A Thermo Ultimate 3000 system equipped with an ACQUITY UPLC® HSS T3 (150 × 2.1 mm, 1.8 µm, Waters, Milford, DE, USA) column was employed to separate the metabolites. The temperatures of autosampler and column were maintained at 8 °C and 40 °C, respectively. The sample volume was 2 µL. Mobile phase in negative-ion mode was solvent A (5 mM ammonium formate in water) and solvent B (acetonitrile). Mobile phase in positive-ion mode was solvent C (0.1% formic acid in water) and solvent D (0.1% formic acid in acetonitrile). The elution procedure was as follows: 2% B/D (0~1 min), 2%~50% B/D (1~9 min), 50%~98% B/D (9~12 min), 98% B/D (12~13.5 min), 98%~2% B/D (13.5~14 min), 2% D in positive model (14~20 min) or 2% B in negative model (14~17 min). The flow rate was set as 0.25 mL/min. The separated metabolites were then analyzed on a Thermo Q Exactive mass spectrometer with electrospray ionization (ESI) system [24 (link)]. The spray voltages applied were 3.8 kV in positive mode and 2.5 kV in negative mode. Sheath gas and auxiliary gas were set as 30 and 10 arbitrary units, respectively. The capillary temperature was maintained at 325 °C. The m/z scan range was 81 to 1000 for full scan at a mass resolution of 70,000 [24 (link)]. Data-dependent acquisition (DDA) and dynamic exclusion were performed according to the procedures as described [24 (link),25 (link)].
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