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

Importance of the topic

Quantitative profiling of bile acids in blood is analytically demanding but clinically important. Bile acids are central to lipid digestion, host–microbiome signaling and inflammatory processes; subtle changes in circulating bile acid composition can reflect or drive pathologies such as Crohn’s disease. Reliable, high-throughput and sensitive assays that resolve structurally similar isomers and detect conjugated and sulfated forms are therefore essential for large cohort studies, biomarker discovery and translational research.

Objectives and study overview

This study aimed to demonstrate a rapid, sensitive and robust targeted LC–MS workflow that quantifies 32 bile acids (including 16 sulfated species) from small-volume samples using the MoveKit BA quantitative kit combined with the Thermo Scientific Stellar MS platform. Goals included: developing a streamlined sample-prep and short chromatographic method (<10 min), validating limits of detection and quantitative performance, testing fragmentation strategies (CID and HCD) for different analytes, and applying the assay to compare serum bile acid profiles between Crohn’s disease patients and age-matched healthy controls with multi-site reproducibility testing.

Methods and sample preparation

• Sample volumes and matrices: 20 µL serum (same protocol applied to urine; fecal extracts diluted 1:10 prior to prep).
• Preparation workflow: protein precipitation by dilution into high-organic solvent, chilling at −20 °C for 10 min, transfer to glass filter plate, centrifugation, and direct injection of 5 µL of the cleared extract onto a reversed-phase C18 column.
• Calibration and QC: each 96-well plate included matrix-matched calibration samples, high and low QCs, and a pooled sample QC derived from the plate. Quantitation used matched mass-labeled internal standards for every analyte and one-point calibration was used for the reported workflow.
• Acquisition strategy: targeted parallel reaction monitoring (PRM) in negative ion mode, combining HCD and CID fragmentation to maximize fragment ion intensity for different bile acids; MS2 fragment ions were used for quantitation for 31 of 32 targets.
• Data handling: acquisition via Xcalibur with MoveApp for sequence generation, real-time quality monitoring, and integrated processing/statistics.

Instrumentation used

• Thermo Scientific Stellar MS (hybrid quadrupole–linear ion trap) operated in targeted PRM mode.
• MoveKit BA quantitative kit (Move Analytical LLC) providing reagents, sample-prep consumables and assay protocols.
• Mass-labeled internal standards from Cambridge Isotope Laboratories.
• Chromatography: C18 reversed-phase columns (note: slightly different C18 columns were used between sites, affecting some matrix separation performance).
• Software: Thermo Xcalibur and MoveApp for acquisition control, QC monitoring and data processing.

Main results and discussion

• Panel performance: the method quantified 32 targeted bile acids (including 16 sulfated forms) with limits of detection in the low nM range for many analytes. Quantitation was achieved using MS2 fragments for 31/32 bile acids; 15 analytes produced stronger fragments with CID optimization while others were best with HCD.
• Throughput and input: the assay uses a 9.5-minute LC–MS method and requires only 20 µL of serum, supporting higher throughput for cohort studies.
• Multi-site reproducibility: pooled samples analyzed at two independent labs (Thermo Fisher San Jose and UNC MAP) showed good agreement across matrices (serum, urine, feces). Slightly higher fecal concentrations were observed at one site, likely attributable in part to column differences affecting chromatographic resolution of matrix interferences.
• Crohn’s disease application: comparison of serum from Crohn’s disease patients and matched healthy controls revealed significant alterations in bile acid composition. Notably, multiple conjugated secondary bile acids exhibited high fold changes; depletion of TDCA in Crohn’s patients matched prior literature reports. Importantly, several sulfated bile acids not previously described in Crohn’s serum were observed as significant, suggesting additional metabolic or microbiome-linked modifications associated with disease.
• Analytical considerations: the combined HCD/CID strategy enabled better coverage of structurally diverse bile acids (some compounds require higher-energy HCD for sulfated species fragment ions at m/z 97, while others fragment more effectively under CID). Use of isotope-labeled internal standards for each analyte supported accurate relative quantitation using a streamlined calibration approach.

Benefits and practical applications

• Small sample volume and short run time make the method suitable for large cohort and multi-site studies where sample throughput and limited specimen volumes are constraints.
• Comprehensive coverage—including sulfated bile acids—expands the biochemical information accessible from serum, improving biomarker discovery potential for inflammatory bowel disease and other metabolic conditions.
• Use of matched isotopic internal standards and plate-based QC improves quantitative rigor and reproducibility across batches and sites.
• The combined fragmentation strategy and MSn-capable Stellar platform deliver both sensitivity and selectivity needed for isomer separation and confident identification.

Future trends and potential applications

• Expanding panels: addition of more conjugated or microbial-derived bile acid derivatives and inclusion of phase II metabolites could improve mechanistic insights.
• Standardization and clinical translation: further inter-laboratory standardization and validation against clinical endpoints could support routine clinical testing or companion diagnostics in inflammatory bowel disease.
• Integration with omics: combining quantitative bile acid profiling with microbiome, lipidomics and proteomics datasets will help link microbial metabolism to host pathophysiology in Crohn’s disease.
• Automation and throughput: further automation of sample handling and adoption of short LC gradients will accelerate large-scale studies and longitudinal sampling.
• Structural confirmation: employing additional MSn experiments and orthogonal separation (e.g., ion mobility) can resolve challenging isomers and confirm sulfation sites.

Conclusion

The presented workflow couples a commercial quantitative kit with the Stellar MS to deliver a rapid, sensitive and reproducible targeted assay for 32 bile acids across serum, urine and fecal extracts. The approach balances throughput and analytical rigor, enabling reliable detection of conjugated and sulfated species from small sample volumes. Application to Crohn’s disease serum identified multiple significantly altered bile acids, including previously unreported sulfated species, illustrating the method’s utility for clinical and translational metabolomics.

References

• 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;5476.
• 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.