Bile Acid Profiling by LC-MS/MS

Bile acids were extracted from cells using the liquid-liquid extraction method and analyzed through high-throughput liquid chromatography–mass spectrometry (LC–MS/MS) described previously^{59 ,60 (link)}. The pooled sample was used as quality control. The extracted bile acids were separated through the ACQUITY UPLC BEH C18, 1.7 μm (2.1 × 100 mm) HPLC column with 45 °C of column temperature. The mobile phases A and B were water:methanol (80:20) in 10 mM ammonium acetate (A) and acetonitrile:water (90:10) in 10 mM ammonium acetate (B). Gradient flows were 0–0.5 min 5% B, 0.5–12 min 98% B, 12–13 min 98% B and 13 min 5% B, followed by re-equilibration until the end of the gradient, 15 min from the initial starting condition of 5% B. The flow rate of the solvents used for analysis was 0.2 ml min⁻¹ and the injection volume was 20 μl. MS data were acquired in the negative ionization mode via multiple reaction monitoring using a 6495 Triple Quadrupole MS coupled to an HPLC system (Agilent Technologies) and operated by Agilent Mass Hunter Software (v.11.0)^{59 ,60 (link)}. The peak integration and data analysis were performed using Agilent Mass Hunter Quantitative Analysis software.