DEVELOPMENT OF THE METHOD FOR QUANTITATIVE DETERMINATION OF ENROFLOXACIN AND CYPROFLOXACIN IN CHICKEN SERUM USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH FLUOROMETRIC DETECTION

Abstract

Since Ukraine is a member of the World Trade Organization, so it has necessitated the transition of the entire veterinary drugs pharmacy industry to European and world levels and quality standards. Thus, a bill was approved this year which amended the process of state registration of veterinary drugs in the country. Therefore, the developed method is intended for clinical and pharmaceutical studies of veterinary drugs based on the active substances enrofloxacin and its main metabolite ciprofloxacin. Target analytes were extracted from the sample using the extraction by dichloromethane for 15 minutes, concentrated by drying and degreased with hexane/carbon tetrachloride. The procedure of sample preparation of fortified blood serum to construct calibration graphs is described in the manuscript. The mobile phase in the chromatographic separation consisted of acetonitrile and citrate buffer solution. The gradient mode of eluents was used during 16 min at a flow rate of 1.5 ml/min. Ciprofloxacin retention time is 8.80 min, and enrofloxacin retention time is 10.45 min. The validation parameters of the method were considered in accordance with the criteria of Council Directive 2002/657/EC and the Eurachem Guide. The specificity of the analytical technique was checked by chromatographic separation of serum sample spiked with enrofloxacin and ciprofloxacin mixture at the concentration of 20 μg/l and blank serum sample. The method is linear in the concentration range of 5.0 - 50.0 μg/l 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 serum samples. 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 enrofloxacin and ciprofloxacin was assessed on three different days of analysis. The main advantages of the developed method are high selectivity and high sensitivity. The limit of detection for enrofloxacin is 0.05 μg/l, and for ciprofloxacin it is 0.02 μg/l, which competes with previously published HPLC/FLD methods for the determination these quinolones.