We dissolved the isolated metabolites with 10 μL acetonitrile and water (1:1). We then vortexed the solution, centrifuged it for 15 min at 14,000 rpm and 4°C, and collected the supernatant liquid for target metabolite examination. In this study, we adopted ultra-high-performance liquid chromatography coupled to triple-quadrupole linear ion-trap tandem mass spectrometry (UHPLC-QTRAP-MS/MS) to measure 346 targeted metabolites in samples (Shanghai Applied Protein Technology, Shanghai, China). In brief, we used an Agilent 1290 Infinity II Multisampler (Agilent Technologies, Santa Clara, CA, USA) to pump the prepared samples through HILIC (temperature: 35°C, flow rate: 0.3 mL/min, sample volume: 2 μL) and C18 (temperature: 40°C, flow rate: 0.4 mL/min, sample volume: 2 μL) columns for metabolite separation and signal recording. We then examined the metabolites with the QTRAP 6500 mass spectrometry system (SCIEX, Framingham, MA, USA) under a multi-reaction monitoring model (sheath and dry gas temperature: 350°C, sheath and dry gas flow: 11 and 10 L/min, respectively).
We used MultiQuant Software (version 3.0.2) to isolate the raw peak intensities of the targeted metabolites from the output data according to their retention times and calculated the quantities of the targeted metabolites based on the peak intensity ratios between the targeted metabolites and internal standards.
We used MultiQuant Software (version 3.0.2) to isolate the raw peak intensities of the targeted metabolites from the output data according to their retention times and calculated the quantities of the targeted metabolites based on the peak intensity ratios between the targeted metabolites and internal standards.