An automated method for the determination of subnanogram concentrations of eprinomectin in bovine plasma

Eprinomectin is a potent anthelmintic compound that kills certain parasitic nematodes and arthropods of cattle. A sensitive and automated bioanalytical assay was developed for quantitation of eprinomectin in bovine plasma in support of clinical development of eprinomectin for use in all classes of cattle. This assay determined the concentration of eprinomectin in plasma by reversed-phase high performance liquid chromatography (HPLC) with fluorometric detection. Plasma sample preparation included liquid extraction performed by the Packard MultiPROBE robotics workstation, followed by solid phase extraction performed by the Gilson ASPEC XL automated workstation. The HPLC assay included automated pre-column derivatization with a fluorogenic reagent system which included trifluoroacetic anhydride and N-methylimidazole as the catalyst. This reversed-phase chromatographic analysis was based on the fluorescence detection of derivatized eprinomectin and an internal standard, L-648 548, which was similarly derivatized by the fluorogenic reagents. The assay was automated and validated for two concentration ranges of 0.05-10 and 0.5-200 ng ml-1. The lower limit of quantitation of eprinomectin in plasma was 0.05 ng ml-1. The %RSD of the assay was 10% or better at all concentrations. This automated analysis of eprinomectin was used for high-throughput clinical assays with acceptable accuracy and precision.