Advancing bile acid quantitation: High-specificity MS2 and MS3 analysis using the Stellar Mass Spectrometer

Importance of the Topic

Bile acids are central to lipid digestion, micronutrient absorption and host–microbiome communication. Accurate profiling of these structurally related molecules is essential for elucidating disease mechanisms, monitoring metabolic disorders and advancing personalized therapies, especially where minor isomeric differences can impact biological interpretation.

Objectives and Study Overview

This work presents the development of a unified targeted-MS2/MS3 assay on the Thermo Scientific Stellar mass spectrometer. The main goals were to achieve high specificity for bile acid isomers, extend dynamic range across five orders of magnitude, and apply the method to human serum samples, including a clinical study of children with autism spectrum disorder (ASD) before and after vitamin‐mineral supplementation.

Instrumentation

• Stellar mass spectrometer with MS2 and MS3 capabilities
• Vanquish Horizon UHPLC system
• Reversed‐phase LC column
• Skyline, Chromeleon CDS 7.3.2 and TraceFinder 5.2 software for data processing

Methodology

Unlabeled and isotope-labeled bile acid standards were diluted in 50% methanol to generate calibration curves. Human plasma (NIST 1950) and serum samples were extracted with 0.1% formic acid in isopropanol, dried and reconstituted in 50% methanol. Chromatographic separation lasted nine minutes using a reversed-phase column. Data acquisition employed both targeted-MS2 (tMS2) and targeted-MS3 (tMS3) scan modes, combining HCD and CID fragmentation to maximize selectivity. Transition lists were optimized in mzVault and Skyline, then transferred to Chromeleon CDS for automated acquisition and quantitation.

Main Results and Discussion

Calibration curves for primary bile acids and conjugates were linear from 0.2 to 1,000 nM with correlation coefficients above 0.99 and residuals within ±20%. Limits of quantitation ranged from 0.2 to 10 nM for most analytes, with intra-run precision (CV) below 15%. MS3 fragmentation proved critical for resolving isomeric pairs (e.g., GDCA vs. GCDCA), enhancing specificity beyond MS2 alone. Quantification in NIST plasma matched expected reference values, confirming accuracy. In the clinical application, total bile acid levels in ASD participants decreased by more than 30% after vitamin-mineral supplementation in 11 of 16 cases, with consistent changes observed for primary and conjugated species.

Benefits and Practical Applications

• High specificity for isomer differentiation via combined MS2/MS3
• Broad dynamic range suitable for trace-level to high-abundance bile acids
• Robust performance in complex biological matrices
• Clinical utility demonstrated in ASD biomarker profiling

Future Trends and Opportunities

Emerging applications may include large‐scale microbiome–metabolome studies, integration with ion mobility for further isomer resolution, and automated high‐throughput workflows. Advancements in data‐driven acquisition and machine learning for fragment confirmation could further streamline bile acid panels in clinical diagnostics and nutritional research.

Conclusion

A combined targeted-MS2/MS3 method on the Stellar mass spectrometer delivers sensitive, selective and reproducible quantitation of bile acids and conjugates across a five-order magnitude range. Its successful application to human serum, including ASD cohort analysis, underscores its potential for clinical and research laboratories focused on metabolic and microbiome‐related disorders.

References

• Remes P. M. et al. Hybrid Quadrupole Mass Filter–Radial Ejection Linear Ion Trap and Intelligent Data Acquisition Enable Highly Multiplex Targeted Proteomics, J. Proteome Res. 2024, 5476.