液相微萃取-色谱联用检测儿茶酚胺类神经递质及在疾病诊断中的应用

The concentration of catecholamine neurotransmitters is one of the important index of several diseases clinical diagnosis such as pheochromocytoma, paraganglioma and so on, which can be lower to level of nM. Therefore, extraction and enrichment are ugently requried for trace analyses of catecholamine neurotransmitters. This project focused on developing new liquid phase microextraction (LPME) method to extracting catecholamine and their metabolites in plasma and urine sample, exploring the effect of various LPME methods on extraction and enrichment, selecting the optimal micrioextraction method and determing the catecholamine and their metabolites after extraction by chromatography-mass spectrometry. The project aims to make the effective and trace analyses of catecholamine and their metabolites in biological sample, and further provide scientific basis for pheochromocytoma, paraganglioma and neuroblastoma in clinical diagnosis and cure. LPME methods can efficiently reduce agents and time consumption, moreover, eliminate matrix interference when extracting the target analytes, and thus increase the efficiency of extraction a lot. Therefore, LPME method is very suitable for complex biological sample preparation. On the other hand, chromatography-mass spectrometry also provides excellent tools for catecholamine neurotransmitters separation and analysis. The successful study of this project will bring in vital theoretical and application value for biological sample preparation standard method, determination method and clinical laboratory of catecholamine neurotransmitters.

儿茶酚胺神经递质的含量是嗜铬细胞瘤和副神经节瘤等疾病诊断的重要指标，但其在生物样品中的浓度低至nM级，因此痕量检测需要对其进行萃取及富集。本项目拟研究新型的液相微萃取方法萃取富集血清和尿液中的儿茶酚胺及其代谢产物，探索各种液相微萃取方法对萃取富集效率的影响，选取最佳的微萃取方法并与色谱-质谱联用对富集后的目标分析物进行特异灵敏测定，最终实现临床生物样品中儿茶酚胺及其代谢产物的高效、痕量分析，为嗜铬细胞瘤、副神经节瘤和成神经细胞瘤等疾病的诊断及防治提供科学依据。液相微萃取方法能够有效地减少试剂的使用、缩短萃取时间，而且在萃取目标分析物的同时消除基质干扰，从而大大提高了萃取的效率，特别适用于复杂生物样品前处理。色谱-质谱联用也为儿茶酚胺神经递质的分离分析提供了有效的手段。本研究的顺利实施将对生物样品中儿茶酚胺神经递质的萃取富集标准方法、测定方法及临床检验等都具有重要的理论意义和应用价值。

由于对方法可靠性、灵敏度、分析速度和样品通量方面的要求非常苛刻，环境和生物样品分析方法的开发在过去几年中变得越来越具有挑战性。本项目重点研究了各种液相微萃取方法，包括盐析辅助液液萃取（Salting-out assisted liquid-liquid extraction, SALLE）、分散液液微萃取（Dispersive liquid-liquid microextraction, DLLME）、浊点萃取（Cloud point extraction, CPE）及微萃取方法联用来萃取和富集多介质环境样品中的污染物（如空气、水、土壤、人尿和血液），并通过液相色谱—质谱（Liquid chromatography-mass spectrometry, LC-MS）和气相色谱—质谱（Gas chromatography-mass spectrometry, GC-MS）测定污染物。该项目综述了各种生物样品的样品前处理方法及环境和生物样品的固相萃取方法。同时，该项目成功建立了SALLE-紫外，SALLE-DLLME-GC-MS，SALLE-GC-MS和CPE-HPLC方法用于测定尿液、血液、空气、土壤和环境水样中的己烯雌酚、2，4-D、拟除虫菊酯类杀虫剂和磺胺类抗菌药物。在最佳萃取及测定条件下，方法具有低的检测限（ng/mL）、高的回收率（70%-120%）和宽的线性范围（2-3个数量级），而且方法简单、快速、灵敏。项目结果表明微萃取-色谱联用方法可以成功用于多介质环境样品中各种环境污染物的测定。该项目将为环境污染物的生物和环境样品前处理方法、测定方法和临床实验室的标准方法带来重要的理论和应用价值。