Validation of R/S-Warfarin Analysis Method in Human Blood Plasma Using HPLC with Fluorescence Detection and its...

Warfarin is extensively used for venous thromboembolism and other coagulopathies. In clinical settings, warfarin is administered as a mixture of S-warfarin and R-warfarin, and both enantiomers are metabolized by multiple cytochrome P450 enzymes into many hydroxylation metabolites. Validation of analysis method and estimation of warfarin plasma levels are important, especially in narrow-index drugs such as warfarin.

This study aimed to obtain a validated method for analyzing warfarin in patient plasma according to the European Medicines Agency (EMA) guidelines.

A total of 77 patients were enrolled in this study. Five millimeters of venous blood was collected using sodium ethylenediaminetetraacetic acid (EDTA) tubes for pharmacokinetic analysis. Samples were prepared by the protein precipitation technique using acetonitrile. The optimum conditions for the analysis of warfarin in human plasma were tested using fluorescence detector (FLD) high-performance liquid chromatography (HPLC) with Chiralcel OD-RH column (4.6 × 150 mm i.d., 5 μm), Chiralcel OD-RH guard column (4.0 × 10 mm, 5 μm), and a column temperature of 45°C. The mobile phase used was acetonitrile: phosphate buffer pH 2 (40:60), with an isocratic flow rate of 1 ml/min and an injection volume of 20 μl. Excitation and emission wavelengths were 310 and 350 nm (warfarin) and 300 and 400 nm (griseofulvin). The retention time of griseofulvin was 6-7.5 minutes; R-warfarin was 10-11.5 minutes; and S-warfarin was 14-16 minutes.

The result of this validation obtained the optimum conditions. This method yielded the limit of detection (LOD) values of 0.0674 ppm (R-warfarin) and 0.0897 ppm (S-warfarin). The limit of quantification (LOQ) values were 0.225 ppm (R-warfarin) and 0.298 ppm (S-warfarin). Linearity existed at concentrations of 0.2-3 ppm with the line equation y = 0.0705x + 0.0704 with R² = 0.978 for R-warfarin and y = 0.0513x + 0.0297 with R² = 0.9924 for S-warfarin. At least 75% of the seven concentrations met the reverse concentration requirements, which were below ± 15%. This method met the requirements of accuracy and precision within and between runs, selectivity, and carryover where the %RSD and %diff values were below ± 15%. The mean plasma concentrations of R-warfarin and S-warfarin were found to be 0.76 ± 1.87 (SD) μg/ml and 0.59 ± 0.81 (SD) μg/ml, respectively. The mean standard dose group plasma concentration from the analysis of 77 samples was 0.68 ± 0.61 μg/mL for R-warfarin and 0.52 ± 0.42 μg/mL for S-warfarin.

Based on these results, this analytical method can be declared valid and can be used for sample measurement in warfarin pharmacokinetic studies.