Sciex qtrap 4500 md triple quadrupole mass spectrometer

Plasma bile acid species were quantified by liquid chromatography-mass spectrometry. To 25 μl of plasma, we added a mixture of internal standards (isotopically labeled bile acids). Samples were centrifuged at 16,000×g for 10 min in a tabletop centrifuge (Eppendorf, Nijmegen, the Netherlands) and the supernatant was transferred and evaporated at 40°C under a stream of N₂. Samples were reconstituted in 200 μl methanol:water (1:1), mixed and centrifuged at 1,800×g for 3 min. The supernatant was filtered using a 0.2 μm spin-filter at 2,000×g for 10 min. Filtrates were transferred to vials and 10 μl was injected into the LC-MS/MS system. The LC-MS/MS system consisted of a Nexera X2 Ultra High Performance Liquid Chromatography system (SHIMADZU, Kyoto, Japan), coupled to a Sciex Qtrap 4500 MD triple quadrupole mass spectrometer (SCIEX, Framingham, MA, USA). Data were analyzed with Analyst MD 1.6.2 software.

For serum bile acid analysis 25 µL serum was used and to each sample 250 µL internal standard solution was added, vortexed for 60 s, centrifuged at 15800 × g and the supernatant poured into a clean glass tube. After evaporation under nitrogen at 40 °C the samples were reconstituted in 200 µL 50% methanol in water, vortexed for 60 s and centrifuged for 3 min at 1800 × g. The supernatant was transferred into a 0.2 µm spin-filter and centrifuged at 2000 × g for 10 min. After filtering, 10 µL of samples was injected on an Acquity UPLC BEH Column (1.7 µm × 2.1 × 100 mm) containing an Acquity UPLC BEH C18 VanGuard Pre-Column (1.7 µm × 2.1 × 5 mm) (Waters, Milford, MA, USA). The quantitative determination of bile acids was performed using a Nexera X2 Ultra High Performance Liquid Chromatography system (SHIMADZU, Kyoto, Japan), coupled to a SCIEX QTRAP 4500 MD triple quadrupole mass spectrometer (SCIEX, Framingham, MA, USA) (UHPLC-MS/MS). The LC-MS/MS system was controlled by Analyst MD 1.6.2 software. Concentrations of bile acid species measured below the linear range of 0.05 µM were indicated as 0.025 µM.