Quantitative Analysis of Metabolites

Chromatographic separation was carried out on a 5 m C18 column (50 2.0 mm internal diameter; Bohus, Sweden) fitted with an Agilent LC-ESI-MS System (Agilent Technologies, Palo Alto, CA, USA). The temperature of the column was maintained at 40°C. The whole movie lasted for 70 minutes. The mobile phase, which was acetonitrile-water (50 : 50, v/v) containing 0.1% trifluoroacetic acid, was administered at a flow rate of 0.3 ml/min. When the mass spectrometer was in the positive ion mode, both of its quadrupoles were set to 0.7 full width at half height (FWHM, unit resolution). In addition, the spray voltage was set at 4500 V and the ion tube temperature at 210°C. At 49, 2.0, and 14 arbitrary units, the nitrogen sheath, ion sweep, and auxiliary gases, respectively, were all set. Through infusion experiments, the ESI-MS/MS parameters were altered to create the most deprotonated molecules and the most effective generation of unique fragment ions for all compounds. MS-DIAL V. 3.70 software was used to identify and calculate the relative percentage of molecules [23 (link)].