Synapt g2 si high definition mass spectrometer

30 mg of accurately weighted BAT or iWAT were homogenized in 200 μL of methanol:water (3:1) mixture under cold conditions. The tissue homogenates were vortexed for 10 min at 4 °C and centrifuged at 13,000 rpm (Heraeus Fresco 21, Thermo Fisher Scientific, Hamburg, Germany) for 15 min at 4 °C. The supernatant was carefully collected and dried under nitrogen gas, resuspended with 100 μL of acetonitrile and centrifuged at 13,000 rpm for 10 min at 4 °C before UPLC–QTOF-MS/MS analysis at the pharmaceutical core facility of Capital Medical University. The chromatographic separation of different metabolites was performed on an Acclaim RSLC C18 column (ThermoFisher Scientific). Mass spectrometry analysis was conducted using the SYNAPT G2-Si high-definition mass spectrometer (Waters) equipped with an electrospray ionization source. The acquired data were imported into Progenesis QI software for data normalization using peak alignment and baseline correction. The processed data were then searched against compound databases (KEGG, HMDB) using ChemSpider to obtain accurate molecular weight and fragmentation information of the compounds. Differentially enriched metabolites (P < 0.05, fold change >1.2, variable importance in projection score >1, and those including mass spectrometry fragments) were selected.