| In recent years, the issue of the quality and safety of agricultural products is a widespread concern in China. Improving the quality of agricultural products and ensuring the quality and safety of agricultural products, is not only to improve the quality of life of people and meet the needs of enhancing the international competitiveness of agricultural products, but also to improve agricultural efficiency and increase farmers’ income. Because of their special effects on the prevention and control of diseases in poultry and livestock industries, antibiotics are widely used in farm animals and aquaculture. However, the abuse of these antibiotics can cause residues in food animals, which could directly do harm to human health, therefore antibiotics maximum residue limits in import and export of animal tissues as well as related products were strictly limited in countries all over the world. The EU has totally baned every antibiotic being added to animal-derived food. The use procedure of antibiotics in China is also clearly defined in documents of "Veterinary Regulations" and "animal derived food regulations". The establishment of detection method for antibiotics residue limits in food animals is very important. In this study, we established liquid chromatography-tandem mass spectrometry (LC-MS/MS) with isotope-labled internal standard method to detect the residues of tiamulin in animal derived food.First, we optimized the conditions of extracting tiamulin from animal origin samples. The protocol was as follows. The tissue samples firstly extracted with phosphate buffer which could convert tiamulin salt binding with tissue protein to tiamulin alkali in molecular state and then easily soluble in organic phase. Then ethyl acetate was added to extract and dred in a water bath under nitrogen flow at50℃. The residues were redissolved with2%formic acid solution. After removing fat by water-saturated n-hexane, the samples were cleaned by MCX mode SPE. Then MCX were washed by5%ammoniated methanol and eluent were dried in water bath under nitrogen flow at50℃and the residue dissolved using mobile phase and filtered over0.22μm microporous membranes and the liquid were used for LC-MS/MS detection.The results of LC-MS/MS detection showed that the limit of detection (LOD) and the limit of quantification (LOQ) of tiamulin in animal derived food were0.25and0.5μg/kg, respectively. The standard curve showed a good linear relationship with the correlation coefficient R2>0.999in the range of2.5～100ng/mL. In pork, when the adding concentration of tiamulin was at0.5,1.0and5.0μg/kg, the average recovery rates were in range of71.0%～95.4%, within-group RSD were in range of2.9%～7.2%, and between-group RSD were in range of1.1%～7.5%. In swine liver, the average recovery rates were in the range of72.3～92.3%, RSD were in range of4.9～8.1%and9.2%～12.6%within group and between group, respectively. In chicken, the average recovery rates were in the range of90.2～103.4%and RSD were in the range of4.2～8.1%and2.9%～8.9%within group and between group, respectively. In chicken livers, the average recovery rates were in the range of70.9～101.3%and RSD were in the range of3.1～8.3%and4.2～9.4%within group and between group, respectively. In eggs, the average recovery was89.2～100.6%and RSD were in the range of3.9～8.3%and4.7～9.4%within group and between group, respectively. In rabbit meat, the average recovery was in the range of81.9%～99.2%and RSD were in range of2.7%～9.6%and3.4%～9.2%within group and between group, respectively. All the results indicate that the LC-MS/MS with isotope-labled internal standard method is suitable to detected tiamulin residues in kinds of tissues origined from animals. |
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