复杂基质样品的电喷雾萃取电离质谱分析理论与应用研究

英文题名：Study on the Theory and Application of Extractive Electrospray Ionization Mass Spectrometry for the Analysis of Samples with Complex Matrix
作者：丁健桦
论文级别：博士
学科专业名称：化学
中文关键词：复杂基质样品 ; 电喷雾萃取电离质谱(EESI-MS) ; 中性解吸(ND) ; 装置 ; 机理 ; 方法 ; 应用
英文关键词：samples with complex matrix ; extractive electrospray ionization mass spectrometry (EESI-MS) ; neutral desorption (ND) ; device ; mechanism ; method ; application
学位年度：2011
导师：任玉林 ; 陈焕文
学科代码：070302
学位授予单位：吉林大学
论文提交日期：2011-12-01

摘要

质谱(MS)是目前发展非常迅速的一种不可替代的分析工具。而现代质谱分析是一类高灵敏度、高特异性、高通量的普适性快速分析方法,它以原位、实时、在线、非破坏、高通量、低耗损为特征,是质谱发展的一个重要趋势,也是近十年来国内外质谱界,乃至整个分析化学领域的研究前沿和热点。
     现代质谱研究主要包括基于各种新型离子源的理论、装置、技术与方法的研究,而新型离子源的研制与开发对于推动质谱技术的发展具有特别重要的意义。使用常规离子源如电子轰击电离(EI)源、化学电离(CI)源、快原子轰击(FAB)源、基质辅助激光解吸电离(MALDI)源、电喷雾电离(ESI)源等的质谱,在对复杂基质样品进行分析之前必须进行样品的预处理,因而无法满足对样品进行原位、实时、在线、非破坏、高通量、低耗损分析的实际需要。2004年Purdue大学的Cooks教授等发明了电喷雾解吸电离(DESI)源,使无须样品预处理的直接质谱分析成为了可能。之后,国内外掀起了新型离子化技术的研究热潮,几十种各具特色的新型离子源得以研制和开发,使复杂基质样品的质谱分析向实时、在线、原位、活体、无损分析方向快速发展。
     电喷雾萃取电离(EESI)技术是2006年发明的一种新型离子化技术。EESI-MS能对液体、气体、气溶胶、悬浊液、固体表面及粉末、胶体等各种形态的复杂基质样品进行无需样品预处理的快速直接分析,首次为实现原位、活体、实时、在线的质谱分析提供了可能。目前,EESI已经逐步应用于食品和生活用品质量监测、药物检测、环境监测、过程分析、毒品和爆炸物检测、生物大分子分析、活体分析、代谢组学等研究领域。本文则对EESI的装置和电离机理进行了研究和探讨,并从复杂基质样品的形态出发,建立了液态、气态和粘性样品的EESI-MS快速直接分析方法,综述了固态表面样品的EESI-MS直接分析的研究进展,为进一步加深人们对EESI-MS的认识,以及继续加强EESI-MS相关研究,拓展其应用范围打下了基础。本论文的主要研究内容为:
     1)进行了EESI的机理研究。EESI是复杂基质样品分析的理想电离方法。为了提高对EESI过程的可控性以及保证EESI分析结果的可靠性,本文对EESI喷雾进行了荧光成像研究,并对不同电喷雾溶剂和样品溶剂体系下待测物的信号变化进行了研究,结果表明,EESI过程是发生在液相中而不是气相中的,且电喷雾雾滴和中性样品雾滴之间发生的是选择性液-液萃取而不是液滴融合。
     2) EESI-MS在复杂基质液态样品直接分析中的应用。采用自制的EESI源,优化了EESI的工作条件,建立了复杂基质液态样品的EESI-MS分析方法,并将其用于牙膏提取液中二甘醇(DEG)和土壤溶液中芹菜素的检测:对牙膏提取液中的DEG进行了定量检测,发现检测的线性范围宽(可达4个数量级),线性关系良好(R2=0.997),灵敏度高(检测限可达0.1μg/L),重现性好(RSD<4.4%);对芹菜素进行了多级串联质谱分析,根据所得到的特征碎片离子,探讨了芹菜素在EESI-MS中的裂解规律和裂解的可能机理,在此基础上,对京大戟根际土壤溶液进行了分析,确定了其中含有芹菜素。由此可见,EESI-MS法可以在无需样品预处理的情况下对复杂基质液态样品进行直接、快速和灵敏的定性和定量分析。
     3) EESI-MS在复杂基质呼吸气体样品直接分析中的应用。采用自制的EESI源,构建了基于活体呼吸气体分析的新型EESI串联质谱平台,并以正辛胺作为参考物质,对该平台工作条件进行了系统优化。在优化的条件下,人体的代谢变化可通过呼出气体中代谢产物的活体指纹谱图迅速得到反映,证明了呼出气体可作为代谢组学研究样品的观点;分别对呼吸气体中尼古丁和乙腈等进行活体检测则发现:对呼吸气体中尼古丁进行定量检测时线性范围可达5个数量级,LOD可达0.05 fg/mL(S/N=3),RSD<5.0%(n=12),同时还获得了呼吸气体中尼古丁及其代谢物可替宁的动态代谢曲线;通过在EESI中引入离子/分子反应而建立的新型R-EESI-MS可对呼吸气体中乙腈进行活体定性和定量分析,在此基础上还对主动吸烟者和被动吸烟者呼吸气体中乙腈的浓度和降解情况进行了研究,研究结果将有助于呼吸气体中乙腈的产生机理研究。可见,EESI-MS是一种可用于呼吸气体活体代谢组学研究的切实方便的分析工具。
     4) EESI-MS在复杂基质粘稠样品直接分析中的应用。采用自制的ND装置和EESI源,优化了ND和EESI的技术参数,建立了复杂基质粘性样品的无需任何样品预处理的ND-EESI-MS分析方法,并将之用于粘稠的牙膏和痰液的直接快速分析:对牙膏中的DEG进行了直接检测,发现ND技术可将混合在凝胶型混合物(如牙膏)中的非挥发性化合物(如DEG)释放出来,然后进入EESI-MS进行检测,且检测的速度快,灵敏度高、准确度高、重现性好;对非小细胞肺癌(NSCLC)患者和非肺癌患者的痰液进行了直接分析,获得了NSCLC患者与非肺癌患者的质谱指纹图谱,结合主成分分析(PCA)模式识别方法,能建立NSCLC患者与非肺癌患者的识别模式,为NSCLC的快速诊断、早期筛查和临床肿瘤复发的监测提供一个新思路和新方法。由此可见,ND-EESI-MS是快速分析和表征高粘性复杂基质样品的有利工具。
     5)对固体表面样品EESI-MS分析研究进展进行了综述。将中性解吸(ND)装置耦合到EESI-MS中建立的ND-EESI-MS方法,不但可用于各种常规固体表面样品的直接分析,还可用于生物表面复杂样品的活体分析以及极端条件下(如,高温、低温、生物或放射性危害等)固体表面样品的远程分析。本文根据ND装置的发展和改进,对敞开式ND-EESI、密闭式ND-EESI、独立结构式ND-EESI和探针式ND-EESI在各种固体表面样品直接分析中的应用进行了综述,并对ND-EESI-MS的应用前景进行了展望。
     通过对液态、气态(呼吸气体)、粘性和固体表面等不同形态样品的分析研究表明,EESI-MS对样品无毒害、无污染,适用于对各种形态的复杂基质样品进行无需样品预处理的原位、活体、实时、在线和远程、无损、直接分析,可对样品中的极性和非极性物质进行检测,是一种具有广阔应用前景的现代质谱方法。
Mass spectrometry (MS) is currently a irreplaceable tool of analysis which develops very rapidly. Modern mass spectrometry is a type of fast analytical method with high sensitivity, high specific, high throughput and universal applicability. It has the features of in-situ, real-time, online, non-destructive, high throhghput and low loss, and is the important trend for the development of mass spectrometry. It is also the research frontier and hot spot in the area of mass spectrometry both at home and abroad, and even in the whole field of analytical chemistry.
     The research of modern mass spectrometry mainly involves the theories, equipments, technologies and methods on developing the new ion sources, which is especially important and significant for driving the development of mass spectrometry. Sample pretreatment must be undertaken before the analysis of samples with complex matrix using the conventional ion source such as electronic bombardment ionization (EI) source, chemical ionization (CI) source, fast Atomic bombardment (FAB) source, matrix secondary laser solutions suction ionization (MALDI) source, and electric spray ionization (ESI) source, which cannot meet the actual needs of sample analysis on in-situ, real-time, online, non-damage, high throughput, and low loss. After desorption electrospray ionization (DESI) source was invented by Professor Cooks of Purdue University in 2004, direct mass spectrometric analysis without sample pretreatment was made possible. Then, the research of new ionization technique boomed all over the world, and dozens of new ion sources were invented, which makes MS analysis of samples with complex matrix develop rapidly in the direction of real-time, online, in-situ, in vivo, non-destructive analysis.
     Extractive electrospray ionization (EESI) is a novel ionization technology appearing in 2006 which makes in-situ, in vivo, real-time, and online analysis possible for the first time. Samples with complex matrix in various forms such as liquids, gases, aerosols, suspension liquid, solid surface, powder, colloids and other forms can be rapidly and directly analyzed without sample pretreatment using EESI-MS. Currently, EESI has been gradually applied to many research fields such as foods and supplies quality monitoring, drug detection, environmental quality monitoring, process analysis, dopes and explosives detection, biological macromolecular analysis, in vivo analysis, and metabolomics. In this thesis, we have studied the devices and mechanism of ionization involved in EESI, and the methods of EESI-MS for direct analysis of liquid, breath and viscous samples with complex matrix. The development of direct analysis of solid surface using EESI-MS has also been reviewed. These study laid a foundation for further deepening the understanding of EESI-MS, continuing to strengthen the research of EESI-MS, and expanding its application scope.
     The main research content of this dissertation involves as following:
     1) The mechanism of EESI. EESI is an ideal ionization method for the analysis of samples with complex matrix. In order to improve the controllability of the process and ensure the reliability of the results of EESI analysis, we have studied the fluorescence imaging of EESI spray, and the effect of different solvent systems of electrospray solvents and sample solvents. The results showed that, the process of EESI occurs in the liquid phase rather than the gas phase, and there is selective liquid-liquid extraction between the electrospray droplet and the neutral sample droplet.
     2) The application of EESI-MS for the analysis of liquid samples with complex matrix. Using the home-made EESI source and optimizing EESI working conditions, the EESI-MS method for analysis of liquid samples with complex matrix was established and applied to the detection of diethylene glycol (DEG) in toothpaste extraction and apigenin in soil solution. For the quantitative detection of DEG in toothpaste extraction, the method has a wide linear range (up to four orders of magnitude), a good linear relationship (R2 = 0.997), high sensitivity (detection limit is 0.1μg/L) and a good reproducibility (RSD<4.4%); and for the analysis of apigenin through multi-stage tandem mass spectrometry, the character and mechanism of the dissociation of apigenin in EESI-MS were discussed according to the various characteristic fragments formed. Then, on the basis of the EESI-MSn of apigenin, the rhizosphere soil solution of Beijing Euphorbia was analyzed and apigenin was found in it. Thus, EESI-MS can be successfully applied in the direct, rapid and sensitive qualitative and quantitative analysis of liquid samples with complex matrix without sample pretreatment.
     3) The application of EESI-MS for the analysis of breath samples with complex matrix. Using the home-made EESI source, the EESI tandem mass spectrometry platform for in vivo breath analysis has been built, and was optimized systematically using octyl amine. Under the optimized conditions, the body's metabolic changes can be observed quickly through the in vivo fingerprint spectra of the breath, and it showed that breath can be used as samples of metabolomics. According to the results of in vivo breath analysis, for nicotine in breath, the linear quantitative detection range was up to five orders of magnitude, LOD was limited to 0.05 fg/mL, RSD<5.0%, and the dynamic metabolic curve of nicotine and its metabolite cotinine in breath can also be obtained; for acetonitrile in breath, through ion / molecule reactions, a new reactive EESI-MS was established for in vivo qualitative and quantitative analysis of acetonitrile, and in order to reveal the generation mechanism of acetonitrile in breath, the concentration and degradation of acetonitrile in breath of active smokers and passive smokers were also studied. Thus, EESI-MS is a practical and convenient tool for in vivo metabolomics study based on breath analysis.
     4) The application of EESI-MS for the analysis of viscous samples with complex matrix. Using the home-made device of ND and EESI source, under the optimized ND and EESI working conditions, the ND-EESI-MS method for analysis of viscous samples with complex matrix was established, and then it was used in the analysis of viscous toothpaste and Sputum. It showed that non-volatile compounds (such as DEG) can be released from the gel mixture (such as toothpaste) and then be detected by EESI-MS with high speed, high sensitivity, high accuracy and good reproducibility. Through the direct analysis of the sputum of non-small cell lung cancer (NSCLC) patients and non-lung cancer patients, their mass fingerprints were obtained. Combined with principal component analysis (PCA), a method of pattern recognition, the recognition model which used to distinguish NSCLC patients and non-lung cancer patients has been established. It provides an opportunity for the rapid diagnosis and early screening of NSCLC, and clinical monitoring of tumor recurrence. Thus, these results prove that ND-EESI-MS is a useful tool for rapid analysis and characterizing of high viscosity samples with complex matrix.
     5) The development of solid surface analysis using EESI-MS. Neutral desorption extractive electrospray ionization mass spectrometry (ND-EESI-MS) was established by coupling ND device to the EESI-MS. ND-EESI-MS can be used not only for the direct analysis of a variety solid surface samples, but also for in vivo analysis of biological surface samples and remote analysis of solid surface samples under extreme conditions (For example, high temperature, low temperature, biological or radiological hazards, etc.). In this thesis, according to the development and improvement of the ND device, we have reviewed the applications of open-air ND-EESI, air-tight ND-EESI, geometry-independent ND-EESI and probe type ND-EESI in direct analysis of a variety of solid surface samples, and looked forward to the application prospects of ND-EESI-MS.
     Through the research above, we know that EESI-MS is not poisonous and polluted to samples, suitable for in-situ, in vivo, real-time, online and remote, non-destructive, direct analysis of various forms of samples with complex matrix without sample preparation, thus can be used to detect both polar and non-polar compounds in samples. So EESI-MS has broad application prospects in modern mass spectrometry.

引文

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