DEVELOPMENT AND VALIDATION OF HPLC-METHOD FOR SIMULTANEOUS QUANTITATIVE DETERMINATION OF IMIDACLOPRID AND IVERMECTIN IN DROPS AGAINST FLEAS AND TICKS

Abstract

Imidacloprid and ivermectin are selective blockers of metabotropic ion receptors of the nervous system of invertebrates, leading to paralysis and death of ecto- and endoparasites. These active substances are used in insect-acaricidal drops to kill lice, fleas and ticks in cattle, domestic animals and pets. The aim of the work was to develop a method for the identification and simultaneous quantitative determination of imidacloprid and ivermectin in drops for external use. The method was developed and validated by selectivity, linearity and suitability parameters of the chromatographic system. Drops for external use were used as a sample-object for development. The standard sample and the test sample were dissolved in acetonitrile to a concentration of imidacloprid 100 μg/ml and ivermectin 10 µg/ml. The samples were separated on a Dionex Ultimate 3000 chromatography system equipped with a chromatographic column Acclaim C18 150×4.6, 5 μm. The mobile phase was a mixture of acetonitrile and water in a volume ratio of 90:10. Ivermectin and imidacloprid were detected at an absorption wavelength of 242 nm. Under the above-mentioned conditions, it was possible to completely separate imidacloprid and ivermectin (retention time of chromatographic peaks – 2.0 min and 10.0 min) and other components of the studied drug. At the same time, the suitability parameters of the chromatographic system did not exceed the limits specified in the recommendations of the USA Food and Drug Association. For the peaks of imidacloprid and ivermectin, the efficiency of the chromatographic system was 8000–10000 theoretical plates. The relative standard deviation (RSD) for the peak areas of the active substances was ± 0.31%, and the peak separation ratio (RS) of imidacloprid from ivermectin and other drug components was 35.9. The symmetry coefficient of the imidacloprid peak was 1.5, and that of ivermectin was 1.1. The calibration curves were linear in the recommended DFU 2.0 range (80–120% of the nominal concentration of the corresponding active substance). The coefficient of linearity (R2) for imidacloprid was 0.9991, and for ivermectin it was 0.9993.