Exploring Different HPLC Column Chemistries for Optimal Separation of 13 Bile Acids by LC-MS/MS

Significance of the Topic

Bile acid profiling in human plasma by LC-MS/MS is critical for diagnosing liver diseases and monitoring drug-induced hepatotoxicity. Reliable separation of structurally similar bile acids and elimination of matrix interferences are essential for accurate quantitation and clinical decision-making.

Objectives and Article Overview

This study evaluates four HPLC column chemistries—Raptor C18, Biphenyl, FluoroPhenyl, and ARC-18—for the separation of 13 bile acids, including three pairs of isomeric unconjugated and glycine/taurine-conjugated acids. A key goal was to resolve a co-eluting matrix interference affecting the internal standard D4-UDCA.

Methodology and Instrumentation

The LC-MS/MS method employed gradient elution with mobile phase A (5 mM ammonium acetate in water) and mobile phase B (50:50 methanol:acetonitrile). Flow rates ranged from 0.5 to 0.8 mL/min, column temperatures were set at 50–60 °C, injection volumes were 3–10 µL, and the total run time was 9.5 min.

Used Instrumentation

• Raptor C18, 50 × 2.1 mm, 1.8 µm end-capped
• Raptor Biphenyl, FluoroPhenyl, and ARC-18, 100 × 2.1 mm, 2.7 µm
• Mobile phase A: 5 mM ammonium acetate in water
• Mobile phase B: 50:50 methanol:acetonitrile
• LC-MS/MS system with electrospray ionization source

Main Results and Discussion

• The original C18 phase separated all analytes but failed to resolve a matrix interference co-eluting with D4-UDCA, even under a very shallow gradient.
• The Biphenyl phase improved separation of glycine/taurine conjugates but did not resolve unconjugated isomers or the interference.
• The FluoroPhenyl phase resolved the interference from D4-UDCA but lacked selectivity for any isomer pairs, resulting in co-elution of analytes.
• The ARC-18 phase achieved baseline resolution of all three isomeric pairs and fully separated the matrix interference within a 9.5 min cycle.

Benefits and Practical Applications

• Enables robust quantitation of 13 bile acids in clinical and pharmaceutical laboratories.
• Short analysis time supports high-throughput workflows.
• Improved accuracy and confidence for biomarker studies and drug safety assessments.

Future Trends and Applications

• Design of novel stationary phases with tailored selectivity for challenging metabolite panels.
• Integration with high-resolution mass spectrometry for comprehensive bile acid metabolomics.
• Automation and multiplexing in personalized medicine and large-scale screening.
• Extension of methods to other complex biological matrices and emerging biomarkers.

Conclusion

The ARC-18 stationary phase provided superior selectivity for bile acid isomers and fully resolved critical matrix interferences, highlighting the importance of exploring alternative C18 chemistries to meet complex bioanalytical challenges.

Reference

1. Li D. & Carroll F. Rapid Analysis of 17 Bile Acids in Human Plasma by LC-MS/MS. Restek (accessed Jan 17, 2024).