Fecal Bile Acid Analysis

Targeted metabolomics profiling was performed to measure the concentrations of 42 bile acids in fecal samples based on previously described methods (Xie et al., 2013). Briefly, fecal samples (10 mg) were homogenized with 200 μL acetonitrile/methanol (8:2 v/v) mixture, containing 10 μL internal standard, using a Bullet Blender Tissue Homogenizer (Next Advance, Inc., Averill Park, NY, USA), and centrifuged at 13,500 rpm (4°C) for 20 min. After centrifugation, 10 μL of each supernatant was diluted with 90 μL of 1:1 (v/v) mobile phase mixture (where phase A=acetonitrile/methanol/isopropanol (8:1:1 v/v/v); and B=ammonium acetate with 0.25% acetate acid). After centrifugation, 5 μL supernatant was transferred to a 96-well plate for analysis with a UPLC-MS/MS system (ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA, USA), which was used to quantitate bile acids. Data processing was performed using MassLynx software (v4.1, Waters, Milford, MA, USA). The ACQUITY UPLC Cortecs C18 column (1.6 μm, 100 mm × 2.1 mm internal dimensions) (Waters, Milford, MA) was used to achieve chromatographic separation. The UPLC-MS raw data acquired in negative mode were processed using TargetLynx application manager (Waters, Milford, MA) to obtain calibration equations and accurate concentrations of each bile acid in the fecal samples.