Determination Of Benzimidazoles By Liquid Chromatography-Tandem Mass Spectrometry Method And ELISA Method

(BZs) are broad-spectrum, high efficiency and low toxicity? parasiticide that are widely used in veterinary production, mainly for the control of gastrointestinal nematodes, lung worms, liver fluke, etc.; Now majority of them are the key points in testing toxic residues of animal products as they show teratogenic and mutagenic in safety evaluation, In order to monitor BZs residues to ensure safety and enhance our competitiveness on international market, it is necessary to develop a rapid and accurate method for screen animal tissue samples from large quantity, meanwhile to establish a LC-MS/MS method with high precision, specificity and good stability for confirmation.LC-MS/MS is used in this paper to establish a method to detect various drug residues including 12 kinds of BZs in animal tissue, such as precursor medicine and metabolites. Pork, liver, mutton, fish tissue samples use ethyl acetate as alkaline extract, after concentrate to nearly dry, acetonitrile is added for re-dissolve and n-hexane is for losing fat; For milk samples, we use QuEChERS optimized by orthogonal test with 0.5% acetic acid as solvent, PSA as adsorbent and external standard method separately. As a result, five matrix (pork, liver, mutton, fish, milk) show good linearity in 1-200μg/L with recovery between 70% to 110% and RSD less than 14%. By adding 613C - thiabendazole of internal standard method, five matrix (pork, liver, mutton, fish, milk) show good linear relation in 1-200μg/L with correlation coefficient reach 0.995 or more, average recovery rate is 71.4%-108.1% under 5μg/kg(3μg/kg for milk), 50μg/kg, 100μg/kg, 200μg/kg adding levels, relative standard deviation less than 9.63%, detection limit lower than 0.5ppb, limit of quantification below the 1.5ppb. All of these testify our method is qualified for residue analysis by accuracy and precision.By analyzing the structure of benzimidazole compounds, we decide to change the carbamate structure of BZs side chain by hydrolysis, coupling HRP, the result indicates initial success by UV spectra confirmation, then establish ELISA between antigen and antibody, determine working concentration for coated antibody and antigen by phalanx, and optimize temperature and time for coating and sealing, then create ABZ antibody's standard curve, the linear equation is y =-29.949x + 84.234 in 1-500μg/L concentration range. R2 is 0.9946, IC50 is 13.80μg/ L. The average recoveries is 88.6%, 82.7%, 88.0% and 83.1% for liver, pork, mutton and fish tissue samples, RSD% is 8.6%, 6.9%, 4.0%, 5.2%. The result shows that the method can meet rapid screening requirement in large quantity.