Determination of Warfarin in Human Plasma by High Performance Liquid Chromatography and Photodiode Array Detector

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Abstract
A simple liquid chromatographic method for the determination of warfarin in human plasma is described. The method involves precipitation of plasma proteins with acetonitril. No further processing of samples is required. The supernatant was analyzed on a short (10 cm) 3-μm reversed-phase column eluted with 23% acetonitril in 100 mM ammonium formate, pH 3.5. The method takes advantage of the fact that warfarin dissolved in this mobile phase has a characteristic absorbance spectrum with distinct peaks at 271, 281, and 305 nm. Using an online photodiode array detector, the UV spectrum could be recorded during analysis without interrupting the flow of the mobile phase. This spectral information improves identification possibilities and evaluation of the purity of the chromatographic peaks. Warfarin was separated from other UV-absorbing compounds in plasma in < 3 min, and there was no interference from numerous drugs given to patients. The standard curve (305 nm) was linear in the concentration range observed after oral intake of the single dose of warfarin and for a time corresponding to several half-lives. The detection limit of the method was about 0.1 μg/ml when the absorbance was recorded at 305 nm. At this wavelength, the solvent front was small relative to that observed at lower wavelengths. The precision of the method, given as coefficient of variation, was 6.4%. The method was used for the determination of plasma half-lives of R- and S-warfarin in humans.