Sensitive and robust quantitation of serum bile acids using a novel quantitative kit on Stellar MS to study Crohn’s disease

Significance of the topic

Accurate quantitation of bile acids in human serum is analytically demanding because concentrations are low and many isomeric and structurally related species are present. Reliable measurement of bile acid patterns is critical for clinical and translational studies — for example, to reveal metabolic and microbiome-related perturbations in inflammatory bowel disease such as Crohn’s disease. A rapid, sensitive, standardized workflow that captures free, conjugated and sulfated bile acids with high reproducibility across laboratories enables larger cohort studies and can support biomarker discovery, mechanistic investigations and therapeutic monitoring.

Objectives and study overview

This study evaluated a targeted, high-throughput workflow combining the Move Analytical MoveKit BA quantitative kit with the Thermo Scientific Stellar MS (a hybrid quadrupole–linear ion trap instrument) for sensitive quantitation of 32 bile acids, including 16 sulfated species. Key aims were to demonstrate: a fast sample-to-result method suitable for cohort serum samples, low limits of detection and accurate MS2‑based quantitation, chromatographic separation of isomeric bile acids, and multi‑site reproducibility including application to serum from Crohn’s disease patients versus matched healthy controls.

Methodology

Sample preparation and general workflow

• Sample volume: 20 µL serum (same protocol used for urine; feces diluted 1:10 prior to extraction).
• Extraction: protein precipitation by dilution into a high-organic solvent, chilling at −20 °C for 10 minutes, followed by filtration through glass filter plates and centrifugation.
• Injection: 5 µL of cleared extract injected onto reverse-phase C18 LC.
• Calibration and QC: each 96‑well plate contained matrix-matched calibration samples, high and low QCs, and a pooled QC prepared from samples on the plate. Stable isotope-labeled internal standards (exact mass matches for each analyte) were included for all targets.

Chromatography and MS acquisition

• Reverse-phase LC with a C18 column (two laboratories used slightly different C18 columns).
• 9.5‑minute instrument method optimized for throughput.
• Targeted Parallel Reaction Monitoring (PRM) in negative ion mode employing both higher-energy collisional dissociation (HCD) and collision-induced dissociation (CID) to maximize fragment intensity for different analytes.
• Quantitation predominantly used MS2 fragment ions; 31 of 32 bile acids were quantified using MS2 transitions.

Used instrumentation

• Thermo Scientific Stellar MS (hybrid quadrupole–radial ejection linear ion trap) for targeted MSn acquisition.
• Thermo Scientific Xcalibur software for data acquisition and MoveApp/MoveKit software for sequence generation, live data quality monitoring and processing.
• C18 LC columns (slightly different stationary phases used between labs).
• Isotopically labeled internal standards supplied by Cambridge Isotope Laboratories.

Main results and discussion

• Analytical scope and sensitivity: The workflow quantified 32 bile acids (including 16 sulfated bile acids) using a one-point calibration approach. Reported lower limits of detection for many analytes were in the low nanomolar range (representative LLODs: DCA-3S 2.7 nM; CDCA-3S 2.6 nM; several conjugated bile acids at 1–6 nM), demonstrating clinical-grade sensitivity for serum studies.
• Fragmentation strategy: 15/32 analytes produced optimal fragment intensity with CID while others used HCD; tailoring fragmentation mode improved MS2 quantitation for structurally diverse bile acids (e.g., UDCA showed low fragmentation with HCD even at high NCE).
• Chromatographic separation: The method achieved separation of key isomeric pairs (e.g., TCDCA‑3S vs TDCA‑3S; CDCA vs DCA), which is essential to avoid misassignment and to obtain accurate abundance measures.
• Multi-laboratory reproducibility: Measurement agreement between Thermo Fisher Scientific (San Jose) and a second site (UNC MAP) was generally strong across matrices (serum, urine, feces). Some quantitative differences—fecal concentrations higher at San Jose—were attributed in part to different analytical columns and unresolved matrix interferences, underscoring the impact of LC selectivity on absolute values.
• Crohn’s disease cohort findings: Comparative analysis of serum from Crohn’s disease patients versus matched healthy controls revealed statistically significant alterations in bile acid composition. Several conjugated secondary bile acids showed high fold changes; TDCA depletion in Crohn’s serum was consistent with prior literature. Notably, multiple sulfated bile acids not previously reported in Crohn’s disease serum were identified as significantly altered, suggesting sulfation pathways or microbiota-related metabolism may be perturbed.

Benefits and practical applications of the method

• High throughput: 9.5‑minute LC‑MS method enables rapid processing of large cohorts with minimal sample volume (20 µL), suitable for clinical and population studies.
• Comprehensive coverage: Inclusion of free, conjugated and sulfated bile acids provides a broader metabolic picture than assays limited to unconjugated or conjugated bile acids only.
• Robust quantitation: Use of exact‑match isotopic internal standards for every analyte improves accuracy and compensates for matrix effects and sample preparation variability.
• Multi-site reproducibility: Demonstrated transferability between laboratories supports multi-center studies and method standardization efforts.
• Clinical research relevance: The assay’s sensitivity and isomer resolution enable detection of disease-associated alterations (e.g., Crohn’s disease) that may inform biomarker development and mechanistic research.

Future trends and potential applications

• Broader cohort studies: Application to larger and longitudinal Crohn’s disease cohorts and other GI disorders to validate sulfated bile acids as biomarkers or mechanistic indicators.
• Integration with multi-omics: Combining bile acid profiling with metagenomics and host metabolomics could link microbial metabolism, bile acid modification (sulfation, conjugation) and disease phenotypes.
• Method harmonization: Continued inter-laboratory studies and standardized reference materials will improve comparability and support clinical translation.
• Expanded chemical coverage: Further method refinement could extend quantitation to additional bile acid isomers and phase II metabolites beyond 32 targets.
• Automation and data pipelines: Enhanced software-assisted QC, real-time monitoring and automated processing (as exemplified by MoveApp) will accelerate throughput and quality control for clinical studies.

Conclusion

The presented workflow—combining the MoveKit BA sample preparation kit with Stellar MS PRM acquisition and matched isotopic internal standards—provides a fast, sensitive and reproducible approach to quantify a broad set of bile acids, including sulfated species, from small serum volumes. The method resolves critical isomers, achieves low nanomolar detection limits, and transfers reliably across laboratories. Application to a Crohn’s disease cohort revealed significant alterations in multiple bile acids, including previously underreported sulfated forms, highlighting the method’s utility for clinical metabolomics and biomarker discovery.

References

1. Remes PM, et al. Hybrid Quadrupole Mass Filter‑Radial Ejection Linear Ion Trap and Intelligent Data Acquisition Enable Highly Multiplex Targeted Proteomics. Journal of Proteome Research. 2024.
2. Sun R, Jiang J, Yang L, Chen L, Chen H. Alterations of Serum Bile Acid Profile in Patients with Crohn's Disease. Gastroenterology Research and Practice. 2022 Oct 3;2022:1680008.