UPLC-MS/MS for Metabolite Profiling

All samples were analyzed by the UPLC-MS/MS system according to our previous work [32 (link)]. Firstly, all chromatographic separations were performed using an ultra performance liquid chromatography (UPLC) system (SCIEX, UK). An ACQUITY UPLC BEH Amide column (100 mm × 2.1 mm, 1.7 μm, Waters, Milford, MA, USA) was used for the reversed phase separation. The column oven was maintained at 35 °C. The flow rate was 0.4 mL/min and the mobile phase consisted of solvent A (25 mM ammonium acetate and 25 mM NH₄H₂O) and solvent B (IPA:CAN = 9:1, v/v, and 0.1% of formic acid). Gradient elution conditions were set as follows: 0 ~ 0.5 min, 95% of solvent B; 0.5 ~ 9.5 min, 95 to 65% of solvent B; 9.5 ~ 10.5 min, 65% ~ 40% of solvent B; 10.5 ~ 12 min, 40% of solvent B; 12 ~ 12.2 min, 40% ~ 95% of solvent B; 12.2 ~ 15 min, 95% of solvent B. The injection volume for each sample was set at 4 μL.
A high-resolution MS/MS TripleTOF 5600 plus (SCIEX, UK) was used to recognize the metabolites eluted from the column. The TOF was carried out in both positive and negative ion modes. The detail parameters of UPLC-MS/MS analysis were set according to our previous work [32 (link)]. Furthermore, in order to evaluate the stability of the UPLC-MS/MS system during the whole data acquisition process, one quality control sample was detected after every 10 samples.