Robust Metabolomics Workflows Using a Modified Benchtop Orbitrap Mass Spectrometer

Importance of the topic

Metabolomics enables comprehensive profiling of small molecules in biological systems and relies on high-resolution, accurate-mass measurements for confident identification. The benchtop Orbitrap Exploris 240 MS addresses the demand for robust, reproducible data acquisition in diverse metabolomic applications.

Objectives and overview

• Develop and validate metabolomics workflows leveraging the Orbitrap Exploris 240 for reliable quantitation and annotation.
• Demonstrate reproducible sub-ppm mass accuracy and stable signal response over multiple days.
• Employ the AcquireX intelligent acquisition strategy to maximize MS/MS coverage.

Methods and instrumentation

• Liquid chromatography: Thermo Vanquish UHPLC with Hypersil GOLD column (15 cm×2.1 mm, 1.9 μm particle size), 0.3 mL/min gradient (0.1% formic acid in water/methanol), 15 min total run time.
• Mass spectrometry: Orbitrap Exploris 240 operated in full scan (120 000 resolution) and MS/MS (30 000 resolution) modes, H-ESI source (3500 V positive, 3000 V negative), gas flows and temperatures optimized for sensitivity.
• AcquireX Deep Scan workflow for iterative data-dependent acquisition to enrich unique MS/MS spectra.
• Data processing: TraceFinder for targeted quantitation; Compound Discoverer for untargeted profiling, elemental composition and fragmentation matching.

Main results and discussion

• Sustained sub-ppm mass accuracy (<1 ppm) across three instruments over five days.
• Signal response stability with coefficients of variation of 5–10% in positive, negative and polarity-switching modes.
• AcquireX increased MS/MS coverage by over 75%, generating spectra for 1641 compounds vs. 793 in traditional DDA.
• High resolution delivered clear isotope patterns and fine structural details, enabling confident assignment of elemental compositions (e.g., adenosine [M+H]+ m/z 268.10385).
• Extended MS/MS mass range differentiated isomeric metabolites by detecting additional fragment ions.

Benefits and practical applications

• Reliable performance supports large-scale studies, QA/QC workflows and longitudinal experiments.
• Enhanced metabolite and lipid annotation in complex biological matrices.
• Improved discrimination of isobaric and isomeric species enhances data quality and biological interpretation.

Future trends and potential applications

• Integration of AI-driven analytics and automated annotation to accelerate data interpretation.
• Extension of workflows to lipidomics, flux analysis and quality control in clinical research.
• Automation of sample preparation and multiplexed acquisition strategies to increase throughput.

Conclusion

The Orbitrap Exploris 240 benchtop mass spectrometer offers a robust platform for metabolomics, delivering high mass accuracy, reproducible signal response and enhanced MS/MS coverage via AcquireX, thereby facilitating comprehensive and confident metabolome analysis.

Reference

• Schroeder M., Ntai I., Mohring S., Kellmann M., Harder A., Tautenhahn R., Souza A., Huhmer A. Robust Metabolomics Workflows Using a Modified Benchtop Orbitrap Mass Spectrometer. Thermo Fisher Scientific; 2020.