IMPROVEMENTS IN SENSITIVITY FOR BIOANALYSIS OF WARFARIN USING ACQUITY™ PREMIER UPLC SYSTEM AND XEVO™ TQ ABSOLUTE TANDEM QUADRUPOLE MASS SPECTROMETER

Importance of the Topic

Bioanalysis by LC-MS/MS provides essential quantitative information on drug and metabolite levels, supporting ADMET profiling and accelerating decision-making in drug discovery and development.

Objectives and Study Overview

This application note evaluates sensitivity gains in quantifying warfarin and furosemide in human and rat plasma using the ACQUITY Premier UPLC system coupled with the Xevo TQ Absolute mass spectrometer.

Methodology and Sample Preparation

Warfarin and furosemide stock solutions (100 ng/mL) were spiked into plasma to create calibration curves (0.025–100 ng/mL) and QC levels. Samples (100 µL) underwent protein precipitation with three volumes of acetonitrile, followed by centrifugation and transfer of supernatant for analysis.

Instrument Used

• Chromatography: ACQUITY Premier UPLC system with MaxPeak High Performance Surfaces technology
• Column: MaxPeak UPLC HSS T3 (2.1×50 mm) at 60 °C
• Mobile phases: 0.01% formic acid in water (A) and acetonitrile (B); gradient from 5% to 95% B over 1.5 min at 600 µL/min
• Injection volume: 1 µL; sample temperature: 5 °C
• Mass spectrometry: Xevo TQ Absolute tandem quadrupole, ESI negative mode; capillary voltage 0.5 kV

Main Results and Discussion

Lower limits of quantification achieved were 25 pg/mL for warfarin and 100 pg/mL for furosemide in both matrices. Calibration curves were linear across the range with coefficients of variation below 12% at all levels. Representative chromatograms confirmed peak shape and reproducibility.

Benefits and Practical Applications

• Reduced non-specific adsorption via MaxPeak HPS reduces method development complexity
• Avoidance of derivatization enables higher throughput
• Compact design and removable source shield of the Xevo TQ Absolute minimize contamination and downtime
• Robust performance across diverse analytes in discovery bioanalysis settings

Future Trends and Opportunities

• Extension to other challenging analytes with metal-chelating properties
• Integration of automated sample preparation and high-throughput workflows
• Advances in MS source technology to further lower detection limits
• Application of data-driven method optimization and AI for rapid assay development

Conclusion

The combination of the ACQUITY Premier UPLC system and Xevo TQ Absolute mass spectrometer delivers enhanced sensitivity, robustness, and throughput for bioanalysis of warfarin and furosemide, meeting stringent discovery-stage requirements.

References

1. Bateman KP, Cohen L, Emary B, Pucci V. Standardized workflows for increasing efficiency and productivity in discovery stage bioanalysis. Bioanalysis. 2013 Jul;5(14):1783–94.
2. Meng M, Wang L, Voelker T, Reuschel S, Van Horne K, Bennett P. A systematic approach for developing a robust LC-MS/MS method for bioanalysis. Bioanalysis. 2013 Jan;5(1):91–115.