Simultaneous determination of warfarin and 7-hydroxywarfarin in rat plasma by HPLC-FLD

Sa？etak In this study, high-performance liquid chromatography with fluorescence detection (HPLC-FLD) has been used for the first time, for direct determination of warfarin and its major metabolite, 7-hydroxywarfarin, in rat plasma. The simple and sensitive method was developed using Fortis？ reversed-phase diphenyl column (150 × 4.6 mm, 3 μm) and a mobile phase composed of phosphate buffer (25 mmol L–1)-methanol-acetonitrile (70:20:10, V/V/V), adjusted to pH 7.4, at a flow rate of 0.8 mL min–1. The diphenyl chemistry of the stationary phase provided a unique selectivity for separating the structurally related aromatic analytes, warfarin and 7-hydroxywarfarin, allowing their successful quantification in the complex plasma matrix. The method was linear over the range 0.01–25 μg mL–1, for warfarin and 7-hydroxywarfarin, and was found to be accurate, precise and selective in accordance with US FDA guidance for bioanalytical method validation. The method was sensitive enough to quantify 0.01 μg mL–1 of warfarin and 7-hydroxywarfarin (LLOQ) using only 100 μL of plasma. The applicability of this method was demonstrated by analyzing samples obtained from rats after oral administration of a single warfarin dose, and studying warfarin and 7-hydroxywarfarin pharmacokinetics