Multi-panel detection of drugs and drug metabolites in hair samples using a comprehensive extraction method

The aim of this presentation is to demonstrate a robust and sensitive LC-MS/MS-based method for the detection of a wide range of drugs and drug metabolites in hair. The presentation will demonstrate that the use of an optimize extraction method enabled accurate detection and sensitive quantification of a panel of 77 compounds including novel psychoactive substance (NPS) and metabolites with a wide range of physical and chemical properties. A total of 77 compounds were included in the novel psychoactive substance (NPS) panel. The drugs of abuse (DOA) panel consisted of 23 compounds and the third panel consisted of Ethyl glucuronide (EtG) only. Hair samples were washed thoroughly with methanol for 5 minutes then diethyl ether for another 5 minutes, dried and spiked with the various drug calibrator mixtures. 500 μL of a proprietary aqueous extracting solution was added to the spiked hair samples, gently vortexed, centrifuged at 5000 rpm for 1 minute and incubated for 60 minutes at 100 °C. Following the incubation step, the liquid extract was transferred to a new tube and centrifuged at 5000 rpm for 5 minutes. The organic phase was transferred to a sample vial for analysis. Analytes were chromatographically separated at 50 °C using a Phenomenex Synergi 2.5 μm Hydro-RP column (50 × 3 mm, 2.5 μm, P/N 00D-4387-Y0). Mobile phases used consisted of HPLC grade water with 0.1% formic acid (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B). Three separate gradients, flow rates and injection volumes were were used for each of the LC methods. MS data were collected in positive mode on the SCIEX QTRAP 6500+ System. Three separate acquisition methods were created using the Scheduled multiple reaction monitoring (MRM) Algorithm. The acquisition methods consisted of 156 MRM transitions for the NPS panel, 71 MRM transitions for the DOA panel and 3 MRM transitions for the EtG experiment, respectively. The calibration curves resulting from the NPS panel demonstrated excellent linearity for all the analytes included in the panel with R2 values > 0.98. The robustness of the NPS screening method was further investigated by analyzing 10 authentic hair samples in which two synthetic cannabinoids JWH-122 and AM-2201 were detected in an authentic case sample at concentrations of 111.8 and 11.72 pg/mg, respectively. Ketamine, oxycodone, tramadol and zolpidem were detected in another authentic case sample at concentrations of 0.39, 1602.5, 248.4 and 0.04 ng/mg, respectively. The results demonstrate that this robust method enable sensitive quantification of a variety of drugs and metabolites from authentic case samples. A comprehensive workflow combining the sensitivity of the SCIEX QTRAP 6500+ LC-MS/MS System with a simple and optimized extraction procedure enabled detection of a wide variety of drugs and drug metabolites in hair samples. The developed workflow was shown to enable accurate identification and sensitive quantification of a wide range of chemically diverse analytes included in three different panels. A number of analytes were accurately detected in authentic hair samples, demonstrating the broad applicability of the sample preparation procedure for the detection of the analytes included in the panel.