Measurement of free concentrations of highly protein-bound warfarin in plasma by ultra performance liquid chromatography-tandem mass spectrometry and its correlation with the international normalized ratio

Background: A novel method has been developed for the determination of free warfarin in human plasma by ultra performance liquid chromatography combined with tandem mass spectrometry (UPLC-MS/MS). Methods: The plasma filtrate was prepared by a high-speed ultrafiltration (UF), and was then extracted by a liquid/liquid extraction (LLE) with ethyl acetate. The chromatographic separation was performed on an Acquity UPLC (TM) BEH C(18) (2.1 mm x 50 mm, 1.7 mu m, Waters) with a mobile phase consisting of 0.1% formic acid/acetonitrile (50:50, v/v). The analyses were carried out by multiple reaction monitoring (MRM) using the precursor-to-product combinations of m/z 307.3 -> 161.2 for warfarin and m1z 347.2 -> 161.3 for CI-War. Results: The lower limit of quantification (LLOQ) was 0.25 ng/ml and the assay exhibited a linear range of 0.25-16 ng/ml and gave a correlation coefficient (r) of >= 0.9999. Quality control samples (0.5,2,8 ng/ml) in 5 replicates from 3 different runs of analysis demonstrated intra-assay CVs of 3.0-10.2%, inter-assay CVs of 4.2-12.0%, and an overall accuracy of 85-115%. Conclusions: The method can be applied to analyze the correlation of free concentration of warfarin and the international normalized ratio (INR). (c) 2008 Published by Elsevier B.V.