Untargeted Metabolome Analysis by LC-MS/MS

LC-MS/MS analyses were performed using an UHPLC system (Vanquish; Thermo Fisher Scientific, Jiading District, Shanghai, China) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) coupled to Orbitrap Exploris 120 mass spectrometer (Orbitrap MS; Thermo). The mobile phase consisted of 5 mmol/L ammonium acetate and 5 mmol/L acetic acid in water (A) and acetonitrile (B). The auto-sampler temperature was 4 ℃, and the injection volume was 2 μL. The Orbitrap Exploris 120 mass spectrometer was used for its ability to acquire MS/MS spectra on information-dependent acquisition mode in the control of the acquisition software (Xcalibur; Thermo). In this mode, the acquisition software continuously evaluates the full scan MS spectrum. The ESI source conditions were set as following: sheath gas flow rate as 50 Arb, Aux gas flow rate as 15 Arb, capillary temperature 320 ℃, full MS resolution as 60,000, MS/MS resolution as 15,000 collision energy as 10/30/60 in NCE mode, spray Voltage as 3.8 kV (positive) or −3.4 kV (negative), respectively.^{[11 (link)]}