Gc gc tofms

GC×GC-TOFMS (LECO, USA) sample preparation, derivatization, and spectral acquisition were prepared according to published methods [13 (link)]. Briefly, 50 μL of plasma and 300 μL of mixed solvent (methanol : chloroform = 3 : 1, Thermo Fisher, USA) were added into a 1.5 mL centrifuge tube and vortexed for 30 s. Leave the hybrid solution in the refrigerator at −20°C for 10 minutes. After centrifugation at 1000 RPM for ten minutes, transfer 300 μL of the supernatant to the vial and add 10 μL of 0.1 mg mL⁻¹ chlorobenzene (Thermo Fisher, USA), alanine (Sigma-Aldrich, USA), and 1 mg mL⁻¹ heptadecanoic acid. After the sample was freeze-dried, 80 μL of 15 mg mL⁻¹ pyridine (Sigma-Aldrich, USA) dissolved methoxamine and 50 μL BSTFA (1%TMCS, Sigma-Aldrich, USA) were added, respectively. The mixed sample was at 70°C for 60 minutes and then cooled down before injection.

Fasting serum samples were analyzed using two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOF-MS) (Leco Corp., St. Joseph, MI, USA) [15 (link),16 (link)] (Supplementary Methods). In all, 65 circulating metabolites, including fatty acids, carbohydrates, amino acids, and other sub-classes (Table S1), could be semiquantified and were known species out of 466 measured. The 65 semiquantified metabolites were included in the current study.