Determination of fecal metabolites by LC-MS.
Stool samples (20 mg) were weighed and transferred to clean centrifuge tubes. A methanol-water internal standard extract (70%; 400 μL) was added, and the samples were mixed for 3 min, sonicated in an ice water bath for 10 min, further vortexed for 1 min, allowed to stand in a −20°C refrigerator for 30 min at 4°C, and centrifuged at 12,000 rpm for 10 min. The supernatant (300 μL) was transferred to a new centrifuge tube and centrifuged again at 12,000 rpm for 3 min. An aliquot of 200 μL of the supernatant was transferred to an injection bottle for analysis. Chromatographic and mass spectrometry conditions were set according to the conditions described in a previous study (