Simultaneous enantiospecific separation and quantitation of mephenytoin and its metabolites nirvanol and 4′-hydroxymephenytoin in human plasma by liquid chromatography

A high-performance liquid chromatographic method for the enantiospecific quantitation of S- and R-mephenytoin and its metabolites S- and R-nirvanol and S- and R-4′-hydroxymephenytoin in plasma is described. The compounds were separated using a reversed-phase C2 column in tandem with a chiral α1-acid glycoprotein column and were detected using ultraviolet detection at 205 nm. The lower limit of quantification was 10 ng/ml for all compounds using 0.5 ml human plasma (intra-day coefficient of variation <13%, accuracy <±20%). The method was validated for human plasma in the concentration range 10–2000 ng/ml for each of the six compounds. The method allows for the simultaneous characterisation of the metabolic capacity of two human drug-metabolising enzymes, CYP2C19 and CYP2B6, and may be used when investigating polymorphisms or changes in activity of these two enzymes.