 1 mL of water was used to wash samples on the Sep-Pak prior to methanol elution since the interferences in CSF are minimal. Under conditions where concentration is not necessary, the CSF may be injected directly into the HPLC system without extraction. The HPLC conditions used are the same as those in Method A for the plasma samples . A typical chromatogram is presented in Figure 16.
2.00 1
1.50 -
1
-0
-0
Minutes Figure 16. Chrotnatogram of an extracted CSF sample from a patient.

4
10
7.
Stability Degradation
7.1
Solid State Stability
Didanosine drug substance is very stable under normal storage
DIDANOSINE
217
conditions. Chemical and physical stability data for didanosine indicate that the drug is stable for at least 24 months at 30" C. Hypoxanthine, didanosine's major degradation product, forms in only very small amounts when didanosine is exposed to 25" C under high intensity light (400 foot-candles) or 87% relative humidity. No significant loss in activity was observed under these storage conditions. Didanosine retained > 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
10
12
14
PH
Figure 17. The pH-rate profile of didanosine.
218
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 2'3'7. dideoxyribose as shown Figure 18 ( )
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 a (22); 40% for oral doses of 1 0.8-10.2 rng/kg and 23% for oral doses of 15.2-33.0 mg/kg by 1 Dollin et a (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 a (26). For pediatric administration, a lower bioavailability of 1 21 % (range <5 to 89%) was observed when oral doses were given two minutes after the ingestion of 10 to 15 mL of MaaloxB or 15 to 20 mL of an aluminum hydroxide suspension, one-half hour before or one hour after meals (27). Bioavailability of didanosine decreased in the presence of food (28,29). When the buffered powder was dissolved in water and administered with food, the bioavailability dropped from 29% f 6% to 17% f 4% (29). Therefore, it is recommended that all didanosine formulations be administered on an empty stomach. The precise mechanism of this decrease is not clear. One possible cause may be increased acid degradation of didanosine caused by augmented acid secretion in the presence of food. Also, prolonged acid contact as a result of delayed gastric emptying may have contributed to this effect. Since different oral dosage forms are available, data reported by
220
Hartman et al. showed that the oral bioavailability was 41 % f 7% for oral solutions with antacid, 25% f 5% for experimental buffered tablets (not the commercial products), and 36% f 6% for buffered tablets with supplemental antacid (29). Another study by Knupp et ul (25) indicates that two didanosine formulations, oral solution with Maalox and citrate-phosphate buffered powder possess comparable bioavailability. The respective mean values of area under the curve (AUC(,,)) were 3530 and 323 1 hr.ng/mL. Videx Chewable/Dispersible Buffered Tablets were found to be 20% to 25% more bioavailable compared to the oral solution (1).
8.2
Metabolism
Didanosine can be metabolized to hypoxanthine, which can either re-enter the purine metabolic pathways or be degraded hrther to uric acid. Because HIV primarily infects cells expressing the CD4 receptor (i.e., lymphocytes and monocytedmacrophages), nucleoside analogs must gain access into these cells to demonstrate activity. Didanosine penetrates lymphocytes by a passive process, where it exhibits a complex intracellular disposition (2). Like other nucleoside analogs, three phosphorylation steps are required to form the active triphosphate moiety. Initially, intracellular didanosine is converted to the monophosphate form (dd-IMP) by 5'-nucleotidase. The dd-IMP is then converted to ddA monophosphate (dd-AMP) via adenylosuccinate synthetase and adenylosuccinate lyase. Subsequently, dd-AMP is converted to dd-A di- and triphosphates (26) (Figure 19). Incorporation of dd-ATP into viral deoxyribonucleic acid terminates chain elongation. Alternatively, dd-ATP may directly inhibit viral reverse transcriptase activity. The time course and extent of these metabolic reactions have not yet been determined in patients. However, it requires at least five enzymes to activate didanosine to dd-ATP in cell cultures and the conversion is relatively inefficient.
8.3
Pharmacokinetics
Didanosine demonstrated linear pharmacokinetic behavior over the
DIDANOSINE
22 1
2', 3'-dideoxyadenosine5'-triphosphate (dd-ATP)
2', 3'-dideoxy-adenosine-
5'-diphosphate (dd-ADP)
2', 3'-dideoxy-adenosine- 5'monophosphate (dd-AMP)
2', 3'-dideoxyinosine-Smonophosphate (dd-IMP)

5'-nucleotidasel
didanosine (ddI)
purine nucleosid phosphorylase
2, 3-dideoxyribose1-triphosphate
1
7
Uric acid
hypoxanthine
h ypoxanthine-guanine phosphoribosyl transferase
inosine-5'-monophosphate (IMP) adenosine-5'-monophosphate (AMP)
guanosine-5'-monophosphate (GMP)
Figure 19.
Metabolic pathways of didanosine.
222
dose range of 0.4 to 16.5 mg/kg following intravenous administration. The overall total body clearance (CL) was 760 mL/min and renal clearance (CLd) was 406 mL/min. Since CL, exceeds the glomerular filtration rate, active tubular secretion of didanosine is indicated. The overall half-life (tlJ was about 1.36 hours with values in the range of 0.88 to 1.72 hour. The overall volume of distribution (V,,) was 54.0 L with values ranging fiom 47.7 to 61.4 L (25). The average urinary recovery of didanosine ranged from 49.2% to 63.3% of the dose which was recovered within 12 hours of didanosine administration. The majority of didanosine recovered in the urine was excreted within the first 8 hours after drug administration. The CSF/plasma ratio ranged fiom 0.12 to 0.27 at 1 hour after the completion of an intravenous infbsion of didanosine (21, 26). Following oral administration, didanosine demonstrated linear pharmacokinetic behavior over the dose range of 0.8 to 10.2 mg/kg. Pharmacokinetic parameters at steady state were not significantly different fiom values obtained after single oral dose. Mean time to reach peak plasma concentration (t,,,J ranged from 0.50 to 1 . I2 hours. The overall half-life was about 1.43 hour with values ranging from 0.76 to 1.87 hour (25). The average renal clearance ranged from 206 to 688 mL/min. Urinary recovery of didanosine was variable from 6.9% to 41.5%. Similar pharmacokinetic data were also reported by Hartman et a (26, 29). 1 When didanosine was administered intravenously at 12-hour intervals, there was no evidence of accumulation (accumulation factor = 1). However, after oral administration, the accumulation factor ranged from 1.0 to 1.3 (25).
9.
Drug Interactions
Drug interaction studies have demonstrated that there are no clinically significant interactions with didanosine and ketoconazole or ranitidine. Drugs whose absorption can be affected by the level of acidity in the stomach (e.g., ketoconazole, dapsone), should be administered at least 2 hours prior to dosing with didanosine.
DIDANOSINE
223
Co-administration of didanosine with drugs that are known to cause peripheral neuropathy or pancreatitis may increase the risk of these toxicities (1).
As with other products containing magnesium and/or aluminum antacid components, didanosine (Videx) Chewable/Dispersible Buffered Tablets or Videx Pediatric Powder for Oral Solution should not be administered with a prescription antibiotic containing any form of tetracycline (1).
Plasma concentrations of some quinolone antibiotics are decreased when administered with antacids containing magnesium or aluminum. Therefore, doses of quinolone antibiotics should not be administered within 2 hours of taking didanosine. Concomitant administration of antacids containing magnesium or aluminum with didanosine may potentiate adverse effects associated with the antacid components (1).
10.
Toxicity
Evidence of dose-limiting skeletal muscle toxicity has been observed in mice and rats (but not in dogs) following long-term (greater than 90 days) dosing with didanosine at doses that were approximately 1.2 to 12 times the estimated human exposure (1). The major clinical toxicities of didanosine (Videx) are pancreatitis, hepatitis, and peripheral neuropathy (30, 3 1). Pancreatitis must be considered whenever a patient receiving didanosine develops abdominal pain and nausea and vomiting. Didanosine at very high concentrations has been reported to cause bone marrow myelopoiesis and erythropoiesis (32). However, at concentrations of 5 to 10 pM (peak plasma levels and within the range of activity against HIV), the inhibition of bone marrow progenitor cells was minimal. Other reported clinical toxicities for didanosine include hypokalemia (33) and optic neuritis (34). Although the relation
224
between didanosine and optic neuritis can not be asserted, the stabilization of visual loss after interruption of didanosine and its deterioration when its therapy was resumed are consistent with a toxic role of the drug. Results from genotoxicity studies suggest that didanosine is not mutagenic at biologically and pharmacologically relevant doses. At significantly elevated doses in vitro, the genotoxic effects of didanosine are similar in magnitude to those seen with natural DNA nucleosides.
11.
Acknowledgements
The authors wish to express their sincere thanks to the following members of Analytical Research and Development, Bristol-Myers Squibb Co., Syracuse, NY who have provided information and spectra for portions of this chapter: J. H. Medley; B. C. Prosser; D. W. Dodsworth; T. R. Marr; and R. D. Rutkowski. Thanks are also due to D. A. Benigni, Chemical Process Department, BristolMyers Squibb Co., for his contribution to the synthesis section. The authors would also like to thank J. B. Bogardus, R. A. Lipper, J. K. Allison, and H. G. Brittain of Bristol-Myers Squibb Pharmaceutical Research Institute for their valuable comments and advice.
12.
References
1. 2.
3.
Package Insert, Videx (didanosine), October, 1991. M. J. Shelton, A. M. O'Donnell, and G. D. Morse, m a c o t h e r . , 26, 660 (1992).
U.S.A.N. and The U.S.P.Dictionary of Drug Names, United States Pharmacopeial Convention, Inc., Rockville, M.D., 1992, p. 196.
DlDANOSlNE
225
4.
5.
N.C.I. Investigational Drugs - Pharmaceutical Data, U. S. Department of Health and Human Services, Public Health Service, National Institute of Health, National Cancer Institute, Bethesda, M.D., 1990, p.49. The Merck Index, 11th Edition, Merck and Co., Inc., Rahway, N.J., 1989, p.490. American Hosuital Formularv Service Drug Information, American Society of Hospital Pharmacist, Inc., Bethesda, M.D., 1992, p.2345. B. D. Anderson, M. B. Wygant, T-X. Xiang, W. A. Waugh and V. J. Stella, Int. J. Pharm., 45, 27 (1988).
A. P. Cheung and D. Kenney, J. Chromatogr., (1990).
6.
7.
8. 9. 10. 11. 12. 13. 14. 15.
506, 119
G. W. Buchanan and K. Bourque, Mag. Res. Chem., 27(2), 200 (1989).

E. J. Prisbe and J. C. Martin, Svnth. Commun., (1985).
u, 401
J. P. Horowitz, J. Chau, M. Noel, and J. T. Donatti, J. Om. Chem., 32, 817 (1967). J. Them, and D. Rasch, Nucleosides and Nucleotides, 4, 487 (1985).
I. Saito, H. Ikehera, R. Kasatani, M. Watanabi, and T. Matsura, J. Am. Chem. SOC., 108, 115 (1986). 3 R. R. Webb 11, J. A. Wos, J. C. Martin, P. R. Brodhehrer, Nucleosides and Nucleotides, 1,147 (1988).
G. Ray and E. Murrill, Analv. Let., 20(1 l), 1815 (1987).
226
16.
J. H. Richardson and W. E. Barkley, Biosafety in Microbiological and Biomedical Laboratories, Public Health Service, Washington, DC, DHHS Publication No. (CDC) 848395 (1984).
17.
CDC, HTLV-III/LAV: Agent Summaty Statement. Mortality Morbiditv Weeklv Report, 35, 540 (1986). CDC, Recommendations for Prevention of HIV Transmission in Health-Care Settings. Mortalitv Morbiditv Weeklv Report, 36 (Suppl. 2), 3s (1987).
M. E. Carpen, D. G. Poplack, P. A. Pizzo, and F. M. Balis, J. of Chromatogr., 526,69 (1990).
18.
19.
20. 21.
C. '4. Knupp, F A. Stancato, E. A. Papp, R. Barbhaiya, J. Chromatogr., 533, 282 (1990).
and
R. Yarchoan, H, Mitsuya, R. V. Thomas, J. M. Pluda, N. R. Hartman, C. Perno, K. S. Marczyk, J. Allah, D. G. Johns, and S. Broder, Science. 245,412 (1989). M. Kozencweig, C. McLaren, M. Beltangady, J. Ritter, R. Ctmetta, L. Schacter, S. Kelly, C. Nicaise, L. Smaldone, L.Dunkle, R. Barhaiya, C. Knupp, A. Cross, M. Tsianco, and R. R. Martin, Rev. Infect. Dis., a ( % ) , S570 (1990). R. D o h , J. S. Lamber, G. D. Morse, R. C. Reichman, C. S. Plank, J. Reid, C. Knupp, C. McLaren, and C. Pettinelli, Rev. Infect. Dis., 12 (S5), S540 (1990).
R. Yarchoan, H. Mitsuya, J. M. Pluda, K. S. Marczyk, R. V. Thomas, N. R. Hartman, P. Brouwers, C. Perno, J. Allain, D. G. Hohns, and S. Broder, Rev. Infect. Dis., 12 ( S 5 ) , S522 (1990).
C. A. Knupp, W. C. Shyu, R. Dolin, F. T. Valentine, C. McLaren, R. R. Martin, K. A. Pittman, R. H. Barbhaiya, Clin. Pharmcol. Ther., 49 ( 9 , 523 (1991).
22.
23.
24.
25.
DIDANOSINE
221
26.
N. R. Hartman, R. Yarchoan, J. M. Pluda, R. V. Thomas, K. S. Marcqyk,, S. Broder, D. G. Johns, Clin. Pharmcol. Ther., - ( 5 ) , 647 (1990). 47
27.
K. M. Butler, R. N. Husson, F. M. Balis, P. Brouwers, J. Eddy, D. El-Amin, J. Gress, M. Hawkins, P. Jarosinski, H. Moss, D. Poplack, S. Santacroce, D. Vernon, L. Wiener, P. Wolters, P. A. Pizzo, New En@. J. Med., 324 (3), 137 (1991). W. C. Shyu, C. A. Knupp, K. A. Pittman, L. Dunkle, R. H. Barbhaiya,, Clin. Pharmacol. Ther., 50 (9, 503 (1991).
N. R. Hartman, R. Yarchoan, J. M. Pluda, R. V. Thomas, K. M. Wyvill, K. P. Flora, S. Broder, D. G. Johns, Clin. Pharmcol. Ther., 50 (3), 278 (1991).
28. 29.
30.
R. Yarchoan, J. M. Pluda, R. V. Thomas, H. PvZitsuya, P. Brouwers, K. M. Wyvill, H. Hartman, and D. J. Johns, Lancet. 336, 524 (1990).
S. F. LeLacheur, and G. L. Simon, J. Acauir. Immune. Defic. Syndr.. 4, 538 (1991).
31.
32.
33.
J-M. Molina and J. E. Groopman, N. Ena. J. Med., 321, 1478 (1989). C. Katlama, R. Tubiana, M. Rosenheim, M. A. Valentin, E. Caumes, F. Bricaire, and M. Gentilini, Lancet, 337, 183
(1991).
34.
A. Laf'euillade, L. Aubert, P. R. Quilichini, Lancet. 337, 615 (1991).
Chaffanjon,
and
DIPIVEFRIN HYDROCHLORIDE
G. Michael Wall and Tony Y. Fan
Alcon Laboratories, Inc.
6201 South Freeway

Fort Worth, Texas 76134
229
Copyright rD 1993 by Academic Press, Inc All rights of reproduction In any form reserved
230
G. MICHAEL WALL AND TONY Y. FAN
1.
DESCRIPTION Name, Formula and Molecular Weight 1.1 1.2 Appearance, Color, Odor 1.3 History 1.4 Pharmacology 1.5 Phannaceutics SYNTHESIS PHYSICAL PROPERTIES 3.1 spectroscopy 3.1 .1 Infrared Spectrum 3.1.2 Ultraviolet Spectra 3.1.3 Nuclear Magnetic Resonance Spectra 3.1.4 Mass Spectra Thermal Properties 3,2 3.2.1 Melting Range 3.2.2 Differential Scanning Calorimetry (DSC) 3.2.3 ThermogravimemcAnalysis (TGA) 3.3 X-Ray Powder Diffractometry 3.4 Partition Coefficients 3.5 Ionization Constant, pKa 3.6 Solubility Solution Color, Clarity and pH 3.7 TYPICAL METHODS OF ANALYSIS Identity 4.1.1 Infrared Spectrophotometry 4.1.2 Thin-layer ChromatographicIdentity Test 4.1.3 Chloride Identity Test 4.2 Elemental Analysis 4.3 Other Pharmacopeial Tests 4.4 Chromatography 4.4.1 Thin-Layer Chromatography 4.4.2 High-Pressure Liquid Chromatography
4.1
2. 3.
4.
5.
STABILITY -DEGRADATION 5.1 Potential Routes of Degradation 5.1.1 Thin-Layer Chromatography of 3- and 4Monopivd oylepinephrine 5.1.2 Analysis of 3- and 4-Monopivaloylepinephrine 5.2 Solid-stateStability 5.3 Solution Stability
DIPlVEFRIN HYDROCHLORIDE
23 1
6.
7.
ABSORPTION, DISTRIBUTION, METABOLISM AND EXCRETION TOXICOLOGY ACKNOWLEDGEMENTS REFERENCES
1. 1.1
DESCRIPTION
Name, Formula and Molecular Weight
used Dipivefrin hydrochloride is a prodrug of epinephrir~el-~ for the topical ophthalmic treatment of elevated intraocular pressure in patients with chronic open angle glaucoma.7 The generic name is dipivefrin hydrochloride.8 The recommended international nonproprietary name is Dipivefrine.9-l Other chemical names: (a) (1)-2,2-Dimethylpropanoic acid4-[ 1-hydroxy-2-(methylamino)ethyl]1,a-phenylene ester hydrochloride8v12
(b) (1)-3,4-Dihydroxy-ol-[(methylamino)methyl]benzyl alcohol 3,4dipivalate hydrochloride8*12
(c) Epinephrine dipivalatelo

(d) Pro-epinephrinelo
CAS registry number8: 64019-93-8 CAS number for the free base?
52365-63-6
Empirical Formula12: C 1gH29NO5 . HC1 Molecular Weightl2: 387.90
232
G. MICHAEL WALL AND TONY Y.FAN
structure:
0
(CH3)3C-C-O I'
y-CH2-NH-CH3
/ (CH3)3C-C-O
0 1.2 Appearance, Color and Odor
OH
HCI
II
Dipivefrin hydrochloride is a white crystalline powder with a faint 0d0r.l~ 1.3 History
Dipivefrin hydrochloride has been patented with regard to synthesis, pharmaceutical formulation, and use as an antiglaucoma and broncholyric agent: D. Henschler etal., German Patent 2,152,058 corresponding to U.S. patent 4,085,27014 (1973 and 1978 both to Adolf Klinge & Co., Munich, Truelove, German patent 2,343,657 Germany); A. Hussein, and J.E. corresponding to U.S.patents 3,809,71415 and 3,839,58416 (all 1974 to INTERx Research Corporation, Lawrence, Kansas).17 Modifications of the synthesis have been patented by Zupan,l8-19Bodor and YuanFO and Maki and Rosenqvist.21 Dipivefrin hydrochloride has been marketed under a variety of trade names around the ~orld.~(),11,1722 the U.S., In Propinep (dipivefrin HC1) Sterile Ophthalmic Solution, 0.1%, has been marketed since 198022 as initial therapy for the control of intraocularpressure in chronic open angle glaucoma.7 Combinations of dipivefrin with betaxolo1,23* de~amethasone?~ panethidid6 have also been patented for the and treatment of glaucoma. 1.4 pharmacology
Dipivefrin is an ophthalmologic adrenergic agent used for the treatment of glaucoma. General phma~0l0gy,27.28toxicology~7~8 clinical and experience27-29with the use of dipivefrin for the treatment of glaucoma have been reviewed. Kaback et ul.30 evaluated the intraocular pressure lowering effect of dipivefiin in concentrations from 0.005 to 0.5% in 10 patients; at a concentration of 0.1 % instilled twice daily for 1 month, significant reductions were observed for intraocular pressure during diurnal testing on days 2 and 31with no toxic side effects. Studies comparing dipivefiin to epinephrine in the treatment of glaucoma have shown that dipivefrin has a greater potency and fewer side effects.27931 Results of
DIPlVEFRlN HYDROCHLORIDE
233
comparison studies with other glaucoma drugs and receptor binding studies using membranes prepared from homogenized rabbit iris and ciliary body have indicated that dipivefrin binds to the p-adrenergic receptor rather than the a-adrenergic receptor.32933
A number of pharmacological studies have been performed with dipivefrin. Bunag et ~1.34 reported that dipivefrin increased heart rates in anesthetized reported that rats compared to epinephrine. However, Wang et ~1.35 dipivaloyl derivatives of epinephrine (dipivefrin), norepinephrine, and isoproterenol produced cardiovasculareffects that were comparable to the parent catecholamine following intravenous injections in dogs, though these effects were delayed with regard to onset and were of longer duration, findings that would be consistent with initial biotransfomation to the active catecholamine. The pharmacological profile of dipivefrin in rats and mice was found to be qualitatively similar to that of epinephrine bitartrate, though the latter compound was generally more potent.36 The effects o dipivefrin f were thought to be a result of the conversion to epinephrine in vivo.36 Dose-related increases in cyclic AMP have been observed with dipivefrin in the aqueous humor of rabbits.30 The application of dipivefrin to the eyes of rabbits via a soft contact lens delivery system inhibited the transport of Evans Blue dye across the blood-aqueous humor barrier for approximately 2 hours.37
Several in vitro studies with dipivefrin have also been reported. Dipivefiin was shown to accelerate the water transport of retinal pigment epithelial cells from chick embryo on glass slips.38 The growth of human corneal keratocyte trabecular meshwork and endothelial cells was inhibited by M, 2.8 X 1 " M) has 0 dipivefrin HC1 in vitro.39 Dipivefrin (2.8 X been shown to inhibit phagocytosis of latex particles by rabbit corneal fibroblasts.40 Studies with rat conjunctiva demonstrated dipivefrin-mediated anaphylactic action through both an epinephrine-likeactivation of padrenergic receptors and an unknown, non-epinephrine-likemechanism41 1.5 Pharmaceutics
In a study comparing the intraocularpressure lowering and mydriatic effects of dipivefrin and epinephrine in rabbits, adjustment of the pH of ophthalmic rm solutions f o 3 to 5 resulted in a 50-fold increase in the potency of dipivefrin compared to e p i n e ~h r i n e .The sodium chloride equivalents and ~~ freezing point depressions of dipivefiin hydrochloride solutions at various concentrations (0.5, 1,2,3,and 5%) have been reported43
234
G.MICHAEL WALL AND TONY Y. FAN
2.
SYNTHESIS
The synthesis of dipivefrin hydrochloride has been patented: D. Henschler et al., German Patent 2,152,058 corresponding to U.S. patent 4,085,27014 (1973 and 1978 both to Adolf Klinge & Co., Munich, Germany); A. Hussein, and J E Truelove, German patent 2,343,657 corresponding to .. U.S.patents 3,809,71415 and 3,839,58416 ( a l l 1974 to INTERX Research Corporation,Lawrence, Kansas).l7 MMications of the synthesis have been patented by Z ~ p a n , Bodor and Yuan?o and Maki and ~*~~~ Rosenqvist.21 The synthesis of dipivefrin has been described by Sittigz and illustrated by Kleenman and Engel& (Figure 1). After dissolution of 0.27 mole of achloro-3',4'-dihydroxyacetophenone, in 200 mL of methanol with 1, warming, 100 mL of a 40% aqueous solution of methylamine is slowly added and the mixture is thoroughly stirred, first at 50-55OC (2 hours), then at room temperature for an additional 24 hours. The adrenolone,2, is isolated by filtration, washing and precipitation as the HCl salt (mp 242OC). Esterification is performed by treating 25.3 g (0.125 mole) of adrenolone 2 in a mixture of ethyl acetate (250 mL) and perchloric acid (0.125 mole) with an excess of pivaloyl chloride (280 mL). The mixture is heated at reflux for 5 hours, then cooled with stirring. The esterified ketone, 3, is precipitated as the perchlorate salt. Reduction of ketone 3. (20 g) is accomplished by dissolution in 95% ethanol (300 mL) in a Parr reaction vessel with Adams catalyst, platinum dioxide (1.5 g), and agitation under hydrogen (50 PSI) for 1 hour at room temperature (Route A). After filtration, the resulting ol is i crystallized by the addition of ether (1200 mL) to give the free base form of dipivefrin, 4 (mp 146-7T). Hussain and Truelovel5J6 also described the resolution of the levo-isomer of dipivefrin by fractionalrecrystallization of the dibenzoyl-d-bitartratesalt. The traditional preparation of the hydrochloride salt, as reported by Hussein and T ~ u e l o v e ,has~been~ ~ * ~ optimized.20 Originally, the free base of dipivefi, 4, was treated with a stoichiometric amount of hydrochloric acid in a short chain alcohol. However, this procedure led to discoloration and excessive loss of yield (only about 50%) and purity of the final product, especially upon scale-up.20 Bodor and Yuan20 achieved higher yields (6080%) of dipivefrin HCl f n lproduct by the use of cesium chloride (CsC1) ia in methanol to form the HCl salt. This could be accomplishedby either treating the free base of dipivefrin with CsCl/methanol (Route A, Figure 1) or preparation of the HCl salt of ketone 3. with CsCl in methanol followed by hydrogenation to dipivefrin HC1,g (mp 158-9OC)(Route B, Figure 1).
$
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236
3.
PHYSICAL PROPERTIES
spectroscopy
3.1
3.1.1 InfraredSpectrum The inffared spectrum of dipivefrin hydrochloride w s obtained45 A mixa ture of the drug substance and potassium bromide was pressed into a pellet and analyzed using a Perkin-Elmer M d l 1750 FI'IR. The spectrum is oe shown in Figure 2. The major absorption bands for the infrared frequencies and the corresponding assignments are listed in Table I. 3.1.2 Ultraviolet Spectra The ultraviolet absorption spectra of dipivefrin hydrochloride in acetonitrile, absolute ethanol, pH 3 buffer (0.05M phosphate), pH 7 buffer (0.05M phosphate) and water were obtained using a Hitachi U2000 WMS spectrophotometerand 1 cm cells.45 A representative W spectrum in ethanol is shown in Figure 3. Samples of dipivefrin hydrochloride in these solvents were scanned from 200 to 300 nm and the absorption coefficients at wavelengths of maximum absorption were calculated (Table I) I. 3.1.3 Nuclear Magnetic Resonance Spectra The 1H-NMR spectrum (200 mHz) of dipivefrin hydrochloridew s a obtained and the chemical shifts have been assigned in Table IIL45 The lHN M R spectrum of dipivefrin hydrochloride in DMSO-6 was obtained using a Varian XL200 spectrometer (Figure 4). The 13C-NMR spectrum (50 mHz) of dipivefrin hydrochloride in DMSO-dtj was obtained using a Varian XL200 spectrometer (Figure 5 ) and the assignments are listed in Table lII.45 The Attached Proton Test (APT,Figure 5 ) was performed in arder to assign all carbons.
as 3.1.4 M s Spectra M s spectra were obtained for dipivefrinhydrochloride using a Finnegan as

A ml MAT TSQ46 GC/MS/MS ~ n i t . ~ 5 s a l amount of dipivefrin hydrochlorm ride was volatilized by heating at a linear rate of 5 mA/sec f o 0 mA to about 500 mA and ionized by either chemical ionization (CI, Ton pres0.3 I I sure of isobutane) or by electron impact (EI) at 70 eV. The C and E mass spectra were presented in Figures 6 and 7 and the interpretationpresented in Table IV.The fragmentation pattern was consistent with the chemical structure of dipivefrin hydrochloride.
100
80
20 -
- 20
200
220
240
260
280
300
nm
Figure 3. UV spectrum of dipivefrin hydrochloride (in ethanol).
DlPIVEFRlN HYDROCHLORIDE
239
Table I. infrared spectral assignments for d i p i v e h hydrochloride Wavelength (cm-1) 3600-3400 3255,2804,2475, 2397 2974-2875 1761 1614, 1595, 1562, 1504 1481, 1461, 1441, 1397 1368, 1332 1273, 1258-1163 1 124-1028 891,842 Assignment
0-H stretch RflHz+-NH stretch sp3 C-Hstretch phenyl ester C=O stretch aromatic C-Cstretch sp3 C-Hbending and scissoring tert-butyl C-Hbending C-0-C stretch secondary alcohol C-0stretch out-of-plane bending for 1,3,4 substitutedbenzene ring
Table II. Ultraviolet absorption of dipivefrinhydrochloride E (176, 1 cm) Solvent Acetonitrile Ethanol pH 3 Buffer pH 7 Buffer Water 210 nm 267.3 246.8 266.7 257.6 278.0 264 Nn 14.8 14.5 12.4 10.8 18.0 270 nm 13.4 13.1 10.4 8.9 16.2
10
Figure 4. IH-NMR Spectrum (200 m HZ) of dipivefrin hydrochloride (in DMSO-d6).
24 I
242
G. MICHAEL WALL AND TONY Y.FAN
Table m.lH- and 13C-NMRdata for dipivefrin hydrochloride in DMSO-d,j
CH, -NH -CH3 (CHJ3C-t-
0 '
I+ a13C 6 e m ) 32.60* 54.34 67.17 140.58 124.02 123.56 141.59 142.07 120.91 175.28, 175.19 38.60* 26.81
Assignment 1 2 3 4 5 6 7
8
1H 6 e ) r n 2.48 (3H,s) 3.15 (lH,d), 2.98 (lH,s) 4.98 (lH,d) 7.30( 1H,m) 7.22 (lH,m) 7.24 (lH,s) 1.27 (9H,s), 1.26 (9H,s) 6.30 (lH,broad) 9.10 (2H, broad)
9 10, 10' 11, 11' 12, 12' OH
NH
* Determined by Attached Proton Test (APT)
E . Ct: b)
> .a
.d
IW.4
118144.
Y. 4
T t
6
' P
sz
Figure 7. EI Mass spectrum of dipivefrin hydrochloride.
24.5
Table IV. Mass spectral data of dipivefrin hydrochloride Electron Impact (EI) m/z 35 1 250 223 85 57 44
7% re1 abundance
Chemical Ionization (CI) Assignment m/z

% re1 abundance
100
11
18 2 11 20 20 100
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 w s heated from a 25OC to 200OC at a linear rate of 200C/min using a Perkin-Elmer DSC-4.45 a One major endothenn w s 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)
t!
K
t-
K
W
<
241
3.2.3 Thermogravimetric Analysis

A 7-mg sample of dipivefrin hydrochloride was heated using a PerkinElmer System 4 ThermogravimemcAnalyzer from 400C to 4 0 0 T at a
linear rate of 200C/min.45 The drug substance exhibited a gradual weight loss above t e melting range (Figure 9). h 3.3 X-Ray Powder Diffractometry
An X-ray powder diffractogram was obtained for dipivefrin hydrochloride using a Philips X-ray powder diffractometer equipped with a diffracted beam graphite monochr~nometer.~~ (1 = 1S405 ) radiation was CuKa used for obtaining the powder pattern (Table V) (Figure 10). 3.4 Partition Coefficients
Some partition coefficient data for dipivefrin and epinephrine has been previously reported.& Dipivefrin solubility ratios for n-octanol/U.lN acetate @H 4.0); n-octanol/phosphate-buffered saline (pH 7.2); and carbon tetrachloride/phosphate buffered saline @H 7.2) were 0.51,4.89, and 1.07; compared to epinephrine ratios of 0.0032,0.0081, and 0.0090, respectively. Therefore, the partition coefficient of dipivefrin w s shown to a be 100 to 600 times that of epinephrine.4 3.5 Ionization Constant, pKa
t The pKa of dipivefrin hydrochloride was found to be 8.40 a room temperature.45

3.6 Solubility
The solubility of dipivefrin hydrochloride in various solvents at room temperature has been determined,data are tabulated in Table W.45 3.7 Solution Color, Clarity and pH
An aqueous solution of dipivefrin hydrochloride in water (1 in 100)is clear, colorless and exhibits a pH value of about 4.80.45 The pH of dipivefrin hydrochloride ophthalmic solution is between 2.5 and 3.5.12
c .
h
Y
n
t!
w
c
! i E
<
i!
LH913A X
248
o
L D o
a n
I a ,
I
0
I
N
249
250
G . MICHAEL WALL AND TONY Y . FAN
Table V. X-Ray powder diffractiondata for dipivefrinhydrochloride Degrees 20

4.41 8.88 10.10 12.88 13.34 15.15 15.75 16.17 17.33 17.57 17.85 18.20 18.76 19.59 19.78 20.06 20.41 21.08 21.55 23.68 24.67 25.87 26.96 29.65
d-Spacings
20.00 9.95 8.75 6.87 6.63 5.84 5.62 5.48 5.11 5.04 4.96 4.87 4.73 4.53 4.48 4.42 4.35 4.21 4.12 3.75 3.61 3.44 3.30 3.01
Relative Intensity
43 73 78 28 100 40 23 21 39 53 35 57 15 70 58 65 35 74 15 12 15 24 17 10
25 I
Table VI.Solubility of dipivefrin hydrochloride Solvent Water Methanol Chloroform Acetonitde Hexane Solubility (mg/mL)
621 58 1 570 69 0.05
4.
TYPICAL METHODS OF ANALYSIS

Identity
4.1
4.1.1 Infrared Spectrophotometry
The identity of dipivefkin hydrochloride may be determined by comparison of its infrared spectrum (KBr) (see Figure 2) to a USP reference standard.12
4.1.2 Thin-layer Chromatographic Identity Test
The USP describes a TLC method for the identification of dipivefrin hydrochloride drug substance.12 A sample of the drug substance dissolved in water (2 mg/mL) is spotted on a silica gel TLC plate, developed with a mobile phase of chloroform-methanol-formicacid (30 10:1, v/v/v), and visualized with a spray of 1% potassium femcyanide in water-alcoholethylenediamine (50:45:5, v/v/v) and heat. The identity of dipivefrin hydrochloride may be determined by comparison of its Rfvalue to that of an USP reference standard.
4.1 .3 Chloride Identity Test
An aqueous solution of dipivefrin hydrochloride (1 in 100) is treated with 6N ammonium hydroxide to precipitate dipivefrin base. The filtrate then
yields a white, curdy precipitate upon the addition of silver nitrate TS. The precipitate is insoluble in nitric acid but is soluble in a slight excess of 6N ammonium hydroxide.l2
4.2
Elemental Analysis
Elemental analysis of a sample of dipivefrin hydrochloride produced the following resdts:45
252
G . MICHAEL WALL AND TONY Y. FAN
Table VII. Hemental analysis of dipivefrin hydrochloride Element Carbon Hydrogen Nitrogen oxygen Chlorine Theoretical 9% 58.83 7.79 3.61 9.14 20.62 Found%
59.05 7.41 3.61 9.31 20.36
4.3
Other Phamiacopeial Tests
Dipivefrin hydrochloride tested according to the U.S. Phannacopeia must meet the following additional specifications:residue on ignition, not more than 0.3%; loss on drying, not more than 1.0% (60 O X 6 hours over C P2O5); heavy metals, not more than 0.0015%; and iron, not more than 5 ppm.12 4.4 Chromatography
4.4.1 Thin-Layer Chromatography Samples of aqueous and vitreous humor have been assayed for dipivefim content using TLC46and a combination of 2-dimensionalTLC for sample clean-up and liquid scintillation for quantitati0n.l Aqueous humor samples were dried, dansylated with 14C-radiolabelleddansyl chloride, and chromatographed on a polyacrylamide plate using a mobile phase of waterfonnic acid (100: 1.5, v/v) followed by benzene-acetic acid (90.5, v/v). The spots correspondingto epinephrine and dipivefrin visualized using dental roentgenograms were cut out, washed into liquid scintillation vials wt ih chloroform and dissolved in Liquifluor-toluene solution (42: 1,000) for a counting. A mass spectrometer w s used for confirmation. Using dansyl chloride derivatization, the assay sensitivity for dipivefrin was 10-3 moles.1 4.4.2 High-pressure Liquid Chromatography The USP describes the HPLC analysis of dipivefrin hydrochloride drug substance and ophthalmicpreparations using a 10 pn octadecylsilane column (3.9 X 300 mm), a mobile phase of acetonitrile-0.014 M dodecyl sodium sulfate-glacial acetic acid (24: 15:1, v/v/v), a flow rate of 2 r n b i n , and W detection at 254 nm.12
253
A versatile, routine HPLC method was developed for numerous pharmaceutical compounds including dipivefrin eye drops which consisted of a C18 Fondapak reversed-phase column (3.9 mm I.D. X 300 mm), a mobile phase of acetonitrile-0.05 M potassium dihydrogen phosphate (4555, vh), an injection of 20 pL (0.10 mg/mL), a flow rate of 2 mL/min and UV detection at 214 MI^^ 5.
STABILITY-DEGRADATION
Potential Routes of Degradation
5.1
Dipivefrin has been shown to be relatively stable in the solid state and in acidic solution but readily prone to degradation at alkaline pH. Hydrolytic by-products of dipivefrin, 3- and 4-monopivaloylepinephrine,have been isolated by semi-preparative HPLC and identified using thennospray HPLC/MS and CI mass spectrometry48 Complete hydrolysis (both esters rather than just one) would also be a likely possibility, which then would result in the typical degradation pathway for epinephrine49(Figure 11).
5.1.1 Thin-Layer Chromatography of 3- and 4-Monopivaloylepinephrine
The isolation of 3- and 4-monopivaloylepinephrinefrom a degraded dipivefrin solution was attempted using prewashed (necessary), preparative TLC plates (silica gel, 150 pore size, 100 pm silica gel thickness, 20 X 20 cm) a mobile phase of chlorofom-methanol-fomic acid (73:25:2%, and 0.34) and one band for 3v/v/v). A band corresponding to dipivefrin (Rf and 4-monopivaloylepinephrine(Rf 0.22) were identified by HPLC analysis of the scraped bands. However, these compounds decomposed during isolation attempts.* 5.1.2. Analysis of 3- and 4-Monopivaloylepinephrine The dipivefrin degradation products, 3- and 4monopivaloylepinephrhe. have been prepared in situ by exposing dipivefiin HC1 to alkaline pH followed by stabilization with HC148 Isolation by semi-preparativeHPLC followed by mass spectral analysis confirmed the presence of 3- and 4monopivaloylepinephrine.Two types of mass spectral analyses were preformed: CI (isobutane) MS of a drop of the isolated HPLC fraction and correspondingto 3- and 4-monpivaloylepinephrine, direct thennospray HPLC/MS of a degraded solution of dipivefrin. The mass spectral results have been summarized in Table WI.
An HPLC method was developed for the routine analysis of 3- and 4monopivaloylepinephe using a C18 column (10 p 4.6 mm I.D. X 250 mm), a mobile phase of acetonitrile-l% sodium dodecylsulfate-glacial acetic acid (51:46:3%, v/v/v), a flow rate of 2 mL/min, and W detection at
.a a
.C
L.
(j i
f
/ \
DIPIVEFKIN HYDROCHLORIDE
2.55
254 m n 4 8 Two peaks were observed for the pair of degradation products but it could not be determined which peak conesponded to which isomer. Typical retention times were: 3- and 4-monopivaloylepinephrine,3-4 minutes; dipivefrin, 6- 12 minutes (Figure 12)48
The photodiode may U V spectrum of dipivefrin was also obtained and compared to the spectra obtained for 3- and 4-rnonopivaloylepinephrine collecting data from 400 to 200 nm at 3.47 second intervals4* The Lax values were determined 3- and 4-monopivaloylepinephrine, 232 and 275 nm; dipivefrin, 233 and 265 nm (compared to literature value for epinephrine, 235 and 281
Table WII. CI (Isobutane) and Thennospray-HPLC Mass Spectral Assignments for 3- and 4-Monopivaloylepinephrine CI We) Relative Abundance (96) Assignment Relative Thermospray Abundance (9%) HPLC
268 250 121 103
65 12 100 84
[MI+ [M - H201+
100 5
268 250
[(C7H502) i- 11+ [(C8H6) 4- 11+
5.2
Solid-state Stability
Dipivefrin hydrochloride drug substance has been shown to be stable for at least 2 years under the conditions of room temperature, 4OC, 35OC, 40OC at 75% relative humidity, 50OC and exposure to light (1000 foot-candles) at room temperature. The drug substance was found to be non-hygros~opic.~~
No change was observed in the idrared spectra of dipivefrin hydrochloride drug substance stored for 2 years under the previous conditions. Also, the infrared spectra of several lots of drug substance from two manufacturers have been consistent. Therefore, polymorphism is not considered to be a practical problem with dipivefrin hydr0chloride4~
5.3
Solution Stability
The stability of dipivefrin hydrochloride in aqueous buffers @H 4.5,7.0, 9.0) and oxygen-saturatedwater has been evaluated under the conditions of room temperature, 35OC, 55OC, and light exposure for 6 months.45 The
. , . . . . . . .. . .
5
10
10
Figure 12. Typical HPLC chromatograms (HPLC System 1) of dipivefrin (DPE) and degradation peaks 1 and 2: (A) a formulation sample of 0.1 o/o dipivefrin ophthalmic solution containing 0.95 mg/mL DPE and each degradation product at levels of 0.1 mg/niL; (B) a raw sample of 1 m & / d dipivefrin HC1 and both degradation products at concentrations of iigniL (Reprinted from ref.48, Copyright 1992, with permission from Pergamon Press Ltd., Headington Hill Hall, Oxford OX3 OBW, UK.)
251
drug was found to be least stable to elevated pH and heat. The acidic solutions (pH 4.5) were the most stable: after 6 months, about 80% dipivefrin remained at room temperature; 70% remained under light; and only 20% remained at 55OC. The neutral solutions @H 7), though colorless upon preparation, tumed pink within 2 hours, and degraded severely after 4 only 4 weeks: 20% dipivefrin r m i e at room temperature and 0% at eand 55OC. The alkaline solutions @H 9) were the least stable: complete l degradation occurred within one day. A l severely degraded solutions turned a range of dark brown colors.
6.
ABSORPTION, DISTRIBUTION, METABOLISM AND EXCRETION
Dipivefrin hydrochloride is commercially available as a 0.1% topical ophthalmic solution. Several studies have shown that after topical administration to the eye, dipivefrin is metabolized to epinephrine, thereby making it a "prodrug" to epinephrine.1-5.46 After topical administrationto rabbits eyes, dipivefrin has been shown to be largely metabolized to epinephrine within 15 minutes, mainly by the cornea.5 The major metabolite of epinephrine,metanephrine, w s detected as soon as 15 a minutes after application of either dipivefrin or epinephrine. The epinephrine produced was taken up and stored in the iris, ciliary body and comea.5 Systemic absorption of dipivefrin has also been studied and found to be similar to epinephrine.50 The presence of the dipivaloyl side chains on dipivefrin make it about 100600 times more lipophilic than epinephrine.46Radiolabelled studies with 714C- and carboxyl-%labelled dipivefrin hydrochloride have shown that dipivefrin penetrates anterior ocular tissue50951 and distributes in other ocular tissue better than epinephrines1 The hydrolyzed pivalic acid moiety was thought to be eliminated by ocular tissue faster than epine~hrine.~~ Radiolabelled 1q-pivalic acid and carboxyl-lq-radiolabelleddipivefrin revealed similar distribution in ocular tissue after topical administration to rabbits.S2 Analysis by HPLC indicated that no further metabolism of pivalic rm acid occmed following hydrolytic release f o dipivefrin and that elimination of pivalic acid from the eye was rapid with no apparent accumulation in ocular tissues.52 Numerous studies have shown that dipivefrin is enzymatically hydrolyzed in ocular tissue by esterase~.~2*4*53,5~ administered 0.1% Rabbits dipivefrin solutions (q.i.d. X 28 days) had no alteration in comeal esterase In a~tivity.5~ young rabbits, variations in esterase activity with age and iris pigmentation did not alter the fraction of dipivefrin absorbed into the eyes.4 Several studies have focused on inhibition of the action of esterase on d i p i v e b . Mandel et ai. and Anderson et ai.2 found that echothiopate was
258
a competitive, reversible inhibitor of the soluble corneal dipivefrin esterases. However, Mindel et al. 53 suggested that the inhibitory action of echothiopate on dipivefrin-induced adrenergic effects was probably not due to an inhibition of esterases but rather to a masking effect by the echothiopate-induced hypertension. The conversion of racemic dipivefrin to racemic epinephrine in vivo was mediated primarily by enzymic hydrolysis and cholinesterase inhibitors did not affect t i conversion? hs
7. TOXICOLOGY
Potential adverse reactions to ocular administrationof dipivefrin would be expected to be similar to that of epinephrine which include tachycardia, arrythmias and hypertension. The most frequent local side effects reported with dipivefrin hydrochloride ophthalmic solution, 0.1%, USP, have been burning and stinging. Other less common adverse effects have also been reported to include follicular conjunctivitis,mydriasis, and allergic reactions. Epinephrine therapy can lead to adrenochrome deposits in the conjunctiva and cornea. Macular edema has been reported to occur in up to 30% of aphakic patients treated with epinephrine: t i edema is reversible. It hs is not known whether dipivefrin is excreted in milk.7 Reproductive (10 mg/kg body weight daily, p.0.) and teratogenicity (5 mg/kg) studies in rats and rabbits have revealed no evidence of impaired fertility or harm to the fetus.7 A safety evaluation of topical dipivefrin (0.04%, 0.1%, and 0.25%) was conducted by treatment of rabbit eyes for 1 month (bid.): dipivefrin produced neither ocular irritation nor injury, except for hyperemia, probably due to the pharmacological action of epinephrine.55 Rats treated with dipivefrin solutions (0.02- 0.50 m a g , s.c.) before mating and female rats treated during gestation were also evaluated. There was slight loss of hair at the injection site and some weight loss of the adults and offspring, but no embryofetal toxicity, teratogenic effects, or any other adverse effects observed in the pups.56*57,58 The chronic toxicities of dipivefrin (0.03,0.3 and 1.0 mg/kg/day, s.c.) and pivalic acid (0.2 m@g/day, s.c.) were evaluated in rats over a 26-week period. Rats receiving high doses of dipivefrin exhibited hair loss at the site l of injection, depressed food consumption and weight gain, a l of which disappeared after termination of treatment. No adverse effects were observed for pivalic acid.59
A variety of other toxicological tests have been performed with dipivefrin. The effects of dipivefrin on degeneration of corneal epithelium was investigated and compared to the effects of other ophthalmic agents including rinderon, rimolol, befunolol and indomethacin.60 Sasamoto et a1.61 reported swelling of corneal epithelium cells after administration of dipivefrin and epinephrine, though, this effect was reversible. Conjunctival administration of dipivefiin, 0.196, to rabbits produced intercellular space and vacuoles in the corneal epithelium to a greater extent t a observed with hn
259
epinephrine, 1.25%.62 In another study,63 no remarkable changes were observed in the electroencephalograms of rabbits following topical ocular administration or intTavenous injections of dipivefrin or epinephrine: some sedation was noted after intraventricular injection of either.
ACKNOWLEDGEMENTS
The authors express their sincere appreciation to the following persons who have provided data and/or information for this chapter: J. Kenny for W and stability data, P. Buck for W data, M. Ready for pKa data, and G. l Havner for DSC data, al at Alcon Laboratories; and Professor Hugo Steinfink, Department of Chemical Engineering, University of Texas, Austin, Texas, for x-ray powder diffraction data.
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Koch, H. Med. Actual., 1979,15, 537-41. Kaback, M.B .; Podos, S.M.; Harbin, T.S.; Mandell, A.; Becker, B. Am. J, Ophthalmol., 1976,81, 768-72. Eisenburger, R.; Hampel, H.; Hofrichter, G.; Loeser, R. Arzneim.Forsch., 1981,31, 325-34. Neufeld, A.H.; Page, E.D. Invest. Ophthalmol. Visual Sci., 1977, 16, 1118-24. Innemee, H.C.; Van Zwieten, P.A. Graefes Arch. Clin. Exp. Ophthalmol., 1982,218, 297-300. Bunag, R.D.; Riley, E.; Walaszek, E.J. Gen. Pharmacol., 1977, 8 , 269-73. Wang, B.C.; Bloxham, D.D.; Goetz, K.L. J. Phurmacol. Exp. Ther., 1977,203, 442-8. Yamauchi, H.; Nishimura, K.; Sasano, M.; Suda, H.; Iso, T. Oyo Yakuri, 1986.31, 93 1-44.
Ocul.,1982,61, 257-65.
Vimo, M.; Giraldi, J.; Pattavina, L.; Cozza, R.; Malagola, R. Boll.
Kiritoshi, A. Osaka Daigaku Igaku Zasshi, 1989,41,387-96. Samples, J.R.; Binder, P.S.; Nayak, S. Exp. Eye Res., 1989.49, 1-12. Roat, M.; Basu, P.K.; Ranadive, N.S.; Cane, F.; Hasany, S.M. J . Toxicol., Cutaneous Ocul. Toxicol., 1985,4, 27-37.
Iso, T.; Uda, K.; Yamauchi, H.; Nakajima, N.; Suda, H. Invest. Ophthalmol. Vis. Sci., 1980,19, 824-6.
Yamauchi, H.; Sasano, M.; Iso, T. Jap. J . Ophthalmol., 1988,32, 187-95. Hammarlund, E.R. J. Phurm. Sci., 1989,78, 519-20. Kleeman, V.A.; Engel, J. Phurmazeutische Wirkstoffe, Georg Thieme Verlag Stuttgart: New York; 1982, p. 329. Alcon Laboratories, Inc., Unpublished &ta on file. Wei, C.-P., Anderson, J.A.; Leopold, I. Invest. Ophthamol. Vis. Sci., 1978,17, 315-21.
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Sidhu, A.S.; Kennedy, J.M.; Deeble, S. J. Chromatogr., 1987, 391, 233-242. Wall, G.M.; KeMy, J.C.; Fan, T.F.; Schafer, C.; Ready, M.A.; Baker, J.A.; fitter, P.; Scott, B.S. J. Phurm. Biomed. Anal., 1992,10, 465-71. Conner, K.A.; Amidon, G.L.; Stella, V.J. Chemical Stabiliity o f Pharmaceuticals:A Handbookfor Pharmacists,Wiley: New Yo&, 1986, p. 94. Anderson, J.A. Arch. Ophthalmol., 1980,98, 350-3. Kito, H.; Chiba, T.; Hikida, M. Nippon Ganka Kiyo, 1986,37,
664-70.
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Tamaru, R.D.; Davis, W.L.; Anderson, J.A., Arch. Ophthalmol., 1983,101, 1127-9.
Mindel, J.S.; Koenigsberg, A.M.; Kharlamb, A.B.; Goldfarb, J.; Orellana, J. Arch. Ophthulmol., 1982,100, 147-51. Nakamura, M.; Shirasawa, E.; Hikida, M. Atarashii Ganka, 1989, 6 , 239-42. Yamauchi, T.; Okahara, A.; Fukumoto, M.; Sakaguchi, N.; Tanioka, H.; Iso, T. Nippon Ganka Kiyo, 1986,37, 814. Yamamoto, Y.; Cozens, D.D.; Barton, S.J.; Clark, R.; Iso, T. Oyo Yakuri, 1986,31, 1067-8 1. Yamamoto, Y.; Cozens, D.D.; Barton, S.J.; Clark, R.; Iso, T. Oyo Yakuri, 1986,31, 1061-6. Yamamoto, Y.; Cozens, D.D.; Barton, S.J.; Clark, R.; Iso, T. Oyo Yakuri, 1986,31, 1083-91. Watson, M.; Colley, J.; Heywood, R.; Street, A.E.; Prentice, D.E.; Iso, T.; Suda, H. Oyo Yakuri, 1986,3I, 331-55. Itoi, M. Nippon Ganka Kiyo, 1984,35, 1849-52. Sasamoto, K.; Akagi, Y.; Kodama, Y.; Itoi, M. Cornea, 1984,3,
37-41
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Kamei, C.; Kitazumi, K.; Tasaka, K. Oyo Yakuri, 1984,28, 601-7.
LACTIC ACID
Fahad J . Al-Shammary,' Neelofur Abdul Aziz Mian,' and Mohammad Saleem Mian'
( 1 ) <'linical Laboratory Sciences Department College of Applied Medical Sciences King Saud University Riyadh, Saudi Arabia
(2) Pharmaceutical Chemistry Department

College of Pharmacy King Saud University Riyadh, Saudi Arabia
263
264
FAHAD I. AL-SHAMMARY ET AL.
Contents
1. Introduction
2. Description
2.1. Nomenclature 2.1.1. Chemical Names 2.1.2. Generic Names 2.1.3. Proprietary Names 2.2. Formulae 2.2.1. 2.2.2. 2.2.3. 2.2.4. 2.2.5. 2.2.6. 2.2.7. Empirical Structural CAS (Chemical Abstract Service Registry Number) Specific Gravity Optical Rotation Wiswesser Line Notation Refractive Index
2.3. Molecular Weight

2.4. Elemental Composition
2.5. Appearance, Colour, Odour and Taste

3. Physical Properties
3.1. Melting Range 3.2. Solubility 3.4. Hygroscopicity 3.5. Dissociation Constant 3.6. LDa 3.7. Sulphated Ash 3.8. Boiling Point 3.9. Residue on Ignition 3.10 Stereochemistry
LACTIC ACID
265
3.11 Spectral Properties 3.11.1. Ultraviolet Spectra (UV) 3.11.2. Infrared Spectra (IR) 3.1 1.3. Nuclear Magnetic Resonance Spectra 3.11.3.1. Proton Spectra (PMR) 3.11.3.2. IT-NMR Spectra 3.11.4. Mass Spectra
4.
Synthesis
5. Pharmacokinetic
5.1 5.2 5.3
Metabolism and Absorption Uptake of Organic Acids by Pea Plant Uses and Adverse Effects
6. Methods of Analysis
6.1 . 6.2 . 6.3 . 6.4 . 6.5 . 6.6 . 6.7 . 6.8 . 6.9 . 6.10. 6.11.
Identification Methods Titrimetric Colorimetric Spectrophotometric Polarographic Fluorimetric Isotachophoresis EnzymicAssay Flow injection system Automatic Determination Chromatographic Methods
6.11.1. 6.1 1.2. 6.11.3. 6.1 1.4. 6.11.5. 6.11.6. 6.11.7.
Paper Chromatography Column Chromatography Thin Layer Chromatography (TLC) Gas Chromatography (GC) High Pressure Liquid Chromatography (HPLC) Low Pressure Liquid Chromatography (LPLC) Ion Exchange Resin Chromatography
7 Acknowledgments .
8. References
266
FAHAD J. AL-SHAMMARY ET AL.
Lactic Acid
1 Introduction .
Lactic acid (1) is the acid formed in the souring of milk, hence the name lactic, from the latin name for milk, and is discovered by Scheele in 1780. It results from the decomposition of the lactose (milk sugar) in milk. This acid is also present in sauerkraut and pickled cucumber. Lactic acid (2) consists o a mixture of 2-hydroxy-propionic acid, f its condensation products, such as lactoyl-lactic acid and other poly lactic acids, and water. The equilibrium between lactic acid and poly lactic acid depends on the concentration and temperature. In most case lactic acid is in the form of the racemate ((RS)-lacticacid), but in some cases the (+) - (s) - isomers is predominant. Lactic acid (3) has two nonequivalent optical isomers, one being the mirror image "enantiomer" of the other. D(-) lactic acid and L(+) lactic acid. Although enantiomers of lactic acid have the same chemical properties, but physical and essentially all of their physiologic properties are different. Enantiomers rotate the plane of polarized light to an equal extent but in opposite directions. Since enzymes act on only one of a pair of enantiomers, only half of a racemic mixture (a mixture of equal quantities of both enantiomers) DL lactic acid generally is physiologically active. Lactic acid (4) occurs in sour milk as a result of lactic acid bacteria, also found in molasses due to partial conversion of sugars, in apples and other fruits, tomato, juices, beer, wine, opium, ergot, foxglove, and several higher plants, especially during germination. Lactic acid is prepared technically by "lactic acid fermentation" of carbohydrates such as glucose, sucrose, lactose, with Bacillus acidi lacti or related organisms such as Lactobicillus delbrueckii, L. bugaricus, etc. The fermentation is carried out at relatively high temperatures, produced commercially by fermentation of whey, cornstarch, potatoes, molasses. L (+) lactic acid (4) occurs in small quantities in the blood and muscle fluid of man and animals. The lactic acid concentration increases in muscles and blood after vigorous activity L(+) lactic acid is
LACTIC ACID
267
also present in liver, kidney, thymus gland, human aminiotic fluid, and other organs, and body fluids and is obtained by resolution of DL. Lactic acid (5). Lactic acid prepared by fermentation of sugars, is levorotatory, and that prepared synthetically is racemic (5).
2. Description
2.1. Nomenclature 2.1.1. Chemical Names

a. b.
c. d. 2-Hydroxypropanoic acid (1,4) 2-Hydroxypropionic acid (1,5,6) a-Hydroxy propionic acid (4) Propanoic acid-2-hydroxy (1)
2.1.2. Generic Names

Lactic acid
2.13. Properietary Name

Acidum Lacticum, E270, E326, p Hygiene lactovagan, LactaGynecogel, Lachydrin, Michsaure, Milk acid, Tonsillosan, ordinary lactic acid, racemic lactic acid, vagoclyss.
2.2. Form u 1 a e 2.2.1. Empirical
2.2.2. Structural (4)

Lactic Acid COOH I CHOH
I
CH3
268
D(-) Lactic Acid

COOH HCCaOH CH3
L(+) Lactic Acid

COOH
(DL) LacticAcid
0
CH,-CH-C-OH
I " OH
CH3-CH-GO-CH-C0
2.23. CAS (Chemical Abstract Service Registery Number) (1,2,5)

[50-21-51 (Lactic acid) [598-82-31 (DL) Lactic acid [79-33-41L(+) Lactic acid
2.2.4. Specific Gravity (2)

Specific gravity about 1.20
2.25. Optical Rotation (4)

n 5 .
[a1
D(-) Lactic acid

L(+) Lactic acid
5%
nz [a1
546.1
LACTIC ACID
269
2.2.6. Wiswesser Line Notation (6)

QYVQ *D *L V
2.2.7. Refractive Index

nO 2
= 1.4251
=
Lactic acid
D
n20
1.4262 1.4270
DL Lactic acid
L(+) Lactic acid
D
n20 D
2 3 . Molecular Weight (1, 2, 4,s)

90.08
2.4. Elemental Composition

C
0
40.00% 53.29%
6.71 %
2 5 . Appearance, Colour, Odour and Taste

A colourless or slightly yellow, viscous hygroscopic liquid, which is odourless or almost odourless (2,5). Taste in dilute aqueous solution, mildly acid (7).
3.1. Melting Range (4)

16.8" 52.8" 53" (DL-Lactic acid) (D(-) Lactic acid) (L(+) Lactic acid)
3.2. Solubility
Lactic Acid
Soluble in water, alcohol, furfurol, less soluble in ether, practically less soluble in chloroform, petroleum ether, carbon disulfide (4).
270
FAHAD I. AL-SHAMMARY ET AL.
D(-) Lactic Acid

Soluble in water, alcohol, acetone, ether, glycerol. Practically insoluble in chloroform. Forms salts with many metals. Most of these salts are dextrorotatory (4).
L(+)Lactic Acid
Forms salts with many metals. The salts are more soluble in water than the salts of the racemic acid. Most of the salts are levorotatory (4).
3 3 . pH (2)
Acidic to litmus
3.4. Hygroscopicity (1)

Absorb water on exposure to moist air, when a dilute solution is concentrated to above 50% lactic anhydride begins to form in the official acid the anhydride amounts to about 12-15%.
3 5 Dissociation Constant ..
pka = 3.83 ID(-) lactic acid] (5) p b = 3.79 at 25O (L(+) lactic acid] (5)
3.6. LD50 (4)

Orally in rats 3.73 g/kg
3 7 Sulphated Ash (2) ..

Not more than 0.1 per cent.
3.8.
110
Boiling Point (6)
Lactic acid D(-) Lactic acid 1190/12mm L(+) Lactic acid and (DL) Lactic acid
103O
3.9. Residue on Ignition (8)

not more than 3mg, from 5ml portion (0.05%)
LACTIC ACID
27 I
3.10. Stereochemistry
Lactic acid (3) has two non-equivalent isomers. Stereoisomers differ only in the way in which the constituent atoms are oriented in space. Certain spatial relationships are readily visualized using ball and stick atomic models. A compound having asymmetric carbon atoms exhibits optical isomerism. Thus lactic acid has 2 nonequivalent optical isomers, one being the mirror image or enantiomer of the other Fig. 1-1. These structures seems indeed different by changing the positions of either enantiomer by rotation about any axis and attempting to superimpose one structure on the other. Although enantiomers of a given compound have the same chemical properties, certain of their physical and essentially all of their physiologic properties are different enantiomers rotate the plane of polarized light to an equal extent but in opposite directions. Since enzymes act on only one of a pair of enantiomers, only half of a racemic mixture (a mixture of equal quantities of both enantiomers) generally is physiologically active. A number of possible different isomers is Zn, where n = the number of different asymmetric carbon atoms. Therefore in lactic acid contains one asymmetric carbon atom hence, there is Z1 = 2 optical isomers. To represent 3-dimensional molecules in 2 dimensions, projection formulas, introduced by Emil Fischer, are used. The molecule is placed with the asymmetric carbon in the plane of the projection. The groups at the top and bottom project behind the plane of projection. Those to the right and left project equally above the plane of projection. The molecule is then projected in the form of a cross Fig. 1-2.
Figure 1-2. Fischer projection formula of (-).lactic acid. Unfortunately, the orientation of the tetrahedron differs from that of Fig. 1-1. Fischer projection formulas may never be mentally lifted from the plane of the paper and turned over. Since the vertical bonds are really below the projection plane while the horizontal bonds are above it, it also is not permissible to rotate the Fischer projection formula within the plane of the paper by either a 90-degree or a 270degree angle, although it is permissible to rotate it 180 degrees.
212
Fig (1-1)
TETRAHEDRAL AND BALL-AND STICK MODEL REPRESENTATION OF LACTIC ACID ENANTIOMERS.
(+) LACTIC ACID
(-)
LACTIC ACID
LACTIC ACID
273
The optical behaviour of L(+) Lactic acid on addition of alkaline hydroxides was studied by K. Droll et al. (9). The specific rotation at 25" of aq. L(+) lactic acid at const. concn. with increasing amounts of KOH rapidly fell to a neg. value and then showed a steady increase. Comparison of the curves with the titration curve showed that the minimum rotation value occurred in the neutral region (pH 6.5-7.5). Since alkalinization with LiOH and KOH led to the same rotations, the cations had no measurable effect. The rotations of L(+)-lactic acid at various concns. treated with rising amounts of KOH, plotted against the normality showed a min. close to the equivalence point. The gradually rising branch of the curve corresponded to the dissocn. of the alc. H of L(+)-lactic acid. Lactic acid prepared by fermentation of sugars in levoratotory and becomes dextrorotatory on dilution which by hydrolysis L(-) lactic acid lactate to L(+) lactic acid (8).
3.11. Spectral Properties

3.11.1.
Ultraviolet Spectra (UV)
The UV spectrum (10) of Lactic acid in H20 (25 mg%) was scanned from 200 to 400 nm Fig. 2 using LKB 4054 W / v i s spectrophotometer. Lactic acid exhibited the following UV data Table 1.
Table 1.
(n.rn) rnax
UV Data of Lxtic Acid.

Molar Absorbitivity ( E ) crn-1 grn rnol/L
A :
Absorbance
10.8
10.2
210
0.27 0.255 0.169 0.167

~
97.28 91.88 60.89 60.173
212 265 294
6.76 6.68
3.11.2.
Infrared Spectra
The IR spectra (10) of lactic acid was recorded on Perkin Elmer 1310 infrared spectrometer. Fig. 3 shows the infrared spectrum of lactic acid. The structural assignments of lactic acid have been correlated with the following frequencies Table 2.
d
0
m
rn
r+ U w a
m
(v
s
?
n N
U
E;
i l
.rl
214
Transmittance
0 0 W
0 0
I n
0 0 0 0
CJ
0 0 0
0 0 U
27s
216
FAHAD J. AL-SHAMMARY ET AL
Table 2,
Values of infrared absorption.

Tvue of vibration
m e n q a-1
3400
-OH CH (stretch) aliphatic -CH3 -CH stretch
2990
2920
2600
1720 1450
0 II
C (carboxylic) -CH -C-H (deformations)
1370
C-O stretching for C-OH
a20 920
C-H bending
3.11.3.
Nuclear Magnetic Resonance Spectra

3.11.3.1.
Proton Spectra (PMR)
The PMR spectra (10) of (DL)lactic acid in DMSO-d6 was recorded on a varian XL-200 NMR spectrometer using TMS as an internal reference. Intermolecular hydrogen bonding, polymerization, and free carboxylic group, present in the molecule extended a bit difficult spectra to interpret and is presented in Fig. 4. The spectral assignments are presented in Table 3.
1
" " ' I $
' I ' ' ' " ' I ' ' I

I
\2
z
lb
11.1
5.9
lL.L
, ~, "
"
2 46-8
dPPM
278
Table 3.
P M R Characteristics of (DL) Lactic Acid.

1.25 - 128 130 -133 1.41 - 1.44 4.07 -4.10 4.19 -4.28 4.89 -5.u2 5.98 -6.84, broad singlet
CHg
CH -COOH
3.113.2.
X - N M R Spectra
The I3C-NMR spectra (10)of (DL) lactic acid in DMSO-d6 using TMS as an intermal reference is recorded on a varian XL-200 NMR spectrometer and are presented in Figs. 5-8 spectral assignments are listed in Table 4.
Table 4.
Carbon-13 Chemical Shifts of (DL) Lactic Acid.

Chemical Shift 16.55, 16.67,20.31 and 20.38 65.65, 65.73, 65, 84, 68.02 and 68.44
163.11, 171.93, 174.23 and 176.45
W o n Assi-ment
CH3 CH
o=c
3.11.4. Mass Spectra
The mass spectra (10) of lactic acid was obtained by electron impact ionization Fig. 9 and was recorded on a Finnigen MAT 90 spectrometer. The spectrum was scanned from 10 to 160 a.m.a. Electron energy was 70 ev. Emerssion current 1 mA and ion source pressure was torr. The base peak is 45 with a relative intensity 100%. Table 5 shows the most prominent fragments and their relative intensities.
3 PPM
Fig (5)
13C-NMR SPECTRA OF LACTIC ACID IN DMSO-d6 (HETCOR)
280
160
1lO
Fig (7)
120
100
80
60
40
20 PPM
13C-NMR SPECTRA OF
LACTIC ACID
IN DMSO-d6 (APT).
CH3
CH2
N
N
- 1 I .
I
CH
160 Fig (8)
1LO
120
100
80
LACTIC ACID
60
IN DMSO-d6
40
20PPM
13C-NMR SPECTRA. OF
(DEPT).
Fig (9)
MASS
SPECTRA OF
LACTIC
ACID.
284
Table 5.
The most prominent fragments of lactic acid.
45 43
100
COOH
0
15
II
-43
29 28 27 18 28
C-OH
30 20
76
H-0-H
4. Synthesis
1. A solution (1)of glucose or of starch previously hydrolyzed with diluted sulfuric acid is inoculated, after the addition of suitable nitrogen compounds and mineral salts, with Bacillus lactis. Calcium carbonate is also added to neutralize the lactic acid as soon as it is formed, otherwise the fermentation stops when the amount of acid exceeds 0.5%. When the fermentation is complete, as indicated by failure of the liquid to give a test for glucose with Fehling's solution. The solution is filtered and the filtrate is concentrated and allow to stand. The calcium lactate that crystallizes out is decomposed with dilute sulfuric acid and filtered with charcoal. The lactic acid in the filtrate is extracted with ethyl or isopropyl ether, then the ether is distilled off and the aqueous solution of the acid concentrated under reduced pressure. 2. Another method (11) for the manufacture of lactic acid from starch materials by immobilized lactobacillus delbrueckii. L. delbruecki was immobilized in Ca alginate for the production of lactic acid. The substrate containtd corn starch and wheat bran (1:l). 3. Production of lactic acid by rapid fermentation was described by L. Ospiov et al (12). Experiments on the production of lactic acid from disaccharides, glucose, molasses, and hydrolyzate of cellolignin of pine cork were made with use of bacterial cultures 52 and 70 and
LACTIC ACID
285
lactobacillus delbrueckii as fermentation agents. The addition to the fermentation mass of yeast decreased the time of lactic fermentation to 1 day and even less. 4. Continuous fermentation of lactic acid (13) by lactobacillus delbrueckii is described. The possibility of lactic acid production by continuous fermentation in a special apparatus and selection of fermenting liquid compound are described.
5. Pharmacokinetic
5.1. Metabo 1ism and Absorption
Physiological and biochemical studies on Saccharomyces sake and uptake and metabolism of exogenous lactic acid by saccharomyces cultivated in a medium containing lactic acid was studied by Sumino et al. (14). The uptake and metabolism of exogenous lactic acid-1-'4C (I) and lactic acid-2-I4C (11) by S. sake was studied under anaerobic conditions. Under the experimental conditions employed, the amount of lactic acid taken into yeast cells was only approximately 1%of the total lactic acid added. A time-course study on the fate of I and I1 revealed that a greater part of I was lost from the cells at a relatively early stage of the growth. Radioactivity of lactic acid was mainly incorporated into sterols and fatty acids of lipid fractions in the cells and a small part of it was recovered from org. acids of the Kreb's cycle and amino acids. These facts indicated that lactic acid is not easily taken up and metabolized under anaerobic conditions. A small part of the acid taken into the cells was 1st dehydrogenated to yield pyruvic acid by lactate dehydrogenase, then converted to the substances mentioned above via acetyl CoA. Abnormal metabolites, which might have caused coagulation and death of the cells under anaerobic conditions were not detected. Absorption of glucose and lactic acid by rat everted intestinal loops after intraperitoneal injection of alcohol was studied (15). In vitro absorption and metabolism of glucose and the utilization of lactic acid by everted loops of small intestine were increased in loops from rats pretreated at 24 hrs. intervals with 3 EtoH injections (2mg/kg/day i.p.). This increase being due to EtoH-induced metabolic disturbances.
5.2. Uptake of Organic Acids by Pea Plant (16)

Excised pea roots were incubated with aeration in solutions containing 1 mM monopotassium phosphate (pH 5.2) and 1 mM carboxylic acid. Following the attainment of an equil., acetic acid, lactic acid, malic acid, benzoic acid succinic acid, propionic acid, tartaric acid, fumaric acid, a -ketoglutaric acid, aconitic acid, and citric acid were absorbed during several hrs., generally at a steady rate. On the whole the rates of utiliza-
286
tion of monocarboxylic acid were higher than those of the dicarboxylic acid and tricarboxylic acids with the exception of tricarboxylic acid which strongly inhibited and malic which slightly stimulated respiration the acids had no significant effect on the uptake of 0 by root cuttings. Absorption of some dicarboxylic and tricarboxylic acids from a complete nutrient solution was followed during a 7-days period. Uptake of malonic acid was exceptionally slow at first, but was highly accelerated after an adaptation period of 4-5 days. In 7-days, 75-98% of the acids were absorbed from 1mM solutions.
5.3. Uses and Adverse Effects

Lactic acid (1) has a limited usefulness as a spermatocide. It is usually incorporated in a jelly base in the concentration of 1 to 2%. It is also used in body milk formulas and as an acidulant in other food preparations. Lactic acid (5) has actions similar to those of acetic acid it is used in the preparation of lactate injections to provide a source of bicarbonate for the treatment of metabolic acidosis. Vaginal dose forms are used in the treatment of leucorrhoea. It is also employed in the treatment of warts. Lactic acid is also used as a food preservative. The application (5) of a mixture of salicylic acid one part, and lactic acid one part in flexible collodion 4 parts (SAL paint) was a s effective as that of liquid Nitrogen or of a combination of both in the treatment of hand warts. The preparation containing 16.7% salicylic acid and 16.7% lactic acid (17) (Duofilm, lactisol, viranol) is used for the treatment of warts. Lactic acid (5) has the same adverse effects as Hydrochloric acid although it is less corrosive. There was evidence that neonates had difficulty in metabolising D(-) lactic acid and this isomer and the racemate should not be used in foods for infants less than 3 months old (18).
6.1.
Identification Methods
1. The paper or thin-layer chromatogram is dried, then sprayed with fresh 10% ammonium ceric nitrate soln. in methanol or ethanol. It is allowed to dry for 10 mins. and then sprayed with 0.25% indole soh. in either alcohol. Pale spots appear on a dark-brown background which are stable for several weeks. For determination, the spots are removed and centrifuged at low speed with 1 ml of HN03 (1:3); 0.5 ml of the supernatant . soln. is introduced into a Warburg flask and treated with 0 5 ml of 30% ammonium ceric nitrate soln. in HN03 (1:3). The vol. of C02 produced in 20 min. is employed as a measure of the concn. of the organic acid present in the spot. Simple a -hydroxy- and a -oxo-acids, e.g., lactic, malic and a 4x0glutaric acids, release an almost stoicheiometric vol. of gas (19).
LACTIC ACID
287
2. Acidic to litmus (7). 3. 1 gm warmed with 0.1 gm of Potassium permanganate yield acetaldehyde, recognisable by its order (7). 4. When carefully superimpose 5 ml on 5 ml of sulphuric acid in a test tube, ensuring that the temperature does not rise above 15". Maintain 1 5 O for fifteen minutes, not more than a faint yellow colour develops at the zone of contact (7). 5. Dilute 1.0 gm with 10 ml of water, neutralize with sodium hydroxide solution, add 5 ml of Potassium cupri-tartarate solution, and boil for two minutes, no red precipitate is produced (7). 6. Dilute 3 gm with 50 ml of water, then add 50 ml of N Sodium hydroxide, boil gently for five minutes, cool and titrate the excess of alkali with N Hydrochloric acid using phenolphthalein solution as indicator. Repeat the operation with the lactic acid. The difference between the titrations represents the amount of alkali required to convert the lactic acid and its condensation products into sodium lactate. Each ml of N sodium hydroxide is equivalent to 0.09008 gm of C3H603(7).
6.2.
Titrimetric
1. Hayashi et a1 (20) extracted lactic acid along with organic acids. The organic acids were extracted from 50 gm of silage with 200 ml of 0.1N H2SO4, than 30 ml of extract was mixed with 1.5 ml of conc. H2SO4, and the mixture was steamed-distilled to yield 300 ml of distillate; 1.5 ml of 50% Cr03 solution was added to extract, which was again distilled to yield another 300 ml of distillate. Acetic and propionic acids were completely recovered, in the first distillate and lactic acid was completely recovered, as acetic acid, in the second. The total acid content in each distillate was determined by titration with 0.02N-NaOH. 2. The total lactic acid content of yogurt can be determined (21) by titration. About 10 g of sample is diluted to approximately 50 ml with H20 and the mixture is titrated with O.1M-NaOH phenolphthalein a s indicator. The total acidity is calculated lactic acid. 3. Lactic acid in maiz extract can be determined (22) (i) alkali metrically by boiling with aq. NaOH excess of which is titrated with HCl, and (ii) iodimetrically with amperometric indication by two polarised electrodes, or (iii) by oxidation with Kmnoq to acetaldehyde. In each instance it is advisable to remove sugars and proteins by initial precipitation or adsorption on ion exchange resin or charcoal. The acetaldehyde can be determined by distillation from the oxidation mixture into 0.01M hydroxylammonium chloride and potentiometer titration of the liberated HCl. Comparable results are obtained by absorption in Na2S205 reagent is less stable and the procedure is less convenient. Method (iii) with oximation of the acetaldehyde gives good accuracy. 4. Another method (23) for the determination of lactic acid by permanganate in alkaline media.
288
5. Determination of lactic acid in wines was done by J.F. Cases Lucas (24). In this method polyhydroxy acids and polyphenols are removed by precipitation with basic Pb acetate. Volatile organic compounds (ethanol and acetaldehyde) are removed by distillation with aq. H2S04. The sample is then oxidised with Ce(SO4)2 at the boiling point. The acetaldehyde evolved is collected in a solution containing NaHS03 and a phosphate buffer at pH 7 and estimated by neutralizing the excess of bisulphite with 0.02N Iodine solution, adding solid NaHCO3 and titrating the liberated bisulphite with 0.02N Iodine solution. Glycerol or butylene glycol do not interfere. Large amounts of sugar introduce a small error. 6. The method (25) describes the titrimetric determination of lactic acid by use of silver (111) as oxidant. Lactic acid in concentration of approximately 3mM was determined by oxidising it with Agrrr (added in a large excess) at 84O for 90 mins. then titrating the unconsumed iodimetrically . 7. K. Berg et a1 (26) describes the determination of lactic acid in silage and the volatility of lactic acid by steam distillation. After steam distillation of volatile fatty acids, in the special apparatus illustrated, lactic acid in the residue was oxidised with K2Cr20TH2S04 acetic acid to which was steam-distilled and titrated with 0.1 N NaOH. Low results were obtained for lactic acid and light results were obtained for volatile fatty acids, owing to the slight volatility of lactic acid, and the method is not specific as it includes substances other than lactic acid that are oxidised to volatile acids. Only 87% of added lactic acid was recovered from silage. 8. A quantitative (27) determination of some carboxylic acids formic, tartaric, citric, lactic acid and their salts were developed. Dissolve 0.5g of lactic acid or 0.7 g Ca salt in 100 ml of H20; mix 5 ml of the solution with 30 ml of 0.1 N dichromate and 4.4 ml conc. H2SO4. Heat 20 minutes. Cool and titrate.
63.
Colorimetric
1. The method (28) describes the determination of lactic acid in cheese. A finely ground 2 gm of sample was suspended in 10 ml of 0.66NNaOH, then 20 ml of H20, 10 ml of BaCl2solution and 5 ml of ZnS04 solution were added. The volume was adjusted to 50 ml, the suspension was filtered, and the filtrate was diluted 1:4 if its lactic acid content was expected to be less than 12 mg per gm of cheese, or 1.598 if the acid content was expected to be higher. To 10 ml of diluted filtrate, 1 ml of FeCl3 solution was added. Although lactose forms a yellow complex with F$+ the concentration of lactose in diluted filtrates was insufficient to influence results significantly. Average recoveries of added Li lactate were 89.9%. 2. Lawrence A.J. (29) determined lactic acid in skimmed milk powder. Reconstitute 1 gm of milk powder by shaking with 10 ml of H20 (final vol. 10.6 mlE. To 10 ml in a centrifuge tube add 7 ml of H S . Mix and
LACTIC ACID
289
add 1 ml of 25% CuS04.5H20 solution. Remix and leave for 10 mins. Add 1 ml Ca(OH)2 suspension (30 gm of Ca(OH)2in 140 ml of H20) add 6 m of conc. H2m4 dropwise, shake for 1 min. and heat in boiling H20 l for 5 min. Cool add 0.05 ml4-phenylphenol reagent (1%in 0.08N.NaOH) shake for 1 min. leave for 1 hr. and measure the extinction of the solution at 570 nm. The colour developed is stable, and obeys Beer's law. 3. In this method (30) the colour produced by the oxidation of organic compounds by Ce(S04)2in the prescence of ferroin (F$+ -1, 10phenanthroline) in N-H2S04medium is applied to the study of such compounds. 4. When lactic acid (31) is heated in conc. H2SO4, acetaldehyde is formed. Acetaldehyde condenses with P. hydroxydiphenyl to form a colored complex that may be used to estimate lactic acid. A violet colour indicates the presence of lactic acid. 5. Lactic acid (32) in gastric contents is most likely the product of bacterial action and fermentation as a result of food stagnation or abscence of free HCI or both A portion of gastric content is extracted with ether. An aliquot of this ether extract is treated with 10% ferric chloride, which give a slight yellow-greenish colour with low concentration of lactic acid more than 50 mg/100 ml of gastric contents and an intense yellow-green colour with high concentrations more than 100 mg/100 ml of gastric contents lack of colour indicates no lactic acid. 6. For the chemical examination of gastric juice lactic acid (33) give characteristic colour with 10% Ferric chloride. Add two drops of 10%FeCl3 soh. to a test tube full of H20 and mix. The mixture should appear colourless when held before the light but should present a faint yellow colour when examined vertically over a white surface Divide the mixture and put it into two test tubes, and to one add a small amount of gastric contents. Lactic acid gives a characteristic greenish yellow (canary yellow) colour compare with other test tube as control. 7. The 25 ml sample is treated (21) with 10 ml of aq. approximately 10% BaC12.2H20, 10 ml of 0.33M-NaOH and 5 ml of aq. 22.5% ZnS04.7H20 solution. After vigorous shaking, the mixture is filtered, and 1 ml of filtrate is diluted to 100 ml; 10 ml of this solution is treated with 1 ml of colour reagent (5 g of FeC13.6H20 dissolved in 12.5 ml of IM-HCI and diluted to 100 ml with H20) diluted 1:5 with H20, and the absorbance is measured at 400 nm. A blank determination is carried out on 25 ml of fresh milk. The calibration graph is established by using the filtrate from fresh milk to which standard amounts of Zn lactate have been added. 8. Colourimetric determination of total lactic acid in wine was described by Pilone et al. (34). The method utilizes the oxidization of both D(-)- and L(+)-lactic acids to acetaldehyde and subsequent formation of a color complex with phydroxydiphenyl. Tartaric acid, malic acid,
290
citric acid, acetic acid, EtOH, sugar and SOz did not interfere with the reaction. The lactic acid of ethyl lactate contributed to the total acid measured by this method; corrections were made by use of boiled neutralized wine. Free and bound acetaldehyde were also measured by this method; however, their levels in wine are normally too low to require correction. 9. The enzymic determination (35) to L(+) lactic acid in liquids containing sucrose is described. To determine lactic acid in solution with sucrose (15%), the acid is oxidized, in alk. soln., to pyruvic acid with excess DPN, using lactate dehydrogenase as catalyst. The p y ~ v i acid is coupled c with hydrazine and estd. colorimetrically at 366 mp. The standard deviation observed in 60 tests (4.5-2215 ?lactic acid/O.l ml.) was 0.13 Y /0.1 ml. The test takes 2 hrs.
6.4. Spectrophotometric
1. S.R. Elsden et a1 (36)describes to determine lactic acid by using ceric sulphate. In this method oxidation of lactic acid to acetaldehyde by Ce(S04)2is markedly affected by concentration of the oxidising agent and by temperature. In the aeration method for determination of lactic acid, the initial concentration of Ce(S04)2 should be 0.05N, the temp. 5 8 to 6 O OC and the rate of aeration should be 500 to 600 ml of air per min for at least 45 min. A new steam distillation method is described in which the acetaldehyde is removed by steam as fast as it is formed, and the Ce(S0412 is added gradually. The sample is mixed with ION HzSO4and steam is passed through the mixture into a receiver containing aq. NaHS03.Ce(S04)2 is added dropwise to the reaction mixture, and the acetaldehyde is determined by the usual iodimetric method. 2. Lactic acid was determined in natural water by A.G. Shadomskaya et a1 (37). The method is based on the oxidation of lactic acid to acetaldehyde and then reaction with 1-naphthol to form a coloured product. Evaporate 100-500ml of sample containing 25-500pg of lactic acid at pH 8 to 9 to 10 to 15 ml, adjust the pH to 2-3 with HCl (1:l) and extract with butanol (3 x 15ml). Extract the lactic acid with butanol layer into 1 to 3 ml of O.1N.NaOH and dilute the aq. extract to 5 ml. Cool 1 ml of this solution in ice, and 2 ml of conc. H$3040ver 10 to 15 mins. set aside for 30 min, then heat for 10 min at 1300 to 1500. To the hot solution quickly add 0.2 ml of fresh 0.5% ethanolic 1-naphthol, cool, and measure the extinction at 430 nm. 3. In this method (38) to the tissue homogenate add an equal volume of 10% HC104,and centrifuge for 10 mins. at 600 r.p.m. Extract the supernatant liquid with ethyl ether (2 x 20 ml), and evaporate the extract. Dissolve the residue in 3 ml of H20, and to a 1 ml aliquot add 2 ml of HzSO4 and heat for 10 min. at 148 to 158 (to remove pyruvic acid and certain other interfering substances). Then add O.lml of 5% ethanolic
LACTIC ACID
29 1
I-naphthol, and measure the extinction at 430 nm against 5% HCI04 similarly treated. Alternatively, add the H2S04 directly to 1 ml of the original supernatant liquid, and after addition of the I-napthol, cool in ice for 5 min. Then add 12 ml of HzO at 0".After 10 min remove the turbidity by extraction with isopentyl alcohol or by filtration and measure the extinction. 4. The method (39) describes the automated method for the determination of lactic acid in muscle tissue. An aq. extract of the sample is mixed in an Auto-Analyser with solution containing NAD and lactate dehydrogenase buffered at pH 9.6 with glycine:hydrazine.NaOH. After reaction at 3 O for 12 min. the extinction of the reduced NAD at 340 nm is 7 measured. The sample rate is 20 per hour.
6.5. Polarographic
1. The method (40) is used for the determination of lactic acid in milk. The method with the use of the 2-hydroxy quinoxalines obtained by condensation of the 2-oxo-acids with o-phenylenediamine. The aq. test solution 0.1 to M in HCI was made and 0.02 to 0.04M in o-phenylenediamine and add 10%of ethanol to prevent precipitation of the condensates. Allow 20 to 60 min for condensation of the 2-0x0-acid. Cathodic wave lights with a dropping mercury cathode are rectilinear with conc. over the range 0.05 to 2mM, for acids. 2. The (41) sample s o h (deproteinized and centrifuged, if necessary) was treated with lactate oxidase s o h in citrate buffer medium of pH 6.2. After 5 min. at 30" citric acid soh was added to adjust the pH to 4.2 to 4.7, the soh was de-aerated with N, F c + ~was added to reduce H202 formed in the enzymic reaction, and the pyruvic acid produced was determined by differential pulse polarography with use of a dropping - Hg electrode, at Pt auxiliary electrode and a SCE. The modulation amplitude, pulse duration and scan rate were -100 mV, 1 s and 5 mV s1respectively. The calibration graph was redilinear for 1 to 20 pM. L- lactate and the method is used to determine L. lactic acid in beverages and yoghurt and should be applicable in clinical analysis.
6.6.
Fluorimetric
1. The method (42) is based on the reaction between acetaldehyde and 2-phenylphenol in H2S04 medium at room temperature in the prescence of NO2-and is applied to determination of lactic acid and muramic acid which readily liberates acetaldehyde when heated with H2S04. A 200 pl portion of the sample solution (containing 1-2 n mol of acetaldehyde, 1.1-22.5 n mol of lactic acid) is mixed with 5 ml of conc. HzSO4, and to solution (kept at 800for 5 mins. for assay of lactic acid) is added 10 pl of 18% 2-phenylphenol solution. The reaction mixture is kept
292
at 200 for 15 mins. and the fluorescence intensity is the measured at 465 nm within 6 hrs. 2. In this method (43) lactic acid is made to react with 1-naphthol in H2S04 medium. The fluorescence is then measured either with a spectrophotometer by excitation with mercury-vapour lamp at 365 or 436 nm. The intensity of fluorescence depends on the temp. during the reaction, and is proportional to the concentration of lactic acid only in certain ranges, e.g. when a reaction mixture containing 2.5-20 pg of lactic acid per ml is heated at 1450 for 10 min. The sensitivity of the method is approximately 0.05 pg per ml with the use of fluorimeter. 3. Another method (44) used for the determination of lactic is by G.G. Guilbault et al. 4. An improved enzymic fluorometric (45) method for automated analysis of lactate in perchloric acid extracts of capillary blood with flow injection. The concentration of lactate can be measured in as little as 20 pl capillary blood from an exercising person. Within-day and between-day coefficients of variation are about 2%. The recovery of lactate from whole blood is 101%. Lactate is stable in perchloric acid extracts for at least 15 days at room temp. and at 4 at least 30 days. O
6.7.
Isotachophoresis
1. Coffee extract 4 ml was mixed with 1 ml of internal standard (1.634 g 1-' of trichloroacetic acid) and a 5 pl aliquot was subjected to isotachophoresis in a 3 cm x 0.55 mm capillary tube, with mixture of 50 ml of O.1N-HCl plus 300 ml of 1%(hydroxypropyl) methylcellulose plus 2 ml Triton X-100 (each adjusted to pH 3 with 3!, .alanine, made up to 1 liter) as the main electrolyte. The current was maintained at 90 pA for 10 min, then add 45 fl until the acids were detected at 254 nm (46). 2. Small portions of the brine were clarified by filtration through a membrane (sartorius, SMN 111061, diluted fivefold, and subjected to isotachophoresis without further clean-up. An LKB Tachophor was used, the 23-cm capillary tube of which was operated at 15O, the detection was by absorbance measurement at 2 4 nm, a conductivity detector was also used 5
(47).
3. In this method instrumentation for duel-wavelength UV absorption detection in isotachophoresis is described and evaluated. Computerized signal storage and processing allow data reduction on the basis of the ratio of absorption at any 2 of the wavelengths 206,254, 289, and 340 nm. The purity of UV-absorbing spikes or zones is verified by plotting the ratio vs. time, the ratio vs. 1 wavelength, or 1 wavelength vs. the other. The method is illustrated with the analysis of nucleotide extracts, of eggs of Nassarius reticularis (48). 4. I the method developed by Chauvet and Sudraud (49) the n determination of lactic acid along with other acids in wine by Isotacho-
LACTIC ACID
293
phoresis. Leading electrolyte was 0.01M-HCl adjusted to pH 3.27 with fi -alanine and containing 0.7% of hydroxypropylmethyl-cellulose as thickening agent. The terminal electrolyte was 0.0133M -acetic acid adjusted to pH 3.65 with 0 -alanine. Alternatively, 5mM-HC104could be used in the leading electrolyte with the same thickening agent. The PTFE analytical column (61 cm x 0.4 mm) was operated at 20. Conductometric detection was preferred to thermometric and spectrophotometric (256 nm) techniques. Calibration graphs were drawn for H3P04 and for malic, lactic, tartaric and gluconic acids; citric and succinic acids were also detected. Results were similar to those from enzymic methods and often better than those from chromatographic methods.
6.8.
Enzymic Assay
1. Neville et a1 (50) determined lactic acid in blood by enzymic assay. The procedures described are based on the reaction lactate + NAD+ NAD + pyruvate in the presence of lactate dehydrogenase. The lactate analysis is conducted in a glycine buffer s o h of pH 9.5 containing hydrozine hydrate to remove the pyruvate, as the hydrozone, as it is formed. Addition of lactate dehydrogenase drives the reaction to the right with eventual disappearance of the lactate and an equimolar generation of NADH from NAD+. The pyruvate analysis is conducted in triethanolamine buffer soln of pH 7.4 + addition of lactate dehydrogenase converts all pyruvate into lactate with an equimolar loss of NADH. The changes is NADH conc. are followed spectrophotometrically at 340 nm. 2. Lactic acid in beer was determined (51) by taking 0.2 ml of filtered de-gassed beer sample and mixed with 3 ml of glycine-hydrazine reagent (22.8 gm of glycine plus 50 ml of hydrazine in 500 ml; pH 9.0) and 0.2 ml of 3% NAD soln., and after equilibration at 2 O for 30 or 60 mins. (for 4 L- or D- Lactate respectively). The extinction is measured at 340 nm (I-cm cell). Then 0.2 ml of L- or D- lactate dehydrogenase suspension (5 mg per ml) is added and after incubation for 60 min. the extinction is again measured. The lactate conc. (mg per liter) is given by the difference in extinction multiplied by 272 F. Where F is the dilution factor. 3. To determine lactic acid (52) in soln containing sucrose (15%) the acid is oxidised, in alkaline medium, to pyruvic acid with excess of NAD. Lactate dehydrogenase acting as catalyst. The pyruvic acid is coupled with hydrazine and determined colorimetrically at 366 nm. The standard deviation in determinations of 4.5 to 2215 pg of lactic acid per 0.1 ml was - 0.13 pg per 0.1 ml. + 4. Lactic acid in faeces in case of glycosidase deficiency was determined (53). The sample is shaken with 0.1 N- HCI in 30% ethanol, and centrifuged and the supernatant liquid is filtered under pressure through visking tubing to give a clear slightly tinted filtrate. Lactic acid
294
is determined by the reaction with NAD and lactate dehydrogenase to give pyruvate and NADH2. The amount of NADH2 is determined by measurement of the extinction ai A0 nm. 5. A simple and sensitive enzymic determination (54) of lactic acid in which the preparation of lactate dehydrogenase from bakers yeast is described. The FdII - 1,lO-phenanthroline complex is used as an electron acceptor in theenzymic reaction. The FeII complex so formed is pink and absorbs strongly at 510 nm. 6. L. Spandrio et a1 (55) determined lactic acid in blood by deproteinizing 100 pl of blood with 100 pl of 0.06M-HC104 centrifuge and add 50 pl of supernatant soln to 2 ml of 0.5M-glycine-O.4M-hydrazine buffer (pH 9.0). Add 30 pl of lactate dehydrogenase suspension in 2.2M(NH4I2SO4 mg of protein per ml) and 0.2 ml of 0.027M-NAD (disodium (2 salt), mix and incubate for 1 hr. at 25O. Measure theextinctiondue to NADH2 at 340 nm vs. a similarly incubated mixture containing 0.3M.HC104 in place of the sample soln. 7. Lactic acid (56) content of finger-tip blood was determined as in which 0.1 ml blood was stirred with 4% aq. HClO, (0.2 ml) and 0.1 ml of the centrifuged extract was added to 1 ml of glycine-hydrazine buffer soln. of pH 9, 0.1 ml of 0.027 M-NAD and 0.2 ml of lactatedehydrogenase (2 mg of enzyme protein per ml). The mixture was incubated for 1 hr. at 2 5 O and the extinction was measured at 340 nm against blank. 8. The method (57)describes the enzymic determination of lactic acid in sugar factory juices. The L or D lactic acid was oxidized with NAD+ in the presence of L- or D- lactate dehydrogenase, respectively, and alanine amino transferase to yield NADH, which was determined at 340 nm. Lower values were obtained for raw and thin juice than by isotachophoresis. 9. A.C. Hadjivassiliou and S.V. Rieder (58) have reported a definitive procedure for the assay of lactic acid. The method used is based on complete enzyme reaction and the known extinction coefficient of NADH2 at 340 nm, thus avoiding the need for standard solution and determination of reaction rates. Mix buffered hydrazine soln. (O.2M-Tris and OdM-hydrazine prepared from an 85% aq. soh. adjusted to pH 9.0 with HCl) (2.0 ml), 1%human albumin soln. (0.2 ml), aq. NAD soln. (20 mg per ml) (0.2 ml) and lactate dehydrogenase s o h (25 units in 0.01 ml of H20). Read the initial extinction at 340 nm, and start the reaction by adding the original supernatant sample soln. (0.1 ml). Read the extinction at 5 min. intervals until it is constant, or until the change is 0.002 unit or less per 5 min. (normally within 1 hr.). Calculate the lactic acid content of the blood (in mg per 100 m ) from the expression [EF x VF)-(EIXVI)IX l 1800/62.2 where EI and EF are the initial and final extinctions and VI and VF are the initial and final volumes respectively. Contamination of glassware during handling must be avoided. The recovery was 97 to 102%. 10. Electrochemical-enzymic analysis of blood glucose and lactate
LACTIC ACID
295
was done by Williams et al. (59). Blood lactate can be determined by reaction with Fe(CN)63 in the presence of lactate dehydrogenase (cytochrom b)and monitoring the Fe(CN)6kelectro-oxidation. A modi -fied voltammetric membrane electrode is described which has a pt electrode, a porous or jelled layer containing the enzyme, and a dialysis membrane. Linear current-concentration curves were obtained for 60 ml blood containing 1.4 g Na2HP04, 1.13g NaH2P04,0.06 g benzoquinone and 0-20 mM added glucose and for a bovine blood sample diluted 20-fold with alkaline buffered 10 mM K3Fe(CH), and containing 0-1.0 mM L. lactate.
6.9. Flow Injection System

Simultaneous determination of L(+) and D(-) by use of immobilized enzymes in a Flow Injection System: 1. Yao and Wasa (60) have developed a method for the simultaneous determination of L(+) and D(-) lactic acid. The stream carrying injected sample was split to follow parallel channels, each containing an immobilized L(+)- or D(-)-lactate dehydrogenase reactor and a mixing coil of different dimensions, thus providing separate peaks for L(+)-lactic acid and D(-)-lactic acid. The flows were re-merged before reaching the thin-layer amperometric flow-cell detector, which contained a diaphorase - bovine serum albumin membrane covalently attached to one side of a platinum sheet (anode), a silver - AgCl reference electrode and a stainless-steel auxiliary electrode. The carrier soln. was 0.1M pyrophosphate buffer of pH 9.5, 0.5mM in NAD+ and 1 mM in K3Fe(CN)6. At the diaphorase membrane the NADH generated by the lactate dehydrogenase produced Fe(CN)64-which was measured amperometrically at 0.4 V. Calibration graphs were rectilinear for up to 2mM(L(+) lactic acid) or (D(-) lactic acid); detection limits were 2pm for L(+) lactic acid and 5 p for D(-) lactic acid, and coeff. of variation at the 0.1 mM level (n = 10) were 2.3% and 2.0%, respectively. 2. In another method (61) lactic acid was determined by flowinjection analysis with detection of an optical fibre lactic acid biosensor. The sensor is based on an oxygen optrode with lactate oxidase immobilized on carbon black. The carbon black protected the optrode from interference from ambient light and sample fluorescence.
6.10. Automatic Determination

1. In this method (62) lactic acid in blood is determined automatically. The method is based on the conversion of lactic acid into pyruvic acid in the presence of NAD by lactate dehydrogenase, the reduced NAD (NADH and H+) formed being measured by the time required for a pre-selected change in extinction, as indicated by a pre-selected change in
296
the output voltage of a photoconductive circuit, measured automatically. 2. Another semiautomated enzymic micro-method (63) for the determining of lactic acid on a single filtrate for the analysis 0.1-1.15 ml of blood with an autoanalyzer was used.
6.11. Chromatographic Methods 6.11.2. Paper Chromatography

1. Klemet et a1 (64) separated organic acids with lactic acid first by column chromatography on Kieselgel with butanol-benzene and butanol CHC13 as solvents, and the fractions were concentrated and submitted to chromatography on S. and S. paper 2043 b for 40 cm, in 15 to 20 hrs. with 10 solvent systems. For location the paper was sprayed with bromophenol blue solution on one side and then with NaIOq-benzidine reagent on the other. 2. Another method used for the analysis of lactic acid by using paper chromatograms was done by R. Pohloudek-Fabini et a1 (65). Ammoniacal AgMO3 solution in a suitable reagent for lactic acid.
6.11.2. Column Chromatography

Lactic acid (66) along with other organic acids were separated from wine. Column was prepared from Kieselgel (Serva No. 27171) that has been steeped in 6N-HCI, washed and dried at 120O. Grind (12 g) of Kieselgel with successive small volume of 0.5N-H2S04 (total 8-9 ml) and then mix it with benzene to form a liquid paste for transference to a glass tube (30 cm x 1.18 cm). Evaporate a 20-ml sample of wine in vavuo at 500 to a syrup and dry the syrup in vacuo over silica gel. Mix the residue with Kieselgel (1 g) and 6N-H2S04(0.4 ml) with cooling, transfer the mixture to the top of the column with benzene. For gradient elution use mixtures of butano1:benzene (increasing from 5-25% of butanol) and mixtures of butanol:CHCI3 (increasing from 22.5% to 50% of butanol), both previously saturated with 0.5N-H2S04. Collect the elute in 3.3 ml tractions.
6.113. Thin Layer Chromatography (TLC)

Summary of conditions used for the TLC of lactic acid are given in the Table 6 67-76) and also by (77-80).
6.12.4. Gas Chromatography (GC)

Different gas chromatographic methods used for the analysis of lactic acid are summarized in the Table 7 (81-98) and other GC methods are also described by (99-104).
Table (6):
Summary of conditions used for the TLC of Lactic acid.
support
Solvent System
Detection and Extd. Solvent
Ref.
Silica gel Silica gel Silica gel impregnated with Cu2+ by immersion of the layers in aq. 1%Cu acetate Silica gel-cellulose
CHC13:butanol CHC13:methanol:formic acid (153:l)
-bromophenol b1ue:indicator
L -
67
68
69
bromocresol green-95% ethanol 85%ethano1:lMNHg (4:l) and second direction with the lower phase of CHC132.methylbutan-2.01-90% formic acidH20 (681215:40) and again ethylether: 90% formic acid:H20 (7:21)
70
. . . . continued
Table (6):
Summary of conditions used for the TLC of Lactic acid.
support
~
Solvent System
Ref.
0.3mm layer o Merck f Silica gel H
96% aq. ethano1:conc. aq. NH3 (193)
methyl red bromo-thymol blue indicator soln. bright red spots on yellow orange background
71
0.25mm layer o cellulose f MN 300 HR
benzene:ethylether:formic acid (4:4:3)
0.2% bromocresol green soh. in 50% aq. ethanol adjusted to pH 7 with NaOH Yellow zones on blue-greenn background
72
Silica gel column (0.8an x 20cm) Cellulose and on Kiese1guhr:silica gel (1:l)
CHC13.isopentyl alcohol saturated with OSN.H2S04 soh. o benzidine and NaIO4 gives f blue or violet or yellow spots
73
74
. . . . continued
Table (6):
Summary of conditions used for the TLC of lactic acid.
Support
Solvent
System
Silica gel G plates Plates of Kieselguhr or Silica gel
CHC13-ACOH (911) benienetthanol-2-59 aq. (24.1 1
methyl red and bromocresol green bromocrcsol green
Table (7):
Summary of conditions used for GC of Lactic Acid.

Flow Rate
Column Support
Mesh
Temperature
Sample
Fused silica column ( E m x 0.32mm) of OV-101(0.15um), OV-1701(0.15um)or
50-100
2.5d /min. H carrier gas
OV-351(0.25um) (1.5m x 4mm) column packed with 10% D f diethylene glycol succinate on Celite, with f.i.d.
synoviol fluid
(1.8 2m x 1.8 or 4mm) column of or Chromosorb 103 or propak Q

(lm x 3mm) column packed with ppakQ Column (180 cm x 2mm) packed with propak P
80-100
3Oml/min. V as carrier gas !Ornl/min. V as carrier gas jOml/min. V as carrier gas
juices
120 - 150
80 -100
.. ..
cont
Table ( ) 7:

~
Column Support
Mesh
remperature
Flow Rate
Sample
Column (7.83m x 2mm) of chromosorb 101 Glass column (2% x 0.25mm) Stainless steel column (2m x 3mm) of 15%Dexsil300 on chromosorb W AW-DMCS (5Om x 0.3mm) column coated wtih Fluorad FC 431
80-100
2000
N as carrier gas
80-100
150 - 240'
80 - 320"
N as carrier gas
60ml/min. N as carrier gas
--
50 - 200"
30 ml/min. N carrier gas
Stainless steel column ( l m x 3mm) packed with propak Q
--
--
rye sour dough
. . coni
Table (7):
Column Support Mesh Temperature Flow Rate Sample Ref.
Glass column (lm x 2mm) packed with acid-washed carbopak B 10%SE30GC on chromosorb WAWDMCS
W
80 -120
4 carrier gas
plasma
91
100 - 200
blood
92
Column of Amberlite cationexchange resin CG120 Glass column (3 ft. x 4mm) containing 15%of polyexythyleneglycol 20M on zhromosorb W HMDS
silage
93
100 120
40ml/min.
juices
94
J as carrier gas
5 m t r capillary column coated with 0 ee A piezon L.
ml/min. Ie as carrier gas
95
. . . . cont uec
Table () 7:
Column Support
Mesh
remperature
Flow Rate
Sample
Column (6ft. x 3/16in.) of 25%of GP-88 silicon gum rubber SE-30 on Anakrom AB Column (8ft. x IOmm) of 15%diethylene glycol succinate on Gas-Chrom P
70 -80
750
blood
80-100
88 ml/min. Argon as carrier gas

80 ml/min.
Glass column packed with 10%of diethylene glycol succinate
100 - 120
1300
food
He carrier gas
304
6.115. High Pressure Liquid Chromatogvaphy
(HPLC)
A summary o some o HPLC methods used to determine lactic acid f f are given in the table 8 (105-119) and lactic acid is also analysed by (120122).
6.11.6.
Low Pressure Liquid Chromatography (LPLC)
Lunder and Messori(l23)have determined lactic acid in the mixture of other organic acids. They used a Beckman model 120 amino-acid analyzer, with minor modifications, is used with two temp.-controlled cation-exchange columns in series and 5mM-HCI as eluent; the separated acids in the eluate are monitored with use of a differential refractometer. Oxalic, citric, tartaric, malic, succinic, lactic, formic, fumaric, acetic and propionic acids have thereby been determined; detection limits range from 2 to 10pM. Oxalic, citric, tartaric, malic and lactic acids can also be monitored colorimetrically after automatic addition of FeCI3to the eluate; detection limits are 1 to 5pM,
6.11.7. Ion Exchange Resin Chromatography

1. Deacidification of fermented milk by ion exchange resin was done by Herve et a1 (124). Restoration of the normal level the lactic acid content of a fermented milk, without changing the balance of the other components, is effected by passing the milk through a column of a strong basic ion-exchanger (in the C1- form), followed by a passage through a weak basic ion-exchanger (in the OH- form). By varying the flow rate of the milk though the columns the lactic acid level of the milk can be adjusted to any desired level. Thus, a milk with an acidity of 2 2 O Dornic was first passed through a column of strong basic ion-exchanger at an hourly rate of 10 to 15 vols. of milk per vol. of resin; the flow rate through the 2nd ion-exchanger varied from 30 to 40 vols. of milk per vol. of resin. The lactic acid content of the milk thus treated was reduced to 15O Domic, while only about 0.2% of the other anions were absorbed by this treatment. 2. Daniel S.A. (125) describes that the lactic acidity decreases by anion-exchange resins. The method is said to bring back to normal any milk in which lactic acidity has increased beyond a desirable degree, due to delays prior to processing, adverse weather, etc., by means of an anionic resin. The best lactic acid retention, together with good selectivity towards other constituents, were obtained with a resin of sp. gr. 0.75, of acrylic compound, having an exchangeable radical of the tertiary amine type, and a slightly basic reaction. For example: milk having an initial acidity of 2 2 O Dornic was run through a resin column 80 cm. high, at a rate of 30 vols. of milk/vol. of resin/hr., giving a final product of 1 5 O Dornic. About
Table (8):
Summary of HPLC conditions for the determination of lactic acid.
I
Column Mobile Phase Flow Rate mllmin. Detection
~~
Sample Ref.
105
(30cm x 7.8mm) of Aminex HPX-87 H (9Fm) with an RP-18Spheri-5 cartridge) (3cm x 4.6mm) as guard column.
--
210nm
Two columns (20cm x 7.8mm) in series of Aminex HPX-87H
0.01N H2S04
0.6ml/min.
Carbohydration organic acids and sugar alcohols skin lotions
106
p b n d a p a k NH2 column (30cm x 3.9rnm)
3.2rnM-NHqH~P04 in aq. 50% acetonitrite
lml/min.
214 nm
107
. . . . continued
Table (8):

Column Mobile Phase
Ref.
Flow Rate mllmin.
Detection
Sample
(30cm x 8mm) o Shimadzu gel f SCR-101H (10Fm) with a guard column (5cm x 4mm) of TSK gel s ( (6 km) Q
W m 0
8 mM - HClO4
0.4ml/min.
380 nm
dood
108
pbndapak C i s (30cm x 4mm) Column (25cm x 4.6mm) and precolumn (5 cm x 4.6 mm) both f l e with low capacity anion ild exchange resin Column (50 cm x 8 mm) of Shodex Ion pak C-811 0.1 76 H3P04
254 nm
210 nm
seef
109
110
lml/min.
200 nm
:om silage
111
. . . coni iuec
Table (8):
Column
Mobile Phase
Flow Rate ml/min.
Detect ion
Sample
[30 cm x 7.8 mm) of Aminex HPX-87 Column (50 cm x 3 mm) of low capacity surface - modified anion exchanger, a suppressor column (15 cm x 9 mm) and a precolumn (15cm x 3 mm) of &WeX-50-X8 cation exchanger Column of Aminex A-6 or A8 resin or Beckman M-72 resin Aminex-HPX 87
1.013M - H2SO4
--
yastric juice
4 3
H20:Methanol (4:l)
wine and grape must
3.013 N - H2SO4
220 nm
:heese
. . . . coni
Table (8):

=
Mobile Phase
Flow Rate ml/min.
Detection
Sample
Ref.
I _
1 Li Chrosorb C18 column

Column containing :
( i)
0.01M KH2P04:H3P04 at pH of 2.2-3.7 0.lM-NaCl
224 nm
juices and ceders
116
juices and molases
117
Zeo-karb 225 resin (H+ form) (1Oml) and,
(ii) De-Acidite FF resin ( c 0 3 ~ f r )(1Oml) om C18 cartridge columns of an Aminex HPX-87 H 0.013 NH2SO4 0.6ml/min.
uv
juices
118
RP-18column
Phosphate buffer pH 2.25
juices and ceder
119
LACTIC ACID
309
0.2% of other anions were absorbed. About 100 1. of milk can be treated with 1'1. of exchanging resin. The column is regenerated as follows: (a) thorough washing with demineralized water; (b) rinsing with an alk. bactericide agent; (c) rinsing with a 1%HCI solution; (d) regenerating with a 4% NaOH solution; and (e) rinsing with demineralized water until neutral.
7. Acknowledgments
The authors are highly thankful to Liberty S. Matibag, College of Applied Medical Sciences for her professional help in typing this manuscript and to Mr. Babkir Awad Mustafa, College of Applied Medical Sciences for drawing the spectras and figures.
8. References
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Hanusova, J.; and Havlik, 8.; Cesk. Hyg. 24(3) 125-130 (1979). Stamer, J.R., Weirs, L.D., and Mattick, L.R. Food Chem. 10(3), 235238 (1983). Petkovic, Miodrag. Actu Pharm. Tugosl. 26(4) 323-326 (1976). I.A. Gonchavova and A.N. Khomenko. Gidrokhim. Muter. 53 3641 (1970). Alen, R., Jannari, P., Sjostrom, E. Finn. Chem. Lett. (5) 190-192 (1985). Riordan, T., Doyle, D. and Tabaqchali, Soad. 1. Clin. Pathol. 3 5 W , 390-394 (1982).
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87. Tarjan, G. Magy. Kem. Foly 91(5) 229-233 (1985). 88. Perl, I.M., Fabian-Vonsik, V., Szakacs, M.P. Chromatographia 20(7) 421-424 (1985). 89. Littman, Sigrid, Schulte, Erhard, and Acker, Ludwig. Z . LebensmUnters Forsch. 175(2) 101-105 (1982). 90. Holas, J. and Kratochvil, J. Ernaehrung (Vienna) 5(7) 331-334 (1981) (in German). 91. Cosmi, G., D, Corcia, A., Samperi, R., Chromatographia 16 322-324 (1982).
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92. Zhu, Yonghe, He, Jinzhu. Zhonghua Yixue Jianyan Zazhi lO(5) 275276 (19871.. 93. G.W. Goodman, B.C. Lewis and A.F. Taylor. Talanta 2617) 807-812 (1969). 94. J.F.T. Oldfield, R. Parslow and M. Shore. Znt. Sug. I. 72 35-40 (1970). 95. H.J. Langner. Z . Lebensmittelunters. u. forsch. 131(4) 218-221 (1966). 96. J. Savory and A. Kaplan. Clin. Chem. 12(9) 559-569 (1966). 97. 98. 99.
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Boettcher, W. Arch. Tierernaehr. 32(4) 287-304 (1982). Bricknell, K.S., Brook, I. and Finegold, S.M. Chromatographia 1211) 22-24 (1979).
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112. Andersson, Rolf, Hedlund, Bruno. 2 Lebensm-Unters Forsch. 176(6) . 440443 (1983). 113. Ivanov, A.A., Shpigun, O.A., Zolotov, Yu. Zh. Anal. Khim. 4 l W 134-139 (1986). 114. Rapp, A., Zeigler, A. Dtsch. Lebensm-Rundsch 7602) 396-398 (1979). 115. Panari, G., Milchwissenschaft 41 (4) 214-216 (1986). 116. Blanco Gomis, D., Moran Gutierrez, M.J., Gutierrez, Alvarez, M.D., Sanz, Medel, A. Chromatographia, 24, 347-350 (1987). 117. J.F.T., Oldfield and M. Shore. Znt. Sug. J. 72: 3 4 (1970).
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52-55 (1989). 119. Blanco, Gomis, D., Moran Gutierrez, M.J., Gutierrez Alvarez, M.D., Mangas Alonso, J.J. Chromatographia 2.502) 1054-1058(1988). 120. Shatirishvilii, I. Sh., Zakalashvili, G.N. Soobshch Kad. Nauk. G ~ SSR 133(2) 309-311 (1989). z 121. Chong, W.K.,Mills, G.A. Weavind, G.P., Walker, V. J. Chromatogr. 487W 147-53 (1989). 122. Gamo, Keiji. Jpn.Kokai Tokkyo Koho JP62 76. 123. Lunder, T.L., and Messori, F. Chromatographia 2 2 W 716-719(1979). 124. Herve, Daniel, Fr. 1,519,608,05 Apr. 1968, 15 Dec. (1966).
16) 125. Prat, Daniel, S.A. Fr. 1,470;425. Feb. 24,1967,Appl. Jan. 14 ( 9 6 .
METHIXENE HYDROCHLORIDE
Ezzat M. Abdel-Moety, Nashaat A . Khattab. and Mohammad Saleem Mian
Pharmaceutical Chemistry Department College of Pharmacy King Saud University P.O. Box 2457 Riyadh 1 145 I , Saudi Arabia
317
318
EZZAT M. ABDEL-MOETY ET AL.
CONTENTS
1. 2.
3.
4.
5.
6.
INTRODUCTORY DESCRIPTION 2.1 Nomenclature 2.11 Systemic Name 2.12 Other Chemical Names 2.13 P r o p r i e t a r y Names 2.14 CAS R e g i s t r y Number 2.2 Formulae and Molecular Weight 2.3 Appearance, Color and Taste. 2.4 The Cr ystal S t r u c t u r e and t h e Three-Dimensional Projection PHYSICAL CHARACTERISTICS 3.1 Elemental Composition M e l t i n g Range and B o i l i n g P o i n t 3.2 3.3 Thermal Behavior 3.4 Salubility 3.5 Crystallographic Characteristics 3.51 C r y s t a l 1i z a t i o n 3.52 X-ray D i f f r a c t i o n Pa t t e r n 3.6 Spectroscopic Data 3.61 U l t r a v i o l e t (UV) Absorption 3.62 I n f r a r e d ( I R ) Spectroscopy 3.63 Mass Spectrometry (MS) 3.64 Nuclear Magnetic Resonance (NMR) 3.641 1 H-NMR Spectrum 3.642 1JC-NMR Spectrum SYNTHESIS METHODS OF ANLAYSIS 5.1 Qualitative (Identification) 5.11 Color Tests 5.12 M i c r o c r y s t a l Test 5.2 Q u a n t i t a t i v e (Determination) 5.2 1 Col o r 1met r i Determi n a t i o n c 5.22 Fluorometric Determination 5.23 T it r imet r ic Dete r m i n a t i on 5.24 Chromatographic Techniques 5.241 Thin Layer Chromatography (TLC) 5.242 Gas L i q u i d Chromatography (GLC) 5.243 High-Performance L i q u i d Chromatography (HPLC) PHARMACOKINETICS 6.1 Absorption and E xcr etion 6.2 Biotr ansfor m ation
319
THERAPEUTIC CATEGORATION 7.1 Pharmacology 7 . 1 1 G a s t r o i n t e s t i n a l Tract ( G I T ) 7 . 1 2 Antiparkinsonism ACKNOWLEDGMENT REFERENCES

7.
320
1.
INTRODUCTORY Methlxene h y d r o c h l o r i d e i s a t e r t i a r y amine antimuscarinic drug w i t h a r e l a t i v e l y h i g h s e l e c t i v i t y f o r t h e g a s t r o i n t e s t i n a l t r a c t . I t i s used i n t h e management o f c o n d l t i o n s i n w h i c h t h e r e i s h y p e r m o t i l i t y , as i n pylorospasm, b i l i a r y dyskinesia, s p a s t i c colon, duodenitis, and g a s t r i t i s and i n other disorders i n which i t i s d e s i r a b l e t o d i m i n i s h even normal m o t i l i t y , as i n duodenal u l c e r . I t does n o t d i m i n i s h g a s t r i c secretion. Although it i s recommended f o r use i n g a s t r i c u l c e r , a decrease i n m o t i l i t y can r e s u l t i n r e t e n t i o n o f a c i d and hence sometimes exacerbate t h e e r o s i v e process. I n c i d e n c e o f s i d e e f f e c t s i s low w i t h usual doses (1). The d r u g p o t e n t i a l l y has a l l t h e s i d e e f f e c t s o f antimusc a r i n i c s and t h e precautions and c o n t r a i n d i c a t i o n s a r e the same. I t i s a l s o mainly used f o r t h e symptomatic treatment o f a r t e r i o s c l e r o t i c , i d i o p a t h i c and p o s t e n c e p h a l i t i c parkinsonism. I t i s c l a i m e d t o be more e f f e c t i v e i n c o n t r o l l i n g t h e tremors ( 2 ) .
2.
DESCRIPTION
2.1
Nomenclature
2.11
Systemic Name (3)
M e t h i x e n e : l-Methyl-3-(9H-thioxanthen-9-y1met hy 11p i per id ine M e t h i xene hydroch1o r ide : 1-Methy 1- 3- ( 9H- t h i oxant hen-g- y 1-met hy 1) p i per id i ne monohydroch 1o r i de.
2.12
Other Chemical Names
M t h ixene is g- (N-me t hy 1-3- p i per idy 1methy 11t h i e oxanthene ( 3 ) ; 9-(l-Methyl-3-piperidylmethyl)thloxanthene ( 4 ) ; (Methyl-l-piperidinyl-3-methyl)-9thioxanthene.
2.13
ProDrletary (Manufacturer) Name Tablets, scored,
T r e m o n i l ( S a n d o z , UK). methi xene hydrochloride 5 mg.
O t h e r P r o p r i e t a r y ( M a n u f a c t u r e r s ) names; Methyloxan (Jpn), T r e m a r l l (Sandoz, B e l g . , Denm.,
32 1
I t a l . , Neth., S. A f r . , Spain; Wander, Tremoquil (Astra, Swed.); T r e s t (USA) ( 2 ) .
Switz.);
Methixene Hydrochloride USAN S J 1977; A t o s i 1 (Teisan, Osaka, Jpn); C h o l i n f a l l (Tokyo Tanabe, Tokyo, Jpn); M e t h i x a r t (Fuso, Osaka, J p n ) ; Methyloxane (Nippon S h o j i , Osaka, Jpn); Raunans (Kowa Y., Tokyo, J p n ) ; Thioperkin (Hokuriku, Fukui , Jpn); Tremaril (Wander, B e l g . ; Sandoz, Denm., I t a l . 8, N e t h . ) ; T r e m a r i t (Wander, Ger.); Trernonil (Wander, Austral., Sandoz, UK); Tremoquil ( A s t r a ) , Swed.); T r e s t (Dorsey, USA)
(5).
2.14 Chemical Abstract Service (CAS) R e g i s t r y Number (3,4)
4969-02-2 (Methixene); 1553-34-0 (hydrochloride, anhydrous); 7081-40-5 (hydrochloride, monohydrate).

2.2
Formulae and Molecular Weight
C
I
CH,
N-CH,
(Methixene) [(C~OHZ~NS; M.W. ; 309.471
(Methixene.HC1) [ C ~ O H ~ S N S . H C M.W.; ~;
345.91
Methixene i s commercially a v a i l a b l e as t h e base and as t h e h y d r o c h l o r i d e s a l t ; each 100 p a r t s o f methixene base i s a p p r o x i m a t e l y e q u i v a l e n t t o 113 p a r t s o f methixene h y d r o c h l o r i d e anhydrous and 131 p a r t s o f t h e monohydrate s a l t .
322
2.3
w a r a n c e . Color and Taste
Methixene i s s l i g h t l y y e l l o w v i s c o u s l i q u i d , w h i l e t h e HC1-salt i s f l a k e s o r c r y s t a l l i n e powder. Both t h e base and t h e s a l t have b i t t e r t a s t e . The powder o f t h e s a l t i s s t a b l e i n a i r , b u t darkens slowly as a r e s u l t o f l i g h t a c t i o n (1,3). 2.4 The C r y s t a l S t r u c t u r e and t h e Three-Dimensional Projecti o n (6 )
The c r y s t a l s t r u c t u r e o f methixene hydrochloride monohydrate, 9-(N-methyl-3-piperidylmethyl)thioxanthene hydrochloride monohydrate, C2oH23NS.HCl .H20, has been determined by t h e heavy-atom method and r e f i n e d three-dimensionally by t h e a n i s o t r o p i c least-squares method t o g i v e a f i n a l R-value a = 15.320 f 0.003, b = 9.118 f 0.002, c = 13.862 f 0.004 A, and i3 = 94.75 f 0.02. A l l t h e hydrogen atoms were l o c a t e d on d i f f e r e n c e - F o u r i e r syntheses, b u t t h e i r parameters were n o t r e f i n e d . The c r y s t a l c o n t a i n s b o t h enantiomsrphs i n an equal amount. The benzenoid r i n g s are normal, and t h e best planes o f t h e benzene r i n g s make a d i h e d r a l angle o f 137.9. The meso atoms, C ( 9 ) and S, a.re s i g n i f i c a n t l y displaced from t h e benzene ring. The p i p e r i d y l r i n g i s i n a c h a i r conformation. The p i p e r i d y l m e t h y l group i s boat a x i a l w i t h respect t o t h e c e n t r a l t h i o x a n t h e n e r i n g , and b o t h t h e thioxanthen-9-ylmethyl and N-methyl groups are i n an e q u a t o r i a l p o s i t i o n w i t h respect t o t h e p i p e r i d y l r i n g . A l l i n t e r a t o m i c distances and angles are normal. The sulfur-carbon bond distance i s 1.765 f 0.003 A. The average carbon-carbon bond d i s t a n c e i s 1.524 2 0.006 A f o r carbon-carbon s i n g l e bonds, 1.384 f 0.006 A f o r carbon-carbon double bonds I n t h e benzenoid r i n g , and 1.505 k 0.005 A f o r carbon-carbon bonds i n v o l v i n g C(9) and t h e benzenoid r i n g . The mean value o f t h e nitrogen-carbon bond distance i s 1.488 2 0.006 A. Each c h l o r i d e i o n i s associated w i t h t h r e e hydrogen bonds; one l i n k s t o a quaternary ammonium i o n and t h e other two l i n k t o two d i f f e r e n t water molecules. The packing o f t h e molecules i n t h e c r y s t a l i s determined by t h e h y d r o g e n b o n d i n g and v a n d e r W a a l s i n t e r a c t i o n s . Table 1 demonstrates t h e f r a c t i o n a l atomic coordinates and t a b l e 2 shows t h e bond lengths and bond angles. Conformation angles w i t h i n t h e
METHlXENE HYDROCHLORIDE
323
p i p e r i d y l r i n g are presented i n t a b l e 3 . Hydrogen-bond distances and angles are reported i n t a b l e 4 . Table 1: F r a c t i o n a l Atomic Coordinates (6).
Atom
Y lo))*
Atom
Y 103)
(X
(X
Cl
5563 ( 1 ) 3807 ( 1 ) 6622 ( 2 ) 4642 ( 3 ) 4557 ( 3 ) 4268 ( 3 ) 4054 ( 3 ) 2757 ( 3 ) 2413 ( 3 ) 2735 ( 3 ) 3410 ( 2 ) 4550 ( 2 ) 4431 ( 2 ) 4128 ( 2 ) 3427 ( 2 ) 3779 ( 2 ) 5410 ( 2 ) 5782 ( 2 ) 6468 ( 3 ) 6908 ( 3 ) 7302 ( 3 ) 6189 ( 2 ) 6992 ( 3 ) 3860 ( 3 ) 618
7272 ( 1 ) 1811 ( 1 ) 4671 ( 3 ) -811 -2013 -2251 -1077 (5) (5)

(6)
280 ( 1 ) 4625 ( 1 ) 1186 ( 1 ) 2438 ( 3 ) 2776 ( 4 ) 3668 ( 5 ) 4244 ( 4 ) 3917 ( 3 ) 3222 ( 4 ) 2321 ( 3 ) 2128 ( 3 ) 2647 ( 2 ) 2998 ( 3 ) 3903 ( 3 ) 3720 ( 3 ) 2822 (31 3129 ( 3 ) 2592 ( 3 ) 3234 ( 3 ) 2659 ( 4 ) 1794 ( 4 ) 1704 ( 3 ) 291 ( 4 ) -172 93 (3)
485 461 424 38 1 249 185 249 366 463 587 527 529 692 620 646 739 757 777 662 570 652 739 731 454 409
-37 -281 -316 -123 396 557 557 41 1 212 204 313 477 423 528 686 858 615 472 278 306 355 335 480 872 1045
170 23 1 399 484 452 333 183 151 197 313 377 237 349 377 244 303 136 198 190 121 -3 43
9
N C(1) C(2) C(3) C(4) C(5) C(6) C(7) C(8) C(9) C(11) C(12) C(13) C(14) C(15) C(16) C(17) C(l8) C(l9) C(20) C(21)
O(W)
(6)
4023 ( 5 ) 4983 ( 5 ) 4978 ( 5 ) 4052 ( 4 ) 2140 ( 4 ) 600 ( 4 ) 337 ( 5 ) 3067 ( 4 ) 3103 ( 4 ) 2793 ( 4 ) 4079 ( 4 ) 4919 ( 5 ) 6091 ( 5 ) 5457 ( 5 ) 3497 ( 4 ) 4062 ( 6 ) 9389 ( 4 ) 544
0
6 -9
H(N)
The c o n f i g u r a t i o n o f a m e t h i x e n e h y d r o c h l o r i d e monohydrate m o l e c u l e and t h e i d e n t i f i c a t i o n o f t h e atoms are shown i n f i g . 1. The t o r s i o n angles and t h e molecular packing diagram are presented i n f i g . 2 and 3 r e s p e c t i v e l y . The o r i e n t a t i o n o f t h e p i p e r i d i n y l
324
Fig. (1). The configuration of methixene HC1 molecule.
Fig. (2). The structure of one asymmetric unit of methixene hydrochloride.
325
methyl group r e l a t i v e t o t h i o x a n t h e n e r i n g i s a l s o shown in f i g . 4. Table 2: Bond lengths and bond angles ( w i t h estimated standard d e v i a t i o n s i n Darentheses) (6)
C(12) C(13) -N C(19) N C(20) -N C(21) C(l)-C(2) C(l)-C(ll) C ( 2 1C ( 3 C 3 1-c (4) ( C(4)-C(12) C 5 )C 6 1 ( -( C(5)-C(13) C ( 6 1--C ( 7 ) C(7 )-C(8) C ( 8 )C ( 1 4) C(9)-C(ll) C(9)-C( 14) C(9 1 C 1 5) -( C(ll)-C(12) C 13)-C( 14) ( C( 15)-C ( 16) C(16)-C(17) C(16)-C(20) C( 17 )-C( 18) C( 18)-C( 19) c ( 1 2)-s-c ( 1 31 C( 19)-N-C(20) C(19)-N-C(21) C(20)-N-C(21) C(2)-C(l)-C(ll)
S -
1.769 1.761 1.485 1.492 1.487 1.371 1.403 1.364 1.391 1.381 1.374 1.391 1.380 1.378 1.380 1.502 1.508 1.546 1.393 1.397 1.524 1.526 1.521 1.523 1.502 100.5 111.9 111.2 110.8 120.8
(3) A C(l)--C(2)-C(3) (3) C(2)-C(3)-C(4) C(3)-C(4)-C(12) (6) (5) C ( 6 )C ( 5 >-C ( 1 3 ) (6) C(5)-C(6)-C(7) C(6)-C(7)-C(8) (7) C(7)-C(8)-C(14) (6) (8) C(ll)-C(9)-C(14) C( 1 1 )-C(9)-C ( 15) (7) C(14)-C(9)-C(15) (6) (6) C(l)-C(ll)-C(9) (6) c ( 1 )c ( 1 1 1-c ( 12) C(9)-C(ll)-C(12) (6) -S C( 12)-C(4) (6) -S C(12)-C(11) (5) C(4)--C(l2)-C(ll) (5) -S C(13)-C(5) (5) S C(13)-C(14) (5) c ( 5)-c ( 1 3 1-c ( 14) (5) (5) C ( 8)-C ( 1 4)-C ( 9) (5) C(8)-C(14)-C(13) (5) C(9)-C(l4)-C(13) (5) C(9)-C(15)-C(16) C(15)-C(16)-C(17) (7) (7) C(15)-C(16)-C(20) (2) C(17)-C(16)-C(20) C(16)-C(17)-C(18) (3) C(17)-C(18)-C(19) (3) (4) N C( 19)-C( 18) N----C(20)-C(16) (4)
120.4 120.5 119.5 120.5 119.2 120.5 121.3 112.0 110.0 110.9 121.1 118.2 120.7 118.8 120.5 120.8 118.3 121.4 120.4 122.3 118.0 119.7 115.1 111.6 108.8 109.3 111.1 111.9 110.2 112.5
(5)A
(5)
(4) (4) (4) (4) (4) (3)

(3)
(3) (3) (3) (3) (3) (3) (4) (3) (3) (4) (3) (3) (3) (3) (3) (3) (4) (4) (4) (3)
(3)
326
F i g . ( 3 ) . The t o r s i o n a n g l e s about t h e (a) C ( 1 9 ) - N , C ( l S ) and (c) C ( 1 5 ) - C ( 1 6 ) bonds.
(b) C ( 9 ) -
F i g . ( 4 ) . The mol e c ula r packing diagram o f methixene hvdrochloride.
321
Table 3: Conformation angles* w i t h i n t h e D i D e r i d v l
rins
C(16) C(17) C(18) C(19) N C(20)
- C(18) - C(19) -N - C(20) - C(16)
- C(17)
-t -t -t
52.9 56.6 56.1
- 55.5
- 56.8 - 53.9
*The conformation angle o f a d i r e c t e d bond C(17) C(18) i s d e f i n e d as t h e angle t h a t t h e p r o j e c t i o n o f t h e bond C(16) - C ( 1 7 ) makes w i t h r e s p e c t t o t h e p r o j e c t i o n o f t h e bond C(17) - C(18). The angle i s p o s i t i v e i f i t i s measured clockwise. Table 4: Hydrogen-bond d i s t a n c e s and angles.
N N
0
c1 c1 c1
c1
c1 c1 c1 N N N 0
0 Ma)* O(a) C(19) C(20) C(21) C1 (a)
0.051 A 3.051 3.280 3.051 3.051 3.051 3.280
3.280 A 3.203 3.203
3.203
157.5" 128.0 68.7 100.9 120.6 100.6 116.6
328
3.
PHYSICAL CHARACTERISTICS
3.1
Elemental Commsition Methixene Methixene.HC1
Element
[Calculated]
(%I
C
H
c1 N
S
77.62 7.49
4.53 10.36
69.38 6.94 10.26 4.05 9.37
3.2
M e l t i n g Range and B o i l i n g P o i n t M e l t i n g range, " C B o i l i n g p o i n t , 'C

171-175 ( 0 . 0 7 (1)
Methi xene Methixene.HC1
215-217 ( 1 ) 215-216 ( 2 ) 213-217 ( 3 ) 211-213 ( 4 )
W II
Hg)
3.3
Thermal Behavior ( 7 )
The d i f f e r e n t 5 a1 scanning c a l o r i m e t r y (DSC) thermal curve f o r methixene hydrochloride i s shown i n f i g . 5. The scanning has been r u n a t a r a t e o f 10C m i n - l from 180 t o 2 5 0 C . The h y d r o c h l o r i d e s a l t o f methixene m e l t s a t 2 1 7 . 1 " C w i t h AH-value o f 3 4 . 7 Kj.mo1-1 f o r 9 9 . 4 4 mol% p u r i t y . A Dupont TA-9900 Thermal Analyzer attached t o a Dupont Data U n i t were used f o r t h e DSC-running.
Comment: 0.50:
r2!:.c
I
0.00-
'. '.
Y
-216.5
u b "A
-.0 05'
-1.00-
. '
-216.0
n.
J
- so pi.
\
'1 I
A
L
- 215.5
r
m
I
'J:
U
I1
u
L 7
\
a
-215.0
-2.00-
X r. O
?a
rn
u
*
A .
. I
, v
L-2.50-
-214.5
I u-3.00-
-3.50-4.00-4.50t
Purity : Melting P t : Depression : Delta H : Correction : H o l . HElghl: C e l l Const : Onset Slope:
99.45 Hole X 216.9 .C

0.33 'C
33.4 k J / m O l E 6.73 X 345.9 p/Hole 1.191
Fi, D 7&
u
n
- A A A
0
El
-214.0
A
b-
- 213.5
b
-213.0
-i.ii m W ' c
5
1
1.0 ta 1 Area,'Per t I a 1 A r e a
1EO
;.
190
,:
10
200
210
220
5 , ' . I' ,
20
-212.5 250
230
240
330
45
20
Fig. (6). X--ray diffraction lines of methixene hvdrochloride.
METHIXENF.HYDROCHLORlDE
33 1
3.4
Solubility
While t h e methixene base i s water i n s o l u b l e , i t s hydrochloride s a l t i s s o l u b l e i n water, alcohol and chloroform b u t i n s o l u b l e i n e t h e r (1-4).
3.5
Crystallographic Characteristics
3.51
Crystallization
Methixene h y d r o c h l o r i d e c r y s t a l l i z e s as w h i t e f l a k e s f r o m e t h e r , mp. 215-217C ( 1 ) o r f r o m alcohol-ether (1:2, v/v), mp. 211-213C.

3.52
X-Ray D i f f r a c t i o n P a t t e r n ( 7 )
The X-ray d i f f r a c t o m e t r y o f methixene.HC1 has been undertaken on a P h i l i p s PW-1710 d i f f r a c t o m e t e r w i t h s i n g l e c r y s t a l monochromator and copper Ka r a d i a t i o n . The p a t t e r n s were recorded on a P h i l i p s PM-8210 p r i n t i n g recorder. The t a b l e o f 26, d-spacing (A), and count were a u t o m a t i c a l l y o b t a i n e d on a P h i l i p s d i g i t a l p r i n t e r , t a b l e 2 showing t h e c o l l e c t e d data i n summarized form. Fig. 6 shows t h e c h a r a c t e r i s t i c p r i n c i p a l l i n e s o f t h e X-ray powder d i f f r a c t i o n c a r r i e d out on a pure sample o f methixene.HC1 s a l t .
3.6
Spectroscopic Data
3.61
U l t r a v i o l e t (UV) AbsorPtion ( 8 )
The U.V. measurement has been c a r r i e d o u t f o r methixene h y d r o c h l o r i d e s o l u t i o n s i n 95% e t h a n o l , water and 0.1 N HCl against t h e corresponding blank u t i l i z i n g matched 1-cm quartz c e l l s . The A ( l % . , 1 cm)v a l u e s and t h e molar a b s o r p t i v i t i e s o f methixene hydrochloride s o l u t i o n s are c o l l e c t i v e l y summarized i n t a b l e 6. Figure 7 demonstrates t h e UV-spectra o f t h e d r u g substance in d i f f e r e n t solvents. The s p e c t r a scanning has been undertaken on a Varian DMS 90 1-cm quartz c e l l s .
332
N/10 HC1
20 0
i0
F i g . ( 7 ) . UV s p e c t r a o f methixene HC1 i n d i f f e r e n t solvents.
333
Table 5: The X-ray d i f f r a c t i o n a l p r i n c i p a l l i n e s methixene hydrochloride.

26
d(R)
[I/Io x 1001
87.8 50.2 54.6 97.1 16.5 25.8 52.9 31.3 61.6 72.2 39.9 28.8 61.6 48.5 34.5 24.5 100.0 18.3 35.3 53.3 53.4 9.3 8.1 28.6 7.5
2@ 32.496 33.535 33.867 34.598 34.979 35.203 36.190 36.651 37.759 38.505 38.907 39.415 40.280 41.151 41,755 42.855 44,250 45,445 46.462 47.401 48.446 48.935 51.136 51.663 52.48
d(A)
[I/Io x 1001
13.5 9.6 7.4 10.3 11.3 15.1 8.3 10.6 9.4 7.0 6.4 5.3 10.8 8.9 15.6 8.7 8.0 5.8 7.1 6.5 6.8 6.0 5.1 5.4 7.6
3.197 6.332 11.568 12.733 13.607 15.081 16.519 17.414 17.931 19.196 19.907 20.460 21.372 22.675 23.281 23.798 24.510 25.279 25.718 26.524 27.439 28.448 29.883 30.668 31.639
27.6380 13.9593 7.6496 6.9519 6,5072 5.8746 5.3664 5.0924 4.9468 4.6234 4.4600 4.3407 4.1574 3.9213 3.8207 3.7388 3.6319 3.5230 3.4639 3.3605 3.2504 3.1374 2.9899 2.9029 2.8279
2.7553 2.6722 2.6468 2.6925 2.5652 2.5493 2.4821 2.4518 2.3824 2.3380 2.3147 2.2861 2.2389 2.1936 2.1632 2.1102 2.0469 1.9958 1.9544 1.9179 1.8789 1.8613 1.7862 1.7692 1.7508
Table 6:
The UV-spectral c h a r a c t e r i s t i c s o f methlxene hydrochloride. (nm)
Solvent
Xrnax
A(l%,
1 cm)
316 324 325+
(l.mo1-1.cm-1)
95% ethanol Water 0.1 N HC1

+
268 266 267
10930 11194 11241
reported a t 268 n as 324 (3). m
334
Other reported UV-data i n o t h e r solvents (9) are a l s o as follows:
Sol vent.
Acetonitrile Chl o r o f arm
Xmex
390 385
A(t%,
1 em)
(~.mol-l.cm-l) 7458 3105
215.6 89.7
3.62
I n f r a r e d ( I R ) SDectroscoDy (8)
The IR-spectrum o f methixene h y d r o c h l o r i d e as K B r - d i s c was made on a P e r k i n Elmer i n f r a r e d spectrometer. F i g u r e 8, shows t h e o b t a i n e d IR-spectrum, w h i l e t a b l e 7 i l l u s t r a t e s t h e s t r u c t u r a l assignments w i t h t h e recorded frequencies. Table 7: The I R - c h a r a c t e r i s t i c s o f methixene hydrochloride. Group assignment
CH3 ,CH2 ,CH s t r e t c h i n g HN+ , s t r e t c h i n g HN+ CHz ,-CH3 , CH-def ormat i on S-C s t r e t c h i n g . CHz, rocking.
Frequency*, cm2950-2880 ( s ) 2480 (m) 1600-1680 ( W) 1460 (s) 1350-1 220 760 ( s )
*s, m & w means strong, medium and weak, respectively.

3.63
Mass S D e c t r m e t r v (US) ( 8 )
The mass spectra o f methixene i s i l l u s t r a t e d i n f i g u r e 9, where a base peak appears a t m/e 197 due t o Ci3HioS i.e., t h i o x a n t h e n e . The mass spectrum o f methixene i s s a i d t o provide a s e n s i t i v e and s p e c i f i c mean f o r i d e n t i f i c a t i o n and q u a n t i f i c a t i o n i n i n pharmaceutical f o r m u l a t i o n . The mass f r a g m e n t a t i o n p a t t e r n f o r methixene i s shown i n t a b l e 8.
i?
0
u3
V
vr
.d
Ld
vr
c d
0 0 0
4
.r(
Q,
5 0
0
0
L n
4
. c
a >
a ,
a ,
.d
5 a ,
E
w
0
0
0
0
h l
c,
cd k
a a ,
k cd
0 0 0 M
a ,
ru
F:
c . W
M
.d
T R A N S M I T T A N C E
197 /
100
309
165
\
210
/
'
40
rn
e
235
Fig.
(9).
Mass spectrum o f methixene.
337
Table 8:
Mass fragmentation o f methixene.
Empirical structure
C3 Ha C4H10 C3 H i NH C5H10
Mass/charge r a t i o (m/e)
44 58 58 70
[Io/I
68 72 72 8
(%)I
Fragment i o n
CH3 CH2 CH3 CH3 (CH2 CH3 (CH2
) 2 CH3
2 NH
CH3 (CH2 1 4 - 1
CsHi oN
84
10
CsHi 3 N
99
66
N-CH,
C7H15N
112
20
210
14
197
100
C2oH23NS
309
38
M+
338
3.64
Nuclear Magnetic Resonance (NMR)
Both t h e p r o t o n n u c l e a r magnetic resonance (1H-NMR) and c a r b o n n u c l e a r m a g n e t i c resonance (13C-NMR) spectra o f methixene hydrochloride have been run on the same s o l u t i o n i n CDC13.
3.641 lH-NHR SDectrurn (8)
The 200 MHz proton magnetic resonance spectrum o f methixene hydrochloride i s given i n the f i g u r e 10 w h i l e Table 9 summarizes t h e chemical s h i f t and s p e c t r a l assignments o f t h e p r o t o n s o f methixene h y d r o c h l o r i d e . The r u n n i n g o f t h e s p e c t r a was undertaken i n CDCl3 using TMS as an i n t e r n a l standard on a V a r i a n XL-200 s p e c t r o m e t e r a t a m b i e n t temperature. Table 9: Chemical s h i f t s and spectral assignments o f 'H-NMR o f methixene hvd rochl o r ide
1 oCH,
~~~~
~~
Drug
Proton p o s i t i o n (Nr) aromatic; 1-8 ( 8 ) CHBN; 13 ( 3 ) CHz; 12,14-16 ( ) C H 2 ; 10 (2) CH; 9 ( 1 ) +NH (1)
(ppm, TMS)
Multiplicity
Methixene.HC1
7.2-7.6 2.8-2.6 2.2-1.9 3.5-3 4.2-4.1 > 12
m
S
m m
S
M
I +
V n V
E
.r(
a
.d
a ,
5
0
I +
c
a , E a,
.d
5 a ,
E
'U
c,
0
cd k
a ,
2
p:
d
.d
M
L
340
3.642 'SC-NMR
Swctrun ( 8 )
The lJC-nuclear magnetic resonance spectrum o f methixene h y d r o c h l o r i d e was o b t a i n e d i n C D C l s a t ambient temperature using TMS as t h e i n t e r n a l standard on a Varian XL-200 spectrometer. The chemical s h i f t s , and s p e c t r a l assignments are given i n t a b l e 10, w h i l e Figure shows t h e obtained 13C-NMR spectrum. The DEPT and APT spectra o f methixene hydrochloride are given i n f i g u r e s (11-14). Figure 15 shows t h e HOMCOR (pulse seqence 1 H-1 H-NMR) spectrum o f t h e drug substance.
Table 10: Chemical s h i f t s and s p e c t r a l assignments o f 13C-NMR o f methixene hydrochloride.

~~
Carbon pos it i on
Chemical s h i f t (6, ppm)

128.84 132.1 137.24 128.9 46.2 22.54 32.04 59.3 43.96 54.5 28.6 35.8
c2 3 , B , 9
c5,13 c6,11
c1,4,10,7 c12 c14
c19 c15
c20
c16
c17
C18
Fig. (11) 13C-N&lR
s p e c t r a o f methixene HC1 in CDC13.
t i
F i g . ( 1 2 ) . 13C-NMR spectra of methixene HC1 in CDC1, (APT Program).
7 U Sec.
' 4 ' 4
Fig. (13).
15
C-NMR spectra of methixene HC1 in CDC13 (continuation of fig. 14).
CH3
CH2
Fig. (14). 13C-NMR s p e c t r a of methixene HC1 i n CDCl
(DEPT Program).
345
1
2
3
4
6
7
a
9 10 11
12
I
1
I , I
1
1
1 2 1 1 1 0 9
5
PPM
F i g . (15). HOMCOR ( p u l s e sequence 'H-lH-NMR) methixene h y d r o c h l o r i d e .
spectrum of
346
4.
SYNTHESIS
Methixene and i t s s a l t s have been t o t a l l y s y n t h e s i z e d from prepared 9 - t h i o x a n t h y l sodium and N-methyl-3-chloromethylpiperidine by j u s t condensation. a n t h e s i s o f 9-thioxanthyl sodium: (Scheme I) To 4.9 p a r t s o f f i n e l y p u l v e r i z e d sodium i n 50 p a r t s o f abs. benzene add dropwise w i t h s t i r r i n g 12 p a r t s o f chlorobenzene i n 50 p a r t s o f absolute benzene. As soon as t h e exothermic r e a c t i o n begins, m a i n t a i n t h e temperature by c o o l i n g between 30C and 3 5 " C , and continue s t i r r i n g f o r 2 t o 3 hours. To t h e r e s u l t i n g phenyl sodium add dropwise 19.8 p a r t s o f thioxanthene i n 120 p a r t s o f a b s o l u t e benzene. The s l i g h t l y exothermic r e a c t i o n ceases a f t e r about 1 t o If hours (10).
dine:
PreDaratlon o f N - m e t h ~ l - 3 - c h l o r o m e t h ~ l ~ i p e r i (Scheme 11) 3 - P y r i d i n e methanol y i e l d s on r e a c t i o n w i t h methyl i o d i d e t h e quaternary s a l t ; which on h y d r o g e n a t i o n g i v e s t h e corresponding N-methy l -3 -p i p e ri d i n e methanol. T h i o n y l c h l o r i d e c o n v e r t s t h e a l c o h o l t o t h e e q u i v a l e n t N-methyl-3chloromethylpiperidine (4). Coupling o f 9-thioxanthyl sodium w i t h N-methyl3 - c h l o r o r n e t h y l ~ i ~ e r i d n e : To t h e f r e s h l y p r e p a r e d 9-thioxanthyl sodium add dropwise, w i t h s t i r r i n g and c o o l i n g , 131.1 p a r t s o f N-methyl-3-chloromethylpiperid i n e i n 30 t o 40 p a r t s o f a b s o l u t e benzene, t h e n continue s t i r r i n g a t about 25C f o r If hours, and heat subsequently t o 40C. Decompose t h e r e s u l t l n g m i x t u r e by adding c a r e f u l l y a small amount o f water, and then e x t r a c t t h e newly formed base from t h e benzene s o l u t i o n by means o f d i l u t e h y d r o c h l o r i c a c l d . The aqueous h y d r o c h l o r l c s o l u t i o n i s made a l k a l i n e by adding d i l u t e sodium hydroxide, and t h e methixene base i s i s o l a t e d by e x t r a c t i o n w i t h ether. This r e s u l t s i n 22 p a r t s o f a s l i g h t l y yellow, viscous base o f BP 171 t o 175'C (10.07 mm.Hg). The base i s a c i d i f i e d w i t h a l c o h o l i c h y d r o c h l o r i c acid. Alcohol-ether (1:2) i s then added and hydrochloride s a l t i s r e c r y s t a l l i z e d as white f l a k e s m e l t i n g a t 211 t o 213C (11).
z
a , E a ,
c
.I+
+J
a ,
c
M
c
v
a ,
N
a ,
N
ocd
e
M
I
a ,
0
E
0
ln
348
EZZAT M. ABDEL-MOETY ET AL
Synthesis (Scheme 11)
CH3 I
U C H 2 O H 3 - Pyridine methano 1
QCH2OH
I CH3
Hydrogenat ion
I+ @ : !
9-Thioxanthyl sod. alc. HC1 Met hixene
N-methyl-3-piperidine met h an o 1
PCH3 - Qjn
Methixene HC1.
N-CH,.
HCI
349
5.
METHODS OF ANALYSIS
5.1
Qualitative (Identification)
5.11
Color Tests ( 4 )
Some s p e c i f i c reagents g i v e c e r t a i n c o l o r a t i o n w i t h methixene and i t s HC1-salt. The f o l l o w i n g common c o l o r reagents are recommended f o r i d e n t i f i c a t i o n o f t h e drug. Reagent
HCHO-H2 so4 L iebermann s Mandelins
H2 SO4
Color orange red orange orange orange ( f l u o r e s c e s under UV) M i c r o c r y s t a l Test
5.12
The aqueous s o l u t i o n o f methixene hydrochloride has been subjected t o some o f t h e most common reagents f o r m i c r o c r y s t a l l i z a t i o n examination. Only t h e aqueous s o l u t i o n o f ZnCl2 (5% w/v) y i e l d s c l e a r r o s s e t t e s a f t e r about 15 min. The f o l l o w i n g f i g . 16 shows t h e c r y s t a l shapes on r e a c t i n g t h e d r u g w i t h t h e p r e c i p i t a t i n g reagent.
Fig. 16: Microscouic examination o f d i f f e r e n t c r y s t a l forms obtained from r e a c t i n g methixene.HC1 w i t h ZnC12 solution.+ + t r a c i n g has been undertaken by using a L e i t z Camera Lucida (x attached t o a L e i t z p r o j e c t o r ; t h e stage scale micrometer was u t i l i z e d under t h e same m a g n i f i c a t i o n (8).
350
EZZAT M. ABDEL-MOETY ET AL
5 2
Q u a n t i t a t i v e (Determi n a t i o n )
5.21
C o l o r i m e t r i c Determination
Wa'lash e t 87. ( 9 ) have presented a c o l o r i m e t r i c method f o r t h e q u a n t i t a t i v e determination o f methixene alongwith other thioxanthene d e r i v a t i v e s . A weighed amount o f powdered t a b l e t s (about 25 mg) was e x t r a c t e d 3 x 15 m l chloroform i n t o a 50-ml volumetric flask. An a l i q u o t o f the chloroform e x t r a c t equivalent t o 100-400 c(g o f t h e d r u g was p i p e t t e d i n t o a 10 m l f l a s k , evaporated t o dryness and t h e residue dissolved i n 2 m l a c e t o n i t r i l e followed by 1 m l tetracyanoethylene (0.2%) reagent and t h e mixture was d i l u t e d with 5 m l a c e t o n i t r i l e , heated a t 80'C i n water bath f o r 5 m i n u t e s , c o o l e d and c o m p l e t e d t o volume w i t h a c e t o n i t r i l e and absorbance was measured a t 390 nm. against a blank.
5.22
Fluorometric Determination
Hassan e t a7. (12) have presented a f l u o r o m e t r i c method f o r t h e determination o f methixene i n pure and dosage forms. The method i n v o l v e s t h e use of t h e hexamine-cobalt (111) t r i c a r b o n a t o c o b a l t a t e (111) (HCTC) as an oxidant i n aqueous s u l p h u r i c medium t o induce fluorscence. Sample PreDaration Stock s o l u t i o n (1.0 mg/ml) o f t h e methixene i n d l s t i l l e d water was f u r t h e r d i l u t e d w i t h aq. s u l p h u r i c a c i d (20% v/v) t o contain 1 pg o f t h e analyte per m l . Procedure f o r Authentic SamDle T r a n s f e r a 7 i q u o t s o f methixene h y d r o c h l o r i d e s o l u t i o n t o cover t h e concentration range (0.04-0.64 pg/ml), t o 25 m l volumetric f l a s k s , d i l u t e w i t h 5 m l o f aqueous s u l p h u r i c a c i d (20% v / v ) , add 0.1 m l o f HCTC s o l u t i o n ( 5 x M), then d i l u t e t o mark w i t h aqueous s u l p h u r i c a c i d . Leave f o r t h r e e minutes t o complete the reaction. The fluorescence was a t X m a x (368 nm) ( e x c i t a t i o n ) and X m a x (545 nm) (emission), where t h e concentration was read from t h e c a l i b r a t i o n graph.
3s I
Procedure f o r t h e Dosane Forms T r a n s f e r a weighed amount o f t h e powdered t a b l e t s equivalent t o 30 mg o f t h e drug, i n t o a small c o n i c a l f l a s k . E x t r a c t 3 x 30 m l d i s t . H20. Transfer t h e s o l u t i o n t o a 100 m l volumetric f l a s k and d i l u t e w i t h H20. Further d i l u t e t h i s s o l u t i o n w i t h aq. H2S04 (20% v/v) t o give an analyte concentration o f % l pg/ml and analyse as f o r t h e authentic sample. 5.23 T i t r i m e t r i c Determination
B e l a l e t a l . ( 1 3 ) have presented a t i t r i m e t r i c method f o r t h e d e t e r m i n a t i o n o f m e t h i x e n e h y d r o c h l o r i d e . I n t h e procedure HCTC i s used as a t i t r a n t w i t h v i s u a l d e t e c t i o n o f t h e end p o i n t using f e r r o i n as an i n d i c a t o r ( t h e complete disappearance o f orange c o l o r ) . El-Brashy (14) has reported an i n d i r e c t method f o r t h e determination o f methixene i n pure and dosage forms. The procedure involves t h e preparation o f a 1.0 mg/ml s o l u t i o n o f methixene i n 3 M HC1. Add an a l i q u o t o f t h e s o l u t i o n t o a known volume o f 0 . 0 0 5 M 2-iodylbenroate s o l u t i o n i n a glass stoppered Erlenmeyer f l a s k . Shake t h e mixture o c c a s i o n a l l y and a f t e r 15 m i n u t e s add 10 m l o f 100 mg/ml potassium i o d i d e s o l u t i o n and t i t r a t e t h e l i b e r a t e d i o d i n e w i t h 0.02M sodium t h i o s u l p h a t e , using s t a r c h as i n d i c a t o r . Repeat t h e experiment without methixene. The amount o f t h e drug was c a l c u l a t e d from t h e f o l l o w i n g equation.
where VI and V 2 a r e t h e volumes o f t h i o s u l p h a t e s o l u t i o n ( m l ) used i n t i t r a t i o n o f t h e b l a n k and sample r e s p e c t i v e l y . R i s t h e molecular weight o f t h e drug and M i s t h e m o l a r i t y o f t h e t i t r a n t . For t h e assay o f t h e drug i n dosage forms, e x t r a c t a weighed amount o f t h e p u l v e r i z e d t a b l e t s equivalent t o 100 m g o f t h e drug 3 x 20 m l o f 3M HC1. F i l t e r t h e combined e x t r a c t s i n t o a 100 m l standard f l a s k and d i l u t e t o volume w i t h t h e used solvent. Transfer an a c c u r a t e l y measured volume o f t h i s s o l u t i o n ; equivalent t o 5-12 mg o f t h e drug, i n t o i o d i n e f l a s k and proceed as described above.
352
5.24
Chromatographic Techniques
5.241
4 (15)
Thin layer chromatography o f methixene has been done w i t h the f o l l o w i n g conditions: Adsorbent: Precoated s i l i c a gel f 2 5 4 (E. Merck). Developer: Methanol t 25% aq. ammonia (110:1.5, v/v). Detection: Dragendorffs reagent. Reference substance: Bupranolol (relative-Rr: 1.05).
5.242 Gas L i a u i d Chromatography (GLC) ( 1 5 )
Some GLC systems and conditions f o r t h e analysis o f methixene hydrochloride are presented i n t a b l e 11.
5.243 High Performance L i a u i d Chromatography
(HPLC) I s o c r a t i c multi-column HPLC as a technique f o r t h e q u a t i t a t i v e a n a l y s i s o f t h e d r u g w i t h some phenothiazines have been reported (16).
6.
PHARMACOKINETICS
6.1
m o m t i o n and Excretion
Methixene s a l t i s water s o l u b l e and can be e a s i l y absorbed a f t e r o r a l administration. The drug i s excreted i n the urine, p a r t l y unchanged and mostly as f r e e o r conjugated metabol it e s [ 2 I .
6.2
Biotransformation
As most o f t h e thioxanthene s a l t s , methixene hydrochloride s u f f e r s from S-oxidation i n t o i t s two main i s o m e r l c s u l p h o x i d e s . Another p a r t o f t h e S - o x i d a t i o n products may be i n t h e form o f t h e N-demethylated methixene s a l t , i . e . normethixene sulphoxides. The f o l l o w i n g scheme i l l u s t r a t e s t h e metabolic transformation o f methixene. The bio-sulphoxidation o f methixene r e s u l t s i n the formation o f t h e monosulphoxide [ l , a ] ; which can be transformed t o the disulphoxide [ l , b l . Equivalent N-demethylation y i e l d s t h e corresponding normethixene,
Q& j
4
0
G
CH
N -CH,
N-CH,
Excretion (unchanged)
/
N-CH,
methixene
G
0
/+ 0
Normethixene Extretion
354
which can be a l s o s u l p h o x i d i z e d t o t h e monoand/or t h e disulphoxide [2bl. Table 11: Summary o f t h e GLC-conditions used f o r i d e n t i f i c a t i o n and determination o f methixene hydrochloride (15). Col umn
6 f t x 2 mm packed 3% OV-1 on Chromosorb W-HP 100-120 mesh. 6 ft x 2 mm packed 3% OV-17 on Chromosorb W-HP 100-120 mesh. 3 f t x 2 mm packed 3% OV-1 on Chromosorb W-HP 100-120 mesh. 3 f t x 2 mm packed 3% OV-17 on Ch romosorb W-HP 100-120 mesh.
[2al
Temperatures 150-25O'C with t h e rate of 1O'C min-1 150-250 " C w i t h the rate o f 10C min-1 200-280 " C with t h e rate o f 10C min-1 220-280 c with the rate o f 1O'C min-1
O
Reference substance 2-Amino-5c h l orbenazophenone Methaqualone
Re1a t i v e Rf 1.67
1.26
Thioridazine
0.35
Thior i daz i ne
0.42
* **
7.
i n i t i a l time o f 1 min; detection w i t h PND a t 300C. C a r r i e r gas i s N2 a t 50 rnl.min-1 other gases are a i r (120 ml.min-'1 and hydrogen ( 2 ml.min-1).
THERAPEUTIC CATEGORATION
7.1
Pharmacology
7.11
G a s t r o i n t e s t i n a l T r a c t (GIT)
Methixene h y d r o c h l o r i d e i s an a n t i c h o l e n e r g i c agent t h a t may be e f f e c t i v e as an a d j u n c t i n t h e treatment o f g a s t r o i n t e s t i n a l hypermotil i t y and spasm associated w i t h f u n c t i o n a l bowel disorders. There i s
355
no evidence t h a t methixene hydrochloride s i g n i f i c a n t l y decrease g a s t r i c s e c r e t i o n ( 1 7 ) . Although methixene c o n t a i n s t h e same t h i o x a n t h e n e n u c l e u s a s chloroprothixene, a major t r a n q u i l i z e r . I t s pharmacological a c t i o n i s p r i m a r i l y a n t i c h o l e n e r g i c . I t has l i t t l e e f f e c t on CNS. Methixene d i l a t e s t h e p u p i l s and i n h i b i t s s a l i v a r y secreation t o a l e s s e r d e g r e e t h a n t h a t due t o a t r o p i n e . Results o f experiments i n several animal species i n d i c a t e t h a t methixene has an i n h i b i t o r y e f f e c t on g a s t r o i n t e s t i n a l motor a c t i v i t y , w i t h o n l y a minimal e f f e c t on g a s t r i c s e c r e t i o n o f h y d r o c h l o r i c acid. Methixene sometimes produces undesired e f f e c t s t y p i c a l l y o f t h e a n t i c h o l e n e r g i c drugs. Dryness o f t h e mouth, mydriasis c y d o p l e g i a , r a s h and u r i n a r y r e t e n t i o n have been noted, e s p e c i a l l y when l a r g e doses have been given. A l t h o u g h no t e r a t o g e n i c e f f e c t s have been demonstrated i n animal s t u d i e s , t h e p o s s i b i l i t y r i s k t o t h e f e t u s must be weighed a g a i n s t t h e expected t h e r a p e u t i c b e n e f i t s i f methixene i s considered f o r a d m i n i s t r a t i o n t o a pregnant woman. Also methixene i s c o n t r a i n d i c a t e d i n presence o f angl e-closure glucoma, py l o r i c o b s t r u c t i o n p r o s t a t i c hypertrophy, bladder-neck o b s t r u c t i o n o r eardiospasm
(17).
The c h a r a c t e r i s t i c pharmacological a c t i v i t y o f t h e d r u g has been demonstrated i n mice, r a t s and g u i n e a p i g s . The r e l a t i v e p o t e n c y o f m e t h i x e n e hydrochloride compared w i t h a t r o p i n e w i t h t h e respect t o e f f e c t on g a s t r o i n t e s t i n a l m o t i l i t y v a r i e s from 1.0 t o 2.2 (when assayed i n t h e mouse f o r i n h i b i t i o n o f chareoal passage) t o 1.0 t o 1 6 . 7 f o r i n h i b i t i o n o f t h e p e r i s t a l t i c r e f l e x i n guenia p i g s . However, i n t h e i n h i b i t i o n o f s a l i v a t i o n a t r o p i n e i s 32 t i m e s as potent i n t h e mouse, 6 4 . 5 times as potent i n guenia p i g and 87 times as potent i n t h e r a t . I n rnydriatic a c t i v i t y i n mouse, a t r o p l n e is 20 t i m e s as p o t e n t as methixene h y d r o c h l o r i d e . Thus s t u d i e s i n e x p e r i m e n t a l animals i n d i c a t e t h a t t h e parasympatholytic a c t i v i t i e s o f methixene hydrochloride are r e l a t i v e l y greater w i t h respect t o i n h i b i t i o n o f g a s t r o i n t e s t i n a l m o t i l i t y than they are w i t h reference t o i n h i b i t i o n o f s a l i v a t i o n o r
356
p r o d u c t i o n o f a m y d r i a t i c e f f e c t . The dose t h a t r e 1i eve symptoms o f hypermot i1 it y in human b e i ngs might n o t produce t h e s i d e e f f e c t s commonly expected when t h e r a p e u t i c a l l y e f f e c t i v e dose o f p o t e n t sympatholytic agents are employed ( 1 8 ) .
7.12
Antiparkinsonism
I n t r a v e n o u s methixene h y d r o c h l o r i d e has been administered t o 48 p a t i e n t s s u f f e r i n g from a v a r i e t y o f neurological and p s y c h i a t r i c disorders. The r e s u l t s o f such a d m i n i s t r a t i o n i n d i c a t e t h a t methixene i s capable o f a c t i v a t i n g d i a g n o s t i c ECG abnormalities i n p a t i e n t s p r e s e n t i n g w i t h e p i l e p s y o f temporal l o b e o r i g i n . No a c t i v a t i o n o f t h e ECG was d e t e c t e d i n a p a t i e n t s w i t h organic b u t non-epl leptogenic disorders o f t h e c e n t r a l nervous system and i n 10 p a t i e n t s w i t h chronic p s y c h i a t r i c disorders. A l l o f whom has never been s u b j e c t t o e p i l e p t i c s e i z u r e s . The o n l y s i g n i f i c a n t s i d e - e f f e c t observed f o l l o w i n g t h e intravenous i n j e c t i o n o f methixene hydrochloride was appearance o f a m i l d c o r t i c o s p f n a l hemiparesis l a s t i n g f o r a p e r i o d o f 30-60 minutes i n one o f 2 p a t i e n t s l a t e r found t o have a cerebral tumour, m i l d dryness o f mouth and tongue was r e p o r t e d i n almost a l l t h e p a t i e n t s . A c a s e - r e p o r t o f a p a t i e n t w i t h temporal lobe epilepsy o f l a t e r ones was b r i e f l y reported. The o n l y abnormality discovered i n t h s p a t i e n t was i n t h e ECG a f t e r t h e a d m i n i s t r a t i o n o f drug; a t autopsy a s m a l l a s t r o c y t o m a was f o u n d n the epsilateral amygdala (19).
ACKNOWLEDGMENT
The authors a r e h i g h l y t h a n k f u l t o M r . Tanvlr A. B u t t f o r t y p i n g t h e manuscript and M r . Osama Shabaan f o r drawing t h e s p e c t r a , b o t h from College o f Pharmacy, King-Saud U n i v e r s i t y , Riyadh, Saudi Arabia.
357
REFERENCES 1. "Remington's Pharmaceutical Sciences", (16th, ed. ) , Mack P u b l i s h i n g Co. Easton, Pennsylvania 18042 (19801, p. 859. "Martindale, The E x t r a Pharmacopoea" (29th' ed. ) , The Pharmaceutical Press, London (19891, p. 539. "The Merck Index", ( l l t h , ed.), Merck and Co., Rahway, N.J., No. 392-d, p. 539 (1989). Inc.,
2.
3.
4.
" C l a r k ' s I s o l a t i o n and I d e n t i f i c a t i o n o f Drugs" (2nd, e d . ) , The P h a r m a c e u t i c a l Press, London (19861, p. 151-752. "Index Nominum", Swiss Pharmaceutical Society. (19871, p. 695.
S.C. Chu S h i r l e y ; Acta Cryst. (1972).
5.
Zurich
6.
(Sec. B):
28, 3625-3633
7.
R.R. Abu-Shabaan, Pharmaceutics Department, College o f Pharmacy, King-Saud U n i v e r s i t y , Personal Communication (1992).
8.
9.
E . M . Abdel-Moety and N.A.
Khattab; Unpublished data.
M.I. Walash, M. R i z k and A . M . E l Brashy; 3 Pharmazeutische Weekblad ( S c i e n t i f i c E d i t i o n l o : t , 234-238 (1986).
10.
J. Schmutz; U.S. Patent 2,905,590, (assigned t o t h e Wander Company).
September 22,
1959,
11.
" P h a r m a c e u t i c a l M a n u f a c t u r i n g E n c y c l o p e d i a " , (M. S i t t i n g , Ed.), Noyes Data Corporation, Park Ridge N.J. (19791, p. 409.
S.M. Hassan, F. B e l a l , F. I b r a h i m and f . A . Talanta: 3 6 ( 5 ) , 557-560 (1989).
12.
Aly;
13.
F. B e l a l , M . I . Walash, F.A. A l y ; Microchem. J.: 295-299 (1988).
38(31,
14.
A.M.
El-Brashy;
Talanta: 37f111, 1087-1090 (1990).
358
15. 16. 17. 18. 19.
T. Daldrup, F. Susanto and P. M i l c h a l k e ; Fresenius 2 . Anal. Ce ; hm . 308: 413-424 (1981). B.B. Wheals; J. Chromatow.: 187, 65-85 (1980). R . C a v i e z e l , E. E i c h e n b e r g e r , F. Kunzle and J. Schmutz; Pharm. Acta H e l v . : 33, 447-452 (1958). W.M. Abruzzi; The Journal o f New Drugs: M a r c h - A w i l , 109-113 (1965). C.J. Vas, K . A . Exley and M.J. Parsonege; EDeleDsia: 252-259 ( 1967).
8,
SIMVASTATIN
Dean K . Ellison, William D. Moore, and Catherine R . Petts
Merck Sharp & Dohme Research Laboratories

West Point, PA 19486
359
Copyrtghl $ 1993 by Academic Press. InC 1 All rights of reproduction in any form reserved
360
DEAN K. ELLISON ET AL.
1.
History and Therapeutic Properties Description

2.1
2.
2.2 2.3
Nomenclature 2.1.1 Chemical Name 2.1.2 Generic Name (USAN) 2.1.3 Laboratory Codes 2.1.4 Trade Name 2.1.5 Trivial Nanies 2.1.6 Chemical Abstract Services (CAS) Structure, Formula and Molecular Weight Appearance
3.
Synthesis Physical Properties

4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11
4.
Infrared Spectrum Proton Nuclear Magnetic Resonance Spectrum Carbon- 13 Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum Mass Spectrum Optical Rotation Thermal Behavior Solubility Crystal Properties Dissociation Constants Partition Behavior
SIMVASTATIN
36 I
5.
Methods of Analysis
5.1 5.2
5.3
5.4 5.5
Elemental Analysis Chromatography 5.2.1 Thin Layer Chromalography 5.2.2 High Performance Liquid Chromatography G s Chromatographyhlass Spectrometry a Mid-Infrared Spectrometry Identification Tcsts
6.
Stability and Degradation

6.1 6.2
Solid State Stability Solution Stability
7.
Pharmacokinetics and Metabolism 7.1 7.2 Absorption and Distribution Metabolism
8. 9.
References
362
DEAN K. ELLISON ET AL
1.
History and Therapeutic Properties Simvastatin was synthesised (1) by Merck Sharp and Dohme Research Laboratories from lovastatin, which is produced commercially via a multistage fermentation process originating from cultures of a strain of Aspergillus ?errtius(2). The isolation. structural characterization and biochemical properties of lovastatin have been reported (2). Simvastatin is a prodrug. After absorption, it undergoes rapid enzymic hydrolysis of the lactone ring to form the principal metabolite. simvastatin (3-hydroxyacid.
lactone
P-hydroxyacid
The 0-hydroxyacid acts as a potent, reversible, competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase which catalyzes the conversion of hydroxymethyl glutarate to mevalonate. This conversion is an early and rate-limiting step in the biosynthesis of cholesterol. Simvastatin is used in the treatment of primary hypercholesterolemia.
SlMVASTATlN
363
2.
Description
2.1
Nomenclature
2.1.1
Chemical Name
Hexahydro-3,7-dimetyl-8-[2-(tetrahydro-4-hydroxy-6oxo-2H-pyran-2-yl)eibyl]- 1 -naphthalenyl 2.2dimethylbutanoate, or: Butanoic acid, 2.2-dimerhyl-1.2,3,7,8,8a,hexahydro-3,7diniethy1-8-[2-(tetrahydro-4-hydroxy-6-0x0-25 pyran-2yl)ethyl]- I-naphthalenyl ester, [ I 1cu.3a.7 P.8 0(2S*,4s*),841 I.
[IS-[I ~,3a.7P,8p(2S*,4S*),8cr931-1,2,3,7,8.8a-
1s-
2.1.2
Generic Name (USANJ Simvastatin
2.1.3
m Codes
L-644,128-OOOU (MK -0733)

2.1.4
Trade Names
ZOCOR, LIPEX, SINVACOR, ZOCORD, DENAN, LIPOVAS, SIVASTIN

2.1.5
Trivial Nmes Synvinolin
2.1.6
Chemical Abstracls Services (CAS) Registry Number: 70902-63-9
364
DEAN K. ELLISON ET AL
2.2
Structure, Formula and Molecular Weight Structure.:
Molecular Fomiula: Molecular Weight: 2.3 Appearance
C25H3805
41 8.57
Simvastatin is a white, crystalline powder.

3.
Synthesis Simvastatin is ii semi-synthetic derivative of lovastatin (3). Lovastatin is produced comniercially via a multi-stage femientation process which originates from cultures of a strain of Aspergillus terreus (2). The commercial synthetic route to produce simvastatin from lovastatin is shown in Scheme I. This synthesis features a very high conversion in the methylation step (>99.5%typically) and an overall yield of 86%. (1)
SlMVASTATlN
365
1Y
TBSCl a iinidazole
a) 2NNaOH
b) HCI
c) NH,OH
R=H R = CH,
R=H
R = CH,
Scheme I Synthesis of Simvastatin from Lovastatin
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
1267
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 Lactone -C-0-C stretch Ester -C-0-C stretch Secondary alcohol C - 0 stretch Trisubstituted olefinic C-H wag Cis olefinic C-H wag
1225 1166 1056 870 670
0 0
0 0
a3
0
0
cu
co
0 0 0
cu
cu
0
0 a3
cu
0
0
(3
cu
0 0
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 Assignments ()
5.98 5.11 5.50 5.35 4.62 4.36 2.92 2.71-2.57 2.50-2.23 2.01-1.2 1.11. 1.1:. 1.08 0.88 0.82
Notes:
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 overlapping in overlapping ins
s
C5H
C6H
c, ,H c4 +H
C4H ClH
c4#OH c, 8,
C3H. C7H C&. c, .H,. C &
$2,
3 11
6 3 3 3
c8%
clO%
d. J=1.4 Hz d, J=7.0 Hz t, J=6.8 Hz
C2(C!!33)* C,-CH3 C7-% CVH3
(1) (2)
Multiplicity: s=singlet; d=doublet; m=multiplet; t=triplet. Assignments made in reference t numbered structure of o simvastatin shown above.
- 0
r 4
..
370
DEAN K.ELLISON ET AL.
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
c 7 c 3
- 0
..
0 - 0
c\I
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
0
(D ( u
3
(u
m
(u
v)
( u
d
(\I
I n
I n N N
N
7 7-
1
0
0 N N
cq
(9 0
7-
f! \
8
a3ueqJosqv
a J ,-I
d
0 0.
d
d
0 0
! ! n n m
l ' " " ' " ' l ' "'""I

.' .
I '
"I""'"
m
gE
i2
0 0
a 2
0
(D
0
N
, -
SIMVASTATIN
375
m/z 400
-H20
M+ = 418
'
'-120
mlz284
-'C3H5
mlz 199
/ J
mlz 240
I
t m/z 173
\
m/z 159
Figure 6:
Proposed Fragnentation Pattern to Explain the Mass Spectrum of Simvastatin.
376
DEAN K . ELLISON ET AL.
50.
100.
150.
'C
Figure 7:
DSC Thermogram for Simvastatin under Nitrogen.
I.
0
a
0
4
I n
4-
d7
Figure 8:
DSC Thermogram for Simvastatin in Air.
SIMVASTATIN
377
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
0.50
0.40
0.30
0.20 0.1 0
0.0
5.0
10.0
15.0
20.0
Degrees 20
25.0
30.0
35.0
40.0
Figure 9: Powder X-Ray Diffraction Pattern of Simvastatin.
SIMVASTATIN
379
X-Ray Powder Diffraction Pattern Data Of Simvastatin
Peak
Angle
@@
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
VImax
(%)
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
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
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
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): Calculatcd Carbon Hydrogen
71.74 9.15
Found
71.79 8.88
5.2
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).
SlMVASTATlN
38 I
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
Drug Substance Purity
System No. Column
Mobile Phase
nin Detection 238
Rf e
Spherisorb ODS
Isocratic and Gradient A = acelonitrile R = 0.025M NaH2 PO, Isocratic 50:50 A = acelonitrile
Drug Substance Potency
I1
Perkin-Elmer C-18CR
238
B = 0.1% (V/V %)
H3P0, aqueous
11 1
Measurement of Simvastatin and metabolites in plasma Measurement of low levels in fermentation broth
G adie n t I Jones Chromatography A = 0.025M sodium acetate Apex 1. C I l 8 B = ncetonitrile

PRP
Gradient A = aqueous ammonium phosphate pH = 6.1 B = acetonitrile
A = acetonitrile
238
IV
260
Measurement in tablets
Hyprsil ODS
238
B = water (0.025M NaH2P0, pH = 4.5) 60:40 A:B

A = 0.025M NaH2P0,
Measurement in tablets
VI
Hypersil 5 micron ODS
230
pH=4 B = CH3CN C = MeOH 33:55:12 A:R:C
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 10). No degradation products heat conduction calorimetry (4, other than those generated by oxidative processes have been found. Degradation is prevented by storage in an inert atmosphere.
SIMVASTATIN
383
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).
384
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 a drug w s 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
385
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.
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
3x7
9.
References
1.
D. Askin, T.R. Verhoeven, T. M.-H. Liu and I. Shinkai, J . 0rg.Cheni. X , j 4929 (1991). 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). 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). Merck Sharp & Dohme Research Laboratories, Rahway, NJ., unpublished data. R. Reamer, Merck Sharp & Dolune Research Laboratories. personal communication. A.I. Scott Interpretation of the Ultrcr\)iolet Spectra of Natural Products, Pergamon Press, Oxford, 1964. D. Zink, Merck Sharp & Dohme Research Laboratories. personal communication.
2.
3.
4.
5.
6.
7.
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.
388
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. - (9), 577 (1990). 19
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. l, (4) 476-483 (1990). J 22. N. Uchiyama, Y. Kagami, Y. Saitoh. M. Ohtawa. Chent. Pharm. Bull. 3, ) 236-8 (1991). (1 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
389
390
VIJAY K. KAPOOR
1.
D e sc ription
1.1 1.2
Name, Formula and Molecular Weight Appearance, Color and Odor
2.
Physical P rop erties

2. 1 2.2 203
204
2^;5 2a 6 2 07 2.8 2.9
2.10 2.11
3.
4.
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
Synthesis S t a b i l i t y and Degradation Pharmacokinetics and Metabolism P r o t e i n Binding
5.
60
SULFATHIAZOLE
39 I
Bloavailabll i t y
8.
9.
Toxicity 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
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
10.
References
392
VIJAY K. KAPOOR
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 p H 7.5 i s 2 3 5 - a t 3. 7 ' 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 72 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
2.2
D i s s o c l a t i o n Constant pka 7.1 (25O)l
2 . 3
Partition Coefficient
Log P (octanol/pH 7.5),

2 04
Optical A c t i v i t y
- 0.4'
a c ti v i t y
2.5
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
394
VIJAY K. KAPOOR
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 03 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%
-A
10
15
20
25
30
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 )~
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
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 38 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 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 <I 3 7
I I1
d
IV
8.21 7.76 5.94 5.82 5 076 5 047 4.82 4.61 4 043 4.14 4.11 4.00 3.82 3 076 3 065 3.60 3.56 30 5 4 30 4 6 3 042 3.38 3 032 3.27 3.02 2.97 2.94 2.88 2 082 2.76 2.74 2.69 2.6 1
I
3 2 17 24 16 14 16 17 31 26 100 7 4 8 2 1 1 48
41
2 10.39 9.96 41 8.29 9 8.04 2 7.59 9 7.36 13 7.26 4 1 6.71 14 6.50 4 1 6.12 1 5.93 4 1 5.88 < I 5.81 4 1 2 5.73 5.52 100 5.45 5 5.19 3 5.10 t i 4.99 4 1 4.91 12 4.81 (1 4.78 <1 4.66 31 4.63 3 4.34 72 4.31 4 17 4.20 4.09 22 4.03 4 1 3.81 4 2 3 3.68 3.63 41 3.58 3 3.48 14 3.37 7 3.33 (1 3.32 (1 3.29 15 3.26 ( 1
16.2 4 7.78 3 6.86 ( 1 6.25 1 6.08 1 5.88 15 5.77 50 7 5.45 5.39 1 4.79 15 4.60 16 4.43 16 4.37 11 4.32 4 4.29 2 11 4.18 4.10 21 4.05 100 3.99 3 3.88 4 1 3.86 3.83 41 3.79 <I 3.75 10 6 3050 3051 52 3.50 3 3.30 2 3.31 10 3.27 1 3.24 1 3.18 2 5 3.13 3.11 1 2.94 1 2.89 41 2.88 4 2.79 2 2.75 4
2 41
1 1 2 6 2 4 7 5 5 2
396
VI JAY K. KAPOOR
Table I (continued)
Form
I
d
2.58
I1
I11
IV
I
2
I
41
d
2.74 2.73
I
7 2 (1
3.22 3.19
3 000
2.92
2.87
28 .3 2.82
(1 3 6 5
il (1
4 1
2.71 2.70 2.68 2.60 2.57
(1 2
2.78
2-71 2-70 2.68 2.60 2.54
41
tl
2 1
It has been reported*' t h a t crystal s t r u c t u r e s of forms 1,111 and I V a l l belong t o Forms I and t h e monoclinic space group PZi/c. 111 contain molecules each 5 t h e asymmetric u n i t , whlle form LV c o n t a i n s only one, The molecular conformations of a l l three forms i s e s s e n t i a l l y t h e same, The structures d i f f e r only w i t h r e s p e c t t o molecular p a c k l q , The packing i n form I shows very l i t t l e resemblance t o t h a t I n forms 111 and IV. The packlngs i n forms 111 and I V is, however, remarkably s i m i l a r . The r s m d e v i a t i o n s b e t w e n t h e molecular packings of form I and 111, I and I V , and 111 and I V , have been found t o be 4.66, 4.34, and 2.19 A O , respectively.21
In another study22 a high resolution e l e c t r o n microscopy has been c a r r i e d out on samples of s u l f a t h i a z o l e o b t a i n e d by r e c r y s t a l l i z a t i o n a t 0 , 30 and 7 0 . Low magnification e l e c t r o n microscopy s t u d y of t h e c r y s t a l s showed f e a t u r e l e s s morphology, y e t t h e resolved l a t t i c e images showed imperf e c t i o n s such as dislocations , l a t t i c e I r r e g u l a r i t i e s and regions of discontinuity
SULFATHIAZOLE
391
2.6
M e l t i n g Range
The m e l t i n g r a n g e of s u l f a t h i a z o l e U.S.p?3 is between 200 and 204O. The thermal b e h a v i o u r of polymorphs of s u l f a t h i a z o l e has been described18921 ; form X melts a t 201, If a t 196O, and X I X a t 173O. Form 111 can a l s o t r a n s f o r m (and not show any m e l t i n g ) t o forms I and 11, b u t a t low h e a t i n g r a t e s in t h e t e m p e r a t u r e range 150-170. Form IV undergoes sol id-sol i d t r a n s f o r m a t i o n t o form I a t ar0und150~. 2.7 I n f r a r e d Spectrum
The infrared spectrum ( n u j o l mull) ( F i g u r e 2) of s u l f a t h i a z o l e e x h i b i t s p r i n c i p a l peaks a t wavenunbers 1130, 1527, 929, 1274, 1082 and 15921. The i n f r a r e d s p e c t r a of t h e forms I, X 1 If , and IV have been d e s c r i b e d . 19921 I
Wavelength
5
6 7
10
11
12
13
14
15
0
CQ
a ,
5 .-
5 c
c!
I-
2000
1500
1200
1000
900
800
700
Figure 2
I n f r a r e d spectrum of s u l f a t h i a z o l e
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VI JAY K. KAPOOR
2.8
U l t r a v i o l e t Spectrum
Aqueous a c i d , 280 NIL (A1 = 498); aqueous a l k a l i , 256 nm (A: = 716)1!

2 9 ;
13C-Nuclear Magnetic Resonance Spectrum
T h e 13C-nuclear magnetic resonance (WR) spectrum of s u l f a t h i a z o l e has been determined in d e u t e r i o d i m e t h y l sulfo x i d e a t 25.15 MHz employing t h e p u l s e Fourier t r a n s f o r m technique.24 The spectral assignments are shown i n Table XI.
Table I1
1 3 G ~ Chemical S h i f t s of S u l f a t h i a z o l e
2 3
Carbon
Chemical S h i f t , ppm
152 a
4
128 .o 128 .o
112.0
168 2
124.4 107.4
The s o l i d - s t a t e 1 3 C = M s p e c t r a of t h e forms I s XI, 111, and IV of s u l f a t h l a m l e have been determined using t h e crossp o l a r i z a t i o n - m a g i c angle splnnicq t e c h n i q u e on an instrument o p e r a t i n g a t 4.7 T (1% resonance 5 0 MHz)21. The chemical s h i f t s of t h e carbon atoms are tabulated i n Table 111.
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Table I11 13C N N C h e m i c a l S h i f t s (ppm) of h Carbon Atoms i n Polymorphs I , 11, 111, and I V of S u l f a t h i a zole
~~
~-
-~
~~~
Carbon Atom Form I

1 24 15204 115.9 114.3 132.3 130 04 127.3,126.0* 170.2 123.6 109.4,107.9*
Form I1 Form I11 Form I V

154.0 115.1 114.0 129.5 127.8 168.7 123.4 108.1 15 1e 2 120.7 118 06 130.0 127.6 134 .7 169 . 7 125.5 108.8 151.2 120.4 118.9 130.8 127.4 134.0 169.7 126.2 106 08
3,5
4 7
* T w nonequivalent molecules i n t h e asymmetric u n i t

2.10
Raman Spectra
The Raman s p e c t r a of t h e v a r i o u s forms of s u l f a t h i a z o l e have been measured21 on a Spex t r i p 1 8 monochromator s p e c t r o p h o t o m e t e r u s i n g a k r y p t o n i o n l a s e r a t a wavelength of 647 nm. The s p e c t r a are shown i n Figure 3 and t h e frequency s h i f t s are g i v e n in T a b l e IV.51
2.11
Mass Spectrum
The mass spectrum of s u l f a t h i a z o l e e x h i b i t s p r i n c i p a l peaks a t m/z 92,156,65,108, 191,45,39, and 551.
3 .
Synthesis
S u l f a t h i a z o l e has been p r e p a r e d 25-29 30 by reacting a c e t y l s u l f a n i l y l 1 with 2-aminothiazole ( 2 ) ; t h e r e s u l t i n g N a - a c e t y l s u l f a t h i a z o l e (3) is d e a c e t y l a t z d by h y d r o l y s i s t o g i v e s u l f a t h i a z o l e .
400
VIJAY K. KAPOOR
100
loso
A Wavenumber (cm-')
2000
F i g u r e 3- Raman spectra of polymorphs I, 11, 111, and IV of sulf a t h i a z o l e .
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T a b l e IV Raman Shifts (ano1) f o r Polymorphs I, 11,111, and IV of S u l f a t h i a z o l e Form
I
150 18 1 205 05 264 287.5 308.5 32 1 342.5 390.5 406 45 1 505.5 549.5 568.5 629 633.5 652 664 5 683 05 736 05 82 1 834.5 862.5 924.5 1068.5 1074.5 1086 05 1116 1128.5 1137 1184.5 1255.5 1320 1425 1502 1529.5 1596 1624
11
114 152 175 201 05 250 5 291 05 298 312 348 401.5
III
116 133.5 10 1 213 265 299 05 346.5 375 05 394 4 16
IV
117.5 133 137.5 155 169 186 212.5 232 05 268 300 0 5 349 377 394 399 415.5 447.5 505 05 528 566 05 634 645 684 70 1 729 798 821 841 05 850 5 878 1073 1092.5 1128 1176.5 1254.5 1262.5 1324 1528.5 1595
446
44 4
506 05 560 633.5 639 646 685 5 735 8 18 829 8 37 936.5 1065.5 1087.5 1123 1178 1239.5 1247 1324 1502.5 1533 1562.5 1592.5 1644
504.5 550.5 567.5 611.5 629 05 634.5 646 05 682 733 808 8 22 828 839.5 928 939 954 966 1072.5 109105 1132 1177.5 1254.5 1265.5 1293 05 1322.5 1330 1530 1570 1596
402
V1 JAY K. KAPOOR
(3)
Sulfa thiozole
Scheme I- S yn th e s i s of Sulf a t h i a zo l e
An alte r n ate route of s y n t h e s i s (Scheme 11) i s through condensation o f N4-acetylsulf a n i l y l thiourea ( 4 ) with 1,2=dichloroethyl methyl ketone ( 5 ) followed by hydrolysis ,31033
CH,CNH a S O z N H C ,NHz
'
4s
+ CH,COCHCICH,CI
Sulfo t h i or o l e
Scheme 11- Alternate Synthetic Route t o Sulf a t h i a z o l e
SULFATHIAZOLE
403
S u l f a t h i a z o l e has a l s o been p r e p a r e d = by t r e a t i n g carbomethox s u l f a n i l y l c h l o r i d e w l t h calcium d e r i v a t i v e o cyanamide t o g i v e carbometho xysul f a n ily l cyanamide, which was c o n v e r t e d t o carbomethoxysulfanilylthiourea by t r e a t i r g w i t h sodium t h i o s u l f a t e . Condensation of c arbometho xysul f a n i l y t h i o u r e a w i t h chl or0 a c e t a l d e h y d e followed by t r e a t m e n t w i t h sodium hydroxide g a v e s u l f a t h i a z o l e .
R e c e n t l y , a method for t h e r o d u c t i o n of b i s ( a c et y l aminobenzenesul f onylT-2-amino t h i a z o l e ( a n %&mediate i n t h e s y n t h e s i s of s u l f a t h i a z o l e v i a a m n o l y s Is ) by s u l f onyl a t i o n o f 2-aminol c i u m c a r b o n a t e as a base has t h i a z o l e using A method f o r t h e p u r i f i c a t i o n been d e s c r i b e d . f t of s u l f a t h i zole v i a i t s sodium s a l t h a s a l s o 6 been g i v e n , 3
4.
S t a b i l i t y and Degradation
S u l f a t h i a z o l e is t o be s t o r e d i n w e l l c l o s e d , l i g h t resistant c o n t a i n e r s . 2 3 It undergoes h y d r o l y s i s in 1M h y d r o c h l o r i c a c i d a t e l e v a t e d temperature. H y a r o l y s i s p r o d u c t s 2-aminothiazole and s u l f a n i l l c ac i d have been i d e n t i f l e d by high performance l i q u i d chromatography u s i n g P r o z o c l l 5 , MicroPak CN-10,and MicroPak CH-10 as t h e columns.37-39 Photod e g r a d a t i v e s t u d i e s on s u l f a t h i a z o l e have a l s o been done.40941 Thermal b e h a v i o r decomposition pathway and thermal s t a b i l i t y have also been evaluated.42
S u l f a t h i a z o l e sodium ( s o l u b l e s u l f a t h i a m l e ) s l o w l y d a r k e n s on exposure t o l i g h t ; on exposure t o moist aFr it absorbs carbon d i o x i d e and becomes Recently, the i n c o m p l e t e l y s o l u b l e in water. e f f e c t of u r i c a c i d as a p h o t o p r o t e c t i v e agent f o r t h e b u f f e r e d and unbuffered s o l u t i o n s of s u l f a t h i a z o l e sodium has been i n v e s t i g a t e d . 4 3 Uric a c f d solution i n g l y c e r i n enhanced t h e photostability of s u l f a t h i a z o l e sodium. The h i g h e r the c o n c e n t r a t i o n of u r i c a c i d u s e d , t h e g r e a t e r was i t s p h o t o p r o t e c t i v e a c t i o n w i t h i n the c o n c e n t r a t i o n range s t u d i e d . Uric a c i d a l s o demonstrated i t s p h o t o p r o t e c t i v e e f f e c t i n t h e
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VI JAY K. KAPOOR
presence of sodium s u l f i t e or ethylenediaminetetraacetic acid.

5
Pharmacokinetics and Metabolism Pharmacokinetics of s u l f a t h i a z o l e has been
s tud i e d e x t e n s ivel y .44-59 D is pos it ion of s u l f a t h i a z o l e sodium i n plasma and u r i n e of swine was determined by f o l l o w i n g s i n g l e intravenous and oral d o s e s .48 Pharmacokinetics of t h e drug was d e s c r i b e d by a one-compartment open model. The drug was r a p i d l y eliminated, mainly by renal e x c r e t i o n of unchanged s u l f a t h i a z o l e and metabolism t o a c e t y l s u l f a t h i a z o l e w i t h a b i o l o g i c a l h a l f - l i f e of 1.4 hr. The drug g e t s r a p i d l y absorbed a f t e r oral administration. I n another study49 plasma, u r i n e and t i s s u e concentrations of sodium s u l f a t h i a o l e were determined a t v a r i o u s times a f t e r Intravenous a d m i n i s t r a t i o n t o c a t t l e . The average plasma and u r i n e data were c o n s i s t e n t w i t h a two-compartment pharmacokinetic model w i t h a h a l f - l i f e of e l i m i n a t i o n of 1.3 hr, and a t o t a l volume of
d i s t r i b u t i o n o f 0.4 L/kg body weight. Pharmac o k i n e t i c s t u d i e s done on cows showed t h a t t h e drug is a l s o excreted t h r o u g h the ruminal w a l l The e f f e c t of concomitant and s a l i v a r y glands.50 a d m i n i s t r a t i o n of sodium s a l i c y l a t e on t h e pharmacokinetics of s u l f a t h i a z o l i n r a t s has been studied.51 A r e c e n t s t u d y has been made t o determine t h e e f f e c t o f Haemonchus c o n t o r t u s p a r a s i t i c i n f e c t i o n I n lambs on t h e c l e a r a n c e of intravenous a d m i n i s t r a t i o n o f sulf a t h i a a l e . The c l e a r a n c e of t h e d r u g from t h e plasma of lambs was u n a f f e c t e d by t h e i n f e c t i o n . The pharmac o k i n e t i c s of sulf a t h i a z o l e when s t u d i e d i n r a t s w i t h p y e l o n e p h r i t i s showed t h a t t h e e l i m i n a t i o n of t h e drug was slower i n i n f e c t e d animals t h a n i n c o n t r o l r 5 6 T h e plasma h a l f - l i f e of s u l f a t h i a z o l e has been mentioned about 4 hrs1. A study done on h e a l t h y v o l u n t e e r s showed t h a t t h e amount of b u c c a l a b s o r p t i o n of s u l f a t h i a z o l e was higher i n t h o s e who t o o k capsaicin.58
51
S t u d i e s o n t h e metabolism of s u l f a t h i a z o l e have shown t h a t b e s i d e s a c e t y l s u l f a t h i a z o l e , s u l f athiazole-"&ulf o n a t e and s u l f athiazole-N4-
SULFATHIAZOLE
405
g l u c u r o n i d e are t h e o t h e r m e t a b o l i t e s of t h e drug.60-62 A s t u d y o n c o r r e l a t i o n between renal e x c r e t i o n and b i o t r a n s f o r m a t i o n of s u l f a t h i a z o l e has shown t h a t t h e a c e t y l a t e d m e t a b o l i t e had a lower c l e a r a n c e r a t i o t h a n s u l f a t h i a z o l e . 6 3 E f f e c t of n i t r i t e o n t h e metabolism of 14cs u l f a t h i a z o l e i n r a t s has been studied.64 I t was shown t h a t c o n v e r s i o n of 14C-sulf a t h i a z o l e to 14C-desaminosulf a t h i a z o l e was g r e a t 1 increased by n i t r i t e i n meals. Another s t u d y 6 r e v e a l e d t h a t t h e 1e v e l s of s u l f a t h i a z o l e and i t s a c e t y l a t e d m e t a b o l i t e I n t h e u r i n e of p a t i e n t s were much lower when t h e drug was administered r e c t a l l y t h a n when it was given orally or i n t r a d u o d e n a l l y .
60
Protein B i n d 1 9
P r o t e i n b i n d i n g s t u d i e s of s u l f a t h i a z o l e have been carried out.66-71 S u l f a t h l a z o l e showed 83 per c e n t of b i n d i n g t o serum protein.66 Bindirg of s u l f a t h l a z o l e t o bovine serum albumin a t pH 7.4 and 3 7 O was s t u d i e d 6 7 u s i n g e q u i l i b r i u m d i a l y s i s ; t h e d a t a r e p o r t e d is: Log P, 0.05; Log l& 4.01 ( o b s d ) , 2.62 ( c a l c d ) ; r n l l l ~ g r a madsorbed p e r gram of p r o t e i n a t c = 1 mg %, 0.93; p e r c e n t of s u l f a t h i a z o l e sound i n 4% albumin s o l u t i o n a t a free drug c o n c e n t r a t i o n of 1 mg %, 79. F u j l t a 6 8 analyzed t h e a d s o r p t i o n c o n s t a n t s of v a r i o u s sulfonamides i n c l u d i n g s u l f a t h i a z o l e w i t h bovine serum albumin a t v a r i o u s pH p o i n t s w i t h free energy re1a t e d phys l c ochemical parameters t a k i n g i n t o account t h e c o r r e c t i o n for i o n i z a t i o n o f t h e drugs. I t was p o s t u l a t e d t h a t t h e a d s o r p t i o n e q u i l lbrium i s determined by the hydrophobicity of drugs and occurs through t h e b i n d i n g o f t h e n e u t r a l drug molecule w i t h t h e hydrophobic f r a c t i o n of t h e p r o t e i n s u r f a c e , t h e v a r i a t i o n of which is dependent on the s t a t e of t h e d i s s o c i a t i o n e q u i l i b r i u m of b a s i c groups o n t h e bovine serum albumin.
T h e r e l a t i o n s h i p between single high-aff l n i t y b i n d i n g sites f o r s u l f a t h i a z o l e and t h e b i r d i n g r e g i o n s of human serum albumin have been examined by a series of experiments.69 C i r c u l a r d i c h r o i c i n v e s t i g a t i o n i n t o b i n d i n g of sulfonamide t o serum albumin has been done.70 B i n d i r g of
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VIJAY K. KAPOOR
s u l f a t h i a z o l e t o lysozyme has a l s o been studied,?l and fluorescence quenching h a s I n d i c a t e d t h a t s u l f a t h i a z o l e binding t o lysozyme occurred as a 1 :1 i n t e r a c t i o n . S u l f a t h i a z o l e has t h e a f f i n i t y c o n s t a n t of 21,000 M-lS
B i n d i n s t u d i e s of s u l f a t h i a z o l e i n r a t liver c e l l s 2 and t o h o r s e l i v e r alcohol dehydrogenase73 have been c a r r i e d o u t . A study74 on b i n d i n g with b i l i r u b i n has Indicated t h a t t h e sulfonarnlde molecule is bound I n d i r e c t contact with t h e bilhwbin.
7.
Bioavaflability
E f f e c t of some polymeric materials used I n suspension on in vivo absor t i o n of s u l f a t h i a z o l e i n a d u l t s has b e e x u d i e d . 5 The highest blood l e v e l of s u l f a t h i a z o l e w a s achieved w i t h suspensions obtained from h i g h v i s c o s i t y mucllages such as gum tragacanth, showing increased b i o a v a i l a b i l i t y of t h e p r e p a r a t i o n . I n another study76 f l o c c u l a t e d and deflocculated suspensions of s u l f a t h i a z o l e were a d m i n i s t e r e d t o healthy human volunteers. B i e a v a l l a b ~ l i t yfrom t h e s e t w o types of suspensions was s t u d i e d from u r i n a r y free drug excretion. B i o a v a l l a b i l i t y was found t o be s i g n i f i c a n t l y lower from f l o c c u l a t e d suspension.
8.
Toxicity
Non-neoplastic and n e o p l a s t i c changes In kidneys and other organs of r o d e n t s f e d for a l o q t i m e l e a d a c e t a t e and s u l f a t h i a z o l e were studied.77 It produced changes mainly i n t h e pelvis and medullary p o r t i o n s of kidneys, The LD50 S.C. i n mice of s u l f a t h i a z o l e are r e p o r t e d as: 1.45 g/kg and 1.95 g/kg3.
9.
Methods of Analysis
9.1
The elemental composition3 of
SULFATHIAZOLE
407
s u l f a t h i a z o l e is as f 011m : s Element
C H
0
S
Per c e n t
42.34
3.55 16 0 4 6 12.53
25.12
9.2
I d e n t i f l c a t i o n Color Tests
Sulf a t h i a z o l e g i v e s a greyish-purple p r e c i p i t a t e when 10 mg of it d i s s o l v e d i n a m i x t u r e of 10 ml of water and 2 m l of 0.1H sodium hydroxide are t r e a t e d w i t h 0.5 ml of copper s u l f a t e s o l u t l o n . 2 An i d e n t i f i c a t i o n t e s t based on t h e primary aromatic amino group p r e s e n t i n s u l f a t h i a z o l e I s as follows: an organge-red p r e c i p i t a t e is produced when 100 mg of it d i s s o l v e d I n 2 r d of 2M h y d r o c h l o r i c a c i d are t r e a t e d w i t h 02 ml of-sodium n i t r i t e s o l u t i o n . followed a f t e r one t o two minutes by t h e a d d i t i o n of 1 m l of p-naphthol s o l u t i o n . 2 A c o l o r r e a c t i o n of sodium 2,4,6-trinitrobenzenesulfonate with t h e amino g r o u p h a s been suggested a s t h e o t h e r i d e n t i f i c a t i o n t e s t f o r s u l f athiazole.78 A dark-brown p r e c i p i t a t e w i t h v i o l e t t i n g e is obtained when s u l f a t h i a z o l e d i s s o l v e d i n d i l u t e sulfuric a c i d is t r e a t e d w i t h 0.2N i o d i n e solution.79 A s e n s i t i v e and simpTe procedure f o r d e t e c t i o n of sulfonamide drugs on a s o l i d phase of s t r o n g l y a c i d i c cation-exchange r e s i n Donex HCR h a s been described.80 The procedure is based on t h e a d s o r p t i o n of t h e c o l o r e d s c h i f f ' s base formed by t h e r e a c t i o n of sulfonamide w i t h dimethylaminobenzaldehyde. T h e d e t e c t i o n l i m t is w i t h i n 20-40 ng of a n a l y t e . O t h e r i d e n t i f i c a t i o n c o l o r t e s t s have been described. 1
9.3
T i t r i m e t r l c Analysis
Nitrite t i t r a t i o n i s t h e method of choice t o a s s a y s u l f a t h i a z o l e . 2 3 The method involved is as f o l l o w s . Weigh a c c u r a t e l y about 500 mg and t r a n s f e r t o a s u i t a b l e open vessel.
408
VIJAY K. KAPOOR
Add 20 ml of h y d r o c h l o r i c a c i d and 50 ml of water, s t i r u n t l l d l s s o l v e d . Cool t o about 15O, and s l o w l y t i t r a t e with 0.W sodium n i t r i t e s o l u t i o n t h a t h a s been p r e v i o u s l y s t a n d a r d i z e d a g a i n s t USP sulfanilamide. Determine t h e end-point e l ec t r o m e t r i c a l l y u s i r g s u i t a b l e e l e c t r o d e s (platinurrccalomel o r platinum-platinum). P l a c e t h e b u r e t t e t i p below t h e surface of t h e s o l u t i o n t o e l i m i n a t e a i r - o x i d a t i o n of t h e sodium n i t r i t e , and s t h t h e s o l u t i o n g e n t l y , using a magnetic stirrer, w l t h o u t p u l l l n g a v o r t e x of a i r under t h e s u r f a c e , m a i n t a i n i n g t h e temperature a t a b o u t 15O.When t h e t i t r a t i o n is w i t h i n 1 ml of t h e end-mint add t h e t i t r a n t i n 0.1 cnl p o r t i o n s . Each m l of 0;lM sodium n i t r i t e is e q u i v d e n t t o 25.53 of C9H9N302S2.
S e v e r a l o t h e r titrirnetric methods f o r t h e a n a l y s i s of s u l f a t h i a z o l e have been described.81-9 1 A t itrimetric d e t e r m i n a t i o n with 0.lF sodium hydroxide i n t h e presence of hexadecylpyridinium c h l o r i d e is described.8 Use of E-bromsuccinimide as a t i t r a n t i n d e t e r m i n i n g s u l f a t h i a z o l e i n mixtures c o n t a i n i n g o t h e r o r g a n i c compounds u s i n g potassium i o d i d e and s t a r c h or methyl r e d as i n d i c a t o r h a s been described.82 &Brornophthal imide i n p l a c e of N-bromosuccinlmide has a l s o been used.83 Methods csing o-iodosobenzoat& as a n o x i d i m e t r i c t Itran or 0.00W chloramine T87 a f t e r bromination have been descri6ed. Bromine reagent90 h a s been u s e d f o r t h e d e t e r m i n a t i o n of sulfa drugs i n bulk and i n dosage forms by an i n d i r e c t t i t r i m e t r i c method based on the r e a c t i o n of t h e sulfa drug w i t h a n excess of s t a n d a r d bromine water t o form t h e correspondirg N-bromo d e r i v a t i v e , which r e l e a s e s a n e q u i v a e n t amount of i o d i n e when treated with iodide. The released i o d i n e could b e determined with sodium t h i o s u l p h a t e . P o t e n t iometr ic d e t e r m i n a t i o n s using s i l v e r s u l p h i d e 9 2 8 3 o r I o n - p a i r complex of t h e s u l f a drug w i t h q u a t e r n a r y phosphonium and q u a t e r n a r y arsonium94 as e l e c t r o d e s have been suggested. O s c i l l o p o t e n t i o m e t r i c t i t r a t i o n s w i t h b i m e t a l l i c electrades u s i n g l i t h i u m methoxide as t h e t i t r a n t has been reported.95
SULFATH IAZOLE
409
9.4
Bioassay
Several m i c r o b i o l o g i c a l methods f o r t h e d e t e r m i n a t i o n of sulf a t h i a z o l e I n animal t i s s u e s and m i l k have been reported.96-100 I n o m of t h e methods sulf a t h i a z o l e r e s i d u e s i n m i l k were detected by t h e a s s a y based on a c o m p e t i t i o n between u n l a b e l e d drug ( i n t h e sample) and l a b e l e d c o ound f o r b i n d i r g s i t e s on b a c t e r i a l cell walls% L i m i t of d e t e c t i o n for s u l f a t h i a z o l e was 0.002 p g / d . Recently, a simple enzyme ( p e r o x i d a s e ) imnunoassay has been developed t o screen honey samples f o r s u l f a t h i a z o l e a d u l t e r a t i o n . 1 0 1 The t e c h n i q u e was able t o d e t e c t 0.3 ppm l e v e l of s u l f a t h i a z o l e i n honey.
9.5
Polarographlc A n a l y s i s
P o l a r r a h i c s t u d i e s on s u l f a t h i a z o l e A h i g h l y sensitive have been done.lZ-lg6 d i f f e r e n t i a l p u l s e p o l a r o g r a p h i c method h a s been developed for the d e t e r m i n a t i o n of t r a c e l e v e l s o f s u l f a t h i a z o l e a f t e r d i a z o t i z a t i o n and coupl ing w i t h N-( l-naphthyl )ethylenediamine dihydroc h l oryde 106
9.6
Adsorptive S t r i p p i n q Voltammetry
The appl i c a t i o n of a d s o r p t i v e s t r i p p i n g voltammetry t o t h e d e t e r m i n a t i o n of s u l f a t h i a z o l e a f t e r i t s d i a z o t i z a t i o n and coupl irg w i t h 1-naphthol t o form a n azo dye has been d e s c r i b e d .107
9.7
Thermal A n a l y s i s
A method s u i t a b l e for t h e d e t e r m i n a t i o n of 30-100 ang of s u l f a t h i a z o l e by means of c h a r a c t e r i s t i c endothermic d i f f e r e n t i a l thermal a n a l y s i s peaks for which t h e area changes l i n e a r l y with t h e amourrt has been described.108
9.8
Atomic Absorption S p e c t r o s c o p y
S u l f a t h i a z o l e has been determined by atomic a b s o r p t i o n spectroscopy a s i t s Co++ c h e l a t e . 1 0 9 Aliquot of t h e drug s o l u t i o n was
410
VIJAY K. KAPOOR
t r e a t e d n i t h c o b a l t s u l f a t e , t h e formed p r e c i p i t a t e s e p a r a t e d and t h e amount of t h e metal was determined i n t h e p r e c i p i t a t e . The percentage r e c o v e r y was 99.5-100.6.
9.9
Spectrophotornetric Analysis 9.9.1 Colorimetric
A number of c o l o r i m e t r i c methods for t h e d e t e r m i n a t i o n o f s u l f a t h i a z o l e have been r e p o r t e d . 110-121 Various reagents employed for c o l o r i m e t r i c d e t e r m i n a t i o n were 9-chloroacridine, 110 -amino-1-hydrox naphthalene3 , 6 - d i s u l f o n i c a c i d , 1% N- 1-naphthylJethylene~ diamine, 112 chloramine l'T1 3 8 - h y d r o ~ y q u i n o l i n e , l4 syringaldehyde, 115 3-ac43-dicarboxyethylrhodanine~ 16 1,2=naphthoquinone4-~ulfonate1~8,3-methylbenzothiazolln-2-one hydrazone, 119 4-N-methylamino121 phenol, 120 and phenothazlne and n-bromosuccinimide : A new r a p i d , simple, s e l e c t i v e and s e n s i t i v e method f o r d e t e r m i n a t i o n of s u l f a t h i a m l e is based on t h e format on of a yellow complex with M T h e drug complex shows Pd(I1) i n 1 Na0H.l maximum abs5rbance a t 410 nm w i t a a b s o r p t i v i t y of 1.9 x 103 1 mol-9 c J f a r
9.9.2
Fluorometric
n s i t i v e f l u o r o m e t r i c method has been developed. The procedure r e q u i r e s d i a z o t i z a t i o n of t h e amino group followed by coupling w i t h 2,4,6-triaminopyrIdine, and t h e r e s u l t i n g azo compound is o x i d i s e d t o a f l u o r e s c e n t tr i a z o l e
9.9.3
Ultraviolet
U 1 tr a v i o l e t spectropho tometr has been used i n t h e a n a l y s i s of s u l f a t h i a z o l e p l * 6 I t has a l s o been used for d e t e r m i n a t i o n of s u l f a t h i a z o l a i n admixture w i t h s u l f a d i a z l n e and s u l f arnerazine. 127 S u l f a t h i a m l e in ointments has been determined by d i f f e r e n t i a l absorbance a t 251 nm. 128 Dif f e r e n t i a l spectrophotometry has a l s o been used f o r t h e a n a l y s i s of a m i x t u r e of s u l f a t h i a z o l e w i t h s u l f anilamide and
SULFATHIAZOLE
41 I
s u l f athiaro e with sulf adimerazine without p r i o r

separation.\29 Derivative spectmphotometry has
been employed f o r s i m u l t a n e o u s d e t e r m i n a t i o n o f
s u l f a t h i a z o l e and s u l f a n i l a m i d e i n pharmac e u t i c a l s , 130 and s u l f a t h i a z o l e and o x y t e t r a c y c l i n e
i n honey.131
9.9.4
Infrared
I n f r a r e d spec t M pho t omet r y h a s b e e n used for t h e i d e n t i f i c a t i o n of sulfat h i a z o l e . 1329133

9.10 'H-Nuclear Magnetic Resonance Spectroscopy
' ~ - ~ u c l e amagnetic resonance r s p e c t r o s c o p y has been u s e d f o r t h e i d e n t i f i c a t i o n and d e t e r m i n a t i o o f a r i o u s sulfonamides i n phannaceut ical s 734 I 135 .
9.1 1 Raman S p e c t r o m e t r y
resonance Raman s p e c t r o m e t r y a f t e r its conversion t o c o l o r e d d e r i v a t e by d i a z o t i z a t i o n and couplFn9 r e a c t i o n . f% The 1h i t s of d e t e c t i o n were 2xl0'8M. L a s e r Raman s p e c t r o s c o p y has b e e n used t o i n v e s t i g a t e s u l f ath iatole-povidone con j u g a t e . 137
9.12
Mass S p e c t r o m e t r y
S u l f a t h i a z o l e has been determined by
Mass s p e c t r o m e t r y has been employed f o r t h e q u a l i t a t i v e l d e n t i f i c a t i o n of sulfonamides l38 139 The u s e of col1 ision-induced d i s s o c i a t i o n mass anal ysed i o n k i n e t i c energy s p e c t r o m e t r y (CID/MIKES ) for t h e i d e n t i f i c a t i o n of s u l f onamide r e s i d u e i n swine 1i v e r h a s been described. 140
9.13
Chromatographic Analysis 9.13.1
Ion-exchange Chromatography
A simple i n e x p e n s i v e procedure h a s been d e s c r i b e d for r a p i d l y s c r e e n i n g small samples of honey f o r s u l f a t h i a z o l e . 141 The
412
VIJAY K. KAPOOR
The method u s e s tm p l a s t i c tubes a r r a n g e d i n tandem. The upper t u b c o n t a i n s a bed of alumina, which removes some i n t e r f e r i n g pigments. The lower tube c o n t a i n s a very small bed of a n i o n exchange r e s i n i n HSO' form, which t r a p s s u l f a t h i a z o l e . The d&g is e v e n t u a l l y e l u t e d and d e t e c t e d u s i n g Bratton-Marshall d i a z o t i s a t l o n coup1 i n g reagents. 9.13.2
Paper Chromatography
S u l f a t h i a z o l e has been s e p a r a t e d and d e t e c t e d by c i r c u l a r p a p e r Of several s o l v e n t s t r i e d chromatography.142 butanol-3% amnonia (1: 1 ) and b u t a n o l - a c e t i c acid-water ( 5 : 1:4) gave b e s t results. Dlmethylamimbenzeldehyde has been u s e as t h e s p r a y i n g reagent. Other paper chromatographic procedure h a s been described. 143
5-
9 * 13.3
Thin-Layer Chromatography
The following t h i n - l a y e r chromatography (TLC) systems have been recommended f o r t h e i d e n t i f l c a t i o n of s u l f a t h i a z o l e . 1 S o l v e n t System Plate
Rf v a l u e x 100
66
M t h a n o l - S t r o rg e ammonia s o l u t i o n ( 100: 1.5)
S i l i c a gel G , 250 ND t h i c k , dipped i n , or sprayed w i t h 0.1M KOH i n metlianol and dried
C h l orof or-Aceto (4: 1)
ne
S i l i c a gel G, 250 pm t h i c k
09
09
E t h y l acetate-Methanol- S i l i c a gel G, S t r o n g ammonia 250 p t h i c k s o l u t i o n ( 8 5 : 10:5) Ethyl acetate S i l i c a gel G t 250 p a t h i c k
20
SLJLFATHIAZOLE
413
Hex anol
Ac etone-Ammonia s o l u t i o n 25% (80: 15) Chloro f ormdAethanol ( 70: 30)
S i l i c a g e l G, 250 pm t h i c k A l u m i n i u m oxide, 250 p t h i c k Aluminium o xide, 250 p t h i c k
53
40
05
The f o l l o w i n g d e t e c t i n g r e a g e n t s 1 have been used: a c i d i f i e d potassium permanganate, ye1 low-brown spot on v i o l e t background ; m rcur i c c h l or i de-d iphe nyl c a r b a zo ne e r e a g e n t , b l u e spot; and Van U r k r e a g e n t , ye1 ow s p o t . Use of 1,2-naphthoquinone4=sulfonate t44 and 2 ,!+d@loro-pbenzoquinone in dimethylsulfoxide as s p r a y r e a g e n t s f o r v i s u a l d e t e c t i o n of sulfonamides h a s been described. Automated densitometry and TLC have been used f o r d e t e r m i n a t i o n of sulfathiazole!46 TLC on s i l i c a g e l F254/366 i n a dioxane-xylenetoluene-isopropanol-Ammonia, 15 m01/dm3 (1:2: 1:4:2) system has been used t o s e p a r a t e a c t i v e components i n dosage 50 m c o n t a i n i n g s u l f a t h i a z o l e and o t h e r components .45 A TLC method using S i l u f o l UV2% p l a t e s and a developing system c o n s i s t i n g of 1,2-dichloroethane-fsopropanol-amnonia s o l u t i o n 25X-rnethanol ( 130:65: 10:65 or 130:65:8:50) h a s been employed for s e p a r a t i o n and i d e n t i f i c a t i o n of h y d r o l y s i s p r o d u c t s of s u l f a t h i a z o l e . 148 S e v e r a l TLC s e p a r a t i o n methods for s u l f a drugs have been d e s c r i b e d . 149-153 Quantitative thin-layer chromatographic p r o c e d u r e s have been employed for d e t e r m i n a t i o n of sulfa t h i a z o l e r e s i d u e i n honey,154,155 nd i n c a t t l e , s w i n e , turkey and duck t i s s u e . 158
Hi h performance t h i n - l a y e r chromatograph (HPTLC f o r sulfonarnides has been T h e 4 values of s u l f a c a r r i e d out.1g7-159 t h i a z o l e as reported 158 i n d i f f e r e n t s o l v e n t systems on p r e c o a t e d s i l i c a g e l 60 HPTLC p l a t e s are g i v e n as follows.
414
VIJAY K. KAPOOR
S o l v e n t Sys tern
C h l orof orm-ButanolPetroleum ether ( 40-60 )
Rf
value
03 .7
(1:l:l)
Ethyl acetate-MethanolAmmonia (25%)(30: 15:l)
0.41
Dichloromethane-Methanol
(95r5)
0.16
Acet on itril +Chiorof or* Ammonia (25%)(35: 10:0m2)

Dlethyl e t h e r
0.07
0 03 ;
Q u a n t i f i c a t i o n of corresponding f l u o r e s c a m i n e d e r i v a t i v e a t nanogram l e v e l was performed by t h e use of a n TLCIscanner configured o n l i n e wlth a microcomputer. 9.13.4
Gas Chromatoqraphy
The f ollowirg g a s chromatography system has been employed for the a n a l y s i s of s u l f a t h i a z o l e as i t s methyl d e r i v a t i v e : column: 5% OV-17 on 80-100 mesh Gas-Chrom Q, 1.5 m x 2 mn i n t e r n a l d i a m e t e r g l a s s column; column temperature: 250O; c a r r i e r gas: n i t r o g e n a t 30 ml/mln; reference compound: grlseofulvln.1 The r e l a t i v e r e t e n t i o n t i m e f o r t h e methyl d e r i v a t i v e of s u l f a t h i a z o l e i s 0.49. Gas chromatography h a s been used f o r eterrnining s u l f a t h i a z o l e r e s i d u e s I n swine f e e d a f 6 0 The r e s i d u e s of s u l f a t h i a i l i o l e from meat, eggs and milk have been determined a f t e r e x t r a c t i o n , clean up and d e r i v a t i z a t i o n w i t h diazomethane by gas chromatography on a 1 35 m c a p i l l a r y column coated with SE-30/SE-52( 1: Conditions have been described by which s u l f a t h i a t o l e can b e i d e n t l f l e d q u a l i t a t i v e l y and determined q u a n t i t a t i v e l y by pyrolys is-gas chromatography from t h e s e p a r a t e d zone o n t h i n l a y e r chromatogram.162 The s e p a r a t e d zone I s c u t from t h e p l a t e and pyrolyzed a t 780 i n a stream of n i t r o g e n I n a continuous mode p y r o l y s e r . The p y r o l y s i s products are s e p a r a t e d
19.
SLILFATHIAZOLE
41.5
b y Tenax TA i n a g l a s s column held a t 210 and t h e amount of drug r e l a t e d t o a n i l i n e peak h e i g h t is determined by flame i o n i z a t i o n detection'.
Gas chromatography-mass s p e c t r o m e t r i c procedures have been d e s c r i b e d for d e t e r m i n i n g s u l f a t h i a z o l e i n l i v e r and muscle 164 t i s s u e of s w i n e , p o u l t r y and c a t t l e . 163 ,
9.1305 Hiqh Performance Liquid Chromatography
High performance l i q u i d chromatography (HPLC) is one of t h e most widely u s e d techniques for t h e a n a l y s i s of s u l f a t h h zole. A HPLC system c o n s i s t i n g of a s i l i c a column ( S p h e r i s o r b , 5 p m , 25 c x 4 mn i n t e r n a l d i a m e t e r ) m w i t h cyclohexaneethanol-acetic a c i d (85.7:11.4: 2.9) as t h e e l u e n t has been employed1 for t h e a n a l y s i s of s u l f a t h i a z o l e . I n a n o t h e r method165 s u l f a t h i a z o l e was a n a l y s e d b r e v e r s e d phase HPLC with a e o n d a p a k C18 column 30 cm x 4 mn) u s i n g water-methanol-acetic a c i d (85: 15: 0 . 5 ) c o n t a i n i n g 0 1% tetrabutylammonium hydroxide ( i o n - p a i r i r g r e a g e n t ) , and d e t e c t i o n a t 254 nm. A v a r i e t y of HPLC methods have been d e s c r i b e d for s u l f a t h l a zole.166-170 A method f o r d e t e r m i n a t i o n of s u l f a t h i a z o l e i n human plasma and u r i n e has been r e p o r t e d . 171
Various HPLC methods for determining t h e r e s i d u e s of s u l f a t h i a z o l e i n meat, f i s h , eggs and o t h e r animal tissues have been I n one of t h e methods176 reported.1f2-178 s u l f a t h i a z o l e e x t r a c t e d from meat and c l e a n e d u p a t C column was d e t e r m i n e d by HPLC on a TSK9 el d&S 80 TM column w i t h 0 0 .% sodium dihydrogenphosphate (pH 4 . 5 ) - a c e t o n i t r i l e (2: 1 ) as t h e mobile phase. R e c e n t l y , a method f o r simultaneous d e t e r m i n a t i o n of sulfonamides, i n c l u d i n g s u l f ath i a t o l e , i n meat by thermospray 1i q u i d chromatography-mass spectrometry has been developed. 178 The l i q u i d chromatography s e p a r a t i o n was c a r r i e d o u t on TSK-gen OSD 80 TM with 00 .% a m n i u m a c e t a t e (pH 4 . 5 ) - a c e t o n i t r i l e (7:3) as The mobile phase a t a flow r a t e of 0.8 ml/min. The v a p o r i z e r temperature w a s s e t t o
416
VI JAY K. KAPOOR
be 1 5 5 O and i o n source block temperature t o be 280. Methods f o mining sul h a t h i a z o l e r e s i d u e s i n mflk, 77g395 honey1 80-882 and swine feed183 are a l s o r e p o r t e d .
c o n t a i n i n g ephedrine h y d r o c h l o r i d e and sodium benzyl p e n i c i l l in188 have been described; 9.13.6
i n mixtures c o n t a i n i n g o t h e r sulfonamides184-187 i n pharmaceutical f o r m u l a t i o n s , and i n a mixture

Supercritical F l u i d Chromat o q r aphy
HPLC a n a l y s i s of s u l f a t h i a z o l e
Packed-column s u p e r c r i t i c a l f l u i d chromatography h a s been used f o r the s e p a r a t i o n of mixtures of sulfonamides on s i l i c a and amino-bonded s t a t i o n a r y phases u t i l i s i n g carbon d i o x i d e w i t h methanol m o d i f i e r as t h e mobile phase. 189 Packed column s u p e r c r i t i c a l f l u i d chromatography-mass spectrometry (SFC-MS) of t h e s e m i x t u r e s n t i l i z i n g both moving-belt and modif l e d thermospray i n t e r f a c e s h a s a l s o been s t u d i e d . 189 10.
1
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J. Chromatogr.,
221,
419 ( 1980).
0.W. Parks, J. - 632 ( 1982). Assoc. 65,
Off A n a l . Chem.,
173.
Aerts, W.M.J. Beek and U e A o T . Erinkman, J. Chromatogr. , 435, 97 (1988).

E.M.L.
174, A.R. Long, L.C. Hsich, MmSe Mdlbrough, C 4 Short, S.A. B a r k e r , J. A g r i c . Food .. Chem., 38, 423 ( 1990).
SULFATHIAZOLE
429
175
M. Rychener, A.E. Mooser and H. Koch, M i t t ; G e b . L e b e n s m i t t e l u n t e r s Hyg. 8 l , 522 ( 1990)
176.
Eis aku Z a h 3 -e i g2102132 s(s 1 i9,9 0 1 , 171(1990); 113, 7

177.
M. Yamada, K.
, Y. Hoshino, N. Nose, N. Hamada and N. Nakazawa Shokuhin

M. H o r i e , K . S a i t o
C.A.,
Y o I k a i , H O k a , N. Kawamura, J. Hayakawa, . H a r a d a , M. S u z u k i and H Nakazawa, J. Chromatogr., 541, .

393 (1991).
178
M. H o r i e , K. S a i t o , Y. Hoshino, N. Nose, hi. Tera, T Kitsuwa, H Nakazawa and Y . . . Yamane, Eisei Kagaku, 36, 283 (1990);
C.A.,
114,4978v
(1991T
179
Off. Anal. Chem.,
M.D.
Smedley a n d J . D .
73,875
V e b e r , J.
A8soc.
(1990).
iao.
181. 182 183.
RJ ..
d r g a u e r , H. S h i m a n u k i and D.A. E n v i m n . Entomol. ll, 820 ( 1982).
Kmx,
Off. Anal. Chem., 66, 4 (1983).
K.P.
B a r r y a n d G.M. MacEachern,
J. Assoc.
T. Galeano Diazz, A. Guiberteau C a b a n i l l a s and F. Salinas , A n a l Lett. , 23 607 ( 1990).
-,
R.L. S m a l l i d g e , E.J. K e n t z e r , K.R. S t r i n g h a m , EH . . Kim, C Lehe, R.W. . S t r i n g h a m a n d E.C. Mundell J Assoc. . Off. Anal. Chem., 71, 710 (1988).
A.A.
184 185.
F a t m i , D. T a y l o r and S.Z. Masih, Drug Dev. I n d . Pharm., l , (1984). o 31
M. DeSmet, L. Dryon and D.L. Massart, 9 100 (1985); C * A e p J Pharm. Belg., . 103, 5939731 ( 1985
186
A.R. C.R.
Chromatogr.,
Long, L.C. H s i e h , M S Malbrough, .. S h o r t and $.A. Barker, J. L i q .
2, 1601
(1989).
430
VI JAY K. KAPOOR
187,
188.
A.E.H.N.
Aimed and S.M. El-Giza Analyst (London), 114, 571 ( 1 9 8 3 :
USSR SU 1, 5 7 0 , 727;
12318V (1991).
T . S . Maloletkina and L.V.

C.A.9
Pesakhovich,
114,
189. -
J.R. Perkins, D. Games, J.R. S t a r t i n and J. G i l b e r t , J. Chromatogr., 540, 239

(1991).
TENOXICAM
Abdulrahnian Mohammad Al-Obaid and Mohammad Saleem Mian
Pharmaceutical Chemistry Department College of Pharmacy King Saud University P.O. Box 2457 Riyadh 1 145 I , Saudi Arabia
431
Copyright 0 1993 by Academic Press. Inc All rights of reproduction in any form reSeNed.
432
ABDULRAHIMAN MOHAMMAD AL-OBAID AND MOHAMMAD SALEEM MIAN
C o n t e n t s
1.
2.
3.
4.
5.
6. 7. 8.
Introduction Description 2.1. Nomenclature 2.1.1. Chemical Name 2.1.2. Synonym 2.1.3. Generic Name 2.1.4. Trade Names 2.2. Formulae 2.2.1. Empirical 2.2.2. Structural 2.2.3. CAS Registry Number 2.3. Molecular Weight 2.4. Elemental Composition 2.5. Appearance, Color and Odour Physical P r o p e r t i e s 3.1. M a l t i n g Range 3.2. Stability 3.3. Solubility 3.4. pKa 3.5. P a r t i t i o n Coefficients 3.6. X-ray Powder D i f f r a c t i o n 3.7. Spectral P r o p e r t i e s 3.7.1. U l t r a v i o l e t Spectrum 3 ,, 7.2. I n f r a r e d Spectrum 3.7.3. Nuclear Magnetic Resonance Spectra 3.7.3.1. PMR Spectrum 3.7.3.2. 13C-NMR Spectrum 3.1.4. Mass Spectrum Pharmacokinetics 4.1. Absorption and D i s t r i b u t i o n 4.2. Metabolism 4.3. Uses 4.4. Adverse E f f e c t s and Precautions Methods o f Analysis 5.1. Polarographic Methods. 5.2. High Pressure L i q u i d Chromatography Methods Synthesis Acknowledgments References
TENOXICAM
433
Tenoxicarn
I n t r o d u c t i o n (1-4) Tenoxicam i s a non-steroidal anti-inflammatory and analgesic agent belonging t o t h e chemical c l a s s o f oxicams. I t possesses a long h a l f - l i f e which enables i t t o be a d m i n i s t e r e d once d a i l y . Tenoxicam i s i n d i c a t e d i n rheumatoid a r t h r i t i s , o s t e o a r t h r i t i s , enkylosing s p o n d y l i t i s , e x t r a r t i c u l a r inflammation and acute gout. A f t e r o r a l a d m i n i s t r a t i o n , mean peak plasma concentrations occur between 0.5 and 2 hours. The b i o a v a i l a b i l i t y o f tenoxcam i s about 100% a f t e r o r a l and 80% a f t e r r e c t a l administration.
2.
DescriDtion
2.1.
Nomenclature
2.1.1.
Chemical Name
1,2-thiazine-3-carboxamide-l,l-dioxide.
2.1.2.
4-Hyd roxy-2-methyl -N-2-pyri dy l-2H-thi en0 [ 2,3-e I Synonym

Ro- 12-0068 ( 5) 2.1.3.
Generic Name Tenoxicam.
2.2.4.
Trade Names T i l c o t i l (F. Hoffmann-LaRoche). M o b i l f l e x (Roche UK), T i l a t i l , T i l s i t i n .
2.2.
Formulae
2.2.1
EmDirical
434
ABDULRAHMAN MOHAMMAD AL-OBALD AND MOHAMMAD SALEEM MIAN
2.2.2.
Structural
2.2.3.
CAS Registry Number [ 59804-37-41
2.3.
mlecular Weight
337.21 ( 5 ) .
2.4.
Elemental CmDosition
C 46.26%;
H 3.29%; N 12.46%, 0 18.98%; S 19.01%.
2.5.
Appearance. Color. and Odour
Tenoxicam is a yellow crystalline powder, almost odour 1 ess .

3 .
Physical Properties
3.1.
Welting Ran209-13C (with decomposition).
3.2.
Stability Sensitive to light.
33 ..
Solubility
Approximate solubility data obtained at room temperature are given in table I.
TENOXICAM
43.5
Table
I. S o l u b i l i t y data o f tenoxicam.
mg/ml .045 l e s s than 1 l e s s than 1
2
Solvent Water Ethanol Methanol Acetone Dichloromethane Ch 1oroform

DMSO
10
8
63
3.4.
pKa
Tenoxicam i s a weak a c i d w i t h pKa values o f 5.3 and 1.1.
3.5.
P a r t i t i o n Coefficients
The octanol-water p a r t i t i o n c o e f f i c i e n t s are 0.3 a t pH 7.4 and 3 . 5 a t pH 1.2. The drug i s t h e r e f o r e slightly lipophilic.
3.6.
X-ray Powder D i f f r a c t i o n (6)
The X-ray d i f f r a c t i o n p a t t e r n o f tenoxicam was determined (Fig. 1) using P h i l i p s f u l l automated x-ray d i f f r a c t i o n spectrogoniometer equipped w i t h PW 1730/10 generator. Radiation was provided by copper t a r g e t (Cu anode 2000 W ) h i g h i n t e n s i t y x-ray tube operated a t 40 KV and 35 MA. The monochromator was a curved s i n g l e c r y s t a l one (PW 1752). Divergence s l i t and t h e r e c e i v i n g s l i t were 1 and 0 . 1 " , r e s p e c t i v e l y . The scanning speed o f t h e goniometer used was 0.02 - 29 per second. The instrument i s combined w i t h P h i l i p s PM 8210 p r i n t i n g recorder w i t h both analogue recorder and d i g i t a l p r i n t e r . The goniometer was a l i g n e d u s i n g
436
ABDULRAHMAN MOHAMMAD AL-OBAID A N D MOHAMMAD SALEEM MIAN
TENOXICAM
437
s i l i c o n sample before use. Values of 28, i n t e r p l a n n e r 1 distance dll and I/Io x 100 are l i s t e d i n Table 1 . Table 11. C h a r a c t e r i s t i c l i n e s o f t h e X-ray powder d i f f r a c t i o n o f tenoxicam.
2 8
d(A)
11.1904 8.1324 7.6568 7.4000 7.0936 6.9626 6.1218 6.0043 5.5359 5.3032 5.0924 4.7925 4.7169 4.5789 4.4102 4.4102 4.2817 4.2466 4.0800 3,9441
1/10
2 0
d(A)
1/10
7.900 10.879 11.557 11.959 12.478 12.714 14.469 14,753 16.009 16.717 17.414 18.513 18.812 19.385 20.134 20.134 20.745 20.918 21.783 22.543
21.186 28.504 60.593 24.581 20.946 35.676 44.093 26.446 66.905 44.332 29.268 38.354 21.616 19.368 15.351 15.351 19.846 37.446 51.649 13.486
23.327 23.949 24.174 24.446 25.216 25.371 25.781 26.224 26.753 27.225 28.366 29.294 30.000 30.537 32.819 33.320 34.312 34.764 35.384 36.292
3.8132 3.7155 3.6816 3.6412 3.5318 3.5104 3.4556 3.3982 3.3322 3.2755 3.1463 3.0487 2.9785 2.9274 2.7288 2.6889 2.6135 2.5805 2.5367 2.4753
100. 15.255 19.368 15.925 27.164 55.236 60.736 43.137 21.377 11.812 65.949 40.841 15.016 15.016 7.269 7.221 12.625 10.808 16.499 10.856
3.7.
Spectral Properties
3.7.1.
Ultraviolet Spectrum
The UV spectrum o f tenoxicam ( 3 mg%) ( F i g . 2 ) was scanned i n ethanol from 200-500 nm, using 4054 LKB UV/Vis spectrometer. It e x h i b i t e d t h e f o l l o w i n g UV data (Table 111).
438
ABDULRAHMAN MOHAMMAD AL-OBAID A N D MOHAMMAD SALEEM MIAN
Table 111. UV data o f tenoxicam ( i n ethanol) n m A'

I
Molar a b s o r p t i v i t y
(E)
205 265
360
487 300.33 372
16422.12
101 27.42
12544.21
3.7.2.
Infrared SDectrum
I n f r a r e d spectrum o f tenoxicam as KBr d i s c , was s c a n n e d on a P e r k i n E l m e r 580 6 i n f r a r e d spectrophotometer t o which an i n f r a r e d data s t a t i o n .Is attached (Fig. 3). The s p e c t r a l band assignments a r e listed i n table I V . Table I V . IR c h a r a c t e r i s t i c s o f tenoxicam. Frequency
cm-1
Assignment
N-H and 0-H s t r e t c h i n g .
3437 3119, 3092 1638, 1598, 1530 1436 1428 1388 1202 1151
Amide -C
0, C
= N-
-C-H deformations.
Hetrocycl i c r i n g .
-CH3
deformation.
-0-H bending.
C-0 s t r e t c h i n g .
0 Ln
Ln
4
0 0 Ln
5 a ,
.d
0 Ln U
.d
0 0
U
fk -
a , ct
0
x 0 c
Ln
cd
ct
u
0 0
a ,
5?
m
4
c, a , 0 Lo
.rl
hl
cd
>
c,
3
N
e
4
ln
.d
M
L4
E u
0
0 d
0
0
0
d
/-I
i',
rransinittance.
0 0
0 N
0 0 M
TENOXICAM
44 1
3.7.3.
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

N-CH3 C4 -H C5-H CS-H CIO-H CI 1-H C i 2-H
Chemical s h i f t 6 (ppm)
2.90
(s)
8.04
7.46
7.33
(d) (d)
(t) (t)
8.16
7.74
(d) (d)
8.35 d = doublet,
singlet,
= triplet.
12
10
I
L
1
I
8 90.6
-2
63.5
PPM
Fig.
PMR s p e c t r a o f tenoxicam i n DPISO-d6.
TENOXICAM
443
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 f r o m 10 t o 350 a.m.a. E l e c t r o n energy was 70 ev. 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
(wm)
Carbon assignment
C- 6 c- 5 c-4 c- 3 c- 2 c- 1
Chemical s h i f t i (PPm) 3 137.56 118.40 132.70 108.23 163.00 40.70
39.22 143.60 122.80 140.10 116.24 149.30 165.60
444
ABDULRAHMAN MOHAMMAD AL-OBAID AND MOHAMMAD SALEEM MIAN
Table VII. Mass fragments of tenoxicam.
m/z
Relative intensity X
Ions
18
62
83
25
94
44.2
o-"
0
121
50.2
ON"
I
i-co
153
42
bH+
179 100
co
256 12.2
213
337
200
180
Fig.
5
160
1
s p e c t r a o f tenoxicam i n DMSO-d
PPM
I3C-NMR
6'
46
Fig.
LA
I l1
1 1 1 1 1 " l ~ l 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 '
'
'
'
' 1 1 1 1 1 1 1
42
"C-NMR
40
30
36
3 4 PPM
s p e c t r a of tenoxicam (Peak Expansion).
a -cJ
0
! L
-0
0 -cJ
-U
-W
'a3
0 '0
. -
cJ
0
.U
7
0 W
s
0
.O
0 cJ
447
c Ii3
ct x i
I
200
Fig.
8
180
160
140
120
100
80
60
40
20
20 I
PM
I3C-NMR spectra o f tenoxicam (DEPT program).
N
\ '
0 I n
E-
0 0
l
0
I n
9
.?I
0 0
c w w
C
0
(d
0 E a , c,
L: I
c
c, a , C,
v)
m
*C
v-
(d
m
.?I
M
Lr,
.c
LT
8
W
I 0
1 0
0
N
449
450
ABDULRAHMXN MOHAMMAD AL-OBAID AND MOHAMMAD SALEEM MIAN
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 fo rm 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
45 I
4.2
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 <-hydroxytenoxicam whereas e x c r e t i o n v i a t h e b i l e i s i n t h e form o f t h e C-(6)-glucuronide o f tenoxicam (14). Only v e r y s m a l l q u a n t i t i e s o f tenoxicam i s recovered i n t h e u r i n e and o n e - t h i r d i n faeces as i n a c t i v e metabolites. No unchanged /drug i s excreted i n t o b i l e . The major m e t a b o l i t e i s 5-hydroxytenoxicam i n humans, and about o n e - t h i r d o f t h e dose i s recovered as t h i s m e t a b o l i t e i n u r i n e (13). Although two major i n a c t i v e m e t a b o l i t e s have been i d e n t i f i e d i n humans, o t h e r minor metabolites may possible e x i s t s . Up t o 6 metabolites have been found i n animals (15). Some o f which are formed by o x i d a t i o n o f t h i a z i n e r i n g by leucocyte peroxidase ( 1 6 ) .
Tenoxicam i s used i n t h e t r e a t m e n t o f c h r o n i c rheumatic d i s o r d e r s such as rheumatoid a r t h r i t i s , o s t e o a r t h r i t i s and ankylosing s p o n d y l i t i s ( 7 ) and a l s o i n g o u t and v a r i o u s n o n - u r t i c u l a r i n f l a m m a t o r y conditions. Tenoxicam has been most e x t e n s i v e l y s t u d i e d i n rheumatoid a r t h r i t i s and o s t e o a r t h r i t i s ( 1 3 ) . A t t h e dosage 20 mg d a i l y , tenoxicam was generally equivalent t o piroxicam w i t h respect t o analgesic and anti-inflammatory e f f i c i e n c y and i t i s good i n terms o f t o l e r a b i l i t y ( 1 3 ) . I t has been suggested t h a t c u r r e n t l y recommended dosage o f 20 mg once d a i l y p r o v i d e s t h e b e s t b a l a n c e between e f f i c i e n c y and t o l e r a b i l i t y . I n l o n g t e r m t h e r a p y , increasing t h e dosage adds l i t t l e t h e r a p e u t i c b e n e f i t w i t h a greater r i s k o f s i d e e f f e c t s . However, 10 m g once d a i l y may p r o v i d e an a p p r o p r i a t e maintenance dosage i n p a t i e n t s ( 7 ) . The usual recommended dosage f o r tenoxicam i n t h e symptomatic t r e a t m e n t o f rheumatoid a r t h r i t i s , ankylosing s p o n d y l i t i s and e x t r a - a r t i c u l a r d i s o r d e r s ( e . g . tend in i t is , b u r s i t is , per ia r t h r i s , s t r a i n s and t
452
ABDULRAHMAN MOHAMMAD AL-OBAID AND MOHAMMAD SALEEM MlAN
spralns) i s 20 mg once d a i l y administered o r a l l y o r r e c t a l l y . When recommended f o r acute a t t a c k s o f gouty a r t h r i t j s , t h e dosage should be 40 mg once d a i l y for 2 days followed by 20 m once d a i l y f o r f u r t h e r 5 days. g No dosage r e d u c t i o n i s s p e c i f i c a l l y necessary i n p a t i e n t s w i t h r e n a l o r hepatic impairment o r i n t h e e l d e r l y . However, f o r p a t i e n t needing l o n g t e r m treatment a reduction t o a d a i l y dose o f 10 mg may be t r i e d f o r maintenance ( 7 ) . Tenoxicam has a n a l g e s i c and a n t i - i n f l a m m a t o r y p r o p e r t l e s . I t i s used i n rheumatic disorders i n usual doses o f 10-20 mg d a i l y by mouth ( 1 7 ) .
4.4
Adverse E f f e c t s and Precautions
Tenoxicam has been w e l l t o l e r a t e d i n t h e t r e a t m e n t o f rheumatic c o n d i t i o n s and s i d e e f f e c t s have u s u a l l y been t r a n s i e n t and m i l d t o moderate i n i n t e n s i t y (7). Generally, t h e s i d e e f f e c t p r o f i l e o f tenoxicam appeared s i m i l a r t o t h a t seen w i t h other non-steroidal anti-inflammatory drugs. The most common s i d e e f f e c t s are g a s t r o i n t e s t i n a l (e.9. e p i g a s t r i c pain, nausea, dyspepsia, i n d i g e s t i o n , vomiting) occuring i n about 7.6% o f p a t i e n t s r e c e i v i n g tenoxicam 20 mg d a i l y , f o l l o w e d l e s s f r e q u e n t l y by c e n t r a l nervous system c o m p l a i n t s ( e . g . h e a d a c h e , d i z z i n e s s ) . Rash, u t r i c a r i a , and oedema o f t h e lower l i m b s have been reported i n c l i n i c a l t r i a l s (13). There have been one r e p o r t o f p e p t i c u l c e r a t i o n w i t h tenoxicam, r e s u l t i n g i n suspension o f therapy f o r 5 weeks (18). However, t h e p a t i e n t s subsequently t o l e r a t e d tenoxicam when c i m i t i d i n e was administered concomitantly (13). G a s t r o i n t e s t i n a l u l c e r a t i o n and haemorrhage, g a s t r o i n t e s t i n a l discomfort, f l a t u l e n c e followed by cutaneous (rash, p r u r i t u s ) complains changes i n renal and h e p a t i c f u n c t i o n t e s t may occur, and decreased h a e m o g l o b i n , g r a n u l o c y t o p e n i a , s l i g h t oedema, p h o t o d e r m i t i t i s , stvennse-Johnson syndrome and lye11 syndrome have been reported ( 7 ) . Contrai n d i c a t ions against t h e use o f tenox icam and m o n i t o r i n g recommendations d u r i n g t h e r a p y a r e t h o s e u s u a l l y a p p l i e d t o a1 1 NSAIDs ( n o n s t e r o i d a l
TENOXICAM
453
anti-inflammatory drugs). Tenoxicam should n o t be used i n p a t i e n t s w i t h known s a l i c y l a t e o r NSAID-induced asthama, r h i n i t i s o r u r t i c a r i a , o r a h i s t o r y o f severe g a s t r o i n t e s t i n a l d i s e a s e . N e i t h e r i t s h o u l d be administered before anaesthesia o r surgery i n e l d e r l y , n o r i n p a t i e n t s w i t h an i n c r e a s e d r i s k o f r e n a l f a i l u r e o r bleeding. Furthermore, concomitant treatment w i t h s a l i c y l a t e s o r other NSAIDs should be a v o i d e d because o f t h e i n c r e a s e d r i s k o f g a s t r o i n t e s t i n a l adverse r e a c t i o n s ( 7 ) . Renal f u n c t i o n s should be c a r e f u l l y m o n i t o r e d d u r i n g tenoxicam treatment i n p a t i e n t s w i t h c o n d i t i o n s t h a t might increase t h e r i s k o f developing r e n a l f a i l u r e , such as p r e - e x i s t i n g renal disease, impaired r e n a l - f u n c t i o n i n d i a b e t i c s o r t h e e l d e r l y , hepatic c i r r h o s i s , congestive h e a r t f a i l u r e , volume d e p l e t l o n , c o n c o m i t a n t d i u r e t i c t r e a t m e n t and c o n c o m i t a n t treatment w i t h drugs o f known nephrotoxic p o t e n t i a l . L i k e w i s e p a t i e n t s r e c e i v i n g a n t i c o a g u l a n t s and/or a n t i d i a b e t i c drugs should be c l o s e l y monitored i f an N S A I D such as tenoxciam i s added t o t h e i r treatment regimen (7). Meals taken b e f o r e t h e i n g e s t i o n o f tenoxicam d i d not e f f e c t qomplete absorption from g a s t r o i n t e s t i n a l t r a c t ( 1 9 ) . Therefore, tenoxicam may be t a k e n e i t h e r b e f o r e o r a f t e r meals. I t would be a d v i s a b l e f o r tenoxicam t o be t a k e n on an empty stomach when a s i n g l e o r a l dose o f tenoxicam i s used as an analgesic f o r acute pain where prompt r e l i e f i s required (7).
5.
Methods o f Analysis
5.1
Polarographic Methods
El-Mali e t a l . (20) have developed a square-wave and square wave a d s o r p t i v e - s t r i p p i n g v o l t a m m e t r i c c o m p a r i s o n o f p i r o x i c a m and t e n o x i c a m . I n the developed method t h e square-wave voltammograms were recorded w i t h use o f a PAR Model 384 8 Polarographic a n a l y z e r coupled w i t h a PAR 303 A s t a t i c droppingmercury e l e c t r o d e (0.017 cm2), a Ag-AgC1 r e f e r e n c e electrode and a P t counter electrode. The supporting e l e c t r o l y t e was 0.05M - H2SC4 a t pH 2. F o l l o w i n g d e p o s i t i o n a t -0.6V vs. Ag-AgC1 f o r 60 s , a negative
454
scan was applied a t 200 m Vs-1. The mean peak c u r r e n t s f o r 60 nM-piroxicam and 10 nM-tenoxicam were 397 and 375 nA, r e s p e c t i v e l y , w i t h c o e f f i c i e n t o f v a r i a t i o n o f 3.2 and 3.6%, r e s p e c t i v e l y . U t o 0.1 mM-ascorbic a c i d p had no e f f e c t on t h e piroxicam peak, b u t enhanced t h e response t o tenoxicam. The d e t e c t i o n l i m i t s were 0.7 nM-piroxicam and 0.1 nM-tenoxicam. For a n a l y s i s o f urine, d i l u t e d 10-fold i n supporting e l e c t r o l y t e , t h e d e t e c t i o n l i m i t s were 50 nM-piroxicam and 5 nM-tenoxicam.
5.2
Hiah Pressure Liauid ChromatograPhy Methods
Heizmann e t a l . (21) developed a h i g h pressure l i q u i d chromatographic method f o r t h e determination o f tenoxicam i n human plasma. Tenoxicam was e x t r a c t e d from t h e b u f f e r e d plasma (pH 3 ) w i t h dichloromethane and t h e evaporated e x t r a c t s were analysed on a Ctrr r e v e r s e d phase column u s i n g a m e t h a n o l (0.1M)phosphate b u f f e r (pH 5.6) (50:50) mobile phase a t a flow r a t e o f 0.8 ml/min w i t h UV d e t e c t i o n a t 371 nm. The d e t e c t i o n l i m i t was 20 ng/ml u s i n g a 0.5 m l sample. Piroxicam was used as an i n t e r n a l standard. Dennis e t a l . (22) have determined tenoxicam and i t s hydroxy m e t a b o l i t e i n t h e human u r i n e . Tenoxicarn and t h e m e t a b o l i t e were e x t r a c t e d from a c i d i f i e d u r i n e by means o f an e x t r e l u t column w i t h chloroform. The evaporated e l u a t e was then analysed on C i a reversed phase column w i t h methanol-phosphate b u f f e r as mobile phase and UV d e t e c t i o n a t 371 nm. The d e t e c t i o n l i m i t f o r both compounds i n 1 m l sample was 50 ng/ml. Day e t a l . ( 2 3 ) have measured t h e tenoxicam concentrations i n t h e plasma. Plasma ( 1 m l ) was mixed w i t h phosphate b u f f e r ( 1 mf, 1 mol. pH 1.5) and e x t r a c t e d by i n v e r s i o n w i t h 6 m l o f d i e t h y l e t h e r . A f t e r c e n t r i f u g i n g , t h e organic phase was recovered. The aqueous phase was re-exctracted w i t h a f u r t h e r 6 m l o f e t h e r and t h e recovered e x t r a c t s were combined. The combined e t h e r phase was e x t r a c t e d w i t h sod. h y d r o x i d e ( 1 m l , 0.01 M ) and t h e n decanted a f t e r f r e e z i n g t h e aqueous phase i n an acetone and s o l i d carbon d i o x i d e bath. The aq. phase was b u f f e r e d w i t h a c e t i c a c i d (200 pl, 0.25 M ) , warmed t o evaporate traces o f e t h e r and then t r a n s f e r r e d t o auto-sampler v i a l s . The chromatographic separation was performed on
TENOXICAM
455
a reversed phase column heated t o 50C, ( u l t r a s p h e r e ODs-I.P., 15 cm, Beckamn), w i t h a mobile phase (700 m l o f CHBOH, 300 m l 0.02 M phosphate b u f f e r pH 6.0). Flow r a t e was 1 ml/min, and t h e peaks were d e t e c t e d a t 374 n u s i n g UV d e t e c t o r . m Pickup e t a l . (24) have a l s o analysed tenoxicam i n human plasma. The plasma was a c i d i f i e d with 1 ml o f I N HC1 and 8 m l dichloromethane was added. The m i x t u r e was c e n t r i f u g e d . The o r g a n i c l a y e r was d r i e d and t h e r e s i d u e d i s s o l v e d i n 0 . 5 m l o f m o b i l e phase, [methanol-phosphate b u f f e r (0.1 moll pH 5) (60:40)] L i C h r o s o r b RP-18 column was used. UV d e t e c t o r was f i x e d a t 361 nm. C a r l u c c i e t a l . (25) have developed a s e n s i t i v e , s p e c i f i c and r a p i d l i q u i d chromatographic procedure t o s e l e c t i v e l y m o n i t o r t e n o x i c a m i n human plasma. The plasma samples were a c i d i f i e d and e x t r a c t e d u s i n g s o l i d phase e x t r a c t i o n column. The procedure i s l i n e a r from 0.1 t o 10 pg/ml w i t h a d e t e c t i o n l i m i t o f 0.05 yg/ml. The c o e f f i c i e n t o f v a r i a t i o n f o r t h e procedure i s 6.2% and 2.0% f o r t h e range o f c o n c e n t r a t i o n s examined. The s e p a r a t i o n o f tenoxicam was performed on a reversed phase v i o s f e r LC-18, 250 x 4.6 mm I . D . , 10 ym p a r t i c l e s i z e column p r o t e c t e d by a 2 cm p e l l i g u a r d column (40 ym p a r t i c l e s i z e ) . The s e p a r a t i o n s were performed a t t h e room temperature and t h e d e t e c t o r s e t a t 0.1 a . u . f . s . The m o b i l e phase c o n s i s t e d o f a m i x t u r e o f methanol-buffer s o l u t i o n (pH 3) (0.2 M t o 0.1 M c i t r i c a c i d , u n t i l pH 3 , f o l l o w e d by d i l u t i o n t o 1000 m l w i t h d i s t . H20). Flow r a t e o f t h e m o b i l e phase was a d j u s t e d a t 1.2 ml/min. Dixon e t a l . (26) have a l s o developed a method f o r t h e d e t e r m i n a t i o n o f t e n o x i c a m i n plasma u s i n g HPLC.
6.
Synthesis
Binder e t a l . ( 2 7 ) have r e p o r t e d on t h e s y n t h e s i s o f tenoxicam from thiophene c a r b o x y l i c acid, w h i c h was o b t a i n e d by a Fiesselmann t h i o p h e n e s y n t h e s i s , f o l l o w e d by s u b s t i t u i o n o f t h e h y d r o x y group by c h l o r i n e . A s u l p h i t e - e x c h a n g e r e a c t i o n o f 3-chloro-2 thiophenecarboxylic a c i d y i e l d e d t h e potassium s a l t o f 3-sulfo-2-thiophenecarboxylic a c i d
456
ABDULRAHM.AN MOHAMMAD AL-OBAID AND MOHAMMAD SALEEM MIAN
sulphonate, which was transformed i n t o t h e b i s ( a c i d c h l o r i d e ) w i t h phosphorus p e n t a c h l o r i d e (P205) i n phosphorus oxychloride (POC13). S e l e c t i v e methanolysis gave t h e e s t e r (msthyl-3-(chlorosulfonyl)-2-thiophenecarboxylate), which i s t h e key intermediate f o r t h e annulation sequence.
(CHsNHCH2COOCH3)
ethoxycarbonyl)methyl]-N-methylaminolsulfonyl]-2
Reaction o f e s t e r w i t h methyl sarcosinate gave t h e d i - e s t e r methyl 3-t"-
thiophenecarboxylate i n a very good y i e l d , which was then c y c l i z e d t o methyl 4-hydroxy-2-methyl-2H-thieno
[2,3-e]-1,2-thiazine-3-carboxylate-l,l-dioxide.
Treatment o f t h i s c a r b o x y l a t e e s t e r w i t h 2-aminop y r i d i n e yielded tenoxicam. The s y n t h e s i s o f tenoxicam (28) has a l s o been done by a mu1t i s t e p c o n v e r s i o n o f hydroxythiophene c a r b o x y l i c e s t e r I , t o t h e s u l f o n y l c h l o r i d e 11. Reaction o f s u l f o n y l c h l o r i d e I1 w i t h N-methylglycine e t h y l e s t e r gives t h e sulfonamide 111. Base-catalysed C l a i s e n t y p e condensation serves t o c y c l i z e t h a t intermediate t o 8-keto e s t e r I V which on heating w i t h 2-aminopyridine y i e l d s tenoxicam. The l a t t e r synthesis i s shown below (Scheme I). Scheme I. Synthesis o f Tenoxicam Me
(fc'
C0,EtI1
b
I11
I R = OH R = C1 R = S03H
C
IV
TENOX ICAM
Reagents: (a) PCl5; (b) CH3NHCH2C02CH3; (c) NaOCH3; (d) 2-Aminopyridine.
TENOXICAM
457
7.
Acknow 1edsments The authors a r e h i g h l y t h a n k f u l t o Hoffmann-La Roche & Co, B a s l e , S w i t z e r l a n d f o r t h e i r h e l p i n supplying a u t h e n t i c samples, t h e product i n f o r m a t i o n and r e l e v a n t l i t e r a t u r e . The a u t h o r s a r e a l s o v e r y much g r a t e f u l t o M r . T a n v i r A. B u t t , Pharmaceutical Chemistry Department, College o f Pharmacy, King Saud U n i v e r s i t y , Riyadh, Saudi Arabia.
8.
References Wiseman, (1985).

E.H.
1.
Vol.
11, CRC Press Boca Raton,
p.
209
2.
K i r c h h e i n e r , B., Holm, P., Jensen, E.M., Kryger, J., Romberg, 0. and Salveson, A. C u r r . Ther. Research, 32(5), p. 627-632 (1982).
O t t , H. Eur. J. o f Rheumatol. Inflamm.,
3.
8(1), p. 39-46
8(1)
(1985). 4. Standel, W . (1985). and Josenhants,

G.
Ibid,
p.
28-38
5.
6.
Merck Index, 11th Ed.ition, p. 9084 (1989). Abdulrahman M . Al-Obaid unpublished data (1992). and Mohammad Sa eem Mian.
7.
8.
Peter A. Todd; and Stephen, P. C l i s s o l d . Drugs 4(4), 625-646 (1991). Francis, R.J., Dixon, J.S., Lowe, J.R., H a r r i s , P.S., European J. o f Drug Metabolism and Pharmacokinetics, 10, 309-314 (1985). Heintz, R.E., Enrico, J.F., Dubach, U.C., Jeunet, F.S. Presented a t t h e Europaische Tagung der Biopharmazie und pharmacokinetic, salamanca, 23-27 Apr. (1984a). E n r i c o , J . F . , Dubach, H e i n t z , R.C. G u e n t e r t , T.W., U.C. Brandt, R., e t a l . European J. o f Rheumatology and I n f l a m a t i o n 6, 33-44 (1984b). Crevoisier, C., Gerber, N . , O t t , H . , Mayer, J . , Heizmann, P. Presented a t t h e 1 s t World Conference on
9.
10.
11.
458
Inflammation. Antirheumatics, Analgesics, l a t o r s , Venice, 16-18 Apr. (1984).

12.
Immunomodu-
Fukuzawa, H, Tomisawa, H., Ischihara, S . T e t e i s c h i , M . , Yakuri, To Chiryo, 12 (Suppl. 5). 921-936 (1984a). John P. Gonzalez and Peter A. Todd. Drugs, 34, 289-310
(1987).
13.
14.
Tamimura, H., Mukaihara, S . , Mine, Y., Yotsumoto, F., Setoyama, M . , Suzuki, T., Kurma, I NipDon Geka Hokan, .
53, 779-785 (1984).
15.
I c h i h a r a , S., Tisuyuki, Y., Tomisawa, H., Fukazawa, H., Nakagama, N. e t a. X e n o b i o t i c a , 1 4 , 727-739 (1984). I c h i h a r a , S . , Tomisawa, H., T a t e i s h i , M . , J o l y , R. e t a l . Drug Metabolism and D l s p o s i t i o n , 1 7 , 463-468
(1988).
16.
17.
"Martindale", "The Extra Pharmacopeia" 29th Ed. p. 44. The Pharmaceutical Press, London ( 1989). Waterworth, R . F . , Waterworth, S . M . , T a y l o r , K.M. European J. o f Rheumatolosy and Inflammation, 8 , 21-27
(1985).
18.
19.
Goebel, M . M . , Scholz, H.J., Tenoxicam, a new oxicam f o r long-term treatment rheumatoid a r t h r i t i s . Poster, EULAR Symposium, Vienna, 9-12, 10 ( 1 9 8 5 ) . El-Maali, N.A., V i r e , J.C., Patriarche, Ghandour, M . A . , C h r i s t i a n , G.D. Anal. S c i . ,
245-250 ( 1 9 9 0 ) .
G.J.,
20.
m,
374,
21.
P. Heizmann, J. Korner and Zinapold. J. Chromat., 95-102 (1986).
22.
Dennis D e l l , Raymond, J o l e y , Walter M e i s t e r , Wolf Arnold, Charles P a r t o s and B e a t i x Guldmann. JChromat., 317, 483-92 ( 1 9 8 4 ) .
23.
R.O. Day, S. Lam, P. Paul1 and D. Wade. B r . J. C l i n .

Pharmac., 24, 323-328 (1987).
TENOXICAM
459
24.
Pickup, M.E.,
Lowe, J.R.,
Galloway, D.B.
J. Chromat.,
2 2 5 ( 2 ) , 493-7 (1981).
25. G. C a r l u c c i , P. Mazzeo, G. Palumbo. Chromat. 1 5 ( 4 ) , 683-695 (1992).
J.
o f Liauid
J -
26.
J.S. D i x o n , J.R. Lowe and D.B. Chromatogr., 310, 455-459 (1984).
Galloway.
27.
D i e t e r Binder, O t t o Hromatlea, Franz Geissler, K a r l Schmied, C h r i s t i a n R. Noe, Kaspar B u r r i , R u d o l f P f i s t e r , Konard Strub and Paul Z e l l e r . J. Med. Chem., - 678-682 (1987). 30, L e d n i c e r , D., M i t s c h e r , L. and George, G. "Organic Chemistry o f Drug Synthesis", Vol. 4, p. 173 (1990) John Wiley and Sons. I n c . New York, USA.
28.
THIAMPHENICOL
Gunawan Indrayanto,' Dian L.Trisna,' Mulja H . Santosa,' Ratna Handajani,' Tekad Agustono? and Purnomo Sucipto'
( I ) Faculty of Pharmacy A irlangga University Surabaya, Indonesia
(2) PT. New Interbat Pharmaceutical Laboratories

Buduran, Sidoarjo Indonesia
461
Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any form reserved
462
GUNAWAN INDRAYANTO ET AL.
CONTENTS 1. Description 1.1 Nomenclature 1.2 Formula 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance Physical 2.1 2.2 2.3 2.4 propert ies Melting point Solubility Thermal Analyeis Spectral Properties
2.
3.
Pharmacokinetics 3.1 Absorption 3.2 Distribution 3.3 Metabolism 3.4 Excretion Microbial activity Methods of analysie 5.1 Official method of Thiamphenicol analysis 5.2 Spectrophotometric and Colorimetric Me thods 5.3 Reversed-phase H i g h Performance Liquid Chromatography Analysis 5 . 4 Gas Liquid Chromatography A n a l Y d 8 5.5 Microbiological methods
4.
5.
Acknowledgement References
THIAMPHENICOL
463
11.1
DESCRIPTION
Nonenclature
1.1.1
Chemical Names
- 2.2-Dichloro-N-(aR,PR)-P-HydroXY-a-HYdroxy-a-Hydroxymethyl4- Methyleulphonyl-Phenethyll Acetamide ( 1,2). - 2.2-Dichloro-N-[(aR,PR)-~-Hydroxy-a-Hydroxymethyl-4-Mesylphenethyll Acetamide (3). - D-Threo-2,P-Dichloro-N- CP-Hydroxy-a- ( Hydroxymethyl)-p- (Methylsulphonyl) Phenethyll Acetamide (4). - D-d-Threo-2 Dichloroacetamido-1- ( 4-Methyleulphonyl ) -1.3- Propanediol ( 4)
1.1.2
Generic Names
Dextroeulphenidol, Thiamphenicol , Thiamphenicolum, Thiophenicol, Thiamfenicolo (3.41 . 1.1.3 Trade Names
Anicol, Biothicol, Cetathiacol, Deecosin,Flogotisol Glitieol, Hydrazin, Igralin. Kalticol, Hacphenicol, Neomyeon, Opiphen, Phenobiotic, Propacin. Rigelon, Rincrol, Sendicol, Thiambiotic, Thiamcol, Thiamika, Thiamycin, Thianicol, Thiaven, Thiocymetin, Tiacin, Thionicol, Urfamycin, Urfamycine, Urophenyl, Vice mycetin (3,4,5).
1.2
Formula
1.2.1
Empirical
1.2.2 H3C
Structural
- 02S
CHOH
CH
- CHZOH
(1.2.4)
I
NH
- CO - CHC12
464
1.2.3 CAS Registry No 15318-45-3 (1,293)

1.3
Molecular weight
358,2
14 .
Elemntal Composition
C 40.46 X ; 0 22.46 X ;
H 4.24 X
S 9.00 X
C1 19.91 X ; N 3.93 X ;
1.5
Appearance
A fine white to yellowish-white odourlese cryetalline powder or crystale,with a bitter taste. A solution in absolute ethanol is dextrorotatory ; a solution in dimethylforrnamide ie laevorotatory (19 % 3 ) *
2.
2.1
PHYSICAL PROPERTIES
Melting Point
163O
22 .
- 167O
Solubility
The solubility data for Thiamphenicol is listed in Table I.
THIAMPHENICOL
465
TABLE I.
Solvente
Thiamphenicol Solubility Measurements Solubility mg/rnl.

> 100 3.33 12.50 3.85 7.14 2.50 1.25 < 0.33
3OoC
~~~
dimethylformamide acetonitrile methanol ethanol (absolute) acetone water ethyl acetate diethyl ether
23 .
Thermal Analysis
The DSC thermogrp of Thiampheonicol waeoobtained at a heating rate of 10 C/min from 30 C to 175 C on Shimadzu DT-30 Thermal Analyzer. The thermogram is shown In Figure 1. A n endothermic peak occuring at 163OC correeponds to the melting of Thiamphenicol. The absence of a desolvation feature at lower temperatures illuetrate the anhydrous nature of the compound.
2.4
Spectral Properties
2.4.1
Ultraviolet spectrum
The ultraviolet absorption epectrum of Thiam phenicol in various solvents was obtained on a Hitachi Model 0-2000 Double Beam Spectrophotorneter. The spectrum is shown in Figure 2. At high concentration (1 100 mg/L) there are two w a k e , while at low concentration (I 100 mg/L) only one peak can be eeen. The high A peak is due to absorption within the amide group(n --> n 1, while the low A peak represent the n -> n from the same group. Specific abeorbance of Thiamphenicol in varioue ~ o l v e n t eare shown in Table I1
rrl
0 u3
rl
0
I n
4
4
9-4
0 0
rl
0
c1 \
0
9-4 rl
0
0
r(
m
03
0
5
0
P
&
il
u3
0 I n
0
cr)
466
WAVELENGTH
( NII)
Figure 2. Ultraviolet Spectra of Thiamphenicol in water I---0.015 m L : / m 0 . 2 ms/mL)
468
TABLE 11.
W Characteristics of Thiamphenicol
Solvent Water 272.4 nm 265.4 nm 224.2 nm 22.9 26.7 381.2
0.1 N HCl
Methanol 272.6 nm 265.4 nm 224.4 nm 21.9 26.0 379.2
Maximum wavelength
272.6 run 265.4 nm 224.2 nm 22.4 26.4

384.6
1%
A
1 cm
2.4.2
Infrared spectrum
The infrared absorption spectrum of Thiamphenicol obtained in potaeeium bromide pellets which wae recorded on Hitachi 1-2001 Infrared Spectrophotometer and ie ahown in Figure 3. The epectrum in Figure 3 is identical with the one previously published by Dibbern (6).Draguet-Brughmas~et.a1.(7) reported that Thiamphenicol has two polymorphs (I and XI) which can be dietingulshed by their infrared spectra and the figure below showed the characteristics of polymorph I. The characteristic bands of the compound are shown on Table 111, along with the bond assignments
CH
a 0
k 6
CH
1.4
'i
c4
4 -
470
TABLIC 111.
IR C h a r a c t e r i s t i c s of Thiamphenicol
Wavenumber
3512 3264
1
broad H broad H
Ass ignmen t
3464
bonded OH and NH s t r e t c h bonded OH and NH s t r e t c h
3092
1696
; 1566
aromatic C-H s t r e t c h
C
0 e t r e t c h amide I band
N - H bend amide I1 band

1280 1070
; 1146
=2
C
0 s t r e t c h ( primary a l c o h o l )
2.4.3
Nuclear Magnetic Resonance Spectrum

i
2.4.3.1
1
H-Nuclear Magnetic Resonance Spectrum
H-NMR spectrum of Thimphenlcol in DMF-d The recorded on a H i t a c h i FT-NMR R-1900 Spectrometer u s i n g TMS a e t h e i n t e r n a l standard. The spectrum i e , shown i n Figure 4. The proton chemical s h i f t s , m u l t i p l i c i t i e s and aeeignmente are g i v e n i n Table IV.
THIAMPHENICOL
47 I
TABLE IV.
H-NMR Characteriatice of Thiamphenicol
Chemical s h i f t Multiplicity Proton Assignment No. of 6 (Ppm) Proton

3.19
singlet
cH3
(a)
3.67
multiplet
CH2- O H
(b)
4.12
multiplet
!j - C
(c)
I
5.05
triplet
cH2-
OH
(d)
5.23
triplet
- CH - OH I C H
(el
5.95
doublet
OF
(f)
I
6.62
1
7.83
2 doublet
H ! !
8.21
doublet
NH
(i)
412
10.0
80
6.0
La
PP(6)
2.0
0.0
Figure 4. H-NMR (90 MHz) Spectrum of Thiamphenicol in DMF-d
THl AMPHENICOL
473
2.4.3.2.
IS
13
C-Nuclear Magnetic Resonance Spectrum
The C-NMR Spectrum of Thimphenicol in DMF-d ( 200 n\g/mL) were recorded on a Hitachi FT-NMR R-1900 Spectrometer, usin8 TMS as the internal etandard. The spectrum is shown in Figure 5. The carbon chemical shifts and spectral assignment8 are given in Table V. TABLE V.
13
C-NMR Characteristics of Thiamphenicol
Carbon Assignment No.
Chemical shifts 6 (PW) 44.10 58.01 61.49 67.44 70.56 127.20
c4 c3 c5
cS
127.69 140.35 149.81 164.31
c7
0 1
414
I Cl-CrH
CI
I
c=o
!P
I n
W u
rn
Figure 5. Broad Band Proton Decoupled isC-NMR (22.6 MHz) Spectrum of Thiamphenicol in DMF-d7
2.4.4
Mass Spectrum
The mass spectrum of Thiamphenicol - M S ether presented in Figure8 6 and 7 waa obtained by electron Impact (El) and chemical ionization (CI) using methane gas a8 reagent on a Jeol, JMS-DX 303 Mass Spectrometer. The ionizing electron beam energy wae at 70 eV. The main fragment8 are given in Table VI.
THIAMPHENICOL
415
TABLE VI. The main fragments of Thiamphenicol-TMS ether
Important
Species
61
m/z
CI
M+ + 29
M+ + 1
*; ;
* *
;$**
* ;it**
484* 486**
M+ M+
- CH3
TMS
0
484*
486**
r I
13C
0
cH2
CH
- 0 - THS
330
330
CH-
NH
coCHc1 '2
0
p p L
0
- TMS +
- TMS
-1I-
242* 244**
242*
244**
257
257
178
476
2,
330
40
a44
n
C
480
188
200
3e0
400
s w
6 1 2
Figure 6. EI Ma66 spectrum of Thiamphenicol-TMS ether
. . . .
aw
400
a
sw
L?:l
.
,
,.
.
71
600
WZ
Figure 7. CI Has8 spectrum of Thiamphenicol-TMS ether
THIAMPHENICOL
411
3.
31 .
PHARHACOKINETICS
Absorption
The oral administration of a single doee of 1 gram of Thiamphenicol in humans resulted in a serum level of 8 mcg/mL after one to one half hour8 ( 8 ) .
32 .
Distribution
Thiamphenicol is always present in the unchanged form,reaching particularly high and sustained levels in the kidney-urine and liver-bile (9). It circulates freely in the extracellular fluid8 and only small quantity is bound to plasma proteins (0-10%)(8,9).Drug levels in the bile were higher and more prolonged than in plasma ( 8 ) .
33 .
Metabolism
Metabolic pathways of Thiamphenicol in human was studied by Mc.Chesney et.a1.(8), and Nakagawa et.al. (10). and are based on the known metabolic pathways for chloramphenicol. Thiamphenicol was transformed into the glucuronic acid conjugation of Thiamphenicol, and/or hydrolysis of the dichloroacetamido group took place with or without prior dehalogenation, and/or oxidation of the a and Y carbon atoms of the propanediolic side chain (11.12). The metabolic fate of Thiamphenicol was studied in cats, rabbits, and rats after oral and intramuscular administration (8.12-14). All model species excreted Thiamphenicol either as unchanged Thiamphenicol, deacylated Thiamphenicol, or Thiam phenicol glucuronide in urine, faeces and bile. Human studies (6-10.14-16) showed that Thiamphenicol under goes biotransformation after oral and intravenous administration. The metabolites found in the urine and bile of rate, guinea pigs, rabbits and human urine were unchanged Thiamphenicol, the hydrolysis product of Thiamphenicol, and a con3ugate of Thiamphenicol with glucuronic acid. The metabolites are shown on Table VII and a possible metabolic pathway (scheme 1) ha8 been proposed (12)
478
OH
OAc
I R-CH-CH-CHZOC8HS06
NHcocHclz
thiamphonicol alwcuronidetrra)
R-CH-CH-CH20Ac NHcocnc12
D o o c y l o t e d Thiomphonicol(r? h e )
R-CH-CH-CH20H
NH2
hydrolysim
R-CH-CH-CHZOH
NHcoui20H
R-COOH
Scheme 1.
Metabolic Pathway of Thlamphenicol
THlAMPHENlCOL
419
TABLE VII. The Metabolites logical Specimens
of Thiamphenicol in
Metabolite
Bio
No
Species human
Biological spec imene
Reference
8 15 14 10 17 9
TP, Total TP urine plasma,ur ine TP urine TP,TP base,TPG urine TP ,TP base ur ine TP ,TP base plasma and amniotic fluid TP blood,seminal fluid, prostatic, TP tieeue
plasma,urine and tisaue urine, faeces urine urine, bile urine,faeces urine urine.bile urine urine,bile
16
rat
TP TP TP ,TP base TP,TP base TPG
13 8 12 14
dog
TP, Total
Tp
rabbit cat guinea Pie

Note :
TP, Total TP TP ,TP Base,TPG

TP,Total Tp
8
14
8
14
TP ,TP base,TPG
TP = unchanged thiamphenicol TP base = deacylated thiamphenicol = thiamphenicol glucuronide TPG
3.4
Excretion
The absorption of orally administered baRic Thiamphenicol in human was about 50 - 70 X of an oral dose of 500 mg, recovered as an active drug in urine within twenty four hour6 following administration (8.11). The most pronounced difference in the excretion of Thiamphenicol was observed in the amount of metabolites
480
recovered, being about 2.2 X of the total amount excreted as the deacylated form and only about 0.5 X as Thiamphenicol glucuronide ( 1 0 , 1 4 1 .
4.
MICROBIAL ACTIVITY
Limeon et.al.(l8) reported that a clinical trial of ThiampheniCOl in 30 patient8 with typhoid or parathypoid fever showed that Thiamphenicol ie highly effective agent in the treatment of Salmonella enteric fever. The result of in v i t m activity using an agar dilution method with an inocula replicating apparatus showed that Thiamphenicol and Chloramphenicol were equally active against 106 ieolates of Haemphflus and against 40 strains of Bactemfdes fragilfs. However, for the inhibition of Enterobacterf aseae, Thiamphenicol required 2 - 16 times a8 much of that needed by Chloramphenicol ( 1 9 ) . Grady et.al. (20) stated that Thiamphenicol exhibited equal or greater activity against Haemphi1 us f nfl uenzae and Neiaseria meningf tidis than did Chloramphenicol. According to Kitoh et. al. (21). Thiamphenicol is far less in activated by anaerobic bacteria (such a8 Bastemides fragflf~) than Chloramphenicol. The rate of Thiamphenicol inactivation by bacterial acetyl transferase was about onehalf that of Chloramphenicol ( 2 2 ) .
5.
5.1
METHODS OF ANALYSIS
O f f i ci a l method of Thiamphenicol Analysis
The official method of Thiamphenicol analyeis found in B . P . 1988 and D . A . B . IX 1986 (1,Z).
are
The procedure of this method are : Dissolve 0.3 g in 30 mL of ethanol (96%), add 20 mL of a 50 % w/v solution of potassium hydroxide, mix and heat under a reflux condenser for 4 hours. Cool, dilute with 100 mL of water and neutralize with 2 M nitric acid. Add a further 5 m l of 2 M nitric acid and titrate with 0.1 t stlver nitrate, determining the end-point l potentiometrically. Repeat the operation without the substance being examined. The difference between the
THIAMPHENICOL
48 I
titrations represents the amount of silver nitrate is required. Each mL of 0.1 M silver nitrate VS equivalent to 0.01781 g of CI2Hl5Cl2NO5S.
52 .
Spectrophotometric and Colorimetric Methods
Hc. Chesney et.al.(B) reported the colorimetric determination of Thiamphenicol by alkaline hydrolysis following periodate oxidation to form p-methylsulphonyl benzaldehyde. This can be determined colorimetrically a 3 the alkali ealts of its p-nitrophenylhydrazone. t Using similiar hydrolyeis and oxidation methods, Uesugi et.al. (14) determined Thiamphenicol colorimetrically by reacting p-methylsulphonyl benzaldehyde with APHS reagent (diethanolamine salt of azobenzenephenylhydrazine sulphonic acid), which turn to blue colour at an absorbance maximum of 590 nm by adding HCL reagent. A W epectrophotometric method for the determination of Thiamphenicol has been carried out in our laboratory. A linear correlation between abeorbance and concentration was verified over 5.98 - 30.28 cre/mL in methanol (224.4 nm), water (224.2 nm) and 0.1 N HC1 (224.2 nm). The limit of detection in methanol, water and 0.1 N HC1 were 0.12, 0.07, and 0.05 crg/nL respectively. The limit of quantitation in methanol, water, and 0.1 N HCl were 0.42, 0.22, and 0.18 w / m L respectively. Recovery studies and precision were performed in water solution of synthetic capsules. Recovery was found to be 99.96 2 1.17 (mean % recovery f SD). The precision, expresaed a8 relative etandard deviation, wa8 found to be 1.17% (n=10). Spectra of Thiamphenicol in synthetic capsule6 and matrix of synthetic capsules are shown in Figure 8.
482
206)
220
240
260
280
WAVELENGTH (NM)
Figure 8. Ultraviolet spectra of Thiamphenicol in matrix of water : - synthetic capsule6 ; --eynthetic capsules
5.3
Reversed-phase high pressure liquid chromatography
A RP-HPLC method for the determination of Thiamphenicol in pharmaceutical preparations wae carried out in our laboratory uaing a Shimadzu LC-6A with SCL-6A system controller, SPD-6A W spectrophotometric detector, CR 3A integrator and a 7125 Rheodyne injector fitted with a 20 tiL loop. Column : RP-18 LiChrospher (E. Merck), 10 pm particles. Mobile phaee :acetonitrile - acetate buffer pH 6.0 (15 : 85, v/v). Flow rate : 2 ml/min ; temperature : ambient ; detection : 272 nm. Under thie condition, the linear range extended from 0.01 - 4.82 mg/mL (n = 9 , r = 0.9999). The limit of detection wae 0.84 pg/mL. the limit of quantitation WBB 2.80 tig/mL. The precision, eSpre66ed a13 relative etandard deviation wae 0.56% (n = 10) and the % recovery w a 8 101.19 t 0.54 (mean X recovery -+ SD). The chromatogram is shown in Figure 9. According to Nagata and Saeki (231, Thiamphenicol reeiduee in chicken muscle6 can be determined by chromatographic method with W detection at levels as low a8 0.500 m g / l - The average recoveries of the drug added to muscles at 0.2 and 0.1 mg/L were 92.8 X and 90.0 X respectively.
THIAMPHENICOL
483
IS
10 MINUTES
0
l c
12
MINUTES
Figure 9. High performance liquid chromatogram of Thiamphenicol (0.
Figure 10. Gas liquid chromatogram of Thiamphenicol (I) and Chloramphenicol (IS).
5.4
Gas L i q u i d Chromatography Analysis
Several GLC method8 have been employed to determine Thiamphenicol in biological fluids and tiseuee. Some of these method8 are listed on Table VII. In our laboratory, analyeie of Thiamphenicol in pharmaceutical preparation was carried out using a Shimadzu GC 9 A equipped with Flame Ionization Detector and CR 3 A Integrator. Column : 3 m glaee column, 3 mm i.d., 5 X OV-101 on Chromosorb W 60-80 mesh ; %tector: 3OO0C : Injector : 3OO0C Oven : ieothermal 260 C. The procedure of analysis involve8 extraction of Thiamphenicol from pharmaceutical preparation with ethyl acetate after derivatization w i n g TMCS - HMDS reagent. Chloramphenicol wa8 used a6 internal standard and 5.0 p L of aliquot8 in ethyl acetate were indected. A linear correlation between peak area ratio concentration wa5 obtained from 0.04 to 20.00 mg/mL ( r = 0.9810, n = 14). The limit of detection and limit of wantitation were 1.50 pg/mL and 5.00 pg/mL respectively. Precieion, eXpre88ed as RSD wa8 1.12% (n=10) and the X recovery was 99.80 2 0.76 (mean X recovery 2 SD). The chromatogram I s shown in Figure 1 . 0
TAB=
COlWnn
VII : GLC methods for t h e a n a l y s i s of Thiamphenicol - R I S e t h e r

Packing Material
Carrier
Gas
Column Temp
Detector Sample
Ref
15
U-ehaped borosilicate glass column(l50 x 0 - 4 c m ) U-shaped stalnlese-steel column ( 7 5 x 0.4 c m )

%
P
1-5% DBGS on Chromeorb W ( 6 0 - 80 m s h ) 1.5% OV-17 on Shimalite W (80 - 100 mesh)
nitrogen
185OC
3~ ECD
plasma,
urine
plaema,
nitrogen
215OC
3H ECD
urlne
helium
268OC
15
Glass column (10 f t x 2 nm)
6.6% OV-3 and 3.3% OV-17 on Chromosorb W-AW DMCS

(70 - 80 m e s h )
0.16% OV-3 on GLC 110 g l a s e bead6 (100 - 120 mesh)
F I D
plasma, 13 u r i n e and tiesue, homogenat e urine
Glass column (3 m x 2 c m )
nitrogen
238OC
P I D
12
U-shaped g l a s s column ( 1 - 6 m x 2
UEII)
3% OV-17 on Gas-Chrom Q (60 - 80 m e h )
2 0 0 ~ ~83Ni ECD plasma o r

amniotic fh i d
(continued)
TABLE VII (continued)

Column Packing Material Carrier Gas nitrogen Column Temp Detector Sample
Ref
16
3%OV-17 on U-shaped g l a s s column (130 en x 4 uun) Gas-Chrom 8 r ( 6 0 - 80 mesh)
Glass column (2 m x 2 nun)

e
'A
1%SE-30 on Gae-Chrom Q
nitrogen
2 1 0 ~ ~63Ni ECD prostatic t isme, blood and esminal fluid 23OoC MS urine
12
10
Glass column ( 1 m x 3 rum)
W-AW DMCS
(60
1.5% OV-17 on Chromoeorb
80 mesh)
gin) 290 c,
(6
1 8 0 ~ ~MS
urine
5 C/min
direct method; without TMS derivatization
486
5.5. Microbiological Methods
In our laboratory, the microbiological procedure to determine the potency of Thiarnphenicol in pharmaceutical preparations is based on the cylinder-plate method using Sarcfna Lute& ATCC 9341 as the test organism. This method is also used for the assay of Chloramphenicol ( 2 4 ) . This method ie a8 follows : U e e one or more plates for each sample (usually three) and fill three cylinders on each plate with the 50 clg/rnL standard and three cylinders with the sample diluted to an estimated 50 pg/mL with pH 6 phoephate buffer (1% potassium phosphate buffer adjusted to p 6). H alternating standard and s p p l e . Incubate the plates for 16 to 18 hours at 32 to 35 C and measure the diameter of each circle of inhibition. Average the zone readings of the standard and the sample on the plate8 used. The concentration corresponding to the corrected values of zone sizes was read from the standard curve which consisted of three dose levels (24,251. The results of the microbiological assay method for Thiamphenicol when compared to those of W method (See 5.2.) showed a significant linear correlation ( rcalc = 0.42 : rtable= 0.36 for P < 0.05 ) and no significant difference ( tcalc. = 0 . 5 8 ; ttable= 2.75 for P < 0.01 1 between the two methods
ACKNOWLEDGEMENTS
The authors are thankful to P.T. HILAB SCIENCETAMA Jakarta - Indonesia for measuring the NMR spectra; Dre. Moegihhardjo.Laboratorium Dasar Bersama, Airlangga University for for recording the DSC thermogram.
REFERENCES
1.
2.
British Pharmacopoeia, Her Majeetys Stationery Office, London p.567 11988). Deutschee Arzneibuch, 9 Ausgabe : Deutecher Apotheker Verlag Stuttgart Govi-Verlag GmbH Frankfurt p.1382 - 1383 (1986).
THIAMPHENICOL
487
Martin Dale The Extra Pharmacopoeia, Twenty Ninth Edition, J.E.F. Reynolds, Ed, The Pharmaceutical Press, London, p.323 - 324 (1989). 4. The Merck Index, Eleventh Edition, Merck and Co., Inc., R8hway.N.J.. USA p.1465 (1989). 5. Indonesia Index of Medical Specialities Volume 20 Number 3 Cocabo S.C. and Kin P.T. Ed, p.130 - 134 (1991). 6. H.W. Dibbern , W and IR Spectra of some important drugs Lditio Cantor Aulendorf 1978. 7. P.M.Draguet-Brughmane, R. Bouche et C. De Rauter Pharm. Acta Helv. 54 Nr. p.74 - 77 (1979). 8. E.W. Mc. Chesney, R.F. Koss, J.H. Shekosky and W.H. Deitz, J. h e r . Pharm. Ass. 49 p.762 - 766 (1960). 9. T.A. Plomp and R.A.A. Haes, J. Chrom 121 p.243 250 (1976). 10. T-Nakagawa, H. Masada and T. Uno. J. Chrom 111 p.355 - 364 (1975). 11. T.A.Plomp, The distribution o f Thiamphenicol in various human tissues and body fluids : some therapeutical toxicological implications ; Rijkeuniversiteit te Utrecht, Dieertation p.30 36 (1979). 12. F. Cattabeni, A. Gazzaniga ; Postgraduate Medical Journal 50 (euppl.5) p.23 - 27 (1974). 13. A. Gazzanfga, 6. Pezzotti, A.C. Ramusino, J.Chrom. 81 p.71 - 77 (1973). 14. T. Uesugi, H.Ikeda, R. Hori. K. Katayama and T. Arita, Chem. Pharm. Bull. 22 p.2714 - 2722 (1974). 15. T. Aoyama, S . Iguchi, J. Chrom. 43 p. 253 - 256 (1969). 16. T.A. Plomp. J.J. Mattelaer and R.A.A. Maes, Journal of Antimicrobial Chemotherapy 4 p.65 - 7 1 (1978). 17. Dian Lestari Trisna, Analisis Kloramfenikol dan Tiamfenikol beserta metabolitnya dalam kemih subyek normal, M.S. Thesis Faculty of Postgraduate studies, Airlangga University p.139 - 140 (1987) B.M. Limeon, H.D., P.S. Laceon, M.D., M . F . Soto, M.D. and D.R.Martinez,JR, H.D. Current Therapeutic Research Vol. 17 N0.4 p.335 - 339 (1975). 19. D. Van Beers, E. Schoutens, H.P. Vanderlinden and E. Yourassoweky Chemotherapy 21 p.73 - 81 (1975) 20. F.0 Grady, N. J. Pearson and C. ,Dennis, Chemotherapy 26 p.116 - 120 (1980).
3.
488
21.
K. Kitoh, T.
22.
F r a g i l i t i n Y . Nayean, ct al. Ced3 S a f e t y P r o b l e m R e 1 ated to Chl or ampheni col and mi ampheni col Therapy Raven Press, N e w York p. 1 4 ClQ813. L. Dettli, G. K r i s h n a , V. F e r r a r i and D . D . B e l l a , P o s t g r a d u a t e Medi cal J o u r n a l 50 C suppl 5 ) p. 17-22
Nagasu, N. Set0 and ? . lTomoeda, Comparative Studiee on the Inactivation of Thiamphenicol and Chloramphenicol by Bacteroides
C19743.
2. 3
84.
25.
T. Nagata. and M. S a e k i , J. Chromatogr. B i o m e d . A p p l . 5Bs : 471 476 C A p r 193 C l Q Q l 3 . D.C. Grove, and W. A. R a n d a l l . Assay Methods of A n t i b i o t i c A L a b o r ator y Manual Medical Encycl opedia, I n c . p. be 88 Cl-3. W. H e w i t t . , Microbiological Assay, A n I n t r o d u c t i o n to Q u a n t i t a t i v e P r i n c i p l e s and E v a l u a t i o n , Academic P r e s s N e w York S a n F r a n s i s c o London, p. 40-42
C19773.
TOLAZAMIDE
John K . Lee, Kazimierz Chrzan and Robert H . Witt
Rh6ne-Poulenc Central Research
500 Arcola Road

Collegeville, PA 19426-0107
489
490
JOHN K. LEE ET AL.
CONTENTS
I.
A.
Introduction Therapeutic Category B. Appearance Description

A.
11.
B.
C. D. E. F.
111.
Nomenclature I. Chemical Names 2. Generic Name 3. Proprietary Names Formula 1. Empirical 2. Structural 3. Registry Number Molecular Weight Elemental Composition Dissymmetry Appearance, Color, Odor
Physico-Chemical Properties
A.
B. C. D. E.
F.
Properties of the Drug Substance Solubility Stability and Storage Dissociation Constant Spectral Properties 1. Ultraviolet Spectrum 2. Infrared Absorption Spectrum 3. Mass Spectrum 4. Nuclear Magnetic Resonance Spectrum Thermoanalytical Behavior 1. Melting Range 2. Loss on Drying
TOLAZAMIDE
49 I
3.
4.
Differential Thermal Analysis Thermogravimetric Analysis
IV. V.
Synthesis Pharmacokinetics
A.
B.
Absorption Metabolism
VI.
Determination in Pharmaceuticals
A. B. C. D. E. F. G. H.
Dissolution Testing High Performance Liquid Chromatography Thin-Layer Chromatography Spectroscopic Spectrophotometric Non-Aqueous Titration Compiexometric Titration Volumetric
VII.
Determination in Biological Matrices

A.
B.
High Performance Liquid Chromatography Gas-Liquid Chromatography
VIII. References
492
JOHN K.LEE ET AL.
TOLAZAMIDE I. INTRODUCTION
A.
Therapeutic Category
Tolazamide is a member of a C A S S of ora hypoglycemic agents chemically known as the sulfonylureas which includes other compounds such as tolbutamide, chlorpropamide, glyburide and glipizide. All are arylsulfonylureas containing substitution on the urea and benzene groups. They are effective in the treatment of Type I1 diabetes mellitus which usually occurs in later life and in which the pancreas has retained its ability to secrete insulin. Individuals with this type of diabetes are not dependent on insulin nor are they usually prone to ketosis. The typical individual who would benefit from tolazamide therapy would be one whose Type I1 diabetes developed after the age of forty, one who requires less than 4 0 - 5 0 units of insulin daily, has no history of ketoacidosis, and who has had the disease no longer than 5-10 years (1). Tolazamide, as well as the other sulfonylureas, is ineffective in the treatment of Type I diabetes mellitus which usually occurs in children or young adults in whom the pancreas has lost its capacity to secrete insulin. These individuals require injected insulin to control their diabetes. It is also contraindicated in pregnant women as well as in patients with severe liver disease. The mechanism of action of tolazamide, typical of the other sulfonylureas, is its ability to stimulate the secretion of insulin from functioning beta cells of pancreatic islet tissue (2,3). In addition, tolazamide, as well as some of the other sulfonylureas, has been shown to be effective in lowering the blood glucose levels of non-insulin dependent diabetics whose disease does not respond to diet, caloric restriction or physical activity. Treatment with these agents is most effective when combined with a physical regimen and close monitoring of blood and urine glucose.
TOLAZAMIDE
493
Several precautions must be observed in tolazamide therapy as with other oral sulfonylureas. These agents have the potential for inducing mild to severe hypoglycemia which can cause irreversible brain damage and coma. This is possible with chlorpropamide with its long duration of 10-18 hr for tolazamide) and action ( " 6 0 hr compared with its limited metabolism (2,3). In addition to this, the results of one particular study tended to show that the administration of these oral hypoglycemics can increase the risk of cardiovascular mortality as compared to treatment with either diet plus insulin or diet alone (4). However, this cited study has been criticized for several reasons ( 3 ) . Tolazamide should not be administered t o patients with inadequate hepatic or renal function due to the vital role that the liver and kidneys have in the metabolism and excretion of this drug and its metabolites. Major side effects may include convulsions, ringing in the ears, breathing difficulties, tingling in the hands or feet, visual disturbances and blood disorders. Minor side effects include nausea, rash, sun sensitivity, weakness, headache and loss of appetite.
B.
History
As early as 1942, Janbon and co-workers ( 5 ) discovered that p-amino-benzenesulfonamido-isopropylthiadiazole,a sulfonamide, exhibited hypoglycemic properties. It was not until 1957 that Loubatieres concluded from his extensive studies that the hypoglycemic effect was due to the stimulation of the pancreas to secrete insulin ( 6 ) . Subsequently, a host of hypoglycemic agents of the sulfonylurea class were synthesized, notably, tolbutamide (7, 8 ) .
494
JOHN K . LEE ET AL.
In 1962 Wright and Willette synthesized tolazamide ( 9 ) and noted that the hypoglycemic structure-activity relationship as observed in intact male Sprague-Dawley rats was quite specific. Using tolbutamide as a reference hypoglycemic agent, they noted t at (a) an -NR2 group attached to the urea nitrogen (N ) where -NR2 was a cycloalkylamino ring such as pyrrolidino, piperidino or hexamethyleneimino (or their homologs) conferred high activity, (b) methyl substitution on N2 (-CH3 replacing ZH) lowered activity, and (c) steric hindrance relative to N lowered activity. Tolazamide, yith a hexamethyleneimino ring and hydrogen attacpd to N as well as low steric hindrance relative to N was shown to have high antidiabetic activity ( " s i x times that of tolbutamide) (10). Other investigators also obtained similar results in short term studies with normal and diabetic subjects. In addition, long term studies in diabetics have confirmed these same findings. For a complete review on sulfonylurea hypoglycemic agents, the reader is referred to the article by Jackson and Bressler (11). Currently, tolazamide is widely prescribed and is often the drug of choice under certain conditions.
11.
DESCRIPTION Nomenclature Chemical Names
A.
1.
N-[[(Bexahydro-1~-azepin-l-yl)-amino]carbonyl]-4-methylbenzenesulfonamide; l-(hexahydro-1H-azepin-l-yl)-3-(p-tolylsulfonyl)urea: N-(p-toluenesulfony1)-N'-hexamethyleniminourea

2.
3.
Generic Name Tolazamide Proprietary Names Diabewas, Norglycin , Tolinase , Tolonase

@
QJ,
TOLAZAMIDE
495
B.
1.
Formula Empirical C14H21N303S
2.
Structural
3.
Registry Number (12) Chemical Abstracts; 1156-19-0
C.
Molecular Weight 311.41
D.
C 54.00%; H 6.80%; N 13.49%; 0 15.41%; S 10.30%

E. Asymmetry
Tolazamide possesses no asymmetrically substituted carbon atoms and is optically inactive.
F.
Appearance, Color, Odor
Tolazamide is a white to off-white odorless crystalline powder
496
JOHN K. LEE ET AL.
111. PHYSICO-CHEMICAL PROPERTIES

A.
Properties o f the Drug Substance
Figure 1 shows a photograph o f crystals of tolazamide drug substance (USP reference standard Lot G). A few particles of the tolazamide drug substance were dispensed in silicone oil on a clean glass slide. The mixture was examined using a Zeiss Axiophot polarizing microscope with 2OOX magnification
Figure 1. Crystals of tolazamide drug substance in silicone oil.
B.
Solubility
Tolazamide is very slightly soluble in water, slightly soluble in alcohol, soluble in acetone and freely soluble in chloroform (1,131.
C.
Stability and Storage
Tolazamide is stable at ordinary temperatures; however, when heated to decomposition, it emits toxic NOx and SOX fumes. Tolazamide tablets should be stored between 15-3OOC in well closed containers (1.14).
TOLAZAMIDE
491
D.
Dissociation Constant (-SOzNHCO- Group) pKa 3 . 6 (25OC) pKa 5 . 6 8 ( 3 7 . 5 O C ) ( 1 5 )
E.
Spectral Properties
1. Ultraviolet Spectrum ( 1 6 )
run was obtained using a Hewlett-Packard 845QA
U.V.
The ultraviolet spectrum (Figure 2 ) from 220 nm to 340
Spectrophotometer and a matched pair of Fisherbrand Suprasil cells for the reference and sample solutions. As seen, the main absorption maximum is at 227 run with other maxima occurring at 2 5 6 , 2 6 3 , 268 and 274 nm (ethanol). Shilar results have been reported elsewhere (13).
a a0
a70
~
am.
ao s
0.40
0.30
a20
0.10
Ethanol 227, 256, 263, 268, 274

-
ao
Wavelength (nm)
Figure 2. Ultraviolet absorption spectrum of tolazamide in ethanol.

(Reprinted with permission from reference 16; Copyright CRC Press, Inc., Boca Raton, m ] .
498
JOHN K. LEE ET AL.
2.
Infrared Absorption Spectrum
The infrared spectrum of tolazamide taken in a KBr pellet is shown in Figure 3. A Nicolet Model 205 Fourier Transform Infrared spectrophotometer was used to acquire the spectrum. As can be seen, four prin i a1 absorptipn peaks occur between approximately 1160 cm -5 and 1700 cmTable I gives the spectral assignments for these principal bands.
P
o
4000
l.
8800
: . . . . : . . . . : . . . . : . - - . : . . . - :
so00
moo
PO00
Leo0
1000
600
Wavenumber (cm-)
Figure 3. Infrared spectrum of tolazamide (KBr disk).
TABLE I- Spectral Assignments f o r Principal IR Absorption Bands of Tolazamide (17-19) Wavenumber, cm 1703 1456 -1 Intensity Strong Strong Assignment
C=O stretching
vibration
C=C stretching
1350
Strong
1167
Strong
vibration CH -scissoring stretch -SO asymmetric s tretching vibration (when linked to -NH-) -SO symmetric '9 t retching vibration
The infrared spectrum of tolazamide taken in a mineral oil mull is shown in Figure 4. The spectra shown in Figures 3 and 4 are both consistent with the chemical structure of tolazamide.
0
0 r (
0
9
a l
c
2:
.-
$:
I O , 0-
4000
3800
3000
2800
2000
1uoo
1000
uoo
Waveriurnber (cm-')
Figure 4.
Infrared spectrum of tolazamide (mineral o i l ) .
JOHN K. LEE ET AL.
3.
Mass S p e c t m (16)
The electron impact mass spectrum of tolazamide (Figure 5) was obtained on a Hewlett-Packard 5985B GC/MS and plotted on a Hewlett-Packard 9876 graphics plotter. The instrument was operated with an electron energy of 70 eV and with the ms source maintained at 200OC. The sample of tolazamide standard was introduced into the spectrometer by direct insertion probe and the gas chromatographic column was packed with a 3X OV-1 phase. As seen, the spectrum shows an extremely low abundance of a molecular ion peak M at a masslcharge (mlz) ratio of 311 and a base peak at m/z of 91. Other prominent ions in order of decreasing abundance occur with masses at 113, 155, 65, 98, 139 and 197. Figure 6 depicts the possible fragmentation pattern of tolazamide with the mlz ratios of fragment ions indicated.
TOLCIZCIMIDE
--
DIP
'@,I
Figure 5.
Mass spectrum of to1azami.de (electron impact mode ) .
(Reprinted w i t h permlaeion from reference 16; C o p y r i g h t CRC P r e s e . I n c . , Boca Raton, FL).
TOLAZAMIDE
511I
Fragment
MI Z
91 155 171 197 113
98
Figure 6.
Possible fragmentation pattern of tolazamide.
4.
Nuclear Magnetic Resonance Spectrum (16)
The proton N.M.R.spectrum of tolazamide (Figure 7) in CDCl /CD OD (containing 1% tetramethylsilane [TMS] as 3 3 internal reference) was obtained on a Varian T-60A NMR spectrometer ( 6 0 MHz). Before the spectrum was obtained, the sample solution was equilibrated to the probe temperature of 340C. Table I1 lists the chemical shifts (ppm) associated with the type of proton undergoing resonance absorption.
502
JOHN K. LEE ET AL.
\ , . ' ~ " " ~ ' , , ' , , , " ~ " " ~ " " , " . "~ , ' . " ' YSO YO0 350 300 ZSO 200 150 100
TOLAZAflIOE (COCLS/C03001
NflR
, ' I ' I
1 1
so
o nt
PPri
Figure 7.
Proton NMR spectrum of tolazamide.

(Reprinted with permission from reference 16; Copyright CRC P r e s s , Inc., ~ o c a xaton, m).
TABLE 11- Proton Chemical Shifts in Tolazamide (17) Chemical Shift, ppm, Approximate"
1.4-1 - 8
-N
2.4
2.7-3.2
3.8-4.2
-NH
-CO
-NH
Relative to tetramethylsilane (0.0 ppm) The proton undergoing resonance absorption appears in heavy type
I I JI . A / . A V I I >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-
170.70
P.sk
f-
to:
1Bl.M 174.70
Onsat- 160.01 J/g l22.tl.Y
I I
I-----
4.w
hb .o
$.a
lm.mx
1.6 46
Idb.a6-id6.M
Terriperalure ( C )
Pjgure
8.
Differential thermoanalytical behavior of tolazamide.
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
llb.00
190.00
&.w
_______
&.w
Figure
9.
Temperature ( C ) Thermogravimetric behavior of tolazamide.
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.
A.
PHARMACOKINETICS 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.
506
JOHN K.LEE ET AL.
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.
TOLAZAMIDE
507
0
CH3-54 --NH --C
II -NH
-N
TOLAZAMIDE
14hexahydroazepi n-1-yl)-3-p-to1ylsulfon ylurea
\
UNKNOWN
3
Metabolite
2
Identity
4
5 6
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) : a previously isolated J and identified (29).
Figure 11.
SO8
JOHN K. LEE ET AL.
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.
00
N
r-
m
0
crl d d
...
a ,
6
i4
rl
B
0 0
P
d
0
m
00
m
m
Y )
0 W 0 1.1 0 rl
rl W 0
@
H
1.1
0
B 3
@
0
V
r-l
4J
0)
ld
9
ll -
0,
e
V
9 w
v)
I n
t3
rl rl
'A 3
" N rl
d
ld U *4
rl .rl
v)
8"
m
.rl rl .rl
v)
TOLAZAMIDE
511
D.
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.
512
JOHN K.LEE ET AL.
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).
514
JOHN K.LEE ET AL.
3. 4. 5. 6. 7. 8.
Drug Evaluations Annual 1992, American Medical Association, 939-940, 953-959 (1992). Diabetes, 19 (supp. 2). 747-830 (1970).
M. Janbon, J. Chaptal, A. Vedel and J. Schaap, Montpell. med., 21-22, 441-444 (1942).
A. Loubatieres, Ann. N.Y. Acad. Sci., 71, 4-11 (1957). S.L. Mukherjee and A. David Ltd., Indian pat. 59,097 (1958)
H. Ruschig, W. Aumueller. G. Korger, H. Wagner, J.

Scholz and A. Baender, U.S. pat. 2,968,158 (1961 to Upjohn).
9. 10. 11. 12. 13. 14. 15. 16.
J.B. Wright and R.E. Willette, J. Med. Pharm. Chem., 5, 815-822 (1962). W.E. Dulin, H.L. Oster and F.G. McMahon, Proc. SOC. Exptl. Biol. Med., 107, 245-248 (1961). J.E. Jackson and R. Bresisler, Drugs, 22, 211-245 (1981). Merck Index, 11th Edition, Merck h Co., Inc. (1989). Clarkes Isolation and Identification of Drugs, 2nd Edition, The Pharmaceutical Press, London, 1029 (1986). Material Safety Data Sheet, United States Pharmacopeial Convention, Inc., No. 66800 (1985). Remingtons Pharmaceutical Sciences, 17th Edition, Mack Publishing Co., 977 (1985).
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).
J. McMurry, Organic Chemistry, BrookslCole Publishing
17. 18.
Co., Monterey, CA, 470-473 (1984). J.D. Roberts and M.C. Caserio, Basic Principles of Organic Chemistry, W.A. Benjamin, Inc., New York, 101, 773 (1965).
TOLAZAMIDE
515
19.
20.
R . T . Conley, Infrared Spectroscopy, Allyn and Bacon, Inc., Boston, 179-181 (1966).
Ger. pat. 1,186,865 (1965 to Upjohn).
21.
H. Ueda and T. Nagai, Chem. Pharm. Bull. (Japan),

26(5), 1353-1356 (1978).
22.
I. Butula, V. Vela and M.V. Prostenik, Croat. Chem. Acta, 52(1), 47-49 (1979).
Fr. pat. 1,458,907 (1961 to Upjohn). F.J. Marshall and M.V. Sigal, Jr., J. Org. Chem.. 23,
927-929 (1958).
23. 24. 25. 26. 27.

28.
Physicians Desk Reference@ , 45th Edition, Medical Economics Co. Inc., 2259 (1991). M.C. Balodimos and A. Marble, Curr. Ther. Res., 13(1),
6-12 (1971).
C.E. Wright I11 and E.J. Antal, (Abstract 66), DICP,

19, 458 (1985).
R.C. Thomas, D.J. Duchamp, R.W. Judy and G.J. Ikeda, J. Med. Chem., 21(8), 725-732 (1978). A.A. Forist and R.W. Judy, J. Pharm. Phamacol., 26(7),
565 (1974).
29. 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.
32.
United States Pharmacopeia, 22nd Revision, 1384 (1990).

K. T s u j i and R.B. Binns, J. Chromatogr., 253(2),
227-236 (1982).
33. 34.
W.F. Beyer, Anal. Chem., 44, 1312-1314 (1972).
K. Tsuji (Editor), GLC and HPLC Determination of Therapeutic Agents, Chromatographic Science Series, 9(3), Marcel Dekker, Inc., 1080, (1979).
516
JOHN K. LEE ET AL.
35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.
G. Severin, J. Chromatogr., 386, 57-63 (1987). United States Pharmacopeia, 22nd Revision, 6th Supplement, 2916-2917 (1992). P.G. Takla and S.R. Joshi, J. Pharm. Biomed. Anal.,
1(2), 189-193 (1983).
A.A. Al-Badr, Spectrosc. Lett., 16(9), 673-682 (1983). G.A. Saleh and H.F. Askal, J . Pharm. Biomed. Anal.,
9(3), 219-224 (1991)
9
S.A. Hussein, A.M.I. Mohamed and A.A.M. Abdel-Alim, Analyst (London), 114(9), 1129-1131 (1989). S.P. Agarwal and M.I. Walash, Indian J. Pharm., 34(5),
109-111 (1972).
L.O. Guerello and J. Dobrecky, Rev. Asoc. Bioquim. Argent., 33(178-179). 185-188 (1968). P.L. Buldini, V. Rossetti and A. Toponi, Freaenius Z. Anal. Chem., 328, 265-267 (1987). B.J. Starkey, G.P. Mould and J.D. Teale. J. Liq. Chromatogr., 12(10), 1889-1896 (1989). 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
517
Copyright 0 1993 by Academic Press, Inc All rights of reproduction in any form reserved.
518
FARID J. MUHTADI AND ABDUL FATTAH A. A. AFIFY
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
2.1 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
2.8.1 2.8.2 2.8.3 2.8.4
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
VINCRlSTlNE SULFATE (SUPPLEMENT)
519
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
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 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 ( ) . However, the confirmations of the nine 6] 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) . (Vincristine sulfate).
52 1
W f i N > o ;
HFOOC
\\\
H
\
\
\
\
\
\
\
H,CO
1.5
C, 66.97%; H, 6.84%; N, 6.79%; 0, 19.40% (vincristine).

C,
59.86%; H, 6.33%; N , 6.07%; S, 3.47%; 0, 24.27% (vincristine sulfate.
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 ) .
522
FARID I. MUHTADI AND ABDUL FATTAH A. A. AFIFY
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 S p e c i f i c Optical Rotation
+ 17"
[,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 ) .
+ 85 .'
2.4
(c=0.8) f o r t h e s u l f a t e ( 9 ) .
p Range (The s u l f a t e s a l t ) H -
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
2.8
Spectral Properties
2.8.1
U l t r a v i o l e t SDectrum fUV1
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 n u s i n g a Pyem Unicum 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. n m
218 252 285 293
log
A(1%, lcm)
568.75 188.75 165.60 185.6
4.72 4.24 4.18 4.23
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
W
0 0 0
3 3
0 0
-T
c
0 0
0 0
Lo
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
'H-NMR
Spectrum
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 . 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
1
4 H , aromatic p r o t o n s of catharanthine
~ c 9 f , 1 0 f , l l& 121)f
H , aromatic proton of
6.66 ( s ) 6.42 ( s ) 3.913 (s) 3.757 ( s ) 3.650 ( s ) 2.068 (s)
v i n d o l i n e (C,)
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). 3H, methoxy p r o t o n s ( C l l ) . 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 3H, e s t e r p r o t o n s of v i n d o l i n e (C16). 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). singlet.
m = multiplet. s
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+
FIGURE 3 :
' H
- NMR SPECTRUM OF
VINCRISTINE.
528
FARID J. MUHTADI AND ABDUL FATTAH A. A. A F I N
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).
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 - 50C 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
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.
[61*
4.2
Synthesis of Vincristine Two methods are available:
- The preferred method is to oxidize the N-methyl group

-0) low temperature 1 6 ' 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-
of vinblastine with chromium trioxide in acetone at
esis of vinblastine from catharanthine and vindoline has also been reported (40).
530
FARID J. MUHTADI AND ABDUL F A l T A H A . A. A F I N
Scheme I : Synthesis of Vinblastine -
4 !
vindol ine coupling

+
red.
VlNCRlSTINE SULFATE (SUPPLEMENT)
53 I
i) TL(OAC)~
-
i i ) NaBH4
R = vindoline
[51
Oxid.
A
--
CO 2 C H ~
OH
H3C0 H 3co OHC OHC
II
C02CH3
[71
532
Scheme 11. Synthesis of V i n c r i s t i n e
It
II
Microbial
u '
Vinblast'.le
Oxidation cr20,/acetone~
~
Vincristine
[31
[I1
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
[31
Cell-free extracts leaves (7. roseus
or - Cell-free homogenates
of C. roseus cell suspension cultures
COOCH3
H3C0
COOCH3
535
roseus plants have shown that the enzyme catalyzed synthesis of vindoline [l] from tryptamine [ 6 ] and secologanin [7l (44,461.
Further studies with cell-free systems from
Catharanthus
CHO
:-i COOC H
m$ $& YToGlu
4 I
Enzymes from
, 4
C . roseus
OCH3
CH3
! -OH
C02CH3
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 ) .
OCH3
%]
3
1-free extract
L -
[ 11
OH C02CH3
J
14 vindoline [ c ]
/
C . roseus
leurosine Catharine
~41[31
[51
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
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 ) . g 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 m 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 m v i n c r i s t i n e s u l f a t e and 50 m of l a c t o s e . g g 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 infection i s present (1).
539
9.
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
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
BP ( ) 9.
usp (10) The infrared absorption spectrum o f a potassium bromide dispersion of vincristine sulfate, previously dried in vacuum at 4' for 16 hours, exhibits maxima only at the 0 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
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 0 Reference standard previously dried in vacuum at 4 ' 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.
Solvent System Toluene-chloroformdiethylamine (80:40:6) acetonit ri1e (5 :95)
Rf value
Ref.
Silica gel HF 254
2.
0.5
N LiOH/alumina Absolute alcoholMethanol Chloroform-methanol

(95 :5)
0.51
(55)
3.
Silica gel G Silica gel G Silica gel G
4.
5.
Ethy 1acetate- ab solute alcohol ( 3 : l ) n-Butanol-glacial acetic acid-water (4:1 :1)
0.18
0.16
6. Silica gel G
542
FARID I. MUHTADI AND ABDUL FATTAH A. A. AFIFY
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- Dragendorffs reagent (63). 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 ) .
543
9.5.2
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 0' 0' ' from 2 0 to 3 0 at 5 min.-l. Carrier gas:Nitrogen, at a flow rate 30 ml/min.-l Detector: Condition:
FID.
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:
544
System 3 : Column: Mobi 1e phase : Retention time :
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)
-1
Flow r a t e : l . 5 m l min.
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. 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. Gradient e l u t i o n with aqueous 50 t o 85%methanol Mobile 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. 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) p Bondapak C18 (30cm x 3.9mm), r e v e r s e d - p h a s e Column: 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 phase : 7.2) - MeCN (51:49). -1 Flow r a t e : 1 m l min. System 5 : System 4:
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 : 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. 0.01M diammonium hydrogen orthophosphate - MeCN (25:75), pH 7.0.
Column: Mobile phase :
D e t e c t i o n : UV a t 2 5 4 and 280 nm.
545
System 7 :
Column: Mobile phase : I nt erna 1 standard :
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, M C N , e 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 : 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 .
Column: Mobile phase :
D e t e c t i o n : UV a t 298 nm. System 9 : Column: Mobile phase : 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).
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 n m 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 c x 4.6 m ) i s packed with chemm m i 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 ) .
546
FARID J. MUHTADI A N D ABDUL FATTAH A. A. AFIFY
T h e mobile p h a s e : Diethylamine (5 ml) is mixed with 295m1 H water and adjusted with phosphoric acid to p 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 X I p. 1116. X 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
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.
548
FARlD J. MUHTADI AND ABDUL FATTAH A. A. AFIFY
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S.B. Hassam and C . R . Hutchinson, T e t r a h e d r o n L e t t . , ( 1 9 ) , 1681 l(1978).

A . I . S c o t t , F . G u e r i t t e and S.L. Lee, J . Am. Chem. S O C . ,
100 -( 1 9 ) ,
6253 (1978). Scott,
R . L . Baxter,, C . R . D o r s c h e l , S.L. Lee and A . I . J . C . S . Chem. Commun., 257 (1979).
55 1
44.
J . P . Kutney, L.S.L. Choi, T . Honda, N . G . Lewis, T . S a t o , K . L . S t u a r t and B . R . Worth, Helv. Chim. Acta, - 2088 65, (1982).
W.R. McLauchlan, M . Hassan, R . L . 39, T e t r a h e d r o n , - 3777 ( 1 9 8 3 ) .
45. 46.
B a x t e r and A . I . S c o t t ,
J . P . Kutney, B . Aweryn, L.S.L. Choi, T . Honda, P . Kolodzeiczyk, N . G . Lewis, T . S a t o , S.K. S l e i g h , K . L . S t u a r t and B . R . Worth, T e t r a h e d r o n , - 3781 ( 1 9 8 3 ) . 39,
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M a r t i n d a l e , "The E x t r a Pharmacopoeia", 2 9 t h e d . , J . E . F . Reynolds E d i t . , p . 655, The Pharmaceutical P r e s s , London (1989). M.R. Cohen, Hosp. Pharm., - 694 ( 1 9 8 9 ) . 24,
48. 49.
Product I n f o r m a t i o n : Oncovin (R) , V i n c r i s t i n e S u l f a t e , E l i L i l l y and Company, I n d i a n a p o l i s I N (1990). H . W . Van den Berg, Z . R . Desai, R . Wilson and e t a l . , Cancer Chemother. Pharmacol., -, 215 ( 1 9 8 2 ) . 8
V.S. S e t h i , D . V . J a c k s o n , D . R . Cancer Res., - 3551 (1981). 41,
50.
51
W h i t e and et a l . ,
52
"Handbook o f C l i n i c a l Drug Data", J . E . Knoben and P . O . Anderson, E d i t s . , 6 t h e d . , Drug I n t e l l i g e n c e P u b l i c a t i o n s I n c . , Hamilton, I L ( 1 9 8 8 ) .

V.S. S e t h i and J . C . Kimball, Cancer Chemother. Pharmacol., -, 111 (1981). 6
53.
54.
Goodman and G i l m a n ' s , "The Pharmacological Basis o f T h e r a p e u t i c s " , - i x e d . , p . 1289, A . G . Gilman, L.S. S Goodman and A . Gilman E d i t o r s , Macmillan P u b l i s h i n g Co. I n c . , N e w York ( 1 9 8 0 ) .
I.M. J a k o v l j e v i c , L . D . 53, S c i . , - 553 ( 1 9 6 4 ) .
55.
Seay and R . W .
S h a f f e r , J . Pharm.
56.
A . Yaneva and T . Tomova, T r . Nauchnoizsled. Khim-Farm. I n s t . , 11_, 205 (1981); CA, - 14923411 (1982). 96, J . Wang, J . Z a d e i i and M . S . Lin, J . E l e c t r o a n a l . Chem. I n t e r f a c i a l E l e c t r o c h e m . , __ ( 2 ) , 281 ( 1 9 8 7 ) . 237
57.
552
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"The United S t a t e s Pharmacopeia", USP X I X , p . 536, United S t a t e s Pharmacopeial Convention I n c . , R o c k v i l l e , MD (1975). I.M. J a k o v l j e v i c , J . Pharm. S c i . , - 187 (1962). 51, N . R . Farnsworth, R . N . Blomster, D . Damratoski, W.A. Meer and L . V . Ca.mmarato, L l o y d i a , - 302 ( 1 9 6 4 ) . 27,
N . R . Farnsworth and I . M . H i l i n s k i , J . Chromatog., -, 18 184 (1965).
A . N . Masoud, N . R . Farnsworth, L . A . S c i u c h e t t i , R . N . Blomster and W . A . Meer, Lloydia, - 202 (1968). 31,
E . G . C . C l a r k e , " I s o l a t i o n and I d e n t i f i c a t i o n of Drugs", Vol. 1, p . 595, The P h a r m a c e u t i c a l P r e s s , London (1978).
59. 60. 61 62. 63. 64. 65 66 67. 68. 69. 70. 71. 72. 73.
P . Horvath and G . I v a n y i , Acta Pharm. Hung., - 4 ) , 52( 150 (1982); Anal. A b s t . , - 1E15 (1983). 44, M. Gazdag, K . M i h a l g f i and G . S z e p e s i , F r e s e n i u s Z . Anal. Chem., - 105 (1981). 309, M . D Smet, S . J . P . Van B e l l e , 5 . A . Storme and D . L . e Massart, J . Chromatog. , - 309 (1985). 345,
- , 83 424(1)
D.E.M.
Vendrig, J . Teeuwsen and J.J.M. H o l t h u i s , I b i d . , (1988).
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S . A u r i o l a , V . P . Ranta, T . N a a r a n l a h t i and S.P.
L a p i n j o k i , J . Chromatog., 474(1)
, 181 (1988).
49,
S . Mandal and M . L . Maheshwari, I n d i a n J . Pharm. S c i . ,
205 (1987).
T . N a a r a n l a h t i , M . Nordstrom, A . Huhtikangas and M. 410(2) Lounasmaa, J . Chromatog. , - , 488 (1987). J . P . Renaudin, I b i d . ,
291,
165 (1984).
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308,
ss3
74. 75.
D . E . M . V e n d r i g and J.J.M. H o l t h u i s , T r a c e T r e n d s A n a l . Chem., -( 4 ) , 1 4 1 ( 1 9 8 9 ) . 8
Atta u p Rahman, M . B a s h i r , M . H a f e e z , N . P e r v e e n , J . 47, Fatima and A . N . M i s t r y , P l a n t a Med., - 246 ( 1 9 8 3 ) .

G.14.
76.
S . T a f u r , W.E. J o n e s , D . E . Dorman, E . E . Logsdon and Svoboda, J . Pharm. S c i . , - 1 9 5 3 ( 1 9 7 5 ) . 64,
77.
J . D . T e a l e , M . C . J a c q u e l i n e and V . Marks, Br. J . C l i n . P h a r m a c o l . , -( 2 ) , 169 ( 1 9 7 7 ) . 4
78.
79. 80.
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V.S. S e t h i , S . S . B u r t o n and D . V . J a c k s o n , C a n c e r C h e n o t h e r . P h a r m a c o l . , -( 3 ) , 183 ( 1 9 8 0 ) . 4
S . P . Lopinjoke, H.M. Verajaankorva, A . E . Huhtikangas, T . J . L e h t o l a and M . Lounasmaa, J . Immunoassay, -( 1 - 2 ) , 7 113 (1986).
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
555
Copyright Q 1993 by Academic Press, Inc. All rights of reproducllon in any form reSeNed.
556
CHRISTIANAH M. ADEYEYE AND EUGENE BARABAS
CONTENTS
1.
Introduction 1 .l Structure 1.2 Nomenclature The Monomer (N-Vinyl-2-Pyrrolidinone) 2.1 Physical Properties
2.
3. Polymerization
4.
4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8
Description of the Polymer Appearance Molecular Weight MoleciJlar Weight Distribution Glass 'Transition Temperature Hygroscopicity Solubility Viscosity Chemi'cal Nature and Stability
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
551
7.12
7.2
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
7.3
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
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 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 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
7.37
7.5
8.
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
559
8.3 8.4 8.5
Uptake of PVP into Isolated Tissues Metabolism Excretion 8.51 Animal Studies 8.52 Human Studies 8 . 5 3 Biliary Excretion
9.
Toxicity 9.1 Acute Toxicity 9 . 2 Subchronic Toxicity 9 . 3 Chronic 9 . 4 Local Tissue Damage 9 . 5 Carcinogenicity Mutagenicity Cryoprotection Anticarcinogenicity Ackowledgements References
10. 11. 12. 13. 14.
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
___t
HOCH2Cz CCH20H
(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 100.5C (213OF) 98.4OC (213OF) 2.07 cps 1.04 1.51 1
POVIDONE
563
3.
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
The scheme of the free radical polymerization with hydrogen peroxide is as
follows:
Initiation
H202
-*
+HO.
HO.+ HO.
H2C=CH
00
+ n.
HOHC-CH.
Propagation
HOHC-CH.
OHC-CH
Termination
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. ,,
concentration in g/100 ml solution, viscosity of the solution compared to that of the solvent = = 1000 KO
=
566
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)the simple arithmetic mean is 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
1
X X
Mol. Wt.
Relative Weight of Each Fract.

=
2,000,000
1,00,000 Total
2,000,000
1,000,000
10
11
3,000,000
= 273,000amu
Mn
3,000,000
11
Weiaht Averaae Molecular Weiaht Relative Weight 01 Each Fraction Mol. Wt. of Molecules in Each Fraction
X X
Weighted Mol. Wt. of Each Fract.

~~
2
1
2,000,000
100,000
Total
4,000,000 100,000 4,100,000
MW =
4,100,000
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. dg
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.
568
Fiaure 2
100
90 80
70
- t j60 s o
0,
4 40
30
20
10
0 lo3
10'
tos
'10'
Nl
K-value vs viscosity-average molecular weight
Mu
4.3
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
569
Fiaure
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.
570
Table 2 Glass Transition Temperatures of PVP Measured K-value

PVP K-15 PVP K-25 PVP K-30 PVP K-60 PVP K-90 PVP K-120
To. OC
14.9 22.5
27.5
130 160 163 170 174 176
55.5
89.6
122.0
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
s
.$
J3
CQ
40
,-
P
5:
c .
20-
20
40
60
80
Rcktive atmospheric humidity, %
POW DONE
57 I
4.6
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 Methanol Ethanol Propanol Butanol Cyclohexanol Chloroform Dichloromethane Di(ethylene glycol) Poly(ethy1ene glycol) 400 Propylene glycol 1,4-Butanediol Glycerol 2-Vinylpyrrolidone Triethanolamine Formic acid Acetic acid Propionic acid 2-Pyrrolidone N-Methylpyrrolidone y-Butyrolactone PVP is insoluble in: Ethyl acetate Acetone Dioxane Diethylether Pentane Cyclohexane Carbon tetrachloride Light petroleum distillates Toluene Xylene Mineral oil
I ,2-Dichloroethane
N-Methylpyrrolidone Ethylenediamine
572
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 s where the larger exponent indicates that methanol i 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
11IIllll 1tIlllIL
1 0 2 0 3 0 PVP. % 4 0 5 0 ~
EITcct of
PVP conccnlralion on lhe viscosity of aqucous solution
POVIDONE
513
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
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 -
Note:
Kinematic Viscosity (in centistokes)
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
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
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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 Because tablet together and prevents cracking and chipping at the edges (36). 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
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 lymphocytes, erythrocytes and leucocytes) (68). offers high recovery rate, 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.
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PVP is used as a diagnostic tool in gastroenterology and hematology
(85). has been used successfully as articular lubricant in the treatment of PVP
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 5.21 Other ADDlications (891 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.
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.
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5.29
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
1940s. 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
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
0.196
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 12 17 25 30 Molecular Weight (amu) Molecular Size, 1,055 18,594 33,378 41,578
2,900
9,000 29,000 45,000
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. L , D 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, 00). lymph nodes, bone marrow and other storage sites (97-1 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). is advisable t o inject it at alternative It 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. 7.1
ComDliance with PharmacoDoeias and Food Reaulations 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
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
583
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
584
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 -
. . ,
1 3
. ,
,
. ,
I S
, ,
.,
I 5
.. ,
t
.,
0
a 5
1 0
2 0
0 P?U
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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
-Methods 7.11 1 (Potassium Dichromate), 7.1 13 (Iodine) are used by all the Note
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.
586
Table 5
7.2 7.21
SDecificationS 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. 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 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) i s c h e w d
B can be found in
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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
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.
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.
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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
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 N add an additional 3.0 mL of 0.1O 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.
POVIDONE
593
7. Continue the titration with 0.10N sodium thiosulfate solution to a sharp decolorizing endpoint. Calculation
(mLi
% VP =
Ni
mL,,)
(5.555)
Weight of sample
100
where
mL, N, mLrs
= = =
total volume of iodine used in the sample, normality of iodine solution, volume of thiosulfate solution used for back titration normality for thiosulfate solution
N , 7.24 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
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.
D: WZIC:supply funnel. E: Sieamtubc. F: FUMCI for zddition o f

G: Rubbcr
alk21i solution (0 flzsk A. tubiog uitb I
C: Spray trap.
'
The f ~ ~ fina *re mm.
H: A small
clamp.
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.
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 not a tared crucible, ignite at a low temperature (about 500), 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
=
w -w * w, - wo
100
where Wo = W, = W, = 7.26 Heavv Metals
crucible weight sample and crucible before ashing sample and crucible after ashing
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
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 500-600C.

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
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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
The viscosi1:y is expressed either as kinematic viscosity ( y ) in square millimeters per second ( m m z s ~ from the expression )
y
= Kt
(q) in pascal seconds (Pa or as dynamic viscosity
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
602
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
by the formula
1
Orel
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
where In qra, the natural logarithm of q,el (-2.303 x log,, is too, the concentration must be specified. e. Intrinsic Viscosity
q,=,) here,
Estimate the intrinsic viscosity [ql,with the following equation using a single q,elvalue.
[ql
1 - 2(qrei
- 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 KPO ,, , (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
605
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.
606
CHIUSTIANAH M. ADEYEYE AND EUGENE BARABAS
3.
p p m acetaldehyde =
where
W,, W,
C,,, ,,,
= =
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.
POVIDON E
607
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.
608
CHKISTIANAH M . ADEYEYE AND EUGENE BARABAS
7.3 7.31
Other Characteristics 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.
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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
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) 1.6 6.2 7.0
B
BY
Y GY
(brown) (brownishyellow) (yellow) (greenishyellow) (red)
3.0 2.4 2.4 9.6 1.o
3.0 1.o 0.6 0.2 2.0
2.4
0.4 0.0
0.2 0.0
0.0 7.0
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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

Reference solutions BY
25.0 50.0 62.5 75.0 87.5 95.0 97.5 98.5 99.0
Reference solution
Standard solution BY
Hydrochloric acid (1 per cent m/V HCI)
BY, BY2 BY3 BY,
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
612
Reference solutions Y
Volume in milliliters
~ ~~~
Reference solution
Standard solution Y
~~
Hydrochloric acid (1 Der cent m/V HCI)
y 2 y 3
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
Reference solutions GY
II
Reference solution
Volume in milliliters Standard solution GY Hydrochloric acid (1 per cent m/V HCI)
II
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 Standard solution R Hydrochloric acid
11
(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

5.0
0.0 25.0 50.0 62.5 75.0 87.5

95.0
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613
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.
02.
2.
Using the 3 primary solutions prepared under B, a "B" standard is made as follows: "B Standard" Yellow Standard: Red Standard: Blue Standard: 1 % (w/v) HCI: Total:
30 mL 30 mL 24 mL 16 mL 100 mL
3.
From the "B" standard, the following 9 reference solutions are made: mL 1 % ( w / v ) HCI c or gs to 100 mL
mL Std "B"
75.0 50.0 37.5 25.0 12.5 5.0 2.5 1.5 1 .o

4.
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.
614
7.31 12 Determination of APHA Color Atmaratus Hellige Aquatester with APHA color disks. Reaaent Potassium Chloroplarinate 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.
- Cobalt Chloride Stock - 500 units.
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.
POVIDONE
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
I II
Ill
IV
Standard of opalescence (ml) Water (ml)
5.0 95.0
10.0
30.0 70.0
50.0
50.0
90.0
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,
R,H
> ROOH + R,.
616
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, O, S . (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, , per mL solution (100 ppm). O
4. Prepare HO 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.
POVIDONE
617
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) *
m * W
1000
= = = =
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
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 O iodine solution N 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 O sodium thiosulfate to clear N endpoint. Calculations
ppm H,o, =
mL t h i o s u l f a t e * N * 1.7 g sample
* loa
( v/ w )
7.33
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
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 o dilute sulfuric acid (1 in 5 ,2 mL of potassium iodide TS, and f 1 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 100C 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 100-105C 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.
622
C'HRISTIANAH M. ADEYEYE AND EUGENE BARABAS
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 100-105C 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 =
4 - w2 -x
W I
100
7.35
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
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.
626
Table 7 Methods for the Determination of Panicle Sizes
Method
Sieving Light Scattering Electronic Sensing Sedimentation Optical Microscopy
Preferred Size Ranae 50 m or greater 1 - 200 p m
Size Averam weight volume or weight volume weight number or volume
1 - 300 pm 2 - 200 pm
0.5
- 100 p m
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
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 1 0 taps per minute. a tapping action at a rate of 1 5 0
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.
POVIDONE
629
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:
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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63 1
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 Cover Screen Sp = Sample
= =
= Wt%
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
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 7.371 Determination of Bulk Density
400 - 600 gll 400 - 600 gll 400 - 600 g/l

110 - 250 g/l
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
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
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
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
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
powder. Calculation
. X I
by averaging the diameter of the flown out pile of the
1
x2
Angle of Repose ( a
= Arc Sin
Arc tg
X
1-
I
I
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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 In the solvent which gives the highest intrinsic viscosity (138). 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). ,
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
19
Synch-
Mom
Sampling port
Stlrring shaft
Ustcr Jxkttrd y i k ln
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.
640
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
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
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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)
10
Note 1: Note 2:
(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)
644
CHRlSTlANAH M. ADEYEYE AND EUGENE BARABAS
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
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 0 at a temperature between 3' 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
650
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: 0 1 dilution of the specimen, 0 express the results as "less than 1 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
1 0 (1 0 UL) 3 3 3 3 2 2 2 2 1 1 1 1 0 0 0
0
1 (1 pL) 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
3 2 1 0 3 2
1
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.
652
Table 11 MorDholoaic Characteristics of StaDhvlococcus aureus on Selective Aaar Media

Selective Medium
Characteristic Colonial Morpholoav Gram Stain
Vogel-Johnson Agar Medium Black surrounded b y yellow zone
Mannitol-Salt Agar Medium Yellow colonies with yellow zones
Baird-Parker Agar Medium Black, shiny, surrounded b y clear zones 2 to 5 mm Positive cocci (in c Iust e r s)
Positive cocci (in clusters)
Positive cocci (in clusters)

~
Table 12 Momholoaic Characteristics of Pseudomonas aeruainosa on Selective and Diaanostic Aaar Media
.
Medium Cetrimide Agar Medium Pseudomones Agar Medium for Detection of Fluorescin Medium for Detection of
Characteristic Colonial
Generally greenish
to yellowish
Fluorescence in Ultraviolet Light Oxidese Test Gram Stain
Greenish
Yellowish
Blue
~
Positive Negative rods
Piitive Negative rods
Positive Negative rods
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 Brilliant Green Agar Medium Description of Colony Small, transparent, colorless or pink to white opaque (frequently surrounded by pink or red zone) Red, with or without back centers
Xylose-LysineDesoxycholate Agar Medium
Bismuth Sulfite Agar Medium
Black or green
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. 8.1 Pharmacokinetics 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).
656
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 8.21
Distribution and storaae of PVP in the body 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
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
659
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 10C. 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-PVP using 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.
660
8.5 8.51
Excretion 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
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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
662
CHRISTIANAH M. ADEYEYE AND EUGENE BAR.4BAS
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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663
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
CHRTSTIANAH M. ADEYEYE AND EUGENE BARAIiAS
9.4
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
CHRISTIANAH M. ADEYEYE AND EUGENE BARAHAS
13.
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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POVIDONE
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POVIDONE
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250.
251.
252.
253. 254.
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255.
256.
257.
258.
682
259. B. Hulme and J. Hardwicke, Clin. Sci., 34, 515 - 534 (1968) 260. B. Hulme, Contr. nephrol., 1, 3 -8 (1975) 261. J. D. Blainey, Curr. Probl. Clin. Biochem., 2,85 - 100 (1968) 262. M. Robson, A. Elraz and J. B. Rosenfield, Israel J. Med. Sci., 9, 424 - 428 (1 973) 263. H. Campbell, P. 0. Kane, D. F. Muggleton and I. G. Ottewill, J. Clin. Path., 7, 252 - 260 (1954) 264. A. W. Wilkinson and I. D. E. Storey, Lancet, 2, 1269 - 1271 (1954) 265. H. H. Brautigam and J. Gleiss, Zeitschrift fur die gesamte experimentelle Medizin, 126,531 - 536 ( 1 956) 266. D. Morgenthaler, BASF Aktiengesellschaft. Unpublished report.
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POVIDONE
683
275.
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283.
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286.
287.
288.
289.
684
CHRISTIANAH M. ADEYEYE AND EUGENE BARAI3AS
290. Clairol Laboratories, Ames Test performed on six samples of hair spray reins. Unpublished report for GAF (1 978) 291.
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POVIDONE
685
306. 307.
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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, 121 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
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
688
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, 1 , 523 1 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
69 1

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Analytical Profiles of Drug Substances and

ACADEMIC PRESS, INC.

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Copyright 0 1993 by ACADEMIC PRESS, INC.

Academic Press, Inc.

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AFFILIATIONS OF EDITORS AND CONTRIBUTORS

AFFILIATIONS OF EDITORS AND CONTRIBUTORS

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 22

History Therapeutic Category Chemistry and Structure-Activity Re1 ati onshi p

Chemistry and Structure-Activity Relationship

DESCRIPTION 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 )

Figure 1: Structure o f Acetazolamide

Appearance, Color, Odor, and Taste

Pharmaceutical Dosage Forms

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

Figure 2: UV Spectra o f Acetazolamide

Figure 3: pH-Sol ubi 1 ity Prof i1 e of Acetazolamide

S o l u b i l i t y o f Acetazolamide i n Various Solvents a t 25'C

Sol ubi 1 it y (mg/mL)

Pol y e t h y l ene G y c o l 1 Propyl ene G ycol 1 Ethanol Glycerin Water

Table 2 Solubilizing Power of Some Binary Solvents Toward Acetazolamide

Solubilizing Power 5.294 x

Differential Scanning Calorimetry

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

Wave number (cm-l)

Infrared spectra of Acetazolamide Polymorphic Forms (in N u j o l )

X-Ray Diffraction Pattern of Two Polymorphic Forms o f Acetazol ami de

Nuclear Uagnetic Resonance Spectra

were measured. Then, tris-

acetazol ami de. Table 3:

1 5 N hemical s h i f t s and coupling constants J (NH) in acetazol amide

J(15NH) Coupling Constant

-241.2 (CONH) [-241.4b (CONH)] -282.4 (S02NH2) -282.6b (SOzNHz)

-15. 7b [ N=C ( S ) -SO21

a23 ' a 01 . Ob a2 '

UV Spectrum of Acetazolamide i n 0.1 N H2SO4 -,

Nuclear Magnetic Resonance Spectroscopy

Sul famerazine Chl orothi azide Sul fadiazine Propazolamide

12% MeoH, 2% ACN, 0.02 M Phosphate

Chl oroth azide

Chl oroth azide

U1 trasphere 0.05 1 % ACN, 0.05 M 0 -20 Acetate, pH 4.5

cia 2-Acetamido-4methyl-5-thiadiazole sul fonamide

The pH-rate p r o f i l e curve of acetazolamide

Stabi 1 ity in Intravenous Admixtures

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

cal /mol /deg

29.24 26.04 18.27

28.77 28.03 26.28

14.84 18.76 20.10

Effect of Buffer Species, Buffer Concentration, and Ionic Strength

ko + kH (H+) + k0H (OH-)

Stability in Various Solvents

BIOPHARIUICEUTICS Phariacokinetics and Metabolism

L., Nonlinear Model f o r

l9, 363 (1985).

Humeida A. El-Obeid and Abdullah A, Al-Badr