Development Of Methods For Determination Of Tylosin, Tylvaosin And Tilmicosin Residues In Food Derived From Animals

Tylosin, tylvaosin and tilmicosin belong to macrolide antibiotics, which are used extensively to treat the infection of gram-positive bacteria and mycoplasmas, as well as some gram-negative organisms in the veterinary clinic. However, as excessive or inappropriate use of such antibiotics may leave residues in food-producing animals. When the concentration of the antibiotics and their metabolites in the tissues and organs reaches a certain amount, they could lead damages to the vestibular and cochlear nerve, leading to dizziness and hearing loss, and may even cause serious damage to the liver, kidney and hematopoietic system.The aim of this study is to develop a new multiresidue method for the extraction, separation and detection by reversed-phase high performance liquid chromatography of tylosin, tylvaosin and tilmicosin residues in food which were derived from animals.1. A HPLC method was developed for simultaneous separation and detection of tylosin, tylvaosin and tilmicosin in isocratic elution condition. By examining column, temperature, flow rate and mobile phase composition, optimizing the conditions for the chromatography. The HPLC system was the Agilent1100series equipment, with an automatic injection performed on a Agilent SB-C18(250×4.6mm,5μm) stainless steel column, which was programmed to inject a sample of50μL. Thermostatted column compartment was used to maintain the column temperature at30℃. Detection of tylosin, tylvaosin and tilmicosin was performed on the variable wavelength detector at280nm. The mobile phase consisted of a acetonitrile-methanol-0.01mol/L ammonium dihydrogen phosphate solution (45:40:15, v:v:v), the flow rate was1.0mL/min, and the running time was15min. The result showed that, calibration curves were linear and correlation coefficient were more than0.9990at0.5-25μg/mL. The limits of detection (LOD) were0.25μg/mL as well as the limits of quantitation (LOQ) were0.5μg/mL. The intra-day and inter-day coefficients of variation were less than5%.2. The conditions for the extraction, purification and enrichment of tylosin, tylvaosin and tilmicosin from milk, eggs and pork were considered. By examining the extract, solid-phase extraction columns, washing solution, eluent, as well as eluate volume were optimized. Samples are extracted by acetonitrile and cleaned up by SPE with ProElut C18 cartridge.5%acetonitrile-water (5:95, v:v) was selected as washing solution and acetonitrile was selected as eluent. The finally eluate volume was6mL.The result showed that, the mean recovery of tylosin, tylvaosin and tilmicosin is63.81～84.71%, The limits of detection (LOD) was20μg/Kg as well as the limits of quantitation (LOQ) was25μg/Kg. The intra-day and inter-day coefficients of variation were less than5%and10%, respectively.