Simultaneous determination of four designer drugs and their major metabolites by liquid chromatography–mass spectrometry

• LC–MS was applied in the analysis of four designer drugs.
• The in vitro metabolism of four designer drugs was performed.
• The main metabolites of drugs in rat liver microsome S9 fraction were identified.

A sensitive liquid chromatography–electrospray ionization-ion trap mass spectrometry (LC–ESI-ITMS) method was utilized for the simultaneous analysis of four designer drugs and their in vitro metabolites in rat liver microsome S9 fraction. Four designer drugs, including methcathinone (MC), 3,4-methylenedioxymethcathinone (MDMC), 3,4-methylenedioxy-pyrovalerone (MDPV) and 4′-methyl-α-pyrrolidinopropiophenone (MPPP), were individually incubated with rat liver microsome S9 fraction, and the incubation mixtures were pooled together and analyzed by LC–ESI-ITMS simultaneously. Besides four designer drugs, five of their main metabolites were identified via the analysis of protonated molecules and tandem mass spectrometry data. Meanwhile, the quantification analysis of four designer drugs in rat liver microsome S9 fraction was performed, the calibration curves showed good linearity in the range of 0.01–5.0 μg/mL and the detection limits were below 0.03 μg/mL with RSDs less than 5.9% and recovery ratios above 77.4%. The experimental results not only showed that these designer drugs could be easily metabolized in rat liver microsome, and also displayed the superiorities of the method including time and cost saving, high efficiency, sensitivity and selectivity. The studies in this study indicated that the approach could be applied in the determination of illicit drugs and their metabolites in medical, pharmaceutical and forensic investigations.