Improving the annotation of bile acids in fecal samples using a Liquid Chromatography-Ion Mobility-High Resolution Mass Spectrometry method

Significance of the topic

Bile acids play crucial roles in metabolism and are linked to disorders such as diabetes, metabolic syndrome, and colorectal cancer. Their structural similarity poses a challenge for accurate identification in complex matrices like fecal and serum samples. Employing ion mobility separation orthogonal to liquid chromatography enhances confidence in compound annotation.

Objectives and study overview

This study aimed to develop and optimize a UHPLC-TIMS-HRMS method for confident annotation of 32 bile acids in fecal and serum samples. An in-house library integrating m/z, retention time, and collision cross section (CCS) values was constructed to support targeted and untargeted profiling.

Methodology and instrumentation

• Standards and samples
  Bile acid standards were sourced from a commercial library. Samples included lyophilized fecal, aqueous fecal, and human serum matrices.
• Sample preparation
  Extraction protocols employed mixtures of water, isopropanol, acetonitrile, or methanol, tailored for each matrix.
• Chromatography
  UHPLC separation used a C8 column with a gradient of aqueous and organic phases buffered with ammonium formate at pH 4.2.
• Mass spectrometry
  A timsTOF instrument with electrospray ionization in negative mode was operated in trapped ion mobility (TIMS) and high-resolution full-scan acquisition, generating four-dimensional data (m/z, retention time, ion mobilogram, intensity).
• Data processing
  Compass HyStar, DataAnalysis, and MetaboScape software were used for calibration, peak annotation, and CCS evaluation.

Key results and discussion

• Library construction yielded retention times and CCS values for 32 bile acids with RSDs below 0.3%, demonstrating high precision.
• TIMS separation resolved isobaric and co-eluting isomers, improving specificity over conventional LC-MS.
• Profiling in biological samples annotated 12 bile acids in aqueous fecal, 14 in lyophilized fecal, and 7 in serum using targeted lists.
• CCS values compared to literature (drift tube CCS compendium) showed deviations under 1.7%; inter-laboratory reproducibility across two TIMS setups exhibited less than 0.3% variance.

Benefits and practical applications

• Enhanced confidence in bile acid annotation supports clinical and nutritional studies.
• Orthogonal TIMS separation adds value for QA/QC in bioanalytical workflows.
• Untargeted profiling with MetaboScape enables discovery of additional metabolites.

Future trends and possibilities

Emerging directions include expanding libraries to novel bile acid derivatives, integrating machine learning for automated annotation, and combining TIMS-HRMS with imaging mass spectrometry for spatial metabolomics. The approach could be adapted for biomarker discovery in gut microbiome research and personalized diagnostics.

Conclusion

The developed UHPLC-TIMS-HRMS workflow provides a robust and reproducible platform for the confident annotation of bile acids in complex human matrices. Orthogonal ion mobility separation and CCS metrics significantly enhance specificity and inter-laboratory consistency, paving the way for advanced metabolomic applications.

References

1. DOI:10.1007/s00216-019-01869-0
2. DOI:10.1021/jasms.0c00015
3. DOI:10.1016/j.jlr.R001941-JLR200
4. DOI:10.1021/acs.analchem.5b01556
5. DOI:10.1039/c8sc04396e