Analysis of the association between untargeted metabolomic and GM data
-20
Samples stored in a -80°C freezer were thawed on ice. A 400 μL solution (methanol: water = 7:3, V/V) containing an internal standard was added to a 20 mg sample, and the mixture was vortexed for 3 min. Each sample was sonicated in an ice bath for 10 min, vortexed for 1 min, and then placed at -20°C for 30 min. Each sample was then centrifuged at 12000 rpm for 10 min (4°C). The sediment was removed, and the supernatant was centrifuged at 12000 rpm for 3 min (4°C). A 200 μL aliquot of supernatant was obtained for Liquid chromatography–mass spectrometry (LC MS) analysis. All samples were analyzed by the LC MS system following the manufacturer’s instructions. The analytical conditions were as follows: UPLC: column, Waters ACQUITY UPLC HSS T3 C18 (1.8 µm, 2.1 mm*100 mm); column temperature, 40°C; flow rate, 0.4 mL/min; injection volume, 2 μL; solvent system, water (0.1% formic acid): acetonitrile (0.1% formic acid); gradient program, 95:5 V/V at 0 min, 10:90 V/V at 11.0 min, 10:90 V/V at 12.0 min, 95:5 V/V at 12.1 min, 95:5 V/V at 14.0 min.Unsupervised PCA (principal component analysis) was performed by the statistics function prcomp using R software (