Simultaneous quantitation and discovery (SQUAD) metabolomics workflow on the orbitrap IQ-X for the analysis of fecal bile acids

Significance of the topic

Bile acids are key metabolites in lipid digestion and gut microbiota interactions, serving as biomarkers of intestinal health and disease. A comprehensive workflow that enables both precise quantitation and discovery of bile acids and their conjugates is crucial for understanding microbiome-mediated metabolic changes and related pathophysiology.

Objectives and study overview

This study aimed to develop and implement a simultaneous quantitation and discovery (SQUAD) workflow for fecal bile acids and conjugates in mice subjected to dietary interventions. The approach targeted a predefined list of primary and secondary bile acids while also enabling untargeted discovery of structurally related unknowns.

Methodology

Sample preparation involved extraction of fecal samples from mice on different diets (standard, SFA-rich, MUFA-rich) using 80% methanol, followed by reconstitution with labeled internal standards. Calibration curves spanning five orders of magnitude were generated using unlabeled and stable-isotope labeled bile acid standards, achieving a lower limit of quantification of 5 pg on column. AcquireX and real-time library search (RTLS) facilitated selective MS³ fragmentation for unknown compound annotation.

Used Instrumentation

• Liquid chromatography: Thermo Scientific Vanquish Horizon
• Mass spectrometry: Thermo Scientific Orbitrap IQ-X Tribrid
• Chromatographic column: Thermo Scientific Hypersil GOLD C18 reversed-phase
• Software: TraceFinder 5.1, Compound Discoverer 3.4, AcquireX, Real-Time Library Search

Main results and discussion

• Calibration curves showed linear quantitation over five orders of magnitude with a lower limit of quantification of 5 pg and coefficients of variation below 10%, confirming robustness and reproducibility.
• Absolute quantitation revealed distinct bile acid profiles among diet groups, highlighting metabolic shifts due to differing fat sources.
• Untargeted analysis via principal component analysis and heat maps identified clear metabolomic separations and annotated novel bile acid-related compounds.
• RTLS-triggered MS³ enabled structural elucidation of unknowns exhibiting fragmentation patterns similar to deoxycholic acid.

Benefits and practical applications of the method

The SQUAD workflow combines targeted quantitation with untargeted discovery in a single analytical run, improving throughput and confidence in data. Its sensitivity and dynamic range suit studies on microbiome perturbations, dietary interventions, and biomarker discovery in research and quality-control settings.

Future trends and applications

Advancements may include expansion to additional metabolite classes, integration of machine learning for spectral annotation, real-time adaptive acquisition strategies, and broader application in clinical and nutritional metabolomics for personalized health insights.

Conclusion

The developed SQUAD workflow on the Vanquish Horizon LC–Orbitrap IQ-X platform enables reliable simultaneous quantitation and discovery of fecal bile acids. Its robust performance and adaptability position it as a powerful tool for metabolomics research focusing on microbiome and dietary studies.