2.6.3. Metabolomics Analysis via UPLC-QTOF-MS/MS

To determine the changes of metabolites in mice serum, 50 μL serum was mixed with 200 μL methanol, and centrifuged at 13,000 g for 15 min. The supernatant was extracted and filtered with a nylon membrane (22 μm). Sample separation was accomplished using a UPLC system of Agilent 1290 Infinity II series. The UPLC system was equipped with an Agilent Eclipse Plus C18 Rapid Resolution HD column (2.1 mm × 100 mm, 1.8 m). The mobile phases and gradient were set as described [31]. For MS acquisition, an Agilent 6545 ESI-Q-TOF mass spectrometer was employed. The mass spectrum parameters were the same as our previous publication [31]. Reference ions 112.985587 and 1033.988109 were used for real-time calibration during acquisition in negative ionization mode. Metabolites were identified with the METLIN Database (DB). Metabolites with DB scores above 80 and mass error lower than 5 ppm (0.0005%) were screened as biomarkers for statistical analysis. Metabolic pathway analysis was conducted using MetaboAnalyst 4.0 and KEGG online platform.
