Refined LC/Q-TOF Methodology for Identification and Profiling of Bile Acids Produced from the Gut Microbiome

Significance of the Topic

Bile acids serve critical functions in dietary lipid absorption and have emerged as key signaling molecules in immune regulation. Profiling the diverse bile acid pool produced by the gut microbiome offers valuable insights into host–microbiome interactions, disease mechanisms, and therapeutic interventions. High-resolution analytical methods capable of resolving isobaric species and detecting novel conjugates are essential to advance research in metabolism, microbiome-driven pathophysiology, and biomarker discovery.

Objectives and Study Overview

This work presents a refined liquid chromatography–quadrupole time-of-flight (LC/Q-TOF) workflow for the comprehensive profiling and quantitation of 67 bile acids across human and mouse biological matrices. The study aims to:

• Develop chromatographic conditions that fully resolve primary, secondary, and conjugated bile acids, including challenging isobaric pairs.
• Implement a single-point internal standard quantitation strategy covering 35 abundant bile acids.
• Demonstrate method applicability to fecal, cecal, serum, and plasma samples from human subjects and antibiotic-treated mice.
• Leverage high-resolution accurate mass and MS/MS data to discover novel amino acid–conjugated bile acids.

Methodology and Instrumentation

Sample Preparation:

• Fecal and cecal specimens were extracted via methanol-based protein precipitation with 35 isotope-labeled internal standards in 80% methanol.
• Plasma and serum samples were processed with a 7:2:1 acetonitrile:1% formic acid:methanol solvent containing the same internal standards.

Chromatography:

• Two 21-minute reversed-phase methods on an Agilent 1290 Infinity II LC:
  • Method 1: BEH Shield RP18 column (2.1×50 mm, 1.7 µm) separating 51 of 67 bile acids.
  • Method 2: Cortecs T3 column (2.1×50 mm, 1.6 µm) resolving remaining isobaric compounds.

Mass Spectrometry and Data Analysis:

• Detection on Agilent 6546 LC/Q-TOF in negative-ion mode using MS and AutoMSMS acquisition.
• Quantitative data processed in MassHunter Quantitative Analysis and Mass Profiler Professional.
• Metabolite identification supported by Sirius/CSI:FingerID and the GNPS platform for spectral matching.

Main Results and Discussion

Method Performance:

• Quantitation of 35 selected bile acids achieved with a single-point internal standard approach, requiring two sample dilutions.
• Quality control injections (18 pooled QC samples per matrix) yielded coefficient of variation (CV) values mostly below 5%, with a maximum under 15%.
• Retention time drift across three 96-well plates remained minimal, facilitating rapid data review.

Profile Correlation:

• Matched stool and plasma samples from healthy human donors displayed expected distributions of unconjugated primary and abundant secondary bile acids.
• Antibiotic-treated BALB/c mice exhibited elevated tauro-primary bile acids and decreased secondary species, reflecting microbiome-driven enzymatic activity loss (BSH gene and bai operon).

Novel Conjugates Discovery:

• Accurate mass and MS/MS workflows enabled preliminary identification of tyrosine-, phenylalanine-, and leucine-conjugated bile acids in human fecal extracts.

Benefits and Practical Applications

This robust LC/Q-TOF protocol offers:

• Comprehensive coverage of a wide bile acid panel, including isobars.
• High precision and throughput suitable for large cohort studies and clinical research.
• Capability to monitor metabolic shifts in response to microbiome perturbations, antibiotic treatment, and fecal microbiota transplantation.
• Platform adaptability to various biological matrices, enabling cross-matrix comparisons.

Future Trends and Applications

Emerging directions include:

• Expansion of novel bile acid conjugate libraries and structural elucidation via ion mobility and advanced MS/MS strategies.
• Integration with multi-omics datasets to map bile acid–mediated host–microbe signaling networks.
• Applications in personalized medicine for metabolic disorders, inflammatory diseases, and microbiome-based therapies.
• Automation of sample preparation and data processing pipelines to accelerate large-scale studies.

Conclusion

The described dual-method LC/Q-TOF approach achieves full separation and reliable quantitation of 67 bile acids across diverse sample types. Demonstrated precision, robustness, and capacity for discovering novel conjugates position this workflow as a valuable tool for metabolic and microbiome research.

References

1. Guzior DV, Quinn RA. Microbial transformations of human bile acids. Microbiome. 2021;9(1):140.
2. Quinn RA et al. Global chemical effects of the microbiome include new bile-acid conjugations. Nature. 2020;579(7797):123-129.