【作者】 卢巧梅；

【导师】 张兰；

【作者基本信息】 福州大学 ， 药物分析学， 2011， 博士

【摘要】 植物激素（plant hormone, PH）是植株体内合成的一系列小分子有机化合物，几乎能调控植物生长周期的每个生理过程。对PHs含量的准确测定，有助于探究各类激素间的相互关系、认识激素调控植株生长发育及其对环境适应的机制，这对于植物生理、品质调控以及遗传育种等方面的研究具有重要的指导意义。当前，色谱技术的快速发展使其在PHs分析中的应用越来越广泛。鉴于内源激素在植株内含量甚微（通常在ng/g鲜重，甚至pg/g水平上）且干扰物多，因此，寻求廉价、简便、高效的样品前处理新方法与色谱技术联用，以期实现激素的超微定量检测有着很好的应用前景。本论文主要采用多种新型的样品前处理技术对一些影响植物生长的PHs进行纯化和富集，并结合色谱以及质谱分析方法实现了PHs高灵敏度、高通量的检测。论文分为三个部分，共八章。第一部分，即论文的第一章，介绍了PHs的概念和分类，并对其主要样品前处理方法和检测技术进行综述，特别介绍了新型样品预处理技术在激素检测中的最新研究进展，最后概括了本课题的主要研究工作。第二部分，包括论文的第二、三、四章，主要研究了传统样品前处理方法结合液相色谱-质谱技术在PHs分析中的应用。在第二章中，以C18填充柱为分离通道，采用固相萃取-加压毛细管电色谱-紫外检测技术（solid phase extraction-pressurized capillary electrochromatography-ultravioletdetection, SPE-pCEC-UV）对5种内源和外源PHs进行定性和定量分析。在最优实验条件下，方法的线性范围为0.8-100μg/mL，检测限介于0.2-0.8μg/mL之间，平均回收率高于81.0%，并将建立的方法用于拟南芥样品的分析。将pCEC与微柱液相色谱法比较，发现pCEC的分析速度更快、柱效更高。在第三章中，采用SPE前处理技术，结合液相色谱-离子阱质谱法（liquidchromatography-ion trap mass spectrometry, LC-ITMS）实现了三种激素的同时分离分析。为研究内源激素在植物遭遇逆境环境时的含量变化和相互作用，以水仙为实际材料进行盐胁迫试验，分析了三种激素的含量变化趋势。结果显示，随着NaCl浓度的增加和胁迫处理时间的延长，吲哚乙酸和赤霉酸含量减少，而脱落酸（abscisic acid,ABA）的含量明显增加。本章重点探讨了ABA在逆境应答中的作用。论文的第四章，发展了LC-ITMS/MS法用于生长素（Auxin,Aux）的快速分离和高灵敏检测。在优化的实验条件下，4种目标物在7min内完全分离，最低检测限为8.0ng/mL。选择液液萃取（liquid-liquid extraction, LLE）法处理样品。经多步的旋转蒸发和溶剂分配，尽管未能在白菜中检测到内源激素，但方法的加标回收率在77.5-99.8%范围内，结果相对满意。第三部分，即论文的第五、六、七、八章，主要是对新型样品预处理技术在PHs分析中的应用进行研究。重点试验了分散液液微萃取（dispersive liquid-liquidmicroextraction, DLLME）、悬浮固化微萃取（solidification of floating organic dropmicroextraction, SFODME）以及基于悬浮固化的分散液液微萃取法（dispersiveliquid-liquid microextraction based on the solidification of floating organic dropmicroextraction, DLLME-SFO）这三种新技术，为激素的痕量分析提供了新方法和新思路。在第五章中，为克服传统的SPE和LLE存在处理时间长、有机溶剂消耗大等缺点，首次将DLLME方法引入植物样品的预处理中，结合荧光检测器（fluorescencedetector, FLD）强选择性和高灵敏度的特点，建立了4种常见Aux的DLLME-HPLC-FLD分析方法。此法具有富集倍数高（约50倍）、重现性好（相对标准偏差<5.64%）、操作简便（不需辅助设备）、环境友好（仅消耗微升级有机溶剂）及处理时间短（萃取时间<1min）等优点，因而具有广泛的应用性。论文的第六章，为简化植物激素的预处理过程，选择30.0μL氯仿和800μL四氢呋喃的预混溶液，注入5.0mL样品溶液（pH3.0）中，经过相分离，吸取20μL下层有机相。经过DLLME法净化和富集两种激素后，进行LC-ITMS测定。以“越南宁平”桃为试材，揭示了不同生长阶段的桃子果实中两种激素的含量变化，对外源施用类似激素具有指导作用。论文的第七章，使用低毒有机溶剂代替DLLME方法中常用的高毒性卤代烃，将SFODME新技术应用于植物激素预处理中。首次建立了SFODME-HPLC-UV同时检测两类激素的新方法。该技术操作重现性好、浓缩倍数高达130-200。方法的线性范围超过两个数量级，相关系数在0.9985-0.9994范围内，检测限达到0.5ng/mL，并成功用于实际样品的检测。第八章中，将高效DLLME和低毒SFODME方法相结合，发展了DLLME-SFO法。以两种Aux为研究对象，考察了DLLME-SFO萃取技术的影响因素和富集效果。并对三种前处理方式（即DLLME、SFODME、DLLME-SFO）从重现性和富集效果等方面进行了全部的比较。更多还原

【Abstract】 Plant hormones (PHs), a series of low-molecular-weight organic compoundssynthesized in plants, can control almost every physiological process during the plant’s lifecycle. Accurate quantitation of PHs will be favorable for the research on the interactionamong different hormones, the mechanism of PHs in plant’s development and adaption toenvironment. These researches have practical guidance significance to physiologicaltechniques, quality control and genetic breeding in plants.Recently, with the rapid development of chromatography technology, its wideapplication in PHs analysis is involved. Due to very low amounts of PHs (usually ng/gfresh weight, and even to pg/g) and many interfering substances coexisting in plants,therefore, searching for new pretreatment techniques with low cost, simplicity andeffectiveness is of vital importance and has extensive application prospect for tracequantitation of related hormones. In this thesis, some new sample pretreatment methodscoupling with chromatography/mass spectrometry were adopted to purify and enrichmultiple PHs affecting plants growth, then sensitive and high-throughput determination ofPHs were established and realized.This thesis consists of three parts, including eight chapters.The first part is chapter1. In this chapter, the general definition and classifications ofPHs were introduced, respectively. Pretreatment procedures and main detecting techniquesfor hormones analysis were reviewed, especially the latest research progress on applicationof novel sample pretreatment assays. Lastly, main research works of this thesis were alsodescribed.The second part includes chapters2-4. In this part, the applications of traditionalsample pretreatment methods combined with liquid chromatography-mass spectrometrytechnology in analyzing PHs were investigated.In chapter2, solid phase extraction-pressurized capillary electrochromatography-ultraviolet detection (SPE-pCEC-UV) was developed for qualitative and quantitative analysis of five kinds of endogenetic and ectogenic PHs with a packed C18capillarycolumn as the separation channel. Under the optimum conditions, this assay presentedgood linearity (0.8-100μg/mL), with LOD of0.2-0.8μg/mL and mean recoveries higherthan81.0%. The pCEC method was also applied to analysis of Arabidopsis thalianasample. Comparied with microcolumn liquid chromatography, it was found that pCEC hadfaster analyses speed and higher column efficiency.In chapter3, a quantitative method consisting of SPE followed by liquidchromatography-ion trap mass spectrometry (LC-ITMS) was developed for simultaneousseparation of three endogenous hormones. Narcissus was selected as real sample to studythe content variation and interaction of above hormones under salty stress. Researchshowed that with the increase of NaCl concentration and processing time, level ofindole-3-acetic acid and gibberellic acid diminished to some extent, while abscissic acid(ABA) upgraded clearly. ABA was considered to play a central part in stress response andits role was emphasized in this part.In chapter4, fast separation and sensitive detection of auxins (Aux) by LC-ITMS wasperformed. Under optimum experimental conditions, four target compounds werecompetely separated in7min with a minimum detection limit of8.0ng/mL. Liquid-liquidextraction (LLE) was involved in this research for sample preparation. Endogenichormones could not be detected in Chinese cabbage after multiple vacuum concentrationsand solvent distribution, however, the recoveries of spiked samples ranged from77.5%to99.8%, thus relatively satisfactory results were obtained.Chapters5-8consist of the third part of this thesis. This part focused on theapplication of new sample pretreatment methods for PHs investigation. Three types ofassays, namely dispersive liquid-liquid microextraction (DLLME), solidification offloating organic drop microextraction (SFODME), and dispersive liquid-liquidmicroextraction based on the solidification of floating organic drop microextraction(DLLME-SFO) were studied in detail. These researches would provide new approach andstrategy for PHs analyses.In chapter5, traditional LLE and SPE processes are usually time-consuming withconsiderable organic solvent consumption. To overcome these weaknesses, DLLME wasintroduced into the pretreatment of plant matrix for the first time. Combined with the highselectivity and sensitivity of fluorescence detector (FLD), a DLLME-HPLC-FLD methodfor detection of four common Aux was proposed. DLLME had several advantages such aspowerful preconcentration (about50-fold enrichment), good reproducibility (relative standard deviation <5.64%), simple operation (no auxiliary equipments), environmentalfriendly (only a few micro liters of organic solvent), and quick analysis (extraction time <1min), so wide application of this method was obtained.In chapter6, to simplify PHs pretreatment, we still adopted DLLME to extract andenrich two hormones.30μL CHCl3and800μL THF were mixed and injected into5.0mLsample solution (pH3.0). After phase separation, the sedimented organic phase (about20μL) was directly withdrawn for HPLC-ITMS determination. Peach (from Ninh Binh,Vietnam) was used as real sample, and the content changes of two hormones indevelopment of peach were revealed, which would provide a guide for exogenousapplication of similar hormone.In chapter7, highly toxic halogenated hydrocarbons were frequently used asextraction solvents in DLLME procedure. To solve this problem, organic solvents with lowtoxicity were expected to adopt, therefore, SFODME technique was used to plant hormoneanalysis. SFODME-HPLC-UV method was firstly established for simultaneous analysis oftwo types of hormones with good repeatability and high enrichment factor (about130-200).Linear ranges of this method were over two orders of magnitude, with correlationcoefficient in range of0.9985-0.9994and detection limit of0.5ng/mL, respectively.Finally, this method was applied to analysis of real samples successfully.In chapter8, dispersive liquid-liquid microextraction based on the solidification offloating organic drop (DLLME-SFO) combines the advantages of DLLME (shortextraction time) and SFODME (no comsumption of highly toxic solvent). Two auxins wereselected as research abjects; extraction factors and enrichment influence for DLLME-SFOsystem were optimized. Lastly, repeatability and enrichment results of three pretreatmentmethods (DLLME-SFO, DLLME and SFODME) were evaluated and compared,respectively.更多还原