DEVELOPMENT OF METHODS FOR QUANTITATIVE DETERMINATION OF MEBENDAZOLE AND ITS METABOLITES IN BIOLOGICAL TISSUES USING HPLC/ DAD

Abstract

This manuscript presents the results of developed method is intended for clinical and pharmaceutical studies of veterinary drugs based on the active substances mebendazole and its main metabolites: mebendazole amin and mebendazole hydroxide in sheep muscles and liver. Tissue samples were made alkaline with sodium carbonate, extracted twice with acetonitrile and degreased with hexane. The extracts are further purified using a series of liquid-liquid extraction and solid phase extraction. After concentration and drying, the dry residue was recovered in the mobile phase. Separation was performed on an inverted phase Kinetex EVO С18 column using acetonitrile and phosphate buffer as the mobile phase. The gradient mode of eluents was used during 10 min at a flow rate of 1,5 ml/min. The peak retention time of a mebendazole is 3,4 min, mebendazole hydroxide is 4,1 min, and the retention time of mebendazole amin peak is 6,1 min. The specificity of the analytical method was checked by comparing the chromatographic separation of a sample of muscle tissue and liver enriched with a standard solution of a mixture of mebendazole and its main metabolites at the level of MDR and a sample of muscle tissue and liver placebo. The procedure of sample preparation of fortified tissues to construct calibration graphs is described in the manuscript. The validation parameters of the method “recovery” and “coefficient of variation” were considered in accordance with the criteria of Council Directive 2002/657/EC. The mean recovery from fortified muscle tissue in the range of 40.0-60.0 μg/kg mebendazole and its metabolites was 98 %. The average extraction of the studied analytes from the loaded liver in the range of 200.0 - 600.0 μg/kg was 100 %. The average coefficient of variation for each compound was ≤ 10 %.
 The method is linear in the concentration range of 5 – 100.0 μg/kg of each analyte in muscles and 50,0 – 800,0 μg/kg in liver. The results obtained in the study of the linearity of this technique were used to estimate the correctness and convergence. The accuracy of the measurements was evaluated by examining the known amounts of analytes added to the control muscle tissue. Recovery data are acceptable because they are within ± 10% of the target value. The method has sufficient convergence (accuracy). The evaluation of the intermediate accuracy of mebendazole and its main metabolites was assessed on three different days of analysis. The average CV for each compound was <10 %. Selectivity and high sensitivity are the main advantages of the developed technique. The developed HPLC/DMD method can be used to study the deficiency of mebendazole and its metabolites.