| Animal-origin foods are closely related to people’s daily lives, their quality problems with safetyissues, received extensive attention from national governments and food safety agencies. Veterinarydrug residues problem is one of the important factors resulting in a decline in animal-origin foodssafety. Therefore, veterinary drug residues analysis has become an important subject to be solved.The most commonly reported methods for the analysis of veterinary drug residues inanimal-origin foods involve two steps: the extraction of veterinary drugs from animal-origin foodssamples and the detection of the concentration of drugs. At present, the analytical instruments canmeet the requirements of various compounds determination. Therefore, the key problem for accurateand effective detection of veterinary drug residues in animal-origin foods is sample preparationtechnology. This paper provides an overview of the existing extraction technologies for veterinarydrug residues in animal-origin foods, and aims at exploring new environment friendly pretreatmenttechnologies. Fluorescence spectrometry and equilibrium dialysis method were carried out toinvestigate the binding interactions of stilbene estrogens with bovine serum albumin (BSA), stilbeneestrogens with actual sample, respectively, expounding the existing state of drugs in protein matrixsample. All the above studies providing theoretical basises for the extraction of drugs in proteinmatrix samples. Combined with ethanol-K2HPO4-water aqueous two phase system, organic aqueousextraction, weak acid extraction and High Performance Liquid Chromatography (HPLC), thedetection technologies for stilbene estrogens, quinolones (QNs) and tetracyclines (TCs) wereestablished in this paper.The paper can be divided into five sections:Part one: The safety status of animal-origin foods and the harmfulness of veterinary drugresidues were summarized. The detection methods for veterinary drugs in animal-origin foods, alsothe commonly used pretreatment technologies were reviewed. The significance and content of thispaper were introduced.Part two：Fluorescence spectrometry was used to investigate the binding interactions of BSAwith three stilbene estrogens (hexestrol, diethylstilbestrol and dienoestrol). And further research into the binding ratios between stilbene estrogens and actual milk sample was carried out by equilibriumdialysis method. Meanwhile, the effect of protein on extraction efficiency of stilbene estrogens inmilk sample was investigated in detail. The results show that stilbene estrogens strongly bound withmilk sample. In70%(V/V) ethanol-water extracting solution, the protein matrix of sample takesplace a slow but full denaturation, which causes the drugs bonded with protein to be released. Thenappropriate amount of K2HPO4was added to above extracting solution to form a stable aqueous twophase system. Following, the fat-soluble stilbene estrogen residues were extracted into upper phasewith high extraction efficiency, and substrate removal could be realized. Purification steps wereomitted in this work because the fat-soluble impurities were extracted less in70%(V/V)ethanol-water solution than those in hydrophobic solvents as liquid-liquid extraction procedure. Theproposed approach was satisfactorily applied to the quick determination of stilbene estrogen residuesin milk by HPLC. Overall recoveries were83.2%～93.8%with RSD values less than4.5%, and thedetection limits were in the range of11.7～20.7ng·g-1. The sample preparation method wasstraightforward,effcient,economicallyadvantageousandenvironment-friendly.Part three：Amultiresidues determinationm method for the analysis of three stilbene estrogens inbeef, pork and fish meat, based on the use of a new sample preparation approach followed by HPLCmethod, is reported. Equilibrium dialysis method was applied to investigate the strong bindinginteractions of meat sample with three stilbene estrogens. Meanwhile, the effect of protein on theextraction of three stilbene estrogens was investigated by rayleigh scattering spectra and HPLC.Experiment results showed that the strong binding interactions between stilbene estrogens and proteindecreased the extraction effciency of stilbene estrogens from meat samples. However, in the80%(volume fraction) ethanol-water solution, a slow protein denaturation might take place, which wouldcause the unfolding of protein and the release of stilbene estrogens, resulting in high extractioneffciencyofstilbeneestrogens fromproteinmatrixsamples.Inthis work,thepurificationsteps werelargely simplified by adding PSA (primary secondary amine modified silica gel) to the crudeextraction solution because that impurities extracted less in the high polarity of80%ethanol-watersolution. The sample preparation method was used satisfactorily for the determination of threestilbene estrogens in spiked meat samples. The overall recoveries were in the range of81.4%~98.1%with acceptable RSD value of1.3%~4.8%, This paper proposes a straightforward and environmentally friendly strategy by slowing down the protein denaturation and releasing boundstilbene estrogens to enhance the extraction effciency of stilbene estrogens in beef, pork and fishmeat samples.Part four：A kind of environment friendly method for simultaneous separation and determinationof six quinolones (enoxacin, norfloxacin, ciprofloxacin, lomefloxacin, enrofloxacin, gatifloxacin) inmilk by HPLC was established in this paper. The quinolones were separated on a reversed-phaseChromatorex C18column and detected by diode array detector with the wavelength of280nm.Experiment showed that protein denaturation was slow but full in the mixture solution of citric acidand ammonium sulfate with suitable concentrations. Meanwhile the quinolones combined withprotein might be dissociated, which would result in a high efficiency on extraction of quinolonesfrom milk sample. Then the protein was precipitated quickly from the extracting solution by heating,and the extracting solution was purified with PSA subsequently to complete the sample preparationprocedure. This method has been applied to the determination of six quinolones in bovine milksamples. The spiked recoveries were all in the range of82.0％~117.8％, and the limits of detectionwere in the range of34.5~85.0ng·g-1. The pre-treatment method proposed in this paper was reliable,rapid and environment friendly.Part five: The effect of protein on extraction efficiency of three tetracyclines (chlortetracycline,methacycline and doxycycline) in milk sample was investigated in detail. Appropriate kind of weakacid solution was chosen as exactraction reagent. Meanwhile, the extraction efficiency of three drugsin different concentrations of weak acid solution was investigated by HPLC. The result showed thatprotein denaturation was slow but full in appropriate concentration of weak acid solution, the drugscombined with protein might be dissociated, which would result in a high efficiency on extraction ofthree tetracyclines from milk sample. The extracting solution was purified with PSA and CleanertNH2SPE (amino modified silica gel), the purification procedures were greatly simplified. Overallrecoveries were80.6％~95.8％with acceptable RSD values of1.7％~4.4％, and the detectionlimits were in the range of13.4~29.2ng·g-1. The proposed method was simple, reliable, environmentfriendly, and was satisfactorily applied to the quick determination of chlortetracycline, methacyclineand doxycycline residues in milk. |
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