Study On The Novol Sample Pretreatment Methods Coupling With Chromatographic Separation And Analysis Of Plant Hormones

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.