限进分子印迹-固相微萃取活体取样新技术及其药物代谢分析应用研究

Sampling, sample pretreatment and analysis of sample are three key steps in drug metabolic analysis. However, some shortcomings such as high mortality of animals, tedious sample preparation procedures with unsatisfactory protein removal efficiency, low degree of automation, the loss of target drugs and metabolites result in the inaccurate analysis results. Based on our prophase work，this project plans to prepare two kinds of restricted access molecularly imprinted solid phase microextraction (RAMIP-SPME) fibers by using active ingredients of traditional Chinese medicines (TCMs) as the objects. The various factors which influence the selective adsorption performance of MIP will be optimized. In addition, different kinds of hydrophilic monomers and low isoelectric point proteins will be introduced as restricted access constituents for the fibers. The fabricated RAMIP-SPME fibers are expected to have good anti-protein interference ability, good capability for enrichment of target drugs and structurally similar metabolites, and can be applied for in vivo sampling and sample pretreatment. Furthermore，combined with HPLC and UPLC-MS/MS，the in vivo metabolic process of active ingredients of TCMs can be analyzed. In this project, we aim to develop a new in vivo sampling technique with sample pretreatment ability, strong specificity, high sensitivity, strong anti-jamming ability and high degree of automation. The development of the technique will provide a new pathway for in vivo metabolic analysis of drugs. Moreover, it is meaningful to elucidate the metabolic pathways of drugs, discover the active or toxic metabolites and etc.

药物代谢分析的三个关键步骤为取样、样品预处理和样品分析。然而，传统的取样和样品预处理方法存在动物死亡率高、处理步骤繁琐且蛋白不易去除、自动化程度低、目标药物及代谢产物易损耗和丢失等不足，致使样品分析结果不准确。本项目拟在前期研究基础上，采用分子印迹-固相微萃取技术，以中药活性成分为研究对象，优化影响分子印迹材料选择性吸附性能的各项因素，并引入不同亲水单体和低等电点蛋白为限进物质，研发两类蛋白排阻能力强、对中药活性成分及其结构类似代谢产物富集能力强的限进分子印迹-固相微萃取活体取样针；并将新取样技术与液相、液质等结合，用于中药活性成分的活体代谢分析。项目旨在发展一种集取样、样品预处理于一体，专属性强，灵敏度高，抗干扰能力强，自动化程度高的活体取样新技术。该技术的发展为药物活体代谢分析提供了一种新手段，对阐明药物代谢途径、发现其活性或毒性代谢产物等具有重要意义。

本项目将具有大分子排阻性能且选择性吸附性能优异的限进分子印迹材料（RAMIPs）用作SPME萃取针涂层，成功制备了RAMIPs-SPME萃取针；将其与HPLC或UPLC-MS/MS技术联用，成功构建了可用于药物活体代谢分析的新方法，并应用于中药活性成分的活体代谢分析领域。研究首先发展出多种可用于中药成分选择性富集的MIPs材料，包括可用于氧化苦参碱（苦参碱）、栎草亭、马钱子碱（士的宁）选择性富集的MMIPs或MIPs材料，探究并证实了这些材料用于复杂体系中目标活性成分选择性富集的优异效果，为后续研制RAMIPs提供了支撑。其次，通过引入限进介质，制备出对大分子具有优异排阻性能且对目标活性成分同样具有优异选择性吸附能力的RAMIPs；并且进一步探究和证实了RAMIPs在复杂生物样本体系（血液）中的应用潜力，为后续将RAMIPs用于活体代谢分析奠定了基础。随后，将RAMIPs用作SPME萃取针的涂层，构筑出RAMIPs-SPME取样新方法；结合HPLC或UPLC-MS/MS，实现了目标成分马钱子碱（及结构类似物士的宁）的活体代谢分析，并且获得了它们的代谢产物，这对阐明它们的代谢途径、发现其活性或毒性代谢产物具有重要意义，也为其他成分的活体代谢分析提供了技术支持。此外，为进一步研制限进性、选择性吸附性能更为优异的新型“RAMIPs”-SPME萃取针，我们还初步探索了共价有机骨架材料（COFs），一类具有尺寸选择性和蛋白排阻性能的高效吸附材料作为限进介质的潜力，初步探究并证实了 COFs-SPME用于复杂生物样本中黄酮类活性成分及相关代谢产物富集的能力，为后续将COFs用作“RAMIPs”-SPME萃取针中的“RA”提供了依据。最后，我们还初步将COFs与MIPs复合，成功制备了COFs-MIPs复合材料，为后续制备以COFs为限进介质的RAMIPs-SPME萃取针提供了技术支撑。