Three-phase hollow fiber liquid phase microextraction of warfarin from human plasma and its determination by high-performance liquid chromatography

A simple, inexpensive and efficient sample preparation technique, three-phase hollow fiber liquid phase microextraction (HF-LPME), followed by high-performance liquid chromatography–ultra violet detection (HPLC–UV) was used for the analysis of warfarin in human plasma. Warfarin was extracted from 11.0 ml of aqueous solution with pH = 2.3 (donor phase) into 1-octanol immobilized in the wall pores of a porous hollow fiber and then extracted into the acceptor phase with pH = 11.0 located in the lumen of the hollow fiber. After the extraction, the acceptor phase was directly injected into the HPLC system for quantification. Different factors affecting the HF-LPME including nature of organic extraction solvent, pH of donor and acceptor phases, stirring rate, extraction time and salt addition to the sample solution (donor phase) were investigated and optimized. Under the optimized conditions (1-octanol as organic solvent, pHdonor = 2.3, pHacceptor = 11.0, stirring speed of 1000 rpm, extraction time of 30 min, without addition of salt), limit of detection (LOD) of 5 ng ml−1 and enrichment factor of 225 were obtained. The calibration curve was linear within the range of 15–550 ng ml−1 with the square of correlation coefficient of 0.9984. The values of intra-day relative standard deviation (RSD) and inter-day RSD were 4.2% and 11.1%, respectively. The method was applied successfully for the analysis of warfarin in plasma sample from a patient under treatment with this drug and excellent sample clean-up was observed.