Determination of Mass Resolution, Sensitivity, and Linear Dynamic Range of Small Molecules in Human Plasma Using ACQUITY UPLC I-Class and Xevo G2-S QTof

Importance of the Topic

High resolution mass spectrometry (HRMS) enables simultaneous acquisition of quantitative and qualitative information in a single analysis. This approach conserves sample volume, reduces workflow complexity, and speeds decision making in drug metabolism and pharmacokinetics (DMPK) during pharmaceutical research and development.

Objectives and Study Overview

The primary goal was to evaluate the mass resolution, sensitivity, and linear dynamic range of the ACQUITY UPLC I-Class System coupled to the Xevo G2-S QTof for seven pharmaceutical compounds spiked in human plasma. The compounds studied span a molecular weight range from 260 to 530 Da.

Methodology and Instrumentation

Samples were prepared by spiking human plasma with a mixture of seven drugs. Chromatographic separation was performed on an ACQUITY UPLC BEH C18 column (1.7 µm, 2.1×100 mm) using a water/acetonitrile gradient (0.1% formic acid) at 0.6 mL/min. Mass spectrometric detection employed the Xevo G2-S QTof in positive electrospray ionization (ESI+) resolution mode, with data acquisition and processing via UNIFI Software.

• Compounds evaluated: propranolol, alprazolam, warfarin, clopidogrel, buspirone, verapamil, ketoconazole
• Calibration range: 3 to 100,000 pg/mL depending on analyte
• Injection volume: 10 µL; cone voltage: 40 V; capillary voltage: 1.0 kV

Main Results and Discussion

Peak widths at half height ranged from 1.3 to 2.1 seconds under UPLC conditions. Mass resolution measured by full width at half maximum (FWHM) varied between 29,000 and 40,000 across the analytes at a 5 Hz scan rate. All compounds demonstrated a minimum linear dynamic range of 3.0 log units, with buspirone achieving the widest range (4.5 log units). Limits of detection (LODs) spanned from 3.05 pg/mL (buspirone, propranolol) to 24.4 pg/mL (ketoconazole, warfarin). Comparison with a tandem quadrupole platform (Xevo TQ-S) showed the QTof’s sensitivity was only two- to seven-fold lower, yet still adequate for routine quantitation.

Benefits and Practical Applications

• Combines quantitation and structural elucidation in one platform
• Reduces sample consumption by avoiding duplicate analyses on QqQ and HRMS instruments
• Delivers high throughput and reproducibility for routine DMPK assays

Future Trends and Opportunities

• Integration of data independent acquisition strategies for deeper metabolite profiling
• Advancements in instrument sensitivity and resolution for ultra-trace analysis
• Implementation of machine learning for automated data processing and interpretation
• Expansion into clinical diagnostics and personalized medicine applications

Conclusion

The ACQUITY UPLC I-Class coupled to the Xevo G2-S QTof, managed by UNIFI Software, offers robust performance for small molecule quantitation in human plasma. It achieves high mass resolution, sub-10 pg/mL sensitivity, and broad dynamic ranges, making it a fit-for-purpose solution for modern drug discovery and development workflows.