Simultaneous quantitation and discovery (SQUAD) of fecal bile acids and their conjugates in children with autism spectrum disorder (ASD)

Importance of the Topic

Bile acids are central mediators of lipid digestion, microbial signaling and gut–brain communication. Alterations in bile acid metabolism by the gut microbiota have been implicated in gastrointestinal and neurodevelopmental features of autism spectrum disorder (ASD). A robust analytical strategy to quantify both known bile acids and novel microbial conjugates in fecal samples can shed light on microbiome–host interactions and support therapeutic monitoring.

Objectives and Study Overview

This work introduces the SQUAD (Simultaneous Quantitation and Discovery) workflow, a single-injection metabolomics platform combining targeted and untargeted analysis. The method was applied to fecal extracts from children with ASD undergoing microbiota transfer therapy (MTT) to evaluate changes in bile acid profiles and discover new metabolites linked to treatment response.

Methodology and Instrumentation

• Sample Preparation: Fecal material was extracted in a methanol–water blend with isotopically labeled bile acid internal standards.
• Chromatography: Reversed-phase separation performed on Thermo Fisher Vanquish Horizon UHPLC.
• Mass Spectrometry: Thermo Scientific Orbitrap Ascend Tribrid system coupled with a linear ion trap.
• Data Acquisition: Parallel Reaction Monitoring (PRM) in the ion trap for quantitation; high-resolution MS1 in the Orbitrap for annotation; real-time library search to improve spectral matching.
• Data Analysis: TraceFinder 5.2 for absolute quantitation; Compound Discoverer 3.4 for untargeted feature identification and multivariate statistics.

Main Results and Discussion

The SQUAD workflow achieved an LLOQ of 1.8 fmol and linear dynamic range across six orders of magnitude for bile acids. MS3 fragmentation in the linear ion trap enabled selective quantitation of co-eluting isomers (e.g., leucine-LCA vs. isoleucine-LCA). Following MTT, significant shifts in fecal bile acid profiles were observed, with principal component analysis separating pre- and post-treatment samples. Lipid and fatty acid metabolites emerged as key drivers of the metabolic changes, while the placebo group showed no such segregation.

Benefits and Practical Applications

The SQUAD approach streamlines high-throughput profiling by unifying targeted quantitation and exploratory discovery in one run. Its enhanced isomer resolution and deep metabolome coverage make it suitable for clinical and research laboratories monitoring microbiome interventions, biomarker discovery, and drug response.

Future Trends and Potential Applications

Further expansion of bile acid libraries, integration with ion mobility, and adaptation to other biological matrices will broaden SQUAD’s utility. Incorporating advanced data processing algorithms and large-scale clinical cohorts could accelerate translation into diagnostics and personalized treatment monitoring.

Conclusion

This study demonstrates a comprehensive, sensitive and reliable SQUAD workflow for simultaneous quantitation and discovery of fecal bile acids and related metabolites. The method provides valuable insights into microbiota-mediated metabolic shifts associated with ASD therapies and offers a versatile platform for future metabolomics research.

Reference

• Takyi et al., 2025. Gut microbiota and metabolic imbalances in ASD.