A rapid UPLC-MS/MS method for simultaneous determination of flunitrazepam, 7-aminoflunitrazepam, methadone and EDDP...

A simple, high-throughput, sensitive LC-ESI-MS/MS method is presented for the simultaneous determination of methadone (MET), flunitrazepam (FNZ) and their major metabolites, EDDP (2-ethilidene-1,5-dimethyl-3,3-diphenylpyrrolidone) and 7-aminoflunitrazepam (7-AFNZ), respectively, in human, rat and rabbit plasma. The isolation of the selected compounds involved a liquid-liquid extraction with ethyl acetate at a basic pH. Good chromatographic separation was achieved on a HSS T3 column (1.8 μm particle size), with a 3 min gradient elution using a mixture of acetonitrile with 0.1% formic acid (solvent A) and 5mM ammonium acetate (solvent B) as the mobile phase. The tandem mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode with ionization of the analytes in positive mode. The assay was fully validated according to current acceptance criteria for bioanalytical methods validation. It was proved to be linear in the range of 0.5-250 ng/mL, with adequate accuracy and precision over this range. Based on accuracy and CV% values the LOQ and ULOQ values were set at 0.509 ng/mL and 2036 ng/mL for MET, 0.520 ng/mL and 2080 ng/mL for EDDP, 0.524 ng/mL and 2096 ng/mL for FNZ and 0.528 ng/mL and 2114 ng/mL for 7-AFNZ, respectively. The method was tested for potential matrix effects, without observing significant ion suppression. The investigated compounds stability was examined in plasma at room temperature and after three freeze-thaw cycles and in the final extract when maintained at 4 °C in the autosampler. Potential stability issues were observed only for FNZ at room temperature. The method was successfully applied to quantify the selected compounds in human, rat and rabbit plasma samples, after exposure to FNZ or simultaneous exposure to FNZ and MET.