Quantification of flunitrazepam's oxidative metabolites, 3-hydroxyflunitrazepam and desmethylflunitrazepam, in hepatic microsomal incubations by high-performance liquid chromatography.

A high-performance liquid chromatographic assay for the quantification of the oxidative metabolites of flunitrazepam, 3-hydroxyflunitrazepam and desmethylflunitrazepam, in human liver microsomal incubations was developed. Both metabolites were quantifiable in a single assay following a solvent extraction and reversed-phase high-performance liquid chromatography with UV detection. Standard curve concentrations for both metabolites ranged from 0.2 to 10 microM. Assay performance was determined using quality control samples and the intra- and inter-day accuracy and precision as determined by the coefficient of variations which were less than 15% (0.5-6 microM) for both metabolites. This method provides good precision and accuracy for use in kinetic studies of the oxidative metabolism of flunitrazepam in human liver microsomes.