Studies On The Binding Effection Of Protein With Pesticides Or Veterinary Drugs And Their Detective Application

Egg and fish products are the major source of protein for the general people.With the environmental pollution and illegal use of veterinary drugs and pesticides,more and more people pay attention to the safety problems of protein products.Nowadays,we usually use the high performance liquid chromatography（HPLC）,gas chromatography（GC）,high performance liquid chromatography-mass spectrometry（HPLC-MS）and gas chromatography tandem mass spectrometry（GC-MS）as the main instrument to detection the drugs in egg and fish products.Furthermore,those instruments can meet the sensitivity,stability and selectivity detection requirement.Because of the complex matrix of egg and fish products,the sample pretreatment process is the key to the detection.The paper discuss the sample pretreatment and the binding of protein with four tetracyclines（TCs）,nine kinds of phthalates（PEAs）,clopidol and four kinds of carbamate pesticides in egg and fish samples.The research includes the following five parts:Part one:Elaborating the current status of drug residues in animal food and the pretreatment method of complex samples,explaining the research content and significance of this paper.Part two:Using egg albumin（CEA）as the model protein combined with molecular fluorescence spectrometry and liquid chromatography（LC）.The effect of protein on the extraction efficiencies of four TCs（tetracycline,oxytetracycline,chlortetracycline,doxycycline）residues in egg samples have been studied.The results show that there was a strong binding effect between tetracyclines and egg serum protein（CEA）.In the methanol aqueous solution of oxalic acid,a slow but full protein denaturation might take place,which would cause the unfolding of protein and the releasing of tetracycline drugs.Based on the results,a novel sample preparation procedure was developed for the determination of TCs residues in egg samples.Which was using the mixture of methanol and oxalic acid aqueous solution(V _methanol/V^-11 _{0.2 mol Loxalic}（40:60）)as the extractant,combined with a dispersive solid-phase extraction（DSPE）cleanup step.This method is used to determinate the tetracyclines（tetracycline,chlortetracycline,oxytetracycline,doxycycline）residues in egg samples.Part three:We studied the combination of four carbamates（metolcarb,carbaryl,Isoprocarb,ethiofencarb）with fish serum protein（FSA）by fluorescence spectroscopy and equilibrium dialysis.Using Stern-Volmer curve fitting to discuss the quenching mechanism of pesticide residues in fish samples.The binding between protein and drug was further investigated by calculating the binding number,equilibrium constant,binding constant and dissociation constant.On the basis of this study,we use acetonitrile aqueous solution as extraction solution to determinate the carbamate pesticide residues in aquatic products rapidly by HPLC.The effects of protein matrix,organic solvents and purification agents on the extraction rate of carbamate pesticides were investigated.The results showed that the coexistence of protein matrix in aquatic products affected the extraction rate of carbamate pesticides.When used 80%acetonitrile aqueous solution as the extraction solution,the protein will be denaturation slowly and completely to release the carbamate pesticide and improve the extraction efficiency.Compared to the Qu ECh ERS pretreatment method,this method can reach a higher recovery rate.Furthermore,it can also avoid the effect of anhydrous acetonitrile and magnesium sulfate which caused the sample protein excessive denaturation.The strong chemical polarity of acetonitrile water solution can reduce the fat soluble impurities in the matrix.Adding appropriate amount of PSA and C₁₈8 powder can achieve a satisfactory purification effect.Furthermore,the extraction time is reduced and the efficiency of extraction is improved..Part four:Using methanol aqueous solution as extractant to extract the residues of chlorine in egg samples directly.The experiment discuss the effect of extraction agents and cleaning agent on the extraction efficiency of clopidol drug in egg samples.A mild and efficient method has been built by optimizing the suitable extraction conditions.The experimental results show that when using 60%methanol water solution as extraction agent can obtain high extraction efficiency.Part five:Using methanol aqueous solution as extractant,one step to extract the clopidol residue from egg samples was carried out by HPLC.The effects of organic solvents,volume fraction of extracting agent and purification agents on the extraction rate of clopidol were investigated.On the basis of this study,a rapid method for the determination of clopidol residues in egg samples was established.The experimental results show that the sample pretreatment method can obtain high extraction efficiency,the recovery rate is between 83.6%¹02.3%,RSD is 0.7%².3%,the detection limit range is 0.20μg·mL^-1,and the limit range is 0.48μg·mL^-1.