Unlocking the Bile Acid Universe: Advanced Workflows and a Multidimensional Library of 280 Unique Species.

Microbes and bile acids are tightly intertwined, especially in the gut. While the liver produces primary bile acids from cholesterol, gut bacteria transform these into diverse secondary forms which act as powerful signaling molecules, influencing host metabolism and immune function. Since bile acid changes are increasingly linked to health and disease, their accurate measurement in the gut and circulation is essential. Analytical evaluations, however, remain challenging as many bile acids co-elute in liquid chromatography (LC), share identical precursor masses in mass spectrometry (MS), and produce similar tandem mass spectrometry (MS/MS) spectra. As a result, conventional LC-MS/MS workflows struggle to differentiate bile acids, motivating the addition of orthogonal separations such as ion mobility spectrometry (IMS). Here, we assess optimal bile acid extraction parameters for stool, serum, and plasma; compare LC conditions; and assess electrospray ionization performance across polarities. Additionally, we created a multidimensional reference library containing LC retention times, IMS collision cross section values, and accurate precursor masses for 280 unique bile acids (264 endogenous and 16 deuterium-labeled species) including unconjugated, host-conjugated, and microbially conjugated bile acids. This multidimensional library empowers bile acid identification in complex samples and enables a more comprehensive exploration of their biological roles and disease associations.