SQUAD analysis of fecal bile acids and their conjugates in children with autism spectrum disorder

Importance of the Topic

Bile acids play a central role in lipid digestion, host–microbiota interactions, and gut–brain signaling. Alterations in bile acid metabolism have been linked to gastrointestinal and neurodevelopmental disorders, including autism spectrum disorder (ASD). Understanding the shifts in bile acid profiles following microbiome interventions can illuminate mechanisms underlying therapeutic responses and guide future diagnostic or treatment strategies.

Study Objectives and Overview

This study applies a single-injection Simultaneous Quantitation and Discovery (SQUAD) metabolomics workflow to characterize fecal bile acids and their microbial conjugates in children with ASD undergoing microbiota transfer therapy (MTT). Objectives include:

• Establishing a combined targeted and untargeted approach for comprehensive bile acid profiling.
• Assessing metabolic shifts in fecal bile acids pre- and post-MTT.
• Demonstrating the workflow’s sensitivity, dynamic range, and isomer resolution.

Methodology and Instrumentation

Sample Preparation:

• Fecal samples from ASD cohorts were extracted with a methanol–water mixture.
• Isotopically labeled bile acid standards were spiked in for accurate quantitation.

Analytical Platform:

• Thermo Scientific Vanquish Horizon UHPLC coupled to an Orbitrap Ascend Tribrid mass spectrometer.
• Parallel Reaction Monitoring (PRM) in the linear ion trap for targeted quantitation.
• High-resolution MS1 scanning in the Orbitrap for untargeted metabolite discovery.

Data Processing:

• Quantitation and annotation performed in TraceFinder and Compound Discoverer software.
• Real-time library search enhanced spectral matching confidence.

Main Results and Discussion

The SQUAD workflow achieved:

• A lower limit of quantitation of 1.8 femtomoles on-column and a detection limit of 1.8 attomoles.
• A linear dynamic range spanning six orders of magnitude for accurate quantitation.
• High selectivity for co-eluting isomers via MS3 fragmentation, validated using leucine-LCA and isoleucine-LCA mixtures.

PCA revealed clear separation between pre- and post-MTT fecal profiles, with fatty acids and lipid-related metabolites driving the observed metabolic shifts. No such separation was observed in placebo controls, highlighting the specificity of MTT effects.

Benefits and Practical Applications

The SQUAD approach enables:

• Simultaneous high-throughput targeted quantitation and untargeted discovery in a single injection.
• Deep coverage of known and novel bile acid metabolites with robust sensitivity.
• Improved confidence in identification and quantitation of structural isomers.

This workflow can be applied in clinical metabolomics, microbiome research, and QA/QC settings where comprehensive profiling of small molecules is required.

Future Trends and Potential Applications

Emerging directions include integration of SQUAD with multi-omics data, real-time decision support in clinical trials, and expanded libraries of microbial metabolites. Advances in machine learning for spectral interpretation and automation of sample prep could further enhance throughput and data quality.

Conclusion

The developed SQUAD metabolomics workflow offers a powerful platform for simultaneous quantitation and discovery of fecal bile acids and related metabolites. Its high sensitivity, broad dynamic range, and isomer resolution capabilities make it well-suited for studying host–microbiome metabolic interactions and therapeutic interventions in ASD and beyond.

References

Takyi, et al. Alterations in gut microbiota and metabolic imbalances in ASD. 2025.
Amer, B., Melnik, A., Adams, J.B., et al. SQUAD analysis of fecal bile acids in ASD. Thermo Fisher Scientific, 2025.