High-performance liquid chromatography determination of flunitrazepam and its metabolites in plasma by use of column-switching technique: comparison of two extraction columns

A study, using on-line column-switching high-performance liquid chromatography, evaluated two different extraction columns for the determination of flunitrazepam and its major metabolites: 7-aminoflunitrazepam, 7-acetamidoflunitrazepam and desmethylflunitrazepam. The procedure was based on the enrichment of benzodiazepines on the extraction column, followed by transfer of the compounds to the analytical column. The two extraction columns were compared: the first column was a BioTrap 500 MS (hydrophobic polymer), 20×4 mm I.D., and the second was a LiChrospher RP-18 ADS, 25×4 mm I.D. The analytical column used was a LiChrospher select B RP-8, 125×3 mm I.D. with 5 μm particle size. The extraction conditions for the two pre-concentration columns, such as extraction temperature, buffer concentration, buffer pH, acetonitrile percentage and flow-rate, were studied for the extraction from plasma of flunitrazepam and its metabolites mentioned above. The mobile phase of the analytical column was isocratic and composed of acetonitrile–20 mM phosphate buffer at pH 2.1 (35:65, v/v) and at a flow-rate of 0.3 ml/min.