A total of 62 feces samples were categorized into three groups according to clinical types. Internal standards, L-norleucine and L-norvaline were purchased from Sigma-Aldrich (Shanghai, China). For the sample preparation, 80 μL of cold methanol with 5 μg/mL L-norleucine was added to extract metabolites from 20 ul of the supernatant obtained from a mixture of 100 mg feces and 100 ul water. After centrifuging at 4 °C and 14,000 × g for 15 min, 40 μL supernatants and 5 μL L-norvaline (50 μg/mL) were evaporated under a nitrogen stream. The dried products were reconstituted in 35 μL methoxyamine hydrochloride in pyridine (containing 5 μg/mL n-alkanes standards) (37 ℃, 90 min) and derivatized in 35 μL of BSTFA (with 1% TMCS) (70 ℃, 60 min) for GC–MS metabolomics analysis. Additionally, 100 µL of each fecal sample was mixed as a quality control (QC) sample.
Free full text: Click here
