Measuring Bile Acid Levels in Human Plasma Using a Triple Quadrupole Mass Spectrometer

Importance of the Topic

Bile acids are critical for dietary fat absorption and cholesterol homeostasis in humans. Quantifying individual bile acids in plasma provides sensitive markers for liver function and various hepatic disorders. Simultaneous measurement of multiple bile acids enhances diagnostic insight and supports research into gut–liver interactions.

Objectives and Study Overview

This study aimed to develop and validate a robust method to quantify 22 bile acids in human plasma using a triple quadrupole mass spectrometer coupled with high-performance liquid chromatography. Key objectives included achieving high sensitivity, reproducibility, and long‐term stability without frequent instrument tuning.

Methodology and Used Instrumentation

Sample preparation followed a protein-precipitation protocol in microtubes:

• Combine 50 µL plasma with 50 µL water.
• Add 350 µL methanol extract containing nine stable isotope internal standards.
• Mix, incubate on ice, and centrifuge to collect supernatant.
• Concentrate by evaporation, reconstitute in methanol, sonicate, and re‐centrifuge.
• Transfer aliquot to analysis vial.

Chromatographic separation used a reversed-phase column on a Nexera™ X2 system with gradient elution of 0.05 % acetic acid in water (mobile phase A) and acetonitrile/methanol 50:50 (mobile phase B) at 0.3 mL/min. Injection volume was 2 µL. Mass spectrometry employed the LCMS-8060 with negative‐mode electrospray ionization, optimized gas flows (nebulizer: 2 L/min, drying: 10 L/min, heating: 10 L/min), and source temperatures (DL: 250 °C, heat block: 400 °C, interface: 300 °C). Multiple reaction monitoring transitions were set according to a specialized bile acids method package.

Main Results and Discussion

In quality control (QC) experiments, 34 replicates of pooled plasma demonstrated concentration precision (%RSD) of 2.8 % to 12.4 % for 21 bile acids; one minor analyte (GHDCA) showed higher variability (49.9 %). Clinical validation over 19 days (272 samples) without any instrument tuning or column change yielded stable peak areas for internal standards, with %RSD between 4.9 % and 6.0 %. Twenty‐two bile acids were consistently detected, confirming method robustness and suitability for high‐throughput analysis.

Benefits and Practical Applications of the Method

• Comprehensive profiling of primary and secondary bile acids in plasma.
• High sensitivity and selectivity afforded by triple quadrupole MRM.
• Long‐term stability without frequent maintenance.
• Applicability in clinical diagnostics, liver function monitoring, and research on metabolic and gut–liver axis disorders.

Future Trends and Applications

Advances may include expanding analyte panels to novel bile acid derivatives, adopting high-resolution mass spectrometry for untargeted profiling, and automating sample preparation to increase throughput. Integration with metabolomics and systems biology workflows will deepen understanding of bile acid regulation in health and disease.

Conclusion

The described LCMS-8060 method provides reliable, precise, and high‐throughput quantification of 22 bile acids in human plasma. Exceptional long‐term stability supports routine clinical and research applications without frequent instrument adjustments.

References

• Nakanishi T. et al. Effect of a High-Fat Diet on the Small-Intestinal Environment and Mucosal Integrity in the Gut-Liver Axis. Cells. 2021;10(11):3168.