Thermospray ionization liquid chromatography-mass spectrometry: a new and highly specific technique for the analysis of bile acids.

Abstract

A rapid, highly specific and sensitive combined high performance liquid chromatography-mass spectrometric (LC-MS) method is described for the analysis of bile acids in biological fluids. Ionization of polar bile acid conjugates is achieved in the thermospray interface that is used to directly couple the LC column to the mass spectrometer, thereby allowing continuous monitoring of the LC effluent. Maximum sensitivity (4-10 pmol) is achieved by recording the negative ions generated in the ionization process and mass spectra obtained for the principal bile acid conjugates are characterized by intense [M-H]-pseudo-molecular ions and fragment ions due to consecutive losses of water corresponding to a number of hydroxyl groups in the molecule. The mass spectrometer thus provides molecular weight and useful structural information for each compound separated by HPLC. Applications of the LC-MS technique to the analysis of bile acids in bile and serum samples after an initial solid-phase extraction step highlight the potential of the thermospray interface for enhancing the specificity and sensitivity of the HPLC technique for bile acid analysis.