SIMPLE AND SELECTIVE ASSAY OF 4-HYDROXYMEPHENYTOIN IN HUMAN URINE USING SOLID-PHASE EXTRACTION AND HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION AND ITS PRELIMINARY APPLICATION TO PHENOTYPING TEST

A simple and selective HPLC method for the determination of 4-hydroxym ephenytoin (4-OH-M) in human urine, using a controlled potential coulo metric detector equipped with a dual working electrode cell of fully p orous graphite, has been developed. After acid hydrolysis of urine, 4- OH-M and the internal standard (I.S.), 5-hydroxy-1-tetralone, were ext racted from urine by means of a Bond Elut Certify LRC column. The extr acts were chromatographed on a reversed-phase mu Bondapak C-18 column using methanol-50 mM KH2PO4 (pH 4.0) (30:70, v/v) as the mobile phase at a how-rate of 1.0 ml/min. Electrochemical detection at applied pote ntial of 800 mV resulted in a limit of quantitation of 0.76 mu g/ml. T he method showed a satisfactory sensitivity, precision, accuracy, reco very and selectivity. The present method was applied to the phenotypin g test in thirteen Japanese healthy volunteers who received an oral 10 0-mg racemic mephenytoin. The phenotypes determined by the present met hod were found to be in agreement with those obtained with the reporte d customary assay based on gas chromatography.