Liquid chromatographic assay of ivermectin in human plasma for application to clinical pharmacokinetic studies

Abstract

There is a need for an accurate, sensitive and selective high-performance liquid chromatography (HPLC) method for the quantitation of ivermectin in human plasma that separates the parent drug from metabolites. Ivermectin and the internal standard, moxidectin, were extracted from 0.2 ml of human plasma using Oasis HLB solid phase extraction cartridges. After extraction, fluorescent derivatives of ivermectin and moxidectin were made by reaction with trifluoroacetic anhydride and N-methylimidazole. Separation was achieved on a Alltech Ultrasphere C18 5mu column with a mobile phase composed of tetrahydrofuran-acetonitrile-water (40:38:22 v/v/v). Detection is by fluorescence, with an excitation of 365 nm and emission of 475 nm. The retention times of ivermectin and internal standard, moxidectin are approximately 24.5 and 12.5 min, respectively. The assay is linear over the concentration range of 0.2-200 ng/ml of ivermectin in human plasma (r = 0.9992, weighted by 1/concentration). Recoveries of ivermectin are greater than 80% at all concentrations. The analysis of quality control samples for ivermectin 0.2, 25, and 200 ng/ml demonstrated excellent precision with coefficient of variation of 6.1, 3.6 and 2.3%, respectively (n = 6). The method is accurate with all intra-day (n = 6) and interday (n = 12) mean concentration within 10% of nominal values at all quality control sample concentrations. Storage stability for 30 days at -80 degrees C and after three freeze-thaw cycles are within acceptable limits. The method separates ivermectin from multiple less and more polar unidentified metabolites. This method is robust and suitable for clinical pharmacokinetic studies. The analytical procedure has been applied to a pharmacokinetic study of ivermectin in healthy volunteers and to the analysis of plasma specimens from patients with disseminated strongyloidiasis.