2.4. GC–MS-based untargeted metabolomics analysis

Metabolic profiling of the derivative sample was analyzed by using an Agilent 7890B gas chromatography system (Agilent Technologies Inc., CA, USA) equipped with a DB-5MS fused-silica capillary column (30 m × 0.25 mm × 0.25 μm) at a constant helium flow rate of 1 mL/min. The parameters of GC–MS condition were set as follows: injector temperature, 260 °C; injection volume, 1 μL; MS quadrupole temperature, 150 °C; ion source temperature, 230 °C; collision energy, 70 eV; mass range, 50–500 m/z. The QC samples were injected every 10 runs for data quality control.

The raw data of GC–MS analysis were converted to ABF format via the Analysis Base File Converter software, and then imported into the MS-DIAL software for peak detection, deconvolution, alignment, and filtering. Metabolites were identified in the basis of the LUG database (Untargeted database of GC–MS from Lumingbio). All internal standards and pseudo positive peaks were removed and then the data matrix with three dimension datasets including sample information, peak names and intensities was exported as the Excel file with for further analysis.