and in micrograms. An average five-fold decrease in the error over the previous study is observed at concentrations from to 5 X 10-6 M in silver. This improvement in accuracy is presumably due to the partial compensation of the dissolution error which had resulted in significantly higher results. A reduction in the standard deviation of the determinations was also effected.
the standard deviation of the measurements, are achieved by pregeneration of the standard solutions and the establishment of an equilibrium null-point potential to reduce the error caused by dissolution of the indicator electrode in the null-point cell, In this study, 54 ng of silver were determined with an error of less than 2 ng, while at higher concentrations (above 10 p M ) errors of less than 1 can be easily obtained using the improvements in the LNPP technique discussed.
DISCUSSION
The advantages of greater linearity of the titration curves, shorter analysis time, improved accuracy, and a reduction in
RECEIVED for review January 2, 1968. Accepted March 6, 1968.
Microdetermination of Diphenylhydantoin in Biological Specimens by UItraviolet Spectrophotometry Jack E. Wallace Forensic Toxicology Branch, USAF Epidemiological Laboratory, Lackland AFB, Tex. 78236
A NEED EXISTS for a rapid, sensitive chemical procedure capable of determining diphenylhydantoin at therapeutic levels in blood. The lack of a well defined ultraviolet absorption spectrum for diphenylhydantoin makes its determination by direct ultraviolet spectrophotometry difficult ; nevertheless, the literature describes several methods which depend upon the spectrum of the unchanged drug ( I ) . Methods that require the conversion of the drug to benzophenone and recovery of the ketone by steam distillation have been reported (2, 3). For determining elevated levels of the drug, these procedures are adequate, but extensive concentration of the distillate is required for accurate analysis of therapeutic levels of the drug in biologic specimens. With the concentration technique, subtherapeutic to therapeutic levels often appear elevated and exhibit a significant degree of variability. This report describes a method which permits rapid and accurate analysis of even subtherapeutic levels of the drug. The method does not require separation of diphenylhydantoin from other acidic drugs with which it is frequently associated. Hydrolysis of the hydantoin ring in strong alkali is followed by permanganate oxidation of the resulting amide to benzophenone, which is subsequently extracted into heptane. In contrast to diphenylhydantoin, benzophenone has a well defined absorption spectrum and exhibits a high molar absorptivity. EXPERIMENTAL
Apparatus. A Beckman DK-2A ratio-recording spectrophotometer with linear wavelength presentation was used for the ultraviolet absorption measurements. The sample path length was 10 mm. Infrared spectrograms were prepared by means of a Beckman IR-4 spectrophotometer. A BarberColman Model 5000 gas chromatograph with a 6-ft column consisting of 2z carbowax 20 M on gas Chrom Q, 100-120 (1) 0. Svensmark and P. Kristensen, J. Lab. Clin. Med., 61, 501 (1963). (2) J. E. Wallace, J. D. Biggs, and E. V. Dahl, ANAL.CHEM., 37, 410 (1965). (3) J. E. Wallace, J . Forensic Sci., 11, 552 (1966).
978
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mesh, was used for gas chromatographic analysis. The column was operated at 200" C and the carrier gas was nitrogen flowing at a rate of 50 ml per minute. Procedure. Ten milliliters of blood, serum, or urine are adjusted to a pH between 6 and 7 by the dropwise addition of 0.5N HCI and placed in a 250-ml separatory funnel to which is added 100 ml of chloroform. This mixture is shaken vigorously for three minutes. After separation, the chloroform layer is removed by filtration through Whatman No. 541 or equivalent filter paper into a graduated cylinder. The volume of recovered chloroform is recorded. Five millimeters of 1.ON NaOH are added to the chloroform and the mixture is shaken for three minutes. After separation, 4 ml of the aqueous layer are placed in a 250-1111 round bottom flask, which is then connected to a vacuum rotary evaporator assembly and placed in a water bath of 50"-60" C. With vacuum applied, the flask is rotated until the volume is re-
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duced to approximately 1 ml to remove traces of chloroform which destroy the permanganate reagent. The flask is removed from the assembly and 20 ml of 1% K M n 0 4 in 7 N NaOH and 5 ml of spectro-quality n-heptane are added to the flask. Sodium hydroxide, low in carbonate (0.31%), must be used in the preparation of the alkaline permanganate solution. The contents of the flask are then refluxed for 30 minutes with constant magnetic stirring. After cooling, the n-heptane layer is separated from the aqueous permanganate layer and read in the recording spectrophotometer from 220 to 340 mp against a heptane blank. For the most accurate results, the blank should be prepared from a blood sample known to contain no diphenylhydantoin Typical blank values from 10 ml of oxalated whole blood should not exceed an absorbance of 0.04. If absorbance at a single wavelength is required, determine it at 247 mp. A standard curve is prepared from aqueous solutions of the drug carried through the procedure outlined above, with the exception that the absorbance of the standards are determined against a pure n-heptane blank. RESULTS AND DISCUSSION
The capability of the procedure to determine diphenylhydantoin over the range 0 to 20 pg/ml is illustrated in Table I. The absorptivity/Mgratio of 0.075 represents a 15% increase in sensitivity over a recently reported procedure ( 3 ) . An increase in sensitivity of approximately twelvefold over the 0.0065
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Table I. Standard Curve of Diphenylhydantoin Reaction Product Diphenylhydantoin Absorbance Absorbance/ in sample, pg/ml in n-heptanea concentration 20 1.50 0.075 16 1.20 0.075 12 0.89 0.074 8 0.58 0.073 4 0.28 0.070 2 0.14 0.070 Determined at 247 mp. Table 11. Recovery Studies of Diphenylhydantoin Diphenylhydantoin Recovery, mean & std dev-pg/ml added, ccg/ml Whole blooda Urineb 10.0 9.0 3~ 0 . 4 (8)b 9.1 i= 0 . 3 (6) 7.1 -I: 0.3(7) 8.0 6.9 i 0 . 3 (8) 5.3 =IC0 . 2 ( 5 ) 6.0 5.3 =t0 . 2 (8) 4.5 =k 0 . 2 (4) 5.0 4.4 k 0 . 2 (10) 3.7 =k 0.1 (6) 4.0 3.6 i= 0 . 2 (10) 2.0 1.7 + 0.1 (10) 1.8 =k 0 . 1 (6) 1.o 0 . 8 =k 0 . 1 (10) 0.8 =k 0.1 (6) Average Recovery 87.5z 88.7x a Ten ml assayed. No. of determinations. VOL 40, NO. 6, MAY 1960
979
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Table 111. Recovery of Diphenylhydantoin and Phenobarbital Added to Whole Blood Recovery, mean i std dev-pg/ml Conc, qg/ml Diphenylhydantoin Phenobarbital No. of detns. Diphenylhydantoin Phenobarbital 8 9.0 f 0 . 3 10.0 9.2 i 0.3 10.0 4.4 i 0.2 5.0 I 4 . 5 + 0.2 5.0 0.8f 0.1 8 1.0 0.9 =t0.2 1.0
Table IV. Compounds Investigated for Interference with the Determination of Diphenylhydantoin. Ab-
sorbance of heptane*
Compound Trade name Chemical name Dilantin 5,5-Diphenylhydantoin 1.501 0.001 Blank Alurate 5-Allyl-5-isopropylbarbituricacid 0.007 Amobarbital 5-Ethyl-5-isoamylbarbituric acid 0.002 Aspirin Acetylsalicylic acid 0.003 Barbital 5,5-Diethylbarbituricacid 0.002 Celontin N,2-Dimethyl-2-phenylsuccinimide 0.003 Ethotoin 3-Ethyl-5-phenylhydantoin 0.004 Hydantoin 2,4-(3H, 5H)-imidazoledione 0.001 Mesantoin 3-Methyl-5-ethyl-5-phenylhydantoin 0,014 N-methyl-2-phenylsuccinimide 0.001 Milontin 5-Ethyl-5-phenylbarbituricacid 0.001 Phenobarbital Phenurone Phenacetylurea 0.002 Tridione 3,5,5-Trimethyl-2,4-oxazoladinedione0.004 Each compound was determined from aqueous solution to correspond to a concentration in heptane of 20 pg/ml. Each value is the average of three determinations. b Read at 247 mp. I
0
absorptivity/pg ratio of the direct ultraviolet method of Svensmark and Kristensen (1) is obtained. The ultraviolet absorption spectrums of diphenylhydantoin and its oxidized reaction product are shown in Figure 1. The product in n-heptane is well defined and exhibits an absorption maximum at 247 mp and a minimum at 222 mp. The recovery of diphenylhydantoin added in known amounts t o blood and urine is summarized in Table 11. Table I11 shows the recovery of diphenylhydantoin and phenobarbital from whole blood to which the drugs had been added in equivalent amounts. The method of Goldbaum (4) was used for analysis of phenobarbital. Phenobarbital does not interfere with the determination of diphenylhydantoin when the latter is assayed by the procedure of this report. Recovery of diphenylhydantoin compares favorably with that of methods previously described ( I , 2). (4) L.R. Goldbaum, ANAL.CHEM.,24, 1604 (1952).
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ANALYTICAL CHEMISTRY
Other acidic drugs, including several hydantoins, which might be given to patients concomitantly with diphenylhydantoin were investigated for interference. None produced a product with significant absorbance at 247 mp (Table IV). Ultraviolet and infrared spectrophotometric and gas chromatographic analysis were used to establish the identity of the diphenylhydantoin reaction product. The ultraviolet absorption spectrum of benzophenone is identical to that shown for the product in Figure 1. Infrared absorption of both the product and benzophenone are displayed in Figure 2. The spectra suggest that the two are the same compound. The retention time for the product on the gas chromatograph was equal to that observed for benzophenone. If the absorbance/concentration ratio obtained from the ultraviolet absorption spectra of the product is compared to that of benzophenone, it is apparent that approximately 95% of the diphenylhydantoin is converted to the ketone. The sensitivity limit of the method is 0.25 to 0.50 pg/ml of specimen, providing a 10 ml sample is used. Since most therapeutic blood levels of diphenylhydantoin are 4 to 20 pg/ml, more than sufficient sensitivity is available to assay normal levels of the drug. The primary metabolite 5-(p-hydroxyphenyl)-5-phenylhydantoin does not interfere. The alkaline nature of the permanganate solution and the polarity of the hydroxyl group make the phenolic metabolites insoluble in nheptane. In addition, p-hydroxybenzophenone has an absorption maximum at 292 mp in methanol and no absorption at or near this wavelength is observed after analysis of urine specimens. With the permanganate method, the diphenylhydantoin isolated from urine of patients taking therapeutic amounts of the drug yields a product with infrared and ultraviolet absorption curves identical to that observed for benzophenone. ACKNOWLEDGMENT
The author is indebted to John D. Biggs, Raymond L. Sumners, Robert 0. Mankes, and Armando Pajaro for technical assistance. RECEIVED for review December 8, 1967. Accepted February 23, 1968.
