Profiling and quantitation of bile acids in human biofluids by LC-TIMS-MS

Significance of the Topic

Profiling and quantification of bile acids in human biofluids are crucial for understanding metabolic pathways, gut microbiome interactions and disease biomarkers. Bile acids display structural diversity and isomeric complexity, posing analytical challenges that demand high specificity and sensitivity.

Objectives and Study Overview

This study aimed to develop and validate a reversed-phase liquid chromatography coupled with trapped ion mobility spectrometry and mass spectrometry (RP-LC-TIMS-MS) method for comprehensive profiling and accurate quantitation of 71 bile acid standards. The assay was applied to human reference plasma (SRM 1950) to assess annotation confidence, dynamic range and interlaboratory comparability.

Methodology and Instrumentation

Bile acids were extracted from human plasma following established protocols. Chromatographic separation employed a C8 reversed-phase column with optimized gradients. Ion mobility separation was achieved using trapped ion mobility spectrometry, orthogonal to LC, to resolve co-eluting isomers. Data acquisition in negative ion mode used an Elute UHPLC connected to a timsTOF Pro 2 equipped with a VIP-HESI source. Processing and annotation utilized TASQ® 2023 and MetaboScape® 2023 software for targeted quantitation and untargeted profiling.

Instrumentation Used

• Elute UHPLC system
• timsTOF Pro 2 mass spectrometer (Bruker Daltonics)
• VIP-HESI electrospray source operated in negative ion mode
• TASQ® 2023 and MetaboScape® 2023 data analysis platforms

Main Results and Discussion

The method separated all 71 synthetic bile acid standards, including critical isomeric pairs such as lithocholic and allolithocholic acids, through combined LC and TIMS dimensions. In reference plasma, 25 bile acids were confidently annotated. Quantitative performance for taurocholic acid showed a linear dynamic range of 1 nM to 5000 nM (3.7 orders of magnitude), R²=0.9987 and residuals below 20%. Determined concentrations in SRM 1950 aligned with published reference values. Interlaboratory comparison of collision cross section (CCS) values across two sites revealed an average deviation below 0.3%, demonstrating reproducibility and compatibility with drift tube CCS databases.

Benefits and Practical Applications

• Enhanced specificity in bile acid annotation by combining LC, m/z and TIMS separation
• Accurate quantitation across a broad concentration range for clinical and research samples
• High interlaboratory reproducibility of CCS values supports multi-site studies
• Untargeted profiling enables discovery of additional bile acid species beyond targeted lists

Future Trends and Applications

Integration of LC-TIMS-MS workflows with expanded bile acid libraries and advanced bioinformatics will further refine metabolomic studies. Clinical translation may benefit from rapid, high-confidence profiling in diagnostics and personalized nutrition. Combining TIMS with emerging fragmentation techniques could enhance structural elucidation of novel conjugates and minor metabolites.

Conclusion

The RP-LC-TIMS-MS approach delivers robust, high-confidence profiling and quantitation of bile acids in human biofluids. Its broad dynamic range, isomer separation capability and reproducible CCS values make it a powerful tool for metabolic research and clinical applications.

References

[1] Nature 2020; DOI:10.1038/s41586-020-2047-9
[2] Sarafian et al. Anal. Chem. 2015; DOI:10.1021/acs.analchem.5b01556
[3] Bowden et al. J. Lipid Res. 2017; DOI:10.1194/jlr.M079012
[4] McLean Research Group. Unified CCS Compendium; https://mcleanresearchgroup.shinyapps.io/CCS-Compendium
[5] Poland et al. J. Am. Soc. Mass Spectrom. 2020; DOI:10.1021/jasms.0c00015