Simultaneous Quantitation and Discovery (SQUAD) metabolomics: an intelligent combination of targeted and untargeted workflows using the novel Orbitrap Astral mass spectrometer

Importance of the Topic

Metabolomics is essential for understanding biochemical processes and disease mechanisms by profiling small molecules in biological samples. Integrating targeted and untargeted approaches in a single analysis enhances both quantitative accuracy and discovery of novel biomarkers, addressing the need for high-throughput and comprehensive workflows in research and quality control.

Study Objectives and Overview

The primary goal was to develop a single-injection metabolomics method—termed SQUAD—that combines accurate quantitation of predefined metabolites with untargeted discovery of unknown features. This approach leverages the novel Orbitrap Astral mass spectrometer to achieve high sensitivity, broad dynamic range, and deep coverage in a single run.

Materials and Methods

The workflow involved spiking isotope-labeled amino acids into the NIST SRM 1950 plasma reference standard across a dilution series (1 nM–2.5 mM). After extraction with 80% methanol, samples were separated using reversed-phase chromatography on a Hypersil GOLD column coupled to a Vanquish Horizon UHPLC. Data acquisition ran in parallel full-scan high-resolution Orbitrap MS1 and data-dependent MS2 on the Astral analyzer. TraceFinder 5.1 and Compound Discoverer 3.3 software processed targeted quantitation and untargeted feature annotation, respectively.

Used Instrumentation

• Thermo Scientific Orbitrap Astral mass spectrometer
• Thermo Scientific Vanquish Horizon UHPLC system
• Thermo Scientific Hypersil GOLD reversed-phase column

Results and Discussion

The SQUAD method delivered a linear dynamic range spanning five orders of magnitude with limits of quantitation down to 10 femtomoles on-column. Parallel acquisition enabled up to 90% of detected compounds to yield high-quality MS2 spectra, surpassing traditional Orbitrap-Orbitrap modes. Fast Orbitrap MS1 scans provided sufficient data points per peak even at low concentrations, and the combined system maintained signal-to-noise and fragmentation efficiency when the LC gradient was shortened threefold.

Benefits and Practical Applications

• Simultaneous accurate quantitation and untargeted discovery in a single run
• High sensitivity down to femtomole levels with broad linearity
• Enhanced structural annotation through high-quality MS2 fragmentation
• Increased throughput by reducing gradient times without loss of performance
• Reduced sample variability by eliminating multiple injections

Future Trends and Applications

Ongoing developments may include expansion to diverse biofluids and tissue extracts, integration with machine learning for automated feature annotation, and further acceleration of chromatographic and mass spectrometric cycles. The SQUAD concept could also extend to lipidomics and exposomics, facilitating broader systems-level investigations.

Conclusion

The Orbitrap Astral-enabled SQUAD workflow offers a balanced, high-throughput solution that unites targeted quantitation and untargeted metabolite discovery in one injection. Its combination of sensitivity, dynamic range, and fragmentation depth positions it as a powerful tool for advanced metabolomics in research and industrial applications.