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.

M Tanaka, K Kato, K Sudo, H Hakusui, T Ohkubo, K Sugawara, S Kaneko, K Otani, K Chiba, T Ishizaki

Index: J. Chromatogr. B, Biomed. Appl. 676(1) , 87-94, (1996)

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Abstract

A simple and selective HPLC method for the determination of 4-hydroxymephenytoin (4-OH-M) in human urine, using a controlled potential coulometric detector equipped with a dual working electrode cell of fully porous graphite, has been developed. After acid hydrolysis of urine, 4-OH-M and the internal standard (I.S.), 5-hydroxy-1-tetralone, were extracted from urine by means of a Bond Elut Certify LRC column. The extracts were chromatographed on a reversed-phase mu Bondapak C18 column using methanol-50 mM KH2PO4 (pH 4.0) (30:70, v/v) as the mobile phase at a flow-rate of 1.0 ml/min. Electrochemical detection at applied potential of 800 mV resulted in a limit of quantitation of 0.76 micrograms/ml. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity. The present method was applied to the phenotyping test in thirteen Japanese healthy volunteers who received an oral 100-mg racemic mephenytoin. The phenotypes determined by the present method were found to be in agreement with those obtained with the reported customary assay based on gas chromatography.