Optimizing Analytical Conditions and Creating a Multidimensional Reference Library for Over 300 Bile Acids

Bile acids are increasingly recognized as pivotal regulators of macronutrient metabolism (lipids, carbohydrates, proteins) and systemic inflammatory homeostasis. Their metabolic and signaling pathways are essential for maintaining health, with dysregulation contributing to cardiometabolic, inflammatory, and neoplastic disorders. Thus, the accurate and reliable identification of the many bile acid isomers is essential to understand their distinct biological functions and diagnostic potential. To date, conventional analytical workflows combining liquid chromatography and mass spectrometry (LC-MS) struggle to differentiate bile acid isomers due to frequent chromatographic coelution, identical precursor masses and often indistinct fragmentation spectra. Here, we compared different analytical parameters in a multidimensional workflow and created a library to improve the resolution and identification of over 300 bile acids, including unconjugated and conjugated versions such as the recently discovered microbially conjugated bile acids (MCBAs). Specifically, we systematically optimized and evaluated extraction protocols for the bile acids in stool, serum and plasma samples and determined advantages and caveats to different LC conditions and MS source ionization modes.