The Research On Analysis Teconology Of Anticoccidial Drugs In Animial Food And Environment

As the society develops and our living standards improves, more and more people are concerned with food safety issues caused by animal food and environment. Currently, triazine and sulfa drugs are widely used in the prevention and treatment of coccidiosis in the livestock industry due to their high efficiency, low toxicity, and broad-spectrum characteristics. The misuse of anticoccidial drugs can cause drug residues in animal food, resulting in a severe threaten to human health.. At the same time, the anticoccidial drugs can enter the soil through drug delivery and metabolic pathways, thus pollute animals and plants in the environment caused by incomplete degradation and accumulation of drugs in soil. Moreover, anticoccidial drugs will enter and enrich in the human body through the food chain, ultimately affect human health. Therefore, analysis of anticoccidial drug residues in animal food and environment is very necessary.This project is aimed to study the pre-treatment method of triazine drug and simultaneous detection of triazine and Sulfonamides using Diclazuril, Sulfanilamide, Sulfaclozine, Sulfaquinoxaline as research objects. The synthesis of diclazuril molecular imprinted polymer and the preparation of molecular imprinted solid phase extraction (MISPE) column were studied to provide the basis of pre-treatment of trace analytes in the complex matrices. A method of simultaneous determination of two types of anticoccidial drugs by high performance capillary electrophoresis were also developed in this project. Based on the above techniques, the analysis of anticoccidial drug residues can be applied in animal food and environment finally.The specific contents are as follows:1. Preparation and evaluation of MIPs with diclazurilThe diclazuril molecular printed polymer was prepared by precipitation polymerization using diclazuril as template molecule, a-methacrylic acid (MAA) as functional monomer, ethylene glycol dimethacrylate (EDMA) as cross-linking agent, and azobisisobutyronitrile(AIBN) as initiator. By studying the effect of the type of solvent and the ratio between the template molecule, the functional monomer and the cross-linking agent to the performance of polymer, the optimal preparation condition of diclazuril molecular imprinted polymer was investigated that N, N-dimethyl carboxamide was used as solvent and the molar ratio was1:5:30.The prepared MIPs was evaluated by Scachard analysis, the structure and morphology of the polymer were characterized using scanning electron microscopy, and the adsorption property of the polymer was studied by static adsorption experiment. The results showed that the MIPs can recognize the template at two classes of binding sites: the high affinity sites with a dissociation constant (Kd) and an apparent maximum binding capacity (Qmax) of2.93mmol/L and218mol/g, respectively; the low affinity sites with a dissociation contents(Kd) of0.12mmol/L and an apparent maximum binding capacity(Qmax) of44mol/g. And the adsorption of diclazuril by MIPs was significantly higher than that by NMIPs.2. Preparation of the MISPE column with diclazuril and evaluation of the MISPE method The prepared molecular imprinted polymer was mixed with diatomite at different ratios in the empty SPE column to prepare MISPE. By comparing the rate of sample solution passing through the MISPE column as well as the adsorption effect of MISPE column, the ratio between molecular imprinted polymer and diatomite was determined as2:1.The method of MISPE was esteblished by studying the effect of washing agents and eluting agents to the MISPE column. Hexane was chosen as washing agent and acetonitrile was used as eluting agent, yielding a recovery of90%and above.3. Establishment of the method of simultaneous determination of four anticoccidial drugs by HPCEA method of simultaneously separating and determining the four anticoccidial drugs was built by using high performance capillary electrophoresis. The effects of some important factors such as pH, composition and concentration of running buffer, separation voltage and separation temperature were investigated and optimized systematically by three factors and three levels orthogonal test, in which the running buffer were consist of50mmol/L borax-200mmol/L boric acid (pH9.0), separation voltage was23kv, separation temperature was25℃, and the UV detection wavelength was220nm and254mn. Under this optimal chromatographic condition, four anticoccidial drugs can be well separated within7.5min. Moreover, the calibration curves showed good linearity in the range of1.56to50mg/L, and the correlation coefficients (r2) were between0.9947and0.9999. The RSDs of peak areas and migration time were below of6%and0.2%, respectively.4. Application of analysis method in the animal food and environmentThe prepared diclazuril MISPE column was applied to pre-treatment of chicken and soil samples. The results showed that the diclazuril’s recovery in chicken was87.6%and the diclazuril’s recovery in soil was89.2%, indicating good purification and enrichment effect of this method, hence more suitable for the detection of trace analytes in the complex matrices.At the same time, the method of HPCE was applied to determine the four anticoccidial drugs in the soil.