Waters beh c8 column

The samples were processed in the same batch for UHPLC-MS/MS analysis on a hybrid quadrupole-Orbitrap mass spectrometer (Vanquish UPLC-Q Exactive, Thermo Fisher Scientific, Rockford, IL, USA). For positive ion mode, we used Waters BEH C8 column (50 mm × 2.1 mm, 1.7 μm) (Waters, Milford, MA) to separate mixture at 60°C. Phase A: water with 0.1% formic acid; phase B: acetonitrile with 0.1% formic acid. Flow rate was set at 0.4 ml/min with the following gradient: 0–0.5 min, 5%B; 0.5–2 min, linearly increased to 40%B; 2–8 min, linearly increased to 100% B; maintained for 2 min; 10.1 min, decreased back to 5% B; balanced for 2 min. For negative ion mode, we used ACQUITY UPLC HSS T3 (50 mm × 2.1 mm, 1.8 μm) (Waters, Milford, MA) to separate mixture at 60°C. Phase A: 6.5 mm NH4HCO3 added to water; phase B: 95% methanol and 6.5 mM NH4HCO3 aqueous solution. Flow rate was set at 0.4 ml/min with the following gradient: 0–0.5 min, 2%B; 0.5–2 min, linearly increased to 40%B; 2–8 min, linearly increased to 100% B; maintained for 2 min; 10.1 min, decreased back to 2% B; balanced for 1.9 min. The acquisition setting for metabolomics data: full scan mode; capillary temperature, 300°C; sheath gas, 45; auxiliary gas, 10; mass resolution, 7e4. For positive ions: spray voltage, 3.50 kV; m/z range, 80–1,200. For negative ions: spray voltage, 3.00 kV; m/z range, 80–1,200.