| In recent years,animal husbandry shows a good momentum of development and huge growth potential with economic development.Environment pose a threat to security, but also the impact of foreign countries economics and business.However,the veterinary drug residues in animals and their additives can be ingested into the human body and pose potential threat to human health and the ecological environment. Detections of veterinary drug residues are extremely important to protect the ecological environment and human health.A variety of offical regulations and standards have been established. Thus, a more efficient, economical, rapaid and accurate method was in demand.Quinolones are widely used in animal husbandry as antimicrobial agents. However, overuse of antibiotics will not only result in resistance increase, but also caused great threat to human health. There are abundant literature reported about analysis methods of Quinolones, but all of the reports have obvious flaw. Such as long time for the pre-treatment operations, large amount of organic solvent, certain burden on the environment and risk of human body. Cloud point extraction is a new pre-treatment method widely used in recent years which is economical and environmental friendly. Compared with conventional solvent extraction, this method has the advantages of simple operation, less organic solvent consumption, but high extraction efficiency, which could conform well to the trends of modern analytical chemistry. In this paper, we established the conditions of cloud point extraction and high performance liquid chromatography( HPLC) method for analysing quinolones antibiotics in milk.The main results are as follows:(1) The CPE pretreatment conditions for extracting quinolones bactericides in milk were built. The conditions of de-emulsi?cation and cloud point extraction in the experiment were optimized. The best de-emulsification conditions were: 0.15 m L glacial acetic acid and 0.95 g Na2SO4, centrifugation at 10000 r/min, 5°C, 10 min. The optimal cloud point extraction conditions were: 80 g/L concentration of Tween 20, 0.25 g Na2SO4, 0.06 m L strong ammonia, equilibrated at 50 oC for 30 min. In order to validate the proposed method, three different spiked levels( 0.05, 0.1, 0.2 μg/m L) with three replicates were carried out. The recoveries were 71.3%~96.8% with relative standard deviations of 1.24% ~ 6.02 %, which demonstrated that the experimental results were accurate and precise.(2) Combined with high performance liquid chromatography-UV detector, chromatographic conditions were optimized. Reversed phase column used Inertsil ODS-3( 4.6 mm × 150 mm, 5 μm). Mobile phase were consist of acetonitrile( A) and 0.1% aqueous solution of formic acid( B). Detection wavelength was setted to change over time. The flow rate was 1 m L/min, and column temperature was 35 oC. The program of binary high-pressure gradient elution actted as: 12% B at 0 min,18% B at 20 min, 28% B at 20.01 B min, 40% B at 37 min. Under the optimized chromatographic conditions, the six quinolones in the milk could be separated well, and no miscellaneous peak interference. Each antimicrobial standarding in the range of 0.05~2 μg/m L had a good linear relationship, and the LODs were 8.27 ~18.01 μg/L,which were lower than the limit requirements of the U.S. Environmental Protection Agency and the European Commission.This method has the advantages of less organic solvent consumption, high extraction efficiency, environmentally friendly and simple operation. It is efficient and reliable for the determination of quinolones. The experiment can also be used for large sample level analyzing, and the results were in accord with the requirement of determination of veterinary. |
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