Quantification of Bile Acids in Serum

The BA concentrations in serum were measured utilizing ultrahigh-performance liquid chromatography–tandem secondary mass spectrometry (UHPLC-MS/MS) and according to the protocol described in our previous study [18 (link)]. In brief, the serum was mixed with an extract solvent (acetonitrile/methanol, 1:1, containing 0.1% formic acid and an isotopically labeled internal standard mixture), vortexed for 30 s, sonicated for 10 min in an ice-water bath, incubated at −40 °C for 1 h, and centrifuged at 12,000 g and 4 °C for 15 min. Then, the prepared samples were detected using a Q-Exactive Focus mass spectrometer (Thermo Fisher Scientific), in combination with an Agilent 1290 Infinity series UHPLC System (Agilent Technologies, Santa Clara, CA, USA) equipped with a Waters ACQUITY UPLC BEH C18 column (150 × 2.1 mm, 1.7 μm, Waters). The typical ion source parameters were as follows: spray voltage = +3500/−3100 V; sheath gas (N2) flow rate = 40; aux gas (N2) flow rate = 15; sweep gas (N2) flow rate = 0; aux gas (N2) temperature = 350 °C; capillary temperature = 320 °C. All detected BAs were classified into primary BAs (PBAs), secondary BAs (SBAs), free BAs (FBAs), and conjugated BAs (CBAs) according to their synthetic sources and properties.