Fast Profiling of 39 Bile Acids in Plasma, Urine and Feces, by Automated Extraction and LC-MS/MS

Significance of the topic

Bile acids regulate cholesterol metabolism and fat absorption in the small intestine and serve as biomarkers for liver function and disease. Detailed quantification of individual bile acids in plasma, urine and feces supports clinical research and improves differentiation of hepatic injury beyond total bile acid assays.

Objectives and study overview

This work aims to develop and validate a rapid, high-throughput LC-MS/MS method for simultaneous quantification of 39 primary, secondary and conjugated bile acids in human plasma, human urine and mouse feces. The study evaluates linearity, accuracy and precision using both internal calibration and direct isotopic dilution strategies.

Methodology and instrumentation

The sample preparation relies on a fully automated 96-well extraction workflow using Biotage® Extrahera™. Fecal samples undergo hydrolysis with KOH, while plasma and urine are directly processed in 96-well plates. Chromatographic separation is achieved on an ACE Excel C18 Amide column with a 10-minute gradient. Detection uses MRM transitions on a Shimadzu LCMS-8060NX™ triple quadrupole mass spectrometer operated in negative ESI mode.

Used instrumentation

• Biotage Extrahera automated extraction platform
• Shimadzu Nexera X2 UHPLC system
• Shimadzu LCMS-8060NX triple quadrupole MS

Main results and discussion

Calibration curves for all 39 bile acids were linear over 0.5–50 ng/mL (R2>0.98). Automated data integration with LabSolutions Insight eliminated manual reintegration. Accuracy in spiked matrices ranged from 80–120% across feces, urine and plasma. Intra-day and inter-day precision met acceptance criteria, with retention time RSD below 0.5% and area repeatability below 12%. Both quantification approaches showed excellent agreement and strong correlation with standard addition.

Benefits and practical applications

The automated workflow reduces sample preparation time to 0.5 minutes per sample and enhances reproducibility compared to manual methods. High throughput and robust quantification enable routine monitoring of bile acid profiles in clinical research, toxicology and metabolic studies.

Future trends and applications

Integration of larger panels including novel bile acid metabolites, coupling with high-resolution MS for structural elucidation, and application to human gut microbiome studies are anticipated. Further miniaturization and on-line extraction could streamline clinical diagnostics.

Conclusion

This method demonstrates a rapid, reliable and fully automated solution for the quantification of 39 bile acids in biological matrices, meeting stringent validation criteria and facilitating large-scale studies of liver function and metabolic regulation.

References

• Jaffuel A, Kunisawa A, Watanabe J. Fast Profiling of 39 Bile Acids in Plasma, Urine and Feces by Automated Extraction and LC-MS/MS. Shimadzu Application Note.