THE DEVELOPMENT OF THE METHOD FOR TYLOSIN RESIDUES DETERMINATION IN EGG SAMPLES USING THE METHOD OF ENZYME-LINKED IMMUNOSORBENT ASSAY

  • D. Yanovych State Scientific Research Control Institute of Veterinary Medicinal Products and Feed Additives, 11, Donetska str., Lviv, 79019, Ukraine
  • Z. Zasadna State Scientific Research Control Institute of Veterinary Medicinal Products and Feed Additives, 11, Donetska str., Lviv, 79019, Ukraine
  • M. Rydchuk State Scientific Research Control Institute of Veterinary Medicinal Products and Feed Additives, 11, Donetska str., Lviv, 79019, Ukraine
  • S. Plotycya State Scientific Research Control Institute of Veterinary Medicinal Products and Feed Additives, 11, Donetska str., Lviv, 79019, Ukraine
  • S. Kislova State Scientific Research Control Institute of Veterinary Medicinal Products and Feed Additives, 11, Donetska str., Lviv, 79019, Ukraine
  • O. Pazderska State Scientific Research Control Institute of Veterinary Medicinal Products and Feed Additives, 11, Donetska str., Lviv, 79019, Ukraine
Keywords: TYLOSIN, EGGS, ELISA, METHOD VALIDATION.

Abstract

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.

References

Błądek Tomasz, Posyniak Andrzej, Gajda Anna, Gbylik Małgorzata, Żmudzki Jan (2012). Multi-class procedure for analysis of antibacterial compounds in eggs by liquid chromatography-tandem mass spectrometry. Bull Vet Inst Pulawy. 56. 321-327, DOI: 10.2478/v10213-012-0057-6.

Botsoglou, N.A. & Fletouris, D.J. (2001). Antimicrobial drugs. In: Drug Residues in Foods. Pharmacology, Food Safety, and Analysis. Marcel Dekker, Inc., New York, NY, USA. 27-115.

Cantwell, H. & O’Keefee, M. (2006). Evaluation of the Premi Test and comparison with the One-Plate Test for the detection of antimicrobials in kidney. Food Add Contam. 23, 120-125.

CODEX AIIMENTARIUS. (2012). Maximum residue limits for veterinary drugs in foods updated as at the 35th Session of the Codex Aiimentarius Commission, CAC/LMR 2. http://www. codexalimentarius.org/standards/list-of-standards/en/7provide = standards &orderField=fullReference&sort=asc&numl =CAC/MRL. Accessed February 2014

COMMISSION DECISION 2002/657/EC of August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Off J Eur Commun 2002, L 221. 8-36.

COMMISSION REGULATION (UE) 37/2010 of 22nd December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. // Off J Eur Commun 2010, L 15. 1-72.

Cornejo, J., Pokrant, E., Carvallo, C., Maddaleno, A., San Martín, B. (2018). Depletion of tylosin residues in feathers, muscle and liver from broiler chickens after completion of antimicrobial therapy Food Additives & Contaminants: part A, 35, 3. 448–457 DOI: 10.1080/19440049.2017.1401740.

Donoghue, D. (2003). Antibiotic residues in poultry tissues and eggs: human health concerns? Poult. Sci. 82, 618-621.

Furusawa, N. (2001). Transference of dietary veterinary drugs into eggs. Vet. Res. Commun. 25, 651-662.

Hakimzadegan, M., Khalilzadeh Khosroshahi, M., Hasseini Nasab, S. (2014). Monitoring of Antibiotic Residue in chicken eggs in Tabriz city by FPT Inter. J. of Advan. Biol. and Biomed. Res. 2, 1. 132-140.

Hamscher, G., Limsuwan, S., Tansakul, N., Kietzmann, M. (2006). Quantitative analysis of tylosin in eggs by high performance liquid chromatography with electrospray ionization tandem mass spectrometry: residue depletion kinetics after administration via feed and drinking water in laying hens. J Agric Food Chem. 54, 24. 9017-9023.

Hekman, P.G. (2011). Schefferlie Kinetic modeling and residue depletion of drugs in eggs. Br. Poult. Sci. 52. 376-380.

Kan, C.A. & Petz, M. (2000). Residues of veterinary drugs in eggs and their distribution between yolk and white. J. Agric. Food Chem. 48. 6397-6403.

Lewicki Jacek. (2007). Tylosin. A review of pharmacokinetics, residues in food animals and analytical methods 66th JECFA meeting. 1-39.

Munoz Ruben, Cornejo Javiera, Maddaueno Audo, Araya-Jordan Carouina, Iraguen Daniela, Pizarro Nicolas, Betty san Martin. (2014). Withdrawal times of oxytetracycline and tylosin in eggs of laying hens after oral administration. Journal o f Food Protection, 77, 6. 1017-1021. DOI: 10.4315/0362-028X.JFP-13-440.

Vandenberge, V., Delezie, E., Delahaut P., Pierret G., De Backer P., Daeseleire E., Croubels, S. (2012). Transfer of flubendazole and tylosin at cross contamination levels in the feed to egg matrices and distribution between egg yolk and egg white. Poult Sci., 91(5).1248-55. DOI: 10.3382/ps.2011-02071.

Published
2020-06-01
How to Cite
Yanovych, D., Zasadna, Z., Rydchuk, M., Plotycya, S., Kislova, S., & Pazderska, O. (2020). THE DEVELOPMENT OF THE METHOD FOR TYLOSIN RESIDUES DETERMINATION IN EGG SAMPLES USING THE METHOD OF ENZYME-LINKED IMMUNOSORBENT ASSAY. Scientific and Technical Bulletin оf State Scientific Research Control Institute of Veterinary Medical Products and Fodder Additives аnd Institute of Animal Biology, 21(1), 263-274. https://doi.org/10.36359/scivp.2020-21-1.33