APPLICATION OF ELISA AND HPLC-MS/MS METHODS IN CLINICAL TRIALS OF VETERINARY DRUGS FOR THE DETERMINATON OF IVERMECTIN CONTENT

The article presents the results of the development and validation of screening method and chromatographic mass spectrometric method for the determining of ivermectin residual amounts in muscles and parenchymal tissues of pigs and calves, as well as in blood plasma, for clinical trials (for withdrawal period establishing and bioequivalence study of drugs based on ivermectin). EuroProxima Ivermectin test kits (Netherlands) were used to determine ivermectin residues in animal tissues. When carrying out the validation at the levels of ½ MRL, which is 15 and 30 μg/kg for muscle and parenchymal tissues, the LOD for the analyte was established at the levels of 2.7 and 4.1 μg/kg, respectively. To establish the percent of analyte’s recovery from the spiked samples, selected blank samples and samples, spiked with ivermectin solution at ½ MRL and MRL levels, were prepared and analyzed according to the technique provided by the test kit manufacturer. The percentage of analyte’s recovery for muscle tissues spiked at the level of 15.0, 30.0, 100.0 μg/kg was: 100.2, 100.4, 99.9, respectively. For liver tissues, spiked at the level of 50.0, 100.0, 500.0 μg/kg, it was 100.0, 99.95, 99.5, respectively. To establish the bioequivalence of veterinary drugs based on ivermectin, we have developed and validated high-performance liquid chromatography with tandem mass spectrometric detection (HPLC-MS/MS) method. The suggested method of sample preparation includes the liquid-liquid extraction of the analyte with ethyl acetate from an acidic medium and concentration by drying. Analysis of the prepared samples was performed by HPLC-MS/MS (analyte’s retention time 2.64 min, mass scanning parameters: precursor ion 892.8 m/z, product ions 307.2 and 569.4 m/z). Various key parameters were used to validate the chromatographic technique: limit of detection, limit of quantification, working and linear concentration ranges, accuracy, and precision. The results of the performed validation are presented in the article in tables and chromatograms, and conclusions about the fitness-for-purpose of the method are also made.