Quantitative Bile Acid Analysis by HPLC-MS

Bile acid quantification was performed by HPLC-MS as previously described [41 (link)]. Briefly, liver samples were homogenized in ice-cold distilled water, and proteins were precipitated using acetone in the presence of deuterated internal standards. The samples were next centrifuged, the supernatant recovered and evaporated to dryness. The resulting residue was resuspended in methanol and analyzed by HPLC-MS using an LTQ-Orbitrap XL coupled to an Accela HPLC system (Thermo Fisher, Merelbeke, Belgium). Analyte separation was performed on an Ascentis Express C-18 column (2.7 µm, 4.6 × 100 mm) (Sigma-Aldrich, St. Louis, MI., USA). The separation was achieved using a gradient of H₂O-ACN-formic acid 75:25:0.1 (v/v/v) and ACN-formic acid 100:0.1 (v/v). The MS analysis was performed in the negative mode with an ESI ionization source. Calibration curves were prepared using the same conditions. Data are expressed as pmol normalized by the amount of tissue. Values below the LOQ (for data sets with less than 25% of such missing values) were imputed using the function impute.QRILC in the R package imputeLCMD [42 (link)].