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 chromatogra phy, evaluated two different extraction columns for the determination of fl unitrazepam and its major metabolites: 7-aminoflunitrazepam, 7-acetamidoflu nitrazepam and desmethylflunitrazepam. The procedure was based on the enric hment of benzodiazepines on the extraction column, followed by transfer of the compounds to the analytical column. The two extraction columns were com pared: the first column was a BioTrap 500 MS (hydrophobic polymer), 20X4 mm I.D., and the second was a LiChrospher RP-18 ADS, 25X4 min I.D. The analyt ical column used was a LiChrospher select B RP-8, 125X3 mm I.D. with 5 mum particle size. The extraction conditions for the two pre-concentration colu mns, such as extraction temperature, buffer concentration, buffer pH, aceto nitrile percentage and flow-rate, were studied for the extraction from plas ma of flunitrazepam and its metabolites mentioned above. The mobile phase o f the analytical column was isocratic and composed of acetonitrile-20 mil p hosphate buffer at pH 2.1 (35:65, v/v) and at a flow-rate of 0.3 ml/min. (C ) 2001 Elsevier Science B.V. All rights reserved.