Glacial acetic acid rpe

Cholic acid (CA) and other endogenous BAs were purchased from Sigma-Aldrich (St. Louis, MO). The molecules INT-767, its 3-glucuronide conjugates (Glucu-INT-767) and internal standard OCA, INT-747 were supplied by Intercept Pharmaceuticals, Inc. (ICPT). All solvents were of high purity and used without further purification. Acetonitrile for HPLC was from Merck (Darmstadt, Germany); methyl alcohol RPE, ammonia solution 30% RPE, glacial acetic acid RPE were from Carlo Erba Reagent (Milan, Italy); activated charcoal was from Sigma-Aldrich; and ISOLUTE C18 cartridges (500 mg, 6 ml) for the plasma sample pretreatment were purchased from StepBio (Bologna, Italy). Plasma BA free mouse plasma was treated with activated 50 mg/ml charcoal and stirred at 4 °C overnight. After centrifugation at 3000 g for 5 minutes the plasma was filtered through Millipore A10 Milli-Q Synthesis (0.45 µm) and stored at −20 °C.

CA and other endogenous BAs were purchased from Sigma-Aldrich (St. Louis, MO). All solvents were of high purity and used without further purification. Acetonitrile for HPLC was from Merck (Darmstadt, Germany); methyl alcohol RPE, ammonia solution 30% RPE, glacial acetic acid RPE were from Carlo Erba Reagent (Milan, Italy); activated charcoal was from Sigma-Aldrich; and ISOLUTE C18 cartridges (500 mg, 6 ml) for the plasma sample pretreatment were purchased from StepBio (Bologna, Italy). Plasma BA free rat plasma was treated with activated 50 mg/ml charcoal and stirred at 4°C overnight. After centrifugation at 3000 g for 5 minutes the plasma was filtered through Millipore A10 Milli-Q Synthesis (0.45 µm) and stored at −20° C.