Quantitative Determination of Zileuton from Spiked Human Plasma Using Liquid-Liquid Extraction Followed by RP-HPLC and UV Analysis

Abstract

A rapid, cost-eff ective and simple “RP-HPLC method with UV detection” was developed for the determination of zileuton from human plasma. The method involved spiking human plasma and validation. Phenacetin (internal standard) and zileuton samples were prepared using LLE in diethyl ether which was used as solvent for extraction. “HiQsil C18 column (250 mm*4.6 mm* 5 m) was used for separation with tetrahydrofuran: water (45:55, v/v) as mobile phase. The fl ow rate was set at 1-mL/min and UV detection was done at 230 nm. Zileuton showed excellent separation from the internal standard and no interference was observed in plasma samples. A linear calibration curve was obtained in the 500 to 10,000 ng/mL range. The relative error (RE) and relative standard deviation (RSD) were found to be less than 15% for both within the run and between the runs. At lower zileuton concentrations, “weighted least square regression with a weighting factor of 1/X was used to reduce the heteroscedastic eff ect”. Extraction effi ciency of LLE method was confi rmed by the recovery of zileuton from samples. The stability data showed that “zileuton was stable in human plasma for 6 hours at room temperature for 30 days at -20°C after freeze thaw cycles”.