HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR THE DETERMINATION OF IVERMECTIN IN PLASMA

摘要

Abstract. A simple, sensitive, selective and reproducible method based on reversed-phase chromatography was developed for the determination of ivermectin in human plasma. The internal standard (moxidectin) was separated from ivermectin on a Hypersil Gold C18 column (150 x 4.6 mm, 5 um particle size), with retention time of 3.7 and 7.0 minutes, respectively. Fluorescence detection was set at an excitation and emission wavelength of 365 and 475 nm, respectively. The mobile phase consisted of acetonitrile, methanol and distilled water (50:45:5, v/v/v), running through the column at a flow rate of 1.5 ml/minute. The chromatographic analysis was operated at 25degC." Sample preparation (100 ul plasma) was done by a single step protein precipitation with acetonitrite, followed by derivatization with 100 ul of N-methylimidazole solution in acetonitrile (1:1, v/v) and 150 ul of trifluoroacetic anhydrous solution in acetonitrile (1:2, v/v). Calibration curve over the concentration range of 20-8,000 ng/ml plasma was linear with correlation coefficient better than 0.995. The precision of the method based on within-day repeatability and reproducibility (day-to-day variation) was below 15% (coefficient of variation) Good accuracy was observed for both intra-day and inter-day assays, as indicated by the minimal deviation of mean values found with measured samples from that of the theoretical values (below +15%). Limit of quantification was 0.02 ng using 100 pi sample. The mean recovery for ivermectin and the internal standard was greater than 90%. The method was free from interference from endogenous substances and commonly used drugs. The method appears to be robust and has been applied to the investigation of plasma concentration vs time profile of ivermectin in five healthy Thai volunteers following a single oral dose of 200 ug ivermectin/kg body weight.