Study On The Determination Method Of Tilmicosin Residue In Aquatic Products

Tilmicosin, one of Semi-synthetic macrolide antibiotics, which inhibit Gram-positive bacteria, part of Gram-negative bacteria, mycoplasma, spirochete, etc., especially strongly on a variety of mycoplasma and spirochete, so that it has been greatly used in aquaculture to prevent and cure related diseases. However, with the enrichment of the antibiotic in our food chain, long-term consumption of aquatic product would influence the micro-ecological environment of human intestinal flora, leading to the imbalance of human intestinal flora. Therefore, the maximum limits of antibiotics in the aquatic products have been defined clearly in European Union , Japan, and other countries.In this paper, tilmicosin was selected to be detected. On the basis of present research status at home and abroad, the determination methods of Tilmicosin residues in aquatic products were studied. The main research contents were as follows:1) Establish a method of high-performance liquid chromatography (HPLC) for the determination of Tilmicosin residues. In this study, the optimal chromatographic separation parameters were determined by optimization of analytical factors, such as wavelength, column temperature, flow rate and mobile phase. Additionally, the optimum pre-treatment conditions were determined based on extraction and purification. The method was validated to be of wide linear range, high precision and good reliability. And the quantitative limit of this method was 20μg / kg.2) Develop a method of high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) for the determination of Tilmicosin residues in aquatic products. Electrospray Ionization (ESI) and selected reaction ion-monitoring (SRM) in positive mode were used to quantify Ttilmicosin residues. The results showed that, the limit of quantification was 5μg/kg. Under the addition levels of 5μg / kg, 50μg / kg and 100μg / kg, the sample recovery was 86.93 %~103.41%, and the relative standard deviation (RSD) was less than 10%.3) Assess the uncertainty of the above two methods. By analyzing the quality of measurement methods, the dispersion of measurement results, the main source of uncertainty was ascertained. According to assessment results, the uncertainty of HPLC detecting Tilmicosin residues in aquatic products mainly rooted in two aspects between the target recovery and peak area measurement, while the uncertainty of HPLC-MS/MS was mainly caused by the target recovery. Hence, the assessment outcomes would provide some theoretical basis to improve the measurement program. 4) Apply the established method of HPLC-MS/MS on the simultaneous detection of 4 kinds of macrolide antibiotics in aquatic products, based on the study of single-target determination. The preliminary experiment indicated that the method was simple, feasible and applicable to a wide range.5) Compose a standard text"The determination of Tilmicosin residues in aquatic products", assisting to enact the national industry standards or local standards.The methods of HPLC and HPLC-MS/MS for determining Tilmicosin residues in aquatic products were established based on previous researches. The results illuminated that the methods were confirmed to be of several advantages of simplicity, high sensitivity and accuracy, and good common, and be suitable for the determination of Tilmicosin residues in aquatic products.