Sample preparation for bile acid metabolomics

Sample preparation and the LC-MS/MS for bile acid analysis were essentially based on the previously developed method²⁴ with modifications as follows: Cecum contents were lyophilized before extraction. To the dried matter (10 mg), 90% ethanol (2 ml) was added and bile acids were extracted by ultra-sonication at room temperature for 1 h. The supernatant was separated by centrifugation at 2,500 rpm for 5 min and collected in a glass test tube. This procedure was repeated twice and, the second and third extracts were combined to the first extract. Ethanol was evaporated under an N₂ stream. Liver (300–400 mg) was homogenized with cold water (500 µl). To each homogenate, 20 mg/ml Proteinase K solution (50 µl) was added and the liver was completely digested at 55℃ for 16 h. Bile acids were extracted using 90% ethanol by the same manner as described for cecum. Serum (50 µl) was added to acetonitrile (5 ml) and evaporated to dryness under and N₂ stream.

Thus, prepared crude bile acid extracts were resuspended in 90% ethanol (1ml) by ultra-sonication and, deuterium-labeled internal standards, d₄-CA, d₄-GCA, and d₄-TCA were added at each concentration at 100 nmol/ml. For the cecum and liver extracts, an aliquot (100 µl) was diluted with water (900 µl) and then applied on a solid-phase extraction cartridge (InertSep C₁₈-B 100 mg sorbent weight, preconditioned with 1 ml of methanol and 3 ml of water). The column was washed with water (1 ml) and then desired bile acids were eluted with 90% ethanol (1 ml). For the serum extract, the whole suspension (1 ml) was diluted with water (9 ml) and, applied on a solid-phase extraction column. The column was eluted in the same manner. After evaporation of the solvent, the residue was dissolved in 20% acetonitrile (1 ml), and aliquot (5 µl) of the solution was analyzed by the LC/ESI-MS/MS.