(T1330-09-54) Improvements in Sensitivity for Bioanalysis of Warfarin Using a Prototype Tandem Quadrupole Mass Spectrometer

Purpose: Accurate and precise quantification of pharmaceutical compounds is critical to the drug discovery and development process. UHPLC coupled with tandem quadrupole mass spectrometry is the gold standard in such analysis. As the importance quantifying drug candidates and biomarkers at lower concentrations increases, so does the need for more sensitive instrumentation platforms. One such drug that needs constant monitoring is warfarin. Warfarin is an anticoagulant used in the treatment and prevention of thromboembolism. Careful and continuous monitoring of warfarin is essential to ensure that the drug concentration stays within its narrow therapeutic window.Here, we evaluate a novel prototype tandem quadrupole mass spectrometer for the quantification of warfarin from biological matrices.

Methods: Warfarin stock solutions at 100 ng/mL were used to spike rat and human plasma to create a calibration curve from 0.1 – 100 ng/mL. A separate stock solution (100 ng/mL) was used to generate QC samples at LLOQ, LQC, MQC and HQC. 100 uL of all samples were extracted in triplicate using 1:3 protein precipitation with acetonitrile. Samples were centrifuged and the supernatant was transferred to a LC-MS vial for analysis. Sample analysis was performed on a UHPLC system coupled to a novel prototype tandem quadrupole mass spectrometer operated in negative electrospray ionization mode. Samples were separated on a BEH C18 column (2.1x50 mm), at a flow rate of 600 uL/min using a generic gradient of 5-95%B over 2 minutes.

Results: Multiple product ions were monitored for warfarin during method development and qualification. In neat solution, the observed LLOQ for warfarin was 25 pg/mL and the calibration curve was linear over at least 4.5 orders of magnitude for the most sensitive transition. Warfarin extracted from both rat and human plasma had a LLOQ of 100 pg/mL for the most sensitive transition. The calibration lines were linear over the tested range of 0.1 – 100 ng/mL with accuracy and precision for technical triplicates at every point < 10% and a r² value of >0.99. QC samples at all 4 concentrations showed excellent accuracy and precision with single digit %CV values for all technical replicates at every point. The new high performance tandem quadrupole mass spectrometer with a compact design and enhanced negative ion detection may allow for improving sensitivity in bioanalysis, especially in negative ion mode. The LLOQ achieved for warfarin in buffer solution as well as protein precipitated plasma shows a 2.5 – 5 fold improvement in sensitivity over previously published methods.

Conclusion: The new prototype tandem quadrupole mass spectrometer significantly improves the observed LLOQ for warfarin from biological matrix.

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