Quantitative estimation of bile acid conjugates in human bile using HPLC

Abstract

An optimized reversed phase high performance liquid chromatographic method for the simultaneous analysis of the 10 major bile acid conjugates present in human bile is described. A one-step sample purification using solid phase extraction with Maxi-Clean® C-18 cartridges was applied. The reversed phase C-18 analytical column, an isocratic mobile phase with a constant flow rate (1.5 mL/minute), and UV detection at 215 nm were used. The original method of calculation using an internal standard was applied to the recovery study and to the quantitative estimation ofthe bile acid content ofhuman bile. An efficient chromatographic resolution ofthe 10 bile acid conjugates was obtained by modification of the mobile phase composition to 65% methanol and 35% aqueous phosphate buffer (v/v) adjusted to pH 4.10, while preserving a relatively short chromatographic analysis of45 minutes. The method was found to be reliable and convenient for the evaluation of conjugated bile acid in human bile.