Development of a high-performance liquid chromatography method for warfarin detection in human plasma

Aim: Most of the high-performance liquid chromatography (HPLC) methods that have been developed to measure warfarin levels use the preextraction spiked internal standard method. We developed a new liquid-liquid extraction (LLE) and reversed-phase HPLC method that uses a postextraction spiked internal standard for the determination of plasma warfarin. Materials and methods: The effect of varying the 1) mobile phase pH, 2) type and composition of organic solvents, 3) type and concentration of buffer solutions, 4) column temperatures, 5) flow rates, 6) organic modifier, and 7) ultraviolet wavelengths were tested. Results: Optimum separation was achieved by using 30:70 (v/v) acetonitrile and potassium dihydrogen orthophosphate (0.01 M) at pH 6.5 without the addition of an organic modifier at 300 nm. The column temperature was fixed at 30 degrees C and the flow rate at 1.0 mL/min. Phenylbutazone was the most suitable internal standard. For LLE, the optimum plasma pH was 4.5, using 2 volumes of 2.5 mL of diethyl ether. The average retention times of warfarin and phenylbutazone were 7 and 11 min, respectively. Conclusion: The postextraction spiked internal standard method saved a significant amount of development time and gave an excellent recovery of nearly 90%. This method was successfully tested using spiked human plasma. On the whole, the developed method is simple, economical, and suitable for routine application.