Determination Of Tetracycline Antibiotics Residues In Animal-derived Food By HPLC

Tetracyclines were a class of broad-spectrum antibiotics produced by streptomyces or by semi-synthesis, including oxytetracycline, tetracycline, chlortetracycline and doxycycline, etc. They had been widely used in the prevention and treatment of diseases in poultry and livestock because of their broad-spectrum antibacterial activity and pharmacokinetic characteristics.In order to pursuit high profits, tetracyclines were illegally used. On the one hand, this would lead to the emergence of the more and more resistance strains directly. Consequently, not only the effectiveness of tetracyclines was reduced, but also the use of other antibiotics was subjected to constraint. On the other hand, this is also hazardous to people's health due to the drug residues in animal products. Therefore, to monitor the residues of tetacyclines in animal products, the method for determination of these agents must be developed.In view of insufficiency of analytical method for the trace antibiotics in animal tissue, the sample pretreatment procedure of tetracyclines such as oxytetracycline, tetracycline, chlortetracycline, doxycycline, including liquid-solid extraction and solid-phase extraction (SPE) purification was systematically investigated. A high performance liquid chromatography method with UV detector was developed for the simultaneous determination of oxytetracycline, tetracycline chlortetracycline and doxycycline in animal tissue (pig muscle, pig liver, pig kidney, chicken muscle, chicken liver, chicken kidney and egg).Sample pretreatment procedure included liquid-solid extraction and SPE purification were used. Tetracycline residues in animal products were extracted by Na2EDTA-Mcllvaine (pH 4.0), and proteins and fats were removed by sulfuric acid and sodium tungstate. The Oasis HLB cartridge was used to perform a solid-phase extraction and cleanup of the extract. Tetracycline antibiotics were separated by C18 column, eluted by 0.01mol/L trifluoroacetic acid-acetonitrile gradiently, detected by HPLC at 350nm.Under the optimized pretreatment conditions, the limits of detection for oxytetracycline, tetracycline, chlortetracycline, doxycycline residues in pig muscle, chicken muscle and eggs were all 10μg/kg, while that in liver kidney of pig and chicken were both 25μg/kg. The limits of quantification (LOQ) for oxytetracycline, tetracycline and, chlortetracycline and doxycycline residues in pig muscle, chicken muscle and eggs were all 20μg/kg, while that in liver, kidney of pig and chicken were all 50μg/kg. The average recoveries of 4 tetracycline antibiotics in the added concentration ranging from LOQ to 2MRL were all from 60% to 110% ,the intra- and inter- assay CVs were all less than 11%.57 samples from markets were determined by this method. Compared to the national standard method, the recoveries of the positive sample from spiked samples by this method were higher than that by GB/T 21317-2007 and GB/T 5009.116-2003 methods.The results show this method is validated for determination of oxytetracycline, tetracycline, chlortetracycline and doxycycline residues in animal-derived food.