MetaboQuan-R for Bile Acids in Human Serum: A Rapid, Targeted UPLC-MS/MS Method for Metabolomic Research Studies

Significance of the Topic

Bile acids represent a key group of endogenous metabolites synthesized in the liver that regulate cholesterol balance, lipid digestion, and signaling pathways. Accurate quantification of individual bile acids in human serum is critical for metabolic research, biomarker discovery, and understanding disease mechanisms such as liver dysfunction and gastrointestinal disorders.

Objectives and Study Overview

This application note details a rapid, targeted UPLC-MS/MS method for simultaneous quantification of 16 bile acids in human serum using the MetaboQuan-R workflow. The primary goal is to achieve high throughput and reliable separation of isomeric and isobaric bile acids within a single analytical run under three minutes, without the need for chemical derivatization.

Methodology

Sample preparation involves protein precipitation of 100 µL serum with methanol, centrifugation, dilution with water, and direct injection. Chromatographic separation is performed on an ACQUITY UPLC I-Class system with a CORTECS T3 column (2.1 × 30 mm, 2.7 µm) at 60 °C. A rapid gradient from 20 % to 55 % organic modifier over 0.7 min followed by a wash at 98 % B ensures elution of all analytes in under three minutes. Mass spectrometric detection uses a Xevo TQ-S micro in negative electrospray ionization mode with Multiple Reaction Monitoring (MRM) transitions optimized for each bile acid. Informatic processing is managed through MassLynx, Quanpedia, and TargetLynx for method setup and quantitation.

Used Instrumentation

• ACQUITY UPLC I-Class System
• CORTECS UPLC T3 Column (2.1 × 30 mm, 2.7 µm)
• Xevo TQ-S micro Triple Quadrupole Mass Spectrometer
• MassLynx Software with Quanpedia and TargetLynx modules
• MetaboQuan-R Method Package

Results and Discussion

The method achieved baseline or near-baseline separation of 16 bile acids, including challenging isomeric and isobaric pairs such as chenodeoxycholic acid and deoxycholic acid. Retention times ranged from 0.44 to 0.90 min, providing a total run time under three minutes. Standard addition experiments demonstrated detection limits in the low ng/mL range and clear resolution of CDCA from co-eluting compounds. Occasional unidentified peaks correspond to unknown isobaric forms, highlighting the method’s sensitivity to chemical variants.

Contributions and Practical Applications

This workflow offers a streamlined, high-throughput approach for targeted metabolomics studies of bile acids in clinical and research laboratories. Key advantages include:

• Single-run analysis of multiple bile acids without derivatization.
• Rapid turnaround enabling large sample sets.
• Compatibility with a generic LC-MS platform adaptable to other metabolite classes.
• Quantitative performance suitable for physiologically relevant concentration ranges.

Future Trends and Potential Applications

Advancements may include integration with automated sample handling, expansion to additional conjugated or minor bile acid species, and coupling with lipidomic or proteomic assays for comprehensive multi-omics profiling. Clinical adoption could enable routine monitoring of bile acid dysregulation in liver diseases, metabolic syndrome, and gastrointestinal pathologies.

Conclusion

A rapid, targeted UPLC-MS/MS method for 16 bile acids in human serum has been validated, demonstrating high throughput, robust separation of isomeric compounds, and sensitivity to low ng/mL levels. The generic LC-MS configuration and simple workflow make this approach a valuable tool for metabolomic research and biomarker studies.

References

No external references were cited in the original document.