Uplc ms ms system

The Shimadzu UPLC-MS/MS system was used for the PK study. The system consists of high-performance liquid chromatography (LC-30A) and a triple quadrupole mass spectrometer (LCMS-8050) with ESI source. The multiple reaction monitoring (MRM) mode was used both in positive and negative mode. The MS parameters were as follows: flow rate of heating gas 10 L/min, flow rate of drying gas 10 L/min, flow rate of atomizing gas 3 L/min, interfacial temperature 300 ℃, DL temperature 250 ℃. The LabSolution LCMS ver. 5.6 software (Shimadzu, Japan) for data collection and processing.
Separation was performed using a Waters ACQUITY UPLC BEH C₁₈ column (2.1 mm × 100 mm, 1.7 μm). The mobile phase was 0.1% formic acid water (v/v) (A) and acetonitrile (B). The optimal elution conditions were as follows: in positive ion mode, 0–8 min, 15–30% B; 8–10 min, 30–100% B; 10–12 min, 100% B; 12–14 min, 100–15% B; 14–17 min, 15% B. In negative ion mode, 0–2 min, 15–30% B; 2–7 min, 30–60% B; 7–8 min, 60–100% B; 8–10 min, 100% B; 10–12 min, 100–15% B; 12–15 min, 15% B.

Sample analysis was performed on Shimadzu UPLC-MS/MS system (Kyoto, Japan) consisting of two LC-30AD pumps, a DGU-20A degasser, a SIL-20AC autosampler, a CTO-20AC column oven, a CBM-20 controller and an MS-8060 mass spectrometer. Instrument control, data acquisition, and data analysis were performed with LabSolutions software.
A Kinetex Phenyl-Hexyl column (150 × 2.1 mm, 1.7 μm, Phenomenex) was applied for chromatographic separation. Aqueous 2 mM ammonium formate with pH 5.2 (Solvent A) and methanol (solvent B) were used as mobile phases with a flow rate of 0.2 mL/min under gradient elution: 0–2.5 min, 50% B; 2.5–2.6 min, 50% B → 80% B; 2.6–5 min, 80% B; 5–5.1 min 80% B → 50% B; 5.1–8 min 50% B. The column temperature was 30 °C, and the injected sample volume was set at 2 μL. The sample vials were maintained at 4 °C in a thermostatic autosampler. The MS data was monitored using multiple reaction monitoring (MRM) mode. The optimal MS parameters were as follows: capillary voltage, 3.0 kV; nebulizing gas flow rate, 3.0 L/min; heating gas flow rate, 10.0 L/min; drying gas flow rate, 10.0 L/min; source temperature, 300 °C; heat block temperature, 400 °C. The MRM parameters for the precursor ions and product ions of all analytes and IS are shown in Additional file 6: Table S4.