Agilent 1290 liquid chromatograph

An Agilent 1290 liquid chromatograph (Agilent, Santa Clara, CA) and waters Acquity BEH C₁₈ column with 1.7-µm particle size (2.1 by 100 mm; Waters, Milford, MA) was used for chromatographic separation. Mobile phase A (MPA) was 0.1% formic acid in LC-MS-grade water. Mobile phase B (MPB) was 0.1% formic acid in acetonitrile. The mobile phase gradient consisted of 0 to 0.5 min of 5% MPB, 3 min of 25% MPB, 17 min of 40% MPB, and 19 to 21 min of 95% MPB and was run on an Agilent 6490 triple-quadrupole mass spectrometer (Agilent, Santa Clara, CA). Data were processed by MassHunter workstation software, version B.06. Table 1 lists the bile acids detected by name and abbreviation as well as their MRM transitions (i.e., parent m/z→daughter m/z).

Water (200 μL) was added to the homogenized liver samples (0.075 to 0.125 g) in a 15-mL polypropylene centrifuge tube and the sample vortexed to produce a slurry. The samples were spiked with 50 μL of surrogate dissolved in acetonitrile (20.0 μg/mL d4-diphacinone; CDN Isotopes, Pointe-Claire, Quebec, Canada) and extracted with 5 mL of 1% ammonium hydroxide in acetonitrile (Fisher Scientific, Waltham, MA, USA). A solvent/water partition was created by the addition of 200 mg of a Quechers salt packet (Agilent Technologies, Santa Clara, CA, USA). An aliquot (1.5 mL) of the acetonitrile layer was transferred to a dispersive solid phase extraction tube (Agilent Technologies, Santa Clara, CA, USA) containing 25 mg C18 and 150 mg magnesium sulfate. A 1-mL aliquot of the sample was evaporated to dryness in a vacuum concentrator (Vacufuge; Eppendorf, Enfield, CT, USA) and reconstituted in 80% 10-mM ammonium acetate:20% acetonitrile. Analysis was performed using an Agilent 1290 Liquid Chromatograph attached to an Agilent 6470A QQQ Mass Spectrometer (Santa Clara, CA, USA). Separation was achieved using a 50 mm x 2.1 mm ID, 2.5 μm, X-Bridge BEH C18 column (Waters Corporation, Milford, MA, USA). Other instrument parameters are listed in Table 1.