【作者】 张玲；

【导师】 陈玎玎；

【作者基本信息】 安徽农业大学 ， 基础兽医学， 2010， 硕士

【摘要】 头孢类药物具有抗菌谱广、杀菌能力强的特点,在兽医临床上被广泛应用。由于不遵守休药期规定或未正确使用头孢类抗生素,动物源性食品中的头孢类药物残留对人类健康构成严重威胁,因此为保障动物性食品安全必须加强对动物性食品中头孢类药物残留的有效监测。建立高效率、高选择性、操作简便、环境友好的头孢类抗生素残留分析方法成为研究热点。本试验开展了以下工作:1.高效毛细管电泳(HPCE)同时检测头孢氨苄、头孢拉定、头孢哌酮和头孢唑啉方法的建立本试验考察了缓冲液类型、缓冲液离子浓度、pH值及运行电压和温度等因素对四种药物分离的影响。通过设计正交试验,优化分离条件,最终筛选出HPCE最佳分离条件:浓度为40mmol/L的柠檬酸/磷酸氢二钠缓冲体系,pH为7.5,分离电压为22kV,柱温为20℃,检测波长为198nm。在此条件下,四种药物在8min内完全分离,在3.125～100μg/mL范围内线性良好,峰面积的RSD均小于3%,迁移时间的RSD均小于0.5%。2. 7-氨基头孢烷酸分子印迹聚合物的合成与评价7-氨基头孢烷酸(7-ACA)为头孢类抗生素的共有母核,本试验以7-ACA为模板分子、9:1的甲醇:水溶液为溶剂、4-乙烯基吡啶(4-VP)为功能单体、乙二醇二甲基丙烯酸酯(EDMA)为交联剂,合成分子印迹聚合物。试验考察功能单体用量、交联剂用量、聚合温度等因素对分子印迹聚合物(MIP)合成的影响。设计正交实验,优化聚合条件,最终确立最佳的聚合条件:模板分子与功能单体及交联剂的摩尔比例为1:10:23,聚合温度为55℃。通过结合试验和Scatchard分析对制备的MIP进行评价,结果显示:分子印迹聚合物对7-ACA的吸附量明显大于空白聚合物(NMIP),并且MIP对头孢类药物均有较强的选择性。MIP在识别模板分子的过程中存在两类非等价的结合位点,高亲和力位点的平衡解离常数Kd为2.12mmol/L、表观最大吸附量Qmax为89.3 mol/g;低亲和力位点的平衡解离常数Kd为0.0037 mmol/L、表观最大吸附量Qmax为68.8 mol/g。3. 7-氨基头孢烷酸分子印迹固相萃取柱(MISPE柱)的制备及MISPE法的建立本试验以7-ACA分子印迹聚合物为填料,聚丙烯固相萃取柱空管为柱体,制备MISPE柱。通过对淋洗剂、洗脱剂进行筛选,建立MIP法。通过回收率的比较,最终确立正己烷为淋洗剂、50%乙腈为洗脱剂。在此种方法下四种药物的回收率均大于80%。4.检测鸡肌肉组织中头孢类药物的前处理方法的比较本试验采用空白鸡肌肉添加标准品,测定回收率的方法,进行溶剂萃取法(SE法)、固相萃取法(SPE法)、分子印迹固相萃取法(MISPE法)3种前处理方法的比较,并通过高效毛细管电泳图考察其净化效果。结果显示:三种方法处理的样品中,四种药物的回收率范围分别为93.9%～108.2%、74.4%～80.6%、80.5%～82.6%。其中,MISPE法的净化效果最佳、SPE法次之、SE法最差。5.MISPE-HPCE法检测鸡肌肉组织中头孢类药物的应用本试验利用优化后的MISPE-HPCE法对加标鸡肌肉样品进行检测,四种药物的回收率可达80%以上。且背景杂质少,可满足兽药残留分析要求。更多还原

【Abstract】 Cephalosporins have the characteristics of broad spectrum antibacterial and strong bactericidal. They havebeen widely used in the veterinary clinic. The human health is threatened by the cephalosporins residues from food of animal origin, because people breach the rules of the provisions and misuse the cephalosporins.Therefore, The effective monitoring of the drug residues from food of animal origin should be strengthened to protect the food security from animal origin. The research of Cephalosporins residues focuses on the methods of efficient, selective, simple and environment-friendly determination of the residues. This sentence can be deleted.1. Establishing the method of mimultaneous determination of CRD、CLX、CPZ and CEZ by HPCEIn this paper, the effects of separating the four drugs were studied in different conditions buffer concentration,buffer pH,running voltage and operating temperature) The results of the orthogonal experiment analysis showed that the best condition is as follows:40mmol/L Citric acid - Disodium hydrogen phosphate buffer (pH7.5),running voltage 20 kV and operated temperature 20℃, detection wavelength 210nm. The four cephalosporins could be separated completely under this condition.The liner range is 3.125μg/mL ~100μg/mL . RSD for peak area are less than 3%. RSD of migration time are less than 0.5%.2. Preparation and evaluation of MIP with 7-ACA7-ACA is the nucleus of cephalosporins, the molecular imprinted polymer was prepared by Situ polymerization with 7-ACA as the template molecule, MAA as functional monomer and EDMA as the cross linkers. The effects of preparing MIP with different mounts of template molecule, functional monomer and cross linkers were studied. The best polymerization conditions were determined by desiring the orthogonal assay and optimizing the conditions.The results showed that the molar ratio of template molecule and functional monomer and crosslinker was 1:10:23, and polymerization temperature was 55℃in the best condition.Evaluating the MIP by combining experiments and Scatchard analysis. The results showed that: the MIP recogniz the template with two classes of binding sites : The dissociation contents (Kd) and the apparent maximum binding capacity(Qmax) of the high affinity sites was 2.12mmol/L and 89.3 mol/g.The dissociation contents(Kd) and the apparent maximum binding capacity(Qmax) of the low affinity sites is 0.0037 mmol/L and 68.8 mol/g. MIP was significantly higher than NMIP on the adsorption of 7-ACA, and it had strong adsorption on the cephalosporins.3. Preparing the MISPE column with7-ACA and evaluation of the method of MISPEIn this experiment, The MISPE column was prepared with 7-ACA molecularly imprinted polymer as the filler and empty polypropylene SPE tube as the column. Through selecting the eluent and the eluent , establish the method of imprinted solid phase extraction. Establishing the method of imprinted solid phase extraction cephalosporins by screening the eluting reagent and the eluant. In this method ,the recovery of the four drugs were more than 80%.4. The Comparison of Pre-treatment methods with detect cephalosporins in chicken muscle tissueThe test add a standard to blank chicken muscle,then detect the recovery. Compared three kinds of pre-treatment methods with SE,SPE and MISPE.And study the effect of purification by HPCE. The results showed that:in these pre-treatment methods, the recovery of drugs is 93.9%～108.2%,74.4%～80.6%,80.5%～82.6%. MISPE has the best of purification, SPE ranks the second, SE is the worst.5.The application of detect cephalosporins in chicken muscle tissues with HPCEThe test detect chicken muscle sample by the method of MISPE-HPCE. The recovery of four drugs is more than 80%.It has low background impurity. Meet the requirements of veterinary drug residue analysis.更多还原