Validation of the quantitative determination method of spectinomycin in muscle samples by the immunoenzyme analysis method


K.S. Myagka, S.A. Tkachuk, I.V. Yatsenko, K.O. Rodionova*, M.V. Kostiuk, N.V. Liniychuk, V.P. Lyasota, V.M. Zhylina, I.L. Tsivirko and L.B. Savchuk

Antimicrobial residues in raw materials and products of animal origin are regulated by the following EU regulations: Commission Regulation (EC) No 37/2010, Council Directive No 96/23/EEC, Council Regulation (EC) 2377/90, Codex Alimentarius Commission. For spectinomycin in animal muscles, the European Union has set Maximum Allowed Levels (MRLs): 300 μg/kg, according to the Codex Alimentarius Commission-500 μg/kg. A wide variety of spectinomycin methods are used worldwide, including Fluorescence Latex Immunoassay (FLI), Micellar Electrokinetic Capillary Chromatography In Combination With Ultraviolet Detection (MEKC-UVD), Enzyme-Linked Immunosorbent Assay (HPA), high-efficiency electrocardiography, and high-performance liquid chromatography with Fluorescence Detection (FLD) of microbiological inhibition. The purpose of the study was to validate the ELISA method and the liquid chromatography method with the mass spectrometric detector (LC/MS/MS) to determine the residual spectinomycin content in muscle samples (cattle, pigs, chickens, geese, and turkeys). The determination of the spectinomycin residue was performed by ELISA on a Tecan Sunrise spectrophotometer (manufactured by Sunrise, Austria) using a test system for competitive enzyme-linked immunosorbent assay Kwinbon Biotech Spectinomycin (Cat. No.: KA02701H) and liquid chromatography. -spectrometric detector on the device Waters Xevo (USA). The chromatograph was equipped with an ACQUITY UPLC BEH C18 analytical column, a two-quadrupole mass spectrometric detector, a positive ionization spray, and MasLynx results calculation software. Validation characteristics have been established for the detection of spectinomycin residues in muscle samples, such as: detection ability (CCβ) is 40.0 μg/kg, cut-off level is 32.9 μg/kg. The lowest spectinomycin content that can be detected by ELISA using a test system for the competitive enzyme-linked immunosorbent assay Kwinbon Biotech (China) is 3.0 μg/kg. The main validation characteristics were also determined by liquid chromatography with a mass spectrometric detector. CCα is 358.08 and CCβ-459.52.

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