Altis qqq mass spectrometer

Single reaction monitoring mode was used to detect glycocholic acid on a Altis QQQ Mass Spectrometer equipped with a Vanquish LC system (Thermo Fisher Scientific). Separation of metabolites was performed on a Acquity HSS T3 column (Waters; 150 mm by 2.1 mm, 1.8 μm). The mobile phase consisted of solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid) using the following gradient: 0 min 20% B, 8 min 95% B and 10 min 20% B, at a constant flow rate of 0.3 ml min⁻¹. The injection volume was 5 µl. Two transitions were optimized using an authentic standard of glycocholic acid (glycine-1-¹³C, CLM-191-PK, Cambridge Isotope Laboratories), from the negative precursor ion (m/z 465) to product ions (m/z 402 and m/z 75). Total cycle time was 0.8 s and Q1 resolution (FWHM) was 0.7 and Q3 resolution (FWHM) was 1.2. For each transition, the collision energy applied was optimized to generate the greatest possible signal intensity. The optimized source parameters were: spray voltage, 2,500 V; sheath gas, 35; Aux gas, 7; ion transfer tube temperature, 325 °C; vaporizer temperature, 275 °C; and RF lens, 105. Data acquisition was performed using Xcalibur 4.1 software.

Bile acids were targeted and quantified as previously reported⁴ . Briefly, dried aqueous-phase sample plates from hydrophilic interaction chromatography (HILIC) ESI+ analysis were removed from a −80 °C freezer and dissolved in 60 µl bile acid run solvent (1:1 ACN methanol containing 100 ng ml⁻¹ of five isotopically labeled bile acid internal standards). Plates were vortexed for 30 s and sonicated in a water bath for 5 min. All samples were diluted 30-fold in bile acid run solvent. All plates were centrifuged at 708g for 15 min at 4 °C and stored in a Vanquish autosampler at 4 °C until analysis. A nine-point standard curve was prepared between 0 to 1,500 ng ml⁻¹ and analysed with samples. A Vanquish UPLC and Altis QqQ mass spectrometer (Thermo Fisher Scientific) were used for targeted LC–MS/MS analysis. An Acquity BEH C18 column (100 mm × 2.1 mm i.d., 1.7-µm particle size; Waters) was used with mobile phase A of water and B of acetonitrile, both with 0.1% formic acid. Skyline software and custom R scripts were used to process and quantify bile acid analytes.