Abstract
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™ BEH C 18 (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. 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).
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