Development and application of an LC-MS/MS method for the combined quantification of oxysterols and bile acids

Oxysterols and bile acids are interconnected bioactive lipids playing pivotal roles in diverse physiological and pathological processes. For this reason, they are increasingly studied together for their implications in various diseases. However, due to analytical challenges inherent to the nature of these analytes, very few methods have been developed for the simultaneous analysis of these lipids. We here report the development of a sensitive LC-MS/MS method for the combined quantification of 18 oxysterols, 11 unconjugated, 15 conjugated bile acids, and 1 bile acid precursor, using 8 isotope-labeled internal standards, addressing the need for a more comprehensive analysis of these interesting lipid families. During the method development, we investigated different extraction protocols, set up a purification step, and achieved chromatographic separation for these lipids, overcoming challenges such as the large number of analytes, isomers, and wide range of polarity across the analytes. Finally, the method was successfully applied to the analysis of preclinical and clinical samples, quantifying 12 oxysterols and 14 bile acids in human plasma, 10 oxysterols and 18 bile acids in mouse plasma from the vena cava, and 10 oxysterols and 20 bile acids in mouse plasma from the portal vein within a single chromatographic run.