HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD FOR MEASUREMENT OF CYTOCHROME P450-MEDIATED METABOLISM OF 7-ETHOXY-4-TRIFLUOROMETHYLCOUMARIN

An HPLC method for analysis of deethylation of 7-ethoxy-4-trifluoromet hylcoumarin (ETFMC), a substrate of various enzymes of the cytochrome P450 superfamily, was developed. ETFMC was incubated at 37 degrees C w ith human hepatic microsomes or microsomes prepared from a lymphoblast oid cell line that expresses human CYP2B6. Under these conditions, the highly fluorescent metabolite 7-hydroxy-4-trifluoromethylcoumarin (HT FMC) is formed. The metabolite was analyzed by reversed-phase HPLC wit h fluorescence detection. The limits of detection of the metabolite we re 5.0 fmol per injection, a sensitivity at least one order of magnitu de greater than the standard method, which does not involve HPLC. This method will be of great utility when quantities of microsomal protein from cell lines expressing human CYP enzymes are limited. (C) 1998 El sevier Science B.V.