Quantitative Bile Acid Profiling in Feces

Bile acids were analyzed using method validated method adapted from Guillemot-Legris et al.^{49 (link)}. Briefly, lyophilized feces (5 mg) were homogenized in ice-cold distilled water prior to protein precipitation using acetone containing seven deuterated bile acids used as internal standards. Samples were next centrifuged, and the supernatant was evaporated to dryness under nitrogen steam. The resulting residue was resuspended in methanol and injected in the HPLC–MS system consisting of an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) coupled to an Accela HPLC system (Thermo Fisher Scientific). Analyte separation was performed on an Ascentis Express C-18 column (2.7 µm, 100 × 4.6 mm) (Sigma-Aldrich) using a gradient between acetonitrile and water, both containing formic acid. Mass spectrometry analysis was performed using an electrospray ionization source in the negative mode. Calibration curves were prepared in the same conditions.