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After thawing 30 μL of bile samples on ice, the bile acids were extracted with 400 μL of chloroform and 200 μL of methanol. After mixing well, 120 μL of distilled water was added to the mixture. The mixture was vortexed and centrifuged for 5 min, followed by transfer of 100 μL of the supernatant to a 1.5 mL micro tube, and drying in a centrifugal vacuum concentrator. After complete drying, bile acids were dissolved in 100 μL of 50% methanol prior to transfer to HPLC autosampler vials for LC-MS/MS analysis.
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