THE DEVELOPMENT OF THE METHOD FOR TYLOSIN RESIDUES DETERMINATION IN EGG SAMPLES USING THE METHOD OF ENZYME-LINKED IMMUNOSORBENT ASSAY
The article presents the results of development and validation of the method of eggs sample preparation for the determination of tylosin residues by enzyme-linked immunosorbent assay using the kits manufactured by Europroxima (Netherlands). Tylosin is a natural antibiotic, highly effective against gram-positive and selectively active against gram-negative organisms. It is a product of microbiological synthesis of Streptomyces fradiae. Irresponsible use of antibiotics and the non-compliance with the withdrawal period for animals after their use have escalated the problems associated with the presence of antibiotic residues in food. Biologically reasonable maximum permissible levels (MRLs) of residual antimicrobials in animal products, officially approved in all countries by the Commission of the Codex Alimentarius, in the European Union by EU Regulation № 37/2010, and in Ukraine by the Order of the Ministry of Health of Ukraine products.
In order to ensure the compliance with the above standards, it is necessary to have sensitive and specific analytical methods that can rapidly and effectively control the presence of residues at the established levels for routine control of antibiotics in eggs by veterinary and manufacturing laboratories.
The influence of different extraction conditions on the percentage of extraction of the target analyte from the homogenized eggs sample fortified with the standard solution was investigated: pH changes of different buffer solutions, different degrees of sample concentration, the influence of separate reagents for the better separation of aqueous and organic phases. The results of the quantitative analysis of tylosin content in the model samples, determined by the developed screening method, were confirmed by liquid chromatography with mass spectrometric detection. Optimal conditions for extraction of the target analyte from egg were provided by the sample preparation method using the extraction with 0.5 M potassium phosphate buffer with pH 8.0, followed by the analyte transfer into the organic phase, the concentration of the analyte by evaporation of an organic extract aliquot and the reconstitution of the dried residue in the buffer solution, degreasing with hexane. It was found that for the better phase separation in "buffer – ethyl acetate" system the procedure of extraction and phase separation is best carried out at room temperature. The research results are presented in tables and chromatograms.
The proposed screening method was validated, the necessary statistical analysis of the obtained results was performed, and as a result, the limit value or “technical threshold” and the cut-off factor were calculated, and their graphical representation was presented.
The main advantages of the developed method are the rapidity, the simplicity of performance and the sensitivity to the target analyte at the level of 2 μg/kg, which is confirmed statistically by the results of validation tests. The technique is offered to the manufacturer to expand the scope of the kits usage.
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