Measurement of Serum Diphenylhydantoin by Gas-Liquid Chromatography D. H. Sandberg, G. L. Resnick, and C. Z. Bacallao Department of Pediatrics, University of Miami School of Medicine, P . 0 . B o x 875, Biscayne Annex, Miami, Fla. 33152
A gas-liquid chromatographic method for measurement of diphenylhydantoin in serum has been developed. In this procedure the diphenylhydantoin is extracted from acidified serum with chloroform. After formation of the methoxy derivative, separation and measurement are accomplished by gas chromatography using a flame ionization detector. The method has the advantages of considerable specificity and sensitivity. Results obtained by this procedure compared favorably with measurements made by the Svensmark-Kristensen colorimetric procedure. for determination of diphenylhydantoin.
DIPHENYLHYDANTOIN (5,5-diphenylhydantoin) (D) is one of the most useful drugs available for treatment of major convulsive disorders. However, it has now been shown that metabolism of this compound varies markedly in different individuals and that blood concentrations of D are only poorly correlated with drug dosage schedules ( I ) . Because therapeutic effectiveness can, to some degree, be predicted from the concentration of D in blood ( 2 ) , monitoring of serum D is helpful in management of those patients whose seizures are difficult to control. Various colorimetric methods for analysis of serum D have been developed, but there is no simple technique which is sufficiently sensitive and also specific for D (3-7). In particular, phenobarbital interferes with these colorimetric methods unless separation by elaborate extraction procedures or a procedure such as thin-layer chromatography is used prior to D measurement. Therefore, we have devised a method which uses chloroform extraction of serum after acidification with dilute hydrochloric acid, followed by methylation of D and gas-liquid chromatography of the methoxydiphenylhydantoin (MD) using a flame ionization detector. It appears to have adequate sensitivity and specificity. EXPERIMENTAL Reagents. Diphenylhydantoin, a gift from Parke, Davis &
Co. (Detroit, Mich.), was recrystallized from tetrahydrofuran. Androsterone (K&K Laboratories, Plainview, N. Y.)was reacted with acetic anhydride in pyridine and recrystallized from benzene. Chloroform, reagent grade (Fisher Scientific Company, Atlanta, Ga.) was freshly distilled every week. Carbon disulfide, reagent grade, was also obtained from Fisher Scientific Co., and was used without further purification. Diazomethane was prepared as described by De Baer and Backer (8), using Diazald (Aldrich Chemical Co., Inc., Milwaukee, Wis.). (1) E. W. Loeser, Jr., Neurology, 11,424 (1961). (2) F. Buchthal, 0. Svensmark, and P. J. Schiller, Arch. Neurol., 2 , 624 (1960). (3) G. L. Plaa and C. H. Hine, J. Lab. Clin. Med., 47, 649 (1956). (4) W. A. Dill, A. Kazenko, L. M. Wolf, and A. J. Glazko, J . Pharrnacol. Exptl. Therap., 118, 270 (1956). (5) 0. Svensmark and P. Kristensen, J . Lab. Clin. Med., 61, 501 (1963). ( 6 ) J. Wallace, J. Biggs, and E. V. Dahl, ANAL.CHEM.,37, 410 (1965). (7) J. W. Huisman, Clin. Chim. Acta, 13, 323 (1966). (8) T. J. De Baer and H. J. Backer, "Organic Synthesis," Vol. IV, 1963, p 250.
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ANALYTICAL CHEMISTRY
Table I.
Comparison of Two Methods for Measurement of Diphenylhydantoin in Serum
Results of methods (pglml) SvensmarkKristensena GLC I*
2
2 3 4
7
1.7 5.6
2
2.2
1 6
3.0 1.8 3.2 34.0 4.0 31.5 8.4 3.2 14.8 3.9 2.9 2.9 11.4 1.4 9.6 27.0 0.5
5
6 7 8 9 10 11 12 13 14 15 16 17 18 19
20
6 35 7 43 15 4 20 7 6 2 23 1 9 20 0
See reference (5). * All of these patients were also receiving phenobarbital. 5
Gas Chromatography. An F & M (Avondale, Pa.) Model 402 gas chromatograph equipped with dual flame detectors was used for the chromatography. Columns were prepared as described by Homing, Moscatelli, and Sweeley (9) and a 2 z (w/v) XE-60 stationary phase was used for the analyses. Chromatography was performed under the following conditions using the XE-60 column: column temperature, 230-240" C; detector temperature, 250" C; inlet temperature, 250' C; and helium flow 90 ml/minute. A 3.8 % SE-30 column and a mixed phase (1 SE-30/1 NGS) column prepared as described by Nair, Sarlos, and Turner (IO) were also used to demonstrate specificity of the chromatographic analysis. General Procedure. After acidification with 0.5 ml of 0.5N HCI, 0.5-1.0 ml of serum was extracted with 10 ml of chloroform by shaking 5 minutes in a 15-ml glass-stoppered, graduated centrifuge tube. The chloroform should be freshly distilled weekly because on standing chemical alterations occur which may give spurious peaks on the chromatogram. Five milliliters of the chloroform phase were evaporated to dryness under air or nitrogen. Five-tenths milliliter of ethereal diazomethane, prepared as described by De Baer and Backer (8),was added to the residue and after nitrogen evolution stopped, excess reagent was evaporated in a hood under nitrogen. After addition of 100 pl of carbon disulfide, an aliquot of 1-5 p1 was injected into the chromatograph. Methoxydiphenylhydantoin had a retention time of four (9) E. C. Homing, E. A. Moscatelli, and C . C . Sweeley, Chem Ind. (London), 53, 751 (1959). (10) P. P. Nair, I. J. Sarlos, and D. A. Turner, Anal. Biochem., 7, 96 (1964).
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