Study On The Green And Efficient Pretreatment Technology Of Veterinary Drug Residues In Animal-origin Foods

The detecting procedure of veterinary drug residues in animal-origin foods is involved in two steps: Sample preparation and sample determination by instruments.With the technology progress,the sensitivity and selectivity of the instruments can meet the requirements of the general determination.However,the key problem for accurate and effective detection of chemical residue in animal-origin foods is sample preparation.This thesis has reviewed the technology of sample pretreatment and detection methods.The binding interactions of drugs and proteins were studied by Fluorescence spectrometry and equilibrium dialysis method,which provided theoretical basises for the extraction of veterinary drug residues in animal-origin foods.A series of novel methods for the analysis veterinary drug residues(include: sulfonamides,diazepam,perphenazine and quinolones)in animal-origin foods,based on the use of aqueous solution of organic solvent as extraction solvent system combied with liquid chromatography(HPLC),have been developed.This thesis includes five sections.Part one: This section described the situation of security in foodstuffs of animal origin,and reviewed the detection technologies and the sample pretreatment methods for analyzing of veterinary drug residues.Part two: Application of molecular fluorescence spectrometry for investigating the binding interactions of six sulfonamides(SAs)with CEA.Equilibrium dialysis method was used to study the binding ratios of SAs and actual egg samples.The effect of protein on the extraction of SAs residues in eggs was investigated in detail.The results showed that SAs strongly bound with egg sample.The appropriate concentration of acetonitrile aqueous solution can cause slow and full denaturation of protein.The drugs combined with protein might be dissociated,achieve efficient extraction of SAs.The proposed approach was satisfactorily applied to thedetermination of SAs residues in egg sample.Overall recoveries were 78.7% ~ 91.1%with RSD values were 0.8% ~ 4.2%.Thsis sample preparation method was straightforward,quick and efficient.Part three: Equilibrium dialysis method was applied to investigate the binding ratios of three samples with drugs.Experiment results showed that the strong binding interactions between drugs and protein decreased the extraction efficiency of two drugs from three samples.However,in the 80%(volume fraction)ethanol-water solution,a slow protein denaturation might take place,which would cause the unfolding of protein and the release of drugs.This resulted in the high extraction efficiency of two drugs from protein matrix samples.In this work,the purification steps were greatly simplified by adding PSA(primary secondary amine modified silica gel)and C18 to the crude extraction solution because the impurities extracted less in the high polarity of 80% ethanol-water solution.The sample preparation method was used satisfactorily for the determination of diazepam and perphenazine in spiked samples.The overall recoveries were in the range of 71.2%~96.8% with acceptable RSD value of 0.4%~3.7%.Part four: The effect of protein on extraction efficiency of five quinolones in egg samples were investigated in detail.Appropriate kind of organic solvent solution was chosen as extraction reagent.Meanwhile,the extraction efficiency of five drugs in different concentrations of organic solvent solution were investigated by HPLC.The research showed that protein denaturation were slow and full in 70%(volume fraction)methanol-water solution,the quinolones combined with protein might be dissociated,which would resulted in a high efficiency on extraction of five quinolones from egg samples.In this work,the purification steps were greatly simplified by adding PSA(primary secondary amine modified silica gel)and C18 to the crude extraction solution because the impurities extracted less in the high polarity of 70% methanol-water solution.The sample preparation method was used satisfactorily for the determination of five quinolones in egg samples.The overall recoveries were in the range of 81.2%~97.3%with acceptable RSD value of 1.1%~4.1%.Part five: A method for rapid separation and determination of five quinolones(Eenoxacin,Ofloxacin,Ciprofloxacin,Enrofloxacin,Gatifloxacin)in milk sample was builded by one-step extraction and purification.The chromatographic separation was performed on a Chromatorex C18 column(250 mm×4.6 mm,5?m),acetonitrile-0.05 mol/L citric acid aqueous solution(15:85)as mobile phase,flow rate was 1.0 mL/min,and the UV detector detection wavelength was 280 nm.The research showed that appropriate concentration of methanol aqueous solution can cause slow and full denaturation of protein.The quinolones drugs combined with protein might be dissociated,the appropriate concentration of methanol aqueous solution can cause the protein in the sample to be slow and fully denatured,and the drugs was released into the extraction solution,achieve efficient extraction of quinolones drugs.Adding appropriate amount of PSA,C18 in the extraction process can get a good purification effect.The sample preparation method was used satisfactorily for the determination of five quinolones in milk sample.The overall recoveries were in the range of 97.3%~100.5% with acceptable RSD value of 1.1%~4.1%.The detection limit of this method was24.1~83.1 ng·g-1.