Determination of flunitrazepam and its metabolites in blood by high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry

A selective assay of flunitrazepam (F) and its metabolites 7-aminoflunitrazepam (7-AF), N-desmethylflunitrazepam (N-DF) and 3-hydroxyflunitrazepam (3-OHF) with liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (LC–APCI-MS, positive ions) is described. The drugs were isolated from serum, blood or urine using a solid-phase extraction procedure previously applied to various drugs of abuse. F-d₃ and 7-AF-d₃ were used as internal standards. The drugs were separated on ODS column in acetonitrile–50 mM ammonium formate buffer, pH 3.0 (45:55, v/v). After analysis of mass spectra taken in full scan mode, a selected-ion monitoring detection was applied with following ions: m/z 284 (7-AF and F), 287 (7-AF-d₃ and F-d₃), 314 (F), 300 (N-DF and 3-OHF), 317 (F-d₃), 330 (3-OHF). The limits of detection were: 0.2 μg/l for F and 7-AF, 1 μg/l for N-DF and 3-OHF. The method was linear in the range 1–500 μg/l, the recoveries ranged from 92 to 99%. The method was applied for determination of F and metabolites in clinical and forensic samples. LC–APCI-MS seems to be a method of choice for these compounds.