Selective and sensitive assay for the determination of benzodiazepines by high-performance liquid chromatography with simultaneous ultraviolet and reductive electrochemical detection at the hanging mercury drop electrode

An isocratic chromatographic method for the simultaneous determination of 10 benzodiazepines is presented. The selectivity of the assay was optimized by variation of stationary phase, temperature, as well as ionic strength, composition and pH of the mobile phase and the dependence of the detector response on the applied potential was investigated. The best results with respect to resolution at moderate retention times were obtained with a mixture of 0.02 mol/l phosphate buffer (pH 6) and acetonitrile in a volume ratio of 55:45 (v/v) on a LiChrospher-100 RP-8ec column (150×4.6 mm I.D.). Considerable improvement of selectivity was achieved if the column temperature was kept constant at 12°C. Two detection modes were applied, UV detection at 250 nm, inserted upstream to the electrochemical detector, and reductive electrochemical detection at the hanging mercury drop electrode at −1.4 V (vs. Ag/AgCl), which proved to be especially sensitive in case of nitrophenyl-containing benzodiazepine species. The elaborated assay showed to be linear up to at least 2 mg/l for each compound. Detection limits generally were in the range of 6.5–123 ng/ml (130 pg–2.46 ng on-column, using a 20-μl loop) with relative standard deviations between 1.1 and 8.6% depending on the actual benzodiazepine investigated.