Simple and sensitive high-performance liquid chromatographic method for the determination of 1,5-benzodiazepine clobazam and its active metabolite N-desmethylclobazam in human serum and urine with application to 1,4-benzodiazepines analysis

A HPLC–UV determination of clobazam and N-desmethylclobazam in human serum and urine is presented. After simple liquid–liquid extraction with dichloromethane the compounds and an internal standard diazepam were separated on a Supelcosil LC-8-DB column at ambient temperature under isocratic conditions using the mobile phase: CH3CN–water–0.5 M KH2PO4–H3PO4 (440:540:20:0.4, v/v and 360:580:60:0.4, v/v for serum and urine, respectively). The detection was performed at 228 nm with limits of quantification of 2 ng/ml for serum and 1 ng/ml for urine. Relative standard deviations for intra- and inter-assay precision were found below 8% for both compounds for all the tested concentrations. The described procedure may be easily adapted for several 1,4-benzodiazepines.