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

BackgroundA 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).MethodsThe 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™ BEH C18 (2.1 mm × 50 mm, 1.7 μm, 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 m/z 347.2→161.3 for Cl-War.ResultsThe 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%.ConclusionsThe method can be applied to analyze the correlation of free concentration of warfarin and the international normalized ratio (INR).