Fast Profiling of 39 Bile Acids in Plasma, Urine and Feces, by Automated Extraction and LCMS-8060NX

Significance of the Topic

Bile acids are central to fat digestion and cholesterol metabolism and serve as sensitive biomarkers of liver function and gastrointestinal health. Detailed profiling of individual primary, secondary and conjugated bile acids can aid in distinguishing specific hepatic disorders and gut microbial interactions. A rapid, high-throughput and accurate analytical method is therefore critical for both clinical diagnostics and research applications.

Objectives and Study Overview

This study aimed to develop and validate a fully automated extraction protocol combined with a fast LC-MS/MS method for simultaneous quantification of 39 bile acids in human plasma, urine and mouse feces. The approach integrates optimized HPLC separation to resolve structural isomers with high‐speed tandem mass spectrometry, reducing sample preparation time and analysis cost while maintaining high sensitivity and reproducibility.

Methodology and Used Instrumentation

The sample preparation employs the Biotage® Extrahera™ system with Evolute Express ABN 30 mg 96-well plates for automated solid-phase extraction. Plasma and urine aliquots (250 µL) undergo internal standard addition, washing and elution in under 45 minutes for 96 samples (0.5 min per sample). Mouse feces (5–10 mg) are hydrolyzed with KOH (20 min, 80 °C) before neutralization and SPE. Chromatographic separation uses a Nexera™ X2 UHPLC equipped with an ACE Excel C18 Amide column (2.1 × 150 mm, 1.7 µm) at 45 °C, 0.65 mL/min, with a 10.95 min run. Detection is performed on a Shimadzu LCMS-8060NX triple quadrupole in negative ESI MRM mode, with optimized gas flows and temperatures for robust ionization.

Main Results and Discussion

• Calibration curves over 0.5–50 ng/mL exhibited linearity and accuracies of 80–120%.
  
• Repeatability (RT RSD <0.4%) and intermediate precision (RT RSD <0.5%, area RSD <20%) confirmed method robustness.
  
• Three quantification strategies—standard addition, one-point internal calibration and direct isotopic dilution using ten stable-isotope labeled standards—yielded comparable accuracies in plasma (82–120%), urine (82–119%) and feces (80–120%).
  
• Isomeric separation by HPLC ensured baseline resolution (R>1.1) enabling unambiguous MRM detection. Correction factors were applied when internal standard responses differed from analytes due to matrix effects.
  

Benefits and Practical Applications

• High throughput: 96 samples processed in 45 min and a 10 min LC-MS/MS cycle per run.
  
• Automated workflow reduces hands-on time, labor costs and variability.
  
• Comprehensive profiling of isomeric bile acids supports liver disease research, microbiome studies and clinical biomarker discovery.
  
• Cost efficiency achieved by reducing the number of isotope standards while maintaining quantification accuracy.
  

Future Trends and Applications

• Expansion of targeted panels to include additional lipid-derived metabolites and conjugates.
  
• Integration with data analytics and machine learning for predictive diagnostics.
  
• Further miniaturization of sample prep and higher-density plate formats to increase throughput.
  
• Development of standardized workflows for multi-site interlaboratory studies.
  

Conclusion

The Shimadzu LC/MS/MS Bile Acids Ver.2 method package delivers a turnkey solution for rapid, automated extraction and fast, high-resolution LC-MS/MS analysis of 39 bile acids across plasma, urine and feces. The approach combines speed, reproducibility, sensitivity and cost-effectiveness, making it well suited for routine clinical and research laboratories.

References

1. Shimadzu. LC/MS/MS Method Package for Bile Acids version 2; P/N S225-38610-91.
2. Advanced Chromatography Technologies. ACE Excel C18 Amide; P/N EXL-1712-7502U.