MSACL 2024 Abstract

INTRODUCTION: Bile acids (BAs) have diverse biological functions in lipid digestion and cell signaling. Their composition and concentration changes in various acute and chronic health conditions, and in response to therapies. Therefore, accurate quantitation of individual BAs is of clinical significance. Nowadays, it is usually achieved by LC-MS/MS analysis. Clinically relevant bile acids are characterized by several isomeric compounds which need to be separated. There are also BAs that do not fragment in the mass spectrometer (or only poorly so). Due to their chemistry, BA analysis in patient samples can be subject to matrix interferences which cannot easily be removed or avoided, and which can greatly affect quantitation of the analytes.

OBJECTIVES: The main objective of this study was to develop an LC method for quantitative BA analysis that minimizes matrix interferences on 13 analytes and their respective IS in patient samples.

METHODS: Six different LC columns with C18 chemistry from four different manufacturers were used with four different mobile phase systems to analyze patient samples (six total combinations). To test contribution of sample preparation to matrix interference removal, simple protein crash methods (acetonitrile or methanol) were tested and compared to several SPE products. Chromatographic separation of analytes and possible interferences was compared between LC systems using non-matrix and matrix samples.

RESULTS: Regardless of column, neutral/acidic pH mobile phases were superior to basic pH mobile phase in separating bile acids, allowing full baseline separation of the 13 analytes into 13 peaks. Removal of matrix interferences was not improved by engaging SPE sample preparation methods compared to an acetonitrile or methanol protein crash. All tested methods gave good linearity over the desired AMR (r2 ≥ 0.99) with neat standards and performed well with spiked controls. But only two combinations of C18 column and mobile phase system were successful at consistently separating interferences from analyte and IS peaks in matrix samples.

CONCLUSION: Due to the chemistry of bile acids, quantitative LC-MS/MS analysis of BAs requires a specific combination of column and mobile phases to successfully separate analyte and IS peaks from interference peaks.