Quantification of Warfarin and Furosemide in Human and Rat Plasma for Discovery Bioanalysis Using ACQUITY™ Premier UPLC™ System and Xevo™ TQ Absolute Mass Spectrometer

Significance of the Topic

The accurate quantification of pharmaceutical compounds in biological matrices is essential for drug discovery and development. Reliable measurement of warfarin and furosemide concentrations in plasma informs pharmacokinetic and safety assessments, supporting decisions on dosing, efficacy, and toxicity.

Objectives and Study Overview

This study presents a streamlined workflow for the extraction and quantification of two model drugs, warfarin and furosemide, from human and rat plasma. The method leverages a high-performance UPLC system coupled to a tandem quadrupole mass spectrometer to achieve low quantitation limits and robust performance across multiple sample types.

Methodology

• Sample Preparation: 100 µL plasma aliquots spiked with analyte stock solutions, protein precipitation with acetonitrile in a 1:3 ratio, centrifugation, and transfer of supernatant to analysis vials
• Calibration and QC Design: Warfarin calibration 0.025–100 ng/mL and furosemide 0.1–100 ng/mL; QC levels at LLOQ, low, medium, and high concentrations
• LC Conditions: ACQUITY Premier UPLC, MaxPeak UPLC HSS T3 column, 2.1 × 50 mm, 60 °C; mobile phase A 0.01% formic acid in water, B 0.01% formic acid in acetonitrile; gradient from 5 to 95% B over 1.5 minutes, flow rate 600 µL/min, injection volume 1 µL, sample tray 5 °C
• MS Conditions: Xevo TQ Absolute operated in negative ion electrospray mode, capillary voltage 0.5 kV, optimized multiple reaction monitoring transitions for each analyte

Instrumentation Used

• ACQUITY Premier UPLC System with MaxPeak High Performance Surfaces
• Xevo TQ Absolute Tandem Quadrupole Mass Spectrometer
• MaxPeak UPLC HSS T3 2.1 × 50 mm Column
• Waters Total Recovery LCMS Vials

Key Results and Discussion

• Lower limit of quantification of 25 pg/mL for warfarin and 100 pg/mL for furosemide in both human and rat plasma, based on signal-to-noise ratio ≥5
• Excellent linearity across the entire calibration range (R² > 0.99)
• Intra- and inter-day precision and accuracy with %CV below 12% for all QC and calibration levels
• MaxPeak HPS technology minimized metal-induced adsorption, improving signal stability for analytes with metal-chelating properties

Benefits and Practical Applications

• High sensitivity suitable for trace-level quantification in discovery bioanalysis
• Generic workflow adaptable to diverse small molecules without extensive reoptimization
• Reduced sample preparation complexity and faster turnaround times
• Enhanced laboratory throughput and robustness for routine pharmacokinetic studies

Future Trends and Potential Applications

Advances in surface chemistry and source design are expected to further enhance analyte recovery and instrument uptime. Expanding generic UPLC-MS workflows to multiplex panels, automating sample preparation, and integrating high-throughput screening platforms will support increasingly complex bioanalytical demands.

Conclusion

The described UPLC-MS/MS method delivers reliable, sensitive, and precise quantification of warfarin and furosemide in human and rat plasma. ACQUITY Premier UPLC System and Xevo TQ Absolute Mass Spectrometer offer a robust platform for discovery bioanalysis, combining simplified workflows with class-leading performance.

References

1. Bateman KP, Cohen L, Emary B, Pucci V Standardized workflows for increasing efficiency and productivity in discovery stage bioanalysis Bioanalysis 2013 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 5(1):91–115