VALIDATION OF METHOD DETERMINING OF NICARBAZIN CONTENT IN FEEDS

The manuscript presents the validation of the developed high-performance liquid chromatography method for the determination of nicarbazin in animal feeding stuffs. Chromatographic separation was performed by means of HPLC RP columns packed with C18 (250×4.6 mm or 150×4.6 mm, 5 µm) using a mixture of acetonitrile and purified water as a mobile phase. The isocratic mode of elution was carried out for 10-15 min. Nicarbazin retention time depended on the eluent flow rate and ranged from 2.4 to 8.8 min. The repeatability of measurements was assessed by analyzing the calibration curve of nicarbazin standard solution. Feed samples were extracted with dimethylformamide. The samples were concentrated by drying, dry residues were reconstituted by the mobile phase, degreased with hexane and subjected to chromatographic separation. The specificity of the analytical method was tested by comparing the chromatographic separation of the feed sample spiked with nicarbazin standard solution with the control feed sample. The extraction degree of the spiked feed samples in the range of 20.0-50.0 mg/kg of nicarbazin was considered to determine the bias as the practical assessment of the validation parameters: trueness, linearity, repeatability and intermediate precision. The method was linear over this range of nicarbazin concentrations. The average degree of nicarbazin extraction from spiked feed samples is 99.1%. The correlation coefficient of the matrix matched calibration curve was more than 0.99. The evaluation of the intermediate precision of nicarbazin determination was estimated using different chromatographic columns over a long period of time. Validated HPLC-UV technique for the assay of animal feed and veterinary drugs based on nicarbazin has been introduced into the routine laboratory practice.