[analytical profiles of drug substances and excipients] volume 22 || tolazamide
TRANSCRIPT
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TOLAZAMIDE
John K . Lee, Kazimierz Chrzan
and Robert H. Witt
Rh6ne-Poulenc Central Research
500 Arcola Road
Collegeville, PA 19426-0107
ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 22 489
Copyright 0 1993 by Academic Press, Inc All rights of reproduction in any form reserved
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490 JOHN K. LEE ET AL.
CONTENTS
I . Introduction A. Therapeutic Category B. Appearance
11. Description
A . Nomenclature I . Chemical Names 2. Generic Name 3 . Proprietary Names
1. Empirical 2. Structural 3 . Registry Number
B. Formula
C. Molecular Weight D. Elemental Composition E. Dissymmetry F. Appearance, Color, Odor
111. Physico-Chemical Properties
A. B. Solubility C. Stability and Storage D. Dissociation Constant E. Spectral Properties
Properties of the Drug Substance
1 . Ultraviolet Spectrum 2. Infrared Absorption Spectrum 3. Mass Spectrum 4. Nuclear Magnetic Resonance Spectrum
1 . Melting Range 2. Loss on Drying
F. Thermoanalytical Behavior
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TOLAZAMIDE 49 I
IV.
V.
VI.
VII.
3. Differential Thermal Analysis 4. Thermogravimetric Analysis
Synthesis
Pharmacokinetics
A. Absorption B. Metabolism
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
Determination in Biological Matrices
A. High Performance Liquid Chromatography B. Gas-Liquid Chromatography
VIII. References
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492 JOHN K. LEE ET AL.
TOLAZAMIDE
I. INTRODUCTION
A. Therapeutic Category
Tolazamide is a member of a CASS of ora hypoglycemic agents chemically known as the sulfonylureas which includes other compounds such as tolbutamide, chlorpropamide, glyburide and glipizide. 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 40-50 units of insulin daily, has no history of ketoacidosis, and who has had the disease no longer than 5-10 years (1).
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.
All are arylsulfonylureas
Tolazamide, as well as the other sulfonylureas, is
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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 action (" 60 hr compared with - 10-18 hr for tolazamide) and 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 ) .
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JOHN K . LEE ET AL. 494
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 hindrance relative to N was shown to have high antidiabetic activity (" s ix 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.
9
as well as low steric
11. DESCRIPTION
A. Nomenclature
1. Chemical Names
N-[[(Bexahydro-1~-azepin-l-yl)-amino]carbonyl]-4-methyl- benzenesulfonamide; l-(hexahydro-1H-azepin-l-yl)-3-(p-tolylsulfonyl)urea: N-(p-toluenesulfony1)-N'-hexamethyleniminourea
2. Generic Name
3 . Proprietary Names
Tolazamide
@ QJ, Diabewas, Norglycin , Tolinase , Tolonase
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TOLAZAMIDE 495
B. Formula
1. Empirical
C14H21N303S
2. Structural
3. Registry Number (12)
Chemical Abstracts; 1156-19-0
C. Molecular Weight
311.41
D. Elemental Composition
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 .
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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).
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TOLAZAMIDE
a a0
a70
a m .
aso
0.40
0.30
a 2 0
0.10
ao
491
I
~
~
-
- Ethanol 227, 256, 263, 268, 274
-
D. Dissociation Constant (-SOzNHCO- Group)
pKa 3 . 6 (25OC) pKa 5 . 6 8 (37 .5OC) ( 1 5 )
E. Spectral Properties
1. Ultraviolet Spectrum ( 1 6 )
The ultraviolet spectrum (Figure 2 ) from 220 nm to 340 run was obtained using a Hewlett-Packard 845QA Spectrophotometer and a matched pair of Fisherbrand Suprasil U.V. 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).
Wavelength (nm)
Figure 2. Ultraviolet absorption spectrum of tolazamide in ethanol. (Reprinted with permission from reference 16; Copyright CRC Press, Inc., Boca Raton, m].
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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. occur between approximately 1160 cm and 1700 cm- . Table I gives the spectral assignments for these principal bands.
As can be seen, four prin i a1 absorptipn peaks -5
P 8800 so00 moo PO00 Leo0 1000 600
o l . . : . . . . : . . . . : . . . . : . - - . : . . . - : 4000
Wavenumber (cm-’)
Figure 3. Infrared spectrum of tolazamide (KBr disk).
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TABLE I- Spectral Assignments f o r Principal IR Absorption Bands of Tolazamide (17-19)
-1 Wavenumber, cm Intensity Assignment
1350 Strong
1703 Strong C=O stretching vibration
1456 Strong C=C stretching vibration
CH -scissoring stretch
-SO asymmetric s t re tching
vibration (when linked to -NH-)
1167 Strong -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
al
m 2 : c c .-
E $ : I- ,O 0-
0 a
0
4000 3800 3000 2800 2000 1uoo 1000 uoo
Waveriurnber (cm-')
Figure 4. Infrared spectrum of tolazamide (mineral o i l ) .
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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 -- D I P
'@,I
Figure 5. Mass spectrum of to1azami.de (electron impact mode ) . (Reprinted w i t h permlaeion from reference 16; Copyright CRC P r e s e .
I n c . , Boca Raton, FL).
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TOLAZAMIDE 511 I
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
3 3 CDCl /CD OD (containing 1% tetramethylsilane [TMS] as 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.
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502 JOHN K. LEE ET AL.
- N
TOLAZAflIOE (COCLS/C03001 NflR \ , . ' ~ " " ~ ' , , ' , , , " ~ " " ~ " " , " " ~ . " ' , ' . , ' I ' I 1 1 I
YSO Y O 0 350 300 ZSO 200 150 100 so o nt
8 7 6 S s 3 2 1 0 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
2.4
2.7-3.2
3 . 8 - 4 . 2 -NH -CO -NH -
a Relative to tetramethylsilane (0.0 ppm) The proton undergoing resonance absorption appears in heavy type
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I I JI .A/ .AV 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 temperature of ... 169OC. A 3.33 mg sample of tolazamide, USP reference standard, lot G was used.
P.sk f- 1 B l . M to: 174.70
Onsat- 160.01 J/g - l22.tl.Y
I I
Peak- 170.70
I-----
4 . w h.bo $.a lm.mx 14.66 I d b . a 6 - i d 6 . M A Terriperalure ( C )
k
Pjgure 8 . Differential thermoanalytical behavior of tolazamide.
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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 i
Temperature ( C )
Figure 9 . Thermogravimetric behavior of tolazamide.
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IV. SYNTHESIS
Tolazamide has been synthesized by Wright and Willette ( 9 ) according to the general method of Marshall and Sigal ( 2 4 ) . The p-toluenesulfonyl derivative of methyl urethane is reacted with the appropriate disubstituted hydrazine (N-aminohexamethyleneimine) to yield tolazamide ( 5 4 % yield) as illustrated in Figure 10.
Figure 10. Synthesis of tolazamide.
V. PHARMACOKINETICS
A. Absorption
Several reports indicate tolazamide as being rapidly allsorbed (3.25) whereas others indicate a somewhat slower absorption (1,2,11,13,26). The drug is probably best characterized as well absorbed from the gastrointestinal tract after oral administration with peak blood levels occurring between 4 - 8 hr. 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.
It has an elhfnatfon half-life
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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).
for both species. 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. tolazamide. Based on these studies. both metabolites 2 and 4 have greater hypoglycemic activity than tolbutamide.
Figure 11 shows the major pathways of metabolism found Based on a 24 hr urine collection,
Metabolite 6 was 5 % as potent as
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TOLAZAMIDE 507
0 CH3-54 --NH --C II -NH -N
- TOLAZAMIDE
14hexahydroazepi n- 1 -yl)-3-p- to1 ylsulfon ylurea
Metabolite
2
3 4
5 6
Figure 11.
t 3
\ UNKNOWN
3
Identity
dl-l-(4-hydroxyhexahydroazepin-l-yl)-3-p- tolylsulfonylurea p-toluenesulfonamide l-(hexahydroazepin-l-yl)-3-p-(hydroxymethyl- pheny1)sulfonylurea Unknown l-(hexahydroazepin-l-yl)-3-p-(carboxyphenyl)- sulfonylurea
Metabolism of tolazamide in man and the rat [modified from ref. (28) J : a previously isolated and identified (29).
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SO8 JOHN K. LEE ET AL.
VI. DETERMINATION IN PHARMACEUTICALS
A. 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)amino- methane 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. specifies filtration of samples).
(The USP method
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 (500 nun x 1 m I.D., microsphere silica) and RP-8 (250 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.
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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. ten tablets is determined followed by grinding to a fine powder. 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 pg [unpublished data (34)J as well as good resolution and quantitative recovery.
an HPLC method for the determination of N-nitrosohexa- methyleneimine 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.
In the method, the average weight of
The equivalent of - 80 mg of drug is transferred
In another recent investigation, Severin (35) developed
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.
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B rl 0
P 0 0
d
@ 0
W
0
1.1 0
rl
e 0
v)
N
rl
8 m V
.rl rl
.rl v)
0
m B 3 H
rl
V
A
V
00 N
a, 6 6 i4
0
8
r- crl
m
d
...
m m @ 0
W
0
1.1 0
r-l
e
Y)
00
0)
4J ld u 0,
9 9 l-l A
w
0
In
N
t3 rl
d ld U
*4
rl
.rl v)
0
d
'A 3 "
N a
8" rl
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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) . without interferences from excipients, and the method was shown to be specific (38).
Quantitative recoveries were obtained
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
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.
A complexornetric titration method, applicable to tablet
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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. 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.
Recently,
VII. DETERMINATION IN BIOLOGICAL MATRICES
A. 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. this procedure, 0.5 mL of serum is added to 0.5 mL of a chloroform solution of the internal standard, 5-(p-methyl- phenyl)-5-phenylhydantoin. 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.
In
After adjustment to a pH of 4.5,
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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. was achieved with a recovery of at least 93% for all components and a detection limit of - 0.04 pg/mL. advantage of this method is that serum components usually do not interfere at 360 run.
Separation of tolazamide from the other drugs
An
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 pg/mL plasma was obtained. Under these conditions, tolazamide is fragmented to p-toluenesulfon- amide and the internal standard is fragmented to p-chlorobenzenesulfonamide.
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