Stability of the ACQUITY UPLC System with Xevo G2-XS QTof Mass Spectrometer for Metabolic Phenotyping of Human Urine in Large Cohort Studies

Significance of the Topic

Large-scale metabolic phenotyping of human biofluids demands highly reproducible analytical platforms to detect subtle biological variations across diverse populations. Minimizing retention time drift, mass accuracy shifts and peak area fluctuations is critical for reliable statistical analysis and meaningful data interpretation in cohort studies.

Study Objectives and Overview

This work evaluated the reproducibility and suitability of the Waters ACQUITY UPLC System coupled with the Xevo G2-XS QTof Mass Spectrometer for metabolic profiling of human urine in a large cohort setting. Over 1,300 injections were performed on pooled urine samples spiked with stable isotope–labeled standards and interspersed quality controls (QCs) to assess precision over extended runs.

Methodology and Used Instrumentation

Reversed-phase separation was carried out on an ACQUITY UPLC HSS T3 Column (2.1 × 150 mm, 1.8 μm) using a ten-minute gradient from 1% to 55% acetonitrile in 0.1% formic acid at 600 μL/min, followed by a high-organic wash and re-equilibration. The Xevo G2-XS QTof operated in positive-ion electrospray sensitivity mode with continuous lock-mass correction using leucine enkephalin. Samples and QCs were aliquoted into 96-well plates permitting triplicate injections per sample and regular QC injections every seventh run.

Main Results and Discussion

Retention time reproducibility for seven representative compounds across six 96-well plates (over 1,300 injections) yielded relative standard deviations between 0.15% and 0.98%, demonstrating robust chromatographic stability. Mass spectrometric peak area variation for the same analytes in triplicate QC injections showed coefficients of variation ranging from 4.2% to 15.5%, with an average of 8.3%. These performance metrics support confident compound identification and quantification for downstream multivariate analyses such as PCA and PLS-DA.

Benefits and Practical Applications

• High retention time precision ensures minimal drift over large sample sets.
• Consistent peak area response enables detection of subtle metabolic differences.
• Automated 96-well format enhances throughput and reproducibility.
• Suitable for epidemiological and clinical cohort studies requiring stringent QC.

Future Trends and Opportunities

Advances in UPLC-QTof technology will further increase throughput and sensitivity, enabling deeper coverage of the metabolome. Integration with automated sample preparation, real-time QC monitoring and AI-driven data analysis will enhance robustness and speed of large-scale phenotyping. Expanding to multi-omics workflows will provide comprehensive insights into disease mechanisms and biomarker discovery.

Conclusion

The demonstrated precision of retention time (<1% RSD) and peak area (mean 8.3% CV) confirms that the ACQUITY UPLC System with Xevo G2-XS QTof MS is ideally suited for high-throughput metabolic phenotyping of human urine in large cohort studies, providing the analytical consistency required for reliable biomarker discovery.