Sensitive and selective quantitation of bile acids using targeted MS2/MS3 on the Stellar mass spectrometer

Importance of the Topic

Bile acids play a central role in lipid digestion, cholesterol homeostasis and signaling. Their concentrations in blood are typically low but can rise in liver dysfunction and disorders of the gut microbiome. Sensitive and selective measurement of bile acids and their conjugates is critical for biomarker discovery, disease monitoring and therapeutic evaluation, including studies of autism spectrum disorder (ASD) where microbiome alterations may affect bile acid profiles.

Study Objectives and Overview

This study aimed to develop and validate a targeted MS2/MS3 workflow on the hybrid quadrupole–linear ion trap Stellar mass spectrometer for the quantitation of 16 primary, secondary and conjugated bile acids in human serum. The method was applied to compare bile acid levels in typically developing children and children with ASD before and after vitamin–mineral supplementation.

Methodology and Instrumentation

Samples were prepared with phenyl solid-phase extraction (Phree plates) and spiked with stable isotope internal standards. A 9-minute reversed-phase UHPLC gradient on a Waters Acquity BEH C18 column (2.1×100 mm, 1.7 µm) at 50 °C separated bile acids. Detection was performed on a Thermo Scientific Vanquish Horizon UHPLC coupled to a Stellar MS using targeted MS2 (HCD or CID) and MS3 scans. Collision energies were optimized per compound via mzCloud libraries. Calibration curves ranged from 0.1 nM to 1000 nM in 50% methanol. Data acquisition used Thermo Xcalibur 4.7; quantitation and data processing were done in TraceFinder 5.2.

Key Results and Discussion

• Alternative fragmentation: HCD provided superior ion ratios for conjugated bile acids, whereas CID was preferred for core bile acid fragments.
• MS3 advantages: Multi-stage fragmentation of the cholic acid quantifier ion (m/z 343) reduced baseline noise and lowered the limit of quantification (LOQ) by two calibration points compared to MS2-CID.
• Dynamic range: Most bile acids were linear over four orders of magnitude (0.5 nM to 1000 nM) with R2 > 0.99.
• Sensitivity and precision: On-column LOQs ranged from 0.38 pg to 15.7 pg. Intra-method RSDs for internal standards were mostly < 8% and all below 15% over 237 serum samples.
• Cohort profiling: Analysis of 49 serum samples revealed 5 nM to 2100 nM total bile acid levels. In the ASD cohort (n = 16), 11 participants exhibited > 30% decrease in total bile acids after supplementation, consistent with reductions in primary and conjugated bile acids.

Benefits and Practical Applications

The targeted MS2/MS3 approach on the Stellar MS delivers high sensitivity, selectivity and throughput for comprehensive bile acid profiling in complex biological matrices. The method supports robust biomarker studies, clinical research and quality control in pharmaceutical and microbiome investigations.

Future Trends and Opportunities

Expanding the method to additional bile acid conjugates and isomers will enrich metabolic pathway analysis. Integration with broader metabolomics workflows, automation of sample preparation and application to other disease models will enhance understanding of host–microbiome interactions and clinical diagnostics.

Conclusion

The study demonstrates that a targeted MS2/MS3 workflow on the Stellar mass spectrometer achieves low-level, reproducible quantification of bile acids and their conjugates in human serum. Application to an ASD cohort illustrates its potential for clinical biomarker discovery and monitoring metabolic interventions.

References

1. Remes PM, et al. J Proteome Res. 2024;5476.