基于分子印迹技术分离与分析多肽类抗生素及其残留研究

The polypeptide antibiotics for livestock have been widely used because of its excellent antibacterial properties and growth promoting effect. Their pharmacokinetics and residue analysis should be highly focused. On basis of the molecular imprinting theory and related research results as well as our previous studies, endogenous antimicrobial polypeptides such as colistin, bacitracin, nosiheptide are considered as model compounds. Predicting the interactions between the template molecule and functional monomer by Schrodinger molecular software, this project will study the synthesis and recognition of polypeptide antibiotics molecular imprinting polymers and its application. The influence of the reactive matrix and condition, monomer and cross-linker on the structure and properties of the polymers will be studied. The methods of characterization and determination of the imprinted polymers will be built up. The ability to recognize the studied drugs and its mechanisms of separation, purification and enrichment to these drugs will be clarified by optimizing the molecular imprinting solid-phase extraction (MISPE) conditions. A novel method based on the MISPE coupled with LC-MS/MS should be established for polypeptide antibiotics and its residues analysis. Eventually, the preliminary procedures of synthesis and evaluation of polypeptide antibiotics imprinted polymers are put forward. The adsorptive principle of the imprinted polymers are expected to be revealed. The project will provide theoretical guidance to the study of polypeptide drug-imprinted polymers. It is significantly meaningful for the monitoring of polypeptide antibiotics and its pharmacology and toxicology.

兽用多肽类抗生素因其优良的抗菌特性和促生长作用，在临床获得日益广泛的使用，其药动学及残留分析值得高度关注。本项目根据分子印迹理论及相关研究成果，在我们原有工作的基础上，以内源性抗微生物多肽（粘杆菌素、杆菌肽、那西肽等）为模型化合物，通过Schrodinger软件模拟模板与功能单体之间的相互作用和空间取向，开展多肽抗生素分子印迹聚合物的合成、识别和应用研究。探讨反应介质与条件、单体、交联剂等对聚合物结构及性能的影响，研究测定、表征多肽抗生素-印迹聚合物结构、性能的方法，通过优化分子印迹固相萃取条件，分析其分离、识别、净化与富集多肽抗生素的机制，构建基于特异性固相萃取LC-MS/MS测定多肽抗生素及其残留新方法。最终提出合成和表征多肽抗生素-印迹聚合物的基本方法，揭示其特异性吸附的机理，为研究多肽药物-印迹聚合物提供理论指导。该研究对多肽类抗生素药物质量监控及其药理毒理学研究具有重要理论意义。

多肽类抗生素（PPAs）具有优良的抗菌特性和促进动物生长作用，在兽医临床获得广泛的使用。动物组织中与PPAs相似的内源性肽类物质丰富，不有效去除会干扰PPAs残留检测。多粘菌素等PPAs的残留检测技术是研究热点。传统的前处理方法选择性差，严重影响PPAs的定性定量分析。分子印迹聚合物（MIP）具有高度专一识别能力，可从复杂基质中特异吸附目标分析物，达到高选择性净化和富集效果。本项目基于分子印迹理论及相关研究成果，系统开展了PPAs分子印迹聚合物的合成、识别、应用和PPAs的分析技术研究。采用固相萃取及分散固相萃取前处理技术，首次建立了饲料等基质中8种PPAs的液相色谱-蒸发光散射检测和液相色谱-串联质谱（LC-MS/MS）分析测定方法，对PPAs含量测定及饲料中PPAs高通量快速筛查具有重要意义，也为MIP的表征及应用提供基础。以黏菌素、替考拉宁、杆菌肽等PPAs为模板分子，采用沉淀聚合法、硅胶表面接枝法成功合成系列MIPs。沉淀聚合法制备的黏菌素-MIP微球聚集成团，表面粗糙；表面印迹法制备的MIP较NIP表面粗糙，比表面积更大，表现出较好的吸附性能；但因PPAs水溶性强、分子量较大，采用本体聚合法不能制备其高特异性聚合物。MIPs作为分子印迹固相萃取填料和分散固相萃取吸附剂，优化选择性萃取条件，可从各种环境水中特异性富集多黏菌素、替考拉宁等PPAs，并建立快速、灵敏和可靠的HPLC方法。研究成果为减少复杂生物基质中多肽类抗生素检测的杂质干扰，提高PPAs残留分析结果的准确度与精密度具有重要实践意义。此外，基于碱水解策略，制备了具有良好色谱和质谱特性的那西肽分子特征片段，据此，构建了LC-MS/MS测定动物组织中那西肽残留的高灵敏、高选择性和可靠性的分析方法，解决了LC-MS/MS不能够检测那西肽残留的问题，为那西肽残留的日常监督检测提供技术支撑。