Confident Structural Annotation of Bile Acid Isomers Combining Electron-Activated Dissociation or Ion Mobility with High-Resolution Mass Spectrometry in Metabolomics.

Accurate identification of lipid isomers remains a major challenge in metabolomics, particularly for bile acids (BAs), whose biological functions critically depend on the number, position, and stereochemistry of hydroxyl groups as well as on the nature of their conjugation. Conventional tandem mass spectrometry (MS/MS) with collision-induced dissociation (CID) often fails to resolve isomeric lipids differing only in subtle structural features, leading to an underestimation of lipidome complexity. Here, we present an analytical framework integrating electron-activated dissociation (EAD) or ion mobility spectrometry (IM) with high-resolution liquid chromatography-mass spectrometry to improve annotation of BA isomers in untargeted metabolomics. EAD provides nonergodic fragmentation that preserves labile bonds and reveals site-specific details, while IM separates ions by their collision cross section (CCS), supplying orthogonal structural information. Using synthetic standards and human plasma extracts, we show that EAD-derived diagnostic ions markedly enhance structural discrimination and enable consistent and reproducible identification of BA isomers compared to CID-only workflows, while CCS measurements alone lack sufficient robustness to reliably distinguish closely related BA isomers across measurements. The integrated data set enables confident assignment of positional and conjugation variants, contributing to a more complete representation of BA diversity. This multidimensional approach offers a robust platform for structural lipidomics and supports the establishment of transferable spectral and mobility libraries to advance metabolome annotation accuracy.