DEVELOPMENT OF METHODS FOR QUANTITATIVE DETERMINATION OF ALBENDAZOLE AND ITS METABOLITES IN BIOLOGICAL TISSUES USING HPLC / FLD
This manuscript presents the results of developed method is intended for clinical and pharmaceutical studies of veterinary drugs based on the active substances albendazole ([5-(propylthio)-1H-benzimidazol-2yl]carbamic acid methyl ester) and its main metabolites, albendazole sulfoxide, albendazole sulfone, and albendazole-2- aminosulfone in sheep muscles. 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 Acclaim 120 C18 column using acetonitrile and phosphate buffer as the mobile phase. The gradient mode of eluents was used during 12 min at a flow rate of 1,8 ml/min. The peak retention time of albendazole 2-aminosulfoam is 3,0 min, albendazole sulfoxide is 3,9 min, albendazole sulfone is 4,8 min, and the retention time of albendazole peak is 6,6 min. The specificity of the analytical method was checked by comparing the chromatographic separation of a sample of muscle tissue enriched with a standard solution of a mixture of albendazole and its metabolites at the level of MDR (100 μg/kg) and a sample of muscle tissue placebo. 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 procedure of sample preparation of fortified tissues to construct calibration graphs is described in the manuscript. The mean recovery from fortified muscle tissue in the range of 50-150 μg/kg albendazole, albendazole sulfoxide, albendazole sulfone and albendazole 2-aminosulfon was 100.2; 100.9; 100.7 and 100.2%, respectively. The average coefficient of variation for each compound was ≤ 10%.
The method is linear in the concentration range of 25.0 - 200.0 μg/kg of each analyte. 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 albendazole and its metabolites was assessed on three different days of analysis. The limit of detection for albendasole is 0.4 μg/kg. The average CV for each compound was <10%.
The procedure was confirmed and then applied to determination albendazole and its metabolites in the sheep muscle tissue obtained after feeding animals with the veterinary drug albendazole. The HPLC/FLD method can be used for withdrawal time albendazole and its metabolites.
COMMISSION DECISION of 14 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results (notified under document number C(2002) 3044) text with EEA relevance 2002/657/EC // Official Journal of the European Union. – 17.08.2002. – L221. 8.
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