UPLC-QTOF-MS Metabolomic Analysis

Analysis was undertaken using UPLC/Q-TOF MS (SYNAPT G2-Si, Waters, USA) with an ACQUITY UPLC^® HSS T3 chromatographic column (1.8 μm, 2.1 mm × 100 mm, Waters, USA) kept at 40 °C. The method was based on the methods described previously [40 (link)]. Briefly, samples (3 μL) were separated using 0.1% (v/v) formic acid (mobile phase A) and 100% acetonitrile (mobile phase B): 0 min at 30% B, 6 min at 45% B, 18 min at 60% B, 23 min at 90% B, at 0.5 mL min⁻¹ flow rate. Mass detection was conducted by electrospray ionization (ESI) in the positive ion mode. The QTOF-MS conditions were as follows: sample cone, 40 V; source temperature, 120 °C; desolvation temperature, 450 °C; cone gas flow, 50 L h⁻¹; and desolvation gas flow, 800 L/h; capillary voltage, 1000 V. The ramp collision energy was set as 20–65 eV for the high-energy scans. Data analysis was performed using the MassLynx V4.1, Progenesis QI 3.0.3 and EZinfo 3.0 software (Waters). Online database included KEGG, MassBank, Nature Communications, Nature Chemistry, Nature Chemical Biology, and ChemSpider. Variable importance parameter (VIP) value > 1.