DEVELOPMENT OF METHOD FOR QUANTITATIVE DETERMINATION OF CEFQUINOME SULFATE IN PIGLETS BLOOD PLASMA USING HPLC/DMD

Abstract

This manuscript presents a developed method for determining cefquinome sulfate in piglets blood plasma intended for clinical and pharmaceutical research of veterinary drugs based on it. Blood plasma proteins were precipitated twice with a solution of trichloroacetic acid. The supernatant was further purified by a series of solid-phase extraction. Separation was performed on an inverted phase Kinetex EVO С18 column using acetonitrile and 0,1 % trifluoroacetic acide solusion as the mobile phase. The gradient mode of eluents was used during 10 min at a flow rate of 1,4 ml/min. The peak retention time of cefquinome sulfate is 4,2 min. The specificity of the analytical method was checked by comparing the chromatographic separation of a sample of blood plasma enriched with a standard solution of cefquinome sulfate and a sample of blood plasma placebo. The preparing loaded blood plasma samples procedure for building a calibration graph is described in the article. 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 blood plasma to construct calibration graph is described in the manuscript. The mean recovery from fortified blood plasma samples in the range of 0.1-2.0 μg/ml cefquinom sulfate was 102.3 %. The method is linear in the concentration range of 0.1 – 4.0 μg/ml of cefquinome sulfate. The correlation coefficient for the determination method is 0.9998. 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 analyte added to the control blood plasma. Recovery data are acceptable because they are within ± 10% of the target value. The method has sufficient convergence (accuracy). The evaluation of the intermediate precision of cefquinome sulfate determination was assessed on three different days of analysis. The limit of detection for cefquinom sulfate is 0.05 μg/ml and limit of quantification - 0.10 μg/ml. The average CV for each compound was <10%. The procedure was confirmed and then applied to determination cefquinome sulfate in the pig blood plasma obtained during the study of the pharmacokinetics of the veterinary drug. The HPLC/DMD method can be used for study of the pharmacokinetics of the veterinary drug.