Liquid-Chromatography coupled to Tandem Mass Spectrometry for 28 Bile Acids Profiling in Serum Samples

Significance of the Topic

Bile acids serve as essential digestive surfactants and metabolic regulators, linking liver function to gut microbiome activity. Detailed profiling of individual bile acid species offers deeper insights into liver health, metabolic disorders, and host–microbe interactions, surpassing the information obtained from total bile acid measurements.

Objectives and Study Overview

This work presents a turnkey LC–MS/MS method package for simultaneous quantification of 28 bile acids and 10 deuterated internal standards. The method is demonstrated on pooled human plasma, highlighting its precision, sensitivity, and applicability to clinical and research settings.

Instrumentation

• UHPLC System: Shimadzu Nexera X2 coupled to LCMS-8060 triple quadrupole mass spectrometer
• Column: GL Sciences Inertsil ODS-4 HP, 150 × 2.0 mm, 3 µm, maintained at 40 °C
• Mobile Phases: A – water with 0.05% acetic acid; B – acetonitrile/methanol (40/60)
• Flow Rate and Gradient: 300 µL/min; 35→50% B in 0.1 min, 50→85% B in 8.4 min, ramp to 100% B in 2 min (total 17 min)
• Ionization: Negative‐mode ESI (−3 kV); interface 300 °C, desolvation line 250 °C, heat block 400 °C
• MRM Acquisition: Optimized dwell times (7 ms) for 28 bile acid transitions plus qualifiers

Methodology and Analytical Conditions

• Standards Preparation: 100 µM stock solutions in methanol; 1 µM one‐point calibration injected four times
• Sample Preparation: 50 µL plasma mixed with 10 µL internal standard (10 µM), acidified with 30 µL HCl (1 M), precipitated with 910 µL acetonitrile, vortexed, centrifuged (14 000 g, 15 min), supernatant dried and reconstituted in 100 µL methanol
• Injection: 2 µL extracts delivered to UHPLC-MS/MS under MRM mode

Main Results and Discussion

Calibration yielded response‐factor precision below 10% RSD for all bile acids, confirming method robustness. In pooled human plasma, major bile acids were quantified with acceptable reproducibility: taurochenodeoxycholic acid (TCDCA) 82.6 ± 5.08 pmol/mL (6% RSD), glycocholic acid (GCA) 116 ± 0.94 pmol/mL (0.8% RSD), cholic acid (CA) 131 ± 5.17 pmol/mL (4% RSD), among others. Chromatograms demonstrated clear separation of structural isomers, critical for accurate identification and quantification.

Benefits and Practical Applications

• Comprehensive coverage of primary, secondary, and conjugated bile acids in a single run
• High throughput (17 min per analysis) suitable for large clinical and microbiome studies
• Reproducible quantification with low RSD supports QA/QC in regulated environments
• Adaptable to plasma, serum, and tissue matrices for translational research

Future Trends and Applications

Advancements may include integration with high-resolution metabolomics, automation of sample preparation, expansion to noninvasive matrices (e.g., fecal water), and development of data-analysis pipelines for systems‐level interpretation. Linking detailed bile acid profiles with microbiome composition and host genetics will foster personalized medicine and novel therapeutic strategies.

Conclusion

The presented LC–MS/MS method package delivers a reliable, sensitive, and comprehensive approach to bile acid profiling. Its turnkey configuration accelerates adoption in research and clinical laboratories, facilitating deeper insights into liver function, metabolic health, and host–microbiome interactions.