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.