2.2. Bile Acid Analysis

LC-MS/MS analysis was applied for the quantification of the following ten of the most common bile acids (BAs) in a complex matrix such as the bile: cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), taurolithocholic acid (TLCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), and glycodeoxycholic acid (GDCA) as described previously [13]. A detailed description of the analytical procedure is presented in the supplementary material. Briefly, 10 BA reference standards were dissolved in methanol to prepare individual stock solutions. 50 μl of each bile sample from all the patients was collected, mixed to obtain a pool. Bile was 2000-fold diluted using deionized water and incubated with 100 mg/ml activated charcoal for 2 h to strip this matrix of endogenous BAs. The calibration curves were prepared in bile striped from endogenous BAs by treatment with activated charcoal.

For bile samples, C₁₈ solid-phase extraction (SPE) cartridges were used for sample clean-up. Bile samples were diluted 2000-fold with LC-MS grade water; 100 μl of diluted bile samples was spiked with 10 μl internal standards, vortexed, and loaded onto preconditioned SPE cartridges. Loaded cartridges were washed with 2 ml H₂O and eluted with 4 ml MeOH. The eluate was evaporated under vacuum and reconstituted in 100 μl of 50% MeOH. The analysis was carried out by LC-MS/MS using a column in reverse phase in a gradient elution with mobile phases consisted of 0.01% acetic acid in water (mobile phase A) and 0.01% acetic acid in methanol (mobile phase B), at a total flow rate of 0.5 ml/min, in negative ionization mode.