Simultaneous analysis of flunitrazepam and its major metabolites in human plasma by high performance liquid chromatography tandem mass spectrometry.

A sensitive and specific high performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS-MS) method has been developed for the simultaneous determination of flunitrazepam and its major metabolites, 7-aminoflunitrazepam and N-desmethylflunitrazepam, in human plasma. After the addition of a deuterium labelled internal standard of flunitrazepam, plasma samples were extracted using Oasis(R) MCX solid phase extraction cartridges. The compounds were separated on a 5 microm Symmetry C18 (Waters) column (3.0 x 150 mm, i.d.) with a step gradient of acetonitrile-0.1% formic acid at a flow rate of 0.6 ml/min. The overall extraction efficiency was more than 89% for all three compounds. The limits of detection were 0.25 g/l for flunitrazepam, 0.5 microg/l for 7-aminoflunitrazepam, and 2.0 microg/l for N-desmethylflunitrazepam. Within-run accuracies for quality-control samples were between 92.5 and 101.3% of the target concentration, with coefficients of variation <8%. The proposed method enables the unambiguous identification and quantitation of flunitrazepam and its major metabolites in both clinical and forensic specimens.