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 BEH C(18) (2.1 mm x 50 mm, 1.7 microm, 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 > or = 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).
Huang
Chenrong
C
Clinical Pharmacology Research Laboratory, First Affiliated Hospital of Soochow University, Suzhou 215006, China.
Yang
Jian
J
Du
Yonghong
Y
Miao
Liyan
L
D001798:Blood Proteins / Q000378:metabolism*; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D019934:International Normalized Ratio; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014859:Warfarin / Q000097:blood*
D001798:Blood Proteins; D014859:Warfarin