With the development of animal husbandry, the use of antibiotics in animal husbandry production has been increasing, and penicillins, tetracyclines, and aminoglycosides have been widely used in animal husbandry. Antibiotic residues generated by the unreasonable application of antibiotics have become the focus of attention in China and the world. Since China’s accession to the WTO, the problem of antibiotic residues has increasingly affected China’s import and export and the country’s economic interests. In order to meet the needs of residue monitoring, the establishment of antibiotic residue detection methods with sensitivity, accuracy and precision that meet the requirements of residue detection has become the focus of research. Based on the experience of years of residual analysis in domestic and foreign literatures and laboratories, this paper establishes a high performance liquid chromatography method for the determination of multiple residues of penicillins, tetracyclines and aminoglycosides in animal foods. The three methods use different sample processing and detection methods, such as solid phase extraction, pre-column derivatization ion-pair chromatography, post-column derivatization fluorescence detection.
In the first method, solid-phase extraction and pre-column derivatization techniques were used to establish a high performance liquid chromatography method for simultaneous detection of edible tissues in pigs and seven penicillin residues in milk. Seven penicillin antibiotics in muscle tissue were directly extracted with phosphate buffer and then purified by SPE. Seven penicillin antibiotics in pig liver and pig kidney samples were firstly treated with acetonitrile, and the acetonitrile layer was dried with high purity nitrogen gas at 45-50 °C. The tissue residue was extracted with phosphate buffer and mixed with acetonitrile to be purified by SPE. Seven kinds of penicillin antibiotics in the milk sample were directly extracted and evaporated with tetrabutylammonium bromide solution, and then dissolved in phosphate buffer for SPE purification. Purified by SPE solid phase extraction column, eluted with acetonitrile, eluent is dried at 45-50 °C high purity nitrogen, fixed to standard dilution, derivatized with 1,2,4-triazole mercury (II) solution, reversed phase high efficiency liquid Separation by phase chromatography, UV (325 nm) detection. The minimum limit of quantification of amoxicillin, penicillin G, ampicillin, and penicillin V standard solution was 10 μg/kg, and the minimum limit of quantification of oxacillin, phenoxypenicillin, and diclofenac was 30 μg/kg. The milk sample was concentrated, and the minimum detection limit and the minimum limit of quantification of amoxicillin, penicillin G, ampicillin, and penicillin V were 2 μg/kg, and oxacillin, phenoxypenicillin, and diclofenac were 15 μg/kg. The minimum detection limit and the minimum limit of quantification of amoxicillin, penicillin G, ampicillin and penicillin V in tissue samples were 25 μg/kg, and the minimum detection limit and the minimum limit of quantification for oxacillin, phenoxypenicillin and diclofenac were 75 μg/kg. The recoveries of the seven drugs ranged from 64.4% to 88.0% in the range of 2 to 300 μg/kg, and the coefficient of variation CV ≤ 19.4%.
Method 2, based on liquid-liquid extraction and solid phase extraction, establish porcine oxytetracycline hydrochloride, tetracycline hydrochloride, chlortetracycline hydrochloride in pig muscle, pig liver, pig kidney, chicken muscle, chicken liver, egg, fish muscle and milk. And doxycycline multi-residue high performance liquid chromatography analysis. The residual drug in the sample was extracted by Mcllvaine buffer, purified by solid phase extraction, eluted with methanolic acid solution, and the eluent was dried at 45-50 °C with high purity nitrogen. The mobile phase was fixed to volume and separated by UV-HPLC (350 nm). Determination. The minimum limit of quantification for the four drug standard solutions was 25 μg/kg. The minimum and minimum limits of quantitation were 50 μg/cg in pig chicken muscle, pig liver, pig kidney, egg and milk samples. When the concentration was 50, 100, 200 μg/kg, the recovery rate of the four drugs was between 70.4% and 89.9%, and the coefficient of variation CV was ≤16.0%.
In the third method, based on solid phase extraction and post-column derivatization techniques, a multi-residue high performance liquid chromatography method for detection of gentamicin, apramycin, kanamycin and neomycin in pork and milk was established. The residual aminoglycoside in pork is extracted with trichloroacetic acid precipitated protein by water and purified by solid phase extraction; the milk sample is extracted with phosphate buffer, precipitated with trichloroacetic acid, purified by solid phase extraction, and eluted with methanol and eluted with N2. Post-column derivatization fluorescence detection by reversed-phase high performance liquid chromatography. The minimum limit of quantification of gentamicin, apramycin, and kanamycin standard solutions was 312 μg/kg, and the minimum limit of quantification of neomycin standard solution was 625 μg/kg. The minimum limit of quantification of gentamicin, apramycin, and kanamycin standard solutions is 100 μg/kg in pig muscle and milk samples after concentration. The minimum limit of quantification of neomycin standard solution is 200 μg/kg. . In the concentration range of 100-800 μg/kg, the recoveries of the four drugs ranged from 71.1% to 85.4%, and the coefficient of variation CV ≤ 18.9%. The minimum and minimum limits of quantitation for aminoglycosides are 100 μg/kg. The sensitivity, accuracy and precision of the three methods are in line with the requirements of the residue detection method, and it is also the first time to establish a multi-residue detection method for these three types of drugs in China. The establishment of the three methods will provide a powerful technical force for the veterinary drug management department, food hygiene and quarantine department, import and export trade and other departments to monitor the multi-residues of these three types of drugs, and lay a foundation for the establishment of multi-residue analysis methods for veterinary drugs in China.
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