DOXYCYCLINE DETERMINATION IN ANIMAL TISSUES AND BLOOD PLASMA SAMPLES USING SCREENING AND CONFIRMATORY METHODS

Abstract

The article presents the results of validation of sample preparation method for muscle and parenchymal tissues of animals for further determination of doxycycline residual amounts by enzyme-linked immunosorbent assay using Oxytetracycline ELISA test kits from EuroProxima (Netherlands), initially developed for the analysis of other target matrices, viz. honey and shrimp. The results of modification of shrimp sample preparation method for the analysis of muscle and parenchymal tissue samples are showed. In accordance with the European Commission Decision 2002/657/EC, the proposed method was validated taking into account the maximum residue levels (MRLs) of doxycycline in tissues of productive animals: 100 μg/kg for muscle tissue and 300 μg/kg for liver. The suitability of the technique was confirmed on the basis of the established main validation parameters for screening methods (technical threshold and cut-off factor) using control (pure) and spiked with standard doxycycline solution at the level of ½MRL samples of muscle and parenchymal tissues of broiler chickens by means of “added – found” criterion. It was discovered that this approach to sample preparation makes it possible to determine doxycycline residues in poultry tissues at the level of 5-10 μg/kg with the absolute recovery about 54 %. The main advantages of the developed technique are simplicity of performance, rapidity and cost-effectiveness.
 The reliability of the results obtained by the screening method was verified using confirmatory UPLC-MS/MS method, viz. a comparative study of blank and spiked samples of muscle and parenchymal tissues of broiler chickens. The method of sample preparation of the studied specimens, the conditions of analysis carrying out, and the main validation parameters of the confirmatory method are also presented. Based on the obtained results, it was concluded that the developed screening method of enzyme-linked immunosorbent assay is suitable for the determination of doxycycline residual amounts in muscle and parenchymal tissues samples, and it is possible to use it for routine laboratory tests, as well as for the establishment of withdrawal periods of veterinary drugs based on doxycycline from the tissues of productive animals. Also using the same test kit, the sample preparation method for blood plasma samples for doxycycline content assay (at the levels of μg/ml) was suggested to study the bioequivalence of veterinary drugs containing doxycycline as an active pharmaceutical ingredient, which was as well confirmed by UPLC-MS/MS.