Simultaneous quantitation and discovery (SQUAD): an intelligent combination of targeted and untargeted metabolomics workflow for the analysis of fecal metabolites using Orbitrap Astral MS

Significance of the Topic

Fecal metabolomics provides critical insights into gut microbiome function and its impact on host health and disease. A combined targeted and untargeted approach enhances both precise quantitation and the discovery of novel metabolites, addressing limitations of speed, sensitivity, and annotation confidence in traditional workflows.

Objectives and Study Overview

The study aimed to develop SQUAD (Simultaneous Quantitation and Discovery), a unified workflow for absolute quantification of selected fecal metabolites alongside an untargeted survey of the broader metabolic profile. An in vivo dietary intervention in mice—standard, saturated-fat–rich, and monounsaturated-fat–rich diets over 29 days—served as the application model.

Methodology and Instrumentation

Fecal samples were extracted with 80% methanol, dried, and reconstituted in 50% acetonitrile. Targeted and untargeted data were acquired in parallel using high-resolution Orbitrap full scans and fast, high-accuracy Astral MS2 scans under both positive and negative ESI modes. Data processing and compound annotation employed TraceFinder 5.1 and Compound Discoverer 3.4.

Used Instrumentation

• Thermo Scientific Vanquish Horizon UHPLC system
• Thermo Scientific Orbitrap Astral mass spectrometer
• Thermo Scientific Accucore 150 Amide HILIC column
• TraceFinder 5.1 and Compound Discoverer 3.4 software

Main Results and Discussion

Calibration curves built from authentic standards spanned four orders of magnitude, achieving a limit of quantitation as low as 2.85 fmol and a limit of detection of 0.71 fmol on column. Targeted analysis revealed significant diet-dependent variations in bile acids and other key metabolites. Untargeted profiling detected a higher number of quality MS2 spectra via the Astral analyzer, enabling confident annotation of unknown features and clear group separation in PCA and heat-map analyses.

Benefits and Practical Applications

• Simultaneous quantitation and discovery accelerates metabolomics studies.
• High sensitivity and broad dynamic range support low-abundance metabolite detection.
• Deep coverage of both known and unknown fecal metabolites enhances dietary and clinical research.

Future Trends and Opportunities

Advances may include expanded spectral libraries for unknown annotation, real-time high-throughput workflows, application to larger cohorts and diverse sample types, and integration with other omics platforms for comprehensive systems biology analyses.

Conclusion

The SQUAD workflow successfully integrates targeted quantitation and untargeted discovery using Orbitrap and Astral mass analyzers, offering a powerful platform for in-depth fecal metabolomics studies in nutritional and microbiome research.

References

1. Amer B, Pavageau MP, Biraghi S, Deshpande RR, Moehring T, Bird SS. Simultaneous quantitation and discovery (SQUAD): an intelligent combination of targeted and untargeted metabolomics workflow for the analysis of fecal metabolites using Orbitrap Astral MS. Thermo Fisher Scientific; 2025.