Development and validation of an ultra-high-performance liquid chromatography-tandem mass spectrometry method to determine the bioavailability of warfarin and its major metabolite 7-hydroxy warfarin in rats dosed with oral formulations containing different polymorphic forms.

A simple, sensitive and rapid ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed and validated for the quantification of warfarin and 7-hydroxy warfarin in Sprague Dawley (SD) rats. Animals were administered a single dose of warfarin sodium formulations (crystalline and amorphous) at 12 mg/kg via oral gavage and blood was drawn over a 96-h time course. Sample process recoveries, matrix effect and analyte stability were determined. The linearity for warfarin and 7-hydroxy warfarin was from 5 to 2000 ng/mL in blank SD rat plasma. Correlation coefficients (r2 ) for standard calibration curves were >.98 and analytes quantified within ±15% of target at all calibrator concentrations. The average percent accuracy and precision for intra- and inter-day were 93.7%-113.8% and ≤12.1%, respectively, for warfarin and 7-hydroxy warfarin, across the quality control standards (5, 10, 500, 1800 and 2000 ng/mL). Acceptable analytical recovery (>55%) was achieved with process efficiencies >41.5% and matrix effects <139.9% over the analytical range. Both analytes were stable in stock solution, autosampler, benchtop and three cycles of freeze-thaw with percent accuracy ≥90.2% and precision (percent relative standard deviation) ≤14%. The validated method was successfully applied to a pre-clinical bioavailability study of crystalline and amorphous warfarin sodium formulations in SD rats.