Bile acid profiling

Bile acid extractions were performed on ice using 50 mg of each fecal sample. Samples were placed into a tube containing one 4 mm steel ball, 1 mL of ice-cold water:methanol (1:10 v/v), and 10 μL of a mixture of internal standards (10 μM each of d4-GCDCA [glyco-chenodeoxycholic acid], d4-LCA [lithocholic acid], d4-CDCA [chenodeoxycholic acid], d4-DCA, and d4-CA [cholic acid] in 1:1 water:methanol) before mechanical disruption using a TissueLyser II sonication in an ice bath for 10 min, and agitation via a vortex mixer. Samples were then centrifuged at 15,000 x g at 4°C for 15 min. 800 μL of the supernatant was transferred to a new tube and dried in a speed vacuum at room temperature. Samples were resuspended in 100 μL of 50% methanol and stored at −80°C until LC-MS. Ten working standards (2.5–10,000 ng/mL) and 3 quality control samples with replicates were prepared using the identical protocol as was used for experimental samples. Before injecting into the LC-MS, samples were transferred into 0.2 μm Costar Spin-X HPLC microcentrifuge filters (Corning Inc., Corning, NY) and centrifuged at 15,000 x g for 10 min. 5 μL filtrate per sample was injected into the Agilent G6460 (version A.00.07.32) ultra-performance liquid chromatography coupled with mass spectrometry in tandem system (UPLC-MS/MS) combined with a triple quadrupole mass spectrometer via an electrospray ionization interface. Chromatographic separation was performed using a ZORBAX Eclipse Plus C18 analytical column (2.1 × 100 mm; id: 1.8μm). The gradient profile for the LC pump under the final chromatography conditions was 0 min, 95:5; 5 min, 95:5; 14 min, 86:14; 14.5 min, 75:25; 17.50 min, 75:25; 18 min, 50:50; 22 min, 50:50; 22.50 min, 20:80; 24.50 min, 20:80; 25-28 min, 95:5 (A:B, v/v). Column temperature was 45°C; sample tray temperature was 9°C. MS/MS spectra were produced in negative ionization mode.