CYP2C19-Mediated (S)-Mephenytoin 4-Hydroxylation Assayed by High-Performance Liquid Chromatography With Radiometric Detection

A reverse-phase, high-performance liquid chromatography method is described for the quantification of 4′-hydroxymephenytoin formed enzymatically from ¹⁴ C-labeled (S )-mephenytoin following incubation with cDNA-expressed CYP2C19 or human liver microsomes. Analytical separation is achieved using a C₁₈ column developed with a gradient from 10 to 100% methanol, with detection using a scintillation detector. This method is applicable to enzymatic studies for determination of CYP2C19-catalyzed (S )-mephenytoin 4′-hydroxylation activity.