Acquity h class lc system

Ultra‐performance LC‐MS analyses of samples were conducted using a Waters ACQUITY H‐class LC system coupled with a Triple TOF 5600 mass spectrometer (AB SCIEX, MA, USA). Metabolites were separated on a Waters HSS C18 column (3.0 × 100 mm, 1.7 μm) with a 17‐min gradient at a flow rate of 0.3 mL/min. Mobile phase A was 0.1% formic acid in H2O, and mobile phase B was acetonitrile. The gradient was set as follows: 0−2 min, 2% solvent B; 2−5 min, 2−55% solvent B; 5−15 min, 55−100% solvent B; 15−20 min, 100% solvent B; 20−20.1 min, 100−2% solvent B; 20.1−29 min, 2% solvent B. The column temperature was set at 50°C. All samples were full scan analyzed from 50 to 1200 m/z. The full‐scan accumulation time was 0.25 s, the MS/MS accumulation time was 0.1 s, GAS1 and GAS2 were 55, the temperature was 550°C, the ionization spray voltage was 4500 V, and the MS/MS scan collision energy was 35. The top 100 precursors of the full scan were selected for the MS/MS analysis. The dynamic exclusion time was 5 s.

The Waters ACQUITY H-class LC system coupled with an AB Sciex TripleTOF 5600 (AB Sciex, United States) was launched to perform the ultra-performance LC-MS analyses of the plasma samples. We separated the plasma metabolites with a 17 min gradient on a Waters HSS C18 column (3.0 × 100 mm, 1.7 μm), and the flow speed was 0.5 mL/min. Mobile phases A and B were 0.1% formic acid in H2O and acetonitrile, respectively. The gradient was as follows: 0–1 min, 2% solvent B; 1–3 min, 2%–15% solvent B; 3–6 min, 15%–50% solvent B; 6–9 min, 50%–95% solvent B; 9–9.1 min, 95%–100% solvent B; 9.1–12 min, 100% solvent B; 12–12.1 min, 100%–2% solvent B; and 12–17 min, 2% solvent B. The column temperature was 45°C. Data dependent acquisition mode was used to acquire the MS and MS/MS spectra. The 10 most abundant ions were submitted for MS/MS fragmentation with a collision energy of 35+-15 eV.