Uhplc q exactive orbitrap ms

Compounds CDD-1713 and CDD-1976 (2.0 μM) were incubated in mouse or human liver microsomes (0.5 mg protein/mL) at 37 °C. The samples were collected at specific time points, 0, 30, and 60 min, in duplicate. The reactions were terminated by adding an equivalent volume of ice-cold methanol and vortexed. The reaction mixtures were centrifuged at rcf 15,000 for 15 min. Three microliters L of the supernatant was analyzed by UHPLC-Q Exactive Orbitrap MS (Thermo Fisher Scientific) equipped with 50-mm × 4.6-mm column (XDB C-18; Agilent Technologies). The column temperature was maintained at 40 °C. The flow rate was at 0.3 mL/min with a 30% mobile phase (acetonitrile containing 0.1% formic acid). Q Exactive MS was operated in positive mode with electrospray ionization. Ultrapure nitrogen was applied as the sheath (45 arbitrary units), auxiliary (10 arbitrary units), sweep (1.0 arbitrary unit), and the collision gas. The capillary gas temperature was set at 275 °C and the capillary voltage was set at 3.7 kV. Mass Spectrometry (MS) data were acquired from 80 to 1,200 Da in profile mode. JQ1 and alprazolam were used as the short and long half-life control, respectively.

Metabolic profiling of liver samples was conducted using a UHPLC-Q Exactive Orbitrap-MS (Thermo Fisher Scientific) fitted with an electrospray ionization (ESI) source. A Hypersil Gold C18 column (100×2.1 mm, 1.9 μm; Thermo Fisher Scientific) was used for metabolite separation. The column temperature was 40°C. Mobile phase A consisted of 0.1% formic acid in water and mobile phase B consisted of acetonitrile with 0.1% formic acid, the elution gradient program used was described in the previous study (Gao et al., 2018 (link)). The sample injection volume was 3 µl and the flow rate was set at 0.2 ml/min. Data were acquired using Full Scan-ddMS2 scan mode, all samples were analyzed under positive and negative ionization modes, mass parameters of the ESI ion source were set as follows: capillary temperature, 320°C; heater temperature, 300°C; sheath gas flow rate, 35 arb; auxiliary gas flow rate, 10 arb; and scan range, 100–1,500 m/z (Gong et al., 2019 (link); Yu et al., 2019 (link)).

The NMR spectra were obtained using a Bruker AV 300 MHz spectrometer (Burker, Fallanden, Switzerland) in CD₃OD and TMS (tetramethylsilane) as the internal standard. High-resolution electrospray ionization mass spectra (HR-ESIMS) were recorded using a UHPLC–Q Exactive Orbitrap–MS (Thermo Fisher Scientific, Boston, MA, USA). Optical rotations were measured with a Rudolph Autopol I automatic polarimeter. Column chromatography was performed using normal-phase silica gel (200–300 mesh, Branch of Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Thin-layer chromatography (TLC) was conducted on pre-coated silica gel GF₂₅₄ glass plates (200 × 200 mm, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) and RP-18 F₂₅₄S (Merck KGaA, Darmstadt, Germany). All reagents and solvents were of reagent grade or purified according to standard methods before use.