Dog bile samples obtained by cholecystocentesis were pooled across animals (n = 3). Samples were mixed with an equal volume of citrate buffer (100 mmol/L) to make the final buffer concentration of 50 mmol/L. The mixture was mixed by vortexing for 1 minute and then centrifuged at 12 000× g for 15 minutes. An aliquot (5‐10 μL) of the supernatant was injected onto the UHPLC column for LC‐MS/MS‐based analysis.
The metabolite profile was obtained using LTQ‐Orbitrap coupled with Accela UHPLC and PDA (ThermoScientific, San Jose, CA) and a CTC PAL autosampler equipped with a cooling stack that maintained samples at 4°C during analysis. A 20‐minute UHPLC method was developed. The column was Acquity UPLC HSS T3 (Waters, Milford, MA) 2.1 × 150 mm, 1.8 µm. The column was maintained at a temperature of 55°C. The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) at a flowrate of 600 µL/min with the following linear gradient conditions: 5% A for 0.5 minutes, 20‐60% B over 14 minutes, 95% B over 3 minutes, and 5% B isocratic for 2 minutes. Instrument settings were as follows: m/z range: 100 to 1000, capillary temperature was set at 280°C, nitrogen sheath gas flow rate 80 (arbitrary units), auxiliary gas 20, spray voltage 4.0 kV, and tube lens offset 80 V. In addition, UV wavelength was monitored at 254 nm.
The metabolite profile was obtained using LTQ‐Orbitrap coupled with Accela UHPLC and PDA (ThermoScientific, San Jose, CA) and a CTC PAL autosampler equipped with a cooling stack that maintained samples at 4°C during analysis. A 20‐minute UHPLC method was developed. The column was Acquity UPLC HSS T3 (Waters, Milford, MA) 2.1 × 150 mm, 1.8 µm. The column was maintained at a temperature of 55°C. The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) at a flowrate of 600 µL/min with the following linear gradient conditions: 5% A for 0.5 minutes, 20‐60% B over 14 minutes, 95% B over 3 minutes, and 5% B isocratic for 2 minutes. Instrument settings were as follows: m/z range: 100 to 1000, capillary temperature was set at 280°C, nitrogen sheath gas flow rate 80 (arbitrary units), auxiliary gas 20, spray voltage 4.0 kV, and tube lens offset 80 V. In addition, UV wavelength was monitored at 254 nm.