DEVELOPMENT AND VALIDATION OF HPLC-METHOD FOR SIMULTANEOUS QUANTITATIVE DETERMINATION OF FIPRONIL AND S-METHOPRENE IN DROPS AGAINST FLEA AND TICKS

Abstract

Fipronil is a selective blocker of metabotropic ion receptors of the nervous system of invertebrate animals. And S-methoprene is an analogue of the juvenile hormone, which leads to the arrest of the development and reproduction of insects. These active substances are used in insect-acaricidal drops to destroy and prevent the reproduction of lice, fleas and ticks in pets.
 Aim of the work was to develop a method for the identification and simultaneous quantification of fipronil and S-methoprene in drops for external use. The method was developed and validated by indicators of selectivity, linearity and suitability parameters of the chromatographic system. Insecto-acaricidal drops for external use were used as a sample-object for development. The standard sample and the test sample were dissolved in acetonitrile to fipronil concentration – 100 μg/ml and S-methoprene – 90 μg/ml. The samples were separated on a Dionex Ultimate 3000 chromatograph equipped with an Acclaim C18 chromatographic column 150×4.6, 5 μm. The mobile phase was a mixture of acetonitrile and water in a volume ratio of 90:10. Fipronil and S-methoprene were detected at an absorption wavelength of 270 nm.
 Under the above-mentioned conditions, it was possible to completely separate fipronil and S-methoprene (retention time of the chromatographic peaks - 2.3 min and 7.6 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 HPLC methods. For fipronil and S-methoprene peaks the efficiency of the chromatographic system was 8300–13000 theor. plates The relative standard deviation (RSD) for the peak areas of the active substances was ± 0.50%, and the peak separation ratio (RS) of fipronil and S-methoprene and other drug components was 27.6. The symmetry coefficient of the fipronil peak was 1.4, and that of S-methoprene was 1.2. 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 fipronil was 0.9990, and for S-methoprene it was 0.9989.