Separation of Clonazepam and Five Metabolites by Reverse Phase HPLC and Quantitation from Rat Liver Microsomal Incubations

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Abstract
Clonazepam [an anticonvulsant], its nitro-reduced metabolites, and the 3-hydroxylated derivatives, were separated by reverse-phase hplc [high performance liquid chromatography] using a 4 solvent optimization procedure. The separation was used to quantitate the metabolism of 3H-clonazepam in rat liver microsomes under aerobic or anaerobic conditions by collection of HPLC eluents and liquid scintillation counting. The predominant metabolite in aerobic incubations was 3-hydroxyclonazepam. The primary anaerobic metabolite was 7-aminoclonazepam. The identity of the microsomal metabolites was confirmed by chemical ionization mass spectrometry. This separation could be applicable for future metabolic studies of clonazepam.