Quantitation of clonazepam and its 7-amino and 7-acetamido metabolites in plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic method is reported for the determination of clonazepam and its metabolites 7-amino- and 7-acetamidoclonazepam. Extraction from buffered plasma is carried out at pH 9.5 with hexane-ethyl acetate (7:3) for clonazepam and with chloroform for the metabolites. Flunitrazepam, 7-aminodemethylflunitrazepam and 7-acetamidoflunitrazepam are used as the internal standards for clonazepam and its 7-amino and 7-acetamido metabolites, respectively. To prevent decomposition of 7-aminoclonazepam a high concentration of 7-aminomethylclonazepam is added to the plasma. Chromatography is carried out on a reversed-phase column with detection at 254 nm for clonazepam and 240 nm for the metabolites. Using the method it was possible to determine 5 ng/ml clonazepam, 7-aminoclonazepam and 7-acetamidoclonazepam in plasma with coefficients of variation of 9.5%, 5.9% and 8.9%, respectively. This method can be used to measure clonazepam in plasma from patients treated with other antiepileptics. It may also be utilized for in vitro studies on the metabolism of clonazepam in subcellular fractions from the liver.