High-performance liquid chromatographic determination of urinary 4'-hydroxymephenytoin, a metabolic marker for the hepatic enzyme CYP2C19, in humans.

The preferential hydroxylation of (S)-mephenytoin to 4'-hydroxymephenytoin (4'-OH-M) displays a genetic polymorphism of drug metabolism in humans. Thus the excreted 4'-OH-M is considered to be an important marker for the hepatic (S)-mephenytoin 4'-hydroxylase. Accordingly, a mixture of urine containing total 4'-OH-M after enzymatic deconjugation and phenobarbital as internal standard (I.S.) was extracted with absolute diethyl ether. The residue remaining after evaporation was dissolved in 50 microliters of eluate and 20 microliters were injected into the chromatographic system. All components were separated isocratically on a reversed-phase column using acetonitrile-water (24:76, v/v) as the mobile phase at a flow-rate of 1.2 ml/min. The effluent was monitored at 204 nm. The retention times for 4'-OH-M and the I.S. were within 6 min. The absolute recovery was in the range 84-89% for 4'-OH-M and that of the I.S. was 75.9 +/- 4.2%. Quantification was performed by measuring the peak-height ratio compared with the ratio of the amount of 4'-OH-M divided by that of the I.S. The intra- and inter-day variations were less than 8% and 10%, respectively. The proposed method is simpler and more convenient than those reported previously. Its practical applicability was assessed by phenotyping the efficient and deficient hydroxylators among the Chinese minorities and Han Chinese populations.