Lcms 2010

A well-developed metabolomic platform based on the GC/TOF-MS technique was used to profile the metabolites in the serum samples from each group, as previously reported, with a few modifications ^{26 (link)}. The amount of serum was reduced to 30 μL and extracted with 120 μL of methanol. The supernatants were dried and derivatized for GC/TOF-MS analysis. For extraction and analysis of metabolites in cecal content, cecal content (0.5 g) were added with 1000 μL purified water and vigorously vortexed for 3 min (1:2, w). An aliquots of 50 μL mixture was transferred to an Eppendorf tube, and then 200μL methanol (containing internal standard [¹³C₂]-myristic acid 5μg/mL) was added and vortexed for 3 min. An aliquot of 100 μL supernatant was dried and derivatized for GC/TOF-MS analysis in the same way as that of serum samples. The raw data acquired with GC/TOF-MS were processed, and the metabolites were identified as previously reported ^{26 (link)}. The multivariate data were evaluated using the SIMCA P-13 software (Umetrics, Umeå, Sweden). Bile acids, such as cholic acid(CA), glycocholic acid (GCA), taurocholic acid (TCA), deoxycholic acid(DCA), ursodeoxycholic acid(UDCA), lithocholic acid(LCA) and chenodeoxycholic acid (CDCA) were quantitative analysed in Shimadzu LC/MS 2010, equipped with an Aglient ZORBAX SB-Aq column (2.1 × 150 mm, 3.5 μm), Method S1.

All synthetic chemistry and DMPK was performed at WuXi App Tec at their China facilities in Tianjin and Shanghai. Proton NMR spectra were recorded on a Varian 400 MHz NMR with chemical shifts δ recorded in ppm relative to TMS. LCMS were taken on a quadrupole Mass Spectrometer on Shimadzu LCMS 2010 (Column: sepax ODS 50×2.0 mm, 5 μm) or Agilent 1200 HPLC, 1956 MSD (Column: Shim-pack XR-ODS 30×3.0 mm, 2.2 μm) operating in ES (+) ionization mode. All final compounds were greater than 95% purity based on HPLC UV% AUC as determined at 214, 254 and 280 nM wavelengths. Structures were confirmed by ¹H NMR and LC/MS. Final compounds in this manuscript are not known to interfere with the assays herein (i.e., they are not PAINS). Detailed synthetic chemistry and analytical analyses are found in the Supplemental materials.