Testing Methodology Of Residual Sulfonamides And Polypeptide Antibiotics In Animal-derived Food

Sulfonamides and polypeptide antibiotics are the most commonly used veterinary drugs in livestock raising. However, frequent intake of food containing sulfonamides and polypeptide antibiotics could cause imbalance of body microflora, antimicrobial drug resistance, and even toxic side effects such as kidney and ear toxicity if the in vivo accumulation is too high. Many countries have strengthened the supervisory control for sulfonamides and polypeptide antibiotics, and respectively formulated the maximum residue limit (MRL) for sulfonamide and polypeptide antibiotics in animal-derived food (0.01 mg/kg ~ 1 mg/kg). Therefore, the study of testing methods on sulfonamide and polypeptide antibiotics has been seriously concerned. In the past several years, many researches on detection methods for sulfonamide in animal-derived food have been conducted by domestic and foreign scholars successively. Nevertheless, so far, these methods can not completely cover the current sulfonamide involved in production practice, such as sulfanilamide and sulfaethoxypyridazine; more over, the limit of detection of existing detection methods still can't keep up with international demand (0.01 mg/kg) for similar drugs detection. In addition, there's no report about detection method for 5 polypeptide antibiotics in animal-derived food, or standards for related detection methods. Therefore, on account of the lack of current international inspection method, and Chinese food exports affected by foreign technical barrier and the specific situation of domestic veterinary drug use, detection methods for sulfonamide and polypeptide antibiotics are developed. Animal-derived food as the research object and through optimization of liquid chromatography parameters, mass spectrometry parameters, the mobile phase composition, pretreatment methods, liquid chromatography-mass spectrum/mass spectrometry, detection methods for 24 sulfonamide drug and 5 polypeptide antibiotic residues in animal-derived food are established. The main research contents and the results are as follows:(1) The sample pre-treatment of every method was optimized. The simple and traditional extraction method was used, and the matrix of complex samples was clear. There's no interference on the peak of chromatographic. More over, the time-consuming of sample pre-treatment was saved and the purification costs were reduced.(2) UPLC - MS/MS detection method was developed for 24 sulfonamide antibiotics in 6 matrixes -- milk, chicken, beef, pork, mutton, honey. This method completes a sample testing in 10 min, and the concentration of the sulfonamide antibiotics was within the range of 1μg/kg ~ 200μg/kg, showing good linear relationship with the peak area, of which the correlation coefficient was 0.995~0.999. The limits of detection (LOD) were 0.04μg/kg ~1.35μg/kg, sulfaisodimidine and sulfametoxydiazine were 0.04μg/kg, and sulfanitran was 1.35μg/kg. Recoveries ranged from 61.4% ~ 120% for the three different concentrations added (5μg/kg, 10μg/kg, 20μg/kg), and relative standard deviation was less than 18.9%.(3)HPLC - MS/MS detection method for milk, chicken, beef, pork, mutton, and honey matrix was established. This method completes a sample testing in 28 minutes, Instrument limits of detection (LOD) was 0.27μg/kg ~2.86μg/kg. The linear relation was good with sample content in a range of 5μg/kg ~ 300μg/kg. Recoveries ranged from 61.9% to 122% for the three different concentrations added (10μg/kg, 20μg/kg, 50μg/kg) and relative standard deviation was less than 18.55%.(4)HPLC - MS/MS detection method for 5 polypeptide antibiotics in animal-derived food was established, and the results showed that recoveries of 5 polypeptide antibiotics ranged from 75.1% to 120% for the three different concentration added (25μg/kg, 50μg/kg, 100μg/kg), and relative standard deviation was less than 15.67; method detection limit was 0.10μg/kg ~ 5.57μg/kg, in accordance with the present international residue limit requirements.This research can meet the domestic and foreign detection requirements-rapidity and accuracy-for sulfonamide and polypeptide antibiotics in animal-derived food, by developing testing method with fast and easy handling and good repeatability.