Metabolomics Workflow using a Xevo™ MRT Mass Spectrometer

Significance of the Topic

Metabolomics is pivotal for uncovering small-molecule biomarkers linked to disease states, environmental exposures, or treatment effects. High mass resolution and sub-ppm mass accuracy are essential to distinguish structurally similar compounds and ensure reliable identification within complex biological matrices.

Study Objectives and Overview

This work evaluates a metabolomics workflow using a Xevo MRT QTof mass spectrometer coupled with an ACQUITY Premier UPLC system. Human urine from non-smokers, passive smokers, and active smokers was profiled. The goal was to demonstrate rapid, robust data acquisition and statistical separation of phenotypic groups using MARS software for peak picking, multivariate analysis, and putative compound identification.

Methodology

Urine samples representing three nicotine intake levels and a pooled QC were diluted with ultrapure water and spiked with an LCMS reference mix. Five analytical replicates of each sample were injected onto the UPLC system. A reversed-phase gradient (0.1% formic acid in water/acetonitrile) was run over 10 minutes at 0.6 mL/min. Data were acquired in positive ESI mode in MSE continuum between 50–1200 Da at 20 Hz with lockspray calibration and alternating low/high collision energy.

Used Instrumentation

• ACQUITY Premier UPLC FTN with HSS T3 column (2.1×100 mm, 1.7 µm)
• Xevo MRT Mass Spectrometer operated in sensitivity mode with StepWave transmission
• Lockspray reference for sub-ppm mass accuracy
• waters_connect software for acquisition control
• MassAnalytica (MARS) software for data processing against the HMDB library

Key Results and Discussion

Unsupervised PCA achieved clear separation of the three nicotine exposure groups with tight QC clustering, indicating high reproducibility. Supervised OPLS-DA and S-plots facilitated feature selection. Over 1300 features were putatively identified with mass error <1 ppm. For example, acetaminophen glucuronide was detected at ~78,000 FWHM and 0.09 ppm mass error; its fragment at ~58,000 FWHM and –0.46 ppm. Nicotine-1′-N-oxide and hydroxycotinine were confirmed with confidence scores >80. Exogenous compounds such as carbamazepine and its metabolite, and recreational drugs like cocaine derivatives, differentiated smoker groups. Pyridoxine (vitamin B6) was elevated in non-smokers, suggesting dietary or oxidative-stress–related differences.

Benefits and Practical Applications

• Mass resolution up to 100,000 FWHM and routine sub-ppm accuracy improve confidence in putative identifications.
• Fast acquisition rates maintain resolution for shorter gradients, boosting throughput.
• Wide dynamic range (5 orders of linearity) enables simultaneous profiling of low- and high-abundance compounds in a single injection.
• Robust workflow with tight QC groupings supports large-cohort metabolomics, QA/QC, and biomarker discovery studies.

Future Trends and Opportunities

Integration of comprehensive spectral libraries and AI-driven annotation will further elevate identification confidence. Advances in shorter chromatographic methods and higher-flow technologies will enhance throughput. Expanding cohort sizes and combining metabolomics with other omics approaches promises deeper insights into phenotype-related metabolic pathways.

Conclusion

The Xevo MRT Mass Spectrometer with ACQUITY Premier UPLC and MARS software delivers a streamlined, reliable metabolomics workflow. High mass resolution and sub-ppm accuracy enable clear phenotypic discrimination and confident putative biomarker identification, supporting clinical, environmental, and pharmaceutical research.

References

• Lisa Reid et al. Screening Metabolomics of Human Urine using a Waters High Resolution QTof MS and MARS; Waters Application Note 720008397, 2024.
• Xu W., Chen YL. A Clinical Study of Toxication Caused by Carbamazepine Abuse in Adolescents. Biomed Res Int 2018;3201203.
• Hosseini SH et al. Abuse Potential of Carbamazepine for Euphorigenic Effects. Drug Res (Stuttg) 2015;65(4):223–224.
• King A., Riba I., Palmer M., Marsden-Edwards E. High Resolution MS Metabolic Profiling of Human Urine; Waters Application Note 720008296, 2024.
• Vermaak WJ et al. Vitamin B-6 Nutrition Status and Cigarette Smoking. Am J Clin Nutr 1990;51(6):1058–1061.
• Skeie E. et al. Circulating B-vitamins and Smoking Habits are Associated with Serum PUFA in Patients with Suspected CHD. PLoS One 2015;10(6):e0129049.