of SRM-1577 are N, 10.6%; K, 0.97%; Na, 0.243%; and the main trace constituents are Fe, 270 ppm; Cu, 193 ppm; and Zn, 130 ppm. When this sample was analyzed by a working curve which is made by a lead solution of 0.1 N “03, the observed result was 0.342 f 0.07 ppm. The result agreed precisely with the NBS value of 0.34 & 0.14 ppm. Thus, i t shows that accurate results can be obtained by the present method even if the standard additions method is not applied. T h e heated area of the carbon tube must be sufficiently large to prolong the residence time so as to decompose the sample completely. If only a small portion of the central part of the tube was heated to high temperature, the good reproducibility and the high sensitivity were not obtained in the case of a solid sample. The temperature distribution of the carbon tube was important to perform an accurate analysis. We did not observe any noticeable chemical interference in all the samples analyzed. The determination of zinc by the present method was also performed, and good results as well as those of cadmium and lead were obtained under the same conditions as in the case of cadmium. Figure 10. Determinationof lead in the standard reference material of NBS (SRM-1577, Bovine Liver) by Zeeman AAS
(a)
Weight, mg (1) 1.42 (2)2.12 (3)1.65 (4)1.73 (5)1.85
Pb, ppm 0.370 0.307 0.340 0.356 0.341
(b) Result: 0.343f 0.023 ppm. Certified value of NBS: 0.34f 0.08 ppm
The biological standard reference material of NBS was analyzed to confirm the accuracy of this method. Figure 10 shows the results and the procedure when the lead in the SRM-1577 Bovine Liver was determined. Dried and weighed samples of 1to 3 mg were positioned exactly in the center of the carbon tube by the sample holder. The major constituents
ACKNOWLEDGMENT We thank T. Hadeiahi of the University of California and M. Katayama of the University of Tokyo for their useful suggestions. Thanks are also due to K. Ohishi and K. Uchino of Hitachi Ltd. for their great help.
LITERATURE CITED (1) (2) (3) (4)
(5) (6) (7)
(8) (9)
H. Koizumi and K. Uchino, HitachiHyoron, 56, 1037 (1974). H. Koizumi and K. Yasuda, Anal. Chem., 47, 1679 (1975). H. E. White, “Introduction to Atomic Spectra”, McGraw-Hill, New York, 1934. A. C. G. Mitchell and M. W. Zemansky, “Resonance Radiation and Excited Atoms”, Cambridge, 1934. E. E. Whiting, J. Quant. Spectrosc. Radiat. Transfer, 8, 1379 (1968). H. Massmann, Spectrochim. Acta, Parts, 23, 215 (1968). A. G. Shenstone, Phys. Rev., 47, 317 (1935). T. E. Manning, Phys. Rev., 78, 417 (1950). J. P. Matousek, Am. Lab., No. 6, 45 (1971).
RECEIVEDfor review January 19,1976. Accepted March 15, 1976.
FIuorometric Determination of Chlordiazepoxide in Dosage Forms and Biological Fluids with Fluorescamine James T. Stewart” and Jonathan L. Williamson School of Pharmacy, University of Georgia, Athens, Ga. 30602
A fluorometrlc procedure for chlordiazepoxide is reported based upon a fluorophor formed with fluorescamine after acid hydrolysis of the drug. A study of optlmum pH, hydrolysis time, and fluorescamineconcentrationis presented. Fluorescence is linear over the range 0.25-6 pg chlordlazepoxlde hydrochloride per ml of sample. The method is free from interference from chlordiazepoxide’s major metabolites as well as other drugs such as clldinlum bromide, oxazepam, and dextropropoxyphene hydrochlorlde. The procedure Is subject to interference from other amlne-containingdrugs such as amphetamines that might be present In analytical samples. Applicatlon of the procedure to the analysis of chlordiazepoxide in dosage forms, urlne, and plasma gave an accuracy of 1-3 %. 1182
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
Chlordiazepoxide (I), a 1,4-benzodiazepine derivative, has been widely used therapeutically because of its muscle relaxant, taming, sedative, anti-anxiety and anticonvulsant properties (1-3).
NHCH,
I
Methods t o analyze the drug in dosage forms and biological fluids have utilized ultraviolet and visible spectrophotometry ( 4 4 3 , gas chromatography (7,8), radioimmunoassay (9), and fluorometry (20-12). In addition, polarography and titrimetry (13-15)have been employed for the determination of the drug in dosage forms and plasma. Existing fluorometric methods lack the simplicity a n d specificity that usually are encountered in fluorescent analysis. Selective and lengthy extractions, light exposure steps, and analysis utilizing differential fluorescence readings are other disadvantages of these procedures. The use of fluorescamine (11)
I1 in the fluorometric determination of aromatic or aliphatic primary amines suggested that it might also be useful in the assay of chlordiazepoxide via one of its acid hydrolysis products, either 2-amino-5-chlorobenzophenoneor methylamine. Fluorescamine reacts almost instantaneously with primary amines (16-18) at specific pH's t o form stable fluorophors with sensitivity in the picomole range. In addition, the reagent and its hydrolysis products d o not exhibit fluorescence behavior and thus alleviate extensive clean-up or chromatographic separations of t h e derived fluorophor from excess reagent. Thus, fluorescamine offers a versatile, sensitive, and specific assay method for primary aromatic and/or aliphatic amines at pH ranges around 5 and 9, respectively. This paper reports a fluorometric method for chlorodiazepoxide based upon a fluorophor formed with fluorescamine after acid hydrolysis of the drug. Application of the procedure to the determination of chlordiazepoxide in dosage forms, urine, and plasma will be discussed. The method is sensitive in t h e submicrogram range and offers a simple and specific assay for the drug in a variety of samples. T h e proposed method, however, is not sufficiently sensitive for the analysis of biological samples obtained after a single-dose administration of chlordiazepoxide.
EXPERIMENTAL Apparatus. Fluorescence measurements were made with an Aminco-Bowman Spectrophotofluorometer equipped with a xenon arc lamp and photomultiplier microphotometer. For quantitative measurements, the following slit settings were used: 5-mm excitation slit, 4-mm emission slit, and 3-mm photomultiplier shutter slit. The microphotometer sensitivity was set at 45 and the microphotometer meter multiplier positions were varied from 0.3 to 0.1. Clear, fused quartz cells (12.5 X 45 mm) were used as sample cells. Reagents and Chemicals. Forty mg of fluorescamine (Fluram, trade mark of Roche Diagnostics, Nutley, N.J.) was dissolved in 100 ml of spectrograde anhydrous acetone and stored at room temperature. The solution was prepared weekly. A pH 7.4 buffer solution was prepared by adding 0.2 M sodium hydroxide to 0.2 M potassium dihydrogen phosphate using a pH meter. Similarly, borate buffer solutions (pH 5-11.5) were prepared by adding 6 N sodium hydroxide to 0.2 M boric acid. Powdered samples of chlordiazepoxide hydrochloride, N desmethylchlordiazepoxide,demoxepam, diazepam, nitrazepam, and clidinium bromide (Hoffmann-LaRoche, Nutley, N.J.) as well as oxazepam (Wyeth laboratories, Philadelphia, Pa.) and dextropropoxyphene hydrochloride (Eli Lilly and Co., Indianapolis, Ind.) were used in the preparation of standard solutions. All other chemicals utilized were the highest grade of the commercially available materials. Procedures. Analysis of Dosage Forms. The powdered contents of a dosage form containing chlordiazepoxide hydrochloride were
placed in a beaker containing 20 ml of water. The mixture was then filtered into a 50-ml volumetric flask and the beaker rinsed with three 5-ml portions of water. These washings were filtered into the flask, and the filtrate was diluted to volume with water. A quantity of the solution was transferred to a 25-ml volumetric flask and diluted to volume with water to obtain a concentration of 4.76 X 10-5 M chlordiazepoxide hydrochloride. One ml of this solution was placed in a 10-ml volumetric flask followed by the addition of 0.25 ml of 6 N HC1. The solution was heated for 45 rnin in a boiling water bath (96 f 1"C). After cooling, 1 ml of 2 M sodium hydroxide was added followed by the addition of 3 ml of pH 9.25 borate buffer. One ml of fluorescamine solution (40 mg/100 ml acetone) was added quickly and the solution vigorously stirred with the aid of a vortex-type mixer. The solution was then diluted to volume with borate buffer (pH 9.25) and the fluorescent intensity recorded using excitation and emission wavelengths of 390 and 486 nm, respectively. Analysis i n Urine. A sample of human urine containing chlordiazepoxide was placed in a 60-ml separatory funnel. Fifteen ml of phosphate buffer (pH 7.4), 40 ml of 1.5%isoamyl alcohol in heptane solution (water-saturated), and 8 g of sodium chloride were added to the funnel. The contents were mixed manually for 1rnin and the layers allowed to separate. The organic layer was transferred into a clean 125-ml separatory funnel and the urine extraction repeated. The combined heptane extracts were washed with one 10-ml portion of 0.45 N sodium hydroxide and then extracted with 1.5N HCl for 1min. A quantity of the acid extract equivalent to 4.5-53.5 figwas pipetted into a 10-ml volumetric flask and the solution heated for 45 rnin in a boiling water bath. After cooling to room temperature, the fluorophor was formed using 1ml of 2 M sodium hydroxide, 3 ml of pH 9.25 borate buffer, and 1 ml of fluorescamine solution. Upon dilution to volume with borate buffer, the fluorescence was measured using excitation and emission wavelengths of 390 and 486 nm, respectively. Urine samples spiked with known concentrations of chlordiazepoxide hydrochloride were assayed under the same experimental conditions for calibration purposes. A control urine sample was utilized to assess background fluorescence arising from biological matrix. Analysis in Plasma. A sample of human plasma containing chlordiazepoxide was placed in a 50-ml culture tube. Ten ml of phosphate buffer (pH 7.4), 25 ml of 1.5%isoamyl alcohol in heptane solution,and 5 g of sodium chloride were added. The tube contents were shaken for 20 min in an automatic shaker at 180 excursions per min. Then the contents were placed in a 50-ml polypropylene centrifuge tube and centrifuged for 10 min at 9500 rpm. The organic layer was aspirated into a 60-ml separatory funnel, washed with 10 ml of 0.45 N sodium hydroxide, and then extracted with 1.5 N HCl. A quantity of the acid extract equivalent to 4.5-53.5 fig was pipetted into a 10-ml volumetric flask and the fluorophor formed as previously described. Fluorescence was monitored using excitation and emission wavelengths of 390 and 486 nm, respectively. Plasma samples spiked with known concentrations of chlordiazepoxidehydrochloride were assayed under the same experimental conditions for calibration purposes. A control plasma sample was utilized to assess background fluorescence arising from biological matrix.
RESULTS AND DISCUSSION Addition of fluorescamine t o an acid-hydrolyzed solution of chlordiazepoxide hydrochloride whose pH had been readjusted t o 9.0 gave a n intense fluorescence using excitation and emission wavelengths of 390 and 486 nm, respectively. Both methylamine and 2-amino-5-chlorobenzophenone are amino-containing products of the hydrolysis ( 4 , 1 9 )that could potentially react with fluorescamine. desilva and Strojny (20) have reported that n o fluorescence behavior is observed over a wide pH range for t h e reaction between fluorescamine a n d the benzophenone. This result has also been substantiated in these laboratories. Therefore, indications are that methylamine is responsible for t h e fluorescent species formed. Maximum fluorescent intensity of the fluorophor formed from known methylamine and fluorescamine was observed at t h e same excitation and emission wavelengths used in the assay procedure (390 and 486 nm, respectively). Also, published reports (21) concerning reaction of fluorescamine with primary aliphatic amines suggest that maximum fluorescence is obtained around pH 9. T h e determination of chlordiazepoxide via acid hydrolysis followed by reaction with fluorescamine necessitated the exANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
1183
or 0
"
10
'
1
&
'
*
4
0
"
3
0
' s
1
o
'
6
' 3
fluorescamine, mg/ IOOml
pH of borate buffer Figure 1. Effect of pH on fluorescent intensity of fluorophor formed with fluorescamine
Figure 3. Effect of fluorescamine concentration on fluorescent intensity of fluorophor formed with fluorescamine
Table I. Analysis of Chlordiazepoxide Hydrochloride Mixtures Using Fluorescamine Mixtures
I
I1 I11 IV minutes
V
Figure 2. Effect of hydrolysis time on fluorescent intensity of fluorophor formed with fluorescamine
amination of three parameters: (a) the determination of optimum pH; (b) the determination of optimum hydrolysis time; and (c) the determination of fluorescamine concentration needed for maximum sensitivity. The results of the p H study shown in Figure 1indicated that maximum fluorescence was obtained a t p H 9.25. This was in agreement with initial observations that fluorescence was most intense when the acid hydrolysis mixture was adjusted to p H 9 subsequent to reaction with fluorescamine. It is important for maximum sensitivity in the procedure to monitor the p H periodically before addition of fluorescamine t o the samples to ensure that the p H is approximately 9.25. T h e hydrolysis study shown in Figure 2 indicated that 45 min was sufficient time for acid hydrolysis of chlordiazepoxide in order to obtain maximum fluorescence readings. Figure 3 shows data t h a t determined t h a t a minimum of 40 mg of fluorescamine per 100 ml of acetone was necessary for maximum readings. T h e results also indicated that a more concentrated fluorescamine solution had little effect on the fluorescent intensity. T h e linearity of fluorescence, determined by performing t h e fluorescamine reaction with known concentrations of chlordiazepoxide hydrochloride subjected to acid hydrolysis, was found to be 0.25-6 wg/ml for the salt equivalent to 0.22-5.4 pg/ml for the free base. A limited study of the determination of chlordiazepoxide hydrochloride using the newly developed assay in the presence of equimolar quantities of its major metabolites, N 1184
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
VI VI1
Components, concn of 3 X M
Fluorescence (arbitrary unit@
Chlordiazepoxide hydrochloride N-Desmethylchlordiazepoxide Chlordiazepoxide hydrochloride Demoxepam Chlordiazepoxide hydrochloride Clidinium bromide Chlordiazepoxide hydrochloride Oxazepam Chlordiazepoxide hydrochloride Dextropropoxyphene hydrochloride Chlordiazepoxide hydrochloride Diazepam Chlordiazepoxide hydrochloride Nitrazepam
22b
20.5 21 23 20
55 84
3X M Chlordiazepoxide hydrochloride gave a reading of 20. Mean calculated on the basis of four assays.
desmethylchlordiazepoxide and demoxepam, as well as other compounds that might be found with it in dosage forms and/or biological fluids was undertaken. In addition, oxazepam, diazepam, and nitrazepam were used to ascertain any specificity of the method for chlordiazepoxide over other benzodiazepines. The results shown in Table I demonstrate t h a t the procedure allows the analysis of chlordiazepoxide in the presence of its major metabolites as well as other drugs such as clidinium bromide, oxazepam, and dextropropoxyphene hydrochloride. However, the method is not completely specific for chlordiazepoxide as evidenced by data from chlorodiazepoxide solutions containing diazepam and/or nitrazepam. I n both cases, significant increases in fluorescent intensity beyond that due to the chlordiazepoxide present were obtained. Studies using each drug alone showed t h a t the increased readings were due to definite fluorescent species formed under the experimental conditions utilized. Efforts
Table 11. Assay of Chlordiazepoxide Hydrochloride i n Dosage Forms Using Fluorescamine Amount declared Prod- per sample, uct mg Ad
Bf
10 5
Percent recoverya ColorimetFluorometric ricb 100.16 f 0.8ge
.*.
98.74 f 2.56
98.29 k 0.75
Ultraviolet 100.97 f 1.78 116.86 f 1.17
a Calculated on basis of four assays of a single aliquot. Reference 5. Reference 22. Commercial capsule containing chlordiazepoxide hydrochloride. e Confidence limits a t p = 0.05. f Commercial capsule containing chlordiazepoxide hydrochloride plus clidinium bromide.
t o minimize the interference caused by these drugs were not investigated further. Obviously t h e acid hydrolysis of diazepam and nitrazepam results in the possible formation of amineb) or amino acid(s) capable of reacting with fluorescamine a t basic pH. This does not diminish the usefulness of the chlordiazepoxide assay since most benzodiazepines are not administered or present concurrently except in drug abuse samples. The utility of the fluorometric assay for the quantification of chlordiazepoxide was demonstrated in dosage form, urine, and plasma. Two commercially available capsule preparations containing chlordiazepoxide hydrochloride were assayed by the fluorescamine procedure and compared t o existing colorimetric and/or spectrophotometric methods (5,22)as shown in Table 11. T h e fluorometric procedure gives data in agreement with the existing methods. Although the uv method is faster and simpler in the assay of product A, its lack of versatility is demonstrated by its failure t o quantitate the chlordiazepoxide in product B because of the absorbance of clidinium bromide in the same uv region. The fluorescent and colorimetric methods take approximately the same overall analysis time since both utilize acid hydrolysis procedures. However, t h e fluorometric method is more sensitive and the reagents are stable for longer time periods. Furthermore, the fluorophor is stable for a t least 2 h ( 1 7), whereas the azo dye formed in the colorimetric assay begins to decompose in 25-30 min (23). The application of the procedure to the determination of chlordiazepoxide in biological fluids required investigation into extractability of the drug using organic solvents. Different authors (4,6,7) had reported the usage of various solvents for extraction of chlordiazepoxide from biological fluid a t p H 7.4. Among t h e choices were chloroform, diethyl ether, and 1.5% isoamyl alcohol in heptane. After adjusting the p H of urine containing spiked quantities of chlordiazepoxide t o 7.4 with phosphate buffer, extractions using these solvents were performed. In all cases, concentration of drug equal to 170% of t h e added quantity was noted as monitored by the fluorescamine assay. As a result, the extraction procedure was modified in an attempt t o remove any interfering species by washing the organic extract with 0.45 N sodium hydroxide prior to acid reextraction. This step was successful in removing the interference and allowed isoamyl alcohol in heptane to be selected as the solvent of choice since it provided highest percent recovery with minimum number of extractions. No attempt was made, however, t o possibly further improve the procedure by washing the acid solution after the hydrolysis step prior to p H adjustment. Two extractions of urine using t h e heptane mix were performed with a percent recovery of
Table 111. Analysis of Chlordiazepoxide Hydrochloride of Known Quantity in Biological Fluids Biological fluid
Added, udml
Amount found, yg/mln
Accuracy,
Urine Plasma
2.68 2.68
2.63 f 0.02h 2.61 f 0.03
1.87 2.61
%
Calculated on basis of three assays. Standard deviation.
96.35 f 0.90% chlordiazepoxide. Additional urine extractions did not significantly affect the results and therefore were not included in the assay method. T h e modified urine extraction procedure was applied t o plasma samples with an additional modification. Centrifugation of the extraction mixture a t 9500 rpm was necessary in order to disperse the emulsion formed upon shaking. Efforts t o use multiple extractions for plasma with the heptane solvent resulted in additional interferences of an unknown nature. Therefore, only one extraction was performed on plasma with a percent recovery of 82.86 f 0.58%. Sensitivity of the method might possibly be increased by extracting the fluorophor a t p H 5.0-5.5 with ethyl acetate. Tenfold increases in sensitivity have been noted using this modification on such drugs as dopa, dopamine, and adamantanamine (19). Urine and plasma samples spiked with known quantities of chlordiazepoxide hydrochloride were assayed by the fluorescamine procedure and compared to urine and plasma calibration curves run concurrently. Linear regression analysis of the calibration data using a programmable calculator gave slope and intercept constants which were then utilized to solve for drug concentration in the spiked samples. T h e data in Table I11 demonstrate the results obtained for chlordiazepoxide in biological fluids. T h e utility of the method is clearly demonstrated with an accuracy of 1.87-2.61%.
LITERATURE CITED (1)L. 0.Randall, W. Schallek, G. A. Heise, E. F. Keith, and R. E. Bagdon. J. Pharmacol Expt. Therap., 129,163 (1960). (2)J. M. Tobin and N. D. C. Lewis, J. Am. Med. Assoc., 174, 1242 (1960). (3)S.C.Kaim and I. N. Rosenstein, Dis. Nerv. Syst., 21 (Suppl.), 46 (1960). (4)L. 0.Randall, Dis. Nerv. Syst., 22 (Suppl. 7),1 (1961). (5) C. S.Frings and P. S. Cohen, Am. J. Clin. fathol., 56, 216 (1971). (6)J. H. Riddick, Clin. Biochem., 6, 189 (1973). (7)I. A. Zingales, J. Chromatogr., 61,237 (1971). (8)J. A. F. desilva, M. A. Schwartz, V. Stefanovic, J. Kaplan, and L. D'Arconte, Anal. Chem., 36, 2099 (1964). (9)W. R. Dixon, J. Early, and E. Postma, J. fharm. Sci., 64,937 (1975). (IO)B. A. Koechlin and L. D'Arconte, Anal. Biochem., 5, 195 (1963). (11) M. A. Schwartz and E. Postma, J. fharm. Sci., 55, 1358 (1966). (12)J. Braun, G.Cailie, and J. A. Mockle, Can. J. fharm. Sci., 3, 65 (1968). (13)H. Oelschiager, Arch. fharm. Berl., 296,397 (1963). (14)M. R. Hackman, M. A. Brooks, J. A. F. desilva, and T. S. Ma, Anal. Chem., 46, 1074 (1974). (15)I. Grecu and S. Barber, Ann. Pharm. Fr., 26, 405 (1968). (16)M. Weigeie, S.L. DeBernardo, J. P. Tengi, and W. Leimgruber, J. Am. Chem. Soc., 94,5927 (1972). (17)M. Weigele, J. F. Blount, J. P. Tengi, R. C. Czajkowski, and W. Lelmgruber, J. Am. Chem. Soc., 94,4052(1972). (18)J. M. Sterling and W. G. Haney, J. Pharm. Sci., 63,1448 (1974). (19)H. V. Maulding, J. P. Nazareno, J. E. Pearson, and A. F. Michealis, J. fharm. Sci., 64,278 (1975). (20)J. A. F. desilva and J. Strojny, Anal. Chem., 47,714 (1975) (21)S.D. Bernardo, M. Weigeie, V. Toome, K. Manhart, W. Leirngruber, P. Bohlen, S. Stein, and S. Udenfriend. Arch. Biochem. Biophys., 163,390 (1974). (22)"The United States Pharmacopeia," 19th revision, Mack Publishing Co.. Easton, Pa.. 1975,p 79. (23)J P Dux and C Rosenblum, Anal. Chem.,21, 1524 (1949).
RECEIVEDfor review November 13,1975. Accepted April 15, 1976. Abstracted in part from a thesis submitted by JLW to the Graduate Faculty of the University of Georgia in partial fulfillment of the requirements for the degree of Master of Science in Pharmacy.
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