DEVELOPMENT AND VALIDATION OF HPLC-METHOD FOR QUANTITATIVE DETERMINATION OF ETHYLBUTYLACETAMINOPROPIONATE IN SPRAYS

Abstract

Ethylbutylacetaminopropionate is a selective activator of metabotropic ionic G-receptors of the nervous system of invertebrate animals, which leads to overexcitation of insects and their repelling from the source of this substance. Ethylbutylacetaminopropionate is used in insect acaricidal sprays as a repellent against mosquitoes, lice, fleas and ticks for pets. The aim of the work was to develop a method of identification and quantitative determination of ethylbutylacetaminopropionate in a spray for external use. The method was developed and validated by indicators of selectivity, linearity and suitability parameters of the chromatographic system. A spray repellent was used as a sample-object for the development of the technique. The standard sample and the test sample were dissolved in a mixture of acetonitrile and water in a volume ratio of 1:1, to a concentration of ethylbutylacetaminopropionate of 50 μg/ml. The samples were separated on a Dionex Ultimate 3000 chromatograph equipped with an Acclaim C18 chromatographic column 250×3.0, 3 μm. The mobile phase was a mixture of acetonitrile and water in a volume ratio of 60:40. Ethyl butylacetaminopropionate was detected at an absorption wavelength of 210 nm. Under the above conditions, it was possible to completely separate ethylbutylacetaminopropionate (retention time of the chromatographic peak – 4.9 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 ethylbutylacetaminopropionate, the efficiency of the chromatographic system was 15,100 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 ethyl butylacetaminopropionate and other components of the drug was 25.0. The symmetry coefficient of the ethyl butylacetaminopropionate peak was 1.31. The calibration curve was linear in the recommended DFU 2.0 range (80–120% of the nominal concentration of active substance). The coefficient of linearity (R2) for ethyl butylacetaminopropionate was 0.9991.