UHPLC-QTOF MS
The LC-MS/MS system used was an ultra-high performance liquid chromatography (Agilent 1260 Quaternary pump, Agilent 1260 High Performance autosampler, and Agilent 1290 Thermostatted Column Compartment SL, Agilent Technologies) coupled to a quadrupole time of flight mass spectrometer (Q-TOF-MS) (TripleTOF 5600+, Sciex) with an electrospray ionization (ESI) using a DuoSpray ion source. Mobile phase system for UHPLC was water containing 10 mM ammonium acetate and 0.1% formic acid (mobile phase A) and acetonitrile:water at a 95:5 ratio (v/v) containing 10 mM ammonium acetate and 0.1% formic acid (mobile phase B). Human liver microsome extracts were transferred directly to a LC vial for injection. Evaporated supernatant from primary human hepatocytes and whole blood samples were reconstituted in 20 and 50 μL, respectively, of mobile phase at starting gradient (mobile phase A:mobile phase B at ratio 60:40 (v/v)) and transferred to a LC vial for injection. LC vials were kept in the auto-sampler at 6°C during analysis. 5 µL of sample was injected on to a C18 reversed phase column (Acquity UPLC HSS T3, 2.1×100mm, 1.8 µM, Waters), protected by a pre-column (Acquity UPLC HSS T3, 2.1×5mm, 1.8 µM, Waters), for separation by UHPLC at a flow rate of 0.3 mL/min at 40°C. The UHPLC elution gradient was started at 40% mobile phase B for 2 min (0-2.0 min), 40-80%B for 2 min (2.0-4.0 min), 80-100% B for 5 min (4.0-9.0 min), 100%B for 5 min (9.0-14.0 min), 100-40%B for 0.1 min (14.0-14.1), and 40%B for 3.9 min (14.1-18.0 min). The UHPLC-QTOF-MS system, mass ion chromatogram and mass spectra were acquired by AnalystTM Software version 1.7 (SCIEX). The QTOF MS was operated in ESI positive mode at ion source gas 1 (GS1) of 40 psi, ion source gas 2 (GS2) of 40 psi, curtain gas (CUR) of 30 psi, ion spray voltage floating (ISVF) of 4500 V, source temperature (TEM) at 350°C, and declustering potential (DP) of 120 V. Data were acquired in the informative dependent acquisition (IDA) mode composed of a TOF-MS scan and 10 dependent product ion scans in the high sensitivity mode with dynamic background subtraction. Mass range of TOF-MS scan was at m/z 100-1,000 and product ion scan was at m/z 50-1,000. IVM standard solution (100 ng/mL) was injected before and after batch analysis for validating the system performance.