Simultaneous Separation of Diazepam and Its Chiral and Achiral Metabolites by HPLC on a Chiralcel OD-R Column

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Abstract
A reversed-phase HPLC method was developed for the simultaneous separation of diazepam and its achiral (nordiazepam) and chiral (oxazepam and temazepam) metabolites. Chromatographic resolution of the analytes was performed under isocratic conditions using a mobile phase of 0.3 M sodium perchlorate-acetonitrile (57:43, v/v) at a flow rate of 0.5 ml/min on a reversed-phase cellulose-based chiral stationary phase (Chiralcel OD-R column) with UV detection set at 210 nm. Disc solid-phase extraction was carried out for serum sample clean-up prior to HPLC separation. Recoveries were determined for diazepam, nordiazepam, and oxazepam and temazepam enantiomers from human serum at 300 ng/ml levels. The limits of detection were 25 ng/ml each for diazepam and nordiazepam, and about 25 ng/ml for each enantiomer of oxazepam and temazepam (S/N = 2). The method can be applicable to the in vivo analysis of human serum samples containing diazepam and its chiral and achiral metabolites.