UPLC-QTOF-MS analysis
All samples were analyzed with an LC-MS system following the manufacturer’s instructions. First, all chromatographic separations were performed using a UPLC system (SCIEX, Framingham, MA, USA). An ACQUITY UPLC T3 column (100 × 2.1 mm, 1.8 µm, Waters, Milford, MA, USA) was used for reverse-phase separation. The column oven was maintained at 35 °C. The flow rate was 0.4 mL/min, and the mobile phase consisted of solvent A (water, 0.1% formic acid) and solvent B (acetonitrile, 0.1% formic acid). Gradient elution conditions were set as follows: 0–0.5 min, 5% B; 0.5–7 min, 5% to 100% B; 7–8 min, 100% B; 8–8.1 min, 100% to 5% B; 8.1–10 min, 5% B. The injection volume for each sample was 4 µL.
A high-resolution tandem mass spectrometer TripleTOF5600plus (SCIEX) was used to detect the metabolites eluted from the column. The QTOF was operated in both positive and negative ion modes. The curtain gas was set to 30 PSI, ion source gas 1 was set at 60 PSI, ion source gas 2 was set at 60 PSI, and the interface heater temperature was 650 °C. For positive ion mode, the ionspray voltage floating was set to 5000 V. For negative ion mode, ionspray voltage floating was set to −4500 V. MS data were acquired in information-dependent acquisition mode. Dynamic exclusion was set to 4 s. During acquisition, the mass accuracy was calibrated for every 20 samples. Furthermore, to evaluate the stability of LC-MS throughout the acquisition, a quality control sample (pool of all samples) was acquired after every 10 samples.